I have prepared an aqueous extract of a plant by boiling the leaves in a water. I have also carried out the phytochemical analysis of the same by camical methods. To proceed further, Is HPLC an ideal option to know the exact components of aqueous plant extract? if yes, what mobile phase do I need to use for it?
thank you
No. You have to decide the nature of compound you are going to detect. Volatile or non volatile? first get to know the phytochemical profile and decide the class of compound. Every HPLC analysis require a standard to detect the specific compound to compare the peak of your sample.
No. You have to decide the nature of compound you are going to detect. Volatile or non volatile? first get to know the phytochemical profile and decide the class of compound. Every HPLC analysis require a standard to detect the specific compound to compare the peak of your sample.
You didn't mention your target class/classes of compounds. Anyway, I do not suggest to use HPLC directly for crude extract.
First you decide, which class/classes of compounds your going to purify.
Secondly,using solvent extraction, fractionate your extract, then perform column chromatography for these different extracts for further fractionation SO THAT YOUR FRACTION HAVE LESS NUMBER OF COMPONENTS.
Then you can purify these fractions (eluted from the column) using HPLC.
Read the protocol in the following links:
Thank you very much Palani Manasa and Ghazanfar Sultan, for your valuable information. I have carried out Phytochemical screening of the extract and got positive results for Flavonoids, Saponins and Triterpenes. Do i need to separate these components first and then carry out HPLC analysis?
My dear Kailash,
Here we purify our compounds step-wise, (1) Solvent Extraction (2) Column Chromatography (3) Preparative HPLC.
Note: Analytical HPLC is used for identification purpose not for purification. So if you want to identify then use analytical HPLC after purification.
Thank you very much Ghazanfar Sultan. i will consider your points and proceed further. Will get back to you if i get difficulties again.
Depends what the compound is? Sometimes might be good to use RP-HPLC. Look in to sephadex -LH 20
You need to first separate the extract into polar and non-polar components by extracting aqueous extract with an organic solvent like ethyl acetate or chloroform. The non-polar fraction (organic) may be analyzed by GC-MS. The aqueous fraction can then be freeze-dried and dissolved in alcohol or water and analyzed by LC-ESI-MS or any appropriate hyphenated method. However, the boiled extracts may contain artifacts. Modify the method to cold solvent or super-critical CO2 extraction
The brief steps are:
Conduct preliminary analysis using laboratory chemical test methods. Then run TLC to know the type of fractions. After that Column Chromatography with HPLC, after isolated pure fraction run FT-IR, LC-MS, HNMR, CNMR for structure elucidation. You can read my papers too.
You should be really careful with aqueous extracts before analysis. I recommend you to pour the samples after extraction into several small plastic centrifuges tubes and put to the feezer. Before analysis (one day hefore) you should move one to the fridge and it is ready to analyse. Although you do above, you should not lead you HPLC analysis too long. Only if you elaborate you chromatograph method well, use the thawed samples. Too long storage aqueous samples of leaves in the refrigerator can cause the fouling with mold.
thank you Monika Przeor for your thought. I will consider it at the time of analysis...
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