At our laboratory we are analysing amino acids in cell culture supernatants using the Waters AccQ tag method. For the reversed phase separation we use a modified solvent system, described in one of the application notes of Waters. The solvent system and gradient are specially designed for cell culture supernatants. One of the problems we frequently encounter is an increase in retention times within a series of samples. Does anyone have an idea what might cause this? Peak widths remain the same. We have thought about temperature effects, but most likely this is not the reason.

Bart van Montfort
Scientist Analytical Biochemistry

Are you allowing enough time for re-equilibration?

Anonymous,

I guess that if he didn't allow enough time for re-equilibration he should have decreased and not increased retention times... and he says that this increase in retention time is within a series of samples and not in the beginning or at the end...

Kostas

We have used the Accqtag system for amino acid analysis for several years with very few problems.
Yo!
If my memory serves me well are their not 3 distinct solvent steps in the gradient? Is your problem confined to one of the three steps or spread throughout? If it is spread throughout then are you Helium sparging ACN out of solution, this is a problem we have had.
A careful study of the brilliant original papers by Cohen and Van der Valeren may help. Incidentally I alone make up the eluants here, and you really do have to be careful on quality control!. Are you buying Waters eluants?.

Thanks for the suggestions. It seems that indeed the equilibration was the cause of the changing retention times. When we increased the equilibration time the retention times became far more stable.