I am working on the isolation and identification of flavonoids. I have different fractions of ethyl acetate extract of a plant. I got those fractions after doing chromatography on silica gel. The second fraction is a yellowish powder. It is to some extent soluble in methanol but it is more soluble in chloroform and ethyl acetate. It is not 100% soluble in either of the above solvents. I took the dissolved portion of the extract in ethylacetate, and did tlc on silica gel (chloroform and ethyl acetate (9:1) as solvent system). It showed three spots. I am now in a dilemma about how to proceed for isolation. If I do silica then I need to apply the sample in dry form as the sample is not 100% soluble in the solvents I do have. Could anybody suggest a better alternative for the isolation?
Dissolve this part in water with methanol, for example 80% or 60%. Swell Sephadex LH-20 on this solvent and try to divide the amount of flavonoids.
You are explaining that your fraction is not soluble by the extent of 100%, How could you say that it is not soluble by that extent? if the fraction is of ethyl acetate it must be soluble in that solvent, otherwise there might be some error you had made in the process of fractionation and isolation. I suggest you to take HPLC of the fraction and you may compare with the standard. Since you may roughly know the Retardation factor and under same condition there might be some standard compound to compare.....................
Bhakta gaire thank you for your interest to my questions. Generally we think that fraction obtained from the ethyl acetate fraction should be soluble in ethyl acetate. But when we take the fraction for chromatiography doessnt dissolves in ethyl acetate. The main reason for this is that during ratiocination we use large amount of solvent so the solubility is high. But during loading the dried powder we use small amount of solvent so the solubility is less. Thats why we search for solvent in which the fraction has high solubility.
you should take into account the formation of certain artefacts during the isolation process. plz try to use Sephadex LH20 and try several solvents beginning from methanol, dichlormethane, EtOAc , and their mixture. try to monitor the fractions by TLC. another alternative using HPLC (gradient elution)
How did you dissolve the fraction? I think you can add some more solvent, and try to dissolve by sonification and/or vortex mixer. If your fraction was stable, you can try to heat it. Do you want to use preparative-TLC to isolate your compounds? If yes, than I think it is OK if you add some more solvent to dissolve completely.
In your condition, you might choose (i) further fractionation with Sephadex LH-20, or (ii) directly do Preparative HPLC to obtain your purify compound. For solubility, you might do (i) choose the best solvent for you solubilise your fraction (How about MeOH, MeOH+Water, etc,.?) -->sonicator --> put in hot water / centrifuge.
I will suggest that from the fractions obtained after silica gel column chromatography you try the TLC with a solvent systems of n-Hexane/EtOAc (70:30, 65: 35 and 60:40) and n-Hexane/Acetone (80:20, 75: 25 and 70:30). If the separation looks good with some yellow, yellowish, brown, ... compounds under UV lights use the same solvent system in increasing polarity to run a large amount of the same fraction on silica gel column chromatography. Sub-fractions from that column will give you cristal compounds that could be purified by recrystallization (acetone,...), Prep TLC, Sephadex LH-20, or small silica gel CC.
From a small amount of the yellowish fraction, try the Sephadex LH-20 with Chloroform/MeOH (90:10) or CH2Cl2/MeOH (90:10) using a long column and collect very small volume (40 to 60 drops a tube) and luckily you may obtain pure flavonoids or not more than 2 to 3 mixtures for further and quick separations ether with preparative TLC or another LH-20.
Good luck and I am sure that most of your flavonoids are present in the yellowish fractions.
Thank you everybody for the suggestions. As your suggestions i checked the solubitiy of my fraction in dichloromethane. I foud fairly good solubility. Now i think it would be better to go for silica gel chromatography.
the use of LH 20 instead of Silica gel makes the Loss of isolated compounds less
Two other suggestions: I have successfully used C-18 and eluted plant flavonoid fractions beginning with polar and ending with nonpolar solvents. You could also use ammonia fumes on your newly separated TLC plates. The flavonoids will turn darker tan, or brighter orange or yellow.
thank you Angela hoffman for the response..... you told about the c- 18 column and starting the elution with the polar solvent and ending with non polar solvent. can you please specify which solvent system do you prefer fro the begin and end part. than you
For my most recent purification, I used a 5 gram column purchased from Fisher. I added my sample (about 0.75 grams) to the column using a vacuum manifold. I ran the vacuum until the column appeared to be dry (about 20-30 minutes). 1. I rinsed the column with plain water until the effluent ran clear (in batches of 100 mL each, and each fraction was kept separate until I could show by TLC and assay that they should be pooled). Next, I rinse with 25% methanol in water. This took more than 12 rinses to get clear effluent. I increased the concentration of methanol to 50%, 75%, 100 % methanol. Next I began increasing percentages of ethyl acetate (in increments of 25%) until 100% ethyl acetate. I then added hexane in increments of 25% until 100% hexane.
I have just about finished the analysis and have found some of the fractions are pretty clean.
Dissolve this part in water with methanol, for example 80% or 60%. Swell Sephadex LH-20 on this solvent and try to divide the amount of flavonoids.
- Badges
- Science topic