Have you pre-tested the separation on a TLC plate to see what solvent system works best? If not yet, you can try diethylether on silica gel but you have to be careful. But I think it is best to test the separability of the extract by TLC.

First do the TLC of the extract and then see where the spots are coming. You will get the idea of which solvent to be taken. Generally column chromatography starts with a non polar solvent and slowly increase the polarity of the solvent till all your compounds are eluted out from the column

Which type of compound you want to separate?

it is important to know the structure of the compound and from that you can select the best solvent of that compound also do not forget the pH, you can select the appropriate pH to extract your compound

Dear Saleheen,

You have NOT mentioned about the chemical nature of the compound that you want to separate. Generally, TLC and column chromatography like HPLC may be useful for isolation.

You could try RP chromatography, but before that get the complete information about your product.

First of all you must determine what type of compounds do you want to isolate and then we can told you what is the best method for isolation

Before you will go for chromatography, pl fractionate your methanolic extract with hexane, ethyl acetate, chloroform and butanol by dissolving your methanolic extract into water. After drying each extract in different solvent you go for column chromatography.

Ajai, what exactly do you mean by drying each extract in different solvent? Do you mean after drying the different fractions i.e. ethyl acetate, hexane, chloroform and butanol fractions using a rotavapor or something similar?

see the type of F.G of your compound and then decide the buffer w.r.t pka value.

Dear All, actually im facing the same problem too. If I wanted to separate phycobilin compounds such as (phycoerythrin and phycocyanin) from Methanol Extract of a red algae by simple Column Chromatography . Then wat will be the most suitable mobile phase to use?? If posible please suggest any protocols or journals to view. Thank you

Prepare the column in 100% methanol/chloroform and pack it by wet packing technique. Take proper precautions so that the column doesn't break. Now take your extract (about 5 gm) and dissolve it in a little quantity of parent solvent i.e. methanol. Once you get a methanolic solution. add such an amount of silica gel to this solution that it adsorbs all the extract completely and allow to stand for some time. After drying make sure that it has become a free flowing silica gel with the extract adsorbed on it.

Then add this sample to the column which has been prepared beforehand in methanol. Add cautiously so that it forms a uniform layer above the column (of course submerged in methanol). Then after total addtion of sample try to remove the excess methanol from top of the sample using either a pippette or you can also use cotton to absorb the solvent and let it remain there. Then run the column with your solvent system (the best one you found out after TLC of the extract). Collect the fractions and do repeated TLC in the same solvent system to find the nature of fractions. Pool out similar fractions and identify single spot fractions. This will be your isolated compound. Once you get single compound in a fraction, collect all similar fractions and pool them together. Finally concentrate the fractions to minimal volume and leave in a refrigerator for crystallization.

Thank you Mr.Alok .

for the methanol extract, I think you need to use the reverse phase flash column packed with sephadex.