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

Discussion in 'Advanced Marijuana Cultivation' started by PhenoMenal, Nov 16, 2017.

  1.  
    PhenoMenal

    PhenoMenal Well-Known Member

    I don't think soap makes too many other peoples boots tremble either mate :) Soap isn't KOH though.
    KOH obviously isn't crazy dangerous (it's no dramas), but it still has properties that need to be respected (just normal common sense) :)

    KOH Safety Data Sheet:
    http://www.inchem.org/documents/icsc/icsc/eics0357.htm
    snippet:
    KOH pH = 11

    And worth rementioning, always add KOH to the ethanol -- never pour ethanol on KOH

    Just buy it mate, no need to make it especially as it's cheap :)
     
    Last edited: Feb 4, 2018
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  2.  
    raggyb

    raggyb Active Member

    Yeah I saw some of these warnings, including "slowly dissolves glass" LOL. It's good that even though we're potheads we're not so "dumb" we would not have a safety moment. Be careful with chemicals, folks! Shit I think with that tablet form you have some dangers to worry about too. If you spilled water in the tablets you would have some serious problems. Don't store any of this shit near acids, nutrients, paint thinner..etcetera. But hey, check it out, I got some results to share!
     
  3.  
    raggyb

    raggyb Active Member

    Did the Bert's test. Pretty easy! Tested Afghan, Cannatonic and Candida. Mixed info on the Afghan on the net, but saw a lab test showing 0.0%CBD. Other info says it does have CBD but doesn't say how much. THC though is HIGH. Cannatonic is known as having like 6.5%THC and 6.5% CBD, but some claim it's inconsistent. Candida is claimed to be below 1% CBD and has 2 phenos one which is 10%CBD and one is 20%CBD.
     
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  4.  
    raggyb

    raggyb Active Member

    Test 1,
    setb.JPG
    2 mins in, though I didn't mean to put this much solution in, see a little more yellow on #3
    2mins in.JPG
    after 5 mins and stirred,
    afterstirringFlash.JPG
    Repeated the test with more sample less solution,
    take2.JPG
    Afghan,
    Afgan.JPG
    Cannatonic
    Cannatonic.JPG
    Candida
    Candida.JPG
    Fuckin A, results are what you would expect! Love this shit! Thanks Phenomenal!
     
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  5.  
    PhenoMenal

    PhenoMenal Well-Known Member

    yeah thats a LOT of solution! i only use about 10ml - third a standard drinking shot, just enough to cover 1-2 peas worth of plant matter. You should find with CBD strains it will then turn reddish instead of yellow. But yep as you now know it's a very simple and pretty reliable test! Nice addition to any toolkit
    ps. Beam's not Bert's lol
     
    Last edited: Feb 5, 2018
  6.  
    PhenoMenal

    PhenoMenal Well-Known Member

    btw here is a good video of Beam's test, it's of a commercial version of the product tho (i'm not endorsing it one way or the other), but it literally is just the Beam's Test you did and shows how easy it is, how little solution you need (the eppendorf tubes he uses are perfect, i use them for Thin Layer Chromatography extractions also), and the resulting color


    I took these screenshots from it and added labels for clarity:
    [​IMG]

    You can see by the small amount used that your first tests were VERY DILUTED by comparison

    [begin pure speculation] btw, the solution as you know is clear like water initially, but its still common to get a mild yellow tinge ... so I have another question - what causes the yellow tinge?

    i don't think that's a reaction to THC, one because Beam's test "doesn't detect THC", but also because if there was a change i would imagine it would be far more strong/deeper color result seeing as THC is usually present in high levels. So I wonder if it's maybe reacting to one of the lessers like CBG, CBC or CBN (it is not a purely-CBD test, and just from what I've read it seems maybe alkalinity might be a part of the key). Would be interesting to see the result from a no-CBD/no-THC hemp variety but i dont have one. I'll have to do some Beam's tests alongside my TLC's from now on, might be able to isolate what's going on [end of pure speculation]

    Can anyone with a chemistry background please shed some light on Beam's Test?
    (((Potassium hydroxide + Ethanol) ... add CBD-heavy-cannabis) ... = colour change)

    Sadly there is very little info currently available on the web about this, and most international laws prohibit such experiments (due to the apparent "illegal material" that is medical marijuana)

    Cannabis illegality is literally the only thing stopping Beam's Test, because the chems required to do the test (ethanol or potassium hydroxide) are not controlled - and, they're both easily available on ebay etc.
     
    Last edited: Feb 10, 2018
  7.  
    PhenoMenal

    PhenoMenal Well-Known Member

    Thin Layer Chromatography to detect Zika and Dengue virii?

    I have to momentarily hijack this TLC thread from detecting cannabinoids to detecting virii ...

    The amazing breakthrough CRISPR genetic engineering technology ... or specifically, a NEW USE for it ... a DETECTION method, that instantly reminded me of TLC ..... and it seems that might actually be the case.

    https://www.engadget.com/2018/02/15/crispr-detect-hpv-and-zika/

    The paper strip part also suggests to me that it's not just chromatography, but possibly Thin Layer Chromatography???

    And if you watch the first part of the image especially, you will notice CAPILLARY ACTION driving the base liquid up the strips... just as what happens in TLC.

    The animated gif image they released reminded me a lot of Thin Layer Chromatography!
    (DISCLAIMER: I have NO idea what technique they're using).

    Anyway, whatever it is, i find it a very beautiful advancement by our smartest brothers and sisters

    [​IMG]
    Notice the CAPILLARY ACTION, and im guessing silica-based strips...

    The $64,000 question -- IS THIS THIN LAYER CHROMATOGRAPHY? :)
    (i'm not in a position to say, but it has many similar properties!)

    IF this is TLC to detect the viruses, you can clearly tell there's something very "funky" at the base of their TLC plates/strips lol... the green/purple part ... which looks very "custom-made".

    If it is TLC they will i'm assuming need a 'dye' to reveal any non-visible components (just as we need to use Fast Blue B or BB to reveal cannabinoids), so im guessing that green and purple patch is the dye ... fascinating physical structure though! or perhaps its some biomatter

    It seems the blue line highlighted in a red circle in the "Control" strip is where the "sample" was positioned on the plate (our equivalent of putting a small dot of cannabinoids in hexane at the bottom of a TLC plate)
     
    Last edited: Feb 17, 2018
  8.  
    ANC

    ANC Well-Known Member

    Geez, yeah I left a bottle of mimosa bark basified in caustic soda... it ate a hole through the bottle and made a big mess.
     
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  9.  
    raggyb

    raggyb Active Member

    Must be bases then that dissolve glass. The KoH is like pH 14.
     
  10.  
    PhenoMenal

    PhenoMenal Well-Known Member

    the plastic eppendorf tubes i use are polypropylene, they handle hexane for TLC no problems. I've only tried Beam's Test once so far (well, two samples) and used glass shotglasses, and that was fine, though I then let it evaporate in the sun so it didnt have much time to do anything to the glass ... I think I'll keep using shotglasses for Beam's, it's just a bit easier to work with having the wider mouth, but I guess i'll have to ensure I don't leave it in the glass overnight!

    i'm not sure, but i know hydrofluoric acid also quickly dissolves glass. Potassium hydroxide solution MSDS = "Slowly dissolves glass"
     
    Last edited: Feb 21, 2018
  11.  
    PhenoMenal

    PhenoMenal Well-Known Member

    In this update of Me, Beam & TLC ...
    • I make a TLC mistake ... there's a weird twist.
    • I test "CBD Therapy" for the first time... i got a ~1:1 pheno.
    • I test "Dinamed CBD" again (a sativa-dom pheno this time)... and it's not good news.
    • I test to see how early in the veg and flowering periods we can see the cannabinoid profile... never been done before, but it's very good news.
    • I do Beam's Test alongside TLC for comparison ... never been done before.
    • I still lament that more people aren't doing TLC ... i'm just talking to myself here!

    Well, finally I've harvested and tested my latest grow ... this is my first grow and harvest from under [quantum boards], and I couldn't be happier! The efficiency and energy/money savings, the spectrum, the depth penetration, the passive-cooling, the dimmability ... loving it :)

    For this grow I grew a "CBD Therapy" by CBD Crew, and a "Dinamed CBD" by Dinafem. I can only grow 2 plants at a time. I was growing them for a family member with terminal cancer, but sadly they lost their battle over a month ago. There's naturally been a "what's the point" combined with an "i failed" kinda feel to the rest of this grow. However, before they passed away they were really enjoying a Dinamed CBD (which turned out 1:1) from the previous harvest, so I take some solace in that.

    Dinamed CBD by Dinafem ...
    Although Dinamed is advertised as 20:1, the first one I grew turned out to be a 1:1 CBD-to-THC. Also, the one I grew was indica-dominant but it's supposed to be sativa-dominant, so I figured "I didn't get the main sativa pheno but I still got a nice 1:1, so if I grow again and get a sativa pheno it should be even better". That's why I grew a second Dinamed CBD for this latest harvest, and was happy to get a very-sativa-dom pheno.

    Unfortunately and frustratingly, to my surprise this sativa-dom pheno has NO CBD. Now, I know and agree that 2 plants is not a good representation of a strain, but I've had both indica-dom and sativa-dom phenos now, one was 1:1 and the other was zero CBD ... not the 20:1 they claim, nor the stability they claim, and to have one completely CBD-free was especially disappointing, particularly given the timing re our desperate medical situation/race vs cancer against time.

    I doubt I will ever grow my remaining Dinamed CBD seeds, especially as there are still a few other CBD strains i'm still yet to try, and although I've never grown any other seeds by Dinafem before it does leave a sour feeling about them, especially as they responded to my email a few months ago about the 1:1 TLC result basically calling me a liar, even though I clarified that a cancer patient had good success with it. They are also conspicuously absent of any test results, whereas if you're proud of your CBD strain surely you'd want to make such data easily available, to encourage buyers. Maybe they just didn't like me testing their strain.

    CBD Therapy by CBD Crew ...
    CBD Crew, to their credit, have been open about the stability of their CBD Therapy offering. Also to their credit they do publish TEST RESULTS. CBD Therapy is their main attempt at "high CBD, low/no THC", whereas most of their other CBD strains are mor

    I bought my CBD Therapy seeds about a year ago, and it was clear at the time that they haven't mastered stability yet -- but at least they're open and honest about that. Theirs does however seem stable enough to always produce CBD. I wish them success with further stabilisation.

    From https://cbdcrew.org/varieties/cbd-therapy/ ...
    TLC indeed shows that mine is approximately 1:1 but slightly more THC ... probably something like 8% CBD 11% THC. It started out looking very very indica with very fat leaves early on, but ended up looking only slightly indica-dom, and looked a bit unconventional/mongrel.

    It also has very healthy levels of CBG and CBC, I would anticipate it will be a very good medicinal smoke -- it's a sexier-looking spectrum than the boring-looking spectrums produced by hemp-based strains (CBD Therapy is not hemp-based). It didn't yield much though, and was more a bit more sensitive to nutes than the Dinamed growing alongside it in identical conditions.

    Solving a weird problem...
    With my last TLC a few months ago i posted that one of my plates came up with a perplexing problem I still have no understanding about ...
    [​IMG]

    The eluent did rise all the way to the top, yet failed to move the sample spots up the plate. I've never seen this before.

    But now in my latest TLC I had a seemingly related problem, but with a different outcome...
    [​IMG]

    I had a problem with this plate during the mobile phase - the eluent had stopped rising, it had already taken about 70 minutes (it normally takes ~20-40), and it seemed it was almost out of eluent, so I reluctantly took the lid off and poured a little more chloroform in. Chloroform evaporates quickly so maybe the lid wasn't on tight enough. Anyway that extra chloroform was enough to finish the plate, but you can see how bad the result was.

    I'm not sure if it was just the duration (~80 mins) of the mobile phase, or the addition of more eluent that is the cause.

    Anyway I started over with another plate, and I raised my eluent level from 10 mL to 13 mL, that solved that problem.


    Q.
    How early can TLC detect the cannabinoid profile?
    A. VEG-****ING-EARLY!


    During this grow I took small samples from both Dinamed CBD and CBD Therapy, including during veg phase, simply because I was curious to see what if anything would they show in a TLC.

    I was stunned when I saw the result. Even in veg phase they produce a strong resemblance to their final spectrum.

    First let me define the sample lanes ...
    [​IMG]

    A = CBD Therapy - Veg phase, 40 days from seed
    B = Dinamed - Veg phase, 24 days from seed
    C = Dinamed - 2wks into flower phase
    D = Dinamed - 4.5wks into flower phase
    E = CBD Therapy - harvested 55 days into flower
    F = Dinamed - harvested 61 days into flower

    All are in hexane, and all were decarb'd @ 125C for 30 mins.
    Notice how small and weak the A sample is, yet the TLC still gave a good result.

    Here is Sample A (CBD Therapy @ 40 days from seed during veg phase), and Sample E (harvested) ...
    [​IMG]

    .... WHOA! The little seedling was only 40 days old from seed but already her CBD and THC profile seemed relatively established and easily detectable. (see also rows B & F - the same held true with a 24-day old)

    Another way of looking at it: Breeders don't need to wait until flowering or harvest to check for CBD levels, they can check seedlings in early veg phase.


    Q. If CBD is detectable by TLC so early, can Beam's Test also detect it that early?
    A. I don't know! But I would guess Yes!


    Anyway here are all 6 runs ...
    Again, A & B are during veg, C & D are during flower, and E & F are at harvest.
    A & E are CBD Therapy, the rest are Dinamed CBD ... which as it turned out has no CBD in this pheno.

    [​IMG]

    I've finished all the Beam's Tests also and will post them later today or tomorrow
     
  12.  
    raggyb

    raggyb Active Member

    Thanks for all your work Pheno. Very sorry to hear about your family member.
     
  13.  
    PhenoMenal

    PhenoMenal Well-Known Member

    BEAMS TEST
    Apparently only when CBD is present does Beam's Test give the RED blush ... don't let orange fool you! These tests are only ~2 minutes old -- the colour keeps enriching slightly over an hour or so, but you get the answer within a minute or so:

    [​IMG]

    Left: approx 1:1 but slightly more THC than CBD
    Mid: High level THC
    Right: Medium level THC
     
    Last edited: Apr 2, 2018
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  14.  
    raggyb

    raggyb Active Member

    Looks like the decarbed Beam's test worked better than the raw.
     
  15.  
    the podencoid

    the podencoid Active Member

    Hey @PhenoMenal - thanks so much for posting all your experiments, it's a real service. It's inspired me to take up tlc testing again after a long hiatus. I first bought an Alpha-Cat kit 5 years ago and did a few write-ups on a couple of forums but I put it aside and hoped some testing gadget would become available to make the whole process easier. I also blanched at the expense of the Alpha-Cat kits & refills.... anyway, I started to try to determine what was in these kits last year and gathered together the Fast Blue BB, chloroform, plates etc. so I could dodge the costly refills and be independent. Your thread has helped put all the pieces of the jigsaw together and I've had some really successful results recently. So, thanks again! your work is valuable....

    One question: could you give me any tips on testing oil/FECO? I've yet to do this but it would be handy.

    Cheers.
     

    Attached Files:

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  16.  
    PhenoMenal

    PhenoMenal Well-Known Member

    Great!!! i wish there were more TLC'ers and my hope from creating this thread was to stir curiosity, because it's such a fantastic tool, especially for CBD hunters - which includes a lot of medical ppl, and was the reason I had to learn it. I'm still just a student though.

    nice looking TLC runs btw! #3 especially looks a beauty (taking nothing away though from the beautiful 1:1's in #1 & #2!)

    It was indeed a complete JIGSAW trying to figure it all out and took several months to figure out with the help of a couple others. Anyway now the process is documented hopefully it'll make things easier for others to get started. :)

    I've tested oil and paste, no special method required, but because it's concentrated you dont need as much compared to plant matter - just a drop or two of CBD oil in a 0.3mL eppendorf tube half filled (if that) with hexane or chloroform etc. Which is handy because when it comes to oil you don't want to lose too much to testing!
     
    Last edited: Apr 7, 2018
  17.  
    the podencoid

    the podencoid Active Member

    Perfect, thanks ;) I was intimidated by the chemistry and fiddliness to begin with but find it fairly easy and routine now, I hope others have a go too.

    Just fyi, #3 is a Z7 a friend in Ireland found when we shared a packet years ago - now released as 'Therapy' from Shantibaba/CBD Crew. Shantibaba's plants always have a nice terpene profile. The first two are Swamp Serum from Greenpoint and RUNCBD from Twisty Treat - both great plants, especially the RUNCBD's and no high THC in them so far. The last one is Charlotte's Cream from Twisty but all four I grew out were high thc unfortunately.

    Thanks for the tip on the oil, I'll give it a go asap!

    Cheers.
     
  18.  
    PhenoMenal

    PhenoMenal Well-Known Member

    Likewise, very intimidated by the chemistry initially. I work in IT - my idea of something going bad is a software program crashing... I restart the program, and breathe again. When things go bad in chemistry shit can blow up or cause deadly gases etc! But confronted with a family member with terminal cancer, and no legal lab tests in my country, TLC seemed the only option. So I just had to figure it out. I'm just lucky it was 2017 and not 2007.

    Thankfully, as youve found out, it is fairly easy in the end when all the steps are known! So now there's no excuse -- if people want to try TLC and without having to buy expensive kits, they can. :)

    Sweet!!! seven degrees of Kevin Bacon eh!? ... from your friend finding it, to Shantibaba breeding with it, to me getting a pack

    well that was my first CBD Therapy ive popped, three or four seeds left i think. I got a ~1:1 as you can see from prev TLC image (probably slightly more THC though, 4:5 or something), so hopefully i'll find something CBD-dom in the remaining seeds. It's interesting how common ~1:1 seems to be in the various CBD strains ... still a very nice medicinal result though! But I think we all need a high CBD low THC in the medicine cabinet, so that's what Im hunting for now.

    Anyway now that I know i can detect the cannabinoid profile only ~3wks from seed I'll soon be doing a kamikaze run of about 16 seeds from CBD strains, TLC them all at 3wks and cull to the sexiest 2. I'll still have another 17 seeds from CBD strains left to test after that, but they can wait for a second run.

    Charlotte's Cream, im guessing thats Charlotte's Web x Something Cream? to bring THC back to Charlotte i guess lol

    oh and don't use too much extraction solvent if your sample is only a single drop of oil! you only end up putting ~1-4uL on the plate anyway, so too much solvent is just an unnecessary waste and makes the result more diluted
     
  19.  
    the podencoid

    the podencoid Active Member

    Hey PhenoMenal - thanks for the reply. Yes, definitely, I'm still hunting a minimal thc, high cbd mother plant too. So useful, especially as quite a few protocols are suggesting thc and cbd be taken in separate doses. Currently trying Sweet Seeds Pure CBD plus some Pennywise, Zion's Cure, Treasure Island and a Treasure Island x Otto#1 cross I made a couple of years ago. I'll keep you updated. With this testing working out so well I have now been digging out some older beans I had stashed away like the above. So, thanks again for your helpful posts.

    Hope your family member is doing ok.

    Ah! I didn't mean to mislead about the Z7, I meant that Shantibaba was selling Z7 beans from the Mr.Nice website years ago (maybe still does) which were the building block for Therapy and a few other strains, I didn't mean to suggest this was *the* plant we found and that Shanti developed it from. Don't want top start any further myths.

    Haven't tested the oil I made at the weekend yet but hope to very shortly. Because, as you noted, high CBD plants are scarce I used some CBD Isolate to make up a higher CBD ratio. I haven't delved into this too far, it was just an experiment, but the resultant mix of 1:1 FECO with added CBD Isolate seems excellent.

    All the best.
     
  20.  
    fookinel

    fookinel Member

    There's a few things to mention here, but before getting in to that, as raggyb said, sorry to hear about your relative.

    I can see similar posts/threads at other forums and from some of the information you've provided I'm guessing you've seen some of what I'm going to say from yet other forums as well, but anyway, here are some thoughts:

    1) Great user name! Even better avatar, should we call you Poida instead?!

    2) Great information! If only it was possible to walk in to a store and get all of these chemicals etc...

    3) I believe you'll find that Beam's test is not specific to CBD. It may also have a colour change from CBG and possibly CBC. I don't see this as a significant problem in comparing results but it's worth keeping in mind.

    4) Beam's test shouldn't require potassium hydroxide, it should also work with sodium hydroxide. There may be reasons to prefer potassium hydroxide (greater solubility) or sodium hydroxide (cheaper and more common).

    5) I believe you'll find a 5 % solution is 5 % w/v which means 5 g in 100 mls of solvent (not 5 g in 95 mls of solvent). However, as you can see it's not a hard and fast rule, a bit more or a bit less should also work, the main thing is consistency in testing.

    6) You can do more than just check for CBD using Beam's test, at the very least you can get an idea about relative CBD concentrations. If you use consistent plant material/solvent ratios you can add a small quantity of the solvent (with extracted cannabinoids) to a container (like one of the Eppendorf tubes) as well as a small quantity of Beam's reagent (obviously you want to add the same quantity of solvent and reagent in each container to make them comparable). Do this for each sample you want to test and then compare the colours. As always, the darker the purple then the more CBD/CBG/CBC in the sample. If you search for "DIY test to find high CBD plants grown from seeds" (with the quotes) the first result should be from another forum, post #3 has an image illustrating this comparison.

    If you want more details it's easy to go through an example but I'll keep this short as this post will be long enough as it is.

    7) If you search for "CBD Seed Breeders: How to test for CBD or THC at home" (with the quotes) the first result should be from that same forum and describes a Duquenois test for THC using vanillin, ethanol, and hydrochloric acid.

    If you apply similar techniques then I would have thought you could combine these two tests to quickly find out if one plant has more or less of these cannabinoids than another plant.
     

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