58 Purification Solution - Developer's Guide
6
Calibration Procedures
Characterizing the delay volumes: Mixing point to UV detector and Column to UV detector
programmed profile of solvent B (is shown in red; the UV profile of the tracer
is shown in blue. Instrument in analytical flow path of combined analytical
and preparative system with 10 μL sample loop in flow path; flow 2 mL/min.
Note that the current procedure uses a step of 40 – 60 % B.
Preparative system
1 Switch off all pumps.
2 Prepare the flow path:
a Replace the column by a union.
b If the flow path includes a splitter with a UV detector after it in the
make-up flow, bypass the splitter by connecting the following tubing with
a union (predefined combined instrument only):
• HPLC stream inlet tubing from the preparative pump (port 1 in the
MRA manual)
• Make-up outlet tubing to the UV detector (port 4 in the MRA manual).
c Recommended: place a back-pressure regulator (2 – 7 bar, 40 – 100 psi)
after the UV detector.
3 Prepare the solvents (solvents can contain additives):
a Solvent A: water
b Solvent B:
• 1 % acetone in water as a UV tracer for 3 mm and 10 mm UV cells.
• 10 % acetone in water as a UV tracer for 0.3 mm and 0.06 mm UV cells.
c Degas both bottles in an ultrasonic bath for 10 min if the back-pressure
regulator is not used.
d Purge the solvent lines with the new solvents.
4 Set up a method as follows:
• Use the General_Purification method settings as described in Default
Purification Method (“Preparing Default Purification Methods” on
page 9).
For the analytical flow path of the combined analytical and preparative instrument, use the
calculation described in “Calculation of delay volume: Mixing point to UV detector” on
page 52.
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