Hi! It is possible that in the TLC you saw two compounds with the same RF values. In other hand, especially in polar eluents, spots are usually 'stretched' but you can't be sure without other spectral analysis. Also, I suggest visualization by spraying with 50% (v/v) aqueous H2SO4 followed by heating.

Does it crude extract or any fraction? The single spot, it might means that  all compounds are eluted together if you are doing with crude extract (Change the solvent gradient, for silica it will be better to use EA-MeOH-Water as mobile phase). Or if you try to purify your compound from a fraction which have less impurity, this gradient is still not a good condition. Are you using silica or other revers phase TLC stationary phase? The dark spot is the compound and the blue periphery, I think it just the solvent reflected with UV-Vis. Are you continue to do HPLC? 

@ ly lin na. yes i am continuing with HPLC. I m using silica and with this result suppose 2D is an option then what gradient can i use. 

@ Marko. i have scrapped the spot and extracted with ethylacetate and analysed through MALDI - TOF and i got two peaks but with small variation in mass. but when i gave the crude extract as such to MALDI-TOF i did not get the same unique peaks. can you suggest what could have happened?

Initial step for crude extract, does it right? So what is your target compound, flavonoid, carotenoid, saponin or what els? If flavonoid or phenolic (EA-MeOH-Water), Saponin (Hexan-EA-MeOH-Water)..etc.

@ly. it is an antibiotic

In case of crude extract, there is fair possibility of collective elution of components (similar RF value) in view of inappropriate polarity of mobile phase. Differential  migration of components need to be assured for better resolution.

To continue with TLC, I suggest 2D or change of mobile phase with aqueous-organic mobile phase combinations. 

I didn't see which solvent you used to run your TLC in the thread. As the compound could be washed off silica with ethyl acetate (mentioned for MALDI), seems like a less polar solvent system could work.

 I'm going to suggest changing the solvent system to allow for different solvent selectivity, but without knowing the starting solvent system, it is difficult to make a cogent suggestion.

Assuming you used hexane/ethyl acetate, try hexane/dichloromethane, or hexane/ether as the second solvent will have differing selectivity. Hexane/acetone may work although acetone is in the same Snyder selectivity group as ethyl acetate, so I hold out little hope that would work.

Dear Balaji,

Run the sample in HPLC, use a diode array detector (190 - 400 nm), and get the peak purity done (if you still get a single peak).  Your TLC indicates differential absorption/reflection of the spot.  If it is because of two components, then the peak purity check based on UV spectra will clearly indicate co-eluting components and confirm that it is not one compound.

All the best

Dear Balaji,

What is your purpose in this work? Do you want to isolate that antibiotic from which source? As the result you scrapped from TLC --> extraction with EA --> MALDI-TOF in the result you got two peaks. In contrast from the crude extract you got different result. It mean that during the TLC process the condition of your compound had changed, try to observe pH, pKa, how you soluble, sonicate or not, did you heat to assist solubility..etc. Due to my experience that small activities can affect to compound if that compound is sensitive.

Follow the procedure, you have to do the TLC--> HPLC, but you can try with HPLC directly, and check the retention time and try to adjust  the gradient of solvent until you can get the best condition. 

Dear Balaji

If you can temme more information for your separation

is there naturel ou synthetic compounds.

wath weave in?

wath mobile éluant?

wath the color of your front solvant?

wath dimesion of your TLC plaque in cm

you now wath structre of your compounds?

you can have one  compound witn two organic fonction with each absorption in UV

you must take spectre in UV 200 to 400 nm

.....

i wil propuse you  wath you must do only witnthe same  micro TLC  with aout HPLC

mostafa

ERIDDECV

FSSM chemsitry

marrakech

Dear Balaji,

At this point of time, one cannot say anything. However, I am 80% sure that it is mixture. To make it clear, reduce the polarity of the mobile phase and run TLC again. If it does not work, do analytical HPLC.

Dinesh

Dear 

Why dont you run your TLC by adding some non-polar solvent in methanol. Please try to get better separation by mixing two solvent. Which definately works.

@all. this is a compound extracted from an actinomycetes. It gives ethylacetate a yellow tinge. First I tried with hexane and ethylacetate as mobile phase. The rf was was too low but still a single spot was the result. Then I swotched to acetonitrile and methanol. When both at equal ratio was tried I found the rf was .6 and methanol alone it is .7 again a single spot was obtained. And at all time I found the green flurescent centre and a varied blue periphery.

I would run two-dimesional TLC using two different solvent systems. First, I would run the TLC on methanol ( I would spot it towards the edge of the TLC) , then I would let it dry and turn the TLC 90 degrees, and woul run the TLC in a different solvent systems with different polarities. This may help you to differentiate if there are two compounds. Best. 

I am supporting the one compound theory and the difference in colour may be due to higher concentration in the centre. The fact that you tried various solvent systems and it will not separate, as well as the shape (the green spot in the centre and not in fron or trailing) seem to point to this. I think the two-dimensional analysis should provide definitive proof.

Dear Belaji

You take  a smal plaque TL 4 x4 cm in the same plaque   make TLC separation in  two dimesional 90° degré with two solvent in the firt with dicchloromethane and 10% of méthanol in the second return to 90° degre with the same phase mobile but witn 30-40% of méthanol

you compund have very polarity 

other protocl dilu your compund to 50% and take phase mobile dichloromathane witn méthanol 99-80% take you plaque 4x4 cmand tak up the silice lake entenoir lake this

     GOOD LIKE Mahrouz mostafa Marrakech ERIDDECV Chemistry marrakech morcocco

diminuer la polarité par du dichloromethane  verifier la pureté de votre methanol c est a dire faire migrer sur une plaque  chromatographique  et beaucoup plus simple prendre une plaque chromatographique plus large et déposer en trait et non en spot 

see my publication jacques pothier thin layer chromatography sur google

your spot is not good 

@ Jacques. Thanks for your suggestion. Please put the same in English..

 Thank you

various track to solve your problem

decrease polarity methanol must not be majority try dichloromethane80methanol20

if no migrationadd either 2p100 ammonia or 5 p100 acide formique

for derivation try sulphuric anisaldehyde reagent or sulphuric acid reagent

for spotting try this method apply a double quantity in line over 3cm is better that  spot

see my publication on google jacques pothier thin layer chromatography

my mail [email protected]

one solution must be try with another mobile phase (with less polarity) such as ciclohexane/toluene/diethylamine (TB) and the compounds probably will be separated

with best regards

It really depends on what product(s) are you expecting. Anyway, I would also recommend to try various mobile phases, generally lowering the polarity usind dichloromethane, toluene, to have completely non-polar mobile phase, I would use hexane or cyclohexane. You can also add a little bit o ammonia dissolved in methanol, in that case I recommend to use some closable TLC chamber (can be substituted with clean glass jar with a screwable lid from some food, e.g. jam/marmelade) , not the standard ones with the lid only freely put on them - the ammonia is quickly evaporating and it will change the ammonia concentration during the analysis, which will harm the results. Try these mobile phases in various mixtures and you will see.

For derivatization, try KMnO4 solution, anisaldehyde solution, bromocresol green, or solid iodine chamber, etc., then dry the TLC plate with on a moderate heat set heatgun. However, the derivatization agent utilization is depending on the expected product.

DEAR BALAJI

Please if you can informe me if you have tested my solution

THANK YOU 

Pr Mahrouz mostafa

Marrakech

UCA FSSM Chemistry

i

@ mostafa.  I have done a series of TLC with methanol and dichloromethane and with 50:50 propotion there is no difference from the single spot obtained earlier. But there was a little change in Rf. the single spot is still pertaining. So i moving to HPLC. 

@ Gareth. Hello sir,

The extraction procedure is just a two phase extraction with ethylacetate at equal volume. chemical group - the fact is its an unknown compound! And the direction of change was a higher Rf!! I am using the same solvents as you have suggested for HPLC.. 

Thank you

Try other solvent systems and then check the spot, you will find your answer

Dear Balaji

You much  find solution iwith TLC . CLHP is expensive but you can resolve with that technology witn C18 and mobile phase poliar witn acétonitrile /méthanl gradient.

In TLC try mixture witn actétate in hexane ' gradient

Have you effect balayage in UV how weave max you have find

Marrakech Mostafa Marrakech UCAM

Hai..

Try use the combination of the solvent, for example Hexane and Ethyl acetate and start from ratio 5:5 under uv 254nm, and 366nm  , then you can see wether it shows single spot or many. If your highest spot go very high, you can reduce the ratio of Ethyl acetate, EA ( semi polar solvent) and increase Hexane (Hex) non-polar solvent., may be it can be Hex: EA(6:4).  You can play around with the solvent ratio. You also can Spray you TLC Plate with the vanilin solution, and you can see some more spot(if any) which can't see under UV or naked eye.  You also can play around with the combination of Diethyl eter: Hexane or Chloroform: Methanol based on the polarity / type of your extracts.

In my case, my TLC PLATE  also shows single spot and same colour after spray with vanillin  but when I run HPLC, it actually show 2 peaks. So, maybe you can run your sample on HPLC also.

Ok

This is à good solution

Pr mahrouz most-afa

ERIDDECV chemistry

UCA Marrakech Morocco

METHANOL IS TOO MUCH POLAR

use acetate d ethyle 95 /5 methanol

spraying by dragendorff reactive but also spray h2so4 then heat 120 c

If  we Assume  that you have two   concurrently  moving spots  then you can simply change the polarity  of the  solvent and run the chromatograms   you may get  two more separated spots or otherwise you still  get a spot  but with better separation to show overlapping spots .If this does not occur then you have a single spot  and the blue perfery is simply due to scattering   effects 

you must try changin the polarity of mobile phase, and may be you could find two compounds at the same spot (i.e: Ciclohehane/toluene /diethylamine  TB)

finally separated the mixture,

Chloroform and acetone worked. Blue  and green fluorescent were two separate compounds.

Thank you to the person who suggested to use chlorine containing solvents.

Thanks all