I am running ion-pair reverse phase HPLC (C18 column, phosphate buffer, 7% acetonitrile or methanol, tetrabutylammonium bromide). All the nucleotides that I analyze look good but when I load cell extracts there are two peaks which are extremely broad with severe fronting. The nucleotide peaks are still sharp and coming at the right positions, so it is not a general problem. I have tried both gradients (varying phosphate) and isocratic elutions with the same observation. Does anyone have any idea what compounds can cause this effect? If there would be a non-specific interaction, I would assume to see tailing rather than fronting. The peaks do not seem to improve if I load less sample.
True LC peak "Fronting" is most commonly caused by sample overload or failure of the sample to dissolve in the mobile phase. Less likely, but possible would be the pH of the mobile phase. Fronting is generally a sign of bad technique or may indicate a poor quality method. *The contaminates may be eluting (co-eluting) early causing the observed changes. We would need far more detailed HPLC method info and a chromatogram to troubleshoot further.
Here are a few basic questions which may help you troubleshoot the issue.
(1) Did you dissolve your sample in the mobile phase, filter it and insure it is fully soluble BEFORE injecting it? (2) Did you do a loading study to confirm that you are not overloading the column? (3) Did you perform proper sample prep to remove any contaminating matrix or cellular matter which may foul the column? (4) Have you incorporated a proper column-wash method step after each analysis which utilizes a mobile phase wash which is stronger than the analysis mobile phase to wash the column off of any later eluters? (5) What is/are the K primes of your sample (IOW: do you have proper retention, K prime > 2)? If only the first peak is effected by the fronting, maybe you can vary the method to increase the K prime of the peak. (6) The IP reagent may be interacting with the matrix portion. If so, either try to remove more of the matrix material before injection, increase the K prime, or try to reduce the amount of IP used (worth a try).
Thanks for all your answers. I have now tried a few things to address them. Since I have very complex samples and I have finally found conditions which look very good (except for the ugly peak mentioned above), I do not want to completely change method so I have mostly looked at the two latest comments. When it comes to ammonium ions, I make a purification on SPE (OASIS WAX) extraction from using a water-methanol-ammonium hydroxide elution step. I would guess most of the ammonium ions are gone after evaporation but I tried also to evaporate twice with resuspension in 80% methanol in between and it did not make any difference. If I make exactly the same procedure with a mock sample (no cells), the chromatogram is clean.
It is mainly one of the broad peaks I have problem with (the other one is very small and almost insignificant). When it comes to the HPLC procedure, I have tested to load 5 times less concentrated sample (in mobile phase) and it did not make any obvious difference for this peak. Filtering through a 3 kD cutoff filter does not make any difference either. There seems to be no carryover from previous samples (I never see it in the standard even if I load it directly after a sample) and no extra peaks come out if I wash the column extensively with 3 times higher phosphate concentration (the retention times of this peak and all the other peaks are much shorter then as expected). The k prime factor is generally around 10-30 for this peak depending on the mobile phase conditions. The shape of the peak differ a bit when I vary the mobile phase conditions but it is always very broad. When I change the mobile phase conditions I can get this peak to move in relation to the other peaks but I cannot manage to move this peak completely out of the region where the peaks of interest are. It could be worthwhile to also test to use less IP reagent in the mobile phase as you suggest, I can try that tomorrow.
Now, I have also tried different concentrations of ion pairing reagent in the mobile phase (half the concetration and the double concentration) but there was no obvious effect on the broad fronting peak.
Rather than 1/2 the IP concentration, I was thinking more like 1/10th. I would not expect much difference at all for changes of only 2x, but maybe some change for 1/10 or 1/20th. Most people use far too much IP in their methods. If that is the case, then it may help to reduce it. As I said earlier, worth a try.
Regarding sample concentration (loading), this too should be by orders of magnitude (10 x less, 25x less and so on). If these fail to improve the chromatography, then based on your observations, it is a method/sample problem and you will need to develop a better method to resolve the 'offending' material away or remove it via a different sample prep. procedure.
Good to know. However, I am uncertain if it really works reducing the ion pairing agent so much. I am usually using 2.8 mM. Nevertheless, it is easy to perform so I can give it a try.
Diluting the sample more than five-fold is not really meaningful since some of the peaks I am interested in will hardly be visible.
I am in parallel trying to modify the SPE procedure. In my first try it looked better when I switched from OASIS WAX to Varian DEA and my hope is that it will become completely good after some optimization.
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