Chromatography purification

eugeneoregon · Jun 9, 2017

Here are photos of a chromatography run I did to purify medicine. From start to finish, the starting sample, then the sample absorbed onto Celite. Next is the set up used and then the test tube fractions collected top to bottom and left to right in the order they eluted off the column. Then a picture of the first four brown fractions evaporating next to the green fractions evaporating. Finally the brown, green and final red/brown fractions side by side evaporating. Final close up shows brown fraction after solvent purge. The medicine tested nominally at 60% thc in the dispensary, and recovery by weight of total extract in the brown fractions was 51% very high purity cannabinoid. This computes to about 83% recovery of the target compound if the other fractions are not added in. The other fractions will be refined further and I estimate that a total of 90% recovery was achieved. There is much learning yet to be done.

The interesting note here is the final fraction shown in the white ceramic dish evaporating did not elute off the column until the solvent gradient had been replaced with methanol for the mobile phase. The ethyl acetate and hexane gradient used simply did not budge the final fraction off the column. Final fraction appears to be cannabinoid in nature but does not appear to be THC.

I vaped some of the light brown medicine shown in the photo and then promptly knocked the petri dish onto the floor and it broke! Sob, but it is the hazard of being me around glass loz. There is still a pronounced pinene presence (terpene) but it is delightfully attenuated in a way that almost becomes a menthol when vaped. Potency is confirmed to be extreme.

I followed the exact protocol as shown in this excellent video that gave me the idea in the first place;

BobCajun · Jun 9, 2017

I thought of an easy way to isolate fairly pure THCA. This patent application, US 2015/0038567 A1 , describes precipitating THCA as an amine salt. They used an amine which has low solubility in polar solvents so they used isopropyl for the solvent. I don't have access to that particular amine, and it's highly toxic anyway, but I do have a bag of phenylethylamine HCL, which I can easily get the base amine from. I tried it using iso but the amine did not precipitate.

But I was recently thinking what if I use a non-polar solvent, like kerosene? Then the phenylethylamine tetrahydrocannbinolate should precipitate, being that salts are insoluble in non-polars. Then of course it's just a matter of collecting the crystals, putting them in water and adding citric acid, or whatever one you want, HCL or whatever. Citric is cheap and usually the acid employed in recovering THCA from a salt. To make it purer you could recrystallize the THCA amine salt until satisfied, then add the acid. It would then be in the high 90 percents, 97 or so. May be possible to use simple ammonia, if you could find clear. It's mostly water though so that could be a problem.

So the procedure would be to extract the plant material with a non-polar, then do the amine salt part. The solvent doesn't need to be evaporated so it doesn't matter that kerosene is slow drying, but you could use a different non-polar. Kerosene is just easy to get.

Here's a representative quote from the patent app;

Example
[0065] 15.7 g of a ca. 80% mixture of delta 9-THC acids A and
B are dissolved in 150 ml isopropanol with stirring and 8.0 g
of dicyclohexylamine are added with stirring. 24 hrs stirring
at 0° C. produces a thick white precipitate of the dicyclohexy
lamine salts of the cannabinoid carboxylic acids. After suction
filtration, washing with 50 ml cold isopropanol and drying,
18.7 g of dicyclohexylamine salt of the delta 9-THC acids with a
content of 91% are obtained.

Example
[0072] 18.7 g of dicyclohexylamine salt of delta 9-THCA A and
B with a content of 91% delta 9-THC acids in the cannabinoid
content are dissolved with stirring in 150 ml of boiling absolute
ethanol, and cooled immediately after dissolution has
occurred. Stir overnight at 0° C. to crystallize out. Suction
filter the white precipitate formed and wash with 50 ml cold
absolute ethanol. Yield: 15.5 g of pure white salt with a
content of >97% of delta 9-THC acids (A and B) in the cannabinoid
content.

Example
[0078] 15.0 g of recrystallized dicyclohexylamine salt of
the tetrahydrocannabinolic acids A and B are suspended in
200 ml water and overlayered with 200 ml petroleum ether.
3 .0 g of citric acid are now added and the mixture stirred until
the cannabinoid carboxylic acid salt has completely dissolved.
The aqueous phase, which now contains the citrate of
dicyclohexylamine, is separated and discarded. The petroleum
ether phase, which now contains the free tetrahydrocannabinolic
acids, is successively washed once with 50 ml of1% citric acid
and three times with 50 ml portions of water. After evaporation of
the petroleum ether phase on the water bath at 40° C. under
reduced pressure, 9.8 g of amorphous residue of the two
positionally isomeric tetrahydrocannabinolic acids remain.

DaveInCave · Jun 10, 2017

I was thinking, is anyone using antibody based chromatography methods?
In my life whenever we needed to purify something we bonded antibodies to an agarose metrix, you can get super pure extracts like that.
There must be anti-thc antibodies commercially, shouldn't be hard also to immunize some rabbits and take few bleeds off.

Chromatography purification

eugeneoregon

Active Member

BobCajun

Well-Known Member

DaveInCave

Well-Known Member