What is TLC?


TLC is a relatively simple method that any non-chemist can use to actually analyze their own plants, extracts and drugs, to see what they contain.


Why use TLC?

TLC can help you identify if your plants have alkaloids of interest (for example DMT, 5-MeO-DMT, Harmalas, etc),

TLC can help you rule out potentially unwanted alkaloids (Gramine, etc) in a plant or plant extract

TLC can tell if your extracts have a single substance or a mix of substances

TLC can tell if your other recreational drugs are mixed/adulterated or not.

TLC can, with some additional steps, tell you more or less how much of a substance is there.


Where to buy or how to build?

Ready-to-use TLC Kits:


PROTEST KIT SHOP

(Use the PROMO CODE nexus10 for 10% off)

TLC Kit with plates for 60 samples (72,22€) + 5 reagents of choice 21.44€ or 12 reagents full kit 44,91€ = Total 93,66€ or 117.13€

or cheaper option:

Kit with plates for 20 samples (54.16e) + 2 reagents of choice 12,41€) = 64.57€

Free worldwide shipping (and 10% off all orders with nexus10 coupon code)

These TLC kits, apart from testing normal alkaloids, also include another eluent to also test cannabis for cannabinoids, and a ruler to semi-quantify cannabinoids, as well as a ruler to semi-quantify MDMA/ecstasy samples



BUNK POLICE SHOP

TLC kit with plates for 60 samples + 8 reagents = 205 USD

or

TLC kit with plates for 30 samples + 6 reagents = 135 USD


Or

TLC kit with plates for 30 samples + 3 reagents = 95 USD

Does not work for cannabis testing nor includes semi-quantification ruler.

Worldwide shipping but price NOT included.


Self-assembled kits
You could also potentially buy the material separately yourself, it would be a larger investment but would result in material for way more tests. Links below are just examples of the necessary products you could buy, this is not an endorsement to those particular suppliers, feel free to search for alternative suppliers that sell in or ship to your area:
- a bunch of Microcapillary tubes, one per sample tested but you can clean and reuse them.
- Merck TLC plates with 254nm fluorescence (cut each 20x20cm plate into 8 10x5cm plates each testing 4 samples so this packet is good for 800 samples tested)
- a mason jar with wide mouth that can fit a 10x5cm plate vertically.
- a few eppendorf vials for dissolving samples. You can clean and reuse them.
- a 254nm UV light reusable forever just need batteries
- a 365nm UV light (recommended for also using when testing unknown samples and LSD and harmalas but not essential) reusable forever just need batteries
- Eluent - For each test day you need around 20ml eluent (19.5ml methanol + 0.5ml 25% ammonia for most substances except cocaine which you test twice, once 20ml methanol, and separately with 20ml acetone, check cocaine testing section below for more info). You can test many plates a day. After some plates opening and closing the jar, the ammonia in this eluent evaps and the substances form streaks instead of nicely defined spots so you need to prepare some more eluent.
- You’d still need the reagents like marquis mecke ehrlich etc. To know which reagents to buy, check the flowcharts further down this post in each individual substance section. Each ml of reagent = around 20 tests, calculate you might test each sample with 2 different reagents just to cross-confirm the results.


How it works?
In practice it is VERY EASY!

Instructions video:


Other relevant links:
1-Explanation of how/why things work in this testing method
2- TLC instructions manual



Results interpretation

Step 1 - Spot separation / position (Rf).

If in your TLC plate you only see one single spot, this indicates your sample had just one substance. If there are more spots, it means there is a mix of substances inside, each spot being one separate substance.

To identify what each spot is, you need to either:

a- Compare your sample vs a known standard.
Load one lane of the plate with a reference standard (which can be for example pure DMT or whatever substance is being tested, or simply a plant or extract which you know contains DMT or substance of interest), and another lane with your unknown sample (for example an extract of a new plant with suspected DMT). If your reference standard spot appears at the same height as the spot on your unknown sample, this likely means it is the same substance (but very importantly, this needs to be further confirmed by dropping one of the appropriate reagents and see if both your reference standard spot as well as the unknown sample spot change color in the same way)

or if that isn't possible.

b- Compare your standard vs another unrelated substance of known identity you have at hand. For this you can use the Spot height calculator (be sure to select the methanol:ammonia sheet within the excel file for relevant values to the recommended eluent used in the TLC kits linked above)

What this calculator does is tell you at what height in the plate your DMT (or whatever alkaloid of interest) should be, compared to another substance you have at hand (for example some coffee for caffeine) which you load in another lane on the plate.

So for example you test a plant or extract which you think has DMT, but you have nothing to compare it to on a plate. So on one lane of the TLC plate you add your extract with supposed DMT. On the lane next to it, you add another substance which you are sure what it is (for example you have some paracetamol at home, or some caffeine). Then on the calculator spreadsheet, you click the substance name on the "unknown substance" cell, which will give you a drop down menu with all sorts of substances, and you select DMT. On the "known substance" cell you select the confirmed substance you have at home (for example paracetamol). Then after you run the plate, you measure with a ruler how many centimeters the paracetamol spot is from the bottom line where you originally loaded the substance, and you put that value on the calculator spreadsheet next to "known substance". Then it automatically will give you a value for your "unknown substance", in this case, DMT. So with a ruler, you measure the height of your DMT spot and it should be within +-0.3cm of the value the calculator gives. If not, it's probably not DMT. Be sure to use a confirmatory reagent (read below) to confirm identity of spot.

Here are hundreds of images of TLC plates with different substances of all kinds using methanol:ammonia25% , 97.5ml:2.5ml as eluent.

Before this calculator was invented with this comparative/relative spot height value, people used absolute numbers called "Rf", but this has a much larger margin of error because different room temperature, humidity and etc will greatly change the height of a spot. In any case, for those inclined to check older literature, here are published values of Rfs for some substances of interest. I recommend ignoring these Rf values and using the calculator only.

Step 2 - Confirmatory reagent

So after looking at spot height in TLC plate, you confirm the identity of the substance by using reagents on top of the spot, that change their color depending on what substance it is. You then compare the results you get with this
amazing page, or use this
reagent results video database (currently being fixed due to some link issues, you can check vimeo.com/bunkpolice for the individual videos you want to watch)

To know which colorimetric reagents are more useful to use for each substance,, below in the section for individual substances there will be a relevant flow chart and video links to reagent results. The more reagents you use the better confidence you will have that your results are right. You cannot use two or more reagents on top of each other, you need to either run the sample several times in different plates and in each use a different reagent, or you can also scrape a tiny bit of your substance in different parts of a white ceramic plate and in each add a drop of a different reagent. Sometimes its easier to visualize a color change with a reagent on the dry pure substance due to concentrations, as opposed to the tiny amounts present in the TLC plate after running it (but this only works if there's only one main substance otherwise the mix of substances may screw the reagent results of the dry sample when still mixed, instead of separated with the tlc)




Quantifying substances

Size of spot is more or less proportional to the amount of that substance. The simplest use for this is by comparing 2 plants by testing equal amounts of each and then just saying which has more or less alkaloids by seeing which has a bigger or smaller spot.

If you have a pure substance to compare to, you can semi-quantify for example how much DMT a plant has. In this example below one can see using this method and comparing to the serially-diluted DMT standard spots, one can (correctly) estimate Mimosa hostilis has around 2% DMT since at 50mg/ml dilution of mimosa in water, it resembles the size of the DMT spot at 1mg/ml (Look at the semi-quantification section of TLC Manual for step by step instructions)


Also look at the mescaline section below for an example of quantification using software to detect spot size


Relevant info to each substance:

Here are hundreds of images of TLC plates with different substances of all kinds using

Harmalas:

UV
Thread with plate images on 254nm and 365nm UV light where one can see how how easy it is to distinguish between harmine, harmaline and THH (and THH isomers, distinguishing natural vs synthetic THH)

TLC:
A TLC plate comparing harmine to mostly harmaline (with a bit of harmine)
Another TLC plate comparing Harmine vs Harmaline vs THH (synthetic and natural)

Reagents:
Harmine: Marquis, Mandelin, Simons, Hoffman / PDMAB-TS, Ehrlich
Harmaline: Marquis, Mandelin, Hoffman / PDMAB-TS, Ehrlich.

DMT, 5-MeO-DMT, Bufotenine, gramine etc

TLC:
The height of Bufo, DMT and 5-MeO-DMT are close but slightly different. There are some ways to help telling them apart (or noticing when there is a mix of them).

On TLC, after some minutes bufotenine stains the plate so you see a spot even under visible light. DMT and 5-MeO-DMT do not do that.

Plate comparing caffeine vs DMT+NMT vs 5-MeO-DMT+DMT vs 5-MeO-DMT

Another plate comparing Caffeine vs DMT+NMT+Gramine vs 5-MeO-DMT+DMT vs 5-MeO-DMT

Compare with a pure standard use the Spot height calculator to find at what height your substance should be (be sure to select the methanol:ammonia sheet within the excel file for relevant values to the recommended eluent used in the TLC kits linked above)

Reagents:

5-MeO-DMT vs DMT comparison on 2 different reagents on TLC (ehrlich and pdmab-ts aka hoffman reagent )
Gramine vs DMT tested on several reagents as well as a mixture of both in different proportions
Gramine reagent video playlist
DMT (freebase): Marquis, Marquis (on TLC), Mecke, Mecke (on TLC), Liebermann, Froehde,
5-MeO-DMT (freebase): Marquis, Mecke, Hoffman / PDMAB-TS, Mandelin, Ehrlich,
5-MeO-DMT (HCl): Ehrlich, Hoffman / PDMAB-TS, Mecke

TLC+Reagent+UV:
Another way to both detect small amounts of DMT/NMT as well as diferentiate it from 5-MeO-DMT is to drop PDMAB-TS (hoffman) reagent and look with 365nm UV light. Both NMT and DMT shine strong green after adding the reagent, while 5-MeO-DMT doesn't. Check the TLC instructions manual for a picture of this reaction on the semi-quantification section.


Acacias

Read section above for more info on tryptamine results in TLC.

Also, read this one thread on one example of TLC testing acacia and troubleshooting

Another relevant thread with
TLC+GC-MS results of acacia testing


Mescaline

TLC

TLC plate comparing pure mesc fumarate (the top spot is fumaric acid ), vs caffeine, vs 2cb

Compare with a pure standard or use the Spot height calculator to find at what height your substance should be (be sure to select the methanol:ammonia sheet within the excel file for relevant values to the recommended eluent used in the TLC kits linked above)

Quantifying mescaline in cactus using TLC and software.

Reagents:
This chart sums up what confirmatory reagents to use and what results to be expected.
Marquis, Mecke, Froehde, Mandelin, Lieberman, Ehrlich


Mushrooms

Blue stain:
Most psilocybe mushrooms stain blue after being picked or any damage.

TLC:
A lot of work needs to be done here. Preliminary work shows psilocybin/cin seems to shine under UV , not as strong as harmalas but significant nonetheless.

Reagents:
Psilocybin: Ehrlich - Reddish violet to purple (source), Hoffman / PDMAB-TS - Yellow-green (source), Marquis - orange (source)
Psilocin: Ehrlich - Blueish purple, darker than psilocybin's reaction (source), Hoffman / PDMAB-TS - Brown (source)
Baeocystin and norbaeocystin: Turn purple with ehrlich too (source)

LSD

UV:
With LSD it is easy to differentiate between good LSD and some fake LSD like an NBOMe or DOx type substance by the way it shines. Here's LSD under 365nm UV light (normal blacklight) even before running the plates you can already see. And here's on 254nm UV light before running the plate compared to nbomes, and after running the plate.

TLC:
Here there are pics of LSD tested on TLC plates using methanol:ammonia eluent.


Other Substances (for harm reduction's sake)

MDMA

TLC:
If there is any more than one spot the sample should be discarded. Plate comparing pure MDMA to an example of impure MDMA.
Main adulterants or substitutes are usually cathinones, which generally appear toward the top of the plate, some shine under UV (like MDPV) others not.

Compare with a pure standard or use the Spot height calculator to find at what height your substance should be (be sure to select the methanol:ammonia sheet within the excel file for relevant values to the recommended eluent used in the TLC kits linked above)

Reagents:
Also be sure to test with reagents to confirm the one spot is MDMA. Here's a chart on what reagents you should use for MDMA and how those reagents would react with other common impurities too.
Marquis, Mecke, Simons, Mandelin, Ehrlich, Hoffman / PDMAB-TS.

2C-x (2C-B, 2C-I, etc)

TLC:
Here there are pics of 2C-x substances in TLC plates compared to caffeine and other substances using methanol:ammonia eluent mentioned throughout this post.

Compare with a pure standard or use the Spot height calculator to find at what height your substance should be (be sure to select the methanol:ammonia sheet within the excel file for relevant values to the recommended eluent used in the TLC kits linked above)

Reagents:
Several 2C-x substances with marquis reagent (on TLC)
Several 2C-x substances with mecke reagent (on TLC)
2C-B: Marquis, Marquis (on TLC), Mecke, Mecke (on TLC), Mandelin
2C-C: Marquis, Marquis (on TLC), Mecke, Mecke (on TLC), Mandelin, Simons, Ehrlich, Hoffman / PDMAB-TS,
2C-D: Marquis, Marquis (on TLC), Mecke, Mecke (on TLC), Mandelin, Simons, Ehrlich, Hoffman / PDMAB-TS,
2C-E: Marquis, Marquis (on TLC), Mecke, Mecke (on TLC), Mandelin, Simons, Ehrlich, Hoffman / PDMAB-TS,
2C-I: Marquis, Marquis (on TLC), Mecke, Mecke (on TLC), Mandelin, Simons, Ehrlich, Hoffman / PDMAB-TS, Liebermann, Froehde, Robadope

Ketamine

TLC:
Here there are pics of Ketamine plates compared to caffeine and other substances using methanol:ammonia eluent mentioned throughout this post.

Compare with a pure standard or use the Spot height calculator to find at what height your substance should be (be sure to select the methanol:ammonia sheet within the excel file for relevant values to the recommended eluent used in the TLC kits linked above)

Reagent:
Here's the ketamine reagent chart
Ketamine: Mecke, Mecke (TLC), Mandelin, Mandelin (on TLC),

Cocaine

TLC:
To test with TLC, usually 2 different eluents are used separately: pure acetone, and pure methanol. Then for the results you look at both plates, because one of these systems separates some impurities, another separates others. Don't use the standard methanol:ammonia eluent. Acetone dissolves most plastic so make sure the developing chamber/mason jar is made of glass.

Here's a picture of two TLC plates of cocaine and common adulterants, the left plate was run in Acetone as eluent, the right one is Methanol. The substances added to those plates are: benzocaine/cocaine, lidocaine/procaine, fenacetin/levamisol/tetracaine, paracetamol/caffeine randomsample1, randomsample2). In each column/lane, the top spots are the first names on the label. Feel free to ask any question.

Reagents:
Here's a chart to know which reagents to use with cocaine

For cocaine impurities the most dangerous are levamisol and phenacetin, both of which can be easily detected using liebermann. Pure cocaine turns yellow, phenacetin turns brown, levamisol turns red, so anything other than yellow should be avoided.

Heroin/Opiates

TLC:
Heroin usually appears as 2 spots, heroin and 6-MAM (partially converted morphine).

Compare with a pure standard or use the Spot height calculator to find at what height your substance should be (be sure to select the methanol:ammonia sheet within the excel file for relevant values to the recommended eluent used in the TLC kits linked above)

Reagents:
Here's a chart to know which reagents to use with heroin/opiates

Methamphetamine / Other amphetamines

TLC:
Compare with a pure standard or use the Spot height calculator to find at what height your substance should be (be sure to select the methanol:ammonia sheet within the excel file for relevant values to the recommended eluent used in the TLC kits linked above)

Reagents:
Here's a chart to know which reagents to use with amphetamines.



Troubleshooting

The most common issues are related to concentration of the sample. If a spot is too large, it is hard to interpret results because it may have covered other spots (co-eluting/masking). Dilute sample so you see small but visible spot.

If substances are leaving too much of a streak on the plate, it is likely because ammonia from the eluent evaporated. Prepare some more eluent.

If you are testing a crude plant extract/soak and a streak is forming on the plate which makes it hard to see the individual spots, then better make a slightly more thorough extraction of the plant material before adding it to TLC plate.

I wish I knew TLC could be applied for substance analysis earlier, thought it was just a technique used for separating different colored inks in black pen ink, why don't they teach kids this in high school?

Just kidding. Thanks for the summary.

Updated first post!

Very happy to announce that there is a new supplier of TLC kits, they are a harm-reduction group started by a friend of mine from Poland, they offer free worldwide shipping and they are giving 10% discount to all Nexus members.

The really cool thing they are doing is that with their kits, apart from testing normal alkaloids of our interest, also can work for testing cannabis for cannabinoids, and he even includes a ruler to quantify those cannabinoids, and also another ruler to quantify MDMA/ecstasy.


So now we have 2 suppliers, one based in USA, one based in Europe, both ship worldwide.

I hope with all this information it helps people testing plants of interest, maybe even find novel plants with tryptamines, and also be safer in general with the drugs they use by making sure they don't have adulterants or unexpected impurities.

Lastly, I want to mention we are working on a database where we will collect the analysis results of all sorts of plants and drugs, gathering data from NGO's and other labs, peer-reviewed results but also users will be able to add the tests they make themselves with for example TLC kits. So if you do have or plan on having a TLC kit, please keep an eye on this thread where in the next few months we will link our new database so you can share your own test results and help the whole world learning more about plants and drugs

This is really fantastic, thanks for putting it all together. I just discovered this thread today. Yes, TLC can be "tricky" as it seems rare that any two investigators report the same Rf values. So many variables: the exact type, and possibly even manufacturer, of plate used matters, the development chamber used matters, vapor saturation within the development chamber matters, exact eluent used matters and this is where everyone seems to use something different, sometimes the published papers don't even specify the strengths of substances used (like ammonium hydroxide concentration used). Its nice to have your calculator as another solid reference point although already I see that I have slightly different Rf values for DMT and NMT in methanol ammonia but I used a very slightly different ratio for my eluent.

I noticed there is no value for the common mushroom alkaloids in your calculator or serotonin or urea also found in mushrooms. It would be nice to know where to expect to find these. There are so many different spots showing up from a pan cyan plate, I would love some help in identifying all of them! I think it would also be useful for the community. The pan cyan sample (first pic below) used p-butanol + Acedic Acid + water as the eluent becauase this seemed to produce the best separation, and urine was added for comparison purposes in hopes of identifying urea (which seems to have worked). I have not yet identified what is at Rf=0.48 (probably Baeocystin) and Rf=0.84

The purpose of the second plate (second pic) was to determine if a mystery sample that someone sent me was really DMT or not. I did a side by side comparison of known DMT to the mystery sample. It turned out that the mystery sample was mostly DMT but also had a bit more NMT in it than the reference sample.

OK, based on another chart posted in a different thread I think I have all but one compound identified in this pan cyan TLC silicon gel plate (pic has UV and Ehrlich's pics side by side) although the chart had no attribution and it seems like the values for psilocybin/psilocin are wrong so maybe not reliable info here) :

Thank you so much for putting this together. After I absorb the content, I’ll contribute if i have questions or can add some value.

I have used formic acid for TLC analyses, what are your thoughts on using FA wrt mescaline TLC?

No thoughts specifically on FA, but for mescaline TLC I HIGHLY recommend you check out this just published paper:

Validation and exploratory application of a simple, rapid and economical procedure (MESQ) for the quantification of mescaline in fresh cactus tissue and aqueous cactus extracts
Van Der Sypt Frederick A.P.
https://zenodo.org/record/6409376#.YlV5zMgzaUm

Phenomenal work.

Awesome

With TLC there is definitely some variation going on based on a number of factors as you mentioned, but doing relative Rf with another substance as a standard and using the calculator as opposed to absolute Rf, definitely helps.

As for mushroom alkaloids, that´s something I definitely want to experiment this year now that I have the lab set up.

Thanks for the picture of the plates! When Im able to test mushrooms and relevant pure standards, I will definitely come back to this thread



What would be the point of using formic acid for testing mesc with TLC? What eluent are you planning on using? In my experience methanol:ammonia 97.5:2.5 works very well for mescaline, there is no trailing of the spot.

I expect that if you can get good separation and derivatization of alkaloids on the plate, a simplified MESQ-procedure (link above) can give you satisfactory quantification. In the first part of the discussion, an alcoholic extraction is proposed that might be useful for mushroom tissue. Still, specific problems will need to be tackled like the instability of psilocin (versus the indestructible mescaline molecule) and the lack of reference standards. Does anyone know of a procedure for isolating the main alkaloids? Preparative TLC perhaps? Still, even without known reference standards, one could still do a relative quantification of mushroom tissue samples that would allow new research on inter- and intra-specimen alkaloid distribution and quantification. In this way, selective breeding with the most potent (or most stable: psilocybin/psilocin ratio) individuals from a multi-spore grow becomes possible. Interesting stuff, keep up the good work guys !

@Endlessness - In the TLConscious video, it says you can mix whatever you are testing with vodka / alcohol / vinegar rather than the same eluent you will use in the testing chamber, is this correct? I have the bunk police kit and have always mixed the test substance with a small amount of the same eluent.

Does the substance to be tested need to be in freebase form? Or will testing work on an acidic solution? I just tried to test a few dmt acetate samples diluted in vinegar and ended up with the picture attached. I was trying to compare ACRB vs MHRB. Would this have resulted from adding too much of the sample fluid or is the vinegar to blame for the long streaks? (I am pretty sure these are pure extracts)

Seems like your spots are overloaded. Try using a more diluted solution for spotting first.

An1cca is right, it seems the sample being loaded on the plate is way too concentrated. Also, how long have you been using the same eluent? If the solvent bottle has been opened for long, the ammonia will start to evaporate, and when ammonia evaporates, the substances don't form neat spots but rather trail . So id test a way more diluted sample and if that still results in trailing id purchase (or prepare) a fresh eluent with methanol and ammmonia

Keep us informed how its going!

I tested again, diluting the original samples with more vinegar and putting only a drop of each on the test plate (the first time, I dabbed the tube on the plate several times to get everything out). I also tested some ACRB enhanced leaf mixed with eluent instead of vinegar to compare.

ACRB1 is the enhanced leaf sample showing clear NMT and DMT spots.
ACRB2 is from the same extraction as ACRB1 in its original acetate form diluted further with vinegar.
MHRB is the mimosa extract, also from an acetate tincture.

The samples may still be too concentrated but I find it strange that there isn't a more pronounced spot at the NMT mark in ACRB2. The MHRB streak shows a much bigger spot at the DMT mark which I was expecting but I also find it odd that the streaks start from the same point in both ACRB2 and MHRB, since there shouldn't be any NMT showing up in the MHRB sample.

I'm also curious about what is going on towards the top of the plate. Clearly, I am not leaving the plate in the chamber long enough since the top part isn't saturated, but is it just my imagination, or does it look like there is something else going in the upper areas I've circled?

Thanks for posting your further experiments!

From the comparison of ACRB1 with the others, it seems to me that the vinegar/acetic acid is interfering with the resolution of the spot, and streaking the plate instead. I would try instead of diluting in vinegar, using the eluent or dissolving it in ethanol or something else.

The ammonia in the eluent should be enough to freebase compounds that are in salt form but the extra acetic acid might screw things up a bit... Thanks for sharing this, I probably have to add a note on the video for people not to use vinegar to dissolve the sample and load the plate.

Regarding the MHRB seemingly showing NMT spot, it definitely does have NMT, but just not as much as ACRB.

As for the top of the plate, there might be something there, though Im unsure what it could be. You could try adding some reagent on top and seeing if it reacts, might give some hints. Mimosa has been found with minor amounts of other alkaloids, might be one of them.. Id have to check back on ACRB if it has ever been found with anything else. Soon I´ll start testing ethnobotanicals at our lab and I´ll take another look at ACRB to see if we can find any other interesting minor compounds.

Be well!