Thin Layer Chromatography Plates: Use and Best Practices

We usually think of chromatography in terms of columns or capillaries, but separations can also be carried out on a planar surface by means of thin-layer chromatography (TLC).

Advantages of TLC include:

  • Cost;
  • Simplicity;
  • Sensitivity;
  • Sample throughput;
  • Sample volume.

TLC is widely employed in reaction monitoring, screening, sample purification, and compound purity evaluation. It is also used to select and optimize (or predict) chromatographic conditions prior to flash chromatography or HPLC and can be performed on both analytical or preparative scales. Recent advances have seen TLC's speed and ease of use matched with advanced instrumentation capable of identifying samples, such as Fourier transform infrared (FTIR) and mass spectrometry (MS).

Like column chromatography, TLC is a solid–liquid partitioning technique. Spots of sample are applied near the base of the plate, which is typically glass, aluminum, or plastic often coated with silica gel or alumina. The moving liquid phase (mobile phase) traverses the plate (stationary phase) via capillary action. The different affinities of the sample components for the mobile and stationary phase cause them to migrate at different rates across the plate, resulting in their separation.

In normal phase thin layer chromatography, a compound is adsorbed onto the stationary phase (e.g., silica), which is more polar than the mobile phase. During elution, the compound is then solvated and captured. In reversed-phase TLC, the stationary phase is non-polar, and the polar mobile phase contains the compound(s) of interest which are retained and selectively eluted.


SiliCycle has been offering TLC plates for more than 20 years in a wide variety of sizes (plate size, thickness, backings) and chemistries (10-15-20 % silver nitrate, CN, C18, NH2 and so on). Our expertise in silica gel technology led to the development of our SiliaPlate line, which demonstrates high separation efficiency and low background noise due to the narrow particle size distribution of the silica gel and the extraordinary silica layer hardness combined with homogenous coating and layer thickness. SiliCycle TLC plate production undergoes strict quality control measures to ensure consistent lot-to-lot and layer-to-layer reproducibility.

Tips for TLC Analysis

Although thin-layer chromatography is a relatively simple analytical technique, a few tips will help ensure the success of your analyses.

  • TLC TIP #1: Stationary Phase/Plate Selection

    Most separations (> 80 %) can be performed on silica gel plates, but for acid-sensitive compounds, alumina is likely to be a better choice. For standard TLC, particle sizes of 10 – 14 µm are used, whereas smaller particle sizes are used for HPTLC. A pore diameter of 60 Å is used for both. TLC is usually run in normal- or reversed-phase modes.

    In normal phase separations, the mobile phase is less polar than the stationary phase, whereas in reversed-phase TLC, the mobile phase (usually water and organic solvent) is more polar than the stationary phase (if a satisfactory separation cannot be achieved using unmodified silica, other functionalized matrices are available from SiliCycle for specific applications).

    Types of SiliaPlate backings

    • Rigid
    • High chemical resistance
    • High heating stability and charring resistance
    • Transparent
    • Thin
    • Low weight & consequent shipping costs
    • High heating stability
    • Low fragility
    • Possible to cut with scissors
    • Can be stored in notebook
    • Thin
    • Low fragility
    • Possible to cut with scissors
    • High chemical resistance
    • Can be stored in notebook
    • Thick
    • High fragility
    • Impossible to cut with scissors
    • Cannot be stored in lab notebook
    • High weight & consequent shipping costs
    • Large shelf space
    • Low chemical resistance
    • Opaque
    • Medium weight
    • Opaque
    • Heating stability up to 175°C
    • Possible cracking of matrix due to high flexibility
    Thickness (approx.)2.0 - 2.5 mm1.5 - 2.0 mm1.5 - 2.0 mm
    Total WeightHighLowMedium
    Heating StabilityHighHighBelow 175°C
    Cutting with ScissorsImpossibleEasilyPossible
    Chemical ResistanceHighLowHigh


    Plates are available for classical TLC, high-performance TLC (HPTLC), and preparative TLC (PTLC).

    Differences between classical TLC, HPTLC, and PTLC

    PropertiesClassical TLCHPTLCPreparative (PLC)
    ApplicationsQuick, inexpensive, flexible and classical separationsHighly sophisticated separations, complex samplesPurification on a TLC plate
    AnalysisQualitativeQualitative & QuantitativeQuantitative
    DetectionUV - StainsInstrumented analysis (use of scanners for detection)UV
    Distribution [Mean Particle Size]5 - 20 µm [10 - 14 µm]4 - 8 µm [5 - 6 µm]5 - 40 µm [22 - 25 µm]
    Layer Thickness200 - 250 µm150 - 200 µm500 - 2,000 µm
    Typical Sample Volume1 - 5 µL0.1 - 0.5 µL5 - 20 µL

    Samples that absorb short-wave UV at 254 nm (F254) or long-wave UV (F366) light can be viewed under UV light as dark spots. You can use SiliaPlate TLC plates with F254 or F366 UV indicator, or none.

  • TLC TIP #2: Mobile Phase Selection

    The selection of your mobile phase, or solvent system, is critical for successful separations.

    In normal phase TLC, where non-polar solvents such as hexane or pentane are used, non-polar compounds will move up the plate while most polar compounds will stay at the baseline. Polar solvents allow polar compounds to move off the baseline. Look for a solvent system that moves all components off the baseline with retention factor (Rf) values between 0.15 and 0.85 (ideally, close to 0.2–0.4). Retention factor is defined as the distance traveled by the compound divided by the distance traveled by the solvent front.

    A common solvent system to start with is 1:1 ethyl acetate (EtOAc)/hexanes, where EtOAc is more polar than hexanes. Varying the ratio can have a significant effect on the Rf value depending on the polarity of the compounds to be separated. If this mobile phase isn't producing enough separation, you can try methanol (MeOH)/dichloromethane (DCM) (1:99–10:90) or toluene with acetone, EtOAc, or DCM.

    In normal phase TLC, to increase the compound's Rf, you'll want to increase the polarity of the mobile phase; increase the ratio of the polar solvent or choose another solvent. Inversely, to decrease Rf, you should decrease the polarity of the mobile phase.


    Standard compounds: 10%–50% EtOAc/hexane

    Polar compounds: 100% EtOAc or 5%–0% MeOH/DCM.

    Non-polar compounds: 5% EtOAc (or ether)/hexane or 100% hexane.

    For basic compounds (amine- or nitrogen-containing): It could be useful or required to add a small quantity of triethylamine (Et3N) to the solvent mixture (0.1%–2.0% but typically 0.1%) or 1%–10% ammonia (NH3) in MeOH/DCM.

    For acidic compounds: It could be useful to add acetic (AcOH) or formic acid (FA) to the solvent mixture (0.1%–2.0%).

    In reversed-phase TLC, typical solvent systems are mixtures of water or aqueous buffers and water-miscible organic solvents, such as acetonitrile (ACN), methanol, and tetrahydrofuran (THF). Other solvents can be used such as ethanol (EtOH) and isopropanol (IPA). If needed, to improve peak shape in flash chromatography, add 0.1% of acetic, formic, or trifluoroacetic acid (TFA) to the solvent system.

  • TLC TIP #3: Plate Preparation

    Use a pencil (not a pen! The ink will separate and migrate along with the analytes – definitely NOT something you want to happen. A pencil's graphite won't interact with solvents.) to draw a straight line parallel to the width of the plate about 1 cm from the base end of the plate. This is the baseline for sample application.

    For analytical analysis, apply a small quantity of sample using a glass capillary or micropipette for optimal resolution. For preparative analysis, apply a series of small adjacent spots to form a band using a glass capillary or microliter syringe. A spotting guide will facilitate sample application.

  • TLC TIP #4: Plate Development and Visualization

    TLC plates are commonly developed by placing them vertically inside a sealed developing chamber to ensure solvent saturation. Place approximately 0.5 cm of the solvent system inside the chamber. The solvent level needs to be below the baseline – otherwise, the spots will dissolve. Slowly place the TLC plate inside the chamber and allow the eluent to travel up the plate until it arrives at 1 cm from the top of the plate. Immediately remove the plate and draw a line along the solvent front. For optimal solvent saturation, filter paper can be added inside the TLC chamber. This also prevents eluent evaporation.

    Most organic compounds are colorless, so a visualization method is needed to reveal the spots. In a few cases, the components of the analysis are colored, so no visualization is needed.

    “Non-destructive visualization” methods include viewing UV-active compounds under an ultraviolet lamp (for polyconjugated compounds such as benzophenones and anthracenes). TLC plates typically have a fluorescent material (e.g. zinc sulfide) in the silica gel, which causes the spots to appear dark against a green plate. An iodine chamber can be used for thiols, phosphines, and alkenes and works in about 50 % of cases for alkanes. Prior to using destructive visualization methods, circle the spots with a pencil on the TLC plate.

    If compounds are not UV-active, “destructive visualization” methods utilizing various types of stains can be used depending on the functional groups of the compounds of interest. To use a stain, simply dip the TLC plate into the staining solution as quickly as possible and then immediately absorb the excess stain with paper. For some stains, heat carefully with a heat gun or on a hot plate until spots are revealed.

  • TLC TIP #5: Interpreting the Results

    Retention factor (Rf) is used to calculate the success of your separation or to compare different samples.

    Rf =  (distance traveled by the compound from the baseline)
    (distance from the baseline to the solvent front)

    The distance traveled by the compound is determined by its affinity (interaction) with the adsorbent on the plate. Each spot represents a compound. Solvent systems should move all components off the baseline with Rf values between 0.15 and 0.85 (ideal is 0.2 – 0.4). In the example below, Rf is calculated as 4.0 cm/5.5 cm for Rf = 0.73. If spots have the same Rf value – for example, when running a standard (known components) and a sample – they are likely the same compound.

    Calculating Rf value

    Calculating Rf value

Using TLC to Test Solvent Systems for Column Chromatography

Because TLC is simple and uses a minimal amount of solvent, it's useful for optimizing solvent conditions for column chromatography. Solvent systems can be evaluated on TLC plates, and when satisfactory conditions are identified to separate the components of interest, these conditions can be adapted for column chromatography.

The relationship between Rf values from TLC and column volume (CV) can be used to predict column elution. CV is the number of column volumes required to elute the component from the column regardless of column dimensions. The CV required to elute a compound isocratically (with a constant ratio for mixed solvents) in column chromatography using the same solvent system as your TLC separation equals 1/Rf.

The separation quality can be determined by subtracting the values for adjacent compounds (Rf1 and Rf2). ΔCV is a measure of the amount of separation between two components. A bigger ΔCV will allow more sample to be loaded onto the column.

CV = 1/Rf

ΔCV = 1/Rf1 – 1/Rf2

For More Information

Youtube: What is the sample mass loading capacity of preparative TLC plates?

Want to learn more? Check out these TLC resources: