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Introduction to TLC

Thin layer chromatography, or TLC, is a highly effective and budget friendly method of chemotaxonomy. Particularly for lichens, which vary significantly across species by their metabolite profiles, methods such as TLC are essential for effectively determining a lichen’s species, in the absence of genetic data. As such, TLC has become the most widely used method of chemotaxonomy in lichenology, with many lichen taxonomy resources often supplementing species descriptions with descriptive metabolite profiles. TLC is not the most effective method of compound determination, with mass spectrometry as a significantly more sensitive, but far more costly and technically prohibitive method, yet TLC remains in favor with many researchers due to its affordability and simplicity. The following information, consolidated from various resources in lichen taxonomy, provides a guideline for performing TLC on lichen samples and analyzing results.

Materials and Tools

TLC Solvents: Thin layer chromatography can be performed using multiple different solvents, which interact with dissolved metabolites in different ways. When assessing a lichen’s metabolite profile for taxonomic purposes, many lichenologist will utilize multiple different solvents (e.g. performing two rounds of TLC using solvents B and C). The recipe for the most common solvents in TLC are as follows:

Solvent system A: benzene:dioxane:acetic acid (90:25:4; 238 mL)

Solvent system B: hexane:ethyl ether:formic acid (5:4:1; 200 mL)

Solvent system C: toluene:acetic acid (85:15; 240 mL)

Chromatography plates: most chromatography or TLC plates are made of a thin sheet of aluminum or glass onto which a porous packing of silica is attached. Either of these materials work well for TLC, although aluminum plates are cheaper, and easier to cut apart for smaller batches of TLC.

Chromatography capillary tubes: Many chemists have access to capillary tubes that don’t need to be modified to effectively perform TLC. However, many lichenologists, lacking access to or knowledge of these tools, instead take open ended capillary tubes and heat their center over an open flame until it melts. At that point, the ends are pulled apart, and when the glass solidifies, a hard surface or a pair of forceps is used to break off a portion of the drawn glass to expose a more narrow point of the tube.

Methods

Step 1 - Prepare the plate: Using a pencil, draw a line near the edge of the plate (approximately 2 cm from the end of the plate) that runs parallel to the edge. Mark ticks along the line roughly 1-2 cm separated from one another (but equally spaced apart). These ticks mark the “spotting” location where dissolved metabolites will be added to the plate.

Step 2 - Extract the metabolites: take a small portion of the thallus (typically smaller than the size of your pinky fingernail, since only a little material is needed to sufficiently sample for metabolites) and place it in a vial, or on a dimpled surface such as a glass or ceramic dish. Add several drops of a solvent such as acetone, methanol or ethyl acetate to the lichen to dissolve any metabolites contained within.*

*note: metabolites may experience differential solubility with the chosen solvent (e.g. methanol is less effective at dissolving usnic acid compared to acetone). Many lichenologists utilize acetone broadly, but results of TLC for specific species may vary slightly by solvent choice.

Step 3 - Administer metabolites to the plate: Using a capillary tube (see materials), draw small quantities of the solvent from the dish or vial and lightly press the tip into the TLC plate at the tick that denotes the sample you are working with. The solvent will quickly bleed into the plate. Remove the capillary tube from the plate if the growing spot starts to encroach on neighboring spots. Allow the spot to dry completely. Repeat this process several times if necessary or until the solvent in the vial or dish is used up.

Step 4 - Prepare and load the solvent chamber: Take the solvent chamber and add a small quantity of the TLC solvent to the bottom. The volume of the solvent in the chamber should not exceed the height of the line you drew on the TLC plate. Place the TLC plate gently and evenly in the solvent chamber so that the end of the plate with the line drawn on it lies level with the bottom of the chamber. Seal the chamber with a lid and leave in a well-ventilated location, such as in a fume hood or outside. The solvent in the chamber will, through capillary action, climb up through the TLC plate, carrying with it dissolved metabolites. The metabolites move through the plate at varying rates depending on the molecule’s polarity.

Step 5 - Run TLC: Periodically monitor the TLC plate through the chamber. Remove the TLC plate from the chamber when the climbing solvent line reaches no higher than 2 cm from the top of the plate. Leave the plate to dry completely in a well-ventilated area.

Step 6 - TLC plate analysis: TLC plates can be assessed for their contents in several ways, and researchers often employ multiple methods together to fully assess their results:

Place the TLC plate in an ultraviolet lightbox and expose the plate to wavelengths of UV light (“short”, at 254 nm, or “long”, at 366 nm). View the plate under the UV light and through a UV eye protector and mark areas of UV fluorescence or absorbance with a pencil
For fatty acids: Run the TLC plate under a faucet until the plate is soaked. Dab to dry and then place on a hot plate to hasten the drying process. Fatty acids, which have hydrophobic properties, will create spots on the plate that will dry out faster than other areas. Mark these spots when you see them.
Place the TLC plate in a container and spray the plate with a 10% solution of sulfuric acid. Dab dry and then heat the plate with a hairdryer or on a hot plate. The sulfuric acid will react with many lichen metabolites, causing spots of color to form on the plate which can be useful for diagnosis.

Step 7 - Record results: After identifying metabolites, record the results of your plate (examples include Rf value which the formula is shown below) somewhere such as a laboratory notebook or online database. Individual TLC plates may become damaged or eventually unreadable over time, and so either drawing the results of the TLC or photographing the plate itself is essential to preserve the plate’s results for future analysis.

How to calculate Rf= Distance traveled by the compound/ Distance traveled by the solvent front (both measured from the origin)

Tips and Tricks

If you are inexperienced with spotting dissolved metabolites on chromatography paper or want to practice using an unfamiliar capillary tube, you can practice using acetone alone on paper towel (spots may appear larger on paper towel than TLC plate)
While spotting a TLC plate, the spot you add to the plate has to dry completely before you add another spot. You can hasten the drying process by lightly blowing on the plate between spotting, or by loading the TLC plate on a hot plate at the lowest setting. Some lichenologists go as far as to use an electric griddle for pancakes because it achieves a very low heat at its lowest setting - ideal for spotting while avoiding burns.
When labeling your TLC plate, be careful not to write too hard on it or use instruments that are particularly sharp, as the silica on the plate’s surface is easily scraped off. We recommend using a dull pointed pencil (non-mechanical) to do labeling.
Mark each tick with a number. Elsewhere, on a piece of paper or in an excel sheet, note which sample corresponds with which number on your plate, so you do not confuse samples on the plate. Similarly, it’s best to also give the plate itself an identifying number, since often researchers will work on many plates and plates can look quite similar, especially if the numbering schemes per plate are the same.
Marks on a TLC plate can be difficult to read at times, let alone make sense of, especially when you were not the person who made the marks. Decide on a system of notation for your plates (e.g. marking UV fluorescing spots at long wavelengths with an X, while circling spots that fluoresce at short wavelengths) and stick to it.
Do not touch the silica part of the plate with your fingers as it will damage the plate, if you’re worried about handling the plate, use a pair of tweezers to assist in transporting the plate.

From: Culberson, C. F., & Kristinsson, H.-D. (1970). A standardized method for the identification of lichen products. Journal of Chromatography A, 46, 85–93. doi:10.1016/s0021-9673(00)83967-9