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



## PhenoMenal (Nov 16, 2017)

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 ..._


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## PhenoMenal (Nov 16, 2017)

*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:

We can't use water, it turns out we need a more powerful solvent for cannabinoids.
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.
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:

if the sample has a particular cannabinoid (does it have CBD? THCV?)
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"


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## PhenoMenal (Nov 16, 2017)

*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:


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.
We'll put up to 5 tiny 'dots' of our cannabinoid-infused-solvent solution near the base of a silica-coated TLC plate.
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.
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.
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)
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_.
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!_
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.


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## PhenoMenal (Nov 16, 2017)

*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.


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## PhenoMenal (Nov 16, 2017)

*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! ...


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## PhenoMenal (Nov 16, 2017)

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!


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## OldMedUser (Nov 16, 2017)

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.


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## ruwtz (Nov 16, 2017)

PhenoMenal said:


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


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.


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## macsnax (Nov 16, 2017)

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.


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## PhenoMenal (Nov 16, 2017)

OldMedUser said:


> 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..._



macsnax said:


> 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 )


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## PhenoMenal (Nov 21, 2017)

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. *


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## macsnax (Nov 21, 2017)

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.


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## LostInEthereal (Nov 21, 2017)

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.


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## PhenoMenal (Nov 21, 2017)

macsnax said:


> 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! 



LostInEthereal said:


> 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


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## LostInEthereal (Nov 21, 2017)

PhenoMenal said:


> 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!


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## Observe & Report (Nov 21, 2017)

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.


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## PhenoMenal (Nov 21, 2017)

Observe & Report said:


> 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


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## LostInEthereal (Nov 21, 2017)

Observe & Report said:


> 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/


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## PhenoMenal (Nov 21, 2017)

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


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## PhenoMenal (Nov 22, 2017)

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


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## LostInEthereal (Nov 22, 2017)

PhenoMenal said:


> 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


Unfortunately these days I don't dabble too much, but just for lack of trying. I used to source everything through the darknet but situation is pretty sketch and has been for some time. I just scoped out the website I purchased my kit from and it doesn't have any labeling or MSDS information (a bit odd actually) for whatever solvents and other chemicals used. I know the solvent was quite strong and I actually avoided using it because of my roommates cats lol, I didn't want to expose them to any harsh chemicals. I do know the kit was non specific towards MDMA, just that was likely to be the most common use but I wonder if it could differentiate between isomers of ketamine for example, would be interesting.

#hugs'n'glowsticks brother, haha

EDIT - weird formatting and added hashtag


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## Sureshot2 (Nov 22, 2017)

This is fantastic work, greatly appreciated and definitely one of the better advanced section threads on this site. I have most of the materials already, just gotta pick up some of the fast blue, lye and TLC plates and then I'll give this a go. 

Cheers


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## PhenoMenal (Dec 13, 2017)

I guess all labs have their good days and bad days ... well, (sadly), amateurs have more than their fare share of bad days  

I've just done another TLC today, this time on a CBD oil, as well as C99 by FemaleSeeds Co (perhaps the grapefruit pheno or just another pheno thats popped out from whatever theyve crossed C99 with... def not the pineapple/tropical/classic C99 tho, but very much still in the neighborhood).

_It's not my cleanest TLC though!_ As I was only doing 2 samples I decided to cut the silica plate in half so I could do two samples per halfplate, each in a different eluent for the mobile phase.

But, unfortunately there was some minor interaction between the lanes. From now on, I think I will only cut a plate in half when doing 1 sample (1 lane per halfplate) in 2 eluents. The aluminium silica plates are only about $1-2 each anyway, and TLC isn't something I do on a regular basis anyway, so I should stop being a tightass with those!

A little ant also ran across the silica plate, probably leaving a pheromone trail, the little ****! I highly doubt it would affect the TLC in any noticeable way though, but I wouldn't want to bet on it, especially as this was one of my worse TLC runs, although that could just be a coincidence.

Anyway here is the first halfplate (the two lanes on the RIGHT), which I developed with Chloroform as the mobile phase eluent. On the LEFT, the smaller one is just a copy from an image ive already posted here, which was the only other time I TLC'd a CBD oil sample (pretty much all my other testing has been with buds). I'm not sure why the Grapefruit lane isn't straight, perhaps there was a slight bend in the plate being only a half-plate, but I haven't seen that problem before with other halfplates, and even if it was bent you'd think it would still go upright, so perhaps it was just slightly oriented off-center due to being in a cylindrical jar with a raised bottom which might've offset it ever so slightly)







I've also TLC'd an official Grimm Brothers C99, so for comparison you can see that in the last image in [this post] a bit earlier in this thread.

ps. you can see two dark dots to the right of the Grapefruit lane's CBG dot ...they're clumps of Fast Blue BB (i wasn't patient enough with the coffee filter paper and pushed a bit too hard, so some FBBB ended up in the mix, whoops... be very gentle when squeezing the FBBB through the coffee filter paper)

While I can't remember what the Unknown CBD oil i tested is called, and although I only used Hexane : Diethyl Ether as the eluent, you can see that its profile is very similar to the CBD oil I tested today (Hempworx 750). Note: This is not an endorsement, and I'm not saying you should or shouldn't get it. *There's a lot of good/legit CBD oils out there, but sadly a lot of scamming going on, and that's ****ing with peoples lives, and in the case of cancer patients they simply don't have time, OR money, OR health & wellbeing to lose to scammers. *Always do a lot of googling about a CBD oil before you buy it - do some basic background checks on the company, and search the web to see if other people have found it helpful.

This particular CBD oil, Hempworx 750, purports to contain 750mg of CBD (in a 30ml/1oz bottle, so that would be 2.5% CBD, W/V%), and comes infused in hemp seed oil, and peppermint oil for taste. The word from my friend going through cancer who's just tried it said it tasted good, which might be good for people who don't like that _planty_ taste some oils have. Or simply add a drop or two of your own peppermint oil to whatever CBD oil you have, no rocket science there!

I've never seen that "W" shape/effect that can be seen in the CBD oil's CBD spot on the TLC plate ... I'm wondering if that's a component of the peppermint oil just ahead of it that might've caused that (clearly it has SOME other molecular components ahead of it near the top, as you can see how much lower on the plate the CBD dot is... CBD appears _above_ THC, so you can tell where the Grapefruit's CBD dot would exist if it had one). It'd be great to have another try on a full plate etc, but the stuff is expensive and comes in tiny bottles. What's the dark-orange line on the top of the CBD spot? I'm not sure - perhaps its due to the minor interaction with the red THC dot in the lane beside it, because i've never seen such a 'line' before... always DOTS. However, perhaps the molecule dot above it is 'restrictive' in a way, something like a wall, which might lead to a buildup along the edges and therefore resulting in a darker color from the higher density, but this is pure speculation and guessing at possibilities.

So, just judging from those two hemp-derived CBD oils (granted, not a big sample size), my takeaway is this:

CBD oils are a great way to LOAD UP on CBD, without having to worry about THC taking over. As CBD isn't psychoactive obviously we can handle a LOT of CBD, but we can only handle so much THC before we fall asleep, and that's a significant limiting factor. I now I'm not saying anything new there, but is an important aspect for patients. Some medical trials with CBD have used _very_ high levels of CBD, and you simply can't obtain those levels with for example "1:1" CBD:THC strains.
However, a lot of trials also show the SYNERGY that CBD and THC have - THC often seems to positively increase CBD's effects (something the cannabis community has known for a long time, and is now finally getting scientifically affirmed). So, after your medicinal CBD oil drops, perhaps have "a quarter of a cookie" or something to introduce a small but manageable amount of THC. (Or the whole cookie, or four, whatever) 
Anyway, so that was the plate developed with just pure Chloroform as the mobile phase eluent ...
but I also use Hexane : Diethyl Ether (4:1) ... I'm still not sure which eluent I prefer (they both seem to have pros and cons, but also _complement_ each other nicely), and I've had nothing but success with it so far ...
... but NOT TODAY! Just one of those days unfortunately! ...







WHOA! Massive fail ..... the _extract_ didn't even get out of the starting blocks!!! (even though the _eluent_ crossed the finishing line!)

The only saving grace I guess, is that yes we can see the CBD Oil lane is clearly showing a fat orange CBD dot, while the C99 lane is clearly showing a fat red THC dot... it's not much information, but it's better than nothing and still definitely provides a _slightly_ clearer indication than _Beam's Test_, as Fast Blue BB is far more specific in indication of cannabinoids. But, for whatever reason, it failed to SEPARATE the substance into its lesser molecules, so we've basically put a bread in the oven and it's failed to rise due to some problem probably with the yeast.

In case you're wondering, yes the eluent did capillary-action all the way to the top (and you can see some minor staining at the top), so that's not the problem.

THE PROBLEM:

The extract failed to be moved up the plate via capillary action.
So, what's responsible for driving it up? there are two fundamental parts...

first we use Hexane to create the EXTRACT... we need to extract the cannabinoids from the target substance (Hexane is something of a molecular magnet)
then we use the eluent (eg. Chloroform, or Hexane : Diethyl Ether) for the MOBILE PHASE, where capillary action drives the eluent up to the top of the plate, separating molecules into groups in the process
I believe I've made a mistake in either of these two.

POSSIBLE CAUSES: (the only ones i can think of - it's a fairly straightforward procedure afterall)

MY BET: perhaps my hexane : diethyl ether measurement was slightly off (i don't know how sensitive the ratio is). In one of my first posts I showed experiments with different eluents that help show how important it is to use "the right eluent for the job (and there aren't many for this)", but I'm confident I was within 1ml of 8ml of hexane, and pretty sure I was close to the 2ml for diethyl ether (should be 4:1 ratio). I've never had a problem with it before, but measuring this time was trickier as my syringes now no longer reach into the ever-depleting level of eluent in the bottle, so I was happy at the time to be less precise. However, there is obviously a reason why the two are used together, and there's obviously a reason for the 4:1 ratio ... so, perhaps there is no elbow room there. Perhaps we need to be very precise with this mix. But in my previous experiments with different eluents, there was never this "gridlock" problem! Even water had no problem driving forward, albeit without separation. So, if this is the problem, the solution is of course simple: be precise ... it's not difficult, especially now in retrospect haha
UNLIKELY 2ND BET: perhaps I didn't use enough hexane in the extraction phase (possibly resulting in extract which is too 'sticky'), but it didn't affect the Chloroform plate.
UNLIKELY: I actually used 4 drops per lane, usually I just use 3, but surely that 1 extra drop couldnt have this much effect, especially as it didn't affect the Chloroform plate.
UNLIKELY: surely that ant that walked across the plate didn't leave a pheromone trail that could have this much effect, especially as it didn't affect the Chloroform plate.
I might be able to revisit this in the future to try to set up a couple of experiments which can test some of these possibilities to try to rule them out.

Due to lack of publicly available knowledge about this, I basically have no other option but to keep experimenting. Hopefully some of you will too


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## BabyLobsterito (Jan 15, 2018)

I greatly enjoyed reading this. Got me wanting to bust out the lab equipment...
Glad I decided to check in after several months!


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## raggyb (Jan 15, 2018)

I want to start with Beams test. See if I can handle it LOL


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## PhenoMenal (Jan 17, 2018)

raggyb said:


> I want to start with Beams test. See if I can handle it LOL


the beauty of Beam's test is how simple it is, and just two easy-to-source ingredients which are quickly and easily prepared ... if you can't handle Beam's test you'll probably struggle putting butter on toast too


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## raggyb (Jan 17, 2018)

Ia canna butter mya toasta


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## raggyb (Jan 17, 2018)

Just Jim Beam,...no, I mean KOH and Ethanol


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## raggyb (Jan 17, 2018)

But seriously man, this is really a cool post. Thanks for doing all that work and sharing!


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## Stink Bug (Jan 17, 2018)

First class post. Well done.


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## PhenoMenal (Jan 18, 2018)

raggyb said:


> Just Jim Beam,...no, I mean KOH and Ethanol


mate I'm not sure if Jim Beam's Test would work as that is < 50% ethanol, plus it's already coloured not clear so maybe it might work but would be tricky to read the result, but what I am sure of is that it would be a waste of bourbon. I know you were only joking, but I still hope you felt guilty as soon as you pressed Enter!

You can buy 95% ethanol like "Everclear" but its very expensive ... also you can distill your own but very time-consuming and i think 95.6% is the max you can get it to.

I just use *methylated spirits*, which is dirt cheap (~$3 / Litre) and can be sourced from local grocery store - dont even need to go to a hardware store usually, you can also order it online, and it's still ~95% ethanol, and visually clear.... basically just Everclear with another substance to prevent people drinking it... and BANG the price goes down! Gets the job done nicely, cheaply, and very obvious color differences when CBD is present, so I say just use methylated spirits - don't bother with pure ethanol... or whisky.


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## p0opstlnksal0t (Feb 1, 2018)

Have any of you found a chart/overlay for the plates like what alphacat's kit uses to get a "not too accurate" quantitative reading of the terpenes?


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## raggyb (Feb 1, 2018)

I don't know what that is but it looks pretty cool, stinksalot. I'm still messing around with the Beams test idea. Found a recipe for making your own koh. I'm going to try.


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## p0opstlnksal0t (Feb 2, 2018)

raggyb said:


> I don't know what that is but it looks pretty cool, stinksalot. I'm still messing around with the Beams test idea. Found a recipe for making your own koh. I'm going to try.


keep us updated bro. im just now looking at starting up TLC for myself and also charging a small fee for local Care Givers to get cheap tests done. The ability to actually quantify (Alpha-Cat says their tests are quantifiable to 1.0% accuracy) each terpene makeup would be an astronimical benefit as opposed to just seeing each terpenes ratio to the others. I know the only way this could be possible is if each input material is minutely measured to exacting precision. the solvent ratios, dye, mixed media, flower, etc... all have to be perfectly proportioned and measured so that the end resulting dot sizes are quantifiable in the end. I would either need to utilize Alpha-Cats charts for measurement or use some type of software to scan the plates and measure the resulting profiles and create a database of ranges. I would then need to pay to send in a few flowers that ive tested at home to a lab that is accurately quantifying these terpenes so that i could build this online database to use the data points to extrapolate my at home TLC data points.


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## raggyb (Feb 2, 2018)

cool. I'm going to start trying to make KoH this weekend.


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## PhenoMenal (Feb 3, 2018)

raggyb, "make KOH"??? !?!?!?? no! just buy it from ebay or other, it's very cheap, and you don't need much at all to do a Beam's test, so a little goes a long way  KOH is caustic too so it's quite different and more dangerous to say brewing your own ethanol! For TLC, making your own KOH is just unneeded extra time and expense.


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## PhenoMenal (Feb 3, 2018)

p0opstlnksal0t,
my advice is don't bother trying to get % from home TLC, there are too many variables that all need to be exactly 100% just to get semi-accurate readings. *TLC is a qualitative test, not quantitative.* You really need to send it to a lab if you want %'s you can rely on.

It's still a beautiful tool for revealing both the EXISTENCE and RATIO and APPROXIMATE QUANTITY and overall FINGERPRINT of cannabinoids though!!! just not accurate %.

You mentioned selling it as a service, but I feel you would be doing a disservice to your clients by using TLC to provide %'s because you could be off by as much as 5-10%, which is a huge error margin considering strains top out at ~30% THC. If people are willing to pay for such a test they generally want accurate results, and TLC's margin of error simply cannot accommodate that.

For example, in the AlphaCat image you posted for example, look at the 12% and 17% blobs:





... almost identical to the naked eye yet 5% difference. I would encourage you to provide a scan IMAGE of the plates to clients, rather than %'s, because it really is afterall a VISUAL test (hence the need for the special Blue B/BB dye!), a non-digital test, and one that _doesn't_ provide concrete numbers.


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## ANC (Feb 3, 2018)

Here is my favorite method


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## PhenoMenal (Feb 3, 2018)

ANC, smoking is an easy detection method ... but only for THC! not for CBD (or CBG, or THCV, or CBC, or CBN etc)

Let me flip this to you -- "Here's a bud, smoke it and tell me how much (_IF ANY_) CBD it has" ...  You simply cannot answer this question by smoking. (I was drawn to TLC because a family member was diagnosed with terminal cancer and was having a lot of good results with CBD, so we needed a way to detect with confidence)

Even if you give it to somebody in pain for example, they might smoke it and still give it a negative rating even if it has high CBD ... so clearly humans smoking is not a good indicator as to whether or not a strain has CBD etc.
This is where TLC (and to some extent Beam's test) really show their unique value. 

ps. having said all that, mate I have to agree with you though smoking is also my favorite THC detection method haha


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## raggyb (Feb 3, 2018)

PhenoMenal said:


> raggyb, "make KOH"??? !?!?!?? no! just buy it from ebay or other, it's very cheap, and you don't need much at all to do a Beam's test, so a little goes a long way  KOH is caustic too so it's quite different and more dangerous to say brewing your own ethanol! For TLC, making your own KOH is just unneeded extra time and expense.


Ok, stupid question, but how dangerous can it be if it's used for making soap. I found the instructions from soap makers. Soap doesn't exactly make my boots tremble.


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## PhenoMenal (Feb 4, 2018)

raggyb said:


> Ok, stupid question, but how dangerous can it be if it's used for making soap. I found the instructions from soap makers. Soap doesn't exactly make my boots tremble.


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

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


> The solution in water is a strong base. It reacts violently with acid and is corrosive to metals such as aluminium, tin, lead and zinc. This produces a combustible/explosive gas (hydrogen - see ICSC 0001). Reacts with ammonium salts. This produces ammonia. This generates fire hazard. Contact with moisture and water may generate heat.


KOH pH = 11

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

Just buy it mate, no need to make it especially as it's cheap


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## raggyb (Feb 4, 2018)

PhenoMenal said:


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


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


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## raggyb (Feb 4, 2018)

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


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## raggyb (Feb 4, 2018)

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


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## PhenoMenal (Feb 4, 2018)

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


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## PhenoMenal (Feb 10, 2018)

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





I took these screenshots from it and added labels for clarity:






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

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

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

*Can anyone with a chemistry background please shed some light on Beam's Test?
(((Potassium hydroxide + Ethanol) ... add CBD-heavy-cannabis) ... = colour change)
*
Sadly there is very little info currently available on the web about this, and most international laws prohibit such experiments (due to the apparent "illegal material" that is medical marijuana)

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


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## PhenoMenal (Feb 17, 2018)

*Thin Layer Chromatography to detect Zika and Dengue virii?
*
I _have_ to momentarily hijack this TLC thread from detecting cannabinoids to detecting virii ...

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

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



> The lab of another CRISPR pioneer, Feng Zhang, has now improved on a previous system it developed last year. SHERLOCK, as it's called, can detect specific bits of DNA and RNA to determine whether viruses like Zika or dengue are present in a blood sample, identify mutations in tumor DNA and spot the presence of harmful bacteria. In their latest study, the research team describes SHERLOCK version 2.0, which is not only over three times as sensitive as the first version, but can also detect both Zika and dengue in the same sample. Their system uses several CRISPR enzymes, including Cas13 and Csm6, *and can be loaded onto a paper strip*, making it incredibly easy to use. You can see examples of the strips in the GIF below.


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

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

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

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






*Notice the CAPILLARY ACTION, and im guessing silica-based strips...*

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

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

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

It seems the blue line highlighted in a red circle in the "Control" strip is where the "sample" was positioned on the plate (our equivalent of putting a small dot of cannabinoids in hexane at the bottom of a TLC plate)


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## ANC (Feb 17, 2018)

raggyb said:


> Yeah I saw some of these warnings, including "slowly dissolves glass"


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


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## raggyb (Feb 17, 2018)

ANC said:


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


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


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## PhenoMenal (Feb 21, 2018)

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



raggyb said:


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


i'm not sure, but i know hydrofluoric acid also quickly dissolves glass. Potassium hydroxide solution MSDS = "Slowly dissolves glass"


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## PhenoMenal (Mar 27, 2018)

*In this update of Me, Beam & TLC ...*

I make a TLC mistake ... _there's a weird twist._
I test "CBD Therapy" for the first time... _i got a ~1:1 pheno_.
I test "Dinamed CBD" again (a sativa-dom pheno this time)... _and it's not good news_.
I test to see how early in the veg and flowering periods we can see the cannabinoid profile... _never been done before, but it's very good news_.
I do Beam's Test alongside TLC for comparison ... _never been done before_.
I still lament that more people aren't doing TLC ... _i'm just talking to myself here!_

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

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

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

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

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

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

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

From https://cbdcrew.org/varieties/cbd-therapy/ ...


> 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. Some closer to 5:1 even a few 2:1, so CBD Crew did an extensive new round of testing of the latest seed crop to see if the variations occurred often. They found that 50-75% of the CBD Therapy seeds will have very low THC, high CBD, but 25-50% could have higher THC. No seed will produce only high THC, always both CBD/ THC. Never seeds with higher THC than the CBD, but variations from 20:1 to 2:1 can occur. In every package, there will be one or more low THC, high CBD phenos.


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

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

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






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

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






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

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

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

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

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

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

First let me define the sample lanes ...






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

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

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






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

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

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

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







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


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## raggyb (Mar 28, 2018)

PhenoMenal said:


> *In this update of Me, Beam & TLC ...*
> 
> I make a TLC mistake ... _there's a weird twist._
> I test "CBD Therapy" for the first time... _i got a ~1:1 pheno_.
> ...


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


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## PhenoMenal (Apr 2, 2018)

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







Left: approx 1:1 but slightly more THC than CBD
Mid: High level THC
Right: Medium level THC


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## raggyb (Apr 2, 2018)

PhenoMenal said:


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


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


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## the podencoid (Apr 7, 2018)

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

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

Cheers.


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## PhenoMenal (Apr 7, 2018)

the podencoid said:


> It's inspired me to take up tlc testing again after a long hiatus.


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

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



> Your thread has helped put all the pieces of the jigsaw together


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



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


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


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## the podencoid (Apr 7, 2018)

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

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

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

Cheers.


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## PhenoMenal (Apr 7, 2018)

the podencoid said:


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


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

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



> Just fyi, #3 is a Z7 a friend in Ireland found when we shared a packet years ago - now released as 'Therapy' from Shantibaba/CBD Crew. Shantibaba's plants always have a nice terpene profile.


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

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

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



> The first two are Swamp Serum from Greenpoint and RUNCBD from Twisty Treat - both great plants, especially the RUNCBD's and no high THC in them so far. The last one is Charlotte's Cream from Twisty but all four I grew out were high thc unfortunately.


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



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


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


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## the podencoid (Apr 11, 2018)

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

Hope your family member is doing ok.

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

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

All the best.


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## fookinel (Apr 12, 2018)

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

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

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

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

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

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

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

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

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

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

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


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## fookinel (Apr 12, 2018)

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...


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## PhenoMenal (Apr 13, 2018)

fookinel said:


> 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.
> 
> ...


BEAUTIFUL INFO mate


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## PhenoMenal (Apr 13, 2018)

fookinel said:


> 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


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## fookinel (Apr 14, 2018)

PhenoMenal said:


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


Sweeeet mate! 



PhenoMenal said:


> 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...



PhenoMenal said:


> 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



PhenoMenal said:


> 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!



PhenoMenal said:


> 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.



PhenoMenal said:


> 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.



PhenoMenal said:


> [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.



PhenoMenal said:


> 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.



PhenoMenal said:


> 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!


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## PhenoMenal (Apr 14, 2018)

fookinel said:


> 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.


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## Wilksey (Apr 14, 2018)

PhenoMenal said:


> 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.


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## PhenoMenal (Apr 14, 2018)

Wilksey said:


> 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


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## fookinel (Apr 15, 2018)

PhenoMenal said:


> 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.



PhenoMenal said:


> "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.



PhenoMenal said:


> 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?



PhenoMenal said:


> 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.


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## PhenoMenal (Apr 16, 2018)

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!?)


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## PhenoMenal (May 3, 2018)

*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] ...


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## raggyb (May 3, 2018)

PhenoMenal said:


> *TLC to detect TERPENES - just use a different dye!*
> 
> We use a dye like Fast Blue BB or Fast Blue B to visualize _cannabinoids_ ...
> 
> ...


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


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## PhenoMenal (May 3, 2018)

raggyb said:


> 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!


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## raggyb (May 4, 2018)

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


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## PhenoMenal (May 4, 2018)

*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)...


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## PhenoMenal (May 5, 2018)

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


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## PhenoMenal (May 6, 2018)

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


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## PhenoMenal (May 11, 2018)

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.


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## dogcat (May 15, 2018)

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>


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## PhenoMenal (May 16, 2018)

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


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## Sour Wreck (May 16, 2018)

following.

gonna use some of this in the future


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## dogcat (May 16, 2018)

PhenoMenal said:


> 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


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## PhenoMenal (May 16, 2018)

dogcat said:


> 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


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## dogcat (May 21, 2018)

PhenoMenal said:


> 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


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## guerreiroweed (Jun 10, 2018)

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...


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## PhenoMenal (Jun 11, 2018)

guerreiroweed said:


> 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)


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## guerreiroweed (Jun 11, 2018)

PhenoMenal said:


> 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)





PhenoMenal said:


> 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?


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## PhenoMenal (Jun 11, 2018)

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


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## cogitech (Jun 20, 2018)

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.


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## raggyb (Jul 20, 2018)

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.


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## PhenoMenal (Jul 23, 2018)

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


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## raggyb (Jul 24, 2018)

PhenoMenal said:


> 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.


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## PhenoMenal (Jul 26, 2018)

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


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## rocho (Nov 24, 2018)

Thankyou so much fpr sharing!!


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## jjng5 (Dec 23, 2018)

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...


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## OldMedUser (Dec 24, 2018)

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!


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## CannaBruh (Dec 24, 2018)

excellent work and thanks for sharing @OldMedUser


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## PhenoMenal (Jan 2, 2019)

jjng5 said:


> @ 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.


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## febisfebi (Jan 31, 2019)

@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!


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## budchemist (Feb 1, 2019)

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


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## PhenoMenal (Feb 9, 2019)

febisfebi said:


> @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!


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## SSR (Mar 11, 2019)

Quick questions for you PhenoMenal

Have you tried HPTLC plates?
Would they provide a better resolution given the smaller particle sizes and thinner deposition layer?

I know they cost a bit more but tbf sigma has them at relatively good prices comparatively speaking (around 25-30 per plate in 10 packs depending on substrate and coating reqd).

Benefits seem to be better resolving power, faster development times, lower sample diffusion, and reduced solvent consumption


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## PhenoMenal (May 1, 2019)

I've never tried HPTLC plates so i can't comment sorry! I'm not sure how much more advantageous they'd be though in regards to asking the basic question "which cannabinoids does this strain have, and in which (approximate) ratios", so the extra price not be worth it. It'd be awesome to hear from somebody trying them though! btw in case you're not aware, unfortunately Sigma doesn't sell to the general public.


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## Apical Bud (May 23, 2019)

Hey PhenoMenal, over the past year I have tried TLC off and on but I have a simple problem. My cannabinoids bleed. I have extracted with acetone, ethyl acetate, hexane - you name it. And although some eluents have worked better than others, even with chloroform my plates produced a line of large splotches rather than dots. I have never gotten a plate that looks as neat as those in your pictures. What do you think I'm doing wrong?
I've made plates out of corn starch, plaster of paris, alumina; I've bought alumina plates, and I've used lab-grade HPTLC plates (yes they are much much better). I have yet to get a trustworthy result.

I have pictures if you want them. Thanks for reading.


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## PhenoMenal (May 28, 2019)

Apical Bud said:


> my plates produced a line of large splotches rather than dots. I have never gotten a plate that looks as neat as those in your pictures. What do you think I'm doing wrong? I have pictures if you want them


my first guess is you're not filtering ... i got splotchy results before i started using coffee filter papers (Fast Blue BB can be quite clumpy). I'd also avoid using your own homemade plates until you get things sorted out using commercial plates, to ensure the plate isn't the problem (i personally wouldn't bother using homemade plates). But yes you'll need to post pictures/scans if you want any more debugging help with that.

Here's one of my first runs before i started filtering... so splotchy:


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## Apical Bud (May 29, 2019)

View attachment 4342023 View attachment 4342024


PhenoMenal said:


> my first guess is you're not filtering ... i got splotchy results before i started using coffee filter papers (Fast Blue BB can be quite clumpy). I'd also avoid using your own homemade plates until you get things sorted out using commercial plates, to ensure the plate isn't the problem (i personally wouldn't bother using homemade plates). But yes you'll need to post pictures/scans if you want any more debugging help with that.
> 
> Here's one of my first runs before i started filtering... so splotchy:


Top is from a fancy smancy lab plate from long ago. I only posted this to show the cripsness they can provide. The other two were run recently on alumina-based plates from amazon. As you can see, the fancy plates under UV fluoresce very well.

But my main question is about the lower two pictures. Even though I have no indicator on the plates, you can see that there is a ton of smearing. Do you see something similar before you add your indicator?

BTW, I don't have the black light that I used in the top picture anymore, so I used a UV-C germicidal lamp and it doesn't seem to fluoresce cannabinoids. I don't have access to fast black k, because I don't work at a lab anymore so I can't order it from chemical companies. I can buy another cheap uv light on amazon but it won't do any good if I can't get separation. The thing is I'm not using homemade plates anymore, they just aren't the high grade lab plates we were talking about earlier.

Also, I did send in the sample I used in the first lane to a local lab and it came back 7.7% CBD, 4.3% THC.


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## PhenoMenal (May 29, 2019)

doesnt look too bad, you might be applying a bit too much on each spot though. what eluent are you using? i get quite different separation from chloroform than i do from diethyl ether & hexane mix


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## Apical Bud (May 30, 2019)

PhenoMenal said:


> doesnt look too bad, you might be applying a bit too much on each spot though. what eluent are you using? i get quite different separation from chloroform than i do from diethyl ether & hexane mix


I used chloroform in the lower two pictures. Also, in my last post I meant to say Fast Blue B, not Fast Black K. Fast Black K is toxic and doesn't differentiate between cannabinoids.


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## PhenoMenal (May 30, 2019)

Fast Blue B is also toxic -- apparently they created Fast Blue BB because of that. BB is still toxic though, just not as much.

I like the separation chloroform gives, seems to be better than hexane/diethyl ether, and has the simplicity of only needing the one chemical.


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## febisfebi (Jul 8, 2019)

PhenoMenal said:


> just a few  will do my best to answer them
> 
> 
> 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) "
> ...



OK so first off, thank you so much for all your help. I was able to perform the Beam's test, and I am just now finding the time to get the rest of the chemicals together. 
I wanted to check with you that the molecular formula and CAS numbers are both the correct product for the following 3 chemicals from my supplier:

Fast blue BB Salt
*CAS#:* 5486-84-0
*Formula: *C34H36Cl4N6O6Zn

Chloroform
*CAS#:* 67-66-3
*Formula:* CHCl3

Hexane
*CAS#: *110-54-3
*Formula: *CH3(CH2)4CH3
From chemical supplier's website

CAS# 110-54-3 ----same as first one
Formula: C6H14 

I am guessing they are likely technicaly the same formula, since the CAS # is the same. The first one just broken down more.

Hexane is the particularly confusing one because there is all different products containing hexane. The second one is the one I am looking at getting, and is from ebay, and advertised as 99.99% reagent grade.

The first molecular formula was pulled from a lab supply that is much more expensive but also reagent grade.

If you could please check this information against the products you have obtained that you have proven work well, I would really appreciate it before I invest in chemicals. Hopefully the info will help somebody else as well.
Thank you!


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## PhenoMenal (Jul 16, 2019)

the bottle of *Hexane* i used is "Hexane Fraction (AR)" (analytical reagent). 
The formula on the side says: C6 H14
(I simply contacted a local lab supplier and said "i need a small bottle of Hexane for Thin Layer Chromatographyy", to ensure I was getting a suitable type ... basically you need a high purity one like AR grade - something over 98%)

the bottle of *Chloroform* i got is 99.8% pure. Again i simply contacted a local lab supplier and said "i need a small bottle of Chloroform suitable for Thin Layer Chromatography"
the formula on the side says: C H Cl3 (the same as yours)

*Diethyl Ether* isnt required if you have Chloroform, but it does make for another great eluent when mixed with Hexane - it provides a different separation.
It's a seriously hardcore nose assaulter though. You really need to hold your breath when using it. Hexane and Chloroform are quite mild by comparison. Seriously though, for first time TLC i would recommend just using Chloroform instead of Hexane : Diethyl Ether
Mine is 99.5% pure, the formula says: (C2 H5)2 O

the *Fast Blue BB Salt* (the magic ingredient) i got i also got from a local lab supplier who imported it for me from from an Asian manufacturer called "Wako". It's called "Fast Blue BB Salt". It should be delivered to you packed with dry ice - keep it in your freezer. Mine seems to work just as well today as when I got it two years ago, so yes it does seem to keep well in a regular home freezer, and I get the sense my tiny vial will successfully last many years.
Formula C17 H18 CIN3 O3
search for "062-05463" or "CI 37175"
There are 2 different forms of Fast Blue BB i'm aware of, make sure you get the right one. It's a yellow salt. I'm not confident that the one you're referring to is the correct one.

But really, the *best advice* I can give you is simply contact your local lab suppliers and ask them to point you towards exactly what you need  (worked perfectly first time for me!)
Due to the nature of chemicals it's probably a good thing to emphasize you only want a SMALL bottle (which is all that's needed anyway), and that its purpose is simply Thin Layer Chromatography. It actually was that simple for me.


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## Saffasteve (Jul 18, 2019)

Have you had any comparison tests done at a lab to see how accurate this method is ?


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## shn3ak (Jul 23, 2019)

PhenoMenal said:


> 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!
> 
> 
> 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


This was an amazing thread mate! thanks very much for posting, will definitely give this a go! and the level of detail you've gone into is nuts. Many thanks!


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## PhenoMenal (Aug 11, 2019)

Saffasteve said:


> Have you had any comparison tests done at a lab to see how accurate this method is ?


Thin Layer Chromatography (TLC) is not specific to cannabis, it's a scientific procedure with a LOOONG history, and I encourage you to look into it which will also answer your question.
Wikipedia: https://en.wikipedia.org/wiki/Thin-layer_chromatography
(as a simple example, Thin Layer Chromatography can be used to show children how black ink in a pen is actually comprised of multiple inks of different color)


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## Saffasteve (Aug 12, 2019)

PhenoMenal said:


> Thin Layer Chromatography (TLC) is not specific to cannabis, it's a scientific procedure with a LOOONG history, and I encourage you to look into it which will also answer your question.
> Wikipedia: https://en.wikipedia.org/wiki/Thin-layer_chromatography
> (as a simple example, Thin Layer Chromatography can be used to show children how black ink in a pen is actually comprised of multiple inks of different color)


How was that link supposed to answer my question ?


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## PhenoMenal (Aug 12, 2019)

Never mind.


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## cannabisgenetics (Aug 15, 2019)

f


PhenoMenal said:


> the bottle of *Chloroform* i got is 99.8% pure. Again i simply contacted a local lab supplier and said "i need a small bottle of Chloroform suitable for Thin Layer Chromatography"
> the formula on the side says: C H Cl3 (the same as yours)


Cannot thank you enough PHENOMENAL!!!! I read about TLC in an attempt to circumvent the expensive labs in 2012 but once i saw the process, I was quick to write it off as to complicated. This thread demistified it for me and now I feel like this is one of the most valuable tools one could have. I've been testing young genotypes leaf samples (dont have space to allow for mature selections) with chloroform about 3 sets of tests so far and feel like I have the hang of it but wanted to see if you had any tips for me to fine tune.

When testing leaf samples (plants in veg), where is the most potent part of the plant? Pieces to avoid? (stem, leaf tips, leaf base, petiole,etc.)

If you are to run one lane per plant, what is the most informative? Seems like a cold print provides a smaller area to interpert and so I have gravitated to the 4ul hot print - thinking largest spots to identify (more likely small pressences will be noticed?)

Is there a option thats better at showing potency, seems like my veg samples are similiar in potency and just showing a precense of Cbs not really quantifiable. I would love to know which candidates have to highest potential (i know you prefaced with this notion, but was hoping if i can presicion everything (exact wieght, same part of plant, extraction solvent amount - i have a 2ul and a 2ml pipette) What is the most important measurement (or step) to insure accuracy accross the board / higher likelyhood of identifying higher potency varitals?

What are modulators and how do you interpret them? What info do they add?

Occasionally i see a spot at the top above the major group, is this geranyal? What does that usually indicate about potential. 

In an effort to start to create some baseline, i attempted to use the Justquantify site to set some real numbers. Am i doing this correctly? I had to redraw some spots because the auto feature was grouping some Cbs together. Is there any other spots/ data on the test i should be quantifying? Purplish spots at top? Purplish spots on bottom? In this 4ul hot test, should i be trying to quantify the acid chain at all?
Here is a test of 3 females and 1 male of the progeny of a high thcv mother and thc father:




 

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

Again Phenomenal, you are the f*ckn man! If you ever come to cali, i will roll you the biggest fatty you every wittnessed


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## curious2garden (Aug 15, 2019)

@cannabineer figured you might enjoy this thread


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## SmokingCrow (Sep 10, 2019)

For similar health reasons, I'm specifically looking at CBD levels. I was about to do down the "beam's test" wormhole and expect TLC results. Although TLC and the process look complex, this thread has made it really simple. Now all I need to do is get the chemistry set together and do it. 

Thank you @PhenoMenal


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## PhenoMenal (Oct 23, 2019)

cannabisgenetics said:


> I was quick to write it off as to complicated. This thread demistified it for me and now I feel like this is one of the most valuable tools one could have.


Yep, I'm not aware of a more powerful tool that's available for average home users. The process itself as you've found out is also quite simple and doesn't take very long either. (I mean you don't have to get all the items yourself if you dont want as it's also available in KIT FORM specifically for home users afterall!)
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

If you're after some more tips, check out this thread where I was originally carving out the protocol, lots of trial and error - there's a lot of reading, but I guarantee you it'll take less than the ~3 months it took me: https://www.icmag.com/ic/showthread.php?threadid=148375

btw keep in mind im using Fast Blue BB whereas you're using Fast Blue B (i can tell as B is darker, more pastel-like and less-vivid), so keep in mind your results will be slightly different to mine. but they both seem to get the same job done beautifully! but seeing as you're using Fast Blue B, if i were you I'd be inclined to go by alpha-CAT's user manual in regards to which cannabinoids are which, as they also use Fast Blue B. I cant recall which eluent they use though - probably chloroform being one-part. This is an image from their website:






And yes my understanding is that the topmost lightly purple spot are probably waxes like geranyl, whereas down the very bottom are unconverted/carboxylated acids (THCA, CBDA, CBGA, etc)


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## PhenoMenal (Oct 23, 2019)

SmokingCrow said:


> For similar health reasons, I'm specifically looking at CBD levels. I was about to do down the "beam's test" wormhole and expect TLC results. Although TLC and the process look complex, this thread has made it really simple. Now all I need to do is get the chemistry set together and do it. Thank you @PhenoMenal


 btw just in case you haven't seen it, check out this thread where i use TLC on 3-week-old-seedlings to check for CBD:





CBD Blitz: Find a high-CBD low-THC mother, from seed, in only 3 weeks


In just 3 weeks, you can go from ungerminated seeds to finding a mother with the cannabinoid profile of your desire (high CBD/low THC for example). Allow me to explain ... It can be incredibly disheartening to spend several months growing a plant that was advertised as "high CBD low THC" only...



rollitup.org


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## cannabisgenetics (Dec 12, 2019)

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


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


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


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## DrTricoma (Jan 12, 2020)

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.


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## cannabisgenetics (Jan 22, 2020)

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


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## DrTricoma (Jan 22, 2020)

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!!!


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## PhenoMenal (Jan 25, 2020)

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.


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.


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


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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## Leef (Jan 28, 2020)

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


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## DrTricoma (Jan 28, 2020)

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.
> 
> ...


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!


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## PhenoMenal (Jan 28, 2020)

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...





YouTube


Enjoy the videos and music you love, upload original content, and share it all with friends, family, and the world on YouTube.




www.youtube.com


----------



## Leef (Jan 28, 2020)

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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## PhenoMenal (Jan 28, 2020)

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.


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.


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## DrTricoma (Jan 28, 2020)

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!


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## Leef (Jan 29, 2020)

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 8 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.





PhenoMenal said:


> use a vanillin reagent and TLC visualises the terpenes also


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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## DrTricoma (Jan 29, 2020)

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
> ...



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


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## Leef (Jan 29, 2020)

DrTricoma said:


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


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.









MilliporeSigma™ Silica Gel 60 F<sub>254</sub> Coated Aluminum-Backed TLC Sheets | Fisher Scientific


Allows analyzation of any substance when combined with suitable mobile phase




www.fishersci.ca





This place has everything, local pick up.

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









50.79C$ |80pcs/box 25*80mm 25*100mm 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


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## PhenoMenal (Jan 29, 2020)

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.


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## Truckn (Feb 19, 2020)

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.


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## cannabisgenetics (Feb 25, 2020)

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.


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


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## DrTricoma (Feb 25, 2020)

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.


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## ryanrayla (Feb 28, 2020)

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.


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## PhenoMenal (Mar 5, 2020)

ryanrayla said:


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


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.


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## ryanrayla (Mar 6, 2020)

I did try ordering from one supplier (Santa Cruz Biotechnology) and they emailed saying they couldn’t ship anything to a residential address. I’ve since checked with about 10 different companies and everyone advertises that they won’t ship to residential addresses or that they “inform law enforcement of any unusual purchases.” To my knowledge it’s not illegal to posses these chemicals, but I don’t want to work with any company that threatens its customers before even ordering.

I have been using the tiny sample of Fast Blue BB that came with a test kit I bought from cannalyticssupply.com. They are selling a replacement vial that holds 150mg of Fast Blue BB for $15, which I’ve since reluctantly purchased. That’s $0.10/mg from the test kit vendor versus the $0.005/mg from Santa Barbara Biotechnology. I’m still looking for a larger quantity to lower the per unit cost.


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## PhenoMenal (Mar 6, 2020)

You're correct, it's not illegal to possess Fast Blue BB (it is not a drug, it is not a precursor, it is not an explosive, it is not a controlled substance, etc etc, it's essentially just a dye). When you email labs, mention that you want to use the Fast Blue BB "specifically for the visualisation of phenolic compounds in strawberries". [Google: "Fast Blue BB" strawberries]

ps. if you got a test kit, it's more than likely Fast Blue B (not BB) that you received, as B doesn't require being kept in the freezer like BB does (you should receive BB packaged with dry ice). I've never personally used B myself, but I do love the extra vibrancy of BB, and BB was created due to safety concerns about B, so definitely go for BB if you can ... B still makes a very good second option though!


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## ryanrayla (Mar 31, 2020)

Thank you for the reply, @PhenoMenal. I ended up purchasing the 150mg of unrefrigerated "visualization dye" just to keep moving forward.

In almost every test I've ever done, I've had a hard time getting the spots to be round enough to accurately measure using some rulers I purchased that are calibrated for that purpose. I've attached two plates below as an example. Lane 1 on both plates was created from the same sample which was plated and developed under the same conditions 9 days apart. The THC spot on the first plate, resolved beautifully while the spot on the second plate was vertically compressed. I've seen this phenomenon on nearly every plate I've developed but haven't been able to understand why.

Eluent: 100% Chloroform
Solvent: 100% Hexane
Plate: Silica on Glass
Plated with: 2ul Micropipette.

Do you have any thoughts as to what might be causing the vertical compression in my spots?


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## cdsmith12 (Apr 11, 2020)

@PhenoMenal So when Missouri passed medical marijuana and allowed patients to home grow, I built a grow room and started growing, mainly to make RSO/FECO because I have severe chronic back pain, and narcolepsy. Due to the narcolepsy I needed to grow low THC strains so I grew ACDC and Harlequin from regular seeds and had 4 females of each. When I started making FECO, I noticed that it really wasn't helping as much as the Hemp FECO I had been getting so I researched testing and decided to purchase a TLC kit from thctestkits. After being intrigued by the process, but scared to test too many due to the cost of the refill kits, I started doing some research on buying my own supplies and found your thread. Excellent information by the way.

After reading the entire thread, I realized that my kit, as well as Grow Buddy/Montana Bio Tech and Alpha Cat are all about the same. All of them have one bottle of liquid for both the eluent and solvent, and of course no label, but I am assuming its hexane. In one of your earlier post you mentioned that you were going to try hexane without the diethyl either for both, but unless I missed it, I didn't see the result. I was wondering if you ever tried this? Or do you know what they are using that would allow one chemical for both? The plate on the left was soaked in the extraction tubes for about 15 mins in accordance with their instructions for flower. The plate on the right is the same extraction tubes that I tested again the next morning. I screwed up and didn't let it dry before spraying the dye so the result is messy, but the THC and CBD are higher. On each plate, the two on the left are indica strains and the two on the right are Harlequin and ACDC.


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## PhenoMenal (Apr 14, 2020)

> All of them have one bottle of liquid for both the eluent and solvent, and of course no label, but I am assuming its hexane.


There are several variations of chemicals that people can use for TLC of cannabinoids at home, and as I've experienced and documented they all produce unique results.

When "reverse engineering" this TLC process it was helpful that they provided MSDS safety data sheets, which showed which chemicals/substances they were using.

I've already documented everything I've tried (mostly on the icmag TLC thread, as I was there while I was learning the process), so I don't want to waste time rehashing anything, so please check that, and the links in my signature

ps. You're getting some good results there, clearly showing your Harlequin and ACDC have high levels of CBD  (and your indica obviously showing heavy levels of THC)

Your 1st and 2nd lanes are just typical beautiful heavy-THC indica bombs 

Your 3rd lane suggests an approx 1:1 THC:CBD ratio, which I know my bro-in-law found to be a very helpful ratio during chemotherapy.

Your 4th lane suggests a lot more CBD than THC!, which might be preferable to people who want medicine with minimal 'stone effect'

Beautiful plates bro


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## PhenoMenal (Apr 14, 2020)

ryanrayla said:


> Do you have any thoughts as to what might be causing the vertical compression in my spots?


I'm not quite sure what you mean by 'compression', but if you mean it's in regards to the outer lanes having weirder effects than the middle lanes, yes that's the effect I got from using Chloroform rather than Hexane+Diethyl


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## PhenoMenal (Apr 14, 2020)

I just wanted to say CONGRATULATIONS to all the people in this thread who've "taken the plunge" by taking the time and expense to learn Thin Layer Chromatography ... *TLC is literally THE MOST POWERFUL ANALYSIS TOOL available to home growers*, and it gives me warm fuzzies of people who've taken advantage of this tool, and to see people posting their TLC plates with clearly distinguishable CBD and THC etc, and seeing their reactions now that they have this new power or ability, it's beautiful 

ps. Don't forget, if you use TLC you now have the ability to detect the cannabinoid profile of plants when they're only a few weeks old (see my signature - "Find a CBD mother in 3 weeks from seed")


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## cdsmith12 (Apr 15, 2020)

@PhenoMenal I am getting ready to order my own stuff instead of getting a refill from the company I got my kit from and I have read tons of post about Fast BB. I'm not sure if I'm allowed to post a link so I will describe what I'm seeing on Sigma. When I type Fast Blue BB it comes up with several, but under the CAS number you have given as reference it has two. One says "Dye Content >=80%" which is $66.40 for 5g, the other says "microscopy (Hist)", for $27.50 for 10g, but this one also says for Cannabis Analysis in the General Description. Is this the one to get, the price is what made me question it because I've seen many other members post they paid $65 and up for 5g. Thanks for all your doing, and I did read the other post all the way through that you referenced in my past post.


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## PhenoMenal (Apr 15, 2020)

I can't really blindly recommend one or the other, but if the CAS number is correct then it should be good. 5gms goes a long way


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## DrTricoma (May 7, 2020)

PhenoMenal finally I got all items to follow your teachings and here are the very first results. I am very very happy, thank you for your generosity!


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## PhenoMenal (May 19, 2020)

DrTricoma said:


> PhenoMenal finally I got all items to follow your teachings and here are the very first results. I am very very happy, thank you for your generosity!


Congratulations, those are some great first results! And I see you've found some CBD too, as well as some healthy CBG levels in some!


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## DrTricoma (May 19, 2020)

PhenoMenal said:


> Congratulations, those are some great first results! And I see you've found some CBD too, as well as some healthy CBG levels in some!


Thank you! your teachings, PhenoMenal. you are the man!


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## DrTricoma (May 19, 2020)

DrTricoma said:


> Thank you! your teachings, PhenoMenal. you are the man!


All this strains are from my garden, quite interesting to see how the cannabidols evolve with the decarboxilation time and temp


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## PhenoMenal (May 20, 2020)

DrTricoma said:


> All this strains are from my garden, quite interesting to see how the cannabidols evolve with the decarboxilation time and temp


Good luck with your decarboxylation/time tests!
Here are some of mine I prepared earlier lol ... I only tried at 125C/257F and 135C/275F though (which is about as low as my oven can go)
Two different strains (both with interesting cannabinoid profiles), both tested in two different eluents (you can see both eluents have their own unique pros and cons) ...


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## Hlusaf (Dec 19, 2020)

PhenoMenal said:


> Good luck with your decarboxylation/time tests!
> Here are some of mine I prepared earlier lol ... I only tried at 125C/257F and 135C/275F though (which is about as low as my oven can go)
> Two different strains (both with interesting cannabinoid profiles), both tested in two different eluents (you can see both eluents have their own unique pros and cons) ...


Thank you for the outstanding walkthrough, I'm extremely grateful and eager to test some plants. This is one of the best threads I've seen! If you ever need or want anything, please let me know. Cheers.


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## Takudzwa (Jan 6, 2021)

Hey guys, which solvent systems can I use for TLC to separate CBD in MCT oil base?


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## Broos (Jan 16, 2021)

I I am very new to this and have read through the thread, and it is very helpful. I am having a very hard time getting a strong reading from coconut oil. An even harder time with alcohol tincture. 
Does anyone have any specific directions, inclusing amounts of oil and reaction fluid? 

The third and fourth lanes are the oil. I did a hot test on the first two lanes (flower) and no heat on the other two as they were already decarboxylated.

Oil was 0.1ml and solvent was 1ml


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## Robin_hearts_Science (Mar 3, 2021)

Really excellent tutorial! Thank you for taking the time to experiment and to share your results. 

As a chemist, I'd like to add a couple small (hopefully helpful) suggestions. 
1) You can purchase a box of glass pasteur pipettes (available on amazon or other sources) instead of using the micropipette (which is pricey to buy). Just dip the glass end of the pasteur pipette in your cannabis solution and dot it onto your TLC plate (just as you suggested using a metal pin head for).
2) Add your solvent to your TLC jar early and close the lid. Letting the solvent sit in the jar allows it to establish equilibrium with the evaporated vapors in the jar and will give you better/more precise capillary results.
3) You can draw a circle around the separated cannabinoids on your TLC plate and label them to review later if the dye is fading.

There is a lot more information about CBD and THC available now than a few years ago. CBD is present in small amounts in the plant but the majority of it is in an acid form called CBDa while it is in the plant. Heat is required to convert the CBDa to CBD (a process known as decarboxylation or decarb). It might give more accurate results of CBD content if you decarb your plant material prior to extraction for your TLC analysis. (Note that the same decarb process is necessary for THC as well.)


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## PhenoMenal (May 19, 2021)

Robin_hearts_Science said:


> CBD is present in small amounts in the plant but the majority of it is in an acid form called CBDa while it is in the plant. Heat is required to convert the CBDa to CBD (a process known as decarboxylation or decarb).


Yes I always decarb before testing with TLC


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## DancesWithWeeds (May 28, 2021)

Hay, PhenoMenal,

I've been following you for about 6 or 8 months now and really have learned a lot from you.

I was just messing around in my lab and came up with something you might be interested in. I am looking for easier and less expensive ways to visulize the TLC plates. This didn't work on the TLC plates (f254) but I tried it on a filter paper and got unexpected results.

This was just a time passer while I wait on chemicals and no care was taken (on this plate). The paper was spotted with some old oil and put under a UV light after running. Not much happened. I soaked the paper in UV ink, the kind they use to stamp your hand. A little more showed up. THEN, about a half cup of water and about 15 drops of green food coloring and I got what you see in the picture. I think it was about half/half hexane and acetone.

Later experiments showed that the UV ink has to be put on after the plate has been run. After reading what you can with UV dip in in the green dye and the colors seem to come out fairly nice. Another thing, shin the UV light thru from the back and it makes it easier to read.

This may be nothing, but it's worth looking at.


In about a week or so I'll try it again with the right solvents and a lot of TenderLovingCare.

DancesWithWeeds


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## DancesWithWeeds (May 30, 2021)

Sorry about that. I was stoned and had one those "profound revelations ".

What was happening was the dye (food coloring) is in water and didn't soak into the paper where the oil was. The paper got dyed, not the sample. Besides, the ink is organic so it wouldn't work anyway.

DancesWithWeeds


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## PhenoMenal (Jun 19, 2021)

DancesWithWeeds, sorry but I have no experience with UV (f254 etc). I don't think I'd be a fan of it though - a bit of extra equipment required (you're lucky to have a lab but most of us don't), and it looks like you're only getting essentially grayscale images vs the colour images the rest of us are getting, and it's the different colours that help easily distinguish which cannabinoid is which (along with vertical position of course). I'll personally stick to non-UV chromatography! Just get silicone TLC plates, and Fast Blue BB as the dye, and it's like when Dorothy opened the door to Munchkinland in Wizard Of Oz


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## PhenoMenal (Aug 6, 2021)

many of my original images/photos/scans no longer appear, which makes my tutorial tricker to understand, BUT they've been archived by the Wayback Machine! (archive.org)
So you can find them here:








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


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...




web.archive.org


----------



## LukeTW (Jun 25, 2022)

PhenoMenal said:


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


Thanx very much Bro.

This is my run With Clorophorm and wih Metanol:H2O:Acetic. 
your Tutorial its the Best.


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## DancesWithWeeds (Jun 26, 2022)

LukeTW said:


> Thanx very much Bro.
> 
> This is my run With Clorophorm and wih Metanol:H2O:Acetic.
> your Tutorial its the Best.


I see you are a new member. Welcome.

I think this thread is dead now. I hope not. If it is maybe we can hit it started back up. I've been wanting to start a thread "Legit MMJ Home Labs". The lab would be wherever or what ever you use to to make whatever you make. At one time it was a bucket and a wooden patlel. 

I'm not a chemist but I try. If you are interestedlet me know.


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## DancesWithWeeds (Jun 26, 2022)

LukeTW said:


> Thanx very much Bro.
> 
> This is my run With Clorophorm and wih Metanol:H2O:Acetic.
> your Tutorial its the Best.


I've done a little TLC. Just enough to know how. I'm getting ready to start DCVC chromatography. You can check it out on YouTube. This looks like the easiest and cheapest way to go about this.

If you are interested I am, too. It would be nice to have someone to boune ideas off of.

P.S.: I'm writing this without my glasses. I hope it says what I want it to.


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## Chopshop697 (Jul 2, 2022)

Just jumped into TLC with the Cannalytics kit. Pretty cool, but I think my print went off the end and I didn't get a good CBD spot on my Cream & Cheese 1:1 on the left. The 3 right prints are my Critical Purple Kush - 2 mature plants and 1 print for the early larf and trim I took. Oh well, practice makes perfect. Note to self - label sample vials with something that doesn't wipe off easily


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## DancesWithWeeds (Jul 2, 2022)

Chopshop697 said:


> Just jumped into TLC with the Cannalytics kit. Pretty cool, but I think my print went off the end and I didn't get a good CBD spot on my Cream & Cheese 1:1 on the left. The 3 right prints are my Critical Purple Kush - 2 mature plants and 1 print for the early larf and trim I took. Oh well, practice makes perfect. Note to self - label sample vials with something that doesn't wipe off easily
> 
> View attachment 5157696


That's super. I've not tried one of the kits. I am a little into the chemistry of pot, but not a chemist. I would like to start a thread "Ligit MMJ Home Lab". This is the kind of thing that it would be for. I'm afraid it might not happen because I can just someone doing something like blowing up their house from fumes from an invented process. 

You can do really nice things with low flash point solvents or very small amounts of volatile solvents for a lot less money. Besides, to me this is interesting enough to me experimenting


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## PhenoMenal (Jul 6, 2022)

TLC is such an amazing way to analyse buds/marijuana/cannabis at HOME, and it's relatively easy once you've tried it (and quite affordable - the kits are inexpensive but can only be used for limited tests, but if you purchase all the parts yourself its a lot more affordable for a lot more tests), so I commend you all for giving it a go, so we can SHARE this knowledge


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## PhenoMenal (Jul 6, 2022)

Chopshop697 said:


> JNote to self - label sample vials with something that doesn't wipe off easily


Just use a "lead pencil" (actually carbon graphite) for marking the plates. Carbon is an element so it cannot be broken down further to interrupt the chromatography. Do NOT use an ink pen.


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## PhenoMenal (Jul 6, 2022)

LukeTW said:


> Thanx very much Bro.
> This is my run With Clorophorm and wih Metanol:H2O:Acetic.
> your Tutorial its the Best.


It's fascinating trying different eluents... I think Chloroform is my fave so far (its just so easy, doesnt assault your nose, only requires 1 chemical, and provides really good DIVISION of chemicals on the TLC plate). However it is awesome to have a second eluent to use for comparison! I salute you on giving TLC a go


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## DancesWithWeeds (Jul 6, 2022)

PhenoMenal said:


> TLC is such an amazing way to analyse buds/marijuana/cannabis at HOME, and it's relatively easy once you've tried it (and quite affordable - the kits are inexpensive but can only be used for limited tests, but if you purchase all the parts yourself its a lot more affordable for a lot more tests), so I commend you all for giving it a go, so we can SHARE this knowledge


I haven't seem you around for quite a while. My shortcut to RIU used to be through your thread.  

It is easier on your wallet just to set up a small lab and buy little larger quantities. I don't like to keep larger amounts of solvents but now I buy by the gallon. Much less than by the liter, and there is no way I could afford the little bottles. 

I also have Fast Blue BB but don't know how to use it so I'd like to pick your brain a little. I still have some of the graphics you posted.


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## DancesWithWeeds (Jul 6, 2022)

BTW, my lab isn't anything like a real lab. It's a shed with some old kitchen cabinets, a still that is becoming nice, some filtering glassware, hot plate, and some glassware. Nothing fancy but I do add things (within my wife's reasoning) that makes the job a little easier. You really have to use a little imagination to call this a lab.


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## PhenoMenal (Jul 7, 2022)

DancesWithWeeds said:


> I also have Fast Blue BB but don't know how to use it so I'd like to pick your brain a little. I still have some of the graphics you posted.


using Fast Blue BB is pretty simple, see "#6 - Prepare the dye bath for visualisation" in my tutorial


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## DancesWithWeeds (Jul 7, 2022)

PhenoMenal said:


> using Fast Blue BB is pretty simple, see "#6 - Prepare the dye bath for visualisation" in my tutorial


Thanks. 
Right now I am working on beets. Did know that if you do an ethonal wash on beets it comes out amber. Do a second wash with water and its red. The good stuff is in the amber. My solvent will be in today and run a column tonight. This is good for practice and doesn't cost an and leg.

I'm starting to do chromatography and want to run something easy before functioning out RSO. The setup I'm using is "Dry Column Vacuum Chromatography", DCVC. It's supposed to a lot easier, faster, and cheaper than flash. On YouTube you can lookup DCVC.

My RSO has a good record for cancer patients. 2 for 2. Lung cancer and leukemia. Both stable after the doctors wrote them off. It's too bad St. Jude wouldn't let my great grand daughter have RSO.

New project - diabetes. That's why the chromatography right now. I'm after THCV.


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## PhenoMenal (Jul 7, 2022)

@DancesWithWeeds, I just need to clarify that I've only ever done Thin Layer Chromatography (TLC) - i've never done Columnar Chromatography so I can't really help you out there.

Regarding THCV, I've found it in decent levels in the Durban Poison strain using TLC but can't recall finding it in any other of the strains I've tested. Here's a TLC plate of it I did whilst also experimenting with decarboxylation times:

(And there is clearly no CBD in this strain - it would've appeared as an orange circle above the THC circle, but clearly there's very significant levels of THCV, but not much else ... pretty much just THC and THCV in this one)

ps. It's best if you check out the archived version of my tutorial, as it includes various photos etc that are now missing from my initial post:








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


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...




web.archive.org


----------



## DancesWithWeeds (Jul 7, 2022)

PhenoMenal said:


> @DancesWithWeeds, I just need to clarify that I've only ever done Thin Layer Chromatography (TLC) - i've never done Columnar Chromatography.
> 
> regarding THCV, I've found it in decent levels in the Durban Poison strain using TLC but can't recall finding it in any other of the strains I've tested. Here's a TLC plate of it:
> View attachment 5159766
> ...


The strain I'll be using is "Diet Durbin THCV:THC. It's a proprietary strain from Seedsman. It's 1:1 ratio. The genetics are listed as secret. 7% THCV, 7% THC, 2% CBD.


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## PhenoMenal (Jul 7, 2022)

DancesWithWeeds said:


> The strain I'll be using is "Diet Durbin THCV:THC. It's a proprietary strain from Seedsman. It's 1:1 ratio. The genetics are listed as secret. 7% THCV, 7% THC, 2% CBD.


Interesting, I have no experience with "Diet Durban" but my only advice if you're after THCV is to start with a Durban Poison or close relative, so you're on the right track there. I can't recall which Durban Poison i used, but it was just called Durban Poison. I wonder why they named their cross "Diet Durban" though? It'll be interesting if you're also able to detect CBD in Diet Durban, there was none detectable in mine as you can see.


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## DancesWithWeeds (Jul 7, 2022)

I decided on a low THC strain because neither of the two are users. They are applying for their MMJ cards, probably doing it right now.


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## DancesWithWeeds (Jul 7, 2022)

PhenoMenal said:


> Interesting, I have no experience with "Diet Durban" but my only advice if you're after THCV is to start with a Durban Poison or close relative, so you're on the right track there. I can't recall which Durban Poison i used, but it was just called Durban Poison. I wonder why they named their cross "Diet Durban" though? It'll be interesting if you're also able to detect CBD in Diet Durban, there was none detectable in mine.


Diet Durban is a strain made exactly for this. I looked at Durbin Poison first. Diet Durbin has about 1 1/2 times the THCV plus CBD. It's the best balance I could find.


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## PhenoMenal (Jul 7, 2022)

well, hopefully analysis will prove that it is what it says it is. When I was hunting for CBD only 2-3 years ago i bought five seemingly "reputable high-CBD" strains, yet most of them showed ZERO CBD, and only one of those strains resulted in consistent and good levels of CBD in every plant (i was growing approx 5 plants each from the 5 strains).


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