2D TLC Troubleshooting Help

[Micro_TLC]

Hello!

I seem to have developed a problem with running my 2D TLC plates.

(1st dimension: 65 ml chloroform, 25 ml methanol, 4 ml H2O;
2nd dimension: 80 ml chloroform, 12 ml methanol, 15 ml acetic acid, 4 ml H2O)

We use silica backed aluminum plates, and my extracts are dissolved in 2:1 chloroform: methanol.

For some reason, my spots are traveling with the solvent line, so that they are all bundled at the top of the plate, or the spots are shifted all the way to the left of the plate.

I've never had this problem before, it seems to have developed overnight. I have baked the silica plates, make sure the lid on the tank was tight, re-made the solvents many times, and can't seem to figure out what is causing this problem. I used generate the perfect profiles with all the spots in the appropriate spot, and now they are just running all skewed. I would greatly appreciate any troubleshooting advice!

[JMB]

You need to determine whether the problem is with the plate or the developers. As a first step,

1. Run the sample with the first solvent system---check the Rf values.
2. On a different plate run the sample with the second solvent system---check the Rf values.

This may tell you if the plate or the solvent system is at fault (or both).

[GOM]

Hi

Welcome to the forum.

Is there anything at all that has changed (however minor) with your procedure/plates since your Rf values went wrong and you apparently have no separation in the first dimension?

Regards

Ralph

[Micro_TLC]

Hello Ralph and JMB!

I did send my extract to another lab to make sure my extraction was working and when they ran their plate, the profile looked fine.

I haven't done anything differently. Luckily, the protocol is pretty simple. I will try your suggestions and post back with the results! I hadn't thought to run the plates separately! Thank you Ralph and JMB!

[carlo.annaratone]

Maybe a different plate (like C18 or so) sneaked inadvertently in an old box of silica plates?