Thin-Layer Chromatography Process


The TLC Working Principle

What causes a dissolved mixture to separate when applied to a TLC plate? The thin-layer chromatography process relies on capillary forces. During development of the chromatogram, the mixture of substances is first transported by the mobile phase, then resides on the stationary phase for a while, and is carried along again. The process repeats many times, and each substance is slowed down at different rates relative to the velocity of the mobile phase. The more a substance preferentially resides on the stationary phase, the slower its progress will be. Even substances with similar affinities for the two phases demonstrate differences in their chromatographic run, and can be separated. Of course, this is a simplified explanation of the TLC working principle. Continue reading to discover further details and practical advice about the TLC process.

Explore the TLC Process

The diagram below illustrates the complete thin-layer chromatography process. Click on the TLC process steps to learn more.

Sample Preparation

Thin-Layer Chromatography plates are economical, single-use products, so sample preparation is simpler or can be avoided completely and is by this more economical than for HPLC. Still, in some cases, such as trace analysis or for complex matrices, samples will require cleaning and/or enrichment prior to application on plates.

Pre-Rinsing the Layer

Glass plates are usually coated with surface-active sorbents, which absorb moisture and “dirt” from air. These and any soluble binder components should be removed by pre-rinsing the layer. This is performed either by dipping (once or twice, 1-7 minutes) or by blank chromatography of the TLC plate. Dipping results in a uniform layer, but cleaning is not as thorough. Blank chromatography takes longer, but is more effective because “dirt” is concentrated at the top edge of the plate.

Choice of Stationary and Mobile Phases in TLC Process

Stationary Phase

The TLC stationary phase consists of a TLC plate coated with a layer of sorbent material. TLC layers can be prepared in the lab, but they are normally purchased as pre-coated plates. There is a diverse range of options available – from silica, aluminum oxide or cellulose, to modified or mixed-layer plates. The choice should be based on the properties of your sample and your application goals.

Learn more how to choose the right stationary phase in TLC

The backing of TLC plates is usually made of glass, aluminum, or plastic, and each has its specific characteristics. Glass plates are chemically inert and resistant to reactive stains and heat, but fragile and difficult to cut. Aluminum and plastic plates can be cut with scissors, but aluminum may not tolerate strongly acidic or oxidizing stains, and plastic will not survive the high temperatures required to develop many stains. Also, the flexibility of aluminum and plastic plates may result in flaking of the sorbent layer.

TLC Mobile Phase (Solvent System)

The solvent dissolves the sample components from the sorbent layer and transports them across the plate. The more strongly the solvent is adsorbed by the sorbent, the greater will be its eluting strength. Sample components that have a greater affinity for the solvent than the sorbent will be eluted closer to the front.

Roles of solvent system in TLC Mobile Phase:

  • Dissolve the mixture of substances and transport them across the sorbent layer
  • Produce hRf values as close to the medium range as possible
  • Provide adequate selectivity for the substance mixture to be separated

Requirements of solvent system:

  • Adequate purity
  • Adequate stability
  • Low viscosity
  • Linear partition isotherm
  • Moderate vapor pressure
  • Lowest toxicity possible

Learn more about how to choose the right solvent system

Pre-Conditioning the Layer

Unless special precautions are taken during sample application, humidity in the laboratory can diminish the activity of the TLC layer within minutes, as equilibrium is reached between the lab atmosphere and the sorbent. Pre-conditioning the TLC plate helps to avoid its deterioration.

TIP: How to pre-condition TLC plates

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Sample Application

Samples can be applied as spots or bands. In general, broadening of spots in the direction of development is less common with band application than with point application. Solvent polarity is another factor to consider during sample application. When n-hexane is used, substances remain at the application point. However, with more polar solvents (toluene, dichloromethane, methanol), the substances are transported toward the edge of the “wet zone”, and typically form a circular chromatogram. After chromatographic development, peaks with an almost Gaussian distribution are achieved whose width increases with increasing polarity of the solvent (Figure 1. Substance distribution in TLC as a function of the solvent).

Substance distribution in TLC as a function of the solvent

Manual Application

Based on the number of samples being analyzed, select a suitable plate size or cut a larger plate to the needed dimensions. Using a pencil, mark the desired positions of the front and of the application zone by drawing lines across the plate. Other important information, such as the solution number and concentration, should also be written beneath the application zone. Take care not to damage the layer surface as this can lead to errors.

You may now place the starting point. Be careful not to apply the sample too close to the bottom edge of the plate, as this can cause the starting point to spread into the solvent. The sample volume depends on the aim of the analysis and the concentration of the sample solutions. A sample volume of 0.5-2.0 µl is recommended for identity tests, and a maximum of 10 µl for purity testing.

Semi-Automatic Application

In semi-automatic application, special equipment is used to spray sample solutions onto the plate. This method avoids direct contact with the TLC layer, and is typically used for band application.

Fully Automatic Application

In fully automatic application, a steel capillary is connected to a dosing syringe, which is controlled by a stepping motor. Samples are applied from the steel capillary as bands or spots

TIP: How to position samples?

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Chromatogram Development

In the development phase of TLC, the solvent system penetrates the layer (due to capillary forces and sometimes on application of pressure), and transports the sample along the layer. The interactions between the substances, solvent system and TLC layer cause the substances to separate into individual components.

Drying Plates Prior to Development

The solvent used in sample application must be completely removed by drying the plates prior to development. The properties of the applied samples and the solvent system are important considerations when choosing a drying method.

TIP: Avoid errors when drying TLC plates

Development Methods

Development of the TLC plates can be achieved through various techniques, as listed below.

  1. One-dimensional development
    1. Single development
      • Vertical
      • Horizontal, in one direction
      • Horizontal, in opposite directions
      • Circular
      • Anti-circular
    2. Multiple development
      • Separate runs over the same migration distance
      • Stepwise, increasing
      • Stepwise, decreasing
      • Automated multiple development, stepwise with solvent gradient
  2. Two-dimensional development
    1. Two dimensions, one solvent system
    2. Two dimensions, two solvent systems
    3. SRS (separation in 1st dimension  chemical reaction  separation in 2nd dimension)
  3. Development with forced flow
    1. OHLC: overpressure layer chromatography
    2. HPPLC: high-pressure planar liquid chromatography
    3. RPC: rotation planar chromatography

Learn more about development methods

Development Chambers

TLC development is usually performed in chambers. Depending on the application and goals, there are various chambers available, differing in materials and chromatographic results. For instance, chambers for ascending chromatography are made of glass and have glass or stainless steel lids, while those for horizontal TLC are made of PTFE and have glass lids.
 
Learn more about development chambers

TIP: How to saturate development chambers?

Derivatization

When separated compounds are colorless, do not respond to UV radiation, and do not fluoresce, derivatization is used to enhance detection. A detection reagent is applied to the plate (by spraying or dipping) to enable chemical visualization. The detection reagent may be used with the solvent system (in-situ derivatization), before development (pre-chromatographic derivatization) or after development (post-chromatographic derivatization).

In-situ derivatization of applied samples:

  • The detection reagent is incorporated into the solvent system
  • Better distribution of reagent

Pre-chromatographic derivatization:

  • To enhance the selectivity of the solvent for substances of interest
  • For transforming labile compounds into stable ones

Post-chromatographic derivatization:

  • For visualizing the separated substances
  • To enhance sensitivity

Learn more about TLC derivatization methods

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Evaluation

When the solvent residues have been removed from the TLC plate, the chromatogram is ready for qualitative and quantitative evaluation. Depending on your experiment goals, there are a variety of evaluation methods to choose from.

Learn more about evaluation with and without derivatization

TIP: Quantitative evaluation with TLC scanners



Documentation of Results

Careful documentation of chromatograms ensures that TLC results are available for future reference.

Documentation by Description

Since pharmacopoeias do not require hRf values, written descriptions of TLC results are permissible and often used. However, this method is highly reliant on the skills of the technician and can lack accuracy.

Documentation by Drawing, Tracing and Photocopying

A common yet time-consuming approach is to draw or trace thin-layer chromatograms on paper, and color in the zones. Photocopying is faster, but colors may be difficult to match. Despite their inaccuracy and inefficiency, these methods are accepted for official purposes.

Photographic and Video Documentation

The most accurate methods for documenting TLC results are photography and video recordings. Here, it is important to secure the camera to a stand, and align it correctly with the TLC plate to ensure sharp images. Although these methods require somewhat more skill, documentation is fast, highly reliable, and can be stored electronically

Recommended TLC Process Tutorials

Fundamentals of the TLC Method

Paper and Thin-Layer Chromatography

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