<Diagnostic: Measuring dwell volume>

Diagnostic: Measuring dwell volume

The dwell volume (gradient delay volume) of an HPLC system is easy to measure.

1. Remove the column from the system and use a suitable length of capillary tubing to connect the injector directly to the detector. Note that many systems require a certain amount of back pressure for correct pump operation. Consult your pump manual for suggestions as to an appropriate length and diameter of tubing.

2. Fill the "A" reservoir with a suitable UV-transparent solvent (e.g., water, methanol, acetonitrile, etc.).

3. Fill the "B" reservoir with the same solvent doped with a UV-absorbing component to give approximately 1AU of detector signal (e.g., 0.1% acetone). Select an appropriate wavelength (e.g., for acetone, use 265 nm).

4. Run a typical gradient from 0 to 100% B (e.g., 0-100% in 20 minutes at 3 mL/min flow). Record the detector signal during this gradient.

5. Print out or display the "chromatogram" from the gradient run. It should look something like this. Note that the signal stays flat at the beginning of the chromatogram, before the change in composition has washed through the dwell volume. All systems will show some curvature at the transition to the linear ramp (the amount of curvature has been exaggerated in this figure). The gradient ramp should be a straight line; if the ramp deviates significantly from linearity, a step-test should be run to check proportioning accuracy. The dwell volume can be determined in one of two ways:

Graphically. Draw the best straight line fit to the flat portion at the beginning of the plot. Draw the best straight line fit to the linear ramp of the gradient. The time at which these two lines intersect is the dwell time (tD). The dwell volume is the product of the dwell time and the flow rate: VD = tD X F

Computationally. Find the time at which the baseline has increased by 50% (the midpoint of the linear gradient ramp, t50). Find the dwell time by subtracting one-half the gradient time: tD = t50 - (0.5 X tG). The dwell volume is the product of the dwell time and the flow rate.