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- Posts: 6
- Joined: Tue Jan 06, 2015 4:50 pm
Column: Weak Cation Exchange (COOH-)
Buffer System: Sodium Acetate and eluting with calcium chloride
Column Cleaning: Elution Buffer->Water->Phosphate Buffer (Storage buffer)
Over the past year and a half I've gone through about 5 columns that all eventually show a growing peak at about 7 minutes into a method. This peak only shows up when an actual sample is run. The particular profile of the peaks in this area are dependent on the column. It is present in both the standards and the test samples, all of the same protein, but not in the blanks. Also the time in which this peak appears varies. I had two columns that produced this in about 4-5 months where another produced it in only a matter of a couple weeks.
I have tried cleaning the column via forward and back flushing in this progression:
Water (RT)
Hot Water (~40C)
Elution buffer (Acetate buffer with .2M CaCl2)
Stronger Elution Buffer (Acetate with 2M CaCl2)
Acetate with 2M NaCl
guanidine hydrochloride, 1M
However I have had no luck in getting rid of these mystery peaks.
I've managed to minimize it (so far) happening on a new column by 1. adding sodium azide to the storage buffer, 2. adding a guard column, 3. adding a pre column filter (samples are centrifuged as well)
I would like to have a better understanding of what this is so I can maybe reverse it to recover my old columns. the only other thing I've noticed really is that i will see jumps in the size of this peak after storage.
Any Ideas?