64 Purification Solution - Developer's Guide
6
Calibration Procedures
Characterizing the column volume
• 3 mm flow cell: 0.5 mM thiourea or uracil in 75 % acetonitrile.
• 10 mm flow cell: 0.3 mM thiourea or uracil in 75 % acetonitrile.
• 60 mm flow cell: 0.04 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: 80 % acetonitrile or other suitable solution.
• Purge the solvent lines with the new solvents if not yet done.
5 Set up the method:
• Set the StopTime to No Limit in all modules (infinite run time).
• Pump:
• Set Solvents: 75 % B.
• Clear the TimeTable.
• Autosampler: Set the injection volume to 1 μL.
• UV detector:
• Thiourea: Set Signal A wavelength to 242 nm with 4 nm bandwidth
and without a reference.
• Uracil: Set Signal A wavelength to 260 nm with 4 nm bandwidth and
without a reference.
6 Display the relevant UV wavelength profile (242 nm or 260 nm) in the Online
Plot.
7 Equilibrate the system with 75 % B at 1 mL/min (if possible) for 2 min.
8 Set the flow to 0.2 mL/min.
9 Open the Sample Info dialog box.
Use a restriction capillary of known volume to maintain the backpressure above 20 bar if
necessary.
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