Waters XBridge Protein BEH, C4, 300A, 3.5 µm Columns - Manual (Page 11)

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February 2014 WAT715001869 Rev D VW-PDF
In a typical HPLC system, the Bandspreading Volume should be no
greater than 100 μL ± 30 μL (or Variance of 400μL
2 ± 36μL 2 ).
In a microbore (2.1 mm i.d.) system, the bandspreading volume
should be no greater than 20 to 40 μL (or variance no greater
than 16μL
2 to 64μL 2 ).
VIII. MEASURING SYST EM VOLUME
(OR DW ELL VOLUME)
1. Remove column.
2 . Use acetonitrile as A, and acetonitrile with 0.05 mg/mL uracil
as B (eliminates non-additive mixing and viscosity problems).
3. Monitor 254 nm.
4. Use the flow rate in the original method and the intended
flow rate on the target instrument.
5. Collect 100% A baseline for 5 min.
6. At 5.00 min, program a step to 100% B, and collect data for
an additional 5 min.
7. Measure absorbance difference between 100% A and 100% B.
8. Measure time at 50% of that absorbance difference.
9. Calculate time difference between start of step and 50% point.
10. Multiply time difference by flow rate.
Figure 10: Determination of System Volume