I used Three solvent of different polarity ranges from non polar, semi polar and polar, should i need to purify my extract before running HPLC and i need help for the purification protocol.
Thank you.
If your objective is polyphenolic compounds - you can go for a aqueous extract.
TLC will give you a good initial idea, but look into literature for any report on phenolic compounds from the plant you are working with - so that you can compare the Rf values in TLC and Rt values in HPLC by running standards.
Properly filtered (commercial prefabricated syringe filters are available) crude extracts can also be applied to the HPLC- just ensure you have a guard column. Hope you are using a reverse phase C18 or C8 column.
For a good account on extraction and quantification of phenolic compounds (and other plant secondary metabolites) have a look at the following book:
Phytochemical Methods A Guide to Modern Techniques of Plant Analysis, JB Harborne.
You can also have a look at the following paper for cell wall bound - cytosolic free and bound phenolics, HPLC procedure for phenolics in a medicinal plant.
http://www.hindawi.com/journals/jphar/2013/270472/
What is your objective? I mean HPLC is basically for quantification of particular chemical compounds. So describe more about work.
Dear Dr. Padmawar,
Actually, my objective is to identify and quantify some of the phenolic compounds.
Thank you
Yes you should have to purify your sample by using TLC or column chromatographic methods (Isolation methods). You can also read my papers. Hope you get some information from papers. Good Luck!
If your objective is polyphenolic compounds - you can go for a aqueous extract.
TLC will give you a good initial idea, but look into literature for any report on phenolic compounds from the plant you are working with - so that you can compare the Rf values in TLC and Rt values in HPLC by running standards.
Properly filtered (commercial prefabricated syringe filters are available) crude extracts can also be applied to the HPLC- just ensure you have a guard column. Hope you are using a reverse phase C18 or C8 column.
For a good account on extraction and quantification of phenolic compounds (and other plant secondary metabolites) have a look at the following book:
Phytochemical Methods A Guide to Modern Techniques of Plant Analysis, JB Harborne.
You can also have a look at the following paper for cell wall bound - cytosolic free and bound phenolics, HPLC procedure for phenolics in a medicinal plant.
http://www.hindawi.com/journals/jphar/2013/270472/
The most significant reason is that you may end up with your HPLC column clogged if you do not purify your extract. It is because that small particles will inevitably go into the extract if you have not filtered...
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