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MonoQ column

M. Johan Broekman hanbroekman at csi.com
Thu Jan 27 22:06:44 EST 2000


Debora Fontanini wrote:
> 
> Hello!
> 
> I have a problem and hope some of you might have a suggestion or two on how
> to solve it.
> In the process of puryfing a peptidase, I have been separating my partially
> purified protein sample on a MonoQ column.  Buffer A is 20 mM TEA/HCl, pH
> 7.5 and B is A + 1M NaCl.  The gradient goes from 0 to 40% B in 35 minutes.
> For a few injections I was able to collect a peak coming off the column at
> about 20% B.  Then I changed the buffer system to separate a different
> sample and when I switched back to the original system and sample, nothing
> would come off the column.  Thinking that the column was dirty, I cleaned
> it following the manufacturer's instructions (not up to the point of using
> pepsin, yet) and despite an improvement in the baseline, there was no
> improvement in the sample recovery.
> I might add, if it is of any help, that the column is fairly new (used for
> about 10 injections) and I am only injecting 150 µl of sample at the time
> since I am using an HPLC system adapted to work as FPLC and I do not have a
> larger loop available.
> 
> I would greatly appreciate any suggestion
> 
> Thanks in advance,
> 
> Debora
> ---

Debora:

It has been a while since I used our MonoQ column.  I don't know what
size column you are using,but the gradient seems fairly steep to me. 
How long do you keep the column at 40% B? Have you used a 10-15 min step
at 100% B to see whether there is a delay with the elution of your
material?  You are sure that previously you got the material off EVERY
time?  Or just once after a few injections?  Can you recover injected
material when you run the column contimuously at 40% B?  I have no idea
how you detect the sample, or whether you introduced a contaminant onto
the column that digests it.

(I think the info you gave us is incomplete for more advice, unless
someone else has encountered a similar problem,AND solved it)

-- 
Best regards,
Han Broekman
(please answer to the newsgroup)




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