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AKTA FPLC - UV instability

FPLC

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4 replies to this topic

#1 Irina1

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Posted 23 November 2016 - 01:55 PM

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

 

 Attached File  FPLC help.pdf   90.7KB   95 downloads



#2 mdfenko

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Posted 25 November 2016 - 03:34 PM

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


talent does what it can
genius does what it must
i do what i get paid to do

#3 Irina1

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Posted 28 November 2016 - 12:45 PM

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. 



#4 labtastic

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Posted 28 November 2016 - 08:59 PM

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. 



#5 mdfenko

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Posted 01 December 2016 - 08:56 AM

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.


talent does what it can
genius does what it must
i do what i get paid to do





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