I have very polar fractions from TLC which I suspect to be flavonoids. From my findings, I was convinced of the possibility of using a C-18 RP column for the separation. However, I have not used or prepared an RP column before.
Start with chloroform to (chloroform:Methanol ) to methanol for purification of flavonoids in C-18 column chromatography
I suppose that you have an analytical HPLC equipped with an UV-vis detector (or better with a diode array detector). In this case, you can use acetonitrile/acetic acid/water (35:2:63) as solvent A and 2% acetic acid as solvent B and a C-18 analytical coumn.
no, you can use C18 open column with 15% acetic acid and cellulose TLC with the same solvent
here is one method used to quantify quercitin & lutoelin ( Since you have not mentioned the compounds of interest I am recommending a standard proceedure).
HPLC system: column, Kromasil RP-C18 column (250×4.6mm i.d, 5μm);mobile phase, methanol-acetonitrile-acetic acid-phosphoric acidH2O (200:100:10:10:200, V/V); detecting wavelength, 352 nm; flow rate, 0.60 ml/min; sensitivity, 0.05 AUFS; quantity volume of injecting sample, 6.0 μl. I hope this helps
The question is phrased such that it could cover flash C18 or C18 on a prep LC system. Others have answered for HPLC
1) Pack the column in methanol, or whatever organic solvent you will use. This gets the C18 conditioned at the same time the column is packed.- this organic solvent is the "B" solvent.
2) After packing, wash the column with 10% water in your B solvent. The small amount of water prevents "phase collapse". If you are using C18 that isn't end-capped, you can use a lower percent B solvent because the free silanols are polar and reduce phase collapse. Alternately, use a C18 designed for mostly aquaous systems since they are end-capped with something to resist phase collapse. Load your sample, then run a gradient to 100%B. For these compounds, I used 0.1% formic acid, but acetic acid would work too, as would TFA.
You can also use CHP-20 (a polymeric reverse phase).
Here's a poster I did where I used C18 flash chromatography (on a flash system) to purify flavanoid compounds: http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Purification_of_Phenolic_Flavonoids.pdf
With C18, you go from polar solvent (water) to less polar (organic). It is possible to only use organic solvents- this is called non-aqueous reverse phase. You still go from a polar solvent (such as methanol) to a less polar solvent (such as dichloromethane), but flavanoids will be polar enough that you probably don't need to use this technique. I've used non-aqueous reverse phase for compounds such as lycopenes.
If u suspect the fraction may be a flavanoids, u may go with the preparative plate with the solvent system of n-butanol: acetic acid : water. Otherwise if u want to run the column means, u may use chloroform : methanol (9:1 and 8:2, 7:3 etc..).
i do agree with Jack Silver,
i did not have experience of pure flavanoids but i do with polyphenolic purification,
purification on sephadex column is the initial step.
I believe the method described by Chen et al (2001) in Journal of Chromatography A, 913: 387-395 for phenolics will be helpful. I have used the method with very good results for hplc analysis of phenolics/flavonoids in plant extract. It is hereby attached for you. Good luck.
I'm not sure that my suggestion meets your pronblem. The first step, you must chek for the polarity by a mixed solvent system of methanol-chloroform (or dichloromethane). If the TLC patter give a clear and separated spots, I recomend you use silica gel-based column chromatography with various format such as conventional column chromatography (silica gel Arch. No. 7734, Merck), flash CC and so on. By contrast, if the TLC pattern is broad and unclear, I recommend you use C-18 or C-8 column chromatography as suggested by previous other rechearcher in here. If you need for more information about silica gel-based separation, please feel free to ask me.
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