the type of your resin is important as that defines the range of your resolution, so you'll want something like Superdex 75 (3-70 kDa), Superose 12 (1-300 kDa), Sephacryl S-100 or S-200 etc.

the longer the column, the better resolution, but also wider peaks what can be problem especially because of the diffusion in SEC

diameter is important for how much sample you can apply. You should not apply more than 0.5-1% of volume of the column, but the bigger diameter, the more you can apply

if you have such lousy system, I'd recommend having buffer somewhere up (on top of your table) and letting it simply flow by the difference of hydrostatic pressure. You can add some LMW dye to know where all your sample is out

The column should be long and narrow. The size depends upon how much protein and what volume you intend to apply. This handbook may help:

http://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314807262343/litdoc18102218_20140915112126.pdf

Hi there,

The usual size for preparative SEC is 16/60 which gives 120mL of bed volume. Buffer flow should be 1mL per min during running. You can find some instructions from GE for column packing like the following:

http://eclub.biomart.cn/sites/eclub.biomart.cn/themes/aktaclub/Files/56119099AE_UM_Superdex_30,_75,_200_prep_grade.pdf

In your case Superdex 75 (efficient resolution up to roughly 75kDa) should be fine unless the oligomerization state of your protein is more than 3. If more than 3 then Superdex 200 should be used (efficient resolution up to around 200kDa)

Hope it helps in some ways...

Dear Peter,

Gravity flow shouldn't be a problem for you : I used a 2m heigh column for a long time, and it was working perfectly provided you put the buffer bottle over the top of your column. In this case, just measure the flow rate of your column so that you are sure that it doesn't exceed the maximum flow rate recommended by provider (or it could pack your medium too much so that it is clogged), and if necessary limit it with a tap below it.

Second, I don't really understand why you say you need fresh resin for each run ? If it is because you column is clogging, it means you apply a sample not compatible to SEC (too aggregated, too sticky, etc). I would recommend rather to put a filter on the top of your column or before (or if you can't, just removing the first half cm of the medium where the clogging molecules are usually will do the job). And cleaning of the column with 0.5 to 1 M NaOH usually removes most of the sticky proteins (which can be even more efficiently made by a small 10-100mM HCl + Pepsine enzyme cleaning, provided your medium isn't destroyed by extreme pHs, which is most of the time possible). If you have too much lipids : the same, just put a filter, and also add some detergents in you buffer...

Concerning the SEC packing, it must be me made on one shot run, with the flow rate as near as possible as the maximum flow rate applicable to the medium, if you want the column resolution to be the best it can be.

As a reminder to what some said, if you want a highly resolutive column, choose a thin and moire long one (1.6 cm width and 60 cm long is one of the best from GE, but if you go to more longer one fore the same width, you will get better resolution). If you want to apply particularly huge amount of protein in each run (say more than 50mg of protein), you can use a larger column, but it will enlarge also your peaks and then lower the resolution !

Eventually, concerning the medium choice, remember that a medium is always more efficient in the lower range of its resolution (elution volume proportional to the LOG of the MW). Then, a Superdex200 for your 20KDa sample will be more efficient than an Sup75...

Hope this will help you Padawan, Best,

YV