FPLC-SEC PLS HELP

[jane10]

Hi,

I have been trying to pack a 10/30 glass column with superdex 75 prep grade. However when I run standards through the column they all elute out in the 1st 15 ml. The bed volume is around 23 ml. I therefore cant get great resolution:( Does this sound normal?

Thanx

Jane

[jane10]

Also when packing the column (I dont have a reservoir) is it better to pack with a gravity-flow or a constant flow rate under pressure using a pump?

[danko]

It seams to me, your standards are all excluded. Make sure you're using the relevant molecule size standards coresponding to the stationary phase separation range.
As for the packing technique; both gravity driven (with a reservoir) and pump applied pressure driven packing will result in an acceptable stationary phase bed, as long as you observe the recomended procedures.
Also, it is utmost important to achieve as homogeinious packing as possible. Last but not least, both too loose and too firm packing will result in peak asymmetry and lack of efficiency.

Best Regards

[jane10]

Thanks Danko. My standards are not all excluded, they elute after the void volume and are reproducable but they all come out in the first 15 ml's. The void volume is 8 ml and so the separation is extremely bad. I'm using Superdex g75 which has an exclusion range of 3-70 kDa and my standards are 17, 29, 43, 66, and 116 (void volume) kDa.

[danko]

I see, the elution range is OK. But you need a higher efficiency.
In that case you should pack a longer column or connect 2 of the kind in series.
There is a slight chance of achieving a bit higher efficiency, if you manage to pack some more stationary phase in the current column (under a higher pressure). But beware of too high pressure - it might destroy the structure of the gel.

Best Regards

[pelle-plutt]

Also when packing the column (I dont have a reservoir) is it better to pack with a gravity-flow or a constant flow rate under pressure using a pump?

What's the bed height of the packed column?
Since you're not using a packing reservoir I doubt you will get a bed height of 29 cm in the 30 cm column in order to get a 23 ml bed volume. If your bed height is about 19 cm that would explain why all your standards elutes in the first 15 ml.