What affects peak area variation HPLC?

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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I run a method on Waters Alliance 2695 with uv detector. Gradient and flow rate of 1ml per min. Mobile phase A is ammonium phosphate buffer pH 2.9, mobile phase B is 100%Acetonitrile. Needle wash is 80%Acetonitrile 20%Water and seal wash is 90%Water 10% Acetonitrile. I use a 5micron, 4.6x250mm Interstil ODS column. We dont filter our samples which can be very concentrated and are only dissolved in 15mls diluent. Diluent is 70%Methanol 30%Water and 2.5mls phosphoric acid added. Uv wavelength is 228nm.
Here is my problem- although sample injections are very reproducible and consistent in terms of area and retention time, a curious thing is happening with my standard injections. We inject 2 blanks followed by 5 standard injections from the 1 vial. My typical standard area for active is 100000. So 1st injection is 80000 2nd goes up to 87000 3rd is 95000 and the 4th and 5th inje tions are typical at 100000. This creates failing rsds because of the outliers. All subsequent injections of standard in the run are typical and are only 1 injection from 1 vial. So when this happened i changed out the column for a brand new one, equilibrated it with initial conditions overnight then put on a system suit and it was perfect for all areas. I prime out all the lines and injector for a long time prior to each run.
However the 2nd and 3rd runs on the system are again showing low initial areas before the 4th injection which is normal. I changed over to a new system and same thing happened! It cant be the column and its not the standard solution since it has given perfect areas on the first run, and its happening on 2 systems so im not sure what im left with? Could it be lack of equilibration before the standard injection? Overfilling of vial where the 5 injections come from? Run time is 80 minutes starting from 70:30 A:B back again. Im stumped as to whats causing this and if anyone has any suggestions i would like to hear it.
When I was running a method for Anatoxin I would see similar problems. I would have to inject about 5 warmup injections before the RT and Area of the peaks would stabilize. It didn't seem to matter if I let the column rest between runs in high aqueous or high organic mobile phase. It just always took several gradient cycles to bring the system into equilibration again. Not sure if it was a column problem or activity in the lines or what. New columns didn't change it either.
The past is there to guide us into the future, not to dwell in.
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