dwell volume determination and TIC of LC/MS

[Ericz]

I've looked at a few literatures about dwell volumw measeurement for an HPLC system. Is it necessary to make an injection in order to determine it?

Another question related to this topic is the early elution observed from TIC of LC/MS. A dead volume is not observed from TIC. If an early peak with expected m/z was observed, it should be a real peak. Is it true even if the UV signal (already offset in time) in parallel showed longer void time?

Thanks for all your thoughts!

[Uwe Neue]

I believe that you got the definitions correct, but let me restate them so we are clear. The dwell volume is the same as the gradient delay volume, and it the volume between the point where the gradient is mixed and the detector. It is measured without any column in place by doing a gradient with the mobile phase "colored" by a marker.

There is no need to make an injection during this experiment (with the possible exception that you got to do something to get the measurement process started).

The dead volume or void volume is the volume inside a column that is not accupied by particles. It is commonly measured by the injection of a marker that does not interact with the packing material.

If you got an early peak before the elution of the void volume marker with an expected m/z, it is the real peak. Your real peak could be excluded from the pores of the packing.

It may very well be that the situation that you describe is caused by the injection of your sample in an organic solvent such as DMSO. DMSO does carry the sample through the column unretained, possibly even before the void volume.

[Ericz]

Ume,

Thank you very much!

The sample is pure aqueous sample. The UV detector is in parallel with MS. There is a peak (with expected m/z, but no chromphor) in TIC ahead of UV void time.

The question I don't know is what is the void volume/time in TIC of MS because the liquid is evaporated before any signal shows up in TIC.

Ericz

[Uwe Neue]

Now I understand your question.
You can use a void-volume marker such as uracil for most RP applications. In 100% water on a nicely hydrophobic column, uracil is actually a bit retained.

A simpler way to deal with it is to make an estimate of the void volume. I always use 66% of the empty column volume as the void volume. If your peak elutes somwhere around there it is unretained. If it elutes at 40% of the column volume, it is excluded from the pores of the packing. If it elutes at larger than 66%, it is retained.

It appears that you are using a flow splitter, and some of your flow goes into the MS and some goes into the UV. If this is the case, you need to estimate the split ratio in order to get at the retention volume.