johnm214 said:

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?

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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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hi to all.  im new here and really interested in learning.  this topic is over my head ATM but im gonna study up, lol!  i find this very interesting.   i am really interested to see this go on.  i have complited a little bunch of PDFs and made a few too which i hope people will find useful, ill list them and upload them.

AdoreChampignons said:

Greetings Workman,
I was thinking of how the chromatography test could be made more quantitative than qualitative.

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Workman said:

I just got mine as well.  It was back ordered for awhile which slowed down progress.  I am excited to see how this works this weekend.  I'll post the results in my journal.

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Alan Rockefeller said:

I just got some p-DMAB, have not tried it yet.

I have 100 grams which is more than I need, anyone that needs some please PM me.

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hi!  i would love to take up on it, pm on the way.  i dont understand everything yet but im gonna study up like crazy!   once i think workingman posts how he does it i hope to copy him.  how do use plan to do it?  as in the PDF I upped? "THE MUSHROOM ENTHEOGEN - The Measure of the Mushroom"?  can you give me any nOOb hints?  im am really good at following directions and once i do it once i bet i can get the hang of it, tnx so much!  im excited to try this

AdoreChampignons said:

Greetings Workman,
I was thinking of how the chromatography test could be made more quantitative than qualitative.

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Quote:

"Quantitative Analysis of Psilocybin and Psilocin in Psilocybe Baeocytis by High-Performance Liquid Chromatography and by Thin-Layer Chromatography"

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Quote:

"Analysis of psilocybin and psilocin in Psilocybe subcubensis GUZMÁN by ion mobility spectrometry and gas chromatography–mass spectrometry"

--this is like the one above i think its on topic (i hope) but too hard?  anyway i thought itd be good to add?

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Quote:

"The Determination of Psilocin and Psilocybin in Hallucinogenic Mushrooms by HPLC Utilizing a Dual Reagent Acidic Potassium Permanganate and Tris(2,2'-bipyridyl)ruthenium(II) Chemiluminescence Detection System"

i hope to try this and id like opoins on methods.  someone said they are trying this soon, in some log?  where is this? tnx yall!

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Quote:

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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will you please review my method?  i incorported some of your methods and used the lasted and greatest TLC software (i think, haha) with an inexpensive source of good silica plates.  all for about 100-150 startup cost   schweet!

in this post ill list the way i think you mean with parts, etc.  then in next post ill put up some great PDFs about this stuff and one which uses the same TLC scanning software where the authors detail how to use the software etc.  and i have other great PFDs too like a great PDF on methods to do TLC.  i edited that PDF for new info on using scanning software.  i hope you like and can let me know if im on the right tract!  tnx!




A FEW Q's:

1. when you say "resolution solvent solution" you mean what goes into the TLC chamber (where the TLC plate sits, 'development chamber') right?  which would be iso and ammonia?

2. when is the "developing solution" applied?  and how is it applied? and what is it used for?



MATERIALS:  (next post ill list the step by step as i think you mean)


tyvek -
to line one side of developing container. to aid in the saturation of the TLC chamber with solvent vapors


microcaps -
to spot plates.  good size is 1µL.  important for good spotting and is important when using plate for scanning.  the microcap makes good TLC plate spotting so results are more accurate. scanner can read it well and get more accurate images = more accurate results.  good source is $11 for 100 and they can be cleaned and reused a few times! schweet! :

-Drummond model #: 1-000-0010*
http://www.drummondsci.com/products/cm_06.html

-another source of drummond at VWR Labshop #: 53440-001
http://vwrlabshop.com/microcaps-disposable-micropipets-drummond/p/0011607/


pint jars (or maybe quart):
to use as developing container

scissors -
to cut up dried mushrooms


mortar and pestle  -
to pulverize and grind cut up mushrooms into fine powder


99% isoproponol rubbing alchol -
part of the resolution solvent system


5% jantorial grade ammonia -
part of the resolution solvent system. where is this found?


muriatic acid -
developing solution (Concentrated HCL)


HEET gas additive -
solvent solution (methanol for extraction).  what is this?  where is this found?  used to dissolve mushrom powder.  can use white vinegar (5%) as an alternative methanol extraction method.


pDMAB (Ehrlich's reagent) -
to spay on plate to bring out colors.  found on ebay, labx, etc.


flat bed scanner -
to scan plates for TLC quantification.  kinkos office store, mail boxes etc, library, your friends house, lol!  a good one is kind of important.


PC computer -
to run TLC software.  needs to be Win 98 or later but XP and later is better


TLC silica plates -
i want to use the whatman "Diamond Series Silica TLC Plates", model LK6D, as they are the 'best' for scanning densitometry but also VERY expensive! $250 for 75   i want to use the diamond is that nearly all inaccuracies in final results is from quality of TLC plate spotting and scanning.  better plates = more accurate results.
 
-whatman stock #4865-621
http://www.whatman.com/DiamondSeriesSilicaTLCPlates.aspx

-here is a place to buy them also (WTM4865-621) http://www.bestlabdeals.com/SearchResults.asp

SO i think found a good alternative but id like your (or anyones) opinion.  "PureChems" has really good sounding and much less expensive plates though not high reflection like the whatman diamond series.  yet the Purechems plates should be great i hope.  the specs for the Purechems plates are the same as the whatman but the Purechems is smaller (5x10cm) which i like.  AND its only $35 for 40 plates!  

-product # PCTLC-001
http://purechems.com/productinfo2.asp


JustTLC -
scanning software and VERY good!  JustTLC by Sweday is top of the line and there have been 4 accepted research articles which utilized JustTLC for the data, so it seems very trusted.  i will upload one of those articles and it is very good with a lot of detail about JustTLC and its use, etc.

JustTLC is expensive, about $450.  but it has a month free trial and if people need it longer than that and they WILL EVENTUALLY pay for it they could run for maybe an extra month or two if they tried using something like VM so its license wont expire or reinstall their OS after 29 days and then reinstall JustTLC.  BUT people should pay for this, fair is fair.  the sweday site is CHALK FULL of info with great videos of usage and step by step guides, etc. 

-i installed JustTLC and it was a snap!
http://www.sweday.com/Products.aspx

Quote:

JustTLC is the new generation of TLC analysis software that gives you the possibility to run chemistry experiments by using just TLC plates.


JustTLC gives you measuring accuracy suitable for high-throughput screening or analytical investigations and reliable results in minutes rather than hours.
Overview


Thin layer chromatography is a simple, quick and inexpensive procedure that gives the chemist a fast answer as to how many and how much components there are in a mixture. Until now it has been difficult to accurately measure the amount of expressed compound without sophisticated, complicated and very expensive TLC equipment.


JustTLC is a modern, intuitive and easy-to-use analysis software that detects and quantifies spots on the TLC plates. Within minutes you have completed a previously complicated task:


- Reliable and robust quantitative compound analysis using just TLC plates.


Key Benefits

With JustTLC you can:

    * Perform quantitative analysis on TLC plates in minutes.

    * Automatically detect and truly compare chromatograms in three dimensions.

    * Compare data from spots and plates.

    * Display graphs and store TLC plates in projects.

    * Export data into Excel or other third party software for further custom analysis.

    * Print graphs, plates and data sheets.

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Quote:

p-DMAB Test Paper

1. Add 1.0 gram of p-DMAB to 28 mil of ethanol(dena- tured). Stir until dissolved.

2. Add water to the above mix until 60 mil total volume achieved.

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6. what am i missing?  can you just layout the steps for me and i think i can figure the rest out,with my gf big brain, haha.  tnx!!





tnx to all!!   ps.  am i missing anything?

Quote:

TLC is a simple, quick, and inexpensive procedure that gives the chemist a quick answer as to how many components are in a mixture. TLC is also used to support the identity of a compound in a mixture when the Rf of a compound is compared with the Rf of a known compound (preferrably both run on the same TLC plate).

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Quote:

ABSTRACT:

Thin layer chromatography (TLC) using staining reagents is a superior method for analyzing organic compounds without chromophores. It is fast, versatile and sometimes the only viable method. We have investigated quantitative TLC using staining reagents, in combination with modern image analysis software. Our results show that it is possible to get reliable measurements, suitable for high-throughput screening or physical organic investigations. The range of detection and the errors for the different parts of the process are illustrated. We show that the errors are largely due to the staining process and can be diminished by measuring ratios of compounds.

© 2007 Elsevier B.V. All rights reserved.

Keywords: TLC; Image analysis software; Quantification; Staining reagents

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Quote:

ABSTRACT:

The use of a TLC scanner can be regarded as a key step in high performance thin layer chromatography (HPTLC). Densitometric measurements transform the substance distribution on a TLC plate into digital computer data. Systems that allow quantitative measurements have been available for many years for either fluorescence or ultraviolet absorption measurements, while lately the reflection analysis mode for both types is the most common application. New scanning approaches are designed to aid the analyst who has common demands for TLC-densitometry without using special data, such as scanned images. Two examples that have been developed lately in the laboratories of the authors are described in this paper. These approaches were developed on the basis of current needs for analysts who employ TLC as a tool in research, as well as in routine analysis. One approach is aimed to support analysts in economically disadvantaged areas, where cost intensive apparatus is unsuitable but trace analysis by simple means is required. The other system, allows the spectral determination of chromatographic spots on TLC plates covering the ultraviolet and visible range, thus, revealing highly desired information for the analyst.

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Quote:

ABSTRACT:

The TLC analysis of agricultural products, foods, beverages, and plant constituents is reviewed for the period from mid-1995 to mid-1999. Techniques and applications for a wide range of analyte and sample matrix types are covered, with specification of the particular layers, mobile phases, detection methods, and quantification conditions in many cases.

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Quote:

ABSTRACT:

This page is an introduction to chromatography using thin layer chromatography as an example. Although if you are a beginner you may be more familiar with paper chromatography, thin layer chromatography is equally easy to describe and more straightforward to explain.

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Quote:

Thin layer chromatography (TLC) is among the most useful tools for following the progress of organic chemical reactions and for assaying the purity of organic compounds.  TLC requires only a few ng (yes that's right nano grams!) of sample for a successful analysis and can be accomplished in a matter of minutes.  Like all chromatographic methods, TLC takes advantage of the different affinity of the analyte with the mobile and stationary phases to achieve separation of complex mixtures of organic molecules

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Quote:

Many TLC users prefer to use pre-coated TLC plates, but others because of their special needs, or because they need special additives or a special thickness, coat their own TLC plates. This paper is a quick review of what is needed for the process.

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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.

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what do you mean by 'known samples'?  do you mean Rf value?  sorry for all the Qs but i am getting ready to do this in a month so i want to cram it into my pea brain! lol.  i think ive got it down pretty good but im prob missing something.   

in the TLC test below there are Rf values for psilocin and psilocybin but i doubt they are usefull?  that TLC test was with Ehrlich's reagent spray too and the plates used in that test are the same type of silica plate i linked to.  the 'purechems' plates are $35 for 40! 5x10cm silica and glass   but in that TLC test they didnt use iso and ammonia for resolution solvent solution they used: butanol - acetic acid - water (12:3:5)

Plasmid said:

Also, if you're looking for easier methods for psiloc(yb)in detection and quantitative estimation, try looking for articles which are a little broader in scope, like review articles on the analytical chemistry of controlled substances.  Articles like that tend to mention a bunch of analytical methods, with references, and will usually mention at least one simple presumptive field test for each (though Ehrlich's reagent is probably the most common mention).

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haha, duh!  now the light turns on!  haha.


you add the muriatic acid (developing solution) after you spray with Ehrlich's reagent.  so less spots are visable yet still psilocybin and psilocin are visable?  the quote below is from the PDF "Quantitative Analysis of Psilocybin and Psilocin in Psilocybe Baeocytis by High-Performance Liquid Chromatography and by Thin-Layer Chromatography"

SO after i take the plate out of the development container (TLC container with iso and ammonia) i spray with p-DMAB, let dry and then note the spots.  then i dip the plate in the muriatic acid, let air dry.  and then scan in flat bed scanner?  but if im using JustTLC i wonder if the muriatic acid step is necessary?  i mean wont JustTLC compute that extra spots?  or will it mess things up?  i ask as id rather not be around muriatic acid.  tnx !!

well i have free customer service for the first trail month  of JustTLC so i guess ill call sweday tomorrow and ask them.  they should be able to tell us if we need the developing solution or not.  unless im misunderstanding the reasons for the developing solution step of muriatic acid.

Quote:

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.

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Workman said:

Whoa, ok I didn't read everything you posted.

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Quote:

The Ehrlich's reagent has the HCL in the mix already.  The test strip method isn't related to the TLC method and adds the HCL later.  To spray the reagent evenly, I was considering using a hobby airbrush used on plastic models.

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Quote:

To generate a standard you can use TLC to purify each detectable chemical you want to quantify. Heavily spot a TLC plate in a line with psilocybin mushroom extract and run the plate.  Scrape off the silica gel at selected Rf values and extract the chemical from the gel scrapings with methanol.  Evaporate the extraction methanol and precisely weigh (need a precise milligram scale) the residue material which should be very pure.  Place a known weight of the extracted material (e.g. psilocybin) in a known quantity of methanol and thats your standard.  Spot measured amounts of this standard on new TLC plates to calibrate TLC software.

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Quote:

I made some test strips for quick presence/absence of indoles and did a quick 10 minute extraction of a young dried azure with heet (methanol) automotive gas treatment.  I got a positive modest purple spot even though the extraction time was very brief.  A longer extraction time (12 hours at room temp is recommended) and partial evaporation of the methanol to concentrate is pending.  The HCL is very caustic with visible vapors when the bottle is opened which is a bit disturbing.

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Quote:

Place a known weight of the extracted material (e.g. psilocybin) in a known quantity of methanol and thats your standard.

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Quote:

I haven't started my journal entries yet.  I'll put up the test strip results with pictures soon.

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great!  i understand most steps but im a little confused with finding the standard, etc.


heres Rf values from TLC test below.  they are Rf values for psilocin and psilocybin but i doubt they are useful(?) as they used a different resolution solvent solution.  that TLC test was with Ehrlich's reagent spray too and the plates used in that test are the same type of silica plate i linked to,  the 'purechems' plates are $35 for 40! 5x10cm silica and glass   but in that TLC test they didnt use iso and ammonia for resolution solvent solution they used: butanol - acetic acid - water (12:3:5).  which is why i think the resulting Rf values may not be useful for us?

Rf 0.16: Psilocybin

Rf 0.34: Psilocin

gianimon said:

SO i think i understand your method in theory:

muriatic acid is used to dissolve the mushroom powder for plate spotting (a 1% mush power concentration is good).  then after the plate is sprayed with Ehrlich's reagent and dried i scrape off the gel at the Rf vale I want and dissolve with HEET gas additive?  OR is it the other way around and HEET is used to dissolve mush powder and muriatic acid is used to dissolve silica gel at specific Rf value?

im a little fuzzy on the use of the standard.  the standard as you described would be from mushroom X and if mushroom Y spotted higher than mushroom X then mushroom Y is stronger?  (all concentrations and weights being equal)

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hi workman, tnx for your time!

Workman said:

The acid is only part of the Ehrlich's reagent which is used as the last step to make the indole spots visible on the plate and not used in extraction or as the developing solvent.  The acid would destroy the mushroom powder.  Also you wouldn't spray the Ehrlich's reagent on the plate you want to re-extract to make a calibration standard as it alters the chemicals.

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Quote:

I know its confusing but maybe it will be more clear after I work on my method and post the results in a stepwise manner.

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yes im sure it will be and im looking forward to that!

Quote:

But basically the steps are thus.

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Quote:

Dry mushrooms, grind mushrooms, weigh ground mushrooms, soak in methanol (HEET), filter out mushrooms from methanol.

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Quote:

Spray plates with Ehrlichs (acid is in this and only this) to make developed spots visible.

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Quote:

Scan plates on flatbed scanner to preserve record for later analysis with TLC software.

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Quote:

----------------------------
You are correct that the Rf values for this solvent system may not match exactly the other similar solvents but the specific colors generated from the Ehrlichs reagent will help resolve that problem.

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Quote:

Very precise measurements of the standards and amount spotted on the plates will be required to get useful numbers.

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yea i assumed as much.  for accurate spots on plates i am buying 1µL microcaps $11 for 100 (Drummond #1-000-0010).  my digital scale only measures to hundredths so i guess a milligram digital scale is needed.

according to a great study focusing on JustTLC and other TLC software i uploaded ("Evaluation of quantitative thin layer chromatography using staining reagents") nearly all inaccuracies in measurement with software comes from poor spotting and plate procedure and materials.  that is why im using microcaps and id rather use the Whatman "Diamond Series Silica TLC Plates" (model LK6D) instead of the 'regualar' silica plates from  PureChems (product # PCTLC-001)


im really looking forward to you log!