Can you eloberate what was your desired compound in your extract

Kishorebabu T

Alkaloids are basic the other compounds are more neutral, so I would try:

1) Make up some 10% aqueous acetic acid, pick up the extract in dichloromethane and extract into the acid.

2) make the aqueous extract basic and extract back into dichloromethane; the dichloromethane should contain your alkaloids.

The aqueous extract may contain your glycosides, or they may be in the first dichloromethane solution. The xanthones will probably also be in the first dichloromethane solution. I suggest adjusting the pH with acetic (or formic) acid and ammonia because the salt is relatively volatile.

Try this on a small scale first so you see where the different compounds go.

I am to analyse the xanthones and the secoiridoid glycosides from swertia, my plans are to go for the liquid liquid separation of the extract then to the column chromatography....so i dont know the exact protocol for the process. Besides can the HPLC technique be used for the Column chromatography development??????????

With some tweaking, you can probably use the column method for the prep chromatography. You will need to convert your time to column volumes (CV). for a HPLC, the void volume is approximately one CV. If a compound elutes in a time corresponding to twice the void volume time, the compound should elute in roughly 2 CV. Pre-packed flash/MPLC columns often have the CV volume written on them. If packing your own column, you can determine the CV by weighing some media, packing a small column (wet), and allowing this column to drain into a flask completely. The volume of liquid you capture is pretty close to 1 column volume. Weigh the media when you pack the larger column and the CV will be proportion to the small column you packed.

HPLC media is often not the same as prep column media so there will be variation in the peak elution time. Loading techniques on the column also are important (solvent you use to dissolve the sample, wet loading or dry loading).

You didn't say what sort of HPLC- I'm assuming C18.

first of all, rather go for Direct column of your extract, carry fractionation of your extract proceeding from non-polar to polar solvents, for glycosides, check they are extremely soluble in polar solvents, perform the tests, and then check TLC with app. solvent system, (see for each phytoconsituents), at last go for column.

If you want a column method, one could try what I call "wide polarity range" chromatography. This works with C18, diol, and could even be done with silica or alumina.

For C18- start as usual with a water/methanol (or acetonitrile) gradient. When you reach 100% organic solvent, continiue the gradient starting with the methanol (or acetonitrile if you used it) to 100% dichloromethane or acetone. C18 is chemically bound to silica and won't wash off.

For normal phase columns (diol, amine, alumina, or silica) use a hexane/2-propanol (or ethanol, acetone would probably work too, but not on diol columns) gradient to 100% alcohol. From 100% alcohol, go to 50% water in a gradient. The solvents were chosen on the basi of polarity and miscibility as the gradient changes.

This allows a very wide range of compounds to be eluted.

Here's some some things I've done using this method (please note I work for this company):

http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN77_Wide_Polarity_Range_Chromatography.pdf

For the separation of alkaloids firstly it must be use an alkalin medium like NH3 , secondly use the columen phase stationaire silica gel and phase mobile toluene+aceton

for the purification of the fractions you can use TLC.