|
 PTreeDish
Registered: 04/22/18
Posts: 353
Last seen: 1 day, 22 hours
|
 How does one quantify compound concentrates in fruits or LC? 
#26739283 - 06/12/20 10:52 AM (3 years, 9 months ago) |
|
|
I'd like to learn how to analyze the presence of and concentration of various compounds in medicinal mushrooms - compounds like cordycepin, adenosine, polysaccharides, terpenes, etc.
Is there a DIY-friendly method for performing this type of analysis?
|
 bodhisatta  
Smurf real estate agent
Registered: 04/30/13
Posts: 61,891
Loc: Milky way
|
Re: How does one quantify compound concentrates in fruits or LC? [Re: PTreeDish]
#26739463 - 06/12/20 12:04 PM (3 years, 9 months ago) |
|
|
No. You have a lab do the analysis. They use chromatography and mass spectroscopy to determine compounds and concentration. These machines are calibrated with expensive reference standards. These machines cost a fortune.
|
AndyHinton
Registered: 12/05/16
Posts: 434
|
Re: How does one quantify compound concentrates in fruits or LC? [Re: bodhisatta]
#26740287 - 06/12/20 06:45 PM (3 years, 9 months ago) |
|
|
You can do thin-layer chromatography easily enough at home. It's not as accurate as gas chroma–mass spec. But the resolution you can achieve with TLC, relative to the price, is favorable to hobbyists. Note that chromatography itself doesn't quantify the substances, it only reveals their relative concentrations. That's why GC is often used with mass spec.
If you know what you're looking for, and most interesting mushroom compounds are already described, there are some good stains available. UV and iodine are pretty typical and they don't destroy the plate. Otherwise, you can run multiple plates and test out different destructive stains like cerium sulfate (alkaloids), ferric chloride (phenols), ninhydrin (amino acids), etc. There are various stain lists online.
I'm also trying to get a nice little TLC setup to test the mycelium piss of king tubers. The total cost for the plates and maybe 3 good stains is probs around $200. The main stumbling block is acquiring the right chemicals in appropriate quantities. If you published a simple, solid protocol for DIY chroma that yields good spots, that would be huge. Good luck! 
--------------------
Edited by AndyHinton (06/12/20 06:47 PM)
|
PTreeDish
Registered: 04/22/18
Posts: 353
Last seen: 1 day, 22 hours
|
Re: How does one quantify compound concentrates in fruits or LC? [Re: AndyHinton]
#26740383 - 06/12/20 07:30 PM (3 years, 9 months ago) |
|
|
Quote:
Note that chromatography itself doesn't quantify the substances, it only reveals their relative concentrations
That's perfect for a diyer experimenting with strains with the goal of improving the yield of a particular compound. It allows for a lot of iteration before one may need to send the optimized sample out for testing and certification.
The stain list and guide is great. I think this will work nicely for me. There are also some videos on YT for making your own TLC plates that could help bring the price down, depending on the volume of testing of course.
Quote:
The main stumbling block is acquiring the right chemicals in appropriate quantities. If you published a simple, solid protocol for DIY chroma that yields good spots, that would be huge. Good luck!
Was this meant for me or bod? Anyway, what makes it difficult beyond the expected challenge any _individual_ seeking to buy lab-grade chemicals would encounter?
|
AndyHinton
Registered: 12/05/16
Posts: 434
|
Re: How does one quantify compound concentrates in fruits or LC? [Re: PTreeDish]
#26741246 - 06/13/20 07:44 AM (3 years, 9 months ago) |
|
|
The plates are maybe $50 for a 10-pack, and you can spot multiple samples per plate. It only takes a few microliters per spot and from what I understand, there's a real chance of getting junk results if you use too much sample. You could only do one stain per plate, but I think there are plates you can cut with scissors. To allow multiple stain dips per plate. Another option are the pre-cut aluminum/silica plates designed for 1–2 spots each.
A lot of the chemicals are readily available on Amazon. Some of them can be quite dangerous (potassium permanganate is a strong oxidizer that can explode), while others may either be expensive or only available from places like Sigma. Careful stain selection or alternate recipes can probably be sourced cheap and easy.
I'm saying if you wrote a comprehensive TLC protocol with all these factors considered, that would be totally awesome. The same as when I encouraged you to transform GFP into a filamentous fungus, just some higher-order DIYbio work that few or no people in the scene are doing.
Let's keep a line open on this. The BosLab is finally opening up again. Before the plague I had asked about the board about this specific thing: TLC on a bring-your-own-plates basis. Working together, we can do it.
A quick note on spotting broth: be careful you don't end up with a big streak of the compounds in malt extract, that would hide the compounds of interest. If you did a ninhydrin stain on unpurified broth, for example, you'd probs just get the LME peptides instead of the mushroom proteins.
Another quickie: for polysaccharides, it may be worth looking into ethanol precipitation and spectrophotometry as an alternative to chromatography. See Structural diversity requires individual optimization of ethanol concentration in polysaccharide precipitation, Evaluation of the efficiency of ethanol precipitation and ultrafiltration on the purification and characteristics of exopolysaccharides produced by three lactic acid bacteria, etc.
--------------------
Edited by AndyHinton (06/13/20 08:33 AM)
|
panne cyanne
albino queen
Registered: 04/15/20
Posts: 145
|
Re: How does one quantify compound concentrates in fruits or LC? [Re: AndyHinton]
#26742196 - 06/13/20 04:38 PM (3 years, 9 months ago) |
|
|
im with bod on this one.
mail it out to a proper lab.
no fear for medicinals.
|
AndyHinton
Registered: 12/05/16
Posts: 434
|
Re: How does one quantify compound concentrates in fruits or LC? [Re: PTreeDish]
#26742363 - 06/13/20 06:07 PM (3 years, 9 months ago) |
|
|
Quote:
PTreeDish said:
I'd like to learn how
This looks like the money quote. Something I forgot to mention is, you need full PPE to do this work safely. Thick gloves, safety goggles, N95 respirator, long pants, full apron, good ventilation, eye wash station, nonreactive (borosilicate) containers, and a chemical fire extinguisher. Most of the good stains rely on sulfuric acid, which can eat through solid metal.
Chemical safety is very important. Dilute strong acids by adding the acid to ice-cold water and not vice versa; store the chemicals locked up in their original containers in a cool, dry place; read and understand the MSDS fully; develop a plan to contain spills before they happen; don't work alone; keep a poison control number on speed dial; and learn how to properly dispose of chemical waste.
You're smart, PTreeDish. With some caution and respect for the caustic power of the reagents, you'll do fine. It's not something to be flippant about, and I won't dissuade you. Ordering sulfuric acid may put you on a DEA watch list though, so you may want to develop a strong security posture first.
--------------------
Edited by AndyHinton (06/13/20 06:51 PM)
|
PTreeDish
Registered: 04/22/18
Posts: 353
Last seen: 1 day, 22 hours
|
Re: How does one quantify compound concentrates in fruits or LC? [Re: AndyHinton]
#26742374 - 06/13/20 06:11 PM (3 years, 9 months ago) |
|
|
Absolutely. I'm pretty close to formally launching the blog and social media for the project we discussed and this would be a great project to document and share back to the world.
I'm happy to contribute funds and/or time to the development of a diy-friendly protocol so long as we can collaborate and I can publish the process. I have many ideas of how to focus the technique to maximize usefulness to both the community and small commercial growers. There may even be an opportunity to co-author a paper once the protocol is developed and published.
I'm going to do some homework and read-up on current TLC methods and reagents for the fungi-related compounds of interest.
Glad to hear the lab is opening up again. I have another connection that was once a member there and there's a chance you two might know each other - which would be serendipitous. Anyway, I'll check out the papers. Thanks man.
|
AndyHinton
Registered: 12/05/16
Posts: 434
|
Re: How does one quantify compound concentrates in fruits or LC? [Re: PTreeDish] 1
#26742416 - 06/13/20 06:32 PM (3 years, 9 months ago) |
|
|
No probs. Something else I'd advise is to pick up a good general organic chemistry textbook and study it. You really do not want to fuck around with some of these chemicals.
Anyway, I'm probably gonna leave the thread MF Doom style before the AARP meeting lets out. Andy stands for pushing limits, being invisible, staying cool, and always raising the bar cuz herbs only grow close to the ground.
Quote:
Definition "super-villain": a killer who love children
One who is well-skilled in destruction, as well as building
While Sidney Sheldon teaches the trife to be trifer
I'm trading science fiction with my man the live lifer
A pied piper holler a rhyme, a dollar and a dime
Do his thing, ring around the white collar crime
--------------------
Edited by AndyHinton (06/13/20 06:33 PM)
|
PTreeDish
Registered: 04/22/18
Posts: 353
Last seen: 1 day, 22 hours
|
Re: How does one quantify compound concentrates in fruits or LC? [Re: AndyHinton]
#26833435 - 07/19/20 11:25 PM (3 years, 8 months ago) |
|
|
A quick update. I've spent quite a bit of time and energy into learning TLC and performing some tests. Now, I'm now blocked. I'll explain why.
My general procedure was to do the following (generalizing for brevity):
* ground some dried mushroom and placed it in acetone solution for 24 hours
* pipetted some into an epp tube and centrifuged for half hour
* measured and mark the point of origin on the test plates
* used capillary tubes to drop some of the supertanant onto the test plates (real silica plates I cut myself)
* developed the plate using methylene chloride in a glass developing chamber
* let dry and check plate under shortwave UV lamp
Here are the latest, uninteresting results with some other experiments (left to right: ibuprofen, aspirin, aspirin #2, psilocybin #1, psilocybin #2, psilocybin #3)
There are some very light bands but I understand those to be impurities. As you can see, there are no visible spots and therefore no way to calculate Rf factors.
After further research, I learned that:
* Methanol or H20 may be better solvents for extracting psilocin / psilocybin for TLC
* The plates need to be developed with Ehrlich's reagent / DMAB / p-dimethylaminobenzaldehyde sprayed on, left to dry, baked and then checked with a shortwave UV lamp
I don't know how to get a hold of DMAB so I until I can address that, I'm dead in the water. Any idears?
|
Bloo
Stranger
Registered: 02/24/20
Posts: 56
Last seen: 1 year, 11 months
|
Re: How does one quantify compound concentrates in fruits or LC? [Re: PTreeDish]
#26859854 - 08/02/20 10:49 PM (3 years, 8 months ago) |
|
|
See Beug and Bigwood, 1981: Quantitative analysis of psilocybin and psilocin in Psilocybe baeocystis (Singer and Smith) by high-performance liquid chromatography and by thin-layer chromatography, J. Chrom, 207 (1981) 379-385.
They used several different systems for TLC, the best overall seemed to be silica gel plates, with butanol/acetic acid/water (12/3/5). The paper provides Rf values for the analytes in this system.
|
PTreeDish
Registered: 04/22/18
Posts: 353
Last seen: 1 day, 22 hours
|
Re: How does one quantify compound concentrates in fruits or LC? [Re: Bloo]
#26860828 - 08/03/20 01:01 PM (3 years, 8 months ago) |
|
|
I've read this paper and many others. Even in this paper, it states:
Quote:
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 I-IV radiation were encircled with a pencil. Ehrlich’s (or Van Urk’s) reagent [lo % p-dimethylaminobenzaldehyde pDAB) in cont. 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.
Newer papers also use similar development reagents. Going to need pDAB to move forward.
|
Bloo
Stranger
Registered: 02/24/20
Posts: 56
Last seen: 1 year, 11 months
|
Re: How does one quantify compound concentrates in fruits or LC? [Re: PTreeDish]
#26861845 - 08/04/20 12:12 AM (3 years, 8 months ago) |
|
|
Ehrlich reagent is readily had by the DIY-test-kit crowd. Check ebay, Bunkpolice, etc. The sprayer is the expensive part, TLC sprayers make a very controlled fine mist, sometimes you can luck out and get a used one on ebay.
|
GandalfSon
Stranger
Registered: 07/01/20
Posts: 34
Last seen: 3 years, 6 months
|
Re: How does one quantify compound concentrates in fruits or LC? [Re: Bloo]
#26882618 - 08/16/20 01:17 AM (3 years, 7 months ago) |
|
|
Quote:
Bloo said:
Ehrlich reagent is readily had by the DIY-test-kit crowd. Check ebay, Bunkpolice, etc. The sprayer is the expensive part, TLC sprayers make a very controlled fine mist, sometimes you can luck out and get a used one on ebay.
Bloo, have you tried this yourself with psilocybin perchance? I’ve actually been thinking recently about how I’d setup experiments to test the ability of the Erlich reagent test for comparing relative psilocybin densities. If you have tried this I’d love to know what you learned so I can avoid wasting time and harvests to test anything already disproven.
On a slightly differrent note I remember years ago when I was in Afghanistan I had a wealth of these little drug test kits that had some kind of reagents in a tiny ziplock and when you burst the pill capsule inside the contents would change colors based on the drugs inside the body. Maybe if we could identify the right chemicals for 4-ho-DMT detection we could create a DIY test like that and then using sterile technique and still box/flow chamber methods to minimize the influence of contaminants giving a false reading.
|
Bloo
Stranger
Registered: 02/24/20
Posts: 56
Last seen: 1 year, 11 months
|
Re: How does one quantify compound concentrates in fruits or LC? [Re: GandalfSon]
#26882643 - 08/16/20 01:35 AM (3 years, 7 months ago) |
|
|
Quote:
GandalfSon said:
Bloo, have you tried this yourself with psilocybin perchance?
I have no need for TLC and droplet tests as I have chromatographic instrumentation for qualitative and quantitative work.
|
GandalfSon
Stranger
Registered: 07/01/20
Posts: 34
Last seen: 3 years, 6 months
|
Re: How does one quantify compound concentrates in fruits or LC? [Re: Bloo]
#26882654 - 08/16/20 01:50 AM (3 years, 7 months ago) |
|
|
Quote:
Bloo said:
Quote:
GandalfSon said:
Bloo, have you tried this yourself with psilocybin perchance?
I have no need for TLC and droplet tests as I have chromatographic instrumentation for qualitative and quantitative work.
Fair enough.
|
|
Threaded
Previous
Index
Next
Edit 
Reply 
kilgore_trout
1 2 all )
StrophariaShaman
Blue Helix
BigJohnson
ZippoZ
Aiko Aiko