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

[PhenoMenal]

In this tutorial I will try to explain how to use both Beam's Test, and, Thin Layer Chromatography of cannabinoids to do a DIY home analysis of your buds, including everything and how much of everything you need to get, with the main aim being to ascertain whether the sample has CBD.

I'm not a chemist, just a regular person like you (unless you're a chemist), and it took a long time for me to figure all of this out. I WISH somebody who actually knew what they were doing had posted a tutorial like this. I know how daunting it is to even consider using TLC because of the lack of specific information (cannabinoid testing) available, but it turns out it IS EASY, so even if this only helps one person hopefully this is a small step towards making TLC more accessible to those on the hunt for CBD, especially medicinal users.


The need to detect CBD as a medicinal home grower ...

Late last year a family friend was diagnosed with terminal cancer. 1 in 3 of us will go through cancer so this is of course nothing new., but this wasn't "just cancer" - it's terminal cancer... it's effectively Game Over, after this round. It's now just the rest of the low charge in your one remaining battery keeping you alive, which has since been assaulted by chemotherapy chemicals like cytotoxins which are warfare on the body's immune system.

So I did the only thing I knew I could do to help... fill up a USB stick with every comedy series and movie in my collection (laughter is an underrated medicine, and everyone should aim to get at least 30 mins laughter in their daily diet). Oh, and also get them some full spectrum CBD oil to try (cannabis is a pretty amazing medicine too yknow!?) Anyway they had really good success with the CBD oil, so now I'm helping them with their small two-plant grow, with some strains touted to have high CBD/low THC. (THC is also very important, but it's always easy enough to add THC later, so we really, really need high-CBD/low-THC strains first)

... but how the hell can you determine how much of a non-psychoactive substances is in your buds!? Only need to consume/smoke it to figure out if it has THC, but... CBD!? I guess, ultimately, if the patient says it's working well for them that's the ultimate test, but it's still good to know exactly what you're consuming or growing, especially for example if you can find out early for culling or wanting to find a good mother plant.

Of course you can pay for lab analysis, and they are of course excellent because of their accuracy as well as information about all the terpenes as well - they give you a great 'fingerprint'. BUT, lab analysis isn't available in many countries, and it's not cheap - Greenleaf Lab just as one random example charges $40 sampling fee, $75 for potency testing, $90 for terpene testing. If you want 2 strains/samples tested, it's double.

Anyway after several months further reading, to my knowledge, for people wanting to test their small grows at home for CBD, that only leaves 2 DIY options:
Beam's Test, and Thin Layer Chromatography (TLC) ...

I was however very, very frustrated by how little information was available in regards to using TLC for cannabinoids. It shouldn't take months for somebody to have to figure out.

This information should be freely available to everyone.
So why wasn't it?!? I don't know. Let's change that, because TLC is an amazingly valuable tool that IS within easy reach of home users.


Beam's Test for CBD

(Beam's Test isn't TLC, but it has many attractive qualities as a CBD test in its own right)

PROS: Easy. Quick. Affordable. Accessible. Field-friendly. Can be used as a 'filter' to determine if you want to go forward and use TLC on the sample.
CONS: Doesn't tell you much other than Yes/No "does this sample have CBD?"

"In 1911, Dr. W. Beam discovered that the tissue of hemp, which is typically low in THC but high in CBD, gives a purple color when treated with bases."

Beam's is a fantastic little test that I only had the privilege of trying for the first time just recently. It basically gives you a Yes/No answer to the question "does this sample have CBD?". Handily, it does not react to THC.

You only need:

• KOH, aka Potassium hydroxide, aka "lye" - used to make soap - easy to find pellets on ebay etc. If it's being sold as "lye" make sure it's potassium and not sodium hydroxide!
• Ethanol. I used cheap methylated spirits (95% ethanol, denatured), or you can use that "Everclear" 99% stuff but that's probably more expensive. Or distill your own.
• Container for the test: a small glass such as a shotglass is ideal, OR a small (eg. 1.5mL) plastic eppendorf tube is great too - plus you can then use those in the field/mobile.

SAFETY: I'm not sure what other solvents if any can be used, and KOH is very caustic, so you shouldn't experiment if you don't know what you're doing - just use ethanol or methylated spirits. Always use protective eyewear and nitrile gloves when working with KOH. ALWAYS ADD THE KOH TO THE ETHANOL - NEVER ADD ETHANOL TO THE KOH. Use at your own risk.

The standard recipe calls for a stock solution of 5% KOH in ethanol, so about 5gms of KOH pellets to 95 mL of ethanol. So that's enough for 10 tests @ 10mL each, although really you could just use 1.5mL eppendorf tubes which would make for 66 tests - you don't need much at all.

When you've made that stock solution you can put it aside and it'll last many years. Then whenever you want to do a test, simply pour a tiny amount into a shotglass or some other glassware - about 10mL is all you need, so about 1/3rd of a standard shot, and sprinkle in your dried-and-crushed bud sample, you only need a small amount the size of a pea.

If the sample has a good amount of CBD, within a couple minutes you'll see the solution start to take on a red/purple tinge. Try it with a sample that has no CBD, for example just a regular THC-rich strain, and there'll be a slight change like a mild white-yellow blur, but definitely no red/purple tinge. For CBD-rich samples the tinge will be especially darker, but lighter for samples with only a low level of CBD, so in that sense it actually gives you slightly more than just a Yes/No answer, but also a mild indication about how rich it might be, and for accurate comparisons you only need to correctly measure the amount of solution and the weight of the samples used.

Disposal: The cannabis community respects the environment. I wish Trump's Environmental Protection Agency did. Never pour potassium hydroxide solutions down the drain/sewer, and even small amounts are hazardous to aquatic life. Pour unused solution (hey its only 10mL anyway, easy to deal with) into a container of sand or earth or similar absorbent dry material so it's diluted. Seal the container and it can be disposed of.

But it'd be nicer to have more than an approximate CBD - Yes/No? answer ...

 

[PhenoMenal]

Thin Layer Chromatography

Don't let the name scare you - TLC is fundamentally very simple, and very amateur-accessible, yet provides amazing results ...

(Now let me butcher its description...)

Basically, all TLC involves is putting a tiny amount of (ideally liquid-suspended) substance we want to test near the bottom of an absorbent (silica-coated) plate, then stand it it in a small amount of liquid solution... capillary action drives the solution up, and in the process separates the substance into its smaller chemical components, which group together if a suitable eluent was used.


TLC for kids!

TLC really can be very, very basic! For example, CHILDREN are introduced to it in school usually with the ink of a black marker pen as the substance to test, and the liquid solution used is often simply water in that case. They might even just use paper instead of a proper absorbent plate.
It's called the mobile phase, and it looks like this (this is standing nearly perfectly upright in a jar).

You can see in the first frame we start out with just a few dots at the bottom. Then, we stand the plate in a jar of eluent solution (the water), and that starts working its way up, separating the substance in the process - in this case the black marker is clearly made of several different inks.

Each different ink (eg. yellow) is its own molecule, just as THC and CBD are, and because of the magic of TLC (when used with correct solvents!) they all separate nicely and stay grouped together.


TLC for Cannabinoids

PROS: Reveals the full 'fingerprint' of the sample, shows the existance (or lack thereof) of all the different major cannabinoids (including THC, CBD, CBG, CBC, THCV, CBN), and their ratios, eg you can easily see if a strain has a 1:1 CBD:THC ratio, and gives you a true visual representation of the composition of the sample, which you can't get from Beam's Test.
CONS: Takes about an hour. Not mobile/field-friendly. Expensive initial startup.

Ok, now it gets just a little trickier. Compared to the above ink test, testing for cannabinoids has a few twists:

1. We can't use water, it turns out we need a more powerful solvent for cannabinoids.
2. We can't just paint the dots on with an ink pen, so we must first absorb the cannabinoids into a solvent solution - we then use dots of that solution.
3. We can't see them - cannabinoids are not visible per se this way; even if you can see them they're just salts that all look the same color (and you cant see then against the white plate anyway!) - they don't come with their own built-in dye like the ink pen! So we also need a visualiser. You can actually use UV to visualise it with the right TLC plates, but here we will use an actual dye, a very specific one for cannabinoids, with regular TLC plates, allowing us to see the result in vibrant colors - THC red, CBD orange etc.

All three of these twists are easily solved.

But due to 1) the lack of information on the internet specifically about TLC for cannabinoids , 2) the differing and sometimes conflicting results of existing literature due to differing chemicals used etc, and 3) the massive amounts of non-cannabinoid TLC testing i had to wade through, it took me two and a half fricken months to figure it all out. If you're bored you can read my full struggle plus experiments [here] (username sadpanda - hey i was desperate for help and way out of my league! special thanks to G.O. Joe there for his help)

There are some commercial test kits for TLC-for-cannabinoids that you can buy, however they're not cheap and they don't give you many tests. One kit for example is $99 (don't think there's any cheaper) and that only gives you 2 test plates (enough for 10 samples) - after that you pay at inflated prices for refills etc. I haven't used any of them so can't make recommendations, but my feeling is AlphaCat is probably the best one of the few available, and maybe those kits give a good starting point anyway by providing everything you need to get started, but then just source the 'refill' stuff yourself, which really is just the dye and the plates.

Before we start, it's important to note that TLC is a qualitative and only semi-quantitative test. This means you cannot really use it to accurately determine the % of THC/CBD/etc the same might have. You can try, by getting every single measurement precise, and hope the spot is easy to match on the chart, but it's just never going to give you much confidence other than "probably 8-12%", not 8.341%, and you could end up being off by as much as 10%, and there's of course a fair difference between 15% THC and 25% THC! This page [here] explains and shows experimental results regarding that.

But what it IS good for, is detecting:

1. if the sample has a particular cannabinoid (does it have CBD? THCV?)
2. the approximate RATIO of THC:CBD. For example 1:1 strains are very easy to see, but again as it's only semi-quantitative you only get a range not a value ... "this higher-THC one is probably 1:3 - 1:5"

 

[PhenoMenal]

Method - TLC for cannabinoids

• Warning: Requires use of hazardous materials and therefore should only be attempted by adults.
• Safety: It's generally a fairly safe procedure, but you must follow standard safety precautions - always use nitrile gloves and safety glasses, long-sleeve lab-coat doesn't hurt either, and the area must have good ventilation due to the solvents. No open flames - do not smoke or ignite anything during the procedure. Dispose of chemicals responsibly, not down the drain. Read the MSDS of every chemical before you use it.
• Disclaimer: You do this at your own risk. This is only for educational purposes of course. Always follow safety precautions. I am not an expert nor a chemist, and there are probably many improvements that could be made to this tutorial. To the experts: I just wish you'd posted a tutorial.

Full Overview

The entire procedure can be broken down into 8 steps, which require about 1hr of work:

1. First we need to extract the cannabinoids from the bud sample into a solvent (Hexane), so we put a tiny amount of bud in a small plastic disposable tube, then add solvent. Give it a good shake. Best to leave it an hour or even overnight, and regular shakes are good.
2. We'll put up to 5 tiny 'dots' of our cannabinoid-infused-solvent solution near the base of a silica-coated TLC plate.
3. We'll then add 10mL of our eluent (solvent) into a glass jar as the driver of the capillary action - the mobile phase. We can either use 10mL of Chloroform on its own, OR a mix of (2mL Diethyl Ether + 8mL Hexane). There are various other solvent mixes that can be used but I've only used these two, but they're both very good.
4. We'll then sit the TLC plate in the jar, standing vertically or close to, then put the jar lid on and patiently wait, being sure to keep it away from sources of vibration - don't touch the jar until it's finished.
5. We'll patiently wait for 10-30 minutes (or specifically, however long it happens to take for the eluent to work its way up to near the top of the TLC plate), then we'll remove the plate and put it aside standing upright until it's completely dry. (Repeat above steps if we're doing more than 1 plate, topping up eluent if required)
6. We'll then prepare a small bath of 25mL sodium hydroxide 0.1M solution (in distilled water), with a couple tiny scoops of the special cannabinoid-revealing wonder-dye - Fast Blue BB.
7. We'll gently, carefully place the TLC plate face-down in the bath, wait ~5 seconds, then remove the plate and again set it aside to dry, but we're now finished and the results are visible immediately, but continue developing in vibrancy for a short period. Zomg so beautiful!
8. Cleanup. Nothing goes down the drain, but it's easy. Please scan and share your TLC analyses online!

You Will Need ...
Most of the things needed are normal, cheap, easy to obtain things. The others are still easy to obtain, not 'monitored' or 'controlled' etc, but still kinda 'funky' lol:

Funky stuff:

• Extraction Solvent: 250mL-1L of Hexane. You need ~1mL for every sample you want to extract (it's all we use for the extraction phase), and 8mL if using it with Diethel Ether as the eluent (mobile phase).
• Mobile Eluent: 250mL-1L of Chloroform, and/or Diethyl Ether. You'll either use 2mL of Diethyl Ether (with 8mL Hexane) OR 10mL of Chloroform as the eluent. If you get both you can make two very different visualisations. Solvents come in various forms/grades which can be confusing - just ask the lab supplier/shop to get "one which is suitable for TLC please".
• Visualiser Dye: Fast Blue B or Fast Blue BB. Both are fairly easy to find on the web, but also try asking your local lab supplier for a quote. In the case of BB, it's a yellow powder/salt. B is cheaper but seemingly you go through it a lot faster as you need a lot more than BB, which only needs a tiny amount. BB was developed over concerns about B being carcinogenic, and BB produces more vibrant results. BB needs to be stored in the freezer (recommended -20C, but regular home freezer is ok) and protected from light and moisture, but I think B is ok just in the fridge @ 2-8C - and they use it in commercial kits and ship without refrigeration, whereas BB is typically shipped with dry ice.
• Dye bath: Sodium hydroxide - small container of pellets (or sodium hydroxide 0.1M solution in distilled water). Often sold as "lye" for making soap etc, so really it's far from "funky"

Normal stuff:

• Silica-coated TLC plates. The 5cm x 10cm ones im using seem pretty much perfect size for this, but wouldn't want shorter than 10cm. Aluminium-backed ones work great, they're cheaper than glass-backed ones, plus you can cut them easily so you don't have to waste a full plate for 1 sample. Don't need any with UV indicators, just good, standard silica plates. You can do up to 5 samples or 'lanes' per 5cm-wide plate.
• ~50 x plastic (polypropylene) disposable 1.5mL eppendorf tubes. You need 1 for every sample, so if you want to test 5 different samples on 1 plate you'll need 5 eppendorf tubes, but they're very cheap.
  
• ~50 x plastic disposable 1uL pippete. Again you need 1 for every sample, but also very cheap.
• A micro-pipette handheld device, you put the disposable pippete's on the end of this to suck up the tiny amount (1uL or 0.001mL) of cannabinoid-solvent to put as the 'dot' on the TLC plate. NOTE: Because the amount we put on the plate is so small, instead of buying the pipette device and disposable pipettes you COULD simply get away with using the head of a pin a couple times to dunk into the eppendorf. Not very accurate, but we don't really need too much accuracy for this.
• 1 x Regular graphite pencil, for drawing on the plate (being only the element carbon it doesn't interfere like inks do).
• 1 x Safety gloves made from nitrile.
• 1 x Safety glasses.
• 1 x Lab coat is recommended, you shouldn't end up with any chems on you but accidents happen, the chems are a bit funky, and Fast Blue is a dye and will stain.
• Approximately 1-2 "peas" worth of dried marijuana sample to stick in the eppendorf tube. If testing things like CBD oil there's no need to dry it. (I guess this is both no
• Glass jar with lid; widemouth jars can make insertion and removal of the plate easier. Needs to be big enough to fit the ~5x10cm TLC plate.
• Metal tweezers for manoeuvring the TLC plate
• Coffee filter papers
  
• Measuring cup to measure 25mL
• Glass syringe for extracting small specific amounts of liquid
• A tiny glass jar for mixing the Fast Blue
  
• Distilled water
• A teeeny-tiiiny plastic spatula, u know those ones with a spoon-head of only 3mm x 3mm, i use three flat scoops of Fast Blue per bath
• A well-ventilated area, especially if using Diethyl Ether

You might prefer to simply buy one of the commercial kits to get most of the above, then after that's run out of tests you basically only need to buy more solvent, more Fast Blue, more silica plates, and more plastic pipettes and eppendorf tubes.

 

[PhenoMenal]

TLC for Cannabinoids - Detailed Procedure

#1 - Extract cannabinoids from a bud sample into a solvent

The little 'dots' of substance to test that we put near the bottom of the TLC plate need to be in liquid form, so you can't just put a tiny amount of bud on the plate - you need to sit it in a suitable solvent that will suck out all the cannabinoids into a smooth soup, mmm!

It's very simple:

• Pour ~1.0mL of solvent (Hexane) into a 1.5mL-sized plastic eppendorf tube. I'm not sure what other solvents can be used in place of Hexane, I'm pretty sure Pentane can but not sure about any others.
• That leaves enough to put approximately 1-2 'peas worth' of dried bud in - you don't need too much, just 1/3rd to 1/2 of the tube loosely packed.
• Give it a GOOD SHAKE! (Labs use spinning centrifuges to quicken this process, but we don't need that for this - just a few good shakes
• Now just leave it for at least ten minutes, but i usually wait at least one hour and sometimes overnight. Keep away from heat and light to prevent cannabinoid degradation. Shakes are goooood, but seemingly not required - Hexane is a cannabinoid magnet!

Labs would possibly filter the resulting "cannabinoid-solvent" solution - we'd use our coffee paper filter, but Ive not found a need for that.


#2 - Prepare the TLC silica plate

There really isn't any preparation required - the plates are ready-to-go out of their box, but it's handy to make some pencil marks for ID purposes so you can differentiate samples, as well as mark their start position to ensure equal spacing etc and give yourself somewhere to aim when setting the samples.

Using just a regular graphite pencil (which, being elemental carbon, doesn't interfere with the process like inks do), draw a light line approx 13mm from the base of the TLC silica plate. This is the origin line. Then, for each sample or 'lane' you want to try (usually up to 5 fit ok), make another small mark to indicate their start position. You can make comments or other identifying marks below the line, but it's probably best to be minimal.

If you're only planning to do 1 or 2 samples, consider cutting the TLC plate in half if it's aluminium-based.


#3 - Put the 'cannabinoid-solvent' sample onto the TLC plate

We only need a tiny amount of the cannabinoid-solvent to comprise each 'dot' (one per 'lane').

The easiest way to do this is using the handheld micropipette device with 1uL disposable pipettes:

• Attach a 1uL plastic disposable pippette to the handheld micropipette device. (Always use a new pippette for each sample)
• Press and hold the button down into the 1st (but not 2nd) resistance-level of the pipette device
• While holding the button down at the 1st resistance-level, dip the ipette tip 5mms into the sample (into the eppendorf tube)
• Release the button so it sucks up 1uL worth of the 'cannabinoid-solvent', and withdraw from the eppendorf tube
• Go over to the TLC plate and carefully touch the tip down onto where you want to place it on the origin line (eg ~13mm from the base)
• Press down on the button, but this time all the way to the 2nd/final resistance level, ejecting the 1uL of solvent onto the plate
• While still holding the button down, withdraw the pipette device, and then you can release the button, and the plastic pipette can be disposed of.
• If I'm doing 5 lanes I use 2uL (so two applications per dot), and 3uL for 4 lanes or less.

However, because the sample size of each is so small, you could probably also simply dip a clean metal pinhead into the solution and go back and forth maybe ten times per dot.


#4 - Capillary action in a jar: prepare the mobile phase

To our glass jar, the one we plan to put the TLC plate in, we need to add 10mL of eluent, which is the solvent or solvent mixture that will drive the capillary action up the plate. 10mL is perfect for my jar because it works out to about 1-2mm liquid depth, usually enough for 1-2 plates. The entire base of the jar should be covered in the liquid to help ensure an even run upwards.

There's at least a small variety of eluents that can be used, including but not limited to:

• 10mL Chloroform
• (4:1) 8mL Hexane + 2mL Diethyl Ether
• 0.1% (10uL) Glacial Acetic Acid + ((3:1) Methanol 7.5mL + Distilled Water 2.5mL)

I have only tried the first two. Both provide excellent separation. I have also tried water, acetone, isopropyl alcohol... all failed to achieve any separation of the cannabinoids.

All we need to do is extract a small, measured amount of solvent in a syringe, and put it in a jar, and put the lid on the jar - it takes about 10 seconds. I hold your breathe, then quickly walk away after sealing the jar for a proper breath. Everything's back to normal after that.

I'm glad I ended up trying both of the first two options, as they both provide a slightly different picture. When using the Diethyl Ether, BE WARNED its fumes are insaaane. But we only need to hold our breathe for ~10 secs.


#5 - Add the TLC plate to start the mobile phase

• Use a pair of tweezers to gently lower the TLC plate into the jar, so it's leaning but standing relatively upright and pointing towards 12o'clock. Avoid splashing or moving the eluent in the jar.
• Gently put the lid back on, seal it but avoid too much vibration. Sealing the lid minimises eluent loss, minimises disruptive evaporation off the plate, and helps gets the job done faster.
• Ideally the eluent level is at least 5mm below the plate's origin line, so that it has to reach the origin line by capillary action first to give it a 'smooth' start
• WAIT until the front line of the eluent has risen to nearly the very tip (i try to spare only 2mm but its ok if it goes to the very top)
• Use tweezers to gently withdraw the plate. It's still wet so avoid too much movement/vibration.
• Sit the plate somewhere such as against a wall or object so it can remain upstanding while drying
• Wait until the plate is completely dry. You don't have to immediately test (ie. dye bath) at this stage, but is recommended as the cannabinoids are now more vulnerable to degradation.

The plates are done! Now all we need to do is dunk them in our dye bath to visualise them.


#6 - Prepare the dye bath for visualisation
Don't start this phase until 1) your TLC plate(s) are fully dry and 2) you're ready to 'develop' them with the visualiser dye, Fast Blue BB. Fast Blue degrades rapidly from light - you've usually got a good half an hour to work with, so it's fine for a few plates, but wouldn't want to take it much beyond half an hour.

• To a tiny jar, add 25mL of sodium hydroxide 0.1M solution (simple to make, see instructions later)
• Now add 3 flat scoops of Fast Blue BB, using a tiny spatula (3mm x 3mm x 1mm spoon area). This is just enough to very lightly cover a 1cm x 1cm area, so not much at all. You should notice the BB will turn the solution fluro-yellow
• If the Fast Blue is clumpy (it's very hygroscopic), use the end of the tiny spatula to crush some clumps.
• Put lid on the jar and give it a good shake.
• Now pour the solution into the glass container to be used for the bath, but I highly recommend pouring it through a coffee paper first, its just as easy and ensures you dont get clumps - which greatly negatively affect the result.

We want the depth to be about 2mm, and this is enough for 2-3 plates.


#7 - Develop the plate in the dye bath

• Using tweezers again, very gently lower the TLC plate face-down into the dye bath (you might hear a tiny fizz), gently pushing down to ensure the full face has good liquid contact, and wait about 3-5 seconds before gently removing it from the path. You don't want to leave it too long as we don't want cannabinoids coming off the plate back into the solution.
• Remove the plate and stand it up somewhere to dry, eg. absorbant paper towel

That's all! The results are immediately visible on the plate, but will continue gaining in vibrancy for a few more minutes.

Longevity? I have read that you can use a simple solution (i can't remember which) to help 'preserve' the resulting dyed plate, but even my first plates from nearly 1yr ago now are still looking pretty much as good as the day they were developed. I have no idea how long B or BB dyes last before they fade away, but in the case of BB at least they clearly last at least a year (im guessing many more). They're DYES afterall so you wouldn't expect them to fade too quickly anyway i guess.


#Ref - How to make Sodium Hydroxide 0.1M

To make the dye bath we add Fast Blue BB to Sodium Hydroxide (NaOH) 0.1M.

To make Sodium Hydroxide 0.1M, we just need 4.0gms of sodium hydroxide pellets, dissolved in 1.0 litres of distilled water. (I used 3.0gms in 750mL, same thing). Warm water makes the dissolving quick and easy, just keep swishing it around in the bottle until dissolved.

WARNING: NEVER ADD WATER TO NaOH - ALWAYS ADD NaOH TO WATER.

STORAGE: Sodium Hydroxide 0.1M should be stored in HDPE plastic (like many plastic milk/orange juice cartons). Apparently you cannot use PET bottles. Store in cool dark conditions and it's good for many years.

 

[PhenoMenal]

Pick a standard any standard! ...
Initially it was tricky for me to figure out which was which, given conflicting data from various reports...

Some of my experiments ...
(In chronological order. Again these are taken from initial discussions [here] - my username is sadpanda - where it took me 2.5 months to figure out all the variables, equipment, and procedures)

First, having just received my first batch of Fast Blue BB and having never done TLC before, I needed to figure out how much Fast Blue BB to use, because I simply couldn't find any specific info about that. It didn't take too many goes though to realise that Fast Blue BB is very strong - i only use 3 of this tiny blue spatula scoops in 25mL of sodium hydroxide solution to make my dye bath:

Now, time to try some actual substances! ... at this stage I don't have Chloroform, so all my first tests are with Hexane : Diethyl Ether as the eluent...

But notice all those clumpy olive-brown dirty dots? That's because Fast Blue BB is clumpy, and I didn't filter the dye bath through a coffee filter. Lesson learned.

Then i wondered - how long should I be decarboxylating samples, and what effect does that have on their resulting TLC?

We can take them into software like JustTLC for further analysis if you like - no need though...

Then I wondered -- why do we have to use specific eluents like Hexane : Diethyl Ether or Chloroform ... so I tried some others, which, perhaps unsurprisingly, simply confirmed - yes, we need specific eluents!!

Because of that experiment I decided to buy Chloroform to try as an eluent. Really nice separation too - here are 5 different samples compared (each lane on each card is the same sample):

So then i wanted to try decarboxylation again, but with the chloroform eluent, but for whatever reason the shape of the jar seems to affect the Chloroform one (but not the Hexane: Diethyl one), so this result was trickier to read ...

Another decarb run, this is with a high CBG strain (big orange dot under the even bigger red THC dot), first with Hexane: Diethyl ...

Then the same with chloroform...

Decarb of a high-THCV strain...

Most recently we grew out Dinamed CBD and Cinderella 99 ... with the Dinamed CBD one I ended up with an indica pheno, not the main sativa one which is purported to be 20:1 CBD:THC, but this indica one was still 1:1, as can easily be seen. The report back from my friend with cancer who's tried it in the form of cannabutter -- "that stuff's really good" - having seen the TLC result, I had a slightly confident feeling it would be! ...

 

[PhenoMenal]

Sorry for the long length of the tutorial, but as you can see there's just a bit more to it than regular TLC of an ink pen, and that's why it took me two and a half bloody months to figure out haha

Next, I would like to find out how EARLY in a grow can CBD be detected using both methods (Beams + TLC). For the TLC I plan to take a sample every ~2 weeks and keep them in the freezer until I'm deep into flower enough that I know they should be getting easily detected, but for Beams I'll be able to test them straight away. I have read but only once that CBD can be detected fairly on in vegetation...... I really hope so!!! Fingers crossed, and I hope to find out in the coming year.

Hopefully this tutorial will encourage more users to try the Beam's Test and TLC techniques, especially those like my friend on a desperate hunt for CBD for medical reasons. And please share your results, discoveries, experiments, tricks etc!

 

[OldMedUser]

Bookmarking this for sure!

I went back to school in my early 30s and 3 years later got a diploma in environmental chemistry, 63 now and haven't worked in my field for over 20 years so pretty rusty to say the least.

Great stuff man! Thanks.

 

[ruwtz]

Pick a standard any standard! ...
Initially it was tricky for me to figure out which was which, given conflicting data from various reports...

Some of my experiments ...
(In chronological order. Again these are taken from initial discussions [here] - my username is sadpanda - where it took me 2.5 months to figure out all the variables, equipment, and procedures)

First, having just received my first batch of Fast Blue BB and having never done TLC before, I needed to figure out how much Fast Blue BB to use, because I simply couldn't find any specific info about that. It didn't take too many goes though to realise that Fast Blue BB is very strong - i only use 3 of this tiny blue spatula scoops in 25mL of sodium hydroxide solution to make my dye bath:

Now, time to try some actual substances! ... at this stage I don't have Chloroform, so all my first tests are with Hexane : Diethyl Ether as the eluent...

But notice all those clumpy olive-brown dirty dots? That's because Fast Blue BB is clumpy, and I didn't filter the dye bath through a coffee filter. Lesson learned.

Then i wondered - how long should I be decarboxylating samples, and what effect does that have on their resulting TLC?

We can take them into software like JustTLC for further analysis if you like - no need though...

Then I wondered -- why do we have to use specific eluents like Hexane : Diethyl Ether or Chloroform ... so I tried some others, which, perhaps unsurprisingly, simply confirmed - yes, we need specific eluents!!

Because of that experiment I decided to buy Chloroform to try as an eluent. Really nice separation too - here are 5 different samples compared (each lane on each card is the same sample):

So then i wanted to try decarboxylation again, but with the chloroform eluent, but for whatever reason the shape of the jar seems to affect the Chloroform one (but not the Hexane: Diethyl one), so this result was trickier to read ...

Another decarb run, this is with a high CBG strain (big orange dot under the even bigger red THC dot), first with Hexane: Diethyl ...

Then the same with chloroform...

Decarb of a high-THCV strain...

Most recently we grew out Dinamed CBD and Cinderella 99 ... with the Dinamed CBD one I ended up with an indica pheno, not the main sativa one which is purported to be 20:1 CBD:THC, but this indica one was still 1:1, as can easily be seen. The report back from my friend with cancer who's tried it in the form of cannabutter -- "that stuff's really good" - having seen the TLC result, I had a slightly confident feeling it would be! ...

Excellent work and equally excellent contribution to the forum, thank you!

And refreshingly brilliant to finally see something 'advanced' in the advanced section! Take heed people, this is what this area should be about, so go post your nute def queries and defoliation arguments elsewhere!

I will take my time reading this properly, for sure.

Thanks again.

 

[macsnax]

Wow my brain hurts a little. I'm going to have to get in the proper frame of mind and go over this carefully. This must have taken you a while, thanks man.

 

[PhenoMenal]

Bookmarking this for sure! I went back to school in my early 30s and 3 years later got a diploma in environmental chemistry

Very cool, hopefully you can offer some help with the chemistry side of things, and can never have too many people who actually know their chemistry in this thread! bloody embarrassing I don't tried my best in high school though, despite having science teachers that somehow managed to make science boring...

This must have taken you a while, thanks man.

two and a half bloody months mate, biggest jigsaw puzzle i've ever finished, although I still have a million questions and nearly as many experiment ideas (half of which i can't do as i don't know which chemical reactions might occur haha. But this is science, where safety comes first)

Just a couple other tips that sound good but I haven't tried myself yet ...
You'll notice in some of my plates - particularly the ones with Hexane : Diethyl Ether as the eluent, that the main result only fills just over half the plate - it'd be nice if it was more stretched towards the top as that would show clearer separation. Apparently you can take the plate out after the first run, let it fully dry, then do a 2nd or even 3rd run. ie 2-3 mobile phases/capillary action. Not sure if the eluent needs to be changed after each run, but would obviously be better, just a bit more expensive. Definitely hope to try that soon. Only the Hexane : Diethyl Ether one needs it though, the Chloroform ones are clearly taking up pretty much maximum real estate already which is nice.

Also, two other possible eluent solutions to try (again for our target of 10mL). The eluent again is just the solution we sit in the base of the jar that we sit the TLC plate in, which then drives up the plate via capillary action, separating the cannabinoids in the process - if it's a suitable eluent (chloroform is, distilled water isn't).

I haven't tried them yet, but were recommended as good for this by user Oxossi when i was asking:

• Hexane 3.3mL : Ethyl Acetate 6.6mL (Oxossi said "Hex:Et.Ac (20-40)")
• Hexane 9mL : Ethanol 1mL (Oxossi said "Hex:EtOH (10%)")

Possible Poor Man's Hexane : Ethanol eluent then?... (no need to buy either Chloroform or Diethyl Ether if this works, providing a slightly cheaper option):

• Hexane 9mL : Methylated Spirits 1mL (~95% ethanol)

(He also said "Hex - DCM (Varies), Hex-Ether, Hex-DXM-Ether" without giving ratios, so I'll leave those alone, but it shows there are quite a few options that seem to achieve our goal: good separation of the cannabinoids)

I don't think the Ethyl Acetate one will ever be on my radar, though a quick google suggests it's very easy to get and affordable (most nail polish remover is this it seems). Anyone who paints their nails may want to consider this option, but hey it's very affordable too so could be a good one, and hopefully some day somebody will post a result from it.

I don't have Ethanol but I do have methylated spirits (95% ethanol denatured), and that worked fine for Beam's Test so I'm tempted to give it a go. I'd be surprised if it gave a better result than Hexane : Diethyl Ether though, but would be more affordable and wouldn't have the nasty fumes of the Diethyl. I'm not sure what the other 5% in the methylated spirits is either though ... it could just be methanol, or it could be something funkier which might negatively effect the result, but it seems worthy of an experiment!

And I can't remember ever trying Hexane on its OWN, but there's obviously some reason why all the Hexane eluents are at least 2-part, so I'm not sure if i want to risk wasting Hexane on its own to test what would surely be a less-efficient eluent, though I'd still love to see it out of curiosity - and I wonder if it exhibit that 'warping near the sides' effect that Chloroform does - maybe the 2nd part 'balances' the polarity or whatever so everything is nice and even like in the Hexane : Diethyl ones ... (can you see i still have NFI what i'm talking about?)

ps. Automatic ban from this thread to anyone who throws any TLC chemicals down the drain! (we'll figure out the logistics later )

 

[PhenoMenal]

am disappointed by hardly any feedback...

I just want to make it clear that TLC is VERY SIMPLE TO DO, ..... (YES - EVEN WITH CANNABINOIDS) ... as I hope the childrens example with using TLC to reveal that black ink is made of a diverse range of colours.

TLC is an amazing and powerful tool that is accessible to everyone, and especially useful for anyone who:

• is hunting CBD, or any other particular cannabinoid(s)
• or hunting quality father and/or mother plants for breeding purposes

PLEASE do not let the length of my post deter you from giving TLC a go. The length of my post was required to cover the procedure in adequate detail, but at the end of the day, the procedure itself is very simple and straight-forward.

 

[macsnax]

I'm really interested in trying this. I plan on giving it a shot soon. Might be a little over some people's heads or they didn't read it through.

 

[LostInEthereal]

Awesome thread, have much homework to do. I have purchased a TLC kit in the past to check for adulterated ecstasy but never needed it so I have an unfulfilled urge to experiment. It would be cool to do this just for fun but it's great that you are bringing this up to promote awareness for the medical patients.

 

[PhenoMenal]

Might be a little over some people's heads or they didn't read it through.

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!

Awesome thread, have much homework to do. I have purchased a TLC kit in the past to check for adulterated ecstasy but never needed it so I have an unfulfilled urge to experiment.

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

 

[LostInEthereal]

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

Thanks brother. I'm glad your comment was met with intrigue rather than criticism. I've used the full spectrum kits (Marquis, Mecke, Ehrlich, etc) before on my other occasional 'meds' and thought taking it to the next level with a TLC would be awesome. Don't want to plug or sponsor something but the TLC kit was from Bunk Police. Gifted it to a buddy that works the onion fields so hopefully it did him well!

 

[Observe & Report]

This is one of the best threads on the site.

I think the lack of interest is just because not that many people are interested in CBD. Coming up with a relatively easy way to determine the sex of seedlings would be very popular.

 

[PhenoMenal]

I think the lack of interest is just because not that many people are interested in CBD.

wait, what!?

It's also an excellent tool for breeders regardless if they're chasing CBD or not

 

[LostInEthereal]

This is one of the best threads on the site.

I think the lack of interest is just because not that many people are interested in CBD. Coming up with a relatively easy way to determine the sex of seedlings would be very popular.

Phylos Bioscience offers this for around $15 a pop, with it being cheaper in bulk. You can test from the cotyledon of young seedlings and have the results in a week. I haven't used them but will likely pick up 8 tests (must be purchased in multiples of 4 as there are 4 tests per card).

http://www.phylosbioscience.com/

 

[PhenoMenal]

yup, need a genetics test like Phylos' if you want early determination of gender, not something TLC (or any chromatography for that matter such as HPLC, GC etc) can reveal

 

[PhenoMenal]

oh btw LostInEthereal, just in regards to using TLC to test MDMA, the only thing you'll probably need to change is the visualiser dye... (Fast Blue B/BB is very cannabinoid-specific, it does have some other uses - for example, testing strawberries phenolic levels, but it's very very niche). I say probably, mainly because I'm not sure what the best eluents to use for amphetamine TLC are - maybe you can just use Chloroform or Hexane : Diethyl but i have no idea. I'm not sure which dye would be used for MDMA, but i would be very surprised if there wasn't a similar dye that reacts to a variety of amphetamines similar to Marquis etc, but I'll leave figuring out TLC for MDMA to others - hugs'n'glowsticks in advance to whoever does! lol