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Home TLC Thin layer chromatography

PhenoMenal

Hairdresser
Veteran
dongle, ive never made TLC plates so I can't give you any help there, kudos for giving it a go though!
Yes you can also just buy 20cm ones and cut in half etc, another benefit of aluminium over glass! well, you can cut the glass ones with a glass cutter, but only regular scissors required for the aluminium plates.

Roughly how long does it take for your 10cm plates to develop in the chamber?
It takes 20-50 mins for the 10cm plates to develop in my jar, which is about the same size as the plates, usually about 30 mins but it depends on jar/chamber size, temp & humidity, whether you poured 11mL or 12mL etc etc, different solvents might take different times too. I use 12mL because that seems ideal for my jar, from my tests. You may need more or a bit less, but it's not much. Your initial thoughts of "25 mins seems a bit long" is understandable but we're only standing the plate in a very small amount of eluent. If it's getting to 50 minutes though, you might not've put enough eluent in there, and/or the lid might not be screwed on tight enough.

What are your thoughts on a using something like a methanolic solution (9:1 meth:water?) of BB and using it as a cannabinoid presence indicator on alkane (heptane or pentane) solutions? In the same way a liquid pH indicator test would be used. Would that work? I just want the solution to turn color if it contains cannabinoids...
sorry but i'm not a chemist and cannot give advice on any of this. That might sound ironic after all i've said, but i was basically forced to figure out TLC when a family member got cancer but found myself in a position where there was no "DIY TLC for cannabinoids" tutorial available and i didnt wanna buy a commercial kit, so while I now understand the basics of cannabis analysis with TLC, when it comes to mixing chems etc it scares me!

I was super lucky to have G.O. Joe help me figure out the puzzles pieces, especially as it was such a desperate time for me/my family, I don't want to think about how things would've been different without him. The feedback he gave me in this thread will continue to help people for a long time to come.

One thing I can say though is that your idea might be forgetting that Fast Blue B should be kept refrigerated, and Fast Blue BB kept in the freezer (though im not sure if you meant it as a field test or not). They also degrade very quickly, i'm not sure if it's from light or exposure to air or whatever, but I always freshly make the Fast Blue bath, and try to finish using it within 30 about minutes, because then the fluro yellow starts to turn to a rusty reddy brown, and stops giving good reaction.

See also Beam's Test if you're wanting to test for the presence of CBD, that's a "solution turns color" test, and can be portable/field suitable. Info about Beam's test is also in my TLC tutorial. It doesn't give you anywhere near the amount of info TLC gives you though.
 

dongle

Member
I'll report back on the methanolic solution.

Largely interested in quick cannabinoid presence determination on column chromatography fractions, which I'm hoping will save a lot of time on sample evaporation, and potentially subsequent 3rd party lab analysis.

Thanks a bunch for sharing your time & expertise.

Good to see you still around after so many years.

Hope your family member made it and is doing well.
 

PhenoMenal

Hairdresser
Veteran
btw, seeing as you didn't shop around (!) so ended up with plates with fluro indicator in them i wonder if it might be worth buying a $5 light or whatever off ebay to check them out (before Fast Blue bath!)
Hopefully the fluro indicator wont interfere with the Fast Blue, id guess it'll be fine tho. And if youve got a light for it then it might even give you a good enough view of the result that you don't need to do the Fast Blue visualisation phase. The thing i dont like about fluro tho is it's basically grayscale ... dots are just black or white ... whereas Fast Blue BB shows THC as bright red, CBD as bright orange etc, so then you have the COLOR as another indicator as to what the molecule is, as opposed to just its position, and it LOOKS BEWDIFUL!
 

dongle

Member
I have UV LED flashlights in 365nm and 395nm, but not 254nm.

Last time I bought (and returned) a dual 254/365nm UV lamp, I couldn't tell any difference (incorrectly) using 254nm so I returned it. Since then, I haven't been able to find a cheap 254nm UV light the few times I looked (365/395nm is available for about $10), so I didn't bother.
 

dongle

Member
Looks line 254nm isnt good for the eye, so maybe the lowest wattage one mounted in a reflector with UV protective eyewear is probably wise.
 

dongle

Member
I did shop around, but was hoping the f254 indicator won't interfere. Nice thing about Amazon is the awesome return policy.
 

PhenoMenal

Hairdresser
Veteran
altho i have no idea, im sure it'll be perfectly fine. Fast Blue is so specific in its reaction anyway, and f254 isnt a cannabinoid or phenolic or similar so it shouldnt react. It will be another experiment though! cool
 

dongle

Member
Oh yeah, I did note that BB should be stored in the freezer. The packaging didn't make any note of storage temps, although other Sigma products are typically labeled with storage temperature directions.

I'm twisting the cap tight, and then storing inside a mason jar just in case for double moisture protection. I also saw GO Joes recommendation to allow such substances to come to room temp first before opening.

Tomorrow I'll probably throw some rice grains in the outer mason just to be sure
 

PhenoMenal

Hairdresser
Veteran
ideally your FBB would've come packed with dry ice. Im still getting results just as vibrant today as when i first got my vial of FBB, so it seems like it'll last for many many years assuming it's kept in the freezer :) And your results WERE vibrant! so your FBB seems good :)

btw, youve probably noticed how CLUMPY FBB is? to get around that ...
  1. add the sodium hydroxide into a small jar
  2. add the FBB into the same small jar
  3. use something (i use the end of the micro-spatula i use to spoon the FBB in) to 'stomp down' on the clumps in the jar to break them up a bit
  4. put lid back on and give it a really good shake
  5. pour it from the jar into your main bath container through a FILTER (i actually just use coffee filter papers and they work fine with seemingly no interference)

If you don't filter the solution and leave it clumpy you get UGLY CLUMPY SPOTTY results like this... this is one of my first TLC's ... (it was my "how many scoops of FBB do i need?" experiment)

tlc-22254.jpg

btw it also shows the micro-spatula i use -- i only use about 3 tiny scoops of FBB in a 25mL bath; FBB is insanely sensitive so u dont need much. (you might've also overloaded your FBB, especially if using instructions based on FB, which is basically what all the commercial kits use)

ps. you mentioned Sigma - if you got your FBB from Sigma thats sweet! probably best quality available. but were u able to get it directly from them? i thought u need a business account?
 

dongle

Member
I actually saw that specific post, when i was skimming over earlier. (Im a little ashamed how impatient I got yesterday.)

The FBBB from Sigma has none of those solubility issues, and instantly dissolved into neutral pH'd R/O water. It was a very fine, even, brownish-red powder if I remember right.

Yes you do need a business account with Ann associated matching non-residential shipping address and all. It is absolutely true that they are not easy to buy from; I had some help from people with accounts, and it took a little extra time to relay orders and packages.

A little side story is that when I learned how to distill cannabinoids a few years ago before most people knew how it was done, I was able to social-engineer the Sigma gatekeeper into selling me a 1L Sigma Adlrich branded Kugelrohr receiving flask (which is identical to a 1L chromatography reservoir) and having it shipped to my residential address. Since then I've never been able to repeat that feat, though.
 

dongle

Member
I filled a test tube to about 50mL and threw in a tiny little "knife point" with plans of adding more as necessary, but a bright yellow liquid color and instantly dissolved with barely a shake. I'd guess say no more than 0.15g or so, but didn't weigh it.

Poured that into a dish to dip
 

PhenoMenal

Hairdresser
Veteran
(btw i posted this msg before i saw your 50ml message). 50mL MIGHT be a lot, depending on your setup .... i mean i only use 1mL hexane to extract in each eppendorf tube, and 12mL eluent (eg 12mL chloroform), and 25mL sodium hydroxide bath for the Fast Blue.

but very cool! youve probably got the best Fast Blue available if it's from Sigma.

But it would be sad to ruin the best Fast Blue with poor TLC plates though (eg homemade), so even though it's awesome you're making your own plates I'm glad you've got some professionally made plates to also compare against!

Anyway fingers crossed! :)

do some EXPERIMENTS across some plates with differing parameters, to zero in on your ideal setup
 

dongle

Member
Professionally made plates won't be here till Friday afternoon, so I might have another chance to mess with it today with more diluted samples to see where exactly the error was from yesterday.

I'm probably gonna be buried in work after today (been kinda waiting on more materials from Sigma etc, expected in this afternoon), so last real chance to dick around. If I don't get to experiment today, I might not get another chance for another month+

Ill report back
 

dongle

Member
I got a little impatient this morning.

Made some half-assed gypsum binder aluminum plates along with aome traditional 75mm glass ones. Wish I had some polymeric binder on hand.

picture.php


picture.php


The larger ones are aluminum, approx 5cm x 10cm
 

dongle

Member
I guess I kinda neglected this facet of the internet today. Simultaneously updating multiple sites at the same time is a little bit tricky and tedious.

Anyways...

Had a helluva time this morning trying to put an end to the streaking. Diluted the sample down, diluted more and more and more, but still got streaks. Felt like it was closer to 30-40:1 than 10:1.

Also, the solvent front never took more than 5 minutes to reach the top on a full 10 CM plate.

In hindsight, I'm starting to wonder if I used way too much gypsum (calcium sulfate hemihydrate) binder.

At the 10 to 15% gypsum ratio as recommended by various recipes, I had a bit of trouble getting it to stay put on a glass plate after the aqueous visualization dye dip. Even in the chloroform development chamber, I had some issues with parts flaking off if I wasn't extremely gentle in handling the plate.

So I upped the gypsum to 25% and that problem mainly went away, and didn't think much of it since. Now I'm starting to wonder if it's largely the gypsum particles that the compounds are "sticking" to? Doesn't make a *too* much sense since I would expect some streaking at 10-15% as well.

I tried using smaller spots as well... instead of trying to drop it onto the plate, I pulled up an even smaller droplet via capillary action that couldn't be dropped, and touched the plate with it.

I guess I'll know the answer to this in less than 24h, but it's gonna bother me a lot til then.
 
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