TLC:
TLC was carried out on three different types (and sizes) of plates: 5 x 20 cm coated with a 0.25 mm layer of silica gel 60F(256) (Merck, Darmstadt, G.F.R.); 10 x 20 cm coated with a 0.25-mm layer of microcrystalline cellulose (Q2F, Quantum Industries, Fairfield, NJ, U.S.A.); and 5 x 10 cm coated with a 0.25-mm layer of alumina K (Q3F, Quantum Industries). All plates were oven-dried and stored in a dessicator (damp plates resulted in inconsistent results). Psilocybin and psilocin standards were spotted on each plate, together with the methanolic extracts used in the HPLC work.
The plates were developed for 15 cm at room temperature in the dark in either a covered development tank or a half-gallon covered Mason jar with one or more filter-paper wicks to ensure vapor saturation in the tanks. Nine solvent systems were used: (1) butanol-acetic acid-water (12:3:5); (2) butanol-acetic acid-water (2:1:1); (3) pure methanol; (4) 1.5% conc. aqueous ammonia in methanol (1.5 ml of reagent-grade ammonium hydroxide in 98.5 ml of methanol); (5) conc. ammonia-water-n-propanol (12:188:500); (6) methanol saturated with conc. aqueous ammonia in chloroform (5:95); (7) water-saturated n-butanol; (8) pure n-propanol; and (9) ammonia-water- n-propanol (10:50:150). Development times ranged from 1.5 h with methanol to more than 7 h with aqueous ammonia-n-propanol.
Developed plates were air-dried and then placed under a short-wave UV lamp (Mineralight, Ultra-Violet Products). Any spots visible to the naked eye or under short-wave UV radiation were encircled with a pencil. Ehrlich's (or Van Urk's) reagent [10% p-dimethylaminobenzaldehyde (pDAB) in conc. hydrochloric acid] was freshly prepared and sprayed in an acetone solution (1 part of Ehrlich's reagent to 4 parts of acetone), and color was allowed to develop overnight by wrapping the plate in the paper towel upon which it had been sprayed. On other plates, color was developed by dipping the plates ina 20% solution of pure anhydrous toluene-p-sulfonic acid (pTSA) in methanol. The plates were then photographed, and the photographs and plates were stored for future reference.
Plate Type: 20 x 5 cm silica gel w/0.25-mm layer silica gel
Solvent System: butanol-acetic acid-water (12:3:5)
| Rf | Upon Drying | Short-Wave UV | Ehrlich's reagent |
| 0.83 | not visible | visible | yellow-brown |
| 0.76 | not visible | visible | strong light purple |
| 0.61 | not visible | visible | very light blue |
| 0.56 | not visible | visible | orange |
| 0.50 | not visible | visible | pink-red becoming grey |
| 0.48 | not visible | visible | yellowish brown (merged with .50) |
| 0.44 | sky-blue | visible | lavender |
| 0.40 | not visible | visible | aquamarine blue |
| 0.36 | sky-blue | visible | darker purple |
| 0.31 | not visible | visible | light blue |
| 0.28 | not visible | visible | brownish orange becoming pink |
| 0.16 | sky-blue | visible | pinkish brown becoming brownish purple |
| 0.13 | not visible | visible | light brown |
| Rf 0.16: | Psilocybin |
| Rf 0.36: | Psilocin |
When developed the same way but dipped in 20% pTSA, only psilocybin,
psilocin and three other spots (Rf 0.13, 0.34 and 0.76) were visible.
Cellulose plates developed in butanol-acetic acid-water (12:3:5) also
showed ample separation of psilocybin and psilocin, but only resolved four
of the other spots. Psilocybin had an Rf of 0.48, and psilocin migrated to
Rf 0.78.
Alumina plates developed in butanol-acetic acid-water (12:3:5) did not
separate psilocybin from psilocin.
Solvent butanol-acetic acid-water (2:1:1), when used with 20 x 5 cm silica
gel plates, also effectively separated psilocybin (Rf 0.21) from psilocin
(Rf 0.46), as well as yielding several other spots, but gave less effective
separation of other peaks than did butanol-acetic acid-water (12:3:5).
Similarly, good separation of psilocybin (Rf 0.14) from psilocin (Rf 0.45)
was obtained with 1.5% ammonia solution in methanol, but only four other
spots were observed. Conc. ammonia-water-n-propanol (12:188:500) resolved
nine spots in addition to psilocybin (Rf 0.11) and psilocin (Rf 0.58)
.
However, the spots were streaked and were not as cleanly resolved as in
butanol-acetic acid-water (12:3:5). Ammonia-water-n-propanol (10:50:150)
resolved eight spots in addition to psilocybin (Rf 0.16) and psilocin
(Rf 0.82), but also displayed considerable streaking. Methanol, n-propanol,
water-saturated n-butanol and methanol saturated with conc. aqueous
ammonia in chloroform (5:95) did not adequately resolve psilocybin and
psilocin.
The only solvent system/plate type even close to what you are using is
possibly the 1.5% ammonia solution in methanol. My guess is that your
spot #4 is psilocybin Rf: ~0.2 (using your picture of the developed plate
and assuming that each hash-mark is .1 RF), spot #3 is psilocin Rf: ~0.5.
I have no idea what spots 1 & 2 are and the researchers didn't mention what
the other four spots were when 1.5% ammonia solution in methanol was used
as the solvent. Spot #2 Rf: ~0.8, Spot #1 Rf: ~1.0 (if it moved with the
solvent front)
It also appears that you have some streaking between spot #3 and spot #2.
It could be possible that the plate needs to be developed for longer than
12 minutes.
So, who's grading this thing anyway? .. and how'd I do? ;)
the grader is "the honorable gene poole - entheogenic research scientist"
poole says you got an A. hee also looked at the reference you posted earlier, and concluded that sally's #4 was psilocybine and #3 was psilocine with #1, and #2 remaining unidentified.
the answer to the extra credit portion is - reduce the amount of methanol to slow the dot run.
BTW Sally, why are you using ethyl acetate-methanol-ammonia hydroxide for > the solvent? Do you have a ref for this or did you make this one up off the > top of your head? Also, I would suggest using a published solvent system > such as butanol-acetic acid-water (12:3:5) with a 20 x 5 cm silica gel > plate like the one in the above experiment... but that's just my opinion.
the 17/2/6drop solvent was recommended by sasha as little as two months ago. this for isolating psilocybine/psilocine and mescaline... and a chloroform/methanol mixture for lsa. he mentioned it is a good gp indole seperating solvent for liquid column chromotography (and could be used for TLC also).
based on the pop quiz results, and further data from later experiments by sally, gene poole would currently recommend...
- keep the 17/2/6drop solvent for column seperation and TLC work if sally is only interested in seperating out psilocybine/psilocine because it develops quickly, low solvent cost, and easily resolves the two desired compounds. (use ehrlich's reagent in addition too)
- use the (12:3:5 - butanol-acetic acid-water) solvent for exacting laboratory research and analysis of fungal material.
- dot #1 is probably not a compound from the extraction at all, but some artifact of the process (sally has a learning curve to overcome), because nothing came out before dot #2 via column chromotograophy.
- dot #2 was a compound that crystallized into beautiful clear white
thin needles, but was not entheogenic in quantities up to 100 mg
(according to sally).
the next quiz will cover liquid column chromotography, so start studying!
- dot #2 was a compound that crystallized into beautiful clear white thin needles, but was not entheogenic in quantities up to 100 mg (according to sally).
sally continued her experiments, and reported the following :
- with more extacting TLC techniques, two smaller dots were found between the original dot#3 and dot#4.
- dot#3 (sky blue under u.v. light) is not psilocine, since it proved to be inactive in quantities up to 70 mg.
pop quiz - TLC of ps. cubensis.
sally extracts fungal material using methanol, and evaporates off the methanol. the resulting goo is dissolved in a solvent mixture of ethyl acetate/methanol/amonium hydroxide (17ml/2ml/6drops) and TLCed. below is what sally's plate looked like :
|
--(*) - dot #1---- <- solvent run after 12 minutes
| (*) - dot #2
|
|
| (*) - dot #3
|
|
| (*) - dot #4
|
---X - sample----- <- baseline
dot #1 moves at the speed of the solvent run, so it is actually distributed evenly along the entire top edge of the solvent run. in normal light it is a light yellowish colour parcipitate, just visible against the white silica background. under shortwave light, it appears very dark.
dot#2 is of less quantity than dot #1, is not visible in normal light, and appears as a fairly dark spot under shortwave light. it almost looks as if a light blue flourescence is occuring just below the dark spot when illuminated with shortwave light. this blueing dissappears within an hour or two.
dot #3 is the smallest dot, much less than the others, is not visible in normal light, and appears as a light blue flourensing spot under shortwave light. the blue disappears within an hour or two, but a faint dark spot is still visible under shortwave light for several days.
dot #4 is the largest dot, roughtly the quantitly of dot #1. on the dry plate, it appears as a dark blue/green to brown spot easily visible in normal light. under shortwave light it appears as a very dark spot.
quiz question -
sally believe that dot #4 is psilocybine. is she correct, and what are dots #1, #2, and #3?
extra credit -
how should sally adjust the solvent mixture so that all four dots appear below the top edge of the solvent run?
Extraction and analysis of indole derivatives from fungal biomass.
Journal of Basic Microbiology, 1994, 34(1):17-22.
(UI: 94267730)
Abstract: The occurrence 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 simple one-step procedure with methanol for the quantitative extraction is still the safest method to obtain the genuine alkaloids from fungal biomass.
This is the procedure Hoffman used. I found it over on hyperreal. I had a dream a while back that this worked real well.
Dry the mushrooms.