Chromatography Forum

Hello:

I'm trying to duplicate Phenomenex's ad on LC/GC North America. They use buffer of 10mM ammonium bicarb/methanol/acetonitrile pH 10.5 flow rate 1.5 ml/min.

I got good peaks for about 60 injections. After that, two of the peaks starts to merge. I contacted their tech support and basically got back three kinds of answers:

1. column is dirty, needs back flush.
2. buffer not good enough.
3. flow rate maybe too high.

I tried their suggestions but did not help much.

question is: I had good peaks for sixty injections. Is there any one else who use their Gemini column and experienced earlier than expected deterioration?

Is there anyone who can give me some pointers on ammonium bicarb buffer? I'm using ammonium hydroxide to pH it up to 11 now. It seems to give me better peaks.

Maybe you should also try increasing the buffer conentration. Does your chromatogram return to normal if you make fresh buffer?

I made fresh buffer but it didn't help

Was there any increase in column pressure? What's your sample injection solvent?
Since you are working at high pH I guess you have a sample that has amines. Higher pH and more buffering capacity would probably help because you are right at the pKa of aliphatic amines, and therefore the concentration of ionized versus unionized form would be changing a lot with a small change in pH.

I am injecting 80 ul of 0.5 M phosphoric acid containing tricyclic drugs (pka around 9-10). Column pressure has not been changing much.

We have worked with ammonium bicarbonate buffers for years. At the pH that you are working, they work fine. I do not see a problem with the buffer.

I am a bit concerned about the injection of a sample dissolved in 0.5 M phosphoric acid into such a dilute buffer. How about neutralizing the sample, or at least check if a standard sample dissolved in water will do better.

80ul is also a very large injection volume if you are using a standard column (e.g. 4.6mm ID). You could try the effect of reducing this volume to a more normal value e.g 10ul, but try this AFTER the suggestion immediately above, so you can first cut out the variability of your injection solvent and the mobile phase. Injecting such a large volume of sample at a different pH to the mobile phase is bound to compound any problems of incompatibility, since it will upset the mobile phase pH.

My supervisor don't want to change the extraction method so I'm bound by it. Our current extraction method extracts the drugs into phosphoric acid, and I have to inject 80ul to get big enough peaks. But still, my guess is that the Phenomenex Gemini column starts to deteriorates after 60 injections, since I had 60 good ones. If anyone have experience using Phenomenex Gemini column please let me know.

I worry about injecting acid into basic mobile phase too. My only experiment was to add some of the same strength of acid into the mobile phase and looks like the buffer still works. If the buffer does not work, I would not be able to get 60 good injections, right? Please let me know if I'm wrong.

Thanks for all the advice and help.

Did you have to renew mobile phase or extracting solution near the time of the 60th injection?

Your buffer "works", but when you inject that concentration of acid with a large injection volume relative to your column, you are creating a zone of lower pH that migrates through the column. Your analytes are positively charged when they are injected. Then, at some point during your run, they become uncharged and more retained by the column.

You would be better off injecting them at higher pH. As Uwe said, see if you can neutralize your extract before you inject it. You might find that your column lasts longer. All columns deteriorate somewhat when you use them, but the effects may show up faster in a situation like yours when you aren't at the optimum conditions for your type of method.

Is your method a gradient? If not, you might be able to use a gradient with a larger injection volume to focus your analytes at the front of the column.