An Overview on Thin Layer Chromatography


The thin layer chromatography (TLC) is made of glass, plastic or aluminum plate coated with a thin layer of silica gel (this is the focal point). Samples are detected on a plate and the solvent (this phase of travel) is sorted on a plate by capillary action, separating the sample components. The most soluble part in the solvent travels a greater distance to the top of the plate and the soluble portion travels a shorter distance. If the separated objects are colorless, the plate can be sprayed with a visualizing agent or viewed under UV light.

TLC results are represented by Rf values (retention factor) which is a measure of the distance traveled by an object compared to the distance traveled by the solvent, as shown below.

Rf = Distance traveled / Solvent-traveled distance (no units)

When using a TLC plate, negative control (water frequency), positive control (observation standard) and unknown sample should be seen on the same plate with their Rf values in comparison. Under normal circumstances, the Rf value of certain items will be the same.


The technique of chromatography is non- destructive in nature for resolving multiple component mixtures of minor, major and trace components into its individual fractions.

• Different alternatives can be used for solids, liquids and gases.

• Chromatography can be used in both quantity and quality it is primarily a separation tool.


Thin layers Chromatography (TLC) depends on the law of separation. Separation depends on the chemical exposure involved in both phases. Integration in the mobile section goes over the face of the vertical section. The movement occurs in such a way that compounds that have a high affinity with the standing phase move slowly while other compounds move faster. Therefore, the separation of the mixture is available. At the end of the separation process, each part from the assembly appears as spots on the appropriate levels of the plates.


As the forensic operates a variety of evidence found at the scene contaminated or acquired in pure ways. Evidence found to be contaminated needs to be investigated by fragmentation of existing component, so TLC is the best way to classify the various components that exist in the organization. TLC is used to identify and compare drugs, explosives, ink and dyes. This method is limited only by the need for the samples to be melted. TLC is used to identify and compare drugs, explosives, ink and dyes. This method is limited only by the need for the samples to be melted. TLC is one of the hidden operational functions of the forensic function; especially in explosive labs where it can identify an unknown sample or support other evidence suggesting a particular combination.


1. The TLC process requires less time and few amounts of substance.

2. Strong reagents can be used for identification purposes.

3. TLC plates can be heated at high temperatures without damage.

4. Used to separation for non-volatile compound.

5. Any time of mixture can be separated.

6. Automation is possible.

7. Great speed of separation.

8. Wide selection of materials.

9. Multiple analysis is possible and Easy to use and cheap.

10. TLC helps to visualization of separated compound spot easily.


In this way the length of the plate is limited and therefore separation only occurs at a certain distance.

1. Its time consuming.

2. More amount of mobile phase is required.

3. Automation makes the process more expensive and complicated

4. TLC plates do not have long stationary phase.

5. Limited quality of separation.

6. Evaporation of the mobile phase.

7. Limited reproducibility.


TLC has three steps – sight, development, and visualization. Spotting consists of using a micro pipette to transfer a small amount of this dilute solution to one end of the TLC plate. The spotting solvent quickly evaporates and leaves behind a small spot of the material.

Different components in the original spot, having different polarities, will Move different distances from the original spot location and show up as separate spots. When the Solvent has traveled almost to the top of the TLC plate, and the plate is removed, the solvent front marked with a pencil, and the solvent allowed evaporating.

A spot will interfere with the fluorescence and appear as a dark spot on a Glowing background. While under the UV light, the spots can be outlined with a pencil to mark their Locations. A second method of visualization is accomplished by placing the plate into iodine vapors for a few minutes. Most organic compounds will form a dark-colored complex with iodine. It is Good practice to use at least two visualization techniques in case a compound does not show up with one particular method.

The Rf value is used to quantify the movement of the materials along the plate.

Rf is equal to the distance traveled by the substance divided by the distance traveled by the solvent. Its value is always between zero and one.

If a development solvent of too high a polarity is used, all components in the mixture will move along with the solvent and no separation will be observed (Rf’s will be too large). If the solvent is of too low a polarity the components will not move enough, and again separation will not occur (Rf’s will be too small). In practice, different solvents or mixtures of solvents are tried until a good Separation is observed [12].

Rf = Y/X (always ≤ 1)


Plates (Stationary Phase)

Silica gel and alumina are among the most common standing categories, but some others are also available. Support for TLC plates is usually made of glass and aluminum. Glass plates are chemical-free and reactive stains and heat, but they are brittle and difficult to cut glass plates. Aluminum and plastic plates can be cut with scissors. Aluminum and plastic plates are also flexible, which can lead to flaking of the stationary phase. The characteristics of your sample should be considered when selecting the standing category.

Solvent (Mobile Phase)

Choosing the right solvent can be perhaps the most important aspect of TLC. Therefore the appropriate cell category should also be prepared for this ink sample analysis. Take 6 ml of chloroform, 2.5 ml of acetic acid, and 1.5 ml of acetone in a beaker and mix well. This mixture is used as mobile phase for the experiment.


The spots are placed on a plate using small glass pipettes. The capillary should be thin enough to apply a near spot, but not so thin as to prevent the detection of an adequate analyzer. Snap this in half and use the small end to apply the spots.

Authored by


(B.Sc. Forensic Science)

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