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

[dogcat]

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

thanks man, much appreciated! This thread opened my eyes to the world of TLC, and made it possible for me to participate. I can't wait to do it again on some cbd strains. I'm such a newb, I thought the plates we reusable, I wasted space on that first one. Live and learn!

By the way, any chance to explain "forums dont use HTML, you'll need to use the icons that are made available when posting for various formatting options"? Thank you

 

[PhenoMenal]

By the way, any chance to explain "forums dont use HTML, you'll need to use the icons that are made available when posting for various formatting options"? Thank you

see "BB Codes" at https://www.rollitup.org/help/
You can either manually type them, or highlight the text you want to affect and then use the various icons just above the textbox that you type these replies in

And yes the TLC plates are single-use! especially considering cannabinoids from resin are seemingly sticky, and Fast Blue is a staining dye, i'm not sure how easy or possible it would be to get that out of the silica without making an absolute dogs breakfast out of it lol

 

[dogcat]

see "BB Codes" at https://www.rollitup.org/help/
You can either manually type them, or highlight the text you want to affect and then use the various icons just above the textbox that you type these replies in

And yes the TLC plates are single-use! especially considering cannabinoids from resin are seemingly sticky, and Fast Blue is a staining dye, i'm not sure how easy or possible it would be to get that out of the silica without making an absolute dogs breakfast out of it lol

perfect, thank you

 

[guerreiroweed]

Can anybody link me with a place that sells TLC plates and ships internationally?I can find everything easily in my country, but the tlc plates are impossible to obtain, they only sell for juridic persons...

 

[PhenoMenal]

Can anybody link me with a place that sells TLC plates and ships internationally?I can find everything easily in my country, but the tlc plates are impossible to obtain, they only sell for juridic persons...

TLC plates, the ones we use in this thread, are simply silica on an aluminium sheet base, so there is no reason why they would be hard to find in your country, and theyre used for science in all countries. Your google search terms might need refining try "silica plates aluminium"
Also, simply try emailing some labs in your country.
Can probably find on ebay too.
specifically, the ones i use are German-made by Merck, "TLC Silica gel 60", aluminium, 5 x 10 cm. (no fluoro indicator)

 

[guerreiroweed]

TLC plates, the ones we use in this thread, are simply silica on an aluminium sheet base, so there is no reason why they would be hard to find in your country, and theyre used for science in all countries. Your google search terms might need refining try "silica plates aluminium"
Also, simply try emailing some labs in your country.
Can probably find on ebay too.
specifically, the ones i use are German-made by Merck, "TLC Silica gel 60", aluminium, 5 x 10 cm. (no fluoro indicator)

TLC plates, the ones we use in this thread, are simply silica on an aluminium sheet base, so there is no reason why they would be hard to find in your country, and theyre used for science in all countries. Your google search terms might need refining try "silica plates aluminium"
Also, simply try emailing some labs in your country.
Can probably find on ebay too.
specifically, the ones i use are German-made by Merck, "TLC Silica gel 60", aluminium, 5 x 10 cm. (no fluoro indicator)

I found one, pretty expensive but better than importing, there are 2 options, 5x10 with fluoro indicator and 5x20 without it, can I just get the 5x20 and cut them all in half?

 

[PhenoMenal]

yes, aluminium plates are easy to cut with a regular pair of scissors

 

[cogitech]

Love this thread!!! Ordered some KOH and going to be trying the Beam's test on some (supposedly) high CBD weed I am ready to harvest.

 

[raggyb]

Ran a Beam's test on 4 just dried strains plus Afghan which I know has no CBD. I decarbed first. I didn't decarb before but it worked both ways.

I could not get a picture that shows what it truly looks like. I ordered them from clearest to yellowest from left to right. But all you can see in the picture is that the one on the left is different from the other 4. As expected that's Afghan and it confirmed it had no CBD. The 4 have CBD and It's just my guestimate, but I believe the order of increasing CBD is Blue Dream CBD, Blueberry, Candida and my Canafdida cross. So two surprises, one being the Blueberry > Blue Dream CBD, and two Canafdida > Candida. Canafdida = (Cannatonic x Afghan) x Candida.

 

[PhenoMenal]

CBD/CBDA (Beam's doesn't seem to matter if it's decarb'd or not) gives it a red/purple tinge, perhaps there is some CBD in that Afghan afterall. TLC would confirm

 

[raggyb]

CBD/CBDA (Beam's doesn't seem to matter if it's decarb'd or not) gives it a red/purple tinge, perhaps there is some CBD in that Afghan afterall. TLC would confirm

I want to do TLC. But I don't know why my camera makes the one on the left look red, it's actually clear. The others are yellow. But I'm using grain alcohol not the other stuff you said, because I have some I don't need for anything else anyway. Maybe that makes the color indications different too.

 

[PhenoMenal]

yeah thats weird if its clear but looks so red on camera! maybe put some white paper or something behind it when you photo. btw a bottle of methylated spirits is < $5 but id guess grain alcohol would work if it's 50+% ... methylated spirits is usually 95-98% ethanol

 

[rocho]

Thankyou so much fpr sharing!!

 

[jjng5]

I think this is super interesting. Would love to try out. Any recommendations on sourcing the initial supplies? I suppose one could order from thctestkits.com and then source the replacement materials; however it seems like $159+shipping is a steep price for what you're receiving, maybe I'm wrong?

@ PhenoMental -- Am I understanding your earlier experiments, that you've been able to test a strains cannabinoid profile at merely 3 weeks into veg and reproduce nearly the same cannabinoid expression compared with your final harvest product?

I also have a question regarding testing for THC potency and a percentage range, because I believe this is being over looked. No doubt, the "thumb print" of a strain and it's various cannabinoid expression / profile has significant value... but THC potency is nothing to sneeze at either:
@ PhenoMental - you mention that there is a 10% variance in range in estimating THC thru this semi-quantitative process. On thctestkits.com they do acknowledge this 10% variance; but they describe it as a 10% margin of error in testing (which is very different than a 10% swing in THC content. They explain (which makes sense)... that ie: If a strain tested at 20% THC and one used TLC for testing then the results would be 20% THC +/- 10% = 18-22% THC range (10% of 20% = 2%; 20% - 2% = 18% & 20% + 2% = 22%).

Semi-qualitative in science and health are not bad things. An ultrasound of your heart? That's semi-qualitative too. While a cardiac cath angiogram is more "accurate", does the plus or minus 5% EF from the echocardiogram change your treatment of the patient for heart failure...? No...

Therefore, I'm assuming most cannabis users and small op growers would be thrilled with, "I test my product every grow and this exact crop showed a THC range of 18-22 % THC" as compared to "This strain tested at 19.54% THC a year or two ago and I just haven't been able to afford to re-test and wait 21 days for the results, but the product is from the same mother plant. And look I have a lab report from two years ago if you want to see it!".

What good is GC anyways if in some states they can get away with retesting once every six months? What's that percentage mean on the side of a package where the sampling size is 10mg out of every 10,000 pounds? I'd take a less precise "range" within a 10% margin of error over an exact calculation with a ridiculous sample size from a crop several generations ago. GC might be more "precise", but with it's cost/scale limitations is it more "accurate"? In this regard, they are not the same. Food for thought...

 

[OldMedUser]

As I'm planning on breeding my own CBD strains using crosses from regular plants that I like with a high CBD strain I decided to try out testing for the presence of CBD in the few plants I have going now.

0.1g from each of 5 samples of decarbed pot was soaked in 5ml of petroleum ether, (camp stove fuel), for approximately 30 min then 4ml of the pet.ether was drawn off and added to 5ml of a 5% sol'n of potassium hydroxide in 190 proof ethanol, (EverClear). The 5cc glass syringe was rinsed twice with fresh pet. ether between each sample to prevent cross-contamination.

Samples were:

I: Otto#1. Hemp bred for high CBD and less than 1% THC. Was to be my breeding girl. From seed.

II: GoldFish 'A'. Cannatonic x a different Otto#1. Grown from seed.

III: GoldFish 'B'. As above but different seed.

IV: Critical Mass CBD. Bought from Kelowna compassion club Dec. /17.

V: Critical Mass. Clone from a buddy.

Samples soaking in pet. ether.



After transferring 4ml of pet. ether sol'n to vials with 5ml 5% KOH/EtOH sol'n.



To my utter dismay, sample I, the Otto, tested negative for CBD! Glad I found out now before wasting the next year making crosses with her and get no CBD. This strain is supposed to be up to 27% CBD for crying out loud.

If how dark the colour goes indicates how high the CBD levels are then the two GoldFish are pretty close to equal with maybe the 'B' being slightly higher. That's good because it is the better yielder by twice so I'll just get clones off it and skip the 'A' plant.

The store bought Critical Mass CBD looks like the highest in CBD but it's long gone other than a gram or two.

The Critical Mass, (sample 5), I grew was there to act as a control as it shouldn't show CBD and it didn't.

Now I'm going to have to shop around for some suitable CBD breeding stock. Still have a couple of the GoldFish beans and 10 or so of it crossed with Herijuana so will get them sprouting ASAP.

Next project is to make my own silver nitrate and sodium thiosulphate so I can use them to make silver thiosulfate and use it to reverse sex on select girls and make fem seeds. Got everything I need so just need to get cooking!

 

[CannaBruh]

excellent work and thanks for sharing @OldMedUser

 

[PhenoMenal]

@ PhenoMental -- Am I understanding your earlier experiments, that you've been able to test a strains cannabinoid profile at merely 3 weeks into veg and reproduce nearly the same cannabinoid expression compared with your final harvest product?

That is correct, using Thin Layer Chromatography (TLC). I do not think Beam's test (or similar) are sensitive enough (at the insanely early age of 3wks), nor can Beam's show the approximate quantitative difference between THC and CBD, but TLC can do both, and at a VERY early age in veg. It's a game-changer, especially because anyone can do TLC at home.

It's still awesome to see new tweaks on Beam/similar though because Beam is such a simpler test by comparison -- a good Beam's test can help determine whether or not you want to even waste your time doing a TLC test. They're both important tools.

 

[febisfebi]

@PhenoMenal
Been looking around for chemicals and such to eventually do some TLC. Had a few questions.
First off I am guessing that these plates will not work?
https://www.amazon.com/gp/product/B07LBK5GL5/ref=ox_sc_act_title_2?smid=A11MUK3W6A9O7N&th=1
Would be nice as they are pretty cheap, and surprisingly glass backed.
what bothers me is this: (listed in the description)
Activity: 3 color separated (BI-methy1 yellow,sultan red and indigo)
Are these what you mean when you say UV indicators?
Silica coated and silica gel TLC plates are the same thing, yes?

Also It seems that Chloroform while providing nice looking results, is fairly difficult and expensive to source. I am curious as to where you found it, and how much you spent?
It seems like most of your tests are done with 4:1 Hexaneiethyl mix. I am assuming the cost/difficulty of obtaining chloroform is the reason?
The only place I found Chloroform, without seeking out chemical suppliers is a $65 liter which I guess isn't too bad. But since we already need Hexane, the 4:1 Diethyl Ether seems more cost effective.

I was curious why you listed the Solvent and Eluent amounts both as 250ml-1L. It seems that you would only need 2ml Diethyl per test and 9ml of Hexane, assuming you are using the 4:1 mix instead of 10ml chloroform. The reason I ask about the amounts, is it appears you only need 1ml of Hexane for solvent use. Far less than the 10ml of Eluent needed per test.
Was thinking maybe 1L Hexane, and around 125-250ml Diethyl which is fairly inexpensive in those qty's, compared to Chloroform.
Some listings on ebay/amazon are for n-hexane. A quick search tells me that this has a different boiling point than Hexane. I am guessing that n-hexane will not work for solvent or Eluent, but I figured I would ask.

As far as the Beam's test goes, you said that you use cheap methylated spirits. 95% ethanol denatured. This is just regular denatured alcohol you would find at almost any hardware or other store correct?
I understand that sodium hydroxide may work for Beam's but so far untested?

Still working on a good source for fast blue BB. Seems to be out of stock on amazon/ebay as some have noted in this thread. I found one chemical supply place that sells 25g for $740. I am confident that I can find a smaller qty, but I will have to keep calling around. If nothing else, can always get it off of Alibaba I suppose.

Thank you for doing all this hard work, and sharing!

 

[budchemist]

Just discovered this thread.
Fantastic work. Gives me an easy tool to monitor decarboxylation and test oil extractions.

 

[PhenoMenal]

@PhenoMenal
Been looking around for chemicals and such to eventually do some TLC. Had a few questions.

just a few will do my best to answer them

First off I am guessing that these plates will not work?
https://www.amazon.com/gp/product/B07LBK5GL5/ref=ox_sc_act_title_2?smid=A11MUK3W6A9O7N&th=1

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.

Are these what you mean when you say UV indicators?

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.

Also It seems that Chloroform while providing nice looking results, is fairly difficult and expensive to source. I am curious as to where you found it, and how much you spent?

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

It seems like most of your tests are done with 4:1 Hexaneiethyl mix. I am assuming the cost/difficulty of obtaining chloroform is the reason?

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.

The only place I found Chloroform, without seeking out chemical suppliers is a $65 liter which I guess isn't too bad. But since we already need Hexane, the 4:1 Diethyl Ether seems more cost effective.

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

I was curious why you listed the Solvent and Eluent amounts both as 250ml-1L. It seems that you would only need 2ml Diethyl per test and 9ml of Hexane, assuming you are using the 4:1 mix instead of 10ml chloroform. The reason I ask about the amounts, is it appears you only need 1ml of Hexane for solvent use. Far less than the 10ml of Eluent needed per test.
Was thinking maybe 1L Hexane, and around 125-250ml Diethyl which is fairly inexpensive in those qty's, compared to 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.

Some listings on ebay/amazon are for n-hexane. A quick search tells me that this has a different boiling point than Hexane. I am guessing that n-hexane will not work for solvent or Eluent, but I figured I would ask.

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.

As far as the Beam's test goes, you said that you use cheap methylated spirits. 95% ethanol denatured. This is just regular denatured alcohol you would find at almost any hardware or other store correct?

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.

Still working on a good source for fast blue BB.

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.

Thank you for doing all this hard work, and sharing!

Best of luck, and please share anything you learn WE NEED MORE PEOPLE MAKING THIS ACCESSIBLE!