Chromatography Forum

I need to determine the system volume of the HPLC.
I use the following conditions as instruction says.
Mobile phase: A:acetonitril; B. acetonitrile with certain amount of substrate having UV absorption.
A: 100% in 5min, then 100% to 0 in the following 10 min, then keep 0% in the next 5min.

The problem is do I need to inject something like water to start the run?
The instruction just says that "Run a typical gradient from 0 to 100% B, and record the detector signal during this gradient." But how? Without injection, the mobile phase stays at the initial condition, which is 100% A. And no UV profile is shown on the computer.

Check this website:
http://www.lcresources.com/resources/TSWiz/hs410.htm

Best regards

The problem is do I need to inject something like water to start the run?

yes.

make an injection of water or eluent A, or do a 0 µL injection.
It's just to start your system.

I inject 10 micro liter acetonitrile. The mobile phase B is acetonitrile with acetone. I'm not sure about the exact concentration of acetone because the liquid spill out during preparation. But it's definitely larger than 0.1%.
The result doesn't look like the standard curve. The increase of UV absorption occurs very late, at around 17min.
I tried 100% eluent B. It takes more than 10min to reach the maximal UV absorption. Is it because the flow rate is too low?
I was told that I should use the flow rate of the original method, which is 0.7ml/min. During this experiment, the pressure is only around 1MPa, because there's no column connected. Should I use higher flow rate or longer running time?

The pressure of the pump A, however, is a horizontal flip-over of the standard curve. May I use that to calculate the volume?

Several comments here:

First of all, "dwell volume" is not the same thing as "system volume". Dwell volume would have been better named "gradient delay volume"; it is the volume of liquid in the system from the point where the gradient is formed to the point where it enters the column. System volume is actually vague, but I would interpret it to be "dead volume": the volume of liquid in the system from the injector to the detector.

Second, in the dwell volume measurement described on our web site, you should have a capillary restrictor tube connecting the injector to the detector. The length and diameter of the restrictor should be sufficient to provide about the same back pressure you would get from a column.

Third, the composition of the B solvent is non-critical. All you need is something that will give about 1 AU of absorbance when you are pumping 100% B.

Fourth, an actual injection is not necessary unless needed to start the gradient, in which case a minimal volume of the A solvent is probably best.

Thanks Tom for correcting me about the terms.

I think what I want to measure is the dwell volume.

I did use a tube to replace the column, but it couldn't provide similar back pressure as a column. I'll check if we have the tube you mentioned.
I hope I could get the right curve with that tube.

I did use a tube to replace the column, but it couldn't provide similar back pressure as a column.

A meter or so of 0.005"id tubing should be about right, go longer as necessary to provide enough back pressure for the pump to function properly (the idea is to fool the pump into thinking there is a column attached).