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  • Ian Bollinger

TLC, a low-tech method for tryptamine analysis

Scientists (Noriyuki Kato et al) establish procedures for quick, focused, and sensitive thin-layer chromatography (TLC) methods that are able to determine the presence of tryptamines including tryptophan (Trp), the neurotransmitter serotonin (5-HT) and numerous psychoactive tryptamines (PATs). These processes take advantage of the low cost and flexibility of the TLC assay; while at the same time, utilizing far safer and readily accessible chemicals relative to most other methods of tryptamine analysis.

#TLC #tryptamine #serotonin #tryptophan #AMT #DMT #DPT #DIPT

Project Proposal and Solid Takeaways

The Scientists goal was to utilize readily available chemicals, for a college level lab or average citizen scientist, as well as low-tech a procedure as possible to analyze the presence of trace tryptamines. Since TLC is a relatively inexpensive test that can be done with no high-end equipment (see in-depth description below) it was the perfect option. While most tryptamines are not readily visible to the naked eye; through chemical and heat changes to their structure it is possible to increase visibility under the right circumstances. Thus the group propose that it is possible to develop a TLC method capable of fluorescence detection for not only Trp, 5-HT, but also numerous PATs including N,N-Dimethyltryptamine, aka DMT (Tbl 1).

The depth of their tests was quite deep and expansive, however the data presented here-in is paired down to the method that, I presume, is most readily accessible to the average citizen scientist or undergraduate science student. The focus on presenting those procedures and compounds that are as chemically similar to those purchasable in local stores, and supplemented by minor ones from places like Amazon, allow for the possibility of home testing methods to be developed. The items that would not be readily available would be the TLC plates and sample applicators. The Scientists chose to utilize the TLC plats plated with different blends of silica gel to have compounds adhere to the surface, easily purchasable from internet searches. Sample application could come in many forms, from the, likely, use of pipettes to the application by capillary tube; also readily purchasable online.

Shulgin's Legacy: The Expanding Tryptamine Library

One of the more interesting aspects of this research was the inclusion of more exotic tryptamines like DPT and 4-AcO-DIPT. These compounds are typically referred to as "TiHKAL class compounds" due to their most documented, and reliable, source of information coming from a book of underground chemist's 'bible': Tryptamines I Have Known and Loved, which is abbreviated TiHKAL, and in my experience pronounced 'teek-al'. This text is typically paired with its sister PiHKAL book (Phenethylamines...,'peek-al') both masterworks written and compiled by the late Alexander 'Sasha' Shulgin and his wife Anne Shulgin. After earning his PhD in biochemistry, and through decades of work within the industry, Shulgin was able to develop an amazing organic chemistry synthetic skill set. He then plied these skills on entheogens after an experience with mescaline: "I understood that our entire universe is contained in the mind and the spirit. We may choose not to find access to it, we may even deny its existence, but it is indeed there inside us, and there are chemicals that can catalyze its availability." [1] Buttressed by his experience and fueled by his passion, with these works Shulgin not only explores the chemical process of creating these compounds but also the experience of taking them at specified doses. It is from this entheogenic library, that the compounds like DPT and 4-AcO-DIPT have both their synthesis and the subjective experiences of Shulgin described in detail. The compounds themselves are so fundamentally similar to serotonin and other biological PATs that their neurochemical effect was theorized to be same or similar and was the impetus for his pursuit in their synthesis.

History and Theory behind Thin-Layer Chromatography

The purpose and concept of TLC has been well established since the early 20th century. First used in the late 1930's by two Russian scientists, N.A. Izmailov and M.S. Schreiber; they dried a layer of adsorption slurry media two millimeter thick and placed plant ethanol extracts it at the center of and observed compound separation as the ethanol wicked through the surface and evaporated having some color compounds 'travel' out from the center ring at different rates. The method was later refined and published for analytical use in the 1940's by J. E. Meinhard and N. F. Hall. [2] While the method is old, it has amazing variability in applications depending on three major components: Stationary Phase, Mobile Phase, and Post-Run Treatments (Fig 1). A TLC plate is chosen for its properties as a Stationary phase, samples are added a few centimeters from one side and called the 'bottom' of the plate in individual, parallel lanes. Before the TLC is placed into the chamber the solvent wick is used to ensure equal distribution of the gaseous solvent in the space. It is important that the solvent liquid layer NEVER touches the spotted sample, as it would cause the sample to leak into the solvent ruining the plate. Once the chamber is ready, the plate is placed in and the chamber sealed until the solvent moves about ¾ of the way up the TLC plate, this is called the solvent front. The plate is removed and solvent front marked, then exposed to a heat/drying step; sometimes an added resolving spray and UV light exposure are performed to better visualize compounds of interest. Samples are separated based on their chemical interactions with the stationary and mobile phases. Compounds that adhere strongly to the stationary phase are likely not really soluble in the mobile phase and thus do not move very far up from the origin spot; while those compounds that readily dissolve in the mobile phase are less likely to adhere to the stationary phase and move ‘faster’ away from the origin spot. Compounds can be identified from extracts when compared to standards, as compounds that share the same chemistry are 'moved' at the same 'speed'; thus, similar compounds would be detectable based on their distanced traveled relative to the distance of the solvent front, this is called the compound's retention time or retention factors (Rf). So if you had a standard of DMT and compared it to an extract of Psychotria viridis bark, the DMT from the plant extract would have extremely similar Rf values.

Over the following decades, new adherence surfaces were developed and produced, as well as new forms for compound separation. As previously mentioned, the Scientists choose to limit their investigation to those components that should be readily available to any chemistry or biology college undergraduate student or citizen scientist. The most applicable of their choices to the average person centered around methanol and ammonia as the Mobile Phase, used a silica gel TLC plate as the Stationary Phase, and as Post-Run Treatments heated their plates and used a basic (as in low pH) bleach Resolving Spray to make all the tryptamines tested visible with 365nm UV light exposure (Fig 2A). I see these steps as the basis for the utilization of cheaper and relatively safer chemicals that would produce a method for testing that could be easily taught for home cultivators. If you get this far in the article you get to hear about the fact that a project just like this is in the works with the San Francisco Psychedelic Society, so check out their courses and look out for this in the coming year!

Conclusion and Caveats

The methods tested for analysis proved to be quite effective for isolations of the compounds of interest (Fig 2A), but were they robust enough to handle unique samples? How about human urine? Well, the researchers found the answer to be that the method handled human urine quite well (Fig 3)! The most important fact to note is the addition of the compound 5-Methoxy-N-isopropyltryptamine (5-MeO-NIPT) a presumable human metabolite of 5-MeO-MIPT and 5-MeO-DIPT. Thus the work of the Scientists was able to produce a robust method that is applicable to a, hopefully, wide variety of sample types, including crude and refined extracts of flora, funga, and fauna.

The most important caveat to note is the paring down of data again. A lot of the other methods and procedures are extremely similar to the ones described here, however, the slight differences produce data, while valid, does not perform as well as the options in the figures presented. Another important caveat is the lack of color images for this paper, as it would be very interesting to note any visible color differences in compounds like 5-MeO-DIPT and DIPT as their Rf values are quite similar in every experiment performed by the Scientists.

Proposal Success or Failure?

It is possible to develop a rapid, sensitive and selective TLC method capable of fluorescence detection for Trp, 5-HT, and numerous PATs.

Reviewing the data and the conclusions put forth, the hypothesis proposed by the Scientists was supported. The reason for acceptance are quoted below (emphasis and parentheticals mine):

"The proposed method [Stationary Phase: Silica Gel TLC plate; Mobile Phase: Methanol–28% Ammonia (100:1.5, v/v); Resolving Spray: 0.25% NaClO in 0.1 M NaOH] separated Trp, 5- HT and eight kinds of PATs and detected them fluorogenically [under 365nm UV light]. Other indole alkyl amines were also detected by this method."


1) Bennett, Drake (2005-01-30). "Dr. Ecstasy". New York Times Magazine. New York Times. Archived from the original on November 17, 2011. Retrieved 2006-07-08.

2) Millipore Sigma. 2020. History Of Thin-Layer Chromatography. [online] Available at: <,nav> [Accessed 29 November 2020].

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