Thin-Layer Chromatography (TLC)

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Thin-layer chromatography: one of the easiest, most used, low cost and a versatile chromatographic separation tool which finds several applications in the analytical world. This article is another meaningful addition in our article series discussing the different types of chromatography. So, continue reading and explore further the world of chromatography.

What is thin-layer chromatography

Thin-layer chromatography abbreviated as TLC is a chromatographic separation and analysis technique. It is a type of normal phase solid-liquid chromatography. As its name suggests, a thin layer of a solid adsorbent such as silica or alumina coated on aluminium foil, plastic or most preferably a glass surface acts as the stationary phase. Organic solvents of different polarities serve as mobile phase in thin-layer chromatography.

Working principle of TLC

Separation of analyte components in thin-layer chromatography occurs on the relative basis of adsorption onto the stationary phase versus their dissolution in the mobile phase solvents. Thin-layer chromatography thus can also be known as an adsorption chromatographic technique. Silica (SiO2) gel or alumina (Al2O3) provides a polar surface for the attachment of polar analytes from a sample mixture. Relatively less polar or non-polar components from the analyte mixture on the other hand travel up a longer distance with the mobile phase.

Image by istockphoto.com

Hydrogen bonding with OH functional groups of silica, dipole-dipole interaction and Vander Walls forces of attraction are the main interactive forces. These forces control the retention of the solute molecules onto the stationary phase in thin-layer chromatography.  Some acid/base attractions may also be involved for analyte retention in TLC as silica is slightly acidic while alumina is slightly basic in nature.

Stationary phase preparation in TLC

As mentioned earlier, the stationary phase in thin-layer chromatography is coated onto inert surfaces. The end product obtained from this coating is called a TLC plate which is a small slide of dimensions 5 cm x 15 cm at the maximum. The following steps show how a TLC plate can be developed in a laboratory prior to performing any thin-layer chromatographic experiment.

Step I: A slurry of stationary phase is first prepared in an aqueous medium. It is then spread onto the inert support with the help of a spreader to form a uniformly thin layer onto the glass or plastic plate.

Step II: A binder such as Plaster of Paris (CaSO4).2 H2O is then added.

Step III: The TLC plate prepared is then dried and activated by heating it at approx. 150°C in an oven to drive off the excess moisture and in order to get the active sites of the adsorbent free for solute attachment.

The TLC plate obtained by following the steps mentioned above should have a uniform white coating of the stationary phase. These plates can also be bought commercially from laboratory suppliers and look like that shown in the image below.

Image by Sorbtech.com.
You can read more about the specifications of a commercially prepared silica-based TLC plate here.

How to perform thin-layer chromatography

The process of performing an analytical separation with thin-layer chromatography (TLC) is quite similar to that of paper chromatography.

A brief description of analyte separation and analysis with TLC is as described below:

Step I: Prepare the stationary phase as described in the previous section or use a commercially bought TLC plate. Prepare the mobile phase by mixing different organic solvents in a specific ratio.

Step II: Draw a fine base line,1 cm from the lower end of the TLC plate. Place a sample spot onto the base line using a thin capillary tube. Allow sometime for the plate to dry.

Step III: Place the TLC plate into a closed chromatographic tank and immerse it into the mobile phase, just below the base line. An enclosed chromatographic chamber ensures a saturated atmosphere with minimum evaporation form the TLC plate so that no interruption exists in the chromatographic separation.

Step IV: As the mobile phase sweeps up, over the stationary phase, it will draw solute components with it via capillary action.

Result:  As the analyte mixture separates into its constituent components, distinct spots appear at differing positions on the TLC plate. The spots can either be compared with the spots obtained from pure compounds or their Rf values can be calculated in order to carry out their identification.

For Rf value calculation, we recommend: Paper chromatography.

Visualizing agents in thin-layer chromatography

In case of colorless compounds, similar visualizing agents can be used for TLC as those discussed for paper chromatography. These include ninhydrin spray for amino acids obtained from protein hydrolysis, Molisch and Tollens’s reagent for different types of sugars and/or iodide crystals etc. Other than that, ultraviolet (UV) irradiation occupies an integral spot in the analysis of organic compounds by thin-layer chromatography.

Chemical compounds containing aromatic rings or conjugated systems can absorb UV radiations (200-400) nm, transmitting complementary radiations to the ones absorbed thus giving off colour i.e., radiations falling in the visible range (400-800) nm of the electromagnetic spectrum. This concept can be utilized to perform thin-layer chromatography.

Silica or alumina used as a stationary phase in a TLC plate can be impregnated with fluorescent, insoluble compounds that absorb ultraviolet radiations. When placed under a UV lamp, the plate emits a bright light, illuminating even the dark/colourless sample spots otherwise difficult to see from the TLC plate.

Visualizing TLC development in the presence of UV irradiation 
Image by chemligin via chem.libretexts.org

Comparison of thin-layer chromatography with paper chromatography

SimilaritiesDifferences
Thin-layer and paper chromatography both fall under the category of normal phase chromatographyTLC is a solid-liquid chromatographic type while paper chromatography is liquid-liquid chromatography
The process of analyte separation is same in both types of chromatographyAdsorption mechanism is used for separating compounds in TLC while paper chromatography is based on partitioning of solute components
Component identification is done via the calculation of retention factor (Rf) in bothUV irradiation forms a greater part of TLC analyses rather than analysis by paper chromatography
Similar visualizing agents can be used in both casesTLC is more reproducible and offers efficient separations, owing to the small particle size and large surface area of the stationary phase used in TLC as opposed to that used in paper chromatography
Both types of chromatography find similar applications/uses in the chemistry lab and the industryPaper chromatography is a purely qualitative analytical separation technique while TLC can be used for both qualitative and quantitative analysis. The sample spot can be extracted from the TLC plate with a pair of forceps and dissolved in an appropriate solvent for quantification

You can read a detailed study on quantitative analysis by TLC from this source.

Uses of thin-layer chromatography

  • TLC is a popular choice for organic and biochemical analysis, it is usually considered a preparatory protocol in sample preparations.
  • TLC is important in the chemical industry for investigating the progress of a chemical reaction by studying the intermediate components formed.
  • TLC can be reliably applied for a diversity of pharmaceutical, environmental, cosmetic and/or other industrial analysis.

Read more on the uses of chromatography on our special article dedicated for this purpose.

For more specific applications of thin-layer chromatography (TLC), consult this.

References

1. Norris, R., L. Ryan and D. Acaster (2013). Cambridge International AS and A Level Chemistry.

2. Sharma, J. (2005). Handbook of Thin-Layer Chromatography.

Thin-Layer Chromatography (TLC)

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