Paper chromatography, tryptamine quantification using caffeine as reference and ninhydrin alternatives?

[vogo]

Hello,
I'm immensely grateful for endlessness's writeups and all the community efforts (An1cca and many others) to make analysis or at least comparison easily accessible.
I have a few questions, hope somebody could answer them.

a. I know TLC is superior to paper chromatography, but wondering if paper chromatography (assuming blotting paper without too many whiteners added) can still give satisfactory results in comparing concentration of tryptamines between plant extract samples?
b. If using caffeine at a known concentration and using same solvents and paper chromatography setup as the plant extracts, could I say use 0.5% w/v caffeine to somehow calibrate the fluorescent spot area on any imaging program, and then estimate tryptamine % of the plant extract, based on that caffeine spot I use as reference?
c. What are some more easily accessible alternatives to ninhydrin to visualize spots with UV? I noticed one should look into fluorogenic compounds such as fluorescamine or OPA, but they don't seem any more readily available. I can't find the source of an old study apparently using sodium hypochlorite as fluorogenic reagent for chromatography?

Thank you

 

[Transform]

Hi, good questions. Have you seen this thread?:

Simplified Column Chromatography

..this was influenced by a short article in one of the very first editions of Entheogen Review, in the early 90s...i've slightly adapted it, from experience, into what i'll present below...this is to help those pioneering independent research at the Nexus.. Simplified Column Chromatography...

forum.dmt-nexus.me

Untextured, premium grade drawing paper should work with that method. Get the highest GSM you can find, and check it for the absence of coloured flecks coming from recycled paper sources. It's probably necessary to avoid paper which contains optical brighteners, although a TiO2 background may provide a contrast for the tryptamine fluorescence

You should be able to dial in fluorescence estimation through the use of dilutions, but you'll need a known standard to calibrate your tryptamine derivatives with. Like pure recrystallised DMT or similar.

The accessible visualisation reagents thing is something I still need to look into, so I hope some more answers come up here too!

 

[aizoaceous]

You'll find references to old paper chromatography methods in Trout's Some Simple Tryptamines, but TLC has replaced it for good reason. I would buy or make TLC plates if at all possible.

The best way to detect tryptamines is by the natural indole fluorescence, without any stain. @Grasshoppers has extensively documented a method for their Phalaris work, with illumination by UV-C LED. This fluorescence would probably also be present on paper, though perhaps with different intensity or spectrum. Phenethylamines do not generally have this fluorescence, and are best detected by stains or UV absorption (by quenching of the fluorescent indicator in the plate).

The natural fluorescence has the additional benefit of distinguishing between DMT and 5-MeO-DMT by emission color, since they're hard to resolve chromatographically. Xanthydrol stain also distinguishes, but that's harder to get than ninhydrin. If you can't get anything better then permanganate stain would probably work, though I've never tried it.

It's conceptually possible to use a reference of Compound A to quantify Compound B if you know the ratio of the two compounds' response factors, but that ratio isn't easy to determine. It would be better to use a reference of the same compound, or at least an extremely similar one. If you recrystallize a DMT-dominant extract a few times, then you can reach >99% DMT pretty easily.

The hypochlorite stain is perhaps this paper, where an oxidizing stain produces a reddish brown spot by absorption and blue fluorescence when excited at 365 nm. That effect is specific to tryptamines. I think I tried their hydrogen peroxide system and it worked, but that was a long time ago.

 

[vogo]

Thank you for your replies and great advice

Hi, good questions. Have you seen this thread?:

Simplified Column Chromatography

..this was influenced by a short article in one of the very first editions of Entheogen Review, in the early 90s...i've slightly adapted it, from experience, into what i'll present below...this is to help those pioneering independent research at the Nexus.. Simplified Column Chromatography...

forum.dmt-nexus.me

Untextured, premium grade drawing paper should work with that method. Get the highest GSM you can find, and check it for the absence of coloured flecks coming from recycled paper sources. It's probably necessary to avoid paper which contains optical brighteners, although a TiO2 background may provide a contrast for the tryptamine fluorescence

You should be able to dial in fluorescence estimation through the use of dilutions, but you'll need a known standard to calibrate your tryptamine derivatives with. Like pure recrystallised DMT or similar.

The accessible visualisation reagents thing is something I still need to look into, so I hope some more answers come up here too!

I understand, makes sense to use a known standard to calibrate. I was hoping caffeine could behave similarly in terms of concentration/fluorescence/migrated spot size under either physical (UV) or chemical visualization (ninhydrin or other fluorogenic substance).

You'll find references to old paper chromatography methods in Trout's Some Simple Tryptamines, but TLC has replaced it for good reason. I would buy or make TLC plates if at all possible.

The best way to detect tryptamines is by the natural indole fluorescence, without any stain. @Grasshoppers has extensively documented a method for their Phalaris work, with illumination by UV-C LED. This fluorescence would probably also be present on paper, though perhaps with different intensity or spectrum. Phenethylamines do not generally have this fluorescence, and are best detected by stains or UV absorption (by quenching of the fluorescent indicator in the plate).

The natural fluorescence has the additional benefit of distinguishing between DMT and 5-MeO-DMT by emission color, since they're hard to resolve chromatographically. Xanthydrol stain also distinguishes, but that's harder to get than ninhydrin. If you can't get anything better then permanganate stain would probably work, though I've never tried it.

It's conceptually possible to use a reference of Compound A to quantify Compound B if you know the ratio of the two compounds' response factors, but that ratio isn't easy to determine. It would be better to use a reference of the same compound, or at least an extremely similar one. If you recrystallize a DMT-dominant extract a few times, then you can reach >99% DMT pretty easily.

The hypochlorite stain is perhaps this paper, where an oxidizing stain produces a reddish brown spot by absorption and blue fluorescence when excited at 365 nm. That effect is specific to tryptamines. I think I tried their hydrogen peroxide system and it worked, but that was a long time ago.

I agree with you about TLC, I saw some inexpensive and readily sourced methods to produce ones from microscope slides, alumina and plaster (for example this one),

not sure if it's one of the easiest DIY methods or there are better ones people might suggest instead.

For me it was more about getting a more portable, locally sourceable, incospicuous and less fragile option (just an example: traveling to test many living specimens with tiny standardized samples, possibly in the field or in very basic accommodations), hence paper chromatography came to mind, with the bulk of materials needed being possibly sourced locally as even the smallest stores (in the least ideal scenario) have some fuel/spirits and cleaning products. That'd be more like comparing which specimens have less or more amines, but I was hopeful standardized pictures could provide a retrospective comparison to then have a quantitative estimation again using caffeine as a proxy.

Would you say 254nm works better for visualizing than 365nm UV, or both have their own usefulness?

Thanks for the paper, yes that's probably the one I remember getting quoted here on the forums, since I forgot it mentioned hydrogen peroxide as well as sodium hypochlorite.

 

[Transform]

Would you say 254nm works better for visualizing than 365nm UV, or both have their own usefulness?

I would say that 365nm is more accessible and simply works for the kind of compounds we're interested in. 254nm is used for plates doped with activated ZnS or ZnSiO4 as a visualization aid.

Looking at wikipedia, we can see that permanganate or acidified vanillin might be usable as relatively accessible visualization reagents.
Vanillin is roughly equivalent in action to the p-anisaldehyde shown on this site:
TLC stains

 

[Transform]

Permanganate would, of course, be useless for paper chromatography, since it reacts with cellulose.

 

[aizoaceous]

Would you say 254nm works better for visualizing than 365nm UV, or both have their own usefulness?

Tryptamines are not directly detectable by eye at 365 nm. That's the reason why that paper derivatizes/stains, presumably as the N-oxide. Tryptamines probably are detectable by the visible tails of the natural fluorescence at 254 nm (the peak from a mercury lamp), but the visible emission from a mercury lamp without a tight filter makes them impossible to see. That's the reason why @Grasshoppers used a UV-C LED, though take care that many UV-C LEDs are fake. Normal polycarbonate safety glasses will absorb most UV-C and should be worn, but it's still a bad idea to look directly at a UV-C source.

I was hoping caffeine could behave similarly in terms of concentration/fluorescence/migrated spot size under either physical (UV) or chemical visualization (ninhydrin or other fluorogenic substance).

You could spot caffeine (or whatever) and DMT, and use that to determine the ratio of their response factors. That would require a standard for DMT though, at which point it would be easier and more accurate just to use that standard directly. Caffeine also wouldn't necessarily be detected at all by tryptamine-specific methods like the natural fluorescence or the oxidizing stain.

 

[modern]

270-280 nm range works while with dmt and 5-meo like grasshopper mentioned even on the 'diy plate' homescientist shows. I did my plates like that but need to make them thinner... the dmt on my plates only show up at higher concentrations.

254nm I think requires a fluorescence agent which many commercial plates have included. 365 nm alone work good for harmalas.

I bought cheap aliexpress 275nm and works good for IDing dmt as a blue/purple spot when moist.