TLC solvents...

[Sabine86]

Hi,

I tried to run a 1D-TLC with two solvents to analyze phopholipids of a fluid. (silica gel plate, 7,2 cm x 16 cm, first solvent (went up 7 cm): 40:10:10:1 chloroform: methanol:acetic acid: water and second solvent (went up to the top - alltogether 14 cm): 120:46:19:3 chloroform: methanol:acetic acid: water)
The problem is that all standards (cholesterol, sphingomyeline, phosphatidylcholine, phosphatidylethanolamine) run with the solvent front on the same level and the sample in consequence hasn't been split up.
I think, I have to vary the composition of the solvents, but I'm not sure in which way.
Do someone have an idea how it could work better?

My second question. After TLC I've to scrape off the spots in order to measure the phospholipid-content (Bartlett). Is it possible to scrape it off from a bought plate or do I have to make a TLC-plate by myself?

Thanks a lot for your answers!!!

Kind regards

[Jack Silver]

Note that with silica, stronger (more polar) solvents cause soluble compounds to move more quickly up the TLC plate. The fact that all the compounds ran to the top suggest the solvent system is too polar. Note that cholesterol runs on silica with a hexane/ethyl acetate gradient.

What is the reason for running the plate twice over?

for the second question, you can scrape commercial plates. They often use calcium sulfate as a binder or silicic acid.

[Sabine86]

Thank you for your answer! This helps a little bit.
Running the plate twice over was done because of an article I read, which mentioned this method instead of using 2D
TLC. There was no other special reason for that.

Thanks and have a nice weekend!

[Sabine86]

Another question about the determination of the phospholipid-content of the scraped off material: Do you think I can use the method of Stewart (by ammoniumferrothiocyanat complex formation => http://www.sciencedirect.com/science/ar ... 9780902699) instead of that one of Bartlett?
Or are there any arguments against it?

[Jack Silver]

Sabine-

Sorry- I don't currently have access to these articles. I suggest trying them and seeing what works for you.

[Sabine86]

Okay!

[chromatographer1]

You may not have had the chamber saturated with vapors before you inserted the TLC plate.

Try Dr. Stahl's book for a method that should work for you.

ISBN-13: 978-0387047362


Thin Layer Chromatography: A Laboratory Handbook [Hardcover]
Ashworth (Author), Stahl. E. (Editor)

Another good book:

Thin-Layer Chromatography, Revised And Expanded (Chromatographic Science) [Hardcover]
Bernard Fried (Author), Joseph Sherma (Author)