Chromatography Forum

Hi all,

I have a very basic analyte (oxymetazoline) that has a tailing factor of 1.2 using a 15ul injection volume on a Mac-Mod Ace C18 but jumps up to ~1.7-1.8 if I try to increase the injection volume to 40ul. Basically, I need the higher injection volume to meet a DL/QL S/N requirement. The nominal concetration of the RF standard is 0.05mg/ml

Is there any data to support a smaller column ID could potentially reduce peak asymmetry? My inital gut reaction would be "No" because you would have to decrease the flow rate which probably makes the flow velocity identical to a larger ID column with a higher flow rate.

I thought I'd get a concensus from the community, especially before I spend the company's money in getting a smaller ID column "just to try".

Tailing of bases is caused by chemistry. Changing the column hardware will not do anything for you.

I hope that you are flexible with your method. If this is the case, try to increase the ionic strength of your buffer (higher concentration). Even better, use a column that allows you to run your method at alkaline pH. We have demonstrated that the loadability increases 10-fold or even more, when the compound is unionized (pH 10 or so), even compared to very good acidic conditions with a high buffer concentration. This is of course some work...

Thanks for your input. The little pKa information on this compound suggests that its pKA is ~10.6. Is there a buffer you would reccomend in the pH 12 region? I seem to recall bicarbonate has a max around 10, but I could be wrong.

With that high a pKa, you might be better off working at lower pH (to fully ionize your analyte) and using either an ion-pair reagent or one of the "mixed-mode" columns to get retention.

I have worked quite comfortably with compounds with a pKa of 10.6 with an ammonium bicarbonate buffer. The buffer remain stable at the pH that you use in water, while the pK of bases goes to lower values when you add organic. There is no need to try to go to pH 12.

Here is method for your compound on Primesep mixed mode column:
http://www.sielc.com/compound_151.html

Contact me if you have any questios.

Vlad

Uwe, last silly question:

I was under the impression that is was WRONG to attempt to analyze compounds with buffered mobile phases AT the pka of the analyte. I guess as long as I get a single peak, I'm OK? I would image that I'd get two peaks, each representing the ionized and unionized components.

There are all kinds of strange rumors around, including the one that running a compound at its pK will give you two peaks. This is plainly nonsense. Try it, and you will see...

There is a small problem, and that is that you need to make your buffer always to the exact same pH. This requires some care about buffer makeup, but is not a fundamental problem. As you develop the method, check, if the retention of your compound will indeed change with small pH changes. Due to the fact the analyte pK will change in the presnece of the organic solvent, you may not have a problem at all.