I have a complex mixture of phenolics, flavonoids and tannins from a methanol extract of Q suber. I did HPLC/UV in a C18 column using MeOH/Water/formic acid gradients and i don´t see any peak. It is strange because i expect as very complex mixture. And this is happpening with all the others quercus samples. Not with other species. Should i do a column chromatography first with hexane/AcOEt first? What do you suggest?I have about 10 mg of crude extract.
Hi Joana, I agree with Aurea regarding the necessity of the clean up steps. However, as you are not getting any peaks from a complex mixture extracted in MeOH, I feel it is important to get a foothold first. For that I would suggest a quick filtration of the extract using a 0.22 micron filter, injection of 50 ul of the sample at a concentration of not more than 0.1mg/ml and running it in isocratic mode, say with MeOH and Water(acidified with 0.01% Formic Acid) in 60:40 ratio for ~1 hour at a flow rate of 1 ml/min. I assume you are using a UV-Vis detector set at 280 nm. If it doesn't work you may try a gradient mode and subsequent fine tuning of the separation process. Do keep us updated how it worked out! All the Best!!
Select proper wavelength based upon the lembda max of your compounds preferably use PDA detector for full UV scan . And check the solubility of your compound in diluent.
Thank you very much Aurea. I already did a liquid-liquid extraction. I will now inject them in the HPLC-UV system.
Sukanta, i did since the beggining a quick filtration of the extract using a 0.25 micron filter, injected 5 ul of sample in 0.05mg/ml. And nothing. In some samples yes, but the others, wich i think could have more tannins, sugars, polyphenols, no.
But i will try now after the liquid-liquid extraction with different polarity solvents.
Will keep you updated :)
Thank you very much Aurea Eiroa!
I will read carefully the publication we sent me and others from your references.
I did already a liquid-liquid extraction, first with cyclohexane, after dichloromethane and finally ethyl acetate. Injected the 3 samples, and i see peaks already in dichloromethane and ethyl acetate fractions. Now here is the problem...they apear right in the beggining of the run first 10 min, and then nothinh more.
The gradiente i use (A:MeOH+o,1%formic acids; B:Water+0,1%formic acid):
0-10 min --- 10% A in B
10-90 min --- 100% A
90-110 min --- 100% A
110-120 min --- 10% A in B
What shoul i do? An isocratic right in the beggining with 10%A during 20 min?
After that just solvent peaks!!!
I did soxhlet extractions first in CH2Cl2, then MeOH and then Water.
I dryed the MeOH extract to determine the yield of extractions.
Then i dissolved the extract in MeOH/Water (50:50).
First i did a liquid-liquid extraction with cyclohexane (3 times) and kept that less polar fraction.
Secondly, did a liquid-liquid extraction with dichloromethane (3 times) and kept that fraction.
Then a liquid-liquid extraction with ethyl acetate (3 times) and ket the fraction.The 3 fractions were vacuum dried and redissolved in methanol for injection in HPLC system.
Aurea, i am interested in nonpolar extracts as weel, but already studied them in GC-MS. Water extracts, since they did not show a lot of concentation in phenols or in flavonoids (after standard determination with Folin and AlCl3 methods) comparing to the methanol extracts, we took those to analyze.
In HPLC, i injected the hexane, dichloromethane and ethyl acetate fractions...hexane one did not show any peaks, i just saw peaks in dichloromethane and ethyl acetate fractions.
Preparative chromatography, meaning thin layer chromatography in plates? No, But i would not have time becau se i am in a shorto« term scientific meeting that end up in 29 this month.
Yes Aurea...i will be more carefull with possible losses during concentation.
Thank you very much for your help.
Regards
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