peak shouldering in FLD
Posted: Thu Nov 09, 2017 2:58 am
by msjeon
Hi, I got a protein peak shouldering in our Waters 2475 fluorescence detector.
What would be the reason of the shouldering?
The peak is protein, mobile phase is Trizma buffer, column is Mono Q™ 5/50 GL.
Thanks in advance.
Re: peak shouldering in FLD
Posted: Sat Nov 11, 2017 4:01 am
by tom jupille
Peak shape issues are almost never related to the detector. Possible causes:
- a headspace at the top of the column
- a partially-plugged inlet frit
- more than one protein (partially separated)
- multiple conformers of the same protein (partially separated)
- a contaminated column
Did the column ever work for this type of sample? If the problem has been there all along, I'd be inclined to suspect multiple proteins or conformational states. If the problem worked well initially and the peak shape problem showed up abruptly, a partially-plugged frit is more likely; check with the manufacturer to establish whether the column can be back-flushed. If the problem worked well initially and the peak shape problem developed gradually, then a head space or contaminated column are the most likely suspects; check with the manufacturer to see if they have a recommended regeneration/cleanup procedure. If that doesn't help, replace the column.