Principles and Practical Aspects of Preparative Liquid Chromatography

5.4 Equilibrating the
column and optimizing
the flow rate
5.4.1 Equilibrating and
purging the column
5.4.2 Optimizing
the flow rate
• Calculate the elution volume without the column:
• Calculate the average elution time of
data without the column
• Multiply it by the applied flow rate
• Subtract the volume of the restriction capillary
if used and one half of the injection volume
• Calculate the final column volume:
• Calculate the average elution time with the column
• Multiply it by the applied flow rate
• Subtract the elution volume without the column
and one half of the injection volume
Sufficient column equilibration time is required to obtain reproducible
retention times. We recommend an equilibration phase of four column void
volumes and two dwell volumes after each column purge phase. At least
three column void volumes are required for a proper column purge.
What is the best flow rate for analytical scouting on a purification system?
A test mix was injected on a manual scale-up system with a 4.6 by
150 mm analytical column. The total system void volume (dwell and
column void volumes) is approximately 3 mL. Using a flow rate of
1 mL/min and a generic gradient from 2 to 98 % organic solvent,
a gradient slope of 10 %B/min was applied, see Figure 5.5. In two
further experiments we increased the flow rate to 1.5 mL/min and
2 mL/min, see Figure 5.6 and Figure 5.7.
[mAU]
1400
1200
1000
800
600
400
200
0
1.872
5.312
5.758
6.246
7.659
7.917
8.405
9.219
9.965
2 4 6 8 10
13.173
Time [min]
Figure 5.5 Chromatogram at 1 mL/min (4.6 x 150 mm, 5 µm column)
for determination of optimum flow rate.
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