I had performed a hexane extraction on my plant. Through TLC, I had identified several spots. Then, I conducted preparative TLC to extract each spot and test their antimicrobial and antioxidant activities. One particular spot had the antioxidant and antimicrobial properties. Through phytochemical screening, I found that the spot flavonoids and terpenoids. So, now, I want to run a HPLC analysis on the spot extract. Does anyone has any suggestions on how should I run the HPLC analyisis?
A gradient of mobile phases A: Acetonitrile and B: formic acid/water (0.1% v/v) can provide you a good separation of flavonoid and terpenoid compounds.
A gradient of mobile phases A: Acetonitrile and B: formic acid/water (0.1% v/v) can provide you a good separation of flavonoid and terpenoid compounds.
Do you have a diode array detector? If so, select a number of different wavelengths as both terpenoids and flavonoids are massive chemical groups and will absorb at varying wavelengths. If the spots are coloured then it may be best to avoid the visible range.
They will also vary greatly in polarity. I presume from your spot residues that sample size is small so you will want to minimise your testing. Using Victor's mobile phase suggestion try running on a gradient from 5% organic to 95% organic over an hour. This should give you a starting point. Make sure your system and column are completely clean beforehand.
If it is qualitative analysis then it could be very difficult to identify your analytes without standards to compare retention time or mass spectrometry to predict molecular formula.
Knowledge from written papers based on your plant may give an idea of what to expect.
Yes, the HPLC machine in my lab has diode array detector. I will try the suggested method and see how it goes. After I complete my HPLC analysis, I plan to proceed with LC-MS and compare the results with known standards for further identification process. Thanks a lot for your inputs!
I have another question. Should I use hexane to dissolve my extract and then inject into HPLC? Or should I use other solvents?
Ideally, use the solvents you use in the mobile phase. Hexane isn't very miscible with water, acetonitrile, or methanol so you'll likely get funny chromatography.
Something else to consider- if your compounds are very soluble in hexane, I wonder if they'll elute from C18 with aqueous solvent systems. If they fail to elute under those conditions, you may consider normal phase or non-aqueous reverse phase. For examples of non-aqueous reverse phase, see:
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Facile_Isolation_of_Carotenoid_Antioxidants.pdf
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN58_Non-aqueous_Reverse_Phase_with_RediSep_Rf_Gold_C18.pdf
You can chain gradients if needed to perform what I call "wide polarity range chromatography"
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN77_Wide_Polarity_Range_Chromatography.pdf
Please see the article
HPLC-DAD-MS/MS profiling of antioxidant flavonoid glycosides in sea buckthorn (Hippophae rhamnoides L.) seeds.
http://informahealthcare.com/doi/abs/10.3109/09637486.2011.652075?journalCode=ijf
I think the best thing is for you to chose the proper solvent such as hexane and ehtylacetate to simplify your fractions using a simpler chromatogrphic method like VLC before running on HPLC. secondly a polyamide column utilizing acetonitril water mixture as eluents for your HPLC will be ideal.
UV detection for flavonoid is in the range 300-366nm, using gradient solvent, RP column C18 using Acetonitrile/Methanol and 0.1% Formic acid.
Vítor Spínola, Isocratic eution with acetonitrile will also seperate terpenoids in C18 column?
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