Issue with Baseline Drift & Ghost Peaks during SEC

Discussions about gel permeation chromatography / gel filtration chromatography / size exclusion chromatography

6 posts Page 1 of 1
Hi Everyone,

I'm utilizing peptide-level size exclusion chromatography to separate tryptic peptides on an AKTA Fast Protein Liquid Chromatography system with a Superdex Peptide PC 3.2/30 gel filtration column (ideal separation from 100-7000 Da). We utilize an isocratic elution with a mobile phase consisting of 30% Acetonitrile and 0.1% TFA at 50 μL/min (bed volume 2.4 mL). In order to calibrate our separation, we use insulin (5.7 kDa) and angiotensin II as standards. While running standard separations today, I noticed a few ghost peaks and substantial baseline drift.

Normally, our standard run has the following UV chromatogram at 215 nm:
Image

However, my standard runs today looked as follows:
Image
Image

The base peak drift seems to occur after small molecules would typically elute , since glycine (75 Da) elutes at around 2.0 mL.
http://eclub.biomart.cn/sites/eclub.bio ... 3.2-30.pdf

I'm confused as to the possible explanations of these ghost peaks and the substantial baseline drift, as the peaks should not be occur from samples left on the column after a previous run (small molecules elute well within the separation, and these standards are all in high-purity and well above that molecular weight range), I include an extra 2 CV wash between sample runs to fully elute any and all molecules injected onto the column. Furthermore, since the separation is isocratic, the baseline should not drift dramatically for any reason.

Could this potentially be due to an old UV lamp? I doubt it, but I've eliminated many other possibilities and our UV lamp is quite old at this stage.

EDIT:

One thing I am noticing is that these ghost peaks and baseline drifts are identical at 280 nm and 215 nm, suggesting that the solvent or any real elution itself is not to blame. Am I right in suggesting that the UV lamp is the predominant cause?
I guess you are right with your UV lamp.
Did you prepared your Insulin standard freshly or was it 1 day old. Than it could be a degradation product from Insulin.
Good luck.
Gerhard Kratz, Kratz_Gerhard@web.de
Gerhard Kratz wrote:
I guess you are right with your UV lamp.
Did you prepared your Insulin standard freshly or was it 1 day old. Than it could be a degradation product from Insulin.
Good luck.


Thanks. I'm starting to think it's the only possibility.

As for the insulin, the insulin was not prepared fresh (it was prepared about a week earlier at 1 mg/mL, stored at -70°C in 30% acetonitrile/0.1% TFA), but the degradation products I would predict are substantially different from that ghost peak. The ghost peak would have to be a dipeptide or an amino acid, due to the time of elution, while the predicted degradation products of insulin would be the A and B chains:
Image

The protocol we utilize is modified slightly from Leitner et al., 2013 [1], where the A chain is actually a standard in the mixture:
Image
Protein 1 is insulin, 2 is insulin chain A, and 3 is angiotensin II.

[1] 10.1038/nprot.2013.168

Thanks for all the help. Is there any way to definitely characterize the issue as one of the lamp and not of the sample?
To me it looks a bit more like an air bubble in the detector: Does the autosampler (or do you by manual injection) introduce an air bubble? The large peak and instant frop in your upper chromatogram looks like something comes right after the solvent peak. Dry to flush the flow cell with isopropanol and tap the detector with your finger while flushing. This should get out bubbles.
bunnahabhain wrote:
To me it looks a bit more like an air bubble in the detector: Does the autosampler (or do you by manual injection) introduce an air bubble? The large peak and instant frop in your upper chromatogram looks like something comes right after the solvent peak. Dry to flush the flow cell with isopropanol and tap the detector with your finger while flushing. This should get out bubbles.


I use manual injection, and I'm very careful to remove any possible air bubbles. We had a problem before, so I'll be sure to do that whenever I have a characteristic that characteristic signature. Thank you.

We've also had small problems previously that seemed to be characteristic of the detector resetting which seemed to disappear.

Example 1:
Red = 215 nm
Blue = 280 nm
Image
Link to higher resolution

The 2nd peak immediately peaks and then drops, suggesting a detector reset. Afterwards, we have a series of peaks where the intensity quickly builds up before instantly dropping, which I believe is an air bubble in the detector. I flushed the system with water for a while to flush the air bubble out (next time I'll use isopropanol, thanks!).

To confirm that the two peaks aren't related, here's another image with both issues but independently of each other (different column and tubing [Flow rates and ideal back pressures for peptide SEC are very different than those for protein-level affinity purification], same detector):
Image
Link to higher resolution

Thanks for all the help.
It's not a lamp problem.

Check your pump method.

Plot the pressure under the elution. and overlag the trace with the UV signal.

Best Regards
Learn Innovate and Share

Dancho Dikov
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