Principles and Practical Aspects of Preparative Liquid Chromatography

6. Display UV profile at 242 nm in online plot
7. Equilibrate the system with 75 %B using
1 mL/min for 2 minutes
8. Set flow to 0.2 mL/min (if necessary, use a restriction capillary
of known volume to maintain backpressure above 15 bar)
9. Open the sample info dialog:
• Enter the location of the sample vial
• Enter a run name
• Run the method
10. Stop the run after the marker peak has been recorded
11. Repeat the run twice (total 3 runs)
12. Install the target column
13. Equilibrate the column until pressure and UV absorbance are stable
14. Set a suitable flow in the range of 0.2 to 4 mL/min
so that the marker elutes at 1 min or later.
Since the expected column volume is about one half of a geometric
column volume (that is, the cross-sectional area multiplied by the
length) set the flow to be about one half of the geometric column
volume in mL units. For example, a geometric volume of 4.6 x 50 mm
column is (3.14 x 2.3 x 2.3 x 50) / 4000 = 0.83 mL, giving a flow
(1/2 x 0.83) mL / 1 min _ 0.4 mL/min.
15. Adapt the injection volume to the applied flow
(and column volume):
• Flow 0.2 to 0.5 mL/min, inject 1 µL
• Flow 0.5 to 1 mL/min, inject 2 µL
• Flow 1 to 2 mL/min, inject 5 µL
• Flow > 2 mL/min, inject 10 µL
16. Note the applied flow and injection volume.
17. Stop the run after the marker peak has been recorded.
18. Repeat and check for consistency.
19. Evaluate data.
• Record the elution time of all peaks at the apex
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