Thin layer chromatography (TLC) sounds like a relatively simple process. However, in practice, separating the different components can be surprisingly tricky. For more information, read the application note: TLC Troubleshooting with SiliCycle's SiliaPlate.

Whether you're trying to detect the presence of a certain compound or you're evaluating compound purity, challenges can occur with the solvent, sample, stain, and/or plate. This troubleshooting guide lists four of the most common pitfalls of thin layer chromatography, as well as reasons why they may be happening and solutions on how to fix the problem.

Once corrected and the sample has been successfully separated, you can then use a visualization method to identify the presence of compounds. To help, this guide also includes a table to assist you in deciding which stain to use. With this information, you'll have the background you need for a successful TLC experiment.

4 Common Problems & Solutions for Thin Layer Chromatography Samples

  • Problem 1: Your sample is streaking or elongated.

    Solution:

    • Your sample may be overloaded. Run the separation again with a more diluted sample solution.
    • For base-sensitive compounds, try adding acetic or formic acid to the mobile phase (0.1–2.0%).
    • For acid-sensitive compounds, try adding triethylamine to the mobile phase (0.1–2.0%) — or 1–10% ammonia in methanol (MeOH)/dichloromethane (DCM).
    • For highly polar compounds, try using a specialized silica TLC plate such as C18 for reversed phase.

  • Problem 2: The spots on your TLC plate are not visible.

    Solution:

    • Even if your plate contains a fluorescent material your compound may not be UV-sensitive, so try another method – such as the staining methods described further below.
    • Your sample may be too diluted. Concentrate your sample spot by spotting several times in the same location,being sure to let dry between applications.
    • Make sure that the solvent level in the tank is lower than the spotting line to avoid sample dissolution.
    • Another reason may be that the compounds are volatile and may have evaporated from the plate. In this case, visualizing the product by TLC will be difficult.

  • Problem 3: You have spots with the same Rf values in your samples.

    Co-spotting procedure to identify compounds with similar Rf values.

    Solution:

    • Try “co-spotting”. Apply the starting material (standard) and the sample in the same spot and compare the Rf values of the resulting separation.
    • If necessary, change your solvent system. Three classes of solvent mixtures provide significantly different results:
      • Polar/hydrocarbon (e.g., ethyl acetate (EtOAc)/hexane or heptane, ethyl ether/petroleum ether).
      • Polar/dichloromethane (e.g., polar solvents: ether, EtOAc, MeOH)
      • Polar/benzene (or toluene) (e.g., polar solvents: ether, EtOAc, MeOH)

  • Problem 4: Your compounds are too close to the baseline or solvent front.

    Solution:

    • If your compounds are too close to the baseline, your eluent is not polar enough. Increase the proportion of polar solvent or choose a more polar solvent.
    • On the flip side, if your compounds are too close to the solvent front, your eluent is too polar. Decrease the proportion of polar solvent or choose a less polar solvent.
    • In some cases, the only solution may be to change the stationary phase (e.g., use a reversed-phase).

Which Stain Should You Use to Visualize the TLC Spots?

Even after troubleshooting your thin layer chromatography process and successfully separating the compounds, you may face some new obstacles. Most of the separated chemicals will be colorless, meaning you'll need to use a stain or another visualization method to observe them.

The visualization method you choose can be non-destructive (the process does not change the compound) or destructive (the compound is changed by the process). The use of ultraviolet light to visualize the results, for example, is considered non-destructive, while the use of reactive compounds such as chemical stains is destructive, meaning the plate cannot be re-used with another stain. It is therefore critical to choose the most appropriate stain or visualization method. The following table outlines some of the common stains and methods, the compounds they work best on, recipes/instructions to make the stains, in addition to other helpful information you'll need to know to identify compounds.

StainWorks best on...RecipeNotes
UV lightHighly conjugated compounds and aromatic ringsUV light and a pencil to trace the light spots while they are still visible.UV light does not usually change the sample, so other tests can be performed on the same sample afterward.
Iodine vaporOrganic compounds, especially unsaturated and aromatic compoundsCreate an enclosed chamber with some iodine crystals inside. Let the iodine equilibrate and fill the chamber with iodine gas. The entire plate should turn light brown.Thiols and phosphines will immediately show up as white spots, while compounds with an affinity for iodine will appear dark brown. Iodine stains do not last long, so take photos or trace with a pencil to record the spots.
AnisaldehydeMost functional groups, particularly ones that are very strongly or weakly nucleophilic. Great for aldehydes, carbohydrates, and phenols.15 g anisaldehyde + 250 g ethanol + 2.5 mL concentrated sulfuric acidThis stain usually does not work on alkenes, alkynes, or aromatic compounds. The heating process with anisaldehyde may permanently stain your plate.
PermanganateOxidizable functional groups and unsaturated compounds, such as alkenes, alkynes, alcohols, and amines.3 g KMnO4 + 10 g K2CO3 + 300 mL waterSpots will appear bright yellow on a pink or purple background, especially alkenes and alkynes. The plate may require some heating to see sulfides, alcohols, and amines.
Phosphomolybdic acid (PMA)Most functional groups; a great universal stain.10 g phosphomolybdic acid + 100 mL absolute ethanolPMA requires strong heat to work. It works well on most functional groups. Spots show up as shades of green.
NinhydrinAmino acids and primary amines1.5 g ninhydrin + 100 mL n-butanol or acetone + 3 mL acetic acidDip the plate into the stain, let the acetone evaporate, and gently warm the plate. Be careful to not overheat. Secondary amines may also show on the plate as faint yellow spots.
VanillinMost steroids, higher alcohols, and phenols15 g vanillin + 250 mL ethanol + 2.5 mL sulfuric acidVanillin gives a wide range of colors, making it a good general stain.

*Note it could sometimes be appropriate to visualize the TLC plate using an ultraviolet lamp, prior to the treatment of the TLC plate with a destructive method if the sample contains UV-active compounds.*

Selecting the correct stain is important for your experiment because the stain influences the visibility of the results. Once the spots are visible, you can calculate their Rf value, aka the retention factor. To calculate your Rf values, divide the distance the sample traveled by the distance the solvent front traveled, using the initial spotting site as a reference.

Retention factor (Rf) = distance traveled by the compound
distance traveled by the solvent front

Keep in mind that these Rf values can be affected by the solvent you used and the type of TLC plate.

Creating a Successful TLC Experiment

For your thin layer chromatography experiment to yield valid results, it's essential to correctly troubleshoot your TLC experiment and select the most useful stain. Most of the common pitfalls of thin layer chromatography are easily solved by changing the amount of sample you are adding, by trying another stain or by changing the eluent proportions.

The correct visualization method, whether it be UV light or a stain, will quickly give you information about your sample. While it's a lot of work, these steps will create a successful TLC experiment that reveals the presence of compounds in your sample. If you would like to explore more Tips for TLC Analysis, read Thin Layer Chromatography Plates: Use and Best Practices.

Learn More about TLC

Want to learn more about thin layer chromatography strategies and tips? Check out the following TLC resources.

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