Oxygraph System Manual

Operations Manual
Setup, Installation &

Maintenance
Oxygraph System
Hansatech Instruments Ltd

Narborough Road, Pentney, King’s Lynn,
Norfolk PE32 1JL England.
Tel: +44 (0)1760 338877 Fax: +44 (0)1760 337303

Email: [email protected]
Web site: http://www.hansatech-instruments.com
28th February 2006

Version 2.1
© Copyright Hansatech Instruments Ltd. All rights reserved.

Contents
Preface ........................................................................... 5
• Document Conventions ....................................... 5
• Notice ................................................................... 5
Chapter 1: Electrode Preparation & ................. 6

Maintenance
Introduction to the S1 Oxygen Electrode ................... 7
• Electrode Disc Theory ......................................... 7
• Operational Principles ......................................... 7
Electrode Preparation ................................................. 8

• Testing the Response of the Prepared ............... 10
Electrode
Electrode Disc Cleaning & Maintenance .................. 11

• Electrode Maintenance ....................................... 11
• Cleaning Procedure ............................................ 11
• Rinsing & Drying ................................................. 12
• Storage ............................................................... 12
The Importance of Good Electrode Maintenance ..... 13

• Failure to Maintain the Disc ................................ 13
• Disc is Left to Dry Out After Use ......................... 14
• Cleaning with Unsuitable Materials ..................... 14
Chapter 2: DW1/AD Electrode Chamber ........... 15
Introduction to DW1/AD ............................................... 16

• Features of DW1/AD ............................................. 16
• Transmission Properties of DW1/AD .................... 17
Setting Up DW1/AD ...................................................... 17

• Installing the Electrode Disc ................................. 17
• Temperature Control ............................................ 18
• Sample Stirring ..................................................... 18
Care & Maintenance .................................................... 19

• Disassembling DW1/AD ....................................... 19
• Cleaning DW1/AD ................................................ 20
• Reassembling DW1/AD ....................................... 21
DW1/AD Plunger Assembly ......................................... 21
Spare Parts List ............................................................ 22

Chapter 3: Oxygraph Electrode Control Unit ... 23
Introduction to Oxygraph .............................................. 24
Setting Up Oxygraph ...................................................... 24

• Connections to the Oxygraph ................................ 24
• Multi-Channel Systems .......................................... 25
• What Additional Hardware is Required? ........... 25
• Linking Control Units Together ......................... 26
• Pitfalls!! ............................................................. 27
• Clearing a Control Unit ID ................................. 27
• Using Multi-Channel Systems ................................ 27
• Channel Calibration & Configuration ................. 27
• Stirring ............................................................... 27
• Signal Display .................................................... 28
• Disable Boxes .................................................... 28
System Calibration .......................................................... 29

• Liquid-Phase Calibration ......................................... 29
• Air Line ............................................................... 29
• Zero Oxygen Line .............................................. 30
• Performing a Liquid-Phase Calibration .............. 31
• Viewing Calibration Details ..................................... 34
Oxygraph Operation ........................................................ 36

• Oxygraph Plus ......................................................... 36
• Overview ............................................................ 36
• Running Oxygraph Plus .......................................... 36
• Graph Functions ..................................................... 37
• Recording a Signal ................................................. 38
• Setting the Measurement Axes .............................. 38
• Trace Settings ........................................................ 39
• The Data Bar ......................................................... 40
• The Menu Bar ........................................................ 41
• Files Menu ........................................................ 41
• Hardware Menu ................................................ 42
• Calibrate Menu ................................................. 42
• View Menu ........................................................ 42
• Graph Menu ..................................................... 42
• Data Bar Menu ................................................. 43
• Tools Menu ...................................................... 43
• Rate Menu ....................................................... 43
• Help Menu ....................................................... 43
• The Toolbar .......................................................... 43
• System Configuration ........................................... 45
• Overview ......................................................... 45
• Data Acquisition Rate ...................................... 49
• Temperature Control ............................................ 51
• Overview ......................................................... 51
• Temperature Effects on Liquid-Phase ............ 51
Samples
• Temperature Effects on the Electrode Disc .... 52
• Setting Up Temperature Control ..................... 52
• The Magnetic Stirrer ............................................. 53
•Operating the Magnetic Stirrer ......................... 54
• Recording an Auxiliary Signal .............................. 55
• System Diagnostics ............................................. 56
• Control Unit Diagnostics ................................. 56
• Electrode Disc Diagnostics ............................. 58
• Testing the Electrode Connection Cable for ... 59
Short Circuits or Breaks
Data Handling .............................................................. 60

• Event Marking ...................................................... 60
• Editing and Deleting Event Marks ................... 61
• File Information ..................................................... 62
• Rate Measurements ............................................. 63
• Tabulated Data ..................................................... 65
• Data Export ........................................................... 66
• Printing Data ......................................................... 67
• Viewing Previously Saved Files ............................ 67
Accessories ..................................................................... 68
• OXY/PDU - Power Distribution Unit ...................... 68
• OXY/AUX - Auxiliary Input Adapter Cable ............ 68
• OXY/PHA - Ph / Ion-Selective Electrode .............. 69
Amplifier
Support Information ...................................................... 70

• Upgrading Oxygraph Plus Software ..................... 70
Appendix A - Sample Printout of Data File ...................... 71

5
Preface
This manual is designed to highlight the key features, setup procedures and general
maintenance of the Oxygraph System.
Document Conventions
If viewed electronically, blue highlighted text can be clicked in order to link to other parts of
this document or to Internet locations. In order for the external links to be completed, an
Internet connection must be established either via a standard or broadband dial-up
modem or via a LAN (local area network) connection.
Notice
This instrument must not be used in situations where its failure could result in injury or
death.
For applications where failure of this instrument to function correctly would lead to
consequential damage, the analyser must be checked for correct operation and calibration
at intervals appropriate to the criticality of the situation.
This manual is provided to help you install and operate the equipment. Every effort has
been made to ensure that the information contained in this manual is accurate and
complete. Hansatech Instruments Ltd does not accept any liability for losses or damages
resulting from the use of this information.
Hansatech Instruments Ltd equipment warranty is limited to replacement of defective

components, and does not cover injury to persons or property or other consequential
damage.
This manual, and the information contained in it, is copyright to Hansatech Instruments
Ltd. No part of the manual may be copied, stored, transmitted or reproduced in any way or
by any means including, but not limited to, photocopying, photography, magnetic or other
mechanical or electrical means, without the prior written consent of Hansatech Instruments
Ltd.
The Oxygraph System is covered under warranty for one complete year, parts and labour
included. This, of course, is provided that the equipment is properly installed, operated and
maintained in accordance with written instructions contained within this manual.
The warranty excludes all defects in equipment caused by incorrect installation, operation
or maintenance, misuse, alteration, and/or accident.
If for some reason, a fault is covered under warranty, it is the responsibility of the customer
to return the goods to Hansatech Instruments Ltd or an authorised agent for repair or
replacement of the defective part(s). For service, please contact us at the address at the
back of this manual.
Chapter 1
Electrode Preparation
& Maintenance
7
Introduction to the S1 Electrode Disc.

The Hansatech Instruments oxygen electrode disc is a specialised form of electrochemical
cell known as a Clark type polarographic sensor. It comprises a resin bonded central
platinum cathode and a concentric silver anode linked by an electrolytic bridge and
continuously polarised by the oxygen electrode control unit.
The electrodes are set into an epoxy resin disc (A) with the platinum
B cathode (B) at the centre of a dome surrounded by a well that
A contains the silver anode (C). When set up a uniform
electrolyte layer is held over the dome area of the
electrode by a paper spacer and P.T.F.E.
(polytetrafluoroethylene) membrane which is secured
by an O-ring around the dome. The well that houses
the anode also serves as a reservoir with electrolyte
C drawn up to the cathode by the paper spacer acting
as a wick. An outer O-ring groove (D) surrounds the
whole apparatus. The outer O-ring seals the electrodes into
the base of the electrode chamber.
D
Electrode Disc Theory.
The oxygen electrode is a specialised form of electrochemical cell which consists of two
electrodes immersed in an electrolyte solution. Typically, a 50% saturated solution of KCl
is used in oxygen electrode systems. This is prepared by dissolving 17.5g of anhydrous
salt in 100ml of de-ionised water at 25°C. Application of a polarising voltage of 700 mV
ionises the electrolyte and initiates current flow via a series of electrochemical reactions. In
the case of KCl electrolyte the following reactions occur:
Oxygen is consumed during the electrochemistry, thus the magnitude of the current flow is
related to the oxygen concentration of the surrounding media. This type of electrode
sensor was first developed by Clark (1956) to measure oxygen in blood samples. As a
result it is often referred to as The Clark Electrode.
Operational Principles.
The following explanation are based around an electrode disc which is connected to one
of the Hansatech Instruments Ltd PC operated electrode control units. For further details,
please refer to the Oxygraph Electrode Control Unit section.
When the disc is correctly prepared and polarised at 700 mV, the nature of the
electrochemical reactions at the disc cause a current to flow in the presence of oxygen.
8
This current is directly proportional to the amount of dissolved oxygen within the sample
held in the reaction vessel.
The electronics within the control unit must take the current produced by the electrode disc
and convert it into a reproducible unit. Several stages are taken applying various factors to
the signal in order to present the signal from the disc as nmol/ml in liquid-phase
measurements and µmol RTA (relative to air) in gas-phase measurements.
The diagram below shows the processes undertaken by the control unit in order to present
the signal in calibrated units. In this example, the disc is placed in a stirred sample of air-
saturated, de-ionised water held at 25°C at a standard atmospheric pressure of 101.72
KPa.
The current produced by the electrochemical reactions in the presence of oxygen is first
converted into a voltage signal. In air-saturated, de-ionised water, the control unit is
designed to read approx. 2000 mV (approximately half the range of the instrument).
Gain and Back Off are applied to the signal at this point. These values are automatically
set during the calibration. However, these values always remain at Gain = approx. 1.0 and
Back off = 0. In cases where measurements of small changes in oxygen are required,
these values may be manually adjusted.
The voltage signal is then passed through the 12 bit resolution A/D converter where the
signal is digitised.
Once digitised, the calibration offset and factor are applied to the signal allowing the
signal to be displayed in the relevant calibrated units.
Electrode Preparation.
Due to the nature of the reactions taking place, the silver anode oxidises slowly with time.
This is characterised by a black deposit on the electrode which may impair performance. If
this occurs, the electrode should be cleaned. This is best achieved by gentle polishing of
the unit using the materials supplied in the S16 ELECTRODE MAINTENANCE KIT or if
this is unavailable, by using a suitable polishing paste or fine grade aluminium oxide
powder suspended in a drop of de-ionised water placed on a cotton wool bud.
NEVER USE HARSH ABRASIVE AGENTS OR COMMERCIAL

METAL POLISHES AS THESE MAY DAMAGE THE RESIN OR
IRREVERSIBLY POISON THE CATHODE.
9
Further detail on cleaning and maintaining the electrode disc can be found in the Electrode
Maintenance section.
Before use, the electrode disc needs to be prepared in such away that an electrolyte
bridge is formed between the anode and cathode in order for current to flow in the
presence of oxygen. Various different compositions of electrolyte have been used
however, a 50% saturated solution of potassium chloride works well in many different
applications. The solubility of the anhydrous salt is 35g per 100g of water at 25°C. Hence
the electrolyte solution is easily made by dissolving 17.5g of anhydrous salt in 100 ml of
de-ionised water at 25°C.
The disc also requires a protective membrane which will prevent any deposits from the
reaction mixture from settling on the cathode yet allow oxygen to diffuse freely so as not to
jeopardise response time of the disc.
There are four preparation stages:
Stage 1.
Place a small drop of electrolyte on top of the dome
of the electrode disc.
Stage 2.
Place a 1.5 sq. cm paper spacer (cigarette paper works
particularly well as it is manufactured to a very tight thickness
tolerance) over the electrolyte ensuring that at least one corner
of the spacer is in the electrode well to act as a wick. Cover this
with a similar sized piece of PTFE membrane.
Stage 3.
Place the small electrode disc O-ring over the end of the
applicator tool. Hold the applicator vertically over the
dome and slide the applicator shaft down to push the
O-ring over the dome.
Stage 4.
Check that the membrane preparation is smooth and that
there are no trapped air bubbles. Top the reservoir well up
with several drops of electrolyte.
It is vital to ensure that the larger O-ring is placed in position on the

recess around the electrolyte well. If this second O-ring is not in place
when the disc is installed into the electrode chamber, the silver anode
will not be sealed from ambient air and measurements may be affected.
10
Testing the Response of the Prepared Electrode.
The following section uses Oxygraph Plus software in order to demonstrate the tests for
electrode responsiveness. In other systems, the controls may be slightly different,
however, the principles of the tests are identical.
Once the electrode disc has been successfully prepared, it is advisable to check the
response of the disc prior to mounting the disc within the electrode chamber.
To check the response, connect the electrode disc to the rear of the control unit at the
electrode input. Open the recording software and start a recording. The signal will take a
few minutes to stabilise. A new or well maintained disc should read approx. 2000 mV in
air.
Once stable, breathe exhaled air across the disc and observe the reaction on the graph
screen. The signal should be plotted as in the diagram below.
The first deviation in signal after breathing across the disc is induced due to a large
increase in temperature of our exhaled breath compared to ambient air temperature.
Oxygen electrode discs are particularly sensitive to temperature and will show an increase
in signal as a result. Since the temperature increase is only temporary, the oxygen signal
will fall after a short time.
After the temperature related signal increase, a steep drop in signal is observed due to
decreased oxygen levels in exhaled breath (approx 17%). The signal should the begin to
return to the original level as the ambient oxygen begins to equilibrate around the
electrode disc.
If the observed signal is not as shown above, it may be caused by an inadequate

electrode preparation or worst case, a problem with the disc itself. Try cleaning the disc
11
and re-preparing before repeating the test described above. If problems still occur, please
contact Hansatech Instruments.
Once this test has been completed satisfactorily, mount the disc into the base of the
electrode chamber. An additional test may be carried out in order to test the response of
the disc in situ. Please refer to the Operating the Magnetic Stirrer section for further
details.
Electrode Disc Cleaning and Maintenance.

Electrode Maintenance.
Periodic maintenance is required if you are to maintain your electrode in good condition. It
should be cleaned after use and before prolonged storage. It is particularly important that
the electrode is never left to dry out with electrolyte in place, as crystallisation of electrolyte
may cause the platinum/epoxy resin seal to be breached and crystalline electrolyte to be
deposited around the cathode. If this occurs, the electrode will become rapidly
unserviceable and will require replacement.
Cleaning is best achieved by following the procedure outlined below. (An electrode
maintenance kit -ordering code S16- containing the items described is available. Please
contact Hansatech Instruments for further details).
Cleaning Procedure.
It is important to use the correct polishing agent as harsh
abrasive substances will damage the resin in which the
electrodes are set. It should also be noted that many
commercial metal polishes contain ammonia or solvents
which may cause irreversible poisoning of the platinum
cathode.
Silver Anode.
The silver electrode (anode) is subject to electrochemical deposition of chloride and oxide
salts during use. This typically manifests as a brown/black deposit which forms on the
surface of the Silver. Whilst deposition of small amounts of brown Silver Chloride is normal
and desirable, excessive deposits or deposits of black oxides will cause a rapid
deterioration in electrode performance and may result in lower signal levels, signal drift
and increased signal noise.
Select a small cotton bud from the maintenance kit. Cotton buds vary a little in size, so
check to see that the diameter of the cotton bud will allow it to be fully inserted into the well
of the electrode, thus allowing contact with the silver electrode in the base of the well. If
the bud is a little large, carefully reduce the size of the cotton bud by removing a layer of
cotton wool from the tip until the bud will fit the well. Moisten the bud with a little distilled
water.
Apply a small amount of either the Rapid Hansatech Polishing paste (revised cleaning kits
supplied from 01/10/01) or the No. 1 Coarse Hansatech Polishing paste (supplied in earlier
cleaning kits with Coarse and Fine pastes) to the tip of the bud.
12
Insert the bud into the well of the electrode and gripping the bud just above the tip, apply
moderate pressure and gently rotate the bud around the electrode well in a circular motion
6 - 10 times. Observe the Silver anode. Continue to gently polish the silver electrode until
all brown or black deposits on the surface of the silver are removed and the surface of the
silver is highly polished.
Platinum Cathode.
The platinum cathode is not subject to such deposition and all that is necessary is to
maintain a scratch-free highly polished finish using the procedure outlined below.
Apply a small amount of the Rapid Hansatech Polishing paste (revised cleaning kits
supplied from 01/10/01) or the No. 2 Fine Hansatech Polishing paste (earlier 2 part
cleaning kits with Coarse and Fine pastes) to the unused end of the cotton bud moistened
with a little distilled water , or if preferred to a small piece of moist cotton wool. Now, using
a circular motion, gently polish the platinum cathode located in the centre of the electrode
dome.
It is important to restrict the polishing motion to the platinum and avoid as much as
possible contact with the epoxy resin surrounding the platinum.
Excessive polishing of the cathode area is to be avoided as this will eventually lead to a
change of curvature of the electrode dome, which will result in deterioration of electrode
performance. When finished, the platinum cathode should have a “mirror finish”.
If the surface of the platinum has become scratched, repeat the above procedure using
the Rapid Hansatech Polishing paste (revised cleaning kits supplied from 01/10/01) or first
using the No. 1 Coarse Hansatech Polishing paste, followed by the No. 2 Fine Polishing
paste (earlier 2 part cleaning kits with Coarse and Fine pastes).
Rinsing and Drying.
Once the electrodes have been satisfactorily cleaned, it is important to remove all traces of
the polishing paste from the electrode surfaces by rinsing the electrode with a small
volume of distilled water whilst gently scrubbing these areas with a soft bristled brush. (A
toothbrush is ideal). Avoid wetting the electrical connector of the disc during this
procedure. Dry the electrode thoroughly using paper towel.
Storage.
Store the electrode, when not in use, in an air-tight vessel containing a desiccant such as
Silica gel. Periodically replace or rejuvenate the desiccant.
13
The Importance of Good Electrode Maintenance.

Although the oxygen electrode disc is one of the smallest
components of an oxygen measurement system, it is the
most crucial component of the system. Therefore,
maintaining the disc to a high standard is extremely
important.
A significant proportion of technical support requests received by Hansatech Instruments

Ltd relate to oxygen electrode disc performance. Of these requests, the vast majority are
the result of failure to comply with recommended working practices and maintenance
procedures. Some of the more common abuses of electrode discs include:
Failure to Maintain Electrode Disc.
If the electrode disc is not maintained, deposits of silver chloride (soft brown deposit) and/
or silver oxide (hard black deposit) will build up on the silver ring (anode) of the disc. The
formation of silver chloride is normal and desirable in small quantities as it improves the
stability of the sensor. It is easily removed by cleaning and will reform when the electrode
is next polarised. By contrast, deposits of black silver oxide are to be avoided. Silver oxide
is an electrical insulator and its formation will reduce the available surface area of the
silver anode and result in a dramatic reduction or complete loss of electrode signal. The
electrode disc should be maintained in accordance with our recommended procedure.
Please refer to the Cleaning and Maintenance section.
This image shows a typical case of poor maintenance. The

disc has not been cleaned after use and was not stored in a
sealed container with desiccant away from strong sunlight. As
a consequence, the epoxy resin has become prematurely
discoloured due to a combination of strong sunlight and
absorption of moisture from the environment. The silver
anode is almost entirely covered with a deposit of black silver
oxide. The platinum stud and dome are both dirty and badly
scratched. This will cause an uneven layer of electrolyte
beneath the membrane during measurement leading to un-
stable and un-responsive signals. This disc is beyond
economic repair.
In contrast, this disc has been maintained according to our

recommendations and has been stored correctly in an air tight
container with desiccant. The epoxy resin is clear, the silver
anode is smooth and polished and the platinum cathode is
scratch free. The area of the electrode dome surrounding the
cathode is also free from abrasions and pits across the entire
surface. An electrode disc, when kept in this kind of condition
will provide years of high quality measurements.
14
Disc is Left to Dry Out After Use.
After use, the electrode disc should be stripped down, cleaned carefully and stored in a
dry, air-tight container. If the disc is simply left, electrolyte will crystallise under the
membrane and may cause irreversible damage to the electrode. The length of time that an
electrode can be left prepared will depend upon local circumstances such as temperature
and air flow through the laboratory. In general terms, we would recommend leaving a
prepared electrode disc in a liquid-phase measurement system with stirred air-saturated
water in the chamber overnight. The electrode disc can be kept polarised or switched off
and re-polarised the next day dependant upon local working practices and user
experience. In gas-phase measurements, the electrode is likely to dry out more rapidly and
we would recommend stripping down and cleaning the electrode after each measurement
day.
The KCl electrolyte dries out quickly in air and begins to crystallise. KCl is quite aggressive
and will, by capillary attraction, deposit in any minor crevice. If the electrode is left to dry
out several times, it is possible that the KCl will finally break down the seal between either
of the metal electrodes and the epoxy resin surround and seep into the fissure formed
where it will crystallise to further expand the fissure. This is most likely to occur between
the platinum cathode and epoxy resin dome and regrettably will cause most damage at
this point. Damage to the electrode can be observed under low
power microscopy (x10 magnification) as a broad white collar
around the neck of the platinum as shown in the image left. The
effect of this will be to cause instability and drift of the electrode
signal and a large increase in the level of electrical signal
observed in zero oxygen (residual current). This will have the
effect of reducing the ability of the electrode to measure small
changes in oxygen flux and will eventually render the electrode
unserviceable and beyond economic repair. The disc will require
replacement.
Cleaning with Unsuitable Materials.
Hansatech Instruments Ltd offer the S16 cleaning kit for maintenance of the oxygen
electrode disc. The kit contains special cleaning pastes which are recommended for
optimal electrode performance. In the absence of this kit, users have resorted to cleaning
the electrode with various abrasive compounds including commercial metal polishes or
smoker's toothpastes etc. We would caution against the use of inappropriate materials as
they may cause irreversible damage to the electrode. Some metal polishes are harmful to
the disc as they can contain an ammonia base or solvent additive.
These substances can cause irreversible poisoning to the platinum
cathode and could also cause significant damage to the epoxy
resin. Excessive cleaning with abrasive substances can badly
scratch and alter the profile of the electrode dome which may cause
an uneven layer of electrolyte across the cathode leading to
instability, drift and lack of responsiveness of the oxygen signal.
The S16 electrode cleaning kit is supplied with all Oxygraph and Oxytherm Systems and
can also be obtained by contacting us directly. Details of the cleaning procedure can be
found in the Cleaning and Maintenance section.
Chapter 2
DW1/AD Electrode Chamber
16
Introduction to DW1/AD
The DW1/AD oxygen electrode chamber provides a versatile solution to measurements of
dissolved oxygen in liquid-phase. It can be used for a wide range of applications from
basic teaching through to more advanced research assays.
The design and construction of DW1/AD provides excellent sample visibility through a
clear, cast acrylic body and excellent temperature control is achieved when connected to a
thermoregulated circulating water bath. The sample is housed within a borosilicate glass
reaction vessel.
The DW1/AD provides a valuable, reliable tool in any laboratory where dissolved oxygen
measurements are required.
Features of DW1/AD
A B
Figure 1
C
I
D
H
E
A: Lugs for A2 top plate key (see instructions on disassembly of DW1/AD).

B: Top plate.
C: Water Jacket.
D: Borosilicate glass reaction vessel.
E: S1 electrode disc.
F: Standard plunger assembly.
G: Non-gas tight plunger assembly (supplied with DW1).
H: Base ring.
I: Water jacket to circulating water bath connectors.
17
Transmission Properties of DW1/AD.
The water jacket of DW1/AD is constructed from cast acrylic to ensure good transmission
of photosynthetically active radiation (PAR) allowing external light sources to be used in
photosynthesis studies. The borosilicate glass reaction vessel also transmits throughout
the PAR (400 - 700 nm) wavelengths.
Figure 2.
% Transmission
Transmission profiles of 10 mm thickness Acrylic

( ) and borosilicate (_ _) across ultra violet (UV),
photosynthetically active radiation (PAR) and infra-
red (IR) wavelengths.
UVC UVB UVA PAR IR
Wavelength (nm)
Setting Up DW1/AD
Installing the Electrode Disc.
Before installing the electrode disc into DW1/AD, it should be correctly prepared according
to the guidelines set out in the Electrode Preparation & Maintenance section.
Once the disc has been prepared and suitably tested, it is now ready to be installed in the
DW1/AD electrode chamber.
Remove the base ring from the DW1/AD electrode chamber and place the prepared
electrode disc onto the top.
Offer the base ring and disc assembly up into the underside of the DW1/AD so that the
electrode cable connection fits with the key way on the chamber base thread. Gently hold
the base in position while threading the base ring onto the DW1/AD so that it is slightly
more than finger tight.
It is important not to over-tighten the base ring as

this will cause the membrane to stretch over the electrode
dome. During measurement, when the membrane starts to
relax, the measured signal will drift.
Place a small amount of water (0.5 to 1 ml) in the reaction vessel in order to prevent the
electrode from drying out whilst other preparations are made prior to measurement.
18
Temperature Control.
All experimental assays will have an optimum temperature at which the sample will be
most efficient in either evolving or consuming oxygen and it is important that this
experimental temperature is maintained at all times throughout the duration of the
experiment. This includes ensuring that any additions to be made to the reaction vessel
during the experiment are pre-equilibrated to the assay temperature before being added.
According to the studies of G.A. Truesdale and A.L. Downing (The solubility of oxygen in
water, 1954, Nature 173: 1236), at any given temperature and atmospheric pressure, air
saturated, de-ionised water contains a known concentration of dissolved oxygen which
may be calculated mathematically.
Oxygen concentration in a liquid decreases as temperature increases which is why it is

important that the assay temperature is established for both the sample and any additions
prior to the commencement of the experiment. However, in addition to this effect, the
oxygen electrode disc itself is also sensitive to temperature.
The electrode disc signal increases with an increase in temperature. In assays where
higher temperatures are used (such as mitochondrial respiration studies), it is
recommended that the disc is fully equilibrated to the assay temperature before any
calibration or measurement takes place. This can take up to 15 minutes.
The assay temperature needs to be decided as one of the initial considerations of the
experiment as the apparatus is calibrated based on a specified temperature.
DW1/AD provides temperature control to the sample and disc via a water jacket which
encapsulates the reaction / sample vessel. Since the vessel is constructed from precision
bore borosilicate glass, heat transfer between the circulating water and the sample is very
efficient, thus maintaining effective temperature control.
The water jacket should be connected, using the plastic connectors provided, to the flow
and return tubes from a thermoregulated circulating water bath.
Connect the flow tube to the lower water jacket connector (Figure 1, I) and the return tube
to the upper water jacket connector. This ensures that a fresh supply of water is constantly
flowing around the DW1 water jacket and assists in dispersing any air bubbles which will
rise naturally to the upper outlet port.
The tubing lengths between the circulator and the electrode
chamber should be kept minimal in order to maintain good
temperature control. A circulator providing a flow rate of 6
litres/min or more is recommended.
Sample Stirring.
Electrochemical reactions taking place at the cathode of the electrode disc require the
consumption of oxygen in order to produce an electric current. This current is then
digitised by an electrode control unit and presented as a chart recorder emulation in
the Oxygraph Plus software.
19
The nature of this process means that as oxygen is continuously consumed at the
cathode, an oxygen depleted layer is produced immediately above the cathode. As the
rate of consumption by the disc is greater than the rate of oxygen diffusion through liquid,
the measured signal from the electrode disc will continuously decrease during
measurement. This problem is overcome by continuously stirring the sample in order to
replenish the depleted layer at the cathode and to ensure that the dissolved oxygen within
the sample is kept evenly distributed throughout the reaction vessel.
The magnetic stirrer is an integral feature of the Oxygraph control unit and is located on
the top of the system.
Stirring of the sample is achieved by placing a magnetic follower or "Flea" into the reaction
/ sample vessel itself. Activating the stirrer rotates the magnet beneath the stirrer cap at
user definable speeds of between 150 and 900 RPM. The flea tracks the magnetic stirrer,
rotating just above the cathode of the electrode so as not to cause any damage to the
membrane preparation.
2 types of flea are available:

PTFE coated magnet. - This is the standard flea supplied with the systems. It consists of a
small magnetic bar coated in PTFE.
Glass flea. - Glass fleas consist of several fine strands of iron sealed into a thin glass
capillary tube.
Each type of flea has advantages and disadvantages of use. For example, for experiments
where very small changes in oxygen tension are to be measured, some users prefer the
glass fleas as PTFE is known to absorb a very minute amount of oxygen. In the large
majority of applications, this absorption is not an issue. Glass fleas are not magnetic
themselves so they are not as reliable at tracking the rotating magnet in more viscous
samples i.e. the maximum speed of stirring would be lower than that of the PTFE coated
flea. etc., etc......
Care & Maintenance.

The DW1/AD electrode chamber should be periodically dismantled and thoroughly cleaned
in order to maintain good working order as particulates in the coolant will gather in the
water jacket and obscure light transmission. It is also possible that after several years use,
some of the O-rings which seal the reaction vessel (Figure 4, E) from the water jacket may
perish causing leaks.
If a trap or filter is installed in the cooler-circulator outlet

the risk of contaminating the electrode with particulates is
reduced.
Figure 3
Disassembling DW1/AD.
On supply, a custom dismantling kit (A3 Top plate key and alignment jig,
see Figure 3) was included with the DW1/AD. This kit is used to remove
the top plate of the chamber so that the reaction / sample vessel may be
safely removed for cleaning and the inside of the water jacket can be
cleaned.
20
Figure 4 shows a cross-section of an electrode chamber prepared for disassembly with the
alignment tool inserted into the reaction vessel and the disassembly key inserted into the
two indentations in the top plate of the unit. The top plate may be removed by gently turning
the key anti clockwise.
Figure 4.
A Disassembly of DW1/AD.
Remove the electrode disc from the base of the chamber.
Pass the alignment jig (D) through the reaction vessel (C)
B from the top of the chamber. The disassembly key (A) is
C located into two indentations (B) in the top plate of the
chamber. The key is now directly above the alignment tool
thus allowing the top plate to be gently unscrewed in an
E anti clockwise direction. When re-assembling the
electrode chamber, use the alignment jig to ensure that
the cleaned reaction vessel is exactly perpendicular to
both the top and bottom plates before screwing the top
D plate into place. If the vessel is misaligned, it may fracture
during re-assembly or fail to seal correctly against the O-
rings (E) at either end of the vessel causing the chamber
to leak.
Cleaning DW1/AD.
Once the top plate has been removed the components can be extracted and cleaned with
a mild detergent or replaced as necessary. (A list of spares and replacement part
catalogue numbers is given on Page 22).
Do not use harsh abrasives to clean the unit as they may

damage the acrylic water jacket and significantly reduce
light transmission. Organic solvents will react with the
acrylic causing fracturing.
Some applications use powerful respiratory inhibitors such as Rotenone which can prove
very difficult to completely remove from the internal walls of the reaction vessel. This can
lead to unwanted contamination of subsequent reaction mixtures. Conventional cleansing
methods, as described previously, may prove ineffective and an alternative method is
required.
Ethanol is widely considered to be an effective solvent in the removal of Rotenone from the
reaction vessel. Hansatech Instruments strongly cautions against the use of this substance
as any spillage on to the acrylic parts of the DW1/AD will cause irreversible damage to the
main body of the chamber.
Through experience, the most desirable solution has been to use a separate reaction
vessel when performing applications utilising Rotenone and other respiratory inhibitors. A
pack of 2 spare reaction / sample vessels are supplied as standard with the DW1/AD.
However, further spares (S3) can be obtained from Hansatech Instruments (or relevant
International Distributor). Alternatively, if possible, reserve a DW1/AD chamber specifically
for use with Rotenone.
21
Reassembling DW1/AD.
Three O-rings are used to make the unit watertight. The largest seals the top plate to the
body of the water-jacket; the two others seal the reaction vessel from the water-jacket
(Figure 4, E). The smaller of the two reaction vessel O-rings should fitted to the base plate
of the unit whilst the larger one is inserted into the top plate.
The alignment jig and key are required for reassembly:
Slide the top plate (with fitted O-ring) onto the alignment jig so that the tapered end is
pointing away from the upper face of the top plate. Fit the reaction / sample vessel on to
the alignment jig so that it fits securely up to the top plate O-ring.
Place the reaction vessel, top plate and alignment jig together with the main body of the
DW1/AD so that the tapered end of the jig fits through the floor of the water jacket. The
bottom of the reaction vessel should fit securely on to the lower O-ring and the top plate
should locate on to the thread of the water jacket. The top plate and reaction vessel are
then sealed by tightening in a clockwise manner with the key. The alignment jig can then
be removed by pushing it up from the floor of the water jacket.
If the alignment jig is not used the reaction chamber may

not locate into the water jacket vertically causing leakage or
possible fracture of the reaction vessel during tightening.
Incorrect alignment may also prevent insertion of the
plunger.
DW1/AD Plunger Assembly.

Diffusion of atmospheric oxygen into the reaction vessel is prevented by the plunger
assembly. This assembly can be adjusted using the screw thread to give a variable
reaction vessel volume of 0.2 - 2.5 ml. The capillary hole in the plunger allows sampling
and additions to be made to the reaction mixture via a Hamilton syringe with minimal
atmospheric oxygen diffusion into the reaction vessel.
The plunger provides an O ring seal between the plunger and the inner wall of the glass
reaction vessel. A central stopper is also provided to plug the central bore in the plunger.
This stopper should not be fitted until the plunger height has been correctly adjusted. After
adjustment of the plunger assembly to the desired height, the stopper may be refitted to the
central bore. Once fitted the plunger assembly provides a gas-tight seal which allows the
DW1/AD chamber to be used for gas-phase measurement, or used in combination with
special equipment for hydrogen measurement (please ask for details). The plunger
assembly also allows measurements to be made on very small reaction volumes (less than
0.5ml approx.).
22
Spare Parts List.

Figure 5 below shows the spare parts and catalogue numbers that are available for the
DW1 electrode chamber. To order any of these parts, please contact Hansatech
Instruments Ltd.
Figure 5.
Description Catalogue Number

A Plunger Screw 820024
B Plunger Nut 820025
C Borosilicate Glass Reaction Vessel S3 (pack of 2)
D Magnetic Follower S2/P or S2/G
E Base Plate O-ring Part of S5 O-ring Kit
F Electrode Disc S1
G Base Ring 820023
H Water Jacket & Bottom Plate Assembly 941020
I Water Jacket Connectors 820026 (2 required)
J Top Plate 820021
- Top Plate Key & Alignment Jig A3
- Stopper Cap for DW1/AD 716161
Chapter 3
Oxygraph Electrode
Control Unit
24
Introduction to Oxygraph.
The Oxygraph respirometer is designed to provide computer control of Oxygen uptake or
evolution measurements across a broad range of applications from studies of cellular and
mitochondrial respiration to photosynthesis of isolated chloroplast suspensions or leaf-discs.
The system may be configured as a single or multi-channel system in order to make
measurements of oxygen in either liquid-phase or gas-phase. Simultaneous recording of an
optional auxiliary input signal (e.g. temperature, pH, fluorescence, TPP+ or other specific ion
electrodes etc) is also possible using the appropriate apparatus coupled via the OXY/AUX
adapter cable.
A system comprises a minimum of one (maximum of eight) control units linked together in a
chain to the serial port of an IBM® compatible Windows® computer. Each control unit
features a built-in magnetic stirrer and all the electronics required to control and measure the
signal from the oxygen electrode. Oxygraph is compatible with all existing liquid-phase and
gas-phase Hansatech oxygen electrode chambers and accessories. Oxygraph control units
may be freely mixed with Oxytherm electrode control units within a multi-channel system.
A custom Windows® software package, Oxygraph Plus, permits independent control over
the gain and back-off applied to each electrode unit with simple calibration routines for both
liquid and gas-phase measurements. Data from each of the electrodes is plotted as a chart-
recorder emulation in "real-time". Completed experiments are saved to a *.CSV (Comma
Separated Values) which then may be opened in other Windows® applications.
Setting Up Oxygraph.
Connections to the Oxygraph.
Before making any measurements, it is important that the system is set up correctly.
The diagram on the following page shows the various connections that must be made to the
control units.
Ensure that the 12V power supply is connected to the 12V input socket on the control
unit. Confirmation that this connection has been made is shown by an illuminated green
LED on the front panel of the control unit.
Connect the RS232 serial cable to an available serial port on the PC. Connect the other
end of the RS232 cable to the INPUT RS232 connection on the rear of the control unit. If
more than 1 control unit is to be used, connect a second serial cable from the OUTPUT
connection of the first channel control unit to the INPUT connection on the second
channel unit. More details of multi-channel systems can be found in the Multi-channel
systems section.
Connect the prepared electrode disc to the control unit via the electrode connection
cable.
If an auxiliary signal is to be recorded, users will require an OXY/AUX connection cable.
This cable connects to the Electrode Disc Input on the rear of the Oxygraph and has a
flying lead to which the prepared electrode disc and connection cable should be
connected.
25
Connections to the Oxygraph
Multi-Channel Systems.
Oxygraph control units are capable of operating either as stand alone systems or in
complex multi-channel systems consisting of up to 8 control units. These control units may
consist of a mixture of Oxygraph and Oxytherm units linked together via an RS232 serial
chain, all connected to and operated by a single PC.
Each of the control units in the serial chain must be calibrated independently to allow for
differences in electrode performance. As a result the Oxygraph Plus software must identify
each of the units in the chain in order to apply the correct calibration factor to each of the
acquired data sets.
This is achieved by referencing a unique serial number that is written to a memory device
inside each control unit at the factory and a box identification number (ID) that is assigned
by the software and stored to memory within the control unit. The first step during
hardware configuration is to assign box ID numbers to each of the control units within the
chain. New control units are supplied with a box ID setting of zero to indicate that they are
UNASSIGNED. The following guidelines should be used in order to set up a system for
multi-channel measurements.
When running a multi-channel system, all units must be

calibrated. Oxygraph Plus software will not allow a
recording from a system with calibrated and uncalibrated
control units present.
What Additional Hardware is Required?
It is perfectly possible to link 2 Oxygraph control units together in a multi-channel setup

without purchasing any additional hardware. However, there are options available which
will cut the cost of multi-channel systems.
26
Oxygraph control units draw very little power. Consequently, it is possible to run up to 8
individual control units from a single power supply with the aid of the OXY/PDU Power
Distribution Unit.
In addition, it would not be necessary to purchase a full set of spares and accessories with
every control unit. Hansatech Instruments Ltd recommend the purchase of 1 spares and
accessories kit for every 4 control units.
The most cost effective method of purchasing, for example, a system comprising 4 control
units would include the following items:
1 x Oxygraph System complete

Consists of control unit, DW1/AD electrode chamber, S1 electrode disc, S1/ADL
connection cable, RS232 serial cable, A2 membrane applicator, A3 top plate key and
alignment jig, S2/P magnetic followers, S3 spare reaction vessels, S4 reel of
membrane, S7A spare o-rings, S16 electrode cleaning kit, 12V power supply, mains
cable and Oxygraph Plus software on mini CD ROM.
3 x Oxyg2 additional channel Oxygraph control units including 3 x 12V power cable, 3 x
S1/ADL electrode connection cables and RS232 serial cables.
3 x DW1/AD electrode chamber including 3 x S1 electrode disc
1 x OXY/PDU power distribution unit.
For further information, please contact Hansatech Instruments Ltd at the address listed at
the rear of this manual.
Linking Control Units Together.
Multiple control units are simply linked together via standard RS232 serial cables. Please
follow the procedure below to configure a multi-channel system:
Connect the INPUT socket of the first channel control unit to the PC using an RS232
serial cable.
Run the Oxygraph Plus software. A message will be displayed indicating that the
control unit has been found and information regarding control unit type (Oxygraph or
Oxytherm), serial number and calibration data is shown. As mentioned above, the
control unit is assigned a unique identification number so that the software can
distinguish between multiple control units.
Select OK on the BOX ID INFORMATION window to proceed to the main screen.
To attach the next channel control unit, connect the OUTPUT socket of the first channel
control unit to the INPUT of the second channel control unit via an RS232 serial cable.
Ensure that the second channel unit is powered either by it's own supply or using the
OXY/PDU distribution unit.
On the toolbar, select the icon to initiate a scan for new control units.
When the new channels is found, a message is displayed indicating that a new control
unit has been found with an ID number of 0. Oxygraph Plus assigns the next available
number as the new ID. In this case, the ID is 2.
Repeat this process with all remaining control units, adding one unit at a time.
27
Pitfalls!!
In a multi-channel system, it is crucial that all the control unit ID numbers are set to 0
before building the system. When leaving the factory, an ID of 0 is written to the unit
memory by default. However, once the system has been used and new ID numbers have
been assigned, it is important to be aware of which control unit is used as which channel.
If 2 units with identical ID numbers (other than 0) are connected to a serial chain, neither of
these units will be recognised by the software. In this case, the following procedure should
be followed.
Clearing a control unit ID.
Connect the control unit to be reset only to the PC serial port.

Click the icon to recognise the attached control unit.
Once confirmation has been accepted, select HARDWARE > BOX ID - CLEAR from the
menu options.
The software will present a message with the details of the connected control unit to be
reset.
Once cleared, the ID number of the control unit will be reset to 0. This unit may then be
re-connected to the serial chain and is recognised by the software by following the
procedure described above.
Repeat this procedure for any other control units with conflicting ID numbers.
Using Multi-Channel Systems.
Channel Calibration and Configuration.
All control units within the serial chain must be treated as individual control units and may
not be simultaneously calibrated or configured. Any function which requires individual
control mechanisms have in place the ability to select the
required channel number for which actions are to be
performed.
Stirring.
The magnetic stirrer

control dialogue (see
diagram opposite) allows
magnetic stirrers in
individual control units to
be operated and
configured separately or
universally under a single
control.
28
Signal Display.
Each control unit is capable of recording and plotting 2 separate traces on the Oxygraph
Plus graph screen. In addition to the oxygen signal recorded from the electrode disc, an
auxiliary signal of 0 - 4V may also be plotted (please refer to the recording auxiliary signals
section for further details). This means that in a multi-channel system, there is the potential
of plotting up to 16 traces per screen (2 signals from each of the maximum of 8 control
units). For this reason, all traces may be customised in order to differentiate between
signals according to colour and data point style from the window shown below.
Disable Boxes.
The disable box function can be used when data

from particular control units within the multi-channel
system is not required. In these cases, the control
units may simply be disabled to avoid removing the
units from the system completely. The disable boxes
function is accessed by selecting HARDWARE >
DISABLE BOXES from the menu bar. A disabled
control unit can be re-enabled by either selecting
HARDWARE > SCAN FOR BOXES from the menu
bar or by clicking the icon on the toolbar.
29
System Calibration.
Liquid-Phase Calibration.
Before any measurements can take place, the electrode disc must be calibrated so that
the electrical signal received from the disc can be presented as actual calibrated units
(nmol/ml). Calibrating the disc for liquid-phase measurements involves a two step
procedure in which the signal from the oxygen electrode is referenced to 2 known oxygen
concentrations in order to derive an Offset and Calibration Factor.
The 2 calibration steps are:
Air Line.
saturated, deionised water contains a known concentration of dissolved oxygen which may
be calculated mathematically. The following information is used by the Oxygraph Plus
software in order to accurately reference the electrical signal from the electrode for the air
line stage of calibration:
This data is based on measurements of dissolved oxygen in water at the given

temperature and standard atmospheric pressure published by Truesdale & Downing
(Nature 173:1236, 1954).
Temperature (°C) Oxygen (PPM) Oxygen (nmol/ml)

0 14.16 442.5
5 12.37 386.6
10 10.92 341.3
15 9.76 305
20 8.84 276.3
25 8.11 253.4
30 7.52 235
35 7.02 219.4
The formula used in calculating the oxygen values in the table is as follows:
Cs=14.16 - (0.394 * T) + (0.007714 * T^2) - (0.0000646 * T^3)
{Where Cs is the oxygen saturated concentration in ppm and T is temperature in °C}
0.03125 µmol/ml
1 ppm is equivalent to 1µg/ml or (1µg/32g/mol)= or
31.25 nmol/ml
30
At any atmospheric pressure other than standard

atmospheric (760mm Hg or 101.32 KPa), the value obtained
is adjusted by a factor of
Current atmospheric pressure

standard atmospheric pressure.
The calibration factor is therefore calculated as is shown in the diagram below:
In the example calibration above, the signal measured from the electrode in a stirred
sample of air-saturated, de-ionised water was 2000 mV. In the first step of the calibration,
the user is prompted to enter assay temperature and pressure variables. These entries are
referenced against the look-up tables and formula of Truesdale and Downing (see above)
in order to establish the amount of oxygen present within the chamber. This value, in this
example, is 253.4 nmol/ml.
The measured 2000 mV must therefore, be married to the nmol/ml value in order to obtain
the calibration factor.
Zero Oxygen Line.
In a perfect world, an oxygen concentration of zero would produce an electrical signal of

zero from the electrode disc. However, the electrode does have what is known as residual
current which does give a small signal in zero oxygen concentration.
It is necessary to subtract the value of signal caused by residual current from all recorded
data points in order to give accurate results. This is known as the Offset. Zero oxygen
within the reaction chamber can be achieved in 2 ways:
31
Sodium Dithionite. - The addition of a strong oxygen reducing agent such as sodium
dithionite provides a good method of achieving zero oxygen. However, great care must
be taken to remove all traces of the dithionite before commencing measurement as
even a minute amount of remaining dithionite will have a serious effect on the oxygen
concentration of the sample. Once removed, the chamber should be thoroughly rinsed
with distilled water several times.
It is crucial that care is taken, when removing the sodium
dithionite and water from the reaction vessel, that the
membrane of the electrode is not damaged. The ideal
method of removing liquid from the chamber is to use an
aspirator with a soft, rubber tip (mind the flea!!!). If an
aspirator is not available, carefully use a Pasteur pipette to
remove the liquid.
Nitrogen. - A more convenient method of achieving zero oxygen is to bubble nitrogen

gas through the liquid in the reaction vessel in order to displace all the oxygen. This
method is in some ways safer than the use of sodium dithionite as there is no risk of
contaminating the actual sample during the measurement and there is less risk of
damaging the membrane of the disc. However, the use of nitrogen is a slower process
and to achieve zero line is more difficult than dithionite.
Performing a Liquid-Phase Calibration.
Please follow the guidelines below for the liquid-phase calibration sequence:
Prepare the disc as described in the Electrode Preparation and Maintenance section
and install into the base of the electrode chamber.
Connect the electrode disc to the rear of the control unit using the electrode connection
cable.
Place approx. 2ml of air saturated, deionised water into the reaction vessel. Air
saturated water is obtained by vigorously shaking a small quantity of deionised water
(approx. 50ml) in a large conical flask (approx. 1L).
Connect the water jacket of the electrode chamber to the circulating water bath for
temperature control purposes and ensure that the sample and electrode disc equilibrate
to the temperature required by the assay before commencing calibration.
It is important to remember that the sample of air saturated

water should be equilibrated to the assay temperature
before the calibration procedure begins. It can be pre-
equilibrated in a water bath or simply allowed sufficient
time to reach temperature once added to the reaction
vessel.
The liquid-phase calibration sequence is activated either from the CALIBRATE > LIQUID
PHASE CALIBRATION menu option or directly from the icon on the toolbar. Once
activated, the following window is generated.
32
Entering the appropriate temperature and atmospheric pressure into this window allows
the Oxygraph Plus software to accurately reference the air line. Whatever signal is
recorded from the electrode disc during the air line phase will be subjected to the formula
of Truesdale and Downing (see previous section) in order to derive the calibration factor.
Once the correct variables have been entered into this window, proceed by pressing OK.
The following window is generated.
Assay temperature and ambient pressure variable defined in the previous window are
displayed as confirmation. The stirrer setting should provide efficient, smooth stirring of the
sample without causing a noisy signal. Please refer to the Stirrers section for details on
selecting an appropriate stirring rate. Once the stirrer setting has been selected, proceed
by pressing OK. The following window is generated.
33
Observe the mV signal in the window above and wait until the prompt that a plateau has
been reached. Once the signal is deemed to be stable, the OK button should be pressed
in order to proceed.
In the case of a new or well maintained electrode disc, you

would normally expect to see a signal of approx. 1800mV
when measuring air line in air saturated water.
If the signal from the electrode disc does not become stable after several minutes, this
could be indicative of either one or a combination of several problems. For details on how
to isolate the fault to either the electrode disc, the control unit or the electrode connection
cable, please refer to the System Diagnostics section.
The second stage of the calibration procedure is to establish the zero oxygen line in order
to determine the Offset (see previous section). To perform this stage, use either of the
following methods:
Nitrogen gas. Bubble nitrogen gas into the reaction vessel in order to displace all the
oxygen in the sample.
Sodium Dithionite. A few crystals of this strong reducing agent is sufficient to reduce all
the oxygen dissolved in the sample.
In the case of a new or well maintained electrode disc, you

would normally expect to see a signal of less than 1% of the
signal observed during the air line stage. For example, if the
air line signal was 1802 mV, you would expect the zero line
to measure less than 18.02 mV.
34
If the zero oxygen line recorded from the disc seems to be too high, this could be
indicative of one of several things:
Insufficient oxygen reduction/removal from sample. Sodium dithionite has a finite

lifespan before it becomes ineffective. If you are using dithionite that is several months
old, try using a fresh batch. Bubbling nitrogen into the chamber must be performed
carefully in order to ensure that all oxygen has been displaced.
Damaged electrode disc. Unfortunately, most cases of high residual current reported
are due to insufficient electrode maintenance. If the electrode is left to dry out after
measurements have been completed, Kcl crystallisation can irreversibly damage the
disc by penetrating any minute fissures in the seal between the platinum cathode and
the epoxy electrode dome and forcing the seal apart. This problem has a "snowball"
effect as every time the disc is prepared from now on, Kcl will enter the fissures causing
them to widen. Please refer to the Importance of Electrode Maintenance feature for
further details of how to diagnose this fault.
Once the zero oxygen stage has been completed, press OK to proceed. The following
window is generated.
Viewing Calibration Details.
Once the system has been successfully calibrated in either liquid or gas-phase, details of
the calibration may be viewed in a separate window. This window is accessed from the
CALIBRATE > CALIBRATION DETAILS command on the menu bar. All aspects of the
calibration are shown in the window as shown in the diagram below:
35
The Date column is relevant should the apparatus be intended for long measurement
assays lasting more than a day or if the electrode is to be left prepared and in situ (please
note that this should only be performed in liquid-phase electrode chambers as electrodes
in gas-phase "dry out"). A calibration warning interval may be set so that after a given
number of days post calibration, the user is prompted that the calibration has expired when
the software is initially run.
It is important to remember that over time, a polarised disc

builds up a deposit of black silver oxide due to the nature of
the electrochemical reactions taking place. A disc that is
left measuring for more than a day may behave differently
at the end of the assay than at the beginning. Please refer to
the Electrode Preparation and Maintenance section.
36
Oxygraph Operation.
Oxygraph Plus.
Overview.
Oxygraph Plus software has been designed to allow the maximum flexibility possible for
the Oxygraph control unit. These systems are in use world wide for a broad range of
different applications in both liquid and gas-phase measurement environments. The
software must therefore encompass a range of features that may be adapted to the needs
of many different users.
Oxygraph Plus provides a powerful instrument control and data acquisition / analysis tool
for students and researchers alike with the ability to save recorded data as CSV (Comma
Separated Values) file which may be opened directly into Microsoft Excel for advanced
statistical analysis and the comprehensive Print feature which provides printed information
on all aspects of the experiment from calibration and gain settings to marked events during
the course of the experiment in addition to the recorded oxygen trace.
Running Oxygraph Plus.
Oxygraph Plus communicates with the control unit as standard by RS232 serial
communications. However, since many of the modern laptop/notebook computers do not
have serial ports, the HAN/USB USB - RS232 adapter may be used. Please contact
Hansatech Instruments for further details.
Oxygraph Plus assumes communications through serial comm port 1 as default. If a

control unit is not found on this port, the following window is generated.
37
If an incorrect port is selected, the window will be further displayed until communications
are established. If communications cannot be established, check that the serial cable is
properly connected both at the control unit and the PC and if necessary, try a different
RS232 cable. Ensure that the control unit is powered.
Accessory hardware within the computer may already occupy the COMM port even though
there is no apparent connection at the COMM socket. This is often the case with internal
modem cards which typically use one of the serial ports for data transfer. Such devices are
usually configured to load device drivers during computer boot-up. These must be
removed to permit normal serial port operation. Check the System Properties on the PC
for further information.
If communications still cannot be established, please contact Hansatech Instruments for

further advice.
The screenshot below shows the main features of the Oxygraph Plus software.
The different features of the software are explained in the following sections of this
document.
Graph Functions.
The graph area of the Oxygraph Plus software allows the signal from the electrode disc
(and optional auxiliary input) to be plotted on the PC screen as a real-time chart recorder
emulation. Various controls and functions exist to facilitate the way in which part or all of
an experiment is displayed on screen.
38
Recording a Signal.
Recording is activated either by selecting HARDWARE > START RECORDING from the
menu bar or by pressing the icon found on the toolbar. Any unsaved data onscreen will
cause the following warning to appear:
Selecting Overwrite will delete any information currently displayed before creating a new
trace. Append begins the new recording after the final data point of the displayed trace. A
Restart event mark is entered to signify the beginning of the new trace.
The recorded signal is plotted to the graph at the pre-defined data acquisition rate. Once
the trace reaches to right side of the graph, the screen begins to scroll. Data recorded that
is not in view may be easily reviewed by moving the horizontal scroll bar at the bottom of
the graph. This does not affect normal measurement.
During recording, most of the functions of the software may still be used. Any function
used that may have a baring on the displayed data (such as gain/back off adjustment,
stirrer speed alteration) is event marked automatically by the software.
The recording is stopped either by selecting HARDWARE > STOP RECORDING from the
menu bar or by pressing the icon.
Setting the Measurement Axes.
Oxygraph Plus features a comprehensive range of zoom controls in order that the
recorded data may be presented most efficiently on screen. They can be accessed either
by selecting one of the options in the GRAPH menu bar option or by clicking one of the
following icons:
Zoom Window ( ). - Clicking this icon allows a selected portion of the trace to be
enlarged. Clicking this icon changes the mouse cursor to cross hairs. Clicking on the graph
activates the selection window. Drag the window to highlight the required section and click
the mouse again to zoom.
Auto Zoom ( ). - Clicking this icon automatically re-sizes the axes to efficiently present
all recorded data.
Zoom XY ( ). - Clicking this icon opens the manual Set Axes window.
39
Axes are adjusted by entering in the required from and to values for oxygen, time and
optional auxiliary axes. The gridlines on the graph can also be modified in this screen.
Increase Zoom ( ). - Clicking this function zooms in on the center section of the screen.
Decrease Zoom ( ). - Clicking this function zooms out of the screen.
Undo Zoom ( ). - Click this icon to undo the previous zoom command.
Trace Settings.
Each control unit is capable of recording and plotting 2 separate traces on the Oxygraph
Plus graph screen. In addition to the oxygen signal recorded from the electrode disc, an
auxiliary signal of 0 - 4V may also be plotted (refer to the recording auxiliary signals
section for further details). This means that in a multi-channel system, there is the potential
of plotting up to 16 traces per screen (2 signals from each of the maximum of 8 control
units). For this reason, all traces may be customised in order to facilitate differentiation
between signals according to colour and data point style from the window shown below.
40
The Data Bar.
The Oxygraph Plus data bar presents a digital read out of the signals from the control unit.
Data from multi-channel systems will be displayed on individual lines. The information
displayed is as follows:
Trace colours. Both oxygen and auxiliary trace colours are represented in the data bar
so that information can easily be matched to traces on screen in multi channel systems.
Oxygen electrode signal. Presented in mV when uncalibrated and nmols/ml (liquid-
phase) or µmol/RTA (gas-phase) when calibrated.
Calibration indicator. Indicates whether the channel is:
= Calibrated
= Calibration has expired (see View Calibration Details section).
= Uncalibrated.
Live rate. Displays the Live Rate according to the options selected (please refer to the
Rate section for further details).
Auxiliary signal. Displays the signal from an auxiliary input in arbitrary units if enabled
(please see Recording Auxiliary Signals for further details).
Set and actual chamber temperature. Oxytherm Only.
The data bar is positioned by default on the right hand side of the Oxygraph Plus screen
but can however, be move to either top, bottom or left side of the screen. The bar can be
moved by right clicking and selecting MOVE. The data bar will then follow the mouse
cursor around the screen. Clicking the mouse at the required position will place the data
bar.
41
Clicking the right mouse button on the data bar brings up a separate window of options as
shown in the diagram above. This window allows any of the columns (except for the
oxygen signal, this is always visible) to be hidden or shown simply by clicking the
appropriate column heading. The default setting shows the live rate along with the oxygen
signal, trace colour and calibration status which are displayed as standard and are not
selectable.
The data bar also has an autohide (Lock) feature which can be switched on or off either
from the right click menu or by using the small padlock icon in the bottom right corner of
the data bar. This allows the data bar to collapse showing only the oxygen signal.
Hovering the mouse over the data bar expands to reveal any other columns which are
currently selected.
Other features of the data bar are selectable font size (either small or large fonts) and a
flashing recording indicator in the bottom left corner of the data bar.
The Menu Bar.
The Oxygraph Plus menu bar provides access to all the functions and features of the
software. The functions and commands are organised into several distinct menu bar
categories.
Files Menu.
New Creates a new document.

Open Opens a previously save document.
Overlay Opens a previously saved document and overlays on to a file already
displayed on screen.
Save File save functions.
Save As
----------
Print Printing functions.
Print Preview Please refer to the Print
Print Setup Files section for further information.
----------
(Recently viewed /
recorded files. Click
on the relevant title
to open)
----------
Exit Quit the application. Prompts to save the active file.
42
Hardware Menu.
Start Recording Recording controls. Please
Stop Recording refer to the Recording Data section for further information.
----------
Channel Configuration Setup the gain and back-off for each channel. Refer to the
Channel Configuration section for further information.
Auxiliary Channels Auxiliary channel controls. Refer to the Recording Auxiliary
Data section for further information.
Stirrer Speed Sets the stirrer speed. Refer to the Stirrers section for further
information.
Temperature Control Oxytherm only.
----------
Scan for Boxes Scans for control units currently connected to the PC. Once
found, serial number, type (Oxygraph or Oxytherm) and
calibration status are displayed.
Disable Boxes Allows active control units to be disabled. Refer to the Disable
Control Units section for further information.
Box ID - Clear Clears the unique identification number of a control unit. Refer
to the Clearing a Control Unit ID section for further information.
Box Test Performs a range of manual and automatic diagnostic tests on
the control unit. Refer to the System Diagnostics section for
further information.
Calibrate Menu.
Liquid Phase Calibration Launches the liquid-phase calibration routine. Refer to the
Liquid-Phase Calibration section for further information.
Gas Phase Calibration Launches the gas-phase calibration routine.
----------
Calibration Details Displays details of current calibration settings including expiry
Set Warning Interval warning details. Refer to the View Calibration Details section
for further information.
View Menu.
File Information View full details of the active file including filename and path,
date, start time, acquisition rate, version, comment and
information. Refer to the File Information section for further
information.
Toolbar Toggles toolbar on and off.
Status Bar Toggles status bar on and off.
Graph Menu.
Setup Trace Colours Allows trace colour and data point marker to be customised.
Refer to the Trace Settings section for further information.
Display Overlayed Traces Hides any overlayed traces when unchecked.
----------
Zoom Window
Zoom Auto
Zoom XY Axes zoom controls. Refer to the Set Measurement Axes
Zoom Plus section for further information.
Zoom Minus
Zoom Undo
43
Data Bar Menu.

Rate Data
Aux Data
Set Temperature Data bar controls. Refer to the Data Bar section for further
Actual Temperature information.
----------
Small Font
Move
Locked
Tools Menu.
Graph Data Toggles between the graph screen and the tabulated data
Tabulate Data screen. Refer to the Tabulated Data section for further
information.
----------
Add Event Mark
Edit Event Marks Event mark controls. Refer to the Event Marking section for
Delete Event Marks further information.
Rate Menu.
Setup Live Rate Display Allows the number of data points over which the live rate is
calculated to be amended. Refer to the Setup Live Rate
section for further information.
----------
Rate Cursors
Enter Rate Cursor Times
Display Rate Table Rate measurement analysis controls. Refer to the Rate
Add Rate to Table Measurements section for further information.
Select Channels
Setup ‘Line of Best Fit’
Help Menu.
Help Launches the Oxygraph Plus HTML help files.
About Oxygraph Displays Oxygraph Plus version and copyright information.
The Toolbar.
The Oxygraph Plus tool bar provides access to the main functions and features of the
software.
Opens previously saved data files.

Save active data file. Refer to Data Export section for further information.
Opens the printer dialogue. Refer to the Printing Data section for further information.
Recording controls (Start / Stop). Refer to the Recording a Signal section for further
information.
44
Setup the gain and back-off for each control unit. Refer to the System Configuration
Select the required data acquisition rate. Refer to the Data Acquisition Rate section for
Set the stirrer speed. Refer to the Magnetic Stirrers section for further information.
Oxytherm temperature control.
Scans for control units currently connected to the PC. Once found, serial number, type
(Oxygraph or Oxytherm) and calibration status are displayed.
Launches the liquid-phase calibration routine. Refer to the Liquid-Phase Calibration
Launches the gas-phase calibration routine.
View full details of the active file including filename and path, date, start time,
acquisition rate, version, comment and information. Refer to the File Information
Allows trace colour and data point marker to be customised. Refer to the Trace
Settings section for further information.
Shows/hides any overlayed traces.
Toggles rate cursors on/off. Refer to the Rate Measurements section for further
information.
Axes zoom controls. Refer to the Set Measurement Axes section for further
information.
Toggles between graph screen and tabulated data. Refer to the Tabulated Data
Adds an event mark during a measurement. Refer to the Event Markers section for
Displays Oxygraph Plus version and copyright information.
System Configuration.
Overview.
At default settings, the sensitivity of the control unit is sufficient to allow effective
measurement over a range of oxygen concentrations typical of either liquid-phase or gas-
phase assays. However some assays involve small changes in oxygen tension for which
default settings provide inadequate resolution.
Amplification or Gain in combination with signal back off can be applied to the raw,
uncalibrated mV signal from the electrode in order to improve the resolution. The following
diagram describes the use of gain and back off:
45
If gain is applied to the signal without the use of back off, the amount of amplification
available is limited as the signal is likely to overscale. However, if the mV signal recorded
directly from the electrode is first "backed off" by subtracting a defined amount from each
data point, larger amounts of gain may be applied without overscaling the signal.
Although applying back off actually subtracts a given

portion of the signal, the calibrated oxygen value remains
unaffected.
The following flow diagram shows the way in which the signal is conditioned when gain is
applied. Example 1 shows a disc in ambient air with default settings. The mV value is
2000mV and the calibrated value is 253.4 nmol/ml. Example 2 shows the same disc but
with a gain factor of 2.0 applied. The calibrated oxygen value remains 253.4 nmol/ml but
note that the mV value has increased to 4000mV.
46
The Channel Configuration window can be accessed either by selecting HARDWARE >
CHANNEL CONFIGURATION from the menu bar or by clicking the icon on the toolbar.
47
The window is divided into separate areas:
Channel selection.
The buttons at the top of the window correspond to the possible 8 channels of a multi-
channel system. Channels which are actively connected to the system are clickable
whereas inactive channels are grayed out.
Digital display of calibrated and uncalibrated

electrode signal.
These 2 panel meters show the readout from
the electrode disc as a digital value. The
calibrated units are displayed in either nmol/ml
for liquid-phase and µmol RTA for gas-phase
calibrations. When alterations are made to gain
and back off settings either manually or
automatically, the changes will be clearly
displayed in this section of the Configuration
window. The uncalibrated signal will be affected
by any changes whereas the calibrated value
will always remain constant.
Manual gain and back off setting.
Gain and back off can be adjusted manually by checking the checkbox in this section of
the window. The required level of back off should be applied first before the gain is
increased. Any changes in gain and back off will be reflected in the digital display panel
meters. Adjusting the gain and back off will also generate an Adjusting Control Box ....
message in the message window below. Default values can be restored by clicking the
Default button in this section of the window.
48
Automatic gain and back off setting.
Automatic gain and back off setting is a quick, convenient method of optimising
measurement resolution when the probable range of measurement is already known. For
example, in an assay measuring oxygen evolution, the maximum oxygen concentration is
expected to be no more than 25% of the starting concentration. If the starting
concentration of dissolved oxygen is 150 nmol/ml, the maximum amount of oxygen to be
measured would be 150 + 25% or 187.5 nmol/ml.
The upper and lower limits of detection are therefore known and can be set in the
automatic gain/back off settings. Move the LOWER slider to approx 120 and the UPPER
slider to approx 200 (this provides a buffer zone in either direction so that the signal does
not over or underscale). The gain and back off will be adjusted automatically to reflect the
values entered. The applied gain and back off levels are displayed in the window as
shown in the following diagram:
49
These settings will ensure that even though only a small change in oxygen concentration
is being measured, the full resolution capability of the control unit is being used.
At higher gain settings, noise becomes more significant. It

is important to experiment with gain and back off settings in
order to achieve the greatest sensitivity possible without
sacrificing signal quality.
Data Acquisition Rate.
Oxygraph Plus is able to log data from the oxygen electrode and also from an optional
auxiliary input at several different user-defined acquisition intervals. The intervals available
for selection between each recording are:
100 msec
200 msec
500 msec
1 sec
2.5 sec
5 sec
10 sec
The acquisition rate settings are accessed by either selecting HARDWARE >
ACQUISITION RATE from the menu bar or by clicking the icon from the tool bar.
50
The window above shows information regarding the available data acquisition intervals.
The table below shows further information for data acquisition rates that is not given in the
Acquisition rate window:
Acquisition Samples Max. Recording Max. Samples Max. Samples Total Estimated
Rate Per Second Time Per Channel (8 O2, 8 aux) File Size
100 msec 10 100 mins 60,000 960,000 62 MB
200 msec 5 3 hrs 54,000 864,000 56 MB
500 msec 2 8 hrs 57,600 920,000 60 MB
1 sec 1 16 hrs 57,600 920,000 60 MB
2.5 sec 0.4 24 hrs 34,560 552,960 35.9 MB
5 sec 0.2 24 hrs 17,280 276,480 17.9 MB
10 sec 0.1 24 hrs 8,640 138,240 8.9 MB
The acquisition rate should be defined based upon the type of experiment to be
performed. Assays where rapid response in oxygen tension is to be studied should use the
fastest acquisition rate whereas experiments such as light response curves can afford a
slower rate of data acquisition.
Once the acquisition rate has been selected, the Oxygraph Plus software opens the Setup
Live Rate Display window.
51
Temperature Control.
Overview.
Oxygraph control units are designed to perform measurements of oxygen across a broad
spectrum of applications from the biomedical field through to plant physiology in either
liquid or gas-phase. All experimental assays will have an optimum temperature at which
the sample will be most efficient in either evolving or consuming oxygen and it is important
that this experimental temperature is maintained at all times throughout the duration of the
experiment. This includes ensuring that any additions to be made to the reaction chamber
during the experiment are pre-equilibrated to the assay temperature before being added.
The assay temperature needs to be decided as one of the initial considerations of the
experiment as the apparatus is calibrated based on a specified temperature.
Temperature Effects on Liquid-Phase Samples.
saturated, de-ionised water contains a known concentration of dissolved oxygen which
may be calculated mathematically.
This data is based on measurements of dissolved oxygen in water at the given

temperature and standard atmospheric pressure published by Truesdale & Downing
(Nature 173:1236, 1954).
Temperature (°C) Oxygen (PPM) Oxygen (nmol/ml)

0 14.16 442.5
5 12.37 386.6
10 10.92 341.3
15 9.76 305
20 8.84 276.3
25 8.11 253.4
30 7.52 235
35 7.02 219.4
The formula used in calculating the oxygen values in the table is as follows:
Cs=14.16 - (0.394 * T) + (0.007714 * T^2) - (0.0000646 * T^3)
{Where Cs is the oxygen saturated concentration in ppm and T is temperature in °C}
0.03125 µmol/ml
1 ppm is equivalent to 1µg/ml or (1µg/32g/mol)= or
31.25 nmol/ml
52
Temperature Effects on the Electrode Disc.
From the information above, we can say that oxygen concentration decreases as
temperature increases. However, in addition to this effect, the oxygen electrode disc itself
is also sensitive to temperature.
The electrode disc signal increases with an increase in temperature. Therefore, the
response from the disc opposes that of the oxygen signal at different temperatures. In
assays where higher temperatures are used (such as mitochondrial respiration studies), it
is recommended that the disc is fully equilibrated to the assay temperature before any
calibration or measurement takes place. This can take up to 15 minutes and can be
observed as follows:
Prepare the electrode disc and mount correctly into the base of the electrode chamber.
For liquid-phase chambers, add a 1ml, stirred sample of air-saturated, de-ionised water
that is pre-equilibrated to the experimental temperature to the chamber.
Connect the water jacket(s) of the electrode chamber to the supply from the circulating
water bath (Refer to Setting Up Temperature Control section for more information).
Begin a recording from the electrode disc and observe the oxygen signal. If the
experimental temperature is high (e.g. 32°C), the signal from the electrode disc will
initially rise due to the temperature sensitivity of the disc.
Once the disc begins to equilibrate to the temperature, the signal will begin to decline
as the higher temperature effect on the sample is opposite.
Once the signal from the disc has re-stabilised, the disc is equilibrated to the assay
temperature and calibration may be performed.
Setting Up Temperature Control.
The DW1/AD liquid-phase electrode chamber has a single water jacket which is in direct
contact with the outer wall of the reaction vessel for efficient temperature control of the
sample. The water jacket should be connected to a thermoregulated circulating water bath
using the connectors supplied with the chamber.
The tubing lengths between the circulator and the electrode

chamber should be kept minimal in order to maintain good
temperature control. A circulator providing a flow rate of 6
litres/min or more is recommended.
Although the water jacket gives more positive control of sample temperature than many
systems in which control is effected entirely by circulated air, it is, like them, entirely
incapable of rapidly dissipating heat generated within the chamber by light containing a
large infra-red component.
Appropriate filters (water, copper sulphate solution, interference filters, heat-reflecting

mirrors etc) should therefore be employed. Light saturation without heating is, of course,
extremely difficult to achieve but the fact that the electrode is itself very responsive to small
changes in temperature provides an easy method of checking the effectiveness of any
light filtering system which is employed.
Hansatech Instruments white light sources are fitted with a

hot-mirror to reduce the infra-red content of the light.
Alternatively the LED light sources LH36/2R and LH11/2R
will produce red illumination without significant heat input.
53
For example, most samples will cease to give a response in oxygen change if heated to 50
- 60°C and any electrode response which is observed on illuminating a disc which has
been previously subjected to such treatment may be attributed to a temperature change.
The Magnetic Stirrer.
Electrochemical reactions taking place at the cathode of the electrode disc require the
consumption of oxygen in order to produce an electric current. This current is then
digitised and presented as a chart recorder emulation in the Oxygraph Plus software. The
nature of this process means that as oxygen is continuously consumed at the cathode, an
oxygen depleted layer is left.
As the rate of consumption by the disc is faster than the rate of oxygen diffusion through
liquid, the measured signal from the electrode disc would continuously decrease in
response to the oxygen depleted layer.
This problem is overcome by continuously stirring the sample in order to replenish the
depleted layer at the cathode and to ensure that the dissolved oxygen within the sample is
kept evenly distributed throughout the reaction vessel.
The magnetic stirrer is an integral feature of the control unit and is located on the top of the
box as shown in the below diagram. All liquid-phase electrode chambers, including the
Oxytherm Peltier chamber, mount directly on to the black stirrer cap.
Stirring of the sample is achieved by placing a magnetic follower or

"Flea" into the reaction vessel itself. Activating the stirrer rotates
the magnet beneath the stirrer cap at user definable speeds of
between 150 and 900 RPM. The flea tracks the magnetic stirrer,
rotating just above the cathode of the electrode so as not to cause
any damage to the membrane preparation.
2 types of flea are available:

PTFE coated magnet. - This is the standard flea supplied with the systems. It consists
of a small magnetic bar coated in PTFE.
Glass flea. - Glass fleas consist of several fine strands of iron sealed into a thin glass
capillary tube.
Each type of flea has advantages and disadvantages of use. For example, for experiments
where very small changes in oxygen tension are to be measured, some users prefer the
glass fleas as PTFE is known to absorb a very minute amount of oxygen. In the large
majority of applications, this absorption is not an issue. Glass fleas are not magnetic
themselves so they are not as reliable at tracking the rotating magnet in more viscous
samples i.e. the maximum speed of stirring would be lower than that of the PTFE coated
flea. etc., etc......
54
Operating the Magnetic Stirrer.
The stirrer is controlled directly from the Oxygraph Plus software. It can be accessed either
by selecting HARDWARE > STIRRER SPEED from the menu bar or by clicking the icon
on the toolbar. The following window is generated.
The effects of the oxygen depleted layer mentioned above can be clearly seen by
performing a short experiment:
Prepare the electrode disc and mount it into the base of the electrode chamber.
Place the chamber on top of the magnetic stirrer.
Add 2ml of air-saturated, de-ionised water into the chamber and drop a flea into the
sample.
Open the stirrer control window as described above and set the stirrer to a speed of
75%.
Start a recording and allow the trace to stabilise.
Once the trace has stabilised, stop the stirrer. The signal will start to decline steadily.
Re-start the stirrer. The signal will return to it's original level.
The following screenshot shows how the trace should respond to the procedure outlined
above.
55
Recording an Auxiliary Signal.
Both Oxygraph and Oxytherm control units are able to record the signal from an optional
auxiliary input. This signal could be from a range of different devices provided the device
output is analogue between 0 - 4V. Example devices are:
Thermometer
pH electrode
TPP+ electrode (or other ion selective electrode)
Output from fluorimeter
PAR
If an auxiliary signal is to be recorded, Oxytherm users should connect to the separate

auxiliary input. Oxygraph users will require an OXY/AUX connection cable. This cable
connects to the Electrode Disc Input on the rear of the Oxygraph and has a flying lead to
which the prepared electrode disc and connection cable should be connected.
Oxygraph Plus software is configured to accept an auxiliary input device from a window
accessed by selecting HARDWARE > AUXILIARY CHANNELS.
Auxiliary inputs for each active channel are enabled by

checking the appropriate check box in the window shown
in the diagram above. An auxiliary axis is added on the
right side of the graph screen and the signal itself is plotted
in the graph area as a real time chart recorder emulation.
The signal is displayed in numerical format in the Data Bar
(if enabled) along with the corresponding trace colour. All
individual data points can be reviewed in tabulated format
once the measurement has completed.
56
System Diagnostics.
In the event of problems with the electrode system such as drifting or unstable signal,
system over or underscale or lack of response to changes in oxygen tension, it is
necessary to isolate the area of the system that is causing the fault.
The electrode system essentially consists of 3 main areas where faults could occur:
Control unit
Electrode disc
Connection cable
By checking these areas individually on a step by step basis, faults may be relatively easily
diagnosed.
Control Unit Diagnostics.
Oxygraph Plus features a Box Test function which allows the control unit to be subjected
to a series of diagnostic tests with controlled parameters in order to ensure that the
electronics of the control unit are functioning to within specification. This feature can be
accessed by selecting HARDWARE > BOX TEST from the menu bar.
2 types of test are launched from this window:
Stirrer Test - The integral magnetic stirrer is calibrated using a tachometer before
leaving the factory. This function allows the stirrer speed to be verified either manually or
automatically. The automatic routine uses a series of prompts in order to verify that the
stirrer is functioning correctly at fast and slow settings. The manual test provides a set of 3
buttons Fast, Slow and Off. Clicking each of these buttons in turn will set the stirrer to the
57
respective command and ask for confirmation. Provided "Yes" is selected for all settings,
the stirrer will pass the test. If there is a problem with the stirrer, it is advisable to contact
Hansatech Instruments for further advice.
Gain and Back Off Test - This feature allows the electronics within the control unit to be
tested in the event of problems related to unexpected signals from the electrode disc.
Should the trace become unstable, drift or unresponsive, it is necessary to ensure that the
control unit is functioning within normal parameters before looking elsewhere.
It is crucial that the electrode connection cable is removed

from the rear of the control unit for this particular test.
Both the automatic and manual test essentially perform exactly the same series of checks.
The auto test procedure automatically engages the internal Test Resistor within the control
unit. This resistor is designed to imitate a new prepared electrode disc in air. The control
unit records the signal at 3 different levels of gain and back off in order to ensure the
electronics of the unit are functioning within normal parameters.
The manual test allows the user to set the control unit to any required gain and back off in
order to observe the signal changes. In the following example, the automatic test
procedure has been used.
Clicking on the Auto Test button first shows a warning that the electrode disc and lead
should be disconnected from the rear of the control unit. Once accepted, the following
series of windows is generated:
58
Once the test is completed, the results of the test will be displayed in the main Box Test
window in the test status section. If the control unit has passed the test, the following
message will be displayed in the window.
This indicates the control unit is operating within expected parameters and that any fault
that is being investigated does not lie with the control unit electronics. In this case, the
electrode disc and lead should now be tested for faults.
If the control unit is not functioning within expected parameters, the following message will
be displayed
In this case, please contact Hansatech Instruments for advice as the control unit will most
likely need to be returned for repair.
Electrode Disc Diagnostics.
Possible Causes of Signal Drift.
Inadequate electrode preparation. If the membrane and space paper have not been
applied properly, i.e. the membrane layer of the electrode dome is particularly uneven, this
could result in an unstable signal as the barrier of electrolyte trapped between the
membrane and the cathode is inconsistent. In areas where the electrolyte layer is thicker,
oxygen would take longer to diffuse through to the platinum cathode than in areas where
the electrolyte layer is normal. Please refer to the Electrode Preparation section for details
on correct preparation of the electrode.
Improper electrode mounting. When the electrode is mounted into the base of the
electrode chamber, if the base ring is screwed on too tightly, the membrane will become
stretched over the top of the dome. This will result in a higher oxygen diffusion potential
through the membrane to he cathode due to the pores in the PTFE becoming stretched/
enlarged. As the membrane begins to relax, the speed at which oxygen can diffuse
through the membrane to the platinum cathode decreases resulting in a downwards signal
drift.
Temperature control. If the chamber is not correctly temperature controlled or the
sample of air saturated water has not been pre-equilibrated to the correct assay
temperature, a signal drift will occur.
Disc condition. If the disc has not been cleaned, there may be deposits on the silver
anode which are bi-products of the electrochemical reactions taking place on the electrode
disc when polarised. In the first instance, a brown deposit of silver chloride may be
present. This deposit may, in some cases, be desirable as it is an electrical conductor and
may improve the sensitivity of the electrode disc. However, in time, black deposits of silver
oxide may build up. This deposit is an electrical insulator and will therefore decrease the
surface area of silver available for electrochemistry to occur. The response time of the disc
will be greatly affected in worst cases. Please refer to the Electrode Maintenance section
for full details on the cleaning procedures for electrode discs.
Damaged electrode disc. If the disc has not been maintained correctly or there is a
genuine fault with the disc, a drifting signal may indicate a problem more permanent than
those described above.
If the signal still drifts, please contact Hansatech Instruments for further advice.
59
Possible Causes of Excessive Signal Noise.
Noisy signals can be caused by the same issues described in the section above. However,
they can also be caused by a problem with the gain settings of the control unit as
described below.
Even in a good condition, an electrode disc will have a small amount of electrical noise (±2
mV). If the signal appears very noisy with large positive and negative deviations in signal
as shown in the diagram below, first check that the gain setting has not been altered to a
high value. A high gain setting will amplify any noise recorded by the disc.
If the gain settings are at a sensible value, it is necessary to check the electrode disc
according to the guidelines listed above. If the signal is still noisy, please contact
Hansatech Instruments for further advice.
Testing the Electrode Connection Cable for Short Circuits or Breaks.
If the connection cable has an intermittent short circuit, this will be seen on the trace as a
sudden rise to a maximum signal followed by a plateau. Checking the mV signal from the
electrode disc in the Configuration window should show that the signal is overscaled at
4095 mV when plateaued. If the signal remains at his level, and a problem with the
electrode disc and control unit have been ruled out, it may be a dead short in the
connection cable. If the signal only remains at this level temporarily and manipulating the
cable restores the signal, the problem may be an intermittent short caused by a cable
break.
The connection cable may also be "buzzed out" using a multimeter.

60
Data Handling.
Event Marking.
During an experiment, certain events may occur that require documentation. For example,
in a study of mitochondrial response to the addition of ADP would require a marker to
indicate at what point the ADP was added so that the speed at which the mitochondria
responded to the addition can be assessed.
Oxygraph Plus makes provisions for this by allowing the addition of event marks with user-
defined labels and comments. At any point during the measurement, an event mark can be
added either by selecting TOOLS > ADD EVENT MARK from the menu bar or by clicking
the icon on the tool bar. The following window is generated:
Any text typed into the label or comment fields of the Add Event Marker window may be
saved on exiting. The following dialogues are generated allowing the text to be saved or
not:
Saved labels and comments will then be available by selecting from the drop down boxes.
61
Event markers are displayed at the top of the graph area along with
any label that has been assigned. Any comment that has been added
to the event mark is displayed when the mouse cursor is hovered
over the event mark on the graph as shown opposite. Event markers
may be reviewed and edited or deleted at any point during and after
the measurement. Events are also saved with the experiment and are
printed.
For some functions of the software, event markers are automatically added to the graph
and cannot be edited or deleted. These features include:
Start recording - Indicated by a right-facing arrow and the label "Start"

Stop recording - Indicated by a left-facing arrow and the label "Stop"
Stirrer speed adjustment - Indicated by 2 downwards arrows with the label "Stirrers".
The first mark is added when the stirrer window is first opened, the second when the
stirrer window is closed. This indicates that between these 2 points, stirrer settings were
being adjusted.
Gain adjustment - When the gain is altered during a recording, 2 marks are added to
the graph. The first mark is added when a change is made and is labelled "Event", the
second mark is added once the control unit has been adjusted and is labelled "Gain".
New gain settings are displayed in the comments field of this event marker.
Back off adjustment - When the back off is altered during a recording, 2 marks are
added to the graph. The first mark is added when a change is made and is labelled
"Event", the second mark is added once the control unit has been adjusted and is
labelled "Back Off". New back off settings are displayed in the comments field of this
event marker.
Editing and Deleting Event Marks.
Event marks can be reviewed and edited or deleted at any point during and after the
experiment. Selecting TOOLS > EDIT EVENT MARKS from the menu bar opens the
following window.
62
The event to be edited is displayed in a blue box along with the information associated
with it. If any changes are made, the Save button should be pressed before either exiting
the window or moving to another mark using the previous or next buttons. Please note that
only the text associated with the event mark may be changed. The type of event (up or
down) cannot be amended.
To delete an event mark, select TOOLS > DELETE EVENT MARKS from the menu bar.
The following window is displayed:
The event to be edited is displayed in a red box along with the information associated with
it. Select the relevant event mark using the previous and next buttons and press delete to
remove the event mark from the graph.
File Information.
Experiments recorded in the Oxygraph Plus software can be saved along with an
information set giving details of the following:
File name and path

Date of experiment
Start time of experiment
Data acquisition rate used in the experiment
Oxygraph Plus version information
Details of what traces were recorded (i.e. number of channels, auxiliary data)
A text description of the experiment
File information is viewed from the following window:

63
This window is accessed either by selecting VIEW > FILE INFORMATION from the menu
bar or by clicking the icon on the tool bar. A text description is added by typing the
relevant comments into the Comments text box.
Rate Measurements.
The rate of change of oxygen tension is measured by Oxygraph Plus in 2 ways:
Live Rate Display.

During measurement, the oxygen signal is continuously monitored by Oxygraph Plus
software for the rate of change in oxygen tension. A Live Rate measurement is displayed
in the Data Bar giving an indication of the rate at which oxygen is evolving or being
consumed by the sample.
The Live Rate is calculated by performing a least
squares regression over a user-defined duration of
measurement. The duration is defined in the Setup Live
Rate Display window which is accessed by selecting
RATE > SETUP LIVE RATE DISPLAY from the menu
bar.
The rate is continuously re-calculated over the time

period defined and displayed in the data bar in nmol/min
for liquid-phase and µmol RTA/min for gas-phase
measurements. The longer the time duration selected,
the more stable the rate measurement will be.
64
Rate Measurement Function.

Rate measurements are made by either "clicking and dragging" a pair of Rate cursors to
the required positions or by manually entering start and end times on the recorded oxygen
signal in order to define the rate interval. The 2 cursors appears as vertical lines on the
graph screen. The left rate cursor is used to move the rate interval along the trace
whereas the right rate cursor is used to set the rate interval itself.
The rate cursors may be activated either by selecting RATE > RATE CURSORS from the
menu bar or by clicking the icon on the tool bar. The 2 vertical cursors will be displayed
on the screen along with the Rate Table. To manually set the rate interval, select RATE >
ENTER RATE CURSOR TIMES from the menu bar. A window is generated allowing start
and end times to be set.
In a multi-channel system, rates may be measured either on all traces simultaneously or

on individual channel traces. Once the rate cursors have been activated, select RATE >
SELECT CHANNELS from the menu bar. A window is displayed allowing either all traces
or individual channels to be included in the rate measurement.
When the rate interval cursors are moved along the trace, Oxygraph Plus software
automatically draws a line of best fit between the 2 cursors. The line of best fit is calculated
65
by least squares regression and can be displayed or hidden from the RATE > SETUP
LINE OF BEST FIT option in the menu bar.
Once the desired rate interval and

position has been defined, the rate can
then be entered into the rate table by
either selecting RATE > ADD RATE TO
TABLE from the menu bar or by clicking
the right mouse button and selecting
ADD RATE TO TABLE from the pop up
menu. Confirmation of the rate will be
displayed before addition to the main rate
table as shown in the image opposite.
Once the information displayed in the window above has been checked, it may be added
to the main rate table simply by clicking the ADD button. Once added to the main rate
table, rate data can be normalised if required. The normalise window allows a factor to be
entered and also if the normalisation factor is to be multiplied or divided by. Any measured
rates are saved along with the file and are also included in the print out.
Tabulated Data.
Once the experiment is complete, data can be viewed optionally in numerical format as the
individual recorded data points. This data is shown either by selecting TOOLS >
TABULATE DATA from the menu bar or by clicking the icon from the tool bar. The data
is displayed as shown in the image below:
66
If the print function is selected whilst the tabulated data screen is displayed, the raw data
may be printed out.
To return to the graph screen, select either TOOLS > GRAPH DATA from the menu bar or
click the icon from the tool bar.
Data Export.
Data recorded in Oxygraph Plus is saved as a *.CSV (Comma Separated Values) file. This
file type may be opened and reviewed directly in Microsoft Excel so that data may be
subjected to further, more advanced statistical analysis.
The diagram below shows the format in which the data is displayed:
All data saved in Oxygraph Plus software is specially

encrypted. If any amendments are made to data files
outside of the Oxygraph Plus software, notification of
external changes is displayed when the file is reloaded into
the software. This notification is also displayed on printed
experiments.
67
Printing Data.
Oxygraph Plus allows a comprehensive print out of completed experiments. Information

including data acquisition rates, gain and back off settings and calibration information are
included in the print out along with a printed graph, event marker and rate measurement
details. If the data has been modified outside of the Oxygraph Plus software, a warning
message will be printed at the top of each page as notification. If raw numeric data is
required, change the view to the tabulated data screen and select FILE > PRINT from the
menu bar. A sample print out can be found at Appendix A.
Viewing Previously Saved Files.
Previously saved data files may be easily loaded into the Oxygraph Plus software for
review and / or further analysis. The software will function as a stand alone operation when
a control unit is not detected however, some operational functions will be disabled.
To operate Oxygraph Plus in view mode when a control unit is not present, simply run the
software as normal. The software initially scans for connected control units and when none
are found, the following window is displayed.
Pressing the VIEW button opens Oxygraph Plus allowing data files to be loaded and
analysed using the integral Data Analysis functions. Obsolete commands and functions
are disabled during view mode. These functions include:
Recording controls
Stirrer controls
Gain / Back off settings
Normal function of the software may be re-established by reconnecting a powered control

unit to the PC via the RS232 serial cable and pressing the icon from the toolbar or by
selecting HARDWARE > SCAN FOR BOXES from the menu bar. This function looks for
control units currently connected to the PC. Once found, the serial number, type
(Oxygraph or Oxytherm) and calibration status are displayed.
68
Options.
The Options dialogue is accessed by selecting TOOLS > OPTIONS from the software
menubar. The following window is displayed.
Accessories.
OXY/PDU - Power Distribution Unit.
The OXY/PDU Power Distribution Unit is available as an accessory to the Oxygraph

control unit for multi-channel use. Please contact Hansatech Instruments for more details.
69
OXY/AUX -
The OXY/AUX cable is available as an accessory to the Oxygraph control unit. Please
contact Hansatech Instruments for more details.
OXY/PHA
The OXY/PHA unit connects to either the Oxygraph or Oxytherm control unit providing the
ability to plot pH, TPP+ or other ion-selective electrode signals directly to the Oxygraph
Plus software.
70
Support Information.
Purchasers of Hansatech Instruments Ltd products can be assured
of ongoing support and prompt and efficient attention to enquiries
at all times.
All products supplied by Hansatech Instruments are guaranteed for

12 months from the time of despatch against manufacturing faults
or defective materials. The guarantee does not cover damage
caused by misuse or unauthorised attempts to repair.
If difficulties are experienced with the equipment, please contact

Hansatech Instruments for advice. If necessary, equipment may be
returned for repair/replacement during the warranty period. No
charge will be made for parts/labour under warranty but we reserve
the right to charge for customs clearance and return carriage if
appropriate.
For repairs outside of the warranty period, please contact us at the

address below for an estimate of cost and instructions regarding
the return of equipment.
If the product was purchased via a distributor, please contact them

for advice in the first instance.
Upgrading Oxygraph Plus Software.
Updates for the Oxygraph Plus software can be found in the

Technical Support section of the Hansatech Instruments web site.
Users will be required to complete a short registration form in order
to apply for a password to the Software Upgrades page.
Narborough Road, Pentney, King’s Lynn,

Norfolk PE32 1JL England.
Tel: +44 (0)1760 338877 Fax: +44 (0)1760 337303

Email: [email protected]
Web site: http://www.hansatech-instruments.com
Oxygraph / Oxytherm Results
C:\oxygraph plus\light response.csv
Date 19/12/03 Experiment Start Time 10:22

Acquistion Rate 0.1 sec Duration 36m 12s
Current View 0m 0s to 36m 13s
comp
Stop
19.82 100
Start
light -
18.53 90
17.25 80
Oxygen (umol Relative To Air)
15.96 70
14.67 60
Aux
13.39 50
12.10 40
10.81 30
9.52 20
8.24 10
6.95 0
0m 0s Time 36m 13s
Signal 1
Aux 1
Page 1 of 3
Oxygraph / Oxytherm Results Cont.
Event Markers
Label Trace Type Comment
Start All Restart Started at:10:22:58
light - 1 Up Arrow adjust light
comp 1 Up Arrow
Stop All Pause Stopped at:10:59:11
Rates Of Change Table

# (Index) Channel Rate Normalised Start Time End Time
1 1 0.2 0.2 0m 44.4s 3m 36.7s

4 1 0.6 0.6 9m 21.3s 12m 13.6s
5 1 0.6 0.6 12m 13.5s 15m 5.8s
6 1 0.7 0.7 15m 5.8s 17m 58.1s
7 1 0.6 0.6 17m 58.1s 20m 50.4s
8 1 0.4 0.4 20m 50.4s 23m 42.7s
9 1 0.1 0.1 23m 42.7s 26m 35.0s
10 1 -0.2 -0.2 26m 35.0s 29m 27.3s
11 1 -0.2 -0.2 26m 35.0s 29m 27.3s
Page 2 of 3
Oxygraph / Oxytherm Results Cont.
Gain, Back Off, Calibration and Temperature Data
Channel Gain Back Off Cal. Offset Cal. Factor Set Temp.
1 1.00 0 1919 0.0253 20.00
Page 3 of 3

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Operations Manual

Setup, Installation &

Hansatech Instruments Ltd

Tel: +44 (0)1760 338877 Fax: +44 (0)1760 337303

28th February 2006

© Copyright Hansatech Instruments Ltd. All rights reserved.

Chapter 1: Electrode Preparation & ................. 6

Electrode Preparation ................................................. 8

Electrode Disc Cleaning & Maintenance .................. 11

The Importance of Good Electrode Maintenance ..... 13

Chapter 2: DW1/AD Electrode Chamber ........... 15

Introduction to DW1/AD ............................................... 16

Setting Up DW1/AD ...................................................... 17

Care & Maintenance .................................................... 19

DW1/AD Plunger Assembly ......................................... 21

Spare Parts List ............................................................ 22

Introduction to Oxygraph .............................................. 24

Setting Up Oxygraph ...................................................... 24

System Calibration .......................................................... 29

Oxygraph Operation ........................................................ 36

Data Handling .............................................................. 60

Support Information ...................................................... 70

Appendix A - Sample Printout of Data File ...................... 71

Hansatech Instruments Ltd equipment warranty is limited to replacement of defective

Introduction to the S1 Electrode Disc.

Electrode Disc Theory.

NEVER USE HARSH ABRASIVE AGENTS OR COMMERCIAL

There are four preparation stages:

It is vital to ensure that the larger O-ring is placed in position on the

Testing the Response of the Prepared Electrode.

If the observed signal is not as shown above, it may be caused by an inadequate

Electrode Disc Cleaning and Maintenance.

Rinsing and Drying.

The Importance of Good Electrode Maintenance.

A significant proportion of technical support requests received by Hansatech Instruments

Failure to Maintain Electrode Disc.

This image shows a typical case of poor maintenance. The

In contrast, this disc has been maintained according to our

Disc is Left to Dry Out After Use.

Cleaning with Unsuitable Materials.

A: Lugs for A2 top plate key (see instructions on disassembly of DW1/AD).

Transmission Properties of DW1/AD.

Transmission profiles of 10 mm thickness Acrylic

UVC UVB UVA PAR IR

It is important not to over-tighten the base ring as

Oxygen concentration in a liquid decreases as temperature increases which is why it is

2 types of flea are available:

Care & Maintenance.

If a trap or filter is installed in the cooler-circulator outlet

Do not use harsh abrasives to clean the unit as they may

The alignment jig and key are required for reassembly:

If the alignment jig is not used the reaction chamber may

DW1/AD Plunger Assembly.

Spare Parts List.

Description Catalogue Number

Connections to the Oxygraph

When running a multi-channel system, all units must be

What Additional Hardware is Required?

It is perfectly possible to link 2 Oxygraph control units together in a multi-channel setup

1 x Oxygraph System complete