Hello everyone

I use the Varian ProStar HPLC system with a fluorescent detector to analyze the content of OPA derivatives of sphigoid bases in biological samples. I use 150mm C-18 RP column with a column oven set at 30 C, the mobile phase is acetonitrile/water 9:1 (isocratic program). Samples are dissolved in 95% ethanol. The problem is that in every injected sample (tissues/standards mixtures) I got a huge, strongly retained impurity. The width of this "peak" is about 30 minutes! It's not a baseline drift, because it never appears in a blank injections. I've found that this impurity propably consists of unreacted phthaldialdehyde and/or mercapthaethanol. But the strangest thing is how the retention time of this impurity is shifting from injection to injection. For example from 90 to 30 minute overnight. The retention times of peaks of interest vary only slightly. What could be the reason of such huge changes in the retention time? Any theories would be greatly appreciated.

Marcin

Ps. Sorry for my english. I hope you'll manage to understand my post.

Actually, the only surprise for me is that you do not seem to have trouble with your analytes. We had so much trouble with OPA (and amino acids) that I wonder how anybody gets anywhere with it. How about FMOC, etc.....?