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  3. 3. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] INTRODUCTION:- Chromatography is the separation of two or more compounds or ions by the distribution Between two phases, one which is moving and the other which is stationary. These two phases can be solid-liquid, liquid-liquid or gas-liquid. Mobile Phase, m v A Stationary Phase, s Figure shows Schematic presentation of a chromatographic system with partition of analyte ‘A’ between the phases; v mobile phase velocity. The molecules of the analytes are distributed between the mobile and the stationary phase. When present in the stationary phase, they are retained, and are not moving through the system. In contrast, they migrate with the velocity, v, of the mobile phase when being there. Due to the different distribution of the particular analytes the mean residence time in the stationary phase differs, too, resulting in a different net migration velocity. This is the principle of chromatographic separation. The position of the distribution equilibrium determines the migration velocity. It reflects the intermolecular interactions of the analyte with the stationary and the mobile phase. However, during migration of the analytes through the separation capillary broadening of the original sample zone takes place. This broadening is greater the longer the migration distance is. Two 3|Page
  4. 4. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] effects are therefore counteracting in the chromatographic system: the different migration is the base of separability by introducing separation selectivity. In contrast, peak broadening effects lead to a potential overlap of the peaks migrating with different velocity. They occur in parallel with a dilution of the initial sample zone with the mobile phase. THIN LAYER CHROMATOGRAPHY:Thin layer chromatography, or TLC, is a method for analyzing mixtures by separating the compounds in the mixture. TLC can be used to help determine the number of components in a mixture, the identity of compounds, and the purity of a compound. By observing the appearance of a product or the disappearance of a reactant, it can also be used to monitor the progress of a reaction. TLC is a sensitive technique - microgram (0.000001 g) quantities can be analyzed by TLC and it takes little time for an analysis (about 5-10 minutes). HISTORY OF TLC:The development of modern thin-layer chromatography has its beginnings in liquid chromatography. Like most scientific methods, chromatography evolved from initial phenomenological observations, through early empirical research and the study of the underlying theoretical principles, and finally, advancements in technique. While the Russian botanist Tswett is generally credited with the discovery of chromatography for his work in the separation of plant extracts on a column of sorbent, others observed the phenomenon of separation before Tswett. For example, in 1844 C. Matteucci observed the rings left by a drop of chocolate on a piece of paper. In 1850 the German dye chemist F.F. Runge recognized the possibility of separating inorganic ions when he observed their migration through paper. He initially described the forces responsible for the separation as the "living forces," but later attributed the phenomenon to capillary forces (D 'Ascenzo and Nicolini1990). Tswett's contribution to chromatography was the understanding of adsorption, which led to chemical separation by liquid column chromatography. Although chromatographic techniques were little used for the next thirty years, in the 1930’s they were reintroduced by biochemists. In 1941, A.J.P. Martin and R.L.M. Synge introduced partition chromatography. They found that they could separate amino acids successfully if a water phase was held stationary by adsorbing it on silica gel, while permitting a mobile chloroform phase to flow over it. Thus they described the use of a liquid stationary phase with a liquid mobile phase and suggested 4|Page
  5. 5. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] that a gas might also be used as the mobile phase. I n 1952, Martin and Synge were awarded a Nobel Prize in chemistry for their theoretical development of partition chromatography (Brooks 1987). The development of TLC can be divided into three eras: the early years (1938-1951), the classical period (1956-1980), and the modern period (1981 to the present). Figure shows a time line for the history of TLC. 5|Page
  6. 6. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] Figure shows one of the first TLC plates developed by Stahl in 1956, showing a valerian oil separation. The plate was sprayed with SbCl3 solution in CHCl3 and subsequently heated 6|Page
  7. 7. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] THEORY:In thin layer chromatography, a solid phase, the adsorbent, is coated onto a solid support as a thin layer (about 0.25 mm thick). In many cases, a small amount of a binder such as plaster of Paris is mixed with the absorbent to facilitate the coating. Many different solid supports are employed, including thin sheets of glass, plastic, and aluminium. The mixture (A plus B) to be separated is dissolved in a solvent and the resulting solution is spotted onto the thin layer plate near the bottom. A solvent, or mixture of solvents, called the eluant, is allowed to flow up the plate by capillary action. At all times, the solid will adsorb a certain fraction of each component of the mixture and the remainder will be in solution. Any one molecule will spend part of the time sitting still on the adsorbent with the remainder moving up the plate with the solvent. A substance that is strongly adsorbed (say, A) will have a greater fraction of its molecules adsorbed at any one time, and thus any one molecule of A will spend more time sitting still and less time moving. In contrast, a weakly adsorbed substance (B) will have a smaller fraction of its molecules adsorbed at any one time, and hence any one molecule of B will spend less time sitting and more time moving. Thus, the more weakly a substance is adsorbed, the farther up the plate it will move. The more strongly a substance is adsorbed, the closer it will stay near the origin. Several factors determine the efficiency of a chromatographic separation. The adsorbent should show a maximum of selectivity toward the substances being separated so that the differences in rate of elution will be large. For the separation of any given mixture, some adsorbents may be too strongly adsorbing or too weakly adsorbing. Table 1 lists a number of adsorbents in order of adsorptive power. Table 1. Chromatographic adsorbents. The order in the table is approximate, since it depends upon the substance being adsorbed, and the solvent used for elution. 7|Page
  8. 8. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] The eluting solvent should also show a maximum of selectivity in its ability to dissolve or desorbs the substances being separated. The fact that one substance is relatively soluble in a solvent can result in its being eluted faster than another substance. However, a more important property of the solvent is its ability to be itself adsorbed on the adsorbent. If the solvent is more strongly adsorbed than the substances being separated, it can take their place on the adsorbent and all the substances will flow together. If the solvent is less strongly adsorbed than any of the components of the mixture, its contribution to different rates of elution will be only through its difference in solvent power toward them. If, however, it is more, strongly adsorbed than some components of the mixture and less strongly than others, it will greatly speed the elution of those substances that it can replace on the absorbent, without speeding the elution of the others. Table 2 lists a number of common solvents in approximate order of increasing adsorbability, and hence in order of increasing eluting power. The order is only approximate since it depends upon the nature of the adsorbent. 8|Page
  9. 9. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] Mixtures of solvents can be used, and, since increasing eluting power results mostly from preferential adsorption of the solvent, addition of only a little (0.5-2%, by volume) of a more strongly adsorbed solvent will result in a large increase in the eluting power. Because water is among the most strongly adsorbed solvents, the presence of a little water in a solvent can greatly increase its eluting power. For this reason, solvents to be used in chromatography should be quite dry. The particular combination of adsorbent and eluting solvent that will result in the acceptable separation of a particular mixture can be determined only by trial. Table 2. Eluting solvents for chromatography If the substances in the mixture differ greatly in adsorbability, it will be much easier to separate them. Often, when this is so, a succession of solvents of increasing eluting power is 9|Page
  10. 10. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] used. One substance may be eluted easily while the other stays at the top of the column, and then the other can be eluted with a solvent of greater eluting power. Table 3 indicates an approximate order of adsorbability by functional group. Table 3. Adsorbability of organic compounds by functional group 10 | P a g e
  11. 11. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] STEPS OF THIN LAYER CHROMATOGRAPHY: Thin Layer Chromatography is a three step process which involves spotting of TLC plate, development of that plate and visualization under UV rays. So, steps of TLC are as follows:- SPOTTING THE TLC PLATE:- One advantage TLC has over other separation methods is that it is truly a micro scale technique. Only a few micrograms of material in solution is necessary to observe the solute on a TLC plate. Dissolve a few milligrams of material in a volatile solvent creating a dilute solution. Choose a volatile solvent that completely dissolves the sample. However, if it is partially soluble, since such only low concentrations are needed, normally you will be able to observe the compound. Once the sample is prepared, a spotting capillary must be used to add the sample to the plate. The spotting capillaries must be extremely small. In fact, the opening at the end of a regular Pasteur pipette is too big for spotting a TLC plate. CG capillary columns donated by Restek, a chromatography company located in Bellefonte, are used to spot the plates. These columns are very small bore and can be cut into three inch sections to provide very good TLC spotting tubes. The solution can be drawn up the tube by capillary action (hence the name) and spotted on the plate at the hash mark labelled in pencil. This is known as the origin and is shown in Figure. Since a TLC plate can run three, if not four mixtures at one time, it is very important to properly label the plate. Notice that pencil is always used to mark a TLC plate since the graphite carbon is inert. If organic ink is used to mark the plate, it will chromatograph just as any other organic compound and give incorrect results. To spot the plate, simply touch the end of the capillary tube to the coated side of the plate. The solvent should evaporate quickly leaving your mixture behind on the plate. You may have to spot the plate a couple of times to ensure the material is present, but do not spot too much sample. If too much solute is added to the plate, a poor separation will result. Smearing, smudging and spots that overlap will result making identification of separated components difficult. 11 | P a g e
  12. 12. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] DEVELOPMENT:Once the dilute solution of the mixture has been spotted on the plate, the next step is the development. Just like paper chromatography, the solvent must be in contact with the stationary phase. Figure shows a wide-mouth bottle commonly used to develop TLC plates. 12 | P a g e
  13. 13. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] The bottle is filled with a small amount of the mobile phase and capped with a cork. In addition, a piece of filter paper is put in the bottle to help create an atmosphere saturated with solvent. Use your tweezers to place the plate in the development chamber; oils from your fingers can sometimes smear or ruin a TLC plate. Also make sure the origin spots are not below the solvent level in the chamber. If the spots are submerged in the solvent, they are washed off the plate and lost. Once the solvent has run within a centimetre of the top of the plate, remove it with tweezers. Using a pencil, immediately draw a line across the plate where the solvent front can be seen. The proper location of this solvent front line will be important for later calculations. VISUALIZATION:Some organic compounds are coloured. If you are fortunate enough to be separating organic molecules that are coloured such as dyes, inks or indicators, then visualizing the separated spots is easy. However, since most organic compounds are colourless, this first method does not always work. In most cases observing the separated spots by UV light works well. TLC plates normally contain a fluorescent indicator which makes the TLC plate glow green under UV light of wavelength 254 nm. Compounds that absorb UV light will quench the green fluorescence yielding dark purple or bluish spots on the plate. Simply put the plate under a UV lamp, and the compounds become visible to the naked eye. Lightly circle the spots, so that you will have a permanent record of their location for later calculations. Figure shows two ways of using UV light to visualize the spots 13 | P a g e
  14. 14. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] Another useful visualizing technique is an iodine (I2) chamber. Iodine sublimes and will absorb to organic molecules in the vapour phase. The organic spots on the plate will turn brown and can be easily identified. Also circle these observed spots, since the colour stain will eventually fade from the plate. Sometimes, a combination of both a UV lamp and iodine is needed to observe all the spots. Some compounds are not “UV active”, that is, they do not absorb light at the wavelength of 254 nm. Using both methods will ensure correct identification of all the spots on the TLC plate. Rf Value:In addition to qualitative results, TLC can also provide a chromatographic measurement known as an Rf value. The Rf value is the “retardation factor” or the “ratio-to-front” value expressed as a decimal fraction. The Rf value can be calculated as: This number can be calculated for each spot observed on a TLC plate. Essentially it describes the distance travelled by the individual components. If two spots travel the same distance or have the same Rf value then it might be concluded that the two components are the same molecule. For Rf value comparisons to be valid; however, TLC plates must be run under the same exact conditions. These conditions include the stationary phase, mobile phase, and temperature. Just as many organic molecules have the same melting point and colour, many can have the same Rf value, so identical Rf values doesn’t necessarily mean identical compounds. Additional information must be obtained before this conclusion can be made. It is important to restate that this number is only significant when the same chromatographic conditions are used. Figure shows a diagram of a typical TLC plate and how the distances are measured to calculate the Rf value. 14 | P a g e
  15. 15. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] Factors Affecting the Rf value:1. Nature of Adsorbent Different adsorbents will give different Rf valve for same solvent. Reproducibility is only possible for given adsorbent of constant particle size and binder. Plates should be the stored over silica gel in a desiccators before use and the sample should be applied quickly so that the water vapour in the atmosphere is not adsorbed by the 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. 2. The Mobile phase The purity of solvents and quantity of solvent mixed should be strictly controlled. It should make freshly for each run if one of the solvents is very volatile or hygroscopic, for example acetone. 3. Temperature Although precise control of temperature is not necessary, the tank should be kept away from draughts, sources of heat, direct sunlight, etc. As the temperature is increased, volatile 15 | P a g e
  16. 16. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] solvents evaporate more quickly, solvents run faster, and Rf values generally decrease slightly. 4. Thickness of Layer Standard plates approximately 250 µm is preferable thickness of layer. Below 200 µm the Rf values vary considerably. The layers may be of higher or lower thickness in individual compounds. 5. Developing Tank It is important that saturated conditions are attained for running TLC plates. This is best accomplished by using small tanks with filter paper liners and sufficient solvent, and by leaving the tank to equilibrate for at least 30 minutes before running the plates. A well-fitting lid is essential. 6. Mass of Sample Increasing the mass of sample on the plate will often increase the Rf of a drug, especially if it normally tails in the system. However, if a plate is grossly overloaded, this too will give a tailing spot and will have the effect of apparently decreasing the Rf value. The two situations are normally easy to distinguish by the intensity of the spot. 7. Chromatographic Technique Depending upon the development technique used i.e. ascending, descending, horizontal etc. the Rf value changes for the same solvent system. 16 | P a g e
  17. 17. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] MOLECULAR INTERACTIONS IN TLC:There are three components in TLC: The TLC plate (stationary phase), the development solvent (mobile phase), and the sample to be analyzed (solute). In our experiment the TLC plate consists of a thin plastic sheet covered with a thin layer of silica gel, a portion of the structure of which is shown here. Silica gel consists of a three-dimensional network of thousands of alternating silicon and oxygen bonds, with O-H groups on the outside surface. Silica gel is simply very finely ground very pure sand. It should be noted that silica gel is highly polar and is capable of hydrogen bonding. Consider the side-on view of the development of a TLC plate below. As the solvent travels up the plate, over the spot, equilibrium is set up, as development solvent competes with the TLC plate for the solute. The silica gel binds to the solute and the development solvent tries to dissolve it away, carrying the solute(s) along as the solvent travels up the plate. 17 | P a g e
  18. 18. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] A balance of intermolecular forces determines the position of equilibrium and thus the ability of the solvent to move the solute up the plate. In other words, would the spot prefer to be stuck on the plate or would it prefer to move along with solvent. The balance depends upon (1) the polarity of the TLC plate (constant and high), (2) the polarity of the development solvent (can be varied by using different solvents), and (3) the polarity of the compounds in the spot (this varies depending upon what compounds are in the spot). For example, if a sample consists of two components, one more polar than the other, the more polar will tend to stick more tightly to the plate and the less polar will tend to move 18 | P a g e
  19. 19. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] along more freely with the solvent. Using a more polar development solvent would cause both to move along further. If the approximate structures of the solutes are known, it is possible to make an educated guess as to what solvent or mixture of solvents to use. In practice though, for a given mixture of compounds to be analyzed, a solvent or mixture of solvents is chosen by trial and error to give the best separation. (A caveat: the polarity argument is helpful in understanding the principles of TLC. Because most compounds have some polarity the argument works well. For compounds having very low polarity however, a lower-polarity solvent may be more effective in moving the solute up the plate.) This interaction can be understood more easily using the following example illustrated here below:- The polarity of molecules, solutes and solvents alike, is ordered as follows, from least to most polar: Alkanes (least polar), alkyl halides, alkenes, aromatic hydrocarbons, ethers, esters, 19 | P a g e
  20. 20. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] ketones, aldehydes, amines, alcohols, and carboxylic acids (most polar). Note however that many molecules contain multiple functional groups and that the overall polarity would be determined by all of the groups. Applications and Importance of Thin Layer Chromatography:1. Pharmaceuticals and Drugs Identification, purity testing and determination of the concentration of active ingredients, auxiliary substances and preservatives in drugs and drug preparations, process control in synthetic manufacturing processes. 2. Clinical Chemistry, Forensic Chemistry and Biochemistry Determination of active substances and their metabolites in biological matrices, diagnosis of metabolic disorders such as PKU (phenylketonuria), cystinuria and maple syrup disease in babies. 3. Cosmetology Dye raw materials and end products, preservatives, surfactants, fatty acids, constituents of perfumes. 4. Food Analysis Determination of pesticides and fungicides in drinking water, residues in vegetables, salads and meat, vitamins in soft drinks and margarine, banned additives in Germany (e.g. sandalwood extract in fish and meat products), compliance with limit values (e.g. polycyclic compounds in drinking water, aflatoxins in milk and milk products). 5. Environmental Analysis Groundwater analysis, determination of pollutants from abandoned armaments in soils and surface waters, decomposition products from azo dyes used in textiles. 6. Analysis of Inorganic Substances 20 | P a g e
  21. 21. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] Thin layer chromatography technique is also used in the determination of inorganic ions (metals). Applications Related To Organic Chemistry:1) It has widely used for checking number of other separation processes. TLC has also been applied successfully in various purification processes, checking of distillation fractions and for checking the progress of purification by molecular distillation. 2) TLC has been used as an analytical tool in organic chemistry due to its high speed of separation and its applicability in a large number of chemical compounds. Its important use is in the separation and isolation of individual components of a mixture, but in organic chemistry it has also been used for: · Checking the purity of samples, · As purification process, · For identification of organic compounds, · For studying various organic reactions, · In characterizing and isolating a number of compounds such as acids, alcohols, glycols, amides, alkaloids, vitamins, amino acids, antibiotics, food stuffs etc. · Examination of reaction. 3) High sensitivity of TLC is used to check purity of sample, because high sensitivity enables impurities to be observed in so called pure samples. With the help of TLC it is possible to know whether a reaction is complete and had followed the expected course. The nature of byproducts can also be ascertained by using TLC. If the reaction does not proceed as desired or expected, then an examination of the behaviour of the spots with standard reagents may sometimes give information for the rapid identification of the products. 21 | P a g e
  22. 22. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] Examination of Reaction The reaction mixture is examined by TLC to assess whether the reaction is complete or otherwise. The method is also used in checking other separation processes and purification processes like distillation, molecular distillation etc. Application of TLC for Separation of Inorganic Ions:Nowadays, TLC has been used for separating cationic, anionic, purely covalent species and also organic derivatives of the metals. In order to carry out TLC of groups of cations, silica gel is first washed with acid and water to remove impurities of sodium, magnesium, calcium and iron. But this treatment removes the calcium sulphate binder. Therefore, calcium sulphate must be replaced by starch or some other suitable binder. After washing and drying of TLC plate, the spots of cations or anions to be separated are applied on this plate. The plate is then kept in a close chamber and the lower part of the plate is then dipped into a solvent. After that it is removed from chamber and dried visualized for spots by suitable visualizing reagents. Examples 1. Amino acids, proteins and peptides A mixture of 34 amino acids, proteins and peptides has been successfully separated and isolated from urine using silica gel plates. All these substances were found to be ninhydrin positive. The development were carried out first with chloroform-methanol-20%ammonium hydroxide (2:2:1) and then with phenol-water. 2. Alkaloids TLC has been used for the isolation and determination of alkaloids in toxicology where the 30-60 minute runs give a great advantage in comparison to the 12-24 hours required for paper chromatography. Purine alkaloids have been separated by TLC on silicic acid, silica gel and aluminium oxide. The spots are visualized by spraying first with an alcoholic iodinepotassium iodine solution followed by 25% HCl – 96% ethanol (1:1). 22 | P a g e
  23. 23. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] 3) Antibiotics Penicillines have been separated on silica gel ‘G’ by using the two solvents, acetonemethanol (1:1) and iso-propanol-methanol (3:7). As the detecting agent, the iodine-azide reaction was employed by spraying the dried plates with a 0.1 iodine solution containing 3.5% of sodium azide. Applications of Thin Layer Chromatography in Analysis of Heavy Petroleum Product Thin-layer chromatography (TLC), which is commonly used in the analysis of complex mixtures, is seldom used in the investigation of petroleum products, maybe the most complex objects. In particular, with respect to heavy petroleum products, no such information has been found in the literature. At the same time, the simplicity, economy, and efficiency of this technique in comparison with column chromatography are advantages that are widely known. TLC technique used (in the preparative variant) for a rapid determination of the group composition of heavy petroleum products (asphalts, pitches, resids), and in connection with spectroscopic studies of the chemical composition of the fractions obtained. Thin-layer chromatographic separation of aromatic amines Cationic and non-ionic surfactant-mediated systems have been used as mobile phases in thinlayer chromatographic separation of aromatic amines on silica gel layers. The effect of surfactant concentration below and above its critical micellar concentration on mobility of amines was examined. Software for TLC Thin layer chromatography (TLC) relies upon polarity and the strength of intermolecular interactions between the stationary phase (silica gel), mobile phase (solvent), and chemical sample to separate the components of a mixture. The rational selection of a proper solvent is one of the most important factors in achieving good resolution and unambiguous results. Thus, by investing some time in understanding the structure and properties of organic solvents more fully, you will be better equipped to carry out TLC experiments. Rather than examining solvents in the “wet” laboratory, by using Spartan ’02, a software package that calculates the structure and properties of compounds. 23 | P a g e
  24. 24. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] SUMMARY:Thin layer chromatography involves the use of thin layer of some adsorbent as stationary phase coated on some inert support (such a glass or plastic sheet), while the solvent, the mobile phase percolates through the layer. Mixture to be analysed is deposited near the bottom of plate. The solvent is made to move up the plate. As it passes over the deposit various components of the mixture are carried up and deposited as separate spots on the plate. The adsorbent commonly used are silica gel, alumina, Kieselguhr, Cellulose powder etc. while the choice of solvent depends on the solubility's of constituents of the mixture. The Rf values of various constituents may be calculated from the distances traversed by the spot and the solvent from the place of origin or the point where the mixture is deposited. Thin layer chromatography, is therefore, similar to paper chromatography. Instead of filter paper, a thin layer of some adsorbent is used as the stationary phase. Thin layer chromatography has many advantages over paper chromatography. The technique is less time consuming and convenient. There is a greater sharpness of separation and a higher sensitivity. The spots can be scrapped off with the help of a knife dissolved and analysed quantitatively by using suitable analytical techniques. Thin layer chromatography has proved to be very useful in the analyses of adulterated food stuffs, determination of organomercurials in fishes, phenolics and aromatic alcohols in beer, organ chlorine and carbamate pesticides in fruits and vegetables. 24 | P a g e
  25. 25. November 8, 2013 [THIN LAYER CHROMATOGRAPHY] REFERENCE: A.I. Vogel, A.R. Tatchell, B.S. Furnis, A.J. Hannaford, and P.W.G. Smith. Vogel's Textbook of Practical Organic Chemistry (5th Ed.).  Scott, R. P. W. Techniques and Practices of Chromatography; 2nd Ed.; Marcel Dekker, 1995.  Joseph Sherma, Bernard Fried (1991): Handbook of Thin-Layer Chromatography Marcel Dekker, New York.  Plant drug analysis: A Thin Layer Chromatography Atlas by Hildebert Wagner, 2nd Edition. 25 | P a g e