Chromatography Forum

I am developing a method for a drug using plasma using HPLC and I see a sudden decrease in the intensity of the peak, or there is change in the peak shape. I tried using a new column, also tried increasing the concentration of the derivatizing agent but still I am facing the same problem. Still the peak shape keeps on changing its not constant. Could you help me with this.

OK, we need more details in order to help.

How do you define "sudden"? Do you mean that one injection was OK and all subsequent injections were bad? Or did the deterioration occur over the span of an hour, or a day, or a week?

When you changed the column, did the response and shape improve and then get worse again, or did the new column make no differenc?

first I had used a new column on which I got a good peak shape and accuracy too. But then there was increase in pressure and my system was shutting down due to it and this was because of clogging of the filter so when the filter was changed the pressure problem was solved but then the peak area reduced nearly by half and this was in about 1 days gap from the previous run.
Also I saw some contamination in my mobile phase (methanol) so i made a new one . So my question is how can I get the same peak shape now? has my mobile phase affected the column?

If I understand correctly, the peak shape was OK until you changed the filter and was bad immediately afterward. If that's the case, then the most likely problem is a dead space somewhere in the plumbing; perhaps the filter incorrectly hooked up or a fitting not seating properly.

do you know your sample solution stability? what kinds of function groups you have in the molecule? what is the buffer/pH in the mobile? did you store at ambient or fridge?

My compound is stable at ambient tempt I do store it overnight in fridge at around 20 degree celcius. The compound has a amino acid group. The mobile phase is phosphate buffer with pH 7.5.