Chromatography Forum

Hello,

I need to determine the dead volume of my HPLC system prior to use the "system suitability" option in the software.

Can someone tell me how can I determine it? Is there any formula? What must I do?

My system (a little complicated) consists in:

A column switch valve + tubing 1/16" from valve to the column inlet + tubing from the column outlet to the valve +
tubing from valve to the detector 1 (DAD) +
tubing from DAD to a "Kobra" cell +
tubing from Kobra to Fluo detector

Thank you in advance!

Inject an unretained component like uracil.
From this retention time you can calculate your dead volume:
RT * flow rate = dead volume

The last reply suggested to do a measurement with a column in place. I am not sure if this is what you asked for.

You can measure the true instrument (=system?) dead volume by disconnecting the column and injecting an unretained compound at a suitable flow rate. You measure the time between the injection and the maximum of the detector response. You multiply this number with the flow rate to obtain a dead volume.

You will get different numbers for your two detectors. Actually, I do not know what your "Kobra" cell is, but it sounds like a post-column derivatization thingy, maybe an irradiated coiled tube? The difference in the volumes between both detectors would give you the volume of the Kobra...

In case the column void volume shall be included the injection of a non-retained component is a pragmatic and sufficient way. However, care must be taken in the selection.

Uracil may be retained on several -polar- reversed phase columns and other more hydrophilic phases.

Dead volume is considered the total volume from the point of mixing to the inlet of the column. Hence if you have mixer or pulse dampener in your configuration, all these will add to your volume.

Do be reminded to set the sampling rate of your detector to 10pts/sec to improve the peak resolution.

I was assuming that there was a column on the system because the use of the dead volume is system suitability.

This dead volume can be used to calculate the k factor, and it's most likely that you do some system suitability on retained peaks.

thank you everybody
I'm going to think of all your answers
Carmen

This is why I *hate* some of the standard terminology!

"Dead volume", unfortunately can be used in several different contexts, and I'm not sure which one the original post referred to:

• - officially, "dead volume" accoring to USP or ASTM/IUPAC refers to Vm; the volume of mobile phase contained in the column (I really wish they had called this "column internal volume"; that would be much clearer). This is measured from the retention of an unretained peak. There have been several discussions of this on the Forum over the years. Go to the archives and do a keyword search.
  - many people refer to this unofficially as the "void volume" of the column. Unfortunately, this begs for confusion with a "void" space at the head of a column.
  - "dead volume" is also often used for "dwell volume" (another bad name, IMO), which is also called "gradient delay volume" (a more descriptive name). This is the volume of liquid in the system from the point where the gradient is formed to the inlet of the column.
  - "dead volume" is often also used as a synonym for "extra-column" volume. This is what would be measured by direct injection of a probe with no column installed.

Have I missed any?