Chromatography Forum

I am using a Waters C18 column to quantify Pyridoxal 5' Phosphate in serum, with fluorescent detection at 418nm. Although I do get an ok separation, I find that the peak occasionally splits. When I prep samples in duplicate, this splitting (or shoulder) happens in both relicates; but, if I repeat the sample prep and re-run it, I often can obtain just the single (ideal) peak for PLP. I know it's a problem of splitting rather than contamination, because when i do get just the single peak for PLP, its area corresponds to the sum of the two split parts.

I have replaced the column and all reagents in the sample prep, and taken measures to keep air out of the system.

Has anyone else had this problem - could it be considered NOT a problem but an acceptable feature of the chromatogram?

A bit more information would be useful. Specifically, column dimensions, flow rate, mobile phase, injection volume, and sample diluent.

Peak shape problems (tailing, shoulders, splitting) sometimes result from the injection of a diluent which is as stronger solvent than the mobile phase. If you're on the "ragged edge", the problem can be intermittent. The remedy is usually a smaller injection or a diluent that more closely matches the mobile phase.

Other possibilities: your sample pH is far from the mobile phase pH, and the mobile phase buffer can't deal with it? sample ionic strength is too high?