SEC Column Cleaning

[mant0]

I'm having problem with residual protein sticking to a TSK G3000 SWXL column. Ive tried washing the column with 6M Guanadine injections through the autosampler which did not work well. I also tried a 0.1% TFA/40% ACN wash which worked but caused a shift in my retention time (possibly due to the low pH changing pore size?) Anyhoo, I'm a bit perplexed at what I could try next. I was thinking 30% MeOH with some Acetic Acid (pH 3) to help solubilize the MeOH. Any suggestion?

Thanks in advance!

[danko]

The low pH hasn’t changed the pore size. But you have TFA stuck to your column material.
I personally would never use TFA on a SEC column.
But here is a suggestion for you: Prepare an acidic phosphate buffer (0.01 M, pH 2.2). Dissolve NaCl (0.1 M) in that buffer, add 15 – 20 % isopropanol and wash the column with the mixture for 3 - 4 hours (flow rate: 0.6 mL/min, column temperature 35 degrees Celsius)
Finally, flush the column with a mixture of 10 % isopropanol and 90 % pure water for an hour (same flow rate and column temperature).
There’s a good chance that this procedure will help you out.

Best Regards

[mant0]

Sounds like a plan. I will give that a try. Just one question. For column cleaning, I was told that you should run at a reduced flowrate. Is your suggested flowrate of .6 ml/min reduced from a normal flowrate of 1.0 ml/min. If so, my normal flowrate is 0.5 ml/min, should I scale this down to 0.3 ml/min as well as increase the run time for equivalent column volumes?

Thanks

[danko]

Hi again,

It is often suggested that cleaning should be undertaken at reduced flow rate and this seems logical when one is dealing with a clogged column (i.e. massive precipitations or just huge amounts of retained matter) which often results in unreasonably high backpressure.
So if you experience high pressure (for this kind of columns it would be > 1200 - 1300 psi) it would be a good idea to set the flow rate down to say 0.3 mL/min. But if the pressure is something like 500 – 600 psi (which is quite normal for this column at flow rate around 0.5 – 0.6) then you can just use your normal flow rate (e.g. 0.5).
I suggested 0.6 mL/min because I usually find that flow rate to be the optimal for this type of columns, but the type of analyte has something to say too. So I’d suggest 0.5 mL/min in your situation. And yes, maybe you should prolong the washing step a bit – say 5 – 6 hours.

Regarding a potential advantage of reduced flow rate in case of column cleaning (other than pressure considerations) I will allow my self to disagree with the notion. But it may be the subject of a different or derived discussion.

Best Regards

[mant0]

Thanks, for the advice danko! The cleaning method worked pretty well to strip the residual protein. As for my retention time shift, it seems that it was due to a leak in the pump since we changed the check valves the the other day and a fitting was loose.

Thanks again for the help!

[HW Mueller]

Though I don´t remember what the main differences between your and my column (TSKgel Super SW 3000) is, but it might be instructive for you to know that I have mentioned, many times here, a problem with perfluoro acids on this column. In short: These permanently changed the resolution of an antibody and its Fab fragment.

[mant0]

Thanks HW Mueller, I will give that search in the forum.

[hplcuser]

Sepax Technologies, for less secondary interaction and smaller particle size: http://www.sepax-tech.com/SEC.php