Chromatography Forum

Hello,

I have an ion chromatograph for separating amino acids using Na exchange. The system is very close to validation except for one little detail. I can't get the peaks to stay at a consistent retention time and they sway back and forth within their window. There is variability with cysteic acid, found at the void volume. So my problem is, I have shifting RT at the VV.

The most obvious cause is a leak somewhere. Ladies and Gents, I swear to you I have looked this system up and down and cannot find a leak anywhere.

My best guess is bubbles in the eluents. There is no degassing system, although the manufacturer has sent me an upgrade that I will have to install myself. Right now the eluent are in inverted IV bottles that I've had to puncture with a pinhole just to equalize pressure (a design problem). I think air is dissolving into the eluents where later they fiendishly wreak havoc on my system.

If anyone has any other suggestions of where I can look, much appreciated. If anyone has questions I'll be happy to clarify as much as I can safely elaborate.

Thanks in advance

I had this kind of problem many years ago. It turned out that the proportioning valve had a slight leak and the pH of the eluent was dancing around. It only affected the first couple of peaks. This was a brand new HPLC too! I have also seen even minor leakage in a checkvalve cause wandering retention times, but it affects all peaks, not just the first one. These kind of leaks are internal and don't drip where you can see them.

I doubt that you have a bubble. This would cause erratic pressures, bad baselines and affect all peaks. Even so, the degasser upgrade is a good idea.

By the way, cysteic acid is not the void volume marker. It is actually separated by ion exclusion. Void volume = excluded volume + pore volume. Cysteic acid elutes near the excluded volume; urea elutes near the void.

Nuts. Not exactly the best news I could have hoped for.

Thanks for pointing out an erratic check valve. This might be the case, but often when I reprime each eluent I get some very small bubbles coming from the valve, so I think I'll look at this first.

By the way, cysteic acid is not the void volume marker. It is actually separated by ion exclusion. Void volume = excluded volume + pore volume. Cysteic acid elutes near the excluded volume; urea elutes near the void.

I did not know that! Thank you for that tip. I guess what you're saying is that cysteic acid actually elutes before the void. That would make sense; my baseline dip follows my component and I just assumed that CYS-Ac was at the head of the slug.