Chromatography Forum

Could anyone recommand some quality SEC column for protein separation? We are using G4000SWxl currently with MAL detector and would like to explore other column (MW of target protein is around 500KDa) for possible better separation.

Thanks,

I use Phenomenex range of SEC with great success.
For your application I would use the BioSep SEC s4000 with a separation range up to 1500kD.

we use Sepax Zenix 300A pore size
so far it always gives the best result in comparison to the other column we have which are the TSKs, Bioseps and dionex columns

I really appreciate your valuable input. I'll check their specifications and try some.

Thanks and Happy Holidays to you all.

I use Phenomenex range of SEC with great success.
For your application I would use the BioSep SEC s4000 with a separation range up to 1500kD.

Pelle-plutt,

Have you any experience with regenerating / cleaning up BioSep Phenomenex columns? We have several used BioSep columns, which deteriorated after only a few (< 100) injections.

The resolution and peak shape became much worse.

There was no improvement after prolonged washing with 20% ACN or other buffers recommended by the column brochure.

I have worked with the Tosohaas columns, and they usually give good separation at first but the don't last long.

The Superdex columns are by far the most durable SEC columns I have tried. I still have the first one I bought, and it is still working perfectly after thousands of injections.

We have also seen deterioration with the Phenomenex BioSep SEC columns after 80 or so injections. The same loss in performance doesn't seem to happen to the TSK or Waters columns. Lately we have been using the Waters BioSuite column because the resolution is slightly better for proteins than the TSK. Washing with NaSO4 and GCl at the end of each run has helped keep our SEC columns pretty clean.

We have also seen deterioration with the Phenomenex BioSep SEC columns after 80 or so injections. The same loss in performance doesn't seem to happen to the TSK or Waters columns. Lately we have been using the Waters BioSuite column because the resolution is slightly better for proteins than the TSK. Washing with NaSO4 and GCl at the end of each run has helped keep our SEC columns pretty clean.

Vickig,
Thanks for the tip. Do you wash with guanidine hydrochloride because you test denatured proteins?

Mattias,

Thanks for the recommendation.. How is the resolution of the sephadex column? is it comparable to the tosoh column?

I use Phenomenex range of SEC with great success.
For your application I would use the BioSep SEC s4000 with a separation range up to 1500kD.

Pelle-plutt,

Have you any experience with regenerating / cleaning up BioSep Phenomenex columns? We have several used BioSep columns, which deteriorated after only a few (< 100) injections.

The resolution and peak shape became much worse.

There was no improvement after prolonged washing with 20% ACN or other buffers recommended by the column brochure.

Can I ask if your protein sample is highly hydrophobic? It is quite sample dependent. Sometimes you will find different sample property may not always work by one single column (surface chemistry).

@biochemist
In my experience the Tosoh supersw3000 blows the superdex 10/30 out of the water when it comes to resolution and speed (15 minutes vs 1 hr). However, as Mattias said, the superdex columns last much longer than the Tosoh columns, and suffer from less non-specific binding as well. With the superdex column you can always strip everything off with NaOH and it works like new, but with silica based columns such as the Tosoh, you have to rely on other methods to clean them. I've found that in certain situations no amount of cleaning will restore the resolution of one of these columns, sometimes after as few as 200 injections. And they're not cheap either.

We have seen very good results with the YMC-Pack Diol-200. It gives a great resolution, a fairly quick run time (30 minutes) and it very durable. It is much more affordable than the Tosoh or Superdex and yields similar results.