Recently my pepsin column got some blockage, supposedly because of the precipitation of aggregated protein. Multiple washes with 0.1% FA or pepsin wash (1.5M GuHCl + AcN + FA 95.2/4/0.8), as well as reverse flushing didn't help, the pressure is still too high. It seems like the column cannot be used anymore anyways, so I was wondering if there's any other washing method that I could try?
Even if it's somewhat dangerous to use it on the pepsin column.
You did not say exactly which type of HPLC column was used, but this article may provide some useful advice.
- "Notes on Cleaning bound Protein from RP HPLC columns"; https://hplctips.blogspot.com/2016/10/notes-on-cleaning-bound-protein-from-rp.html
William Letter Thank you for your advice! My problem is with Waters' Enzymate Protein Pepsin Column (300Å, 5 µm, 2.1 mm X 30 mm) rather than RP HPLC column, so strong wash solutions used for HPLC columns are not suitable for it, and I have already tried all the methods recommended by the manufacturer.
The article referenced offers advice for many types of HPLC columns. However, columns are consumable items. After a suitable amount of use, the time and materials required to regenerate them may cost more than the purchase of a replacement column. Dispose of the column.
Contact an experienced chromatographer to assist you in the use of the column and method. Ask them to review your method, sample prep, instrument settings and sample solubility to insure you are not overloading or fouling the column as well as checking the method meets good chromatography fundamentals.
Dear Bolonova,
Considering your column was clogged due to the protein fouling which was induced by a lower pH environment requested by immobilized pepsin activity in Enzymate column. The best option is to buy a new one and lower concentrations of injection for analysis.
I was also faced with a similar problem.
Any cleaning attempt will also cause pepsin activity loss. Traditional protein column wash applications such as urea wash, and alkaline wash probably cause pepsin denaturing and therefore activity loss... You may try ambic wash at pH 8-9 overnight to solubilize clogged proteins but I am not sure that option can both clean the column and able to keep the pepsin fully active.
I never tried but non-denaturing detergent (e.g. triton x-100) flow without introducing the column effluent to mass spec, and using an individual pump (only used for cleaning purposes) may do the job...
If you are about to put the column in the waste bin then it will be worth trying.
Good luck
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