The crystallization of psilocybine hydrochloride is much easier than previously thought. The most important existing techniques are at the end of this post. Problem of those is that they use chromatography as final purification step.
But that can be omitted!
The magic step probably was in the addition of the few drops of hydrochloric acid to the alcoholic extract. This produces the psilocybin HCl salt, which is insoluble in acetone and which forms nice big crystals.
It is also important to give each step in the protocol enough time. This is most obvious in the last step, the double acetone wash. The dark residue crystallizes by itself in the acetone after 12 hours!
I looked into this method to see if the green-blue-dark part of the extract can be separated from the psychoactive part. In this method that is not possible. The growing crystals absorb the pigment and then become transparent. The identity of the blueing reaction of psilocybian mushrooms still is a mystery to me.
Summary of the (tested&proven) method:
- harvest&dry the mushrooms/mycelial tissue
- turn it into powder (blender works fine)
- extract overnight with a mixture of ethanol and water (denatured 140 proof is fine)
- filter (vacuum filtration or 0.2um filter mounted on syringe)
- add a few drops of hydrochloric acid (aim at pH3)
- evaporate down to 1/10th of the volume (can be done in a large tupperware container) and put extract in a (tall) vial
- remove fats&resins with a solvent which is not miscible with water (cigarette lighter gas, paint thinner, naphta etc.). Just add the solvent to the extract, mix (slowly, not vigorous) and let it stand for a couple of hours. The solvent floats on top of the extract and can be removed by syringe or by freezing the water and pouring off the solvent.
- remove the rest of the gunk by adding acetone to the extract. Mix slowly and let stand. Again two layers will form. On the bottom of the vial is a dark layer. On top of that floats the acetone which is yellowish to greenish. Remove the toplayer by syringe or pipette.
- add new acetone. Mix slowly. Now the dark layer becomes real sticky. Let stand for a few hours and remove the acetone.
Final step: collect all the dark sticky residue and dry it slowly. Large transparent crystals will form. The more slowly the evaporation the bigger the crystals. The potency of the mushrooms can now be determined by weighing the crystals.
What a relief - Albert Hofmann's approach to use chomatography for the crystallization can be omitted. It is not a difficult technique but it needs a large volume of solvents and very specialized ingredients and reagens. But now I am absolutely sure that large crystals will form after a double acetone wash and simply allow the residue to stand overnight.
Further reading:
American patents 3183172 and 3192111.
Can be viewed at http://patft.uspto.gov/netahtml/srchnum.htm (http://patft.uspto.gov/netahtml/srchnum.htm)
Popularized version:
Gottlieb, THE PSILOCYBIN PRODUCERS GUIDE (1976, 1997)
URL: http://nepenthes.lycaeum.org/Plants/shrooms/shroom1.html (http://nepenthes.lycaeum.org/Plants/shrooms/shroom1.html)
Albert Hofmann's
article 'History Of The Basic Chemical Investigations On The Sacred
Mushrooms Of Mexico', which appeared in the now hopelessly difficult to
obtain book TEONANACATL of 1976 (cut, paste&see
http://dogbert.abebooks.com/abe/BookSearch?AID=8244485&PID=453119&an=ott&sn (http://dogbert.abebooks.com/abe/BookSearch?AID=8244485&PID=453119&an=ott&sn)
=
&tn=teonanacatl&ph=2
Quote:
"In order to preserve the possibly very sensitive, active principles, we
used only neutral solvents and the extractions were carried out at room
temperature. After the extraction of the finely-ground mushrooms with
chloroform, with benzene, and with acetone, the whole activity was still in
the mushroom material. The active principles were easily and completely
extracted with methanol. From the residue of this extract, inactive
constituents could be eliminated by treatment with chloroform. The remaining
easily water-soluble preparation was purified by precipitation of a
concentrated solution in water with ethanol. The activity remained in the
filtrate. The residue of the evaporated filtrate contained the active
principles enriched a hundred fold compared to the dried mushrooms. A
further concentration of the active constituents was possible by paper
chromatography. Using Whatman-I-paper with water-saturated butanol as
solvent, four zones were obtained, the nature of which was determined by
cutting the chromatograms into small strips, extracting the single strips
with methanol, and weighing the residues. In one of the four bands, the
whole activity was found in the form of an easily water-soluble,
halogen-containing powder. After treatment with silvercarbonate, elimination
of silver ions with H2S, and concentration of the aqueous solution in vacuo,
the substance crystallized in fine white needles. With the few milligrams
obtained in this way we made several tests. The new psychotropic principle,
which was named 'psilocybin,' elicited a violet color with Van Urk-reagent,
characteristic for indole derivatives.
For the subsequent isolation experiments, we could rely on this color test.
When paper chromatograms prepared as described above, were sprayed, after
having been dried, with a solution of p-dimethylaminobenzaldehyde in benzol
and put in an atmosphere of dry HCl gas, psilocybin produced a violet spot
with Rf 0.1. A weaker spot with a blue color and Rf 0.5 was observed,
corresponding to a second active principle, which we named 'psilocin.'"[end
After reading this:
http://www.tacethno.com/info/psilocybe/gartz3.txt (http://www.tacethno.com/info/psilocybe/gartz3.txt)
I think that the salty crystals might be urea. I can't imagine that no one came up with the described crystallization method for psilocybine HCl if it were that simple. And there is twice as much urea as alkaloids in the mushrooms.
So What is a good method to separate urea from psilocybine? Can urea be a/b'd?
Yachaj
This was posted by Yachaj elsewhere, and moved here by me /Rhodium
Indeed it seems that the dark pigment in the mushrooms is associated with psilocine, which can be isolated by A/B.
PDF file, see:
http://www.tacethno.com/info/psilocybe/casale_1985_jfs_30_247.pdf (http://www.tacethno.com/info/psilocybe/casale_1985_jfs_30_247.pdf)
At page 77 of Trout's Notes On Some Simple Tryptamines , someone with the alias theobromus proposed two molecular structures of the dark pigment. They were called Psindigo and Leucopsindigo.
Leucopsindigo is drawn as a combination of two psilocine molecules which are attached to eachothers 5th position.
Psindigo looks the same, but the two molecules are attached with a double bond and their 4-OH group is replaced by a double bond & oxygen or 4=O
Yachaj
"The occurence and extraction of indole derivatives in six species from four genera of higher fungi were investigated. By using pure methanol for extraction of the mushrooms analysis revealed the highest concentrations of psilocybin and baeocystin. The psilocin content of the species was higher by using aqueous solutions of alcohols than with methanol alone but was an artificial phenomenon caused by enzymatic destruction of psilocybin. The extraction with dilute acetic acid yielded better results than with the water containing alcohols. The simlpe one-step procedure with methanol for the quantitative extraction is still the safest method to obtain the genuine alkaloids from funghal biomass."
aqueous solutions of alcohols aren't the best route.
http://www.erowid.org/plants/mushrooms/mushrooms_journal1.shtml (http://www.erowid.org/plants/mushrooms/mushrooms_journal1.shtml)
MENH2, I mentioned the A/B of PSOH in this thread already! Read before you post!
See http://www.tacethno.com/info/psilocybe/casale_1985_jfs_30_247.pdf (http://www.tacethno.com/info/psilocybe/casale_1985_jfs_30_247.pdf)
As you can see, this method uses a lot more solvents than the PSOP crystallization method by M/P (mixed phase) extraction which I am proposing in this thread.
Urushibara wrote:
>I don't know what kind of psilocybin mushrooms you are >referring to saying 10mg/g dry, afaik, dry cubensis (afaik >amazonian strain) only contain about 5mg/g
That is the potency on bulk substrates. But see this:
Cubensis on rye:
http://jeremybigwood.net/JBsPUBS/JBScientific/VariationOfPsi/pages/Variation3.htm (http://jeremybigwood.net/JBsPUBS/JBScientific/VariationOfPsi/pages/Variation3.htm)
Similar results have been reached on a brown rice medium. Two grams of dried young cubensis will most certainly knock many people out of their socks.
> if we are dealing with absolute ethanol (ie ~96% ethanol, > the rest water)
I think 140 proof (70 percent) extracts the PSOP faster
> what about doing the extraction at -18 to -20°C?
No idea. But I don't see a concrete problem which is solved by doing so. I think it is more useful to compare results of the extraction at 4 centigrade to an extraction at roomtemperature. If the latter leads to the same results faster I think I prefer a rt extraction above one which needs to be done in a freezer.
The less equipment you need the better!
After a long time lost, a bird flies up through the long awaited crowd of onlookers...
A document was read by this chemist who went to school for a long time that SWIM ran into.
Psilocybin:
Crystals from Boiling water, mp 220-228°; from boiling methanol, mp 185-195°. uv max (methanol): 220, 267, 290 nm (log E 4.6, 3.8, 3.6). pH 5.2 in 50% aq ethanol. Sol in 20 parts boiling water, 120 parts boiling methanol; difficultly sol. in ethanol. Practically insol in chloroform, benzene. LD50 in mice, rats, rabbits (mg/kg): 285, 280, 12.5 i.v. (Usdin, Efron).
Would that not suggest that a bee would merely use a distillation apparatus with vacuum with some methanol (after separating the retail grade methanol from all the water that's added)? What's the trouble with that? Whay all the hubub over A/B's and ethanol? According to the notes above, use water...and if a bee is afraid of damaging the active ingredient because of heat, use a vacuum.
Psilocin:
Plates from methanol, mp 173-176°. Amphoteric substance. Unstable in soln, esp. akaline soln. Very slightly sol in water. uv max: 222, 260, 267, 283, 293nm (log E 4.6, 3.7, 3.8, 3.7, 3.6).
According to the document read, and some of the above information posted by several bees, it seems that this active ingredient isn't worth fighting for.
However, there is concern to SWIM that Baeocystin and Norbaeocystin may be concentrated just like the desired ingredient...is this a factor? SWIM doesn't really want to die right now.
SWIMs' not too sure about chromatography (in red above), does that have to do with all the hubub? If a bee doesn't have a chromatography column?
Please correct SWIM if there's a problem with the above logic.
Looks like this
Maybe sugars, but SWIM tried a very simular process (unresearched, just giving it a try). The residu after a MeOH pull was very sticky and gooie apon evaping the MeOH. A few ml of Vodka were added to the goo along with a drop of HCl. A Acetone flash was then done 2x followed by the addition of a few more ml of MeOH. Neadle like crystals formed after the MeOH evaped off the pie pan. A tine sample (when scraped with a razor blade, 1/3 the lenth of the blade was used as the sample) was bio-assayed. A very nice experance was noted for a slightly longer time frame compared to injestion of just caps/stems.
If there is no h2o pressent in any of the solvents during the extraction, then there should be no sugars present in the final product as MeOH and sugars dont like each other.
seems to have solved the sugar and urea problem as urea is soluble in water (big time...Solubility in Water: 1,193 g/L at 250C), and it's toxic to the gonads...yea yea cool...are you thretening me? I am cornholio!
Is that right?
Lilienthal wrote:
"your product is a mixture of everything soluble in aqueous MeOH / EtOH and unsoluble in ethanol"
True. But the undesired components do not interfere enough with the solubility of the major psychoactive component (PSOP) to make the standardized extract unreliable.
But hey - look at this. It is about the use of 4-DMCA instead of 4-DMBA as main ingredient in Ehrlich's reagent. 4-DMCA is OTC and most likely useful as indicator when a simple piece of typing paper is used as TLC surface.
From the paper then all different components can be separately recognized, -extracted and purified with only alcohol as solvent.
Of course the results will probably be better (less losses)with more difficult to obtain solvents and real TLC paper, but given the fact that the desired alkaloids can readily be biosynthesized from brown rice or malt agar, a crude typing paper TLC with 4-DMCA as OTC color indicator can be the finishing touch.
(newbees who have no idea about TLC and mushrooms may also read the text http://www.erowid.org/plants/mushrooms/mushrooms_article2.shtml (http://www.erowid.org/plants/mushrooms/mushrooms_article2.shtml)
)
from:
Occurence of 5-hydroxylated indole derivatives in Paneolina foenescii (Fries) Kuehner from various origin. T. Stijve, C. Hischenhuber, D. Ashley. Z. Mycol. 50: 361 (1984)
Ehrlich's reagent (Révélateurs pour la chromatographie en couches minces et sur papier, E. Merck, Darmstadt 1975, no 91, p. 32) was initially used because it yielded brightly coloured spots with most of the compounds of interest. However, detection of psilocybin required a few minutes heating at 100 deg. C and under these circumstances co-extracted urea yielded a brightly yellow zone which interfered with the evaluation of serotonin, a major constituent. In addition, sensitivity for tryptophan was poor.
Better results were obtaines using 4-dimethylamino cinnamaldehyde (DMCA) (Fluka no 39421) which was used as a solution of 0,5 g in 10 ml fuming concentrated hydrochloric acid, mixed with 50 ml methanol. This reagent was more sensitive than its benzaldehyde analogue and did not need heating to react with the various indoles. In addition, it produced a different shade of colour with each compound. For example, psilocin turned greenish gray, psilocybin reddish, bufotenin violet, 5-hydroxyindole acetic acid green, serotonin and 5-hydroxytryptophan bright blue, tryptophan purple and tryptamine purple red.
It should be pointed out that these colours may vary with the chemical nature of the TLC adsorbent. For example, psilocybin spots are reddish on Si02 and on SilCel layers, but violet on cellulose.
The reagent proved tobe remarkably sensitive: the detection limitfor serotonin and 5-hydroxytryptophan was 10 ng and for psilocybin and tryptophan 25 ng. At room temperature psiocybin was the last of the indolic ompounds to become visible. Usually, optimal visibility was obtained after 10-15 mm. The reaction could be accelerated by slightly heating with a stream of warm air from a hair-dryer.
Interestingly, the DMCA reagent reacted only very slowly with urea, which yielded a reddish spot only after a few hours, and thus did not interfere with the determination of serotonin.
I'm inclined to agree yachaj - sure, in the sum total of the contents of the solvent before precipitating it there is oodles of junk, there's all sorts of junk (the major crap in there is urea, you can smell it when it's evaporated down), but the use of ethanol is selective for precipitating the psilocybin. most everything that was named is much more soluble in ethanol than psilocybin.
on another note, rhodium just posted on another thread about a method of doing an acid/base extraction on psilocybin that dephosphorylates the psilocybin into psilocin using acetic acid and a little heat. Post 449383 (https://www.thevespiary.org/talk/index.php?topic=12923.msg44938300#msg44938300)
(Rhodium: "Mushroom Aqueous-Organic Psilocin Extraction", Tryptamine Chemistry) it's an acid/base, and the article quoted claims that it is pure enough to fully characterise with it's IR spectra etc.
I put an idea forward in a response there too - would making the psilocin into the ascorbate salt help allieviate stability problems?