Teledyne ACCQPrep User Manual

Global Calibration Function of
Analytical to Different Column

®

Chemistries on the ACCQPrep

Abstract

Chemists often evaluate different column chemistries
for compound purification during analytical scouting
runs. For example, it is common for chiral purification,
for several column types to be screened on an analytical
HPLC. Despite very different types of columns, with
widely varying chemistry, the ACCQPrep can calculate
focused gradients from them with only a single
calibration on one column type. Although the ACCQPrep
uses a global calibration for all columns installed on the
system, that calibration is not limited to a single column
chemistry. The global calibration can also be used for
different column chemistries, so long as the analytical
columns have the same dimensions and use the same
gradient method table and flow rate, allowing fast and
easy column and method screening.

Background

Many labs, such as those performing chiral purifications,
do method development using many types of columns
and solvents. The method development starts with
a scouting run which allows an initial evaluation of
retention and resolution. The scouting runs can then
be used to create focused gradients for the ACCQPrep
preparative HPLC system. The ACCQPrep preparative
HPLC has a built-in calibration for the scouting method
calculated by PeakTrak® software when columns are
installed. This built-in calibration works for all columns,
regardless of manufacturer. The external HPLC
calibration in the ACCQPrep can likewise be used for a
variety of column chemistries provided the analytical
columns are the same dimensions, and the same
gradient table is used for all columns.

Experimental and Results

C18 runs with a C8 calibration

Universal Test Mix (PN 60-5234-835) was run on a
RediSep® Prep C8 column (20x150 mm, 5µ, 200Å, PN
69-2203-858) to get a retention of 6 minutes for the first
eluting peak using a methanol/water solvent system, as
described in Technical Note 52 (TN52), Calibration of
Analytical LC systems available on the Teledyne ISCO
web site.

Figure 1—Calibration of Teledyne ISCO C8 columns on the
ACCQPrep (top), and UHPLC (bottom ).

A matching C8 UHPLC column (2x50 mm, PN 69-2203-

853) was run in a water/methanol gradient (5 to 100%
B over 5 minutes, with an isocratic hold for 2.5 min at
100% methanol). The peak eluting at 3.757 minutes,
corresponding to the peak eluting at 6 minutes on the
ACCQPrep, was used for the calibration into PeakTrak
in the ACCQPrep.

Preparative runs

The C8 analytical column was replaced with an
analytical RediSep Prep UHPLC C18 column (2x50
mm, PN 69-2203-854) and test mix was run in a water/
methanol gradient using the same gradient as used for
the C8 column. As expected, the compounds eluted later
in the gradient due to the increased retention of the C18
column, with 100 Å pores.

Chromatography Technical Note

TN54

Chromatography Technical Note TN54

The data from the C18 analytical run was used to
calculate the preparative gradients in Figure two while
using the calibration from the C8 columns in Figure 1.
The preparative purifications utilized a RediSep Prep
C18 column (20x150 mm, 5µ, 100Å, PN 69-2203-810) In
both cases, the compounds eluted close to the expected
6-minute elution time.

The test mix was also run on the C8 preparative HPLC
column with the C8 calibration; the analytical data in
Figure 1 was used to calculate the gradient for the second
eluting peak at 4.451 minutes.

Figure 2—Analytical scouting runs and focused gradients
on a C18 column, using the C8 column calibration. The first
preparative run was focused on the compound eluting at

4.324 minutes in the analytical run, while the peak at 4.953
minutes was used to calculate the second preparative gradient.

Figure 3—Preparative run on a RediSep Prep C8 column
using the second compound eluting from the analytical run
in Figure 1.

The chromatogram appears almost the same as the C18
column, the only difference is the solvent composition
at the start and end of the focused gradient due to the
differences in retention between the two columns.

Silica runs with a C18 calibration

A 4.6x150 mm RediSep Prep C18 column was calibrated
on an Agilent analytical HPLC system as described in
TN52 using a gradient from 5 to 100% methanol over 6
minutes, followed by 100% methanol for 6 minutes at 1.0
mL/min. The solvent was changed to hexane/ethyl acetate
and the column replaced with a 4.6x150 mm RediSep
Prep silica column. The same gradient method was used
for the new solvent and column, 5 to 100% ethyl acetate
over 6 minutes. A sample of methyl paraben was injected,
and the retention time used for the focused gradient
shown in Figure 4 on a 20x150 mm RediSep Prep silica
column run in hexane/ethyl acetate. The sample eluted
near the middle of the calculated gradient.