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A Brief Introduction to Thin Layer Chromatography from an Amateur Scientist

Let’s dive into the basics of thin-layer chromatography and how this useful tool can make meaningful connections to many other areas of science.

I was hanging around some mycology forums one day and a fellow diyBio scientist suggested that I look into learning something called thin-layer chromatography. The idea came as a response to my question to the community: how does one quantify the amount of a particular compound in a mushroom?

This question has been on my mind for some time, especially as the popularity and the science behind the medicinal benefits of consuming mushrooms becomes more mainstream. It’s been my experience that the majority of amateur mushroom growers have no means to detect and quantify the presence of these medicinal compounds. As a result, mushroom growers which are interested in creating unique strains are restricted to using physical phenotypes alone in their selective breeding process.

Imagine if one could detect the amount of cordycepin or terpines in two separate strains. That information could then be used to select for the strains which express the highest yields of these compounds and immediately give themselves a competitive advantage over other breeders focused on gourmet and medicinal mushrooms.

So I started to look into thin-layer chromatography (TLC) in a quest to explore if it would be possible for me to develop this technique to benefit the amateur mycology community.

What’s in a Name?

Let’s look at the word chromatography. Chroma means color and –ography is the study of something. From this, we can infer that TLC has something to do with the study of color…and that isn’t far off.

The general idea behind chromatography is to take a substance, separate out the compounds using a solvent and then interpret the color-based result in order to determine what compounds are present in the analyte (the substance being analyzed).

Here, let’s take a look a little drawing I made of the components.

Thin-Layer Chromatography Drawing
Thin-Layer Chromatography Drawing

The components of TLC are:

  • developing chamber: a glass jar with a lid to hold the solvent and stationary phase. I found using an empty seasoning bottle or a glass salsa jar worked just fine. Preferably, the jar has a flat bottom.
  • mobile phase: this is a solvent like acetone or methylene chloride, both of which can be obtained off Amazon or eBay for minimal cost. The mobile phase is what is drawn up the stationary phase, separating out the analyte on its way up.
  • origin: the origin indicates where on stationary phase the analyte was deposited. It’s used in the calculation for the Rf factor and so usually is drawn with a carbon-based pencil onto the TLC strip a couple of cm from the bottom.
  • stationary phase: this component is typically known as a TLC plate, a silica dioxide coated strip of glass or clear plastic. The solvent moves up this plate via capillary action.
  • solvent front: this is where the solvent stopped at the end of the procedure. It needs to be marked soon after the plate is removed from the chamber as its used in the Rf calculation and will eventually disappear as the solvent evaporates completely from the plate.

Here’s a video explainer of the process with a demo.

As mentioned in the video, the less polar a compound is, the higher the compound will travel up the plate and the more polar compound is, the lower on the plate will stay.

Here is what my very first setup looked like and the results under a UV-A lamp.

TLC Setup
First TLC Setup
Thin-Layer Chromatography with Paper, Acetone and Highlighters
Thin-Layer Chromatography with Paper, Acetone and Highlighters

On the left, we see the results of TLC run using a couple of dabs of yellow highlighter. Note how the acetone was drawn up by the chromatography paper, separating out the highlighter dye as it went up the paper, revealing the blueish hue seen near the solvent front.

On the right, we see several trials using various highlighters, including the yellow one on the left.

Note that I used a UV-A light. UV lamps come in various wavelengths and important to note that only some compounds fluoresce and those that do may only fluoresce under specific wavelengths.

Chart Showing The Different Wavelength Bands of UV Light

And as I learned, some compounds require that you spray the plate afterwards with an additional reagent in order to induce fluorescence. This is the case with psilocybin, for example, which requires Ehrlich’s reagent to properly develop the plate in order to make it fluoresce. And in the case of psilocybin, a short wave uv (UV-C) light is required.

When you do your own TLC experiments, you will be faced with a few questions that I’m still trying to work through myself. These questions include:

  • Which solvent or solvent mixture should I use for my mobile phase?
  • Is there an additional development step required to make the compounds visible?

Obtaining the answers to these questions is still a challenge. A biologist friend of mine told me most folks can find the right solvents to use but must go through a series of trials and errors to narrow-in on the right parameters that best optimize the experiments for compound visibility.

How to find TLC methods and reagents for your subject compound

Here’s my current approach to finding out what solvents, development steps and techniques are used for a compound I wish to analyze:

  1. Head over to scholar.google.com
  2. Search for: your_compound thin layer chromatography (ex: cordycepin thin layer chromatography)
  3. Look for a research paper and read through it.

That’s pretty much of it. The only caveat here is that you might only find a paper that was written over 30 years ago and uses an antiquated procedure or reagent that is impossible for the average person to obtain.

For example, I was unable to develop my psilocybin TLC plate correctly because I am currently unable to obtain p-dimethylaminobenzaldehyde (DMAB).

TLC Setup Outside
My TLC Setup Performed Outside

How I setup TLC for Mushroom and Common Analgesic Extract Experiment

Note: Although my test results were inconclusive due to not having the proper developing reagents, the analyte preparation and testing procedures don’t change much.

Here was my general procedure:

  • Use a mortar and pestle to grind ibuprofen, aspirin and 1.5g dried mushroom fruit body
  • Mix each analyte with 2ml acetone in individual air-tight containers and leave for 24 hours
  • Pipette 1mL of each into eppendorf tubes and centrifuge at 10K rpm for 1 hour
  • Measure and mark the point of origin on the test plates
  • Use seperate capillary tubes to drop some of the supertanant onto each of the test plates (real silica plates I cut myself), being sure to mark the top of each plate with the name of the analyte
  • Develop the plate using methylene chloride in a glass developing chamber
  • Once the solvent front has reached a few cm from the top of the plate, remove from the chamber and mark the location of the solvent front
  • Let the plate dry and check plate under shortwave UV lamp
EverymanBio Josh TLC
Pipetting Extracts for Placement on TLC Plates

What is Rf factor and how to calculate it?

Rf means retardation factor. It is the ratio of how far the compound traveled up the plate over the total migration distance of the solvent front.

Most compounds have a published Rf factor which can be used as a reference to determine whether the spot on your plate is indeed the compound in question.

But in a real-world scenario, a scientist would be running TLC alongside a control containing a pure sample of the compound being analyzed. So if the control had an Rf factor of 0.4, you can be reasonably sure that the compound you are looking for will have the same Rf value all things being equal.

Closing Remarks on Thin-Layer Chromatography

Most folks I’ve spoken to who have taken an organic chemistry lab at a college or university speak fondly of their experience in using TLC. It’s really fun to run an experiment where you can watch a physical observable result happen in near real-time.

Aside from the fun, it’s also an incredible useful technique for identifying the presence or absence of a compound. Learning TLC has opened up some new connections to other organic chemistry concepts like polarity, solubility, solvents and qualitative experimentation in a way that I would not have learned had I been presented the information in abstract as is often the case in formal education systems.

Although my final tests (shown in the big green image above) did not yield the clear definitive results I was hoping for, I am confident that once I obtain the correct arrangement of solvents and developing reagents, I’ll be a TLC pro in no time.

So there you have it. There’s TLC from an amateur scientist who went from never having heard of it to running experiments in just two weeks time. Give it a shot – it’s really a lot of fun.

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Quick procedural test of the fungal chromatography kit I’m developing to help mushroom farmers improve production of medicinal compounds in their proprietary strains. Different compounds travel up and settle on the plate at various heights and colors based on their respective polarity and solubility. A cheap and easy TLC kit will give growers a competitive advantage over breeders who do selective breeding using physical phenotypes alone. #chromatography #thinlayerchromatography #labiotech #diybio #scienceexperiments #diyscience #mycology #mushroomgrowers #medicine #biotechnology #startupcompany #startup #lifescience #biohacking #amateurchemist #organicchemistry #selectivebreeding #phenotypes #geneticengineering

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