What is the specific problem? What solvent system? Analytical or preparative TLC?

If the issue is that the compounds elute at the solvent front in very low concentrations of organic solvent, you may need another technique. Note that C18 TLC won't work with 100% water as the C18 chains are "collapsed" and won't interact with your compounds.

If no resolution, you may need to change your solvent or pH, depending if the compounds are basic or acidic.

You may also need to run column chromatography using a HILIC system- normal phase TLC plate binders often fail with HILIC solvent systems.

There isn't enough information in your question to give a good answer.

More on HILIC- although pre-packed columns are used, the information is applicable to glass columns as well:

http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/RediSep%20Rf%20Gold%20Silica%20and%20Highly%20Polar%20Solvents%20App%20Note.pdf#search=highly%20polar

http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Posters/Strategies%20for%20Flash%20Purification%20of%20Highly%20Polar%20Compounds%20Poster.pdf#search=highly%20polar

I am running a full phytochemical analysis of a plant leaf sample. I am using hexane, dichloromethane, ethyl acetate, methanol and water to dissolve the phytochemicals sequentially. I have successfully performed column chromatography and TLC with normal phase silica of hexane, dichloromethane, ethyl acetate and methanol. For water fraction, I used reverse phase silica column to separate them. After separation, now I am using reverse phase TLC. But the problem is it's too strong to separate them. I am using water and methanol to separate them and gradient ranged from 95% water-5% methanol to 5% water-95% methanol. But unfortunately no luck.

The specific question is 'how can I separate the water fraction'?

Jack Silver

I see this part- I assume this is how the column was run:

" For water fraction, I used reverse phase silica column to separate them. After separation, now I am using reverse phase TLC. But the problem is it's too strong to separate them. I am using water and methanol to separate them and gradient ranged from 95% water-5% methanol to 5% water-95% methanol. "

What solvent system are you running for the TLC?

Also, make sure the TLC chamber is saturated with vapor. For C18 TLC, the chamber may need to sit for several hours to become "equilibrated". The chamber needs to be sealed.

The solvent system for TLC is just like the ones I used to separate the chemicals, methanol and water.

Thank you very much for your suggestions. I will try TLC after equilibrizing, as per your suggestion

Jack Silver

Use dianion to seperate 4 fractions of 25, 50, 75, and 100 MeOH in water. Check TLC and chose fraction to continous. Usually 50, 75% for my work

Thank you for your valuable suggestion Khanh Tran Huynh Nguyen

I got another question. is there any alternative method to separate the water fraction without using both C18 and dianion?

you can use solvent system of 3 or 4 solvent: Chloroform: Methanol: Water (or acetic acid) with ratio of 5:1: 0.1 (0.05) of example

Ok, but I use Buchi Fraction Collector, C-660 ,to perform separation, which has only two pumps. That's the problem, that's why I can't use three or more solvents to perform separation.

I don't have other instrument available to perform separation.

Khanh Tran Huynh Nguyen

You can apply open colunm for normal phase, with 3-4 solvents, it is easy.

Thank you very much Khanh Tran Huynh Nguyen