DIY Thin Layer Chromatography (TLC) of cannabinoids at home - tutorial

SSR

SSR

Well-Known Member
Quick questions for you PhenoMenal

Have you tried HPTLC plates?
Would they provide a better resolution given the smaller particle sizes and thinner deposition layer?

I know they cost a bit more but tbf sigma has them at relatively good prices comparatively speaking (around 25-30 per plate in 10 packs depending on substrate and coating reqd).

Benefits seem to be better resolving power, faster development times, lower sample diffusion, and reduced solvent consumption
 
PhenoMenal

PhenoMenal

Well-Known Member
I've never tried HPTLC plates so i can't comment sorry! I'm not sure how much more advantageous they'd be though in regards to asking the basic question "which cannabinoids does this strain have, and in which (approximate) ratios", so the extra price not be worth it. It'd be awesome to hear from somebody trying them though! btw in case you're not aware, unfortunately Sigma doesn't sell to the general public.
 
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Apical Bud

Well-Known Member
Hey PhenoMenal, over the past year I have tried TLC off and on but I have a simple problem. My cannabinoids bleed. I have extracted with acetone, ethyl acetate, hexane - you name it. And although some eluents have worked better than others, even with chloroform my plates produced a line of large splotches rather than dots. I have never gotten a plate that looks as neat as those in your pictures. What do you think I'm doing wrong?
I've made plates out of corn starch, plaster of paris, alumina; I've bought alumina plates, and I've used lab-grade HPTLC plates (yes they are much much better). I have yet to get a trustworthy result.

I have pictures if you want them. Thanks for reading.
 
PhenoMenal

PhenoMenal

Well-Known Member
Apical Bud said:
my plates produced a line of large splotches rather than dots. I have never gotten a plate that looks as neat as those in your pictures. What do you think I'm doing wrong? I have pictures if you want them
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my first guess is you're not filtering ... i got splotchy results before i started using coffee filter papers (Fast Blue BB can be quite clumpy). I'd also avoid using your own homemade plates until you get things sorted out using commercial plates, to ensure the plate isn't the problem (i personally wouldn't bother using homemade plates). But yes you'll need to post pictures/scans if you want any more debugging help with that.

Here's one of my first runs before i started filtering... so splotchy:
 
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Apical Bud

Well-Known Member
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PhenoMenal said:
my first guess is you're not filtering ... i got splotchy results before i started using coffee filter papers (Fast Blue BB can be quite clumpy). I'd also avoid using your own homemade plates until you get things sorted out using commercial plates, to ensure the plate isn't the problem (i personally wouldn't bother using homemade plates). But yes you'll need to post pictures/scans if you want any more debugging help with that.

Here's one of my first runs before i started filtering... so splotchy:
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Top is from a fancy smancy lab plate from long ago. I only posted this to show the cripsness they can provide. The other two were run recently on alumina-based plates from amazon. As you can see, the fancy plates under UV fluoresce very well.

But my main question is about the lower two pictures. Even though I have no indicator on the plates, you can see that there is a ton of smearing. Do you see something similar before you add your indicator?

BTW, I don't have the black light that I used in the top picture anymore, so I used a UV-C germicidal lamp and it doesn't seem to fluoresce cannabinoids. I don't have access to fast black k, because I don't work at a lab anymore so I can't order it from chemical companies. I can buy another cheap uv light on amazon but it won't do any good if I can't get separation. The thing is I'm not using homemade plates anymore, they just aren't the high grade lab plates we were talking about earlier.

Also, I did send in the sample I used in the first lane to a local lab and it came back 7.7% CBD, 4.3% THC.
 

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PhenoMenal

PhenoMenal

Well-Known Member
doesnt look too bad, you might be applying a bit too much on each spot though. what eluent are you using? i get quite different separation from chloroform than i do from diethyl ether & hexane mix
 
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Apical Bud

Well-Known Member
PhenoMenal said:
doesnt look too bad, you might be applying a bit too much on each spot though. what eluent are you using? i get quite different separation from chloroform than i do from diethyl ether & hexane mix
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I used chloroform in the lower two pictures. Also, in my last post I meant to say Fast Blue B, not Fast Black K. Fast Black K is toxic and doesn't differentiate between cannabinoids.
 
PhenoMenal

PhenoMenal

Well-Known Member
Fast Blue B is also toxic -- apparently they created Fast Blue BB because of that. BB is still toxic though, just not as much.

I like the separation chloroform gives, seems to be better than hexane/diethyl ether, and has the simplicity of only needing the one chemical.
 
febisfebi

febisfebi

Well-Known Member
PhenoMenal said:
just a few :) will do my best to answer them


I'm not sure if they'd work... I would assume they would, but the description has this part which I find confusing: "Activity: 3 color separated (BI-methy1 yellow,sultan red and indigo) "
To me it suggests they may be pre-treated, but i'm not sure.
I'd keep searching.


There are two main ways to view the results of TLC plates -- UV light, or provide your own 'light' in the form of a stain (like Fast Blue BB). I'm not sure if that 3-color-activity thing is in relation to UV or not sorry, but it's not the sort of thing you find in descriptions of regular plates. When you buy plates they should state if theyre UV or regular.


I just contacted a local lab supplies company and asked "do you have a small bottle of chloroform suitable for Thin Layer Chromatography?"


No, it's because I didn't get chloroform until later on. I actually prefer just using chloroform, because it's easier than measuring and dealing with 2 liquids, plus you can then avoid the horrid nasal assault of diethyl ether. Having said that though, it is awesome to have TWO different separation methods (chloroform, and diethyl+hexane) because they both help make each others results clearer to interpret.


Yes i think i paid about $40 for half a litre of chloroform.


You'll go through quite a bit of hexane because you also use that for the extraction phase (which is the most expensive phase for eluents), but yes hardly any diethyl ether is used. I only say "250mL - 1L" because those are generally the smallest sizes available.


it's called "Hexane Fraction (AR)". i'm not sure if n-hexane is different to hexane sorry. If youre not sure just ask your lab supply company for "hexane suitable for Thin Layer Chromatography". I don't know which other eluents are suitable for the extraction phase, but hexane works awesomely.


correct. The original recipe for Beam's test asks for pure ethanol, but it turns out that the ~2-5% of adulterants added to methylated spirits (simply to prevent us drinking it) doesn't seem to interfere with or affect the result of Beam's test. If this is indeed holds true (as it seems to be) it means Beam's test is an incredibly inexpensive and accessible test anyone can do at home.


be smart about the keywords you search for. It's out there. Easiest way might actually be to simply ask your local lab suppliers to see if they can source it for you - worked for me. I think i paid about $250ish for 5gm, which is actually quite a lot because you only use a few pinheads worth per test (which is good for a few plates). Just keep in mind you should be receiving it packed in dry ice, and will need to keep it in your freezer.


Best of luck, and please share anything you learn :) WE NEED MORE PEOPLE MAKING THIS ACCESSIBLE!
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OK so first off, thank you so much for all your help. I was able to perform the Beam's test, and I am just now finding the time to get the rest of the chemicals together.
I wanted to check with you that the molecular formula and CAS numbers are both the correct product for the following 3 chemicals from my supplier:

Fast blue BB Salt
CAS#: 5486-84-0
Formula: C34H36Cl4N6O6Zn

Chloroform
CAS#: 67-66-3
Formula: CHCl3

Hexane
CAS#: 110-54-3
Formula: CH3(CH2)4CH3
From chemical supplier's website

CAS# 110-54-3 ----same as first one
Formula: C6H14

I am guessing they are likely technicaly the same formula, since the CAS # is the same. The first one just broken down more.

Hexane is the particularly confusing one because there is all different products containing hexane. The second one is the one I am looking at getting, and is from ebay, and advertised as 99.99% reagent grade.

The first molecular formula was pulled from a lab supply that is much more expensive but also reagent grade.

If you could please check this information against the products you have obtained that you have proven work well, I would really appreciate it before I invest in chemicals. Hopefully the info will help somebody else as well.
Thank you!
 
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PhenoMenal

PhenoMenal

Well-Known Member
the bottle of Hexane i used is "Hexane Fraction (AR)" (analytical reagent).
The formula on the side says: C6 H14
(I simply contacted a local lab supplier and said "i need a small bottle of Hexane for Thin Layer Chromatographyy", to ensure I was getting a suitable type ... basically you need a high purity one like AR grade - something over 98%)

the bottle of Chloroform i got is 99.8% pure. Again i simply contacted a local lab supplier and said "i need a small bottle of Chloroform suitable for Thin Layer Chromatography"
the formula on the side says: C H Cl3 (the same as yours)

Diethyl Ether isnt required if you have Chloroform, but it does make for another great eluent when mixed with Hexane - it provides a different separation.
It's a seriously hardcore nose assaulter though. You really need to hold your breath when using it. Hexane and Chloroform are quite mild by comparison. Seriously though, for first time TLC i would recommend just using Chloroform instead of Hexane : Diethyl Ether
Mine is 99.5% pure, the formula says: (C2 H5)2 O

the Fast Blue BB Salt (the magic ingredient) i got i also got from a local lab supplier who imported it for me from from an Asian manufacturer called "Wako". It's called "Fast Blue BB Salt". It should be delivered to you packed with dry ice - keep it in your freezer. Mine seems to work just as well today as when I got it two years ago, so yes it does seem to keep well in a regular home freezer, and I get the sense my tiny vial will successfully last many years.
Formula C17 H18 CIN3 O3
search for "062-05463" or "CI 37175"
There are 2 different forms of Fast Blue BB i'm aware of, make sure you get the right one. It's a yellow salt. I'm not confident that the one you're referring to is the correct one.

But really, the best advice I can give you is simply contact your local lab suppliers and ask them to point you towards exactly what you need :) (worked perfectly first time for me!)
Due to the nature of chemicals it's probably a good thing to emphasize you only want a SMALL bottle (which is all that's needed anyway), and that its purpose is simply Thin Layer Chromatography. It actually was that simple for me.
 
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S

shn3ak

Active Member
PhenoMenal said:
It's only "over some people's heads" for those people who don't understand the "childrens TLC" example with the ink pen :) .... It really is VERY SIMPLE. I'm just worried I've scared people off by the length of my thread by going into detail etc. Though, I thought I was concise enough with Beam's Test! :)


That is very cool/fascinating mate! ppl use those quick Marquis tests etc, but sadly not much TLC with MDMA :( - but it provides a SEPARATION of the constituent molecules, so yes - you should get a MUCH better picture about any ecstasy tablet/MDMA etc via TLC than via Marquis etc :)
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This was an amazing thread mate! thanks very much for posting, will definitely give this a go! and the level of detail you've gone into is nuts. Many thanks!
 
PhenoMenal

PhenoMenal

Well-Known Member
Saffasteve said:
Have you had any comparison tests done at a lab to see how accurate this method is ?
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Thin Layer Chromatography (TLC) is not specific to cannabis, it's a scientific procedure with a LOOONG history, and I encourage you to look into it which will also answer your question.
Wikipedia: https://en.wikipedia.org/wiki/Thin-layer_chromatography
(as a simple example, Thin Layer Chromatography can be used to show children how black ink in a pen is actually comprised of multiple inks of different color)
 
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Saffasteve

Saffasteve

Well-Known Member
PhenoMenal said:
Thin Layer Chromatography (TLC) is not specific to cannabis, it's a scientific procedure with a LOOONG history, and I encourage you to look into it which will also answer your question.
Wikipedia: https://en.wikipedia.org/wiki/Thin-layer_chromatography
(as a simple example, Thin Layer Chromatography can be used to show children how black ink in a pen is actually comprised of multiple inks of different color)
Click to expand...
How was that link supposed to answer my question ?
 
C

cannabisgenetics

Member
f
PhenoMenal said:
the bottle of Chloroform i got is 99.8% pure. Again i simply contacted a local lab supplier and said "i need a small bottle of Chloroform suitable for Thin Layer Chromatography"
the formula on the side says: C H Cl3 (the same as yours)
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Cannot thank you enough PHENOMENAL!!!! I read about TLC in an attempt to circumvent the expensive labs in 2012 but once i saw the process, I was quick to write it off as to complicated. This thread demistified it for me and now I feel like this is one of the most valuable tools one could have. I've been testing young genotypes leaf samples (dont have space to allow for mature selections) with chloroform about 3 sets of tests so far and feel like I have the hang of it but wanted to see if you had any tips for me to fine tune.

When testing leaf samples (plants in veg), where is the most potent part of the plant? Pieces to avoid? (stem, leaf tips, leaf base, petiole,etc.)

If you are to run one lane per plant, what is the most informative? Seems like a cold print provides a smaller area to interpert and so I have gravitated to the 4ul hot print - thinking largest spots to identify (more likely small pressences will be noticed?)

Is there a option thats better at showing potency, seems like my veg samples are similiar in potency and just showing a precense of Cbs not really quantifiable. I would love to know which candidates have to highest potential (i know you prefaced with this notion, but was hoping if i can presicion everything (exact wieght, same part of plant, extraction solvent amount - i have a 2ul and a 2ml pipette) What is the most important measurement (or step) to insure accuracy accross the board / higher likelyhood of identifying higher potency varitals?

What are modulators and how do you interpret them? What info do they add?

Occasionally i see a spot at the top above the major group, is this geranyal? What does that usually indicate about potential.

In an effort to start to create some baseline, i attempted to use the Justquantify site to set some real numbers. Am i doing this correctly? I had to redraw some spots because the auto feature was grouping some Cbs together. Is there any other spots/ data on the test i should be quantifying? Purplish spots at top? Purplish spots on bottom? In this 4ul hot test, should i be trying to quantify the acid chain at all?
Here is a test of 3 females and 1 male of the progeny of a high thcv mother and thc father:

THCV_small.jpg

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Green - CBD
Red - THC
Yellow - THCV
Pink - CBG
Blue - CBC

Again Phenomenal, you are the f*ckn man! If you ever come to cali, i will roll you the biggest fatty you every wittnessed ;)
 
SmokingCrow

SmokingCrow

Active Member
For similar health reasons, I'm specifically looking at CBD levels. I was about to do down the "beam's test" wormhole and expect TLC results. Although TLC and the process look complex, this thread has made it really simple. Now all I need to do is get the chemistry set together and do it.

Thank you @PhenoMenal
 
PhenoMenal

PhenoMenal

Well-Known Member
cannabisgenetics said:
I was quick to write it off as to complicated. This thread demistified it for me and now I feel like this is one of the most valuable tools one could have.
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Yep, I'm not aware of a more powerful tool that's available for average home users. The process itself as you've found out is also quite simple and doesn't take very long either. (I mean you don't have to get all the items yourself if you dont want as it's also available in KIT FORM specifically for home users afterall!)
ps. how good is that feeling when you lift your first ever TLC plate out of the Fast Blue bath and see the colored spots developing lol :) jaw-dropper

If you're after some more tips, check out this thread where I was originally carving out the protocol, lots of trial and error - there's a lot of reading, but I guarantee you it'll take less than the ~3 months it took me: https://www.icmag.com/ic/showthread.php?threadid=148375

btw keep in mind im using Fast Blue BB whereas you're using Fast Blue B (i can tell as B is darker, more pastel-like and less-vivid), so keep in mind your results will be slightly different to mine. but they both seem to get the same job done beautifully! but seeing as you're using Fast Blue B, if i were you I'd be inclined to go by alpha-CAT's user manual in regards to which cannabinoids are which, as they also use Fast Blue B. I cant recall which eluent they use though - probably chloroform being one-part. This is an image from their website:


And yes my understanding is that the topmost lightly purple spot are probably waxes like geranyl, whereas down the very bottom are unconverted/carboxylated acids (THCA, CBDA, CBGA, etc)
 
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PhenoMenal

PhenoMenal

Well-Known Member
SmokingCrow said:
For similar health reasons, I'm specifically looking at CBD levels. I was about to do down the "beam's test" wormhole and expect TLC results. Although TLC and the process look complex, this thread has made it really simple. Now all I need to do is get the chemistry set together and do it. Thank you @PhenoMenal
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:) btw just in case you haven't seen it, check out this thread where i use TLC on 3-week-old-seedlings to check for CBD:

CBD Blitz: Find a high-CBD low-THC mother, from seed, in only 3 weeks

In just 3 weeks, you can go from ungerminated seeds to finding a mother with the cannabinoid profile of your desire (high CBD/low THC for example). Allow me to explain ... It can be incredibly disheartening to spend several months growing a plant that was advertised as "high CBD low THC" only...
rollitup.org
 
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