AKTApure

ÄKTA™ pure
User Manual
Table of Contents
Table of Contents
1 Introduction ...................................................................................................................................................... 6
1.1 Important user information ............................................................................................................................................ 7
1.2 ÄKTA pure overview ........................................................................................................................................................... 9
1.3 ÄKTA pure user documentation .................................................................................................................................... 11
2 The ÄKTA pure instrument ............................................................................................................................. 13

2.1 Overview illustrations ........................................................................................................................................................ 14
2.2 Liquid flow path ................................................................................................................................................................... 25
2.3 Instrument control panel ................................................................................................................................................. 27
2.4 Instrument modules ........................................................................................................................................................... 32
2.4.1 System pumps ...................................................................................................................................................................................................... 33
2.4.2 Mixer ......................................................................................................................................................................................................................... 37
2.4.3 Valves, overview .................................................................................................................................................................................................. 38
2.4.4 Inlet valves ............................................................................................................................................................................................................. 39
2.4.5 Mixer valve ............................................................................................................................................................................................................. 46
2.4.6 Injection valve ....................................................................................................................................................................................................... 49
2.4.7 Loop valve .............................................................................................................................................................................................................. 52
2.4.8 Column valves ...................................................................................................................................................................................................... 55
2.4.9 Versatile valve ....................................................................................................................................................................................................... 59
2.4.10 pH valve ................................................................................................................................................................................................................... 60
2.4.11 Outlet valves .......................................................................................................................................................................................................... 64
2.4.12 Pressure monitors ............................................................................................................................................................................................... 67
2.4.13 UV monitors ........................................................................................................................................................................................................... 69
2.4.14 Conductivity monitor ......................................................................................................................................................................................... 72
2.4.15 Flow restrictor ....................................................................................................................................................................................................... 74
2.5 Installation of internal modules .................................................................................................................................... 76
2.6 Accessories ............................................................................................................................................................................ 79
3 ÄKTA pure external modules .......................................................................................................................... 86

3.1 External air sensors ............................................................................................................................................................ 87
3.2 Fraction collector F9-C ..................................................................................................................................................... 89
3.2.1 Function .................................................................................................................................................................................................................. 90
3.2.2 Fraction collector F9-C illustrations ............................................................................................................................................................ 92
3.2.3 Cassettes, Cassette tray and racks ............................................................................................................................................................. 96
3.2.4 Connect tubing to the ÄKTA pure instrument ......................................................................................................................................... 102
3.3 Fraction collector F9-R .................................................................................................................................................... 103
3.4 Sample pump S9 and S9H .............................................................................................................................................. 107
3.5 I/O-box E9 ............................................................................................................................................................................... 111
3.5.1 Overview of the I/O-box ................................................................................................................................................................................... 112
3.5.2 Analog connector and signals ....................................................................................................................................................................... 114
3.5.3 Digital connector and signals ........................................................................................................................................................................ 116
3.5.4 Connect external equipment to the I/O-box ........................................................................................................................................... 118
3.6 Connection of external modules .................................................................................................................................. 121
4 System configuration ...................................................................................................................................... 123

4.1 Configuration overview .................................................................................................................................................... 124
4.2 Configure modules ............................................................................................................................................................. 130
4.2.1 Configuration of inlet valves .......................................................................................................................................................................... 131
4.2.2 Configuration of Mixer valves ........................................................................................................................................................................ 132
4.2.3 Configuration of Loop valves ......................................................................................................................................................................... 133
4.2.4 Configuration of column valves .................................................................................................................................................................... 134
2 ÄKTA pure User Manual 29119969 AC

Table of Contents
4.2.5 Configuration of Versatile valves .................................................................................................................................................................. 136

4.2.6 Configuration of pH valves ............................................................................................................................................................................. 137
4.2.7 Configuration of outlet valves ....................................................................................................................................................................... 138
4.2.8 Configuration of UV monitors ........................................................................................................................................................................ 139
4.2.9 Configuration of Conductivity monitor ...................................................................................................................................................... 140
4.2.10 Configuration of external air sensors ......................................................................................................................................................... 141
4.2.11 Configuration of fraction collectors ............................................................................................................................................................ 142
4.2.12 Configuration of I/O-box .................................................................................................................................................................................. 144
4.3 General system settings .................................................................................................................................................. 145
5 Operation .......................................................................................................................................................... 148

5.1 Before you prepare the system .................................................................................................................................... 149
5.2 Prepare the flow path ........................................................................................................................................................ 150
5.3 Start UNICORN and connect to system .................................................................................................................... 157
5.4 Prime inlets and purge pump heads .......................................................................................................................... 160
5.4.1 System pumps ...................................................................................................................................................................................................... 161
5.4.2 Sample pump ....................................................................................................................................................................................................... 167
5.5 Connect a column ............................................................................................................................................................... 172
5.6 Pressure alarms ................................................................................................................................................................... 176
5.7 Sample application ............................................................................................................................................................. 179
5.7.1 Sample application using direct injection onto the column ............................................................................................................. 180
5.7.2 Sample application using a Superloop™ ................................................................................................................................................... 182
5.7.3 Sample application using a sample loop ................................................................................................................................................. 189
5.8 Fractionation ......................................................................................................................................................................... 192
5.8.1 Prepare Fraction collector F9-C .................................................................................................................................................................... 193
5.8.2 Prepare Fraction collector F9-R .................................................................................................................................................................... 199
5.8.3 Fractionation overview ..................................................................................................................................................................................... 206
5.9 Create a method and perform a run ......................................................................................................................... 210
5.9.1 Create a method .................................................................................................................................................................................................. 211
5.9.2 Prepare and perform a run ............................................................................................................................................................................. 216
5.9.3 Monitor a run ........................................................................................................................................................................................................ 218
5.9.4 After run procedures ......................................................................................................................................................................................... 220
6 Performance tests ........................................................................................................................................... 224

6.1 General performance test actions .............................................................................................................................. 225
6.2 Air sensor A and Inlet valve A tests ............................................................................................................................. 228
6.3 Air sensor B and Inlet valve B tests ............................................................................................................................. 230
6.4 Air sensor S and Sample inlet valve tests ................................................................................................................ 232
6.5 Column valve C tests ......................................................................................................................................................... 234
6.6 Fraction collector F9-C test ............................................................................................................................................ 236
6.7 Fraction Collector F9-R Test ........................................................................................................................................... 239
6.8 Sample pump tests ............................................................................................................................................................ 241
6.9 System Test UV U9-L (fixed) ............................................................................................................................................ 243
6.10 System Test UV U9-M (variable) .................................................................................................................................... 247
7 Maintenance ..................................................................................................................................................... 251

7.1 Maintenance Manager ..................................................................................................................................................... 252
7.2 Maintenance program ...................................................................................................................................................... 258
7.3 Weekly maintenance ......................................................................................................................................................... 261
7.3.1 Change pump rinsing solution ...................................................................................................................................................................... 262
7.3.2 Replace the inline filter ..................................................................................................................................................................................... 266
7.3.3 Clean the Fraction collector F9-C sensors ................................................................................................................................................ 267
7.4 Monthly maintenance ....................................................................................................................................................... 269
ÄKTA pure User Manual 29119969 AC 3

Table of Contents
7.5 Semiannual maintenance ............................................................................................................................................... 272

7.5.1 Clean the UV flow cell ........................................................................................................................................................................................ 273
7.5.2 Replace the pH electrode ................................................................................................................................................................................. 275
7.6 Maintenance when required .......................................................................................................................................... 276
7.6.1 Clean the instrument externally ................................................................................................................................................................... 277
7.6.2 Perform System CIP ........................................................................................................................................................................................... 278
7.6.3 Perform Column CIP .......................................................................................................................................................................................... 284
7.6.4 Clean Fraction collector F9-C ......................................................................................................................................................................... 288
7.6.5 Clean Fraction collector F9-R ......................................................................................................................................................................... 290
7.6.6 Storage of the pH electrode ........................................................................................................................................................................... 291
7.6.7 Clean the pH electrode ..................................................................................................................................................................................... 293
7.6.8 Clean the pump head check valves ............................................................................................................................................................ 296
7.6.9 Wipe off excess oil from the pump head .................................................................................................................................................. 299
7.6.10 Clean the Conductivity flow cell .................................................................................................................................................................... 300
7.7 Calibration procedures ..................................................................................................................................................... 302
7.7.1 Calibrate the pH monitor ................................................................................................................................................................................. 303
7.7.2 Calibrate the pressure monitors ................................................................................................................................................................... 305
7.7.3 Calibrate the Conductivity monitor ............................................................................................................................................................. 308
7.7.4 Calibrate the UV monitors ............................................................................................................................................................................... 312
7.8 Replacement procedures ................................................................................................................................................ 316
7.8.1 Replace tubing and connectors .................................................................................................................................................................... 317
7.8.2 Replace internal tubing in Fraction collector F9-C ............................................................................................................................... 319
7.8.3 Replace the Mixer ................................................................................................................................................................................................ 322
7.8.4 Replace the O-ring inside the Mixer ............................................................................................................................................................ 323
7.8.5 Replace the UV monitor U9-M flow cell ..................................................................................................................................................... 325
7.8.6 Replace the UV monitor U9-L flow cell ...................................................................................................................................................... 328
7.8.7 Replace Flow restrictor ..................................................................................................................................................................................... 330
7.8.8 Replace the inlet filters ..................................................................................................................................................................................... 331
7.8.9 Replace the pump head check valves ........................................................................................................................................................ 332
7.8.10 Replace pump piston seals of Pump P9 or P9H .................................................................................................................................... 335
7.8.11 Replace pump piston seals of Pump P9-S ............................................................................................................................................... 344
7.8.12 Replace pump pistons ...................................................................................................................................................................................... 351
7.8.13 Replace pump rinsing system tubing ......................................................................................................................................................... 352
8 Troubleshooting ............................................................................................................................................... 355

8.1 Introduction to troubleshooting ................................................................................................................................... 356
8.2 Troubleshooting: General Checklist ............................................................................................................................ 358
8.3 Troubleshooting: Monitors .............................................................................................................................................. 360
8.4 Troubleshooting: Valves ................................................................................................................................................... 375
8.5 Troubleshooting: Fraction collector ............................................................................................................................ 377
8.6 Troubleshooting: Pumps .................................................................................................................................................. 387
8.7 Troubleshooting: Other components ......................................................................................................................... 394
8.8 Troubleshooting: Instrument communication ....................................................................................................... 401
8.9 Troubleshooting: Method development ................................................................................................................... 403
8.10 Error codes ............................................................................................................................................................................. 404
9 Reference information .................................................................................................................................... 410

9.1 System specifications ........................................................................................................................................................ 411
9.2 Module specifications ....................................................................................................................................................... 413
9.3 Tubing and connectors ..................................................................................................................................................... 420
9.4 Recommended tubing kits for prepacked columns ............................................................................................ 428
9.5 Chemical resistance guide .............................................................................................................................................. 431
9.5.1 General information about biocompatibility and chemical resistance ....................................................................................... 432
9.5.2 Chemical resistance specifications ............................................................................................................................................................. 433
9.6 Wetted materials ................................................................................................................................................................. 436

Table of Contents
9.7 Predefined methods and phases ................................................................................................................................. 438

9.7.1 Predefined purification methods .................................................................................................................................................................. 439
9.7.2 Predefined maintenance methods .............................................................................................................................................................. 445
9.7.3 Predefined phases .............................................................................................................................................................................................. 446
9.8 System settings .................................................................................................................................................................... 448
9.8.1 System settings - UV .......................................................................................................................................................................................... 449
9.8.2 System settings - Conductivity ...................................................................................................................................................................... 450
9.8.3 System settings - pH .......................................................................................................................................................................................... 451
9.8.4 System settings - Pressure alarms .............................................................................................................................................................. 452
9.8.5 System settings - Air sensor ........................................................................................................................................................................... 454
9.8.6 System settings - I/O-box ................................................................................................................................................................................ 456
9.8.7 System settings - Fraction collection .......................................................................................................................................................... 457
9.8.8 System settings - Tubing and Delay volumes ......................................................................................................................................... 459
9.8.9 System settings - Wash settings .................................................................................................................................................................. 461
9.8.10 System settings - Watch parameters ........................................................................................................................................................ 462
9.8.11 System settings - Advanced ........................................................................................................................................................................... 463
9.8.12 System settings - Data collection ................................................................................................................................................................. 464
9.9 Manual instructions ........................................................................................................................................................... 465
9.9.1 Manual instructions - Pumps ......................................................................................................................................................................... 466
9.9.2 Manual instructions - Flow path .................................................................................................................................................................. 470
9.9.3 Manual instructions - Monitors ..................................................................................................................................................................... 472
9.9.4 Manual instructions - Fraction collection ................................................................................................................................................. 473
9.9.5 Manual instructions - I/O-box ........................................................................................................................................................................ 477
9.9.6 Manual instructions - Alarms ......................................................................................................................................................................... 478
9.9.7 Manual instructions - Wash settings .......................................................................................................................................................... 481
9.9.8 Manual instructions - Watch parameters ................................................................................................................................................ 482
9.9.9 Manual instructions - Advanced ................................................................................................................................................................... 483
9.9.10 Manual instructions - Other ............................................................................................................................................................................ 486
9.10 Available Run data .............................................................................................................................................................. 487
9.11 Available Curves .................................................................................................................................................................. 490
9.12 Injection volumes and peak broadening .................................................................................................................. 492
9.13 Delay volumes ...................................................................................................................................................................... 493
9.14 Component volumes ......................................................................................................................................................... 498
9.15 Pressure control ................................................................................................................................................................... 500
9.16 Node IDs .................................................................................................................................................................................. 502
10 Ordering information ...................................................................................................................................... 505

Index ................................................................................................................................................................... 513

1 Introduction
1 Introduction
Purpose of the User Manual

The User Manual provides you with instructions and information to run the ÄKTA pure system. It also
includes relevant guidance for practical handling and maintenance of instrument components.
In this chapter
This chapter contains the following sections:
Section See page
1.1 Important user information 7
1.2 ÄKTA pure overview 9
1.3 ÄKTA pure user documentation 11

1 Introduction
1.1 Important user information

Read this before operating
ÄKTA pure
All users must read the entire ÄKTA pure Operating Instructions before installing, operating, or
maintaining the instrument. Always keep the ÄKTA pure Operating Instructions at hand when operating
ÄKTA pure.
Do not operate ÄKTA pure in any other way than described in the user documentation. If you do, you
may be exposed to hazards that can lead to personal injury and you may cause damage to the
equipment.
Intended use
ÄKTA pure is intended for purification of bio-molecules, in particular proteins, for research purposes
by trained laboratory staff members in research laboratories.
ÄKTA pure shall not be used in any clinical procedures, or for diagnostic purposes.
Prerequisites
In order to operate the system according to the intended purpose, it is important that:
• you have a general understanding of how the computer and the Microsoft® Windows® operating
system work.
• you understand the concepts of liquid chromatography.
• you have read and understood the Safety instructions chapter in ÄKTA pure Operating Instructions.
• a user account has been created according to UNICORN™ Administration and Technical Manual.
Safety notices
This user documentation contains safety notices (WARNING, CAUTION, and NOTICE) concerning the
safe use of the product. See definitions below.
WARNING
WARNING indicates a hazardous situation which, if not avoided, could result in death
or serious injury. It is important not to proceed until all stated conditions are met
and clearly understood.

1 Introduction
CAUTION
CAUTION indicates a hazardous situation which, if not avoided, could result in minor
or moderate injury. It is important not to proceed until all stated conditions are met
and clearly understood.
NOTICE
NOTICE indicates instructions that must be followed to avoid damage to the product
or other equipment.
Notes and tips

Note: A Note is used to indicate information that is important for trouble-free and optimal use of the
product.
Tip: A tip contains useful information that can improve or optimize your procedures.

1 Introduction
1.2 ÄKTA pure overview

Introduction
ÄKTA pure is intended for purification of bio-molecules, in particular proteins, for research purposes
by trained laboratory staff members in research laboratories.
This section gives an overview of the ÄKTA pure instrument and the UNICORN software. For detailed
information about UNICORN, see the UNICORN manuals listed in UNICORN user documentation, on
page 12. For detailed information about the instrument, see Chapter 2 The ÄKTA pure instrument, on
page 13.
Main features
The main features of ÄKTA pure are listed below.
• ÄKTA pure is a flexible system that allows the user to configure both hardware and software to
meet many purification needs.
• The instrument can be easily extended with additional valves, detectors and fraction collectors.
• There are a large number of different hardware modules to choose from. The user can adjust, for
example, the number of columns, inlets, outlets and detectors and choose between different ways
to apply and collect samples.
• Purification and maintenance methods are easily created using predefined methods and method
phases. A method phase refers to a specific step/part in the method, such as column wash or elution.
Method phases are described in Phase Properties and are displayed graphically in a method outline.
This makes methods and phases easy to understand and edit.
• ÄKTA pure is controlled by the UNICORN software: a complete package for control, supervision and
evaluation of chromatography instruments and purification runs.
• UNICORN has different software licence options, such as Design of Experiments (DoE) and Column
logbook, to further add user support.
UNICORN modules overview

UNICORN consists of four modules: Administration, Method Editor, System Control and Evaluation.
The main functions of each module are described in the following table.
Module Main functions
Administration Perform user and system setup, system log and database adminis-
tration.

1 Introduction
Module Main functions
Method Editor Create and edit methods using one or a combination of:
• Predefined methods with built-in application support
• Drag-and-drop function to build methods with relevant steps
• Line-by-line text editing
The interface provides easy viewing and editing of run properties.
System Control Start, monitor and control runs. The current flow path is illustrated
in the Process Picture, which allows manual interactions with the
system and provides feedback on run parameters.
Evaluation Open results, evaluate runs and create reports.

The default Evaluation module includes a user interface optimized
for workflows like quick evaluation, compare results and work with
peaks and fractions.
To perform operations like Design of Experiments, users can easily
switch to Evaluation Classic.
When working with the modules Administration, Method Editor, System Control and Evaluation it is
possible to access descriptions of the active window by pressing the F1 key. This can be especially
helpful when editing methods

1 Introduction
1.3 ÄKTA pure user documentation

Introduction
This section describes the user documentation that is delivered with ÄKTA pure.
User documentation
The user documentation listed in the table below is delivered with ÄKTA pure. It is also available on the
user documentation CD.
Document Main contents
ÄKTA pure Operating Instructions Instructions needed to install, operate and maintain ÄKTA
pure in a safe way.
Fraction collector F9-C and F9-R Instructions needed to install, operate and maintain Fraction
Operating Instructions collectors F9-C and F9-R in a safe way.
Sample pump S9 and S9H Operating Instructions needed to install, operate and maintain Sample
Instructions pump S9 and S9H in a safe way.
ÄKTA pure User Manual Detailed instrument and module descriptions and instruc-
tions on how to run, maintain and troubleshoot the system.

1 Introduction
UNICORN user documentation

The user documentation listed in the following table is available from the Help menu in UNICORN or
from the UNICORN Online Help and Documentation software accessed by pressing the F1 key in any
UNICORN module.
Documentation Main contents
UNICORN Help Descriptions of UNICORN dialog boxes (available from the Help
menu).
Getting started with Evaluation • Video clips showing common workflows in the Evaluation
Note: module.
Available in UNICORN 7.0 and lat- • Overview of features of the Evaluation module.
er.
UNICORN Method Manual 1 • Overview and detailed descriptions of the method creation
features in UNICORN.
• Workflow descriptions for common operations.
UNICORN Administration and • Overview and detailed description of network setup and
Technical Manual1 complete software installation.
• Administration of UNICORN and the UNICORN database.
UNICORN Evaluation Manual1 • Overview and detailed descriptions of the Evaluation Classic
module in UNICORN.
• Description of the evaluation algorithms used in UNICORN.
UNICORN System Control Manual1 • Overview and detailed description of the system control
features in UNICORN.
• Includes general operation, system settings and instructions
on how to perform a run.
1 Current UNICORN version is added to the title of the manual.
Additional literature
For practical tips on chromatography, refer to ÄKTA Laboratory-scale: Chromatography Systems Instru-
ment Management Handbook (product code 29010831).

2 The ÄKTA pure instrument
About this chapter

This chapter provides an overview of the ÄKTA pure instrument. It also describes the internal instrument
components and how these are installed in the instrument.
In this chapter
Section See page
2.1 Overview illustrations 14
2.2 Liquid flow path 25
2.3 Instrument control panel 27
2.4 Instrument modules 32
2.5 Installation of internal modules 76
2.6 Accessories 79

2.1 Overview illustrations

Introduction
This section provides an overview of the system and its available modules.
Core module configurations

ÄKTA pure is available with two core module configurations, one for flow rates up to 25 ml/min and
one for flow rates up to 150 ml/min. In this manual they are referred to as ÄKTA pure 25 (25 ml/min)
and ÄKTA pure 150 (150 ml/min).
Illustrations of the main parts of the instrument

The illustrations below show the location of the main parts of the instrument.
1 2 3
Part Function
1 Wet sides
2 Buffer tray
3 Holder rails
4 Instrument control panel
5 Power switch
6 Ventilation panel

Example of a typical configuration of the wet side

A typical configuration of ÄKTA pure is illustrated below.
11 12
10
9 1
8
2
7
6 5
Part Function
1 Multi-module panel
2 Inlet valve
3 Pump rinsing liquid tube
4 System pump B
5 Pressure monitor
6 System pump A
7 Mixer
8 Outlet valve
9 Injection valve
10 Conductivity monitor
11 Column valve
12 UV monitor

Available modules
The modular design allows the user to customize ÄKTA pure in multiple ways. The system is always
delivered with the core modules of the selected configuration, but optional modules may be added to
the flow path.
The table below lists the available modules forÄKTA pure 25 and ÄKTA pure 150. Core modules are in-
dicated with an asterisk (*).
Note: The valves for ÄKTA pure 25 and ÄKTA pure 150 are compatible with both systems but for the
best performance the specific valve type should be used. The narrow channels in the valves
for ÄKTA pure 25 will give too high back pressure if used above 50 ml/min. The larger volumes
in the “H” valves for ÄKTA pure 150 may decrease resolution and increase peak broadening if
used in ÄKTA pure 25.
Module Label in
ÄKTA pure 25 ÄKTA pure 150
System pump A* P9 A P9H A
System pump B* P9 B P9H B
Pressure monitor* R9 R9
Mixer* M9 M9
Injection valve* V9-Inj V9H-Inj
Inlet valve A V9-IA V9H-IA
Inlet valve B V9-IB V9H-IB
Inlet valve AB V9-IAB V9H-IAB
Inlet valve IX V9-IX V9H-IX
Sample inlet valve V9-IS V9H-IS
Mixer valve V9-M V9H-M
Loop valve V9-L V9H-L
Column valves V9-Cs V9H-Cs
V9-C V9H-C
V9-C2 V9H-C2
pH valve V9-pH V9H-pH
Outlet valves V9-O V9H-O
V9-Os V9H-Os
Versatile valve V9-V V9H-V

Module Label in
UV monitors U9-L U9-L
U9-M U9-M
Conductivity monitor C9 C9
External air sensor L9-1.5 L9-1.5
L9-1.2 L9-1.2
Fraction collectors F9-C F9-C
F9-R F9-R
I/O-box E9 E9
Sample pump S9 S9H
Illustration convention
In the valve illustrations below, the following convention is used to point out the location of the ports
on the valve head. Loop valve V9-L is used as an example.
V9-L
2E
3E
4E • Ports located on the valve head rim are indicated outside the black
ring (e.g., 1E, 2E, etc.).
1E
5E
• Ports located on the pivot part of the valve head are indicated on
the inside of the black ring (e.g., 3E and 3F).
1F
5F
• Ports located on the valve head front are indicated by an arrow

F
E
3F
4F 2F
(e.g., E and F).
Core modules
Core module Description
System pump P9 A or P9H A A high precision pump, which delivers buffer or sample in purification runs.
For further information, refer to Section 2.4.1 System pumps, on page 33.

Core module Description
System pump P9 B or P9H B A high precision pump, which delivers buffer in purification runs.
For further information, refer to Section 2.4.1 System pumps, on page 33.
Pressure monitor R9 Reads the system pressure after System pump A and System pump B.
For further information, refer to Section 2.4.12 Pressure monitors, on page 67.
Mixer M9 Mixes the buffers delivered from the system pumps to a homogeneous buffer
composition.
Three Mixer chambers are available for ÄKTA pure 25, their volumes are: 0.6
ml, 1.4 ml (mounted at delivery) and 5 ml.
Three Mixer chambers are available for ÄKTA pure 150. Their volumes are:
1.4 ml (mounted at delivery), 5 ml (included in delivery), and 15 ml.
CAUTION
Risk of explosion. Do not use Mixer chamber 15 ml with
an ÄKTA pure 25 system configuration. The maximum
pressure for Mixer chamber 15 ml is 5 MPa.
For further information, refer to Section 2.4.2 Mixer, on page 37.
Injection valve V9-Inj or V9H-Inj Directs sample onto the column.

For further information, refer to Section 2.4.6 Injection valve, on page 49.
V9-Inj
LoopE
W
1
S yr
Loo
W2
pF
SaP

Optional modules
Module Description
Inlet valve V9-IA or V9H-IA Inlet valve for System pump A with seven inlet ports and integrated air sensor.
For further information, refer to Section 2.4.4 Inlet valves, on page 39.
V9-IA
A3 A2
A4
A1
A5
Q
A7
A6
Out
Inlet valve V9-IB or V9H-IB Inlet valve for System pump B with seven inlet ports and integrated air sensor.
V9-IB
B2 B3
B1
B4
Q
B5
B6
B7
Out
Inlet valve V9-IAB or V9H-IAB Inlet valve with two A inlet ports and two B inlet ports. No integrated air
sensor.
A2 B2
A1 B1
A B
Sample inlet valve V9-IS or V9H-IS Inlet valve with eight inlet ports (seven sample inlets and one buffer inlet)
and integrated air sensor. The Sample inlet valve requires an external Sample
V9-IS pump module.
S2 S3
S1
S4
Buff
S5
S7
S6
Out

Module Description
Inlet valve V9-IX or V9H-IX Inlet valve with eight inlet ports. No integrated air sensor.
V9-IX
2 3
4
1
8
5
7
Out
Mixer valve V9-M or V9H-M Directs the flow to the Injection valve, bypassing the Mixer, or to the Injection
valve via the Mixer.
V9-M Re-Inj For further information, refer to Section 2.4.5 Mixer valve, on page 46.
Out
In
Mixer
Loop valve V9-L or V9H-L Enables the use of up to five loops connected to the instrument.
For further information, refer to Section 2.4.7 Loop valve, on page 52.
Column valve V9-C or V9H-C V9-C or V9H-C can connect up to five columns to the instrument. Up to ten
and V9-C2 or V9H-C2 columns can be connected by installing the optional column valves V9-C2
or V9H-C2.
The column valves direct the flow to one column at a time and feature two
integrated pressure sensors.
The valves allow the user to choose flow direction through the column, or
to bypass the column.
For further information, refer to Section 2.4.8 Column valves, on page 55.

Module Description
Column valve V9-Cs or V9H-Cs Connects a single column to the instrument.

Allows the user to chose flow direction through the column, or to bypass the
V9-Cs
A column.
For further information, refer to Section 2.4.8 Column valves, on page 55.
Out
In
pH valve V9-pH or V9H-pH Enables the pH electrode to be included in the flow path or bypassed during
a run. The pH electrode may be calibrated when installed in the pH valve.
For further information, refer to Section 2.4.10 pH valve, on page 60.
Outlet valve V9-O or V9H-O Directs the flow to the Fraction collector, Fraction collector 2 (out 10), any
of the ten outlet ports, or waste.
V9-O
Fra
c Ou
t1 In
For further information, refer to Section 2.4.11 Outlet valves, on page 64.
W
t10
Ou
Ou
t2
Out9
Out3
Ou
t4
t8
Ou
Ou Out6 t5
t7 Ou
Outlet valve V9-Os or V9H-Os Directs the flow to the Fraction collector, Fraction collector 2, the outlet port,
or waste.
V9-Os In For further information, refer to Section 2.4.11 Outlet valves, on page 64.
Frac
Out

Module Description
Versatile valve V9-V or V9H-V A 4-port, 4-position valve, which can be used when adding extra features
to the flow path.
V9-V 1 For further information, refer to Section 2.4.9 Versatile valve, on page 59.
2
4
UV monitor U9-L Measures the UV absorbance at a fixed wavelength of 280 nm.

For further information, refer to Section 2.4.13 UV monitors, on page 69.
UV monitor U9-M Measures the UV/Vis absorbance at up to three wavelengths simultaneously

in the range 190-700 nm.
For further information, refer to Section 2.4.13 UV monitors, on page 69.
Conductivity monitor C9 Measures the conductivity of buffers and eluted proteins.

For further information, refer to Section 2.4.14 Conductivity monitor, on
page 72.

Module Description
External air sensor L9-1.5 or L9-1.2 Prevents air from being introduced into the flow path.
For further information, refer to Section 3.1 External air sensors, on page 87.
Fraction collector F9-C Flexible fraction collector that can collect up to 576 fractions.
Up to two fraction collectors can be connected at the same time, of which
only one (the primary) can be a Fraction collector F9-C.
For further information, refer to Section 3.2 Fraction collector F9-C, on page 89.
Fraction collector F9-R Round fraction collector that can collect up to 175 fractions.
Up to two fraction collectors can be connected at the same time.
For further information, refer to Section 3.3 Fraction collector F9-R , on
page 103.
I/O-box E9 Receives analog or digital signals from, or transfers analog or digital signals
to, external equipment that has been incorporated in the system.
For further information, refer to Section 3.5 I/O-box E9, on page 111.

Module Description
Sample pump S9 or S9H A high precision pump with an integrated pressure monitor. The sample
pump delivers buffer or sample in purification runs.
For further information, refer to Section 3.4 Sample pump S9 and S9H, on
page 107.

2.2 Liquid flow path

Introduction
ÄKTA pure is a liquid chromatography system with a flexible flow path.
This section provides an overview of the liquid flow path, and its possibilities.
Example of a typical liquid flow path

The illustration below shows the flow path for a typical system configuration. The individual instrument
modules are presented in the table below. The configuration of the system is defined by the user.
1 3
2
6
4 7 9
5 8
10
W1 W2
16
W
11 12 13 14 15
17
Part Description
1 Pressure monitor
2 Sample pump
3 Sample inlet valve

Part Description
4 Inlet valve
5 System pump B
6 System pump A
7 Pressure monitor
8 Mixer
9 Injection valve
10 Sample loop or Superloop
11 Column valve
12 Column
13 UV monitor
15 Flow restrictor
16 Outlet valve
17 Fraction collector
W, W1, W2 Waste

2.3 Instrument control panel

Introduction
This section describes the design and main function of the Instrument control panel B9.
Function of the Instrument control panel

The Instrument control panel shows the current state of the system. The Pause and Continue buttons
can be used to control an ongoing run. It is possible to lock and unlock the Instrument control buttons
from UNICORN.
Location and illustration

The illustration below shows the location and detailed view of the Instrument control panel.
Part Function
1 1 Power/Communication indicator (white)
2 2 Continue button with a green light indicator
3 Pause button with an orange light indicator

3
4 Alarm and error indicator (red)
4

Lock/Unlock function
Follow the instruction below to lock or unlock the Pause and Continue buttons of the Instrument
control panel from UNICORN.
Step Action
1 In System Control, select System:Settings.

Result: The System Settings dialog opens.
2 In the System Settings dialog:

• Select Advanced:Instrument control panel.
• Select Locked or Unlocked.
• Click OK.
Buttons
The Instrument control panel includes the following buttons:
Part Function
Resumes instrument operation from the following states:

• Pause
• Wash
• Hold
Pauses the run and stops all pumps.

Status indications
The light indicators on the Instrument control panel indicate the current status of ÄKTA pure.
The table below describes the different states that can be displayed.
Display State Description
All light indicators are off. Off The instrument is turned off.
Off
The Power/Communication indicator Power-on The instrument has no communication

flashes slowly. with the Instrument server.
Power-on
The Power/Communication indicator Connecting The system is starting up.

flashes quickly.
Connecting
The Power/Communication indicator Ready The instrument is ready to use.

displays a constant light.
Ready

Both the Power/Communication indi- Run A run is ongoing.

cator and Continue button display a
constant light.
The Power/Communication indicator Wash A wash instruction or a pump synchro-

displays a constant light and the Con- nization is ongoing.
tinue button flashes slowly.
Hold A run has been put on hold.
Both the Power/Communication indi- Pause A run has been paused.

cator and Pause button display a
constant light.
The Power/Communication indicator Alarms and er- The system has been paused due to
displays a constant light. and the rors an alarm. To resume the run, acknowl-
Alarm and error indicator flashes. edge the alarm and continue the run
in UNICORN.

The Power/Communication indicator Power-save The system is in power-saving mode.

displays a pulsating light.
All indicators are lit in a wave pattern. Re-programming A module is being re-programmed to
be compatible with the current instru-
ment configuration.

2.4 Instrument modules

Introduction
This section describes the design and main functions of the instrument modules.
In this section
This section contains the following subsections:
Section See page
2.4.1 System pumps 33
2.4.2 Mixer 37
2.4.3 Valves, overview 38
2.4.4 Inlet valves 39
2.4.5 Mixer valve 46
2.4.6 Injection valve 49
2.4.7 Loop valve 52
2.4.8 Column valves 55
2.4.9 Versatile valve 59
2.4.10 pH valve 60
2.4.11 Outlet valves 64
2.4.12 Pressure monitors 67
2.4.13 UV monitors 69
2.4.14 Conductivity monitor 72
2.4.15 Flow restrictor 74

2.4.1 System pumps
2.4.1 System pumps
Introduction
This section describes the design and main functions of the system pumps, and also the pump piston
rinsing systems. The system can also be equipped with an external, optional sample pump, see Sec-
tion 3.4 Sample pump S9 and S9H, on page 107.
Function of the system pumps

The ÄKTA pure instrument is fitted with two high precision system pumps, System pump A and System
pump B. The system pumps can be used individually, or in combination to generate isocratic or gradient
elution in purification methods.
Each pump module consists of two pump heads that work alternately to give a continuous, low pulsation,
liquid delivery. To ensure delivery of correct liquid volume, the pumps must be free from air. Each pump
head is equipped with a purge valve that is used for this purpose. See Section 5.4 Prime inlets and purge
pump heads, on page 160.
The table below contains the operating limits and labels of the system pumps of ÄKTA pure 25 and
ÄKTA pure 150, respectively.
Configuration Label Pump type Flow rate Max. pressure
ÄKTA pure 25 P9 A and P9 B P9 0.001 – 25 ml/min 20 MPa

Note:
When running the
Column packing
flow instruction, the
maximum flow rate
is 50 ml/min.
ÄKTA pure 150 P9H A and P9H B P9H 0.01 – 150 ml/min 5 MPa
Note:
When running the
Column packing
flow instruction, the
maximum flow rate
is 300 ml/min.

2.4.1 System pumps

The illustration below shows the location of System pump A and System pump B, together with a detailed
view of a system pump.
1 2 3
8 7 6 5 4
Part Description
1 Purge valve: Used to remove air from the pump
2 Outlet port with check valve
3 Connections to pump piston rinsing system: Tubing is connected between the pumps
and the Pump piston rinsing system tube (6)
4 Inlet port with check valve
5 Pump head: Encapsulates the inner parts of the pump
6 Pump piston rinsing system tube
7 System pump B
8 System pump A
The pump piston rinsing system

A seal prevents leakage between the pump chamber and the drive mechanism. The seal is continuously
lubricated by the presence of liquid. The pump piston rinsing system continuously flushes the low
pressure chamber behind the piston with a low flow of 20% ethanol. This prevents any deposition of
salts from aqueous buffers on the pistons and prolongs the working life of the seals.
The pump piston rinsing system tubing is connected to the rearmost holes on the pump heads.
For instructions on how to fill the rinsing system, see Prime the system pump piston rinsing system, on
page 263.

2.4.1 System pumps
Illustration of the pump piston rinsing system
5 4
Part Description
1 Rinsing system tube holder, top
2 Rinsing system tube
3 Rinsing system tube holder, bottom
4 Outlet tubing
5 Inlet tubing
System pump rinsing systems flow path

The illustration below shows the tubing configuration of the pump piston rinsing system of the system
pumps.
1 2

2.4.1 System pumps
Part Description
1 System pump A
2 System pump B

2.4.2 Mixer
2.4.2 Mixer
Function of the Mixer

Mixer M9 is located after System pump A and System pump B, and before the Injection valve. The
Mixer is a dynamic mixer for high-performance gradients. It is used to make sure that the buffers from
the System pumps are mixed to give a homogenous buffer composition.
The Mixer has a built-in filter that prevents particles from reaching the column.
Note: Replace the sealing ring of the mixer with a highly resistant O-ring if the system is going to be
exposed to organic solvents or high concentrations of organic acids for longer periods of time.
Refer to Section 7.8.4 Replace the O-ring inside the Mixer, on page 323 for how to change the
ring.

The illustration below shows the location, together with a detailed view of Mixer M9.
Part Description
1 Outlet
2 Mixer chamber (0.6, 1.4, 5 or 15 ml)
CAUTION
Risk of explosion. Do not use Mixer chamber 15 ml with
an ÄKTA pure 25 configuration. The maximum pressure
for Mixer chamber 15 ml is 5 MPa.
3 Inlet

2.4.3 Valves, overview
2.4.3 Valves, overview
General design and function of rotary valves

The valves of the ÄKTA pure instrument allow flexibility in the liquid flow path.
All valves used in the ÄKTA pure instrument are rotary valves. The motorized rotary valve consists of
a Valve connection block with a number of defined bores with channels to the inlet and outlet ports
of the valve. The Rotary disc, mounted on the motor, has a number of defined channels. The pattern
of channels of the Rotary disc together with the pattern and location of the ports of the Valve connection
block, define the flow path and function of each type of valve. When the Rotary disc turns, the flow
path in the valve changes.
Illustration of inlet valve components

The illustration below shows the components of a disassembled Inlet valve A or a disassembled Inlet
valve B.
Inlet valve AB is built up by the same parts but have another configuration of defined bores.
Part Description
1 Valve connection block (stator)
2 Rotary disc (rotor)
3 Defined channel(s) in the Rotary disc
4 Defined bores in the Valve connection block
Note: Inlet and outlet ports are not visible in the picture. They are located on the opposite side of the
Valve connection block.

2.4.4 Inlet valves
2.4.4 Inlet valves
Function of the inlet valves

The inlet valves are used to select which buffers or samples to use in a run. The inlet valves available
for ÄKTA pure and their functions are described in the table below.
Inlet valve Label in Function
Inlet valve A V9-IA V9H-IA Enables automatic change between different buffers
and wash solutions (seven inlet ports) .
Can be used together with Inlet valve B to generate
gradients by mixing buffer from System pump A and
buffer from System pump B.
Inlet valve B V9-IB V9H-IB Enables automatic change between different buffers
and wash solutions (seven inlet ports).
Can be used together with Inlet valve A to generate
gradients by mixing buffer from System pump A and
buffer from System pump B.
Inlet valve AB V9-IAB V9H-IAB Enables automatic change between different buffers
and wash solutions (two A and two B inlet ports).
Can be used to generate gradients by mixing buffer
from System pump A and buffer from System pump
B.
Sample inlet valve V9-IS V9H-IS Enables automatic loading of up to seven samples
when used together with a sample pump.
Inlet valve X V9-IX V9H-IX Increases the total number of inlets to the system.
Can be used in two different configurations X1 and
X2. The configurations are called V9-X1 and V9-X2
for ÄKTA pure 25 and, V9H-X1 and V9H-X2 for ÄKTA
pure 150.
The modular design of ÄKTA pure allows the use of several combinations of inlet valves.
The possible combinations of Inlet valve A, Inlet valve B and Inlet valve AB are:
• one Inlet valve A
• one Inlet valve B
• Inlet valve A and Inlet valve B,
• Inlet valve AB together with Inlet valve A or Inlet valve B,
• one Inlet valve AB,
or

2.4.4 Inlet valves
• no installed inlet valves.

The sample inlet valve can be used together with any of the combinations listed above.
The air sensors integrated in Inlet valve A, Inlet valve B, and Sample inlet valve detect the presence of
air and prevent the air from entering the pump.
Inlet valve AB and Inlet valve IX lack built-in air sensors, but can be used together with external air
sensors.
Location of inlet valves

The locations of the inlet valves are described in the following table:
Inlet valve Location
Inlet valve A Before System pump A
Inlet valve B Before System pump B
Inlet valve AB Before both System pump A and System pump B
Sample inlet valve Before the sample pump
Inlet valve IX For example, before another inlet valve
Illustration of Inlet valve A and Inlet valve B

The illustration below shows a detailed view of Inlet valve A and Inlet valve B, in this example with labels
V9-IA and V9-IB.
1 2
Part Description
1 Integrated air sensor of Inlet valve A (located under the plug)
2 Integrated air sensor of Inlet valve B (located under the plug)

2.4.4 Inlet valves
Ports of Inlet valve A and Inlet valve B

The illustration below shows the ports of Inlet valve A and Inlet valve B, in this example with labels V9-
IA and V9-IB.
Out Out
Port Description
A1-A7 Buffer inlets of Inlet valve A
B1-B7 Buffer inlets of Inlet valve B
Q Not used for ÄKTA pure
Out To the respective System pump
Illustration of Inlet valve AB

The illustration below shows a detailed view of Inlet valve AB.
1 2
4 3

2.4.4 Inlet valves
Part Description
1 A inlet ports
2 B inlet ports
3 Outlet port to System pump B
4 Outlet port to System pump A
Note: Inlet valve AB does not have any integrated air sensor.
Ports of Inlet valve AB

The illustration below shows the ports of Inlet valve AB.
Port Description
A1-A2 Inlet ports A1 and A2 of Inlet valve AB are used when buffers or samples
should be delivered to System pump A
B1-B2 Inlet ports B1 and B2 of Inlet valve AB are used when buffers or samples
should be delivered to System pump B
A Outlet port A of Inlet valve AB leads to System pump A
B Outlet port B of Inlet valve AB leads to System pump B

2.4.4 Inlet valves
Illustration of Sample inlet valve

The illustration below shows a detailed view of the Sample inlet valve.
Part Description
1 Integrated air sensor (located under the plug)
Ports of Sample inlet valve

The illustration below shows the ports of Sample inlet valve, in this example labeled V9-IS.
Port Description
S1-S7 Sample inlets
Buff Buffer inlet
Out To Sample pump

2.4.4 Inlet valves
Ports of Inlet valve IX

The illustration below shows the ports of Inlet valve IX.
Port Description
1-8 Inlets
Out For example, to another inlet valve
Note: Inlet valve IX does not have an integrated air sensor.
Connect tubing
The table below shows the tubing and connectors that is delivered together with the optional inlet
valves.
Tubing Connection Tubing Connector Tubing

label length
ÄKTA pure 25 ÄKTA pure 150 ÄKTA pure 25 ÄKTA pure 150 (mm)
A1-A2 Inlets to FEP, o.d. 1/8", FEP, o.d. 3/16", Tubing connec- Tubing connec- 1500
and Inlet valve AB i.d. 1.6 mm i.d. 2.9 mm tor, 5/16" with tor, 5/16" with
B1-B2 Ferrule (yellow), Ferrule (blue),
1/8" 3/16"
A1-A7 Inlets to FEP, o.d. 1/8", FEP, o.d. 3/16", Tubing connec- Tubing connec- 1500
Inlet valve A i.d. 1.6 mm i.d. 2.9 mm tor, 5/16" with tor, 5/16" with
Ferrule (yellow), Ferrule (blue),
1/8" 3/16"
B1-B7 Inlets to FEP, o.d. 1/8", FEP, o.d. 3/16", Tubing connec- Tubing connec- 1500
Inlet valve B i.d. 1.6 mm i.d. 2.9 mm tor, 5/16" with tor, 5/16" with
Ferrule (yellow), Ferrule (blue),
1/8" 3/16"

2.4.4 Inlet valves

label length
InA From Inlet valve FEP, o.d. 1/8", FEP, o.d. 3/16", Tubing connec- Tubing connec- 300
A or i.d. 1.6 mm i.d. 2.9 mm tor, 5/16" with tor, 5/16" with
Inlet valve AB to Ferrule (yellow), Ferrule (blue),
System pump A 1/8" 3/16"
InB From Inlet valve FEP, o.d. 1/8", FEP, o.d. 3/16", Tubing connec- Tubing connec- 300
B or i.d. 1.6 mm i.d. 2.9 mm tor, 5/16" with tor, 5/16" with
Inlet valve AB to Ferrule (yellow), Ferrule (blue),
System pump B 1/8" 3/16"
S1-S7 Sample inlets to FEP o.d. 1/8", FEP, o.d. 3/16", Tubing connec- Tubing connec- 1000
Sample inlet i.d. 1.6 mm i.d. 2.9 mm tor, 5/16" with tor, 5/16" with
valve Ferrule (yellow), Ferrule (blue),
1/8" 3/16"
Buff Buffer inlet to FEP o.d. 1/8", FEP, o.d. 3/16", Tubing connec- Tubing connec- 1000
Sample inlet i.d. 1.6 mm i.d. 2.9 mm tor, 5/16" with tor, 5/16" with
valve Ferrule (yellow), Ferrule (blue),
1/8" 3/16"
InS Sample inlet FEP o.d. 1/8", FEP, o.d. 3/16", Tubing connec- Tubing connec- 580
valve to i.d. 1.6 mm i.d. 2.9 mm tor, 5/16" with tor, 5/16" with
Sample Pump Ferrule (yellow), Ferrule (blue),
1/8" 3/16"
Note: Narrow inlet tubing is available for S1-S7. Refer to Section 9.3 Tubing and connectors, on
page 420 for more information.

2.4.5 Mixer valve
2.4.5 Mixer valve
Function of Mixer valve

Mixer valve (V9-M or V9H-M) allows the user to bypass the mixer. It is intended to be used when the
System pump is used for sample application or when a sample is re-injected.
Note: Mixer valve (V9-M or V9H-M) cannot be used together with Sample pump S9 or Sample pump
S9H.
Location and illustration of Mixer valve

The illustration below shows the recommended location, together with a detailed view of the Mixer
valve.
Ports and flow paths of the mixer valve

The illustration and tables below describe the ports of and different flow paths through the Mixer valve.
The Mixer valve has two available flow paths; By-pass, and Mixer (default). If the Mixer valve is installed
in the recommended location before the Mixer, By-pass allows the flow to bypass the Mixer, and Mixer
directs the flow to the Mixer.

2.4.5 Mixer valve
Primary flow path
Alternative flow path
Port Description
In Port in which the flow enters the valve. Should be connected to the System pressure
monitor outlet.
Out Port from which the flow leaves the Mixer valve and bypasses the Mixer. Connect to the
injection valve SaP port.
Re-inj Port for advanced configurations including re-injection.
Mixer Port from which the flow leaves the valve and is directed to the Mixer.
Connect tubing
The table below shows recommended tubing and connectors.

label length
ÄKTA pure 25 ÄKTA pure 150 (mm)
3-1 Pressure monitor R9 PEEK, o.d. 1/16", PEEK, o.d. 1/16", Fingertight connector, 160
to i.d. 0.75 mm i.d. 1.00 mm 1/16"
Mixer valve V9-M port
In
3-2 Mixer valve V9-M port PEEK, o.d. 1/16", PEEK, o.d. 1/16", Fingertight connector, 330
Mixer i.d. 0.75 mm i.d. 1.00 mm 1/16"
to Mixer M9
3-3 Mixer valve V9-M port PEEK, o.d. 1/16", PEEK, o.d. 1/16", Fingertight connector, 260
Out to i.d. 0.75 mm i.d. 1.00 mm 1/16"
Injection valve V9-Inj
port SaP

2.4.5 Mixer valve
Flow paths through Mixer valve

The Mixer valve (V9-M, V9H-M) has two available flow paths; By-pass, and Mixer. If the Mixer valve is
installed in the recommended location before the Mixer, By-pass allows the flow to bypass the Mixer,
and Mixer directs the flow to the Mixer.
The illustration below shows the different flow paths through the Mixer valve V9-M.
Primary flow path
Flow path Description
By-pass The flow from the system pumps bypasses the mixer.
Mixer The flow from the system pumps is directed to the Mixer. Mixer is the default flow path.

2.4.6 Injection valve
Function of the Injection valve

The Injection valve is used to direct sample onto the column. The valve enables usage of a number of
different sample application techniques.
The injection valve is labeled V9-Inj for ÄKTA pure 25 and V9H-Inj for ÄKTA pure 150.
A sample loop or a Superloop can be connected to the injection valve and filled either automatically,
using a Sample Pump or System pump A, or manually, using a syringe. The sample can also be injected
directly onto the column using a Sample pump, or System pump A together with the mixer valve.
For instructions on how to connect and use loops, see Section 5.7 Sample application, on page 179.
Location and illustration of the Injection valve

The illustration below shows the location, together with a detailed view of the Injection valve, in this
exampel labeled V9-Inj.
Ports and flow paths of the Injection valve

The illustration and tables below describe the ports of and different flow paths through the Injection
valve.
The Injection valve can be set to different positions that give different flow paths through the valve.

Primary flow path
Flow path for manual load
Closed flow path
Port Description
SaP Inlet from

• sample pump, or
• system pump via the Mixer valve Out port.
SyP Inlet from the System pumps via the Mixer
Syr Syringe connection
Col Outlet to one of the Column valves or to the column.
LoopF Port for connection of a loop. Used to fill the loop.
LoopE Port for connection of a loop. Used to empty the loop into the flow path.
W1 Loop and System pump waste
W2 Sample flow waste

Manual load - Default posi- The system flow is directed onto the column or column valve. Sample
tion of the valve can be manually injected into the loop.
Excess sample leaves the valve through waste port W1.
Inject The system flow is directed through the loop and onto the column
or column valve. If a mixer valve or the sample pump is used, the
flow entering the SaP port is directed to waste port W2.
System pump waste The system flow is directed to waste port W1. If a mixer valve or the
sample pump is used, the flow entering the SaP port is directed to
the column or the column valve.
Direct inject The flow entering the SaP port is directed to the column or the col-
umn valve. This position is used with:
• the sample pump, or
• a mixer valve and System pump A, bypassing the mixer.
Flow entering the SyP port is directed to waste port W1.
Sample pump load The flow entering the SaP port is directed to the loop. This position
is used with:
• a mixer valve and System pump A, bypassing the mixer.
Excess sample leaves the valve through waste port W1. The flow
entering the SyP port is directed to the column or the column valve.
Sample pump waste The flow entering the SaP port is directed to waste port W2. This
position is used with:
• a mixer valve and System pump A.
The flow entering the SyP port is directed to the column via the loop.
Note: • In order to avoid sample carry-over when switching techniques for loading samples, wash
the injection valve with buffer between the loading of two different samples. For example,
when switching from loading sample onto the loop to loading sample directly onto the
column with the valve in Direct inject position.
• Make sure that the SaP port is plugged with a stop plug if neither the sample pump nor
the mixer is used.

2.4.7 Loop valve
2.4.7 Loop valve
Function of the Loop valve

The Loop valve allows the user to connect several loops simultaneously to the instrument. It can for
example be used for storing intermediate fractions in multi-step purifications, for storing samples to
be used in scouting runs, or for storing eluents needed in low volumes. The valve also has a built-in
bypass function that enables bypassing all loops. The Loop valve is labeled V9-L or V9H-L.
Location and illustration of Loop valve

The illustration below shows the recommended location, together with a detailed view of Loop valve
V9-L.
Ports and flow paths of the Loop valve

The illustration and tables below describe the ports and different flow paths through the Loop valve.
In the Position 4 example, the

loop is connected to loop posi-
tion 4 and the loop is being
emptied.

2.4.7 Loop valve
Port Description
F Port connected to the LoopF port of the Injection valve.
1F and 1E Ports for connection to loop 1.
E Port connected to the LoopE port of the Injection valve.
Note: Ports denoted by the letter F are used for filling the loop and ports denoted by the letter E are
used for emptying the loop.
Position 1-5 The flow direction depends on the Injection valve position.
By-pass The flow bypasses the loop(s). By-pass is the default flow path.
Connect tubing

label length
L1 Injection valve posi- PEEK, PEEK, Fingertight connector, 160

tion LoopF to o.d. 1/16", o.d. 1/16", 1/16"
Loop valve position F i.d. 0.50 mm i.d. 0.75 mm
L2 Injection valve posi- PEEK, PEEK, Fingertight connector, 160

tion LoopE to o.d. 1/16", o.d. 1/16", 1/16"
Loop valve position E i.d. 0.50 mm i.d. 0.75 mm

2.4.7 Loop valve
Connect a Loop valve

The Loop valve is connected to the Injection valve instead of a loop, as described below.
Step Action
1 Connect port E on the Loop valve to port LoopE on the Injection valve.
2 Connect port F on the Loop valve to port LoopF on the Injection valve.
3 Connect one or many loops to the Loop valve. See Section 5.7 Sample application, on
page 179.
Note:
Always use the the first positions of the valve for the connected loops (e.g., if three loops
will be used, use port 1F-3F and the corresponding ports 1E-3E) to avoid cross-contamina-
tion.
Note: It is possible to place the Loop valve in other positions in the flow path than the one described
above. However, the volume used for washes will then be incorrect, just as the system config-
uration shown in the process picture.

2.4.8 Column valves
2.4.8 Column valves
Function of the Column valves

The Column valves are used to connect columns to the system, and to direct the flow onto the column.
The Column valves available for ÄKTA pure and their functions are described in the table below.
Label in Function
V9-C V9H-C Connects columns to the system and allows the user
to choose column, flow direction through the column,
or to bypass the columns.
Up to five columns can be connected to the valve.
V9-C2 V9H-C2 Optional column valve allows up to ten columns to

be connected when combined with V9-C or V9H-C.
The user can choose column, flow direction through
the column, or to bypass the columns.
V9-Cs V9H-Cs Connects one column to the system and allows the
user to choose flow direction through the column,
or to bypass the column.
The inlet and outlet ports of Column valves V9-C, V9H-C and V9-C2, V9H-C2 have built in pressure
sensors that measure the actual pressure over the column. For further information on the pressure
sensors, see Function of pressure monitors integrated in Column valves V9-C or V9H-C, on page 68.
Column valves V9-Cs and V9H-Cs have no pressure sensors. See Section 5.5 Connect a column, on
page 172 for information on how to set the pressure alarm to protect the column when using V9-Cs or
V9H-Cs.
Location and illustration of Column valves

ÄKTA pure with one column valve
The illustration below shows the recommended location of the column valve when only V9-Cs or V9-
C are installed.
1 2

2.4.8 Column valves
Part Function
1 Column valve V9-Cs or V9H-Cs (no integrated pressure sensors)
2 Column valve V9-C or V9H-C (integrated pressure sensors)
ÄKTA pure with two column valves

The standard configuration for ÄKTA pure is with one column valve. Valves V9-C or V9H-C have 5
column positions. To increase the number of column positions to 10, V9-C or V9H-C must be combined
with a second column valve (either V9-C2 or V9H-C2).
Use positions 8 and 9 to install two column valves with the shortest possible flow path. Install the first
column valve (V9-C or V9H-C) in position 8. Install the second column valve (V9-C2 or V9H-C2) in position
9.
By-pass
Ports and flow paths of the column valves

The illustration and tables below describe the different ports of and flow paths through Column valves
V9-C, V9H-C, V9-C2 and V9H-C2. In the example below the column is connected to column position 4
and the valve is labeled V9-C.
By-pass
Port Description
In Inlet from Injection valve via a built-in pressure monitor.

2.4.8 Column valves
Port Description
1A-5A Ports for connection to the top of columns.
1B-5B Ports for connection to the bottom of columns.
Out Outlet to UV monitor via a built-in pressure monitor.
By-pass The flow bypasses the column(s). By-pass is the default flow path.
Down flow The flow direction is from the top of the column to the bottom of the column.
Down flow is the default flow direction.
Up flow The flow direction is from the bottom of the column to the top of the column.
Ports and flow paths of Column valves V9-Cs and

V9H-Cs
The illustration and tables below describe the different ports and flow paths of Column valve V9-Cs.
1A 1A 1A
1B 1B 1B
Port Description
In Inlet from Injection valve.
1A Port for connection to the top of a column.
1B Port for connection to the bottom of a column.
Out Outlet to UV monitor.
Flow Description
path
By-pass The flow bypasses the column. By-pass is the default flow path.

2.4.8 Column valves
Flow Description
path
Down The flow direction is from the top of the column to the bottom of the column. Down flow
flow is the default flow direction.
Up flow The flow direction is from the bottom of the column to the top of the column.
Connect tubing
Tubing Tubing
Tubing
Connection Connector length
label ÄKTA pure 25 ÄKTA pure 150 (mm)
Injection valve to
5 100
Column valve
Standard column PEEK, o.d. 1/16", PEEK, o.d. 1/16",

Fingertight connector,
5C2 valve and extra col- i.d. 0.50 mm (or- i.d. 0.75 mm 100
1/16"
umn valve ange) (green)
Column valve to
6 160
UV monitor
Follow the instructions below to connect tubing to the column valves.

Step Action
1 If no Column valve is installed, remove the Union F/F between tubing 5 and tubing 6
2 Connect tubing between Injection valve, Column valve and UV monitor according to the
table above.
Note: The built-in pressure sensors for column valve(s) V9-C, V9-C2,V9H-C and V9H-C2 have to be
re-calibrated after installation. See Calibrate the monitors, on page 305.
Note: When using two column valves, only the pre-column pressure sensor on the first valve and
the post-column pressure sensor on the second valve are used.

2.4.9 Versatile valve
2.4.9 Versatile valve
Function of the Versatile valve

The Versatile valve is a 4-port, 4-position valve, which can be used to add extra features to the flow
path. For example, the valve can be used to connect external equipment to the flow path during parts
of a run. The versatile valve can be installed in any position. The versatile valve is labeled V9-V or V9H-
V.
It is possible to install up to four versatile valves simultaneously in ÄKTA pure. The configuration is defined
by the module's Node ID.
Ports and flow paths of the Versatile valve

The illustration and table below describe the different ports of and flow paths through the Versatile
valve. The valve has four ports (1-4).
The Versatile valve has four available sets of flow paths; two where a single flow channel is used and
two where the flow can be directed through two different channels simultaneously.
Flow Description
path
1-3 A single flow channel where the flow is directed between port 1 and port 3.
2-4 A single flow channel where the flow is directed between port 2 and port 4.
1-4 and Two simultaneously used flow channels where the flow is directed between port 1 and
2-3 port 4 and between port 2 and port 3.
1-2 and Two simultaneously used flow channels where the flow is directed between port 1 and
3-4 port 2 and between port 3 and port 4.

2.4.10 pH valve
2.4.10 pH valve
Function of the pH valve

The pH valve is used to direct the flow to a pH electrode when inline monitoring of pH is desired during
a run. The pH valve is labeled V9-pH for ÄKTA pure 25 and V9H-pH for ÄKTA pure 150.
The pH valve has an integrated flow cell in which the pH electrode can be installed.
It is recommended to connect a Flow restrictor to the pH valve. The flow restrictor is used to generate
a back pressure high enough to prevent the formation of air bubbles in the UV flow cell.
The valve directs the flow to the pH electrode and to the flow restrictor, or bypasses one or both.
Note: The Flow restrictor is normally included in the flow path after the Conductivity monitor. When
installing the pH valve on ÄKTA pure, the flow restrictor should be moved from its normal po-
sition on the conductivity monitor to the pH valve.
Location and illustration of the pH valve

The illustration below shows the recommended location, together with a detailed view of
the pH valve.
Part Description
1 pH valve
2 pH electrode
3 pH flow cell
4 Flow restrictor

2.4.10 pH valve
Ports and flow paths of the pH valve

The illustration and table below describe the different ports of and flow paths through the pH valve,
in this example labeled V9-pH.
By-pass
Primary flow path
Flow path for calibrations
Closed flow path
Port Description
In From Conductivity monitor
ToR To Flow restrictor
FrR From Flow restrictor
Out To Outlet valve
Cal Calibration port
W3 To Waste

2.4.10 pH valve
By-pass Both pH electrode and Flow restrictor are bypassed.
Restrictor Flow restrictor is in use and pH electrode is bypassed.
Restrictor Both pH electrode and Flow restrictor are in use.

and pH
pH pH electrode is in use and Flow restrictor is bypassed.
Calibration Flow path used when calibrating the pH monitor and when filling the pH flow cell
with storage solution. The Cal port is used to inject solution into the flow cell using
a syringe.
Excess solution leaves the valve through port W3.
pH monitor
The pH monitor continuously measures the pH of the buffer and eluted proteins when the pH electrode
is inline. A pH electrode can be installed in the pH flow cell. For instruction on how to install the pH
electrode, see Section 7.5.2 Replace the pH electrode, on page 275. The pH electrode should not be ex-
posed to more than 0.5 MPa during a normal run, but can withstand pressure spikes of 0.8 MPa. It is
therefore important to place it after the column.
The illustration below shows the location of the pH flow cell and a pH electrode installed in the pH
valve.
Part Description
1 pH flow cell
2 pH electrode

2.4.10 pH valve
Connect tubing
The table below shows recommended tubing.

label length
8pH Conductivity monitor PEEK, o.d. 1/16", PEEK, o.d. 1/16", Fingertight connector, 180
to port In i.d. 0.50 mm i.d. 0.75 mm 1/16"
1R Flow restrictor to PEEK, o.d. 1/16", PEEK, o.d. 1/16", Fingertight connector, 80
port ToR i.d. 0.50 mm i.d. 0.75 mm 1/16"
2R Flow restrictor to PEEK, o.d. 1/16", PEEK, o.d. 1/16", Fingertight connector, 80
port FrR i.d. 0.50 mm i.d. 0.75 mm 1/16"
9pH Port Out to PEEK, o.d. 1/16", PEEK, o.d. 1/16", Fingertight connector, 160
Outlet valve i.d. 0.50 mm i.d. 0.75 mm 1/16"
N/A to Calibration port Cal N/A N/A N/A N/A
W3 Port W3 to Waste ETFE I.D. 1 mm ETFE I.D. 1 mm Fingertight connector, 1500

1/16"
Note: The dimensions of the PEEK tubing depends on which tubing kit that is used. See Section 9.3
Tubing and connectors, on page 420 for more information.

2.4.11 Outlet valves
Function of the outlet valves

The outlet valve is used to direct the flow to the fraction collector, to an outlet port, or to waste. The
table below shows the labeling of the outlet valves for ÄKTA pure 25 and ÄKTA pure 150.
Label in Description
V9-O V9H-O 10 outlet ports
V9-Os V9H-Os 1 outlet ports
Location and illustration of outlet valves

The illustration below shows the recommended location, together with a detailed view of the outlet
valves. In this example the valves are labeled V9-Os and V9-O.
Ports of Outlet valves V9-O and V9H-O

The illustration below shows the ports of Outlet valve V9-O and V9H-O, in this example labeled V9-O.
Port Description
In Inlet port

Port Description
Out1 - Outlet ports 1 - 10

Out10
Frac Port to Fraction collector

Note:
If a secondary Fraction collector F9-R is used it should be connected to port Out10.
W Waste port
Ports of Outlet valves V9-Os and V9H-Os

The illustration below shows the ports of Outlet valve V9-Os.
In
Frac Out1
W
V9-Os
Port Description
In Inlet port
Out1 Outlet port
Frac Port to fraction collector

Note:
If a secondary Fraction collector F9-R is used it should be connected to port
Out1.
W Waste port

Connect tubing

label length
9 Flow restrictor to PEEK, o.d. 1/16", PEEK, o.d. 1/16", Fingertight connector, 135
9pH pH valve to PEEK, o.d. 1/16", PEEK, o.d. 1/16", Fingertight connector, 160
Out1 - Outlets from the ETFE, o.d. 1/8", ETFE, o.d. 1/8", Fingertight connector, 1500
Out10 outlet valves i.d. 1 mm i.d. 1 mm 1/16"
Note: If ÄKTA pure previously has been configured without an Outlet valve, remove the Union F/F
between tubing 9 and tubing W before installation of the Outlet valve. Then connect tubing 9
to the In port on the Outlet valve and the waste tubing W to the W port on the Outlet valve.

2.4.12 Pressure monitors
Introduction
This section describes the location and function of the pressure monitors.
Up to four pressure monitors are included in ÄKTA pure.
Function of the system pump and the sample

pump pressure monitors
Up to four pressure monitors are included in ÄKTA pure. One pressure monitor is always connected
to the system pumps and one pressure monitor is part of the Sample pump (S9 or S9H). The monitors
of the pumps are both labelled R9.
The System pump pressure monitor measures the pressure after the system pumps, the system
pressure. The pressure monitor of the sample pump measures the pressure after the sample pump,
the sample pressure. To provide a reading of the highest pressure in the system there are connections
to all pump cylinders.
The pressure monitor also contains pump flow restrictors in order to stop siphoning effects. Siphoning
effects can occur if tubing in the system is disconnected while the inlet tubing is immersed in buffers
that are placed on a higher level than the pump.
Location and illustration of System pressure

monitor
The illustration below shows the location, together with a detailed view of System pressure monitor.
Part Description
1 Pressure monitor
2 Pump flow restrictors

Function of pressure monitors integrated in

Column valves V9-C or V9H-C
Pressure monitors are integrated into the column valves: V9-C, V9H-C, V9-C2 , and V9H-C2. Column
valves V9-Cs and V9H-Cs do not have pressure monitors.
Pressure monitors are integrated in the inlet and outlet ports of the column
valve to measure pre-column pressure (A), and post-column pressure (B).
The Delta column pressure (C), or pressure drop, is the difference between
the pre- and the post-column pressure.
Pressure alarms can be set for both the pre-column pressure and the
Delta-column pressure. Pressure control of flow can use either the pre-
column pressure or the Delta-column pressure.
The table below shows the pressure monitor stettings when the instrument
is configured with one or two column valves. Set the pre-column and post-
column pressure monitors by turning the arrows on the switches on the
left and right sides of the module with a screwdriver. Set Node ID for the
column valve by turning the arrows in the two rotating switches at the
back of the module.
Note:
Column valves V9-Cs and V9H-Cs do not have pressure monitors.
Settings
Module Label Pressure
Node ID
monitor
First column valve V9-C or V9H-C 5 NA
Pre-column pressure monitor in column NA NA 2

valve V9-C and V9H-C
Post-column pressure monitor in column NA NA 3

valve V9-C and V9H-C
Second column valve V9-C2 or V9H-C2 6 NA
Pre-column pressure monitor in column NA NA 4

valve V9-C2 and V9H-C2
Post-column pressure monitor in column NA NA 5

valve V9-C2 and V9H-C2
Column valve V9-Cs and V9H-Cs V9-Cs or V9H-Cs 7 NA

2.4.13 UV monitors
2.4.13 UV monitors
Introduction
This section describes the design and function of UV monitors U9-M and U9-L.
The modules include a monitor unit and a detector with a UV flow cell.
Function of UV monitor U9-M

UV monitor U9-M measures the UV absorbance at a wavelength range of 190 to 700 nm.
A flip-mode enables measuring of UV/Vis absorbance at three wavelengths simultaneously during a
run. The second and third wavelength can be turned off or on in method phase properties, by manual
instructions or in system settings. The UV lamp can be shut off manually if not needed during a run.
The lamp starts automatically for next run.
Note: Installation of UV monitor U9-M should only be performed by GE Service personnel.
Note: The resolution is decreased when more than one wavelength is used simultaneously due to
lower sampling frequency per wavelength. Do not use more wavelengths than necessary.
Location and illustration of UV monitor U9-M

The illustration below shows the location of UV monitor U9-M, together with a detailed view of the
monitor unit and detector. The monitor unit is labelled U9-M and the detector U9-D.
Note: When UV monitor U9-M is used, the entire Multi-module panel shown in the illustration is re-
placed by U9-M.
1 2
4 3
Part Description
1 Inlet
2 UV flow cell. Three different path lengths are available:

0.5 mm, 2 mm (default) and 10 mm
3 UV detector
4 Outlet

2.4.13 UV monitors
Function of UV monitor U9-L

The UV monitor U9-L measures the UV absorbance at the fixed wavelength of 280 nm.
It is not possible to vary the wavelength. Therefore it is not shown in the Phase Properties pane in
Method Editor. The UV lamp can be shut off manually if not needed during a run. The lamp starts au-
tomatically for next run.
Location and illustration of UV monitor U9-L

The illustration below shows the recommended location of UV monitor U9-L, together with a detailed
view of the monitor and detector. The UV monitor U9-L requires that the Multi-module panel is installed,
see Example of a typical configuration of the wet side, on page 15. The UV monitor can also be installed
in an Extension box, see Extension box, on page 85.
1 2 3
5 4
Part Description
1 Inlet
2 UV monitor U9-L
3 UV flow cell. Two different path lengths are available:

2 mm (default) and 5 mm
4 Outlet
5 UV detector
Using two UV monitors

It is possible to use two UV monitors in ÄKTA pure, in two combinations. The configuration is defined
by the module's Node ID.
• UV monitor U9-M together with UV monitor U9-L, 2nd
• UV monitor U9-L together with UV monitor U9-L, 2nd

2.4.13 UV monitors
Note: When using two UV monitors, the signal from the first UV monitor is by default used for peak
fractionation. This can be changed by editing the text instruction Fraction Collection:Peak
fractionation parameters:Signal source and choosing UV 2nd as Signal source.
Note: When using two UV monitors with different cell lengths to increase the UV absorption dynamic
range, the U9-L signal comes from the real cell length and has to be calibrated for exact calcu-
lations. The U9-M signal is automatically calibrated to nominal cell length.
UV monitor U9-L, 2nd can be located anywhere in the flow path and is therefore shown in the Process
Picture in UNICORN as a component without a fixed place. This means that it is possible to place U9-
L, 2nd before the other UV monitor in the flow path.
Note: If U9-L 2nd is placed on the high pressure side of the column, pressure limits have to be con-
sidered. See UV monitor options, on page 416
Connect tubing
The table below shows the tubing and connectors to be used with UV monitor U9-L.
Note: If a second UV monitor is used, the tubing for this has to be cut manually.

label length
6 Tubing to PEEK, o.d. 1/16", PEEK, o.d. 1/16", Fingertight connector, 160
UV monitor U9-L i.d. 0.50 mm i.d. 0.75 mm 1/16"
7 Tubing from PEEK, o.d. 1/16", PEEK, o.d. 1/16", Fingertight connector, 170
UV monitor U9-L i.d. 0.50 mm i.d. 0.75 mm 1/16"
Note: To perform a run with the flow in reverse direction through UV monitor U9-L, a longer tubing
7 is needed. Replace the 170 mm tubing from the UV monitor with tubing that is 210 mm long
and adjust the delay volume accordingly. For example, changing from 170 mm to 210 mm for
0.5 mm i.d. tubing increases the delay volume with 8 µl.

2.4.14 Conductivity monitor
Function of the Conductivity monitor

The Conductivity monitor continuously measures the conductivity of buffers and eluted proteins. The
monitor is labelled C9.
The Conductivity flow cell has two electrodes positioned in the flow path of the cell. An alternating
voltage is applied between the electrodes and the resulting current is measured and used to calculate
the conductivity of the eluent.
The conductivity is automatically calculated by multiplying the measured conductance by the cell
constant of the flow cell. The cell constant is factory-calibrated on delivery but can be re-calibrated if
needed, see Section 7.7.3 Calibrate the Conductivity monitor, on page 308.
As variation in temperature influences conductivity readings, the conductivity flow cell is fitted with a
temperature sensor that measures the temperature of the eluent. A temperature compensation factor
is used to report the conductivity in relation to a set reference temperature.
Location and illustration of the

Conductivity monitor
The illustration below shows the recommended location of Conductivity monitor C9, together with a
detailed view of the monitor.
Part Description
1 Inlet
2 Conductivity flow cell
4 Outlet

Connect tubing

label length
7 UV monitor U9-L to PEEK, o.d. 1/16", PEEK, o.d. 1/16", Fingertight connector, 170
Conductivity monitor C9 i.d. 0.50 mm i.d. 0.75 mm 1/16"
8 Conductivity monitor C9 to PEEK, o.d. 1/16", PEEK, o.d. 1/16", Fingertight connector, 95
Flow restrictor i.d. 0.50 mm i.d. 0.75 mm 1/16"

2.4.15 Flow restrictor
Function of Flow restrictor FR-902

The Flow restrictor is included in the flow path to generate a steady back pressure of approximately
0.2 MPa, to prevent formation of air bubbles in the UV flow cell.
Note: Do not remove the flow restrictor to lower the pressure in the system. There is a risk that air
bubbles cause large disturbances in the UV flow cell. Use the automatic pressure control
function to avoid pressure alarms, see Recommended pressure control parameters, on page501
Location and illustration of Flow restrictor FR-902

The Flow restrictor is normally included in the flow path after the Conductivity monitor. The Conductiv-
ity monitor is equipped with a special holder for the Flow restrictor.
The illustration below shows Flow restrictor FR-902 fitted on the Conductivity monitor.
4 3
Part Function
1 Flow restrictor
2 Holder
3 Inlet
4 Outlet
If ÄKTA pure is fitted with pH valve V9-pH or V9H-pH the Flow restrictor has to be moved from the
Conductivity monitor to the pH valve.

The illustration below shows Flow restrictor FR-902 fitted on the pH valve.
2
3
1
Part Function
1 Flow restrictor
2 Flow restrictor inlet connection from pH valve ToR port
3 Flow restrictor outlet connection to pH valve FrR port

2.5 Installation of internal modules

Introduction
Optional modules and valves are easy to install in the instrument. The existing module or Module
Panel is removed with a Torx T20 screwdriver and the cable is disconnected. The cable is then connected
to the optional module, which is subsequently inserted into the instrument. The newly installed module
is then added to the System properties in UNICORN.
Node ID
All of the available optional modules are preconfigured to give the desired function. However, the
function of a module or valve can be changed by changing its Node ID. Node ID is a unit number des-
ignation that is used by the instrument to distinguish between several units of the same type.
In a troubleshooting situation it may be useful to check a valve's or module's Node ID. Refer to Sec-
tion 9.16 Node IDs, on page 502 for a list of Node IDs for valves and modules.
Note: The function of a valve or module is defined by its Node ID, not by its physical position.
The Node ID is set by positioning the arrows of the one or two rotating switches at the back of the
valve. Use a screwdriver to set the arrows of the switches to the desired number.
The illustration shows an example of a valve module with

A B two rotating switches.
• The first rotating switch, labeled A, sets the tens
• The second switch, labeled B, sets the units.
Hardware installation of a module

The instruction below describes how to install a module in the instrument.
Note: The illustrations show the principle how to install an optional module. The position of the
module on the instrument and the used type of module will depend on the module being in-
stalled.
CAUTION
Disconnect power. Always switch off power to the ÄKTA pure instrument before
replacing any of its components, unless stated otherwise in the user documentation.

Step Action
1 Disconnect power from the instrument by switching off the instrument power switch.
2 Loosen the connectors and remove the tubing from the existing module.
Note:
This step does not apply for a Module Panel.
3 Loosen the module with a Torx T20 screwdriver.
4 Remove the module.
5 Disconnect the cable and secure it in the slit.

Step Action
6 Connect the cable to the module to be installed.
7 Insert the module.
8 Fasten it with a Torx T20 screwdriver.
Note: A warning message is displayed at start up if a module has been installed in the instrument
but not added to the current system configuration in UNICORN.
Install internal modules in the Extension box

The Extension box can be used to install extra modules on the ÄKTA pure instrument. See Extension
box, on page 85 for more information.

2.6 Accessories
2.6 Accessories
Introduction
This section describes the holders and other available accessories. These are used to attach and organize
columns, tubing and bottles to the ÄKTA pure instrument. The holders are attached to the instrument
using the holder rails on the left side and the front of the instrument.
Rail extension
The Rail extension rod can be used to attach accessories, eg., column holders or a Multi-purpose
holder. The rod has extra rails on both sides. Push the button of the rod to attach it to a holder rail.
The illustration below shows the Rail extension rod.
1 2
Part Function
1 Button
2 Extension rod
Multi-purpose holder
The Multi-purpose holder can be used to attach accessories, eg., a Loop holder or a cassette. Attach
the holder to a holder rail.
The illustration below shows the Multi-purpose holder.
1 2
Part Function
1 Attachment point for accessories
2 Snap-in to holder rails
3 Attachment points for tubing hold-

ers
4 Tab
3 4 3

2.6 Accessories
Loop holder
The Loop holder can be used to attach up to five 10 ml sample loops. Use two Multi-purpose holders
to attach the holder to a holder rail.
The illustration below shows the Loop holder.
Part Function
1 Upper attachment to multi-purpose holder
2 Lower attachment to multi-purpose holder
Column holder
The Column holder has one position for medium sized columns and one position for small sized columns.
The Column holder can also be used for bottles. Use two holders to attach long columns.
The illustration below shows the Column holder.
1 2 3
Part Description
4 1 Position for a medium sized column

or bottle
2 Position for a small sized column
3 Tab

2.6 Accessories
Column clamp
The column clamp can be used to attach small sized columns. Use two clamps to attach long columns.
The illustration below shows the Column clamp.
Part Description
1 Position for a column

1
2 Inner end tabs
Column holder rod

The Column holder rod can be used to attach several HiTrap™ columns. The holder has threaded ports
for HiTrap columns and tubing connectors. Push the button of the holder to attach the holder to a
holder rail.
The illustration below shows the Column holder rod.
2 3
Part Description
1
2 Button
3 Column holder rod

2.6 Accessories
Flexible column holder

The Flexible column holder can be used to attach, for example, HiScreen™ columns.
The illustration below shows the Flexible column holder.
Part Function
5 6
1 Lower tubing
2 Lower part
3 Snap-in-strips
7 4 Attachment part
4 5 Upper tubing
8 6 Lever
3 7 Upper part
8 Upper connector
9 Lower connector
9
1
Tubing holder spool

The Tubing holder spool is used to hold and arrange tubing.
The illustration below shows the Tubing holder spool.
2 3
Part Description
1 1 Positions for tubing
2 Tab

2.6 Accessories
Tubing holder comb

The Tubing holder comb is used to hold and arrange tubing.
The illustration below shows the Tubing holder comb.
2
Part Description
3 1 Positions for tubing
1
2 Tab
Bottle holder
The Bottle holder is used for holding bottles. For example, the Bottle holder can be attached to the
holder rails to hold a sample bottle.
The illustration below shows the Bottle holder.
Part Description
2
1 1 Position for bottle
Adapter for air sensor

The adapter for air sensor is used to hold an optional air sensor.
The air sensor with adapter is connected to the Bottle holder, see illustrations below.
3 Part Description
2 1 Air sensor
1
2 Air sensor adapter
3 Bottle holder

2.6 Accessories
Module Panel
Description
All positions in ÄKTA pure must be occupied. Positions not used for core or optional modules must be
fitted with a Module Panel.
Module Panels are installed in the same way as the other optional modules and the cable inside must
be connected to the Module Panel, see Hardware installation of a module, on page 76.
When an optional module is replaced by a Module panel, the removed module has to be deselected
in the software configuration, see Edit system properties, on page 127.
Illustration
The illustration shows the Module Panels installed in the example configuration used in this manual.

2.6 Accessories
Extension box
Description
The Extension box can be used to install extra modules on the ÄKTA pure instrument outside the system
chassis when the positions on the chassis are filled. It is possible to install up to six Extension boxes
with extra modules when using ÄKTA pure. See the Extension box instruction for more information.
Location
The illustration below shows an Extension box with a module mounted on the side of ÄKTA pure.
The Extension box can be mounted in three ways.

• On the side of ÄKTA pure.
• Standing on top of or next to ÄKTA pure.
• On a Rail extension rod (29-0113-52) on ÄKTA pure.

3 ÄKTA pure external modules
About this chapter

This chapter provides an overview of the external modules that can be connected to the ÄKTA pure
instrument. A brief description of how to connect external modules is also provided.
In this chapter
Section See page
3.1 External air sensors 87
3.2 Fraction collector F9-C 89
3.3 Fraction collector F9-R 103
3.4 Sample pump S9 and S9H 107
3.5 I/O-box E9 111
3.6 Connection of external modules 121

3.1 External air sensors

Introduction
Up to four external air sensors can be added to ÄKTA pure, and there are two different versions to
choose from. They differ in internal diameter and optimal position on the instrument.
The air sensors can be attached to the instrument using the rails and holders, see Adapter for air sensor,
on page 83. No Module Panels need to be removed.
In addition to be used for preventing air from entering the system, the external air sensors can be used
together with System pump A, or an Sample pump to load the entire sample volume.
Air sensor L9-1.5

L9-1.5 has a 1.5 mm inner diameter and is designed for i.d. 1.6 mm FEP, ÄKTA pure 25, and for i.d. 2.9
mm FEP tubing, ÄKTA pure 150, at the low pressure side before the pumps. It is installed in the flow
path before the system pumps or the sample pump and is used to prevent air entering the subsequent
module.
Air sensor L9-1.2

L9-1.2 has a 1.2 mm inner diameter and is designed for o.d. 1/16" tubing at the high pressure side after
the pumps. It is installed after the injection valve and is used to prevent air entering the column.
Note: L9-1.2 can be used in the same fashion as L9-1.5 if O.D. 1/16" ETFE tubing is used between
the air sensor and the inlet valve.
Tubing connections
Air sensor L9-1.5
Connection Tubing Connector Tubing

between... length
L9-1.5 and inlet FEP, o.d. 1/8", i.d. FEP, o.d. 3/16", i.d. Tubing connector, Tubing connector, See note
valves 1.6 mm 2.9 mm 5/16" + Ferrule 5/16" + Ferrule
(yellow), 1/8" (blue), 3/16"
Note: When sample is loaded at high flow rate and the external air sensor is placed before the pump
that is used for loading the sample, it is necessary to use longer tubing to ensure that no air
reaches the pump. Use tubing with the minimum lengths given below between the valve located
before the pump and the external air sensor. The length applies for maximum flow rate, but
shorter tubing can be used at lower flow rates.
• Sample pump S9: 40 cm.
• Sample pump S9H: 20 cm.

Air sensor L9-1.2
Connection Tubing Connector Tubing

between... length
(mm)
Injection valve and L9-1.2 PEEK, o.d. 1/16" Fingertight connector, 100
1/16"
L9-1.2 and Column valve/the PEEK, o.d. 1/16" Fingertight connector, 100
connected column 1/16"

3.2 Fraction collector F9-C

About this section
This section shows an overview of Fraction collector F9-C.
Technical details are found in the ÄKTA pure User manual.
In this section
Section See page
3.2.1 Function 90
3.2.2 Fraction collector F9-C illustrations 92
3.2.3 Cassettes, Cassette tray and racks 96
3.2.4 Connect tubing to the ÄKTA pure instrument 102

3.2.1 Function
3.2.1 Function
Introduction
Fraction collector F9-C can collect fractions in deep well plates, tubes of different sizes or bottles. Up
to six cassettes for deep well plates and tubes can be used. The cassettes can be used in any combi-
nation and are placed on the Cassette tray. A rack for 50 ml tubes and a rack for 250 ml bottles are
also available.
Scanner functions detect which types of trays, racks, cassettes and deep well plates that are used in
each run.
The Cassette tray or one of the racks is placed inside the fraction collector. A height exclusion bar en-
sures that the bottles, tubes and deep well plates are correctly positioned and cannot damage the
Dispenser head. The Tray catch and positioning discs on the floor of the fraction collector guide the
Cassette tray or the rack into the correct position.
Scanning of Cassettes
When the door of the fraction collector is closed automatic scanning is performed. There are two types
of scanning procedures:
• Full scan: Scanning of Cassette type codes to determine which types of Cassettes are used, and
scanning of rows and columns in deep well plates to identify which types of plates are used (24,
48, or 96 wells). Full scan is performed only when the system is in state Ready.
• Quick scan: Scanning of Cassette type codes to determine which type of Cassettes are used. Quick
scan is performed during the run to ensure that correct Cassettes are placed in the Fraction collector.
Fractionation modes to avoid spillage

Three fractionation modes are available, all of which avoid spillage between wells or tubes during
fractionation:
• Accumulator: The accumulator is used to collect liquid during movement between wells, tubes or
bottles. The liquid is then dispensed in the next well or tube. Fractionation with accumulator can
be used at all flow rates.
• DropSync: When using DropSync the sensors in the Dispenser head detect when a drop is released
from the nozzle. The Dispenser head moves to the next well or tube just after a drop is released.
Fractionation with DropSync can be used at flow rates up to 2 ml/min. If the cassettes are placed
near the waste funnel the DropSync mode can be used at higher flow rates. Volatile solutions and
solutions with low surface tension may require a lower flow.
• Automatic: The fraction collector uses the Drop Sync mode for flow rates up to 2 ml/min and auto-
matically switches to Accumulator mode for higher flow rates.

3.2.1 Function
Fractionation arm positions

• Home position: The home position is used when the fraction collector is idle. The Fractionation arm
is positioned in the front of the interior of the fraction collector and the Dispenser head is positioned
over the waste funnel. This position is called Waste (Frac) in UNICORN.
• Frac cleaning position: The Frac cleaning position is used for convenient cleaning of the Dispenser
head. The Fractionation arm is positioned in the front of the interior of the fractionation collector
and the Dispenser head is moved to the center of the Fractionation arm.

3.2.2 Fraction collector F9-C illustrations
Introduction
This section provides illustrations of Fraction collector F9-C. The main features and components are
indicated.
Front view
The illustration below shows the main parts of the exterior of Fraction collector F9-C.
1 2 3 4 5 6
Part Description
1 Fractionation indicator
Symbol indicating that fractionation is ongoing. Do not open the door while the indicator
is lit.
2 Door
3 Window
4 Door handle
5 Tubing connector for outlet valve tubing
6 Vents

Rear view
The illustration below shows the rear view of Fraction collector F9-C.
3 2
Part Description
1 Vents
2 UniNet-9 D-type connector (for communication and power supply)
3 Waste tube

Interior
The illustration below shows the main parts of the interior of Fraction collector F9-C.
3 4 5 6 7 8
2 9
10
11
12
14 13
Part Description
1 Fractionation arm guide rail
2 Fractionation arm main rail
3 Lamp
4 Tubing guide
5 Tubing connection
6 Dispenser head
7 Fractionation arm
8 Tubing guide
9 Height exclusion bar
10 Waste funnel
11 Waste tube
12 Tray catch
13 Waste groove, in case of overflow
14 Tray guides

Dispenser head
The illustration below shows the Dispenser head of Fraction collector F9-C.
1 2 3
6 5
Part Description
1 Dispenser head
2 Nozzle
3 Dispenser head cover
4 Accumulator (back part of Dispenser head)
5 Drop sync sensor
6 Type code reader

3.2.3 Cassettes, Cassette tray and racks
Introduction
Fractions can be collected in deep well plates and in tubes of different sizes. A number of cassettes
and racks for different tubes and deep well plates are available. The cassettes are placed on a rack
with six cassette positions. The Cassette type codes are scanned by the Cassette code reader to deter-
mine the type of Cassette.
Available cassettes, trays and racks

The following Cassettes and racks are available:
• Cassette 3 ml tubes (for 40 tubes)
• Cassette for deep well plate (24, 48, 96 wells)
• Cassette tray (for six cassettes)
• Rack for 50 ml tubes (for 55 tubes)
• Rack for 250 ml bottles (for 18 bottles)
For information on dimension requirements for tubes and deep well plates to be used in the fraction
collector, see Fraction collector tubes and bottles, on page 99 and Deep well plates, on page 100 respec-
tively.
Illustrations of Fraction collector F9-C tray and

racks
The illustrations below show the Cassette tray, the Rack for 50 ml tubes and the Rack for 250 ml bottles.
The fronts of the tray and the racks are marked with the GE-logotype.
In the Cassette tray, the cassette positions are marked 1 to 6.
Cassette tray Rack for 50 ml tubes Rack for 250 ml bottles

Note: The tray and racks are inserted into the fraction collector with the GE-logotype facing outwards.
Note: Do not use the Cassette tray when a rack for tubes or bottles is placed in the fraction collector.
Illustration of Cassettes on the Cassette tray

The illustration below shows Cassettes placed on the Cassette tray.
Part Description
1 Cassette tray
2 Cassette position number
3 Cassette
4 Cassette type code
5 Tubes or deep well plates placed in a Cassette

QuickRelease function
The Cassettes for the smaller tube sizes (3, 8, and 15 ml) have a built-in QuickRelease function. The
QuickRelease function enables easy handling of tubes in the Cassettes. With the QuickRelease device
in lock position the tubes are fastened in the Cassette and can easily be emptied. With the QuickRelease
function in release position, the Cassette can easily be loaded with tubes and used tubes can easily
be discarded.
Step Action
1 Load the Cassette with tubes before fractionation:

• Pull the QuickRelease device to the release position.
• Load the Cassette with tubes, and press the QuickRelease device to the locked position.
Release position Locked position
3 After fractionation, pull the QuickRelease device to the release position and remove the
tubes containing the fractions of interest.

Step Action
4 Empty and discard the remaining tubes:

• Press the QuickRelease device to the lock position, and empty the remaining tubes.
• Pull the QuickRelease device to the release position, and discard the tubes.
Empty the tubes Discard the tubes
Fraction collector tubes and bottles

The tubes and bottles used in Fraction collector F9-C must fulfill the requirements listed in the table
below. Examples of manufacturers are also listed in the table.
Tube or bottle Diameter (mm) Height (mm) Examples of manufac-

size (ml) turers
Min. Max. Min. Max.
3 10.5 11.5 50 56 NUNC™
5 10.5 12 70 76 VWR™
8 12 13.3 96 102 BD™ Biosciences, VWR
15 16 17 114 120 BD Biosciences
50 28 30 110 116 BD Biosciences
250 ml bottle L: 55 L: 64.5 - 121 Nalgene™, Kautex™

W: 55 1 W: 641
1 Length and width of the rectangular bottle base

Maximum flow rate

Fraction collection can be performed at different maximum flow rates depending on the size of the
tubes that are used. The table below lists the maximum flow rates for the Fraction collector tubes.
Tube size [ml] Maximum flow rate [ml/min]
3 15
8 25
15 50
50 150
250 150
Deep well plates

Requirements
The deep well plates used in Fraction collector F9-C must fulfill the requirements listed in the table
below.
Property Specification
No. of wells 24, 48, or 96
Shape of wells Square, not cylindrical
Well volume 10, 5, or 2 ml
Approved deep well plates

The plates listed in the table below are tested and approved by GE to be used with Fraction collector
F9-C.
Plate type Manufacturer Part no.
96 deep well plate GE 7701-5200 (Whatman™)
BD Biosciences 353966
Greiner Bio-One 780270
Porvair Sciences 219009
Seahorse Bioscience™ S30009
Eppendorf™ 951033405/ 0030 501.306
48 deep well plate GE 7701-5500 (Whatman)
Seahorse Bioscience S30004

Plate type Manufacturer Part no.
24 deep well plate GE 7701-5102 (Whatman)
Seahorse Bioscience S30024
Maximum flow rate

Fraction collection can be performed at different maximum flow rates depending on what type of deep
well plates that are used. The table below lists the maximum flow rates for the different plate types.
Plate type Maximum flow rate (ml/min)
96 deep well plate 10

3.2.4 Connect tubing to the ÄKTA pure instrument
3.2.4 Connect tubing to the ÄKTA pure instrument
Connect tubing
Fraction collector F9-C is delivered with all internal tubing in place. The tubing between the fraction
collector and purification instrument need to be installed.
Follow the instructions in the table below to connect the tubing from the ÄKTA pure instrument to the
fraction collector.
Step Action
1 Connect the tubing Frac to the Frac port on the outlet valve on the ÄKTA pure instrument.
2 Connect the other end of the Frac tubing to the inlet port on the fraction collector.
3 Adjust the delay volume setting in UNICORN, see Section 9.8.8 System settings - Tubing
and Delay volumes, on page 459 for more details.

3.3 Fraction collector F9-R

About this section
This section shows an overview of Fraction collector F9-R.
Technical details are found in the ÄKTA pure User manual and ÄKTA avant User manual.
Function
The fraction collector collects fractions from ÄKTA pure purification runs.
The fraction collector can be used for:
• Fixed volume fractionation
• Peak fractionation
• Combined fixed volume fractionation and peak fractionation
Fraction collector F9-R has the following function for reducing sample spill during fractionation:
• Drop Sync
Front view illustration

The illustration below shows the main parts of the Fraction collector.
3 4
5
2 6
1 7

Part Function
1 Lock knob
2 Stationary part of delivery arm
3 Delivery arm
4 Tubing connector
5 Tube sensor
6 Collection tubes
7 Tube rack
8 Base unit
Connector panel illustration

The illustration below shows the main parts of the connector panel on the fraction collector.
1 22
Part Function
1 Node ID switch
2 UniNet-9 F-type connector (for communication and power supply)

Available tubes
For Fraction collector F9-R the fractions are collected in tubes of different sizes.
Tubes with the following diameter can be used with Fraction collector F9-R:
• 12 mm
• 18 mm
• 30 mm
The tubes can have a tube length between 50-180 mm.
Illustration of the Fraction collector F9-R Tube

rack
Each tube rack is made up of a combination of a Bowl, Tube support, Tube guide and Tube holder. For
more information on the assembly of the tube rack, see Assemble the Tube rack, on page 199. For infor-
mation on which Tube rack to use, see Tube rack inserts, on page 200.
1
2
6
5 3
4
Part Function
1 Single cutout
2 L-shaped cutout
3 Bowl
4 Tube support
5 Tube guide
6 Tube holder
Note: Note that the tube guide has both single and L-shaped cutouts, while the tube holder only has
single cutouts. See Single and L-shaped cutouts, on page 200 for more information.

Connect tubing to ÄKTA pure

Step Action
1 Lift out the Tubing holder (4) from the Delivery arm (1).
3
5
4
2 Loosen the nut of the Tubing holder. Do not remove the Tubing holder nut (5) from the
Tubing holder.
3 Insert the tubing through the Tubing holder.
4 Place the Tubing holder with the tubing over the Tube adjustment cavity (2) of the Delivery
arm. Push the tubing down against the bottom of the Tube adjustment cavity, and then
fingertighten the Tubing holder nut. This ensures the correct length of the exposed tubing
end (6).
5 Re-install the tubing holder in the Delivery arm.
6 For Fraction collector F9-R: Connect the tubing from the fraction collector to the port
Frac on the outlet valve.
For Fraction collector F9-R, 2nd: Connect the tubing from the second fraction collector
to:
• port Out 10 on Outlet valve V9-O or V9H-O
• port Out 1 on Outlet valve V9-Os or V9H-Os
7 Adjust the delay volume setting in UNICORN to the volume of the tubing, see Section 9.8.8
System settings - Tubing and Delay volumes, on page 459 for more details.

3.4 Sample pump S9 and S9H

Introduction
This section describes the design and function of Sample pump S9 and S9H.
Function of the Sample pump

The Sample pump is dedicated to direct loading of sample onto a column, or to filling of sample loops
or Superloops. The pump consists of two pump heads that work alternately to give a continuous, low
pulsation, liquid delivery. There are two sample pump configurations available, one for ÄKTA pure 150
and one for ÄKTA pure 25. To ensure delivery of correct liquid volume, the pumps must be free from
air. Each pump head is equipped with a purge valve that is used for this purpose.
The Sample pump also comprises a pressure monitor.
The table below contains the operating limits and labels of the sample pump configurations.
Configuration Pump label Pump type Flow rate Max. pressure
Sample pump S9, P9-S P9-S 0.001 to 50 ml/min 10 MPa

ÄKTA pure 25
Sample pump S9H, P9H S P9H 0.01 to 150 ml/min 5 MPa

ÄKTA pure 150
Front view
3 4 5
2 6
9 8

Part Function
1 Pump head: Encapsulates the inner parts of the pump
2 Pump rinsing liquid tube holder
3 Outlet port with check valves
4 Purge valve: Used to remove air from the pump
5 Sample pump outlet port
6 Sample pressure monitor
7 Vents
8 Sample pump inlet port
9 Inlet manifold
Rear view
Part Function
1 UniNet-9 type D port
2 IP cover: Protects pump electronics from liquid spill
3 Vents

Sample pump piston rinsing system

A seal prevents leakage between the pump chamber and the drive mechanism. The seal is continuously
lubricated by the presence of solvent. The pump piston rinsing system continuously flushes the low
pressure chamber behind the piston with a low flow of 20% ethanol. This prevents any deposition of
salts from aqueous buffers on the pistons and prolongs the working life of the seals.
For instructions on how to fill the rinsing system, see Section 7.3.1 Change pump rinsing solution, on
page 262
Illustrations of the sample pump piston rinsing

system
The illustrations below show the parts, tubing and flow path of the sample pump piston rinsing system.
4 5
Parts and tubing Rinsing system flow path
Part Function
3 Inlet tubing, from the rinsing liquid tube to the left pump head (lower rinsing system
connection) .
4 Tubing between the left pump head (upper rinsing system connection) and the right
pump head (lower rinsing system connection).
5 Outlet tubing, from the right pump head (upper rinsing system connection) to the rinsing
liquid tube.

Connect tubing to the ÄKTA pure instrument


label length
ÄKTA ÄKTA ÄKTA pure 25 ÄKTA pure 150 (mm)
pure 25 pure 150
InS Sample inlet valve port FEP FEP Tubing connector Tubing connector 580
Out to sample pump o.d. 1/8", o.d. 3/16", 5/16" with Ferrule 5/16" with Ferrule
inlet manifold i.d. 1.6 i.d. 2.9 (yellow), 1/8" (blue), 3/16"
mm mm
No Sample container to FEP FEP Tubing connector Tubing connector optional

label sample pump inlet mani- o.d. 1/8", o.d. 3/16", 5/16" with Ferrule 5/16" with Ferrule
fold (sample inlet valve i.d. 1.6 i.d. 2.9 (yellow), 1/8" (blue), 3/16"
not used) mm mm
3S Sample pump pressure PEEK, PEEK, Fingertight con- Fingertight con- 530
monitor to Inlet valve o.d. 1/16", o.d. 1/16", nector, 1/16" nector, 1/16"
port SaP i.d. 0.75 i.d. 1 mm
mm
Note: The recommended location of the sample pump is on the bench to the left of the ÄKTA instru-
ment. If the sample pump is placed elsewhere, longer tubing may be needed.

3.5 I/O-box E9
3.5 I/O-box E9
About this section
This section describes the design and the function of the I/O-box E9
In this section
Section See page
3.5.1 Overview of the I/O-box 112
3.5.2 Analog connector and signals 114
3.5.3 Digital connector and signals 116
3.5.4 Connect external equipment to the I/O-box 118

3.5 I/O-box E9
3.5.1 Overview of the I/O-box
Function of the I/O-box

The I/O-box E9 is used to interface other equipment in order to measure parameters such as refractive
index, light scattering and fluorescence. See Requirements on connected equipment, on page 118 for
information on requirements of the equipment that can be connected to ÄKTA pure. The I/O-box can
control external equipment by a digital output signal, as well as detecting the state of them by digital
inputs. It is also possible to send out internal detector signals to external equipment.
Using two I/O-boxes

It is possible to install up to two I/O-boxes when using ÄKTA pure. If two I/O-boxes are to be used, the
second I/O-box has to be configured as I/O-box E9, 2nd. The configuration is defined by the Node ID
of the I/O-box. The Node ID is set by positioning the arrow of two rotating switches at the back of the
I/O-box, see Connectors, on page 113 and Section 9.16 Node IDs, on page 502.
Location
The illustration below shows the I/O-box, its recommended location and connection.
Part Description
1 I/O-box
2 UniNet-9 F-type cable
3 Multi-purpose holder
4 Clip

3.5 I/O-box E9
Connectors
Part Description
Analog in/out Signal connector for analog input and output signals.
UniNet-9 Connector used to connect the I/O-box to the ÄKTA pure instru-
ment.
Status Status indicator for service purposes.
Node ID Switches used to configure I/O-box E9 as I/O-box E9 or I/O-box

E9, 2nd.
Digital in/out Signal connector for digital input and output signals.

3.5 I/O-box E9
3.5.2 Analog connector and signals
Analog connector pins

9 8 7 6
5 4 3 2 1
Part Function
1 Analog in signal 1 +
2 Analog in signal 1 - (or signal ground)
3 Shield, analog in (both ports)
4 Analog in signal 2 +
5 Analog in signal 2 - (or signal ground)
6 Calibration pin for service purposes

Analog out signal (1.9 V)
Note:
Do not use for other purposes.
7 Analog out signal 1
8 Signal ground, analog out (both ports)
9 Analog out signal 2
Analog signals
All analog input and output signals are confined to the same Analog in/out connector.

3.5 I/O-box E9
Analog input signals

There are two analog input channels from which analog input signals can be used for peak detection,
or data collection in UNICORN. It is possible to auto-zero the input signals, which means that the current
value will be displayed as 0 V in UNICORN. This can be done individually for the two analog input
channels. The auto-zero value is saved between runs and power-offs. The auto-zero value can be reset.
Parameter Description
Input signal range -2000 to 2000 mV
Analog output signals

There are two analog output channels from which analog output signals and system parameters, that
is, UV, cond, conc B, temperature and pH, are transferred to the external connected equipment, for
example, light scattering detectors or plotters.
Output signal range -1000 to 1000 mV
Default output The user defines the default output level for the selected signal.
Full scale output The user sets the desired output signal value of, e.g., mAU, % or mS, which
will corresponds to the full scale output voltage 1000 mV.
Negative full scale out- The corresponding negative full scale output value is set automatically.
put For example, if the full scale output is set to 2000 mAU, a UV value of 500
mAU will give an output voltage of 250 mV, and -100 mAU will give -50
mV output voltage. A signal value of 0 mAU, 0% or 0 mS will always give
an output voltage of 0 mV.
Fix point Zero is always a fix point. A desired output signal of, i.e., 0 mAU, 0% or 0
mS corresponds to an output voltage of 0 mV. At power-on, an output
signal of 0 mV is transferred to the connected equipment, until the output
signal range values are set.
Note: No warning will be displayed in UNICORN if the analog output signal exceeds the set full scale
output value or is less than the set negative full scale output value.

3.5 I/O-box E9
3.5.3 Digital connector and signals
Digital connector pins

9 8 7 6
5 4 3 2 1
Part Function
1 Digital in signal 1
5 Signal ground
6 Digital out signal 1
Digital signals
All digital input and output signals will be confined to the same D-sub connector and have a common
ground. The four input signals will be scanned synchronously, and the outputs will be set synchronously.
Digital input signals

The digital in-signal can be used to monitor external equipment by registering, for example, error signals
or event marks. An event mark can be used as a trigger for watches. The measured digital signals can
be shown as a curve in UNICORN. The unit will handle both open/closed circuit and TTL-type voltage
signals. An open circuit is interpreted as logical 1 and a closed circuit as logical 0.
Note: A closed circuit is always closed against signal ground.

3.5 I/O-box E9
Input connection UNICORN interpretation
Open circuit Logical 1
Applied voltage 3.5 to 5.0 V
Closed circuit Logical 0
Applied voltage 0 to 0.8 V
Digital output signals

The digital output signal can be used to control external equipment that can receive digital signals,
such as pumps or fraction collectors. The digital output signals define an open or closed circuit, where
a logical 1 will result in an open circuit and a logical 0 will give a closed circuit. The default level, 1 or
0, is set by the user. The level can be changed by instructions either manually, in System Settings or
by a method. It is possible to send pulses from the current level, with a pulse length of 0.1 s to 10 s.
Note: A closed circuit is always closed against signal ground.

3.5 I/O-box E9
3.5.4 Connect external equipment to the I/O-box
Requirements on connected equipment

The signal characteristics for the connected equipment are described in the following tables. All con-
nected equipment must have a common ground.
Analog input
Parameter Value
Channels 2
Range ±2000 mV
Input impedance 1 MΩ
Accuracy ±(0.1% + 0.2 mV)
Analog output
Parameter Value
Channels 2
Range ±1000 mV
Input impedance 100 kΩ
Accuracy ±(0.3% + 1 mV)
Digital input
Parameter Value
Channels 4
Compatibility TTL, open/closed circuit
Digital output
Parameter Value
Channels 4
Compatibility Open/closed circuit
Required material
The following material is required:
• Flat-blade screwdriver, 2 mm
• Shielded cable with 9 conductors, 4 to 8 mm diameter

3.5 I/O-box E9
• Wire stripping tool
Instruction
Follow the instructions to connect one or two external cables to the supplied D-sub connectors.
Step Action
1 Open the connector housing by removing housing screw and unlatch the housing top
shell using a flat-blade screwdriver.
2 Locate the connection block mounted on the PCB board. The screw terminals has numbers
corresponding to the connector contacts.
1 2 3 4 5
6 7 8 9
3 Strip the signal cable.

• Strip-off 50 mm of the shield insulator.
• Strip-off 4 mm of the single conductor insulation.
4 Loosen the strain relief clamp and insert the cable with the shield under the strain relief
clamp. Fasten the strain relief clamp over the cable shield.
Tip:
The connection block can be rotated inside the housing in order to position the screw ter-
minals for left side or right side cable entry.
5 Insert and fasten the single conductors in the screw terminals.
6 Close the housing top shell with the latch and screw the housing together.
System properties
Follow the instruction to update the system properties.
Step Action
1 Open the system properties Edit dialog box.
2 In the Component types list, click Other.
3 Select the I/O-box (E9) or I/O-box 2 (E9) check box in the Component selection list. Then
click OK.

3.5 I/O-box E9
System settings
Default values for digital out ports, noise reduction and configuration of analog out ports can be set.
Instruction name Description
Digital out X Sets the value of the signal sent out by digital port number X to
either 0 or 1. The default value is 1.
Noise reduction analog in X Filters the noise in the analog signal in port number X.
Alarm analog in X Enables or disables the alarm for the analog signal in port number
X. When enabled, it sets the alarm limits for the analog signal. If
the alarm is enabled and the analog signal falls outside the set
limits, an alarm will be triggered and the method will be paused.
Alarm digital in X Enables or disables the alarm for the signal in digital port number
X. The alarm can be triggered by either of the signal values, 0 or
1. If the alarm is enabled and the condition set in ‘Value’ occurs,
an alarm will be triggered and the method will be paused.
Configure analog out X Enables the user to send one of the pre-defined signals (UV signal,
conductivity, temperature, pH or concentration of eluent B) to the
analog out port number X, and also to set the range of that signal.
Note: The delay volume has to be updated if an external component is added to the flow path.

3.6 Connection of external modules

Introduction
The external modules are not installed in the instrument cabinet, but are connected via a UniNet-9
cable at the back of the system. it is possible to install up to six external modules with F-type connectors
and up to two external modules with D-type connectors at the same time.
Some of the external modules are also connected to the ÄKTA pure instrument by tubing. See the ap-
propriate section in Chapter 3 ÄKTA pure external modules, on page 86 for detailed instructions on
how to install this tubing, if applicable.
F-type and D-type connectors

The illustrations below show the F-type and D-type connectors.
F-type D-type
Constraints
The table below indicates usage constraints for the different external modules.
External module Connector Constraints
I/O-box E9 F-type I/O-box E9 has no constraints.
I/O-box E9, 2nd F-type I/O-box E9, 2nd requires I/O-box E9.
External air sensor L9 F-type External air sensor L9 has no constraints.
External air sensor L9, 2 F-type External air sensor L9, 2 requires External air sensor L9.
External air sensor L9, 3 F-type External air sensor L9, 3 requires External air sensor L9,
2.
External air sensor L9, 4 F-type External air sensor L9, 4 requires External air sensor L9,
3.
Fraction collector F9-C D-type Fraction collector F9-C requires an Outlet valve.
Fraction collector F9-R F-type Fraction collector F9-R requires an Outlet valve.

External module Connector Constraints
Fraction collector F9-R, 2nd F-type Fraction collector F9-R, 2nd requires Fraction collector
F9-R or Fraction collector F9-C.
Sample pump S9 and S9H D-type Cannot be used at the same time as Mixer valve V9-M
or V9H-M.
Note: To optimize signal quality, the total cable length connecting all external modules to the ÄKTA
pure instrument should not exceed 10 m.
ÄKTA pure connector plate

The connector plate with the UniNet-9 connectors is located on the back of the ÄKTA pure instrument.
Connector Connector name Function
9 Test Connector point for service.
NOTICE
Do not connect any module to the connector Test on the ÄKTA pure instrument.
Note: Plug all unused UniNet-9 ports (ports 1 to 8) on the ÄKTA pure instrument with jumpers.
Note: The connector Test should be protected by a plastic lid. Do not plug the connector with a
jumper.

4 System configuration
About this chapter

This chapter describes hardware configuration of the ÄKTA pure instrument and how to install optional
modules and add them in UNICORN.
In this chapter
Section See page
4.1 Configuration overview 124
4.2 Configure modules 130
4.3 General system settings 145

4.1 Configuration overview

Introduction
ÄKTA pure is a flexible system that allows the user to configure both hardware and software to meet
many purification needs. The instrument can be easily extended with additional valves, detectors and
fraction collectors. There are a large number of different hardware modules to choose from in order
to customize the number of columns, inlets, outlets, detectors and ways to apply and collect samples.
System configuration steps

The system configuration consists of four main steps:
1 Select modules and positions
2 Install the module(s)
3 Edit system properties
4 Edit system settings
Select modules and positions

Optional modules can be placed in any of the available positions in the cabinet. Its function is determined
by the module type and node ID. However, to achieve an optimal flow path for a purification run, most
modules have a dedicated location in the cabinet. The dedicated location for all modules and, if appli-
cable, any constraints are described in the table below. Each location is marked with a number, which
can be seen in the illustration below.
8 9 11 12 13
6 7 10
1 2 3 4 5
All positions in ÄKTA pure must be occupied. Positions not used for core or optional modules should
be fitted with a Module Panel.

Recommended module position
Module Recommended location Constraints
Column valve V9-C or 9 Column valve V9-C or V9H-C cannot be

V9H-C used at the same time as Column valve
V9-Cs or V9H-Cs.
Column valve V9-C or 8 (V9-C or V9H-C) These positions give the shortest possible
V9H-C with 9 (V9-C2 or V9H-C2) flow path.
Second column valve V9-
C2 or V9H-C2
Column valve V9-Cs or 9 Column valve V9-Cs or V9H-Cs cannot

V9H-Cs be used at the same time as Column
valve V9-C or V9H-C.
Conductivity monitor C9 7 Conductivity monitor C9 has no con-

straints.
Injection valve V9-Inj or 6 Core module.

V9H-Inj
Inlet valve V9-IAB or V9H- • 4 Possible combinations of inlet valves:

IAB
• 5, if Inlet valve V9-IAB • Inlet valve A and Inlet valve B.
or V9H-IAB is used in • Inlet valve AB together with Inlet valve
combination with Inlet A or Inlet valve B.
valve V9-IA or V9H-IA.
• One Inlet valve AB.
Inlet valve V9-IA or V9H-IA 4 or
Inlet valve V9-IB or V9H-IB 5 • No installed inlet valves.
Sample inlet valve V9-IS or 8 Cannot be used at the same time as Mixer
V9H-IS valve V9-M or V9H-M.
Inlet valve V9-X1 or V9H- None Inlet valve V9-X1 or V9H-X1 has no con-
X1 straints, and has no support in the
method phases.
Inlet valve V9-X2 or V9H- None Inlet valve V9-X2 or V9H-X2 requires Inlet
X2 valve V9-X1 or V9H-X1, respectively. It has
no support in the method phases.
Loop valve V9-L or V9H-L 8 Loop valve V9-L or V9H-L has no con-
straints, but is connected with the injec-
tion valve by replacing the loop.
Mixer M9 0 Core module.
Mixer valve V9-M or V9H- 3 Cannot be used at the same time as

M Sample pump S9 or S9H.

Module Recommended location Constraints
Outlet valve V9-Os or V9H- 1 Cannot be used at the same time as

Os Outlet valve V9-O or V9H-O.
Outlet valve V9-O or 1 Cannot be used at the same time as

V9H-O Outlet valve V9-Os or V9H-Os.
pH valve V9-pH or V9H-pH 2 pH valve shall always be connected after

the column due to pressure constraints
and fractionation control. The delay vol-
ume calculations will be effected if the
valve is located elsewhere. Normally it is
situated between the monitor and the
Outlet valve.
Versatile valve V9-V or None • Versatile valve V9-V or V9H-V has no

V9H-V constraints.
Versatile valve 2 • Versatile valve 2 requires Versatile
valve V9-V.
Versatile valve 3
• Versatile valve 3 requires Versatile
Versatile valve 4 valve 2.
• Versatile valve 4 requires Versatile
valve 3.
UV monitor U9-M 10 UV monitor U9-M cannot be used at the

same time as UV monitor U9-L.
Note:
UV monitor U9-L, 2nd can be used at the
same time as UV monitor U9-M.
UV monitor U9-L 11 UV monitor U9-L:

• Cannot be used at the same time as
UV monitor U9-M.
• Requires the Multi-module panel.
Note:
UV monitor U9-L, 2nd can be used at the
same time as UV monitor U9-L.
UV monitor U9-L, 2nd None UV monitor U9-L, 2nd requires UV monitor

U9-L or UV monitor U9-M.

Install the modules

Detailed information about installation of the modules is found in Chapter 2 The ÄKTA pure instrument,
on page 13 and Chapter 3 ÄKTA pure external modules, on page 86.
Below is a quick guide of how to perform a module hardware installation.
Step Action
1 Switch off the power.
2 Check/set node ID. See NodeID list.
3 Install the module.
4 Connect tubing.
Edit system properties

When a new module has been installed, the system properties have to be updated in UNICORN. The
system will restart automatically when the configuration has been changed in System Properties and
the system can be reconnected.
The following instruction gives a general description of how to update the system properties in UNICORN.

Step Action
1 • In the Administration module, choose Tools:System Properties or click the System

Properties icon to open the dialog.
Result: The System Properties dialog is displayed.
• Select the system of interest in the System Properties dialog.
• Click the Edit button.
Note:
Only active systems can be edited.
Result: The Edit dialog is displayed.
2 Select the component type of interest from the Component types list.
Result: All available components are shown in the Component selection list.
• Click the checkbox to select the added component.
• When applicable, choose the appropriate Property.
Note:
Instrument modules are referred to as Components in UNICORN.
3 Click the OK button to apply the changes.

There are five main types of modules (named components in UNICORN) to select from:
• Valves and pumps
• Monitors and sensors
• Fraction collectors
• Other (e.g., I/O-box)
• Core components (always present)
Multiple choices are not shown unless a component has been selected. The second component is only
available when the first has been selected. The selection made is reflected in which instructions and
phase properties that are available.
Edit system settings

It may be necessary to edit the System Settings when the configuration of the system is changed. For
example, if the change in configuration affects the delay volume following the UV monitor (or other
monitor connected via the I/O-box) the appropriate system settings for Tubing and delay volumes
have to be updated. This is to ensure that the fractions marked in the chromatogram corresponds to
the actual collected fractions.
Other system settings might also need to be edited for some optional modules.
The Edit dialog in which to edit the system settings is shown below.
In this dialog it is possible to set the parameters for the available instructions.
All system settings available for ÄKTA pure are found in Section 9.8 System settings, on page 448.

4.2 Configure modules

In this section
This section describes the software configuration that must be set for the individual modules that are
to be used for a specific run.
A general description of how to update the system properties are found in Edit system properties, on
page 127.
A general decription of how to edit system settings are found in Edit system settings, on page 129.
Section See page
4.2.1 Configuration of inlet valves 131
4.2.2 Configuration of Mixer valves 132
4.2.3 Configuration of Loop valves 133
4.2.4 Configuration of column valves 134
4.2.5 Configuration of Versatile valves 136
4.2.6 Configuration of pH valves 137
4.2.7 Configuration of outlet valves 138
4.2.8 Configuration of UV monitors 139
4.2.9 Configuration of Conductivity monitor 140
4.2.10 Configuration of external air sensors 141
4.2.11 Configuration of fraction collectors 142
4.2.12 Configuration of I/O-box 144

4.2.1 Configuration of inlet valves
4.2.1 Configuration of inlet valves
System properties
Follow the instruction below to update the system properties.
Step Action
1 Open the system properties Edit dialog.
2 Select Valves and pumps from the Component types list.
3 Select components and properties according to the following table:
Valve Component selection Property
Inlet valve V9-IA Inlet A V9-IA (7-ports)
Inlet valve V9H-IA V9H-IA (7-ports)
Inlet valve V9-IB Inlet B V9-IB (7-ports)
Inlet valve V9H-IB V9H-IB (7-ports)
Inlet valve V9-IAB or V9H- Inlet A V9-IAB part A (2-ports)

IAB used for inlet A or
V9H-IAB part A (2-ports)
Inlet valve V9-IAB or V9H- Inlet B V9-IAB part B (2-ports)

IAB used for inlet B or
V9H-IAB part B (2-ports)
Sample inlet valve V9-IS Sample inlet V9-IS N/A
Sample inlet valve V9H-IS Sample inlet V9H-IS N/A
Inlet valve V9-IX Inlet valve X1 (V9-IX) N/A
Inlet valve V9H-IX Inlet valve X1 (V9H-IX) N/A
Inlet valve V9-IX, 2nd Inlet valve X2 (V9-IX) N/A
Inlet valve V9H-IX, 2nd Inlet valve X2 (V9H-IX) N/A
System settings
There are no system settings available for the inlet valves.

4.2.2 Configuration of Mixer valves
4.2.2 Configuration of Mixer valves
System properties
Step Action
3 Select Mixer valve (V9-M) or Mixer valve (V9H-M) in the Component selection list.
System settings
The flow rate for Mixer by-pass wash can be set.
Mixer by-pass wash settings Sets the flow rate used during Mixer by-pass wash and defines
wash volumes for mixer by-pass wash options.
Note:
The flow rate should not exceed 10 ml/min if narrow inlet tubing
(i.d. 0.75 mm) is used.

4.2.3 Configuration of Loop valves
4.2.3 Configuration of Loop valves
System properties
Step Action
3 Select Loop valve (V9-L) or Loop valve (V9H-L) in the Component selection list.
System settings
The flow rate for Loop wash can be set.
Loop wash settings Sets the flow rate used during Loop wash.
Note:

4.2.4 Configuration of column valves
System properties
Step Action
Column selection valve V9-C Column valve V9-C (5-columns)
Column selection valve V9H-C Column valve V9H-C (5-columns)
Second column selection valve Column valve V9-C2 (5-columns)

V9-C2
Second column selection valve Column valve V9H-C2 (5-columns)

V9H-C2
Column control valve V9-Cs Column valve V9-Cs (1-column)
Column control valve V9H-Cs Column valve V9H-Cs (1-column)
System settings
After selecting Column valve V9-Cs, V9H-Cs or no column valve, in UNICORN, the tube length has to
be defined.
The instructions Tubing: Injection valve to column and Tubing: Sample pump to injection valve are
used in calculations of pre-column pressure when Column valve V9-C or V9H-C (5-columns) is NOT
mounted onto the system. In such cases, there is no pre-column pressure sensor present. The estimated
pre-column pressure is calculated using the measured system pressure and the length of the tubing
between the system pressure sensor and the Column.
The instructions are available only when the Column valve V9-C or V9H-C (5-columns) is not selected
in the component list. The instruction Tubing: Sample pump to injection valve is in addition only
available if the sample pump is selected in the list.
Follow the instructions below to set the tube length between the Injection valve and the Column and
between the sample pump and the injection valve.
Step Action
1 • In the System Control module, choose System:Connect to Systems or click the Con-
nect to Systems icon.
Result: The Connect to Systems dialog opens.

Step Action
2 • Select a system.
• Select Control mode.
• Click OK.
Result: The selected instrument can now be controlled by the software.
3 • Select Tubing and Delay Volumes and select Tubing: Injection valve to column.
• Select the I. D. from the drop-down list.
• Type in the length of the tube in the Length field and click OK.
4 Perform step 3 for the instruction Tubing: Sample pump to injection valve.
Note: The built-in pressure sensors for Column valves V9-C and V9H-C have to be re-calibrated after
installation. See Calibrate the monitors, on page 305.
Note: A Pre-Column pressure alarm shall always be set to protect the column, see Section 5.6 Pressure
alarms, on page 176. Column valve V9-Cs and V9H-Cs does not contain pressure monitors.
See Section 5.6 Pressure alarms, on page176 for how to protect columns when not using Column
valves V9-C and V9H-C.

4.2.5 Configuration of Versatile valves
4.2.5 Configuration of Versatile valves
System properties
Step Action
3 Select Versatile valve (V9-V), Versatile valve 2 (V9-V), Versatile valve 3 (V9-V) or Versatile
valve 4 (V9-V) in the Component selection list.
System settings
If the valve is placed in the flow path between the UV monitor and the outlet valve, the delay volume
must be set. See Check/Set delay volume, on page 145.

4.2.6 Configuration of pH valves
4.2.6 Configuration of pH valves
System properties
Step Action
3 Select pH valve (V9-pH) or pH valve (V9H-pH) in the Component selection list.
System settings
If the valve is placed in the flow path between the UV monitor and the outlet valve, the delay volume
Note: It is recommended not to alter the default values for restrictor and pH cell delay volumes when
standard modules and standard tubing for flow restrictor are used.

4.2.7 Configuration of outlet valves
4.2.7 Configuration of outlet valves
System properties
Step Action
Outlet fractionation valve Outlet valve V9-O (10-outlets)

V9-O
Outlet fractionation valve Outlet valve V9H-O (10-outlets)

V9H-O
Outlet control valve V9-Os Outlet valve V9-Os (1-outlet)
Outlet control valve V9H- Outlet valve V9H-Os (1-outlet)

Os
System settings
If the valve is not placed in the recommended position using the standard tubing kit, the delay volume

4.2.8 Configuration of UV monitors
4.2.8 Configuration of UV monitors
Using two UV monitors

The UV monitor U9-L module can be used in two configurations, UV monitor U9-L and UV monitor U9-
L, 2nd. The configuration is defined by the module's Node ID.
It is possible to use two UV monitors in ÄKTA pure, in the following combinations:
• UV monitor U9-M together with UV monitor U9-L, 2nd
• UV monitor U9-L together with UV monitor U9-L, 2nd
UV monitor U9-L, 2nd can be located anywhere in the flow path and is therefore shown in the Process
Picture as a component without a fixed place. This means that it is possible to place U9-L, 2nd before
the other UV monitor in the flow path.
Note: If U9-L, 2nd is placed on the high pressure side of the column, pressure limits have to be con-
sidered. See UV monitor options, on page 416 for pressure limits.
System properties
Step Action
2 Select Monitors and sensors from the Component types list.
Monitor Component selection Property
UV monitor U9-L UV monitor U9-L (fixed)
UV monitor U9-M UV monitor V9-M (variable)
UV monitor U9-L, 2nd UV monitor 2nd (U9-L) N/A
System settings
If the monitor is not placed in the recommended position using the standard tubing kit, the delay volume
For UV monitor U9-L the flow cell length must be set. This is done as a calibration. See Perform the
calibration, on page 313.

4.2.9 Configuration of Conductivity monitor
4.2.9 Configuration of Conductivity monitor
System properties
Step Action
3 Select Conductivity monitor (C9) in the Component selection list.
System settings
If the monitor is placed in the flow path between the UV monitor and the outlet valve, the delay volume

4.2.10 Configuration of external air sensors
4.2.10 Configuration of external air sensors
System properties
Step Action
3 Select External air sensor (L9) in the Component selection list, or the appropriate com-
ponent if multiple external air sensors are used.
4 Select Property according to where the air sensor is placed.

Note:
The available system properties are the same, regardless of which type of air sensor that
is used, L9-1.2 or L9-1.5.
System settings
The sensitivity of the air sensor can be set.
Parameter Air volume detected Usage
Normal (default) 30 μl 100 μl Detect empty

buffer/sample vessels
High 10 μl 30 μl Detect even small air

bubbles
Note: The sensitivity should be set to Normal when the air sensor is located before the System pump
(Air sensor ext - Before pump A or Air sensor ext - Before pump B). Due to higher pressure
and risk of small air bubbles, the sensitivity should be set to High when the air sensor is located
after the Injection valve (Air sensor ext - after Injection valve).

4.2.11 Configuration of fraction collectors
System properties
Step Action
2 Select Fraction collectors from the Component types list.
Fraction collector Component selection Property
Fraction collector F9-C Fraction collector Fraction collector F9-C
Fraction collector F9-R Fraction collector Fraction collector F9-R
Fraction collector F9-R, 2nd Fraction collector 2 (F9-R) N/A
Note: Fraction collector 2 is only available in the Component selection list if Fraction collector is
already selected.
System settings
If non-standard tubing is used between the outlet valve and the fraction collector, the delay volume
Fraction settings and numbering mode can be set.
Fraction collector F9-C
Fractionation settings Fractionation settings comprises fractionation mode and frac-

tionation order.
Fractionation mode (Automatic, Accumulator or DropSync).
Fractionation order (Row-by-row, Column-by-column, Serpen-
tine-row, Serpentine-column). For fractionation mode DropSync,
only the Serpentine option is available.
Last tube filled Last tube filled: Action when last tube is filled (pause, direct the
flow to one of outlets or direct the flow to waste.
Cassette configuration Cassette configuration: Automatic or Manual configuration.

If Automatic is selected, a Quick scan or a Full scan will be per-
formed when the door of the fraction collector is closed to deter-
mine which type of cassettes and plates are used. If Manual is
selected, used plates and tubes in each tray position are entered.

Fraction collector lamp Fraction collector lamp: Lamps in the fraction collector chamber
on or off.
Peak fractionation parameters The Peak fractionation parameters set the detection parameters
for peak collection, that is they decide when a peak starts and
ends. This information is used by the instructions Peak fractiona-
tion and Peak frac in outlet valve in order to start/end the peak
collection.
Fraction collector F9-R
Fractionation settings Drop sync synchronises tube change to drop release. The available
settings are on or off. It is recommended to use Drop sync for
flow rates below 2 ml/min. Higher flow rates can however be used,
depending on the properties (for example viscosity) of the liquid.
Fractionation numbering mode Determines whether the fraction number is reset at the end of a
method or not.
Note:
The default setting is Reset.
Fractionation settings frac 2 Drop sync on or off. It is recommended to use this setting for flow
rates below 2 ml/min. Higher flow rates can be however be used,
depending on the properties (e.g. viscosity) of the liquid.
Fractionation numbering mode Determines whether fraction number for the second fraction col-
frac 2 lector is reset at the end of a method or not.
Note:
Peak fractionation parameters Peak fractionation parameters sets the detection parameters
for peak collection, i.e. it determines when a peak starts and ends.
This information is used by the instructions Peak fractionation,
Peak fractionation frac 2 and Peak frac in outlet valve in order
to start/end the peak collection.

4.2.12 Configuration of I/O-box
4.2.12 Configuration of I/O-box
System properties
Step Action
3 Select I/O-box (E9) or I/O-box 2 (E9) in the Component selection list.
System settings
Default values for digital out ports, noise reduction and configuration of analog out ports can be set.
Digital out X Sets the value of the signal sent out by digital port number X to
either 0 or 1. The default value is 1.
Noise reduction analog in X Filters the noise in the analog signal in port number X.
Alarm analog in X Enables or disables the alarm for the analog signal in port number
X. When enabled, it sets the alarm limits for the analog signal. If
the alarm is enabled and the analog signal falls outside the set
Alarm digital in X Enables or disables the alarm for the signal in digital port number
X. The alarm can be triggered by either of the signal values, 0 or
1. If the alarm is enabled and the condition set in ‘Value’ occurs,
an alarm will be triggered and the method will be paused.
Configure analog out X Enables the user to send one of the pre-defined signals (UV signal,
conductivity, temperature, pH or concentration of eluent B) to the
analog out port number X, and also to set the range of that signal.
Note: The delay volume has to be updated if an external component is added to the flow path.

4.3 General system settings

Check/Set delay volume
When a module has been installed after the UV monitor in the flow path, the delay volume has to be
adjusted in the System Setting dialog in UNICORN, to make sure that the collected fractions correspond
to the fractions indicated in the chromatogram.
Delay volumes can be set for the options Monitor to outlet valve, Monitor to frac, Monitor to frac 2,
and pH valve. Depending on the system configuration used, different delay volume options will be
available for selection in the System Setting dialog. The delay volume has to be set for all displayed
options.
Delay volumes for modules and standard tubing configurations are found in Section 9.13 Delay volumes,
on page 493.
Follow the instructions below to check/set the delay volumes:
Step Action
1 • In the System Control module, choose System:Connect to Systems or click the Con-
nect to Systems icon.
2 • Select a system.
• Select Control mode.
• Click OK.
Result: The selected instrument can now be controlled by the software.
3 • When the system is in state Ready, select System:Settings.

Result: The System Settings dialog is displayed.
4 • Select Tubing and Delay Volumes and select the delay volume option of interest.
• Check the delay volume in the Volume field and enter a new value if necessary.
• Click OK.

Lock/Unlock function
Follow the instruction below to lock or unlock the Pause and Continue buttons of the Instrument
control panel from UNICORN.
Step Action
1 In System Control, select System:Settings.


• Select Advanced:Instrument control panel.
• Select Locked or Unlocked.
• Click OK.
Power-save
ÄKTA pure has a power-save mode. The instrument enters Power-save after having been in the Ready
state for a set period of time. Power-save can be used both in room temperature and in cold room
temperature. The system enters the Ready state when a method run, a method queue or a manual
run ends.
To enable Power-save, a system must be connected and in state Ready.

Follow the instructions below to activate Power-save.

Step Action
1 Select System:Settings in the System Control module.

2 • Select Advanced
and
• select Power-save
3 • Select On in the Mode field

and
• type the number of minutes in the Time field.
Note:
This is the time the instrument will be in state Ready before power-save mode is entered.
• Click OK.

5 Operation
5 Operation
About this chapter

This chapter describes the steps involved when operating ÄKTA pure.
In this chapter
Section See page
5.1 Before you prepare the system 149
5.2 Prepare the flow path 150
5.3 Start UNICORN and connect to system 157
5.4 Prime inlets and purge pump heads 160
5.5 Connect a column 172
5.6 Pressure alarms 176
5.7 Sample application 179
5.8 Fractionation 192
5.9 Create a method and perform a run 210

5 Operation
5.1 Before you prepare the system
5.1 Before you prepare the system

Introduction
It is important to prepare the system in accordance with the settings in the method to be run. Before
preparing the system, check the settings in the Method Editor and make sure that all accessories to
be used are available.
Checklist
Make sure the system is prepared in accordance with the settings in the method to be run. Depending
on configuration, remember to check:
• which valve ports to use for inlets and outlets
• which column type to use
• which column position to use
• which buffers and samples to prepare
• which sample application technique to use
• that the pH electrode is connected and calibrated

5 Operation
5.2 Prepare the flow path

Introduction
The flow path is defined by the user and may contain tubing, valves, pumps and monitors. This section
gives an overview of a flow path and describes how to prepare the flow path before a run.
Illustration of the flow path

The illustration below shows the flow path for a typical system configuration. The individual instrument
modules are presented in the table below. The configuration of the system is defined by the user.
1 3
2
6
4 7 9
5 8
10
W1 W2
16
W
11 12 13 14 15
17
Part Description
1 Pressure monitor
2 Sample pump
3 Sample inlet valve

5 Operation
Part Description
4 Inlet valve
5 System pump B
6 System pump A
7 Pressure monitor
8 Mixer
9 Injection valve
10 Sample loop or Superloop
11 Column valve
12 Column
13 UV monitor
15 Flow restrictor
16 Outlet valve
17 Fraction collector
W, W1, W2 Waste
Select Mixer chamber

To obtain a homogeneous buffer composition, it is important to use a Mixer chamber suitable for the
flow rate of the method. The tables below show what Mixer chambers to use in ÄKTA pure at different
flow rates.
If the liquids are difficult to mix, use a larger Mixer chamber to achieve optimal mixing. However, note
that a larger Mixer chamber distorts and delays the gradient.
The default mixer size is 1.4 ml for both systems. ÄKTA pure 150 also includes a 5 ml mixer. Other
mixer sizes are available as accessories.
Mixer chamber volume [ml] Flow rate [ml/min], Binary gradient
0.6 0.1-5 Not recommended
1.4 0.5-15 0.5-15
5 2-25 2-50
15 Not compatible 15-150

5 Operation
CAUTION
Risk of explosion. Do not use Mixer chamber 15 ml with an ÄKTA pure 25 configura-
tion. The maximum pressure for Mixer chamber 15 ml is 5 MPa.
Note: In ÄKTA pure 25 at low flow rates (below 0.2 ml/min) a gradient of sufficient quality may be
achieved with the mixer bypassed. Similarly, for low flow rates in ÄKTA pure 150 the 0.6 ml
mixer might be used.
Note: The 1.4 ml mixer in ÄKTA pure 25 or 5 ml mixer in ÄKTA pure 150 might work up to maximum
flow rate provided that the buffers are easily mixed.
For information on how to install a Mixer chamber, refer to Section 7.8.3 Replace the Mixer, on page 322.
Select UV flow cells

General considerations
Flow cells with shorter path lengths are suitable to use when high protein concentrations are used.
Flow cells with longer path lengths are suitable to use when low protein concentrations are used.
UV flow cells for UV monitor U9-M

UV flow cells are available with three different path lengths; 0.5 mm, 2 mm (default) and 10 mm.
The real cell path length of the UV cell is automatically recognized by the monitor when a cell is fitted.
The UV data is normalized to the nominal path length. This allows UV data from runs made with different
UV flow cells (but with the same nominal path length) to be directly compared.
UV flow cells for UV monitor U9-L

UV flow cells are available with two different path lengths; 2 mm (default) and 5 mm. When replacing
a UV flow cell, the path length must be set in the System Control module, in System:Calibrate. Use
the nominal flow cell length if the UV flow cell is replaced but not calibrated. See Update the cell path
length , on page 314 to set the flow cell path length.
The path length of the UV flow cell might differ from the nominal length, which leads to incorrect results
in the calculation of protein concentration in the eluate. The UV data is normalized to the nominal path
length. This allows UV data from runs made with different UV flow cells (but with the same nominal
path length) to be directly compared. To achieve normalized absorbance, the path length of the UV
flow cell must be calibrated and the calculated flow cell path length set manually. See Calibration of
the UV monitor U9-L flow cell length, on page 312.
Prepare the inlet tubing

Connect inlet tubing to the inlet ports that are to be used, and place all inlet tubing that is to be used
during the method run in the correct buffers.
Note: When using high viscosity buffers/samples in combination with high flow rates it is recom-
mended to increase the tubing I.D. and/or shorten the length.

5 Operation
Prepare the outlet tubing

Connect outlet tubing to the outlet ports of Outlet valve that are to be used. If a fraction collector is to
be used, make sure that tubing is connected as described in Connect tubing, on page 102 and Connect
tubing to ÄKTA pure, on page 106, and prepare the fraction collector. Otherwise, place the outlet tubing
in suitable tubes or flasks.

5 Operation
Waste tubing overview

The table below lists the waste tubing of the instrument and where it is located. Make sure that the
waste tubing is connected to the correct positions on the modules.
Module Tubing connections Location of tubing
Injection valve Waste ports W1 and W2 Front of the ÄKTA pure instrument.
pH valve (optional) Waste port W3 Front of the ÄKTA pure instrument.
Outlet valve (optional) Waste port W Front of the ÄKTA pure instrument.
Buffer tray (Rescue drainage) Drainage hole of the Buffer Rear of the ÄKTA pure instrument.
tray
Fraction collector F9-C Fraction collector waste outlet Rear of the fraction collector.
(optional)
Prepare waste tubing

Follow the instructions below to prepare the waste tubing.
CAUTION
Fasten the waste tubing. During operation at high pressure the ÄKTA pure instrument
may release bursts of liquid in the waste tubing. Securely fasten all waste tubing to
the ÄKTA pure instrument and to the waste vessel.
CAUTION
Make sure that the waste vessel will hold all the produced volume of the run. For
ÄKTA pure, a suitable waste vessel should typically have a volume of 2 to 10 liters.
NOTICE
The maximum level of the waste vessel must be lower than the bottom of the ÄKTA
pure instrument.

5 Operation
Step Action
1 Insert the waste tubing from all installed modules, in this example Injection valve, Outlet
valve (W, W1 and W2) and the fraction collector, in a vessel.
2 Make sure that the tubing is securely fastened to the ÄKTA pure instrument:
• Fasten waste tubing from the valves with the clips on the front of the system.
• Fasten waste tubing from the Buffer tray with the clips on the rear of the system.

5 Operation
Step Action
3 Cut the waste tubing to appropriate length. It is important that the tubing is not bent and
will not be submerged in liquid during the run.
Note:
If the tubing is too short, replace it with new tubing. Do not lengthen the tubing as this
might cause obstruction of the tubing.
4 Fasten all waste tubing securely to the waste vessel.
Plug unused valve ports

It is recommended to plug all unused valve ports with stop plugs before starting a run. See Tubing
connectors, on page 421 for information about connectors.

5 Operation
5.3 Start UNICORN and connect to system

Introduction
This section describes how to start and log on to UNICORN and how to connect the instrument to
UNICORN.
Start UNICORN and log on

Follow the instructions to start UNICORN and log on to the program. A valid e-license must be available
for the workstation. See UNICORN Administration and Technical Manual for more information about
e-licenses.
Step Action
1 Double-click the UNICORN icon on the desktop.

Result: The Log On dialog box opens.
Note:
If there is no connection to the database it is still possible to log on to UNICORN and control
a running system. The Log On dialog box will give the option to start System Control
without a database. Click Start System Control to proceed to the next Log On dialog box.
2 In the Log On dialog box:

• Click a user name in the User Name list
and
• enter the password in the Password field.
Note:
It is also possible to select the Use Windows Authentication check box and enter a
network ID in the User Name box.
• select which UNICORN modules to start.

• click OK.
Result: The selected UNICORN modules open.

5 Operation
Connect to system
Follow the instructions to connect the instrument to UNICORN.
Step Action
1 In the System Control module,

• Click the Connect to Systems button,
or
• click Connect to Systems on the System menu.

5 Operation
Step Action
2 In the Connect to systems dialog:

• Select the checkbox in front of the system name.
• To control the selected system, click Control.
• Click OK.
Result: The instrument control panel displays a white, rapidly flashing light followed by
a steady white light when the system is ready. The selected instrument can now be
controlled by the software.
Note:
Instruments that are turned off or disconnected from the network appear dimmed and
cannot be connected.
Tip:
To view the users currently connected to systems, either in control or view mode, click the
Connected Users button.
Tip:
Result: UNICORN states Connection = Connected in control or Connection = Connected
in view in the status bar in the lower part of the System Control window.

5 Operation
5.4 Prime inlets and purge pump heads

About this section
Before usage of a pump, it is important to:
• Prime the inlets (fill the buffer inlets with liquid).
• Purge the pump (remove air from the pump heads).
This section describes how to prime inlets and purge the pump heads of the system pumps and the
sample pump (the sample pump is an optional module).
Note: Note that the procedures described in this section may have to be adapted if your system
configuration differs from the one described in this manual.
In this section
Section See page
5.4.1 System pumps 161
5.4.2 Sample pump 167

5 Operation
5.4.1 System pumps
5.4.1 System pumps
Introduction
This section describes how to prime inlets and purge the System pumps.
Overview
The procedure consists of the following stages:
Stage Description
1 Prime all inlet tubing to be used during the run
2 Purge System pump B
3 Validate purge of System pump B
4 Purge System pump A
5 Validate purge of System pump A
6 End the run
Tip: The procedures for purging the pump heads and priming the inlets using the Process Picture,
are described below. It is also possible to perform the procedures from the Manual instructions
dialog.
Prime inlet tubing

Follow the instructions below to fill all A and B inlet tubing to be used in the run with appropriate
buffer/solution.
Step Action
1 Make sure that all inlet tubing that is to be used during the method run is placed in the
correct buffer.
2 Open the System Control module.

5 Operation
5.4.1 System pumps
Step Action
3 In the Process Picture:

• Click on the buffer inlets.
• Select the position of the inlet to be filled. Select the positions in reverse alphabetical
order and start with the highest number. For example, if all the four inlets in Inlet valve
AB are to be filled, fill them in the following order: B2, B1, A2, A1.
Result: The inlet valve switches to the selected port.
4 Connect a 25 to 30 ml syringe to the purge valve of one of the pump heads of the pump
that is being prepared. Make sure that the syringe fits tightly into the purge connector.
5 Open the purge valve by turning it counter-clockwise about three quarters of a turn.
Draw liquid slowly into the syringe until the liquid reaches the pump.
6 Close the purge valve by turning it clockwise. Disconnect the syringe and discard its
contents.
7 Repeat steps 3 to 6 for each piece of inlet tubing that is to be used during the run.
Purge System pump B

Follow the instruction below to purge both pump heads of System pump B.
Step Action
1 Make sure that the piece of waste tubing connected to the Injection valve port W1 is
placed in a waste vessel.

5 Operation
5.4.1 System pumps
Step Action

• Click on the Injection valve and select System pump waste.
Result: The Injection valve switches to waste position. This is necessary to achieve a
low back pressure during the purge procedure.

• Click on the pumps.
• Set Conc % B to 100% B.
• Click Set % B.
Result: Only System pump B is active.

5 Operation
5.4.1 System pumps
Step Action

• Click on the buffer inlets.
• Select the position of one of the inlets that will be used at the beginning of the run.
Result: The inlet valve switches to the selected port.

• Click on the Pumps.
• Set the System flow to 1.0 ml/min for ÄKTA pure 25 or 10.0 ml/min for ÄKTA pure 150.
• Click Set flow rate.
Result: A system flow starts.
6 Connect a 25 to 30 ml syringe to the purge valve of the left pump head of System pump
B. Make sure that the syringe fits tightly into the purge connector.
7 Open the purge valve by turning it counter-clockwise about three quarters of a turn.
Draw a small volume of liquid slowly into the syringe (with a rate of about 1 ml per second).
contents.

5 Operation
5.4.1 System pumps
Step Action
9 Connect the syringe to the purge valve on the right pump head of System pump B, and
repeat steps 6 to 8. Keep the system flow running.
Validate purge of pump B

Follow the instructions below to check that there is no air left in the pump after performing a purge.
Step Action

• Click on the Injection valve and select Manual load.
Result: The Injection valve switches to manual load position.
2 Make sure that the pump flow is on.
3 In the Chromatogram pane:

Check the PreC pressure curve.
If the PreC pressure do not stabilize within a few minutes there may be air left in the
pump. Refer to Section 8.6 Troubleshooting: Pumps, on page 387.

5 Operation
5.4.1 System pumps
Purge System pump A

Purge both pump heads of System pump A by following the same procedure as in Purge System pump
B, on page 162, but replace step 3 with the following actions:
In the Process Picture:
• Click on the pumps.
• Set Conc % B to 0% B.
• Click Set % B
Result: Only System pump A is active.
Validate purge of pump A

Follow the procedure described in Validate purge of pump B, on page 165 to check if there is air left in
the pump.
End the run

Click the End button in the System Control toolbar to end the run.

5 Operation
5.4.2 Sample pump
5.4.2 Sample pump
Introduction
This section describes how to prime inlets and purge Sample pumps S9 and S9H.
Overview
The procedure consists of the following steps:
1 Prime all sample inlet tubing to be used during the run
2 Purge the Sample pump
3 Validate purge
4 End the run
Prime sample inlets

Follow the instructions below to fill all sample inlet tubing, to be used in the run, with appropriate buffer
or solution. Skip steps three and four if no sample inlet valve is used.
Step Action
1 Make sure that all sample inlet tubing that is to be used during the method run is immersed
in the correct buffers.
2 Make sure that the waste tubing connected to Injection valve port W2 is immersed in a
waste vessel.

5 Operation
5.4.2 Sample pump
Step Action
4 In the Process Picture :

• Click on Sample inlet valve.
• Select the position of the inlet to be filled. Start at the inlet position with the highest
number and end at the position with the lowest number.
Result: the sample inlet valve switches to the selected port.
5 Connect a 25 to 30 ml syringe to one of the purge valves of the pump heads of the
Sample pump. Make sure that the syringe fits tightly into the purge connector.
6 Open the purge valve by turning it counter-clockwise about three-quarters of a turn.

Draw liquid slowly into the syringe until the liquid reaches the sample pump.
contents.
8 Repeat steps 2-5 for each sample inlet that is to be used in the method run.

5 Operation
5.4.2 Sample pump
Purge the Sample pump

Follow the instruction below to purge both the pump heads of the Sample pump.
Step Action
1 Make sure that all sample inlet tubing that is to be used during the method run is immersed
in the correct buffers.
2 Make sure that the waste tubing connected to Injection valve port W2 is immersed in a
waste vessel.
4 In the Process Picture :

• Click on Injection valve and select Sample pump waste .
Result: The injection valve switches to waste position. This is necessary to achieve a
low back pressure during the purge procedure.
5 In the Process Picture: Click on Sample pump.

• If Sample inlet valve V9-IS or V9H-IS is used: Click on the Sample inlet valve icon (if
the valve is not used, the icon will not be present):
Select the Buffer inlet.
• Click on the Sample pump icon: Set the Sample flow to 1.0 ml/min for ÄKTA pure 25
or 10.0 ml/min for ÄKTA pure 150.
•
Click and close the pop-up window.
Result: a sample pump flow starts.

5 Operation
5.4.2 Sample pump
Step Action
6 Connect a 25 to 30 ml syringe to the left purge valve of the Sample pump. Make sure
that the syringe fits tightly into the purge connector.
7 Open the purge valve by turning it counter-clockwise about three-quarters of a turn.

Draw 5-10 ml of liquid slowly into the syringe with a rate of about 1 ml/s.
contents.
9 Connect the syringe to the right purge valve on the Sample pump, and repeat step 6 to
step 8.

5 Operation
5.4.2 Sample pump
Validate purge
Follow the instructions below to check that there is no air left in the pump after performing a purge.
Step Action

• Click on the Injection valve and select Direct inject.
Result: The Injection valve switches to direct inject position.
2 Make sure that the pump flow is on.
3 In the Chromatogram pane:

Check the PreC pressure curve.
If the PreC pressure does not stabilize within a few minutes there may be air left in the
pump. Refer to Section 8.6 Troubleshooting: Pumps, on page 387.
End the run

Click the End button in the System Control toolbar to end the run.

5 Operation
5.5 Connect a column

Introduction
This section describes how to connect a column to the instrument using a column holder and without
introducing air into the flow path. Several types of column holders are available for ÄKTA pure.
WARNING
To avoid exposing the column to excessive pressure, make sure that the pressure
limit is set to the specified maximum pressure of the column. Before connecting a
column to the ÄKTA pure instrument, read the instructions for use of the column.
Methods automatically include a pressure alarm based on the specifications of the chosen column
type. However, when running manual runs you have to set the pressure limits yourself. Also, to protect
the column media, special settings are needed. See Section 5.6 Pressure alarms, on page 176 for more
information on pressure alarms.
Note: Do not overtighten when connecting columns. Overtightening might rupture the connectors
or squeeze the tubing and thereby result in high back pressure.
Note: If no column valve is used, remove the column from the system before running a system wash.
The pressure during a system wash may become too high for the column.
Attach a column holder and connect a column

Follow the instructions below to connect a column to the instrument. Always use a column holder. If
a column valve is used, connect the column to the appropriate A and B ports on the valve. If no column
valve is used, connect the column directly to the flow path tubing. Use appropriate tubing and connec-
tors. The instructions below show a system configured with Column valve V9-Cs.
Step Action
1 Attach an appropriate column holder to the rail on the instrument.

5 Operation
Step Action
2 Attach the column to the column holder.
3 Connect a suitable tubing to a Column valve port, in this example port 1A.

• Click on the Column.
• Select Column down flow.
Result: The Column valve switches to position 1.

5 Operation
Step Action

• Enter a low System flow (e.g., 0.2 ml/min).
Result: A system flow of 0.2 ml/min starts.
7 When buffer leaves the tubing in a continuous mode and the top part of the column is
filled with buffer, connect the tubing to the top of the column.
8 Connect a piece of tubing to the bottom of the column.

5 Operation
Step Action
9 When buffer leaves the tubing at the bottom of the column in a continuous mode, connect
this piece of tubing to the Column valve. Use the port opposite to the one already con-
nected to the column, in this example port 1B. If no column valve is used, connect the
tubing to the next module in the flow path.
10 Click the End icon in the System Control toolbar to end the run.

5 Operation
5.6 Pressure alarms
5.6 Pressure alarms

Introduction
The columns can be protected by two different types of pressure alarms:
• The pre-column pressure alarm protects the column hardware
• The delta-column pressure alarm (only available when V9-C or V9H-C is installed) protects the column
media
Column valves V9-C and V9H-C have built-in pressure sensors that automatically measure the pre-
column and delta-column pressure. If Column valve V9-C or V9H-C is not used (column is connected
without a Column valve or to Column valve V9-Cs or V9H-Cs), the pre-column pressure is calculated
from the system pressure and tubing dimensions.
See the instructions below to set the pressure alarm for the column to be used in the run and, if appli-
cable, to set the parameters for the tubing dimensions.
Set tubing dimension parameters

to calculate pre-column pressure
For instruments where there is no pre-column pressure sensor, i.e. the column is connected without
a Column valve or to Column valve V9-Cs or V9H-Cs, the pre-column pressure is calculated from the
system pressure and tubing dimensions. Follow the instructions below to set the tubing dimension
parameters.
Step Action

2 • Select Tubing and Delay Volumes

and
• select Tubing: Injection valve to column

5 Operation
5.6 Pressure alarms
Step Action
3 • Select the inner diameter of the tubing between the injection valve and the column
from the I.D. drop-down list.
• Type in the tubing Length.
4 If the sample pump is used:

• Select Tubing: Sample pump to injection.
• Set tubing i.d. and length, see step 3.
5 Click OK.
Note:
The system now calculates the Pre-column pressure.
Pre-column pressure alarms

It is important that the pre-column pressure alarm is set during all runs where a column is used. The
pressure alarm can be set in:
• the method to be run,
• the System Settings dialog , or
• during a manual run
Pre-column pressure alarm limits are automatically set in the method when a column from the column
list is selected in the method. Refer to UNICORN Method Manual for more information on pressure
alarms.
For some columns the max delta-column pressure (media) is significantly lower than the max pre-
column pressure (hardware). To protect the media if a delta-column pressure measurement is not
available (that is, when column valve V9-C or V9H-C is not used), the pre-column pressure alarm must
be manually set to the value in the column list that is the lowest of the max pre-column pressure and
the max delta-column pressure.
Delta-column pressure alarms

If column valve V9-C or V9H-C is installed the delta-column pressure will be measured, but the alarm
must be set manually if needed.
Set pressure alarms

Pressure alarm limits may be set manually in System Control. The example below describes how to
set the high pressure limit for the column. Other alarms are set in a corresponding way.
Step Action
1 Select Manual:Execute Manual Instructions... in the System Control module.

Result: The Manual instructions dialog opens.

5 Operation
5.6 Pressure alarms
Step Action
2 • Select Alarms
and
• select Alarm pre column pressure.
3 Select Enabled in the Mode field.
4 • Type the high pressure limit in the High alarm field.

• Click Execute.

5 Operation
5.7 Sample application

Introduction
This section describes the different sample application techniques that can be used with ÄKTA pure.
The table below shows the alternatives for sample application available in the Sample application
phase of a method.
Sample application Via Compatible loops
Inject sample directly • Sample pump • Not applicable

onto column
• System pump A
• System pump A and Air sensor
Inject sample from • Syringe • Sample loop

loop
• Sample pump • Superloop, 10 ml
• Sample pump and Air sensor • Superloop, 50 ml
• System pump A • Superloop, 150 ml
• System pump A and Air sensor
Note: In order to avoid sample carry-over when switching techniques for loading samples, wash the
valve with buffer between the loading of two different samples. For example, when switching
from loading sample in the loop to loading sample directly onto the column with the valve in
Direct inject position.
When using a pump for sample application, it is important to prime inlets and purge the pump before
using the pump to load the sample:
• Sample pump, see further instructions in Section 5.4.2 Sample pump, on page 167
• System pump A, see further instructions in Section 5.4.1 System pumps, on page 161
When loading sample using System pump A and an external air sensor, the sensor should be installed
according to Adapter for air sensor, on page 83.
Note: When sample is loaded at high flow rate and the external air sensor is placed before the pump
that is used for loading the sample, it is necessary to use longer tubing to ensure that no air
reaches the pump. Use tubing with the minimum lengths given below between the valve located
before the pump and the external air sensor. The length applies for maximum flow rate, but
shorter tubing can be used at lower flow rates.
• Sample pump S9: 40 cm.
• Sample pump S9H: 20 cm.

5 Operation
5.7.1 Sample application using direct injection onto the column
Introduction
There are two ways to load sample directly onto a column:
• a fixed volume is loaded, or
• all the sample is loaded.
To inject all the sample, one of the following configurations is required:
• system pump, external air sensor, inlet valve A and the mixer valve, or
• sample pump and sample inlet valve.
Minimize sample loss

To minimize sample loss during direct injection of sample onto the column, sample remaining in the
flow path will be pushed onto the column with buffer from the inlet valve. This step is called Finalize
sample injection in the text instructions of the sample application phase of the method to be used.
Refer to Section 5.9 Create a method and perform a run, on page 210 for more information on methods
and phases.
When preparing to inject... Then...
a fixed volume of sample • manually prime the sample inlet tubing with sample, see Prime
sample inlets, on page 167.
• in the Method editor, make the following selections for the
Sample Application phase of the method to be run:
- select Inject sample directly onto column,
- select Inject fixed sample volume and set the volume to be
injected.
• make sure that the flow path from the sample inlet valve up to
the injection valve will be filled with an appropriate buffer:
- make sure that the buffer inlet tubing of the inlet valve is im-
mersed in buffer, and
- enable the function Wash sample flow path with buffer in
the Sample application phase.

5 Operation
When preparing to inject... Then...
all the sample • in the Method editor, make the following selections for the
Sample Application phase of the method to be run:
- select Inject sample directly onto column, and
- select Inject all sample using air sensor.
• manually prime the sample inlet tubing with sample or buffer,

see Prime sample inlets, on page 167, and make sure that the
tubing is immersed in sample before starting the run.
• make sure that the flow path from the sample inlet valve up to
the injection valve is filled with sample or an appropriate buffer
and that the buffer inlet tubing is immersed in buffer.
Maximize precision and accuracy

To achieve full precision and accuracy when a volume of sample is injected directly onto the column,
make the following selections in the Sample Application phase of the method to be run (refer to Sec-
tion 5.9 Create a method and perform a run, on page 210 for more information on methods and phases):
• select Inject sample directly onto the column,
• select Inject fixed sample volume and set the volume to be injected,
• enable the function Prime sample inlet with and set the volume to be used for priming.
Result: the step Finalize sample injection is automatically deactivated in order to maximize precision
and accuracy. See Minimize sample loss, on page 180 for more information.
Note: If manual priming of the flow path up to the injection valve is preferred, enable Prime
sample inlet with but set the volume to 0 ml.
• If the system pump is used to load the sample: enable the function Wash sample flow path with
buffer after sample application in order to ensure that the correct sample volume is loaded onto
the column.

5 Operation
5.7.2 Sample application using a Superloop™
Introduction
A Superloop allows injection of large sample volumes onto the column. A Superloop can also be used
for multiple injections, for example in a scouting experiment when the same application conditions
are required. Superloop models are available in 10 ml, 50 ml and 150 ml sizes.
A superloop can be connected to either the Injection valve or the Loop valve. When using the Loop
valve, up to five loops can be connected simultaneously.
Note: After loading a Superloop, always plug the Syr port on the Injection valve with a Stop plug.
With a Superloop connected to the valve, an over-pressure may be created during injection.
Prepare the Superloop

To avoid injecting air into the system flow path, the Superloop should be prefilled with buffer manually,
before fitting the Superloop to the system.
Note: Read the instruction for the Superloop to be used.
Connect the Superloop

Follow the instruction below to connect the Superloop to the Injection valve or to the Loop valve.
Step Action
1 Attach the Superloop to the instrument using a Column holder.
2 Connect a piece of tubing from the top of the Superloop to:

• port LoopE on the Injection valve
or
• a E port, eg., 1E, on the Loop valve

5 Operation
Step Action
3 Connect a piece of tubing from the bottom of the Superloop to:

• port LoopF on the Injection valve
or
• the F port corresponding to the connected E port, eg., 1F, on the Loop valve
Fill the Superloop using a syringe

Follow the instruction below to fill the Superloop using a syringe.
Step Action
1 Check if the system is in state Ready.

• If yes: The Injection valve is in position Manual Load per default. Continue to step 3.
• If no: Continue to step 2 to position the valve.

• Click the Injection valve and select Manual Load.
Result: The Injection valve switches to Manual Load position.

5 Operation
Step Action
3 If the loop is connected to:

• the Injection valve, continue to step 5.
• the Loop valve, continue to step 4.

• Click the Loop valve.
• Select the position the loop is connected to, for example Position 1.
Result: The Loop valve switches to the selected position.
5 Fill a syringe with sample.
6 Connect the syringe to Injection valve port Syr.
7 Load sample into the Superloop by emptying the syringe into the Injection valve.
8 Disconnect the syringe and plug the Syr port with a Stop plug.
Fill the Superloop using the Sample pump

Follow the instruction below to fill the Superloop using the Sample pump.
NOTICE
Glass tube splinter. Make sure to set the sample pressure below the max pressure
of the Superloop before executing a flow in the Manual instructions dialog when the
Superloop is connected.
Tip: The Superloop can also be filled as part of a method run, as set in the Sample Application
phase in the Method Editor. For multiple injections, it may be more convenient to fill the Super-
loop once, as described in the instruction below.

5 Operation
Step Action
1 In the Manual instructions dialog:

• Select Flow path:Injection valve.
• Select Sample pump load from the Position drop-down list.
• Click .
Result: The Injection valve switches to Sample pump load position.
2 Make sure that the sample inlet tubing from the sample vessel is connected to the Sample
inlet valve.

• Select Alarms:Alarm sample pressure.
• Set Mode as Enabled.
• Set a High alarm level that is below the maximum pressure of the Superloop.
• Click .

• Select Pumps and pressures:Sample flow.
• Set Flow rate to an appropriate value for the Superloop size, in this example 10 ml/min.
• Click .
Result: A sample flow starts, in this example of 10 ml/min.
5 When the Superloop is filled with as much volume as is needed, click the End icon in the
System Control toolbar to end the run.
6 Plug the Syr port on the Injection valve with a Stop plug.

5 Operation
Fill the Superloop using System pump A

Follow the instruction below to fill the Superloop using the System pump A. Note that Mixer valve has
to be installed for this loop filling technique.
NOTICE
Glass tube splinter. Make sure to set the system pressure below the max pressure
of the Superloop before executing a flow, when the Superloop is connected.
Tip: The Superloop can also be filled as part of a method run, as set in the Sample Application
phase in the Method Editor. For multiple injections, it may be more convenient to fill the Super-
loop once, as described in the instruction below.
Step Action

• Select Alarms:Alarm system pressure.
• Set Mode as Enabled.
• Set a High alarm level that is below the maximum pressure of the Superloop.
• Click .

• Click the Injection valve and select Sample pump load.
Result: The Injection valve switches to Sample pump load position.

5 Operation
Step Action

• Click the Mixer valve and select By-pass.
Result: The Mixer valve switches to By-pass position.



• Click on the buffer inlet valve A.
• Select the inlet position to be used for sample injection, for example A2.
Result: Inlet valve A switches to the selected port.

5 Operation
Step Action

• Set the System flow to an appropriate value for the Superloop size, in this example
10 ml/min.
Result: A system flow starts, in this example of 10 ml/min.
8 When the Superloop is filled with as much volume as is needed, click the End icon in the
System Control toolbar to end the run.
9 Plug the Syr port on the Injection valve with a Stop plug.

5 Operation
5.7.3 Sample application using a sample loop
Introduction
A sample loop is recommended for injection of smaller sample volumes onto the column.
A sample loop can be connected to either the Injection valve or the Loop valve. When using the Loop
valve, up to five loops can be connected simultaneously.
Note: Sample loop is called capillary loop in UNICORN.
How to fill a sample loop

Follow the instructions below to fill the sample loop with sample.
Step Action
1 Connect a suitable sample loop to Injection valve ports LoopF (fill) and LoopE (empty).
2 Fill a syringe with sample.
3 Connect the syringe to the Injection valve port Syr.

5 Operation
Step Action

• Click on the Injection valve and select Manual load.
Result: Injection valve is set to manual load.



5 Operation
Step Action
7 Load sample into the sample loop. To avoid sample loss due to siphoning, leave the syringe
in the port until the sample has been injected onto the column during the run.
Tip:
It is recommended to overfill the loop to make sure that the loop is completely filled. Excess
of sample will leave the valve through port W1.
Fill the sample loop using the Sample pump

Most often the sample loop is filled using a syringe. However, to fill it using the sample pump, follow
the instructions for filling the Superloop, see Fill the Superloop using the Sample pump, on page 184.
Note: • It is not necessary to set the Alarm Sample Pressure when filling a sample loop.
• It is recommended to overload the loop to make sure that the loop is completely
filled.
• Set Flow rate to an appropriate value for the loop size.
• After loading, plug the Syr port on the Injection valve with a Stop plug.
Fill the sample loop using System pump A

Most often the sample loop is filled using a syringe. However, to fill it using System pump A, follow the
instructions for filling the Superloop, see Fill the Superloop using System pump A, on page 186.
Empty the loop

General considerations
During the method run, the sample is automatically injected onto the column. The loop is emptied and
washed out using buffer from the system pumps. The total buffer volume to be used for emptying and
washing the loop is set in the Method Editor.
Using a sample loop connected to Loop valve V9-L or V9H-L
For maximum reproducibility, use complete loop fill when loading the loop, that is, overfill the loop with
a sample volume of up to 3-5 times the volume of the loop. For minimum sample loss, use partial loop
fill, that is, fill only up to 50% of the loop volume. Empty the loop with 3-5 times the volume of the loop.
Volume used to empty a loop connected to Loop valve V9-L or V9H-L
To minimize the risk for carry over and to make sure that the complete sample volume reaches the
column, the loop should be emptied with an excess of buffer. The tubing between the Loop valve port
E and the Injection valve port E holds a small volume. If the loop is emptied with a volume equal to, or
less than the loop volume this needs to be taken into account. It is also important to use a low flow
rate to ensure that the correct volume is added to the column when injecting a small volume.
Note: Partially emptying the loops that are attached to Loop valve V9-L or V9H-L can increase the
risk for carry over from one loop position to the next.

5 Operation
5.8 Fractionation
5.8 Fractionation
Introduction
Fraction collector F9-C and Fraction collector F9-R collect fractions from ÄKTA pure purification runs.
The fraction collectors are connected to ÄKTA pure and controlled by UNICORN. Control of the fraction
collector can be achieved automatically in a method run, or manually.
In this section
Section See page
5.8.1 Prepare Fraction collector F9-C 193
5.8.2 Prepare Fraction collector F9-R 199
5.8.3 Fractionation overview 206

5 Operation
5.8 Fractionation
5.8.1 Prepare Fraction collector F9-C
About this section

This section describes how to prepare and assemble Fraction collector F9-C before a run.
The fraction collector is connected to ÄKTA pure and controlled by UNICORN. Control of the fraction
CAUTION
Fire Hazard. Do not fractionate flammable liquids using Fraction collector F9-C.
When running RPC methods, or other procedures using organic solvents, collect
fractions through the outlet valve or Fraction collector F9-R.
Note: The tray and racks can tilt slightly when not fully inserted into the fraction collector and may
harm the fractionation arm. The tilt is due to the height difference between the door and the
floor of the fraction collector. The tendency to tilt is affected by the placement and weight of
the cassettes, tubes or bottles
Prepare the fraction collector

Before starting to prepare Fraction collector F9-C, check the fractionation settings in the method to
be run. Perform the steps described below according to the settings in the method.
• Insert the Cassette tray or a rack for tubes or bottles.
• Change the System Settings in UNICORN to set the fractionation mode and other settings for
fraction collection.
How to insert a tray or a rack is shown below.
For information on how to change the System Settings before a run, see UNICORN System Control
Manual. The available System Settings are described in the ÄKTA pure User manual.

5 Operation
5.8 Fractionation
Prepare and insert the Cassette tray

Follow the instructions below to add cassettes to the Cassette tray and insert the tray into the fraction
collector.
Step Action
1 If you are to use cassettes with the QuickRelease function, open the cassettes. For more
information on the QuickRelease function see QuickRelease function, on page 98.
2 Place the tubes and deep well plates in the cassettes. Make sure that the deep well plates
are rotated so that the well marked A1 is positioned above the A1 marking on the cassette.

5 Operation
5.8 Fractionation
Step Action
3 Close the cassettes that have the QuickRelease function.
4 Place the cassettes on the Cassette tray. Make sure that the cassette type code (see illus-
tration below) faces the front of the tray marked with the GE logo.
5 Open the door of the fraction collector using the handle.

5 Operation
5.8 Fractionation
Step Action
6 Insert the tray into the fraction collector:

• make sure that the front of the tray (marked with the GE logo) faces outwards
• position the tray and slide it into the fractionation collector until it reaches the end
• make sure that the Tray catch snaps into closed position, as shown below.
Note:
• The tray can tilt slightly when not fully inserted into the fraction collector and may harm
the fractionation arm. The tilt is due to the height difference between the door and the
floor of the fraction collector. The tendency to tilt is affected by the placement and
weight of the cassettes.
• A height exclusion bar ensures that the tubes or deep well plates are correctly positioned
and cannot damage the Dispenser head.
7 Close the door. Make sure that it closes properly.

Result: After the door has been closed, the fractionation arm scans the cassette type
code of each cassette to identify the cassette types. If deep well plates are used, the in-
strument also identifies the types of deep well plates.
Note:
If the tray is inserted with the front of the tray facing the wrong way it will not be possible
to close the door.

5 Operation
5.8 Fractionation
Prepare and insert a rack for tubes or bottles

Follow the instructions below to insert a rack for tubes or bottles into the fraction collector.
Step Action
1 Place the tubes or bottles in the rack.
2 Open the door of the fraction collector using the handle.
3 Insert the rack into the fraction collector:

• make sure that the front of the rack (marked with the GE logo) faces outwards
• position the rack and slide it into the fractionation collector until it reaches the end
• make sure that the Tray catch snaps into closed position, as shown below.
Note:
• The rack can tilt slightly when not fully inserted into the fraction collector and may harm
the fractionation arm. The tilt is due to the height difference between the door and the
floor of the fraction collector. The tendency to tilt is affected by the placement and
weight of the tubes or bottles.
• A height exclusion bar ensures that the tubes or bottles are correctly positioned and
cannot damage the Dispenser head.
• Do not use the Cassette tray when the Rack for 50 ml tubes or the Rack for 250 ml
bottles is placed in the fraction collector.

5 Operation
5.8 Fractionation
Step Action
4 Close the door. Make sure that it closes properly.

Note:
If the rack is inserted with the front of the rack facing the wrong way it will not be possible
to close the door.

5 Operation
5.8 Fractionation
5.8.2 Prepare Fraction collector F9-R
Introduction
This chapter describes how to prepare and assemble Fraction collector F9-R before a run.
Fraction collector F9-R is connected to ÄKTA pure and controlled by UNICORN. Control of the fraction
Prepare the fraction collector

Before starting to prepare the Fraction collector F9-R, check the fractionation settings in the method
to be run. Perform the steps described below according to the settings in the method.
• Assemble the Tube rack
• Insert collection tubes
• Adjust the Delivery arm
• Update System Settings in UNICORN
Assemble the Tube rack

Illustration of the Tube rack
Part Function
1
1 Single cutout
2
2 L-shaped cutout
3 Bowl
4 Tube support
5 Tube guide
6
5 3
6 Tube holder
4
Note: Note that the tube guide has both single and L-shaped cutouts, while the tube holder only has
single cutouts.

5 Operation
5.8 Fractionation
Tube rack inserts

The Fraction collector F9-R is delivered with the 18 mm tube rack mounted. Each tube rack is made
up of a combination of a Bowl, Tube support, Tube guide and Tube holder. Change the Tube holder
and the Tube guide to collect fractions in 12 mm tubes or 30 mm tubes. The 12 mm tube rack is delivered
with ÄKTA pure and the 30 mm tube rack is available as an accessory. The table below describes inserts
and corresponding fraction collection tubes.
Inserts Maximum number of tubes Tube diameter Tube length
12 mm Tube holder 175 12 mm 50 - 180 mm

12 mm Tube guide

18 mm Tube guide

30 mm Tube guide
Single and L-shaped cutouts

When assembling a tube rack, different cutouts are used for the various inserts depending on the
length of the collection tubes. Which cutouts to use are summarized in the tables below.
12 mm and 18 mm Tube rack inserts
Inserts 50 - 85 mm tubes 85 - 180 mm tubes
Tube support L-shaped cutout Not required
Tube guide Single cutout L-shaped cutout
Tube holder Single cutout Single cutout
30 mm Tube rack inserts
Inserts 30 - 50 mm tubes 1 50 - 85 mm tubes 85 - 180 mm tubes
Tube support Single cutout L-shaped cutout Not required
Tube guide Single cutout Single cutout L-shaped cutout
Tube holder Single cutout Single cutout Single cutout
1 For 30-50 mm tubes, first insert the tube guide from the 18 mm rack using the single cutout, before inserting the
tube support for the 30 mm rack.

5 Operation
5.8 Fractionation
Assembly instructions
Follow the instructions below to assemble the Tube rack.
Step Action
1 Insert the Tube support (4), if required, into the bowl (3). The circular marks on the Tube
support should face down.
1
2
6
5 3
4
Note:
When assembling a Tube rack, Single cutouts (1) and L-shaped cutouts (2), are used for
various inserts depending on the length of the collection tubes. See Single and L-shaped
cutouts, on page 200 for detailed information.
2 Insert the Tube guide (5) with the tube position numbers upwards. The Tube guide should
rest about 1 cm above the Tube support.
3 Insert the Tube holder (6) with the tube position numbers upwards:
Check that tube position 1 (7) is directly above tube position 1 (7) of the Tube guide.

5 Operation
5.8 Fractionation
Step Action
4 • Push the flexible bowl out at each rib and snap the Tube holder under the top lip of
the rib (8).
Note:
Do not force the tube holder into place as this may damage the lip.
• Check that the surface of the Tube holder is level.
5 Gently move the Delivery arm (10) out to the outer stop.
6 Place the Tube rack (9) over the Central spindle (11) and pull the spring loaded Drive sleeve
(12) out so the Tube rack comes to rest.
9
10
12
11

5 Operation
5.8 Fractionation
Insert collection tubes

Insert a sufficient number of collection tubes in to the Tube rack, starting at position 1, pushing each
one down as far as they will go. All the tubes must be of the same length and diameter and there
should be no spaces in the sequence.
Adjust the Delivery arm

Follow the instructions in the table below to adjust the height of the Delivery arm.
Step Action
1 Lift and then lower the Delivery arm (1), and allow it to move in so the Tube sensor (2)
touches the collection tubes of the outer track.
1
2 Loosen the lock knob (3)

5 Operation
5.8 Fractionation
Step Action
3 • Adjust the height so that the horizontal mark (5) on the Tube sensor (6) is at the same
level as the top of the flat collection tubes and approximately 2 mm over the top of
the flanged collection tubes (4).
• Lock the Delivery arm at this height with the lock knob.
4 Check that the Tube sensor (9) is in the correct position for the tubes used (10). The eluent
tubing (7) should be above the center of the collection tube.
7
8
10
5 Use the Sensor control (8) to position the Tube holder over the center of the collection
tube.
NOTICE
Never lift Fraction collector F9-R by the Delivery arm. This may damage the Fraction
collector.

5 Operation
5.8 Fractionation
Sensor control
The sensor control can be switched between the two positions "small tubes" and "large tubes", indicated
in the illustration below.
The position for large tubes is used for tubes of approximately 18 mm i.d. and larger. The position for
small tubes is used for tubes smaller than 18 mm i.d.
Note that this is a rough approximation. Always check that the eluent tubing is centered above the
collection tube.

5 Operation
5.8 Fractionation
5.8.3 Fractionation overview
Fractionation types
The table below lists the types of fractionation that the fraction collectors can be used for.
Type Description
Fixed volume fractiona- During fixed volume fractionation the fraction collector continuously
tion switches tubes according to the set volume throughout the entire fraction-
ation. This type of fractionation is also known as straight fractionation.
Peak fractionation Peak fractionation can be used to further increase the purity of the collect-
ed protein peaks and minimize the number of tubes used. The monitor
signal is used to determine when to switch the tubes. See Section 9.8.10
System settings - Watch parameters, on page 462 for information about
different watch options.
Combined fixed vol- The two fractionation types listed above can be used in combination.
ume fractionation and Combination of fixed volume and peak fractionation allows fractions col-
peak fractionation lected by fixed volume fractionation and fractions collected by peak frac-
tionation to be directed to different collection tubes.
To be able to analyze different parts of the peak, the fraction size during elution is usually set to a value
smaller than the expected peak volume.
Illustration
The illustration below shows examples of fractionation using fixed volume fractionation and fraction-
ation using peak fractionation.
A) Straight/fixed fractionation
Absorbance
Volume
B) Peak fractionation
Absorbance
Volume

5 Operation
5.8 Fractionation
Delay volume
Description
The delay volume settings are used to make sure that the fractions collected during fractionation, using
the outlet valve or the fraction collector, correspond to the fractions indicated in the chromatogram.
The delay volume is the volume between the UV monitor, and the fraction collector or outlet that is
used, see the illustration below.
Valve
UV/Vis
Conductivity pH Fraction collector
Delay volume
The time when a peak is detected The peak reaches the fraction
by the UV/Vis detector is called T0 collector at time T1
As the delay volume is affected by the length and diameter of the tubing, it should be set according
to the tubing and modules used.
Illustration of fraction marking using fixed volume fractionation

The illustration below shows the fractions collected, and the numerical marking of fractions, when
fixed volume (straight) fractionation is used with Fraction collector F9-R.
Absorbance
Volume
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Fractionation marks shown in chromatogram
When fixed volume fractionation is used the delay volume is collected at the beginning of fractionation.
Fraction collector F9-R saves the delay volume in the first fraction (fraction 1), see the example illustration
above. Delay volumes are not indicated with a numerical fraction mark on the chromatogram.
Fraction collector F9-C does not save the delay volume as a fraction, instead the delay volume is dis-
posed of in the waste funnel.
Note: If two fraction collectors are used, the fractions collected by Fraction collector F9-R, 2nd are
indicated by the prefix 2: (i.e. fractions 2:1, 2:2, 2:3 etc.).

5 Operation
5.8 Fractionation
Illustration of fractions and fraction marking using peak fractionation

The illustration below shows the fractions collected, and the numerical marking of fractions, when
peak fractionation is used with Fraction collector F9-R.
When peak fractionation is used, Fraction collector F9-R collects the delay volumes in the fractions
preceeding each peak. In the chromatogram above, delay volumes are collected in fractions 1, 5 and
8. The numerical fraction marks for the delay volume fractions are not indicated on the chromatogram.
Fraction collector F9-C does not save the delay volumes as fractions, instead the delay volumes are
disposed of in the waste funnel.
Note: If two fraction collectors are used, the fractions collected by the second Fraction collector are
indicated by the prefix 2: (i.e. fractions 2:1, 2:2, 2:3 etc.).
Fractionation modes for Fraction collector F9-C

There are three fractionation modes for Fraction collector F9-C, Automatic, Accumulator and DropSync.
Spillage between collection vessels during a run is avoided with all three fractionation modes.
• Automatic: The fraction collector uses the Drop Sync mode for flow rates up to 2 ml/min and auto-
matically switches to Accumulator mode for higher flow rates.
• Accumulator: Liquid is collected during movement between tubes or wells. The liquid is then dis-
pensed in the next well or tube. Fractionation with accumulator can be used at all flow rates.
• DropSync: When using DropSync , the sensors in the Dispenser head detect when a drop is released
from the nozzle. The Dispenser head moves to the next well or tube just after a drop is released.
Fractionation with DropSync can be used at flow rates up to 2 ml/min. If only one Cassette is used
and it is placed near the waste funnel, DropSync can be used at higher flow rates. Volatile solutions
and solutions with low surface tension may require a lower flow.
Fractionation settings for Fraction collector F9-R

There are two fractionation settings for Fraction collector F9-R, DropSync off or DropSync on .
Dropsync off: No synchronization of collection.

5 Operation
5.8 Fractionation
DropSync on: When using DropSync the sensors in the Tube sensor detect when a drop is released.
The Tube rack moves and positions the next tube under the Tube sensor just after a drop is released.
Fractionation with DropSync can be used at flow rates up to 2 ml/min. For water and solutions with
higher surface tension, a higher flow rate can be used. Volatile solutions and solutions with low surface
tension may require a lower flow.
Missing tubes in Fraction collector F9-R

If a tube is missing, Fraction collector F9-R will continue the fractionation on the tube row located
closer to the center of the fraction collector. The fractionation marks in the chromatogram will then
not reflect the tubes in which the sample is collected.
If the fraction collector runs out of tubes, the delivery arm moves to the fraction collector center position
while ÄKTA pure pauses and displays an error message.
Missing tubes or plates in Fraction collector F9-C

When automatic cassette configuration is selected in the system settings the fraction collector auto-
matically detects which types of cassettes and plates that are present. The fraction collector will
however not detect if tubes or bottles are missing in the cassettes. Make sure that the cassettes to be
used are occupied by appropriate types and numbers of tubes or bottles before starting a run.
It is not possible to change the cassette configuration during a run. When the system state is set to
Pause it is possible to take out cassettes or plates from the fraction collector only if they are replaced
by cassettes or plates of the same type and are placed in the same positions.
The action of the system when the last tube in the fraction collector is filled is set in the instruction
Last tube filled in the system settings. The flow can be directed to waste or to any of the outlets or
the run can be paused. If the action is set to Pause, the system automatically pauses when the last
tube is filled and prompts the user to replace the filled tubes.
Peak broadening
The width of peaks at the fraction collector is influenced by the properties of the column and the di-
mensions of tubing connecting the components. Initial sample volume affects the peak width in gel
filtration (GF) chromatography. A sample zone is broadened during passage through a GF column so
that the sample is diluted and the resolution decreases with increasing sample volume. Sample volume
does not however affect the resolution in adsorption chromatography techniques such as affinity
chromatography (AC), ion exchange chromatography (IEX), and hydrophobic interaction chromatography
(HIC). The effect of peak broadening in the system from sample injection to peak detection (including
dilution on the column) is apparent in the chromatogram from the UV monitor, but broadening from
the UV monitor to fraction collection is not visible in the chromatogram. This "hidden" effect can
sometimes be dramatic, especially for high-resolution columns.
To minimize peak broadening, use narrow and short tubing connections as far as possible. Remember
that using narrow tubing will increase the back pressure in the system.

5 Operation
5.9 Create a method and perform a run

Introduction
This section provides an overview of how to create a method in UNICORN and how to perform manual
and method runs on ÄKTA pure.
It also contains advice on things to be considered during a run.
In this section
This section contains the following sub-sections:
Section See page
5.9.1 Create a method 211
5.9.2 Prepare and perform a run 216
5.9.3 Monitor a run 218
5.9.4 After run procedures 220

5 Operation
5.9.1 Create a method
Introduction
The predefined methods are built up using phases, where each phase corresponds to a step in a
chromatography run with a number of properties associated with that phase.
See UNICORN Method Manual for more information about method structure, definitions and concepts
of methods in UNICORN.
Predefined methods
There are several predefined methods to choose from. All the predefined methods are listed below.
The predefined methods available for each system are defined by the Instrument Configuration. Refer
to Section 9.7 Predefined methods and phases, on page 438 for more information about each method.
Purification methods
• Affinity Chromatography (AC)
• Affinity Chromatography (AC) with Tag Removal
• Anion Exchange Chromatography (AIEX)
• Cation Exchange Chromatography (CIEX)
• Chromatofocusing (CF)
• Desalting
• Gel filtration (GF)
• Hydrophobic Interaction Chromatography (HIC)
• Manual Loop Fill
• NHS-coupling
• Reversed Phase Chromatography (RPC)
Maintenance methods
• Column CIP
• Column Performance Test
• Column Preparation
• System CIP
• System Preparation
Main steps when defining a new method

The main steps when defining a method are:
1 Create/open a method
• Create a Predefined method (including a set of phases that may be edited)

5 Operation
or
• Open an existing method that can be edited and saved with a new name or overwritten
2 Build/edit the Method Outline and/or edit the Phase Properties for the appropriate phases
3 Save the method
Create a new method

Follow the instructions below to create a new method.
Step Action
1 In the Method Editor:

• click the Create a new method icon in the Toolbar
or
• select File:New Method...
Result: The New Method dialog opens.

5 Operation
Step Action
2 In the New Method dialog:

• select a System
• select a Predefined Method
• click OK
Result: The Method Outline pane shows the included phases for the chosen method and
the Phase Properties pane shows the default settings for the currently highlighted phase.
See UNICORN Method Manual for more information about methods and method creation in UNICORN.
Note: Sample loop is called capillary loop in UNICORN.
Text Instruction mode

In most cases methods can be edited using the Phase Properties pane in the Method Editor module.
However, modules without a recommended position are not supported by Phase Properties and have
to be edited in the Text Instruction mode.
The following modules require that the method is created using the Text Instruction mode:
• Inlet valve IX
Note: When using Inlet valve IX (V9-IX or V9H-IX) connected to another inlet valve in a run, it is
necessary to use the instructions for both Inlet valve IX and the other inlet valve, respectively.

5 Operation
• Versatile valve
• I/O-box
• UV monitor, 2nd
Note: It is possible to reset the UV monitor U9-L, 2nd using Phase Properties. All other UV monitor
U9-L, 2nd instructions need to be edited in the Text Instruction mode.
Note: If installing more than one external air sensor, it is necessary to create the methods for the
additional air sensors using the Text Instruction mode.
Open and save methods for different systems

New ÄKTA pure methods are always created for the set of components of the current system. Methods
can be shared between systems, with the restriction that a method created for an ÄKTA pure 150
configured system cannot be used with an ÄKTA pure 25 configured system, and vice versa.
When a method is opened for a system that has been changed or if a method is saved for a system
with a different flow configuration, a dialog is opened where the user can choose to either adapt the
method or to keep the text method unchanged.

5 Operation
If you se- Then..

lect..
Adapt the method will be adapted to the new set of components. All functions and positions
method that are still available will be unchanged. For example, valve positions present on
both the original and the new system will be unchanged in the adapted method.
Non-compatible settings will be adjusted in order to properly adapt the method for
new components. Settings that have been changed are described and saved in
Method Notes, which are displayed after the method has been adapted.
Change the settings in Phase Properties if required.

Note:
Some predefined methods require certain components to be functional. Adapting
these methods to systems that do not include the required components is not possible.
Keep text the text method will be kept and nothing will be changed. Non-compatible settings
method will still be present in the method but they will not be functional. All Phase Properties
of the method will be replaced by tables of Phase Variables.

5 Operation
5.9.2 Prepare and perform a run
Introduction
This section describes how to start a run using a previously created method and how to perform a
manual run. For further information about the capabilities of the System Control module, refer to
UNICORN System Control Manual.
Choose and start a method

The following instruction describes how to open a method and start a run.
Step Action
1 Open the System Control module and click the icon Open Method Navigator.
Result: The Method Navigator pane opens.
2 Select the method to run, and click the Run icon.
Result: The Start Protocol dialog opens.
3 Step through the displayed pages in the Start Protocol, add requested input and make
appropriate changes if necessary. Click Next.
4 Click Start on the last page of the Start Protocol.

Result:
• If column logging was chosen during installation of UNICORN and a column type was
selected when the method was created, the Select Columns dialog opens. For further
information on column handling, please refer to UNICORN Method Manual and
UNICORN System Control Manual.
• If column logging was not chosen during installation of UNICORN and/or no column
type was selected when the method was created, the run starts directly.

5 Operation
Perform a manual run

Manual runs can be convenient for procedures such as filling tubing with buffer or packing a column
with media.
Step Action
1 On the Manual menu, click Execute Manual Instructions.
2 Select instruction group and instruction.
3 Select or enter parameter values.
4 Click Insert to have several instructions performed at the same breakpoint.

Note:
Manual runs are only stored temporarily. However, you can choose to store them perma-
nently in a selected directory. To save results in a chosen directory, click Browse before
the run is started.
Note:
If a method run is started during a manual run, the results from the manual run are not
stored.
5 To perform the instructions, click Execute.

5 Operation
5.9.3 Monitor a run
5.9.3 Monitor a run
Introduction
During the run, the System Control module will display the run progress of the method being executed.
This section describes how to interact with the run from the process picture or by executing manual
instructions.
To find an overview of the System Control user interface, see UNICORN System Control Manual.
Monitor the run

To interrupt a method during a run you may use the Hold, Pause or End icons in System Control. A
held or paused method run can be resumed by using the Continue icon. See the instructions in the
table below.
If you want to... then...
temporarily hold the method, with current flow rate and

valve positions sustained click the button.
temporarily pause the method, and stop all pumps

click the button.
resume, for example, a held or paused method run.

click the button.
Note:
An ended method cannot be resumed.
permanently end the run

click the button.
Note: When ending a method run in advance, it is possible to save the partial result.
More information regarding UNICORN capabilities during the method run is available in the UNICORN
System Control Manual.

5 Operation
5.9.3 Monitor a run
Actions in the process picture pane

It is possible to manually interact with an ongoing method through the Process Picture, see UNICORN
System Control Manual.
Using UNICORN to control the pressure during a

run
The function Pressure control allows a method to be run with the set flow rate without the risk of a
method stop due to pressure alarm. Pressure control is enabled in the instructions System flow and
Sample flow. Refer to Section 9.15 Pressure control, on page 500 for more information.
The system configuration affects the pressure

Using narrow tubing between components will improve resolution but will lead to increased back
pressure in the system. Narrow tubing after the column will increase the pressure in the column at a
given flow rate. Make sure that the sensor limits for the pre-column pressure are set so that the maxi-
mum pressure for the column in use is not exceeded.
Note: If Column valve V9-C or V9H-C is mounted, the integrated pressure sensor of the valve allows
the system to monitor the post-column pressure. The limit for the pressure sensor in Column
valve V9-C or V9H-C is automatically set so that the UV monitor and the pH monitor are pro-
tected from high pressure. If Column valve V9-C or V9H-C is not mounted, make sure to keep
the pressure in the system after the column below the pressure limits for the modules in the
flow path.

5 Operation
5.9.4 After run procedures
Introduction
This section describes how to clean the instrument and columns after a chromatographic run, and
how to prepare the system for storage.
The instrument and the columns should be cleaned between the runs. This will prevent, for example,
sample contamination, protein precipitation and column clogging. If the instrument is not going to be
used for a couple of days or longer, the instrument, columns and the pH flow cell should be filled with
storage solution. For further information about cleaning and maintenance procedures, see Chapter 7
Maintenance, on page 251.
Tip: To clean and fill the instrument and columns with storage solution, use System CIP and Column
CIP either as separate, predefined methods or as phases included in a chromatographic
method.
CAUTION
Hazardous chemicals and biological agents. Before maintenance, service and
decommissioning, wash the ÄKTA pure instrument with a neutral solution to make
sure that any hazardous solvents and biological agents have been flushed out from
the system.
System cleaning
After a method run is completed, perform the following:
• Rinse the instrument with one or several cleaning solution(s) (e.g., NaOH, buffer solution or distilled
water) using System CIP.
valve V9-C or V9H-C is automatically set so that the UV monitor and the pH monitor are
protected from high pressure. If Column valve V9-C or V9H-C is not mounted, make sure
to keep the pressure in the system after the column below the pressure limits for the modules
in the flow path.
• If applicable, empty the fraction collector.
• Clean all spills on the instrument and on the bench using a moist tissue.
• Empty the waste vessel.
• Clean the manual injection port of the injection valve, see Section 7.6.2 Perform System CIP, on
page 278 for detailed instructions.
• If applicable, clean the pH electrode manually and make sure to leave it in an appropriate buffer.
See Section 7.6.7 Clean the pH electrode, on page 293 for detailed instructions.

5 Operation
System storage
If the instrument is not going to be used for a couple of days or longer, also perform the following:
• Fill the system and inlets with storage solution (e.g., 20% ethanol) using System CIP.
valve V9-C or V9H-C is automatically set so that the UV monitor and the pH monitor are
protected from high pressure. If Column valve V9-C or V9H-C is not mounted, make sure
to keep the pressure in the system after the column below the pressure limits for the modules
in the flow path.
• If applicable, prepare the pH electrode for storage as described in Section 7.6.6 Storage of the pH
electrode, on page 291.
Column cleaning
After a method run is completed, perform the following:
• Clean the column with one or several cleaning solution(s) using Column CIP.
Column storage
If the column is not going to be used for a couple of days or longer, also perform the following:
• Fill the column with storage solution (e.g., 20% ethanol) using Column CIP.
pH electrode storage
If pH monitoring will not be used for a week or longer, perform one of the following actions:
• Inject new storage solution into the pH flow cell. Refer to Section 7.6.6 Storage of the pH electrode,
on page 291 for instructions.
• Replace the pH electrode with the dummy electrode that is installed in the pH valve on delivery.
In the following situations, in order to increase the lifetime of the pH electrode, use the By-pass position
and store the electrode in storage solution inside the pH flow cell:
• pH monitoring is not needed during the run.
• Organic solutions are used.
• Extremely acidic or extremely basic solutions are used.
For further information on how to prepare the pH electrode for storage, refer to Section 7.6.6 Storage
of the pH electrode, on page 291.

5 Operation
Log off or exit UNICORN

Follow the instructions to log off or exit UNICORN. This can be performed from any of the UNICORN
modules.
If you want to... then...
log off UNICORN on the File menu, click Log off.
Result: All open UNICORN modules close and the Log On dialog box
opens.
exit UNICORN on the File menu, click Exit UNICORN.
Result: All open UNICORN modules close.
Note: If an edited method or result is open and not saved when you try to exit or log off UNICORN,
you will see a warning. Click Yes to save, No to exit without saving, or Cancel to stay logged
on.

5 Operation
Shut down the instrument

Switch off the instrument by pressing the power switch to the O position.

6 Performance tests
6 Performance tests
About this chapter

This chapter provides information about performance tests.
Performance tests should be run after installation to check the function of the ÄKTA pure system. Dif-
ferent tests are available and the tests to perform depend on the system configuration used.
Performance tests can also be used at any time to check the condition of the system, for example,
after a prolonged stop. This chapter describes how to prepare, run, and evaluate the different perfor-
mance tests available.
In this chapter
Section See page
6.1 General performance test actions 225
6.2 Air sensor A and Inlet valve A tests 228
6.3 Air sensor B and Inlet valve B tests 230
6.4 Air sensor S and Sample inlet valve tests 232
6.5 Column valve C tests 234
6.6 Fraction collector F9-C test 236
6.7 Fraction Collector F9-R Test 239
6.8 Sample pump tests 241
6.9 System Test UV U9-L (fixed) 243
6.10 System Test UV U9-M (variable) 247

6 Performance tests
6.1 General performance test actions

Introduction
Some actions are identical for each performance test. These actions are described in this section.
Start the performance tests

Follow the instructions to start a performance test.
Step Action
1 In the System Control module, on the System menu, click Performance Test and Report.
Result: The System Performance Test and Report dialog opens.
2 In the System Performance Test and Report dialog, click one of the following tests:
Air sensor A and inlet valve V9-IA test Air sensor A and inlet valve V9H-IA test
Air sensor B and inlet valve V9-IB test Air sensor B and inlet valve V9H-IB test
Air sensor S and Sample inlet valve Air sensor S and Sample inlet valve
V9-IS test V9H-IS test
Column valve V9-C test Column valve V9H-C test
Fraction Collector F9-R Test Fraction Collector F9-R Test
Fraction Collector F9-C Test Fraction Collector F9-C Test
Sample pump S9 test Sample pump S9H test
System Test UV U9-L (fixed) System Test UV U9-L (fixed)
System Test UV U9-M (variable) System Test UV U9-M (variable)
3 Click Run Performance Method.

Result: The Start Protocol dialog of the selected test opens.
4 Click Next in the Start Protocol dialog to open the next page. The pages are described
in the table Overview of the Start Protocol dialog, on page 226.
5 In the last page, Result name and location, click Start.

Result: The selected test starts.

6 Performance tests
Overview of the Start Protocol dialog

The following table describes the pages of the Start Protocol.
Page Description
Notes Displays the Method Notes of the method. The Method Notes con-
tains a method description and instructions on how to run the
method. This dialog box also allows the user to enter Start Notes.
Evaluation Procedures Allows the user to select to save the report to file (recommended)
and/or to print the report.
Result Name and Location Allows the user to change result name and result location.
During the run

A Message dialog box opens during the run. Read the messages in the dialog, and make sure that
necessary preparations have been performed.
• Click Confirm and Continue in the Message dialog box to change system state from System Pause
to Run and proceed with the test.
• Alternatively, click Confirm in the Message dialog box and click the Continue button on the Instru-
ment display.

6 Performance tests
Automatic evaluation
The system automatically generates a report when the test is finished. The report can be printed in
two ways:
• It is recommended to select Save the report to file in the Evaluation Procedures page of the Start
Protocol dialog box when starting the test. The report is saved in the folder Temp in your UNICORN
installation folder. For example C:\Program Files\GE Healthcare\UNICORN\UNICORN 7.0\Temp.
• If the option Print report was selected in the Evaluation Procedures page of the Start Protocol
dialog box when starting the test, the report is also automatically printed on the system printer.
Refer to UNICORN Administration and Technical Manual for information on how to install a printer.
Print the report and check the status of the tests. For each of the tests the report states "The test
passed" or "The test failed".
Note: The fraction collector test is evaluated manually and no report is generated.

6 Performance tests
6.2 Air sensor A and Inlet valve A tests

Air sensor A and Inlet valve A tests
The following Air sensor A and Inlet valve A tests are available, for ÄKTA pure 25 and ÄKTA pure 150,
respectively:
• Air sensor A and inlet valve V9-IA test
• Air sensor A and inlet valve V9H-IA test
Method description
The Air sensor A and inlet valve A tests switches inlet valve A, labeled V9-IA or V9H-IA, to an empty
position and checks if the air sensor detects air.
The method run takes approximately 1 minute.
Required configuration
A correctly installed Inlet valve A is required to run the test.
Required material
The following materials are required:
• Syringe, 25-30 ml
• Distilled water
Prepare the test

Follow the instructions below to prepare the system before method start.
Step Action
1 Direct the outlet tubing marked W to a waste container.
2 Place inlet tubing A1 into distilled water.
3 Prime the A1 inlet and purge the pump. See Section 5.4.1 System pumps, on page 161.
4 Disconnect the inlet tubing connected to the A3 inlet valve position. During the test
method air is introduced into the inlet valve through that inlet port to test the function
of the air sensor.

6 Performance tests
Run and evaluate the test

Follow the instructions described in Section 6.1 General performance test actions, on page 225 to start,
run and automatically evaluate the performance test.
Possible causes of a failed test

The tables below describe possible causes of a failed test. When possible sources of error have been
checked and corrected, repeat the test.
Faulty air sensor A test
Cause Action
Faulty air sensor For further information, see Section 8.4 Troubleshooting: Valves, on
page 375.
Incorrect preparation of Make sure that the tubing was correctly prepared, see Prepare the
tubing test, on page 228.
Faulty inlet valve A test
Cause Action
The valve did not switch For further information, see Section 8.4 Troubleshooting: Valves, on
page 375.

6 Performance tests
6.3 Air sensor B and Inlet valve B tests

Air sensor B and Inlet valve B tests
The following Air sensor B and Inlet valve B tests are available, for ÄKTA pure 25 and ÄKTA pure 150,
respectively:
• Air sensor B and inlet valve V9-IB test
• Air sensor B and inlet valve V9H-IB test
Method description
The Air sensor B and inlet valve B test switches the inlet valve B, labeled V9-IB or V9H-IB, to an empty
position and checks if the air sensor detects air.
A correctly installed Inlet valve B is required to run the test.
Required material
• Distilled water
Prepare the test

Step Action
1 Direct the outlet tubing marked W to a waste container.
2 Place inlet tubing B1 into distilled water.
3 Prime the B1 inlet and purge the pump. See Section 5.4.1 System pumps, on page 161.
4 Disconnect the inlet tubing connected to inlet valve position B3. During the test method
air is introduced into the inlet valve through that inlet port to test the function of the air
sensor.

6 Performance tests


Faulty air sensor B test
Cause Action
page 375.
Faulty inlet valve B test
Cause Action
page 375.

6 Performance tests
6.4 Air sensor S and Sample inlet valve tests

Air sensor S and Sample inlet valve tests
The following Air sensor S and Sample inlet valve tests are available, for ÄKTA pure 25 and ÄKTA pure
150, respectively:
• Air sensor S and Sample inlet valve V9-IS test
• Air sensor S and Sample inlet valve V9H-IS test
Method description
The Air sensor S and Sample inlet valve test checks the functionality of the Sample inlet valve, labeled
V9-IS or V9H-IS, and the integrated air sensor in ÄKTA pure.
Core components of the ÄKTA pure and correctly installed Sample Inlet valve and Sample pump are
required to run the test.
Required material
• Distilled water
Prepare the test

Step Action
1 Open System Control and update the system setting Tubing: Sample pump to injection
valve (found in System Settings:Tubing and Delay volumes).
Note:
The system setting Tubing: Sample pump to injection valve is available only if Column
valve C is not included in the system.
2 Immerse waste tubing W in a waste container
3 Immerse sample inlet tubing Buffer in distilled water.
4 Prime inlet tubing A1 and sample inlet Buffer and purge the sample pump. See Section 5.4
Prime inlets and purge pump heads, on page160. No air shall be left in the pumps or tubings.

6 Performance tests
Step Action
5 Disconnect the inlet tubing connected to the sample inlet valve position S3. During the
test method air is introduced into the sample inlet valve through that inlet port to test
the function of the air sensor.


Faulty air sensor S test
Cause Action
page 375.
Faulty Sample inlet valve test
Cause Action
page 375.

6 Performance tests
6.5 Column valve C tests

Column valve C tests
The following Column valve C tests are available, for ÄKTA pure 25 and ÄKTA pure 150, respectively:
• Column valve V9-C test
• Column valve V9H-C test
Note: No tests are available for V9-Cs or V9H-Cs.
Method description
The Column valve C test checks the functionality of Column valve C, labeled V9-C or V9H-C, and of the
integrated pressure sensors that measure pre-column and post-column pressure. If two valves are
used, the test will check the pre-column pressure sensor in the first valve and the post-column pressure
sensor in the second valve.
A correctly installed Column valve C is required to run the test.
Required material
• Distilled water
• Ref 1 tubing, see Reference capillary, on page 426.
Prepare the test

Step Action
1 Connect the Ref 1 tubing between Column valve C ports 1A and 1B.
2 Make sure the Flow restrictor is inline.
3 Direct outlet tubing W to a waste container.
5 Prime the inlet A1 and purge the pump. See Section 5.4.1 System pumps, on page 161.

6 Performance tests


The table below describes possible causes of a failed test. When possible sources of error have been
Cause Action
Faulty Column valve For further information, see Section 8.4 Troubleshooting: Valves, on
page 375.
The integrated pressure Calibrate the pressure monitors, see Section 7.7.2 Calibrate the
monitors are not calibrated pressure monitors, on page 305.
Incorrect preparation of the Make sure that the reference tubing is connected between column
system ports 1A and 1B and that the flow restrictor is in-line.

6 Performance tests
6.6 Fraction collector F9-C test

Method description
The Fraction Collector Test checks the functionality of Fraction collector F9-C.
The method run takes approximately 6 minutes.
The core components of ÄKTA pure, an outlet valve and a correctly installed Fraction collector F9-C
are required to run the test.
Required material
• Distilled water
• Two deep well cassettes
• Two 96 deep well plates for collecting the fractions
Prepare the test

Step Action
1 Immerse waste tubing W in a waste container
2 Immerse inlet tubing A1 in distilled water.
3 Prime inlet tubing A1 and purge System pump A. See Section 5.4.1 System pumps, on
page 161. No air shall be left in the pump or tubing.
4 Place two 96 deep well plates in positions 1 and 6 in the fraction collector. Make sure
that no other cassettes are present in the fraction collector during the run.
5 In the System Control module, select System:Settings.


• Select Fraction collection:Fractionation settings.
• In the Fraction mode field, select Automatic.
• In the Fractionation order field, select Row-Automaticby-Row.
• Click OK.

6 Performance tests
Step Action

• Select Tubing and Delay volumes.
• Select the instruction Delay volume: Monitor to frac
• Set the correct delay volume, see Section 9.13 Delay volumes, on page 493
• Click OK.

Follow the instructions described in Section 6.1 General performance test actions, on page 225 to start
and run the performance test.
During the run

• Visually check that the fraction collector wash is performed.
• Several messages will be displayed on the screen during the startup of the run. Read the messages
in the dialog and make sure that necessary preparations have been performed. Click the "Confirm
and Continue" button in the message dialog to proceed with the test.
Evaluate the result

To evaluate the results, do the following:
• Check that correct volumes, 2 ml/well, are collected in the three first wells in row A (A1-A3) of both
plates.
• Check if the fractionation marks in the chromatogram are in accordance with the filled wells. For
further information on delay volumes and fractionation marks, see Delay volume, on page 207.

6 Performance tests

Cause Action
Incorrect volumes collected Air in pumps: Make sure to prime inlet tubing A1 and purge System
in the tubes, and distur- pump A before method start, see Section 5.4.1 System pumps, on
bances of system pressure page 161.
curves: Faulty pump: See Section 8.6 Troubleshooting: Pumps, on page 387.
• Air trapped in System
pump A
• Faulty System pump A
Incorrect preparation of Make sure that the system was correctly prepared, see Prepare the
buffer and tubing test, on page 236.

6 Performance tests
6.7 Fraction Collector F9-R Test

Method description
The Fraction collector F9-R test checks the functionality of Fraction collector F9-R.
A correctly installed Outlet valve, and a correctly installed Fraction collector F9-R are required to run
the test.
Required material
• Distilled water
• 7 tubes for collecting the fractions.
Prepare the test

Step Action
1 Direct outlet tubing W to a waste container.
3 Prime inlet A1 and purge System pump A. See Section 5.4.1 System pumps, on page 161.
4 Place 7 tubes in the Fraction collector, in positions 1 to 7.


• Select Fraction collector:Fractionation settings.
• In the Drop sync field, select On.
• Click OK.

Follow the instructions described in Section 6.1 General performance test actions, on page 225 to start
and run the performance test.

6 Performance tests
Evaluate the result

Check that the correct volumes have been collected in the tubes. The tubes should contain the following:
• Tube 1: The delay volume
• Tube 2-4: 2 ml
• Tube 5-6: 1 ml
Also, check that the fractionation marks in the chromatogram correspond to the filled tubes and that
spillages are kept to a minimum.
For further information on delay volumes and fractionation marks, see Delay volume, on page 207.

Cause Action
Incorrect volumes collected Air in pumps: Make sure to prime inlet tubing A1 and purge System
in the tubes, and distur- pump A before method start, see Section 5.4.1 System pumps, on
bances of system pressure page 161.
curves: Faulty pump: See Section 8.6 Troubleshooting: Pumps, on page 387.
• Air trapped in System
pump A
• Faulty System pump A
Liquid collected in wrong Make sure the fraction collector delivery arm is positioned above
tubes: tube number 1 before starting the test.

6 Performance tests
6.8 Sample pump tests

Sample pumps tests
The following Sample pump tests are available, for ÄKTA pure 25 and ÄKTA pure 150, respectively:
• Sample pump S9 test
• Sample pump S9H test
Method description
The Sample Pump test checks the functionality of the sample pump, labeled S9 or S9H.
The core components of ÄKTA pure and a correctly installed Sample pump are required to run the test.
Required material
• Distilled water
• 1% Acetone and 1.00 M NaCl in distilled water
Prepare the test

Step Action
1 Connect a tubing between Injection valve port column and the UV inlet, preferably the
Ref 1 tubing included with the system at delivery.
2 Immerse waste tubing W, W1 and W2 in a waste container.
3 Immerse inlet tubing A1 in distilled water.
4 Immerse sample inlet tubing Buffer in 1% Acetone and 1.00 M NaCl in distilled water.
5 Prime inlet tubing A1 and sample inlet Buffer and purge the pumps. See Section 5.4.2
Sample pump, on page 167 and. No air shall be left in the pumps or tubings.

6 Performance tests


Cause Action
The two injected peaks of liq- Air in pump: Make sure to prime inlet tubing Buffer and purge sample
uid are not delivered as pro- pump before method start, see Section 5.4.2 Sample pump, on
portional peaks: page 167.
• Air trapped in Sample Faulty pump: See Section 8.6 Troubleshooting: Pumps, on page 387.
pump
• Faulty Sample pump
The delivery of liquid is pulsat- Air in pump: Make sure to prime inlet tubing Buffer and purge sample
ing (unstable pump pressure) pump before method start, see Section 5.4.2 Sample pump, on
page 167.
Faulty pump: See Section 8.6 Troubleshooting: Pumps, on page 387.
The pump pressure is too Folded, twisted or blocked tubing: Check the tubing.
high (at a flow rate of 25 The Sample pump pressure monitor is not calibrated: Calibrate the
ml/min or 150 ml/min for pressure monitor, see Section 7.7.2 Calibrate the pressure monitors,
ÄKTA pure 25 and ÄKTA pure on page 305.
150, respectively)
Faulty pump: See Section 8.6 Troubleshooting: Pumps, on page 387.

6 Performance tests
6.9 System Test UV U9-L (fixed)

Method description
The System test with UV monitor U9-L checks the functionality of the solvent delivery, the pumps, the
System pump pressure sensor, UV monitor U9-L and the Conductivity monitor.
A correctly installed UV monitor U9-L and a correctly installed Conductivity monitor are required to
run the test.
Required material
• Distilled water
• 1% acetone and 1.00 M NaCl in distilled water
• Mixer, 1.4 ml
Prepare the test

Step Action
1 Direct outlet tubing W and W1 to a waste container.
3 Place inlet tubing B1 into a solution of 1% acetone and 1.00 M NaCl in distilled water.
4 Connect the Ref 1 tubing between Injection valve port Col and the UV monitor U9-L inlet,
to generate a back pressure.
5 Make sure that the Mixer with a chamber volume of 1.4 ml is installed.
For further information, see Section 7.8.3 Replace the Mixer, on page 322.
6 Prime the buffer inlets and purge System pump A and System pump B. See Section 5.4.1
System pumps, on page 161.
Note: If the test is performed at cold room temperature, select Conductivity:Cond temp compensa-
tion in the System Settings dialog, and set the Compensation factor to 2.1.

6 Performance tests

Illustration of chromatogram
The illustration below shows a chromatogram from a System test with UV monitor U9-L.

The following tables describe possible causes of a failed test. When possible sources of error have
been checked and corrected, repeat the test.
Faulty Gradient Test Result

Cause Action
Disturbances caused by air Make sure to prime the buffer inlets and to purge the System pumps
trapped in any of the pumps before method start. See Section 5.4.1 System pumps, on page 161.
Disturbances caused by Replace piston seals. See Chapter 7 Maintenance, on page 251.
damaged pump piston seals
Unstable or incorrect UV sig- See Section 8.3 Troubleshooting: Monitors, on page 360.
nal, or drifting base line:
• Faulty UV monitor
Wrong Mixer chamber size Replace the Mixer. See Chapter 7 Maintenance, on page 251.
or faulty Mixer

6 Performance tests
Faulty Step Response Result

Cause Action
If all values are faulty: Air in pumps: Make sure to prime the buffer inlets and to purge the
System pumps before method start. See Section 5.4.1 System pumps,
• Air in the pump or a faulty
on page 161.
pump
Faulty pump: See Chapter 7 Maintenance, on page 251.
Faulty values at 5%: Replace piston seals. See Chapter 7 Maintenance, on page 251.
• Damaged pump piston
seal in System pump B
seal in System pump A
Faulty UV Absorbance Test

Cause Action
Incorrectly prepared acetone Make sure that the acetone solution is 1% and that no solution has
solution evaporated.
Wrong UV cell path length See, Calibration of the UV monitor U9-L flow cell length, on page 312.
set in UNICORN
Faulty Pulsation Test

Cause Action
Air trapped in the pumps Make sure to prime and purge the system pumps before starting
the test, see Section 5.4.1 System pumps, on page 161.
Faulty Conductivity Test Result

Cause Action
Faulty Conductivity monitor See Conductivity monitor, on page 367.
Incorrectly prepared NaCl Make sure the NaCl solution is 1.00 M.

solution
The value set for the Cond If the test is performed at cold room temperature, open the System
temp compensation factor is Settings dialog, select Conductivity:Cond temp compensation and
not optimal set the Compensation factor to 2.1

6 Performance tests
Faulty UV Noise Test

Cause Action
Air or dirt in the UV flow cell Flush or clean the UV cell, see Section 7.5.1 Clean the UV flow cell,
on page 273.
Impure buffers Check the buffers.
Faulty Pressure Check Test

Cause Action
Folded, twisted or blocked Check the tubing.

tubing
The inline filter is dirty Replace the inline filter, see Section 7.3.2 Replace the inline filter, on
page 266.
The System pressure monitor Calibrate the pressure monitor, see Section 7.7.2 Calibrate the pres-
is not calibrated sure monitors, on page 305.

6 Performance tests
6.10 System Test UV U9-M (variable)

Method description
The System test with UV monitor U9-M checks the functionality of the solvent delivery, the pumps, the
System pump pressure sensor, UV monitor U9-M and the Conductivity monitor.
A correctly installed UV monitor U9-M and a correctly installed Conductivity monitor are required to
run the test.
Required material
• Distilled water
• 1% acetone and 1.00 M NaCl in distilled water
• Mixer, 1.4 ml
Prepare the test

Step Action
1 Direct outlet tubing W and W1 to a waste container.
3 Place inlet tubing B1 into a solution of 1% acetone and 1.00 M NaCl in distilled water.
4 Connect the Ref 1 tubing between Injection valve port Col and the UV monitor U9-M inlet,
to generate a back pressure.
5 Make sure that the Mixer with a chamber volume of 1.4 ml is installed.
For further information, see Section 7.8.3 Replace the Mixer, on page 322.
6 Prime the buffer inlets and purge System pump A and System pump B. See Section 5.4.1
System pumps, on page 161.
Note: If the test is performed at cold room temperature, select Conductivity:Cond temp compensa-
tion in the System Settings dialog, and set the Compensation factor to 2.1.

6 Performance tests

Illustration of chromatogram
The illustration below shows a chromatogram from a System test with UV monitor U9-M.

The following tables describe possible causes of a failed test. When possible sources of error have
been checked and corrected, repeat the test.
Faulty Gradient Test Result

Cause Action
Disturbances caused by air Make sure to prime the buffer inlets and to purge the System pumps
trapped in any of the pumps before method start. See Section 5.4.1 System pumps, on page 161.
Disturbances caused by Replace piston seals. See Chapter 7 Maintenance, on page 251.
damaged pump piston seals
Unstable or incorrect UV sig- See Section 8.3 Troubleshooting: Monitors, on page 360.
nal, or drifting base line:
• Faulty UV monitor
Wrong Mixer chamber size Replace the Mixer. See Chapter 7 Maintenance, on page 251.
or faulty Mixer

6 Performance tests
Faulty Step Response Result

Cause Action
If all values are faulty: Air in pumps: Make sure to prime the buffer inlets and to purge the
System pumps before method start. See Section 5.4.1 System pumps,
• Air in the pump or a faulty
on page 161.
pump
Faulty pump: See Chapter 7 Maintenance, on page 251
seal in System pump B
seal in System pump A
Faulty UV Response Test Result

Cause Action
Faulty UV monitor Restart the instrument to calibrate the UV monitor.

See Section 8.3 Troubleshooting: Monitors, on page 360.
Faulty UV Absorbance Test

Cause Action
Incorrectly prepared acetone Make sure that the acetone solution is 1% and that no solution has
solution evaporated.
Faulty Pulsation Test

Cause Action
Air trapped in the pumps Make sure to prime and purge the system pumps before starting
the test, see Section 5.4.1 System pumps, on page 161.

6 Performance tests
Faulty Conductivity Test Result

Cause Action
Faulty Conductivity monitor See Conductivity monitor, on page 367.
Incorrectly prepared NaCl Make sure that the NaCl solution is 1.00 M.
solution
The value set for the Cond If the test is performed at cold room temperature, open the System
temp compensation factor is Settings dialog, select Conductivity:Cond temp compensation and
not optimal set the Compensation factor to 2.1
Faulty UV Noise Test

Cause Action
Air or dirt in the UV flow cell Flush or clean the UV cell, see Section 7.5.1 Clean the UV flow cell,
on page 273.
Impure buffer Check buffers for impurities.
Faulty Pressure Check Test

Cause Action
Folded, twisted or blocked Check the tubing.

tubing
Dirt in inline filter Replace the inline filter, see Section 7.3.2 Replace the inline filter, on
page 266.
System pressure monitor not Calibrate the pressure monitor, see Section 7.7.2 Calibrate the pres-
calibrated sure monitors, on page 305.

7 Maintenance
7 Maintenance
About this chapter

This chapter describes the maintenance program for ÄKTA pure and provides instructions for mainte-
nance and replacement of spare parts.
In this chapter
Section See page
7.1 Maintenance Manager 252
7.2 Maintenance program 258
7.3 Weekly maintenance 261
7.4 Monthly maintenance 269
7.5 Semiannual maintenance 272
7.6 Maintenance when required 276
7.7 Calibration procedures 302
7.8 Replacement procedures 316

7 Maintenance
7.1 Maintenance Manager

Introduction
Maintenance Manager allows the user to display general information about the system and its modules,
and also operational statistics of the modules. Notifications for maintenance actions of the system
and its modules are predefined. The user can add automated maintenance notifications for the system.
Maintenance notifications are based on calender periods of system use, and for some systems also
on operational statistics for the modules.
Open Maintenance Manager

In the System Control module, on the System menu, click Maintenance Manager to open the Mainte-
nance Manager dialog box.

7 Maintenance
View general information and statistics

In the left pane of the Maintenance Manager dialog box, select the system of interest to view general
information of the selected system. For some systems it is possible to view information for a module
of interest. When modules are selected, operational statistics are also displayed.

7 Maintenance
View maintenance notifications

Click the plus symbol (+) of the system of interest to expand the list of related maintenance notifications.
For some systems it is possible to view maintenance notifications for a module of interest. Select a
notification to view notification details.
Note: Modules with no plus symbol (+) have no related maintenance notifications.

7 Maintenance
Edit a maintenance notification

Follow the instruction to edit a maintenance notification.
Step Action
1 In the left pane of the Maintenance Manager dialog box, select a maintenance notification.
Result: Details of the selected maintenance notification are displayed in the dialog box.
2 Edit the maintenance notification as desired:

• Select the Enable Event check box to activate the notification. If the box is unchecked,
the notification is not issued.
• Enter a new interval after which the new notification is issued.
• Click Restart to reset the counter and add a complete interval before the next notifi-
cation.
• Edit the message that is shown in the maintenance notification.
• Click Default to restore the default settings for maintenance notifications.
3 Click Apply to save the changes.
Add a new system notification

The user can add new system notifications to the list of system events.
Follow the instructions to add a new system notification.

7 Maintenance
Step Action
1 In the Maintenance Manager dialog box, click New System Notification.

Result: The NewNotification field appears in the Maintenance Manager dialog box.
2 In the NewNotification field:

• Enter a name for the new notification.
• Select a time interval after which the new notification is issued.
• If desired, write a message that is shown for the maintenance notification.
3 Click Apply to save the changes and apply the notification settings.
Delete a user defined system notification

To delete a user defined system notification, select the notification in the Maintenance Manager dialog
box and press the Delete button.
Note: Module notifications are predefined and cannot be deleted. If desired, they can be disabled.

7 Maintenance
Handle a maintenance notification

Each maintenance notification has a time interval after which the notification is issued. When this time
interval has been reached, a Maintenance Notification dialog box appears.
Note: The possibility to add maintenance notifications for modules is only available for some systems,
but all systems can add maintenance notifications for the complete system.
Follow the instruction to handle the notification.
Click... to...
Acknowledge reset the counter for a new maintenance notification period.

Note:
Make sure that the maintenance action is performed as instructed
after the notification is acknowledged, otherwise the system perfor-
mance can deteriorate.
Ignore close the dialog box without action.

Note:
The Maintenance Notification is displayed each time the System
Control module is opened until the notification is acknowledged.
Note: The predefined maintenance notifcation periods use average values. The actual service interval
for a specific module can be shorter or longer.

7 Maintenance
7.2 Maintenance program

Introduction
This section lists the periodic maintenance activities that should be performed by the user of ÄKTA
pure, as well as maintenance activities that should be performed when required.
Maintenance is divided into:
• Daily maintenance
• Weekly maintenance
• Monthly maintenance
• Semiannual maintenance
• Maintenance when required
WARNING
Hazardous biological agents during run. When using hazardous biological agents,
run System CIP and Column CIP to flush the entire system tubing with bacteriostatic
solution (e.g., NaOH) followed by a neutral buffer and finally distilled water, before
service and maintenance.
CAUTION
Hazardous chemicals during run. When using hazardous chemicals, run System
CIP and Column CIP to flush the entire system tubing with distilled water, before
CAUTION
Always use appropriate personal protective equipment when decommissioning the
equipment.
Periodic maintenance program

The following periodic maintenance should be performed by the user of ÄKTA pure.
Interval Maintenance action See section
Daily Calibrate the pH monitor Section 7.7.1 Calibrate the pH monitor,

on page 303
Weekly Calibrate pressure monitors Section 7.7.2 Calibrate the pressure

monitors, on page 305

7 Maintenance
Interval Maintenance action See section
Weekly Change pump rinsing solution Section 7.3.1 Change pump rinsing so-
lution, on page 262
Weekly Clean fraction collector diodes Section 7.3.3 Clean the Fraction collec-
tor F9-C sensors, on page 267
Monthly Check the Flow restrictor Check flow restrictor
Semiannually Clean the UV flow cell Section 7.5.1 Clean the UV flow cell, on
page 273
Semiannually Replace pH electrode Section 7.5.2 Replace the pH electrode,

on page 275
Maintenance when required

The following maintenance should be performed by the user of ÄKTA pure when required.
Maintenance action See section
Clean the instrument externally Section 7.6.1 Clean the instrument externally, on page 277
Perform System CIP Section 7.6.2 Perform System CIP, on page 278
Perform Column CIP Section 7.6.3 Perform Column CIP, on page 284
Clean Fraction collector F9-C Section 7.6.4 Clean Fraction collector F9-C, on page 288
Clean Fraction collector F9-R Section 7.6.5 Clean Fraction collector F9-R, on page 290
Replace tubing and connectors Section 7.8.1 Replace tubing and connectors, on page 317
Storage of pH electrode Section 7.6.6 Storage of the pH electrode, on page 291
Clean the pH electrode Section 7.6.7 Clean the pH electrode, on page 293
Clean the Conductivity flow cell Section 7.6.10 Clean the Conductivity flow cell, on page300
Calibrate the Conductivity monitor Section 7.7.3 Calibrate the Conductivity monitor, on
page 308
Calibrate the UV monitor Section 7.7.4 Calibrate the UV monitors, on page 312
Replace Mixer Section 7.8.3 Replace the Mixer, on page 322
Replace inline filter Section 7.3.2 Replace the inline filter, on page 266
Replace O-ring in Mixer Section 7.8.4 Replace the O-ring inside the Mixer, on
page 323

7 Maintenance
Maintenance action See section
Replace UV flow cell Section 7.8.5 Replace the UV monitor U9-M flow cell, on
page 325 and Section 7.8.6 Replace the UV monitor U9-L
flow cell, on page 328
Replace the Flow restrictor Section 7.8.7 Replace Flow restrictor, on page 330
Replace inlet filters Section 7.8.8 Replace the inlet filters, on page 331
Wipe off excess oil from the pump head Section 7.6.9 Wipe off excess oil from the pump head, on
page 299
Clean the check valves Section 7.6.8 Clean the pump head check valves, on
page 296
Replace check valves Section 7.8.9 Replace the pump head check valves, on
page 332
Replace pump piston seals, pump P9 and Section 7.8.10 Replace pump piston seals of Pump P9 or
P9H P9H, on page 335
Replace pump piston seals, pump P9-S Section 7.8.11 Replace pump piston seals of Pump P9-S,
on page 344
Replace pump pistons Section 7.8.12 Replace pump pistons, on page 351
Replace pump rinsing system tubing Section 7.8.13 Replace pump rinsing system tubing, on
page 352

7 Maintenance
7.3 Weekly maintenance

Introduction
This section provides instructions for weekly maintenance activities.
In this section
Section See page
7.3.1 Change pump rinsing solution 262
7.3.2 Replace the inline filter 266
7.3.3 Clean the Fraction collector F9-C sensors 267

7 Maintenance
7.3.1 Change pump rinsing solution
Maintenance interval
Replace the pump rinsing solution in the system pumps and the sample pump (optional module) every
week to prevent bacterial growth.
Required material
The following material are required:
• 20% ethanol
Illustration of the system pump piston rinsing

system
The illustrations below show the parts and tubing of the system pump piston rinsing system
5 4
Part Description
4 Outlet tubing
5 Inlet tubing

7 Maintenance
Prime the system pump piston rinsing system

Follow the instructions below to fill the pump piston rinsing system with rinsing solution. See the tubing
configuration of the rinsing system in the illustration above.
Step Action
1 Remove the rinsing system tube from the holder.
2 Fill the rinsing system tube with 50 ml of 20% ethanol.
3 Place the rinsing solution tube back in the holder.
4 Insert the inlet tubing to the System pump piston rinsing system into the fluid in the
rinsing solution tube.
Note:
Make sure that the inlet tubing reaches close to the bottom of the rinsing solution tube.
5 Connect a 25 to 30 ml syringe to the outlet tubing of the System pump piston rinsing
system. Draw liquid slowly into the syringe.
6 Disconnect the syringe and discard its contents.
7 Insert the outlet tubing into the fluid in the rinsing solution tube.
8 Fill the rinsing solution tube so that the tube contains 50 ml of 20% ethanol.

7 Maintenance
Illustrations of the sample pump piston rinsing

system
4 5
Part Function
connection) .
liquid tube.

7 Maintenance
Prime the sample pump piston rinsing system

Follow the instructions below to fill the pump piston rinsing system with rinsing solution. For the tubing
configuration of the rinsing system, see the illustration above.
Step Action
1 Unscrew and remove the rinsing liquid tube from the holder.
2 Fill the rinsing liquid tube with 50 ml of 20% ethanol.
3 Put the rinsing liquid tube back in the holder and fasten it by screwing it into place.
4 Insert the inlet tubing to the piston rinsing system into the fluid in the rinsing liquid tube.
Note:
Make sure that the inlet tubing reaches close to the bottom of the rinsing liquid tube.
5 Connect a 25 to 30 ml syringe to the outlet tubing of the System pump piston rinsing
system. Draw liquid slowly into the syringe.
Note:
The outlet tubing is colored orange for clarity in the above illustration.
6 Disconnect the syringe and discard its contents.
7 Insert the outlet tubing into the fluid in the rinsing liquid tube.
8 Fill the rinsing liquid tube so that the tube contains 50 ml of 20% ethanol.

7 Maintenance
7.3.2 Replace the inline filter
7.3.2 Replace the inline filter
Replace the inline filter that is located in the top section of the Mixer every week, or when required, for
example when the filter becomes clogged.
Required material
• Online filter kit
• Forceps
• Gloves
Instruction
Follow the instructions below to replace the inline filter that is located in the top of the Mixer.
Tip: Use forceps and gloves during the replacement procedure to avoid contaminating the Mixer
components.
Step Action
1 Unscrew the top section of the Mixer.
2 Remove the old filter using forceps. Replace the support net if this is damaged. Fit the
new filter.
3 Check the O-ring of the Mixer. If the O-ring is damaged, replace it according to Section 7.8.4
Replace the O-ring inside the Mixer, on page 323.
4 While holding the Mixer upright, screw the top section back onto the Mixer.

7 Maintenance
7.3.3 Clean the Fraction collector F9-C sensors
Clean the fraction collector sensors every week, or when required, for example if the fraction collector
fails to read the tray ID or do not collect fractions correctly.
Required material
• Wash bottle
• Water or 20% ethanol
• Cloth
Instructions
Follow the instructions below to clean the fraction collector diodes. See Section 3.2.2 Fraction collector
F9-C illustrations, on page 92 for the locations of the components of the fraction collector.
Step Action
1 In System Control, select Manual:Execute Manual Instructions:Fraction collection:Frac

cleaning position. Click Execute.
Result: The Dispenser head moves to cleaning position, and the Instrument display states
System pause.

7 Maintenance
Step Action
2 Wipe off the Dispenser head and the Drop sync and Type code reader sensor windows
using a wash bottle with water or 20% ethanol and a cloth.
3 Let the Dispenser head dry completely before starting a run.
4 Close the fraction collector door.

Result: Automatic scanning is performed.
5 In the System Control module, press the End icon in the toolbar.
Result: The Dispenser head moves to home position, and the Instrument display states
Ready.

7 Maintenance
7.4 Monthly maintenance

Introduction
This section provides instructions for monthly maintenance actions.
Check the back pressure for the Flow restrictor every month.
Location of the Flow restrictor

The recommended positions for Flow restrictor FR-902 are:
• Connected between the Conductivity monitor C9 and the Outlet valve.
or
• Connected to the ToR and FrR ports of the pH valve.
Check Flow restrictor connected to Conductivity

monitor and Outlet valve
Follow the instructions below to check the back pressure of Flow restrictor FR-902, when this is con-
nected between the Conductivity monitor and the Outlet valve.
Step Action
1 Immerse the piece of inlet tubing marked A1 in distilled water, and insert the piece of
tubing from Outlet valve port W into a waste container.
2 In the System Control module, select Manual:Execute Manual Instructions.


• Select Flowpath:Injection valve, and select Manual Load. Click Insert.
• Select Flowpath:Column valve, and select By-pass. Click Insert.
• Select Flowpath:Outlet valve, and select Out-Waste. Click Insert.
• Select Pumps:System flow and set the Flow rate to 2.5 ml/min. Click Insert.
• Click Execute.
4 Note the PreC pressure displayed in the Run Data pane.

Tip:
If PreC pressure is not displayed, click the Customize icon. In the Customize dialog, under
the Run Data Groups tab, select PreC pressure.

7 Maintenance
Step Action
5 Click on the Pause icon to stop the flow.
6 Replace the Flow restrictor with a female/female union connector.

Tip:
The female/female union connector is included in the accessory kit.
7 Click the Continue icon to restart the flow.
9 Calculate the difference between the two pressure values.
10 Check that the pressure difference is within the range 0.2 ± 0.05 MPa.
If this is not the case, the Flow restrictor should be replaced, see Section 7.8.7 Replace
Flow restrictor, on page 330.
Check Flow restrictor connected to pH valve

Follow the instructions below to check the back pressure of Flow restrictor FR-902.
Step Action
1 Immerse the piece of inlet tubing marked A1 in distilled water, and insert the piece of
tubing from Outlet valve port W into a waste container.


• Select Flowpath:Injection valve, and then select Manual Load. Click Insert.
• Select Flowpath:Column valve, and then select By-pass. Click Insert.
• Select Flowpath:pH valve, and set the pH electrode to Off-line and the Restrictor to
In-line. Click Insert.
• Select Flowpath:Outlet valve, and then select Out-Waste. Click Insert.
• Select Pumps:System flow and set the Flow rate to 2.5 ml/min. Click Insert.
• Click Execute.

Tip:
If PreC pressure is not displayed, click the Customize icon. In the Customize dialog, under
the Run Data Groups tab, select PreC pressure.

7 Maintenance
Step Action

• Select Flowpath:pH valve, and set the pH electrode to Off-line and the Restrictor to
Off-line.
• Click Execute.
7 Calculate the difference between the two pressure values noted in step 4 and step 6.
8 Check that the pressure difference is within the range 0.2 ± 0.05 MPa.
If this is not the case, the Flow restrictor should be replaced, see Section 7.8.7 Replace
Flow restrictor, on page 330.

7 Maintenance
7.5 Semiannual maintenance

Introduction
This section provides instructions for semiannual maintenance activities.
In this section
Section See page
7.5.1 Clean the UV flow cell 273
7.5.2 Replace the pH electrode 275

7 Maintenance
7.5.1 Clean the UV flow cell
Clean the UV flow cell every six months, or when required.
NOTICE
Keep UV flow cell clean. Do not allow solutions containing dissolved salts, proteins
or other solid solutes to dry out in the flow cell. Do not allow particles to enter the
flow cell, as damage to the flow cell may occur.
Required material
• Luer connector
• Waste container
• 10% surfactant detergent solution (e.g., Decon™ 90, Deconex 11, or RBS 25)
• Distilled water
Instruction
Follow the instructions below to clean the UV flow cell for UV monitor U9-M or UV monitor U9-L. The
UV flow cell can be mounted or not mounted on the instrument during the cleaning procedure.
The illustrations in the instructions below show UV monitor U9-M. UV monitor U9-L is cleaned in a
corresponding way.
Step Action
1 Disconnect the tubing from the top of the UV flow cell, and replace the fingertight con-
nector with a Luer connector.
2 Disconnect the tubing from the bottom of the UV flow cell, and connect a piece of waste
tubing to the UV flow cell. Insert the waste tubing into a waste container.

7 Maintenance
Step Action
3 Fill a syringe with distilled water, and connect the syringe to the Luer connector.
4 Squirt the distilled water through the UV flow cell in small amounts. Disconnect the syringe.
5 Fill a syringe with a 10% surfactant detergent solution, such as Decon 90, Deconex 11,
RBS 25 or equivalent, and connect the syringe to the Luer connector.
Tip:
Heat the 10% surfactant detergent solution to 40°C to increase the cleaning effect.
6 Squirt the detergent solution through the UV flow cell about five times.
7 Leave the detergent solution in the flow cell for at least 20 minutes.
8 Inject the detergent solution remaining in the syringe into the flow cell. Disconnect the
syringe.
9 Fill a syringe with distilled water. Connect the syringe to the Luer connector.
10 Inject the distilled water into the UV flow cell to rinse the flow cell. Disconnect the syringe.
11 Disconnect the Luer connector from the top of the UV flow cell. Reconnect the piece of
tubing from the Column valve to the top of the UV flow cell.
12 Disconnect the waste tubing from the bottom of the UV flow cell. Reconnect the piece
of tubing from the Conductivity monitor to the bottom of the UV flow cell.

7 Maintenance
7.5.2 Replace the pH electrode
7.5.2 Replace the pH electrode
Replace the pH electrode every six months, or when required.
Required material
• pH electrode
• Deionized water
• Standard buffer pH 4
Instruction
CAUTION
pH-electrode. Handle the pH-electrode with care. The glass tip may break and cause
injury.
Follow the instructions below to replace the pH electrode.

Step Action
1 Disconnect the pH electrode cable of the used pH electrode from the connection on the
front of the pH valve.
2 Unscrew the nut of the pH electrode by hand, and pull the used electrode away.
3 Unpack the new pH electrode. Remove the cover from the tip of the new pH electrode.
Make sure that the electrode is not broken or dry.
4 Prior to first use of the electrode, immerse the glass bulb in deionized water for 30 minutes
and then in a standard buffer, pH 4, for 30 minutes.
5 Carefully insert the new pH electrode into the pH flow cell. Tighten the nut by hand to
secure the electrode.
6 Connect the pH electrode cable of the new electrode to the connection on the front of
the pH valve.
7 Calibrate the new pH electrode, see Section 7.7.1 Calibrate the pH monitor, on page 303.

7 Maintenance
7.6 Maintenance when required

Introduction
This section gives instructions for maintenance activities to be performed when required.
In this section
Section See page
7.6.1 Clean the instrument externally 277
7.6.2 Perform System CIP 278
7.6.3 Perform Column CIP 284
7.6.4 Clean Fraction collector F9-C 288
7.6.5 Clean Fraction collector F9-R 290
7.6.6 Storage of the pH electrode 291
7.6.7 Clean the pH electrode 293
7.6.8 Clean the pump head check valves 296
7.6.9 Wipe off excess oil from the pump head 299
7.6.10 Clean the Conductivity flow cell 300

7 Maintenance
7.6.1 Clean the instrument externally
7.6.1 Clean the instrument externally
Clean the the instrument externally when required. Do not allow spilled liquid to dry on the instrument.
Required material
• Cloth
• Mild cleaning agent or 20% ethanol
Instruction
Follow the instructions below to clean the instrument externally.
Step Action
1 Check that no run is in progress.
2 Switch off the instrument.
3 Wipe the surface with a damp cloth. Wipe off stains using a mild cleaning agent or 20%
ethanol. Wipe off any excess.
4 Let the instrument dry completely before using it.

7 Maintenance
7.6.2 Perform System CIP
Perform a System cleaning in place (System CIP) when required, for example between runs where dif-
ferent samples and buffers are used. This is important to prevent cross-contamination and bacterial
growth in the instrument.
Required material
• Appropriate cleaning solutions (e.g., NaOH, buffer solution or distilled water).
Introduction
The System CIP method is used to fill the instrument and the selected inlets and outlets with cleaning
solution.
WARNING
CAUTION
Hazardous substances. When using hazardous chemical and biological agents,
take all suitable protective measures, such as wearing protective glasses and gloves
resistant to the substances used. Follow local and/or national regulations for safe
operation, maintenance and decommissioning of the equipment.
CAUTION
Explosion hazard if flammable liquid leaks during cleaning of the flow path. When
cleaning the flow path of Fraction collector F9-C with a flammable liquid like ethanol,
carefully inspect the flow path, including the waste tubing, to make sure there will
be no leakage.
Tip: If hazardous chemicals are used for system or column cleaning, wash the system or columns
with a neutral solution in the last phase or step.

7 Maintenance
Create a System CIP method

Follow the instruction below to create a System CIP method.
Step Action
1 In the Method Editor module,

• click the New Method icon
or
• select File:New Method.
2 In the New Method dialog, select System and then System CIP in the Predefined Method
drop-down list. Click OK.
Result: One Method Settings phase and three System CIP phases show in the Method
Outline pane. Each System CIP phase uses one cleaning solution.
3 If desired, add additional System CIP phases to the method using the Phase Library.

7 Maintenance
Step Action
4 In the Phase Properties tab of each of the System CIP phases:

• Enter a note for the first solution (optional).
• Select values for Flow rate, Volume per position and Incubation time.
• Define the extent of cleaning by checking the check boxes.

Note:
For complete cleaning of the Injection valve, select at least one of the sample inlets and
clean the manual injection port using a syringe (see Clean the manual injection port of
the Injection valve, on page 282).
Note:
• The pH electrode and the pH valve are not included in the system CIP. Refer to Sec-
tion 7.6.7 Clean the pH electrode, on page 293 for instructions on how to clean the pH
electrode.
• If the fraction collector is included, a lower flow rate might need to be set in the instruc-
tion Fraction collector wash settings.
• Make sure not to exceed the pressure limits for any of the modules that are part of the
flow path during the CIP.

7 Maintenance
Step Action

• click the Save the method icon
or
• select File:Save As
Result: The Save As dialog opens.
6 In the Save As dialog:

• Select a target folder to enable the Save button.
• Type a Name for the method.
• Select a System from the list.
•
Click .
Result: The created method is saved in the selected folder.
Perform a System CIP

Follow the instructions below to run a System CIP method.
Step Action
1 In the Method Editor module, create a System CIP method according to the instructions
above.
2 Connect bypass tubing to all selected column positions and loop positions if a loop valve
is used.
3 Prepare cleaning solutions and immerse the selected inlet tubing in the solutions.
Note:
Note that each phase uses one solution. All inlets selected in one phase should be immersed
in the same cleaning solution.
4 In the System Control module, select the created method and start the run.
5 For complete cleaning of the flow path, clean the manual injection port of the Injection
valve and the pH valve manually, see the instructions below.

7 Maintenance
Clean the manual injection port of the Injection

valve
Follow the instructions below to manually clean the Manual load position of the Injection valve.
Step Action

2 In the Manual instructions dialog, select Flowpath:Injection valve, and select Manual
Load. Click Execute.
3 Connect a suitable sample loop to Injection valve ports LoopF (fill) and LoopE (empty).
Note:
Do not use a Superloop when cleaning the Injection valve.
4 Connect tubing to Injection valve port W1, and direct this tubing to a waste container.
6 Fill a syringe with approximately 10 ml of an appropriate cleaning solution (e.g., NaOH

or buffer solution). Connect the syringe to Injection valve port Syr, and inject the cleaning
solution.
6 Fill a syringe with distilled water. Connect the syringe to Injection valve port Syr, and inject
the distilled water.

7 Maintenance
Clean the pH valve

Follow the instructions below to clean the pH valve. The calibration function is used to switch the valve
position. However, no calibration is performed.
Step Action
1 Connect tubing to pH valve port W3, and direct the other end of this tubing to a waste
container.
2 Unscrew the pH electrode from the pH valve, and replace it with the dummy electrode.
3 Open the System Control module and select System:Calibrate.

Result: The Calibration dialog opens.
4 In the Calibration dialog, select pH from the Monitor to calibrate drop-down list.
5 Press the Prepare for calibration button.
Result: The pH valve switches to the calibration position.
7 Fill a syringe with approximately 10 ml of 1 M NaOH. Connect the syringe to the pH valve
port Cal, and inject the solution.
8 Fill a syringe with distilled water. Connect the syringe to the pH valve port Cal, and inject
the distilled water.
9 Press the Close button.
Result: The pH valve switches back to the default position and the Calibration dialog
closes. No calibration is performed.

7 Maintenance
7.6.3 Perform Column CIP
Perform a Column cleaning in place (Column CIP) when required, for example between runs where
different samples are used.
Required material
• Appropriate cleaning solutions. Please refer to the instructions for use of the column.
Introduction
The Column CIP method is used to clean the column after purification runs, to remove non-specifically
bound proteins and to minimize the risk for carry-over between different purification runs.
WARNING
CAUTION
Hazardous substances. When using hazardous chemical and biological agents,
take all suitable protective measures, such as wearing protective glasses and gloves
resistant to the substances used. Follow local and/or national regulations for safe
operation, maintenance and decommissioning of the equipment.
Tip: If hazardous chemicals are used for system or column cleaning, wash the system or columns
with a neutral solution in the last phase or step.

7 Maintenance
Create a Column CIP method

Follow the instruction below to create a Column CIP method.
Step Action

• click the New Method icon
or
• select File:New Method.
2 In the New Method dialog, select System and then Predefined Method and Column CIP.
Click OK.
Result: One Method Settings phase and one Column CIP phase will be displayed in the
Method Outline pane.
3 In the Phase Properties tab of the Method Settings phase, select Column type and Col-
umn position.

7 Maintenance
Step Action
4 In the Phase Properties tab of the Column CIP phase:

• Click Add Step to add a step.
• Select the step and click Remove Step to remove a step.
• To enter a value, select the cell and enter or select a new value.
5 In the Phase Properties tab of the Column CIP phase, click the Get Suggested Steps
button to get a suggested procedure for the selected column type. Note that this function
is not available for all column types.
Result: Suggested cleaning steps for the selected column type are displayed.
6 If several cleaning solutions are used, change settings for Inlet A and/or Inlet B . Select
one inlet for each solution. If Inlet B is used, remember to edit the values in the %B column.

7 Maintenance
Step Action

• click the Save the method icon
or
• select File:Save As
Result: The Save As dialog opens.
8 In the Save As dialog:

• Select a target folder to enable the Save button.
• Type a Name for the method.
• Select a System from the list.
• Click the Save button.
Result: The created method is saved in the selected folder.
Perform a Column CIP

Follow the instructions below to run a Column CIP method.
Step Action
1 In the Method Editor module, create a Column CIP method according to the instruction
above.
2 Prepare cleaning solutions and immerse the selected inlets in the solutions.
3 Connect the column to the selected column position.
4 In the System Control module, select the created method and start the run.

7 Maintenance
7.6.4 Clean Fraction collector F9-C
Clean the Fraction collector when required, for example if liquid has been spilled in the Fraction collector
chamber. The internal tubing of the fraction collector may need to be replaced for maintenance or for
process purposes. Information on how and when to replace the internal tubing can be found in the
ÄKTA pure User manual.
Required material
• Wash bottle
• Cloth
Instruction
Follow the instruction below to clean the interior of the Fraction collector. The locations of the compo-
nents of the fraction collector are shown in Section 3.2.2 Fraction collector F9-C illustrations, on page 92.
Step Action
1 Perform a fraction collector wash:

In System Control, select Manual:Execute Manual Instructions:Fraction collection:Frac-
tion collector wash. Click Execute.
NOTICE
• If no column valve is used, make sure to replace any columns in
the flow path with tubing before a fraction collector wash is per-
formed.
• Lower the flow for the fraction collector wash in the instruction
Fraction collector wash settings if the system back pressure is el-
evated during the wash.
• Make sure not to exceed the pressure limits for any of the modules
that are part of the flow path.
2 In System Control, select Manual:Execute Manual Instructions:Fraction collection:Frac

cleaning position . Click Execute.
Result: The Dispenser head moves to cleaning position, and the Instrument display states
System pause.
3 Open the door of the fraction collector and remove the rack

7 Maintenance
Step Action
4 Wash the Cassette tray or rack and the Cassettes (if applicable), with water and a mild
cleaning agent.
5 Lift off the Waste funnel and wash it with water and a mild cleaning agent.
Refit the Waste funnel.
6 Wipe off the interior of the fraction collector using a damp cloth. Wipe off stains using a
mild cleaning agent or 20% ethanol.
7 Wipe off the Dispenser head and its diode windows (the Drop sync sensor and the Type
code reader) using a wash bottle with water or 20% ethanol and a cloth.
8 Let the fraction collector dry completely before starting a run.
9 Close the door of the fraction collector.

Result: Automatic scanning is performed.
10 In the System Control module, press the End icon in the toolbar.
Result: The Dispenser head moves to home position.

7 Maintenance
7.6.5 Clean Fraction collector F9-R
7.6.5 Clean Fraction collector F9-R
Clean the Fraction collector when required, for example in case of liquid spill.
Required material
• Cloth
Clean the instrument

Follow the instructions below to clean the instrument externally.
Step Action
3 Wipe the surface with a damp cloth. Wipe off stains using a mild cleaning agent or 20%
ethanol. Wipe off any excess.
4 Let the Fraction collector F9-R dry completely before restart.
Clean DropSync sensor

Clean the drop sensor photocell located above the tube sensor (see Front view illustration, on page 103)
with a damp cloth.

7 Maintenance
7.6.6 Storage of the pH electrode
When pH monitoring is not used, the pH electrode can be stored in storage solution inside the pH flow
cell. If pH monitoring is not used for a week or longer, inject new storage solution into the pH flow cell
or replace the pH electrode with the dummy electrode that was installed in the pH valve on delivery.
Required material
• Syringe, 25 to 30 ml
• Storage solution (1:1 mixture of standard buffer, pH 4, and 1 M KNO3)
Instruction
Follow the instructions below to fill the pH flow cell with storage solution. The calibration function is
used to switch the position of the pH valve. However, no calibration is performed.
Step Action
1 In the System Control module, on the System menu click Calibrate.

2 In the Calibration dialog, click pH on the Monitor to calibrate drop-down list.
3 Click Prepare for Calculation.

4 Prepare at least 10 ml storage solution by mixing equal volumes of a standard buffer,

pH 4, and a 1 M Potassium Nitrate (KNO3) solution.

7 Maintenance
Step Action
5 Fill a syringe with approximately 10 ml of the storage solution. Connect the syringe to
the pH valve port Cal, and inject the storage solution.
6 Click Close.

7 Maintenance
7.6.7 Clean the pH electrode
Clean the pH electrode when required. The pH electrode can be cleaned either when it is installed in
the pH valve or when it has been removed. The pH electrode has a limited longevity and should be
replaced every six months or when the response time is slow, see Section 7.5.2 Replace the pH electrode,
on page 275. After cleaning has been performed, re-calibrate the pH monitor, see Section 7.7.1 Calibrate
the pH monitor, on page 303.
Required material
• Distilled water
• 0.1 M HCl and 0.1 M NaOH
or
• Liquid detergent
or
• 1% pepsin solution in 0.1 M HCl
or
• 1 M KNO3
Cleaning agents
Clean the pH electrode using one of the following procedures:
Salt deposits
Dissolve the deposits by immersing the electrode for a five minute period in each of the following solu-
tions:
• 0.1 M HCl
• 0.1 M NaOH
• 0.1 M HCl
Rinse the electrode tip in distilled water between each solution.
Oil or grease films
Wash the electrode tip in liquid detergent and water. If the films are known to be soluble in a particular
organic solvent, wash with this solvent. Rinse the electrode tip in distilled water.
Protein deposits
Dissolve the deposit by immersing the electrode in a solution of 1% pepsin in 0.1 M HCl for five minutes,
followed by thorough rinsing with distilled water.

7 Maintenance
If these procedures fail to rejuvenate the electrode, try the following procedure.
Note: This procedure can be performed only when the pH electrode is not installed in the pH valve.
Step Action
1 Heat a 1 M KNO3 solution to 60°C–80°C.
2 Place the electrode tip in the heated KNO3 solution.
3 Allow the electrode to cool while immersed in the KNO3 solution before re-testing.
If these steps fail to improve the electrode, replace the electrode, see Section 7.5.2 Replace the pH
electrode, on page 275.
Clean a pH electrode installed in the pH valve

Follow the instructions below to clean a pH electrode installed in the pH valve. The calibration function
is used to switch the position of the pH valve. However, no calibration is performed.
Step Action
1 Open the System Control module and select System:Calibrate.

2 Set Monitor to calibrate by selecting pH from the list.
3 Press the Prepare for calibration button.

7 Maintenance
Step Action
4 Fill a syringe with approximately 10 ml of chosen cleaning solution. Connect the syringe
to the pH valve port Cal. Inject the liquid and wait for 5 minutes. Disconnect the syringe.
5 If several cleaning solutions are to be used, repeat step 4 with distilled water and then
with the next solution.
6 As the last step in the cleaning procedure:

• Fill a syringe with distilled water.
• Connect the syringe to the pH valve port Cal.
• Inject the water.
• Disconnect the syringe.
7 Press the Close button.

7 Maintenance
7.6.8 Clean the pump head check valves
Clean the check valves when required, for example if solids in the check valve cause irregular or low
flow. The cleaning procedure is the same for the system pumps and the sample pump.
Required material
• Adjustable wrench
• Methanol
• Distilled water
• Ultrasonic bath
Instruction
Follow the instructions below to remove and clean the pump head check valves.
Step Action
2 Disconnect the tubing from the pump head and disconnect the pump inlet tubing. Dis-
connect the tubing of the pump rinsing system.
3 Unscrew the purge valve by turning it counter-clockwise, and lift off the metal ring.

7 Maintenance
Step Action
4 Unscrew the plastic nut of the upper check valve using an adjustable wrench, and gently
lift off the upper check valve.
5 Unscrew the two white plastic screws located below each pump head. Pull the plastic
connectors to the sides to release the inlet manifold.
6 Unscrew the lower check valve using an adjustable wrench.

7 Maintenance
Step Action
7
CAUTION
Hazardous substances. When using hazardous chemical and biolog-
ical agents, take all suitable protective measures, such as wearing
protective glasses and gloves resistant to the substances used. Follow
local and/or national regulations for safe operation, maintenance and
decommissioning of the equipment.
Immerse the valves completely in methanol and place them in an ultrasonic bath for a
few minutes. Repeat the ultrasonic bath with deionized water.
8 Refit the check valves.
9 Tighten the nut until fully finger-tight and then use the adjustable wrench to tighten a
further 90 degrees.
10 Refit the inlet manifold and reconnect the tubing to the pump head.

7 Maintenance
7.6.9 Wipe off excess oil from the pump head
7.6.9 Wipe off excess oil from the pump head
During the first months of use it is normal that excess oil leaks out of the drain hole below the System
pump. The function of the pump is not in any way affected by this.
Required material
• Cloth
• Mild cleaning agent or 20% ethanol
Instruction
Follow the instructions below to clean the System pumps externally.
Step Action
3 Wipe off the excess oil from the pump head with a damp cloth. Wipe off stains using a
mild cleaning agent or 20% ethanol.
4 Let the pump dry completely before using the instrument.

7 Maintenance
7.6.10 Clean the Conductivity flow cell
Clean the Conductivity flow cell when required.
Required material
• Luer connector
• Waste container
• 1 M NaOH
• Distilled water
Instruction
Follow the instruction below to clean the flow cell of the Conductivity monitor.
Step Action
1 Disconnect the fingertight connector and the piece of tubing from the top of the Conduc-
tivity monitor, and attach a Luer connector.
2 Disconnect the piece of tubing from the bottom of the Conductivity monitor, and connect
a piece of waste tubing to the Conductivity monitor. Place the waste tubing in a waste
container.

7 Maintenance
Step Action
3 Fill a syringe with distilled water, and connect the syringe to the Luer connector.
4 Squirt distilled water through the Conductivity flow cell in small amounts. Disconnect the
syringe.
5 Fill a syringe with 1 M NaOH, and connect the syringe to the Luer connector.
6 Squirt 1 M NaOH through the Conductivity flow cell about five times.
7 Leave the liquid in the flow cell for at least 15 minutes.
8 Fill a syringe with distilled water. Connect the syringe to the Luer connector.
9 Inject the distilled water into the Conductivity flow cell to rinse the flow cell. Disconnect
the syringe.
10 Disconnect the Luer connector from the top of the Conductivity flow cell, and reconnect
the fingertight connector with tubing.

7 Maintenance
7.7 Calibration procedures

Introduction
This section provides instructions for calibration procedures that can be performed using the System
Control module in UNICORN software.
In this section
Section See page
7.7.1 Calibrate the pH monitor 303
7.7.2 Calibrate the pressure monitors 305
7.7.3 Calibrate the Conductivity monitor 308
7.7.4 Calibrate the UV monitors 312
Menu command in UNICORN

Open the System Control module and select System:Calibrate to open the Calibration dialog.

7 Maintenance
7.7.1 Calibrate the pH monitor
Calibrate the pH monitor once a day, when the pH electrode has been replaced, or if the ambient
temperature has changed by more than ± 5°C.
Required material
Use two pH calibration buffers with a difference of at least one pH unit. Preferably use a pH standard
buffer, pH 4 or pH 7, as the first calibration point, and a pH standard buffer close to the lowest or
highest pH you need to measure as your second point. Allow the buffers to equilibrate to ambient
temperature before use.
Instruction
CAUTION
pH-electrode. Handle the pH-electrode with care. The glass tip may break and cause
injury.
Follow the instructions below to calibrate the pH monitor.

Step Action
1 In the Calibration dialog, select pH from the Monitor to calibrate drop-down list.
2 Click the Prepare for calibration button.
3 Enter the pH of the first pH standard buffer in the pH for buffer 1 field.

7 Maintenance
Step Action
4 Fill a syringe with approximately 10 ml of the first pH standard buffer. Connect the syringe
to the Luer connector of pH valve port Cal, and inject the buffer.
5 When the Current value is stable, click the Calibrate button.
6 Wash the pH flow cell by injecting water into pH valve port Cal using a new syringe.
7 Enter the pH of the second pH standard buffer in the pH for buffer 2 field.
8 Repeat steps 4-6 using the second pH standard buffer.

Result: The calibration date and time are displayed in the dialog, along with values for
Calibrated electrode slope and Asymmetry potential at pH 7.
9 Is the Calibrated electrode slope ≥ 80% and the Asymmetry potential at pH 7 inside
the interval ± 60 mV?
• If Yes: Click the Close button to switch the pH valve back to the default position and
to close the Calibration dialog.
• If No: Clean the pH electrode and repeat the calibration procedure. If this does not
help, replace the electrode.

7 Maintenance
7.7.2 Calibrate the pressure monitors
ÄKTA pure can have up to four pressure monitors: System pump pressure monitor, Sample pump
pressure monitor, Pre-column pressure monitor and Post-column pressure monitor. Check the pressure
monitors every week, or when the the ambient temperature has changed by more than ± 5°C. Calibrate
the monitor if the zero pressure reading is outside the range ± 0.02 MPa.
Check the monitors

Follow the instructions below to check the pressure monitors. The procedure is the same for each
monitor.
Step Action
1 Disconnect the relevant tubing from the pressure monitor to obtain zero-pressure, see
table Tubing and pressures, on page 306.
2 Click the Customize icon to open the Customize dialog. In the Customize dialog, under
the Run Data Groups tab, select the relevant pressure to display, see table Tubing and
pressures, on page 306. Click OK to close the Customize dialog.
3 In the Run Data pane in the System Control module, check what pressure is displayed.
4 If the zero pressure reading is outside the range ± 0.02 MPa, calibrate the pressure
monitor according to the instruction below.
Calibrate the monitors

Follow the instructions below to calibrate any of the pressure monitors.
Step Action
1 Disconnect the relevant tubing from the pressure monitor, see table Tubing and pressures,
on page 306.
2 In the Calibration dialog, select the pressure monitor to calibrate from the Monitor to
calibrate drop-down list.
3 Click the Reset pressure button.

Result: The atmospheric pressure is defined as zero. The date and time of the most recent
calibration, and the current pressure value are displayed.
4 Reconnect the tubing to the pressure monitor.

7 Maintenance
Tubing and pressures

The table below shows the tubing to disconnect when checking and calibrating the pressure monitors.
The UNICORN names of the pressures measured by the monitors are also shown.
Pressure monitor Tubing to disconnect Pressure in UNICORN
System pump pressure Tubing from the System pump pressure monitor System pressure
monitor
Sample pump pressure Tubing from the Sample pump pressure monitor Sample pressure
monitor
Pre-column pressure Tubing to Column valve port In (V9-C, V9H-C or PreC pressure
monitor V9-C2, V9H-C2).

7 Maintenance
Pressure monitor Tubing to disconnect Pressure in UNICORN
Post-column pressure Tubing to Column valve port Out (V9-C, V9H-C PostC pressure
monitor or V9-C2, V9H-C2).

7 Maintenance
7.7.3 Calibrate the Conductivity monitor
Introduction
Two types of calibrations can be performed:
• Conductivity monitor - factory calibration: Restores the conductivity cell constant to the factory
default value.
• Conductivity monitor - user calibration: Calibrates the conductivity cell constant.
Recommended maintenance intervals for the two types of calibrations:
• Conductivity monitor - factory calibration: Perform calibration to override an incorrect user cali-
bration.
• Conductivity monitor - user calibration: The conductivity cell is factory calibrated, and should not
require recalibration under normal usage. Perform calibration when the signal is unstable or you
suspect that it is incorrect. It is also recommended to recalibrate the Conductivity monitor after
cleaning.
Conductivity monitor - factory calibration

Follow the instruction below to restore the conductivity cell constant to the factory default value.
Step Action
1 In the Calibration dialog, select Conductivity monitor - factory calibration from the
Monitor to calibrate drop-down list.
Result: The time for the new calibration and the current value are displayed.
2 Click Restore.
Result: The conductivity cell constant is restored to the factory default value. The conduc-
tivity cell constant is written on the packaging of the Conductivity monitor.
Conductivity monitor - user calibration

Follow the instruction below to calibrate the Conductivity flow cell constant.
Step Action
1 Make sure that the instrument has been switched on for at least one hour.


7 Maintenance
Step Action
3 In the System Settings dialog, select Conductivity and Cond temp compensation. Set
the Compensation factor to 0%, and click OK.
4 Wash the whole flow path and fill it with deionized water using a suitable inlet and the
System pump, until the conductivity value reaches 0.00 mS/cm.
5 Connect the tubing (labelled 3) from the system pump pressure monitor directly to the
conductivity cell inlet, by-passing the Mixer, Injection valve, Column valve and UV-monitor,
see image below. Ensure that the Flow restrictor is inline.
6 Prime and purge the inlet used in step 4 with the 1.00 M NaCl calibration solution. Fill the
conductivity cell with the calibration solution at 1 ml/min. Pump in at least 15 ml of the
calibration solution and wait until the conductivity signal and the temperature have
stabilized before continuing the calibration. See Section 5.4.1 System pumps, on page 161.
7 Under continuous flow, read the current Conductivity temperature from Run Data with
an accuracy of one decimal place.
8 In the Calibration dialog, select Conductivity monitor - user calibration from the Mon-
itor to calibrate drop-down list.

7 Maintenance
Step Action
9 In the Run Data pane of System Control, read the current Cond temp.
Tip:
If Cond temp is not showing, click the Customize icon. In the Customize dialog, under the
Run Data Groups tab, select to view Cond temp.
10 In the Calibration dialog, enter the theoretical conductivity value at the current conduc-
tivity temperature in the Enter theoretical conductivity value input field.
• If a certified conductivity standard solution is used, use the supplied theoretical con-
ductivity value.
• If a manually prepared 1.00 M NaCl calibration solution is used, see the graph for
conductivity value at the current temperature on page 311.
11 In the Calibration dialog, click Calibrate.

Result: The new conductivity cell constant is displayed in the Conductivity cell 1 con-
stant/cm box. The new constant should normally be 40 ± 10 cm-1. The date and time for
the calibration are also displayed.
12 In the System Control toolbar, click the End icon to end the run.
13 In the System Settings dialog, select Conductivity:Cond temp compensation and set
the Compensation factor back to desired value, default 2.0%. Click OK.
Note:
When using NaCl, the optimal compensation factor is 2.1%.

7 Maintenance
Graph for conductivity value

The graph below shows the conductivity value at the current temperature when 1.00 M NaCl calibration
solution is used.
Conductivity of 1.00 M NaCl at 20–30°C
97
95
90
Conductivity (mS/cm)
85
80
77
20 25 30
Temperature (°C)

7 Maintenance
7.7.4 Calibrate the UV monitors
Automatic calibration of the UV monitor U9-M

The wavelength is automatically calibrated every time the instrument is switched on. If the instrument
has been switched on for a couple of days, and the ambient temperature and/or the humidity has
changed, restart the instrument using the power switch to calibrate the UV monitor.
Calibration of the UV monitor U9-L flow cell length

The path length in the UV flow cell or the UV 2nd flow cell might differ from the nominal length, which
leads to incorrect results in the calculation of protein concentration in the eluate. To achive normalized
absorbance, the path length in the UV flow cell or the UV 2nd flow cell must be calibrated. The calibration
procedure that is described below is the same for both UV flow cell and the UV 2nd flow cell, unless
otherwise stated.
Note: The flow cell path length must be registered or updated in UNICORN, when the flow cell is re-
placed.
Equipment needed
To perform the calibration, a calibration kit containing test solutions, syringes and accessories is
needed. A specified kit is available for each cell length.
If using a UV flow cell with Then use calibration kit...

the theoretical path
length...
2 mm UV-900 2 mm calibration kit (Product Code 18-6324-02)
5 mm UV-900 5 mm calibration kit (Product Code 18-6324-04)
To calculate the real path length of the UV flow cell, use the following software:
• UV-900 cell calibration Excel-file (Product Code 18-6324-06)
Prepare for calibration

Follow the instructions below to prepare for the calibration of the UV monitor U9-L.
Step Action
1 Ensure that the flow restrictor is inline in the flow path after the UV flow cell.
2 Mount the union Luer female/1/16” male, included in the test kit, in the upper inlet of the
UV flow cell.
3 Open the software UV-900 cell calibration Excel-file.

7 Maintenance
Step Action
4 The solution bottles are labelled with the concentration value and the reference ab-
sorbance value for each solution. Enter the concentrations of the solutions in ascending
order into the column UV Test kit Concentration (mg/l). Enter the corresponding ab-
sorbance values into the column UV Test kit Absorbance (AU/cm).
Perform the calibration

Follow the instructions below to calibrate UV monitor U9-L and UV monitor U9-L, 2nd.
Step Action
In the Manual Instructions dialog:

• Select Pumps:System flow and set the Flow rate to 0.0 ml/min.
• Click Execute.
Result: The absorbance can now be monitored.
2 Fill one of the supplied syringes with 1.5-2 ml of the first solution (0 mg/l). Ensure that
there are no air bubbles in the syringe.
3 Fit the syringe in the union Luer connector and inject the solution. DO NOT remove the
syringe.
Note:
Air trapped in the UV cell causes inaccurate measurements. To avoid introducing air into
the UV cell, gently fill the union Luer up to the edge with test solution that is to be introduced,
using the syringe. Then insert the syringe into the union Luer.
4 Wait until the monitored absorbance value has stabilized.

7 Maintenance
Step Action
5 In the Manual Instructions dialog:

For UV monitor U9-L:
• Select Monitors:Auto zero UV
• Click Execute.
For UV monitor U9-L, 2nd:
• Select Monitors:Auto zero UV 2nd
• Click Execute.
Result: The UV absorbance is set to zero.
6 Remove the syringe.
7 Repeat the injections with the remaining four test solutions in increasing concentration
order. Use a new syringe for each solution.
8 After each injection, wait for a stable absorbance value. Note the measured absorbance
values for each solution.
9 Enter the measured absorbance values into the table in the column UV-900 Absorbance
(AU).
Note:
The values should be converted from mAU to AU.
10 When all absorbance values have been entered into the table, the real UV flow cell path
length is shown at the bottom of the table.
Note:
The regression coefficient R2 should be larger than 0.999. If this is not the case, one or
more measured values are faulty.
Update the cell path length

Follow the instructions below to define the UV cell path length or the UV 2nd cell path length. The
flow cell path length should be updated when the flow cell has been replaced or calibrated.
Step Action
1 In the System Control module, select System:Calibration.
2 In the Calibration dialog, select

• UV cell path length
or
• UV 2nd cell path length
from the Monitor to calibrate drop-down list.
3 Enter the nominal flow cell path length in the Nominal length input field and click Set.

7 Maintenance
Step Action
4 • If a calibration has been performed: enter the calculated flow cell path length, obtained
in the calibration procedure, in the Real length input field and click Set.
• If no calibration has been performed: enter the nominal flow cell path length in the
Real length input field and click Set.
Result: The UV flow cell path length is updated.

7 Maintenance
7.8 Replacement procedures

Introduction
This section gives instructions for the replacement procedures to be performed by the user of ÄKTA
pure.
WARNING
Disconnect power. Always disconnect power from the instrument before replacing
any component on the instrument, unless stated otherwise in the user documenta-
tion.
In this section
Section See page
7.8.1 Replace tubing and connectors 317
7.8.2 Replace internal tubing in Fraction collector F9-C 319
7.8.3 Replace the Mixer 322
7.8.4 Replace the O-ring inside the Mixer 323
7.8.5 Replace the UV monitor U9-M flow cell 325
7.8.6 Replace the UV monitor U9-L flow cell 328
7.8.7 Replace Flow restrictor 330
7.8.8 Replace the inlet filters 331
7.8.9 Replace the pump head check valves 332
7.8.10 Replace pump piston seals of Pump P9 or P9H 335
7.8.11 Replace pump piston seals of Pump P9-S 344
7.8.12 Replace pump pistons 351
7.8.13 Replace pump rinsing system tubing 352

7 Maintenance
7.8.1 Replace tubing and connectors
Replace tubing and connectors when required, for example when a tubing has clogged or has been
bent so that the flow is stopped.
Required material
• Tubing and connectors
• Tubing cutter
• Fingertight wrench
Instruction
Follow the instruction below to replace tubing and connectors.
Step Action
1 Make sure that no run is in progress on the instrument.
2 Unscrew the connectors, and disconnect the tubing.
3 If the tubing has labels, remove the labels to be used with the new tubing later. Discard
the tubing and connectors.

7 Maintenance
Step Action
4 Cut the new tubing to the same length as the old tubing. Use a tubing cutter to get a
straight angle cut.
CAUTION
Cut injuries. The tubing cutter is very sharp and must be handled with
care to avoid injuries.
Note:
When replacing system tubing, use the original inner diameter and length to ensure that
the correct delay volumes are maintained. Inlet and outlet tubing may be shortened if re-
quired.
5 Put the old labels on the new tubing.
6 Mount the connectors on the tubing.

For fingertight connectors:
• Slide the connector onto the tubing.
For tubing connectors 1/8":
• Slide the connector onto the tubing.
• Slide the ferrule onto the tubing with the thick end towards the end of the tubing.
7 Insert the tubing with connector into the port. Make sure to insert the tubing all the way
into the bottom of the port.
8 Tighten the connector fully. For areas difficult to access, use the fingertight wrench in-
cluded in the accessory kit.

7 Maintenance
7.8.2 Replace internal tubing in Fraction collector F9-C
Replace tubing and connectors when required, for example when the tubing has clogged or has been
bent so that the flow is stopped.
Required material
• Tubing and connectors
• Tubing cutter
• Fingertight wrench
Instructions
Follow the instructions below to replace tubing and connectors. See Section 3.2.2 Fraction collector
F9-C illustrations, on page 92 for the locations of the components of the fraction collector.
Step Action
1 Switch off the power to the fraction collector by switching off power to the ÄKTA pure
instrument.
2 Remove the connector for the internal tubing that is attached to the inlet port:
• Unscrew the connector from the inlet port.
• Remove the connector from the tubing.

7 Maintenance
Step Action
3 Remove the tubing connector from the dispenser head:

• Open the fraction collector and gently move the fractionation arm forward.
• Unscrew the connector from the dispenser head and remove the tubing.
• Gently move the fractionation arm all the way back.
4 Cut the new tubing to the same length as the old tubing. Use a tubing cutter to get a
straight angle cut.
CAUTION
Cut injuries. The tubing cutter is very sharp and must be handled with
care to avoid injuries.
Note:
Use tubing with the original inner diameter and length to ensure that the correct delay
volume is maintained.

7 Maintenance
Step Action
5 Attach the new tubing to the inlet port:

• Thread the tubing from the interior of the fraction collector to the inlet port.
• Slide the fingertight connector onto the tubing.
• Insert the tubing with connector into the port. Make sure to insert the tubing all the
way into the bottom of the port.
• Tighten the connector fully.
6 Thread the tubing into the fraction collector and through the tubing guide in the ceiling
of the fractionation chamber.
7 Position the new tubing:

• Gently move the fractionation arm forward while holding the tubing.
• Thread the tubing through the second tubing guide.
8 Attach the new tubing to the dispenser head:

• Slide the fingertight connector onto the tubing.
• Insert the tubing with connector into the port. Make sure to insert the tubing all the
way into the bottom of the port.
• Tighten the connector fully.
9 Make sure that the tubing not is twisted or bent and that it does not dip downwards.

7 Maintenance
7.8.3 Replace the Mixer
7.8.3 Replace the Mixer
Replace the Mixer when a different Mixer chamber is desired, or when the Mixer is damaged.
Required material
The following is required:
• Mixer
Instruction
Follow the instruction below to change the Mixer.
Step Action
1 Disconnect the tubing from the top and bottom of the Mixer.
2 Pull the Mixer away from the instrument.
3 Attach the new Mixer.
4 Reconnect the tubing to the top and bottom of the new Mixer.

7 Maintenance
7.8.4 Replace the O-ring inside the Mixer
Replace the O-ring inside the Mixer if it is damaged.
Required material
One of the following O-rings are required:
• O-ring 13.1 x 1.6 mm (for Mixer chambers 0.6, 1.4, and 5 ml)
• O-ring 13.1 x 1.6 mm (highly resistant, for use when the system is exposed to organic solvents or
high concentrations of organic acids such as acetic acid or formic acid for longer periods of time).
• O-ring 22.1 × 1.6 mm (for Mixer chamber 15 ml)
Instruction
Follow the instruction below to replace the O-ring inside the Mixer.
Tip: Use a forceps and gloves during the replacement procedure to avoid contaminating the Mixer
components.
Step Action
1 Loosen the top section of the Mixer.
2 Unscrew the top section of the Mixer and pull apart the Mixer in two halves.
3 Remove the outer locking O-ring from the top section.

7 Maintenance
Step Action
4 Lift up the top section of the Mixer and pull away the old O-ring inside.
5 Wet the new O-ring with 20% ethanol and fit it in position. Make sure that the inline filter
is still in position.
6 Reassemble the Mixer components and, while holding the Mixer upright, screw the top
section back onto the Mixer.

7 Maintenance
7.8.5 Replace the UV monitor U9-M flow cell
Replace the UV flow cell when it is desired to use a flow cell with a different path length, or if the cell
is damaged. Clean the optical fiber connectors if they have accidentally been touched.
Required materials
For replacement of flow cell
• UV flow cell
For cleaning of the optical fiber connectors
• Lens paper
• Isopropanol
Replace the flow cell
CAUTION
Hazardous chemicals or biological agents in UV flow cell. Make sure that the entire
flow cell has been flushed thoroughly with bacteriostatic solution (e.g., NaOH) and
distilled water, before service and maintenance.
Follow the instruction to replace the UV flow cell.

Step Action
2 Disconnect the tubing from the UV flow cell.
3 Push the latch on the UV detector to disconnect the detector.
Note:
While the UV detector is disconnected, the UV lamp becomes inoperable so no UV light
can be emitted from the instrument.

7 Maintenance
Step Action
4 Pull off the detector and the flow cell from the monochromator. Be careful not to damage
the UV flow cell.
Note:
Make sure that the flow cell does not come into contact with any liquid, and that no liquid
enters the UV detector or monochromator.
Note:
While the UV detector is disconnected, protect the fiber connectors from dust or other im-
purities by mounting the rubber protective caps onto them.
Note:
Do not touch the optical fiber connectors as this will result in poor monitor performance.
If you accidentally touch the optical fiber connectors, clean them according to Clean the
optical fiber connectors, on page 327.
5 Pull off the UV flow cell from the detector.
6 Pull off the black protective caps from the new UV flow cell, and connect the new UV flow
cell to the detector.
7 Connect the detector, with the new flow cell connected, to the monochromator. Pull the
latch upwards to fasten the detector.
8 Connect the tubing to the new flow cell.
9 Switch on the instrument.

Result: The flow cell path length is automatically recognized by the monitor when a new
flow cell is connected.

7 Maintenance
Clean the optical fiber connectors

Follow the instruction to clean the optical fiber connectors.
WARNING
Hazardous substances. When using hazardous chemicals, take all suitable protective
measures, such as wearing protective clothing, glasses and gloves resistant to the
substances used. Follow local and/or national regulations for safe operation and
maintenance of the product.
Step Action
1 Wipe the optical fiber connectors with isopropanol on lens paper.
2 Wipe the optical fiber connectors dry with lens paper.

7 Maintenance
7.8.6 Replace the UV monitor U9-L flow cell
Replace the UV flow cell when it is desired to use a flow cell with a different path length, or if the cell
is damaged.
Required material
• UV flow cell
Replace the flow cell
CAUTION
Hazardous chemicals or biological agents in UV flow cell. Make sure that the entire
flow cell has been flushed thoroughly with bacteriostatic solution (e.g., NaOH) and
distilled water, before service and maintenance.
Follow the instruction below to replace the UV flow cell.

Step Action
2 Disconnect the tubing from the UV flow cell.
3 Unscrew the knurled wheel at the bottom of the UV monitor (1). Press the wheel upwards
to release the flow cell (2).
1 2

7 Maintenance
Step Action
4 Pull the flow cell upwards out of the monitor. Hold the flow cell by the top part with the
O-ring: do not touch the optical surfaces of the flow cell.
Note:
Make sure that the flow cell does not come into contact with any liquid, and that no liquid
enters the monitor.
5 Insert a new flow cell into the monitor.
6 Tighten the knurled wheel firmly.
7 Connect the tubing to the new flow cell.
8 Switch on the instrument and log on to UNICORN.
9 Update the UV flow cell path length in the Calibrate dialog, in System Control.
Note:
The flow path cell length can be:
• updated with the nominal value, see Update the cell path length , on page 314 to set
the nominal flow cell path length
or
• calibrated and updated with the calculated value, see Calibration of the UV monitor
U9-L flow cell length, on page 312 to calibrate the flow cell path length and to update
the software with the calculated value.

7 Maintenance
7.8.7 Replace Flow restrictor
7.8.7 Replace Flow restrictor
Replace the Flow restrictor when required, for example when the back pressure of the Flow restrictor
is outside the range 0.2 ± 0.05 MPa.
Required material
• Flow restrictor FR-902
Instruction
Follow the instruction to replace the Flow restrictor.
Step Action
1 Disconnect the tubing connected from the used Flow restrictor, and discard the used
Flow restrictor.
2 Connect the tubing to the new Flow restrictor. Make sure that the Flow restrictor connector
marked IN is connected to the pH valve port ToR (To Restrictor), and that the Flow restrictor
connector marked OUT is connected to the pH valve port FrR (From Restrictor).
3 Check the back-pressure of the new Flow restrictor, see Section 7.4 Monthly maintenance,
on page 269.

7 Maintenance
7.8.8 Replace the inlet filters
7.8.8 Replace the inlet filters
Replace the inlet filter when required, for example when the filters are clogged.
Required materials
• Inlet filter set
Instruction
Follow the instruction to replace an inlet filter and a support net.
Step Action
1 Pull off the inlet filter and the support net from the inlet filter holder.
2 Fit the new support net and inlet filter, and press the filter into position.

7 Maintenance
7.8.9 Replace the pump head check valves
Replace a check valve when required, for example if the check valve is damaged or clogged. The fol-
lowing instructions are valid for the system pumps and the sample pump.
Required materials
• Check valve kit
Instruction
NOTICE
Handle the check valves with care when they have been removed from the pump
heads, to prevent loss of any internal components.
Follow the instruction to replace the check valves of a pump.

Step Action
1 Disconnect the tubing from the pump head and disconnect the pump inlet tubing.
2 Unscrew the purge valve by turning it counter-clockwise, and lift off the metal ring.

7 Maintenance
Step Action
3 Unscrew the plastic nut of the upper check valve using an adjustable wrench, and gently
lift off the upper check valve.
4 Replace the upper check valve with a new one.
further 180 degrees.
6 Place the new metal ring onto the new upper check valve, and screw the new purge
valve.
7 Unscrew the two white plastic screws located below each pump head. Pull the plastic
connectors to the sides to release the inlet manifold.
8 Unscrew the lower check valve using an adjustable wrench.

7 Maintenance
Step Action
9 Replace the lower check valve with a new one.
further 180 degrees.
11 Refit the inlet manifold and reconnect the tubing to the pump head.

7 Maintenance
7.8.10 Replace pump piston seals of Pump P9 or P9H
Replace the O-rings and piston seals and rinse the membranes of the pumps if they are damaged.
After replacement, perform a run to break in the new piston seals.
The instructions in this section apply to the following pumps.
Configuration Label Pump type
System pump A, ÄKTA pure 25 P9 A P9
System pump B, ÄKTA pure 25 P9 B P9
System pump A, ÄKTA pure 150 P9H A P9H
System pump B, ÄKTA pure 150 P9H B P9H
Sample pump S9H, ÄKTA pure 150 P9H S P9H
NOTICE
Advanced maintenance. Read the instruction carefully before disassembly of the
pump head.
Required material
• For Pump P9: Star screwdriver, T20
• For Pump P9H: Star screwdriver, T10 and T20
• Ultrasonic bath
• Ethanol, 20%
• For Pump P9: Tubing giving a back pressure of 6 to 8 MPa (60 to 80 bar).
• For Pump P9H:Tubing giving a back pressure of 2 to 3 MPa (20 to 30 bar)
• For Pump P9: P9 Seal kit, 25 ml
• For Pump P9H: P9H Seal kit, 150 ml

7 Maintenance
Illustrations
The illustrations below show the parts of the pump heads of the pumps P9 and P9H.
Pump P9
8 9 10
1 2 3 4 5 6 7
11
12
13
Pump P9H
8 9 10
1 2 3 4 5 6 7
11
12
13

7 Maintenance
Part Description Part Description
1 Piston 7 Piston seal
2 Return spring 8 Outlet check valve
3 Pump membrane housing 9 O-ring
4 Star screws 10 Purge valve
5 O-ring 11 Star screws
6 Support washer 12 Inlet check valve
13 Star screws
Introduction
Follow the instructions below to replace the O-rings, piston seal, and pump membrane housing of
pumps P9 and P9H.
Note: Always replace the O-rings, piston seals, and pump membrane housing of both pump heads
of a pump at the same time.
Disassemble the pump head

Step Action
2 Disconnect the tubing from the pump head, and disconnect the pump inlet tubing.
3 Unscrew the two white plastic screws located below each pump head by hand. Pull the
plastic connectors to the sides to release the inlet manifold.
4 Disconnect the tubing of the pump piston rinsing system.

7 Maintenance
Step Action
5 Unscrew the two screws of the front section of the pump head using a star screwdriver
T20, and pull off the front section.
6 Place the front section of the pump head face down on the bench. For Pump P9, unscrew
the two screws of the support washer using a star screwdriver, T20. For Pump P9H, un-
screw the four screws of the support washer using a star screwdriver, T10. Discard the
O-ring (1) on the support washer, and the discard the piston seal (2) located in the front
section of the pump head.
Note:
Be careful not to scratch the metal surfaces.
Pump P9 Pump P9H
2
1
2 1

7 Maintenance
Step Action
7 Unscrew one of the two screws securing the pump membrane housing using a star
screwdriver, T20. Unscrew the second screw, and at the same time push firmly on the
front of the pump membrane housing to compensate for the pressure of the piston return
spring.
8 Carefully pull off the pump membrane housing together with the piston and return spring.
9 Inspect the piston and return spring for signs of damage. If damaged, discard the piston
and return spring and use a new piston and return spring when assembling the pump
head.
10 Clean the pump head and pump membrane housing in an ultrasonic bath. If there are
particles on any surfaces, the check valves should be removed and cleaned separately,
see Section 7.6.8 Clean the pump head check valves, on page 296.

7 Maintenance
Replace O-rings, piston seal and pump membrane

housing
Step Action
1 Unscrew the purge valve of the pump head. Replace the O-ring on the purge valve with
a new O-ring, and screw the purge valve back into the pump head.
Note:
Always use Lubricant 56686700 when exchanging the O-ring 3 x 1 mm.
2 Wet a new seal with 20% ethanol. Place the new seal in the hole in the front section of
the pump head and press it into position.
Pump P9 Pump P9H

7 Maintenance
Step Action
3 Place the support washer on top of the new seal in the front section of the pump head.
Screw the two or four screws of the support washer. Make sure to tighten the screws
fully.
Pump P9 Pump P9H
4 Wet a new O-ring, 21.4 x 1.6 mm, with 20% ethanol. Fit the O-ring around the support
washer.
Pump P9 Pump P9H

7 Maintenance
Assemble the pump head

Make sure to assemble the pump head correctly. Refer to Illustrations, on page 336.
Step Action
1 Insert the piston into the return spring. Insert piston and return spring into hole in the
pump module.
Note:
Do no touch the ceramic or glass part of the pump piston.
2 Wet the membrane in the hole with 20% ethanol before mounting.
3 Place the pump membrane housing onto the locating pins on the front of the pump
module.
4 Screw one of the two screws securing the pump membrane housing using a star
screwdriver, T20. Push firmly on the front of the pump membrane housing to compensate
for the pressure of the piston and then screw the second screw.
5 Make sure that the new seal is wetted with 20% ethanol and then tighten both screws
fully.
6 Reconnect the tubing of the pump piston rinsing system.
7 Reconnect the inlet manifold.
8 Reconnect the tubing to the pump head, and reconnect the pump inlet tubing.
Break in the new pump piston seal

Follow the instruction below to run in the new pump piston seal of pumps P9 and P9H.
Step Action
1 Fill a buffer vessel with 20% ethanol in water. Immerse the inlet tubing, for example A1
for System pump A, or B1 for System pump B, in the buffer vessel. Place the buffer vessel
on the Buffer tray.
2 Prime the inlets and purge the pump, see Section 5.4.1 System pumps, on page 161.
3 • For pump P9: Connect tubing that gives a back pressure of 6 to 8 MPa to one of the
column positions of the Column valve or between the Injection valve and the UV
monitor.
• For pump P9H: Connect tubing that gives a back pressure of 2 to 3 MPa to one of the
column positions of the Column valve or between the Injection valve and the UV
monitor.
3 Immerse the waste tubing in the buffer vessel to recirculate the liquid.

7 Maintenance
Step Action
4 • For a system pump, in the Manual instructions dialog:

- Select Flow path:Column valve, and select the position of the tubing connected
to the Column valve, in this example Position 1. Click Insert.
- Select Flow path:Inlet A (for System pump A) or Flow path:Inlet B (for System pump
B) and select a Position, in this example A1 or B1. Click Insert.
- Select Pumps:Gradient and set Target to 0% B (for System pump A) or 100% B
(for System pump B).
- For pump P9: Select Pumps:System flow and set the Flow rate to 5.0 ml/min. Click
Insert.
- For pump P9H: Select Pumps:System flow and set the Flow rate to 25.0 ml/min.
Click Insert.
- Click Execute.
Result: A system flow starts.
• For a sample pump, in the Manual instructions dialog:
- Select Flow path:Column valve, and select the position of the tubing connected
to the Column valve, in this example Position 1. Click Insert.
- Select Flow path:Sample inlet and select a Position, in this example S1. Click Insert.
- Select Flow path:Injection valve and select Direct inject from the Position drop-
down list. Click Insert.
- Select Pumps and Pressures:Sample flow and set the Flow rate to 25.0 ml/min.
Click Insert.
- Click Execute.
Result: A sample flow starts.
5 Run the flow for 2 hours.
6 Discard the used buffer.

7 Maintenance
7.8.11 Replace pump piston seals of Pump P9-S
Replace the O-ring and the piston seal, and rinse the membrane of the pump if they are damaged.
After replacement, perform a run to break in the new piston seals.
The instructions in this section apply to the following pump.
Configuration Label Pump type
Sample pump S9, ÄKTA pure 150 P9-S P9-S
NOTICE
Advanced maintenance. Read the instruction carefully before disassembly of the
pump head.
Required material
• Star screwdriver, T20
• Cross-headed screwdriver
• Hex wrench
• Ultrasonic bath
• Ethanol, 20%
• Reference capillary Ref 1
• P9-S Seal kit, 65 ml

7 Maintenance
Illustration
The illustration below shows the parts of the pump heads of Pump P9-S.
7 8 9
1 2 3 4 5 6
10
Part Description Part Description
1 Piston 6 Piston seal
2 Return spring 7 Outlet check valve
3 Drain plate 8 O-ring
4 Rinse membrane 9 Purge valve
5 Rinse chamber 10 Inlet check valve
Introduction
Follow the instructions below to replace the o-ring, piston seal, and rinse membrane of pump P9-S.
Note: Always replace the o-rings, piston seals, and rinse membranes of both pump heads of a pump
at the same time.
Disassemble the pump head

Step Action
2 Disconnect the tubing from the pump head, and disconnect the pump inlet tubing.

7 Maintenance
Step Action
3 Unscrew the two white plastic screws located below each pump head by hand. Pull the
plastic connectors to the sides to release the inlet manifold.
4 Disconnect the tubing of the pump piston rinsing system.
5 Unscrew one of the two screws of the pump head using a hex wrench. Unscrew the
second screw, and at the same time push firmly on the front of the rinse chamber to
compensate for the pressure of the piston return spring.
6 Place the pump head face down on the bench. Pull out the piston together with the return
spring.

7 Maintenance
Step Action
7 Inspect the piston and return spring for sign of damage. If damaged, discard the piston
and return spring and use a new piston and return spring when assembling the pump
head.
8 Unscrew the two screws securing the drain plate and the rinse chamber. Lift off the drain
plate, and discard the membrane located between the drain plate and the rinse chamber.
9 Lift off the rinse chamber. Gently pull off the piston seal. Discard the used seal.
10 Clean the pump head, rinse chamber and drain plate in an ultrasonic bath. If there are
particles on any surfaces, the check valves should be removed and cleaned separately,
see Section 7.6.8 Clean the pump head check valves, on page 296.

7 Maintenance
Replace O-ring, piston seal, and rinse membrane

Step Action
1 Unscrew the purge valve of the pump head. Replace the O-ring on the purge valve with
a new O-ring, 3x1 mm, and screw the purge valve back into the pump head.
Note:
Always use Lubricant 56686700 when exchanging the O-ring 3 x 1 mm.
2 Wet a new seal with 20% ethanol. Place the new seal in the hole in the front section of
the pump head and press it into position.
3 With the pump head facing downwards on the bench, place the rinse chamber onto the
front section of the pump head with the rinse ports in line with the check valves. The
conical depression in the rinse chamber shall be facing upwards. Wet a new membrane
with 20% ethanol, and place the membrane into the rinse chamber with the conical face
upwards.

7 Maintenance
Assemble the pump head

Make sure to assemble the pump head correctly. Refer to Illustrations, on page 336.
Step Action
1 Place the drain plate on top of the assembly. Screw the two screws through the drain
plate and the rinse chamber using a cross-headed screwdriver.
2 Wipe clean the piston and remove all finger prints. Wet the piston with 20% ethanol, and
insert the piston into the return spring. With the pump head facing downwards on the
bench, insert the piston into the pump head by pushing it gently but firmly vertically
downwards into the seal.
3 Place the complete pump head over the locating pins on the front panel of the sample
pump module. Turn the pump head so that the text UP on the drain plate is facing up-
wards. Push firmly on the front of the pump head, and at the same time screw one of
the screws to fasten the pump head onto the front of the module using a hex wrench.
Screw the second screw of the pump head. Make sure to tighten both screws fully.
4 Reconnect the tubing of the pump piston rinsing system.
5 Reconnect the inlet manifold.
6 Reconnect the tubing to the pump head, and reconnect the pump inlet tubing.
7 Break in the new pump piston seal, see instruction below.
Break in the new pump piston seal

Follow the instruction below to break in the new pump piston seal of pump P9-S.
Step Action
1 Fill a buffer vessel with 20% ethanol in water. Immerse a piece of sample inlet tubing, for
example S1, in the buffer vessel. Place the buffer vessel on the Buffer tray.
2 Prime the inlets and purge the pump, see Section 5.4.2 Sample pump, on page 167
3 Connect the reference capillary Ref 1 (or equivalent tubing that gives a backpressure of
2-3 MPa) to one of the column positions of the Column valve (e.g., ports 1A and 1B).
4 Immerse the waste tubing in the buffer vessel to recirculate the liquid.

7 Maintenance
Step Action

• Select Flow path:Column position, and select the Position of the tubing connected
to the Column valve, in this example 1. Click Insert.
• Select Flow path:Sample inlet and select a Position, in this example S1. Click Insert.
• Select Flow path:Injection valve and select Direct inject from the Position drop-down
list. Click Insert.
• Select Pumps:Sample flow and set the Flow rate to 25.0 ml/min. Click Insert.
• Click Execute.
Result: A sample flow of 25 ml/min starts.
6 Run the flow for 2 hours.
7 Discard the used buffer.

7 Maintenance
7.8.12 Replace pump pistons
7.8.12 Replace pump pistons
Replace the pump pistons if they are damaged.
Required material
• Star screwdriver, T20
• Piston kit
Replace pump pistons of Pump P9 and P9H

If a damaged piston has been in operation, the piston seal will be destroyed and should also be replaced.
To replace the piston and the seal of a system pump, see Section 7.8.10 Replace pump piston seals of
Pump P9 or P9H, on page 335.
Replace pump pistons of Pump P9-S

If a damaged piston has been in operation, the piston seal will be destroyed and should also be replaced.
To replace the piston and the seal of Pump P9-S, see Section 7.8.11 Replace pump piston seals of Pump
P9-S, on page 344.

7 Maintenance
7.8.13 Replace pump rinsing system tubing
Replace the pump rinsing system tubing when required , for example if the tubing is clogged or damaged.
Replacement instructions for the system pumps and the sample pump (external module) are given
below.
Required material
• Rinsing system tubing
System pump piston rinsing system

The illustrations below show the parts and tubing of the system pump piston rinsing system
5 4
Part Description
4 Outlet tubing
5 Inlet tubing

7 Maintenance
Sample pump piston rinsing system

4 5
Part Function
connection) .
liquid tube.
Connect new tubing

Step Action
1 Disconnect the used tubing.
2 Cut the new tubing to desired length.
3 Connect the new tubing according to the illustrations above.
4 Fit all pieces of tubing into the tubing holders on the pump modules.

7 Maintenance
Prime the rinsing systems

Before usage, prime the pump rinsing system tubing. Refer to Section 7.3.1 Change pump rinsing solution,
on page 262 for detailed instructions.

8 Troubleshooting
8 Troubleshooting
About this chapter

This chapter describes troubleshooting and corrective actions for ÄKTA pure.
In this chapter
Section See page
8.1 Introduction to troubleshooting 356
8.2 Troubleshooting: General Checklist 358
8.3 Troubleshooting: Monitors 360
8.4 Troubleshooting: Valves 375
8.5 Troubleshooting: Fraction collector 377
8.6 Troubleshooting: Pumps 387
8.7 Troubleshooting: Other components 394
8.8 Troubleshooting: Instrument communication 401
8.9 Troubleshooting: Method development 403
8.10 Error codes 404

8 Troubleshooting
8.1 Introduction to troubleshooting

Introduction
This section describes troubleshooting procedures for ÄKTA pure and how to generate a System error
report for service purposes. Subsequent sections in this chapter present general troubleshooting
checklists, module-specific problems and corrective actions.
Troubleshoot the software

For software related troubleshooting, see the following table and the list of literature below:
Problem Possible cause and action
Text in the Process Picture The operating system of the computer does not have the text font
pane in the System control Calibri True Type installed. For example, Windows XP does not
module looks strange include this font by deafault.
Install the font Calibri True Type or switch to an operating system
that includes the font.
• UNICORN Method Manual,

• UNICORN Evaluation Manual and
• UNICORN Administration and Technical Manual
Troubleshooting procedure
To troubleshoot ÄKTA pure:
Step Action
1 Always start by checking the General checklist. See Section 8.2 Troubleshooting: General
Checklist, on page 358.
2 In this document, search for solutions in the section corresponding to the problem.
3 Make the recommended corrective actions.
4 If problems remain after corrective actions, generate a System error report and contact
your local GE representative.
Generate a System error report

A System error report can be generated during a troubleshooting case with information about the
problem and can also include methods, logs, and results. The report can then be sent to Service for
action.
To generate a System error report for information to Service:

8 Troubleshooting
Step Action
1 In the System Control module, on the System menu, click Create System Error Report.
Result: The first page of a wizard is displayed.
2 • Click Next and start to enter information about the problem, click Next.
• Choose to enclose methods, logs or result files.
• Select location for the report and click Finish to generate the report. The filename of
the zip file will be Report_YYYYMMDD.zip and the default folder location is: C:Program
Files\GE Healthcare\UNICORN.
3 E-mail the report to GE Service department.

8 Troubleshooting
8.2 Troubleshooting: General Checklist

Introduction
Check the items in the following topics before starting more in-depth troubleshooting work.
System checks
• Is the correct system selected in UNICORN System Control? For more details, see Section 5.3 Start
UNICORN and connect to system, on page 157.
• Are the fans blowing at the back and at the right side of the system?
Monitor checks
• Is the UV monitor set to the correct wavelength? Check the wavelengths that are used in the method.
For the predefined methods the wavelengths are set in the Method Settings phase. For more details,
see UNICORN Method Manual.
• Is the air sensor sensitivity set to normal in UNICORN System Control:System Settings to avoid
unnecessary stops due to minor air bubbles?
Instrument communication
• Have Node IDs been set correctly for all instrument modules? To check and change Node IDs, see
Node ID, on page 76.
Flow path checks

• Is all tubing connected correctly? See Section 9.3 Tubing and connectors, on page 420 and Section 5.2
Prepare the flow path, on page 150.
• Is there leakage at any of the connections?
• Is any tubing folded or twisted?
• Is the inlet tubing correctly immersed in the buffer solution, beneath the liquid surface but not too
close to the flask bottom?
• Have Fraction collector F9-R and Fraction collector F9-C been correctly prepared? For more details,
see relevant sections in Chapter 3 ÄKTA pure external modules, on page 86 and Chapter 4 System
configuration, on page 123.
• Are the inlet and inline filters clean or are they generating a back pressure higher than normal? If
this is the case, change the inline and inlet filters. For more details, see Section 7.3.2 Replace the
inline filter, on page 266 and Section 7.8.8 Replace the inlet filters, on page 331.

8 Troubleshooting
• Does the positioning of the columns correspond to the selections made in the method? For more
details, see Section 5.5 Connect a column, on page 172.
Purification checks
• Have all columns been cleaned and prepared according to the column recommendations?
• Have the samples been adjusted to binding buffer conditions?
• Have the samples been clarified by centrifugation and/or filtration prior to sample loading?
• Are the correct buffers used for the chosen columns and proteins?
• Check buffers for precipitations. Adjust to room temperature.
• Are the chosen columns suitable for the chosen target proteins?
• Do the buffers have correct pH? The pH of some buffers changes with the temperature.
• Are the UV-wavelengths used by the method appropriate with respect to used buffers and proteins?
For more details see the method handbooks available from GE.

8 Troubleshooting
8.3 Troubleshooting: Monitors

In this section
• UV monitor and UV detector
• Conductivity monitor
• pH monitor and pH valve
• Pressure monitors
UV monitor U9-M and UV detector unit

The UV module is not found • Communication problem

by the instrument
Contact Service.
• The cable between the UV module and the ICU is not connected
Remove the UV module and make sure that the cable is connect-
ed.
• Wrong Node ID
Check the module´s Node ID. If necessary, change the Node ID.
See Node ID, on page 76.
No UV signal • The lamp is turned off.

Turn the lamp on with the Manual instruction Monitors – UV lamp.
Too low UV lamp intensity • The detector is not correctly fitted

Contact Service.
• Unclean optical fiber connectors
Clean the connectors. See Section 7.8.5 Replace the UV monitor
U9-M flow cell, on page 325.
• Dirt on optical sensors in detector
Remove visible dirt on detector photo diodes.
Use lint free lens paper winded around a thin wood splinter (e.g
a match or toothpick). Clean the sensors with Isopropanol through
the small hole of the metal plate which covers the sensors. Dry
the sensors with clean and dry lint free lens paper.
• Worn-out or broken lamp
Contact Service.

8 Troubleshooting
No light transmission • Wrong wavelength for current buffer

through the UV cell
Change wavelength or buffer.
• Dirt in the UV flow cell
Clean the UV cell. See Section 7.5.1 Clean the UV flow cell, on
page 273.
• Broken UV flow cell
Replace UV flow cell. See Section 7.8.5 Replace the UV monitor
Autozero out of accepted • Wrong wavelength for current buffer

range
Change wavelength or buffer.
The internal temperature of • The air intake on the rear or on the left side of the instrument
the UV monitor is too high is covered
Make sure that none of the air intakes on the instrument are
covered.
• Hot surroundings
Decrease the room temperature. Maximum operating tempera-
ture is 35°C.
• Hardware error
Switch off the instrument and wait until the temperature has
decreased. Restart the instrument. If this error is recurrent, gen-
erate a System error report and contact Service.
UV cell path length unread- • No UV flow cell is attached

able
Attach UV cell. See Section 7.8.5 Replace the UV monitor U9-M
flow cell, on page 325.
• UV flow cell is not correctly installed.
Verify that the UV cell is correctly installed. See Section 7.8.5 Re-
place the UV monitor U9-M flow cell, on page 325
• The UV flow cell is broken
Replace the cell. See Section 7.8.5 Replace the UV monitor U9-M

8 Troubleshooting
Ghost peaks • Air in the UV flow cell

Use the Flow restrictor. Use the pH valve instruction to manually
set the Flow restrictor inline (Flow path:pH valve:Restrictor in-
line), or select the Flow restrictor in the Method Settings phase
of a method.
Remove the air by flushing the cell with water or buffer. If persis-
tent, clean the UV cell. See Section 7.5.1 Clean the UV flow cell,
on page 273.
• Air in buffers
De-gas if necessary.
page 273.
• Dirt in the flow path
Clean the system in accordance to Section 7.6.2 Perform System
CIP, on page 278. Clean the column in accordance to Section 7.6.3
Perform Column CIP, on page 284.

8 Troubleshooting
Baseline drift or noisy signal • Flow restrictor in off-line position

of a method.
• Air in the UV flow cell
Use the Flow restrictor.
Remove the air by flushing the cell with water or buffer.
If persistent, clean the UV cell. See Section 7.5.1 Clean the UV flow
cell, on page 273.
• Air in buffers
• Make sure that both the instrument and the buffers have reached
the ambient temperature.
• Impure buffers
Check if the signal is noisy with water.
Perform a System CIP. See Section 7.6.2 Perform System CIP, on
page 278.
If necessary, manually clean the UV cell. See Section 7.5.1 Clean
the UV flow cell, on page 273.
• Wrong type of UV cell is used
If a UV cell with 2 mm path length is to be used, use only cells
marked U9-2.

8 Troubleshooting
Unstable signal • Bad pump function

Check that the pump is operating properly. See Select Mixer
chamber, on page 151 for example of pump pressure curves.
• Poor mixing function
Check the mixer chamber size and change the chamber if neces-
sary. See Select Mixer chamber, on page 151.
Check the function of the mixer. Place a stirrer bar in the palm
of your hand. Hold the hand above the mixer. The stirrer should
move when the mixer is activated.
Check that the mixer chamber is free from solids. To open the
mixer, see Section 7.3.2 Replace the inline filter, on page 266.
The UV curve shows a gradi- • Large difference in refractive index between buffer A and buffer
ent that is inverted com- B
pared to the expected gradi-
Due to light spreading effects in the UV cell, the buffer with the
ent
highest UV absorption shows the lowest UV absorption in the
chromatogram, and the buffer with the lowest UV absorption
shows the highest UV absorption. This can occur if there is a large
difference in refractive index between buffer A and buffer B and
the UV is run at high sensitivity.
UV monitor U9-L
No UV signal • The lamp is turned off.

Turn the lamp on with the Manual instruction Monitors – UV lamp.
Autozero out of accepted • Wrong UV flow cell for current buffer

range
Change to a shorter UV flow cell or change buffer.
• The UV flow cell is not correctly installed
Check that the UV flow cell is fitted correctly, see Section 7.8.6
Replace the UV monitor U9-L flow cell, on page 328.
Replace the cell, see Section 7.8.6 Replace the UV monitor U9-L

8 Troubleshooting
The internal temperature of • The air intake on the rear or on the right side of the instrument
the UV monitor is too high is covered
covered.
ture is 35°C.
• Hardware error
Maximum absorbance that • Wrong UV flow cell for current buffer

can be measured by the de-
Change to a shorter UV flow cell or change buffer.
tector is reached
• The UV flow cell is not correctly installed
Check that the UV flow cell is fitted correctly, see Section 7.8.6
Replace the UV monitor U9-L flow cell, on page 328.
Clean the UV flow cell, see Section 7.5.1 Clean the UV flow cell, on
page 273.
Replace the cell, see Section 7.8.6 Replace the UV monitor U9-L
The UV cell is not correctly • Check that the UV flow cell is fitted correctly, see Section 7.8.6
installed Replace the UV monitor U9-L flow cell, on page 328.

8 Troubleshooting
Ghost peaks • Air in the UV flow cell

of a method.
Remove the air by flushing the cell with water or buffer. If persis-
tent, clean the UV cell. See Section 7.5.1 Clean the UV flow cell,
on page 273.
• Air in buffers
page 273.
• Dirt in the flow path
Clean the system in accordance to Section 7.6.2 Perform System
CIP, on page 278. Clean the column in accordance to Section 7.6.3
Perform Column CIP, on page 284.
The UV lamp is broken or • Contact Service

worn out

8 Troubleshooting
Distorted protein peaks in • Rapid changes of the refractive index

IEX gradients (for example
The refractive index of the buffer changes rapidly in quick IEX
step gradients).
gradients. The rapid change may cause light spreading effects
and disturb the shape of the protein peaks in the U9-L 2 mm flow
cell.
Run with reversed flow direction through the 2 mm cell: connect
the inlet tubing at the bottom and the outlet tubing at the top of
the flow cell.
Note:
The standard tubing (7) between the UV monitor and the conduc-
tivity monitor is too short (170 mm) for mounting the monitor for
reversed flow direction. Perform the following actions:
1 Replace the standard tubing with a tubing that is 210 mm long.
2 Update the total delay volume. The increase in volume depends
on the inner diameter (i.d.) of the tubing:
- i.d. 0.25 mm : 2 µl
- i.d. 0.50 mm : 8 µl
- i.d. 0.75 mm: 18 µl
- i.d. 1.0 mm: 32 µl
The conductivity part of the Test was performed at cold room temperature and the conductiv-
system performance test ity temperature compensation factor was not optimized.
failed In the System Settings dialog, select Conductivity:Cond temp
compensation and set the Compensation factor to 2.1%.
The cell constant measure- • Internal errors

ment has been aborted
See error log. Restart instrument and retry. If this problem recurs,
generate a System error report and contact Service.
Unstable conductivity • Air in the Conductivity flow cell

Flush the Conductivity flow cell with water.
• Solids in the Conductivity flow cell
Clean the Conductivity cell. See Section 7.6.10 Clean the Conduc-
tivity flow cell, on page 300.

8 Troubleshooting
Temperature out of range • This error can only occur when the temperature compensation
for calibration is turned on. The error will occur when the temperature is
outside the range 2°C to 40°C.
Make sure the temperature of the calibration solution is within
2°C and 40°C.
Baseline drift of noisy signal • Air in the Conductivity flow cell

Remove the air by flushing the flow cell with water or buffer.
• Leaking tubing connections
Tighten the connectors. If necessary, replace the connectors.
• Unclean Conductivity flow cell
Clean the Conductivity flow cell. See Section 7.6.10 Clean the
Conductivity flow cell, on page 300.
Check the Mixer chamber size and change chamber if necessary,
see Select Mixer chamber, on page 151.
Check the motor operation of the Mixer. Place a magnet close to
the Mixer chamber during run. The magnet should vibrate.
Check that the Mixer chamber is free from solids. To replace the
inline filter, see Section 7.3.2 Replace the inline filter, on page 266.
Conductivity measurement • Unclean conductivity flow cell

with the same buffer ap-
Clean the Conductivity cell, see Section 7.6.10 Clean the Conduc-
pears to decrease/increase
tivity flow cell, on page 300.
over time.
• The ambient temperature may have decreased/increased
Use a temperature compensation factor. The temperature com-
pensation factor is found in System Control:System Settings:Con-
ductivity. Instruction regarding the factor is also found in Sec-
tion 7.7.3 Calibrate the Conductivity monitor, on page 308.
• The Conductivity monitor needs to be calibrated
Check calibration with a solution with known conductivity.
Calibrate the Conductivity monitor, see Section 7.7.3 Calibrate
the Conductivity monitor, on page 308.

8 Troubleshooting
Waves on the gradient • Bad pump function

Check that the pump is operating properly. See Examples of pump
pressure curves, on page390 for example of pump pressure curves.
• Air in the flow path
Purge the pumps. See Section 5.4.1 System pumps, on page 161.
Check that the correct Mixer chamber size is used. See Select
Mixer chamber, on page 151 for recommendations. To change the
Mixer, see Section 7.8.3 Replace the Mixer, on page 322.
Check the motor operation of the Mixer. Place a magnet close to
the mixer chamber during run. The magnet should vibrate.
Check that the Mixer chamber is free from solids. To open the
Ghost peaks appear in the • A charged particle has been detected

gradient profile
Prepare the sample so that charged particles are eliminated.
• Air bubbles are passing through the flow cell
Check for loose tubing connections.
Non-linear gradients or • Dirt in the tubing

slow response to %B
Make sure that the tubing has been washed properly.
changes
• Bad pump function
Make sure that the pump operates properly. See Examples of
pump pressure curves, on page390 for example of pump pressure
curves.
• The Mixer chamber is too large
Change to a Mixer chamber with a smaller volume. See Select

8 Troubleshooting
Incorrect or unstable read- • The temperature compensation factor is not properly set
ing
Use a temperature compensation factor. The temperature com-
pensation factor is found in System Control:System Settings:Con-
ductivity. Instruction regarding the factor is also found in Sec-
tion 7.7.3 Calibrate the Conductivity monitor, on page 308.
• The column is not equilibrated
Equilibrate the column. Use the method phase Equilibration.
If necessary, clean the column. Use the predefined method Col-
umn CIP. See Section 7.6.3 Perform Column CIP, on page 284.
Check that the correct Mixer chamber size is used. See Select
Check the motor operation of the mixer. Place a magnet close
to the Mixer chamber during run. The magnet should vibrate.
Check that the Mixer chamber is free from solids. To open the
pH monitor and pH valve

The pH module is not found • The cable between the pH valve and the ICU is not connected
by the instrument
Remove the pH valve and make sure that the cable is connected.
See Hardware installation of a module, on page 76.
The internal valve tempera- • The air intake on the rear or on the left side of the instrument
ture is too high is covered
covered.
ture is 35°C.
• Hardware error

8 Troubleshooting
Unstable pH signal • Calibration time out

Check the connections between pH electrode and pH monitor.
Regenerate the pH electrode. Place the electrode in deionized
water for 30 minutes followed by 30 minutes in a buffer with pH
4.
If persistent, replace the pH electrode. See Section 7.5.2 Replace
the pH electrode, on page 275.
• Bad pH electrode
4.
Clean the pH electrode. See Section 7.6.7 Clean the pH electrode,
on page 293.
If persistent, replace the pH electrode. See Section 7.5.2 Replace
the pH electrode, on page 275.
• Wrong mixer size for the used flow rate
Use the recommended mixer size for the used flow rate. See
Drift of pH signal when the • Decreasing salt concentration in the electrode membrane due
pH electrode has been re- to osmosis to buffer
moved from storage solu-
tion
4.
Temperature reading error • The temperature compensation of the pH monitor is turned

off
Contact Service.
It is not possible to inject • Waste tubing is twisted or blocked

calibration solution
Untwist the tubing.
Perform System CIP to clean waste tubing. See Section 7.6.2
Perform System CIP, on page 278.
Change the tubing.

8 Troubleshooting
Alarm in UNICORN: (Alarm) • High pressure in the pH cell

The pH cell can only be run
Decrease the flow rate.
at pressures below 0.8 MPa.
Please check the tubing or Bypass the pH electrode (see Ports and flow paths of the pH valve,
lower the flow through the on page 61) and measure pH in fractions manually.
pH cell.
Note: The pressure limit 0.8
MPa is for the post column
pressure.
Pressure monitors
Pressure offset • The monitors have lost their calibration

Calibrate the pressure monitors. See Section 7.7.2 Calibrate the
pressure monitors, on page 305.
• The temperature has changed
Wait until the temperature has stabilized and calibrate the pres-
sure monitors.
Excessively high pressure • Unclean inline filter in the Mixer

values
Replace the inline filter in the Mixer. See Section 7.3.2 Replace the
inline filter, on page 266.
• Solids in the flow path
To use the predefined method System CIP to clean the flow path,
see Section 7.6.2 Perform System CIP, on page 278.
To clean the column, see Section 7.6.3 Perform Column CIP, on
page 284. If persistent, replace the column.
• 0.25 mm .i.d. tubing kit is mounted
A smaller tubing inner diameter gives higher back pressure. The
back pressure for the 0.25 mm i.d. tubing kit is normally 16 times
higher than for the 0.50 mm i.d. tubing kit used at the same run-
ning conditions.
Maximum system flow rate for 0.25 mm i.d. tubing is 10 ml/min
The pressure monitors are • The cable between the Pressure monitors and the ICU is not
not found by the instrument connected.
Remove the monitor and make sure that the cable is connected.

8 Troubleshooting
The internal temperature of • The air intake on the rear or on the left side of the chromatog-
the pressure monitor is too raphy instrument or the sample pump is covered
high
covered.
ture is 35°C.
• Hardware error

8 Troubleshooting
Irregular pressure curves • Air bubbles are passing through or are trapped in the pump
Check that there is a sufficient volume of buffer present in the
flasks.
Check all connections for leaks.
Check pump pressure curves. See Examples of pump pressure
curves, on page 390 for examples of pump pressure curves.
Purge the pumps, see Section 5.4.1 System pumps, on page 161
or Section 5.4.2 Sample pump, on page 167
• The check valve does not function correctly
Remove any solids in the valves by cleaning the check valves
according to the instructions in Section 7.6.8 Clean the pump
head check valves, on page 296.
• Piston seal is leaking
Replace the piston seal of a system pump according to the in-
structions in Section 7.8.10 Replace pump piston seals of Pump
P9 or P9H, on page 335. Replace the piston seal of the sample
pump according to the instructions in Section 7.8.11 Replace
pump piston seals of Pump P9-S, on page 344
• Blockage of flow path
Use the predefined method Prepare System to flush through to
clear blockage.
If necessary, replace tubing. See Section 7.8.1 Replace tubing and
connectors, on page 317.
Check the mixer inline filter. It can be clogged if unfiltered buffers
or samples are applied. See Section 7.3.2 Replace the inline filter,
on page 266 for instructions how to replace the mixer inline filter.
Check the inlet filters. They can be clogged if unfiltered buffers
or samples are applied. To replace the filters, see Section 7.8.8
Replace the inlet filters, on page 331.
Check the column. It can be clogged if unfiltered buffers or
samples are applied. To clean a column, see Section 7.6.3 Perform
Column CIP, on page 284.

8 Troubleshooting
8.4 Troubleshooting: Valves

General
The following table lists the general problems that may occur for the different valves.
The valve is not found by • The cable between the valve and the ICU is not connected
the instrument
Remove the valve and make sure that the cable is connected.
See Hardware installation of a module, on page 76.
• Wrong Node ID
Check the valve Node ID. If necessary, change the Node ID. See
Node ID, on page 76.
The internal valve tempera- • The air intake on the rear or on the left side of the instrument
covered.
ture is 35°C.
• Hardware error
The valve is not switching • Hardware error

or is switching to wrong po-
Generate a System error report and contact Service.
sition
External leakage • Hardware error

Internal leakage • Broken valve

Replace the valve. See Section 2.5 Installation of internal modules,
on page 76.

8 Troubleshooting
Inlet valves
The Inlet valves include Inlet valve V9-IA, V9H-IA, V9-IB, V9H-IB, V9-IAB, V9H-IAB, V9-IX and V9H-IX, as
well as Sample inlet valve V9-IS and V9H-IS.
Faulty air sensor in the • Hardware error

valve
Restart the instrument with the power switch. If this error is re-
current, generate a System error report and contact Service.
pH valve
Leaking pH valve • The dummy electrode was dry when it was installed in the
valve.
1 Remove the dummy electrode.
2 Wet the dummy electrode properly with distilled water.
3 Insert the dummy electrode into the pH valve.
4 Rotate the dummy electrode before securing it with the nut.
Module Panel
The instrument is unable to • A Module Panel is missing and the position is left empty
find some of the modules
Install the missing Module panel.
• The cable between the Module Panel and the ICU is not con-
nected
Remove the Module Panel and make sure that the cable is con-
nected.

8 Troubleshooting
8.5 Troubleshooting: Fraction collector

In this section
• Fraction collector F9-C
• Fraction collector F9-R

The fraction collector can- • The cable between the fraction collector and the ÄKTA pure
not be found by the instru- instrument is not connected
ment
• A fuse in the instrument ICU is broken
The ICU needs to be changed. Generate a System error report
and contact Service.
Fraction collector arm is • Obstruction inside the fraction collector

blocked or internal fault in
Switch off the instrument and check for obstruction inside the
the fraction collector
fraction collector. Try to move the fractionation arm by hand.
Switch on the instrument. If this error is recurrent, generate a
System error report and contact Service.
The accumulator is jammed • Salt crystals or protein residuals block the accumulator
or there is an internal error
Restart the instrument and perform an accumulator wash.
in the instrument
• Mechanical error
If this error is recurrent, generate a System error report and
contact Service.
Fraction collection tube or • Too many commands are pending in the fraction collector
well is overfilled and frac-
The reason could be that too many fraction collector instructions
tionation movements are
have been sent. Wait for a while and try again.
lost.

8 Troubleshooting
The fraction collector failed • Cassette tray or tube rack is inserted in the wrong direction
to detect the code on the
Take it out and insert it in the correct direction.
Cassette
• Unclean Cassette type code reader
Clean the dispenser head and its four diode windows using a
cloth and a mild cleaning agent or 20% ethanol. See Section 7.6.4
Clean Fraction collector F9-C, on page 288 for more information.
• Unclean Cassette type codes
Clean the Cassette type codes. See Section 7.6.4 Clean Fraction
collector F9-C, on page 288 for more information.
If this error is recurrent, set the Fraction collector configuration
manually in UNICORN. In System Control, select System:Settings.
Navigate to Fraction collector:Cassette configuration and select
Manual.
The Cassette tray is loose in • The Tray guides in the fractionation chamber are missing
the fraction collector
Replace the Tray guides. See Section 7.6.4 Clean Fraction collector
F9-C, on page 288.
• The Tray catch is broken
Contact service.
Calibration of the Drop sync • Unclean Drop sync sensor diode windows
sensor failed
Clean the Drop sync sensor diode windows. See Section 7.6.4
Clean Fraction collector F9-C, on page 288 for location of the Drop
sync sensor diode windows and cleaning instructions.
contact Service.
Drop sync does not work • Liquid spatter in the fraction collector
Change to another type of tubes or deepwell plates or collect
fraction volumes of maximum two thirds of the volume of the
tubes. Clean the diode windows of the drop sync sensor more
frequently than once a week, using a cloth and a mild cleaning
agent or 20% ethanol.

8 Troubleshooting
The Dispenser head failed • Air in the flow path

to detect a drop
contact Service.
Check the flow path for air. Fill system and purge pumps accord-
ing to Section 5.4.1 System pumps, on page 161.
• Unclean Drop sync sensor diode windows
Clean the diode windows of the Drop sync sensor. See Sec-
tion 7.6.4 Clean Fraction collector F9-C, on page 288. If this error
is recurrent, generate a System error report and contact Service.
• Too high flow rate
The Dispenser head failed • Air in the flow path

to detect the flow properly
and has switched to tube
change with reduced accu-
mulator functionality If this error is recurrent, generate a System error report and
contact Service.
• Unclean diode windows in the Drop sync sensor
The Dispenser head failed • Too high flow rate

to detect a drop and has
switched to tube change
without Drop sync • Air in the flow path
contact Service.
• Unclean diode windows in the Drop sync sensor
Tubes do not fit in the Cas- • Wrong tube dimensions are used
sette
Check that the used tubes have the right dimensions. See Fraction
collector tubes and bottles, on page 99 for information about
tubes and Cassettes.
• QuickRelease function is worn out
Order a new Cassette. See Chapter 10 for ordering information.

8 Troubleshooting
Deep well plate does not fit • Unsupported deep well plate model
in the Cassette
Check that the deep well plates are supported. See Deep well
plates, on page 100.
The Cassette does not fit • The Cassette is turned in the wrong direction
the Cassette tray
See Prepare and insert the Cassette tray, on page 194 for informa-
tion of how to place the Cassettes.
• Objects or dirt under the Cassette
Remove the object or dirt.
Cassette tray in wrong posi- • The Tray guides in the fractionation chamber are missing
tion
F9-C, on page 288.
• The Cassette tray is facing the wrong direction
Make sure that the front of the tray (marked with the GE-logotype)
is facing outwards. See Prepare and insert the Cassette tray, on
page 194.
• Dirt under the Cassette tray
Remove the dirt.
The loaded Cassette tray • The Cassette tray is facing the wrong direction
cannot be inserted into the
Check that the GE-logotype is facing outwards when the tray is
fractionation collector
inserted into the fraction collector.
• Some of the tubes or plates are incorrectly placed in the Cas-
settes
Check that all tubes and plates are correctly inserted in the Cas-
settes. See Prepare and insert the Cassette tray, on page 194 .
• Some of the tubes or plates have the wrong dimensions
Check that the deep well plates and the tubes used are of the
right type. See Fraction collector tubes and bottles, on page 99
and Deep well plates, on page100 for information about supported
tubes and plates.

8 Troubleshooting
Quick scan or Full scan does • The Tray guides in the fractionation chamber are missing
not work
F9-C, on page 288.
• The Cassette tray is facing the wrong direction
Check that the GE-logotype is facing outwards.
• The Cassette type codes are unclean
Clean the the Cassette type codes.
• The QuickRelease of a Cassette is in open position
Close the Cassette. See Prepare and insert the Cassette tray, on
page 194.
• The Cassette code reader diode window is unclean
Clean the Cassette code reader diode window. See Section 7.6.4
Clean Fraction collector F9-C, on page 288.
• The automatic scanning is turned off in UNICORN
Make sure that the automatic scanning is turned on in UNICORN
software. In System Control, select System:Settings. Navigate
to Fraction collector:Cassette configuration and select Auto-
matic.
• Wells in deep well plates are prefilled to a volume above 25%
of the total well volume
Full scan will not work with prefilled wells during these conditions.
• Hardware error
The interior of the fraction • The light has been turned off in UNICORN
collector is dark
Turn on the light in UNICORN. In System Control, select Sys-
tem:Settings. Navigate to Fraction collector:Fraction collector
lamp and select Lamps On.
• The lamp is broken
Contact Service.

8 Troubleshooting
The waste or the interior of • The waste tubing is positioned so that the flow is obstructed
the fraction collector is
Untwist the waste tubing.
flooded
• The waste container is placed in a position higher than the
waste outlet
Place the waste container in a position lower than the waste
outlet.
• The waste tubing is blocked.
Clean or replace the waste tubing.
The liquid leaving the nozzle • Check the position of the waste funnel
does not strike the waste
Refit the waste funnel.
funnel
The tubing in the fraction • Salt residuals in the tubing

collector is blocked
Perform a Fraction collector wash, see Section 7.6.4 Clean
Fraction collector F9-C, on page 288.
If persistent, contact Service.
The fraction volume found • Leakage on the wet side of the instrument
in the tubes or wells are
Localize the leakage and take care of the leakage, for example
smaller than expected
by tightening connectors.
• Air in pumps
Purge pumps. See Section 5.4.1 System pumps, on page 161.
• Bad pump function
See troubleshooting of pumps in Section 8.6.
• Leakage inside the Frac chamber
Contact Service.
Liquid on the floor of the • The fingertight connector on the Dispenser head is not tight
fraction collector enough
Tighten the connector. Replace the connector if the leakage is
persistent.
• The waste tubing or the waste funnel is blocked.
Make sure that there is no blockage or clogging and that the
waste tubing not is bent.
• Leakage in the Frac arm
Contact service.

8 Troubleshooting
Spillage by the fraction col- • Unclean diode windows in the Drop sync sensor or unclean
lector during fractionation nozzle
Clean the Drop sync diode windows and the nozzle. See Sec-
tion 7.6.4 Clean Fraction collector F9-C, on page 288. Use the
Fraction collector cleaning position.
• Too high flow rate during usage of Drop sync
Use a flow rate below 2 ml/min.
• One or more Cassettes have empty positions
Make sure that all Cassette positions contain tubes or plates.
• The tubes are flooded
Make sure that the fraction volume is adapted to the tube volume.
Fraction tubes have not been replaced by empty tubes when the
Fraction collector was opened.
It is not possible to fraction- • The door of the fraction collector is not properly closed
ate
Close the door.
The fraction collector frac- • Quick scan has not detected the correct Cassette
tionates in the wrong well
Clean the Cassette type code of the Cassette. See Section 7.6.4
or tube
Clean Fraction collector F9-C, on page 288.
• Unsupported deep well plate is used.
Make sure that approved deep well plates are used. See Deep
well plates, on page 100.
• Wells in deep well plates are prefilled to a volume above 25%
of the total well volume
Full scan will not work with prefilled wells during these conditions.
• The deep well plate is not correctly positioned in the Cassette
See Prepare and insert the Cassette tray, on page 194 for informa-
tion of how to place the deep well plate.
• Dirt on the nozzle or Drop sync sensor diode windows. Dirt
may effect where the drops fall.
Clean nozzle and Drop sync sensor diode windows. See Sec-
tion 7.6.4 Clean Fraction collector F9-C, on page 288.
Error message when the • Some parts in a method require that the door of the fraction
door of the fraction collec- collector is closed.
tor is opened during a run
Do not open the door when the Fractionation indicator is lit.

8 Troubleshooting
Fraction collector wash is The Fraction collector wash instruction is aborted when the fraction
reported as Completed collector door is opened during state pause. When selecting to
when it has been aborted. continue the run the instruction is reported as Completed, it should
state Cancelled.
Action: Do not open the door during the Fraction collector wash
instruction. If the door has been opened, restart the Fraction collec-
tor wash instruction to make sure that the Fraction collector wash
has been performed as recommended.
The instrument does not • The Detector - Outlet valve delay volume has been set to zero.
fractionate via the Outlet
1 In System Control, select System:Settings:Fraction collec-
valve ports
tions:Delay volumes.
2 Correct the Detector - Outlet valve delay volume.
When starting a method run Action: End a manual run before starting a method run.
during a manual run, the
fraction collector generates
an error message indicating
that the wrong Cassettes
are in place, even though
the correct Cassettes are
present in the Fraction col-
lector.
High pressure alarm when Action:

collecting fractions with the
• Decrease the flow rate or use Outlet valve fractionation,
fraction collector
or
• replace the following tubing with tubing of larger inner diameter
(i.d.)
- the tubing between the outlet valve and the fraction collector
and
- the internal tubing of the fraction collector.
Note:
Update the delay volume. In the System Control software module,
select System:Settings:Tubing and Delay volumes and update
the value for Delay volume: Monitor to frac.
Spillage in Fraction collector Action: If possible, place the Cassettes close to waste, i.e., position
when the Frac arm is mov- 5 or 6 during fractionation.
ing from Cassette placed in
position 1 and 2 to waste

8 Troubleshooting
The fractionation starts in • The door of the fraction collector has been opened between
the first row again two runs. When the instrument is in state Ready and the door
is opened and closed, the Fraction collector content is reset.
If fractionation will continue after the last fraction of the first run,
do not open the door of the fraction collector between two runs.
Spillage between fractions • The combination of high flow rates and liquids with low surface
tension might lead to spillage in the Fraction collector.
If possible use Cassette positions 5 and 6. Lower the flow rate
when using liquids with low surface tension.

The fraction collector can- • The cable between the fraction collector and the ÄKTA pure
not be found by the instru- instrument is not connected
ment
Make sure that the cable is connected.
• Wrong node ID
Check the Node ID of the fraction collector. If necessary, change
the Node ID. See Section 9.16 Node IDs, on page 502.
The internal temperature of • Hot surroundings

the fraction collector is too
high
ture is 35°C.
• Hardware error
erate a System error report and contact service.

8 Troubleshooting
The fraction collector failed • To high flow rate for drop sync
to detect a drop and has
Decrease the flow rate or disable drop sync.
changed tube without drop
sync. • Air in the flow path
Check the flow path for air. Fill system and purge pumps. If this
error is recurrent, generate a System error report and contact
service.
• Unclean drop sync sensor
Clean the drop sensor photocell located above the tube sensor
with a damp cloth.
• The tubing is not correctly mounted in the tubing holder nut
Check that the tubing is correctly mounted, see Connect tubing
to ÄKTA pure, on page 106.
The tube sensor has not de- • The fraction collector movement is blocked
tected a new tube.
Make sure that the fraction collector can move and is free from
obstructions.
The delay queue is full or • The flow rate is too high

there is a tube change
Reduce the flow rate.
overload.
• The fraction volume is too small
Collect larger fractions.
• Too many fraction collector instructions have been sent
Wait for a while and try again.
Fraction numbering does Fraction numbering continues from where it was at the time of
not start at 1 when the the No tube error
fractionation is restared af- Manually reset the fraction number in the System settings menu.
ter a No tube error

8 Troubleshooting
8.6 Troubleshooting: Pumps

In this section
• Troubleshooting for System pumps and Sample pumps
• Example of pump pressure curves
• Remove persistent air bubbles
Pumps
Liquid is leaking between • Piston seal or rinsing membrane incorrectly fitted or worn
the pump head and the side
Replace or reinstall the seal or the membrane. For System pumps
panel
and Sample pump S9H, see Section 7.8.10 Replace pump piston
seals of Pump P9 or P9H, on page 335. For Sample pump S9, see
Section 7.8.11 Replace pump piston seals of Pump P9-S, on
page 344
Low eluent flow and noise • Air in pumps

Purge the pumps. See Section 5.4 Prime inlets and purge pump
heads, on page 160 or Section 5.4.2 Sample pump, on page 167.
• Bad piston spring
Disassemble the pump head and examine the piston spring. If
the spring is corroded, check piston seal and rinse membrane.
Make sure that the pump piston rinsing system is always used
when working with aqueous buffers containing salt.
• Bad pump piston
If the piston is damaged, replace it according to Section 7.8.12
Replace pump pistons, on page 351.
• Bad pump piston seal
Replace the piston seal and rinse membrane according to Sec-
tion 7.8.10 Replace pump piston seals of Pump P9 or P9H, on
page 335 or Section 7.8.11 Replace pump piston seals of Pump P9-
S, on page 344 .
Leakage around a connec- • Leaking connection and/or crystallized material around a

tor connector
Unscrew the connector and check if it is worn or incorrectly fitted.
If so, replace the connector. Gently finger tighten the connector.

8 Troubleshooting
Erratic pump pressure • Air trapped in the pump heads

• Partially blocked solvent filters
• Leaking connections
• Piston seal leakage
• Check valve malfunction
• Piston damaged
See Examples of pump pressure curves, on page 390 for examples
of pump pressure curves.
The pump is not found by • The cable between the system pump and the ICU is not con-
the instrument nected.
Contact Service.
• The cable between the sample pump and the ÄKTA pure instru-
ment is not connected.
Connect the cable.
• A fuse in the instrument ICU is broken.
Contact Service.
The internal temperature of • The air intake on the rear or on the left side of the instrument
the pump is too high is covered
covered.
ture is 35°C.
• Hardware error
High pressure alarm • The pressure has increased due to increased viscosity
The viscosity increases in cold room. Lower the flow when per-
forming runs in a cold room.

8 Troubleshooting
High pressure alarm when • The parameter selected for pressure control is not the most
pressure control is activat- appropriate one
ed
The pressure control is based on either the Pre column pressure
or the Delta column pressure. To change the parameter for
pressure control, select Pre column pressure or Delta column
pressure from the Pressure control drop-down list in the Instruc-
tion box of the instruction of interest.
• The flow is too high
Lower the flow.
Abnormal difference in sys- • Clogged inline filter

tem pressure compared to
Replace the inline filter, see Section 7.3.2 Replace the inline filter,
pre column pressure
on page 266.
Internal pump error com- • The flow path is blocked

bined with high pressure
Remove obstructions in the flow path. For example, remove stop
plugs and replace constricted tubing.
Internal pump error at nor- • Blocked pump restrictor

mal pressure
Contact Service.
Too slow pressure build up • Too low I factor in the Pressure control parameters instruction
when pressure control is
In the Manual Instructions dialog, increase the I factor of the
active
Pressure control parameters instruction.
Too slow pressure build up • Too low I factor in the Constant pressure flow parameters in-
when using constant pres- struction
sure flow
In the Manual Instructions dialog, increase the I factor of the
Constant pressure flow parameters instruction.
Pressure overshoot or oscil- • Too high I factor in the Pressure control parameters instruction
lating pressure when pres-
In the Manual Instructions dialog, decrease the I factor of the
sure control is active
Pressure overshoot or oscil- • Too high I factor in the Constant pressure flow parameters in-
lating pressure when using struction
constant pressure flow
In the Manual Instructions dialog, decrease the I factor of the
Constant pressure flow parameters instruction.

8 Troubleshooting
Examples of pump pressure curves

The table below shows some examples of pump system pressure curves obtained when errors have
occurred. The examples can be useful in troubleshooting of the system pumps and the sample pump.
The system pressure monitor R9 has higher resolution than the other pressure monitors, and is therefore
recommended for troubleshooting purposes.
Incident: 30 μl of air enters pump.

Action: Purge pump. See Section 5.4 Prime inlets and purge pump heads, on page 160. If the problem
is persistent, see Remove persistent air bubbles, on page 392.
Incident: A large volume of air enters pump.

Action: Purge pump. See Section 5.4 Prime inlets and purge pump heads, on page 160.

8 Troubleshooting
Incident: Blocked outlet check valve

Action: Clean the check valve, See Section 7.6.8 Clean the pump head check valves, on page 296.
Incident: Inlet check valve is loose.

Action: Tighten the check valve. See Section 7.8.9 Replace the pump head check valves, on page 332.
Incident: Leaking inlet check valve.

Action: Replace the check valve. See Section 7.8.9 Replace the pump head check valves, on page 332.

8 Troubleshooting
Incident: Leaking outlet check valve.

Actions: Replace the check valve. See Section 7.8.9 Replace the pump head check valves, on page 332.
Incident: One inlet is blocked,

Action: Clean inlet tubing. For example, perform a System CIP.
Remove persistent air bubbles

After purging the pump (see Section 5.4 Prime inlets and purge pump heads, on page 160), check that
all air bubbles have been removed by analyzing the pre-column pressure curve (see examples above).
If the pressure curve indicates that there are still air bubbles present, repeat the purging process. If
the problem persists, follow the instructions below to purge the pump with methanol (see Section 5.4
Prime inlets and purge pump heads, on page 160 for detailed instructions for the purge procedure).
1 Make sure that the pump contains water.
2 Use a syringe to draw 100% methanol into the pump (see Section 5.4 Prime inlets and purge pump
heads, on page 160 for details).
3 Set the pump flow to 2.5 ml/min for ÄKTA pure 25 or 10 ml/min for ÄKTA pure 150.
4 Let the flow run until the disturbances in the pressure curve disappear.
5 Remove the methanol:
• Stop the pump and move the inlet tubing to water. Make sure that no air is introduced into the
system.
• Set the pump flow to 1 ml/min for ÄKTA pure 25 or 5 ml/min for ÄKTA pure 150.
• Run for 5 minutes.

8 Troubleshooting
6 Purge the pump again using an appropriate buffer.

8 Troubleshooting
8.7 Troubleshooting: Other components

In this section
• General Hardware: All modules
• Mixer
• Superloop
• Cabinet
• Power and ICU
• External air sensors
• I/O-box E9
• Instrument control panel
General hardware: All modules

Modules cannot be found by • The cable between a module and the ICU is not connected
the instrument
Remove the module and make sure that the cable is connected.
• Two similar modules have been added to the instrument, for
example two Inlet valve A
Remove one of the modules with the same Node ID.
• The Node ID for one or more of the modules is incorrect, for
example an Inlet valve A2 has the same Node ID as Inlet valve
A. The instrument then considers them to have the same
identity.
Remove Inlet valve A2 and change the Node ID according to
Section 9.16 Node IDs, on page 502.
An unknown instrument • The Node ID for one or more of the modules is incorrect
module is connected to the
Check Node ID and change the Node ID according to Section 9.16
system
Node IDs, on page 502.

8 Troubleshooting
Mixer
Leakage • Leaking tubing connections

Check the tubing connections. Tighten or replace if necessary.
Check the O-ring. Replace it if it is damaged. See Section 7.8.4
Replace the O-ring inside the Mixer, on page 323.
Check the Mixer chamber. Replace it if the liquid has penetrated
the Mixer chamber walls and sealings. See Section 7.8.3 Replace
the Mixer, on page 322.
See Chapter 10 Ordering information, on page 505.
Unstable gradients • Bad mixing of eluents

Check the function of the Mixer. Place a stirrer bar in the palm of
your hand. Hold the hand above the Mixer. The stirrer should
move when the Mixer is activated.
Check the Mixer chamber size and change chamber if necessary.
See Section 7.8.3 Replace the Mixer, on page 322.
Noisy UV signal • Bad mixing of eluents

Check the function of the Mixer. Place a stirrer bar in the palm of
your hand. Hold the hand above the Mixer. The stirrer should
move when the Mixer is activated.
Check the Mixer chamber size and change chamber if necessary,
see Select Mixer chamber, on page 151.
The Mixer chamber was not • The Mixer chamber is not correctly installed
recognized
Verify that the Mixer chamber is correctly installed. See Sec-
tion 7.8.3 Replace the Mixer, on page 322. If the error is recurrent,
replace the Mixer chamber. See Chapter 10 Ordering information,
on page 505.
The internal Mixer tempera- • The air intake on the rear or on the left side of the instrument
covered.
ture is 35°C.
• Hardware error

8 Troubleshooting
Superloop
Overpressure during filling • The Superloop is filled to the max. Pressure is not released
anywhere.
Manually turn Injection valve to Manual load position.
Cabinet
The temperature of the in- • The air intake on the back or on the left side of the instrument
strument or an instrument is covered
component is too high
covered.
• Broken fans
Contact Service.
ture is 35°C.
Liquid from the Buffer tray • The waste tubing from the Buffer tray is loose
is flowing onto the table
Contact Service.
Power and ICU

The instrument cannot be • The power cord is not connected

turned on
Connect the power cord to the wall outlet and to the electrical
inlet on the instrument. Make sure that the cord is attached using
the clip, thereby preventing the cord from coming loose.
• No electric current in the wall outlet
Make sure that there is electric current in the wall outlet.
• A fuse in the instrument ICU is broken
Contact Service.
• The instrument is overheated

8 Troubleshooting
One or more modules are • One or some of the minor modules use too much current. Minor
automatically turned off modules include all modules except the Fraction collector, the
UV monitor and the pumps.
The current is cut off by a temperature sensitive component. The
instrument can be restarted when the temperature has de-
creased. If persistent, generate a System error report and contact
Service.
The instrument cannot be • The network cable is not connected

seen in UNICORN
Connect the cable, see UNICORN Administration and Technical
Manual.
One or more module(s) is • The cable between a module and the ICU is not connected.
not found by the instrument
Remove valves and check that cables are connected.
• One or more jumpers are loose or missing.
Check the connections on the back of the instrument. Empty
positions for UniNet-9 connectors 1 to 8 must have connected
jumpers. See the illustration below. The connector 9 should be
protected with a plastic lid and must never have a jumper con-
nected to it.

8 Troubleshooting
The internal instrument • The air intake at the rear or on the right side of the instrument
temperature is too high is covered
covered.
ture is 35°C.
• Hardware error
External air sensors

The external air sensor is • The cable between the external air sensor and the ICU is not
not found by the instrument connected
Check the back of the instrument and make sure that the cable
is connected.
• The external air sensor has not been selected in UNICORN
Select the external air sensor in UNICORN. Refer to the installation
instructions delivered together with the external air sensor.
Air is introduced into the • One of the connections is not tight enough
flow path
Tighten the connectors.
Liquid is leaking from the • One of the connections is not tight enough
external air sensor
Tighten the connectors.

the air sensor is too high
ture is 35°C.
Hardware error

8 Troubleshooting
I/O-box E9

the I/O-box is too high
ture is 35 C.
• Hardware error
The I/O-box is not detected • Wrong Node ID

by the system
Make sure that the Node ID is (00) for the primary box, and (01)
for a potential secondary box.
The system does not detect • Cables incorrectly connected

Digital signals correctly
Make sure that the cables are connected correctly, earth to earth
etc.
Digital In always ”0”, regard- • Digital Out connections switched

less of input signals
Check that the digital Out cables are correctly connected, earth
to earth etc.
External digital equipment • Cables incorrectly connected

does not respond to set
Make sure that the cables are connected correctly, earth to earth
changes in Digital Out
etc.
To isolate the problem:
1 Connect Digital Out (e.g. pin 6) to Digital In (pin 1,2,3, or 4).
2 Change the Digital Out 1 signal between ”1” and ”0”. Verify that
Digital out follows.
3 If not: contact Service.
If it does: the problem probably lies within the connected equip-
ment.
Analog In does not measure • Auto-zero on the wrong level

expected voltage
Reset Auto-zero.
• Digital Out connection error
Make sure that Digital Out is connected correctly, earth to earth
etc.
Noisy analog signal • Too long cable between the external equipment and E9
Use as short cable as possible. Use shielded cable. Connect the
cable shield to the D-sub connector’s shield.
Analog In does not measure • Analog In is not calibrated

expected voltage
Contact Service.

8 Troubleshooting
Analog Out does not gener- • Analog Out is not calibrated

ate expected voltage
Contact Service.
Instrument control panel

The internal temperature of • The air intake on the rear or on the right side of the instrument
the instrument control pan- is covered.
el is too high
Make sure that none of the air intakes of the instrument are
covered.
ture is 35 C.
• Hardware error

8 Troubleshooting
8.8 Troubleshooting: Instrument communication

Scenario Possible cause and action
Multiple error messages in • A cable to a module (including Module Panels) is not connected
UNICORN: Lost modules
Connect the cable:
2 Check all modules and connections.
4 In the displayed dialog in UNICORN, select the option Restart
the system only and click OK.
• A UniNet-9 connector is not plugged
Check that all UniNet-9 connectors that are not in use are
plugged.
UNICORN has lost communi- • The UNICORN client has lost connection to the instrument
cation with the instrument server during a temporary overload of the processor
server
Restart the UNICORN client to regain control. The run continues
and no data will be lost.
Warning message in • The module is not functioning properly

UNICORN: Instrument mod-
In the displayed dialog in UNICORN, select the option Restart the
ule is missing
system only and click OK.
If the problem still remains, replace the module or contact Service.
Warning messages in • Two or several modules have the same Node ID

UNICORN: (Warning) Two
Change to correct Node IDs:
instrument modules have
the same Node ID 1 Switch off the instrument.
2 Check the Node ID for all modules, see Section 9.16 Node IDs,
on page 502.

8 Troubleshooting
Warning messages in • One module has an incorrect Node ID

UNICORN: (Warning) Gate
Change the Node ID:
(12): Internal instrument er-
ror 1 Switch off the instrument.
2 Check the Node ID for all modules, see Section 9.16 Node IDs,
on page 502.

8 Troubleshooting
8.9 Troubleshooting: Method development
8.9 Troubleshooting: Method development

Phase properties or text in- • The module required for the instruction has not been enabled
structions are not available in the component selection in the administration module
in the method editor as ex-
Review the component list.
pected
• The wrong system has been selected when creating the new
method
Make sure that the right system has been selected in the "new
method" dialog.
A method has been created • Action:

for a system that now has a
1 Open the method in the method editor.
different configuration
2 Select to either adapt the method to the new configuration
OR
Keep the text method unchanged and manually replace the
text instructions that are not available for the new configura-
tion.
Refer to Open and save methods for different systems, on page214
for more information.
An old method is to be • Action:

transferred to a new system
1 Open the method in the method editor.
configuration.
2 Save as and select the new system.
OR
If the configuration is different on the new system, select to
either adapt the method to the new configuration or to keep
the text method unchanged and manually replace the text
instructions that are not available.
Refer to Open and save methods for different systems, on page214
for more information.

8 Troubleshooting
8.10 Error codes
8.10 Error codes

Introduction
This section describes the error codes that can appear for the different modules, together with corrective
actions.
All modules
Error code Description Action
0 - 19 Internal instrument error Restart the instrument. If recurrent please con-

tact Service.
Instrument control unit

21 - 69 Internal instrument error Restart the instrument. If recurrent please con-

tact Service.
Valve
20, 24 Internal instrument error Restart the instrument. If recurrent please con-
tact Service.
22 Valve not finding position Restart the instrument. If recurrent please con-
tact Service.
23 Faulty air sensor Restart the instrument. If recurrent please con-

tact Service.
25 High temperature See Section 8.4 Troubleshooting: Valves, on

page 375
Pressure monitor
20, 21, 24- Internal instrument error Restart the instrument. If recurrent please con-
27 tact Service.
23 High temperature See Pressure monitors, on page 372.

8 Troubleshooting
8.10 Error codes
Air sensor
20 High temperature See External air sensors, on page 398.
Mixer
25 Mixer motor error Restart the instrument. If recurrent please con-

tact Service.
26 Internal instrument error Restart the instrument. If recurrent please con-

tact Service.
27 High temperature See Mixer, on page 395
pH monitor
tact Service.
25 No factory calibration Contact Service
26 High temperature See pH monitor and pH valve, on page 370.
20-27 Internal instrument error Restart the instrument. If recurrent please con-
tact Service.
28 High temperature See Conductivity monitor, on page 367
29 Temperature data error Restart the instrument. If recurrent please con-

tact Service.
32-34 No factory calibration Contact Service

8 Troubleshooting
8.10 Error codes
Pump
51-53 Internal pump error Check that there is no blockage of the pump
outlet. Restart the instrument. If recurrent please
contact Service.
54 High temperature See Section 8.6 Troubleshooting: Pumps, on

page 387
UV monitor U9-M
21, 25, 26, Grating error Restart the instrument. If recurrent please con-
31 tact Service.
22, 23, 32 Block filter error Restart the instrument. If recurrent please con-
tact Service.
24 Internal instrument error Restart the instrument. If recurrent please con-

tact Service.
27 Spectrum calibration error Restart the instrument. If recurrent please con-

tact Service.
28, 29 Lamp error Restart the instrument. If recurrent please con-

tact Service.
30 High temperature See UV monitor U9-M and UV detector unit, on

page 360.
UV detector U9-D
24, 26, 28, Internal instrument error Restart the instrument. If recurrent please con-
29, 31, 33 tact Service.
25 Too low light intensity Check that the detector and flow cell are fitted
correctly. If warning reappears, contact Service.
27 Too high light intensity Check that the detector and flow cell are fitted
correctly. If warning reappears, contact Service.
30 Too high light intensity, R channel Check that the detector is fitted correctly. If
warning reappears, contact Service.

8 Troubleshooting
8.10 Error codes
32 Too high S light intensity, S chan- Check that the flow cell is fitted correctly. If
nel warning reappears, contact Service.
34 No light detected Check optical pathway and restart the instru-

ment. If recurrent please contact Service.
35 Too low light intensity, R channel Check that the detector is fitted correctly. If
warning reappears, contact Service.
36 Too low light intensity, S channel No light through flow cell. Check solution absorp-
tion and that the cell is fitted correctly.
UV monitor U9-L
51 High temperature See UV monitor U9-L, on page 364.
52, 55 Low lamp intensity Contact Service.
54 Autozero out of range Autozero requested when AU value is larger than

2.
58 Too low light intensity, S channel No light through flow cell. Check solution absorp-
tion and that the cell is fitted correctly.
tact Service.
61 Measurement error Restart the instrument. If recurrent please con-

tact Service.

20 - 22 Internal instrument error. Restart the fraction collector with the power
switch on the ÄKTA pure instrument. If recurrent,
generate a system error report and contact ser-
vice.
32 The fraction collector failed to Restart the fraction collector with the power
operate the accumulator. switch on the ÄKTA pure instrument. If recurrent,
vice.

8 Troubleshooting
8.10 Error codes
33 - 35, 37 - Fraction collector configuration The instrument restarts. If the error is recurrent,
40, 55 error. generate a system error report and contact ser-
vice.
47 - 48 The fraction collector failed to Clean the cassette code reader, the cassette type
detect the code on the cassette code on the cassette and the plate.
or the type of plate.
53 Calibration of the dropsync sen- Clean the Drop sync sensor, see user documen-
sor failed. tation for the location of the sensor. If this error
is recurrent, please contact service.
54 Fraction collector movement is Close the door of the fraction collector.

blocked. Either the door is open
or there are communication
problems.
57 - 59 Internal fraction collector error. The instrument restarts. If the error is recurrent,
vice.
63 Tube change is too fast. Lower the flow rate or increase the fractionation
volume.
68, 72 Fraction collector arm movement The instrument restarts. If the error is recurrent,
is blocked. generate a system error report and contact ser-
vice.

20 High temperature See Section 8.5 Troubleshooting: Fraction collec-

tor, on page 377
21 Drop sync warning Clean the sensor and remove air bubbles in the
flow path.
22 Tube sensor error Check that the tube sensor is adjusted properly.
23 Delay queue full Increase the fraction size. Fraction size must be
greater than 1/10 of the delay volume.
tact Service.
25, 28 Too fast tube change Increase the fraction size or lower the flow rate.

8 Troubleshooting
8.10 Error codes
27 Drop sync error Clean the drop sensor

Error code
Instrument control panel

51 High temperature See Instrument control panel, on page 400
I/O-box
20 High temperature See section "Troubleshooting"
21 Analog in signal below -2V Check the external equipment connected to the
I/O-box.
22 Analog in signal above 2V Check the external equipment connected to the

I/O-box.
23-28 Internal instrument error Restart the instrument. If recurrent please con-
tact Service.

9 Reference information
About this chapter

This chapter lists the allowed environmental and operational ranges for ÄKTA pure.
Refer to ÄKTA pure Product Documentation for detailed technical specifications.
In this chapter
Section See page
9.1 System specifications 411
9.2 Module specifications 413
9.3 Tubing and connectors 420
9.4 Recommended tubing kits for prepacked columns 428
9.5 Chemical resistance guide 431
9.6 Wetted materials 436
9.7 Predefined methods and phases 438
9.8 System settings 448
9.9 Manual instructions 465
9.10 Available Run data 487
9.11 Available Curves 490
9.12 Injection volumes and peak broadening 492
9.13 Delay volumes 493
9.14 Component volumes 498
9.15 Pressure control 500
9.16 Node IDs 502

9.1 System specifications

System specifications
Parameter Data
System configuration Benchtop system, external computer
Control system UNICORN 6.3 or other compatible version
Connection between PC and instrument Ethernet
Dimensions (W x D x H) 535 x 470 x 630 mm
Weight (excluding computer) up to 53 kg
Power supply 100-240 V ~, 50-60 Hz
Power consumption 300 VA (typical)

25 VA (power-save)
Enclosure protective class IP 21
Tubing and connectors ÄKTA pure 25:

• Inlet: FEP tubing, i.d. 1.6 mm, Tubing connec-
tor 5/16" + Ferrule (yellow), 1/8"
• Pump to Injection valve: PEEK tubing, i.d. 0.75
mm, Fingertight connector, 1/16"
• After Injection valve: PEEK tubing, i.d. 0.50 mm,
Fingertight connector, 1/16"
• Outlet and waste: ETFE tubing, i.d. 1.0 mm,
Fingertight connector, 1/16"
• Optional tubing kits: i.d. 0.25 mm, i.d. 0.75 mm,
i.d. 1.0 mm

Parameter Data
Tubing and connectors ÄKTA pure 150:

• Inlet: FEP tubing, i.d. 2.9 mm, Tubing connec-
tor 5/16" + Ferrule (blue), 3/16"
• Pump to injection valve: PEEK tubing, i.d.
1.0 mm, 10-32 UNF connections
• After Injection valve: PEEK tubing, i.d. 0.75 mm,
10-32 UNF connections
• Outlet: FEP, i.d. 1.6 mm, 5/16-24 UNF connec-
tions
• Waste: ETFE tubing, i.d. 1.0 mm, Fingertight
connector, 1/16"
• Optional tubing kits: i.d. 0.5 mm, i.d. 1.0 mm
Environmental ranges
Parameter Data
Storage and transport temperature range -25°C to +60°C
Chemical environment See the relevant purification instrument User

manual.
Operating ranges
Parameter Data
Operating temperature range 4°C to 35°C
Relative humidity 20% to 95%, non-condensing
Equipment noise level

Equipment Acoustic noise level
ÄKTA pure instrument < 60 dB A

9.2 Module specifications

Introduction
This section specifies the operating data of the components in ÄKTA pure. For general data for the
system see System specifications, on page 411.
System pumps
Parameter Data
Pump type Piston pump, metering type
Flow rate range ÄKTA pure 25:

0.001 to 25 ml/min
(up to 50 ml/min during column packing)
ÄKTA pure 150:
0.01 to 150 ml/min
(up to 300 ml/min during column packing)
Pressure range ÄKTA pure 25:

0 to 20 MPa (2900 psi)
ÄKTA pure 150:
0 to 5 MPa (725 psi)
Viscosity range ÄKTA pure 25:

0.35 to 10 cP (5 cP above 12.5 ml/min)
ÄKTA pure 150:
0.35 to 5 cP
Flow rate ÄKTA pure 25:

specifications
• Accuracy: ± 1.2%
• Precision: RSD < 0.5%
(Conditions: 0.25 to 25 ml/min, < 3 MPa, 0.8 to 2 cP)
ÄKTA pure 150:
• Accuracy: ± 1.5%
(Conditions: 1.0 to 150 ml/min, < 3 MPa, 0.8 to 2 cP)

Sample pump
Parameter Data
Pump type Piston pump, metering type
Dimensions (W x D x H) 215 x 370 x 210 mm
Weight 11 kg
Flow rate range ÄKTA pure 25:

0.001 – 50 ml/min
ÄKTA pure 150:
0.01 to 150 ml/min
Pressure range ÄKTA pure 25:

0 to 10 MPa (1450 psi)
ÄKTA pure 150:
0 to 5 MPa (725 psi)
Viscosity range 0.7 to 10 cP
Flow rate ÄKTA pure 25:

specifications
• Accuracy: ± 2%
(Conditions: 0.25 – 50 ml/min, < 3 MPa, 0.8 – 3 cP)
ÄKTA pure 150:
• Accuracy: ± 2%
(Conditions: 1.0 – 150 ml/min, < 3 MPa, 0.8 – 3 cP)
Valves
Parameter Data
Type Rotary valves
Number of valves Up to 12
Functions Standard: Injection

Options: Inlet A, Inlet B, Sample inlet, Extra inlet, Mixer by-
pass, Loop selection, Column selection, pH, Outlet, Versatile

Inlet options
Parameter Data
Inlet A 1, 2 or 7 inlets
Inlet B 1, 2 or 7 inlets
Sample inlet Up to 7 sample inlets and 1 buffer inlet
Outlet options
Parameter Data
Number of outlets 1 or 10
Mixer
Parameter Data
Mixing principle Chamber with magnetic stirrer
Mixer volume ÄKTA pure 25:

0.6, 1.4 or 5 ml
ÄKTA pure 150:
1.4, 5 or 15 ml
Gradient formation
Parameter Data
Gradient flow rate range ÄKTA pure 25:

0.1 to 25 ml/min
ÄKTA pure 150:
0.5 to 150 ml/min
Gradient composition accuracy ÄKTA pure 25:

±0.6%
(Conditions 5 to 95% B. 0.5 to 25 ml/min, 0.2 to 2 MPa, 0.8
to 2 cP)
ÄKTA pure 150:
±0.8%
(Conditions 5 to 95% B. 2 to 150 ml/min, 0.2 to 2 MPa, 0.8
to 2 cP)

Pressure monitors
Parameter Data
Number of sensors Up to 4
Placement of sensors Standard: The System pressure monitor is located after the
System pump
Options:
• The Pre-column pressure monitor and the Post-column
pressure monitor are integrated in Column valve V9-C
or V9H-C.
• The Sample pressure monitor is located after the Sample
pump.
External air sensor options

Parameter Data
Number of sensors Up to 7
Placement • Integrated in inlet valve A, inlet valve B and sample inlet

valve
• After the injection valve
• Before the system pumps
• Before the sample pump
Sensing principle Ultrasonic
UV monitor options
Parameter Data
Number of monitors Up to 2
Wavelength range U9-L: 280 nm

U9-M: 190 to 700 nm in steps of 1 nm, up to 3 wavelengths
Absorbance range -6 to 6 AU
Resolution 0.001 mAU
Linearity U9-L: within ± 5% at 0 to 2 AU

U9-M: within ± 2% at 0 to 2 AU
Drift U9-L (2 mm cell): ≤ | 0.2 mAU | AU/h

U9-M (2 mm cell at 280 nm): ≤ | 0.2 mAU | AU/h

Parameter Data
Noise U9-L: < 0.1 mAU

U9-M: < 0.08 mAU
Operating pressure 0 to 2 MPa
Lamp operating time U9-L: > 10000 h

U9-M: > 5000 h
Flow cells: U9-L Standard:

Optical path length 2 mm
Cell volume 2 µl
Total volume: 30 µl
Option:
Cell volume 6 µl
Total volume 20 µl
Flow cells: U9-M Standard:

Cell volume 2 µl
Total volume: 11 µl
Option:
Cell volume 8 µl
Total volume 12 µl
Optical path length 0.5 mm
Cell volume 1 µl
Total volume 10 µl
Conductivity monitor options

Parameter Data
Conductivity reading range 0.01 to 999.99 mS/cm
Accuracy ± 0.01 mS/cm or ± 2%, whichever is greater,

(within 0.3 to 300 mS/cm)
Operating pressure 0 to 5 MPa
Flow cell volume 22 μl
Temperature monitor range 0°C to 99°C
Temperature monitor accuracy ±1.5°C within 4°C to 45°C

pH monitor option
Parameter Data
pH reading range 0 to 14
Accuracy ± 0.1 pH unit

within pH 2 to 12, temperature within ±3°C from calibration
temperature
Operating pressure 0 to 0.5 MPa
Flow cell volume ÄKTA pure 25:

76 μl
ÄKTA pure 150:
129 μl
Outlet valve fractionation option

Parameter Data
Number of outlets 10
Fraction volumes 0.01 to 20 000 ml
Delay volume (UV – outlet valve) ÄKTA pure 25:

125 µl
66 µl with optional tubing kit (i.d. 0.25 mm)
ÄKTA pure 150:
296 µl
245 µl with optional tubing kit (i.d. 0.5 mm)
Fraction collector options

Parameter Data
Number of fraction collectors Up to two.

The second fraction collector must be an F9-R.
Number of fractions F9-C: Up to 576

F9-R: Up to 175

Parameter Data
Vessel types F9-C:

• Deep well plates, 96, 48 or 24 wells
• Tubes 3, 5, 8, 15, 50 ml
• Bottle, 250 ml
F9-R: 3, 5, 8, 15 or 50 ml tubes
Fraction volumes F9-C: 0.1 to 250 ml

F9-R: 0.1 to 50 ml
Spillage-free mode F9-C: Automatic, Drop sync or Accumulator

F9-R: Drop sync
Flammable liquids F9-C: no

F9-R: yes
Delay volume (UV – dispenser head) ÄKTA pure 25:

F9-R: 205 µl, 86 µl with optional tubing kit (i.d. 0.25 mm)
F9-C: 435 µl, 214 µl with optional tubing kit (i.d. 0.25 mm)
ÄKTA pure 150:
F9-R: 473 µl, 278 µl with optional tubing kit (i.d. 0.5 mm)
F9-C: 876 µl, 508 µl with optional tubing kit (i.d. 0.5 mm)
Dimensions (W x D x H) • F9-C: 390 x 585 x 320 mm

• F9-R: 320 x 400 x 250 mm
Weight • F9-C: 21 kg
• F9-R: 5 kg
I/O box
Parameter Data
Number of ports 2 analog in, 2 analog out

4 digital in, 4 digital out
Analog range In +/- 2 V

Out +/- 1 V

9.3 Tubing and connectors

Tubing types
The table below shows the tubing types used in ÄKTA pure.
Description Color Scope of use Volume/cm
PEEK, o.d. 1/16", i.d. 0.25 Blue High pressure tubing 0.5 μl
mm Reference capillary 1
Tubing Kit 0.25
PEEK, o.d. 1/16", i.d. 0.50 Orange High pressure tubing 2.0 μl
mm Tubing kit 0.5 (stan-
dard)
PEEK, o.d. 1/16", i.d. 0.75 Green High pressure tubing 4.4 μl
mm Tubing kit 0.75
PEEK, o.d. 1/16", i.d. 1.0 mm Beige High pressure tubing 7.8 μl
Tubing kit 1.0
FEP, o.d. 1/8", i.d. 1.6 mm Transparent Inlet tubing 20.0 μl
FEP, o.d. 3/16", i.d. 2.9 mm Transparent Inlet tubing for high 66.0 μl
flow rate and high vis-
cosity
ETFE, o.d. 1/16", i.d. 0.75 Transparent Narrow inlet tubing 4.4 μl
mm (optional)
ETFE, o.d. 1/16", i.d. 1.0 mm Transparent Outlet and waste tub- 7.8 μl
ing
Silicone, o.d. 12 mm, i.d. 8 Transparent Waste tubing from 0.3 ml

mm Buffer tray
Pump rinse solution

tubing
Note: • Many different sizes/types of tubing can be connected to a chromatography system. Tubing
with a smaller inner diameter (i.d.) holds less delay volume and will therefore generate less
dilution of the protein peak. Narrow tubing, however, increases the system pressure, espe-
cially when running at high flow rates. The tubing used should match the application needs.
See Section 9.4 Recommended tubing kits for prepacked columns, on page 428 for more
information.
• When using the high pressure tubing kit with i.d. 1.0 mm to allow high flow rates in combi-
nation with high viscosities in the pumps, inlet tubing with a larger i.d. than standard might
be needed to avoid outgassing.

Tubing connectors
The table below shows the tubing connectors used in ÄKTA pure.
Description Use with tubing...
Fingertight connector, 1/16" • PEEK, o.d. 1/16", i.d. 0.25 mm

• PEEK, o.d. 1/16", i.d. 0.50 mm
• PEEK, o.d. 1/16", i.d. 0.75 mm
• PEEK, o.d. 1/16", i.d. 1.0 mm
• ETFE, o.d. 1/16", i.d. 1.0 mm
Tubing connector 5/16" + ETFE, o.d. 1/16", i.d. 0.75 mm

Ferrule (blue), 1/16"
Tubing connector 5/16" + ferrule (blue) FEP o.d. 3/16" i.d. 2.9 mm
3/16"
Tubing connector 5/16" + FEP, o.d. 1/8", i.d. 1.6 mm

Ferrule (yellow), 1/8"
Other connectors
The table below shows other connectors used in ÄKTA pure.
Description Scope of use
Stop plug 1/16" Stop plug for valve ports
Luer female Syringe connector for pH valve and Injection valve

Tubing labels
The illustration below shows the tubing labels for for a typical system configuration.
1A1 1S2
4
1S1
InA 1A2 2B
3S
1B1
1B2 2A 2S
A2 B2 InS S1
A1 B1 3
W1 W2
InB
6 7 8 9 W
Out1 Frac
Inlet tubing
The table below shows the labels, standard diameters, and standard lengths of the inlet tubing.
Label Description Tubing Length Volume (ml)

(mm)
ÄKTA pure 25 ÄKTA pure 150 ÄKTA pure 25 ÄKTA pure 150
A1-A2 Inlets to Inlet valve FEP, o.d. 1/8", FEP, o.d. 3/16", 1500 3.0 9.9
and AB i.d. 1.6 mm i.d. 2.9 mm
B1-B2
A1-A7 Inlets to Inlet valve A FEP, o.d. 1/8", FEP, o.d. 3/16", 1500 3.0 9.9
i.d. 1.6 mm i.d. 2.9 mm
B1-B7 Inlets to Inlet valve B FEP, o.d. 1/8", FEP, o.d. 3/16", 1500 3.0 9.9
i.d. 1.6 mm i.d. 2.9 mm
InA From Inlet valve A or FEP, o.d. 1/8", FEP, o.d. 3/16", 300 0.6 2.0
Inlet valve AB to Sys- i.d. 1.6 mm i.d. 2.9 mm
tem pump A
InB From Inlet valve B or FEP, o.d. 1/8", FEP, o.d. 3/16", 300 0.6 2.0
Inlet valve AB to Sys- i.d. 1.6 mm i.d. 2.9 mm
tem pump B

Label Description Tubing Length Volume (ml)

(mm)
S1-S7 Inlets to Sample inlet FEP, o.d. 1/8", FEP, o.d. 3/16", 1000 2.0 6.6
valve V9-IS or V9H-IS i.d. 1.6 mm i.d. 2.9 mm
Buff Inlet to Sample inlet FEP, o.d. 1/8", FEP, o.d. 3/16", 1000 2.0 6.6
InS From Sample inlet FEP, o.d. 1/8", FEP, o.d. 3/16", 580 1.2 3.8
to Sample pump S9
or S9H, respectively
Note: Narrow inlet tubing, ETFE, o.d. 1/16", i.d. 0.75 mm, is available for S1-S7.
High pressure tubing

The tables below shows the labels, standard diameters, and standard lengths of the standard high
pressure tubing and the optional high pressure tubing.
Standard high pressure tubing
Label Description Tubing Length Volume (μl)

(mm)
1A1 System pump A left PEEK, o.d. PEEK, o.d. 340 150 270
to Restrictor A 1/16", i.d. 0.75 1/16", i.d. 1.0
mm mm
1A2 System pump A right PEEK, o.d. PEEK, o.d. 340 150 270
to Restrictor A 1/16", i.d. 0.75 1/16", i.d. 1.0
mm mm
2A Restrictor A to Pres- PEEK, o.d. PEEK, o.d. 115 50 90

sure monitor 1/16", i.d. 0.75 1/16", i.d. 1.0
mm mm
1B1 System pump B left PEEK, o.d. PEEK, o.d. 340 150 270
to Restrictor B 1/16", i.d. 0.75 1/16", i.d. 1.0
mm mm
1B2 System pump B right PEEK, o.d. PEEK, o.d. 340 150 270
to Restrictor B 1/16", i.d. 0.75 1/16", i.d. 1.0
mm mm


(mm)
2B Restrictor B to Pres- PEEK, o.d. PEEK, o.d. 115 50 90

sure monitor 1/16", i.d. 0.75 1/16", i.d. 1.0
mm mm
1S1 Sample pump left to PEEK, o.d. PEEK, o.d. 340 150 270
Restrictor S 1/16", i.d. 0.75 1/16", i.d. 1.0
mm mm
1S2 Sample pump right PEEK, o.d. PEEK, o.d. 340 150 270
to Restrictor S 1/16", i.d. 0.75 1/16", i.d. 1.0
mm mm
2S Restrictor S to Sam- PEEK, o.d. PEEK, o.d. 115 50 90

ple pressure monitor 1/16", i.d. 0.75 1/16", i.d. 1.0
mm mm
3S Sample pressure PEEK, o.d. PEEK, o.d. 530 230 420

monitor to injection 1/16", i.d. 0.75 1/16", i.d. 1.0
valve. mm mm
3 Pressure monitor to PEEK, o.d. PEEK, o.d. 400 180 310

Mixer 1/16", i.d. 0.75 1/16", i.d. 1.0
mm mm
4 Mixer to Injection PEEK, o.d. PEEK, o.d. 200 90 160

valve 1/16", i.d. 0.75 1/16", i.d. 1.0
mm mm
5 Injection valve to PEEK, o.d. PEEK, o.d. 160 30 70

Column valve 1/16", i.d. 0.50 1/16", i.d. 0.75
mm mm
6 Column valve to UV PEEK, o.d. PEEK, o.d. 160 30 70

monitor 1/16", i.d. 0.50 1/16", i.d. 0.75
mm mm
7 UV monitor to Con- PEEK, o.d. PEEK, o.d. 170 30 80

ductivity monitor 1/16", i.d. 0.50 1/16", i.d. 0.75
mm mm
8 Conductivity monitor PEEK, o.d. PEEK, o.d. 95 20 40

to Flow restrictor 1/16", i.d. 0.50 1/16", i.d. 0.75
mm mm
9 Flow restrictor to PEEK, o.d. PEEK, o.d. 135 30 60

Outlet valve 1/16", i.d. 0.50 1/16", i.d. 0.75
mm mm


(mm)
Frac Outlet valve to Frac- PEEK, o.d. PEEK, o.d. 400 180 310
tion collector F9-R 1/16", i.d. 0.50 1/16", i.d. 0.75
mm mm
Outlet valve to Frac- PEEK, o.d. PEEK, o.d. 350 70 160

tion collector F9-C 1/16", i.d. 0.50 1/16", i.d. 0.75
mm mm
Note: Tubing of different inner diameter can be used for tubing Frac and tubing 5 to 9. An inner di-
ameter (i.d.) of 0.5 mm is standard, but tubing kits of 0.25 mm i.d., 0.75 mm i.d. and 1.0 mm
i.d. are also available. Tubing 3S is not included in the kits, but should be changed to 1.0 mm
i.d. if the Tubing kit 1.0 is used.
Optional high pressure tubing

(mm)
3-1 Pressure monitor to PEEK, o.d. PEEK, o.d. 160 70 130

Mixer valve 1/16", i.d. 0.75 1/16", i.d. 1.0
mm mm
3-2 Mixer valve to Mixer PEEK, o.d. PEEK, o.d. 330 150 260
1/16", i.d. 0.75 1/16", i.d. 1.0
mm mm
3-3 Mixer valve to Injec- PEEK, o.d. PEEK, o.d. 260 120 200
tion valve 1/16", i.d. 0.75 1/16", i.d. 1.0
mm mm
8pH Conductivity monitor PEEK, o.d. PEEK, o.d. 180 40 80

to pH valve 1/16", i.d. 0.50 1/16", i.d. 0.75
mm mm
9pH pH valve to Outlet PEEK, o.d. PEEK, o.d. 160 30 70

valve 1/16", i.d. 0.50 1/16", i.d. 0.75
mm mm
L1 Injection valve port PEEK, o.d. PEEK, o.d. 160 30 70

LoopF to Loop valve 1/16", i.d. 0.50 1/16", i.d. 0.75
port F mm mm
L2 Injection valve port PEEK, o.d. PEEK, o.d. 160 30 70

LoopE to Loop valve 1/16", i.d. 0.50 1/16", i.d. 0.75
port E mm mm


(mm)
1R From pH flow cell to PEEK, o.d. PEEK, o.d. 80 20 40

flow restrictor 1/16", i.d. 0.50 1/16", i.d. 0.75
mm mm
2R From flow restrictor PEEK, o.d. PEEK, o.d. 80 20 40

tp pH flow cell 1/16", i.d. 0.50 1/16", i.d. 0.75
mm mm
Note: Tubing of different inner diameter can be used for tubing 8pH, 9pH, 1R and 2R. An inner diam-
eter (i.d.) of 0.5 mm is standard, but tubing kits of 0.25 mm i.d., 0.75 mm i.d. and 1.0 mm i.d.
are also available.
Reference capillary
The table below shows the label, diameter, and standard length of the reference capillary. The capillary
is used during the System performance tests.
Label Description Tubing Length Volume

(mm) (μl)
Ref 1 Reference capillary PEEK, o.d. 1/16", i.d. 0.25 mm 400 20
Outlet tubing
The table below shows the labels, diameters, and standard lengths of the outlet tubing. The tubing is
not mounted on delivery.

(mm) (ml)
Out Outlets from Outlet valve ETFE, o.d. 1/16", i.d. 1.0 mm 1500 1.2
V9-Os or V9H-Os
Out1 - Outlets from Outlet valve V9-O: ETFE, o.d. 1/16", i.d. 1.0 mm 1500 1.2
Out10 V9-O or V9H-O V9H-O: FEP o.d.1/8", i.d. 1.6 mm 3.0
Waste tubing
The table below shows the labels, diameters, and standard lengths of the waste tubing. The waste
tubing is mounted on delivery.

(mm) (ml)
W1 System pump waste ETFE, o.d. 1/16", i.d. 1.0 mm 1500 1.2


(mm) (ml)
W2 Sample pump waste ETFE, o.d. 1/16", i.d. 1.0 mm 1500 1.2
W3 pH valve waste ETFE, o.d. 1/16", i.d. 1.0 mm 1500 1.2
W System waste ETFE, o.d. 1/16", i.d. 1.0 mm 1500 1.2
N/A Top tray waste Silicone, o.d. 12 mm, i.d. 8 mm 1500 80
N/A Fraction collector F9-C Silicone, o.d. 12 mm, i.d. 8 mm 1350 70

waste

9.4 Recommended tubing kits for prepacked columns

This section specifies recommended tubing kits for prepacked columns used with ÄKTA pure.
Note: Where alternative tubing kits are listed for a given column, the Pressure control option may
be used instead of changing the tubing kit to adapt to different run conditions. Refer to Sec-
tion 9.15 Pressure control, on page 500 for details.
Column name Recommended Tubing Kits Comments
Standard sys- Advanced system 2

tem 1
All HiTrap 1 ml 0.5 0.5 with Pressure

(including HisTrap™, GSTrap™, control or 0.75
StrepTrap™)
All HiTrap 5 ml 0.5 or 0.75 0.75 0.75 with Pressure control for
(including HisTrap, GSTrap except maximum flow rate combined
4B, StrepTrap) with high viscosity.
GSTrap 4B, 5 ml 0.5 or 0.75 0.5 with Pressure

controlor 0.75
All HiScreen 0.5 or 0.75 0.5 with Pressure

control or 0.75
RESOURCE™ Q, S, ETH, ISO, PHE and 0.5 0.5

RPC 1 ml
RESOURCE RPC 3 ml 0.5 0.5
RESOURCE Q, S 6 ml 0.5 or 0.75 0.75 0.75 with Pressure control for

maximum flow rate combined
with high viscosity.
SOURCE™ 15RPC ST 4.6/100 0.5 0.5 Avoid combination of column

valve V9-C and pH position in
valve V9-pH.
Resolution may be improved with
0.25.
SOURCE 15Q. S, PHE 4.6/100 PE 0.5 0.5 Avoid combination of column

valve V9-C and pH position in
valve V9-pH.
0.25.
Mini Q™, Mini S™ 4.6/50 PE 0.25 or 0.5 0.25 or 0.5 Use 0.25 for maximum resolution.
Avoid column valve V9-C and pH
position in V9-pH.


tem 1
Mono Q™, Mono S™, Mono P™ HR 0.5 0.5 Avoid combination of column
5/5 valve V9-C or V9H-C and pH posi-
tion in valve V9-pH or V9H-pH.
0.25.
Mono P HR 5/20 0.5 0.5
Mono Q, S HR 10/10 0.5 0.5
Mono Q, Mono S HR 16/10 0.5 or 0.75 0.5 or 0.75
Superdex™ Peptide, 75, 200 0.5 0.5

Superose™ 6, 12 HR 10/30
Mono Q, Mono S 4.6/100 PE 0.5 0.5 Avoid combination of column

valve V9-C or V9H-C and pH posi-
0.25.
Mono Q, Mono S, Mono P 5/50 GL 0.5 0.5 Avoid combination of column

valve V9-C or V9H-C and pH posi-
0.25.
Superdex 200 5/150 GL 0.25 0.25 Avoid column valve V9-C and pH
position in V9-pH.
Mono P 5/200 GL 0.5 0.5
Mono Q, Mono S 10/100 GL 0.5 or 0.75 0.5 or 0.75
Superdex Peptide, 75, 200, Superose 0.5 0.5

6, 12 10/300 GL
All HiPrep™ 16/10 (including GSTPrep 0.5 or 0.75 0.75

FF, HisPrep FF)
HiPrep 16/60 Sephacryl™ S 100-500 0.5 or 0.75 0.5 with Pressure

HR control or 0.75
HiPrep 26/10 Desalting 0.5 with Pres- 0.75 with Pressure

HiPrep 26/10 Sepharose™ 6 FF sure Control or control or 1.0
0.75


tem 1
HiPrep 26/60 Sephacryl S 100-500 0.5 or 0.75 0.5 with Pressure

HR control or 0.75
HiLoad™ 16/10 Phenyl Sepharose 0.5 or 0.75 0.5 with Pressure

HP, Q Sepharose HP, SP Sepharose control or 0.75
HP
HiLoad 16/60 Superdex 30 pg, 75 pg, 0.5 or 0.75 0.5 with Pressure
200 pg control or 0.75
HiLoad 26/10 Phenyl Sepharose HP, 0.5 or 0.75 0.75

Q Sepharose HP, SP Sepharose HP
HiLoad 26/60 Superdex 30 pg, 75 pg, 0.5 or 0.75 0.5 with Pressure
200 pg control or 0.75
1 Standard system configuration.

Normal flow rates.
Moderate sample and buffer viscosities.
2 Complex system configuration (for additional tubing, use the same tubing dimension as the Tubing Kit).
Maximum flow rates.
High viscosities (≥3 cP).

9.5 Chemical resistance guide

Introduction
This section provides general information about biocompatibilty and detailed information about
chemical resistance of the ÄKTA pure instrument.
In this section
Section See page
9.5.1 General information about biocompatibility and chemical resistance 432
9.5.2 Chemical resistance specifications 433

9.5.1 General information about biocompatibility and chemical resistance
9.5.1 General information about biocompatibility and chemical resistance
Biocompatibility
The ÄKTA pure instrument is designed for maximum biocompatibility, with biochemically inert flow
paths constructed mainly from titanium, PEEK and highly resistant fluoropolymers and fluoroelastomers.
Titanium is used as far as possible to minimize contribution of potentially deactivating metal ions such
as iron, nickel and chromium. There is no standard stainless steel in the flow path. Plastics and rubber
materials are selected to avoid leakage of monomers, plasticizers or other additives.
Cleaning chemicals
Strong cleaning works well with 2 M sodium hydroxide, 70% acetic acid or the alcohols methanol,
ethanol and isopropyl alcohol. Complete system cleaning using 1 M hydrochloric acid should be
avoided in order to not damage the pressure sensors. If you are cleaning separation media using 1 M
hydrochloric acid, use loop injections of the acid and make sure that the column is not mounted on
the Column Valve V9-C. The Column Valve V9-C contains a pressure sensor which can be damaged
by 1 M hydrochloric acid.
If sodium hypochlorite is used as sanitizing agent instead of 2 M sodium hydroxide, use a concentration
up to 10%.
Organic solvents
Reversed phase chromatography of proteins works well with 100% acetonitrile and additives trifluo-
roacetic acid (TFA) up to 0.2% or formic acid up to 5%.
Strong organic solvents like ethyl acetate, 100% acetone or chlorinated organic solvents should be
avoided. These might cause swelling of plastic material and reduce the pressure tolerance of PEEK
tubing. For this reason, flash chromatography and straight (“normal”) phase chromatography is gen-
erally not recommended on the system
Assumptions made
The ratings are based on the following assumptions:
• Synergy effects of chemical mixtures have not been taken into account.
• Room temperature and limited overpressure is assumed.
Note: Chemical influences are time and pressure dependent. Unless otherwise stated, all concentra-
tions are 100%.

9.5.2 Chemical resistance specifications
Introduction
This section provides detailed information about chemical resistance of the ÄKTA pure instrument to
some of the most commonly used chemicals in liquid chromatography. Regarding exposure to solutions
not covered by this information, contact your GE representative for recommendations.
Note: A user can be exposed to large volumes of chemical substances over a long time period. Ma-
terial Safety Data Sheets (MSDS) provide the user with information regarding characteristics,
human and environmental risks and preventive measures. Make sure that you have the MSDS
available from your chemical distributor and/or databases on the internet.
Aqueous buffers
The specified aqueous buffers are suitable for continuous use.
Chemical Concentration CAS no/EC no
Aqueous buffers N/A N/A

pH 2-12
Strong chemicals and salts for CIP

The following chemicals are suitable for up to 2 h contact time at room temperature.
Acetic acid 70% 75-05-8/ 200-835-2
Decon 90 10% N/A
Ethanol 100% 75-08-1/ 200-837-3
Methanol 100% 67-56-1/ 200-659-6
Hydrochloric acid 1 0.1 M 7647-01-0/ 231-595-7
Isopropanol 100% 67-63-0/ 200-661-7
Sodium hydroxide 2M 1310-73-2/ 215-185-5
Sodium hydroxide/ethanol 1 M/40% N/A
Sodium chloride 4M 7647-14-5/ 231-598-3
Sodium hypochlorite 10% 7681-52-9/231-668-3
1 If hydrochloric acid, HCl, is used as a cleaning agent when columns are connected to the system, the HCl concen-
tration should not exceed 0.1 M in the pressure sensors. Remember that the ÄKTA pure system has pressure sensors
in the column valve V9-C.
For other parts of the system up to 1 M HCl is acceptable for short periods of use. See Cleaning chemicals, on page 432

Solubilization and denaturing agents

The following chemicals are suitable for continuous use, as additives in separation and purification
methods.
Guanidinium hydrochloride 6M 50-01-1/ 200-002-3
Sodium dodecyl sulfate (SDS) 1% 151-21-3/ 205-788-1
Tween™ 20 1% 9005-64-5/ 500-018-3
Urea 8M 57-13-6/ 200-315-5
Chemicals used in reversed phase

chromatography (RPC)
The following chemicals are suitable for continuous use.
Acetonitrile 1 100% 75-05-8/ 200-835-2
Acetonitrile/Tetrahydrofuran1 85%/15% 109-99-9/ 203-726-8
Acetonitrile/water/Trifluoroacetic Max 0.2% TFA N/A

acid (TFA) 2
Ethanol 100% 75-08-1/ 200-837-3
Isopropanol 100% 67-63-0/ 200-661-7
Methanol 100% 74-93-1/ 200-659-6
Water/organic mobile Max 5% formic N/A

phase/formic acid acid
1 Organic solvents can penetrate weaknesses in PEEK tubing walls more easily than water based buffers. Special
care should therefore be taken with prolonged use of organic solvents close to pressure limits.
Depending on pressure, tubing between pump head and pressure monitor needs to be changed. See Section 7.8.1
Replace tubing and connectors, on page 317 for more information.
2 Mobile phase system.
Note: It is recommended to replace the mixer sealing ring with the highly resistant O-ring (product
code 29-0113-26) if the system is to be exposed to organic solvents or high concentrations of
organic acids, such as acetic acid and formic acid, for a longer period of time.

Salts and additives for hydrophobic interaction

chromatography (HIC)
Ammonium chloride 2M 12125-02-9/ 235-186-4
Ammonium sulfate 3M 7783-20-2/ 231-984-1
Ethylene glycol 50% 107-21-1/ 203-473-3
Glycerol 50% 56-81-5/ 200-289-5
Reducing agents and other additives

Arginine 2M 74-79-3/ 200-811-1
Benzyl alcohol 2% 100-51-6/ 202-859-9
Dithioerythritol (DTE) 100 mM 3483-12-3 / 222-468-7
Dithiothreitol (DTT) 100 mM 3483-12-3 / 222-468-7
Ethylenediaminetetraacetic acid 100 mM 60-00-4/ 200-449-4

(EDTA)
Mercaptoethanol 20 mM 37482-11-4/ 253-523-3
Potassium chloride 4M 7447-40-7/ 231-211-8
Other substances
Acetone 10% 67-64-1/ 200-662-2
Ammonia 30% 7664-41-7/ 231-635-3
Dimethyl sulphoxide (DMSO) 5% 67-68-5/ 200-664-3
Ethanol for long-term storage 20% 75-08-1/ 200-837-3
Phosphoric acid 0.1 M 7664-38-2/ 231-633-2

9.6 Wetted materials

Material definitions
The tables below list the materials that come into contact with process fluids in the ÄKTA pure system.
Primary flow path: Material

Material Abbreviation
Ethylene ChloroTriFluoroEthylene ECTFE
Ethylene TetraFluoroEthylene ETFE
Fluorinated Ethylene Propylene FEP
Fluorinated Propylene Monomer FPM/FKM
Fully Fluorinated Propylene Monomer FFPM/FFKM
PolyChloroTriFluoroEthylene PCTFE
PolyEtherEtherKetone PEEK
PolyPropylene PP
PolyTetraFluoroEthylene PTFE
PolyVinylidene DiFluoride PVDF
UltraHighMolecularWeightPolyEthylene UHMWPE
Aluminum oxide
Elgiloy™
Hastelloy™ C-276
Quartz glass
Ruby
Sapphire
Titanium grade 2
Titanium grade 5

Pump rinse system: Material

Material Abbreviation
EthylenePropyleneDiene M-class rubber EPDM
PolyEtherEtherKetone PEEK
PolyPropylene PP
PolyPhenylene Sulfide PPS
PolyVinylidene DiFluoride PVDF
Silicone

9.7 Predefined methods and phases

Introduction
A predefined method contains a set of phases, each phase reflecting a specific stage of a chromatog-
raphy or maintenance run. You can select additional phases from the phase libraries and add these
to an existing method, or remove phases that are not required.
The predefined purification methods have default values with suitable running conditions for the
chosen column type such as flow and pressure limits. Other settings (e.g., sample application technique,
sample volume, elution profile and fractionation) are set on the Phase Properties pane in the appropriate
phases.
This section describes the predefined methods and phases.
A method is built up by a number of phases. Each phase represents a major process step in the method,
for example, equilibration or elution. Predefined methods, that include all the phases necessary to run
the system, are available for different chromatography techniques and also for system cleaning.
This sections contains the following subsections:
Section See page
9.7.1 Predefined purification methods 439
9.7.2 Predefined maintenance methods 445
9.7.3 Predefined phases 446

9.7.1 Predefined purification methods

The Method Editor has predefined methods for different separation techniques. The methods include
a number of relevant phases.
The table below describes the available predefined purification methods and which phases that are
included.
Predefined purification Principle Included phases

method
Affinity Chromatography (AC) After equilibration and sample application, the

protein of interest is adsorbed to the column
ligand. After a wash to remove unbound sample,
elution is performed either by using a buffer
containing a competitor to displace the protein
of interest, or by changing the pH or ionic
strength. Finally, the column is re-equilibrated
with start buffer.


method
Affinity Chromatography (AC) After equilibration, the sample of tagged protein

with Tag Removal is applied. After washing to remove unbound
sample the column is equilibrated with cleavage
buffer, a protease is applied and the flow is
stopped. The cleaved protein and the protease
are eluted by starting the flow. The next step
regenerates the column, eluting the tag, un-
cleaved protein and protease (if tagged). Finally,
the column is re-equilibrated.
Required components
The required components for this method are
Inlet valve (2-ports or 7-ports) and Mixer valve,
or the sample pump.
Required solutions
The method phases are pre-configured to use
the following solutions in the following inlets and
loop:
Inlet A1: Equilibration buffer
Inlet A2 or sample inlet: Sample
Inlet B1: Cleavage buffer
Inlet B2: Affinity Regeneration buffer
Loop: Protease
Note:
• Select Inject sample directly onto the col-
umn in the sample application phase.
• The protease application phase is preconfig-
ured to empty the loop with the protease with
a volume of 0.7 ml. The recommended pro-
tease volume is 0.7 CV (column volume). If a
column volume different than 1 ml is used,
this value needs to be changed to correspond
to 0.7 CV.


method
Anion Exchange Chromatogra- After equilibration and sample application, neg-

phy (AIEX) atively charged proteins are adsorbed to the
column ligand. After a wash, to remove unbound
sample, elution is performed using a gradient of
increasing salt concentration (of e.g. NaCl). Final-
ly, the column is washed and re-equilibrated
with start buffer.
Cation Exchange Chromatog- After equilibration and sample application, posi-

raphy (CIEX) tively charged proteins are adsorbed to the col-
umn ligand. After a wash, to remove unbound
sample, elution is performed using a gradient of
increasing salt concentration (of e.g. NaCl). Final-
ly, the column is washed and re-equilibrated
with start buffer.
Chromatofocusing (CF) After equilibration and sample application, elu-

tion is performed using a pH gradient. The pro-
teins separate and elute according to their iso-
electric points. Finally, the column is re-equilibrat-
ed.


method
Desalting After equilibration and sample application, the

proteins are eluted isocratically. This technique
is commonly used for buffer exchange.
Gel filtration (GF) After equilibration and sample application, pro-

teins separate and elute according to their size
(largest first).
Hydrophobic Interaction After equilibration and sample application (use

Chromatography (HIC) a buffer containing a high salt concentration,
for example 2 M ammonium sulfate) hydropho-
bic proteins are adsorbed to the column ligand.
After a wash to remove unbound sample, elution
is performed using a gradient of decreasing salt
concentration. Finally, the column is washed and
re-equilibrated with start buffer.
Manual Loop Fill The sample application loops of the loop valve
are manually filled with samples. The method
will guide the user through the process by
pausing and displaying on-screen instructions.
Up to five loops can be filled with different sam-
ples. The loops are filled in descending order.
Partial or complete loop fill can be chosen. Auto-
matic washing of loops and flow path are inte-
grated in the method.
The required components for this phase are a
column valve and a loop valve.


method
NHS-coupling A column packed with NHS-activated Sepharose

is washed with 1 mM HCl, followed by immediate
application of the protein for covalent coupling
onto the column. After incubation the non-bound
protein is washed out and the remaining active
groups are deactivated with ethanolamine
buffer, followed by further washes.
Required components
The required component for this method is an
Inlet valve (2-ports or 7-ports).
Required solutions
The method phases are preconfigured to use
the following solutions in the following inlets and
sample loop:
Inlet A1: Coupling buffer, for example 0.2 M
NaHCO3 + 0.5 M NaCl pH 8.3.
Inlet A2: Activation solution, for example 1 mM
HCl
Inlet B1: High pH buffer, for example 0.5 M
ethanolamine, 0.5 M NaCl pH 8.3
Inlet B2: Low pH buffer, for example 0.1 M Sodi-
um acetate, 0.5 M NaCl pH 4.0
Sample loop: Ligand in coupling buffer
Note:
The NHS coupling phase is preconfigured to
empty the loop with the ligand with a volume of
1 ml. The recommended ligand volume is 1 CV
(column volume). If a column volume different
than 1 ml is used, this value needs to be changed
to correspond to 1 CV.


method
Reversed Phase Chromatogra- After equilibration and sample application, hy-

phy (RPC) drophobic proteins adsorb to the column ligand.
After a wash to remove unbound sample, elution
is performed by generating a gradient of a non-
polar, organic solvent such as acetonitrile. Final-
ly, the column is washed and re-equilibrated.

9.7.2 Predefined maintenance methods
9.7.2 Predefined maintenance methods

A number of predefined methods for preparation and cleaning are available. These maintenance
methods are used to prepare the system, clean the system, and to fill the system with storage solution.
The table below describes the available predefined maintenance methods.
Predefined maintenance Principle Included phases

method
Column CIP The column is filled with a cleaning solution. Se-

lect inlet positions. Enter the solution identity,
volume, flow rate and incubation time. By adding
steps, several cleaning solutions can be used.
Suggestions for cleaning steps are available for
a number of column types.
Column Performance Test After equilibration of the column, sample is in-

jected via a loop and eluted isocratically. A non-
adsorbing sample like acetone or salt should be
used. After the run, calculate column perfor-
mance in the Evaluation module. The efficiency
of the column is determined in terms of height
equivalent to a theoretical plate (HETP), and the
peak asymmetry factor (As). The result is logged
in the column logbook.
Column Preparation The column is filled with buffer solution. Select

inlet positions. Enter the solution identity, vol-
ume, flow rate and incubation time. By adding
steps, several preparation solutions can be used.
System CIP The system is filled with cleaning solution. Select

for example inlets, outlets and column positions
to be cleaned. Three System CIP phases are in-
cluded in the method to facilitate the use of
three different cleaning solution. Additional
System CIP phases can be added from the
Phase Library if desired.
System Preparation The system is filled with preparation solution.

Select for example inlets, outlets and column
positions to be prepared. Two System Prepara-
tion phases are included in the method. Addition-
al System Preparation phases can be added
from the Phase Library if desired.

9.7.3 Predefined phases

The table below describes the predefined phases.
Phase Name Description
Method Settings The first, and mandatory, phase in any method. Defines common parameters
used in the subsequent phases.
The Method Settings phase defines:
1 Column type
Note:
The Column type list can be filtered in two steps:
• Select the chromatography technique to be used in the list Show by
technique
• Select Show only suggested columns to show the columns that are
suggested for the selected chromatography technique.
2 Pressure limit
3 Flow rate
4 Option to control the flow to avoid overpressure
Note:
Default values for pressure limits and flow rate are given for the selected column
type.
• Column position
• Flow restrictor use
• Unit selection for Method base and Flow rate
• Monitor settings:
- pH monitor
- Air sensor alarm settings
- UV monitor
Note:
The first wavelength of U9-M and the fixed wavelength for U9-L or U9-
L 2nd is always turned on. The second and third wavelengths for UV
monitor U9-M can be set on or off.
• Settings for Column Logbook
• Start Protocol
• Result name and location
Note:
Some of these options may not be required by certain methods.

Phase Name Description
Equilibration Equilibrates the column before purification, or re-equilibrates the column after
purification.
Sample Application Applies sample to the column. Defines the sample application technique, the
sample volume, and the handling of flowthrough.
Column Wash Washes out unbound sample after sample application or removes strongly
bound proteins after elution.
Elution Elutes the sample from the column. Defines parameters for the elution and
fractionation settings.
Column Prepara- Prepares the column before use by removing the storage solution and equili-
tion brating the column. By adding steps, several preparation solutions can be
used sequentially.
Column CIP Cleans the column after purification runs by rinsing the column with a
cleaning solution to remove nonspecifically bound proteins. By adding steps,
several cleaning solutions can be used sequentially.
System Prepara- Prepares the system before a run by removing storage solution and filling
tion the system and inlets with buffer solution. One preparation solution is used
per phase.
System CIP Cleans the system after purification runs by rinsing the system with a cleaning
solution. One cleaning solution is used per phase.
Column perfor- Tests the efficiency of a packed column in terms of height equivalent to a
mance test theoretical plate (HETP), and the peak asymmetry factor (As).
Manual Loop Fill Is used to manually fill the additional sample application loops mounted on
the loop valve. The filling options are:
• Partial loop fill
• Complete loop fill
Miscellaneous Can be added to any method at suitable places. The instructions can help
the user to better organize the graphical output of the results or introduce a
controlled delay in the method run.

9.8 System settings
9.8 System settings

Introduction
The System Settings function is used to set the parameters for the available instructions.
The Edit dialog in which to edit the system settings are shown below.
The following subsections list the system settings available for ÄKTA pure.
Section See page
9.8.1 System settings - UV 449
9.8.2 System settings - Conductivity 450
9.8.3 System settings - pH 451
9.8.4 System settings - Pressure alarms 452
9.8.5 System settings - Air sensor 454
9.8.6 System settings - I/O-box 456
9.8.7 System settings - Fraction collection 457
9.8.8 System settings - Tubing and Delay volumes 459
9.8.9 System settings - Wash settings 461
9.8.10 System settings - Watch parameters 462
9.8.11 System settings - Advanced 463
9.8.12 System settings - Data collection 464

9.8 System settings
9.8.1 System settings - UV
9.8.1 System settings - UV

The table below describes the UV related system settings available for ÄKTA pure.
Alarm UV Alarm UV enables or disables the alarm for the UV signal. When
enabled, it sets the alarm limits for the UV signal from UV monitor
U9-L. When the UV signal falls outside the set limits, an alarm will
be triggered and the method will be paused.
Alarm UV1 Alarm UV 1 enables or disables the alarm for the UV 1 signal from
UV monitor U9-M. When enabled, it sets the alarm limits for the
UV 1 signal. When the signal falls outside the set limits, an alarm
is issued and the method will be paused.
Note:
It is not possible to set an alarm signal for the UV 2 or UV 3 signals
from UV monitor U9-M.
Alarm UV 2nd Alarm UV 2nd enables or disables the alarm for the UV signal from
U9-L, 2nd. When enabled, it sets the alarm limits for the UV signal
from UV monitor U9-L, 2nd. When the signal falls outside the set
Wavelength Wavelength sets the wavelengths for UV monitor U9-M. The cor-
responding curves in the chromatogram are marked with the set
wavelengths.
Note:
• The instruction is available when UV monitor U9-M (variable) is
selected in the component list. For best performance, do not
use more wavelengths than necessary for the application.
• At low wavelengths, the eluent can have significant absorbance
of its own.
Noise reduction UV Noise reduction UV filters the noise in the UV signal from U9-M
or U9-L (depending on the configuration). A column-specific aver-
aging time is set automatically when a column is defined in a
method run and Averaging time is set as a variable.
Noise reduction UV 2nd Noise reduction UV 2nd filters the noise in the UV signal from U9-
L, 2nd. A column-specific averaging time is set automatically when
a column is defined in a method run and Averaging time is set
as a variable.

9.8 System settings
9.8.2 System settings - Conductivity
9.8.2 System settings - Conductivity

The table below describes the conductivity related system settings available for ÄKTA pure.
Alarm conductivity Alarm conductivity enables or disables the conductivity alarm.

When enabled, it sets the alarm limits for the conductivity signal.
When the conductivity falls outside the set limits, an alarm will be
triggered and the method will be paused.
Relative scale cond Relative scale cond facilitates monitoring of a gradient, for which
the user sets the conductivity values for 0% and 100%. The Rela-
tive scale cond can be set in ascending manner (0% for low and
100% for high conductivity) or in descending manner (0% for high
and 100% for low conductivity).
Note:
The Relative scale cond in descending manner is especially useful
for conductivity visualization in RPC and HIC, where the conductiv-
ity curve is reversed compared to the concentration curve (i.e., high
conductivity at 0% B and low conductivity at 100% B).
Cond temp condensation Cond temp compensation is used to adjust the conductivity values
to a reference temperature in order to compare conductivity val-
ues between runs that have been performed at different temper-
atures.
Setting the compensation factor to 0% turns this function off.

9.8 System settings
9.8.3 System settings - pH
9.8.3 System settings - pH

The table below describes the pH related system settings available for ÄKTA pure.
Alarm pH Alarm pH enables or disables the pH alarm. When enabled, it sets

the alarm limits for the pH signal. When the pH falls outside the
set limits, an alarm will be triggered and the method will be
paused.

9.8 System settings
9.8.4 System settings - Pressure alarms

The table below describes the pressure alarm related system settings available for ÄKTA pure.
Alarm system pressure Alarm system pressure sets the alarm limits for the system
pressure. When enabled and the system pressure falls outside
the set pressure limits, an alarm will be triggered and the method
will be paused. Default values for the alarm limits are set by the
values in the column list when a column is selected in the method
and Alarm system pressure is set as a variable. Low alarm is
only triggered if the pressure first exceeds the Low alarm limit for
ten seconds continuously and then falls below the Low alarm
limit.
Note:
Setting the Low alarm or the system flow rate to 0 deactivates the
low pressure alarm.
Alarm sample pressure Alarm sample pressure sets the alarm limits for the sample
pressure. When enabled and the sample pressure falls outside
and Alarm sample pressure is set as a variable. Low alarm is
limit.
Note:
low pressure alarm.
Alarm delta column pressure Alarm delta column pressure sets the alarm limits for the delta
column pressure (pre-column pressure minus post-column pres-
sure). When enabled and the delta column pressure falls outside
will be paused. Low alarm is only triggered if the pressure first
exceeds the Low alarm limit for ten seconds continuously then
falls below the Low alarm limit.
Note:
• Setting the Low alarm to 0 deactivates the low pressure alarm.
• Instruction Alarm delta column pressure is available only when
Column valve V9-C or V9H-C (5-columns) is selected in the
component list.

9.8 System settings
Alarm pre column pressure Alarm pre column pressure sets the alarm limits for the pre col-
umn pressure. When enabled and the pre column pressure falls
outside the set pressure limits, an alarm is issued and the method
and Alarm pre column pressure is set as a variable. Low alarm
is only triggered if the pressure first exceeds the Low alarm limit
for ten seconds continuously then falls below the Low alarm limit.
Note:
Setting the Low alarm to 0 deactivates the low pressure alarm.

9.8 System settings
9.8.5 System settings - Air sensor

The table below describes the air sensor related system settings available for ÄKTA pure.
Normal (30 µl) is used to detect when a buffer or sample vessel is empty. High (10 µl) is used to detect
small air bubbles
Alarm inlet A air sensor Alarm inlet A air sensor enables or disables the air sensor alarm
for the built-in air sensor at inlet A. If the alarm is enabled and air
is detected, an alarm will be triggered and the method will be
paused.
Alarm inlet B air sensor Alarm inlet B air sensor enables or disables the air sensor alarm
for the built-in air sensor at inlet B. If the alarm is enabled and air
paused.
Alarm inlet S air sensor Alarm inlet S air sensor enables or disables the air sensor alarm
for the built-in air sensor at the sample inlet. If the alarm is enabled
and air is detected, an alarm will be triggered and the method will
be paused.
Alarm external air sensor X Alarm external air sensor X enables or disables the alarm for the
optional air sensor number X. If the alarm is enabled and air is
detected, an alarm will be triggered and the method will be
paused.
Sensitivity inlet A air sensor Sensitivity inlet A air sensor is used together with the Alarm inlet
A air sensor instruction and sets the sensitivity of the built-in air
sensors at inlet A.
Normal (30 µl) is used to detect when a buffer or sample vessel
is empty. High (10 µl) is used to detect small air bubbles
Sensitivity inlet B air sensor Sensitivity inlet B air sensor is used together with the Alarm inlet
B air sensor instruction and sets the sensitivity of the built-in air
sensors at inlet B.
Sensitivity inlet S air sensor Sensitivity inlet S air sensor is used together with the Alarm inlet
S air sensor instruction and sets the sensitivity of the built-in air
sensors at the sample inlet.

9.8 System settings
Sensitivity external air sensor X Sensitivity external air sensor X is used together with the Alarm
external air sensor X instruction and sets the sensitivity of the
optional air sensor number X. The optional air sensor can be locat-
ed either before any of the inlets A or B or after the injection valve.
- When located before an inlet, the default sensitivity is Normal.
- When located after the injection valve, the default sensitivity is
High and the pump currently pumping onto the column is used
for calculating the air volume for the external air sensor.
Note:
Using an air sensor after the injection valve is only useful when
running at lower pressures. High pressure dissolves any small air
bubbles present.

9.8 System settings
9.8.6 System settings - I/O-box
9.8.6 System settings - I/O-box

The table below describes the I/O-box related system settings available for ÄKTA pure.
Digital out X Digital out X sets the value of the signal sent out by digital port
number X to either 0 or 1. The default value is 1.
Noise reduction analog in X Noise reduction analog in X filters the noise in the analog signal
in port number X.
Alarm analog in X Alarm analog in X enables or disables the alarm for the analog
signal in port number X. When enabled, it sets the alarm limits for
the analog signal. If the alarm is enabled and the analog signal
falls outside the set limits, an alarm will be triggered and the
method will be paused.
Alarm digital in X Alarm digital in X enables or disables the alarm for the signal in
digital port number X. The alarm can be triggered by either of the
signal values, 0 or 1. If the alarm is enabled and the condition set
in ‘Value’ occurs, an alarm will be triggered and the method will
be paused.
Configure analog out X Configure analog out X enables the user to send one of the pre-
defined signals (UV signal, conductivity, temperature, pH or con-
centration of eluent B) to the analog out port number X, and also
to set the range of that signal.

9.8 System settings
9.8.7 System settings - Fraction collection
The tables below describe the fraction collection

related system settings available for ÄKTA pure.
Fractionation settings Fractionation settings comprises fractionation mode and frac-

tionation order.
Fractionation mode (Automatic, Accumulator or DropSync).
Fractionation order (Row-by-row, Column-by-column, Serpen-
tine-row, Serpentine-column). For fractionation mode DropSync,
only the Serpentine option is available.
Last tube filled Last tube filled: Action when last tube is filled (pause, direct the
flow to one of outlets or direct the flow to waste.
Cassette configuration Cassette configuration: Automatic or Manual configuration.

If Automatic is selected, a Quick scan or a Full scan will be per-
formed when the door of the fraction collector is closed to deter-
mine which type of cassettes and plates are used. If Manual is
selected, used plates and tubes in each tray position are entered.
Fraction collector lamp Fraction collector lamp: Lamps in the fraction collector chamber
on or off.
Peak fractionation parameters The Peak fractionation parameters set the detection parameters
for peak collection, that is they decide when a peak starts and
ends. This information is used by the instructions Peak fractiona-
tion and Peak frac in outlet valve in order to start/end the peak
collection.
Fractionation settings Drop sync synchronises tube change to drop release. The available
settings are on or off. It is recommended to use Drop sync for
flow rates below 2 ml/min. Higher flow rates can however be used,
depending on the properties (for example viscosity) of the liquid.
Fractionation numbering mode Determines whether the fraction number is reset at the end of a
method or not.
Note:

9.8 System settings
Fractionation settings frac 2 Drop sync on or off. It is recommended to use this setting for flow
rates below 2 ml/min. Higher flow rates can be however be used,
depending on the properties (e.g. viscosity) of the liquid.
Fractionation numbering mode Determines whether fraction number for the second fraction col-
frac 2 lector is reset at the end of a method or not.
Note:

9.8 System settings
9.8.8 System settings - Tubing and Delay volumes

The table below describes the system settings related to tubing and delay volumes, available for ÄKTA
pure.
Tubing: Injection valve to col- The instruction is used in calculations of pre-column pressure
umn when Column valve V9-C or V9H-C (5-columns) is not mounted
onto the system. In such cases, there is no pre-column pressure
sensor present. The estimated pre-column pressure is either cal-
culated by using the measured system pressure and the length
of the tubing between the system pressure sensor and the column
valve or by using the measured sample pressure and the length
of the tubing between the sample pressure sensor and the column
valve depending on the position of the injection valve.
The instruction is available only when Column valve V9-C or V9H-
C (5-columns) is not selected in the component list.
Tubing: Sample pump to injec- The instruction is used in calculations of pre-column pressure
tion valve when Column valve V9-C or V9H-C (5-columns) is not mounted
onto the system. In such cases, there is no pre-column pressure
sensor present. The estimated pre-column pressure is either cal-
culated by using the measured system pressure and the length
of the tubing between the system pressure sensor and the column
valve or by using the measured sample pressure and the length
of the tubing between the sample pressure sensor and the column
valve depending on the position of the injection valve.
The instruction is available only when Column valve V9-C or V9H-
C (5-columns) is not selected and a sample pump (S9 or S9H) is
selected in the component list.
Delay volume: pH valve Delay volume: pH valve is used to calculate the delay volume
between the monitor and the Outlet valve. The instruction is used
to make sure that the collected fractions correspond to the frac-
tions indicated in the chromatogram. It is recommended not to
alter the default values for restrictor and pH cell delay volumes
when standard modules and standard tubing for flow restrictor
are used.
Delay volume: Monitor to outlet Delay volume: Monitor to outlet valve is used to define the delay
valve volume between the monitor and the Outlet valve. The instruction
is used to make sure that the collected fractions correspond to
the fractions indicated in the chromatogram. The delay volume
must be changed when changing tubing to another I.D. or length
or when removing or adding components.

9.8 System settings
Delay volume: Monitor to frac Delay volume: Monitor to frac is used to define the delay volume
between the monitor and the Fraction collector. The instruction
the fractions indicated in the chromatogram. The instruction is
available only when the Fraction collector is selected in the com-
ponent list.
Delay volume: Monitor to frac 2 Delay volume: Monitor to frac 2 is used to define the delay volume
between the monitor and the second Fraction collector. The in-
struction is used to make sure that the collected fractions corre-
spond to the fractions indicated in the chromatogram. The instruc-
tion is available only when the second Fraction collector is selected
in the component list.

9.8 System settings
9.8.9 System settings - Wash settings
9.8.9 System settings - Wash settings

The table below describes the wash related system settings available for ÄKTA pure.
System wash settings System wash settings sets the flow rate used for System wash.
Note:
• The volume for system wash is set in the System wash instruc-
tion.
• The flow rate should not exceed 10 ml/min if narrow inlet tubing
• Adjust the flow rate during the system wash so that the system
pressure does not exceed 2 MPa.
System pump wash settings System pump wash settings sets the flow rate and the wash
volume used during system pump washes.
Note:
Sample pump wash settings Sample pump wash settings sets the flow rate and the wash
volume used during sample pump washes.
Note:
Fraction collector wash settings Fraction collector wash settings sets the flow rate during Frac-
tion collector wash.
Mixer by-pass wash settings Mixer by-pass wash settings sets the flow rate used during Mixer
by-pass wash and defines wash volumes for mixer by-pass wash
options.
Note:
Loop wash settings Loop wash settings sets the flow rate used during Loop wash.
Note:
• The volume for system wash is set in the Loop wash instruction.

9.8 System settings
9.8.10 System settings - Watch parameters
9.8.10 System settings - Watch parameters

The table below describes the watch parameter settings available for ÄKTA pure.
Watch UV parameters Watch UV parameters sets the accepted signal fluctuation and
Delta peak limit of the UV signal for some of the tests in the Watch
and Hold until instructions.
Watch UV 2nd parameters Watch UV 2nd parameters sets the accepted signal fluctuation
and Delta peak limit of the UV 2nd signal for some of the tests in
the Watch and Hold until instructions.
Watch cond parameters Watch cond parameters sets the accepted fluctuation and Delta
peak limit of the conductivity signal for some of the tests in the
Watch and Hold until instructions.
Watch pH parameters Watch pH parameters sets the value for the accepted fluctuation
of the pH signal used for the test Stable signal in the instructions
Watch and Hold until.
Watch flow parameters Watch flow parameters sets the value for the accepted fluctua-
tion of the flow rate signal used for the test Stable signal in the
instructions Watch and Hold until with signal System flow.
Watch pressure parameters Watch pressure parameters sets the value for the accepted
fluctuation of the pressure signals used for the test Stable signal
in the instructions Watch and Hold until.
Watch analog in parameters Watch analog in parameters sets the accepted signal fluctuation
and Delta peak limit of the analog signal for some of the tests in

9.8 System settings
9.8.11 System settings - Advanced
9.8.11 System settings - Advanced

The table below describes the advanced system settings available for ÄKTA pure.
Power-save Power-save sets the instrument into power saving mode. When
the function is enabled, the instrument enters power-saving mode
after having been in state Ready for a certain time period. The
instrument turns into state Ready when a method run, a method
queue or a manual run ends. The time interval before the instru-
ment enters power-saving mode is defined by the user.
Instrument control panel Instrument control panel locks/unlocks the control panel located
on the front side of the instrument. When unlocked, the buttons
on the Instrument control panel are active and can be used to
control a few basic functions of the instrument. When the Instru-
ment control panel is locked, no functions are available.
Pressure control parameters By using Pressure control the method can be run with the set
flow rate without the risk of method stop due to pressure alarm.
Pressure control is enabled in the instruction System flow or
Sample flow. Pressure control parameters provides the P and I
factors used in the regulator and can be adjusted for different
columns.
Constant pressure flow param- Constant pressure flow parameters sets the values for the P and
eters I factors needed to keep a constant pressure by varying the flow
rate. The signal used for pressure control is set in the instruction
Constant pressure flow.
Max flow during valve turn Max flow during valve turn sets the maximum flow rate used
during the turning of the injection and outlet valve in order to
avoid high pressure alarms.
Method progressing flow Method progressing flow sets which flow (automatic, system
flow, sample flow) the progress of the method is calculated after.
In automatic mode, the position of the injection valve determines
if the system flow or the sample flow is used.
Sample pump setting Sample pump setting enables sample pump flow while the injec-
tion valve is in manual load position.

9.8 System settings
9.8.12 System settings - Data collection
9.8.12 System settings - Data collection

The table below describes the Data collection related system settings available for ÄKTA pure.
Data collection The Data collection settings determine the maximum number of
data points collected for a given curve. Data reduction occurs if
the maximum number of data points is exceeded. To avoid data
reduction, set the maximum number of data points to be collected
to 180000 or insert a New Chromatogram instruction in the
method.
Note:
The default setting is 54000 data points, which corresponds to 1.5
h for a signal of 10 Hz.

9.9 Manual instructions

It is possible to manually interact with an ongoing method using Manual instructions.
Step Action
1 In the System Control module:

• select Manual:Execute Manual Instructions
or
• use the shortcut Ctrl +M.

• Click the + symbol to show the instructions for the instruction group that you want to
modify.
• Select the instruction that you want to modify.
• Enter the new values for the instruction.
3 To execute several instructions at the same breakpoint, select and edit an instruction
and click Insert. Repeat for several instructions.
4 To update parameter fields during method run, check the Auto update... box.
5 To perform the instructions, click Execute.
All available manual instructions are described in the following subsections.
Section See page
9.9.1 Manual instructions - Pumps 466
9.9.2 Manual instructions - Flow path 470
9.9.3 Manual instructions - Monitors 472
9.9.4 Manual instructions - Fraction collection 473
9.9.5 Manual instructions - I/O-box 477
9.9.6 Manual instructions - Alarms 478
9.9.7 Manual instructions - Wash settings 481
9.9.8 Manual instructions - Watch parameters 482
9.9.9 Manual instructions - Advanced 483
9.9.10 Manual instructions - Other 486

9.9.1 Manual instructions - Pumps

The table below describes the pump related manual instructions available for ÄKTA pure.
System flow System flow defines the system flow rate.

Flow rate can be set either as volumetric or as linear flow. A col-
umn type must be selected before using linear flow.
Sample flow Sample flow defines the system flow rate. Flow rate can be set
either as volumetric or as linear flow. A column type must be se-
lected before using linear flow.
Gradient Gradient sets a gradient (linear or stepwise) using the system

pumps A and B.
Note:
Set gradient length value to 0 to perform a step gradient.
Pump A wash Pump A wash is used to change buffers in the inlet tubing, pump
and mixer.
Note:
• Pressing End during Pump wash will terminate both the wash
and the run immediately.
Pressing Continue during Pump wash will terminate the wash
and the run will continue from the point at which the pump
wash instruction was executed.
• An instruction issued when a Pump wash is in progress will not
be executed until the wash is completely finished and all valves
have turned back to the previous positions.
• Pump wash cannot be executed when the system is in state
HOLD.

Pump B wash Pump B wash is used to change buffers in the inlet tubing, pump
and mixer.
Note:
• Pressing End during Pump wash will terminate both the wash
Pressing Continue during Pump wash will terminate the wash
and the run will continue from the point at which the Pump
• An instruction issued when a Pump wash is in progress will not
• Pump wash cannot be executed when the system is in state
HOLD.
Sample pump wash Sample pump wash is used to change buffers in the sample inlet
tubing and sample pump.
Note:
• Pressing End during Sample pump wash will terminate both
the wash and the run immediately.
Pressing Continue during Sample pump wash will terminate
the wash and the run will continue from the point at which the
Sample pump wash instruction was executed.
• An instruction issued when a Sample pump wash is in progress
will not be executed until the wash is completely finished and
all valves have turned back to the previous positions.
• Sample pump wash cannot be executed when the system is
in state HOLD.
(I.D. 0.75 mm) is used.

Mixer by-pass wash Mixer by-pass wash is used to wash or fill the flow path (mixer
valve and tubing) that is used for sample application with system
pump. It is possible to choose a more extensive or a less extensive
wash option.
Note:
• Pressing End during Mixer by-pass wash will terminate both
the wash and the run immediately.
Pressing Continue during Mixer by-pass wash will terminate
the wash and the run will continue from the point at which the
Mixer by-pass wash instruction was executed.
• An instruction issued during a Mixer by-pass wash will not be
executed until the wash is completely finished and the valves
• Mixer by-pass wash cannot be executed when the system is
in state HOLD.
Loop wash Loop wash is used to wash the Loop valve. It is possible to wash
a single sample application loop, all loops or only the by-pass
position. Loop wash instruction is not available, if a Column valve
is not mounted on the system.
Note:
• Pressing End during Loop wash will terminate both the wash
Pressing Continue during Loop wash will terminate the wash
and the run will continue from the point at which the Loop wash
instruction was executed.
• An instruction issued when a Loop wash is in progress will not
be executed until the wash is completely finished and the valves
• Loop wash cannot be executed when the system is in state
HOLD.

System wash System wash is used to fill the system with the selected buffer
composition. The flow can be directed to the waste position of
either the injection valve or the outlet valve. The flow is directed
to the end of the flow path if outlet valve is not present.
Note:
• Pressing End during System wash will terminate both the wash
Pressing Continue during System wash will terminate the wash
and the run will continue from the point at which the System
• If System wash is performed during a Gradient operation, the
current component B concentration is maintained during the
wash.
• An instruction issued during a system wash operation cannot
• System wash cannot be executed when the system is in state
HOLD.

9.9.2 Manual instructions - Flow path

The table below describes the flow path related manual instructions available for ÄKTA pure.
Inlet A Inlet A turns Inlet valve A to the selected position.

Note:
Positions A1 - A7 are available, if Inlet valve V9-IA or V9H-IA (7-
ports) is used. Only positions A1 and A2 are available, if Inlet valve
V9-IAB or V9H-IAB (2-ports) is used.
Inlet B Inlet B turns the Inlet valve B to the selected position.

Note:
Positions B1 - B7 are available, if Inlet valve V9-IB or V9H-IB (7-
ports) is used. Only positions B1 and B2 are available, if Inlet valve
V9-IAB or V9H-IAB (2-ports) is used.
Sample inlet Sample inlet turns the Sample inlet valve to the selected position.
Injection valve Injection valve sets the Injection valve to the selected position.
The instruction gives an injection mark in the chromatogram when
the inlet valve switches to Inject or Direct Inject.
Note:
Sample flow refers to the flow that enters the injection valve via
SaP port.
Column position Column position turns the Column valve to the position specified
in the parameter Position.
pH valve pH valve sets the pH cell and the 0.2 MPa restrictor in positions
inline or offline.
The pH valve also has a calibration position. This position is only
available when performing calibration of the pH monitor (In Sys-
tem control select System:Calibrate).The calibration position can
also be used to fill the pH cell with storage solution since the pH
valve is in open position.
Note:
• It is not possible to turn the pH valve during any type of fraction-
ation as it affects the delay volume.
• The pH valve instruction can be given during the delay volume
of the different stop fractionation instructions, but it is executed
only after the set delay volume has been collected.
Outlet valve Outlet valve turns the Outlet valve to the selected position. The
instruction gives a mark in the chromatogram when the valve is
switched to the selected position.

Loop valve Loop valve turns the Loop valve to the selected position.
Mixer valve Mixer valve turns the Mixer valve to the selected position.
Note:
Setting Mixer valve in by-pass position makes it possible to use
System pump A for direct loading of the sample onto the column.
Versatile valve X Versatile valve X turns the Versatile valve number X to the selected
position.
Note:
Four sets of positions are available. In positions 1-3 and 2-4 only a
single flow channel can be used. In positions 1-4 & 2-3 and 1-2 &
3-4 the flow can be directed through two channels simultaneously.
Inlet valve (X1) Instruction Inlet valve X1 turns the extra valve to the selected
position. The extra valve is a basic 8 port valve, without air sensor,
to be used for general applications.
Inlet valve (X2) Instruction Inlet valve X2 turns the extra valve to the selected
position. The extra valve is a basic 8 port valve, without air sensor,
to be used for general applications.
Injection mark Injection mark sets an injection mark in the chromatogram at

the point where this instruction is executed.
Note:
The instruction is useful when the sample is loaded onto the column
by the system pump.

9.9.3 Manual instructions - Monitors
9.9.3 Manual instructions - Monitors

The table below describes the monitor related manual instructions available for ÄKTA pure.
Auto zero UV Auto zero UV sets the UV signals from U9-M or U9-L to 0 mAU.
Auto zero UV 2nd Auto zero UV 2nd sets the UV signal from UL-9, 2nd to 0 mAU.
Wavelength Wavelength sets the wavelengths for UV monitor U9-M. The cor-
responding curves in the chromatogram are marked with the set
wavelengths.
Note:
• The instruction is available when UV monitor U9-M (variable) is
selected in the component list. For best performance, do not
use more wavelengths than necessary for the application.
• At low wavelengths, the eluent can have absorbance of its own.
Noise reduction UV Noise reduction UV filters the noise in the UV signal from UV
monitor U9-M or U9-L. A column-specific averaging time is set
automatically when a column is defined in a method run and Av-
eraging time is set as a variable.
Noise reduction UV 2nd Noise reduction UV 2nd filters the noise in the UV monitor signal
from U9-L, 2nd. A column-specific averaging time is set automat-
ically when a column is defined in a method run and Averaging
time is set as a variable.
UV lamp Sets the UV lamp ON or OFF. Default is ON. The UV lamp is turned
ON when the system changes state to RUN, HOLD, or WASH.
UV lamp 2nd Sets the UV lamp 2nd ON or OFF. Default is ON. The UV lamp 2nd
is turned ON when the system changes state to RUN, HOLD, or
WASH.
Relative scale cond Relative scale cond facilitates monitoring of a gradient, for which
the user sets the conductivity values for 0% and 100%. The Rela-
tive scale cond can be set in ascending manner (0% for low and
100% for high conductivity) or in descending manner (0% for high
and 100% for low conductivity).
Note:
The Relative scale cond in descending manner is especially useful
for conductivity visualization in RPC and HIC, where the conductiv-
ity curve is reversed compared to the concentration curve (i.e., high
conductivity at 0% B and low conductivity at 100% B).

9.9.4 Manual instructions - Fraction collection

The table below describes the fraction collection related manual instructions available for ÄKTA pure.
Fractionation Fractionation is used when collecting fractions with a fraction

collector.
Stop fractionation Stop fractionation ends the fractionation after the set delay vol-
ume (specified in System Settings:Tubing and Delay volumes)
has been collected. The outlet valve is then turned to position
Waste.
Note:
If Stop fractionation is issued when both Fractionation and Peak
fractionation are active, fractionation is stopped after the set delay
volume has been collected. The outlet valve remains in position
Frac and peak fractionation continues.
Peak fractionation Peak fractionation enables collection of only those peaks that
fulfill the conditions set in the Peak fractionation parameters in-
struction.
Stop peak fractionation Stop peak fractionation ends the peak fractionation after the set
delay volume (specified in System Settings:Tubing and Delay
volumes) has been collected. The outlet valve is then turned to
position Waste.
Last tube filled Only for Fraction collector F9-C. Last tube filled sets the action
to perform after the fraction collector fills the last tube of the run:
pause the fractionation, direct the flow to one of the outlet ports
of the outlet valve or direct the flow to waste.
Reset frac number Only for Fraction collector F9-R. Sets fraction numbers to restart
from 1. The restart occurs when the instruction is issued. The in-
struction overrides the continuous numbering mode if Fractiona-
tion numbering mode is set to Continue in System Settings.
Feed tube Fraction collector F9-C: Feed tube moves the fractionation arm
to the position specified by the parameter Start position, after
the set delay volume has been collected. A fraction mark is given
in the chromatogram.
Fraction collector F9-R: Feed tube moves the tube rack forward
one tube after the set delay volume has been collected and a
fraction mark is set. When fractionation or peak fractionation is
not ongoing, Feed tube moves the rack instantly and no fraction
mark is set.

Fractionation numbering mode Only for Fraction collector F9-R. Fractionation numbering mode
determines whether the fraction number is reset at the end of a
method or not.
Note:
Fraction collector wash Only for Fraction collector F9-C. Fraction collector wash is used
to wash the fraction collector with the current solution present in
the system. The wash flow rate is set in the instruction Wash
settings:Fraction collector wash settings and the current inlet
positions are used. After the wash, the flow rate and the valve
positions automatically go back to their previous settings.
Note:
Fraction collector wash cannot be executed during any type of
fractionation.
Reset tube type Only for Fraction collector F9-C. Reset tube type resets all the
tube types in the fraction collector.
Note:
It is not allowed to execute the Reset tube type instruction during
fractionation.
Frac cleaning position Only for Fraction collector F9-C. Frac cleaning position enables
manual cleaning of the dispenser head. The system is paused and
the fractionation arm is moved to the middle front of the interior
of the fraction collector. It is then possible to open the door of the
fraction colelctor and manually clean the dispenser head.
Note:
The Frac cleaning position instruction cannot be executed during
fractionation.
Fraction collector lamp Only for Fraction collector F9-C. Fraction collector lamp turns the
light in the fraction collector on or off.
Cassette configuration Only for Fraction collector F9-C. Cassette configuration is set to
either automatic or manual:
• Automatic: the fraction collector automatically detects the
cassette types present in the fraction collector.
• Manual: The fraction collector content is manually set.
Fractionation frac 2 Fractionation frac 2 is used when collecting fractions with the
second Fraction collector.

Stop fractionation frac 2 Stop fractionation frac 2 ends the fractionation after the set delay
volume for the second Fraction collector (specified in System
Settings:Tubing and Delay volumes) has been collected. The
outlet valve is then turned to position Waste.
Note:
If Stop fractionation frac 2 is issued when both Fractionation
frac 2 and Peak fractionation frac 2 are active, fractionation is
stopped after the set delay volume has been collected. The Outlet
valve V9-O or V9H-O remains in position Outlet 10 / Frac 2 (or
Outlet 1 if Outlet valve V9-Os is mounted on the instrument) and
peak fractionation in the second Fraction collector continues.
Peak fractionation frac 2 Peak fractionation frac 2 enables collection of only those peaks
that fulfill the conditions set in the Peak fractionation parameters
instruction.
Stop peak fractionation frac 2 Stop peak fractionation frac 2 ends the peak fractionation in
second Fraction collector after the set delay volume (specified in
System Settings:Tubing and Delay volumes) has been collected.
The outlet valve is then turned to position Waste.
Reset frac number frac 2 Sets fraction numbers to restart from 1 for the second Fraction
collector. The restart occurs when the instruction is issued. The
instruction overrides the continuous numbering mode if Fraction-
ation numbering mode frac 2 is set to Continue in System Set-
tings.
Feed tube frac 2 During fractionation or peak fractionation the instruction Feed
tube frac 2 moves the second Fraction collector tube rack forward
one tube after the set delay volume has been collected and a
fraction mark is set. When fractionation or peak fractionation is
not ongoing, Feed tube frac 2 moves the rack instantly and no
fraction mark is set.
Fractionation in outlet valve Applicable if no fraction collector is used. Fractionation in outlet

valve enables fractionation via the outlet valve. When the set
fraction size/outlet has been collected, the outlet valve turns to
the next position. A fraction mark is set in the chromatogram for
each new outlet position. If using Outlet valve V9-Os or V9H-Os
(1-outlet), only one fraction can be collected via Outlet 1 position.
Stop frac in outlet valve Applicable if no fraction collector is used. Stop frac in outlet valve
ends the fractionation in outlet valve after the set delay volume
(specified in System Settings:Tubing and Delay volumes) has
been collected. The outlet valve is then turned to position Waste.

Peak frac in outlet valve Applicable if no fraction collector is used. Peak frac in outlet valve
enables collection of only those peaks that fulfill the conditions
set in Peak fractionation parameters. When the set fraction
size/outlet has been collected, the outlet valve turns to the next
position. A fraction mark is set in the chromatogram for each new
outlet position. If Outlet valve V9-Os or V9H-Os (1-outlet) is used,
only one peak can be collected via Outlet 1 position.
Stop peak frac in outlet valve Applicable if no fraction collector is used. Stop peak frac in outlet
valve ends the peak fractionation in outlet valve after the set delay
volume (specified in System Settings:Tubing and Delay volumes)
has been collected. The outlet valve is then turned to position
Waste.

9.9.5 Manual instructions - I/O-box
9.9.5 Manual instructions - I/O-box

The table below describes the I/O-box related manual instructions available for ÄKTA pure.
Auto zero analog in X Auto zero analog in X sets the value of the analog signal in the
analog port number X to 0 mV.
Reset auto zero analog in X Reset auto zero analog in X sets the signal in analog port number
X to its current value, i.e. the actual voltage in the analog port
number X.
Noise reduction analog in X Noise reduction analog in X filters the noise in the analog signal
in port number X.
Digital out X Digital out X sets the value of the signal sent out by digital port
number X to either 0 or 1. The default value is 1.
Pulse digital out X Pulse digital out X generates a pulsed signal in digital port number
X. The signal changes from the initial state (0 or 1) to the opposite
state and returns to the initial state after the defined length of
time.
Configure analog out X Configure analog out X enables the user to send one of the pre-
defined signals (UV signal, conductivity, temperature, pH or con-
centration of eluent B) to the analog out port number X, and also
to set the range of that signal.

9.9.6 Manual instructions - Alarms

The table below describes the alarm related manual instructions available for ÄKTA pure.
Alarm system pressure Alarm system pressure sets the alarm limits for the system
pressure. When enabled and the system pressure falls outside
and Alarm system pressure is set as a variable. Low alarm is
limit.
Note:
low pressure alarm.
Alarm sample pressure Alarm sample pressure sets the alarm limits for the sample
pressure. When enabled and the pressure falls outside the set
pressure limits, an alarm will be triggered and the method will be
paused. Low alarm is only triggered if the pressure first exceeds
the Low alarm limit for ten seconds continuously then falls below
the Low alarm limit.
Note:
Alarm delta column pressure Alarm delta column pressure sets the alarm limits for the delta
column pressure (pre-column pressure minus post-column pres-
sure). When enabled and the delta column pressure falls outside
will be paused. Low alarm is only triggered if the pressure first
exceeds the Low alarm limit for ten seconds continuously then
falls below the Low alarm limit.
Note:
• Setting the Low alarm to 0 deactivates the low pressure alarm.
• Instruction Alarm delta column pressure is available only when
Column valve V9-C or V9H-C (5-columns) is selected in the
component list.

Alarm pre column pressure Alarm pre column pressure sets the alarm limits for the pre col-
umn pressure. When enabled and the pre column pressure falls
outside the set pressure limits, an alarm is issued and the method
and Alarm pre column pressure is set as a variable. Low alarm
is only triggered if the pressure first exceeds the Low alarm limit
for ten seconds continuously then falls below the Low alarm limit.
Note:
Alarm UV Alarm UV enables or disables the alarm for the UV signal. When
enabled, it sets the alarm limits for the UV signal from UV monitor
U9-L. When the UV signal falls outside the set limits, an alarm will
be triggered and the method will be paused.
Alarm UV1 Alarm UV 1 enables or disables the alarm for the UV 1 signal from
UV monitor U9-M. When enabled, it sets the alarm limits for the
UV 1 signal from UV monitor U9-M. When the UV signal falls out-
side the set limits, an alarm is issued and the method will be
paused.
Note:
It is not possible to set an alarm signal for the UV 2 or UV 3 signals
from UV monitor U9-M.
Alarm UV 2nd Alarm UV 2nd enables or disables the alarm for the UV signal from
UV monitor U9-L, 2nd. When enabled, it sets the alarm limits for
the UV signal from U9-L, 2nd. When the UV signal falls outside the
paused.
Alarm conductivity Alarm conductivity enables or disables the conductivity alarm.

When enabled, it sets the alarm limits for the conductivity signal.
When the conductivity falls outside the set limits, an alarm will be
triggered and the method will be paused.
Alarm pH Alarm pH enables or disables the pH alarm. When enabled, it sets

the alarm limits for the pH signal. When the pH falls outside the
paused.
Alarm inlet A air sensor Alarm inlet A air sensor enables or disables the air sensor alarm
for the built-in air sensor at inlet A. If the alarm is enabled and air
paused.

Alarm inlet B air sensor Alarm inlet B air sensor enables or disables the air sensor alarm
for the built-in air sensor at inlet B. If the alarm is enabled and air
paused.
Alarm sample inlet air sensor Alarm sample inlet air sensor enables or disables the air sensor
alarm for the built-in air sensor at the sample inlet. If the alarm is
enabled and air is detected, an alarm will be triggered and the
Alarm external air sensor X Alarm external air sensor X enables or disables the alarm for the
optional air sensor number X. If the alarm is enabled and air is
detected, an alarm will be triggered and the method will be
paused.
Alarm analog in X Alarm analog in X enables or disables the alarm for the analog
signal in port number X. When enabled, it sets the alarm limits for
the analog signal. If the alarm is enabled and the analog signal
falls outside the set limits, an alarm will be triggered and the
Alarm digital in X Alarm digital in X enables or disables the alarm for the signal in
digital port number X. The alarm can be triggered by either of the
signal values, 0 or 1. If the alarm is enabled and the condition set
in ‘Value’ occurs, an alarm will be triggered and the method will
be paused.

9.9.7 Manual instructions - Wash settings
9.9.7 Manual instructions - Wash settings

The table below describes the wash related manual instructions available for ÄKTA pure.
System wash settings System wash settings sets the flow rate used for System wash.
Note:
• The volume for system wash is set in the System wash instruc-
tion.
System pump wash settings System pump wash settings sets the flow rate and wash volume
used during system pump washes.
Note:
Sample pump wash settings Sample pump wash settings sets the flow rate and wash volume
used during sample pump washes.
Fraction collector wash settings Fraction collector wash settings sets the flow rate during Frac-
tion collector wash.
Mixer by-pass wash settings Mixer by-pass wash settings sets the flow rate used during Mixer
by-pass wash and defines wash volumes for mixer by-pass wash
options.
Note:
Loop wash settings Loop wash settings sets the flow rate used during Loop wash.
Note:
• The volume for system wash is set in the Loop wash instruction.

9.9.8 Manual instructions - Watch parameters
9.9.8 Manual instructions - Watch parameters

The table below describes the watch parameter instructions available for ÄKTA pure.
Watch UV parameters Watch UV parameters sets the accepted signal fluctuation and
Delta peak limit of the UV signal for some of the tests in the Watch
and Hold until instructions.
Watch UV 2nd parameters Watch UV 2nd parameters sets the accepted signal fluctuation
and Delta peak limit of the UV 2nd signal for some of the tests in
Watch cond parameters Watch cond parameters sets the accepted fluctuation and Delta
peak limit of the conductivity signal for some of the tests in the
Watch and Hold until instructions.
Watch pH parameters Watch pH parameters sets the value for the accepted fluctuation
of the pH signal used for the test Stable signal in the instructions
Watch and Hold until.
Watch flow parameters Watch flow parameters sets the value for the accepted fluctua-
tion of the flow rate signal used for the test Stable signal in the
instructions Watch and Hold until with signal System flow.
Watch pressure parameters Watch pressure parameters sets the value for the accepted
fluctuation of the pressure signals used for the test Stable signal
in the instructions Watch and Hold until.
Watch analog in parameters Watch analog in parameters sets the accepted signal fluctuation
and Delta peak limit of the analog signal for some of the tests in

9.9.9 Manual instructions - Advanced

The table below describes the advanced manual instructions available for ÄKTA pure.
Pressure control parameters By using Pressure control the method can be run with the set
flow rate without the risk of method stop due to pressure alarm.
Pressure control is enabled in the instruction System flow or
Sample flow. Pressure control parameters provides the P and I
factors used in the regulator and can be adjusted for different
columns.
Pressure control min flow rate can be set either as volumetric or
as linear flow. A column type must be selected before using linear
flow.
Constant pressure flow Constant pressure flow enables column packing at constant
pressure. The system pump automatically adjusts the flow rate
within the specified Minimum allowed flow rate – Maximum al-
lowed flow rate range. The goal is to reach and keep the set
Pressure at the selected Pressure sensor using the P and I factors
set in the Constant pressure flow parameters instruction. The
total volume is continuously updated using the actual flow rate.
Both pressure control flow rates can be set either as volumetric
or as linear flow. A column type must be selected before using
linear flow.
Note:
• When Constant pressure flow is used, the P and I factors set
in the Constant pressure flow parameters instruction are used
to control the pressure, instead of the P and I values set in the
• Pressure sensor Delta column pressure is available only when
column valve V9-C or V9H-C (5-columns) is selected in the
component list.
Constant pressure flow param- Constant pressure flow parameters sets the values for the P and
eters I factors needed to keep a constant pressure by varying the flow
rate. The signal used for pressure control is set in the instruction
Constant pressure flow.

Column packing flow Column packing flow is used to set flow rates over 25 ml/min and
150 ml/min for ÄKTA pure 25 and ÄKTA pure 150, respectively.
Both A and B pumps are used to generate the flow, making it
possible to set flow rates up to 50 ml/min and 300 ml/min for
ÄKTA pure 25 and ÄKTA pure 150, respectively. Flow rate can be
set either as volumetric or as linear flow. A column type must be
selected before using linear flow.
Before executing the Column packing flow instruction it is impor-
tant to:
• Immerse inlet tubing A1 and B1 in the same buffer
• Disconnect the column outlet tubing from the Column valve
and place the tubing in a waste vessel
Note:
When running Column packing flow only isocratic runs can be
performed, gradients cannot be generated.
Delay volume: Monitor to outlet Delay volume: Monitor to outlet valve is used to define the delay
valve volume between the monitor and the Outlet valve. The instruction
the fractions indicated in the chromatogram. The delay volume
must be changed when changing tubing to another i.d. or length
or when removing or adding components.
Delay volume: Monitor to frac Delay volume: Monitor to frac is used to define the delay volume
between the monitor and the Fraction collector. The instruction
the fractions indicated in the chromatogram. The instruction is
available only when the Fraction collector is selected in the com-
ponent list.
Delay volume: Monitor to frac 2 Delay volume: Monitor to frac 2 is used to define the delay volume
between the monitor and the second Fraction collector. The in-
struction is used to make sure that the collected fractions corre-
spond to the fractions indicated in the chromatogram. The instruc-
tion is available only when the second Fraction collector is selected
in the component list.
Start volume count Start volume count starts the volume counter function. The
counted volume is saved into a memory.
This instruction is best used in combination with Watch instruc-
tions.

Stop volume count Stop volume count stops the volume counter function. The
counted volume is stored in the memory and can be recalled with
the instruction Hold counted volume. The counted volume can
also be recalled in following runs and is stored until a new Stop
volume count instruction is issued.
This instruction is best used in combination with Watch instruc-
tions.
Hold counted volume Hold counted volume sets the system to Hold. The system will
remain in the state Hold until the accumulated volume reaches
the volume stored by the instructions Start volume count / Stop
volume count.
Method progressing flow Method progressing flow

defines the flow from which the volume base is calculated. When
set to Automatic, the position of the injection valve determines if
the system flow or the sample flow is used.

9.9.10 Manual instructions - Other
9.9.10 Manual instructions - Other

The table below describes the other manual instructions available for ÄKTA pure.
Set mark Set mark inserts a mark into the current chromatogram with the
text entered for the parameter Mark text .
Timer Timer sets the system to pause or end after a set volume or time
has passed. Select base sets the base to either accumulated time
or accumulated volume. Timeout sets the volume or time. Action
sets the action to perform (pause or end)

9.10 Available Run data

The table below lists all available Run data for ÄKTA pure.
Run Data Range/Unit Description
System state N/A Status of connection and run.
Acc. Volume ml Total accumulated volume in the current

method or manual run.
Block volume ml Accumulated volume in the current block

(method run only).
Acc. Time min Total accumulated time in the current method

or manual run.
Block time min Accumulated time in the current block

(method run only).
Scouting no N/A The current scouting number in the scouting

scheme.
System flow 0.001 – 50.000 ml/min The set flow rate of the system pumps.
(ÄKTA pure 25)
0.01 – 300.00 ml/min (ÄKTA
pure 150)
System linear flow cm/h The set flow velocity of the system pumps.
Only available if a column is selected.
Sample flow 0.001 – 50.000 ml/min The set flow rate of the sample pump.
(ÄKTA pure 25)
0.01 – 300.00 ml/min (ÄKTA
pure 150)
Sample linear flow cm/h The set flow velocity of the sample pump.
Only available if a column is selected.
Inlet A A1 - A7 The set position of the inlet valve A.
Inlet B B1 - B7 The set position of the inlet valve B.
Inlet S S1 - S7, buff The set position of the sample inlet valve.
Conc B 0.0 – 100.0 %B The set concentration B or the current value

during a gradient.
Mixer valve N/A The set position of the Mixer valve.
Injection N/A The set position of the Injection valve.
Loop position N/A The set position of the Loop valve.

Column position N/A The set position of the Column valve.
Column flow direction N/A The set flow direction position of the Column
valve V9-C, Column valve V9H-C, Column
valve V9-Cs and V9H-Cs.
System pressure -1.00 – 20.00 MPa (ÄKTA The system pressure signal (at the system
pure 25) pumps).
-1.00 – 5.00 MPa (ÄKTA
pure 150)
Sample pressure -1.00 – 10.00 MPa (ÄKTA The sample pressure signal (at the sample
pure 25) pump).
-1.00 – 5.00 MPa (ÄKTA
pure 150)
PreC pressure -1.00 – 20.00 MPa (ÄKTA The pre-column pressure signal.
pure 25)
-1.00 – 5.00 MPa (ÄKTA
pure 150)
DeltaC pressure -1.00 – 20.00 MPa (ÄKTA The delta-column pressure signal.
pure 25)
-1.00 – 5.00 MPa (ÄKTA
pure 150)
PostC pressure -1.00 – 20.00 MPa (ÄKTA The post-column pressure signal.
pure 25)
-1.00 – 5.00 MPa (ÄKTA
pure 150)
UV -6000.000 – 6000.000 mAu The UV absorbance signal of the U9-L monitor.
UV 1 -6000.000 – 6000.000 mAU The first UV/Vis absorbance signal of the U9-
M monitor.
UV 2 -6000.000 – 6000.000 mAU The 2nd UV/Vis absorbance signal of the U9-
M monitor.
UV 3 -6000.000 – 6000.000 mAU The 3rd UV/Vis absorbance signal of the U9-
M monitor.
UV 2nd -6000.000 – 6000.000 mAU The UV absorbance signal of the second U9-
L monitor.
Cond 0.00 – 999.99 mS/cm The conductivity signal.
% Cond 0.0 – 100.0 % The conductivity signal as a percentage of a

set range.

Cond temp 0.0 – 99.0 °C The temperature signal (in the conductivity
flow cell).
pH 0.00 – 14.00 The pH signal.
pH valve N/A The set position of the pH valve.
Outlet N/A The set position of the outlet valve.
Frac position N/A The current tube position of the fraction col-
lector.
Frac 2 position N/A The current tube position of the fraction col-
lector 2.
Inlet valve X1 N/A The set position of the X1 valve.
Inlet valve X2 N/A The set position of the X2 valve.
Versatile valve N/A The set position of the versatile valve.
Versatile valve 2 N/A The set position of the versatile valve 2.
Air inlet A No air, Air The current state of the air alarm for the inte-
grated air sensor in inlet valve A.
Air inlet B No air, Air The current state of the air alarm for the inte-
grated air sensor in inlet valve B.
Air inlet S No air, Air The current state of the air alarm for the inte-
grated air sensor in inlet valve IS.
Ext. air sensor, to No air, Air The current state of the air alarm for the ex-
Ext. air sensor 4 ternal air sensors.
Analog in 1, to -2000.0 – 2000.0 mV The I/O-box analog input signals.

Analog in 4
Digital in 1, to 0, 1 The I/O-box digital input signals.

Digital in 8
Digital out 1, to 0, 1 The set value of the I/O-box digital output

Digital out 8 signals.

9.11 Available Curves

The table below lists all available Curves for ÄKTA pure.
Curve Range Sampling Description

frequency
UV 1 -6000.000 – 6000.000 mAU 10 Hz The first UV/Vis absorbance signal

of the U9-M monitor.
UV 2 -6000.000 – 6000.000 mAU 2 Hz The 2nd UV/Vis absorbance signal

UV 3 -6000.000 – 6000.000 mAU 2 Hz The 3rd UV/Vis absorbance signal

Cond 0.00 – 999.99 mS/cm 5 Hz The conductivity signal.
% Cond 0.0 – 100.0 % 1 Hz The conductivity signal as a percent-

age of a set range.
Conc B 0.0 – 100.0 % 1 Hz The set concentration B or the cur-

rent value during a gradient.
System flow 0.001 – 50.000 ml/min (ÄKTA 1 Hz The set flow rate of the system
pure 25) pumps.
0.01 – 300.00 ml/min (ÄKTA
pure 150)
System linear cm/h 1 Hz The set flow velocity of the system

flow pumps. Only available if a column
is selected.
Sample flow 0.001 – 50.000 ml/min (ÄKTA 1 Hz The set flow rate of the sample
pure 25) pump.
0.01 – 300.00 ml/min (ÄKTA
pure 150)
Sample linear cm/h 1 Hz The set flow velocity of the sample

flow pump. Only available if a column is
selected.
PreC pressure -1.00 – 20.00 MPa (ÄKTA 1 Hz The pre-column pressure signal.
pure 25)
-1.00 – 5.00 MPa (ÄKTA pure
150)
DeltaC pressure -1.00 – 20.00 MPa (ÄKTA 1 Hz The delta-column pressure signal.
pure 25)
150)

Curve Range Sampling Description

frequency
pH 0.00 – 14.00 1 Hz The pH signal.
UV -6000.000 – 6000.000 mAU 10 Hz The UV absorbance signal of the

U9-L monitor.
UV 2nd -6000.000 – 6000.000 mAU 10 Hz The UV absorbance signal of the

second U9-L monitor.
System pressure -1.00 – 20.00 MPa (ÄKTA 10 Hz The system pressure signal (at the
pure 25) system pumps).
150)
Sample pressure -1.00 – 10.00 MPa (ÄKTA 10 Hz The sample pressure signal (at the
pure 25) sample pump).
150)
PostC pressure -1.00 – 20.00 MPa (ÄKTA 1 Hz The post-column pressure signal.
pure 25)
150)
Cond temp 0.0 – 99.0 °C 0.5 Hz The temperature signal (in the con-
ductivity flow cell).
Analog in 1, to -2000.0 – 2000.0 mV 10 Hz The I/O-box analog input signals.

Analog in 4
UV cell path 0.2, 0.5 cm 1 Hz The nominal cell path length of the
length U9-L monitor.
UV cell path 0.05, 0.2, 1.0 cm 1 Hz The nominal cell path length of the
length U9-M monitor.
UV cell path 0.2, 0.5 cm 1 Hz The nominal cell path length of the
length 2nd 2nd U9-L monitor.
Digital in 1, to 0, 1 10 Hz The I/O-box digital input signals.

Digital in 8
Digital out 1, to 0, 1 10 Hz The I/O-box digital output signals.

Digital out 8

9.12 Injection volumes and peak broadening
9.12 Injection volumes and peak broadening

The width of peaks at the fraction collector is influenced by the following:
• the properties of the column,
• the dimensions of the tubing,
• the dimensions of the modules in the flow path, and
• fluid dynamics.
Initial sample volume affects the peak width in gel filtration (GF) chromatography and other isocratic
techniques. A sample zone is broadened during passage through a GF column so that the sample is
diluted and the resolution decreases with increasing sample volume. Sample volume does not however
affect the resolution in adsorption chromatography techniques such as affinity chromatography (AC),
ion exchange chromatography (IEX), and hydrophobic interaction chromatography (HIC) if the retention
factor k is high.
The effect of peak broadening in the system from sample injection to peak detection (including dilution
on the column) is apparent in the chromatogram from the UV monitor, but broadening from the UV
monitor to fraction collection is not visible in the chromatogram. This "hidden" effect is more pronounced
for smaller peak volumes.
Narrow and short tubing reduces peak broadening. However, narrow tubing also increases the back
pressure. See for recommendations on tubing dimensions for prepacked columns.

9.13 Delay volumes
9.13 Delay volumes

Introduction
A number of methods, both theoretical and experimental, exist for determining the delay volume of a
system. The easiest and recommended method is to perform a theoretical determination. Delay volumes
for standard configurations are listed in Standard delay volumes, on page 495.
Explanation of delay volume

The delay volume is the volume between the detector and the fraction collector or outlet that is used.
The illustration below shows and example of the delay volume between the UV/Vis monitor and the
fraction collector.
Valve
UV/Vis
Conductivity pH Fraction collector
Delay volume
The time when a peak is detected The peak reaches the fraction
by the UV/Vis detector is called T0 collector at time T1
Theoretical determination of delay volumes

A theoretical determination is performed as described in the steps below:
Step Action
1 Identify all components in the system flow path that contribute to the delay volume of
interest.
2 Determine the internal volumes of all hardware modules and tubing, see Section 9.14
Component volumes, on page 498 for information about theoretical module volumes and
Section 9.3 Tubing and connectors, on page 420 for information about tubing lengths and
dimensions.
3 To obtain the total delay volume, sum up half of the flow cell volume of the monitor used
(that is, the UV or UV/Vis monitor) with all volumes of tubing and modules that are located
after the monitor in the flow path.
Note:
For pH-valve V9-pH and V9H-pH always use the volume for the valve in bypass position.
The system automatically adds the volumes for the flow restrictor and the pH flow cell
when if they are part of the system.

9.13 Delay volumes
Set the delay volume in UNICORN

Follow the instructions below to set the delay volume between the UV monitor and the Outlet valve
and between the UV monitor and the Fraction collector.
Step Action

2 • Select Tubing and Delay Volumes and select Delay volume: Monitor to outlet valve.
• Type in the volume in the Volume field and click OK.

• Select Delay volume: Monitor to outlet valve or Monitor to frac according to the
configuration you are using.
• Type in the volume in the Volume field and click OK.
Note:
The system will use the delay volume appropriate to the configuration used and ignore
other settings (e.g. the value for Monitor to outlet valve will be ignored if you are using a
fraction collector). It is however recommended to set all delay volumes so that the volumes
remain correct if you change fractionation method.

9.13 Delay volumes
Standard delay volumes

The table below lists the delay volumes for standard configurations using the available tubing kits with
and without a pH valve and using tubing of standard length from the outlet valve to the fraction col-
lector. The values are based on the flow cells for UV monitor U9-M. When using U9-L, add 9 µl for the
2 mm flow cell and 4 µl for the 5 mm flow cell. The values for delay volume Monitor to frac F9-C assume
that the internal tubing of Fraction collector F9-C has the same inner diameter as the tubing kit.
Delay volumes ÄKTA pure 25
Tubing kit pH Delay vol- Delay volume UV monitor to point of collection of eluent
valve ume pH
V9-pH valve re- Delay volume Delay volume Delay volume
strictor Monitor to outlet Monitor to frac Monitor to frac
valve F9-R F9-C
0.25 No N/A 66 µl 86 µl 214 µl
0.25 Yes 25 µl 77 µl 96 µl 225 µl
0.5 (stan- No N/A 125 µl 205 µl 435 µl

dard)
0.5 (stan- Yes 48 µl 152 µl 231 µl 462 µl

dard)
0.75 No N/A 223 µl 400 µl 803 µl
0.75 Yes 88 µl 277 µl 454 µl 857 µl
1.0 No N/A 361 µl 675 µl 1319 µl
1.0 Yes 143 µl 452 µl 766 µl 1410 µl
Delay volumes ÄKTA pure 150
Tubing kit pH Delay vol- Delay volume UV monitor to point of collection of eluent
valve ume pH
V9H-pH valve re- Delay volume Delay volume Delay volume
strictor Monitor to outlet Monitor to frac Monitor to frac
valve F9-R F9-C
0.5 No N/A 198 µl 278 µl 508 µl
0.5 Yes 60 µl 245 µl 324 µl 555 µl
0.75 (stan- No N/A 296 µl 473 µl 876 µl

dard)
0.75 (stan- Yes 100 µl 370 µl 547 µl 950 µl

dard)
1.0 No N/A 434 µl 748 µl 1392 µl
1.0 Yes 155 µl 545 µl 859 µl 1503 µl

9.13 Delay volumes
Note: • Always set the delay volume from the UV monitor to the point of collection of eluent. De-
pending on the system configuration, this will be Delay volume: Monitor to outlet valve
or Delay volume: Monitor to frac in the system settings.
• If the pH valve is mounted, make sure to set the value for the delay volume of the pH valve
restrictor. The system automatically adds the volumes for the flow restrictor and the pH
flow cell if they are part of the flow path.
Experimental method for determination of delay

volumes
Delay volumes can be determined experimentally for Fraction collector F9-R, but not for Fraction col-
lector F9-C. Instructions for measuring the delay volume using the UV monitor U9-M or UV monitor
U9-L are provided below.
Two volumes are used to perform experimental delay volume measurements:
• V1: the volume between the Injection valve and the UV monitor used
• V2: the volume between the Injection valve and the fractionation collector tubing tip
The delay volume is then measured using the following procedure.
Step Action
1 Check that a flow rate of 1 ml/min is delivered by the pump.

Note:
If the flow rate is not correct, check if air bubbles may have entered the pump. Make sure
to correct any problems before continuing.
2 Fill a small sample loop, for example 20 μl, with a 1% to 5% acetone solution.
3 Set all components between the monitor and the fraction collector to by-pass mode.
4 Fill the system with water. Run the pump at a flow rate of 1 ml/min and inject the acetone
solution as a sample.
5 Note the volume from the point of injection to the maximum of the peak in the chro-
matogram. This volume is volume measurement V1.
6 Reconfigure the system as follows to include the fraction collector in the flow path before
the monitor:
• Disconnect the two tubing segments from the UV monitor flow cell and connect these
with a low-dead-volume connector, a 1/16'' female to a 1/16'' female union connector.
• Mount the fractionation collector tubing tip on the top of the UV monitor flow cell.
Connect tubing to the bottom of the UV monitor flow cell and lead this tubing to waste.
It is preferable if the waste tubing gives some back pressure as the flow restrictor
now is placed before the UV flow cell.
7 Set the Frac size to a large volume, for example 100 ml, so that the Outlet valve is in the
Frac position during the entire run.

9.13 Delay volumes
Step Action
8 Start the pump at a flow rate of 1 ml/min and inject the acetone solution.
9 Note the volume from the point of injection to the appearance of the peak in the chro-
matogram. This volume is volume measurement V2.
10 To obtain the delay volume:

• Subtract V1 from V2 and
• add half of the volume of the UV flow cell.
Use of different monitors for peak fractionation

in the same method
If different monitors or detectors are used for peak fractionation in different parts of the same method,
the delay volumes have to be set as method instructions for each of the method parts. For example,
both an external fluorescence detector and the UV monitor module can be used for peak fractionation.

9.14 Component volumes

The table below shows the component volumes of ÄKTA pure.
Component Volume (μl)
Inlet valve V9-IA, V9-IB 88
Inlet valve V9H-IA, V9H-IB 212
Inlet valve V9-IAB 95
Inlet valve V9H-IAB 116
Sample inlet valve V9-IS 88
Sample inlet valve V9H-IS 212
Inlet valve V9-IX 88
Inlet valve V9H-IX 212
Air sensor L9-1.2 20
Air sensor L9-1.5 35
Pump P9 (P9-A, and P9-B) 549

(total volume for two heads including T-connector and check valves)
Pump P9H (P9H A, P9H B, P9H S) 2163

(total volume for two heads including T-connector and check valves)
System pump pressure monitor R9 45
Sample pump pressure monitor R9 45
Sample pump S9 1392
Mixer, 0.6 ml 600
Mixer, 1.4 ml 1400
Mixer, 5 ml 5000
Mixer, 15 ml 15000
Mixer valve V9-M 14
Mixer valve V9H-M 31
Loop valve V9-L 17
Loop valve V9H-L 76
Versatile valve V9-V 14
Versatile valve V9H-V 31
Injection valve V9-Inj 10

Component Volume (μl)
Injection valve V9H-Inj 23
Column valve V9-C 110
Column valve V9H-C 190
Column valve V9-C2 110
Column valve V9H-C2 190
Column valve V9-Cs 14
Column valve V9H-Cs 31
UV monitor U9-M: Flow cell 0.5 mm 10
UV monitor U9-M: Flow cell 2 mm 11
UV monitor U9-M: Flow cell 10 mm 12
UV monitor U9-L: Flow cell 2 mm 30
UV monitor U9-L: Flow cell 5 mm 15
Conductivity cell 22
Flow restrictor FR-902 10
pH valve V9-pH, in By-pass position 15
pH valve V9H-pH, in By-pass position 35
pH flow cell 76
Flow restrictor FR-902 and tubing when mounted on pH valve V9-pH 48
Outlet valve V9-O 9
Outlet valve V9H-O 82
Outlet valve V9-Os 9
Outlet valve V9H-Os 28
Fraction collector F9-C internal tubing (0.5 mm i.d.) 147
Fraction collector F9-C Dispenser head 94
Note: The given values for the component volumes of the valves are average values. Depending on
the chosen flow path the actual component volume may differ somewhat.

9.15 Pressure control

Introduction
By using the function Pressure control to regulate the run, the method can be run with the set flow
rate without the risk of method stop due to pressure alarm. If the pressure approaches the pressure
limit, for example if the sample has higher viscosity than the buffer, the flow rate is automatically
lowered. Pressure control is enabled in the manual instructions Pumps:System flow or Pumps:Sample
flow. The default setting for Pressure Control is Off. To enable the function, set what pressure signal
to use. It is recommended to use the pre-column pressure. The instruction Advanced:Pressure control
parameters provides the P and I factors used in the regulator and can be adjusted for different columns,
see information further down.
In the Method editor, pressure control is enabled by selecting Control the flow to avoid overpressure
in the predefined phase Method settings.
Pressure control parameters

The table below describes the factors used for pressure regulation.
P factor Proportional component in PI pressure regulation. Reduces the error between

actual and requested target pressure, but may leave a permanent error.
I factor Integrating component in PI pressure regulation. Eliminates the stationary

error from the P factor, but introduces a slight instability that may lead to os-
cillation in the pressure and the actual flow rate. Set I = 0 to disable the I factor.
As a general guide use a small I factor for high pressure columns and a large
I factor for low pressure columns, see p. 501 for more recommendations.
Target value for Sets the target value for the PI pressure regulation as a percentage of the
pressure control pressure limit. If the target pressure is too close to the pressure limit there is
a risk that a short pressure spike will trigger the pressure alarm. The pressure
limit is set in the Alarm pressure instruction. The Alarm pressure used for
pressure control depends on the settings in the System flow instruction.
Pressure control If the flow rate is reduced below the value set in Pressure control min flow
min flow rate rate, the method is paused and the system is set to state ALARMS AND ER-
RORS.
Pressure control min flow rate can be set either as volumetric or as linear
flow. A column type must be selected before using linear flow.

Recommended pressure control parameters

The table below contains the recommended values for P and I parameters for different media types.
Column/Media Recommend- Recommended Additional information

ed P factor I factor
Default 8 40 N/A
Small soft media 8 40 N/A

columns
Large soft 1 media 8 300 - 600 A higher I value than the default val-
columns ue is needed to speed up pressure
ramp-up times.
Small rigid 2 media 8 15 A lower I value than the default value

columns is needed to avoid large fluctuations
20 40
in pressure or flow rate. As an alter-
native, try increasing P.
1 Soft media is defined as all GE separation media, except silica and MonoBeads.
2 Rigid media is defined as GE separation media that is based on silica and MonoBeads.
Back pressure
Using narrow tubing between components will improve resolution but will lead to increased back
pressure in the system. Narrow tubing after the column will increase the pressure in the column at a
given flow rate. Make sure that the pressure sensor limits in the system are set so that the maximum
pressure for the column used is not exceeded.
Additional instructions for avoiding pressure

alarms
The instruction Max flow during valve turn sets the maximum flow rate used during the turning of
the Injection valve and Outlet valve in order to avoid high pressure alarms. If the flow rate passing
through the Injection valve or the Outlet valve is higher than the set max flow rate, the valves will only
turn after decreasing the flow to the specified flow rate. After the valves have turned, the previous flow
rate will be restored. The instruction is found in System Control:System settings:Advanced.

9.16 Node IDs
9.16 Node IDs

Node ID for core modules
The table below lists the Node ID for the core modules.
Core module Label Node ID
System pump A P9 A / P9H A 0
System pump B P9 B / P9H B 1
Pressure monitor R9 0
Mixer M9 0
Injection valve V9-Inj / V9H-Inj 4
Node ID for optional modules

The table below lists the Node ID for the optional modules.
Note: The Node IDs of Sample pump S9 and Fraction collector F9-C cannot be changed by the user.
Note: The Node IDs for UV monitor U9-M and UV monitor U9-D should only be changed by service
personnel.
Module Label Node ID
Inlet valve A V9-IA / V9H-IA 0
Inlet valve B V9-IB / V9H-IB 1
Inlet valve AB V9-IAB / V9H-IAB 3
Inlet valve X1 V9-IX / V9H-IX 15
Inlet valve X2 V9-IX / V9H-IX 16
Mixer valve V9-M / V9H-M 22
Sample inlet valve V9-IS / V9H-IS 2
Loop valve V9-L / V9H-L 17
Column valve (5-columns) V9-C / V9H-C 5
Pre-column pressure monitor N/A 2
Post-column pressure monitor N/A 3
Second column valve (5-columns) V9-C2 / V9H-C2 6
Pre-column pressure monitor N/A 4
Post-column pressure monitor N/A 5

9.16 Node IDs
Module Label Node ID
Column valve (1-column) V9-Cs / V9H-Cs 7
pH valve V9-pH / V9H-pH 11
Outlet valve (10-outlets) V9-O / V9H-O 8
Outlet valve (1-outlet) V9-Os / V9H-Os 19
Versatile valve V9-V / V9H-V 20
Versatile valve 2 V9-V / V9H-V 21
UV monitor (fixed) U9-L 0
UV monitor 2nd U9-L 1
UV monitor (variable) U9-M 0
UV detector U9-D 0
Conductivity monitor C9 0
External air sensor L9 0
External air sensor 2 L9 1
Fraction collector (cassettes) F9-C 0
Fraction collector (round) F9-R 0
Fraction collector (round), 2nd F9-R 1
Sample pump P9-S / P9H S 2
I/O-box E9 0
I/O-box, 2nd E9 1
Check/Change Node ID
Step Action
1 Remove the module according to the instruction in Section 2.5 Installation of internal
modules, on page 76.

9.16 Node IDs
Step Action
2 The Node ID of a module is set by the position of an arrow on a rotating switch at the
back of the module.
Valve modules have two rotating switches, as shown in the image below:
• the first switch, labeled A, sets the tens and
• the second switch, labeled B, sets the units.
For example, to set the Node ID to 6 for a valve module,
switch A is set to 0 and switch B is set to 6.
A B
3 Check the Node ID and compare it with the listed Node IDs in the tables above.
4 To change the Node ID, use a screw driver to set the arrows of the switches to the desired
number.
5 Re-install the module in the instrument, if applicable.

10 Ordering information
Introduction
This chapter lists accessories and user replaceable spare parts available for ÄKTA pure.
Mixer
Item Code no.
Mixer chamber 0.6 ml 28956186
Mixer chamber 1.4 ml (mounted at delivery) 28956225
Mixer chamber 5 ml (included with ÄKTA pure 150) 28956246
Mixer chamber 15 ml 28980309
O-ring 13.1 × 1.6 mm 28953545

Note:
For Mixer chamber 0.6, 1.4, and 5 ml.
O-ring 13.1 × 1.6 mm (highly resistant) 29011326

(can be used as an alternative to 28953545)
O-ring 22.1 × 1.6 mm 28981857

Note:
For Mixer chamber 15 ml.
Online filter kit 18102711
Tubing
Item Code no.
Reference capillary 1 28950749
Reference capillary 2 28950750
Tubing Kit 0.5 mm standard, ÄKTA pure 25 29011327
Tubing Kit 0.5 mm, ÄKTA pure 150 29051669
Tubing Kit 0.75 mm standard, ÄKTA pure 150 29048242

Item Code no.
Tubing Kit 1.0 mm 29034551
Tubing kit 10×1.0 m, ETFE ID 1.0 mm OD 1/16 28980995
Tubing kit for sample inlet valve V9-IS (7-ports) 29035331
Tubing kit for sample inlet valve V9H-IS (7-ports) 29051166
Sample tubing kit for 7 inlets, i.d. 0.75 mm 28957217
Inlet tubing kit 2+2 29011330
V9-pH tubing kit 29011331
V9H-pH tubing kit standard 29051674
Tubing kit for inlet valve V9-IA (7 ports) 29011332
Tubing kit for inlet valve V9H-IA (7 ports) 29051197
Tubing kit for inlet valve V9-IB (7 ports) 29011333
Tubing kit for inlet valve V9H-IB (7 ports) 29051189
Outlet tubing kit, ÄKTA pure 25 29011334
Outlet tubing kit, ÄKTA pure 150 29048611
Rinse system tubing 29011348
Union 1/16 male/male, i.d. 0.5 mm (5-pack) 28954326
Tubing cutter 18111246
Inlet filter holder kit 11000407
Inlet filter set 11000414
Holders
Item Code no.
Adapter for air sensor 28956342
Bottle holder 28956327
Column clamp o.d. 10–21 mm 28956319
Column holder 28956282
Column holder rod 28956270
Flexible column holder 28956295
Loop holder 29011350

Item Code no.
Multi-purpose holder 29011349
Rail extension 29011352
Tube holder (5-pack) 28954329
Tubing holder comb 28956286
Tubing holder spool 28956274
Inlet filter holder kit 11000407
Screw lid GL45 kit 11000410
UV monitor
Item Code no.
UV monitor U9-L (Fixed wavelength) 29011360
UV flow cell U9-0.5 0.5 mm for U9-M 28979386
UV flow cell U9-2 2 mm for U9-M 28979380
UV flow cell U9-10 10 mm for U9-M 28956378
UV flow cell 2 mm for U9-L 29011325
UV flow cell 5 mm for U9-L 18112824
I/O box
Item Code no.
I/O box E9 29011361

Item Code no.
Fraction collector F9-C 29027743
Tubing kit for F9-C 29033632
Cassette tray 28-954209
Cassette, for deepwell plate (2-pack) 28954212
Deep well plate, 96 x 2 ml 77015200

Item Code no.
Cassette, for 50 ml tubes (2-pack) 28956402
Rack, for 50 ml tubes 28980319
Rack, for 250 ml bottles 28981873
Cable 2.5 m, UniNet-9 D-type 29032425


Item Code no.
Fraction collector F9-R 29011362
Tube Rack Complete, 175 x 12 mm 19868403
Tube Rack Complete, 95 x 10-18 mm 18305003
Tube Rack Complete, 40 x 30 mm 18112467
Bowl 18305103
Tube support 18305402
Tube holder 18646401
Tube rack upgrade kit, 175 x 12 mm 19724202
Drive sleeve 19606702
Valves
Item Code no.
Column valve kit V9-C 29011367
Column valve kit V9H-C 29050951
Column valve V9-C2 28957236
Column valve V9H-C2 28979330
Column valve V9-Cs 29011355
Column valve V9H-Cs 29090693
Inlet valve V9-X1 28957227
Inlet valve V9H-X1 28979326
Inlet valve V9-X2 28957234
Inlet valve V9H-X2 28979328
Inlet valve kit V9-IA 29012263
Inlet valve kit V9H-IA 29050945
Inlet valve kit V9-IB 29012370

Item Code no.
Inlet valve kit V9H-IB 29050946
Inlet valve kit V9-IAB 29011357
Inlet valve kit V9H-IAB 29089652
Sample inlet valve kit V9-IS 29027746
Sample inlet valve kit V9H-IS 29050943
Loop valve kit V9-L 29011358
Loop valve kit V9H-L 29090689
Mixer valve kit V9-M 29011354
Mixer valve kit V9H-M 29090692
Outlet valve kit V9-O 29012261
Outlet valve kit V9H-O 29050949
Outlet valve kit V9-Os (1 outlet) 29011356
Outlet valve kit V9H-Os (1 outlet) 29090694
pH valve kit V9-pH 29011359
pH valve kit V9H-pH 29051684
Versatile valve V9-V 29011353
Versatile valve V9H-V 29090691
Note: All valve kits include the necessary tubing.
Injection valve accessories

Item Code no.
Sample loop 10 µl 18112039
Sample loop 100 µl 18111398
Sample loop 500 µl (mounted at delivery) 18111399
Sample loop 1 ml 18111401
Superloop 10 ml 19758501

Item Code no.
Fill port 18112766
Injection kit 18111089
Connector 1/16" male and Luer female 28985812
External air sensors

Item Code no.
Air sensor L9-1.2 mm 28956502
Air sensor L9-1.5 mm 28956500
pH monitor
Item Code no.
pH electrode 28954215
O-ring 5.3 × 2.4 mm 28956497
Item Code no.
Conductivity monitor C9 29011363
Flow restrictor
Item Code no.
Flow restrictor FR-902 18112135
Module components
Item Code no.
Module Panel 29011364
Multi-module front 29011351
Extension box 29110806

Cables
Item Code no.
Jumper 1 IEC 1394 (F-type) 28956489
Jumper D-SUB (D-type) 29011365
External module cable, short (F-type) 29012474
External module cable, long (F-type) 29011366
Cable 2.5 m UniNet-9 D-type 29032425
System Pumps and Sample pump S9H

Item Code no.
P9 Seal kit 25 ml 28952642
P9 Piston kit 25 ml 28952640
P9H Seal kit 150 ml 28979373
P9H Piston kit 150 ml 28979368
Check valve kit 28979364
Sample pump S9H 29050593
Sample Pump S9
Item Code no.
Sample pump S9 29027745
P9-S Seal kit 28960250
P9-S Piston kit 18111213
Check valve kit 28979364
Cable 2.5 m UniNet-9 D-type 29032425
UNICORN
There are different UNICORN products and licenses available for different purposes, for example licenses
for use with a workstation or for working remotely. Contact your local GE salesperson for more infor-
mation on UNICORN products and licenses and how to order.

Index
Index
A storage, 221
Affinity chromatography Column CIP
predefined method, 439–440 maintenance method, 445
Air sensors predefined phase, 447
air sensor adapter, 83 Column performance test
A test, 228 maintenance method, 445
B test, 230, 232 predefined phase, 215, 447
error codes, 405 Column preparation
external air sensors, 87 maintenance method, 445
inlet valves, 40 predefined phase, 447
troubleshooting, 398 Column valve
ÄKTA pure description, 55
description, 13, 86 flow paths V9-C, 56
illustrations, 14 flow paths V9-Cs, 57
main features, 9 V9-C test, 234
maintenance, 251 Column wash
overview, 9 predefined phase, 447
troubleshooting, 356 Component volumes, 498
Anion exchange Conductivity monitor
predefined method, 441 calibration, 308
clean the conductivity flow cell, 300
C description, 72
Cabinet error codes, 405
troubleshooting, 396 troubleshooting, 367
Cassettes, 96 Connectors
quickrelease function, 98 for ÄKTA pure, 421
Cation exchange replace connectors, 317
predefined method, 441 D
Chromatofocusing
predefined method, 441 Delay volume
CIP reference information, 493
column CIP, 284 settings, 207
column maintenance method, 445 Delta column pressure
column predefined phase, 447 description, 68
system CIP, 278 Desalting
system maintenance method, 445 predefined method, 442
system predefined phase, 215, 447 Documentation, 11–12
Cleaning E
column, 221
Elution
system, 220
predefined phase, 447
Column
Equilibration
attach column holder, 172
predefined phase, 447
cleaning, 221
Extension box, 85
column CIP, 284
column holder, 80 F
column valve, 55 Flow cells
connect column, 172 conductivity, 72
pressure alarm, 176

Index
pH, 60 ordering information, 507

UV, U9-L, 70 rail extension, 79
UV, U9-M, 69 tubing holder comb, 83
Flow path tubing holder spool, 82
component volumes, 498 Hydrophobic interaction chromatogra-
illustration, 25, 150, 422 phy (HIC)
prepare, 150 predefined method, 442
specifications, 25
Flow restrictor
I
check function, 269 I/O-box E9
FR-902 description, 74 connectors, 113
replace flow restrictor, 330 description, 112
Fractionation error codes, 409
assemble tube rack, 105, 199 Fraction collector F9-C installa-
delay volume, 207 tion, 112
fractionation types, 206 specifications, 419
prepare, 199 troubleshooting, 399
Fraction collector Injection valve
cleaning, 267, 288, 290 description, 49
description, 90 flow paths, 49
error codes F9-R, 408 Inlet tubing
F9-R test, 236, 239, 241 prepare, 152
function, 90 prime inlet tubing B, 161
preparation, 199 inlet valves
quickrelease function, 98 V9-IA test, 228
replace internal tubing, 319 V9-IB test, 230, 232
specifications, 418 Inlet valves
troubleshooting, 377, 385 air sensors, 40
Fraction collector F9-C description, 39
Cassettes and racks, 96 inlet valve A, 39
connect tubing, 102 inlet valve AB, 39
requirements on deep well plates, 100 inlet valve B, 39
requirements on tubes, 99 inlet valve X1, 39
Tubes, 105 inlet valve X2, 39
troubleshooting, 376
G Installation
Gel filtration prepare waste tubing, 154
predefined method, 442 Instrument control panel
error codes, 409
H function, 27
Hardware installation location, 27
of a module, 76 status indications, 29
Holders troubleshooting, 400
air sensor adapter, 83 Instrument control unit
bottle holder, 83 error codes, 404
column clamp, 81 Instrument overview
column holder, 80 Fraction collector F9-R, 103
column holder rod, 81 Instrument configurations, 14
Extension box, 85 main parts, 14
flexible column holder, 82 modules, 16
loop holder, 80 wet side, 15
multi-purpose holder, 79

Index
Ion exchange reverse phase chromatography

predefined method, 441 (RPC), 444
Method settings
L predefined phase, 446
Log on Miscellaneous
UNICORN, 157 predefined phase, 447
Loop valve Mixer
description, 52 choose mixer chamber, 151
flow paths, 52 description, 37
M error codes, 405
mixer valve, 46
Maintenance replace mixer, 322
calibration procedures, 302 replace the inline filter, 266
clean the instrument, 277 replace the o-ring, 323
Maintenance Manager, 252 specifications, 415
maintenance program, 258 troubleshooting, 395
maintenance when required, 276 Mixer valve
replacement procedures, 316 description, 46
semiannual maintenance, 272 flow paths, 46, 48
weekly maintenance, 261 Module panel
Maintenance manager description, 84
handling maintenance notifica- troubleshooting, 376
tions, 254 Module Panel Assembly, 124
Maintenance methods Modules
column CIP, 445 column valve, 55
column performance test, 445 conductivity monitor, 72
column preparation, 445 flow restrictor, 74
system CIP, 445 injection valve, 49
system preparation, 445 inlet valves, 39
Manual instructions, 465 loop valve, 52
Manual loop fill mixer, 37
predefined phase, 447 mixer valve, 46
Manual Loop Fill outlet valve, 64
predefined method, 442 pH valve, 60
Methods pressure monitor, 67
affinity chromatography, 439–440 pumps, 33, 107
anion exchange, 441 recommended positions, 124
cation exchange, 441 UV monitor, 69
chromatofocusing, 441 versatile valve, 59
create method, 211 Monitors
desalting, 442 conductivity, 72
gel filtration, 442 external air sensors, 87
hydrophobic interaction chromatog- pH, 62
raphy (HIC), 442 pressure monitor, 67
maintenance methods, 211 specifications, 417
manual loop fill, 442 troubleshooting, 360
NHS-coupling, 443 UV, 69
predefined maintenance meth-
ods, 445 N
predefined methods, 211, 439 NHS-coupling
purification methods, 211 predefined method, 443

Index
Node ID sample application, 447

change Node ID, 503 system CIP, 215, 447
check Node ID, 503 system preparation, 447
description, 76 pH monitor
list, 502 calibration, 303
clean the pH electrode, 293
O description, 62
Optional configurations error codes, 405
install optional modules, 76 replace the pH electrode, 275
Ordering information storage of the pH electrode, 221, 291
cables, 512 troubleshooting, 370
conductivity monitor, 511 pH valve
dummy module, 511 description, 60
external air sensors, 511 flow paths, 61
flow restrictor, 511 troubleshooting, 370, 376
fraction collector, 509 Power and ICU
holders, 507 troubleshooting, 396
I/O-box E9, 507 Power save, 146
mixer, 505 Prepare the system
pH monitor, 511 connect column, 172
pump, 512 flow path, 150
tubing, 506 fraction collector, 199
UV monitor, 507–508, 511 inlet tubing, 152
valves, 510 mixer, 151
Outlet tubing outlet tubing, 153
prepare, 153 sample application, 179
Outlet valve Start UNICORN, 157
description, 64 UV monitor U9-L, 152
V9-O ports, 64 UV monitor U9-M, 152
V9-Os ports, 65 waste tubing, 154
P Pressure alarm
set, 176
Performance tests Pressure control
air sensor A and inlet valve V9-IA, 228 system configuration, 219
air sensor B and inlet valve V9- Pressure monitor
IB, 230, 232 error codes, 404
column valve V9-C, 234 Pressure monitors
fraction collector F9-R, 236, 239, 241 calibration, 305
overview, 225 check the pressure monitors, 305
UV U9-L system, 243 description, 67
UV U9-M system, 247 troubleshooting, 372
Phases Prime inlet tubing B, 161
column CIP, 447 Process picture
column performance test, 215, 447 actions in the process picture, 219
column preparation, 447 Pump piston rinsing system
column wash, 447 flow path, 35
elution, 447 illustration, 35, 109, 262, 264, 352–353
equilibration, 447 prime, 263, 265
manual loop fill, 447 Pumps
method settings, 446 change pump rinsing solution, 262
miscellaneous, 447 clean check valves, 296
predefined phases, 446

Index
description, 33 sample loading, 189

description, sample pump, 107 Sample pump
error codes, 406 description, 107
purge sample pump, 167 Software overview
purge system pumps, 161 software modules, 9
replace check valves, 332 Storage
replace pump pistons, 351 column, 221
replace pump piston seals of pump pH electrode, 221
P9, 335 system, 221
replace pump piston seals of Pump Superloop
P9-S, 344 sample loading, 182
replace pump rinsing system tub- troubleshooting, 396
ing, 352 System CIP
specifications, 413 maintenance method, 445
troubleshooting, 387 predefined phase, 215, 447
System cleaning, 220
Q System configuration
QuickRelease function, 98 control pressure, 219
R installation of modules, 76
modules and positions, 124
reference information overview, 124
injection volumes and peak broaden- system properties, 127
ing, 492 system settings, 129
Reference information, 410 System Control module
back pressure, 501 manual instructions, 465
chemical resistance guide, 431 System preparation
delay volumes, 493 before preparation, 149
module specifications, 413 maintenance method, 445
pressure regulation, 500 predefined phase, 447
system specifications, 411 System pumps
Reverse phase chromatography (RPC) operating ranges, 33
predefined method, 444 System storage, 221
Run System test
after run procedures, 220 UV U9-L, 243
available curves, 490 UV U9-M, 247
available run data, 487
choose method, 216 T
monitor run, 218 Technical specifications
perform manual run, 217 module specifications, 413
perform run, 216 Trays, 96
prepare run, 216 Troubleshooting
S error codes, 404
fraction collector, 377
Safety notices, 7 Fraction collector, 385
Sample application instrument communication, 401
flow paths, 49 monitors, 360
injection valve, 49 other components, 394
predefined phase, 447 pumps, 387
sample loading, 179 Tube rack
sample loop, 189 assembly, 105, 199
superloop, 182 Tubing
Sample loop

Index
for ÄKTA pure, 420 error codes, 406

replace tubing, 317, 319 system test, 247
U
UNICORN V
connect to system, 158 Valves
Log on, 157 column valve, 55
Start, 157 error codes, 404
User information, 7 extra Column valve, 56
UV detector U9-D extra inlet valves, 39
error codes, 406 injection valve, 49
UV monitor inlet valves, 39
calibration, 152, 312 loop valve, 52
clean UV flow cell, 273 mixer valve, 46
description, 69 outlet valve, 64
installing two monitors, 139 overview, 38
replace UV flow cell, 325, 328 pH valve, 60
UV monitor U9-L specifications, 414
description, 70, 152 versatile valve, 59
error codes, 407 Versatile valve
replace the flow cell, 328 description, 59
system test, 243 flow paths, 59
UV monitor U9-M
W
description, 69, 152 Waste tubing, 154
prepare, 154

GE, the GE Monogram, ÄKTA, AxiChrom, GSTrap, HiLoad, HiPrep, HiScreen, HiTrap, HisTrap, Mini Q, Mini
For local office contact information, visit S, Mono Q, Mono S, Mono P, RESOURCE, Sephacryl, Sepharose, SOURCE, StrepTrap, Superdex, Superloop,
www.gelifesciences.com/contact Superose, UNICORN, and Whatman are trademarks of General Electric Company.
Decon is a trademark of Decon Laboratories, Limited. Elgiloy is a trademark of Elgiloy Limited Partnership.
GE Healthcare Bio-Sciences AB Eppendorf is a trademark of Eppendorf AG. Hastelloy is a trademark of Haynes International Inc. Kautex
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All other third party trademarks are the property of their respective owners.
www.gelifesciences.com/AKTA
Any use of UNICORN is subject to GE Healthcare Standard Software End-User License Agreement for
Life Sciences Software Products. A copy of this Standard Software End-User License Agreement is
available on request.
© 2012-2018 General Electric Company
All goods and services are sold subject to the terms and conditions of sale of the company within GE
Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact
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29119969 AC 02/2018 a859

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ÄKTA™ pure

2 The ÄKTA pure instrument ............................................................................................................................. 13

3 ÄKTA pure external modules .......................................................................................................................... 86

4 System configuration ...................................................................................................................................... 123

2 ÄKTA pure User Manual 29119969 AC

4.2.5 Configuration of Versatile valves .................................................................................................................................................................. 136

5 Operation .......................................................................................................................................................... 148

6 Performance tests ........................................................................................................................................... 224

7 Maintenance ..................................................................................................................................................... 251

ÄKTA pure User Manual 29119969 AC 3

7.5 Semiannual maintenance ............................................................................................................................................... 272

8 Troubleshooting ............................................................................................................................................... 355

9 Reference information .................................................................................................................................... 410

4 ÄKTA pure User Manual 29119969 AC

9.7 Predefined methods and phases ................................................................................................................................. 438

10 Ordering information ...................................................................................................................................... 505

ÄKTA pure User Manual 29119969 AC 5

Purpose of the User Manual

Section See page

1.1 Important user information 7

1.2 ÄKTA pure overview 9

1.3 ÄKTA pure user documentation 11

6 ÄKTA pure User Manual 29119969 AC

1.1 Important user information

ÄKTA pure User Manual 29119969 AC 7

Notes and tips

8 ÄKTA pure User Manual 29119969 AC

1.2 ÄKTA pure overview

UNICORN modules overview

Module Main functions

ÄKTA pure User Manual 29119969 AC 9

Module Main functions

Evaluation Open results, evaluate runs and create reports.

10 ÄKTA pure User Manual 29119969 AC

1.3 ÄKTA pure user documentation

Document Main contents

ÄKTA pure User Manual 29119969 AC 11

UNICORN user documentation

Documentation Main contents

1 Current UNICORN version is added to the title of the manual.

12 ÄKTA pure User Manual 29119969 AC

2 The ÄKTA pure instrument

About this chapter

Section See page

2.1 Overview illustrations 14

2.2 Liquid flow path 25

2.3 Instrument control panel 27

2.4 Instrument modules 32

2.5 Installation of internal modules 76

ÄKTA pure User Manual 29119969 AC 13

2.1 Overview illustrations

Core module configurations

Illustrations of the main parts of the instrument

4 Instrument control panel

14 ÄKTA pure User Manual 29119969 AC

Example of a typical configuration of the wet side

3 Pump rinsing liquid tube

ÄKTA pure User Manual 29119969 AC 15

ÄKTA pure 25 ÄKTA pure 150

System pump A* P9 A P9H A