Increasing Peptide Areas

[Eric Moore]

More peptide questions.

What would cause increasing area of multiple injections of a peptide standard (C18, AcN/H2O/Tfa mobile phase)? I know evaporation could cause this, but my standard (prepped in 2% AcN, so not very volatile) increased by 50% in 90 minutes. That was the end of a trend of area increase over 5 injections from the same vial. This seems extreme for evaporation.

Could it be due to junk on my column blocking active sites, then the junk washing away, improving the retention of my analyte? We haven't done a great job of washing the column (early stage R&D).

Could the vial caps be an issue? We've seen some variability before, but nothing this extreme, and the only difference (besides a dirtier column nowadays) is the vial caps are different. [Which, again, could indicate evaporation, but 50% in 90 minutes, 2% AcN, at room temp??]

I'm going to wash the column today and see if it helps.

EM

[unmgvar]

from my experience it is many times a carry over issue.
what HPLC model do you use and does it have some sort of wash solvent?

[Eric Moore]

Thanks, unmgvar. I checked for carryover, and it is negligible. I also tried adding a high %age organic flushout at the end to wash the column.

I also checked the injector reproducibility by weight, and the injector is working just fine.

EM

[HW Mueller]

Do you know how big your peaks should be? Could it be that your initial peaks are small, because some peptide gets hung up on the column ("active sites")?

[Eric Moore]

Thanks, HW Mueller. I don't know how big the areas are supposed to be, but I know they were smaller the first few times this peptide was run. For example, samples that a few weeks ago were running about 200 AUC are now approaching 450.

I washed the column. We'll see what happens. These samples have had who-knows-what in them alongside the peptide, so I'm hoping the column just needed a bath.

EM

[danko]

The most probable cause for this observation is – as HW Mueller anticipates – a progressive saturation of the stationary phase with sample.
If that is the case, then you’ll observe decrease in the observed tendency ending in a stable level, following a number of injections 5 – 10? (depending on the sample load).
Another confirmation of that would be a repetition of the above experience following column cleaning (an effective one anyway) or/and introducing a new column.

Best Regards

[Eric Moore]

Thanks, everyone.

I did a column wash as described in the column booklet (0.1% TFA followed by 0.1% TFA in IPA/AcN) and my peak shape improved, my areas got bigger than they had been (AUC ~780), and my reproducibility for 3 injections was <0.3. Then I went home.

Thanks for all your help, everyone!

EM

[HW Mueller]

Did your peaks improve in shape in the experiment described in your first post? Was that injection series one of a kind?

[Eric Moore]

In the first experiment, the peak shape was not great, just acceptable. As the peak areas varied, the shape remained "okay."

The peak shape did improve after cleaning. The reproducibility was better (but not exceptional) throughout an entire run (2.3% RSD for 6 injections across a 5 hour run).

However, the next day the peak areas were all over the place again. I think I just have to replace the column. The improvement I saw with cleaning was pretty good evidence that the column was the issue. And knowing the junk that is in some of these samples, I'm not surprised.

EM

[HW Mueller]

Doesn´t make sense unless some of the injections you mentioned were not clean standards (mentioned in the first post?), but rather gunky samples. Maybe you need to clean up the act before hitting the column?

[Eric Moore]

Hi, HW -

The standards in the first post were "clean," but they came after repeated use of the column, running samples with a variety of filth. I think the effect I saw on the original standards was a result of what had come before.

EM