Similar Rf spots are always a problem during chromatographic analysis. You can always separate the two spots together using prep-TLC (I recommend this).

Then once you have two spots separated form the parent extract or mixture, you should try solvent combinations ranging from chloroform:MeOH 1:1 to 10:1. Even then you dont get separation, then add toluene to the solvent system. Their is every possibility that the spots will move.

Good Luck.

Dear Sudhir,

The answer to your question which has been given by Jhon Galvis was actually given by me in response to some other question.

I would send the same to you as follows and it also pertians for your question.

Similar Rf spots are always a problem during chromatographic analysis. So you should not panic and do some very conventional techniques for their separation. You can always separate the two spots together using prepTLC. Then once you have two spots separated form the parent extract or mixture, you should try solvent combinations ranging frm chloroform:methanol 1:1 to 10:1. Even then you dont get separation, then add toluene to the solvent system. Their is every possibility that the spots will move.

DO this for now and if still dont get the result, try to find out/ inform the nature of compound , I will help you out then.

Regards and gud luck

Try different columns, like C18, Silica, Sephadex LH-20.

I used Amberlite XAD16 to isolate two flavones from the DCM-soluble fraction and was able to do it in just one run of chromatography

Inrease the lenght of your separation column. Beforehand, determine a solvent system using TLC which ideally have the following properties: Rf of the highest spot around 0.3 (same stationary phase, eg Si60); solvent system should not contain minute amounts of water (because it might break during separation), solvent system should be easily evaporable (to recover separated fractions).

Hope this helps,

kind regards,

Kristian

well, the other suggestions are right. Depending on the quantity of the matrix you have, you can re-extract the mixture in each of the solvent system. the try the TLC once again. Finally if still the same Prep TLC can be a way out, you can also try Prep HPLC. i wish you the best.

Try to run HPLC (c18 column) for your fraction by using Solvent A - (ACN -45%+ MeoH-55%) and solvent B - (1% formic acid in H20). in different gradient level, you will find a good separation after that using preparative column try to separate the compounds as per the corresponding peaks

you need sephadex LH20 for column chromatography.

By using sephadex LH20 for column chromatography. You can always separate the two spots together using prepTLC. Then once you have two spots separated form the parent extract or mixture, you should try solvent combinations ranging frm chloroform:methanol 1:1 to 10:1.

regards- Vijay Nigam

One can separate a mixture of two compounds having very close Rf values by using Flash chromatography. Kindly read the article W. Clark Still, Michael Kahn, and Abhijit Mitra Rapid Chromatographic Technique for Preparative Separations with Moderate Resolution Org. Chenz., Vol. 43, No. 14, 1978. This article might be very useful.

Preparative TLC

Probably you will need more than one column and do stepwise purification

Thank you all for giving me good suggestions.......!

Preparative and flash chromatography

2D Chromatography is also good when Rf is very close

In this case Sephadex column is useful , you can search sephadex which one quality will be best for the purpose you are intrested.

Sephadex its a good option as it separates based not in the samples polarity but their molecular size/ weight. Also, if your sample is formed by a mixture of few compounds you can try recrystalization.

Like others mentioned, Sephadex LH-20 is a good clean-up step. Polyamide is good too.

Here's some work I've done: http://192.168.100.5/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Purification_of_Phenolic_Flavonoids.pdf

U can run column with silica gel-230-400 mesh size. The elution will be very slow but effective separation can be obtained.

You may follow Teodoro Kaufman idea or Jack Silver Idea. But you can also use HPLC using reverse phase column LH-80

First of all partitioned it in different solvents. Then proceed with each fraction on silica gel column. If still fractions contains more than one compds, use each fraction on sephadex column (size exclusion chromatography).

Run a slow(er) gradient

Yes, slow the gradient.

I agree with all suggestions given above that appear to be suitable methods to separate close substances on TLC. However, if you do not have HPLC or Sephadex or other options which usually are more expensive, you can try carry out your column chromatography along a very long column, so it increases the number of theoretical plates and then all substances will get more distance among them when reach the final along column chromatography.

If sephadex LH20 is not an option then use prep TLC.

If none of the previous suggestions work then consider making more non-polar derivatives. This might help to better separate your compounds and improve your chromatography. Methylation (with Diazald for example), acetylation or others might do the trick for you. Just ensure the process goes to completion. A preliminary NMR of your mixture might help you to decide.

One thing that you have to consider for your flavonoid compounds is that using Silicagel as a stationary phase. Your polar compounds will be absorbed by Silicagel. I prefer using ODS or gel filtration w/ Sephadex LH-20. I agree with Ronan suggestion.

You can use Sephadex LH-20 eluting with methanol as the easiest way in the absence of HPLC, i have done that severally when i have Flavonoid rich extracts especially the N-butanol,however, not all Flavonoids can be separated using Sephadex, you might need Reverse Phase HPLC to separate highly polyglycosylated ones.

Please try to separate them with polyamide column chromatography or Sephadex LH-20.column chromatography .

It may be ultimately faster to obtain clean separations by running a second, shorter column on some of the fractions obtained from an initial column. Why would we think that a single efficient method exists to separate a number of closely eluting compounds, especially when resources are limited. Run the first column and collect a number of fractions, some with a single component, some with several. Then run TLC on each fraction to optimize a separation for that specific fraction. Then run another column on each fraction. A person could spend a lifetime trying to come up with the perfect separation.

Dear Sudheer,

From your question , I suppose your extract is a butanolic extract. If you have Hplc, then it is the best solution. Of course a few fractionations may be needed and then pure compounds may be obtained from the different fraction.

However, if you do not have a Hplc or if the queue for it is very long, what can be done?

My suggestion is as follows:

First you do a solvent- solvent extraction, dissolve your sample in water and add minimal amount of MeOH if needed. Then extract, using separatory funnel, with solvents from non polar to polar for example hexane, then dichloromethane or acetone and then finally you may freeze dry the aqueous portion. So now you have partitioned your compounds into non polar, semi polar and polar fractions.

For non polar and semi polar fractions you may separate the compounds by column chromatography or tlc with silica gel as the stationary phase. Run tlc first to check the profile of your separation. If rf values of compounds are too close, tlc is a better choice than column. You may fractionate first using column then purify the compounds from the fractions using tlc. You may have to repeat this a few times until you get the pure compound.

For the polar fraction and the more polar part of the semi polar fraction, separation using C-18 is the most practical. If you use silica you may have a low yield as many sample will be trapped in the stationary phase. The longer the column and the longer the time taken to elute, meaning the compounds are left for longer time in the column, then more compounds will be degraded. So the best solution for these polar compounds is to use c-18 column or Hplc or recycle hplc.

I hope these info will be of help, all the best.

Some suggestions, which could be tested isolated or combined:

- decrease mesh size of sillica

- test other stationaty phase (e.g. allumina instead of sillica)

- chose a solvent mixture that gives a rf = 0.3 in TLC for your desired compound(s) (e.g. CHCl3/MeOH 80:20)

- use a solvent gradient (using the example aboove, start elution with CHCl3 100ml, the CHCl3/MeOH 95:5 100 mL, then CHCl3/MeOH 90:10 100 mL, then CHCl3/MeOH 85:15 100 mL, CHCl3/MeOH 80:20 100-300 mL; this volume of eluent is for a sample with 100-150 mg, and 100-150 g of sillica, packed in a 20 cm x 2.5 cm collum)

I hope these info will be of help, all the best.

I am not saying to adopt much more technique, just try to separate out in solvent mixture in which your are getting your TLC resolution.

Based on your mobile phase for TLC (EtOAc:MeOH:H2O = 8:1:1), polar compounds are rich in your extract. Therefore, isolation of those compounds can not be effective by CC using silica gel as stationary phase. The isolation will be effective using C-18 CC (reverse phase column) as well as TLC analysis. If possible for you to use C-18, then apply MeOH:CHCl3 (9:0.1) as mobile phase. You can also re-use the column since it can be flushed by MeOH/CHCl3/H2O similar to reverse phase HPLC column. Good luck.

The best way to do the separation is to follow what Dr. Carla sabandar said using C-18 CC (reverse phase column) as well as TLC analysis. with this you get the flavonoid I have have used gel filtration w/ Sephadex LH-20 to get flavonoids, so you can go ahead.

Thanks

Get a good eluent system (try Hex/EA with increasing gradient) ensuring that the compounds migrate and separate well . Use the same eluent system in prep. TLC

Everyone seeking help, such as in this thread regarding a separation, really needs to explain their sample, their previous attempts to solve the problem, and more detail. Rereading the question (which btw is from 2012), I am uncertain of where the poster is at in his separation in his question. Oh, I can speculate, but that is just it, speculation. These vague questions are frequent on this site.

What kind of TLC plate did he/is he using? What resources (C18, silica, etc) does he have available? Analytical TLC or prep? How much sample, grams, milligrams? How was the plant initially extracted (yes, it was butanolic, but) for how long and how was it handled afterwards, dried down? Still in solvent? What solvent was it dissolved in when it was TLC'd? What's he mean, "I repeated the column?" TLC? An actual column? Were there any TLC spray reagents used to visualize different classes of chemistry?

Plus, the more data, the more fun working on the problem!

And, incidentally, some flavanoids will have acidic phenolic protons and some will not. Then there are all the possible lignans/lignins (I don't remember which one) that could interfere with a separation.

Why not pop the crude sample over a weak anion exchange resin (the ligands on the solid support are tertiary amines (very inexpensive and with high loading capacity) and stick the more acidic plant compounds. Catch the flow through for neutral and very weakly acid compounds. Then elute with just a few washes of stronger and stronger acidic solutions (protonating the bound weak acids and eluting them as neutral compounds) or stronger and stronger and stronger alkaline solutions (deprotonating the column ligands and breaking the ionic attraction between the compounds and the column and eluting the acidic compounds in their negatively charged forms/aqueous). The eluting solutions do not have to be very stongly acidic or basic, though the final elution could be at a pH of 1 or 12. The flavanoids are going to be stable at those pH's.

Or, use a strong or weak cation exchange resin and capture any amines/bases in the extract and catch the flow through with the acids and neutrals. Then knock off the amines with a base.

Just another thought.

Fun discussion.

Sephadex is best absorbent for the isolation of constituents having almost similar polarity because, it separate the compounds on the basis of molecular weights.

The butanolic extract can be completely dried and dissolved in methanol:water (50:50) and column chromatography with the synthetic reversed phase resins (PS-DVB based) can be done using step gradient elution with increasing percentage of methanol from 50 to 100 with intervals of 5 or 10%. Each step gradient may give a pure component or a mixture of one or two which can be further separated in subsequent crystallization.

first of all try to develop a solvent system for TLC and then start column from non-polar solvents followed by solvents of increased polarity very slightly, so that there must not be overlapping of the peaks..

Good Luck

You have to be careful with your flavonoid compounds (butanolic) if using silica gel as stationary phase. I think you have tailing spot(s) in silica gel TLC so far. I agree with Dr. Carla W Sabandar and Dr. Garima Bhardwaj. It will be better you separate the butanolic fraction with VLC methode using polarity gradient solvent system from Hexane to DCM then Methanol, prior to CC with C-18.

Sorry, I mean RP-18 not C-18. Thank you

Please do use C-18 reverse phase column. Tq

Please do try it in prparative TLC to separate the exact fraction and then tried solvent in coulmn based on polarity for separation of pure compound.

You can try long column with larger diameter (i.e. 100 cm length and diameter 50 mm) and silica gel of small partial size (mesh size 230-400 or more) this will increase the Rt of the sample as well as of solvent. Hence help in better separation. Also used combination of solvents with step by step increase in polarity by small factor. if there is no proper separation then go for preparative TLC.

You can use flash vaccum column chromatography for separation using suitable solvent system which gives better separation. The solvent system should have enough polarity .