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FPLC is behaving strangely


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12 replies to this topic

#1 Luria Bertani

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Posted 21 August 2009 - 05:22 AM

My FPLC is behaving strangely.
It overpressures at very predictable intervals- every 10 mLs or so.

I'm using a HiPrep 16/60 Sephacryl S-200 (Code 17-1166-01). I'm only running distilled water to flush the column; The UV and conductivity readings seem stable.

The maximum flow rate I can go to is about 0.20 ml/minute (the recommended flow rate for this particular column is 0.5ml/min). Higher than 0.20 ml/min, and the column overpressures more readily.

#2 genehunter

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Posted 21 August 2009 - 07:19 AM

It seems your column is blocked with fine particles. Do you see anything color difference on the top of the column? Be sure to filter your samples with 0.22 filter before loading.

You may want to clean it up with one of the following: high salt solution (like 2 M NaCl); denaturing solution, like 4 M Gu Cl; Strong base, 0.1 N NaOH, or Trypsin ( incubate for a while before washing).

#3 HomeBrew

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Posted 21 August 2009 - 02:07 PM

I agree with genehunter -- your column probably needs to be cleaned. See here for recommended cleaning procedures.

#4 mdfenko

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Posted 21 August 2009 - 05:00 PM

running pure water through sephacryl, superose or superdex will cause the gel to swell and the system will overpressure.

also, i find that the inline filter (between the pumps and the injector in the akta system) tends to clog at the most inopportune times.
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#5 Luria Bertani

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Posted 23 August 2009 - 04:12 AM

running pure water through sephacryl, superose or superdex will cause the gel to swell and the system will overpressure.

also, i find that the inline filter (between the pumps and the injector in the akta system) tends to clog at the most inopportune times.


Thank you for the suggestions. The column specifications for the S-200 say I can use 0.5M NaOH, so I will do!
If that doesn't work, I'll try the pepsin treatment. It may have been that some other undesired proteins have come out of solution...

The inline filters- are these difficult to clean/replace? (Ill get advice from the manufacturer before I do anything).

#6 mdfenko

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Posted 23 August 2009 - 08:54 PM

washing with naoh will clean the column. make sure you clean out the residual naoh when you re-equilibrate the column with buffer.

changing the inline filter is easy, but you need a fresh filter for replacement. get that first. then you remove the housing from the line, open it, pop out the frit, replace it and close the housing. then connect it to the feed line, clear it of air and connect to the other line.

you may be able to reuse the old one if you clean it in a sonicator bath.
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#7 sudhakar mutyala

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Posted 25 August 2009 - 01:09 AM

My FPLC is behaving strangely.
It overpressures at very predictable intervals- every 10 mLs or so.

I'm using a HiPrep 16/60 Sephacryl S-200 (Code 17-1166-01). I'm only running distilled water to flush the column; The UV and conductivity readings seem stable.

The maximum flow rate I can go to is about 0.20 ml/minute (the recommended flow rate for this particular column is 0.5ml/min). Higher than 0.20 ml/min, and the column overpressures more readily.



Hi

Unpack the column, Define the Resin and Pack the column

Regards
Sudhakar

#8 T C

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Posted 25 August 2009 - 04:14 AM

Hey,

Maybe its good to add this:

B4 doing anything to the column, just chk if the blockage is really in the column by disconnecting it and running buffer thru the machine.

Best,
TC

#9 mdfenko

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Posted 25 August 2009 - 02:36 PM

sometimes the blockage is found in the filter on top of the column. the filter needs to be removed and replaced.

the parts come with the procedure and the tool should have come with the column (or you can purchase it).
talent does what it can
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i do what i get paid to do

#10 Luria Bertani

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Posted 25 August 2009 - 11:16 PM

sometimes the blockage is found in the filter on top of the column. the filter needs to be removed and replaced.

the parts come with the procedure and the tool should have come with the column (or you can purchase it).


Hello, thank you all for your suggestions.

I have disconnected and run back pressure tests (I did this after calling GE). Everything is fine there.

I have run 0.2M NaOH through the column. To my amazement, the pressure went right down, and I was able to increase the flow rate from 0.2 mL/min to 0.5 mL/min. The A280 didn't register any large peaks, so I suspect that it might have been DNA or other proteins.

It is still flushing, but the column still overpressure almost exactly every 9.5mL. Hopefully this will go away once the NaOH is flushed through the whole column. I have pepsin on standby should it require a more vigorous clean/protein digest.

Unfortunately, I can't open the column. It's a pre-packaged column with "Do not open the column" written through the specifications sheet.

Once again, thank you. I'll let people know how it goes once it's done...

Edited by Luria Bertani, 25 August 2009 - 11:19 PM.


#11 Luria Bertani

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Posted 05 September 2009 - 01:25 AM

We'll after cleaning it with NaOH wash protocol, I equilibrated with my buffer and ran a prep.
And I increased the pressure alarm... to 0.3 MPa (under supervision).

The prep worked out fine, and I got a crisp peak. No annoying overpressures every 10 mL.
You could still see tiny pressure spikes, but these instantaneously return to normal baseline pressures. Must be something quirky with the machine or resin.

Finally. It works. I'm very happy :rolleyes:
Thanks for the advice everyone.

LB

* Always call up the manufacturer and work under supervision before doing. I can't accept any liability if you follow this advice, damage results or guarantee that it is correct.*

Edited by Luria Bertani, 05 September 2009 - 01:29 AM.


#12 HomeBrew

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Posted 05 September 2009 - 02:51 AM

Finally. It works. I'm very happy :rolleyes:


Congrats, LB!

#13 Must_Grow_Bugs

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Posted 12 January 2010 - 10:04 AM

I don't doubt that your column had some blockage, but if you are using an Akta FPLC and the spikes are about every 10ml you also may want to turn pulse compensation off. This is especially true if you are running columns with max pressure of 0.5MPa or less.




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