Since you are working with crude extract, There's a lot of stuff you probably don't want in the mixture. Do you know what sort of compounds you are looking for? Are you using assay directed fractionation? Or are you looking for a particular class of compound? I ask these questions because the answers will guide you to some extent.

For example, I noticed that an assay I used for assay directed fractionation always had alkaloids as the final purified products. I would extract the crude into acidic water, make the aqueous layer basic, and back-extract the alkaloids into organic solvent and submit the extracts for assay (both the final extract and the intermediate extracts); I found that the activity was greatly purified.

If you are purifying a class of compounds, you can perform extractions (like I described for the alkaloids above). If you are interested in phenolic compounds, an ion exchange column may work well.

If you are starting completely from the beginning with no idea what gives the activity you are looking for, I'd run TLC in a systematic fashion and see what gives the best separation of spots. I like to start from less polar and go to more polar: Hexane/ethyl acetate (0, 25, 50, 75, 100% ethyl acetate), dichloromethane/methanol (0, 15, 30% methanol; of your TLC plate binders can handle it, 0, 25, 50, 100% methanol). There are also solvent systems with methanol/DCM/water for more polar compounds.

I've had good luck with "wide polarity range" chromatography- this is a column technique where I change the solvent system during the run.

Also, research what compounds have already been isolated from your plant, and see how those were purified.

Here are some examples of some of what was discussed above, many involve plant extracts:

Ion exchange purification:

http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN40_Purification_of_Alkaloids_with_RediSep_Rf_SCX_Columns.pdf

http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Isolation_and_Purification_of_Alkaloids_with_SCX_Columns.pdf

http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN87_RediSep%20Rf_SAX_Column_Applications.pdf

http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Purification_of_Phenolic_Flavonoids.pdf

Wide Polarity Range Chromatography:

http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Wide%20Polarity%20Range%20Flash%20Purifications.pdf

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

first try to optimize the solvent system by TLC using two different kinds of solvents. One must be less in polar and another with a polar solvent. Once you have optimized, pack the column using less polar solvent what you used in TLC and do gradient elution by slowly increasing the polarity. But solvent should be miscible solvents. But care should be taken during column development. Maintain a constant flow around 1 ml per min. Bit hectic job, but try to work on it. Later you will like it.

Good luck.

Since you are working with crude extract, There's a lot of stuff you probably don't want in the mixture. Do you know what sort of compounds you are looking for? Are you using assay directed fractionation? Or are you looking for a particular class of compound? I ask these questions because the answers will guide you to some extent.

For example, I noticed that an assay I used for assay directed fractionation always had alkaloids as the final purified products. I would extract the crude into acidic water, make the aqueous layer basic, and back-extract the alkaloids into organic solvent and submit the extracts for assay (both the final extract and the intermediate extracts); I found that the activity was greatly purified.

If you are purifying a class of compounds, you can perform extractions (like I described for the alkaloids above). If you are interested in phenolic compounds, an ion exchange column may work well.

If you are starting completely from the beginning with no idea what gives the activity you are looking for, I'd run TLC in a systematic fashion and see what gives the best separation of spots. I like to start from less polar and go to more polar: Hexane/ethyl acetate (0, 25, 50, 75, 100% ethyl acetate), dichloromethane/methanol (0, 15, 30% methanol; of your TLC plate binders can handle it, 0, 25, 50, 100% methanol). There are also solvent systems with methanol/DCM/water for more polar compounds.

I've had good luck with "wide polarity range" chromatography- this is a column technique where I change the solvent system during the run.

Also, research what compounds have already been isolated from your plant, and see how those were purified.

Here are some examples of some of what was discussed above, many involve plant extracts:

Ion exchange purification:

http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN40_Purification_of_Alkaloids_with_RediSep_Rf_SCX_Columns.pdf

http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Isolation_and_Purification_of_Alkaloids_with_SCX_Columns.pdf

http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN87_RediSep%20Rf_SAX_Column_Applications.pdf

http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Purification_of_Phenolic_Flavonoids.pdf

Wide Polarity Range Chromatography:

http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Wide%20Polarity%20Range%20Flash%20Purifications.pdf

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

If you want start column chromatography of crude extract. it better to your crude extract partition with different polartity of solvents such as hexane, chloroform, DCM , n-butanol , methanol after that you will start your column according to that polartity and choose your mobile based your partition fraction ( partition hexane, partition chloroform ..etc). other method is start with no-polar solvent (hexane, chloroform and slowly increase polar solvents percentage(ethyl acetate, methanol) .

all the best

Dear Rajesh,

Thank you for your kind suggestions, I will apply in my future experiments.

Dear Dr. Silver,

My plant is rich of indole alkaloids and my target to isolate maximum out of that (in literature more than 30 are reported). How can I go for further experimentation.

We don't have sophisticated instruments like flash chromatography or preparative chromatography. I have to go through the conventional methods only.

Kindly advice me for future experiments.

Sincerely yours.

Since you are looking for alkaloids, I'd run the extraction I mentioned above.

- Partition the crude extract between dichloromethane (DCM) and water containing 0.1 N HCl, wash with DCM

- Set the DCM extract aside to test for activity, then make the aqueous layer basic with ammonium hydroxide (pH 8 or 9), then extract again with DCM, wash a couple of times with DCM and combine the washings with the extract. Your alkaloids should be in the DCM layer as free bases, and you can purify with silica, CHP-20, or whatever you feel best.

-Evaporate the aqueous layer and submit for assay; ammonium chloride is a volatile salt and will go away after some time.

My experience with some indole alkaloids suggest some of them (like harmine and harmaline) are not completely soluble in organic solvents as a free base, but are very soluble in acidic methanol.

Alternatively, you can still do ion exchange as a batch rather than a column.

-Dissolve your crude extract in acidic methanol

-Mix with a Strong Cation Exchange resin (SCX), let mix for a few hours- you may need to wash the resin with acidic water prior to using it since the hydrogen cation is more easily exchanged for your compound and they will bind better.

- Filter the resin, wash with water. Submit the washings (do not combine the aqueous washings with the methanol- some alkaloids may have come off here since, under neutral pH, some alkaloids elute in water) and the methanol supernatant for assay

-Wash the resin with water containing ammonium hydroxide, submit this for assay.

-Wash one last time with methanol and submit this for assay. Sometimes the free-bases are not soluble in water, but are washed off the ion-exchange media in methanol.

Look up work done by Barbara Timmermann and her people at University of Kansas; they presented a poster describing the use of ion exchange resins in batch mode at the same time I presented my poster (link in my earlier reply). I think some of their published papers will list the procedure.

You digest your extract in slica and then start extraction with using different solvent with increasing polarit. The extract you obtain by this confirm by tlc and then tried to elut it with column using solvent used in TLC

First optimize your solvent system by TLC, always start from less polar to more polar and then try this for PCC

Based on TLC, I can detrmine the suitable eluent system which increases in polarity gradually for CC