Here are some preliminary TLC results of different kinds of Acacia foliage.

All foliage was seed grown indoors in northern continental Europe with only natural light coming through regular windows.

Thus these results might not be conclusive for native habitats, but I assume it to be very interesting and practical to get some results on which Acacias do well in European indoor settings.

These plates are also my first TLC plates ever and having only very limited chemistry knowledge and experience, I want to first establish some useful parameters for further experimentation.


PLATES ARE NUMBERED 1 to 5 FROM LEFT TO RIGHT


PREPARATION


- Reference:

A single Coffee arabica bean was crushed and placed into a test tube, testing liquid was added. The preparation was done at the same time as the first 2 of the following plates.

This Coffee reference was transferred to the middle column of each shown plate.




- Plate 1 + 2 : Acacia melanoxylon

Dried A. melanoxylon foliage from a plant roughly one year old and which was harvested around early September 2016 was placed into a test tube, testing liquid was added, and the tube was shaken for about 30sec.

Then, the liquid was transferred (repeating 3 times with the microcapilars) to the two outer columns of the TLC card.


- Plate 3: Acacia melanoxylon

After 24h since preparing the test tube for plates 1 and 2, the testing liquid had changed in colour to a very deep green. Another TLC card was prepared as before, but repeating the microcapilar transfer 5 times.

Note: also the Coffee reference had 24h to soak at this point.



- Plate 4: Acacia maidenii

Dried A. maidenii foliage from a plant roughly one year old and which was harvested around early September 2016 was placed into a test tube and testing liquid was added.

The preparation was given 2+ days to soak.

Then, the liquid was transferred to a TLC card as described above, again repeating the transfer 5 times.

Note: the Coffee preparation had about 3+ days to soak at this point.



- Plate 5: Acacia floribunda

Dried A. floribunda foliage placed into a test tube and testing liquid was added. The actual time of harvest for this sample is unknown. The plant at time of harvest was anywhere between 2 and 4 years old, therefore the foilage might be between 0 and 2 years old.

Previous extraction of about 10g of foliage that was collected over the first 1-2 years of multiple plants (early growth was ok, but not very fast) yielded 340mg of yellowish-green oil by a simple method of repeated boiling in vinegarized water with added vodka, the combined liquid reduced to a very low volume, which was basified with calcium hydroxide and dried. Once dry, pulls were done with isopropyl alcohol, which was evaporated to leave the described yellowish-green oil. The psychoactivity of that extract was successfully established. Also smoking small amounts of foliage have noticeable (but short and subtle) effects. It has quite some "body" feel, early trials were "tingly" and felt a bit spikey (like if you would use Pine tree needles as acupuncture tools), later trials felt more smooth and in a Changa preparation with B. Caapi "white", there is a noticeable enhancement of the harmala space with quite pleasant energetic chills. "High" doses have not been tested, but low doses give subtle and short lived CEVs consistently at below 10mg of extract.

The low dose (even of foliage) needed to notice anything led to suspect beta-carbolines, the further effects above suggest a DMT/NMT combination with being healthy on the NMT side.

The preparation was done during the same timeframe as processing the A. maidenii foliage and given 2+ days to soak as well.

The liquid was transferred to a TLC card as described above, again repeating the transfer 5 times.

Note: the Coffee preparation had about 3+ days to soak at this point.




QUESTIONS

- Plate 1 + 2: Acacia melanoxylon

Clearly, the amount of actives on the TLC card was too low. The cards are included here only for completeness and for demonstrating that when foliage is to be tested without prior extraction then it is not enough to let the testing liquid soak for just a minute or two.


- Plate 3: Acacia melanoxylon

Not related to the Acacia per se, but which of the marked points of the Coffee reference would be the Caffeine spot to use for the spot height calculator? Looking at the other cards as well, I would guess it should be the third marked one from the top, because it looks the most "pure", i.e. not like a streak or blurred out, but I'm not sure.

The lower one of the two marked spots looks fairly yellow-green in daylight, so I would assume that to be chlorophyll or some other plant oil. Is the "green" of plants generally creating such a "green spot" ?

The top point, by its height, looks similar to the spots found in the Acacia mangium thread.

It would thus hint, that A. melanoxylon doesn't contain DMT as was also found in other threads previously. However, it grows strong and fast in northern europe climate, so if any interesting substance is to be found in it, it is a very good plant to grow.

As the A. mangium thread was inconclusive in regards to reagents, which reagents would be good to use on the spot, next to Ehrlich? I'm thinking something that indicates beta-carbolines or phenethylamines would be useful. But I'm not sure. Maybe also the Hofmann+blacklight test for 5-meo ?



- Plate 4: Acacia maidenii

Now that's an interesting one. There is some separation in the upper part, whose relation could imply NMT/DMT. However, in comparison to the A. mangium plate, it happens at quite a high level on the card. On the other hand, the A. mangium plate had no caffeine reference.

Is it possible (and why?) to have such a big difference in height on the card? The DMT/NMT reference in the A. mangium thread shows up around middle and below, this card shows it in the upper third.

On again another hand, the upper point could be the same stuff as the stuff in the A. melanoxylon sample, relative to the Coffee spots, it's seems to have the exact same height.

The sample then has more interesting features: spots at the bottom just above the starting line. That also looks separated, i.e. two compontents. What could these be?

Again the question: Which reagents would be most interesting for which of these spots ?

On a further note, this A. maidenii result is quite surprising. It looks like it has the highest alkaloid concentration of the three tested samples and is also the one with the highest number of clearly identifiable spots. Prior to the tests, I expected the A. maidenii to be the least interesting of the samples. I hope I didn't accidentally switch the A. maidenii with the A. floribunda, but I tracked which test tubes are which and if they should turn out to be switched, I have no clue how that could possibly have happened.

On a further note as well: The amount of foliage used was not weighted. It was just "some" foliage stuffed into the test tube until deemed "enough". Maybe 50mg to 100mg foliage for each sample.



- Plate 5: Acacia floribunda

This result is also quite surprising and a bit disappointing. The high spots look (in relation to the Coffee reference) to be the same as with A. maidenii and A. melanoxylon. So either that stuff turns out to be DMT (which would be great but surprising as well, especially for A. melanoxylon), or my beloved Floribundas don't have DMT. Which is weird given its psychoactivity.

In addition, no obvious signs of possible separation between DMT/NMT, while I would have been certain that it contains more NMT than DMT based on its subjective effects.

So, it's testing to the rescue of truth. Any input is highly appreciated.

I cant see the pics, can you please try to reupload so I can check them out ?

Now it should be there, I can see it. I assumed before that someone might need to approve uploads, but it was probably some issue with the jpg itself, multiple uploads didn't help, but re-converting to jpg did the trick.

Did you use any reagents on top of those spots?

Also, I highly suggest you label under each column with a pencil to make it easier to see what is what.

Do you have any DMT or a plant that you know contains DMT to test next to your acacia?

The DMT/NMT should be more towards the middle of the plate (like this), not so high like near the caffeine spot, so I suspect either none of your plants tested have DMT (maybe the lower spots on the third fourth and fifth plates are NMT but you gotta use reagent to confirm), or they just have another alkaloid in larger quantity and you need to concentrate the sample more to see the dmt/nmt spots better, hard to say. You can see here the TLC plates of a few acacias that also have some substance(s) with a higher RF (spots towards the top of the plate), but I didnt know what it was.

Did you use the TLC calculator to compare the caffeine spot and then see where your DMT spot should be? Then you can drop your reagent there and see if it turns the right color (for example pink/purple with ehrlich).

Also, for the plates that you cant see much, just concentrate the sample further.. Use less of the testing liquid to soak the plant material, that way you have a more concentration and might see other spots that for the moment are hidden.

Do you by any chance also have a normal UV light ? Some beta carboline shine with long wave (normal) UV light and using it can be an extra interesting test.

Anyways these are just a few comments and questions, feel free to ask me whatever or to keep this discussion going in general.

Thanks a lot for testing and sharing the results, this is excellent

Nice Work plantatron very interested to see the refined results.

No reagents were used on the above plates yet, I first wanted to get a bit strategic about that.

Next to Ehrlich, is any of the other reagents of particular help? If it doesn't matter much, I would go with Hofmann for now. I can also run more plates for other reagents, but maybe first start somewhere.

Regarding the Coffee reference: I marked three points in the middle plate, which one of these three (top, middle, bottom) is the Caffeine spot to use for calculating the heights ?

Thanks and I'll keep you updated.

Interesting post thanks plantatron..

regarding floribunda.. its a tricky one and I think may depend on area/strain. Trees from certain areas are consistent in yielding dmt.. other areas the trees seem to be devoid of it. I've done a lot of tests on it and found it to be highly variable.. sometimes it yields extracts that are probably almost entirely tryptamine, nmt or betacarbolines (as was the case with a recent test)

the other thing to take into consideration is that young plants may not have dmt.. in the "trying to improve acacia information" thread (a link in my signature) I remember nen mentioning a test on a young a.phlebophylla that was void of dmt and just contained simple tryptamine so that may apply to other acacias.

good experimental work and intent, plantatron..

i'll look more closely at TLC pics when time.. solvent system can affect the distance or spread of spots slightly, from experience, when comparing..so yes dmt (and nmt) confirmed references run through same system helps..

and good point and yes, acacian - age is a factor...tests of A. phlebophylla (by researcher 'JJ', who 'found' a number of species) found that, under one and a half to 2 years age, there was no dmt, with tryptamine as the predominant alkaloid - above that age it is absolutely consistent in its dmt profile..
similarly, tests run on A. mucronata showed at 18months old, nmt and tryptamine predominant (almost no dmt), this was endlessness' test on material i sent him, whereas older material i had tested a while back (reported by Snu Voogelbreinder) had dmt..

lastly, if it's the right sub-type of A. maidenii then it's one of the 'best' yielding plants (especially in phyllodes) you'll find..see that thread acacian referred to..

keep up the good work

plantatron, yeah you definitely have to use a reagent. If you have ehrlich, use that on all spots, otherwise use hofmann if you have that instead. With ehlrich it should turn pinkish with tryptamines, or with hofmann it will turn yellow and then eventually green (or fast green if its 5-meo-dmt). So if you have more than one column repeated with the same plant, you can use one reagent in one column (for example the ehrlch/hofmann) and another in another (for example mecke or marquis). The more reagents and repeated tests the better, but first start with just ehrlich or hofmann to see if there's some good indication, and then you can plan whats next after that.

As for what spot it is in the coffee beans, I suspect its the middle one but it would be good to confirm it by running some pure caffeine sample. As asked, do you have any pure dmt or plant that knowingly contains dmt, to test there too? Otherwise do you have any known drug with you, psychedelic or otherwise?

ADDITIONAL PREPARATION

In the spirit of science, two more plates were prepared:

- Plate 6 + 7:

First column: A. floribunda

Dried A. floribunda foliage placed into a test tube and testing liquid was added. The sample was recently harvested from 4 year old plants early October 2016 and dried for two weeks. More foliage than for plate 5 was used. It had about 10 hours to soak. Liquid was transferred 5 times to the TLC plate.


Second column: A. confusa root bark extract

A. confusa root bark was previously extracted by the same method as described above in the section about Plate 5. Early pulls yielded a more white-yellow-brownish gooey crystal, later pulls yielded a darker red-brown oil. This oil from later pulls was used for the test. About a match head size was put in a test tube and testing liquid added. It had about 20 min to soak and dissolve. Liquid was transferred 5 times to the TLC plate.


Third column: Coffee reference

The same Coffee preparation as used for the earlier plates was used again. Liquid was transferred 5 times to the TLC plate.



Results and full labeling key to follow below.

TLC PLATES UNDER GENERIC BLACKLIGHT

All plates were viewed again using a standard issue blacklight "energy saving" light bulb.

Nothing of particular interest was found, no additional fluorescence was noted except for a very faint shine around the Coffee reference of plates 6 and 7.

TLC PLATES UNDER THE BUNKPOLICE BLACKLIGHT

- Plate 4 + 5

For reference, the plates are shown again under the bp blacklight, with labeling. For details and key see below.


- Plate 6 + 7

First column of A. floribunda looks similar as on plate 5.

Second column of ACRB extract shows nice spots

Third column shows Coffee reference.

So plates 6+7, the middle column is acacia confusa? Those spots are the nmt/dmt ones. The one closer to the bottom is NMT, the one in the middle is DMT, and the fainter spot towards the top im unsure..

You should run your acacias always next to that acrb extract, and then try to see if you can see spots at the same heights. In case you dont see anything you can concentrate those acacia samples by using less solvent per plant matter before the tlc (or evapping what you had already soaked to a more concentrated amount) , and do another tlc plate/card with the concentrated one.

It seems that column C on the plates 6+7 might have a bit of NMT in that lower streak but it could also just be another substance on the same height. The hofmann/ehrlich test would be a good idea to test, drop the reagent in the acacia confusa column and then on column C at that NMT height and see if you see a similar (albeit softer) color change

TLC PLATES WITH EHRLICH AND MARQUIS REAGENTS


FULL KEY

- Plate 1

Column A: A. melanoxylon - no reagent
Column B: Coffee - no reagent
Column C: A. melanoxylon - no reagent



- Plate 2

Column A: A. melanoxylon - Ehrlich at calculated height of DMT
Column B: Coffee - no reagent
Column C: A. melanoxylon - no reagent


- Plate 3

Column A: A. melanoxylon - Ehrlich at spots and calculated heights of DMT and NMT
Column B: Coffee - no reagent
Column C: A. melanoxylon - Marquis at spots and calculated heights of DMT and NMT


- Plate 4

Column A: A. maidenii - Ehrlich at spots and calculated heights of DMT and NMT
Column B: Coffee - no reagent
Column C: A. maidenii - Marquis at spots and calculated heights of DMT and NMT


- Plate 5

Column A: A. floribunda - Ehrlich at spots and calculated heights of DMT and NMT
Column B: Coffee - no reagent
Column C: A. floribunda - Marquis at spots and calculated heights of DMT and NMT


- Plate 6


Column A: A. floribunda - Ehrlich at spots and calculated heights of DMT and NMT
-> DMT height shows some slight brown reaction?
-> NMT height shows slight pink reaction - YAY


Column B: ACRB extract - Ehrlich at DMT and NMT spots
-> Nice pink reaction, not bad for the oil from later pulls
-> no reagent at the third top spot, maybe that should be done as well

Column C: Coffee, no reagent




- Plate 7


Column A: A. floribunda - Marquis at spots and calculated heights of DMT and NMT
-> I'm not sure, how to interpret the results

Column B: ACRB extract - Marquis at DMT and NMT spots
-> Yellow-brown reaction
-> no reagent at the third top spot, maybe that should be done as well

Column C: Coffee, no reagent






RESULT DISCUSSION

- The A. floribunda seems to have at least some NMT, not sure how to interpret the slight brown around the DMT height.

- The A. floribunda NMT was only detectable in the stronger extract, thus the A. melanoxylon and A. maidenii results are probably meaningless.

- Generally, just soaking foliage in the testing liquid does not seem to extract enough actives for conclusive testing.

Yeah by the reagent results it does seem like the floribunda is more likely to have NMT.. Id concentrate it a bit more and test again if you get a clear pink on that height

Thanks a lot for sharing the results!

that's the spirit plantatron (apologies my spell correct got your name wrong earlier)

phyllode material crushed and straight into ethanol:methanol 90:10, or commercial 'denatured' alcohol (methylated spirits) for 15-20 mins, yielded clear results (if actives present) from 2-3 grams of plant material in past TLC tests i observed..
personally also i think finding nmt alone is quite a buzz

thanks for the work
(good advice endlessness too)

Thanks everyone for all the feedback, especially endlessness for being super fast

I will respond to the other comments as well in due time. I can squeeze the tests in, but my time during the week is still a bit limited.

Guesstimating 1% actives of dried material and requiring 5mg for the test, my follow-up plan was to create a crude extraction (with the testing liquid) of 0.5g of dried foliage.

That amount was cut to small pieces with scissors (not done in the tests above, the foliage was stuffed as is into the test tubes), final weight in the tube yielded 543mg which will soak for the next day or two.

Thanks nen, its very helpful to know that 2-3g have proven successful before. If the 0.5g turn out to be not enough, the follow-up will be to add 2.5g more

Regarding solvents: I'm not fully sure what the "testing liquid" is, my guess is its the methanol:ammonia mix endlessness mentioned a few times around, but I don't recall the exact parameters.

I think, when time permits, I'll compile a small FAQ with additional questions I had (and partial answers I already have) regarding the TLC kit to have that in one place. The kit definitely has a learning curve (especially regarding handling the solvent/chamber, how strong to push the capillaries, etc.), but once you start to get the hang of it, it's not finicky at all. Thanks again for making these kits available, they're great fun and the insights they provide are great.

ADDITIONAL PREPARATION (2)

The above mentioned 543mg of A. floribunda were cut with scissors and soaked in one of the "big" test tubes that comes with the reagent bottles. The test tube was filled about 1/3 of its height. The preparation was left to soak for about 10 hours. The liquid was combined with the remaining liquid from the A. floribunda preparation that was tested above.

The liquid was left to evaporate and the remaining oil was transferred to a small test tube. The volume of the oil was approximately equivalent to the volume of 2-3 match heads. That test tube was filled only to 1mm below the end of the conical bottom section. It was shaken several times over a period of 30 min to allow the extracted oil to dissolve completely.

The A. floribunda liquid was transferred to Columns A and C of "Plate F". Transfer per column was repeated 8x.
The middle Column B received 2 transfers of the ACRB extract that was used above as reference.


Two more Plates "I" and "II" were prepared just using the ACRB extract for two objectives:
1) Provide a reference result for more reagents.
2) Figure out what the third spot above the DMT spot is.

Each column of Plates "I" and "II" received 2 transfers of the ACRB extract preparation.



FULL KEY

Note: The labelling of the plates was done pretty late in the process, all pictures show the plates in the same order.


*Plate I* (left)

- Column A: ACRB extract - Ehrlich
- Column B: ACRB extract - Liebermann
- Column C: ACRB extract - Hofmann


*Plate II* (middle)

- Column A: ACRB extract - Mandelin
- Column B: ACRB extract - Froehde
- Column C: ACRB extract - Mecke


*Plate F* (right)

- Column A: A. floribunda - Ehrlich
- Column B: ACRB extract - No reagent
- Column C: A. floribunda - Ehrlich on NMT spot (forgot to switch the reagent), Froehde on all other spots above

Note: Froehde was chosen, because most reagents behaved similar for DMT and NMT. The Froehde reaction on NMT was slower, thus allowing to distinguish between NMT and DMT. The effect is shown in the earlier, non-labelled reagent result picture.




QUESTIONS / DISCUSSION

1) What could the third (top) spot in the ACRB extract be ? Any why ?

2) In the blacklight picture, there seem to be spots in the lower area of the "green plant oil flame", in the A column on the left, in the C column on both sides, but a bit smaller. In the "early" picture, the Ehrlich might show a faint reaction, but it's hard to interpret. The spot is higher than DMT and with Froehde it seems to have turned brown. The spot is somewhat higher than the DMT reference. What could that be? The wikipedia page on Froehde mentions brown for ephedrine and opiates, so this can only be a start.

3) There seems to be a clear "band" at the NMT height.

4) There's the skewed upper "band" which in the early pictures only shows a lighter Ehrlich reaction, but in the several hours late picture, it can be seen a bit more towards the left side of the card and the band seems to stop at the exact height of the DMT reference. So could this upper band be DMT? I guess so, but why is it skewed? I'm thinking transferring the liquid 8x was too much, but can too much material lead to such skewed bands ?

5) There is the two big pinkish areas between the NMT and DMT bands. Approx. the center is right in the middle between DMT and NMT. I was wondering whether that could be also an artifact of using too much material, but it's weird that it stays and extends around that height instead of intensifying the DMT band, so I guess it can't be DMT. Further, it's bit unclear what Froehde on the C column did. It might show a super slight browning on the picture but nothing like on the ACRB DMT reference. With bare eyes, this was not really observable, I first thought it didn't do anything. Inspecting the HDR photo, it shows a slight blueish hued halo, which is also present around the top mystery spots around the "plant oil flame". So, not sure if that's just the Froehde under HDR or if this indicates a reaction of sort.


In total, I think transferring 8x was too much esp. with all the plant oil present, so I'll repeat with less once I'm back home (currently traveling).

However, it would be really great to get feedback on the above points.

No clue what the top acacia spot is but we can try to look into it. One possibility is to instead of loading the substance in one single spot on a plate, load it as a line all over the plate. This will make the spots be like horizontal streaks. Then you can scrape silica where the horizontal unknown spot is, off the TLC plate. Then you get the scrapped silica and soak it in the methanol/ammonia solution, filter the silica away, and this methanol solution you put in a vial and send me a message and we get it sent to Energy Control to test what it was.

As for the acacia green stuff, yes it could potentiall affect the elution of DMT or other substances screwing up their height a bit (though you would have probably seen it turn clearly pink with ehrlich anyways). One possibility of what you can do is to evap the methanol/ammonia solution where your plant was soaking (which should have freebased the alkaloids that were there), and then use a non polar solvent like xylene/toluene (or even naphtha but that might leave behind some alks... ), and load the plate with that instead. That should make the plate cleaner and the real non-chlorophyl spot easier to see. Or from the beginning, instead of soaking the plant material in the methanol/ammonia solution, you can do a small extraction with a non polar solvent on it, and then use the results on the plate, should also be cleaner as opposed to simply soaking in the methanol solution.

These are just some ideas, as you will be noticing, many experiments can be done and you learn with practice and find tweaks and tricks over time.

Thank you so much for sharing all these experiments, im very happy to see that, this was just what I hoped for when I was working on the TLC kits

Ah, this is great! I remember you mentioning the possibility to use TLC to separate substances, but never really figured how that would work. Thanks for sharing the method.

Regarding professional testing: I think it's a great option, but I'd like to wait with that until I come across some really interesting stuff. I'm not much concerned regarding the ACRB atm as it's widely used and the extract procedure was also not aimed at yielding super clean stuff, but getting a smokeable extract for changa with kitchen methods and without any smelly chemicals.

I'm way more interested in the Floribunda, as I have 3 plants and they are finally old enough to produce some useful amounts of foliage. Took 1.5+ years to get the first 10g together, but over the last year I got something useful. Still not super much, I need to weigh it once I'm back, but enough to potentially become fully self-sustainable in the mid-term future once my current ACRB stash is exhausted. And it grows great indoors in northern Europe, very resilient plants, very easy to take care of.

I, however, had hoped that someone who already did Acacia analysis might recognize these spots and know what they are. With none of the reagents doing something obvious (maybe the Liebermann getting white says something?), I guess it's some plant oils.


Regarding extraction: I will follow up with your suggestions once I'm back and post results. I had hoped that the TLC separation would allow to skip extraction steps, but apparently that has been wishful thinking.

Anyway, I think posting negative results/non-optimal steps will provide some help to others to avoid them. The floribunda is also a great training case, as it's not super straight-forward. I have several further testing projects already in mind after that, so any gained insights will be quite helpful. Maybe also more folks will chime in posting their TLC results.


Regarding the Froehde brown-ish spots in the "plant oil flame": I'm thinking it might be some kind of sugar. The nexus thread mentions Froehde on sugar as "brownish black", which would fit. Yet, wikipedia says "brilliant yellow". Googling some TLC results for sugar suggests it could be fructose. At the very least there are some sugars that would be consistent in terms of spot height, see Ghebregzabher et al. 1976, doi: 10.1016/S0021-9673(00)80168-5

I'm looking forward to the next plates, hope they will provide a clearer picture