Home TLC Thin layer chromatography

[PhenoMenal]

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

Only lanes A and E are showing CBD (the large orange dot above the red dot near the top of the plate)

The Dinamed CBD was a disappointing result -- no CBD at all from this particular phenotype. The first Dinamed i grew was 1:1.

 

[dybert]

View Image
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

Only lanes A and E are showing CBD (the large orange dot above the red dot near the top of the plate)

The Dinamed CBD was a disappointing result -- no CBD at all from this particular phenotype. The first Dinamed i grew was 1:1.

Nice separation!

 

[PhenoMenal]

cheers, yeah i usually get excellent separation from chloroform, as you've also personally experienced! ... its just a bit less hassle than measuring out the 20:80 diethyl ether : hexane (and chloroform doesn't assault the nostrils as bad as diethyl ether, that stuff is hardcore!), but as i also use hexane for the extraction phase i'm starting to run low on hexane!

so now I usually just do chloroform separations, but when I do some seed runs soon (~12-16 at a time, then destroy all but the two with the best CBD profiles after ~3 weeks) I'll do just chloroform initially, but then use both methods on the two 'winners', as it gives a slightly different 'viewing angle':

Apparently it will be even better separation if i let the plate fully dry and then re-do the mobile phase - it should use most of the plate then (no need to do that with chloroform though which already uses the whole plate). Hope to try the 'double-dip' soon.

It's weird how the shape of the base of the jar (eg having more eluent depth around the edge than the middle of the jar) effects the shape of the TLC with a slight curve of the results ... or at least, it does with chloroform, but that doesn't happen with hexane : diethyl ether. *shrug!*

 

[dybert]

cheers, yeah i usually get excellent separation from chloroform, as you've also personally experienced! ... its just a bit less hassle than measuring out the 20:80 diethyl ether : hexane (and chloroform doesn't assault the nostrils as bad as diethyl ether, that stuff is hardcore!), but as i also use hexane for the extraction phase i'm starting to run low on hexane!

so now I usually just do chloroform separations, but when I do some seed runs soon (~12-16 at a time, then destroy all but the two with the best CBD profiles after ~3 weeks) I'll do just chloroform initially, but then use both methods on the two 'winners', as it gives a slightly different 'viewing angle':
View Image
Apparently it will be even better separation if i let the plate fully dry and then re-do the mobile phase - it should use most of the plate then (no need to do that with chloroform though which already uses the whole plate). Hope to try the 'double-dip' soon.

It's weird how the shape of the base of the jar (eg having more eluent depth around the edge than the middle of the jar) effects the shape of the TLC with a slight curve of the results ... or at least, it does with chloroform, but that doesn't happen with hexane : diethyl ether. *shrug!*

For sure... I always used chloroform for both the extraction and mobile phase.

 

[PhenoMenal]

ahh, cool! ive only ever tried hexane for the extraction phase... i'll definitely try to remember to try chloroform on the next run, as well as diethyl ether on its own (might as well, i dont use much of it), so i can compare ...

I've done a side-by-side comparison before but that was just of the mobile phase eluent (all extractions were hexane) ...

that's pretty cool though that chloroform can be used for both stages, simplifies things a bit for those wanting to get started with TLC! "just get a bottle of chloroform, some Fast Blue B or BB, some TLC plates, and you're good to go"

 

[dybert]

ahh, cool! ive only ever tried hexane for the extraction phase... i'll definitely try to remember to try chloroform on the next run, as well as diethyl ether on its own (might as well, i dont use much of it), so i can compare ...

I've done a side-by-side comparison before but that was just of the mobile phase eluent (all extractions were hexane) ...
View Image

that's pretty cool though that chloroform can be used for both stages, simplifies things a bit for those wanting to get started with TLC! "just get a bottle of chloroform, some Fast Blue B or BB, some TLC plates, and you're good to go"

Way simpler, as long as you can get a hold of chloroform!

 

[PhenoMenal]

Way simpler, as long as you can get a hold of chloroform!

yeah, im a bit fuzzy about which chemicals are 'monitored' etc, or if quantities are important etc, and its different in each country, but you should be able to email your local lab supplies company "im after 250ml to 1L of (chloroform/whatever) suitable for Thin Layer Chromatography" ... this way they give you the right grade eg AR/analytical (sometimes they have half a dozen different grades of the one chemical, and I dont know wtf i should be getting!), and it probably sets off less flags saying outright you only want a small amount and it's for a specific scientific use. You might also be able to find them on ebay, and some lab supplies shops sell them online.

Fortunately a small bottle goes a long way with Thin Layer Chromatography! I have my fingers crossed the chloroform will prove to be as good at extraction as hexane when i get to do the side-by-side tests, and your TLC plate a few posts back showed it seems to work just fine

 

[PhenoMenal]

my latest TLC test results in [this icmag link]
16 seedlings from 6 strains, ended up with 4 that are high-CBD/no-detectable-THC

 

[PhenoMenal]

TLC Hack -- I don't have a centrifuge, and I don't enjoy standing around manually shaking eppendorf tubes, but I've found that my reservoir's air pump (ie. the one most indoor growers already have) provides good vibrations! ... then I have a conveniently shaped piece of polystyrene foam that I stick the eppendorf tubes into, which sits perfectly to vibrate away all day and night.

Alas, a still photo fails to capture the beauty of this hack ...

 

[theJointedOne]

Top Gun quality thread mate

 

[G.O. Joe]

this way they give you the right grade eg AR/analytical

HPLC maybe. It's all LC. Chloroform is easy to purify or make from acetone and hypochlorite if you're set for distillation and you have a big reactor with a condenser on it. It's sold stabilized typically with ethanol. The stabilizer is usually but not always left in for TLC, even though ethanol is easy to remove. Going through a column of active basic alumina will do a lot but it has to be used or stabilized or then phosgene etc. starts to form a little over weeks. Many analysts historically have liked chloroform a lot for extracting, but not all by itself for TLC. So I wonder what the deal is.

From the 60's like me but if you want TLC tips
b-ok.xyz/book/2304503/37f9e9

 

[PhenoMenal]

yep mine is "Chloroform AR stabilised with ethanol", ~US$30 for 500mL/17 fl oz. Min assay 99.8%, so i guess only a few drops of ethanol are needed to stabilise it!
I only use 12mL for the mobile phase so it's gonna last quite a while!

Many analysts historically have liked chloroform a lot for extracting, but not all by itself for TLC.

yeah i dunno. It seems to get the job done fine for our canna-specific TLC purposes though? *shrug*
I'm still using hexane for the extraction phase, haven't gotten around to trying chloroform for that yet but hope to in my next run

From the 60's like me but if you want TLC tips
b-ok.xyz/book/2304503/37f9e9

FANTASTIC!!!!!!!!!!! Thankyou!!!!!!!!! 53mb free pdf download

Where was this a year ago haha

 

[PhenoMenal]

searching the pdf for "cannab" shows a few mentions
Including this interesting sentence, which makes no mention of Fast Blue BB (im guessing the book was written just before BB was known/available), but it does seem to offer yet another alternative to Fast Blue B ...

The chief substances in Fig. 201 can be quantitatively determined by either spraying the chromatogram with alkali and the Fast Blue B salt mentioned or by visualisation with blue tetrazolium (Firm 88)

Which im guessing is https://en.wikipedia.org/wiki/Nitro_blue_tetrazolium_chloride
No idea how it compares to B/BB but always good to have options!

Also, their mobile phase was "carried out 2-3 times with cyclohexane to a height of 10 cm" ... I've been using n-hexane and diethyl ether (only 1 run though, will try 2 and 3 soon) ... do you know if there'd be any advantage with cyclohexane?

 

[PhenoMenal]

wow, this book is a beauty, thanks again Joe!
page 66 solves the mystery of that "edge effect" which has had me perplexed!
The reason: Insufficient chamber saturation.
The solution: use filter paper (wet from the mobile eluent) to line the chamber.

But, we're still getting the job done with insufficient chamber saturation, and the results are still easy to read, so at this stage I don't think i'll bother adding filter paper.

It's just nice to have a better understanding about what was going on

 

[SkyHighLer]

HPLC maybe. It's all LC. Chloroform is easy to purify or make from acetone and hypochlorite if you're set for distillation and you have a big reactor with a condenser on it. It's sold stabilized typically with ethanol. The stabilizer is usually but not always left in for TLC, even though ethanol is easy to remove. Going through a column of active basic alumina will do a lot but it has to be used or stabilized or then phosgene etc. starts to form a little over weeks. Many analysts historically have liked chloroform a lot for extracting, but not all by itself for TLC. So I wonder what the deal is.

From the 60's like me but if you want TLC tips
b-ok.xyz/book/2304503/37f9e9

5. Constituents of Hashish
KORTE and co-workers [28, 123-126] in particular have worked on the TLC of the narcotic drug hashish, also known as marihuana. This drug is the resinous constituent of the female inflorescence of Cannabis sativa, a plant which occurs in various varieties or chemical races. The solution for investigation is obtained by extraction at high rotation speed with petrol ether (BP 40-60°C). Separation is carried out on silica gel G layers impregnated by dipping in dimethylformamide-carbon tetrachloride (60 + 40) and allowing the mixture to ascend 15 cm in a suitable chamber. After 45 min, when the solvent and some of the impregnation liquid have evaporated, 1-5% solutions in n-hexane of the cannabis and hashish extracts are applied. Ascending development is carried out 2-3 times with cyclohexane to a height of 10 cm, at chamber saturation. Fast Blue B salt (Rgt. No. 100) is the most suitable reagent for visualising the separated substances, permitting detection of amounts down to 0.01 fLg. Fig. 201 shows that TLC is just as suitable for distinguishing hashish extracts of different origins and possibly chemical races as it is for ascertaining the pyrolysis products arising when hashish is smoked. M rn A S and co-workers [146] also have worked on this and established that only the cannabidiolic acid is decomposed during smoking of hashish.

Fig. 201. TLC of hashish extracts and CBD pyrolysis products [124].
THC tetrahydrocannabinols; CBN cannabinol; CBD cannabidiol; CBDA cannabidiolic acid; 1-4 hashish of oriental origin; 5 Cannabis non indica, cultivated in Karlsruhe, Germany, 1956; 6 as 5, but 1957; 7 Cannabis indica, Karlsruhe 1957; 8 Cannabis non indica, Karlsruhe 1962; 9 pyrolysis product of CBD; the zones with dotted outlines are only faintly visible

The chief substances in Fig. 201 can be quantitatively determined by either spraying the chromatogram with alkali and the Fast Blue B salt mentioned or by visualisation with blue tetrazolium (Firm 88) [125, 126]; in each case the zones are than scraped off, eluted with acetic acid-methanol (1 + 1) and the solution evaluated photometrically in the visible spectral region.

Egon Stahl, Thin-Layer Chromatography A Laboratory Handbook

pages 715-716
(Fig. 201 is attached)

 

[G.O. Joe]

Many of the old published TLC procedures for cannabinoids did not get positive reviews. Stahl was mentioned for the fundamentals. Nothing else appears to qualify as a TLC bible, although there are other big works. Honorable mention to lighter fare also translated from German:

Applied Thin-Layer Chromatography: Best Practice and Avoidance of Mistakes (2007)
b-ok.xyz/book/510038/03ec07
Thin-Layer Chromatography: Reagents and Detection Methods (1990)
b-ok.xyz/book/1292771/351068

Why not terpenes too?

 

[PhenoMenal]

Applied Thin-Layer Chromatography: Best Practice and Avoidance of Mistakes (2007)

yep give me that link now that I've used trial-and-error to figure the bloody process out haha j/k, it's sweet to have some quality literacy on it!

Why not terpenes too?

coincidinky! i was wondering about that only last week ("can we just use a different dye to detect terpenes?" because eg the cannabinoid profile tells us nothing about why C99 is pineapple) ... and as terpenes aren't exclusive to cannabis there's obviously a lotttt more info out there compared to TLC for cannabinoids. Not that i'm telling you anything you don't know!

This link was one of the many pages i came across, and "vanillin reagent" seemed very common as a dye to use for visualisation of terpenes.

They list the vanillin reagent ingredients (solution to be stored in fridge at 4C/39F), but no method given:

• 1.4 g vanillin
• 40 mL methanol
• 250 µL sulfuric acid

Seems easy and accessible and affordable enough, but i don't have any of those ingredients so it's not currently on my radar, though I would love to add it to the toolkit some day.

Some of its examples...

 

[G.O. Joe]

Stahl mentions vanillin. There's a less interesting volume 1b (1994) to the third link that has an entry for it, and some more stuff. In Stahl, under terpenes it says anisaldehyde, which can be bought or made from anise oil, and it's in the third book. Both come up in the second book. Hazekamp et al. (Chromatographic and Spectroscopic Data of Cannabinoids from Cannabis sativa L.) used anisaldehyde and other pot analysts have too.

 

[PhenoMenal]

sweet, again always nice to have options! any idea if anisaldehyde is better than vanillin?

like vanillin, anisaldehyde seems inexpensive enough, eg https://www.alfa.com/en/catalog/A15364/ lists 50g for $23 up to 1000g for $95.

A solution of para-anisaldehyde with acid and ethanol is used as stain in thin layer chromatography (TLC), which provides easy identification of different compounds.

So they say ethanol with that one, and the previous vanillin one uses methanol ... I have neither ethanol or methanol, but I wonder if I could just use methylated spirits @ 95% ethanol ... it works fine in Beam's CBD test anyway!

Vanillin is easy to get, it seems anisaldehyde might be too, not sure about sulfuric acid tho. Damnit I am tempted.

btw, any idea if hydrochloric acid (which i do have) could be used instead of sulfuric acid? don't wanna chlorine gas myself or anything

 

[G.O. Joe]

HCl will end up as heavy acidic water vapor that will condense on and corrode your shiny metal surfaces if you allow it. Sulfuric and phosphoric acids concentrate with moderate heating without much acid in the vapor. Battery acid is around 30-35% concentration I think. Alcohols and acetic acid are just solvents for the aldehydes.