Home TLC Thin layer chromatography

[Chimera]

I use plates designed for acidic cannabinoids, rather than decarbing the material prior to extraction, or worse yet on the plate.

Mobile phase is methanol and water with 1% glacial acetic.

Spot identification by UV, forget fast-blue or vanillin. It's a more expensive initial investment by a few hundred bux, but worth the hassles it saves in the long run.

I'll see if I can find and post some pics.

 

[sadpanda]

one thing i really like about Fast Blue though is its color differentiation as well... even though the CBD spot is always going to be in that same spot above the THC spot i find it nice to be able to see exactly which color is which - where one stops and the other starts... im guessing you can't really get that with UV, plus i dont think i'd be able to do a flatbed scan of the results?

Would love to see any pics you have anyway, thatd be great thanks!

i was intrigued when you mentioned vanillin alongside Fast Blue so i did some googling there and came across this interesting mention of three different cannabinoid tests:

The Beam test is relatively specific. It gives a purple color with 5% ethanolic KOH, based on the oxidation of CBD, CBG, etc., and their acids to hydroxyquinones. However, THC does not react to the Beam test. Only two plants (Rosemary and Salvia) out of 129 common species tested give a weakly positive reaction. Among some 50 pure vegetable substances such as mono- and sesqui-terpenes, aromatics, etc., only juglone, embelin, and alkyl dioxyquinone develop a color reaction close to that of Cannabis. The reaction is not always dependable; it can be absent if the ethanol is hot.

A modification of the Beam test uses absolute ethanol saturated with gaseous hydrogen chloride. When added to an extract of suspect material, it gives a cherry red color which disappears if water is added. However, the test also gives more or less similar red color reactions with pinene, tobacco, julep, sage, rosemary, and lavender, etc..

The colorimetric test of Duquenois and Moustapha is not so specific as the Beam test, but it is very sensitive. The test reacts to CBN and CBD, but not to THC:

Vanillin (0.4 gr, acetaldehyde (0.06 gr) and 20 ml 95% ethanol is stored in a bottle. Extract the plant material with petroleum ether, then filter it and evaporate the solvent. Add exactly 2 ml of reagent and 2 ml concentrated hydrochloric acid. Stir the mixture; it turns sea-green, then slate gray, followed by indigo within 10 minutes. It turns violet within 30 minutes and becomes more intense.

The Duquenois-Negm hydrogen peroxide/sulfuric acid test is suitable for following the development of the resin and its potency. Macerate cannabis in chloroform or light petroleum ether for several hours. Evaporate 0.2 ml of the extract in a porcelain dish. Add 2 drops 30% hydrogen peroxide and 0.5 ml concentrated sulfuric acid. Rotate the dish gently, and observe the color of the liquid after 5 minutes. A pink color indicates CBD; blood-red color indicates a high concentration of THC. Violet or strong brown indicates THC. CBN produces a green color which quickly turns green-brown.

src: http://www.hempbasics.com/hhusb/hh6thc.htm

 

[G.O. Joe]

I use plates designed for acidic cannabinoids, rather than decarbing the material prior to extraction, or worse yet on the plate.

Mobile phase is methanol and water with 1% glacial acetic.

Are these C18-type reverse-phase plates?

 

[sadpanda]

yes i'd too love to hear more about these "plates designed for acidic cannabinoids" please!

 

[Chimera]

Are these C18-type reverse-phase plates?

Those are the ones, Aluminum backed C18-W Silica, uv254

"Designed for cannabinoid-acids" were not well chosen words, call them suited for the purpose. Either way, they work well.

THCa and CBDa separate out fine, you can play with the ratio of methanol : H20 and length of time in the mobile phase, which provides for better resolution between spots. i will typically just make a 'standard' of known THC and known CBD plants, then blot these for reference points on either side of the plate - unknown samples go in between.. Pics forthcoming.

It'a very suitable method for discriminating between type 1,2 and 3 plants from a CBD segregating generation. Quick and dirty and gets the job done with little prop, no machine time, and minimal processing. I have TLC also for CBDv with colorimetry (both fast blue, & vanillin), but to be honest for those populations I would rather use the GC/HPLC due the sensitivity and quantification. I really just use TLC for screening and narrowing down larger populations as seedlings - no point in growing Bt/Bt plants in CBD breeding programs!

 

[sadpanda]

wow thats very cool!!!
btw what size TLC plate do you recommend for cannabinoids?
[update] it seems all the commercial kits use 5cm x 10cm plates

 

[sadpanda]

Specifically for testing cannabinoids using TLC...
Q1. Which solvent(s) are recommended for the extraction (ie. bud/resin/oil/etc -> solution)?
Q2. Which solvent(s) are recommended for the mobile eluent phase to carry the solution up by capillary action?

It seems there's actually quite a lot of options, which is good in that it makes more options available in a world where getting some chems can be tricky, but also makes it initially more complicated for newbs like me who don't really know the underlying chemistry too well!

Anyway i put together a list of what some have used/recommended (specifically for cannabinoid testing by Thin Layer Chromatography), just scratching the surface, in no particular order:
_____

http://www.sirchie.com/thin-layer-chromatography-of-marijuana-kit-with-6-tests.html
Extract Solvent: Hydrocarbons C>=5, C5-6-rich (petroleum ether).
Mobile Eluent: 6:4 chloroform : Hydrocarbons C>=5, C5-6-rich (petroleum ether).
(This is a LEO-only kit, i don't know if solvents are chosen for being the most mobile/field-use friendly, or if simply because they produce best results, or both, or...)

https://www.alchimiaweb.com/blogen/make-cannabinoid-analysis/
Extract Solvent: hexane
Mobile Eluent: chloroform
(Shows it can be nice and easy, and the photos of the result still look good, but i get the feeling the separation may not be quite as good as with the two-part eluents?)

http://www.medicinalgenomics.com/wp-content/uploads/2011/12/Chemical-constituents-of-cannabis.pdf
https://www.researchgate.net/public...Planar_Chromatography_Techniques_TLC_AMD_OPLC
All the standard solutions were prepared in 0.5mg/1mL in methanol.
Cannabis resin (0.1g) extracted by shaking for 20 min with 10mL of hexane.
Hemp sample (0.5g) was extracted for 10 min with 20 mL of hexane.
The filtrate was evaporated to dryness and the residue dissolved in 1mL of toluene.
For Classical TLC and TLC-MAT, the eluent used was 4:1 hexane : diethyl ether.
... it also goes on to say this interesting comparison ...
In the literature, the eluents which are mostly used are:
eluent A: isooctane-ethyl acetate-acetic acid 30:10:1 v/v;
eluent B: petroleum ether-diethyl ether 90:10;
eluent C: acetone-methylene chloride-diisopropyl ether-hexane 1:1:3:20 v/v;
eluent D: toluene-chloroform-methanol 100:10:1 v/v;
eluent E: hexane-dioxane 90:10 v/v double migration;
eluent F: hexane-diethyl ether 80:20.
The eluents A and B result in a clean separation between ∆9-THC and CBN, but not between ∆9-THC and CBD.
With eluent C, the main Cannabinoids are separated but the spots are stretched.
The best results were obtained with eluent F, hexane-diethyl ether 80:20 v/v which allowed a clean separation of ∆8-THC, ∆9-THC, CBN and CBD.
Different eluents were tested: isooctane, heptane, hexane, pentane with diethyl ether with a ratio of 90-10 v/v. The comparison between these four alkanes showed that the separation capability decreases when the carbon-bearing chain lengthens.
(so at this stage i think this is what i'll personally try to get, and its handy that the hexane can be used as both extract + mobile, so only the two chems needed, yet still with a two-part mobile phase for excellent separation!)

https://www.icmag.com/ic/showpost.php?p=2934216&postcount=19
Extract Solvent: petroleum ether
Mobile Eluent: 4:1 hexane : ether

https://www.icmag.com/ic/showpost.php?p=7592261&postcount=62
UNODC document: 4:1 petroleum ether : diethyl ether
Perhaps ethyl acetate and many other things can substitute for the diethyl ether polar part.
A nonflammable option is chloroform with just a little bit of methanol.
1:1 hexane : methylene chloride.

http://www.botanicalauthentication.org/index.php/Cannabis_spp._%28pistillate_inflorescence_and_leaf%29 (Uni Mississippi)
Mobile Eluent: 0.1% glacial acetic acid in a mixture of methanol and water (75:25)
Extract Solvent: 100mg powdered plant material in 10ml dichloromethane, evaporated, dissolved in 10ml methanol.

https://www.icmag.com/ic/showpost.php?p=7592274&postcount=63
Mobile Eluent: methanol and water with 1% glacial acetic. (*with TLC plates designed for acidic cannabinoids)

http://www.patentstorm.us/applications/20080057117/fulltext.html
Chromatography on silica gel (0.035-0.070 mm, chromatography with a mobile phase mixture of 1% by weight ethyl acetate in petroleum ether) resulted in a cannabinoid fraction which was detected by TLC [silica gel 60 F254 (HPTLC pretreated plate), hexane/diethyl ether 8:2; detection: 125 mg of fast blue B salt in 30 ml of 1 N sodium hydroxide solution, dist. water ad 300 ml, evaluation under daylight].

http://www.sciencedirect.com/science/article/pii/S0021967301953476
Extract Solvent: light petroleum (BP 40-60°)
Mobile Eluent: two systems
1) 15:1 chloroform (ethanol-free)-1 : 1-dichloroethane
2) 19:1 xylene (mixed isomers)-1 : 4,dioxan

http://scholarlycommons.law.northwestern.edu/cgi/viewcontent.cgi?article=5832&context=jclc
Extract Solvent: .3ml (6 drops) petroleum ether for a few minutes
Mobile Eluent: toluene, "however benzene or xylene could probably be used with similar results"

https://www.unodc.org/unodc/en/data-and-analysis/bulletin/bulletin_1985-01-01_4_page011.html
Toluene for elution in one direction (first front) and 9:1 hexane : dioxane for elution in the perpendicular direction (second front).

https://montanabiotech.com/2011/05/...noid-fingerprint-test-kit-by-montana-biotech/
Mobile Eluent: 15:10 chloroform (ethanol-free) : 1,1-dichloroethane

https://www.google.com/patents/US20070077660
Good results are obtained with a developing solvent with at least 25 vol. % chloroform and at least 25 vol. % 1,2-dichloro ethane.
Extraction solvent comprises at least 50 vol. % chloroform, relative to the total volume of the extraction solvent.

http://www.thctalk.com/cannabis-for...-level-of-THC-CBD&p=1069722795#post1069722795
Extract Solvent: hexane
Mobile Eluent: 4:1 hexane : diethyl ether

https://www.realhemp.com/wp-content...y-System-Of-Cannabis-Sativa-L-Cannabaceae.pdf
either a polar (ethyl acetate-methanol-aqueous 2% acetic acid; 67:7:1)
or more non-polar (toluene-ethyl acetate-formic acid; 17:10:1) solvent.

http://ww.asianforensic.net/documents1/ForensicAsia Issue 7/ForensicAsia_Issue7_Final.pdf
Thin layer chromatography (TLC) plates should be silica gel or equivalent and sufficient to resolve the three major cannabinoids. Sample A was spotted on the TLC plate with the cannabinoid standards. Mobile phase was petroleum ether:ether (80:20). The visualisation spray used in this method was Fast Blue BB salt. Approximately 50 mg of fast blue BB was dissolved in 20 mL of 0.1 N sodium hydroxide. This solution was freshly prepared. Results obtained from this analysis showed that the three cannabinoids migrated and developed in the following order;
• Top spot – Cannadiol (CBD)-orange
• Middle spot – Tetrahydrocannabinol (THC)-red
• Lower spot – Cannabinol (CBN)-purple

 

[SkyHighLer]

Do you really need optimum? Some of those solvents are of the type usually reserved for real chemists... diethyl ether is not something I keep around the house.

Both diethyl (ethyl) ether, toluene, and acetone are on the DEA precursor watch list, and if the only source of chloroform at eBay is outside the country, something's up, and I would look into why before attempting to purchase it.

"List II chemicals

These chemicals are designated as those that are used in the manufacture of the controlled substances.

Acetic anhydride
Acetone
Benzyl chloride
Ethyl ether
Potassium permanganate
2-Butanone (or Methyl Ethyl Ketone or MEK)
Toluene
Hydrochloric acid (including anhydrous Hydrogen chloride)
Sulfuric acid
Methyl isobutyl ketone (MIBK)
Sodium permanganate"

https://en.wikipedia.org/wiki/DEA_list_of_chemicals#List_II_chemicals

 

[G.O. Joe]

AA and BnCl are really the only suspicious chemicals for domestic delivery on that list. The truth is ether is not a big deal to work with, separate from starting fluid, whatever.

 

[sadpanda]

is "Fast Blue BB salt" just zinc chloride? could i just buy 1gm zinc chloride salt?
[edit] oops guess not ... "(Fast Blue BB) salt is a zinc chloride complex of diazotized 5-amino-2-benzoyl-amino-1,4-diethoxy-benzene"

[edit] lol, actually Joe when i asked that question you probably should've stopped helping and told me to go find another hobby. I think i was having a long week...

 

[sadpanda]

Fast Blue B salt: store at 2-8C
Fast Blue BB salt: store at -20C
Hmmm, might have to end up going with Fast Blue B, shipping might be an issue with BB

 

[sadpanda]

glass vials are expensive and im not sure about cleaning for reuse? dont want contamination issues spoiling the TLC results. So i was wondering what disposable plastic vials would be safe to hold my solvent mix? (hexane : diethyl ether 4:1) - only need for ~1hr, to mix the bud or oil etc into the solvent before extracting 2ul for the start spot on the TLC plate.

It turns out you shouldn't just blindly get random "plastic vials" off ebay for holding solvents, lol - well, at least not without checking what type of plastic it is. Anyway I came across this cool little chemicals & containers compatibility list
http://www.calpaclab.com/chemical-compatibility-bottles-containers/

Here's the part of the list relevant to my mix:

E = No damage after 30 days of constant exposure. (best!)
G = Little or no damage after 30 days of constant exposure.
F = Some effect after 7 days of constant exposure.
N = Immediate damage may occur. Not recommended for continuous use. (worst. this is the only one i really need to avoid)

So for my hexane : diethyl ether mix, i guess it shows that clearly the plastic containers i want to be using are FEP, TFE, PFA, or ETFE, all of which are also suitable for longterm storage. And i definitely do NOT want to use LDPE, PPCO, PMP, PC, or PS.

[edit] actually i just realised that my extraction solvent (which is all i need the disposable vials for) is simply the hexane on its own - I'm only using the diethyl ether with the hexane in the mobile eluent part, and i have the glass jar for that. However it would still be good to be able to reuse any unused diethyl-ether:hexane mix afterwards, so all of the above is still applicable but i only need 1 large bottle for that, whereas i only need lots of tiny plastic vials for the hexane only.

 

[Chimera]

glass vials are expensive and im not sure about cleaning for reuse?

Methanol, 99% isopropyl, or most cheap organic solvents from home depot will suffice.

dont want contamination issues spoiling the TLC results.

A double rinse wth just a few ml of solvent is enough to clean your vials; it's easy to check if your vials are clean, run a blank in your cleaned 'used' tube (I use 50 ml Falcon tubes), and see if your TLC plate shows cannabs. Those little squeeze bulb disposable pipettes are perfect for dosing a few mls of solvent to clean the vials.

With a good solvent extraction rinse you should be fine. Do NOT rinse your tubes with H2O after extracting a sample, your cannabs will crash out and 'jump' onto your extraction vessel (aka the falcon tube) making cleaning more difficult. I simply rinse the falcon tube with 2-3 ml of iso, then shake 'dry' and re-rinse, that's all it takes

 

[sadpanda]

Chimera thanks again for some excellent advice! i guess a cheap solvent to clean glass vials is probably the way to go instead of disposable plastic vials, because when i was looking for those plastics i couldnt really find any suitable and affordable, lol

Now im just waiting anxiously to see if Sigmawill let me create an account with them (im not a uni or science company, just individual wanting to do home TLC, but it says they do 2 days 'screening' to check im not meth lab or whatever, sigh), otherwise it might be very tricky to get this Fast Blue B/BB

 

[G.O. Joe]

Now im just waiting anxiously to see if Sigmawill let me create an account with them (im not a uni or science company, just individual wanting to do home TLC, but it says they do 2 days 'screening' to check im not meth lab or whatever, sigh), otherwise it might be very tricky to get this Fast Blue B/BB

They're so famous for not selling things. It would be a big surprise if they would send you anything. A VWR is probably about the biggest size of supplier that might sell chemicals to individuals and/or deliver to residences.

Google gave me a few sources for the BB last week - additional search terms might bring up more. It will not arrive cold and may have been made in India and never refrigerated. Google just might give you a source for the B salt on the first hit.

Oh and what I said about ether is after learning everything you need to know about dealing with peroxide formation and the ease of ignition. Once you've got that down it's no big deal. Except for the penetrating smell freaking out neighbors.

 

[sadpanda]

so the bad news is that creating an account with Sigma is basically impossible for an individual like me wanting to do home TLC, lol ... its got fifty or so non-optional fields, and you have to give them your business info (so, no individuals to begin with), and even if you have a business it has to be in the chem/bio kinda scientific field, and even then you also need to provide info of four other companies that've dealt with you, etc etc etc blah blah BLAH, ARGH! lol

The good news - Sigma arent the only ones that sell the Fast Blue dye salt, and the salt itself is not a restricted or listed/watched product (and you can use it to visualize strawberry phenolics for a non-cannabinoid example), so yes it just means shopping around a lot because its just relatively rare i guess. Plus the BB is a frozen item and B a chilled item, so there's that freight issue too.

There are quite a few manufacturers in China and India, but fortunately i was able to find a good source in my native non-US country. Fast Blue BB too so there's a bit of extra peace of mind being non-carcinogenic (just toxic lol), it comes packed in dry ice and the lab said it doesnt come with expiry so "probably good for 5+ years", and surprisingly and fortunately the freight surcharge is very inexpensive! ~$20. i guess because dry ice is pretty straightforward as far as freight goes.

SO IM NEARLY THERE!!! whew!!! Finally i just need to work out exactly how much Fast Blue BB to use ... it seems either 1% or 0.1%. And what to mix it in ... it seems distilled water is fine, but i'll also have isopropyl so maybe that'll be better? Im also going to try to find 10 or so really cheap ebay TLC plates, just to use for testing to get comfortable with the process and mixes first, before i use good plates.

Getting excited now will be so beautiful and powerful to see cannabinoid thumbprints and see exactly how much CBD-to-THC my friend can get in her fight against cancer - now she won't be fighting blind, thanks to the people that've made information about cannabinoid testing with Fast Blue via TLC available, such as the very generous people in this thread and I can't thank you all enough!!!

 

[sadpanda]

comparing the MSDS for Fast Blue B and Fast Blue BB, my conclusion is they should both be treated as if they are equally carcinogenic, and surely should be BATHED not aerial sprayed like most cannabis test kits do!

Fast Blue BB:
GHS Classification:
Acute toxicity, Oral (Cat 4 = LD50 is >2000mg/kg)
Carcinogenicity (Cat 2 = suspected human carcinogens)
Signal word: Warning
Hazard statement(s)
H302 Harmful if swallowed
H351 Suspected of causing cancer
Prevention
P201 Obtain special instructions before use
P202 Do not handle until all safety precautions have been read and understood.
P264 Wash skin thoroughly after handling.
P270 Do not eat, drink or smoke when using this product.
P281 Use personal protective equipment as required.

Fast Blue B:
GHS Classification:
Carcinogenicity (Cat 1B = known or presumed to have carcinogenic potential for humans, based primarily on animal evidence)
Signal word: Danger <- Danger is more severe than Warning (http://www.chemsafetypro.com/Topics/GHS/GHS_signal_word.html)
Hazard statement(s)
H350 May cause cancer.
Prevention
P201 Obtain special instructions before use.
P202 Do not handle until all safety precautions have been read and understood.
P281 Use personal protective equipment as required.

 

[sadpanda]

would 1 milligram of Fast Blue BB added to 100mls distilled water (so, 1% or 1:100) be sufficient to in-and-out bathe a 10cm x 5cm TLC plate? (in a dish of approximately the same size but obviously a bit larger)

"The most commonly used reagent is 1% Fast Blue BB in water, which has replaced the previous use of the carcinogenic Fast Blue B." - 'The Analysis of controlled Substances' by Michael D. Cole

im trying to get the maths right.... 1mm depth seems sufficient, surely no need for more than 2mm depth. 1mm depth x 100mm x 50mm = 5000mls, wait what, 5 liters? lol ok i failed there

A cola can is like ~350mls and im visualising in my mind that 1/3rd of that would be more than enough, so 100mls sounds about right to me?

In which case i could get 1000 x 'TLC plate baths' from the 1gm Fast Blue salt?

perhaps im being optimistic though, because although ive read in many officialy papers to use 1% in water, i just checked out Alpha-Cat instructions and it seems they're using 30mgs of Fast Blue B salt in 25mls water (ok cool so at least now we know 25mls is enough depth ie 1mm or so for the 10cm x 5cm TLC plates all the cannabinoid test kits seem to use). And they only use 2mls of the eluent solvent in the developing jar! so it looks like my 500ml bottle will go a long way hehe, nice

btw can we use aluminium TLC plates??? or do they have to be glass

 

[Chimera]

As per your formula, 1 mg should be added to 90 ml of water and then brought up to 100 ml.

This is different from 1mg added to 100 ml, or 1:100.

Think about it this way, if you had a 1 ml stock solution containing 1 ml of solute, and you added this to 99 ml of water, then you would have 1mg in 100 ml.

If you added the same stock solution (1 mg dissolved in 1 ml) and added this to 100 ml, you would have 1mg of solute in 101 ml.

Always add your solids to a lesser amount of your solvent, then top it up using a properly graduated measuring device like a graduated cylinder, or preferably a volumetric flask.

Small difference, but a difference nonetheless. Accuracy counts in chemistry.

~

Aluminum backed plates are fine.

 

[sadpanda]

wouldnt it be easier/the same to add 1mg dye to 99mls water then? i'll be mixing in a glass jar i can shake before pouring into the bath dish to get a good mix, seemed better than a glass stirring rod

Seems like i might only need 25mls though, so its gonna be interesting trying to accurately get 0.25mgs lol, my scale is accurate to 1mg so i might have to divide down

That'll be so cool getting ~1000 baths out of just the 1gm of Fast Blue salt i wont have to worry about sourcing some more for a couple decades lol, probably even enough spare to sell to a strawberry farmer

Aluminum backed plates are fine.

awesome!!! looks like i can make some great $aving$ then, thankyou from what i read before your post it seemed aluminium plates were fine, but just "shouldnt be used with certain acids" or something - which didnt seem to be an issue with my hexane:diethyl ether solvents, so i had my fingers crossed, great to have the confirmation though and even if there's slight curl the flatbed scanner will straighten that out lol. Also that means i can CUT them as needed to reduce wastage!!! yay!!! (i dont have a cutter for glass)

I cannot thankyou again enough for all your help!!! its been good being able to make purchases with confidence, because there are soooo many options lol - thats why its taken me a good 2-3 weeks research!

btw i wonder if itd be worthwhile getting another dye to also be able to examine terpenes? just thinking out loud lol, and probably gonna be hard to find a Standard for them although im assuming i could find their RF values, but then there are so many of them id imagine itd be tricky on the small 10cm x 5cm plates