Chromatography Forum

Using silica base SEC columns for protein analysis with pH 6.8 phosphate buffered saline (generic buffer), we encounter poor performance as follows:
1. After long-term storage in methanol-water, wash in 100% water before column use, then equilibrate in run buffer, the column needs about 10-15 column volumes before reaching an equilibrated state( as indicated by dimer/monomer ratios to historical performance). Wouldn't expect this if just size was controlling the separation.
2. Number of injections per column typically 150-200 before column completely dies ( loss of resolution, response suppressed) making SEC analysis very expensive. We have tried 2M guanidine HCL washes of the columns to restore performance, but this doesn't work on the older columns usually.

Any thoughts on more robust SEC with faster equilibration and longer column life?

I have had the same experiences as you with SEC on both silica and polymer based columns. They are not very stable, and 200 injections is not that bad (seen worse).

A couple of things: an inline filter just before the column could extend the lifetime.

Also, air in the system can cause severe damage to the column. It is a good idea to run the column backwards for a minute or two, to be sure that all air is removed from the top of the column.

The equilibration time seems quite normal too.

Actually, washing with guanidine HCl may be counterproductive. Years ago (late 70's), when working with silica-based SEC columns, we had to make several large injections of BSA on a new column in order to get decent recovery. Over time, the recovery would deteriorate and the BSA injections had to be repeated. The assumption was that the BSA was being adsorbed and therefore blocking residual active sites. Granted that column technology has improved in the past couple of decades, but it might be worth a try.

Oldtimer,

I have this feeling; you’re washing the stationary phase out. The reason I’m thinking of it is the lost resolution and recovery. The first could be due to the fewer and fewer pores for the analytes to go in and out. The second (poor recovery) could be due to more and more free silanol groups get available for the analyte to be retained non-specifically and irreversibly under the running conditions.
If the theory is true, the guanidine treatment will not do any good.
I can give you 2 suggestions:

1. Dilute some NaCl (1 to 2 mol) in a portion of your phosphate buffer and wash the column for an hour or so. Try and monitor the UV signal while washing. Maybe you’ll se “a forestâ€

Let us know about your typical number of runs and for how long you store the column.

Assuming that you commonly just make a few runs, maybe 10 or so, followed by storage, it could be that the storage is the problem. If this is the case, I would store the column in 100% acetonitrile for long-term storage.

Those who have been participting here for a long time know that I have this "standing" problem of why TFA and probably other perfluoro acids permanently changed the resolution of antibodies on a Toso TSKgel Super SW 3000 column. The proprietary surfaces of these silica columns seem to be quite touchy. The problem could be alleviated somewhat by using a buffer (without TFA, of course) which keeps the proteins happy (well solvated, etc.).

Hans,

Oldtimer uses the very same mobile phase as you’re suggesting, i.e. phosphate buffer (although no concentration is mentioned, which can make a huge difference). So how does TFA fit in this particular situation?

Best Regards

The original idea was that maybe TFA would help in preventing Mab-stationary phase interaction. Having had some bad experience with TFA before this (with peptides on RP), I thought I should be fair and give it another go. Bad mistake.