What would cause high AREAS?

[EmpowersBane]

I have 2 UPLCs that I run a gradient method for related substances. Machines were both working fine and reporting consistent areas, amounts and retention times. However, I did not use Machine 2 for about 5 months and when I went to use it again last week, I washed it down with hot water and Acetonitrile before use.

But when I set up a new column, new mobile and washes etc my peak areas are about 20% HIGHER than usual. The peak shape is bang on and precision isn't a problem, just the areas being too high and also retention time was off by about 2.5 minutes for each peak (total run time 42 mins).

I changed mobile phase and column- still the same. I tripled checked injection volumes, detector wavelength, tubing and loop amounts, all normal. Any idea what could be causing this?? Maybe ingrained drug on the tube to detector or somewhere in the injector?

All suggestions welcome!

[tkubowicz]

Hello

First of all you shouldn't judge if method is working or not based only on peak area.
Compare std/sample concentration or ratio betwen two peaks.
If you change lamp in detector or flowcell you can have different peak height and area so peak area as "value" will not give you useful information about method.

Other thing is retention time drift - in your case I'd start with mobile phase checking (composition, pH etc) and also pump diagnostic (leak test, pressure test, valve test etc)

Regards

Tomasz Kubowicz

[Peter Apps]

Are the peaks earlier or later than they were before ?

Peter

[pepterpol]

Hello

Pressure curves would be also useful if your software has option and you storage such data.

[James_Ball]

Are the peaks earlier or later than they were before ?

Peter

If the flow is slower you can get larger peak areas at the expense of peak heights because of slightly broader peaks.

[Peter Apps]

Are the peaks earlier or later than they were before ?

Peter

If the flow is slower you can get larger peak areas at the expense of peak heights because of slightly broader peaks.

And with concentration sensitive detectors a slower moving peak generates more area for the same quantity.

Peter

[EmpowersBane]

Thanks for the replies guys, I managed to find out what it was. I ran a test injection of a standard then went in and watched the pumps, injector etc for anything amiss. I noticed there was a leak in the Accumulator B Check valve and therefore the wrong amounts were in the final A1/B1 gradient causing the RT to come off 2 mins early. However I was surprised with a leak like that, that the peak shape wasn't affected, I thought any void in the pump system would affect retention but maybe because the fittings were before the injection it wasn't an issue? I had lower pressure than usual so that is indicative of leaks.

I tightened up and re-sat the valve and primed back up the system, now areas and RTs are as normal.

[James_Ball]

Thanks for the replies guys, I managed to find out what it was. I ran a test injection of a standard then went in and watched the pumps, injector etc for anything amiss. I noticed there was a leak in the Accumulator B Check valve and therefore the wrong amounts were in the final A1/B1 gradient causing the RT to come off 2 mins early. However I was surprised with a leak like that, that the peak shape wasn't affected, I thought any void in the pump system would affect retention but maybe because the fittings were before the injection it wasn't an issue? I had lower pressure than usual so that is indicative of leaks.

I tightened up and re-sat the valve and primed back up the system, now areas and RTs are as normal.

Correct, leaks before the injector will not cause void problems for the peaks, only flow and gradient discrepancies.