I use ethylacetate:hexane solvent system, and is giving me tailing separations. My aim is to isolate flavonoids from the n-butanol fraction. Regards
As flavanoids are generally acidic, you could consider adding a little acid (acetic or formic acid) to the mobile phase to keep them as the free acid. Also consider dichloromethane/ methanol solvent systems.
Here is an overview for these sort of compounds:
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN85_Purification_Strategies_for_Flavones_and_Related_Compounds.pdf
You should use petroleum ether, acetone and dichloromethane as 1:1:3 ratio or choose any other combination for separation.
The comments ad suggestion of Jack Silver is reasonable and practical. You should add 70% ethyl alcohol in different rations to the solvent mixture. Flavonoids are mostly soluble and extractable by 70% EtOH.
A mobile phase you could use is Ethyl acetate-formic acid-acetic acid-water 100:11:11:27, this system is used for TLC of glycosylated flavonoids and similar kinds of phenolics, you can reveal flavonoids with the NP reagent or phenolics in general with ammonium phosphomolybdate
For glycolysated flavonoids You could also use as mobile phase chloroform/methanol/water: 8/2/0.2; 7/3/0.5 or 6/4/0.7
The CAMAG company can supply you with a wealth of information on TLC. Since you already use butanol, I would suggest butanol/acetic acid/water for your normal phase plates. It is very slow development. Crude but effective as far as analysis goes. But since you want to isolate the pure compound(s) you may need to pursue Dr. Khan's suggestion above or go to lipophilic Sephadex LH-20 or CCC.
You can use the system Ethyl acetate: Formic acid: Acetic acid: water (100:11:11:26).
This is from Wagner and generally used for flavanoids. Also there is a reagent named Natural Product-PEG reagent used only for detection of flavanoids on TLC
n-BuOH extract contains many glycosides of aglycons such as C15 hydroxylated flavonoids. For TLC you can use EtOAc-MeOH-H2O-HCOOH of various percentages depending on the qualities of the TLC plates. In addition, for purification of sub-fractions coming from the fractionation of the extract you can use sephadex LH-20 column chromatography eluted with MeOH or silica gel CC eluted with EtOAc-MeOH-H2O of increasing polarity.
Dear Asekunowo Kelechi,
¿How complex is your mixture? ¿Do you expect for flavonoid glycosides or simple flavonoids with no sugar moiety? Something important to obtain a good TLC is concentration of your sample, be sure you apply a very well dissolved sample and not too concentrated.
You can try with EtAcO:MeOH for TLC and use ceric ammonium sulphate for develop. If you have a complex mixture and you want to do a previous separation, I suggest to obtain the ethyl acetate fraction of your actual extract, In which you can obtain mainly not glycosilated flavonoids.
Good luck!
Thanks all for your responds, I finally used EtOAc fraction of my crude extracts, the fractions collected from open column on solvent system of EtOAc: MeOH have pecipitate but are tailing on TLC on CHCl3: MeOH, DCM: MeOH, looking for a better way of separating them. Regards
.
CT
Hello Kelechi, am currently facing this challenge. Please, how did you finally succeed in this? Thanks alotCT
Hello Kelechi, am currently facing this challenge. Please, how did you finally succeed in this? T
Hi.
you should use the combination of methanol and chloroform (1:9 or 2:8) or ethyl acetate and methanol mixer.
I am facing the same problem as Kelechi with choosing a TLC solvent system for butanol extract of my sample. I will try solvent mixtures you suggested. Thank you.
Hello Aman and kelechi,
I recommend to try reverse phase with solvent systems 25 % MeOH/H2O, 50 % MeOH/ H2O or 75 % MeOH/H2O. You might need to do repeated chromatography on each fraction you get from the above solvent systems.
Best of luck
Thank you all for your assistance.
CHCl3/MeOH:H2O (8:1.5.0.5) gave me a good TLC profile.
We use the following systems for the butanol fraction to identify flavonoids using TLC:
1. Chloroform: methanol: acetic acid: water in a ratio of 7:3:0.5:0.5 (v/v/v/v)
2. n-butanol: acetic acid: water in a ratio of 4: 1: 2 and 4: 1: 5(v/v/v). PS^ after cooking the system is left to saturate, then the non-displaced water must be removed
We use the following systems for the butanol fraction to identify flavonoids using TLC:
1. Chloroform: methanol: acetic acid: water in a ratio of 7:3:0.5:0.5 (v/v/v/v)
2. n-butanol: acetic acid: water in a ratio of 4: 1: 2 and 4: 1: 5(v/v/v). PS^ after cooking the system is left to saturate, then the non-displaced water must be removed
I use reverse phase TLC for the polar fraction/part, butanol part of methanol extract. Then I use methanol:Water in different ratio for reverse phase TLC.
For Flavonoid isolation chloroform:hexane as solvent system for 8:2 composition is good.
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