Decrease in Peak Shape Quality/Area over course of HPLC run

[mhart]

Good morning,

I am new to this forum.

I am producing inconsistent peak area values over the course of a run. Generally speaking, the peak areas decrease over time (although I do not suspect sample degradation) when injecting from either the same vial or multiple vials of the same solution. The peak shape is also slightly decreasing in quality (i.e., getting wider but maintaining acceptable asymmetry).

Additionally, I have linearity (r2 = 0.9999) over a wide range (50% - 150%), but I am not meeting my y intercept bias specification (spec = 2.0%, I am getting ~2.8%). I am using an Agilent 1290 DAD single wavelength with reference wavelength. Further, a simple peak area comparison is showing ~48.8% recovery of the 50% standard (multiple preparations), while the 150% has shown ~151.0% recovery, when in the past I've seen 50.0% and 150.0% ± 0.2% in previous validations.

I am leaning towards a detector/system issue, since I have spoken with the column manufacturer and column overload is not an issue at these conentrations. I believe I have also eliminated sample preparation as a potential issue since I've demonstrated this issue multiple times with multiple preparations and multiple diluents.

Please advise. Thank you.

[KM-USA]

This belongs in HPLC section

Done
-mod

[AnalMD]

One possibility of the decreased area count could be the growing presence of an air bubble in the injector system or in the syringe. Purge the injector system and if possible, move the syringe barrel rapidly. This could displace any hidden or bound air bubbles.

For the peak shape, are your standard injections and secondary standards (standard checks) consistent throughout the run? I've seen on occasion the sample matrix affect the peak shape of components in samples, but the standard checks were fine and consistent.

Is your procedure isocratic or gradient? If isocratic, maybe something in your mobile phase is accumulating in the column resulting in a change in peak shape.

[mattmullaney]

Hi mhart,

Another injector-related possibility is the autosampler syringe. Just had to swap out one of mine because I observed the same symptom, and it was a quick-and-easy cure.

Good Luck!