Thin-Layer+Chromatography

Thin-Layer Chromatography Basic Description:

The technique of Chromatography involves spotting a small initial zone on a stationary phase. Thin-Layer Chromatography (TLC) is a type of liquid chromatography in which the sample is spotted in a small amount on a thin sorbent later. The technique can be performed on foil, glass, or plastic. Absorbent materials such as silica gel can be used. Once the sample is spotted on the stationary phase, the mobile phase involves the movement of the stationary phase by capillary action. The components of the mixture are carried along up the plate. Usually gravity or pressure aids the movement of the sample as well. TLC separations give each different component the same amount of time to move up the plate, but the distance varies per component. An Rf value can be calculated using the equation Rf= Distance from Baseline traveled by Solute/ Distance from Baseline traveled by Solvent. The less polar compounds of a mixture will move higher up the plate which will result in a higher Rf value (Fried & Sherma, 1999).

Figure 1: Shows the spotting of a mixture and the compounds moving upward on the plate along with the solvent. Image from http://www.waters.com/waters/nav.htm?cid=10048919&locale=en_US

What is the purpose of this technique?

Thin-Layer Chromatography can reveal many characteristics of a mixture or solution (Fried & Sherma, 1999). It specializes in separation, isolation, identification, and quantification of the mixture’s components. The main task of this technique is to separate a mixture to see what is it made of. Various components like amino acids, drugs, carbohydrates, vitamins, peptides, antibiotics, and lipids can be identified in samples such as blood, saliva, cerebrospinal fluid, body tissues, etc…The different sorbents and mobile phases allow very efficient separations between polar and nonpolar compounds. In addition, the use of precise instruments and tools allows for accurate quantitative analysis. TLC is a detection method that provides a vast range of simultaneously separating and identifying spotted samples on the same plate in an inexpensive way (Sherma & Fried, 2005). TLC can be performed as a preparation for another technique. It can be used and characterized through other techniques like gas chromatography, high-performance liquid chromatography, ultraviolet, nuclear magnetic resonance (NMR) and mass spectrometry (Fried & Sherma, 1999).

Origin & History:

Mikhail Tsvet was the inventor of chromatography. Thin-Layer Chromatography is a fairly new discipline of chromatography and most historians date modern TLC back to 1958. Izmailov and Shraiber separated certain medicinal compounds using alumnia spread on glass plates. The solvent was dropped on the glass plate which contained the sample and sorbent. This procedure was called drop chromatography. In 1948, Meinhard and Hall used alumnia plates on microscope slides to help separate inorganic ions using the drop chromatography method. Between 1950 and 1954, Kirchner developed TLC at the U.S Department of Agriculture. The sorbents were utilized on the glass plate with the assistance of a binder for the plates to be developed. The term “thin-layer chromatography” was primarily coined by the German, Egon Stahl in the late 1950s. In the early years of TLC, workers had to coat their own plates which became tedious and messy and thus resulted in many rejected plates. However, in the early 1960s, precoated plates became available commercially. Kirchner and colleagues were the first to describe quantitative TLC of biphenyl in citrus fruits (Fried & Sherma, 1999).

Utilization  of Thin-Layer Chromatography:


 * 1) Zhang, Z., Xiao, Z., and Linhardt, J. (2009). Thin-layer chromatography for the separation and analysis of acidic carbohydrates. // Journal of Liquid Chromatography & Related Technologies. // 32:1711-1732.
 * 2) Pehlic, E., Nuhanovic, M., Sapcanin, A., Banjanin, B., Nanic, H., Redzic, S., Poljakovic, M. (2012). Characterization of acetylsalicylic acid with thin-layer chromatography and hot-stage microscopy depending on solvent system. // Healthmed. // 6(11): 3878-3884.
 * 3) Sharma, O., Kumar, N., Singh, B., Bhat, T. (2012). An improved method for thin-layer chromatographic analysis of saponins. // Food Chemistry. // 132(1): 671-674.

1. This experiment performed ten TLC separations of simple carbohydrates in urine. Sensitivity and resolution varied per procedure to analyze the importance of simple sugars. Eleven simple sugars were analyzed by TLC, including glucuronic aicd. The TLC shows was sugars are identified in urine samples. The TLC separated acidic carbohydrates. This experiment showed substantial progress in the separation of unmodified acidic disaccharides and oligosaccharides.

2. Synthesized aspirin has to go through TLC to identify the given compounds. By using TLC, one gets a better ability to identify acetylsalicylic acid, also known as aspirin. TLC separates other analgesics as well. The aim of this experiment was to evaluate different mobile phase composition on acetylsalicylic acid identification in the presence of salicylic acid by TLC.

3. Saponins are constituents found in foods that are anti-inflammatory, antimicrobial, antioxidant, immunostimulant, etc… In this experiment, TLC uses its quick analysis and the comparison of different samples simultaneously. Haemolysis of saponins can be detected by TLC. The method of TLC on haemolytic properties of erythrocytes is applied to ten saponin-rich plants and compared. TLC provides a simple, specific, and convenient procedure to analyze saponins for purification and chemprofiling of plants.

Bibliography:
 * 1) Fried, B. & Sherma, J. (1999). Thin-layer chromatography (4th Edition). New York: Marcel Dekker
 * 2) Pehlic, E., Nuhanovic, M., Sapcanin, A., Banjanin, B., Nanic, H., Redzic, S., Poljakovic, M. (2012). Characterization of acetylsalicylic acid with thin-layer chromatography and hot-stage microscopy depending on solvent system. //Healthmed//. 6(11): 3878-3884.
 * 3) Sharma, O., Kumar, N., Singh, B., Bhat, T. (2012). An improved method for thin-layer chromatographic analysis of saponins. //Food Chemistry//. 132(1): 671-674.
 * 4) Sherma, J. & Fried, B. (2005). Thin layer chromatographic analysis of biological samples. A review. //Journal of Liquid Chromatography & Related Technologies.// 28(15) p 2297-2314.
 * 5) Zhang, Z., Xiao, Z., and Linhardt, J. (2009). Thin-layer chromatography for the separation and analysis of acidic carbohydrates. //Journal of Liquid Chromatography & Related Technologies//. 32:1711-1732.