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Thin Layer Chromatography: a Tool of Biotechnology for Isolation of Bioactive Compounds from Medicinal Plants

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Thin Layer Chromatography: a Tool of Biotechnology for Isolation of Bioactive Compounds from Medicinal Plants Int. J. Pharm. Sci. Rev. Res., 18(1), Jan – Feb 2013; nᵒ 18, 126-132 ISSN 0976 – 044X Review Article Thin Layer Chromatography: A Tool of Biotechnology for Isolation of Bioactive Compounds from Medicinal Plants Sanjeet Kumar*1, K. Jyotirmayee2, Monalisa Sarangi2 1. Department of Botany, Ravenshaw University, Cuttack - 75 3003, Odisha, India. 2. MITS School of Biotechnology, Bhubaneswar -75 1024, Odisha, India. Accepted on: 25-11-2012; Finalized on: 31-12-2012. ABSTRACT In this study, an attempt was made to review the basic principles and the importance of Thin Layer Chromatography (TLC) in research in general and in phytochemistry in particular. Thin layer chromatography is a simple, cost-effective, and easy-to-operate planar chromatographic technique which has been used in general chemistry laboratories for several decades to routinely separate chemical and biochemical compounds. Traditionally, chemical and optical methods are employed to visualize the analyte spots on the TLC plate. Also it has a wide application in identifying impurities in a compound. Study highlights the review on TLC and its application of qualitative and quantitative estimation of bio-active compounds from medicinal plants. Keywords: Thin Layer Chromatography, Principle of TLC, Advantages of TLC, Applications of TLC. INTRODUCTION different adsorbents. .Thin layer chromatography (TLC) is among the most useful tools for following the progress of hromatography is the collective term for a set of organic chemical reactions and for assaying the purity of laboratory techniques for the separation of organic compounds in phytochemistry and Biotechnology. mixtures into their components. All forms of C Like all chromatographic methods, TLC takes advantage of chromatography work on the same principle. They all the different affinity of the analyte with the mobile and have a stationary phases to achieve the separation of complex stationary mixtures of organic molecules. A TLC plate is a sheet of phase (a solid glass, metal or plastic which is coated with a thin layer of or a liquid a solid adsorbent. A small amount of the mixture to be supported on a analyzed is spotted near the bottom of this plate. The TLC solid) and a plate is then placed in a shallow pool of a solvent in a mobile phase developing chamber so that only the very bottom of the (a liquid or a plate is in the liquid1 . A TLC plate is a sheet of glass, gas). The metal, or plastic which is coated with a thin layer of a mixture is solid adsorbent (usually silica or alumina). A small amount dissolved in a of the mixture to be analyzed is spotted near the bottom fluid called the of this plate. The TLC plate is then placed in a shallow mobile phase, pool of a solvent in a developing chamber so that only the Figure 1: Inventor of Chromatography which carries it very bottom of the plate is in the liquid. This liquid, or the through a eluent, is the mobile phase, and it slowly rises up the TLC structure holding another material called the stationary plate by capillary action. To determine the best solvent or phase. The mobile phase flows through the stationary mixture of solvents (a "solvent system") to develop a TLC phase and carries the components of the mixture with it. plate or chromatography column loaded with an The various constituents of the mixture travel at different unknown mixture, vary the polarity of the solvent in speeds, causing them to separate. The separation is several trial runs: a process of trial and error. Carefully based on differential partitioning between the mobile and observe and record the results of the chromatography in stationary phases. There are different types of each solvent system. You will find that as you increase the chromatography such as Column chromatography, Paper polarity of the solvent system, all the components of the chromatography etc. among them Thin layer mixture move faster (and vice versa with lowering the chromatography (TLC) is a widely employed laboratory polarity). The ideal solvent system is simply the system technique and is similar to paper chromatography. that gives the best separation. TLC elution patterns However, instead of using a stationary phase of paper, it usually carry over to column chromatography elution involves a stationary phase of a thin layer of adsorbent patterns. Since TLC is a much faster procedure than like silica gel, alumina, or cellulose on a flat, inert column chromatography, TLC is often used to determine substrate. Compared to paper, it has the advantage of the best solvent system for column chromatography. For faster runs, better separations, and the choice between instance, in determining the solvent system for a flash International Journal of Pharmaceutical Sciences Review and Research 126 Available online at www.globalresearchonline.net a Int. J. Pharm. Sci. Rev. Res., 18(1), Jan – Feb 2013; nᵒ 18, 126-132 ISSN 0976 – 044X chromatography procedure, the ideal system is the one The solvent is allowed to rise until it almost reaches the that moves the desired component of the mixture to a top of the plate. That will give the maximum separation of TLC Rf of 0.25-0.35 and will separate this component from the dye components for this particular combination of its nearest neighbour by difference in TLC Rf values of at solvent and stationary phase. least 0.20. Therefore a mixture is analyzed by TLC to PRINCIPLE OF TLC determine the ideal solvent(s) for a flash chromatography procedure.Thin layer chromatography can be used to Thin layer chromatography uses a thin glass plate coated Monitor the progress of a reaction, identify compounds with either aluminum oxide or silica gel as the solid present in a given substance, and determine the purity of phase. The mobile phase is a solvent chosen according to a substance. Separation of compounds is based on the the properties of the components in the mixture. The competition of the solute and the mobile phase for principle of TLC is the distribution of a compound binding places on the stationary phase. For instance, if between a solid fixed phase applied to a glass or plastic normal phase silica gel is used as the stationary phase it plate and a liquid mobile phase, which is moving over the can be considered polar. Given two compounds which solid phase. A small amount of a compound or mixture is differ in polarity, the more polar compound has a applied to a starting point just above the bottom of TLC stronger interaction with the silica and is therefore more plate. The plate is then developed in the developing capable to dispel the mobile phase from the binding chamber that has a shallow pool of solvent just below the places. Consequently, the less polar compound moves level at which the sample was applied. The solvent is higher up the plate. If the mobile phase is changed to a drawn up through the particles on the plate through the more polar solvent or mixture of solvents, it is more capillary action, and as the solvent moves over the capable of dispelling solutes from the silica binding places mixture each compound will either remain with the solid and all compounds on the TLC plate will move higher up phase or dissolve in the solvent and move up the plate. the plate. Practically this means that if you use a mixture Whether the compound moves up the plate or stays of ethyl acetate and heptane as the mobile phase, adding behind depend on the physical properties of that more ethyl acetate results in higher Rf values for all individual compound and thus depend on its molecular compounds on the TLC plate. Changing the polarity of the structure, especially functional groups. The solubility rule mobile phase will normally not result in reversed order of “Like Dissolves Like” is followed. The more similar the running of the compounds on the TLC plate1,2,4,6,7. physical properties of the compound to the mobile phase, the longer it will stay in the mobile phase. The mobile Chromatogram phase will carry the most soluble compounds the furthest As the solvent slowly travels up the plate, the different up the TLC plate. The compounds that are less soluble in components of the dye mixture travel at different rates the mobile phase and have a higher affinity to the and the mixture is separated into different coloured particles on the TLC plate will stay behind16. spots. Rƒ values The behavior of an individual compound in TLC is characterized by a quantity Known as Rƒ and is expressed as a decimal fraction. The Rƒ is calculated by dividing the distance the compound traveled from the original position by the distance the solvent travelled from the original position (the solvent front). Nature of adsorbent: Different adsorbents will give different Rƒ value for same solvent. Reproducibility is only possible for given adsorbent of constant particle size and binder. Plates should be stored over silica gel in desiccators before use and the sample should be applied quickly so that the water vapor in the atmosphere is not adsorbed by the Figure 2: Chromatogram plate. Because of the difficulties associated with activation procedures, it is far better to use plates stored at room temperature and not to activate them. Measuring Rf values Measurements are often taken from the plate in order to help identify the compounds present. These measurements are the distance travelled by the solvent, and the distance travelled by individual spots. When the solvent front gets close to the top of the plate, the plate is removed from the beaker and the position of the solvent Figure 3: Developing Tank International Journal of Pharmaceutical Sciences Review and Research Available online at www.globalresearchonline.net 127 Int.
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