In order to transfer a gradient method from one instrument to another, it is often necessary to know the gradient delay time to account for differences in the dwell volume. If you know your system’s dwell volume, you can easily calculate delay time using the following formula:
tD = VD / F
where tD is the gradient delay time (min), VD is the dwell volume (mL), and F is the flow rate (mL/min). You know the flow rate because this parameter can be set using your instrument’s method control software. Hence, you simply solve for tD.
Let’s try an example: Suppose you know the dwell volume is 1.0 mL and the flow rate is set at 1.5 mL/min. What is the delay time?
tD = VD / F = 1.0 mL / 1.5 mL/min
tD = 0.667 min
In most cases, the dwell volume is not known but you have the flow rate and measure the gradient delay time. This can be accomplished by running a gradient method where the gradient begins at the start of the injection method (at t = 0 min). Use a zero-dead-volume connector instead of an HPLC column. At the point where the detection baseline begins to slope, you call this time tD. Then you simply solve for VD:
VD = (tD ) (F)
Here is a chromatogram showing what you might observe in the baseline for a tD measurement:
Another example: You set the flow rate at 0.5 mL/min and measure tD to be 2.5 min. What is the dwell volume? This time, solve for VD:
VD = (0.5 mL/min) (2.5 min)
VD = 1.25 mL
Tip: In the gradient method to measure tD, it is best to choose a low UV wavelength such as 210 nm to see a significant slope in the baseline. You will not see much change at 320 nm, for example, which makes tD hard to measure. For A and B solvents, it is best to use A = DI water and B = acetonitrile.