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Author: Subject: Need help interpreting this TLC plate
alchemizt
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[*] posted on 8-3-2022 at 21:56
Need help interpreting this TLC plate


Here is a TLC plate I just stained with iodine:



The stained line at the stop is exactly where the solvent front was. And the faint spots at the bottom are where I spotted the sample. Both spots are the same sample. The solvent is 100% ethyl acetate and I couldn't fully dissolve the sample in ethyl acetate, its not extremely soluble.

This is an alkaloid extract from the plant banisteriopsis caapi. Compounds that should be present in high concentration are harmine and tetrahydroharmine. And to a lesser extent harmaline.

What does it mean that I got this huge, merged together line of compound at the solvent front? Why did it run so close to the solvent front? Does this mean the compound is highly soluble in ethyl acetate? Why did it spread into a line like that, it means the sample is too concentrated right?

Also why are there two faint spots where I spotted the sample? Is this a compound thats almost completely insoluble in ethyl acetate (and thus doesn't run with it)? Maybe this is the solvent that the sample was dissolved in? I should mention that both dots are the same sample, one on the left dissolved in ethyl acetate, one on the right dissolved in ethanol.

Now heres what happened when I let the iodine evaporate and sprayed the plate with dragendorff reagent:




So here you can see the streak at the top didn't turn bright orange, so its not an alkaloid, its something else. The two spots in the middle turned orange, so they are alkaloids. Could it be the streak at the top is ethanol+water from the sample which I didnt give sufficient time to evaporate? If iodine doesn't bind to ethanol, then this streak must be a non alkaloid impurity?

[Edited on 9-3-2022 by alchemizt]

[Edited on 9-3-2022 by alchemizt]
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Tsjerk
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[*] posted on 8-3-2022 at 22:58


You should downsize the images, I can't even zoom out far enough to get everything into one screen.
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alchemizt
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[*] posted on 9-3-2022 at 08:19


Images downsized
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[*] posted on 9-3-2022 at 08:48


That's a nice clear spot you obtained. The easiest way to tell if it is one of the compounds you are interested in is to buy samples of the pure compounds and use them as references. You can put a spot of reference on the left, a "co-spot" that has a spot of the reference and the unknown in the middle, and a spot of your unknown on the right. If the spot in the unknown is the same compound as the reference, then you should get one spot in each lane. If they are different, you would probably get two spots in the middle lane.

If the solvent you use to elute the TLC is too polar, it will flush the spots you are interested in all the way to the solvent front. To bring them down, a less polar solvent must be used to elute the TLC. One way to do this is to dilute the ethyl acetate you are using with a non-polar solvent like hexane.
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[*] posted on 18-3-2022 at 23:18


Have you tried running an "empty" run on your plates? I mean a TLC run in the same solvent without actually putting anything on the start line...

I found that my old TLC plates behave similarly: they may absorbed something from the air during the past few decades since their manufacture, or maybe the fluorescent dye started to decompose, but I also have that kind of strong line at the solvent front this way. (I also use iodine as developer.)

Actually, I could wash it out by having an empty run. The solvent carried away this stuff to the top of the plate and then I dried the plate, and re-run with real spots and it was fine... :) Maybe worth a shot.

The spots at the start are usually junk. Tar. Crap.
Definitely not the solvent you used. Something (or a mix of somethings) that are highly polar and bind to the silica gel very strongly.
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Texium
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[*] posted on 19-3-2022 at 06:25


Quote: Originally posted by Pumukli  

The spots at the start are usually junk. Tar. Crap.
Definitely not the solvent you used. Something (or a mix of somethings) that are highly polar and bind to the silica gel very strongly.
Usually. Sometimes it can be that the solvent mixture you used is just nowhere near polar enough and your product is stuck to the baseline. In the case of amines and carboxylic acids, you’ll often have to add 1% or so of triethylamine or acetic acid, respectively, to your eluent to suppress ionization of your compound on the silica gel in order to get it off the baseline. I don’t think that’s the case here, since the baseline spot is so faint, but I wanted to mention that caveat.



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Serybva
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[*] posted on 15-4-2022 at 00:06


What you have is a characteristic consequence of "I used only one solvent as eluent", most of the compounds present in your extract were eluted too fast because they have more affinity with ethyl acetate than your plate material.

The 2 spots in the middle are some less polar compound that aren't very soluble in ethyl acetate.

From my little experience I can tell you can never obtain good results at TLC using only one solvent, your eluent should always be a blend of polar and less polar solvents.

Here's the mixture I use most of the time and gives very good results: pentane/ethyl acetate, hexane will also work in place of pentane.

Proportions are usually 70% to 90% pentane/hexane.
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[*] posted on 15-4-2022 at 08:25


Highly recommend cleaning and trimming finger nails.
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