I want to purify crude alpha-amylase with column chromatography. I am using a spehadex 75, but for some reason I can't find any information on how to make the slurry.

I can quickly find a lot of information on how to pack and store a column, but what I really want to know is how much powder and buffer do I mix together?


1 Answer 1


Sephadex resins swell as they absorb buffer, so the amount of buffer you need to add is dependent on the amount that the specific resin you are using is expected to swell. It is also dependent on the final thickness of slurry that you want to achieve. Too thick - the resin will not be suspended well enough and will pack poorly. Too thin - the resin at the bottom will settle before enough buffer has flowed through to allow you to add more, and again you will not get good packing (and it will take a lot longer to pour).

Sigma Aldrich provides technical documents online with recommendations for preparing Sephadex slurries from dry powder, which you can find here.

Here are some the the key points. The expected swelling of the Sigma Aldrich resins is provided in units of mL per 1 g dry resin:

Sephadex G-10 Medium: 2-3 mL/g

Sephadex G-25 (all grades): 4-6 mL/g

Sephadex G-50 Fine: 9-11 mL/g

In all cases, add buffer and allow the resin to swell (1-3 hours at room temperature), then top off the buffer so that the settled swollen resin is ~75% of the total volume. That will give you a slurry of appropriate thickness when stirred.


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