Problems with Sephadex G-200 column

[Sabine86]

Hi,

I`m using a Sephadex G-200 column with a Gradi-Frac-machine or actually I try to use it. Its the first time that I`m working on chromatography. Today after the equilibration with Tris-NaCl-buffer overnight at a very low flow rate, I find the Sephadex gel of the column flowing out of the column through the rest of the system.
I cant explain this because actually I have both a nylon net and a filter to close the bottom of the column. Do someone know, whats the reason for that? Can it be because of too high flow rate? I`m also using a pump (P1), of which I dont know the operating pressure - is it possible that it is to high for Sephadex G-200 (max. pressure: 0,016 bar) ?

Thanks a lot for your answers! I`d be glad, if you could help me!

Have a nice weekend!

[ScottHorn]

What brand of column do you have? What length and diameter? Do you know the porosity of the filter you used for the bottom of the column? For Sephadex G-200 it should be smaller than 20 micron. I assume, since you mentioned the "p-1" pump that you're working with GE equipment. If you have a GE XK series column as well, you may wish to remove the bottom end piece and make sure that the red plastic ring that retains the nylon filter is seated firmly on the rest of the endpiece. You may have to press pretty hard, but eventually it will snap on. You should also check it for tears or holes.

Just out of curiousity, what are you using Sephadex G-200 for?

[Sabine86]

Thank you very much for your help!!! Its great!!! The pore size of the filter could really be my problem, I`ve to check it, when I`m at the university again on Monday. (I think, its 1000µm - what means too high- but Im not sure).
I dont know the brand of the column, because its a very old one (of the university - they actually dont use it anymore, but its the only one they have) and I couldnt find a name on it.But it has no red plastic rings. the rings are white with a space around the column to be able to cool it. the column itself, its about 33cm (length) x 1,5 cm (diameter). I use it to separate some vesicles (out of a fluid) from an amorphous protein fraction. its for my doctoral thesis.
I think the rest of the equipment is a GE equipment - the p1-pump, Gradifrac and so on.

Thanks a lot! Kind regards, Sabine

[ScottHorn]

I'm glad to help. I've had to learn what little I know through trial and error as well. 1000um sounds like a pretty coarse frit. It would have 1mm holes in it, which isn't really going to hold much back. The reason I gave the 20 micron figure is because the only information I could find for Sephadex G-200 was that particle size for the "superfine" grade. If it's anything like the rest of the Sephadex media there will be a "superfine", "fine", "medium" and "coarse" grade, each one with a larger average particle size than the last. So you may be able to get away with a larger porosity on the filter if you have the coarse grade for example. Some manufacturers have strange ways in which they install the frit into the column endpiece, so if you can't find another frit around your lab you may have to contact the manufacturer to get one. Also, are you sure it's "Sephadex G-200" and not "Superdex 200"?

[bioelemic]

hi every one
in deed i advice you to choose another sephdex grad such as sephdex G150 if you can , because the sephdex g200 has a low flow rat and other disadvantages .
by the way i think you need to activate the sephdexG200 powder by incubation in water bath (100C) for 2 hours or in room temperature for 48 hours before the putting in the column .
best regards