Sciencemadness Discussion Board

TLC Reference paper

GreenD - 31-5-2013 at 10:16

Need help understanding something again; odd description of developing TLC, in context they are trying to separate
monoacylglycerides/diacyglycerides/tri- of long-chain fatty acids.

Five/ul (50 units) of enzyme solution was spotted onto the pre-adsorbent area of a silica gel plate (Type LK6, Whatman Inc., Clifton, N J, USA) impregnated with 3.0% boric acid and dried for 2 h at 150°C, and I gl of the substrate mixture containing 76 wt% glycerol, 19 wt% of oleic acid and 5 wt~ of water was then layered. After 20 min at room temperature, the plate was pre-developed in the pre-adsorbent area twice with diethylether and then; developed with CHCl3/acetone (96.4, v/v). The spots were visualized by spraying 50~0 sulfuric acid followed by heating for 20 min at 150°C.

confused about the bold

DJF90 - 2-6-2013 at 08:57

They're using plates with a pre-concentration area. You spot below the baseline, where the pre-concentration area is, and when you elute it the compounds migrate quickly up to the baseline, giving a tight spot. This is then developed with the desired solvent system to run the actual TLC. It looks like they pre-eluted twice, just to get everything up to baseline. Diethyl ether is only weakly polar, and so I assume that it will only carry material through the pre-adsorbent zone to the TLC baseline.

If you want to do this, and don't have pre-adsorbent plates, just spot normally and run the TLC in 4 %v/v acetone in chloroform.

GreenD - 3-6-2013 at 10:52

Ok, thanks. I don't see the necessity of doing this, but understand it now, at least.

DJF90 - 3-6-2013 at 11:44

If the extract is dilute it allows you to apply a reasonable amount of material to the plate without it all spreading out with the repeated applications. Pre-elution then gives a nice tight spot on the baseline, ready for elution with the chosen solvent system.

Peach is fine by the way...