The dwell volume is equal to the time between the injection and half height of the detector trace, multiplied by the flow rate. Where optimal injection volumes are directly related to the cylinder volume of your column.
Dwell Volume Calculation
If the dwell volume used during the elaboration of the monograph is given in the monograph, the time points (t min) stated in the gradient table may be replaced by adapted time points (tc min), calculated using the following equation:
tc = t−[(D−D0)/F]
Where,
- D = dwell volume (mL)
- D0 = dwell volume used for development of the method (mL)
- F = flow rate (mL/min)
Injection Volume Calculation
When the column dimensions are changed, the following equation may be used for adjusting the injection volume:
- Vinj1 = injection volume indicated in the monograph (µL)
- Vinj2 = adjusted injection volume (µL)
- L1 = column length indicated in the monograph (cm)
- L2 = new column length (cm)
- dc1 = column internal diameter indicated in the monograph (mm)
- dc2 = new column internal diameter (mm)
This equation may not be applicable to changes from TPP columns to SPP columns.
Even in the absence of any column dimension change, the injection volume may be varied provided system suitability criteria remain within their established acceptability limits. When the injection volume is decreased, special attention is given to (limit of) detection and repeatability of the peak response(s) to be determined. An increase is permitted provided, in particular, linearity and resolution of the peak(s) to be determined remain satisfactory.
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Reference: USP 〈621〉
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