HPLC can help you purify your target compound. The target compound must have a characteristic peak in order for you to choose optimal conditions for the separation process (mobile phase, flow rate, detectors, and columns )

What is your target substances? You can use handmade silica column. And by passing your extract from that, you can take impurities and analyze your sample by HPLC.

If your target is alkaloids, after extraction by methanol and evaporation of that, you can use water-TFA (trifluoro acetic acid) (pH=2) for extraction of the alkaloids from the plate, then by alkalization of the mixture (pH=10) by NaOH, you can sediment the alkaloid. This stage just use chloroform for extraction of alkaloids. Know, you can evaporate chloroform, and use methanol for dissolving of the residue and analyze it by HPLC.

I'm assuming you are doing assay directed fractionation, since one could look up how a known compound was previously purified. First, do a literature search on the plant you extracted, look at the genus level. This tells you what compounds have already been found. Your active compound may be one of those. Also, research the biology where your extract shows activity since some biological assays tend to show activity with certain compound classes such as alkaloids, or phenolics, for example. You can then purify according to those types of compounds.

If it seems like you have an unknown compound, I like to run a column screen. This allows me to determine the following:

• Column chemistry likely to purify the extract such as reverse phase, normal phase silica, normal phase alumina, ion exchange, or something else
• An estimate of the compound’s polarity which suggests solvents for chromatography

The initial column screen includes “wide polarity range chromatography”, essentially chained gradients. Although the links below used an automated flash system, one can use glass columns with step gradients.

http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Posters/New%20Techniques%20in%20Extract%20Column%20Screening%20Poster.pdf#search=column%20screening

http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Posters/New%20Techniques%20in%20Extract%20Column%20Screening%20II%20Poster.pdf#search=column%20screening

After the conditions (column and solvent) are determined from the column screening, the scale-up is simplified and optimized to purify the active compounds by using the column and solvent system which elutes the desired peaks.

Thank you for the valuable suggestions.

@Mahdi Yahyazadeh I target flavonoids and tannins

what would you suggest as a method for purification?

Preparative thin-layer chromatography will equally be of great help.

Regards.

@Sara-

Generally, ion exchange, silica, and reverse phase should work for flavanoids and tannins. Also consider polyamide resin.

http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/RediSep%20Rf%20Strong%20Anion%20Exchange%20Column%20App%20Note.pdf#search=flavanoid

http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/Purification%20Strategies%20for%20Flavones%20and%20Related%20Compounds%20App%20Note.pdf#search=flavonoid

http://www.teledyneisco.com/en-uk/liquidChromatography/Chromatography%20Documents/Posters/Purification%20of%20Phenolic%20Flavonoids%20with%20FC%20Poster.pdf#search=flavonoid

Many thanks for your reply

Dear Sara

I think there is lot of suggestion and answer already written on this question. I just want to notice you that Extract derived by 80% Methanol will not fully soluble in water.If you filter it, the clear solution contains all hydrophilic compounds and precipitate or insoluble residue also have different profile of compounds.

See first of all you must run a preparative column chromatography of the plant extract once using a polar solvent system and then with a non polar solvent system and separate the fractions