Fractionation of mushroom extract

[seamoro]

hello, i am currently fractionating a mushroom extract (ethyl acetate fraction). I have the sample aborsbed on silica and packed in a column and i am now passing acetone/hexane through the column and collecting fractions. I have started off using 0.5% acetone/99.5% hexane and i am continuing increasing acetone by 0.5% after every couple of fractions collected. My main question is how to interpret the TLC of the fractions!! obviously single band indicates pure compound but when i have a number of bands it is unlikely i will get pure compounds if i see 2 bands in a TLC for a fraction...just some advice would be great!!

[tom jupille]

Pretty much the same as any other form of chromatography:
- if you see two or more spots, you have two or more compounds
- if you see only one spot, you have one or more compounds.

A single spot by TLC is no guarantee of purity. Conventional TLC is (usually) a relatively low-efficiency technique (i.e., spots are wide relative to their migration distance), so you only have a room to fit in a dozen or so spots. On top of that, if you are using silica gel, the selectivity is probably similar to what you have on your fractionation column, so that coeluting compounds in one system will probably coelute in the other as well.