Unmodified silica is the most widely used sorbent in tlc. A simple thin layer chromatography method for separation of selected natural steroid hormones 626 synthetic steroids nandrolone, dromostanolone and etc are often used illegally as doping agents in sport, since they. Chromatography is used to separate mixtures of substances into their components. Reverse phase thin layer chromatography and high performance liquid chromatography are used to evaluate the lipophilicity of a series of organic compounds.
Tlc can be used to help determine the number of components in a mixture, the identity of compounds, and the purity of a compound. Chromatography is a unique method which can yield a great amount of quantitatively comparable. The mobile phase is an organic solvent or solvent mixture. Reversedphase chromatography is a technique using alkyl chains covalently bonded to the stationary phase particles in order to create a hydrophobic stationary phase, which has a stronger affinity for hydrophobic or less polar compounds. Reversed phase thin layer chromatography of five co. Thin layer chromatography tlc is a method of separating mixtures from several compounds using a stationary phase and a mobile phase through thin layers 18. Jevri1 1department of general and inorganic chemistry, faculty of technology, university of novi sad, bulevar. Solids most commonly used in chromatography are silica gel sio 2 x h 2 o and alumina al 2 o 3 x h 2 o. Introductory experiments of chromatography are often conducted by separating colored samples, such as inks, dyes, and plant extracts, using filter paper, chalk, or thin layer chromatography tlc plates with various solvent systems. Thinlayer chromatography tlc is widely used particularly for. While tlc is not a novel technique, its use as a method for resolving stereoisomeric controlled substances has not been widely explored. Thin layer chromatography selection of the mobile phase.
Factors that affect rf values in thin layer chromatography. They all have a stationary phase a solid, or a liquid supported on a solid and a mobile phase a liquid or a gas. This constitutes an important difference to hplc where most separations are carried out on chemically bonded phases by partition chromatography in the reversedphase mode. What is the mobile phase in thin layer chromatography. The use of a hydrophobic stationary phase is essentially the reverse of normal phase chromatography, since. Normal and reversed phase thinlayer chromatography of new. Other materials used as stationary phase were alumina, zirconium oxide. The solid support in a reversephase column is coated with a greasy, nonpolar substance usually a longchain hydrocarbon mix tethered to the support through a. The principle of tlc is known for more than 100 years 11. Stationary phase is cove red with water layer, while the mobile phase contains less water analyte partitions between the water layer and the mobile phase. Thin layer chromatography, or tlc, is a method for analyzing mixtures by separating the compounds in the mixture. Tlc is used routinely to follow the progress of reactions by monitoring the consumption of starting materials and the appearance of products. As with the other types of chromatography, tlc involves an adsorbent the stationary phase and a solvent the mobile phase. The tlc plate coating can serve two purposes in the separation process.
Quantitative thinlayer chromatography a practical survey. The separation ability and retention behaviour of normal and reversed phase tlc, with nonaqueous and aqueous organic mobile phases, have been studied by measuring the retention constants of a series of variously substituted 2,4dioxotetrahydro1,3thiazoles. Chromatography significant liquidsolid chromatography procedure is reversephase chromatography, in which the liquid mobile phase is water combined with an organic solvent such as methanol or acetonitrile and the stationary phase surface is nonpolar or hydrocarbonlike. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent. Sorbent technologies provides c18 silane for our reversed phase tlc plates, but with varying levels percentages of silanization. Thinlayer chromatography tlc is a chromatography technique used to separate nonvolatile mixtures. Chem 344 thin layer chromatography thin layer chromatography tlc is a useful technique for the separation and identification of compounds in mixtures. Thin layer chromatography tlc stationary phases and supports.
Tlc, thin layer chromatography linkedin slideshare. Conceptually, the technique is very simplethere are only two components. Thin layer chromatography tlc is a solidliquid technique in which the two phases are a solid stationary phase and a liquid moving phase. In this laboratory, you will experiment with thin layer chromatography tlc, in which the stationary phase is coated on a plate of glass or plastic. Phases in which substance will be separated the stationary phase stationary phase. All forms of chromatography work on the same principle. This chromatographic technique can be coupled to flame ionization detectors fid to characterize sara fractions or even separate the aromatic fraction into three groups. The relationship between normal chromatography and reversedphase chromatography is, however, seldom illustrated with. Retention factor values in thin layer chromatography are affected by the absorbent, the solvent, the chromatography plate itself, application technique and the temperature of the solvent and plate. The kinetic properties of the silica gel thinlayer and the new tlc plates have been presented.
The sample mixture is applied near the bottom of the plate as a small spot, then placed in a jar containing a few ml of solvent. It may be used to determine the extent of a reaction, the purity of a compound, or to ascertain the presence or absence of materials in fractions from column chromatography. Development of a thin layer chromatography method for the. It is a micro scale technique that can detect as little as 104 grams of material. Be like a chemistry special agent learn the method of thin layer chromotography and its applications.
Thinlayer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide alumina, or cellulose. Thin layer chromatography tlc is a chromatography technique used to separate mixtures. Chapter 27 basic principles of chromatography 477 271 table characteristics of different chromatographic methods method mobilestationary phase retention varies with gasliquid chromatography gasliquid molecular sizepolarity gassolid chromatography gassolid molecular sizepolarity supercritical. Shaise jacob faculty, nirmala college of pharmacy muvattupuzha kerala, india slideshare uses cookies to improve functionality and performance, and to provide you with relevant advertising. Chem 211 thin layer chromatography wellesley college. Analyze your caffeine using thin layer chromatography tlc and compare it to. Tlc is an established method for the separation of all major phospholipid classes. High performance thin layer chromatography hptlc has several advantages over tlc. Thinlayer chromatography tlc serves for the analytic separation of single.
The mobile phase has different properties from the stationary phase. This demonstration describes how normal and reverse phase chromatography can be illustrated using only chromatography paper for the separation of extracts of dandelions. It is also used to determine the proper solvent system for performing separations using column chromatography. Tlc is a quick, inexpensive microscale technique that can be used to. Reversedphase thinlayer chromatography behavior of. Thin layer chromatography tlc figure 2 illustrates thin layer chromatography tlc, the simplest and fastest liquid chromatography technique. Normal and reversed phase thin layer chromatography of green leaf extracts.
Thin layer chromatography selection of the mobile phase 2 to return to solute retention by polar interactions in the example employing silica gel as the stationary phase, to reduce retention, the polar interactions between the solutes and the mobile phase must be increased to compete with the polar interactions between the solutes and the. Solvents or solvent mixtures the mobile phase or eluent. Uncoated silica gel is the dominant stationary phase used in thinlayer chromatography tlc. Thinlayer chromatography tlc is a simple technique for separating nonvolatile compounds.
The mobile phase is a suitable liquid solvent or mixture of solvents. Thin layer chromatography is an important method in police investigation work and you. The method is rapid and separations can be completed in less than one hour. For example, with silica gel, a very polar substance, nonpolar mobile phases such as. Normal and reversephase paper chromatography of leaf. The mobile phase flows through the stationary phase and carries the components of the mixture with it. This layer of adsorbent is known as the stationary phase. Thinlayer chromatography an overview sciencedirect topics. Solidphase extraction spe is gradually replacing the traditional lle method.
Tlc uses a stationary phase, usually alumina or silica, that is highly. A tlc plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent usually silica or alumina. Thinlayer chromatography or tlc, is a solidliquid form of chromatography where the stationary phase is normally a polar absorbent and the mobile phase can be a single solvent or combination of solvents. Thin layer chromatography chemistry 6 thin layer chromatography tlc is among the most useful tools for following the progress of organic chemical reactions and for assaying the purity of organic compounds. The stationary phase may be a solid as in thin layer chromatography, tlc or either a liquid or solid on a solid support as in gas chromatography, gc. Definition thinlayer chromatography tlc is a sensitive analytical technique that is used commonly during organic experiments. Thinlayer chromatography chromatography represents the most versatile separation technique readily available to the chemist.
Tlc requires only a few ng yes thats right nano grams. Thin layer chromatography thin layer chromatography tlc is an analytical technique for determining the composition of a mixture. Thin layer chromatography tlc is an extremely useful technique for monitoring reactions. Normal and reversed phase thinlayer chromatography of new 16,17secoestrone derivatives marijanam.
Types of liquid chromatography 2 3 we focus on the stationary phase chemistry. Pdf theory and mechanism of thinlayer chromatography. Transfer of a mobile phase through the thin laye r is induced by capillary forces. The retention factor for a chemical during thin layer chromatography is a measure of how far it moves up the plate in response to. Liquidliquid extractionthin layer chromatography lletlc has been a common and routine combined method for detection of drugs in biological materials. The silica gel remains the most important adsorbent for tlc separation. The surface of the coated material can made to contain chemical groups that actually interact directly with the solutes themselves and, thus, determines the extent of their retention and the selectivity of the phase system in which case the material acts as the stationary phase proper. Normal and reversedphase thin layer chromatography of.
Normal and reversed phase thinlayer chromatography of. Calculations for molecular biology and biotechnology third edition, 2016. Thinlayer chromatography tlc is a method for separating compounds by their rate of movement through a thin layer of silica gel the stationary phase coated on a glass plate. Thin layer chromatography is available from thin layer chromatography is available from. At the tlc, a mobile phase flows by a stationary phase in which the mixture. Fast and precise thin layer chromatography by merck millipore. The addition of a small volume of aqueous ammonia in a nonaqueous mobile phase miscible with water, generally increased. Reversed phase chromatography is a technique using alkyl chains covalently bonded to the stationary phase particles in order to create a hydrophobic stationary phase, which has a stronger affinity for hydrophobic or less polar compounds. Thin layer chromatography tlc is a quick, sensitive, and inexpensi ve technique used. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminum oxide, or cellulose blotter paper. In chromatography the stationary phase maybe solid, liquid, gel or a solid liquid mixture and the mobile phase may be liquid or gas tlc is a.
580 1518 1470 399 717 1176 1076 410 824 270 52 154 53 645 298 1097 863 1149 1304 1510 227 1142 655 602 514 563 39 1153 968 278 374 375 28