You can use preparative TLC, but it is up to how close spots they are.Can you see them as two different spots?Moreover, the solvent mixture which you use as mobile phase is important. Could you indicate which solvent system you use?Are your compounds visible by using UV lamb without spraying any reagent?

Very close, I mean one spot on the other, I used different solvent mixture but they still move together on the TLC plate. Out of many, the best solvent mixture is Dichloromethane/Methanol (9:1).

Yes, they are visible under UV lamp at 254 nm without spraying

Actually Dichloromethane/Methanol (9:1) mixture can be used as mobile phase for prep-TLC. But could you clarify it, can you see at least a line between these spots, please?

You can do prep TLC with this system as it seems that you can resolve the spots, but the plate capacity will be limited- you won't be able to load much compound.

I suggest trying some other solvents to change the selectivity of the compounds.

If you can get a copy of Synder and Kirland's "Introduction to Modern Liquid Chromatography", they discuss how to change solvents to alter selectivity in chromatography systems.

I would try substituting acetone, acetonitrile, or ethyl acetate for DCM, for example to see if the compounds have a better separation.

Some of the same information is described in a free book "Effective Organic Compound Purification" from Teledyne Isco (not that I work for them). You can order a free copy of the book here:

http://www.isco.com/flashguide/default.asp?url=/flashguideLit/Default.asp

Thanks Jack and all contributors for your time and professional suggestions to move this work forward. I will try following those suggestions and get back to you here.

No clear line in between the two spots. one spot is atop the other.

Is it possible to do a 2D TLC? I feel that it will help in your problem. Then the spots will separate out in your second run, while Rf will remain same.

Did you try silver nitrate impregnated silica plates

M.P.dobhal Jaipur ,India

You have this TLC system Dichloromethane/Methanol (9:1).can you use ethyl acetate-DCM-acetone system, by changing the ratio accordingly.

I would suggest that:

First do an analytical 2D TLC, just to check if one spot isn't a degradation product of the other.

Second, swap methanol for ethanol, it won't solubilse your silica.

If you have aromatic compounds to separate, adding a few percents of toluene to your mobile phase might help.

Finally, use longer TLC plates

Good luck.

You may pl try

1. Multiple development with different solvents. Develop with one solvent system - allow solvent to evaporate without heating - blow gentle stream of nitrogen and then with another. It depends on your patience.

2. Derivatise the mixture and separate. Acetylation at RT if you have hydroxyl etc.

By any chance do you have idea of the class of compound, if not run the NMR and find out, I will give you very effective and specific answer.

any idea of the general nature of the compounds you want to separate ? have you tried to add few drops (

1. addition of NH3/amine; acetic acid are normally to reduce tailing of spots.

2. 2D TLC cannot be used for prep TLC

@ Bapuji ... yes normally it is to reduce very polar tailing of acidic or basic compounds BUT ... try and separate two idendical molecules that one has amidine group and the other has in the same plase amidoxime group withoud the aid of a base or and acid !! What i mean is that if it is there are any difference in pKa (or pKb) a base or an acid can enhance separation of already untailed TLC spots !

Thank you Nano. I agree.

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

You should try hydrolysis.

Jusr add some drops of acetic acid to your TLCsolvent system

Sometimes acids like formic and acetic acid may help resolve the problem of tailing and provide good resolution. But the quantity added should be very small. Just 2-4 drops are enough for getting a clue.

Tailing can be reduced using either organic acids or amines depending on the nature of compounds.

if fraction is reasonably good in quality "Flash Chromatography " can resolve the mixture very effectively.

Its not about tailing, but that 2 spots on one another in the TLC which could not be resolved using a very lot s of different solvent systems.

You can use two directional tlc. You run the tlc in one direction when that has developed, you remove and change the direction in the same solvent system. at this point they should move separetely.

Thanks Prof. I will try it and get back.

Use repeated elutions with a lower strength solvent mixture or 2D TLC.

Use formic acid or acetic acid 2-3 drop in solvent system is useful

in case of two close spots in TLC, the mild acid like acetic acid should be added in a very small proportion. It will resolute the spots and finally the preparative-TLC can help you to separate both the constituents. you can refer the following document.

The best solution for this problem is Two Dimension TLC only

It depends on the nature of your compound. With so little detail about the nature of your compounds, it is difficult to suggest a method to resolve your problem. It will be helpful if you can also provide information on the relative polarity of your products. E.g. what is the current solvent system that you are using to run your TLC plates.

The previous advice on the use of additives like formic acid, acetic acids or base like triethylamine may help if your side product contains basic or acidic groups that can be correspondingly deprotonated or protonated by the additives.

Thanks researcher, i too even faced the same problem once in separating two closely related sesquiterpenes. Yes adding HCOOH a drop or two to the solvent system will do wonder but at the same time depending on the nature of compounds and as per my experience you run the spot for a longer distance in the plate ( hope you are using alumina plates) i.e. solvent front should be more or solvent migration should occur for a larger distance.

i would suggest using ammmonia in jar of TLC

Keep trying. You may change from hexane to benzene or toluene (good for aromatics); and EtOAc to Et2O or other ethyl esters; also chlorinated solvents. If this does not work and your compounds have acid-base properties, add some Et3N or AcOH depending on the circumstances.

Alternatively, if there is at least some separation, use repeated TLC runs, this will separate them. Once you get a good system, then you can go for CC or prep-TLC.

How about use three system solvent like I did, such as hexane:toluene:ethyl acetate (6:2:2) or other combinations to separate very close Rf of triterpenes in TLC plate.

Yes, I too agree with Carla. Using a mixture of three solvents also can help resolve the spots. I also used Chlorofrm: methanol: toluene (8:1:1) for ethanolic extract in my studies and got good resolution.

I have also experienced using 2-3 drops of acetic acid / formic acid / amines like triethyl amine or pyridine (depending upon the nature of components to be separated) help in separation of compounds with close Rf.

Yes you can use preparative TLC for separation

The old way is to run your tlc plate more than once in the same system, that is, do your first run, dry the tlc plate and take it back into your tank and run it again until such time that the separation appears. Normally this happens if the compounds you are trying to separate are isomers.

you have not mentioned the nature (polarity, i .e. polar or non polar) have you tried the followinfg type solvent system if not u can try.

Hexane -EthylacetateAcetone (8+1+1 or 8+2+0.5 or u can change quantity)

Hexane -Acetone(3.5+1.5 ---)

Methanol(4%in DCM)-hexane-Ethylacetate)

8+4+2).good luck

You can try by changing stationary phases silical gel coated Aluminium sheets may be used. Cellulose tlc may be used. Also try 2 dimensional or 3D chromatography.

If your compound having isomers you can try different propotions of IPA and Hexanes combination. I can provide you the better solution if you provide the nature of the compound is either acidic or basic

When you have such problems 3 solvent system work great for separation. Butanol or iso propyl alcohol can be used as a third solvent based on polarity of compounds with normally used solvent. 2D TLC is also one of effective method for purification of such compounds.

I have the same problem too.

Pl try the following

1. run with a solvent where the spots move to about 25%(approx) of the length of the plate. remove, dry in inert atmosphere and again dip in the same solvent. Repeat and check if you get any improvement.

2. run with one solvent system (single or two solvent system- rarely we need 3 solvents) to move the spot up by 25% (approx) and then run with a different solvent system. Alternate . Dry the plate carefully between the changes of solvent system

These two resolve most overlapping spots.

Try two dimensional TLC. The spots will surly seperate. Google it !! :)

If you want to see them separate- you can try 2D

If you want them on hand - no 2D.

Change the ratio of the solvents.

You can use the cross chromatography technique, very effective in these cases. Put your plate in the Jar with solvent system, when migration reach the front line take your plate, then rotate the plate 90 degree and put it again in the same solvent system...

Prep TLC is just to confirm the compound. You could not have sufficient gram for net step. you have to choose a low polarity solvent system to isolate it. Atleast by first chromatographic run, you can able to purify it by atleast 60-70%. Then once again pach the column and purify it to increase purity to >90%

Or else if you have Preparative HPLC, just submit it and get the compound.

you can try the following system solvents if your compound are nitrogenated: CHCL3: MeOH: NH4OH (83:14:3) or a different ratio of this system.

Good luck !!

Try FLASH COLUMN CHROMATOGRAPHY . It is an old technique but works very well.

try to change the PH of the mobile phase, try the same system but add 1-2 drops of acetic acid or formic acid, otherwise add 1-2 drops of ammonia, if the spots continue to be very close you have to use HPLC to separate them.

Yes u try with prep TLC, try fraction method.

I have separated two closely related spots like that with Sephadex LH-20 using methanol as eluting solvent especially if the compounds are polar. Prep. TLC will not effectively separate them.

You can try with a low polar solvent system and run the tlc several times till you see both compounds

for qualitative separation, use 2D-TLC, for quantitative separation use RP-8 TLC (Merck)

If you have enough of your sample to go on column, then your best bet is to use size exclusion with Sephadex LH-20 as suggested bu Ahmadu. They will be separated according to molecular weight.

If you want to separate it on normal phase TLC plate., spot the very dilute sample. try different combination of solvent (Hexane : Ethyl acetate OR Chloroform: Methanol).

But if you want to isolate it by chromatographic technique, normal phase system will not work. On reverse phase system using Acetonitrie:water- C18 column of 5 micron column will give better separation.

Use hcl deactivated silica gel on tlc plate for coating

the two spots on TLC, sitting atop meaning that the mobile phase used has powerful elution that take all the sample with it. reduce the elution power of the mobile phase. study the nature of your sample acidic/basic to make use of amonia or acetic acid in the sepration

Separation is completely depend on structure of the molecules. If your compound is steroisomer, normal phase system will not work at all and then you have to use reverse phase system.(C18-acetonitrile/Methanol and water with presence of suitable buffer).

Additionally try helix column it will work for midpolar compound in wide range of solvent in aqueous as well as non aqueous mobile phase. but for limited molecule.

On C18 resin, add a little (0.01-0.05%) trifluoroacetic acid, TFA, to your solvent system to sharpen the chromatography. If the compounds tail, try adding a little triethyl amine in place of the TFA.

How do you know if you have two spots if they elute on top of one another?

Run your TLC plate several times with a lower elution power solvent system.

Use RP-8 pre-coated TLC using a developing solvent either water and acetonitrile or water and methanol in appropriate composition.

trying 2D TLC with solvents of varying polarities in each case will help you out.

 It depends on the composition of binary solvent system. I would say, try to compose tri-solvent system as it changes the individual:spot RF value, as specific spot affinity differs to a particular solvent. although you will have fair chances to get separation through this way. Hope it will work for you!! 

Try to use the longer plate, or deep twice. After the solvent reach finish line, take it from the jar and deep once more.

This is a very old thread. 

DID SOMETHING WORK?  Would the original poster please update the thread.

I vote for 2D with a completely different 2nd phase solvent system. Then to try Reverse Phase. 

The OP has certainly moved on, though.

Please collect the spot compound try sequential extraction or collect fractions from the spot compound and analyze further.

2D is not for preparative separation. Trying different solvent systems, running repeatedly with lower polar solvent systems are better options. RP TLC is not readily available in several developing countries. Patience pays very well. I separated cis-trans cinnamates of three diterpene molecules by this method- six compounds close in structures could be separated (HPLC instrument was out of order that time). HPLC is better for semi prep/prep separations.

How much material do you need? Is prep TLC going to give you enough material for your next steps?

I have not had to go the prep TLC route and am interested in how much material you are able to obtain.

5-10mg each is enough for spectroscopic identification purpose- also for antibiotic activty screening. Each 20x20cm plate takes about 100mg of solid mixture dissolved in a solvent (if gummy/liquid,we may load less.) Preparation of sample for prep TLC will help. Reduce the number of spots in the sample by trituration or rough separation on a short column. 

Please try to separate the compounds by using preprative HPLC. Already u know ur mobile phase.