66 Purification Solution - Developer's Guide
6
Calibration Procedures
Characterizing the column volume
• Multiply it by the applied flow rate.
• Subtract the elution volume without the column and one half of the
injection volume.
Combined Analytical and Preparative System
1 Prepare a sample vial containing thiourea or uracil at the following
concentration, and place it in the autosampler (if the sample cannot be
dissolved in 75 % acetonitrile under the given conditions, use pure water):
• 3 mm preparative flow cell: 0.5 mM thiourea or uracil in 75 %
acetonitrile.
• 0.3 mm preparative flow cell: 5 mM thiourea or uracil in 75 %
acetonitrile.
• 0.06 mm preparative flow cell: 25 mM thiourea or uracil in 75 %
acetonitrile.
2 Recommended: Filter the sample before use with the regenerated cellulose
syringe filter.
3 Replace the column by a zero-dead-volume union.
4 Prepare the solvents:
• Solvent A: water (optionally with 0.1% formic acid).
• Solvent B: acetonitrile (optionally with 0.1% formic acid).
• Needle wash solution (degas in ultrasonic bath): 80 % acetonitrile or
other suitable solution (methanol should not be used with dual-loop
autosampler due to peristaltic pump tubing – check the dual-loop
autosampler manual).
• Purge the solvent lines with the new solvents if not yet done.
5 Set up the method:
• Use the Analytical_Purification method settings (see “Preparing Default
Purification Methods” on page 9 for details).
Do not use the combined preparative system to determine the column volume of columns
with ID smaller than 4.6 mm (column volume too low) and particle sizes below 3.5 µm
(back pressure too high).
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