Hi!

Sometimes we have seen that HPLC analyses can give increasing peak areas for the same standard solution injected several times during a series. The drift in the peak area is approx. 10% (over a period of 3 hours and 20 runs). The retention times are not drifting. The analysis is simple (C18, MeOH/water, UV, only one analyte). This problem seems to appear independantly of instrument and sample. What may be the reason for this problem?

Is there any chance that your standard could be evaporating ? Perhaps you might consider using 1 injection from 5 different vials rather than 5 injections from one vial as a small test to check this.

The only other situation that comes to mind would perhaps pertain to the detector, but as you say, the problem occurs independently of the instrument. What evidence do you have suggesting this independence ?

Do you use an internal standard? If you do and it also gives larger area as time goes on, I'd say that Mike's observation that it's probably evaporation is right on. (You could add an internal standard and check this out rather easily). If you don't use an internal standard, you could maybe try keeping samples cold until analysis time and see if that helps :)

Hello

for my experience the most possible reason for increasing of peak area is caused by evaporation of diluent, which often happends, if just Teflon stepta are used.
I recommend to repeat the test using 1) Silicone Rubber Teflon septa and also without septa.

Beside this it should be concidered, if the sample itself is stabile. In case of fluorescence temperature and oxigene quenching could cause a change of peak areas.

I hope this helps