First steps with Chromatography

[Tannenberg]

Hello,

i wanted to try out some simple chromatography and tested different sorts of paper with "Textmarkers".. The results varied greatly. (from: completly no effect (hard paper used to package bottles), to a pretty blurry mess with the sample just soaked through all of the paper (kitchen roll paper)).. I got the best results with a tissue, but´s still far from any noticable lines / dots. (see attached picture).

How do i improve this results? Is specially made chromatography paper usable, as i could use it to really analyze stuff? Or do i need silica?

b.t.w. i used tap water as the solvent

edit: second picture is rotated and the gamma / saturation is adjusted.

 

[endlessness]

hmmm I only have experience with TLC silica chromatography, maybe nen (or other experts) can help out since he worked with paper chromatography. Any particular alkaloids you want to work with?

Also you're gonna need a better eluent than water depending on what youre gonna test... Often it can be a mixture of alcohols and organic solvents, or for tryptamines/phenetylamins you can add a bit of ammonia (like 100:2.5 ethanol or methanol to ammonia) to reduce trailing of alkaloid spots, if thats what youre working with.

Are you gonna be spraying any visualizing reagent? If so, which? Or do you literally just want to work with marker pens?

 

[Tannenberg]

hmmm I only have experience with TLC silica chromatography, maybe nen (or other experts) can help out since he worked with paper chromatography. Any particular alkaloids you want to work with?

Well yes DMT but, not right now, just want to get used to the technique.

Also you're gonna need a better eluent than water depending on what youre gonna test... Often it can be a mixture of alcohols and organic solvents, or for tryptamines/phenetylamins you can add a bit of ammonia (like 100:2.5 ethanol or methanol to ammonia) to reduce trailing of alkaloid spots, if thats what youre working with.

So i can use 100:2.5 Ethanol : Ammonia to work with DMT? Is it possible to use acetic acid as well? I saw a line in your big table with Rf values where Acetic Acid was used, and the Rf of DMT was 0.5?

Are you gonna be spraying any visualizing reagent? If so, which? Or do you literally just want to work with marker pens?

Yes, later. But i first need to get that "reagent", i have to look for sources. I realized that it is pretty difficult to get lab stuff at all. I mean silica plates just literally cost like ~250€ for 25 plates.. (ebay, big ones with fluorescent). I think i will make my own ones, because they are just too expensive for trying out stuff just for fun.

 

[endlessness]

Im gonna add a warning to that table because for me, the acetic acid system didnt work at all. Substances did not separate well and just formed some amorphous blurs, the silica soaked the water very unevenly, etc. Ethanol:ammonia worked great.

As for the silica plates, those 20x20cm plates with fluorescence are enough for many tests. Each plate fits 19 columns (meaning 19 tests), but you can cut them in half so each plate is 38 tests x 25 = 950 tests. That's 30 cents a test in the price that you pay (and it should be possible to get those plates even cheaper), not expensive at all, totally worth the investment.

I can send you pictures and Rfs of how different standards look, we've been testing a lot of phens and tryptamines, specially if you can also use methanol or ethanol:ammonia system.

As for reagent, I hear xanthydrol and draggendorf are often used but I have no experience with them. Ehrlich is typically used for tryptamines but im not sure how well it diferentiates tryptamines in colors. We use PDMAB-TS which is a bit different formulation of Ehrlich, using Ferric Chloride and sulfuric acid, since it seems to differentiate some tryptamines better (for example 5-MeO-DMT turns greener/darker, while DMT and NMT are yellow). Im not sure if you can buy anywhere easily thouhg, maybe you can check out just normal ehrlich which should be easier to get and check it out how it works.

Im currently trying to help finding sources for chromatography equipment/kits to help people out, Ill let you know if there are advances in my plans.

 

[Tannenberg]

I can send you pictures and Rfs of how different standards look, we've been testing a lot of phens and tryptamines, specially if you can also use methanol or ethanol:ammonia system.

That would be nice! How do you take the pictures b.t.w.? with a DSLR and a UV-Lamp? Or do you just use a scanner?

Im currently trying to help finding sources for chromatography equipment/kits to help people out, Ill let you know if there are advances in my plans.

In terms of plates, i found out that sometimes on ebay there a some old packs for aprox. 60€, but there is only one seller. Other sources are commercial lab supply shops, but most of them require a authentification process with scanned identity documents and so on..

For Ethanol, is it fine to use that Bio-Ethanol used in little stoves? I think it is 95-97% Ethanol, but they add some bitter substance, that you don´t drink it (for tax reasons i believe). Is 95% fine or would someone need analyses grade ethanol?

 

[endlessness]

I doubt bitrex would affect chromatography for these these purposes, so 95-97 should be fine, I used 96% ethanol. I can try with denatured ethanol next time, see what thats like.

As for the pics, I just take with normal camera, with 254nm UV light (not sure if the normal blacklight for parties works). Im still just beginning to experiment with spraying reagents... Since with the UV light you can already easily see the spots, I only drop some PDMAB-TS later to confirm... But in the case of people who dont have UV, the drops arent good enough because it's too much liquid, you wont see distinct spots, you need a fine-spraying bottle. There are these more expensive bottles that you pump so it comes with a more homogeneous pressure and very fine, but I want to find something cheap that works. I tried some china dollar store spray bottle but the drops were a bit too thick, looks kinda messy.

In a couple of minutes I can attach the pictures I took in my last test with ethanol:ammonia, a couple of plants extracts and some standard substances. I plan on doing a more organized collection of these.

 

[benzyme]

365 nm works fine. longwave UV, blacklight blue.

tryptophan is the parent compound for tryptamines, its max abs is at 280 nm. with
increasing pi-bond conjugation, the wavelength of max abs increases

 

[Tannenberg]

I doubt bitrex would affect chromatography for these these purposes, so 95-97 should be fine, I used 96% ethanol. I can try with denatured ethanol next time, see what thats like.

it´s pretty cheap (arround 1€ / L) and easy to get, it would be ideal

As for the pics, I just take with normal camera, with 254nm UV light (not sure if the normal blacklight for parties works).

I think the "party lights" are ~365nm. But there are also some UV-Lightsources out there which are used to sterialize water and things like that. Maybe they are more useful. Maybe someone could also get a very old UVC Light source on ebay, they were sold as "room air fresheners" before someone realized they cause skin cancer.

I don´t know it exactly, but in my opinion it is easier / cheaper to get a UVC ligth source, before messing with spraying bottles / gettting the reagents.

Another solution i could think of, is a Xenon-Flash combined with a optical grating / slit. The Xenon flash also generates UVC light and the grating could spread the spectrum, so only uvc hits the sample. The flash can also easily be synchronised with the photo trigger. (maybe to complicated at all..., or not bright enough) [FLD detectors in LC-Equipment use Xenon Flash lamps too]

For spray bottles i could think of this spray bottles in which you first build up pressure with a pump to water your flowers. But they are pretty big. Another thing would be a Ultra Sonic "fog" generated which is used as medical equipment to inhale things, when you catched a cold.

 

[Tannenberg]

365 nm works fine.

So DMT and MeO-5-DMT would be visible with 365nm?

 

[Tannenberg]

B.t.w. i made another one out of a newspaper , it worked a littlebit better, but took like 10 times longer.
The blue Color of the green marker has a Rf value of 52%

 

[endlessness]

but anyways getting the reagent is essential because you need to confirm that the spots at the right height is what you think and not just something else with same/similar Rf. Maybe you can get away if you're analysing a more limited variety of substances that have significantly different Rfs, but if it's plant mixtures (like say, you're analysing phalaris or other plants with possible unknown alkaloids at different Rfs), you would be increasing your probabilities of being wrong if you dont confirm with a reagent.

I think you can order ehrlich and other similar reagents online internationally, maybe check dancesafe.org or eztest.com.

Regarding the spray bottles, the one for flowers, the potential problem I see is that its too big, you'll need a lot of reagent to use that, no? Maybe once the level is too low, it doesn't spray well? As for the sonic fog, how does it disperse, is it directional? You dont want to be breathing sulfuric or hydrochloric acid, or formaldehyde (in the case of marquis and others), sounds like bad idea.

 

[benzyme]

365 nm works fine.

So DMT and MeO-5-DMT would be visible with 365nm?

yes.

I can't imagine a 254 nm UVC light being cheaper than a commercial 365 nm GE bulb.
and with the former, you'll want to wear eye protection

Spectroline sells dual-wavelength lamps, 254/365, but those suckers are not cheap, even on e.bay

 

[endlessness]

Yes, we have these UV chambers, for it.

 

[benzyme]

and consider using ethyl acetate in a tertiary solvent mixture.
butanol, EtOAc, NH4OH. 4.5:3:1.5

 

[Tannenberg]

yes.

Well thats nice so no problem at all these UV-Party lights are sold under 10€ here IMHO.

Thanks for the links endlessness, they sell some drops of sulfuric acid and formaldehyd for 25$, pretty expensive but hey, they at lease have it.

 

[Tannenberg]

Hm, when i searched a littlebit i found out, that those testers for money / stamps, should emit 254nm, at least a lot of sellers of these products state that.
At ebay, you could also get a "dedicated" 254nm UV light for 20-25€

http://www.ebay.de/itm/UV-C-254nm-Germicidal-100mm-by-12mm-Light-Lamp-Bulb-12V-DC-Kit-/320883241809?pt=LH_DefaultDomain_0&hash=item4ab621b351#ht_861wt_1399

 

[benzyme]

Hm, when i searched a littlebit i found out, that those testers for money / stamps, should emit 254nm, at least a lot of sellers of these products state that.

then they are misrepresenting what they are selling.
254 nm is used for germicidal purposes, and exciting amide bonds in proteins. it's also
used in mutagenic experiments with microbes.

fluorescent illumination of stamps, minerals, or forensic investigation, is typically done with 365 or 385 nm lamps

 

[Tannenberg]

Hm, when i searched a littlebit i found out, that those testers for money / stamps, should emit 254nm, at least a lot of sellers of these products state that.

then they are misrepresenting what they are selling.
254 nm is used for germicidal purposes, and exciting amide bonds in proteins. it's also
used in mutagenic experiments with microbes.

fluorescent illumination of stamps, minerals, or forensic investigation, is typically done with 365 or 385 nm lamps

Yes, but they are also selling some lamps for germicidal usage, which have a transparent body. I think they are 254nm. It´s just mercury inside there.. so it´s not that expensive..

http://www.ebay.de/itm/UV-254nm-Germicidal-U-shape-75x8x75mm-Dual-Light-Lamp-Bulb-5V-DC-Kit-/320832801725?pt=LH_DefaultDomain_0&hash=item4ab3200bbd#ht_832wt_1399

is one example.

 

[benzyme]

here are a couple pictures of 253.7 nm (254) UVC, and 365 nm UVA light, respectively.
the second pic contains an alcoholic solution of ergolines.

 

[Tannenberg]

So the second picture uses the 365nm light source?

Does it show diferences in color between different tryptamines with 365 too?