Teledyne ISCO HPLC User Manual
HPLC to Flash
Abstract
Teledyne ISCO analytical columns work very well to
create methods for ACCQPrep® HPLC systems from a
simple scouting run. Aside from matching the RediSep®
Prep stationary phase, these columns also match
RediSep Gold® flash media, which means they can be
used to develop methods for flash chromatography
as well. Although C18 columns from other vendors
may work for this purpose, there may be differences
in selectivity as compared to RediSep columns, which
means peaks may not elute at the expected time. The
scale-up technique described in this application note
works because a compound elutes at the same time at
a particular solvent composition, so long as the time is
defined in column volumes. An alternative method uses
the ACCQPrep HP150 with RediSep Prep columns and
the Focused Gradient Generator built into the system
to create a focused gradient from a scouting run (see
Application Note AN119).
Materials required
Development of the flash method requires an analytical
HPLC system and one of the columns listed in Table 1.
Typical Column
Column Type Part # flow rate Volume
(mL/min) (mL)
RediSep Prep UHPLC C8 2x50 mm 69-2203-853 0.4 0.1
RediSep Prep UHPLC C18 2x50 mm 69-2203-854 0.4 0.1
RediSep Prep UHPLC C18AQ 2x50 mm 69-2203-855 0.4 0.1
RediSep Prep HPLC C8 4.6x150 mm 69-2203-856 1.0 1.5
RediSep Prep HPLC C18 4.6x150 mm 69-2203-800 1.0 1.5
RediSep Prep HPLC C18AQ 4.5x150 mm 69-2203-801 1.0 1.5
Table 1–RediSep Prep analytical columns
Chromatography Application Note
AN118
A three-segment linear gradient works well. The first
segment is 12 CV long, with the desired compound
eluting in the middle, at about 6 CV. The starting and
ending solvent composition for this segment are whatever
is needed to elute the compound. There is no need for
an isocratic hold at the start of the run because one
naturally occurs due to the HPLC system dwell volume.
Depending on the size of the HPLC dwell volume relative
to the column volume, peaks can elute earlier or later
when running the CombiFlash® NextGen gradient. There
is a step 0.01 minutes long followed by a wash at 100% B
to purge retained compounds from the column.
Each segment length in minutes is calculated by
multiplying the gradient length by the column’s volume
followed by dividing by the flow rate. The gradient table
is made by adding the segment length to the sum of the
previous times. In Table 2, segment lengths in column
volumes are converted to time, and the segment lengths
added to form a gradient table.
Segment Segment 2x50 mm Segment 4.6x150 mm
length length (Minutes) column length (Minutes) column
(CV) 2x50 mm column gradient 4.6x150 mm column gradient
(0.4 mL/min) table (1.0 mL/min) table
12 3 3 18 18.0
0 0.01 3.01 0.01 18.01
3 (100% B) 0.5 3.5 4.5 22.5
Table 2– Suggested segment lengths for analytical columns
in column volumes, converted to minutes and gradient
tables that can be entered to create HPLC methods.
Once you have a working method with an acceptable
resolution, it is easy to create a flash system gradient
using the same gradient table, but in units of column
volumes, for the CombiFlash instrument.
If solvent modifiers are needed, the same modifiers
should be used on the HPLC and the flash system.
Volatile modifiers are easier to remove from the sample
after purification.
Experimental & discussion
For flash chromatography, an elution time of about 6
column volumes (CV) is preferred. As it is easy to scaleup from one column to another in units of CV, convert
the uHPLC gradient to units of CV.
Results
The runs shown below used the gradient segments listed
in Table 2. One run used a 2 x 50 mm C8 column, while
the other used a 4.6 x150 mm C18 column. Benzophenone
was run with a water/acetonitrile gradient.
Chromatography Application Note AN118
0 5 10 15 20
0
100
200
300
400
500
Time (Minutes)
Absorbance (220 nm)
0
10
20
30
40
50
60
70
80
90
100
% B (Acetonitrile)
0 2 4 6 8 10 12 14
0.0
0.2
0.4
0.6
0.8
1.0
Time (CV)
Absorbance (254 nm)
0
10
20
30
40
50
60
70
80
90
100
% B (Acetonitrile)
0 1 2 3 4
0
200
400
600
800
1000
1200
Time (Minutes)
Absorbance (220 nm)
0
10
20
30
40
50
60
70
80
90
100
% B (Acetonitrile)
0 2 4 6 8 10 12 14
0.0
0.2
0.4
0.6
0.8
1.0
Time (CV)
Absorbance (254 nm)
0
10
20
30
40
50
60
70
80
90
100
% B (Acetonitrile)
C8, 2x50 mm example
A method was developed on a RediSep Prep C8 2 x 50 mm
column. The gradient was 40% to 50% B over 3 minutes,
followed by a step to 100% B to purge contaminants from
the column using a water/acetonitrile gradient.
Figure 1–Benzophenone run on a RediSep Prep C8 column,
2x50 mm, 0.4 mL/min.
Figure 3–Benzophenone run on a RediSep Prep 4.6 x150 mm
C18 column, 1.0 mL/min
The compound eluted at 11.9 minutes, or 7.9 CV. The
run was transferred to a 50 g RediSep Rf Gold C18
column (PN 69-2203-336) in water/acetonitrile.
The compound was found to elute at 2 minutes or 7.9 CV.
The sample was then run on a 50 g RediSep Rf Gold C8
column (PN 69-2203-712) using the same gradient,
defined in column volumes.
Figure 2–Benzophenone run on a RediSep Rf Gold C8 column,
50 g, run with the same gradient used for Figure 1
The elution from the flash column was very close to that
from the uHPLC column, showing that a method can be
transferred in this manner.
C18, 4.6 x150 mm example
A method was developed on a RediSep Prep C18
4.6 x 510 mm column using water/acetonitrile. The
gradient was 50% to 60% B over 18 minutes, followed
by a step to 100% B to wash contaminants from the
column. C18 is more retentive than C8, so the solvent
system needs to be stronger to cause elution at the
same retention time in column volumes.
Figure 4– Benzophenone run on a RediSep Rf Gold C18
column, 50 g, run with the same gradient used for Figure 3,
in column volumes.
Again, the flash column has excellent agreement
compared to the analytical HPLC retention.
It may be noted that the flash run derived from the
uHPLC column eluted slightly earlier than the predicted
retention, while the HPLC predicted a slightly earlier
elution than the actual flash run. This is because system
dwell volumes were not considered in the calculations.
The uHPLC and HPLC columns were run on the same
analytical instrument. The analytical system dwell
volume was large compared to the uHPLC column, but
small relative to the 4.6 mm column, so the gradient
was delayed to a different extent for the different
runs. It is best to cause the desired compound to elute
towards the middle of the gradient in the analytical
run to allow for some variation in retention from dwell
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