Analytical to Preparative HPLC

Method Transfer
An easy way to scale up from UHPLC to preparative
HPLC using focused gradients

Technical Overview

Author Abstract
Pierre Penduff Synthesizing novel compounds or isolating natural products can be a laborious
Agilent Technologies, Inc. and time-consuming process. After analyzing the precious sample on an analytical
Waldbronn, Germany UHPLC system, the crucial step is to transfer the method to a preparative system
with a minimal risk of losing valuable work or collecting impure compounds.

This Technical Overview describes a practical way to optimize the scale-up

process for reversed-phase chromatography from an analytical UHPLC system to
preparative LC systems using a focused gradient to increase the sample load on
the preparative column to achieve optimum purity.

764 µL
98 % Purity
93 % Recovery
2.5 µL

2.0 µL

1.5 µL

1.0 µL

0.5 µL

Focused Analytical Scale-up transfer

gradient column volume on preparative
overloading column and
concentration
overloading
Introduction In this Technical Overview, an approach All optimization steps occur on the
with focused gradients on target peak analytical system. After obtaining the
Generic gradients are suitable to cope was used. This approach generates first chromatographic information of
with a large variety of sample types then a unique and dedicated method to the crude mixture by using a generic
when there is no capacity or time to a concerned target peak which has the gradient, the resolution is optimized by
optimize the separation. Each gradient advantage to increase the resolution flattening the slope followed by a loading
can be divided into four different steps. better than the time slices method. study to determine the maximum column
After the injection, an isocratic hold load before scaling up to the preparative
step can be applied to remove the column dimensions.
injected solvent from the column and to
improve resolution especially for polar
compounds. The second step is a linear Area of interest Solvent B composition (%)
slope, which will be applied to separate
effectively based on the chromatographic
properties of the target compounds,

Column reconditioning
followed by a purge phase. In the last
Generic gradient
UV absorption

step, the column is re-equilibrated at

Isocratic hold

Purge phase
the initial solvent composition for the
next sample analysis or purification run.
To improve resolution around the target
compound, the linear slope has to be
modified.

In preparative chromatography, most

often the goal is to isolate, efficiently,
a large amount of one or a few target Time
compounds out of a crude mixture.
Figure 1. Schematic view of a generic gradient with the desired product peak (highlighted in green).
Ideally, the chromatographic resolution
around the target peak and the column
load are increased without significantly
increasing the separation runtime. From Solvent B composition (%)
the crude sample, an optimized method
can be generated with the goal to extend
the resolution between the target peak

Column reconditioning
(green peak, Figures 1 and 2) and its
Focused gradient
UV absorption

Isocratic hold

Purge phase

neighbor compounds.

A basic approach to generate optimized

preparative methods can be to divide the
linear generic gradient method into time
slices (described in the Technical Note
5991-3070EN1). This approach generates
a set of preparative methods that can be
used in any further sample purification
simply by identifying the time slice where Time
the target peak elutes.
Figure 2. Schematic view of a focused gradient.

2
Experimental Columns
• Agilent ZORBAX RRHD SB-C18,
Instrumentation
2.1 × 50 mm, 1.8 µm (857700-902)
Analytical System
Agilent 1290 Infinity Binary LC: • Agilent ZORBAX SB-C18, Prep HT
Cartridge 21.2 × 150 mm, 5 µm
• Agilent 1290 Infinity Binary Pump (870150-902) with end fittings
(G4220A) (820400-901)

• Agilent 1290 Infinity Autosampler

Software
with Thermostat (G4226A, G1330B)
Agilent OpenLAB CDS ChemStation
Edition for LC & LC/MS Systems, Rev.
• Agilent 1290 Infinity Thermostatted
C.01.05 [36]
Column Compartment (G1316B)
Solvents and Samples
• Agilent 1290 Infinity Diode-Array
Detector, including a Max-Light Solvent A
Water + 0.1 % formic acid
standard cartridge cell with 10-mm
path length, V(σ) 1 µL (G4212A) Solvent B
Acetonitrile + 0.1 % formic acid
Preparative System
Purification mixture for preparative runs
Agilent 1260 Infinity Preparative-scale O-acetyl salicylic acid (0.07 g/mL)
Preparative System: Salicylic acid (0.175 g/mL) in
DMSO:acetonitrile 3:1
• Agilent 1260 Infinity Preparative
Binary Pumps (G1361A, G1391A) Purification mixture for analytical runs
(dilution by 50 in water:acetonitrile 1:2)
• Agilent 1260 Infinity Preparative O-acetyl salicylic acid (1.4 µg/µL),
Autosampler (G2260A) Salicylic acid (3.5 µg/µL)

All solvents used were LC grade. Fresh

• Agilent 1260 Infinity Multiple ultrapure water was obtained from a
Wavelength Detector (G1365D) Milli-Q Integral system equipped with a
equipped with a Quartz flow cell 0.22-μm membrane point-of-use cartridge
0.06-mm path length for MWD (Millipak)
(G1365D#026)

• Column organizer module (G1383A)

• Agilent 1260 Infinity Fraction

Collector PS (G1364B)

3
Results and Discussion 4. Replace the column by a zero dead 7. The Dwell volume is then:
volume connection. VDwell = tDwell × Flow,
A multistep process has been developed
that ensures consistent purification 5. Run the same gradient at the same and the column volume is:
results. The first step is only done once flow rate as in the previous run at VColumn = tColumn × Flow.
for a purification system. The further 270 nm detection wavelength.
steps are to be repeated for each sample. On the preparative flow path/system
6. Calculate the time differences at 8. Repeat Steps 3 to 7 on the
Step 1 50 % of the maximum absorption, preparative flow path using the
between the programmed gradient typical preparative flow rate (for
Determination of dwell volume and and the obtained signal with a example, 25 mL/min for a 21.2 mm
column volume bypass (tDwell), and between the id preparative column).
The scale-up process starts with a signals obtained with bypass and
characterization of all void volumes with column (tColumn).
from the analytical and the preparative
systems.
%B Monitored wavelength: 254 nm Programmed solvent composition (% B)
The total system void volume (dwell Effective solvent composition (% B)
volume, column volume, and extra column
volumes caused by the flow path after the
column) has to be determined first on the
analytical and the preparative LC system.
UV Absorption

These void volumes generate a delay

between the programmed gradient 50%
and the effective gradient (Figure 3).
To determine the effective solvent
30%
composition at a certain retention time of
a compound, for example, the percentage
of organic solvent, in this example
corresponding to 50 %, needs to be
corrected due to the dwell volume (153 µL 0 0.50 1.00 1.50 2.00
for the 1290 Infinity Binary LC) and the Time (min)
column volume (147 µL for this ZORBAX Figure 3. Sample mixture with the programmed solvent composition (red), and the effective gradient
RRHD 2.1 × 50 mm, 1.8 µm column). For composition (blue) after consideration of the system void volume.
the desired compound shown, Figure 3,
the effective elution composition, or
Monitored wavelength: 270 nm
elution point of the sample corresponds
Programmed solvent composition (% B)
to 30 % of solvent B. UV signal at 270 nm with a bypass
UV signal at 270 nm with an Agilent ZORBAX RRHD SB-C18, 2.1 × 50 mm, 1.8 µm
If the dwell volume of the system is not
known, it can be measured using the
following procedure:

1. Prepare Solvent A:
25 % water/75 % acetonitrile.
UV Absorption

tDwell Flow 0.5 mL/min

tColumn Gradient Time (min) %B
2. Prepare Solvent B: 0.00 5
1.00 5
25 % water/74 % acetonitrile/ 4.00 95
1 % acetone. 5.00 95
Dwell volume 0.153 µL
Column volume 0.137 µL
On the analytical system/flow path
3. Run a linear gradient from 5–95 % B
in 10 minutes at the usual flow Time (min)
rate used on the analytical column
(for example 1 mL/min) at 270 nm Figure 4. Characterization of the analytical system: Overlays of the programmed solvent composition
(red), UV signal of the tracer with a bypass (blue) and the signal of the tracer with column (green).
detection wavelength.

4
Steps 2 and 3 After the elution point of a target The example presented in Figure 5
compound has been calculated, an area shows the target peak (main peak on
Analyzing the crude mixture by a
around this peak is defined where a linear both analytical chromatograms) with
generic gradient and generation of a gradient with a flat slope will be applied. a non-baseline separated neighbor
focused gradient Table 1 gives typical slopes which can be compound using a generic gradient
The virtual elution point is defined as the used for the focused gradients: (upper chromatogram). The lower
percentage of solvent B determined at chromatogram shows a baseline
The elution point of the target peak in separation after the focused gradient
which the elution of a compound occurs
this example has been identified at 30 % has been applied. The runtime has been
in the applied gradient and by taking into
(Figure 3). A new linear gradient around reduced by a factor of two.
consideration the void volume of the
the elution point from 25 % to 35 %
system.
organic solvent with a slope of 20 %/min
can be applied to improve the resolution
of the target peak.

Table 1. Typical slopes for focused gradients.

Column lengths 50 100 150 250

Slope (%B/min) 10-20 5-10 3-6 2-4

Programmed solvent B composition (% B)

Effective solvent B composition (% B)
Generic gradient
Monitored wavelength: 254 nm
UV Absorption

Flow 1 mL/min
Injection volume 0.5 µL
Gradient Time (min) %B
Gradient 0.00 2
slope: 0.10 2
64 %/min 1.60 98
2.00 98

0 0.50 1.00 1.50 2.00 Time (min)

Focused gradient
Flow 1 mL/min
UV Absorption

Injection volume 0.5 µL

Gradient Time (min) %B
0.00 2
Gradient slope: 20 %/min 0.10 2
0.11 25
Start: Elution point –5 % 0.61 35
End: Elution point +5 % 0.62 98
1.00 98
0 0.50 1.00 Time (min)

Figure 5. Initial generic gradient (above), and focused gradient on target peak (below).

5
Step 4 mAU Monitored wavelength: 254 nm
2.5 µL Flow 1 mL/min
Loading study 800 Resolution = 1.29
(for 2.5 µL injection volume) Gradient Time (min) %B
0.00 2
After identification of the optimal 2.0 µL 0.10 2
method, a loading study was performed 600 0.11 25
0.60 35
to determine the maximum column load 1.5 µL 0.62 98
without losing the chromatographic 400
1.00 98
resolution. The maximum injection 1.0 µL
volume on the analytical column was
200
determined by increasing the injection 0.5 µL
volume stepwise (Figure 6).
0
In this example, the maximum injection
0.2 0.4 0.6 0.8 1 min
volume was experimentally determined
to be 2.5 µL on the ZORBAX SB C-18, Figure 6. Column overloading study: The maximum loading has been determined here by an increment of
2.1 mm column, corresponding to a pure 0.5 µL per injection.
target compound loading mass of 133 mg
on the preparative column (a scale-up
DPREP 2 Dpart, ANA
from 0.5 µL of injection volume would Flow rate calculation: FlowPREP = FlowANA × × (a)
allow purifying 26.6 mg of pure target DANA Dpart, PREP
compound).
DwellANA LPREP, col Dpart, PREP DwellPREP
Isocratic hold: Tini, PREP = Tini, ANA + × × – b)
Step 5 FlowANA LANA, col Dpart, ANA FlowPREP

Application of the method transfer

LPREP, col Dpart, PREP
formulas2 Segment duration: Tgrad, PREP = Tgrad, ANA × × (c)
LANA, col Dpart, ANA
The method transfer formulas between
HPLC and UHPLC systems are well
LPREP, col × DPREP, col2
known from literature. It is an equivalent Injection volume: Vinj, PREP = Vinj, ANA × (d)
process when performing a sample LANA, col× DANA, col2
analysis of the crude mixture on a UHPLC
Figure 7. Scale-up formulas.
column and transfer the results to a much
larger column dimension for purification.
For this method transfer, the goal was to
retain the resolution and the column load Table 2. System characteristics and methods from UHPLC to HPLC preparative system.
from the analytical run. A method transfer Prepative flow path Analytic flow path
without complicated formulas can also
System characteristics
be performed, but the gradient duration
and the injection volumes are parameters DwellPREP = 3.25 mL DwellANA = 0.153 mL
that will change the resolution of the Column characteristics
target peak from the other substances
Dcol, PREP = 21.2 mm Dcol, ANA = 2.1 mm
and might cause collection of impure
fractions. Lcol, PREP = 150 mm Lcol, ANA = 50 mm
Dpart, PREP = 5 µm Dpart, ANA = 1.8 µm
For this purpose, the method transfer
Gradient
formulas could be applied between the
analytical and preparative systems. Obtained from scale-up methods
The Table 2 describes the two system 2.96 min isocratic hold at 2 % 0.10 min gradient from 2 %
characteristics which will be applied on 6.12 min gradient from 25 to 35 % 0.60 min gradient from 25 to 35 %
the method transfers. 4.89 min purge segment at 98 % 0.40 min purge segment at 98 %
Flow rate: 25 mL/min Flow rate: 1 mL/min
Injection volume: 764 µL Injection volume: 2.5 µL

6
The scale-up from the analytical Table 3. Scale-up process.
method to the preparative system was
No. Step
accomplished by applying formulas b, c,
and d to the analytical and preparative 1 Determination of dwell volume and column volume for the system
systems (Figure 7). 2 Sample analysis to determine the elution point of the target compound
3 Creation of a focused gradient to increase resolution for the target compound
A method transfer from the gradient on a 4 Determine the maximum column loadability
UHPLC (1.8 µm) column to a preparative
5 Scaling up to a preparative column using the available formula
(5 µm) column was done by applying
formulas b, c, and d using a flow rate of 6 Preparative injection and fraction collection
25 mL/min on the target LC system. Note: The first step is performed only once for a system to characterize its void volumes.

The injection volume was scaled up to

764 µL, which is equivalent to 187 mg
of sample mixture on the preparative
column.

By applying the method transfer

described in this Technical Overview, the
target compound was purified on the
preparative flow path (Figure 8), and a
purity of 98 % and a recovery of 93 % was
obtained. The resolution was retained
during the scale-up transfer, and a 47 %
reduction of solvent consumption was
achieved.

mAU Monitored wavelength: 254 nm

Resolution = 1.82 Flow 25 mL/min
Injection volume 764 µL
Gradient Time (min) %B
0.00 2
2.96 2
120 3.09 25
9.20 35
9.32 98
13.97 98

Runtime gain: 12 minutes (generic gradient length 26 min)

Solvent saving: 47%

2 4 6 8 10 12 14 min

Figure 8. Preparative chromatogram.

7
Conclusion
A scale-up from a 2.1-mm id column
on a UHPLC system to a 1260 Infinity
Preparative scale system equipped with
a 21.2-mm id column was successfully
developed.

For all scale-up situations, a correct

method transfer was required to keep
the resolution constant. This ensured
maximum purity and recovery from the
precious sample.

The steps in Table 3 summarize the

process.

References
1. Peptides Purification on the Agilent
218 Bio-inert Binary Purification
System, Agilent Technical Overview,
5991-3070EN.

2. Guidelines for the use of UHPLC

Instruments. Requirements for UHPLC
instruments, method development
in UHPLC and method transfer from
regular HPLC to UHPLC, Dr. Davy
Guillarme, Prof. Jean-Luc Veuthey,
Freeware download: http://www.
unige.ch/sciences/pharm/fanal/
lcap/telechargement.htm

3. U. Huber and R.E. Majors, “Principles

in preparative HPLC”, Agilent
Technologies Primer, Publication
Number 5989-6639EN, 2007.

www.agilent.com/chem/purification
This information is subject to change without notice.

© Agilent Technologies, Inc., 2013

Published in the USA, September 1, 2013
5991-2013EN