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Aspects of Thin-Layer Chromatography Report

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Updated: May 13th, 2022

Background Information

TLC stands for thin layer chromatography. This is an analytical technique widely used in the separation of compounds into their constituents. TLC entails the use of a thin, uniform sorbent layer immobilized on a firm, solid support usually made of glass, plastic or aluminium (Wall 2005). The uniform layer is mostly made of silica gel or alumina. TLC is a form of liquid-solid chromatography where the sample is applied as a streak or a tiny spot on the stationary phase. Capillary action aids the motion of the mobile phase, which moves through the stationary phase. The force of gravity and pressure can also play a substantial role in the advancement of the mobile phase (Fried & Sherma 2005). As the mobile phase passes through the stationary phase, it carries the constituents of the analyte with it causing different components to travel at different rates (Thin layer chromatography n.d.). The mobile phase in TLC can either be a single solvent or a mixture of solvents, which can be organic or inorganic solvents (Fried & Sherma 2005).

The rate of movement of substances depends on the extent of their adsorption to the stationary phase and the scope of their solubility in the mobile phase. At the end of the run, the separated components are visualised using ultraviolet light because the TLC plate is usually made of substances that fluoresce. Chemical treatment or derivatisation can also be used in cases where UV light visualization is not feasible (Wall 2005).

“TLC can be used both as an analytical and a preparative technique” (Fried & Sherma 2005, p.1). Preparative TLC entails the separation of substances for further experimental uses whereas analytical TLC involves determining the presence and quantity of a given compound of interest in the analyte. For quantitative purposes, separated spots are scraped from the surface, eluted using a suitable buffer after which they are quantified (off-plate quantification). On-plate quantification is achieved using densitometry or radiochromatograph scanning where the plate is scanned at a fixed wavelength without upsetting it (Wall 2005).

TLC is essential in a wide range of analytical applications such as defining and ensuring the quality of pharmaceutical drugs to guarantee user safety (“Introduction” 2007).


Naphthoquinone (1,4-naphthoquinone) is an organic compound, which is a derivative of the “quinine” family (What is 1,4-Naphthoquinone? n.d.). Vitamin K is a naturally occurring derivative of 1,4-naphthoquinone. Naphthoquinone is utilized as a raw material in the manufacture of pharmaceuticals and agrochemicals among other chemicals. Therefore, it is essential that naphthoquinone is used in its most pure form. A sample of 1,4-naphthoquione purchased from Sigma Aldrich was 95% pure and had a brownish yellow colour instead of a pure yellow colour. A purification step was, therefore, carried out to obtain pure naphthoquinone.


A makeshift column chromatography apparatus was made using a glass sinter funnel packed with 30 grams of silica powder. 15.078 grams of 1,4-naphthoquinone were placed on top of the makeshift column. The quinine was dissolved with dichloromethane (DCM) until the washing reagent was clear. The resulting solution was then monitored for purity using TLC where a 50:50 mixture of DCM and ethyl acetate was used as the solvent.


The solution obtained from the column chromatography was a dull yellow colour. A brown solid was left behind at the end of the column chromatography. The following images indicate the spots obtained from the TLC runs.

Results Results


Column chromatography was the preparative technique used to purify 1,4-naphthoquinone partially before TLC was performed. TLC was used to monitor the purity of the naphthoquinone by checking for the presence of impurities. This was achieved by performing the TLC of the impure naphthoquinone analyte together with a pure 1,4-naphthoquinone sample as the standard. The TLC plates indicated that pure naphthoquinone was a single spot, whereas impure naphthoquinone had three different spots. This indicated that the impure naphthoquinone contained two impurities.

According to Kowalska, Kaczmarski & Prus, the Rf value was the fundamental quantity that expressed solute position on the developed chromatogram (2003, p. 71). This was obtained by finding the ratio between the distance travelled by the chromatographic spot and the distance travelled by the solvent (Kowalska, Kaczmarski, & Prus 2003). Rf values were physically observed. Therefore, large Rf values were visualised as spots with a large distance between the solvent front and the base line. It was observed in the first chromatogram that pure and impure naphthoquinone had one common spot with the same Rf value. This spot indicated the position of 1,4-naphthoquinone in a TLC run using a silica stationary phase and a 1:1 blend of dichloromethane and ethyl acetate as the mobile phase.

The impure substances were less soluble in the mobile phase than 1,4-naphthoquinone, hence exhibited small Rf values. A careful observation of the plates revealed that the number of spots reduced as the number of TLC runs increased. That was because more impurities were adsorbed on the stationary phase. In addition, the colour intensity of the impure spots reduced as the sample was further purified. This made the impure spots appear colourless to the naked eye. The final run had a single spot indicating that the sample was pure (American Chemical Society & Committee on Analytical Chemical Reagents 2006, p.78).


The final chromatogram produced only one spot, whose position coincided with that of pure 1,4-naphthoquinone. This indicated that the final sample was pure. It was, therefore, concluded that TLC was a remarkably efficient technique in the separation of substances.


American Chemical Society & Committee on Analytical Chemical Reagents 2006, Reagent Chemicals: Specifications and Procedures : American Chemical Society Specifications, Official from January 1, 2006,10th edn, Oxford University Press, New York.

Fried, B. & Sherma J 2005, Thin layer chromatography, Marcel Dekker, New York.

“Introduction” 2007, in E Reich & A Schibli (eds), High performance thin layer chromatography for the analysis of medicinal plants, Thieme Medical Publishers, New York.

Kowalska, T., Kaczmarski, K., & Prus, W 2003, “Theory and mechanism of thin layer chromatography,” in J Sherma & B Fried (eds), Handbook of thin layer chromatography, Marcel Dekker, New York, pp. 62-105.

Thin layer chromatography n.d. 2013, Web.

Wall, P. E 2005, Thin layer chromatography: a modern practical approach, The Royal Society of Chemistry, Cambridge.

What is 1,4-? n.d. 2013, Web.

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