Purification Solution - Developer's Guide 71
Calibration Procedures
6
System Configuration and Delay Time Calibration
The following example illustrates the change of the delay time in the UV-to-FC
calibration when the preparative flow is changed and a splitter is used. It is
assumed that the splitter-to-FC flow path volume is 5 mL, with a flow of
30 mL/min, and the splitter-to-UV detector flow path volume is 50 μL, with a
flow of 1.5 mL/min. Under these conditions, sample arrives from the splitter
to the FC in 10 s, and from the splitter to the UV detector in 2 s. The delay time
is then 10 – 2 = 8 s.
Now, if the preparative flow is changed from 30 mL/min to 60 mL/min, the
resulting delay is 5 – 2 = 3 s.
So, in this example, by increasing the preparative flow from 30 mL/min to
60 mL/min, the delay time is reduced from 8s to 3s. Also, there are
combinations of preparative and make-up flows that cannot be used; for
example, if the preparative flow of 60 mL/min (that is, a 5 s delay from splitter
to FC) and a make-up flow of 0.5 mL/min (that is, a 6 s delay from splitter to
detector) are used, the resulting delay time becomes negative.
Running conditions for calibration procedures
Calibration results are unique for each set of preparative and make-up flows,
MSD cycle time, and tubing. If one or more of them changes, the procedure has
to be repeated. Therefore, before starting the procedure decide the final set-up
of:
• Preparative flow
If a flow splitter is used, then the delay calibration is valid only for the specific combination
of preparative and make-up pump flows. If one of the flows is changed, or if tubing after
the splitter is modified, then the delay calibration must be rerun. This is because the
sample signal is split in to two independent flow paths, splitter to FC and splitter to
detectors, that generally have different flows. If one or both flows are changed, then the
resulting delays (UV to FC and UV to MSD) cannot simply be recalculated to the new
conditions.
A similar approach applies for a system without a splitter: it is recommended to
re-measure the delay when either the preparative flow or the delay tubing is changed. The
main reason is a possible change of a hydrodynamic velocity profile in the tubing when the
flow is changed significantly, or the material or shape of the tubing is modified.
To Next Page
Loading...
Need help?
General
