Hello, Nida. HPLC is more universally applicable, so that would be my recommendation for broad separation of the extract. GCMS has some advantages but will only be applicable for the volatile compounds. HPLC is slower but can work well for all classes of compounds. Best of luck.

Hello Shaikh,

as king told it may be applied for separation of a little amounts by Pre-HPLC and more for the analytical purpose to separate, identify and to quantify these techniques are very much help full.

but, Isolation of secondary metabolites from the plants begins with extraction in various solvents depend on the target compounds to separate. For example, alkaloids are extracted by acidic water, and after basification of the extract, they are again extracted with organic solvent such as chloroform. Sometimes, the methanol extract was suspended in water, and re-extracted with successive increase of solvent polarity, from hexane to chloroform. Afterward, each extract is subjected to separation or purification by the other methods such as chromatography.

Depend on the carrier material in chromatography, the isolation method may be changed into the following manner. The methanol extract is made first. After concentration of the extract, the residue is put on the top of silica gel column, then eluted with solvent gradually increasing its polarity, starting from hexane to chloroform-methanol. By this method, the range of compounds for isolation was widened from non-polar hydrocarbons to some water-soluble compounds such as saponins. Later, variety of new packing material for column chromatography such as Amberite XAD2, Diaion HP-20, MCI gel, and Sephadex LH-20 were introduced. Those new materials effected to fractionate highly polar compounds with greater efficacy and improved reproducibility. For example, chromatography on Sephadex LH-20 effected isolation of highly polar tannins.

Please refer these books for further understanding.

all the best

Generally it can be extracted by water, ethanol and pass through the selected column

Solvent extraction can be a first step . Sequential extraction will do.. Where you start washing the crude product with less polar solvent such as Hexane, Ethyl acetate, Dichloromethane, Ethanol, and Methanol. Then check the extract as the diffusion of chemicals will depend on the solubility

Hello, the first step is to specify the compounds that you want to separate. Suppose you want to separate the alkaline compounds, there are several methods of separation, but in general you need a solvent and an acid and a base once you make the medium acidic and again base to separate the largest amount of alkaloids and to make sure that the separation process has taken place one of the lists of alkaloids The second step is to use TLC to determine the number of separated compounds and if you want to separate a specific compound you use a column technique