2D-TLC-Questions

[Sabine86]

Hi,

at the moment I`m preparing for my first 2D TLC to separate phospholipids and there`s something, I dont understand, although it`s probably very obvious, but I dont get it.
If I can applicate only one sample per round on the silica gel and if I -because of this- cant applicate standards at the same time on the same gel, how can I identify the spots in the end? Or do I have to use an extra gel for each sample (unknown and standards) and develop it in the same chamber or -if no space is left- in another one during the same time? But this would mean a huge wastage of gels... I`m a bit confused.

And another question - to separate lipids (phospholipids and cholesterol) of a fluid - is it really necessary to do a 2D TLC? Or does it perhaps also work with 1D?

Thanks a lot for your answers!

Best regards!

[chromatographer1]

I would think that you could spot your standards at the opposite side of the plate for one sample, and at the top of the plate , at the same side corner as your sample.

Then developing the plate at the bottom, your standard and your sample would migrate in parallel.

Comparisons could them be made in 1D.

When turned 90 degrees, the separated components of the sample would then be developed in parallel with a second spot of standards and be compared accordingly.

This means however that the standard will be run twice, each time in a different 1D development.

But would this be useful to you? What doesn't separate among the standards in the first dimension could not be compared. And what doesn't separate on the second development in 1D could not be compared either.

best wishes,

Rod

[Sabine86]

Thank you! I didnt thought about this, but it could work, even if its maybe not the optimum. But on the other hand I cant imagine another way and at least it doesnt need so many gels.

Do you know something about the detection limit for phospholipids/cholesterol, if I´m using a Merck silica gel 60 F 254, where I detect the spots with UV? Is it enough to applicate 5 µg per standard?

Best regards,

Sabine

[chromatographer1]

I used to do sulfuric acid char with UV fl detection possible if you don't over char the plate.

It has been many years since I did TLC routinely. Sorry I can't add more. There was also a spray that did not use acid char.

Time to reread the old standard text on TLC. A German book, if I remember correctly.

Good luck.

Rod

[Sabine86]

Thank you

[chromatographer1]

Also before you use the acid spray on the UV fl plate.

If you place the plate into a Iodine vapor tank briefly and then quickly use the UVab light the iodine improves the detection of the spots by also absorbing the UV light.

best wishes,

Rod

[Johnny Rod]

How do you rate sulfuric compared to PMA (polymolybdic acid) for sensitivity? I haven't used the former but the latter was prefereable over permanganate for sensitivity in my synthetic days.

[chromatographer1]

I found (gosh this was 30 years ago) that my results varied, depending upon the freshness of the reagent and the type of plate used (and probably the humidity and temperature of the lab, among other things).

And it was easy to under or over cook the plate with methanolic H2SO4 to get a good UVfl response.

but 1microliter of a 1g/l was easily detected of lipds, especially if unsaturated for both sprays.

best wishes,

Rod

[chromatographer1]

Stahl's book on TLC

A laboratory handbook on TLC was written by Egon Stahl in 1965.

ISBN-10: 0387047360
ISBN-13: 978-0387047362