Preparation of crystalline THCA

[SkyHighLer]

^^

"CHAPTER 3

Preparative isolation of cannabinoids from Cannabis sativa by centrifugal partition chromatography
•••
Arno Hazekamp, Ruud Simons, Anja Peltenburg-Looman, Melvin Sengers, Rianne van Zweden, Robert Verpoorte
••
Leiden University, Department of Pharmacognosy, Gorlaeus Laboratories Leiden, The Netherlands
•
Published in J. Liq. Chrom. Rel. Technol. 2004, 27(15): 2421-2439

Abstract
A simple method is presented for the preparative isolation of seven major cannabinoids from Cannabis sativa plant material. Separation was performed by centrifugal partition chromatography, a technique that permits large scale preparative isolations. Using only two solvent systems, it was possible to obtain purified samples of the cannabinoids; (-)-∆9-(trans)- tetrahydrocannabinol (∆9-THC), cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), (-)-∆9-(trans)-tetrahydrocannabinolic acid-A (THCA), cannabigerolic acid (CBGA) and cannabidiolic acid (CBDA). A drug-type and a fiber-type cannabis cultivar were used for the isolation. All isolates were shown to be 90-95% pure by gas chromatography. This method makes new cannabinoids available on a large scale for biological testing. The method described in this report can also be used to isolate additional cannabinoids from cannabis plant material."

https://openaccess.leidenuniv.nl/bitstream/handle/1887/12297/Thesis.pdf

 

[goose920]

Pangea- those are amazing!! I really need to give this method a try. Been reading up about it on here all day and I think I could. I have a couple questions I haven't seen asked and I hope it's not because they are silly!

 

[SkyHighLer]

I used to have an Acme 6001 stainless steel centrifugal juicer, seems to me it might be useful for experimental purposes. Either to spin up some high THC-A laden oil to see if the THC-A crystals can be coaxed out, or to do some on the cheap centrifugal partition chromatography by packing the screened basket with your filter material, and squirting measured amounts of solvent into the spinning basket at intervals.

It can be operated with the top off as can be seen in this video,

https://youtu.be/LvBoQNVaLJI

You should be able to wash down the individual partitions with some extra solvent from a lab wash bottle while keeping it running. The switch is the only source of ignition, tape it in the on position, and switch it externally.

You could probably also use it to filter winterized oil, by pre-freezing the basket lined with filter paper.

Pic of the basket

 

[SkyHighLer]

^ Used SS Acme 6001's are cheap at eBay, they're extremely robust, should do you fine, but it's still available under the Waring Commercial brand,

https://www.amazon.com/6001-Juicerator-550-Watt-Extractor-Stainless/dp/B0028RXDFM

At that page I noticed they have filter strips to line the basket,

https://www.amazon.com/Replacement-...9_1?ie=UTF8&psc=1&refRID=GKB79APDXC7JS3CJHJJG


You might want to slow it down, should function fine with a 120V 10A or higher rated Variac, also something you might consider getting used,

https://www.amazon.com/Circuit-Spec...3&sr=8-2&keywords=variac+variable+transformer

If you run it with the top off, use some tape or bungee cords, whatever, to hold the collection pan more firmly in place, you don't want the spinning basket scraping against the pan when working with flammable solvents. I say that because I just know someone's going to try filtering sub zero butane through it, just covering my ass.

 

[weedaholic721]

Talking gibberish there SkyHighler I see.

But seriously, you might be in to something good. It would be great for all the the high terpene extractions goin around. The cannabinoids naturally separate it seems so it seems like a very good route for getting as much if the terpene layer off of the cannabinoid microcrystals.

 

[SkyHighLer]

^ All kinds of things you could try with it, how about thickly packing the basket with "acid-washed see-sand (Sigma) and topped with glass pearls (±1 mm diameter)?"

"3.2.7 Separation of acidic and neutral Cannabinoids

A glass-filter (mesh size 2) of about 5 cm in diameter and 7 cm in height was filled for 2/3 with acid-washed see-sand (Sigma) and topped with glass pearls (±1 mm diameter). Before use the sand was sequentially washed with 200 ml of hexane, ethanol and water. Cannabis hexane extract was concentrated to about 5 ml of hexane, placed drop-wise on top of the sandfilter and evaporated by using a warm air blower. The sandfilter was then placed onto a suction Erlenmeyer and acidic cannabinoids were eluted by washing the sandfilter under vacuum with a 0.1 M NaOH solution. The elution was continued until the eluate turned from deep-orange to colorless. Neutral cannabinoids and other compounds were then eluted with ethanol (200 ml), followed by hexane (200 ml). Acidic cannabinoids were precipitated in the aqueous eluate by adding HCl until the pH reached 2 and then filtered through the (dried) sandfilter. The precipitate that remained on top of the sandfilter was finally eluted with ethanol (200 ml). Neutral and acidic cannabinoid fractions were both concentrated into a small volume by evaporation under reduced pressure and analyzed by GC and HPLC."

https://openaccess.leidenuniv.nl/bitstream/handle/1887/12297/Thesis.pdf

 

[SkyHighLer]

^^ You could even do the initial extraction with an Acme SS centrifugal juicer:

1) Line the basket with a couple of the filter papers I linked to above, wet them with solvent.

2) Grind your herb through the machine like you would fruit or vegetables.

3) (optional) Freeze the basket with the ground herb (it should be a solid 'cake' on the inside of the basket,) and the heavy cutter/shedder plate. Plastic bag them so they don't pick up moisture. Freeze the solvent.

4) You can either pour the solvent through the plunger opening on the lid; or run it without the lid, and squirt solvent with a wash bottle.

The above post used hexane for the initial extraction and elution, why not use pentane, less smell, and a safer Class 3 rather than Class 2 food and drug solvent.


Should be good for a super cold ethanol quick wash? And then hit it again with room temp solvent for oral meds.

 

[SkyHighLer]

^^^ Another, wetted paper filter in the Acme 6001 basket, dump activated carbon (charcoal) powder through the plunger opening to form a thick packed filter, hit it with your oil solvent solution and out comes, near clear?? And with way less solvent/oil loss than a Buchner.

On that drift, line the basket with a one micron sock filter...

Or line the basket with a really 'tight' filter, and you can filter mold without the hassle of syringe filters.


I didn't see anyone using a centrifugal juicer as a DIY centrifuge at YouTube, maybe underground chemists are aware of the idea, has anyone run across it?

 

[SkyHighLer]

The bee keeper/bio diesel guys have a basic centrifuge design, see the video link below, and unless I'm missing something, you'd get the exact same action by blocking the Acme 6001's basket's filter screen with something, and pouring solvent/oil slowly down the plunger opening... the unit is designed to handle overflow, it will catch in the lid bulge, and flow down and out through the spout.

The Acme 6001 runs at 3,600 rpm, and if you use a Variac as mentioned in one my posts above, you can overdrive it by as I recall, about ten percent along with varying the speed.

https://youtu.be/PsLGX-RbKuQ

 

[SkyHighLer]

^ Found a Youtube video of how to do just what I described above to filter waste vegetable oil, the Omega 1000 is very similar to the Acme 6001,

https://youtu.be/rS26K9xt3Zg

 

[adamSmithWealth]

what is RO water?

what is RO water?

what is RO water?

There are many reasons for developing chromatography methods for isolating cannabinoids, I am sure even if GW Pharm were aware of the simplicity involved in a method like ive shared they would still pursue and research other methods, there are utility and merits to different paths.

Ive shared it a few times, but guess I can be more detailed.

The recrystallization for purity part is pretty standard, the initial crystallization part is the first hurdle and just as simple. Its actually easier than making traditional BHO as it involves less steps. Instead of spreading thin and purging, collect it in a container/vessel that allows for slow, controlled evaporation, depth of the solution vs surface area is a important factor as is the headspace and lid for evaporation. A container like a graduated cylinder is ideal or a regular 250ml mason jar filled at least half full work as well. The larger the surface area the easier it is for the solution to crash, and also has other limiting factors. The solution viscosity is important as well, I vac my collection chamber down to -20, there is no liquid tane, I cannot pour out the solution, I scape collect it, but its basically like a No 1. maple syrup consistancy, very liquidy, very terpy, glass baster/droppers work well to transfer.

So recap,

1. Collect bho into proper vessel.
2. Store in proper area; consistent temp, limit light, limit vibrations, limit disturbances.
3. Wait until satisfied with crystallization amount.
4. Pour off un crystallized solution into new vessel to further form crystals(depends on state of solution) or pour/spoon out and treat remaining solution as if it was just extracted, so either vac purge or decarb for edibles.
5. Carefully collect crystals from the bottom of the vessel(probably where a jar has a advantage over a skinny cylinder)

I've been able to grow crystals in little 2.5ml vials with high terp varieties after they have been vac purged, just by leaving the lid loose.

I've had a crystal in a sealed solution of RO water since I first collected them, it hasnt changed. Ive had a few in indirect sunlight for 60 or so days with no visible changes. They are quite stable.

I am excited to see some really large crystals, they're going to look very cool!

 

[sadpanda]

RO reverse osmosis (effectively the same as distilled i guess?)

 

[Thomas@LRL]

what is RO water?

Reverse Osmosis, purified water.

 

[Thomas@LRL]

Skyhigh - the thesis by Arno hazekamp titled "cannabis; extracting the medicine"

His paper covers cannabinoid isolation using CPC, proton NMR quantitative analysis, reverse phase hplc analysis, and thc complexing with beta-cyclodextrins

Hellen Perotin-Brunel also describes CPC as being the ideal method of isolating cannabinoids following Supercritical CO2 extraction in her thesis on Sustainable Cannabinoid Production with Supercritical Carbon Dioxide.

 

[easystephen]

Has anyone tried an acid/base extraction to isolate THCA? I was going to try this for CBDA but then I realized I would probably convert most of my CBDA into THCA.

 

[Old Gold]

Has anyone tried an acid/base extraction to isolate THCA? I was going to try this for CBDA but then I realized I would probably convert most of my CBDA into THCA.

I attempted it, but due to limited access to even my own resources, my trials were cut short and left alone for later experimentation. But I can tell you what I went through...

We briefly mentioned it here:
https://www.icmag.com/ic/showthread.php?t=333935

And some cat "gordonliu" casually threw this at us in 2007, when the subject was cleaning up an edible ISO extract:

a couple things

lipophilic and "idrophylic" - the correct word is hydrophilic (hydro for water, lipo for fat, lipophilic solvents are more commonly referred to as non-polar or hydrophobic)

you will be tough pressed to find food grade hexanes in quantities high enough for what you will be doing without being questioned about its intended used. best to stay away all together.


DO NOT BUY GLASSWARE UNLESS IT IS BRAND NEW!!!! it is extremely difficult to remove any and all traces of contaminants from laboratory glassware. 2 ppm of benzene in an analytical sample is nothing, but it is something you do not want to be ingesting. benzene is the least of your worries when you buy used glassware!

activated carbon works best when it is in contact with the material for longer periods. be careful, however, as activated carbon will absorb anything, it just absorbs polar organics quicker than non-polar organics. also it works best when you heat a solution containing the extract with activated carbon.




as a last note:

you guys are thinking in the right direction to help purify an extract. A little advice, however.

1) dissolve in hexanes (works best in my experienced, methylene chloride, ethyl acetate, ether, all form pretty shitty emulsions)

2) wash hexane solution with 2M HCLx4 with 1/2 the volume of hexanes, discard acid

3)wash hexane solution with 2M KOHx5-10 with 1/2 the volume of hexanes, recover aqueous phase (you can probably still get more cannabinoids out of the organic phase, so save it)

4)acidify aqueous phase until ph~5 (just less than 7 is fine)

5)extractx4 with 1/2 volume of aqueous phase using hexanes, recover organics

6)take like 2 times the volume of hexanes that were recovered from previous step of water, pour a shitload of salt in it. shake it up. let it settle.

use this brine solution to dry the organic phase

7)let the organic phase also dry over a drying agent like Na2SO4

8)concentrate the organics to yield a purified product containing cannabinoids, POSSIBLY other substituted phenols, and in theory nothing else.

AFOAF has done this and the 1H NMR (CDCl3) is pretty clean! (two spots on a TLC using CHCl3:MEOH 10:1)

didnt smoke it as procedure utilized a bunch of used glassware, and yield was low, because THC isnt very water soluble, even as the phenoxide anion, and the starting material was vaporized buds (although it was made from about 224 grams, and the extract was run through washed activated carbon for about 4 hours using a makeshift homemade glass column)

As for my experience, nothing was measured. Everything was eyeballed according to prior knowledge.

I began with the end scrapings (estimated 15-20g) from my collection pot after a very tasty nug run (very heavy on pinene and limonene). Here is the whole run, purged and ready to dab.



And below is the 15-20g of oil dissolved in nonpolar naphtha, after having been winterized very well. This is all experimental, so I didn't spend the money on heptane considering I had this stuff lying around. Also, I planned on washing the final product several times with something like butane and letting it re-evaporate to wash residual nonpolar solvent.



I then made an alkaline solution that was pretty much saturated (NaOH in distilled water). This reaction heats up a lot, so I let the solution cool before mixing it with my thc-laiden naphtha (did not want to catalyze decarboxylation via heat). Here is 20-30 seconds after adding the aqueous NaOH to naphtha, and stirring.



As you can see, three layers formed. This was a great surprise to me. I let it settle, then stirred, let settle, then stirred several times. Every time, these three distinct layers formed. Due to a broken separatory funnel, I threw the jar in the freezer. Before the water had time to freeze, I noticed the thick emulsion solidified as it's own layer in the middle. So I was able to pour off the alkaline water and naphtha.
Here is what was left behind...it looks dark maroon, but upon close inspection, I would honestly say it's purple.



And here are my liquid layers after removing the emulsion via freeze-separation.

 

[Old Gold]

The naphtha remained a gorgeous, rich amber (terptown?!?!), while the previously clear water grabbed a bright, almost neon yellow. I rinsed the alkaline water two more times with clean naphtha. The first of these pulled a rich yellow color (terps?!?!) and the second wash pulled absolutely no color at all. The first picture below is the amber naphtha from above diluted with the yellow naphtha from the first clean naphtha wash. I had to consolidate, and forgot to snap a pic. The second photo below is the second clean naphtha wash, which indicated I was no longer pulling impurities from the alkaline water.




Days later, a new separatory funnel arrived, although it was a small one. I separated my water solution, acidified it dropwise with dilute HCl (again EYEBALLED lol). Seeing no visible precipitate or cloudiness after surely acidifying, I washed the solution several times with again clean naphtha, hoping to extract the goodies from water. The naphtha definitely obtained an off-white color that it didn't have before, which excited me, but after evaporating, a completely clean glass dish remained.

In the future, I would mix the thc/naphtha solution with the initial aqueous NaOH solution several times (5-10 times according to gordonliu!)...I thought I'd be able to play with it more, so I wanted to see what came of the first wash quickly before spending time or resource on any more (I had no separatory funnel for most of the process...)
Also, I would measure things out and use a digital pH meter.

Regarding what gordonliu suggested about washing the initial solution with dilute acid to remove impurities - it is logical, but I am not sure if the presence of a strong acid would catalyze decarboxylation. I think it may.

Anyways, I hope you get the chance to try it. I still have high hopes for the process, and think it's one of the more affordable and time-friendly methods for isolating a 90+% pure THCa product.

 

[Old Gold]

More advancement in my spare time!
This was a very simple approach that I'd like to play with some more. I took a mostly [but not fully] winterized extract (approximately 0.5 g or so) and dissolved it in a small amount of ye' ole naphtha. Separately, I combined less than a mL of acidic water (guessing pH~4 or 5) with a literal few drops of denatured alcohol. After mixing the polar and nonpolar solutions, the polar layer was much darker than the naphtha now was, having definitely grabbed some color.

Both solutions were evaporated down and finished for a few hours under vacuum. Look at the pretty crystal left behind from the naphtha solution. Pictured next to it (on the dab tool) is what the polar alcohol and acid-water extracted. With minimal moisture, the dark stuff is almost stable enough to be slabbed, but spreads thin and oily very easily. I suppose these polar components are flavonoids (perhaps specifically cyanins - which could be quickly figured with color and pH changes?), while more nonpolar cannibanoids stayed in naphtha.

You can see a dark impurity on the crystal (which I'm positive has naphtha locked up in it, and perhaps a tiny amount of wax). I don't have much equipment accesible at the moment, so I ramped my heat pad up to it's maximum temperature of 160F. It certainly didn't fully melt at that temperature, but I noticed it get a little bit hazier than before, and the dark spot began to spread on the crystal. I quickly cooled it back off, and tried washing the impurity off with alcohol, dropwise. It worked, but definitely took some yield with it, as it rounded the edges and made the crystal more spherical and single-bodied.

Then I got curious enough to vacuum it for a few hours and take a dab... Smooth, almost tasteless (sweetness from solvents), and I was able to dab it at the temperature I normally dab my winterized oils at. The nail left hardly any residue, and I took it down in single dab. Crystal size seemed to be about 1 cm at it's longest (before dropwise alcohol wash), and 0.5 cm after the alcohol wash.



Below is the final, washed crystal. Definitely not "pure" but a nice crystallization that I'm sure is somewhere in the 90%+ range.

'

Update: I just ate the dark stuff. It was oily (easily dissolved on tongue) and not sticky, tasted slightly hashy (it came from winterized trim run), but was not bitter. My dumb ass is also realizing as I type this, that I never threw it onto the heating pad at 160F. For next time... (I plan on having a good hotplate and thermocouple - as well as hopefully better solvents and a digital pH meter)

Upon review, I think the major presence of water (potentially the acid as well) was a key factor.

 

[Old Gold]

In upscaling this, I'm coming to some basic chemistry realizations lol
Ethanol solubility is more troublesome, so I am looking for a different choice of solvents. I think the reason I had such easy separation was the high water content as well as high methanol content. Ethanol isn't as friendly in its solubility.
Three layers of [not so beautiful] separation now when using denatured alcohol and naphtha...

I am looking to do this again with water (still trying to determine how helpful the acid was), hexane/heptane, and pure methanol.


This is quite random, but could [or should] octyl acetate (or simply octanol) be used as a laboratory solvent? Very high boiling point, but has otherwise interesting characteristics.

 

[ClandestineChem]

THANK YOU!

THANK YOU!

In upscaling this, I'm coming to some basic chemistry realizations lol
Ethanol solubility is more troublesome, so I am looking for a different choice of solvents. I think the reason I had such easy separation was the high water content as well as high methanol content. Ethanol isn't as friendly in its solubility.
Three layers of [not so beautiful] separation now when using denatured alcohol and naphtha...

I am looking to do this again with water (still trying to determine how helpful the acid was), hexane/heptane, and pure methanol.


This is quite random, but could [or should] octyl acetate (or simply octanol) be used as a laboratory solvent? Very high boiling point, but has otherwise interesting characteristics.

Reply:
After spending days searching multiple blogs, forums, and patents I am so happy that you directed me to this thread. Im also glad to see someone had a breakthrough and actually did it instead of just taking about it. I just wish I saw this sooner. I actually tried the a/b extraction that also didint work for you. I was disappointed when my n-hexane evaporated and nothing remained.

Could you please provide details about the most recent discover[FONT=Arial, Helvetica, sans-serif]y? I am a little lost on how you added only 1mL of acidic water and got separation. Did the THCA remain in the non-polar naphtha while the THC migrated into the polar acidic water?
How does the addition of ethanol or methanol work? Being both miscible with your polar and non-polar solvents wouldn't it just make for a poor separation layer? If not please explain.

Again, thank you for posting this. If possible I would like to direct message you but cant find how on this website (still new to icmag.com).
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