Troubles with SEC retention times.

Discussions about gel permeation chromatography / gel filtration chromatography / size exclusion chromatography

5 posts Page 1 of 1
I'm having some difficulty with my size-exclusion chromatography (SEC).

I have 2 SEC columns. They seem to be working fine (protein standards look good). However whenever I inject a specific protein into the columns, the retention time of the protein exceeds the permeation limit. This result suggests some sort of secondary interaction between this specific protein and the column, but I'm not sure what's causing it. In contrast all my other proteins seem to be flowing through the column at the appropriate retention times, suggesting no secondary interactions. Currently I'm looking through the literature to see if I can find anything about SEC troubles with this protein, but I haven't found anything yet.

Buffer: Phosphate and NaCl at pH 7.0

Any thoughts? Thanks.
Without knowing anything about the type of column or the protein, it's hard to be specific. In general, proties can be retained by either hydrophobic binding or by ion exchange / adsorption. The end result is that different proteins "like" different combinations of pH and ionic strength. Try doubling or halving the ionic strength to see if that helps. Possibly decreasing the pH to suppress silanol ionization if it's a silica-based column.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
Thanks for the suggestion, I'll give it a shot. However I'm concerned that reducing ionic strength (by dropping the NaCl concentration) will reduce the effectiveness of the electric-field screening. Also I typically use 0.3 M to 0.5 M NaCl (that's moderate to high for SEC in my lab), so I'm not sure how effective increasing the salt concentration will be.

The columns are a Tosoh TSKgel G3000 Swxl and a GE Superdex 75 GL. The protein is streptokinase.
But you can't know for sure unless you try. :wink:
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
I agree with Tom and would also recommend to double the concentration.
Gerhard Kratz, Kratz_Gerhard@web.de
5 posts Page 1 of 1

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