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[sadpanda]

interesting patent... http://www.google.com/patents/US4771005

Reagents, test kits and methods for the detection of cannabinoids (US 4771005 A)
Abstract: The invention provides a stable solid reagent for use in a prepackaged test kit for the detection of cannabinoids comprising a diazonium salt which undergoes characteristic color change when combined with a cannabinoid under basic conditions, the salt comprising an anion capable of causing the entire salt including the diazonium cation thereof to dissolve in an organic solvent when combined therewith to form a stable liquid reagent for the detection of cannabinoids. The invention also provides a stable liquid reagent comprising the aforementioned diazonium salt and an organic solvent as well as providing prepackaged reagent test kits containing the aforementioned reagents and methods for detecting cannabinoids using the reagents of the invention.

Sounds like they're making another solid from Fast Blue, which you then add to an organic solvent to create a mix ready for TLC ....... but that's basically already the situation we're in now with adding the Fast Blue to a solvent, so im not sure what its advantages are, or if its better for us to simply leave our dye separate in the fridge(B)/freezer(BB) as they are now

 

[G.O. Joe]

Plastic polyester backed TLC plates would be easy to cut. They're still expensive. You may be a bit optimistic on the desirable concentration of the salt. Where is such a weak solution recommended? I was going to say that this is not so much the kind of chemistry where an order of magnitude makes a difference, but in that concentration it should.

 

[sadpanda]

thats the last thing i still havent worked out yet! how much Fast Blue to use. There's so much variation in the literature, with some people using 0.1%, some using 1.0%, all the way up to for example AlphaCat's kit which seems to be 30mgs of Fast Blue B salt added to 25mls water! will probably have to do some tests to zero in on whats best.
How much do you guys use???

 

[G.O. Joe]

How much do you guys use???

A spatula or a few knife points. If anyone wants to read up on TLC, bookzz.org happens to be a good book site to search for thin-layer chromatography. Applied Thin-Layer Chromatography seems to be OK. Sherma's Handbook of TLC gives a brief mention of terpene testing. This uses silver nitrate impregnation which is also super good at separating CBD and THC, but I didn't mention it in this thread until now because it's silver nitrate. I already have Thin-Layer Chromatography: Reagents and Detection Methods because it's excellent, and Thin-Layer Chromatography: A Laboratory Handbook should be good.

 

[sadpanda]

speaking of spatulas I have a few of these plastic "~3mg spatulas" that you get free when you order a few grams of nutritional powders like nootropics etc, hopefully it'll prove a useful measure for Fast Blue I do have a 0.001 digital scale but while its fine for measuring 20mgs etc its not good for just 1mg

btw lol yes i think i should give the silver nitrate a miss!! but the solution ill be using (4:1 hexane:diethyl ether) was mentioned in one of the literatures (from 2004 too) as being the best (of at least 6 or so various solutions they tested) specifically for the task of separating the cannabinoids, so i dont know if its as good as silver nitrate or not but it's probably the best route for a newb @ home like me to take and conveniently the hexane also doubles-up as the solvent used for extracting, so only those 2 solvents required in the whole process (+ ive gone with isopropyl alcohol for cleaning parts).

I'll post back with my EXPERIMENT RESULTS in a few weeks with the exact Fast Blue:water ratio i find best when i've got all the parts together and have done some experiments! pretty sure i'll have all required components within a few weeks. Also I might try 5:1 and 3:1 eluent mixes also just to help confirm 4:1 as best, it'll result in the waste of a little bit of solvent initially, but its best to figure out the right way straight away. Im so glad i took the extra time to read up about the various solvents though because it's clear that some just aren't particularly suitable for separating cannabinoids. This testing will be possible without being too expensive because i can cut the aluminium TLC plates!

And ive printed out the MSDS for everything im using, and also bought nitrile rubber gloves (same type that comes with AlphaCat) + goggles + lab coat (only need cheap ones - not dealing with hardcore acids/alkali etc!) - because safety is #1 priority in science!

ps. does anyone know if Fast Blue B and BB are supposed to be the same 'strength'? eg should 1mg of B give essentially the same result as 1mg BB? being different chemicals i would hazard a guess that they're at least a bit different, and perhaps that might be the source of some of the conjecture about exactly how much to use, and I had read at least one paper that seemed to stress that its easy to use too much or too little.

 

[sadpanda]

btw how awesome would it be if breeders made TLC plate scans available of their strains, would help to give a lot more to go by than just the current "This strain flowers in xx weeks, has a nice fruity smell, great high" descriptions

"6 of 8 Fruity Jack phenos displaying high CBD"

 

[sadpanda]

found this unique little gem which has some TLC lanes which are individual/isolated cannabinoids (as well as a few of the usual 'full spectrum' ones we've all already seen). The results/data is by Uni of Mississippi, because well, nobody else has been able to because of Schedule I insanity.

source: http://www.botanicalauthentication.org/index.php/Cannabis_spp._%28pistillate_inflorescence_and_leaf%29''

Its interesting to compare lanes 4 (raw) vs 12 (decarboxylated) ... all the THCA/CBDA seems to have been converted/disappeared, but the resulting THC/CBD spots appear pretty much the same

Cannabis spp. (rhizome) HPTLC ID

Lane Assignments
Lanes, from left to right (Track, Volume, Sample):
1 = 2 uL Δ9-Tetrahydrocannabinol (Δ9-THC or THC)
2 = 2 uL Δ9-Tetrahydrocannabinolicacid (THCA)
3 = 5 uL THC-type Cannabis
4 = 5 uL Intermediate-type Cannabis (looks like a ~1:1 CBD:THC ratio)
5 = 5 uL Fiber-type Cannabis (looks like a high-CBD low-THC hemp strain)
6 = 2 uL Cannabidiolic acid (CBDA)
7 = 2 uL Cannabidiol (CBD)
8 = 2 uL Cannabichromene (CBC)
9 = 2 uL Cannabigerol (CBG)
10 = 2 uL Cannabinol (CBN)
11 = 2 uL Tetrahydrocannabivarin (THCV)
12 = 5 uL Intermediate-type Cannabis decarboxylated

Reference Sample(s) Reference Standard Solutions: 1.0 mg/mL of each Δ9-Tetrahydrocannabinol, Δ9-tetrahydrocannabinolicacid, and cannabidiol in methanol.

Reference Sample Preparations: 100 mg of powdered plant material in 10 mL of dichloromethane, sonicate for 1 h, filter, and evaporate the extract to dryness under nitrogen. Dissolve the residue in 10 mL of methanol.

Stationary Phase Stationary Phase: TLC, Silica gel 60RP-18 F254, 150 um (Sorbent Technologies)

Mobile Phase Mobile Phase: 0.1% glacial acetic acid in a mixture of methanol and water (75:25)

Sample Preparation Method Test Sample Preparation: Prepare test sample as described under Reference Sample Preparations and apply 5uL. Derivatization reagent: Fast blue reagent– 5 mg/mL fast blue B salt in water.

Detection Method Development: Saturated chamber, developing distance 70 mm from lower edge of the plate; relative humidity 33%, temperature 25°.

Detection: Heat plate at 70°C for 2 min, dip (time 5, speed 5) in Derivatization reagent, dry, and examine under white light.

I modded it in Photoshop for clarity with the isolated cannabinoid lanes:

But interestingly it's not quite what the Alpha-Cat kit shows:

 

[sadpanda]

Rf values ...
"retention factor", but ive seen "retardation factor" (wiki uses it!) also used a bit. I'll just use retention factor ...

Example overview in case you're wondering what Rf is ... you can kinda think of it as a value between 0 and 1, essentially a percentile if * 100, where 0 is the start base level of your ~2 microgram sample drops, and 1 is the 'solvent front' after the capillary action has reached your targeted top:

It's a beautiful thing to have the multi-color reactions from Fast Blue B/BB, virtually instantly being able to see the orange of the CBD in contrast to the scarlet of the THC but having those colors AS WELL as Rf values = double awesome!!!

source: https://www.researchgate.net/public...c_Data_of_Cannabinoids_from_Cannabis_sativa_L
Leiden University (Netherlands) & Institute Universitaire de Me'decine le'gale (Switzerland) -- 2005

Polar TLC system (silica)
0.65 D9-THC & D8-THC
0.64 CBD
0.62 CBN
0.61 CBG
0.58 CBC
0.39 THCA
0.37 CBDA
0.31 CBGA
0.25 CBCA

Non-polar TLC system (RP-18)
0.68 CBDA
0.67 CBGA
0.59 CBG
0.58 CBD
0.48 CBN
0.44 D9-THC
0.43 D8-THC
0.40 THCA
0.37 CBC
0.35 CBCA

THCV was not included in the paper.

___________________________

BUT!... DO RF VALUES CHANGE WITH DIFFERENT SOLVENTS??? Im guessing yes. Anyway Alpha-Cat's Rf values are a bit (sometimes a lot) different, and the Rf values quoted dont appear to match up with whats shown in the picture:

 

[sadpanda]

So in conclusion, Uni of Mississippi, the Leiden University (Netherlands) & Institute Universitaire de Me'decine le'gale (Switzerland), Alpha-Cat, Cannalytics etc and all the other kits ... ie. EVERYONE ... seems to have their own at least slight variation on 1) which each dot/cannabinoid is meant to be, and 2) what its Rf value should be ... and then even when you compare the Rf's to the dots the same company shows they don't even agree either ..... yeah that's just great In other words... because they're all different, it seems only 1 of them might be correct, with the others putting out at least slightly incorrect information. Plus i have a feeling not all the kits have done their own research, there's probably been at least a bit of copying going on. But, at least CBD/THC or CBDA/THCA seem fairly obvious, that's all most of us are mainly interested in i guess, but yes i would love to know more with confidence.

 

[G.O. Joe]

Results from the petroleum and diethyl ethers system are shown here, with Gaoni and Mechoulam's Rf's in the next post.

 

[sadpanda]

Very handy thread, thankyou!!! Thought I had seen all icmag TLC threads but clearly not, doing another search after this. Hollly shmoly that thread started out like a drama movie, lol... firstly starting with 500mgs as first Fast Blue solution... when like, normally if you just buy 1000mgs youre looking at $100-300ish! (and he swallowed some B aerial spray and said it irritated, ugh!!!) And also getting NO reaction at the start the poor guy, my heart sunk! Lucky he figured it out quick enough and it was very helpful that he documented various ratios and quantities etc. Was a bonus seeing his FBBB results too as most images ive seen have been FBB. Pretty much same system as me too. Looks like i've definitely got some ratio variation experimenting to do to help fine-tune it! I look forward to sharing the scans in ~3 weeks when ive finished getting all supplies.

Just copying over those Rf's you provided in that thread (Gaoni and Mechoulam, 1971):
CBL 62
CBD 58
d8 THC 57
d9 THC 51
CBN 47
CBC 43
CBG 42

I would've loved to have read that original paper but had no luck finding with Google, 1971 factor perhaps

 

[sadpanda]

Pick a Standard any Standard! i mashed this together in Photoshop ...

Cannabinoid ordering:
Alpha-CAT: CBD CBN THC THCV CBG CBC
Cannalytics: CBGM CBD CBN THC THCV CBG CBC CBND
Cannalytics?: CBC CBG THC THCV CBD CBN CBNV CBND
MontanaBio1: CBC CBD THC THCV CBG CBN
MontanaBio2: CBC CBD THC THCV CBG CBN
Mississippi: CBDA THCA THCV CBD CBG CBN THC CBC
LeidenUni: THC CBD CBN CBG CBC THCA CBDA CBGA CBCA
Gaoni/Mech: CBL CBD THC-D8 THC-D9 CBN CBC CBG

 

[G.O. Joe]

I would've loved to have read that original paper but had no luck finding with Google, 1971 factor perhaps

http://dx.doi.org/10.1021/ja00730a036

 

[sadpanda]

I found the full paper that described the system i'm using (hexane : diethyl ether 4:1, which it said was the best of the six systems they tried) - http://chromsci.oxfordjournals.org/content/42/3/130.full.pdf

So unlike the previous text-only versions this one had some good graphics, including (h)Rf's! -- i also learned that hRf is just rF x 100

So in theory my Rf's should match up with those, allowing me to also identify CBN with high degree of confidence. I was also planning to degrade THC to CBN which should also help increase confidence.

btw IS MY HEXANE OK FOR TLC??? there are so many different variations of each solvent, different grades etc... hopefully the ones ive got are ok?
my hexane is "Hexane Fraction", proper shipping name: "HEXANES", "Analytical Reagent" AR Grade. It's 30% color though!?!

Max. limits of impurities:
30% Colour (Saybolt)
0.09% Benzene
0.001% Non-volatile matter
0.001% Sulfur
0.2% Aromatics

Likewise the Diethyl Ether, is "Minimum assay 99.5%", with Max 10% Color, and then a list of about 30 other things with really low like 0.02 - 0.000001 values.

So is this ok for TLC??? *holds breath* (and if i do have to buy again, i can get HPLC grade, is 95% ok or does it have to be 99%?)

 

[SkyHighLer]

I found the full paper that described the system i'm using (hexane : diethyl ether 4:1, which it said was the best of the six systems they tried) - http://chromsci.oxfordjournals.org/content/42/3/130.full.pdf

So unlike the previous text-only versions this one had some good graphics, including (h)Rf's! -- i also learned that hRf is just rF x 100
View Image
So in theory my Rf's should match up with those, allowing me to also identify CBN with high degree of confidence. I was also planning to degrade THC to CBN which should also help increase confidence.

btw IS MY HEXANE OK FOR TLC??? there are so many different variations of each solvent, different grades etc... hopefully the ones ive got are ok?
my hexane is "Hexane Fraction", proper shipping name: "HEXANES", "Analytical Reagent" AR Grade. It's 30% color though!?!

Max. limits of impurities:
30% Colour (Saybolt)
0.09% Benzene
0.001% Non-volatile matter
0.001% Sulfur
0.2% Aromatics

Likewise the Diethyl Ether, is "Minimum assay 99.5%", with Max 10% Color (APHA), and then a list of about 30 other things with really low like 0.02 - 0.000001 values.

So is this ok for TLC??? *holds breath* (and if i do have to buy again, i can get HPLC grade, is 95% ok or does it have to be 99%?)

You're question concerning "hexanes" is confusing, do you have a Certificate of Analysis like this for

"Hexane (hexanes)
>98.5%, mixed isomers, Analytical Reagent ACS
Assay (by GC, sum of 5 isomers total hexanes plus methyl cyclopentane) 99.9%"

https://gtilaboratoriessupplies-com.3dcartstores.com/assets/images/Hexane, MIACS, 992503.pdf

 

[sadpanda]

i dont have a Certificate of Analysis no

 

[SkyHighLer]

Where is the full percentage info on the "hexanes?" I'm pretty sure it's in front of you, and it just didn't get posted up...

 

[sadpanda]

moved to next post
https://www.chemsupply.com.au/documents/HA018.pdf

 

[SkyHighLer]

There it is, Bp 60-70C with the composition being, "Mixture of n-hexane and other isomers," with "Volatile Component 100%."

Bp of hexane 68.5 to 69.1 C

Remember the Bp ranges for petroleum ether? Does '35-65C pet ether' now make sense with pentane's ~36C Bp?


We be in at this awhile, my next door neighbor's way in,

https://archive.org/stream/hashishsmuggling00unit#page/n1/mode/2up

 

[sadpanda]

btw here is the Specs for my Diethyl Ether
(my mobile-phase eluent is 4:1 hexane:diethyl ether, recommended in previously mentioned paper as the best of the 6 mixes they tested in regards to cannabinoid separation)

I'm embarrassed to say I've got no idea what the BP has to do with any of this though sorry!?? apart from evaporation rate!? hehee

so does this mean the ones i've got are fine to use for TLC? (i have no other intended uses - not making extracts or anything, just TLC)