N-Glycan Analysis Problem

[wingol]

Hi, all.

First of all, I'm sorry, I'm not good at English. But, I'd really appreciate it if you read my situation and give your opinion.

We use Shodex Asahipak-NH2P 50 4E column and Waters alliance system with FLR for N-glycan method for an antibody product.
Recently, abnormal chromatogram was observed during the analysis.
We injected 2-AA (2-aminobenzoic acid) standard for system suitability test. A single 2-AA peak is usually observed in 2-AA standard chromatogram.
However, during our recent analysis, 2-AA peak was splitted consistently to two adjacent peaks in all 2-AA standard injection chromatograms (6 injections), and the chromatogram of sample treated with PNgase also showed abnormal pattern. The largest peak (G0F) in the sample chromatogram looks like to be splitted, as splitted 2-AA peaks.
All peaks are well-separated and resolution was also good. However, there were additional spiltted peaks that we did not want.

Could 2-AA be present in dimer or multimer under any specific condition? and if yes, could it affect sample chromatogram? (e.g. when the glycan in sample labeled with AA dimer or multimer)

Anyone know about this situation? or have a similar experience?

Thank you for your patience and consideration.

[Vlad Orlovsky]

I would suggest to inject something else and see if it produces a splitted peak. If it does, most likely, your column bed settled and formed a void. You might need a new column.