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TLC for mono-, di- and triglycerides



I'm new to the practical use of thin layer chromatography and I'm having some issues. In the example in the pictures I've been trying to get a good TLC of a mixture of glycerides. The conditions are fixed, so I'm not supposed to change those: dichloromethane as solvent and a mixture of dichloromethane and aether (90/10) as mobile phase. The visualisation is done with iodine.

So the first set would be the best, but it's not great. Visualisation is a problem. The highest arrow would indicate triglycerides, then the next two arrows would be diglycerides and at the bottom it's monoglycerides.

I think one of the problems I have is that the plate itself got stained too much in the second attempt. I'm not sure why that is, because I used the same tank and amount of iodine as before. But I've been searching for an improvement and I think I will try by adding a layer of powdered silica to the tank in combination with the iodine, which seems to have better results. The silica is then coloured by the iodine and brought into contact with the TLC-plate.

Any tips are welcome, but I have some specific questions:

- Why are there often two tails/distinctive spots for the triglycerides? I would guess this is because I often spotted twice for each sample, but on the other hand these were really in the same spot most of the time, so it doesn't make sense to me. I don't understand why these wouldn't mix when applied on the same spot.

- The origin still shows clear spots. So there's still something there that hasn't moved, right? The sample may contain surfactants. Could that be it?

Thank you

I would apply samples and develop the plates exactly as at present.

However, instead of Iodine vapour, use a spray of 5% H2SO4 in ethanol, followed by heating on a hot plate to detect the spots.

Organic compounds are charred by this process, and give a black spot on the white background; the spots will not fade.

Doing this will enable you to more easily optimize the DCM/Ether developing phase.

Once the separation has been optimized, switch back to iodine vapour per your protocol.

Try loading once, twice and thrice on adjacent sample lanes repeated across the baseline to determine any loading effects.

Good Luck!

Edit: do you have your tank lined with filter paper to provide a saturated atmosphere of the developing DCM/Ether mix?

I can think of one other possible explanation regarding the doubled spots.  Your triglycerides may have different fatty acyl groups that change the Rf slightly.  Fatty acids can differ in the number of carbons and the degree of saturation.  If you have ionic surfactants, they might not move.
I realize that you need to stay to one method.  However, in principle one could to distinguish between saturated and unsaturated fatty acids using color-change reagents that are selective for carbon-carbon double bonds.  Potassium permanganate comes to mind, as well as more exotic reagents that are sometimes used in fingermark analysis, such as RTX.

Thank you for the input.

I tried the development with sulphuric acid, but couldn't get it visualised properly. Probably due to my lack of experience. The plate deformed due to the heat before I got to see much. Or should I use glass plates instead of aluminium? Maybe I should try it in the oven, because I don't have as much control over the temperature of the plate.

And yes, I do have the tank lined with filter paper. It should be saturated. I'm going to try a smaller tank to see if there's a difference.

Interesting remark, Babcock. Whenever there's more time, I would definitely love to try other reagents to see if I can make a distinction.

Anyway, I tried the suggestion of adding it in different ways: I tried different quantities and applying this in a single or several loadings, as indicated. And to me it seems that yes, the doubled spots seem to be related to the substance and not the application. I tried to alter the picture to get it better visualised.


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