Chromatography Forum

Dear Members,
Some manufacturers claim they have column that could retain uracil (for example, Atlantis dC18 – Waters). So, I think we need other substance to know accurately t0 of this kind of column (or maybe common reverse phase column).
What is the most suitable t0 marker in reverse phase column other than uracil?
What are the criteria for a substance could be used as t0 marker other than unretained property?

Regards,
SYX

This has been discussed before, my favorite is T2O (tritiated water). Most people do not have a flow-through counter, however, it should be possible to find someone with a scintilation counter for measuring fractions.

acetone?

Like DR, I was thinking acetone or DMF.

Dear Syx,

I have used aqueous solutions of Nitrate or Chloride ions to make the void volume. Both absorb well at suitable wavelengths, and are very unlikely to be retained by any RP column.

Good Luck,

josebenjamin

someone in my group made a reference to EDTA. On thoughts?

We have looked at various materials as void markers. Nitrate, nitrite or bromide work well as long as you can convince yourself that the column has no ion exchange sites; anions are repelled by ionized silanols and give low values of t0.

One prospective customer asked us to use allantoin as a void marker for their qualification tests.

I have seen thiourea mentioned, but never tried it myself.

Actually, uracil works well enough if the mobile phase has plenty of organic solvent. If you are working in mostly aqueous mobile phase, it isn't as good.

If you have an RI detector, plain water works nicely.

acetone is not a good choice in polar solvents. It is more retained than uracil.

From the standpoint of the average practitioner, you may use several different compounds. It really does not matter, if the compound is really unretained or excluded, as long as it comes out at pretty much the same place.

From the standpoint of the theoretician, one needs to weigh carefully, what one intends to measure to select the most suitable marker for an unretained peak. Sometimes an excluded peak is the best choice (and some of these ionic markers could show some exclusion, depending on the stationary phase and the mobile phase.

On the other hand, I am a strong advocate for the use of something that is well defined. I do not think that the use of the "first perturbance in the baseline" is a meaningful choice, but there are papers out there who that are based on such a choice.

It would still the gossip hunger to know who suggested that "first perturbance" thing: One can get a wiggle shortly after switching the injection valve (in UV, especially if one has a strongly absorbing mobile phase), etc.

As mentioned elsewhere, partially and recently, unless your ion concentration is high enough you will get the following:
Normal ODS or silica: Retardation or complete hangup of cations, exclusion
of anions (weird peak shapes result at intermediate ion conc.)
Hypercarb and Phenomenex´s Gemini: The opposite of ODS, silica.