gel filtration questions

[nulliespooner]

Hello everyone

I am a biology student and in our protein seperation course we did gel filtration seperation on sephacryl s-200 column

in the beginning we collected the void volume eluted from the column

can someone please explain to me why we check the o.d of void volume? what could be there?

also, we loaded different sample volumes - 1.5 ml and 0.5 ml

what could happen if we overload or underload the column?

if I load a very small protein sample, will it not get seperated well on the column?

Thank you!!

[nulliespooner]

[BabcockHall]

Generally one wants to keep the volume of sample low, perhaps around 3% of the column volume for best separation. As the volume increases, the resolution should fall, all else held equal. The buffer in the void volume might possibly have small molecules that contain chromophores (I am just speculating). This establishes a kind of baseline for absorbance values as one's peak elutes.

[CharonY]

The void volume is important in assessing when the unretained i.e. very large molecules pass through. So in certain applications, e.g. for desalting purposes (or group separation in general), your sample should elute at or shortly after the void volume. Checking the void volume is generally to establish that nothing is wrong with the column (e.g. whether channeling occurs) as well as getting a background measurement (e.g. of the solvent itself). In these cases where you want to separate a high MW from a low MW species, the void volume determines how much you can load. In a well packed column this is about 30% if the total column volume.

For high resolution separations a lower volume is required as explained by BabcockHall. I.e. it is the other way round, a small volume increases resolution (usually down to 0.5% can be used). On the other hand, assuming equal concentration, a lower volume will get less signal, so if you approach detection limits you will have to increase the concentration or volume (and sacrifice resolution in the latter case).