Purification Solution - Developer's Guide 77
Calibration Procedures
6
System Configuration and Delay Time Calibration
Delay time calibration for Preparative Systems
If a flow splitter is used, the calibrated values can be used only for runs with
identical tubing and settings of preparative and make-up pump flow. In
addition, a change of the data rate (that is, peakwidth for UV or cycle time for
MSD), of the detector that is used for fraction collection, can cause previously
measured delay times to be insufficient.
Preparation
1 Prepare the solvents:
• Solvent A: water with 0.1 % formic acid
• Solvent B: acetonitrile with 0.1 % formic acid
• Make-up solvent: 80 % acetonitrile in water with 0.1 % formic acid
• Needle purge and wash solution (degas in ultrasonic bath): 80 %
acetonitrile or another suitable solution (methanol should not be used
with dual-loop autosampler due to peristaltic pump tubing – check the
dual-loop autosampler manual).
2 If not yet done, install the preparative column (recommended SB-C18 21.2 ×
50 mm).
3 Prepare a sample:
G1364B, G1364E and G7166A:
• Dilute the Prep LC Standards #2 (5190-6887) sample with water,
depending on the purification instrument setup, and filter the sample
before use with the regenerated cellulose syringe filter (5190-5108):
• Preparative UV or UV/MSD instrument with 3 mm UV cell: Dilute 20×
(1 mL = 50 μL sample + 950 μL water).
• Preparative UV or UV/MSD instrument with 0.3 mm or 0.06 mm UV
cell: Dilute 10× (1 mL = 100 μL sample + 900 μL water).
• Combined preparative instrument: Dilute 5× (1 mL = 200 μL sample +
800 μL water).
G7159B:
• Use undiluted Agilent Delay and Checkout Calibrant (5190-8223). It is
recommended that you filter the sample with the PTFE syringe filter
(5190-5084).
4 Place at least 1000 μL of diluted sample in a vial and place it in the injector.
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