Purification Solution - Developer's Guide 57
Calibration Procedures
6
Characterizing the delay volumes: Mixing point to UV detector and Column to UV detector
Experimental measurement of delay volume: Mixing point to UV
detector
Because of the presence of the damper and degasser in analytical pumps, it is
not recommended that this value is measured by this procedure for any
analytical system. Use calculation based on the known volumes of individual
modules and capillaries as described in “Calculation of delay volume: Mixing
point to UV detector” on page 52.
Experimental characterization of the Mixing point to UV detector tubing
volume is based on a step response of acetone as a UV tracer that is added to
solvent B. The LC system without a column is first preconditioned to a
starting percentage of solvent B and, during a run, a step change of % B is
programmed in the pump timetable. The time offset between the recorded and
programmed positions of the tracer’s step in the UV signal is the Mixing point
to UV detector tubing volume, as shown in the following figure:
The figure shows an example of the measurement of Mixing point to UV
detector tubing volume by step boundary using 1% acetone as UV tracer. The
Because of piece-to-piece variations noted in preparative tubing, the calculated Mixing
point to UV detector tubing volume can deviate from the actual value, typically by 5 – 10 %
. For this reason, consider measuring this value for the preparative flow path
experimentally as described in “Experimental measurement of delay volume: Mixing point
to UV detector” on page 57 to minimize the error.
Because of the presence of the damper and degasser, it is not recommended that this
value is measured experimentally for any analytical system.
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