tk7434

Thanks for your responses. I've since re-examined chromatograms and noticed that for some reason, newer batches of buffer had higher baseline conductivity despite following the same recipe (hence decrease in binding capacity). I'm currently troubleshooting this; I think the increase in the ionic strength was the result of re-using old filters which had embedded salt crystals. And for clarification, the random peaks I was referring to were, in general, not in the UV absorption channel but in conductivity measurement. After running the column through with water excessively (>120 CV) they seemed to have stopped appearing.

I've been having issues with my anion exchange chromatography column on an FPLC that I was hoping someone would be able to help with. The column is a GE ResourceQ 1 mL. I'm using the column as a first step in purification of an enzyme, I've previously done the chromatography (two months ago) with very good results. I was just barely saturating the column with enough input material to generate a main peak of around 1400 mAU (so binding wasn't an issue originally). Since then, I noticed the capacity had decreased, and I was losing a lot of protein in flowthrough and only retaining enough to generate a peak of around 500 mAU. The buffers are the same formula but different batches, but I remade them and checked pH and tried again, still 500 mAU. So then I cleaned the column with GE's recommend procedure (2 M NaCl, 1 M NaOH, 2 M NaCl, H2O, buffer). Now, when I run just filtered deionized water through it the conductivity is around 0.18 mS/cm (it used to be around 0) and randomly will rise as a gaussian peak, sometimes up to 2 mS/cm. Does anyone here have any idea as to what could be happening, and how to fix it?