[aser25]

Hi there

I am analyzing different samples under TLC. Developing the plate with a mixture of chloroform, methanol, glacial acetic acid and water. And staining with phosphomolibdic acid. Please see the attached images.
Do you think the signals highlighted with arrows correspond to actual compounds still soluble in the mobile phase, or might these stains be some sort of artifacts? They migrated exactly as far as the solvent did.

Thanks for any insight.

[rolnor]

Hard to say, you could run the plate with less polar system and see if the spots move less. You can try to stain with iodine-vapour or permanganate as well.

[clarkstill]

I agree with Rolnor, it's hard to tell since the photos aren't very clear. However, the marks you've highlighted look quite inhomogeneous, not how I would normally expect TLC spots to appear, so there's definitely a chance they are artifacts. You could try spotting several times to increase the intensity of the spots?

I'm also a fan of the ceric ammonium molybdate stain, but it often depends on the chemistry of the compounds you are detecting.

[aser25]

Thank you for your answers. You´re right, I need to optimize it!

[Babcock_Hall]

UV-active anomalies are often seen near the solvent front in my experience.  I sometimes use phosphomolybdate staining, and I believe that I have seen markings that were not true spots; however, my memory on this is less certain.

[aser25]

I ran the same samples but dipped the plate in 10% sulfuric acid then heated for 1 hour at 65 Celsius. Please see image. Doesn't this result confirm that there is some compound (right line) migrating as much as does the solvent, something with really low polarity? Or might this still be some sort of artifact? These are expected to be lipids.

[rolnor]

I ran the same samples but dipped the plate in 10% sulfuric acid then heated for 1 hour at 65 Celsius. Please see image. Doesn't this result confirm that there is some compound (right line) migrating as much as does the solvent, something with really low polarity? Or might this still be some sort of artifact? These are expected to be lipids.

Yes, it seems so. Can you run the plate in a less polar system? What is the right-hand sample, is it reference or startingmaterial?

[aser25]

Here you go, I removed the water and methanol, so it is running with chloroform:ethyl acetate (50:50). Same samples.
To answer your query, right-hand sample is starting material.