AKTA FPLC - UV instability - (Nov/23/2016 )

Hi

I'm currently trying to cleanup my protein using the AKTA FPLC, however after my last protein run the UV light is not stabilizing anymore. I've already washed the column with .5M NaOH and then 30% ETOH, and degassed my buffer (50mM Tris-HCL + 300mM NaCl) before running it on the column again. Any and all help is much appreciated!

Thanks

uv instability can be due to a number of reasons. some of them are:

dirty flow cell

leak(s), especially around the flow cell

fluctuating flow rate (caused by leaks in the system, leaking pump seal(s), column compression, etc)

detector lamp near or at end of life

fluctuating line current

Hi!

Thanks for the advice! We just switched to another column and the UV is starting to level out. Any suggestions for cleaning a very dirty column? We have tried .5M NaOH and then ETOH but it doesn't seem to be enough to fully clean it up. 

What kind of column is it?

If you think the issue is due to contamination and, assuming resin is compatible, try washing with 6M guanidine. 

it looks like a rising baseline (and a couple of pressure drops and a systemic rise in pressure).

the column may not have been completely equilibrated (including after washing).

besides washing with naoh and etoh, you may need to replace the filter on top of the gel.