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

[fookinel]

Okay, now for the questions:

1) What tests have you done on plants in the veg phase (the 24-40 days from seed results)? Just TLC or Beam's as well?

2) How did you do those tests? I mean how much dry plant material did you use and how much solvent (since I'm assuming you used leaves in veg which will be much lower in concentration than flowers)?

3) How confident are you that you actually grew Dinamed CBD? The reason I ask is not to be anything like the poor response you got from Dinafem, it just seems like a real statistical anomaly if everything is accurate.

What I mean is, if you really grew Dinamed CBD then there's a real problem with Dinafem's claims. They claim Dinamed CBD is derived from Dancehall. According to Reggae seeds Dancehall has a minimum of a 1:1 ratio. Dancehall is derived from Juanita la Lagrimosa which I believe Cannatonic is derived from and subsquently AC/DC and the like. There seem to be a lot of high CBD genetics that owe their origins to Juanita la Lagrimosa/Dancehall/Reggae seeds.

On top of that, Sweet Seeds say this about the Sweet Pure CBD mentioned in post #59 of this thread:

"This strain is the result of two generations of autopollination (S2) of a CBD-rich clone with ancestors from the Diesel family."

Well guess what? Juanita la Lagrimosa and Kalijah (what Dancehall is a corss between) are both derived from NYC Diesel suggesting that Sweet Pure CBD has a similar background!

BTW I've seen some complaints about CBD Therapy not standing up to the claims they made originally. The CBD Crew website states: "Now after getting a lot of great feedback from growers and medicinal users, CBD Crew noticed that not all seeds came out with very low THC, high CBD." Hopefully they've fixed the issues...

 

[PhenoMenal]

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

OI !!!!... it's ..... POIDA!!!!

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

dont need to, they're all legal and easily available at ebay

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.

yes, it does react to several cannabinoids, not THC though ... calling it a "CBD test" is probably incorrect

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.

BEAUTIFUL INFO mate

 

[PhenoMenal]

Okay, now for the questions:
1) What tests have you done on plants in the veg phase (the 24-40 days from seed results)? Just TLC or Beam's as well?

just TLC during veg phase. I plan to try Beam's during veg in the coming weeks

2) How did you do those tests? I mean how much dry plant material did you use and how much solvent (since I'm assuming you used leaves in veg which will be much lower in concentration than flowers)?

ive provided all data in previous posts if you mean TLC, in regards to concentrations, raw/decarb'd, etc. But you can see in these eppendorf tubes how much plant matter i use (for both TLC and Beams) - just a pea or two's worth...

3) How confident are you that you actually grew Dinamed CBD? The reason I ask is not to be anything like the poor response you got from Dinafem, it just seems like a real statistical anomaly if everything is accurate.

Very fair and good question ... there are many dodgy seed sellers these days, taking advantage of "all seeds look the same", and when ppl post reports we have no idea of whether or not they got their seeds from a legit reseller. I got the seeds from reputable longterm seed vendors.

Here's the high-CBD/low-THC hunt candidates ...

[edit] bonus! - i just found out that the Candida (supposed to have 3 seeds as per the label) has 4 seeds I also got another Candida but that was a single freebie seed in a ziplock so I cant verify it's legit like these 4.

What I mean is, if you really grew Dinamed CBD then there's a real problem with Dinafem's claims. They claim Dinamed CBD is derived from Dancehall. According to Reggae seeds Dancehall has a minimum of a 1:1 ratio. Dancehall is derived from Juanita la Lagrimosa which I believe Cannatonic is derived from and subsquently AC/DC and the like. There seem to be a lot of high CBD genetics that owe their origins to Juanita la Lagrimosa/Dancehall/Reggae seeds.

They've also claimed they've tested out 5000 plants but never posted any proof of that, so I'm not sure how credible they are. I'll be growing at least 1 more Dinamed soon, so maybe 3rd time lucky.

BTW I've seen some complaints about CBD Therapy not standing up to the claims they made originally. The CBD Crew website states: "Now after getting a lot of great feedback from growers and medicinal users, CBD Crew noticed that not all seeds came out with very low THC, high CBD." Hopefully they've fixed the issues...

yes CBD Crew has been fairly honest about Therapy not being as stable as they'd like. That was a couple years ago though but seemingly they still haven't released a more stable version yet. It does sound like Therapy is at least reliable in terms of providing at least some CBD, whereas my second Dinamed had no TLC-detectable CBD, just lots of THC

 

[fookinel]

OI !!!!... it's ..... POIDA!!!!

Sweeeet mate!

dont need to, they're all legal and easily available at ebay

I just had a quick look and can't find Fast Blue BB anymore...

just TLC during veg phase. I plan to try Beam's during veg in the coming weeks

I look forward to seeing any results!

Keep in mind that with Beam's test I believe you'll find there's a reaction taking place and the colour change is dependent on the quantity of reactants. In other words, without a high enough concentration of cannabinoids and hydroxide ions in the final solution you won't be able to see the colour change.

Since the plant material in veg has a lower cannabinoid content then you'll need more plant material without increasing the amount of solvent to get a similar colour change

ive provided all data in previous posts if you mean TLC, in regards to concentrations, raw/decarb'd, etc. But you can see in these eppendorf tubes how much plant matter i use (for both TLC and Beams) - just a pea or two's worth...

So even for the TLC from veg you only needed a pinch to get those results?! I didn't realise TLC would be so sensitive!

Very fair and good question ... there are many dodgy seed sellers these days, taking advantage of "all seeds look the same", and when ppl post reports we have no idea of whether or not they got their seeds from a legit reseller. I got the seeds from reputable longterm seed vendors.

It doesn't even have to be intentional, it could always just be a mistake like someone mislabelling what they think is one strain but is actually a different one.

Here's the high-CBD/low-THC hunt candidates ...

I've haven't seen anything about Harlesin, I've seen some bad reports of Thunderstruck (I'm pretty sure it was here on RIU), Cannatonic is highly variable. Of those I'd be trying Candida next if you really want a high CBD low THC plant. They've published a lot of results you can grab from their site, spoiler alert, they're all good! They had a minimum CBD:THC ratio of 19.5:1 and up to 24.5:1 with a minimum CBD of just under 10% and a maximum THC of just under 1% with those cannabinoids generally rising and falling together.

[edit] bonus! - i just found out that the Candida (supposed to have 3 seeds as per the label) has 4 seeds I also got another Candida but that was a single freebie seed in a ziplock so I cant verify it's legit like these 4.

Did you get the 3 pack from overseas as well or did you happen to get them from a local seedbank? I've seen them available locally (they may not be in stock right now, I'm not sure) but I don't know how trustworthy that seedbank is.

They've also claimed they've tested out 5000 plants but never posted any proof of that, so I'm not sure how credible they are. I'll be growing at least 1 more Dinamed soon, so maybe 3rd time lucky.

I've never seen them claim to have tested 5000 plants. I have seen in a blog post from them that they grew about 2000 seeds of Dancehall or something derived from Dancehall which they then selected a few males/females that they thought would be best. Something like 500 seeds from those selected males/females were then used to get their so called "Pure CBD 4".

Of course, you'd think there'd be testing along the way to determine what cannabinoids are present...

I will agree on one thing, they haven't provided any evidence of their claims and I wouldn't be touching Dinafem genetics if it turns out they've been making everything up.

yes CBD Crew has been fairly honest about Therapy not being as stable as they'd like. That was a couple years ago though but seemingly they still haven't released a more stable version yet. It does sound like Therapy is at least reliable in terms of providing at least some CBD, whereas my second Dinamed had no TLC-detectable CBD, just lots of THC

The problem is that I believe they originally released CBD Therapy as having a much higher CBD:THC ratio than 1:1 yet a lot of the plants turned out closer to 1:1. However, as you say, 1:1 is much better than no CBD at all!

 

[PhenoMenal]

I just had a quick look and can't find Fast Blue BB anymore...

Oh it's definitely out there. Yes it can be a bit tricky finding it, but keep searching, also try the chemical name, also try "Add to cart" in your query, or "lab supplies" etc, and also try emailing some labs in your area/country to ask if they can get some in for you, which is what I did in the end, and they sent it to me Express and in dry ice, ready for me to put in the freezer.

"Fast Blue B" is also fine to use, though BB produces more vibrant results that dare I say are prettier. The switch to BB from B was also made due to carcinogen concerns about B, though you wanna treat BB with respect too ... should treat all dyes with respect, really!

I look forward to seeing any results!

I'll reserve you a seat for 3-4 weeks from now

Keep in mind that with Beam's test I believe you'll find there's a reaction taking place and the colour change is dependent on the quantity of reactants. In other words, without a high enough concentration of cannabinoids and hydroxide ions in the final solution you won't be able to see the colour change. Since the plant material in veg has a lower cannabinoid content then you'll need more plant material without increasing the amount of solvent to get a similar colour change

yeah the eppendorf tubes are only 0.3mL, so even with a small amount of plant matter its still fairly concentrated

So even for the TLC from veg you only needed a pinch to get those results?! I didn't realise TLC would be so sensitive!

I too was blown away by the results from veg phase, especially as i didn't use much plant matter ...
TLC is pretty fricken sensitive alright!!! ... behold ... (each lane is just 2 x 1uL drops from the pipette)

ps. no I don't bother accurately weighing the plant matter, as I'm only looking at each lane independently (not comparing lanes) ... eg im comparing the orange dot to the red dot in the same lane, im never comparing red dots from two lanes. Again, TLC is a qualitative test, it's not accurate enough for quantitative! which is why it's pointless using it to determine "%THC", even if you're super-careful about all the variables and measurements and air temperature etc. But you CAN determine approximate CBD:THC ratio.

I've haven't seen anything about Harlesin, I've seen some bad reports of Thunderstruck (I'm pretty sure it was here on RIU), Cannatonic is highly variable. Of those I'd be trying Candida next if you really want a high CBD low THC plant. They've published a lot of results you can grab from their site, spoiler alert, they're all good! They had a minimum CBD:THC ratio of 19.5:1 and up to 24.5:1 with a minimum CBD of just under 10% and a maximum THC of just under 1% with those cannabinoids generally rising and falling together.

I only have 3 (now 4 since opening the pack to discover a bonus seed) Candida seeds, so I'm only doing 2 this run, but also 5 Thunderstruck, 5 Cannatonic, 1 Dinamed, 1 Harlesin, 2 CBD Therapy and 2 Candida .... why 16? because I like to use 4 lanes per TLC plate And because it's about half of the available seeds ... I don't want to use them all straight away and end up having to throw away good ones, but at the same time I want to get through them relatively quickly. Fingers crossed they all germinate. Then I'll cull to the best 1 or so.

Did you get the 3 pack from overseas as well or did you happen to get them from a local seedbank? I've seen them available locally (they may not be in stock right now, I'm not sure) but I don't know how trustworthy that seedbank is.

all from overseas.

I've never seen them claim to have tested 5000 plants. I have seen in a blog post from them that they grew about 2000 seeds of Dancehall or something derived from Dancehall which they then selected a few males/females that they thought would be best. Something like 500 seeds from those selected males/females were then used to get their so called "Pure CBD 4".

Sorry, yes you're right, it's 2000 not 5000 ... (still a sufficient shitload though)
From https://www.dinafem.org/en/blog/dinamed-cbd-first-100-therapeutic-strain-dinafem/

To achieve this, we worked on a CBD-rich elite line based on the Dancehall strain. The first selection process was carried out with 2000 seeds, from which we selected a tiny handful of elite plants comprising the two best females and the best male. Then, after selecting 500 seeds, Pure CBD 4 came to life, the mother plant producing 12% of CBD and 0.5% of THC that we used for creating Dinamed CBD.

The strength, morphology and yield were the three aspects we mainly took into account when carrying out such processes, which came to an end after conducting several thorough analyses of the chemical composition using a gas-chromatograph that allowed us to obtain detailed information about the selected individuals and the real CBD content of every plant.

Again though I've only tried 2 Dinamed's, not enough to make any verdict from, but to see the one that didn't have any CBD at all was disheartening.

Of course, you'd think there'd be testing along the way to determine what cannabinoids are present...

One would think so! Also would've thought they'd do stability tests before release.

 

[Wilksey]

I would like to find out how EARLY in a grow can CBD be detected

I chopped some plants at about 7 weeks that turned out to be males, made butter with the leaf material, and got high as fuck. If you test modern plants, I think you'll find that they have way more THC available then we once thought, including males.

 

[PhenoMenal]

I chopped some plants at about 7 weeks that turned out to be males, made butter with the leaf material, and got high as fuck. If you test modern plants, I think you'll find that they have way more THC available then we once thought, including males.

Yep, the leaves still have plenty of cannabinoids, seemingly from the very first pair of true leaves too, just not as high in concentration compared to flowers. My relative that recently passed away actually said he just grew for the leaves because the butter from that is still strong enough! The first two TLC lanes above were just a small leaf in each

 

[fookinel]

Oh it's definitely out there. Yes it can be a bit tricky finding it, but keep searching, also try the chemical name, also try "Add to cart" in your query, or "lab supplies" etc, and also try emailing some labs in your area/country to ask if they can get some in for you, which is what I did in the end, and they sent it to me Express and in dry ice, ready for me to put in the freezer.

I was referring to it being available on eBay

I've seen it at chemical suppliers but at least some of them won't sell to an individual.

"Fast Blue B" is also fine to use, though BB produces more vibrant results that dare I say are prettier. The switch to BB from B was also made due to carcinogen concerns about B, though you wanna treat BB with respect too ... should treat all dyes with respect, really!

I believe there are other ways to visualise the results as well, such as UV which I guess isn't carcinogenic but still has some safety issues.

I too was blown away by the results from veg phase, especially as i didn't use much plant matter ...
TLC is pretty fricken sensitive alright!!! ... behold ... (each lane is just 2 x 1uL drops from the pipette)

ps. no I don't bother accurately weighing the plant matter, as I'm only looking at each lane independently (not comparing lanes) ... eg im comparing the orange dot to the red dot in the same lane, im never comparing red dots from two lanes. Again, TLC is a qualitative test, it's not accurate enough for quantitative! which is why it's pointless using it to determine "%THC", even if you're super-careful about all the variables and measurements and air temperature etc. But you CAN determine approximate CBD:THC ratio.

Since you're not going for the most accuracy (I.e. not carefully measuring plant material etc) have you tried making your own TLC plates?

Again though I've only tried 2 Dinamed's, not enough to make any verdict from, but to see the one that didn't have any CBD at all was disheartening.

Assuming they were Dinamed CBD

Just in case you haven't seen it I stumbled on another forum post regarding these tests (Beam's/Duquenois/TLC) and they might have even used Beam's on plant material in veg but I'm not too sure if they did or not as I've only looked at it briefly...

If you search for "In Home and DIY Testing your Cannabis for THC and CBD Potency" (with the quotes) it should be the first result. On page 2 post #24, possibly #28, and #32 refers to plant material in veg and then continue through to page 3 to end up with a little TLC (that sounds weird with the other things TLC can stand for...)

Unfortunately, it looks like the plant material in veg didn't show a colour change. Most likely because they needed to use a lot more plant material from veg than from the flowers.

 

[PhenoMenal]

fookinel -- "tried making my own TLC plates"? hell no! ... here's my take on that

• inaccuracies in the plate can mess with the TLC, mobile process especially, but also development phase etc
  
• home users don't need many plates, so why waste time making them
  
• we only need to use aluminum ones (not more expensive glass ones)
• they're relatively inexpensive
• we comfortably get 4 test lanes per plate

I'd recommend you keep enquiring for Fast Blue B or BB mate, it IS OUT THERE, UV won't give you the same satisfaction! (and how do you take photos or scan it!?)

 

[PhenoMenal]

TLC to detect TERPENES - just use a different dye!

We use a dye like Fast Blue BB or Fast Blue B to visualize cannabinoids ...

Simply by changing to a different dye we can also visualize terpenes too...
It would be a great way to fingerprint the pineapple of Cinderella 99, the blueberry in Blueberry, etc!

And because terpenes aren't specific to cannabis there's a lot more info out there, compared to the limited info about cannabinoid detection.

It's not something I've done yet, but the main dye people seem to use for terpenes is a vanillin reagent (store in fridge at 4C):

• 1.4 g vanillin
• 40 mL methanol
• 250 µL sulfuric acid

Clearly it's the vanillin that's the main part of the dye ... I wonder if we could just use the same sodium hydroxide solution that we use with Fast Blue BB/B (instead of the methanol and sulfuric acid?). Again I'm no chemist though, so I have no idea about that one! just thinking out loud

The plates might need to be heated briefly to 100C for the vanillin reagent to take effect, but im not 100% sure. Still easy either way.

Some examples from [biomedcentral link] ...

 

[raggyb]

TLC to detect TERPENES - just use a different dye!

We use a dye like Fast Blue BB or Fast Blue B to visualize cannabinoids ...

Simply by changing to a different dye we can also visualize terpenes too...
It would be a great way to fingerprint the pineapple of Cinderella 99, the blueberry in Blueberry, etc!

And because terpenes aren't specific to cannabis there's a lot more info out there, compared to the limited info about cannabinoid detection.

It's not something I've done yet, but the main dye people seem to use for terpenes is a vanillin reagent (store in fridge at 4C):

• 1.4 g vanillin
• 40 mL methanol
• 250 µL sulfuric acid

Clearly it's the vanillin that's the main part of the dye ... I wonder if we could just use the same sodium hydroxide solution that we use with Fast Blue BB/B (instead of the methanol and sulfuric acid?). Again I'm no chemist though, so I have no idea about that one! just thinking out loud

The plates might need to be heated briefly to 100C for the vanillin reagent to take effect, but im not 100% sure. Still easy either way.

Some examples from [biomedcentral link] ...

another cool find Phenomenal! I'm just going to have to make one joke though- smegmatis! LOL, don't want to smell that!

 

[PhenoMenal]

smegmatis! LOL, don't want to smell that!

oh the Smegmatis column lol, i'm not sure what that is ..... I thought you were referring to the vanillin reagent recipe lol -- which does sound like a good recipe for vanilla-scented rotten eggs!

 

[raggyb]

Maybe one could zero in on the elusive skunk this way.

 

[PhenoMenal]

TLC Life Hack - Good Vibrations

When waiting for the cannabinoids to leach out into the extraction solvent, the natural vibrations from the reservoir's air pump make for a great centrifuge substitute ... (eppendorf tubes stuck into polystyrene foam)...

 

[PhenoMenal]

CBD Blitz: How to find a high-CBD/low-THC, from seed, in only 3 weeks. [Rollitup Link]

 

[PhenoMenal]

i was just made aware of this "Thin Layer Chromatography" book written by E. Stahl, by G.O. Joe (who was instrumental in helping me get started with TLC), it's a 53mb free PDF

http://b-ok.xyz/book/2304503/37f9e9

 

[PhenoMenal]

btw, the correct CAS number for Fast Blue BB Salt is 5486-84-0.
(There are at least two variations of Fast Blue BB, and it seems one of them (the base, not the salt) isn't suitable for our needs as somebody else has inadvertently found out, so make sure you check the CAS number matches the above). You'll probably be offered 1gm, 5gm, and 25gm ... 5gm is what I went with - it's a vial the size of your thumb, and considering how we only use tiny microscoops it'll last a loooong time. Actually I think 1gm would easily be more than enough for 50 plates. Keep stored in your freezer.

 

[dogcat]

I finally got a chance to try out the TLC kit as a first test. I tested a blue dream at 5 weeks of veg, and some cbd oil. Basically just a test to see if I can get it to work, and sure enough, as you said, it's not all that hard. It appears I used a little too much of the dye powder when making the dye, though it was less than what was indicated in the instructions. Also, I photographed it from the wrong side. I didn't realize the results are visible cleanly from the back. Not sure what the lower cannabinoid is on the blue dream side (left side). I did the blue dream twice, once with 2 ul, and once with 4ul, as I was a bit mixed up with the instructions. No CBD in that one, but wasn't expecting it. I will pop the 3 dinameds tonight, and get on with the search.

<a href="http://www.freeimagehosting.net/commercial-photography/"><img src="https://i.imgur.com/90Us5bL.jpg" alt="Commercial Photography"></a>

<a href="http://www.freeimagehosting.net/commercial-photography/"><img src="https://i.imgur.com/q0e0OgV.jpg" alt="Commercial Photography"></a>

 

[PhenoMenal]

congrats, welcome to the party! just keep tweaking and experimenting and you'll have it dialed in in in just a few plates
ps. forums dont use HTML, you'll need to use the icons that are made available when posting for various formatting options

 

[Sour Wreck]

following.

gonna use some of this in the future