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

Thank you for directing me to the T&E thread, although I don't have the option to fine-tuning ideal eluent ratios or make supplies at home, i did pick up a few tips.

PhenoMenal said:

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

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Dude, so awesome. But i actually spray my plates (was instructed in the starter kit I purchased) It seems like a more accurate process - less stirring or agitation of the separated cbs. Your thoughts? ( i do wear a standard dust mask)

What do you think about OXOSSI 's advice:
1.) Decrease the load by 50 %
2.) Run it 2-3 times, when the eluent reaches the top of the plate, dry it and do it again
Are you now running 1ul samples or still 2-3?

PhenoMenal said:

And yes my understanding is that the topmost lightly purple spot are probably waxes like geranyl

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Found this online:
THCV. The story behind this recently appreciated cannabinoid goes all the way back to the first steps in cannabinoid synthesis: substrate binding and prenylation. Recall that the first cannabinoid described in part 1 was formed by the combination of the substrates olivetolic acid and geranyl pyrophosphate. Well, there is another molecule that is very similar to olivetolic acid but rather than having a C5H11 (n-pentyl) group coming off of the aromatic ring, it has a C3H7 (n-propyl) group (see Figure 2A).

This derivative of olivetolic acid is called divarinolic acid and it is formed through the same pathway as olivetolic acid but using a different substrate. When the substrates divarinolic acid and geranyl pyrophosphate bind to the GOT prenyltransferase, CBGVA is formed (see Figure 2B and the illustration at the top). CBGVA can cyclize in the same manner as CBGA and can thus lead to 3 more classes of cannabinoids, the THCVAs, the CBDVAs, and the CBCVAs. Each of these decarboxylate as described in part 1 and lead to THCV, CBDV, and CBCV (see figure 2B showing THCV). For every class of cannabinoid structure there are members bearing the C3H7 (n-propyl) group that have been observed, indicating that CBGVA reacts with all of the same enzymes as CBGA.


Recall the THCV rich Leaf sample test results above. I had flower samples of 2 and 3 HPLC/DAD tested by SC Labs. Notice the THCV levels are well below the TLC reports. I did let the plants run late to mature the seeds but I cannot find what THCV degrades into like CBD>THC. What could cause this inconsistency?


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

I have a very bad experience with a "CBD Oil" and my Mom ( 75 years old) also trip heavily with some home made RSO, so my interest in TLC is to test oils and edibles. Have any used the proposed protocol to test oils?

What dilution rates do you recomend to get a "CBD Oil" sample ready to drop in the TLC Plate??? 1:1- CBD oil/hexane in a 1ml microcentrifuge.

vial sounds good?

About the suppliers... https://www.sigmaaldrich.com/ online site has all you need and more, including the BB Dye.

PhenoMenal, You are a generous and patient teacher. I have learn more reading your lecture that in any other way. Thank you for your time and resources! ( If you ever come to Los Cabos, I have a bucket of cold beer with your name on.

Stumbled upon this in the United Nations' "Recommended Methods for the Identification and Analysis of Cannabis and Cannabis Products" manual

Thanks! the question remains in the solvents. As per Phenomenal's records, I will try with chloroform and keep you guys posted. for now i have to wait 6 weeks for the Fast Blue BB. Cheers!!!

DrTricoma said:

What dilution rates do you recomend to get a "CBD Oil" sample ready to drop in the TLC Plate??? 1:1- CBD oil/hexane in a 1ml microcentrifuge.

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It doesn't really matter the exact dilution rate, because the end result will basically be the same (unless you want to compare 2+ vials), but I would probably use something like 1 part CBD oil to 9 parts hexane for the extraction phase (CBD oil is a very concentrated substance with relatively high viscousity, so you want to ensure it's diluted enough). I'd encourage you to have a play around with the dilution rates ... 1:1, 1:10 etc, to see and learn for yourself - and please share your results so we can learn too!

btw, remember that you only need enough for one tiny droplet on the TLC plate, so you dont need to fill up the 1mL extraction vial, and you don't need to waste lots of CBD oil to test it - just one or two drops of CBD oil should be suffice.

About the suppliers... https://www.sigmaaldrich.com/ online site has all you need and more, including the BB Dye.

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Most people cant order from Sigma though as they only sell to accredited labs etc, not random individuals like me. No problem though - I just asked a local lab to order some Fast Blue BB in for me, and a few weeks later it arrived packed in dry ice to keep it cold, it was literally as simple as that. Congrats if you're able to order from Sigma though, that would be super handy!

As per Phenomenal's records, I will try with chloroform

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btw just to clarify I've only ever used chloroform for the mobile phase, never for extraction phase (i've only ever used hexane for extraction). Chloroform is awesome for the mobile phase though, and a bit easier to use than the Hexane & Diethyl ... and avoiding the overpowering smell of the Diethyl is also a bonus! But they're both awesome, and both provide unique separations as you've seen in my previous photos. It's good to have options.

Last but not least, CONGRATULATIONS on giving Thin Layer Chromatography a go - I know it can sound a bit daunting initially, but really it's quite a simple process at the end of the day, and it's the most powerful tool us amateurs have practical access to do from home. You will become empowered, and will not be disappointed!

I love this...starting with a Beam. EXCELLENT information. Was going to order a kit. So glad I didnt.

PhenoMenal said:

It doesn't really matter the exact dilution rate, because the end result will basically be the same (unless you want to compare 2+ vials), but I would probably use something like 1 part CBD oil to 9 parts hexane for the extraction phase (CBD oil is a very concentrated substance with relatively high viscousity, so you want to ensure it's diluted enough). I'd encourage you to have a play around with the dilution rates ... 1:1, 1:10 etc, to see and learn for yourself - and please share your results so we can learn too!

btw, remember that you only need enough for one tiny droplet on the TLC plate, so you dont need to fill up the 1mL extraction vial, and you don't need to waste lots of CBD oil to test it - just one or two drops of CBD oil should be suffice.


Most people cant order from Sigma though as they only sell to accredited labs etc, not random individuals like me. No problem though - I just asked a local lab to order some Fast Blue BB in for me, and a few weeks later it arrived packed in dry ice to keep it cold, it was literally as simple as that. Congrats if you're able to order from Sigma though, that would be super handy!


btw just to clarify I've only ever used chloroform for the mobile phase, never for extraction phase (i've only ever used hexane for extraction). Chloroform is awesome for the mobile phase though, and a bit easier to use than the Hexane & Diethyl ... and avoiding the overpowering smell of the Diethyl is also a bonus! But they're both awesome, and both provide unique separations as you've seen in my previous photos. It's good to have options.

Last but not least, CONGRATULATIONS on giving Thin Layer Chromatography a go - I know it can sound a bit daunting initially, but really it's quite a simple process at the end of the day, and it's the most powerful tool us amateurs have practical access to do from home. You will become empowered, and will not be disappointed!

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Ordering N-Hexane in 3,2,1, Done! jajajaja Already pay about 400 US for One liter of Chloroform, 20 Aluminum Silica Plates and 5 grams of Fast Blue BB. All from a local Lab Supplier, not Sigma Direct. All the other materials will sum up to 100 USD from Amazon. Fast Blue BB takes 6 weeks to ship, so for now all I can do is wait, and collect samples (try not to smoke them jajaja). Nothing like new learning to keep the creative sinapsis firing in all cylinders! Thank you again for your feedback!

btw there are several videos on youtube demonstrating the TLC process, which can help get your head around things before trying it for the first time

The TLC process itself is all that's needed to separate the sample into its different molecules, which is why you can simply use water to separate black ink from a texta/pen into the various coloured inks it's comprised of. This is generally how school children are introduced to TLC.

However, when separating something like cannabinoids, we need a stronger extraction solvent than water (like hexane), a stronger mobile phase (like chloroform), and the result cannot be seen with the naked eye, which is why we need to use a stain/dye ... Fast Blue B and especially Fast Blue BB are the two best dyes to visualise cannabinoids.

Enjoy...

I am enjoying this read. Got a question. Does the plant show all of its cannabiniods in their ratios as soon as it develops trichomes? AND Is the complete terpene profile, as well, testable with a vanillin reagent, at three weeks? Seems they develop later but I would have thought that of cannabiniods as well.

Leef said:

I am enjoying this read. Got a question. Does the plant show all of its cannabiniods in their ratios as soon as it develops trichomes? AND Is the complete terpene profile, as well, testable with a vanillin reagent, at three weeks? Seems they develop later but I would have thought that of cannabiniods as well.

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See https://www.rollitup.org/t/cbd-blitz-find-a-high-cbd-low-thc-mother-from-seed-in-only-3-weeks.963846/

sorry but I have zero experience with vanillin reagents.

Those are some of the many many questions I am planning to solve some time in late March when I get the solvents and plates. Maybe we can work in a protocol that many of us use and share the data, this way we can move forward faster... Keep in mind that Phenomenal has already made great progress for all. I have already posted the 4 supplies to get from a lab supplier next to you. Saludos!

^^^^^ These? Best price I could find. Not sure If I can cut them up. I asked vendor what the back is made of. They come 2.5cm X 10cm, same amount of overall surface for 8 bucks more.


^^^^^ This for beam test? Also for the Beam test, will methanol work instead of ethanol? I can make the ethanol if I need to. Not sure how high above 90% I can get it though.

PhenoMenal said:

use a vanillin reagent and TLC visualises the terpenes also

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I was asking about vanillin because of this. Stoned musings on my part. Far down the road. Thank you again PhenoM.

Leef said:

42.56C$ |10pcs/box 100mm*200mm Hsg Highly Active Tlc Silica Gel Plate Silica Gel Precast Slab Free Shipping - Educational Equipment - AliExpress

Smarter Shopping, Better Living! Aliexpress.com

www.aliexpress.com

^^^^^ These? Best price I could find. Not sure If I can cut them up. I asked vendor what the back is made of. They come 2.5cm X 10cm, same amount of overall surface for 20 bucks more.

Potassium Hydroxide - (Caustic Potash) (90%) (Flakes) (4oz to 20pound) (10 Pound): Amazon.ca: Health & Personal Care

Potassium Hydroxide - (Caustic Potash) (90%) (Flakes) (4oz to 20pound) (10 Pound): Amazon.ca: Health & Personal Care

www.amazon.ca

^^^^^ This for beam test? Also for the Beam test, will methanol work instead of ethanol? I can make the ethanol if I need to. Not sure how high above 90% I can get it though.





I was asking about vanillin because of this. Stoned musings on my part. Far down the road. Thank you again PhenoM.

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this are the aluminum plates by Sigma, this are the ones that can be cut with scissors

DrTricoma said:

this are the aluminum plates by Sigma, this are the ones that can be cut with scissors

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Yes, but those are 300$ plus ship. I have that price locally. I am looking for more budget friendly alternatives. Did you say you had all your needs for around 400$? We are getting very different prices from Sigma.


This place has everything, local pick up.

I am asking about these in 10x20cm sheet or these cut.

regarding TLC plates:

• you only need TLC plates, not the more expensive HPTLC plates.
• aluminum/aluminium is the way to go, easy to cut with regular scissors if needed, and cheaper than glass.
• 5cm x 10cm (50mm x 100mm) is ideal and fits perfectly in a sealed jar (which it needs to!). Or get 10cm x 10cm and cut in half.
• ensure they're coated with silica gel (not cellulose fiber or anything else).
• should be well under $100 for ~20pack, i encourage you to shop around for a good deal.
• don't get ones with fluorescent indicator (eg. "F254" - they're for use under UV light, which we're not using).
• you can run up to 5 lanes (ideally 4) on each 5cm-wide plate. That's 80 to 100 lanes for a 20pack.

Thank you PhenoMenal for the informative guide.

Can TLC be used to approximate the percentage of THC in distillate? Of the references I've found online they seem to max at 25%. Would there be a way to recalibrate the test for concentrates opposed to oils? If I had a tested lab sample already I could make a standard sort to speak.

Or my concern is that over a certain percentage the growth of the dot is indistinguishable.

Leef said:

I am enjoying this read. Got a question. Does the plant show all of its cannabiniods in their ratios as soon as it develops trichomes? AND Is the complete terpene profile, as well, testable with a vanillin reagent, at three weeks? Seems they develop later but I would have thought that of cannabiniods as well.

Click to expand...

Can you use vanilla to TLC test terpenes? How are you doing it/ heard it can be done?

I am still here and waiting for my chloroform to arrive. As soon as i get it ( is the one thing missing from my list) I will get into "chromatographing" all kinds of weed infused samples... patience is my name for now.

Can anyone suggest a place to buy Fast Blue BB online? I haven't had any luck here or with any local lab supplier. My biggest challenge has been getting a supply of all the chemicals needed to run the test. Right now I'm stuck looking for Fast Blue BB. I'm in the Washington, DC area and can't find a local lab supplier and haven't yet found a website that will sell it to me directly.

By the way, @PhenoMenal, your tutorial has been immensely helpful. I reference it nearly each time I run a TLC. So far, with your guidance, I've run TLC on nearly 100 samples. Thank you from the bottom of my heart! I am so grateful for your time and the information you posted.

ryanrayla said:

So far, with your guidance, I've run TLC on nearly 100 samples.

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Im confused! how have you run 100 TLC's without Fast Blue B/BB?

btw you don't need to necessarily find a LOCAL lab supplier, just one able to order it in for you in your country. There are plenty of such labs in America, so you just need to keep emailing.