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 AdoreChampignons
Mycophilic One
Registered: 08/10/08
Posts: 313
Loc: Seti Alpha 5
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 Testing For Psilocybin Potency Using TLC
#8853761 - 08/30/08 07:57 PM (3 months, 3 days ago) |
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In testing for the potency of a mushroom, Ive read in previous posts that thin layer chromatography(aka TLC) or paper chromatography was used. Im fairly familiar with the chromatography technique. Ive read in a couple places that the chromatogram is very characteristic of psilocybic species. Has anyone seen what the chromatogram looks like of P. cubensis? Is it just one big blue band, multiple bands, or are there other more prominent features?
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There is no such thing as a dumb question. There are just curious people trying to learn something new.
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 agmotes165
I only wish i was this good!
 
Registered: 12/30/07
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wow yea i would love to know about this. I could use some of this info to get a more pure form of psilocybin/psilocin extract 
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all things are relative, its either a tv or an x-ray tube depending on where ur standing...
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AdoreChampignons
Mycophilic One
Registered: 08/10/08
Posts: 313
Loc: Seti Alpha 5
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 Re: Testing For Psilocybin Potency Using TLC [Re: agmotes165]
#8853925 - 08/30/08 09:06 PM (3 months, 3 days ago) |
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Quote:
agmotes165 said:
wow yea i would love to know about this. I could use some of this info to get a more pure form of psilocybin/psilocin extract
Oh, you're looking for a purification technique. Check out this web page at www.erowid.org/archive/rhodium/chemistry/psilocin.extraction.html
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There is no such thing as a dumb question. There are just curious people trying to learn something new.
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agmotes165
I only wish i was this good!
Registered: 12/30/07
Posts: 377
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all things are relative, its either a tv or an x-ray tube depending on where ur standing...
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AdoreChampignons
Mycophilic One
Registered: 08/10/08
Posts: 313
Loc: Seti Alpha 5
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Re: Testing For Psilocybin Potency Using TLC [Re: agmotes165]
#8854039 - 08/30/08 09:43 PM (3 months, 3 days ago) |
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My pleasure. The good thing about the extraction process is that the chemicals are readily available. If you have trouble getting a hold of nitrogen gas, I have a feeling you could substitute helium. They're just using nitrogen as an inert gas to collect the isolate under, and there's nothing more inert than a noble gas.
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There is no such thing as a dumb question. There are just curious people trying to learn something new.
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Workman
Psilocybe Microscopist
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Re: Testing For Psilocybin Potency Using TLC [Re: AdoreChampignons]
#8868389 - 09/02/08 08:21 PM (3 months, 12 hours ago) |
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This plate is sideways with the initial spotting on the left. Its not Psilocybe cubensis but you get the idea. All of the colored spots are not visible before developing with pDMAB (Ehrlich's reagent) spray.
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Moodion
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Re: Testing For Psilocybin Potency Using TLC [Re: Workman]
#8868448 - 09/02/08 08:30 PM (3 months, 12 hours ago) |
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What are those TLC plates called, where you don't have to view them under a UV?
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fastfred
Registered: 05/17/04
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Re: Testing For Psilocybin Potency Using TLC [Re: Moodion]
#8868563 - 09/02/08 08:50 PM (3 months, 12 hours ago) |
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Quote:
What are those TLC plates called, where you don't have to view them under a UV?
Fluorescent plates.
Here is some more TLC data using different solvent systems.
-FF
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AdoreChampignons
Mycophilic One
Registered: 08/10/08
Posts: 313
Loc: Seti Alpha 5
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Re: Testing For Psilocybin Potency Using TLC [Re: Moodion]
#8869165 - 09/02/08 10:41 PM (3 months, 10 hours ago) |
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Quote:
Moodion said:
What are those TLC plates called, where you don't have to view them under a UV?
Depending on the stain, the plates can be viewed under normal lighting. There are others that need to be viewed under UV-light
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There is no such thing as a dumb question. There are just curious people trying to learn something new.
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AdoreChampignons
Mycophilic One
Registered: 08/10/08
Posts: 313
Loc: Seti Alpha 5
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Re: Testing For Psilocybin Potency Using TLC [Re: Moodion]
#8869290 - 09/02/08 11:11 PM (3 months, 10 hours ago) |
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Quote:
Moodion said:
What are those TLC plates called...?
They're called chromatograms.
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Alan Rockefeller
Mycologist
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Re: Testing For Psilocybin Potency Using TLC [Re: AdoreChampignons]
#8869639 - 09/03/08 12:45 AM (3 months, 8 hours ago) |
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AdoreChampignons
Mycophilic One
Registered: 08/10/08
Posts: 313
Loc: Seti Alpha 5
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Quote:
Alan Rockefeller said:
...I wonder what else I would need to make this work...
I'd check the previous posts on the type of solvent system that you want to use. You want to use of course some thing that is easily available and fairly non-toxic. The next step after all the materials have been secured is to develop a very systematic way to ensure consistent results. For example, you'd want to set up your own testing standards and limit each test to some amount of dried mushroom that you can always obtain for sampling. The mushroom has to be first completely dried, ground to a powder then weighed before being put through the testing procedure. Consistency is important because you'll be comparing dots or bands on one chromatogram to another. You want to be able to say that this chromatogram has a larger band, therefore it must have a higher content of psilocybin.
What you don't want to say to yourself is: "Gee, this band is larger than the other one. Was this sample drier than the previous sample and causing me to weigh out more dried mushroom?" Or, "Gee, the band on this one is smaller. Is there less psilocybin because this sat around for a few days before I was able to dry it?" There can be many variations that could skew your results. Therefore, it's best to initially write out a flow chart for your procedure. Later, write out a table that you could write down your results in. I myself haven't performed test using psilocybin, but have a lot college lab experience. In knowing that, recording results as quickly as possible is important. Also, it may be a good idea to scan your chromatogram into your computer and store as a Gif file as well as storing your actual chromatogram in a cool dry place. Experience has shown me that those chromatograms aren't very stable and often shift their color or fade in a few days.
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There is no such thing as a dumb question. There are just curious people trying to learn something new.
Edited by AdoreChampignons (09/03/08 11:10 PM)
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Workman
Psilocybe Microscopist
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Posts: 2,403
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Re: Testing For Psilocybin Potency Using TLC [Re: AdoreChampignons]
#8876010 - 09/04/08 09:43 AM (2 months, 29 days ago) |
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I've been gathering information on this for a few years and have already started gathering supplies. I am going to practice using filter paper (cut up filter disks) just to get the feel for the method, then invest in some silica gel plates. The easiest/cheapest good resolution solvent system I've found is 99% isopropranol rubbing alcohol and 5% janitorial grade ammonia (5:2). I'll document my method and use as many household products as possible. Concentrated HCL for the developing solution is available as Muriatic acid from the hardware store. Methanol for extraction is available as HEET gas additive. So far, only the pDMAB is not readily available from a non-chemical supplier. Household white vinegar (5%) is an alternative for methanol extraction.
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Research funded by the patrons of
The Spore Works
Exotic Spore Supply
Reinvesting 25% of Sales Towards Basic Research and Species Identification
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AdoreChampignons
Mycophilic One
Registered: 08/10/08
Posts: 313
Loc: Seti Alpha 5
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Re: Testing For Psilocybin Potency Using TLC [Re: Workman]
#8879460 - 09/04/08 09:04 PM (2 months, 29 days ago) |
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Quote:
Workman said:
.... So far, only the pDMAB is not readily available from a non-chemical supplier...
You're quite right. pDMAD is not readily available. There is also a reagent called Ninhydrin. They use in forensics to enhance the visibility of finger prints. Ninhydrin is widely available. However, it's pretty toxic. I'm pretty sure it'll work. It's labeled as one of the reagents used to develop chromatography plates for nitrogenous functional groups found in the psilocybin structure. However, I haven't found a website that can confirm this. If you know someone who is into forensics, see if you could borrow their can of ninhydrin spray and spray it on a chromatogram of a mushroom isolate. The pattern that should appear should look very similar to the one you posted earlier. However, I don't believe that the ninhydrin stain is as definitive as the pDMAD reagent. Different compounds don't express themselves with different colors. Ninhydrin pretty much stains all nitrogenous compound purple. Therefore, identification has to be done by the position of the band.
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There is no such thing as a dumb question. There are just curious people trying to learn something new.
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AdoreChampignons
Mycophilic One
Registered: 08/10/08
Posts: 313
Loc: Seti Alpha 5
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An Atempt at Quantifying Chromatographic Bands [Re: Workman]
#8931345 - 09/15/08 12:09 AM (2 months, 19 days ago) |
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Quote:
Workman said:
I've been gathering information on this for a few years and have already started gathering supplies. I am going to practice using filter paper (cut up filter disks) just to get the feel for the method, then invest in some silica gel plates....
Greetings Workman,
I was thinking of how the chromatography test could be made more quantitative than qualitative. Normally, a chromatogram is purely quantitative. It’s designed to show that certain compounds are present but not how much of that compound. However, there might be a way of making it more quantitative.
One way of making it more quantitative is to first make a color photocopy of the chromatogram at the original size. Next, make a blow up of the chromatogram to about five times the original size. Blowing up the chromatogram may not fit on the entire sheet of paper but that doesn’t really matter. The only two bands that need to fit on the paper are the bands for psilocin and the bands for psilocybin. Once the corresponding bands have been blown up, place a sheet of transparent plastic grid over the enlarged copy. To make it easier for counting, use a grid that is graduated in square centimeters. Next, to get a roughly quantitative measurement from the chromatogram, all one has to do is count the squares that the band for psilocin and the band for psilocybin encompass.
By counting the square centimeters of each band, what we’re actually doing is saying that the square centimeters, or area of each band, is equivalent to the concentration of psilocin or psilocybin in the sample mushroom. For example, the first sample may come out with 10 square centimeters of psilocin and 20 square centimeters of psilocybin. The second sample mushroom may have a figure of 20 sq. cm of psilocin and 40 sq. cm of psilocybin. What you can now say is that the second mushroom has practically twice the content of active compounds. You couldn't say that before. You cannot say what the concentration is. All you know is that the second sample mushroom has twice the active compounds. Therefore, you can then conclude to select spores from the second mushroom to breed more potent shrooms from.
The beauty of this technique is that now something that was originally quantitative now has hard numbers assigned to it. Originally, one could only say that one band looks larger than the other. Therefore, it must have more of the active compound. Now, in enlarging the chromatogram and measuring the area of the band itself, one can at least have firm numbers. This technique becomes even more helpful in attempting to assess the area of chromatographic bands that at times show up not as circles, but as ellipses, tear drop shapes, irregular lines, and so on.
There are major assumptions to this procedure though. The three major assumptions are that both psilocin and psilocybin both spread out consistently and uniformly across the filter paper when they are put through chromatographic separation process. They must not only spread only consistently and uniformly during the first chromatogram, but to every chromatogram it is compared to. The second major assumption is that each sample of mushroom tissue tested is always at the same amount, having the same moisture content, and from the same part of the mushroom. The third assumption is that the amount of mushroom extract applied to the chromatography paper, is always the same amount and at the same concentration.
There are an incredible number of variables to deal with but it is the cheapest way I can think of to make quantitative measurements of mushroom extracts. One of my college lab profs said that I always seem know what I’m doing, its just that I come up with the quick and dirty way of doing things. I’m not exactly sure what he meant by that. Never the less, it’s way cheaper to do it this way than to buy a gas chromatograph that’ll costs thousands of dollars.
It sounds like you're better prepared to begin testing samples than anyone I know. Best of luck to you. Please keep us posted of your results.
Mycophilic One
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There is no such thing as a dumb question. There are just curious people trying to learn something new.
Edited by AdoreChampignons (09/15/08 12:17 AM)
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Workman
Psilocybe Microscopist
Registered: 03/01/01
Posts: 2,403
Loc: Washington, USA
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Re: An Atempt at Quantifying Chromatographic Bands [Re: AdoreChampignons]
#8932950 - 09/15/08 11:58 AM (2 months, 18 days ago) |
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Actually, there is software and freeware available to analyze flatbed scanned plates automatically. Some quantify spot color density and some the area of the spot (similar to your suggestion). Calibration with known samples should give some pretty decent numbers on the cheap.
I'll fully document the method in my journal as time allows.
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Research funded by the patrons of
The Spore Works
Exotic Spore Supply
Reinvesting 25% of Sales Towards Basic Research and Species Identification
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AdoreChampignons
Mycophilic One
Registered: 08/10/08
Posts: 313
Loc: Seti Alpha 5
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Re: An Attempt at Quantifying Chromatographic Bands [Re: Workman]
#8935263 - 09/15/08 07:07 PM (2 months, 18 days ago) |
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Quote:
Workman said:
Actually, there is software and freeware available to analyze flatbed scanned plates automatically. Some quantify spot color density and some the area of the spot (similar to your suggestion). Calibration with known samples should give some pretty decent numbers on the cheap.
I'll fully document the method in my journal as time allows.
Wow!!! That is incredible!!! I didn't know they had software that could do that.
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Hotnuts
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Re: An Attempt at Quantifying Chromatographic Bands [Re: AdoreChampignons]
#8997778 - 09/28/08 11:57 AM (2 months, 5 days ago) |
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I had considered attempting this as well using this technique. See TL chromatography sketch and quick read in the link.
http://leda.lycaeum.org/?ID=16311
Edited by Hotnuts (09/28/08 11:58 AM)
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AdoreChampignons
Mycophilic One
Registered: 08/10/08
Posts: 313
Loc: Seti Alpha 5
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Re: An Attempt at Quantifying Chromatographic Bands [Re: Hotnuts]
#8998095 - 09/28/08 01:39 PM (2 months, 5 days ago) |
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Quote:
Hotnuts said:
I had considered attempting this as well using this technique. See TL chromatography sketch and quick read in the link.
http://leda.lycaeum.org/?ID=16311
Gee..., thanks. The information was very useful. By the way, I was thinking about how someone can begin their own testing. While I'm sure the testing procedure works, how does one know if the concentration of psilocybin is considered low or high? There's no reference point. Therefore, before any real testing can be done, a baseline concentration needs to be established. One can establish their own baseline by first getting a mixture of different shrooms of the same species and grind them up into a fine powder. Next, one needs to put that powder through the chromatographic separation procedure. The chromatogram that is made from this mixture of shrooms should express the average concentration of the strain that you're working with. This chromatogram can now serve as the baseline to compare future chromatograms against. Essentially, when you start producing chromatograms of individual mushrooms, one can compare it to the baseline chromatogram to determine if the concentration of psilocybin is considered low, high, or average.
Edited by AdoreChampignons (09/28/08 11:18 PM)
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johnm214
Registered: 05/31/07
Posts: 7,022
Loc: Americas
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Quote:
AdoreChampignons said:
In testing for the potency of a mushroom, Ive read in previous posts that thin layer chromatography(aka TLC) or paper chromatography was used. Im fairly familiar with the chromatography technique. Ive read in a couple places that the chromatogram is very characteristic of psilocybic species. Has anyone seen what the chromatogram looks like of P. cubensis? Is it just one big blue band, multiple bands, or are there other more prominent features?
tlc doesn't give you much information on the quantity of a substance, its more useful to determine the presence of a substance.
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