TLC

jtm · Jul 7, 2015

I am trying to set up my own cromatography kit (TLC), because the ones available (though some of them are very good, I must say), are too expensive. Much overpriced, like many things in this ... field of activity.

So I've got some 50x100mm silica on glass plates, isopropanol for extraction (even this may be a little too polar), diethyl ether and petroleum ether for eluents, and Fast B Blue salt for staining.

The method is:
-extraction with isopropanol, I did 1g material/5ml ISO, (not standard)
-charging the plates on 2 lanes with 3 microliter cappilary (also not standard, usually is done with 2).
-Letting dry for 30min at 110C in a oven, and after that charging other two lanes with the same samples (for seeing the differences).
-Developing the plates, in said eluent mixture (80% petroleum ether and 20% diethyl ether, usual for cannabinoids, but not mandatory). Here I can see clear migration and separation. To much green though, I should add a picture, but I forgot today. Maybe I dripped too much on the plates, or I shouldn't make the extraction with ISO.
Also, this may be because I let the product too much in the extraction vial (days).
-Drying plates at room temperature
-Staining. Now, this is the moment when I cant't figure what is wrong. I can clearly see migartion on the plates, separation between compounds too, and I know that Fast B Blue should stain in red/pink/purple/yellow/orange, depending of cannabinoid present.

Nada. I can't see anything. I am doing staining like that ( I tried other variants too): Dissolve 0.5g Fast B Blue in 50ml of 9:1 acetone: water, and also making a 0.1M NaOH solution (this is one of the standard procedures). Spraying first with the dye (Fast B Blue) and them with the sodium hydroxide solution.

I also tried to dissolve the dye in NaOH 0.1M, but it didn't make it. I mean I couldn't dissolve it. Maybe I put too much (0.5g/50ml), I should try again with 50mg/50ml.

I will post photos of the las plate, maybe someone can help with this.

Thank you very much in advance.

G.O. Joe · Jul 8, 2015

Does the dye work without the TLC? The dye should be mucho sensitive, and THC - or THCA if this wasn't decarboxylated - gives a notable red color even without the NaOH. A knife point mg. of FBB in a few ml. of water should give a strong reaction after shaking in a vial with a drop of a weak IPA extract of a few mg. of fan leaf - in seconds - orange, red, or orange then red.

jtm · Jul 8, 2015

G.O. Joe said:
Does the dye work without the TLC? The dye should be mucho sensitive, and THC - or THCA if this wasn't decarboxylated - gives a notable red color even without the NaOH. A knife point mg. of FBB in a few ml. of water should give a strong reaction after shaking in a vial with a drop of a weak IPA extract of a few mg. of fan leaf - in seconds - orange, red, or orange then red.

Thank you Joe for your feedback.

Indeed, I tried the dye without plates, and I haven't noticed anything. Still, I will try it only in water today, again. The procedure says that it must be dissolved in acetone plus water (it is much more soluble), or methanol plus water but I will give it a try again today, the way you sugested.

I will let you know the results.

jtm · Jul 8, 2015

Here are yesterday's tests.

[/IMG]

As you can see, it stained all but what needed. Now, I will try to make a decarbed extract with some nonpolar solvent, for eliminating clorofill. Maybe the stain is masked by green? However, I am doubting this...

I tried today using FBB directly in NaOH 0.1M. It dissolved, in small quantity. On filter paper I placed a drop of extract (green too), and tried to stain, but ... no use, it didn't stain.

FBB is fresh, bought a month ago, stored at 4C, and solution is prepared fresh too.

Well, somewhere I am wrong...

G.O. Joe · Jul 9, 2015

Ridiculously small weights of FBB and cannabinoids make ridiculously intense azo dye solutions. If in my last post a ml. of IPA extract is added to the aq. FBB instead of just a drop of it, the red goes to the point of opaque. No one would instead send you Fast Blue B base 3,3'-dimethoxybenzidine I hope.

jtm · Jul 9, 2015

I hope too...

Well, these long names are a little tough for me, but there it is an identical product:

http://www.sigmaaldrich.com/catalog/product/sial/d9805?lang=de&region=AT

I thought it is the right one but... maybe not.

G.O. Joe · Jul 9, 2015

That is the correct product. What color is yours?

jtm · Jul 9, 2015

Grey. Unfortunately. But if (?) I remember right, it is the same like in AlphaCat kits. However, I noted in a datasheet, that it should be yellow or dark brown.

However, this one:

Fast Blue BB Salt hemi(zinc chloride) salt, CAS Number 5486-84-0, shouldn't be a better option? I've heard that in terms of storage and toxicity it is... hugh... better than FBB salt.

G.O. Joe · Jul 10, 2015

jtm said:
Grey.

chemicalbook.com: Grey fine crystals.

For FBB base. Does your product dissolve much better in acid than it did in base? And melt at 137-138 °C?

I looked for all the other substitutes a few years ago, but could only find FBB available to me, because I'm just some guy and Alfa would have said no way we're selling you FBBB - which I'm not sure is much more stable than FBB, which is of dubious toxicity. That toxicity is based on the weakly toxic and carcinogenic in animals FBB base which may or may not be mixed in the salt, and possible generation of FBB base in the body from the salt - which hasn't been shown to occur. Really, FBB salt would need polonium toxicity - that it doesn't have - to be a worry. If FBB salt was as carcinogenic as the FBB base parent chemical benzidine, it still wouldn't be an actual hazard to use.

The Indian FBB salt that was sold to me is a golden brown olive green powder. After standing a few minutes in tap water it starts giving color, and I'm more worried about drinking my tap water now than using the salt.

jtm · Jul 10, 2015

You are funny with your water

Interesting the fact that FBBB was not sold to you while FBB yes. Or maybe I understood a little wrong and nor FBBB or FBB was sold to you by a company, but you managed to grab some FBB from somewhere...

Well, maybe the salt is not so toxic in itself, when loking at it

, but I sprayed it and it was pretty irritant, as I missed to put it under a vent or something. Because of this I kept my breath every time I was spraying.

I contacted the supplier and them offered to send me another vial if I will answer them to some (normal) questions, wich I will not, because I don't want another vial with the same substance, hehe...

I ordered another one from another supplier, and I will post if I will make some progress. Maybe this will be helpfull to somebody else too.

G.O. Joe · Jul 11, 2015

FBB is a useful biological stain, so someone willing to send chemicals to residential addresses was bound to have it. Only the big names had FBBB. Maybe some free samples from China would have arrived by now if I'd asked for some.

So it is an actual hazard to use if you use it like that, but in a lab this would all be done in the hood or under some approximation of one - I've seen local-area air-suckers installed but don't know what they're called.

Thought you might like to know that tea reacts with FBB and gives a positive test for cannabinoids, if you feel like squeezing a wet tea bag. Isn't this the test used by all police in the US? The color is slightly more purple than the color with THC, but THC + CBN might look exactly like that. Other kitchen phenols were tested - no dice with vanilla or nutmeg in IPA.

jtm · Jul 11, 2015

Indeed, working with it other than under the hood was wrong. However, one may use a tray for staining, but at the moment, I was following the "protocol" of some stupid video from a company selling such kits.

Yes, this is the classical test used everywhere, but I was not aware of using it beside for cannabinoids, even I do know that FBB is staining other substances too.

Now we will wait...

jtm · Jul 20, 2015

I received FBB and FBBB. Now, these are good (FBB is reddish, FBBB is yellow). I used them the same like before (50mg FBB dissolved in 100ml 0.1M NaOH, sprayed onto the TLC plate) but... the results are the same.

The procedure is: extracting 100mg resin in 1ml ISO (so not a fan leave or something weak), then place 3microliter of extract on the first lane, decarboxilating in a oven 30min at 110C, then again place 3 microliter of extract on the second lane, let it dry, and then developing with diethyl ether and petroleum ether mixture. When ready, let it dry a couple of minutes, and then spraying with the dye solution (mentioned before). Nada. Zero. Niente. Nicht. Nothing. And I am sure that I have some cannabinoids there (tested, guess how?).

At this point I am considering buying a kit from AlphaCat. Somewhere I am wrong, but however I don't know where. I barely can stain some filter paper, but not a TLC plate...

G.O. Joe · Jul 27, 2015

It doesn't make any sense - that's why I mentioned looking for a reaction in a vial or with tea. If there is no reaction with tea phenols, I don't think you have FBB - if there is a reaction with tea and not extract, you don't have natural phenolic cannabinoids, since the LOD should be in the nanogram area.

Petroleum ether extract of decarboxylated cannabis is the cleanest for cannabinoids if you're not using plates and development technique for the natural acids. The FBB solution can be made by dissolving in a small amount of water and then diluting with fast drying solvent like methanol. A good place to saturate the plate with spray would be inside a plastic bag. NaOH overspray or ammonia vapors is totally optional for color intensification - I don't use either since what I am using is the 10% silver nitrate impregnation (dissolved in water and diluted with methanol like the FBB, but dipped not sprayed) and toluene system, which gives particularly good separation of CBD, THC, and CBN, with only a little silver staining of you and everything else if you're extremely careful.

Cutting down big plates to microscope slide size really nice or even half way nice has been the real challenge.

jtm · Jul 28, 2015

Thank you again Joe for your useful feedback.

About the substances, I think that they are ok now, being from some big names (FBB and FBBB). I am almost sure that I am doing something wrong.

About the plates, thay are regular silica on glass, not for UV, 0.2mm thickness, 50x100mm. With these, I am not very familiar, but I can pm you the vendor link to see yourself.

The developing technique is not for acids, from theory, being the one mentioned somewhere up.

I've read several times to be sure I understood, and these days, when I will have time, I will try to check your methods. INteresting methods you have, developing with toluene and staining with AgNO3. Common and cheap I would add.

But I can't really understand why would somebody cut the plates to such slides. For economy reasons?

G.O. Joe · Jul 29, 2015

jtm said:
I am almost sure that I am doing something wrong.

If THC and not excessive FBB solution is applied to silica gel, the only reason why it wouldn't show up is because it's in the developer. That means low spotting, tall solvent mixture, and the green part being something more polar that stayed. I doubt you did that.

If I had someone to charge for my plates things would be different but being a mostly stoner stoner chemist and not a professional entity means that I want my free 3 kg. of plates to last forever.

There's no need for tall plates even though this toluene system is done in a open tank beaker, because the CBD is stopped dead and all I want is good THC and CBD separation. This was applied to column chromatography over 40 years ago with benzene instead of toluene.

jtm · Jul 29, 2015

It worked. I am a little tired right now, and I will not give too much details, but I will do in later posts.

Basicaly, it seems that the FBB and FBBB will not stain acids, but only decarbed substances. I thought I was doing this directly onto the plates, but it was not so.

So I got a stained plate, weak staining but... staining. The material was some feral hemp (ruderalis), decarbed at 110-120C in a oven, and then extracted with petroleum ether, for avoiding the clorophyl extraction. The ether was evaporated, and replaced with ISO. This was placed onto the TLC plate, and developed in a mixture of petroleum ether and diethyl ether. After that, the plate was stained with a unknown (hehe, lost my patience!) mixture of water, FBBB and ISO, by spraying it. This last phase sucked, but it worked, even so.

I was trying to decarboxilate the plate in the oven, after I placed the acids on it but, it seemed it was not a good idea. Don't know why. So I did it before. Maybe the substances evaporated from the TLC plate, I don't really know.

I will come back with a thorough flowchart, eventually, these days.

Thank you G.O. In fact, if this would be possible, I would send you some flowers. O.G., of course

Maybe some pictures though, when they will grow a little...

jtm · Jul 31, 2015

Thank you.

Thank you.

Now, this is the result:

[/IMG]

The flowchart was like this:
-extraction with ISO
-placing extract on plate (silica on glass, 0.2mm 50x100 plates, non fluorescent, 3uL each point
-decarbing directly on plate in the owen, set at 120C 30-40min (not really know wich was the plate temperature, maybe lower)
-developing with petroleum and diethyl ether in the mentioned proportions
-staining with FBBB, 50mg/50ml deionised water. I given up at NaOH, as it does not seem needed at high concentration of cannabinoids.

Now we are in business. So to speak.

G.O. Joe · Aug 2, 2015

Nice. Those are interesting results - maybe some CBC, CBG, and incomplete decarboxylation. At higher temperatures for a longer time the THC is isomerized from the acidity of the silicic acid maybe, so the decarboxylation is best done beforehand.

That looks good. But trying the NaOH that's often added in the FBBB can't hurt if it might help you use a lower loading for better resolution. The truth is I've been using tap water and the pH is like 8.5.

Of the many thread viewers, none are saying they need any explanations of that picture. Everyone knows that CBG, CBGA, CBD, and CBDA are yellow-orange and the other common cannabinoids are pink-red-purple-violet, and that Gaoni and Mechoulam's Rf's x100 for this system from 1971 were, not including the acids at the bottom,
CBL 62
CBD 58
d8 THC 57
d9 THC 51
CBN 47
CBC 43
CBG 42

jtm · Aug 2, 2015

Well, as you know, most of the readers are interested in a good smoke, not in a good TLC, hehe!

Nice numbers though, I was not aware of them. Very useful. Now I can see clearly that I was not wrong in my supposition about CBC. I was not very sure of it.

In fact the left plate is done with NaOH. However, the resolution is poor mainly because it was overdeveloped.

However, the important thing is that... it is done and repeatable.

TLC

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