FPLC is behaving strangely - Slow running (Aug/21/2009 )

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.

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).

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

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.

mdfenko on Aug 22 2009, 11:00 AM said:

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).

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.

Luria Bertani on Aug 21 2009, 06:22 AM said:

Hi

Unpack the column, Define the Resin and Pack the column

Regards
Sudhakar

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

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).

mdfenko on Aug 26 2009, 08:36 AM said:

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...