Chromatography, thin layer

Ion chromatography separation of anions. (Courtesy of Hewlett-Packard Company.) 

Thin-layer chromatography (TLC) is a planar form of chromatography useful for wide-scale qualitative analysis screening and can also be used for quantitative analysis. The stationary phase is a thin layer of finely divided adsorbent supported on a glass or aluminum plate, or plastic strip. Any of the solids used in column liquid chromatography can be used, provided a suitable binder can be found for good adherence to the plate. 

Development times of 5 min can be accomplished by using small microscope slides for TLC plates, and preliminary separations with these can be conveniently used to determine the optimum developing conditions. Typically, sample sizes range from 10 to 100 /xg per spot (e.g., 1 to 10 /xL of a 1% solution). Sample spots should be 2 to 5 mm in diameter. 

If the solutes fluoresce (aromatic compounds), they can be detected by shining an ultraviolet light on the plate. A pencil line is drawn around the spots for permanent identification. Color-developing reagents are often used. For example, amino acids and amines are detected by spraying the plate with a solution of ninhydrin, which is converted to a blue or purple color. After the spots are identified, they may be scraped off and the solutes washed off (eluted) and determined quantitatively by a micromethod. 

Frequently, colorless or nonfluorescent spots can be visualized by exposing the developed plate to iodine vapor. The iodine vapor interacts with the sample components, either chemically or by solubility, to produce a color. Thin-layer plates and sheets are commercially available that incorporate a fluorescent dye in the powdered adsorbent. When held under ultraviolet light, dark spots appear where sample spots occur due to quenching of the plate fluorescence. 

Thin-layer LC has been used for the determination of alkylated cresols and amine antioxidants [130, 131] in polybutadiene, phenolic antioxidants in polyethylene [132-134] and PP [134], dilauryl and distearyl thiodipropionate antioxidants in polyolefins, ethylene-vinyl acetate copolymer, acrylonitrile-styrene terpolymer and PS, UV absorbers and organotin stabilisers in polyolefins [130], and accelerators such as guanidines, thiazoles, thiurams, sulfenamides, dithiocarbamides, and morpholine disulfides in unvulcanised rubber compounds. 

Thin-layer chromatography (TLC) has passed its heyday as an analytical procedure and has been surpassed by more sensitive and automatic methods, but it still has some advantages in the analysis of complex lipids. Despite its decrease in usefulness, because of the simple realization and low-cost apparatus, some examples for TLC are given in this chapter. 

Thin-layer chromatography (TLC) as an easy to carry out analytical technique was evolved more than 30 years ago. The method has found wide-ranging applications in the separation and semiqantitative determination of both organic and inorganic compounds present in very low quantities in complicated accompanying matrices. In recent years the use of different TLC techniques has been markedly enhanced. This development may be 

Thin Layer Chromatography is a valuable analytical technique. It is cheap, fast and simple. Optimization of TLC is therefore of the highest importance and subject of many studies. A review of optimization methods is given by Nurok [1]. The aim of such optimizations is to find a mobile phase composition at which a good separation of all solutes is possible. However, not only the mobile phase has influence on the retention time, but also the temperature and the relative humidity. 

Temperature and relative humidity cause problems because they are difficult to control except when special equipment is used. The variation depends on the weather and the quality of the climate control, but they do vary. Their effect on the retention is different for different solutes. Therefore the resolution can change. 

Thin Layer Chromatography (TLC) is methodological simple. The solutes travel different distances with a mixture of solvents, the mobile phase, along 

TLC differs in many aspects from High Performance Liquid Chromatography (HPLC). The first difference is that the solutes are not separated over a fixed length (the separation column) but during a fixed time (the development time). Therefore, the chromatographic behaviour is not characterized by the time needed to traverse the column, but by distance travelled within a certain time span. A second difference is that the composition of the mobile phase may vary over the length of the plate. More volatile components may vaporize causing a different composition at different places on the TLC plates. 

Since the retention is measured as distance in stead of time, the / y-value, which expresses the position of a substance on a developed plate, should be calculated differently than retention measures are calculated in HPLC. First we introduce the distances which are of importance. These are Zp the distance between the solvent source and the solvent front, Zg the distance between the solvent source and the place where the solutes start and the distance between the start and final places of the solutes. From these distances it follows that  

Thin-layer chromatography (TLC), sometimes also called planar chromatography, employ a stationary phase immobilized on a glass or plastic plate and an organic mobile phase. It is a rather old technique whose application in residue analysis has been limited in the past by poor chromatographic resolution, inadequate selectivity, and insufficient sensitivity (49). This was due to inherent problems in the quality of the available stationary phase materials and in the uniformity of the layers prepared. Today, the availability of affordable, precoated plates with acceptable performance and consistency has led to the general acceptance of TLC as an efficient procedure for residue analysis (50). The method is used preferentially when analysts must process large numbers of samples in a short period of time (51). 

In its conventional mode, capillary action TLC is a simple but versatile procedure that does not require expensive equipment. Therefore, TLC has a particular potential as a reliable technique for laboratories with very limited resources for instrumental equipment. The sample, either liquid or dissolved in a volatile solvent, is deposited as a spot on the stationary phase. Standards are also applied on the layer to be simultaneously run with the unknown sample for identification purposes. Volume precision and exact positioning are ensured by the use of a suitable instrument. The bottom edge of the plate is placed in a solvent reservoir, and the mobile phase moves up the plate by capillary action for a predetermined distance. In this process, the different components of the sample migrate up the plate at different rates due to differences in their partitioning behavior between 

Two-dimensional TLC uses the same liquid chromatographic procedure twice to separate spots that are unresolved by only one process. After a sample is run in one solvent, the TLC plate is removed, dried, rotated 90 degrees, and run in another solvent. Any of the spots from the first run that contain mixtures can now be separated. The finished chromatogram is a two-dimensional array of spots. 

Normal-phase adsorption on silica gel with a relatively less polar mobile phase is the most widely used mode in conventional TLC. To improve separations, silica gel may be impregnated with various solvents, buffers, and selective reagents. Other commercial precoated layers include alumina, florisil, polyamide, cellulose, and ion exchangers. 

Conventional TLC should be considered for applications in which many samples are to be analyzed because it is cost-effective and environmentally friendly. On a per sample basis, it uses typically 5% of the solvent consumption of LC. Sample cleanup is usually simple or not required at all. Because stationary phases are used only once, it is often possible to apply relatively crude samples including those containing irreversibly sorbed impurities. However, impurities that comigrate with the analyte can adversely affect its detection and thus should be removed prior to TLC (53). 

Thin layer chromatography, often called film-development chromatography, is the simplest, quickest to perform, and cheapest type of chromatography, in comparison to gas chromatography and liquid chromatography. However, this method can only be used for qualitative analysis. This type of chromatography is very popular with crude oil geologists, because it can be used conveniently for 

Marcel Dekker, Inc. 270 Madison Avenue, New York, New York 10016 

Thin layer chromatography differs from all other types of chromatography discussed before by the simplicity of the technique used. Thin layer chromatograph has a stationary phase and a mobile phase just like every other chromatographic method. However, the stationary phase for thin layer chromatography is not located in the column as in gas or liquid chromatography. Instead, it is fixed on a glass, aluminum or plastic plate as a thin layer. 

Thin layer chromatography is very similar to paper chromatography. Thin layer chromatography has a wide variety of possibilities depending on the choice of the stationary phase. Adsorption, distribution and ion chromatography can be carried out in thin layer chromatography. 

The humidity that comes with the mobile phase filter paper ensures the evenness of the vapor pressure in the analysis chamber and acts as an accelerator for the achievement of saturated vapor with the mobile phase. The filter paper can also act as an indicator if the solvent front reaches the upper limit of the filter paper, it is usually postulated that the analysis chamber is ready for the commencement of the analysis. 

Thin-layer chromatography (TLC) is commonly used for the determination of radiochemical purity in nuclear medicine. TLC was described as early as 1967 for testing radiopharmaceuticals (Hoye 1967). Since the introduction of TLC (Izmailov and Shraiber 1938 Stahl 1956), a variety of modifications and new applications have been reported (Fairbrother 1984). 

The principle of this analytical method is that a mobile phase (solvent) moves along a layer of adsorbent (stationary phase) due to capillary forces. Depending on the distribution of components between the stationary and the mobile phase, a radioactive sample spotted onto the adsorbent will migrate with different velocities, and thus, impurities are separated. The distance each component of a sample migrates is expressed as the Rf value. The Rf is the relative migration of a component in relation to the solvent front (SF)  

The Revalues range from 0-1. If a component migrates with the SF, the Rf is 1. If a component remains at the point of application (origin), the Rf is 0. For a given TLC system, which is defined by the mobile and the stationary phases, the Rf value of a pure chemical compound is specific and reproducible. 

The main principles of separation are adsorption (electrostatic forces), partition (solubility), and ion exchange (charge). Information on the theoretical background of TLC is presented elsewhere (Miller 2004). Depending on the movement of the mobile phase, TLC may be ascending or descending in the nuclear medicine laboratory, ascending TLC is the method of choice (Robbins 1983). 

For the analysis of radiopharmaceuticals, techniques should be fast and safe. TLC offers reliable separation properties with easy and rapid performance. The applied sample remains quantitatively on the plate, and therefore, no losses of radioactivity during analysis occur. The commercially available ready-to-use stationary phases combine adsorbent with ionic or hydrophobic properties and are suited for separation of a variety of molecules using polar or nonpolar solvents. This chapter emphasizes methods for routine use in nuclear medicine and describes materials, techniques, and methods for quantification (Carpenter 1986 Hammermaier et al. 1986 Thoebald 1984). 

Thin-layer Chromatography. — A t.l.c. method for the analysis of eleven carbohydrates commonly found in foodstuffs has been described using silica gel plates, which are eluted three times with acetonitrile-water (85 15). The spots were visualized using a diphenylamine dip and estimated spectrophoto-metrically.  

Malonamide has been recommended as a spray reagent for the fluorimetric detection of reducing sugars on t.l.c. plates, and o-aminobenzene sulphonic 

likegami, A. Shono, T. Mega, T. Ikenaka, and Y. Matsushima, Anal. Biochem., 1979, 97,166 Chem. Abs., 1979, 91, 189018c). 

Thin-layer chromatography is frequently used to separate pyridine alkaloids from tobacco extracts. This method is frequently used by tobacco breeders to screen plant populations and individual tobacco plants to insure seed purity. Thin-layer is considerably faster than paper chromatography and can also be used as a preliminary cleanup for more sensitive methods for analytical analysis such as capillary gas chromatography (GC). Semi- 

Gas chromatographic analysis of tobacco alkaloids does not require derivation. The general procedures for GC analyses are as follows (1) use the smallest sample applicable for analysis (2) utilize preparations that clean up the sample without loss of alkaloids (3) pre-extract the sample prior to alkaloid extraction with hexane (removes pigments and lipids) (4) extract alkaloids from tobacco with an aqueous acid solution, filter, partition the aqueous acid extract with organic solvent (hexane, methylene chloride, chloroform, etc.) (5) increase pH of the aqueous extract to pH 10 (5N NaOH) and partition the basic solution with chloroform (6) dry the chloroform solution over Na2S04, and (7) concentrate the sample or analyze as is. 

Standardization of GC parameters (injector temperatures, linear gas flows, column temperature, detector temperature) are required before analysis is performed. Detector response should be determined for each 

The oven temperature is programmed from 170 to 200°C at 4°C/min. Column linear gas flows were calibrated to 20cm/s and analysis was complete after 15 min. Anabasine and myosmine were not completely resolved. Myosmine was not present in many samples and usually occurs in concentrations too low to be detected. This method is applicable to green or cured samples. 

The use of HPLC for separation of tobacco alkaloids has been limited to few studies (Court 1986). Saunders and Blume (1981) developed a procedure for analyses of nicotine, nornicotine, anabasine, and anatabine in tobacco. This method utilized a pBondapak Cig reversed-phase column. The mobile phase of 40% methanol containing 0.2% phosphoric acid was adjusted to pH 7.25 with triethylamine. Retention times with 0.5ml/min flow rates were 23 min for nicotine, 13.9min for anatabine, 12.2min for anabasine, and ll.Omin for nornicotine. This method is capable of analyzing samples containing 40-50 ng of alkaloid, and quantitative results were reliable. The column was stable for 800-1000 injections before it showed appreciable degradation in separation but was still capable of separating nicotine and nornicotine. 

Thin-Layer Chromatography.- A separation of 2-deoxy-2-fluoro-D- 

Thin-layer chromatography (TLQ is closely related to column chromatography, in that the phases used in both techniques are essentially identical. Alumina and siHca gel are typical stationary phases, and the usual solvents are the mobile phases. There are, however, some distinct differences between TLC and column chromatography. The mobile (liquid) phase descends in column chromatography the mobile phase ascends in TLC. The column of stationary-phase material used in column chromatography is replaced by a thin layer (100 (xm) of stationary phase spread over a flat surface. A piece of window glass, a miCTOscope sUde, or a sheet of plastic can be used as the support for the thin layer of stationary phase. It is possible to prepare your own glass plates, but plastic-backed thin-layer plates are only commercially available. Plastic-backed plates are particularly attractive because they can easily be cut with scissors into strips of any size. Typical strips measure about 1X3 in., but even smaller strips can be satisfactory. 

Thin-layer chromatography has some distinct advantages it needs little time (2- 5 min) and it needs very small quantities of material (2-20 xg). The chief disadvantage of this type of chromatography is that it is not very amenable to preparative scale work. Even when large surfaces and thicker layers are used, separations are most often restricted to a few milLigrams of material. 

Do not touch the active surface of the plates with your fingers. Handle them only by the edge. 

Step 1. Draw a light pencil line parallel to the short side of the plate, 

5-10 mm from the edge. Mark one or two points, evenly spaced, on the line. 

Thin-layer chromatography (TLC), like GC, comes into its own when dealing with mixtures of substances. TLC using plates coated with 250 pm absorbent is an excellent technique for separating quantities of up to 20 mg of additive mixtures into their individual components 

Thin Layer Chromatography.—Silica gel compacted to a density comparable with that of h.p.l.c. columns allowed good separations of a range of malto- 

Thin-layer Chromatography.— Improved or more-rapid separations by t.l.c. have been reported for mono-, di-, tri- and oligo-saccharides, naturally occurring pentitols and hexitols, iridoid D-glucopyranosides, methylated nucleosides, and D-arabinosyl-, o-ribosyl-, and 2-deoxy-D-ery Aro-pentosyl-nucleotides.  

A simple system for the computerized storage of information obtained by t.l.c. and paper chromatography has been devised.  

Thin-layer Chromatography.—A useful discussion of the factors (e.g. solvents and sorbents) affecting the t.l.c. of carbohydrates has appeared.  

on cellulose has been used to separate mixtures of monosaccharides. Other separations accomplished by t.l.c. include those of the steroidal glycosides (saponins) from Agave americana L., [ H]inositol and [ H]inositol monophosphates (on sheets of silica gel-glass fibre), purine nucleoside 3, 5 -phosphates from their respective nucleoside 5 -phosphates and nucleosides [on plastic-backed sheets of (polyethyleneimine)cellulose], 2 -deoxythymidine and the corresponding 5 -mono-, 5 -di-, and 5 -tri-phosphates [on plastic-backed sheets of (polyethyleneimine)cellulose], 2 -deoxy-5-halogenouridines, and erythromycin stearate and allied antibiotics.  

Thin layer chromatography (TLC) has been used in the identification of isoflavonoids. Precoated polyamide 6 TLC plates have been used for the initial fractionation of isoflavones and phenolics from soybeans and soy products (Pratt and Birac, 1979). 

TLC-silica (straight phase) and rechromatography on RP octadecylsilica column HPLC have been used to fractionate isoflavonoids from diethyl ether extracts of beer (Lapcik et al., 1998). The fractions are analyzed by radioimmunoassay specific for daidzein/formononetin and genistein/biochanin A. The diethyl ether extract is chromatographed on a Merck PSC silica plate along with standards of daidzein, formononetin, genistein, and biochanin A in separate tracks. The plate is developed 

Compound TLC System VRf TLC System R( HPLC System U Retention Time (min) 

Source From Lapcfk, O., Hill, M., Hampl, R., Wahala, K., and Adlercreutz, H., Steriods, 63, 14-20, 1998a. 

the chromatographic mobility as the ratio of the distanee of the center of the spot from the start to the distance between the start and the solvent front. 

Thin Layer Chromatography.- The first separation of all the constituent sugars (seven neutral, two acidic) of plant cell-wall polysaccharides on a single chromatogram was achieved by two-dimensional t.l.c. on cellulose. The values for 12 sugars in 15 solvent 

Thin-layer chromatography is a technique where the components of mixtures separate by differential migration through a planar bed of a stationary phase, the mobile phase flowing by virtue of capillary forces. The solutes are detected in situ on the surface of the thin-layer plate by visualizing reagents after the chromatography has been completed. 

A variety of finely-divided particulate sorbents are used as thin-layer stationary phases. These include silica-gel, cellulose powder, ion-exchange resins, restricted pore-size materials, and chiral selectors. 

Single solvents or blends of two or more solvents having the appropriate overall polarity necessary to achieve the required separation are used as mobile phases. They range from nonpolar hydrocarbons to pxrlar alcohols, water, and acidic or basic solvents. 

Methods of visualizing solutes include spraying the surface of the thin-layer plate with a chromogenic reagent, or viewing it under a UV lamp if the sorbent has been treated with a fluorescent indicator. 

Alternative development procedures aimed at improving chromatographic performance have been introduced, and new stationary phases are becoming available. 

3 Thin Layer Chromatography. - Complex mixtures of phosphorus-32 postlabelled DNA adducts have been analysed by high-resolution anion-exchange and partition thin-layer chromatography.  

5 Thin Layer Chromatography-.f This name (abbreviated TLC) is applied to the thin layer versions of the column techniques described in Secs. 2.6.1 to 2.6.3, other than paper chromatography. The stationary phases (except cellulose) mentioned in these sections lack the strength of paper and require the support provided by a glass plate or polyester film. 

The commonest stationary phases for TLC are silica gel and alumina, the latter usually mixed with calcium sulfate (plaster) as a binder. Ion-exchange and gel filtration beads are available in finer mesh sizes for TLC. 

Rolls and sheets of thin layers on polyester films can be purchased ready to use, or glass plates can be coated in the laboratory. For small scale experiments, a microscope slide can be coated by simply dipping it into a slurry of stationary phase (e.g., 30% silica gel in chloroform). To coat large plates uniformly on one side, a spreader—either commercial or constructed by putting two collars of adhesive tape or rubber tubing around a glass rod—is used (Fig. 2-35). The spreader is drawn or rolled along in one motion to produce a uniform layer of stationary phase about 0.3 mm thick. The coated plate is in most cases air dried and then oven dried before use. 

Samples are spotted on the plate, which is developed in a solvent chamber as described for paper chromatography. The solvents used for TLC are the same ones used in column chromatography on the same stationary phase. 

To permit visualization of the spots, the plate is often sprayed with something e.g., silica gel plates show spots for most organic substances when the plates are sprayed with a methanol solution of sulfuric acid and then baked in an oven. A permanent record can be made by photographing the plates in color before the colors fade. An alternative method for detecting spots consists of mixing about 1% of a fluorescent or phosphorescent substance (sold by companies which handle TLC accessories) with the adsorbent 

Technique of thin-layer chromatography. Preparation of the plate. In thin-layer chromatography a variety of coating materials is available, but silica gel is most frequently used. A slurry of the adsorbent (silica gel, cellulose powder, etc.) is spread uniformly over the plate by means of one of the commercial forms of spreader, the recommended thickness of adsorbent layer being 150-250 m. After air-drying overnight, or oven-drying at 80-90 °C for about 30 minutes, it is ready for use. 

Ready to use thin-layers (i.e. pre-coated plates or plastic sheets) are commercially available the chief advantage of plastic sheets is that they can be cut to any size or shape required, but they have the disadvantage that they bend in the chromatographic tank unless supported. 

care should be exercised in handling the plate to avoid placing fingers on the active adsorbent surface and so introducing extraneous substances  

pre-washing of the plate is advisable in order to remove extraneous material contained in the layer, and this may be done by running the development solvent to the top of the plate. 

Development of plates. The chromatogram is usually developed by the ascending technique in which the plate is immersed in the developing solvent (redistilled or chromatographic grade solvent should be used) to a depth of 0.5 cm. The tank or chamber used is preferably lined with sheets of filter paper which dip into the solvent in the base of the chamber this ensures that the chamber is saturated with solvent vapour (Fig. 8.6). Development is allowed to proceed until the solvent front has travelled the required distance (usually 10-15 cm), the plate is then removed from the chamber and the solvent front immediately marked with a pointed object. 

Fried and J. Sherma, Thin-Layer Chromatography Techniques and Applications, M. Dekker, New York, NY (1999). 

Soczewinski (ed.), Planar Chromatography (TLC) - Handbook of Thin Layer Chromatography, Harwood Academic Publishers, Amsterdam (1998). 

Koch and S. Hofstetter-Kuhn, Dunnschicht-chromatographie, Springer-Verlag, Berhn (1996). 

Fischer and H. Wimmer, Thin-layer Chromatography, Vol. lb, Reagents and Detection Methods, VCH, Weinheim (1994). 

The solvents used in thin layer chromatography (TLC) can be classified by the same methods as used for stationary phases in gas chromatography. The separation characteristics of parallel TLC runs with different solvents should differ as much as possible. Massart et. al. C67, 68, 1873 applied pattern recognition methods (numerical taxonomy and cluster analysis) for an objective characterization of the solvents. 

One application deals with the selection of optimum solutes for a TLC separation of 26 synthetic food dyes. Hierarchical clustering was employed to reduce the number of solutes from ten to four C1873. 

Commercially available thin-layer chromatography equipment is shown in Fig. 4.2S. It allows the determination of absorbent and/or fluorescent products after a small part of the photosolution has been extracted and applied onto a thin-layer plate which is developed by a suitable eluent. This is an interactive process comparable with early techniques in kinetics where small portions of the reaction solution were frozen and conventionally analysed afterwards off-line. 

Since the determination of peak areas, peak detection, and baseline correction are well established methods, the determination of concentration is a smaller problem and results in values with small standard deviations. Nevertheless, TLC is not suitable for automation in its application to photokinetics since the photoactivation of the sample by the chromatographic material is not negligible [108]. The light paths for reflection and fluorescence measurements are given in principle in Fig. 4.27. 

The result of an examination using a TLC device with spectral detection using a photodiode array is given in Fig. 4.27. The advantage is the combined observation of absorption and fluorescence spectra using a xenon light 

Laser dyes are assumed to be relatively photostable. However, as Fig. 4.28 shows, the spectra change within a few seccmds. Under these conditions 7,7 -diethylamino-4-methylcoumarine (DEMQ undergoes jJiotodegrada-tion. The different photodegradation products demonstrate a change in absorption and fluorescence maxima. In addition, their fluorescence quantum yields differ. 

This specific technique allows the determination of the different reactants and metabolites for different reaction conditions. However, in many cases the rates of photoreaction jM ocesses are not compar le to those in solution due to the photocatalytic behavicmr of the soibent matmal. Detectivity and limit of determination can be optimised by pixel bunching of the diode array. It measures concentraticms as low as in the case of monochromatic detection using commercial equipment [108]. 

As described in Sec. 5.3.3, thin layer chromatography could be used in the purification of HP-j8-CDs. Nevertheless, thin layer chromatography could be used in qualitative analysis of hydroxypropyl derivatives of CDs including composition and DS. Thin layer board is a sihca gel plate which can be purchased or prepared in lab. The formula for developing are propanol, ethyl acetate, aqua ammonia and water are mixed at a ratio of 6 1 1 3, or acetonitrile, water, aqua ammonia are mixed at a ratio of (6 3 1). The latter formula has a shorter developing time. The color development reagent cerous sulfate 1 g, ammonium molybdate 24 g, concentrated sulfuric acid 50 mL are dissolved in 450 mL deionized water. 

The principle of what we now know as thin layer chromatography (TLC) was proposed first by N. Izmailov and 

Shaiber in 1938. They dusted AljOj (alumina) on glass plates. The particles were uniform and inert to acids, but the dust would blow or wash away unless extreme care was taken, so a photograph of the results had to be obtained. There was no way to store the plates. 

The mechanism for the separation is most likely a combination of adsorption and partition. If essentially round spots form that are separated from each other, then the separation is primarily partitioning. If tailing occurs, then adsorption is a major factor. 

Most commercially prepared plates are 20 cm x 20 cm and are coated with a 0.25-mm-thick layer of adsorbent. The plate backing is usually glass, plastic, or aluminum. The coating is usually silica gel (Si02) or alumina (AljOj) cellulose, modified cellulose, kieselghur, ion exchange resins, and octyldecylsilane on glass beads are used less often. 

Aluminum plates are cut easily with a pair of scissors into any size, and alumina appears to bind well to this 

Currently, most planar chromatography is based on the thin-layer technique, which is faster, has better resolution. and is more sensitive than its paper chro- 

In terms of theory, types of stationary and mobile phases, and applications, thin-layer and I.C are ro markably similar. TLC techniques in fact have been used to develop conditions for IfPl.C separations. At one time TLC methods were widely used in the pharinaccuiical industry, Today, such techniques have largely been replaced by I.C methods, which are readily automated and faster. Thin-layer chromatography has found wide.sprcad use in clinical laboratories and is the backbone of many biochemical and biological studies. It also (inds extensive use in industrial laboratories. fk cause of these many area.s of application, TLC remains a very important technique. 

A thin-layer plate is prepared by spreading an aqueous slurry of the finely ground solid on the clean surface of a gla.ss or plastic plate or microscope slide. Often a binder is incorporated into the slurry to enhance adhesion of the solid particles lo the glass and to one another The plate is then allowed to stand until the layer has set and adheres tightly to the surface for some purposes. it may be heated in an oven for several hours. Several chemical supply houses offer precoaied plates of various kinds. C osis arc a few dollars per plate. The 

FtGURC 28-29 (a) Ascendrng-flow developing chamber, (b) Horizonlal-flow developing chamber, in which samples are placed on both ends of the plate and developed toward the middle, Ihus doubling ihe number of samples that can be accommodated. 

Sample application is perhaps the most critical aspect of thin-layer chromatography, particularly for quantitative measurements. Usually, the sample, as a 0.01% lo 0.1 % solui ion, is applied as a spot I to 2 cm from the edge of the plate. Tor best separation efficiency, the spot should have a minimal diameter — about. 5 mm for qualitative work and smaller for quantitative analysis. For dilute solutions, three or four repetitive applications are used, with drying between. 

Patrikeev et al. also employed imprinted silicas for thin layer chromatography. The silica was mixed with plaster and immobilised on a plate for use in the separation and identification of gramines [31] and for the resolution of amino acid derivatives [29]. In the latter experiment it was found that the influence of the amino acid protective group, dinitrophenol, was too dominant to allow the separation of 

Independently, Erlenmeyer and Bartels performed similar studies using starch as the immobilising agent [56]. They demonstrated that imprinted silicas could be used to distinguish between dimethyl- and diethyl-aniline, a distinction that the native silica was unable to make in the solvent mixture used (ethyl acetate/methanol/ HO Ac (5 M) 60 30 10). 

Although not a very widely used technique in this application, thin-layer chromatography (TLC) has been used to analyze evolved gaseous products and also for kinetics studies. Permanent-type gases, of course, cannot be handled by this technique, but high molecular weight compounds, which may be difficult to identify by other methods, can be separated and characterized. In addition, the equipment required for TLC is much less expensive than that required for any of the other methods. 

A developed TLC plate for TNT (93) showed that the TNT and volatile impurities begin to vaporize and appear on the TLC plate between 125 and 135°C, corresponding to the first appearance of gas in the pyrolysis curve. Most of the TNT vaporizes, and is collected undecomposed since it is a relatively stable compound thermally. Within the temperature range where TNT dissociates exothermally, as indicated by the DTA curve, the following products appear 2,4.6-trinitrobenzyl alcohol ITNB-OH) 4.6-dinitroan-thranil (DNA) 1.3.5-trinitrobenzene (TNB) 2.4,6-trinitrobenzoic acid (TNB-a) and a trace of an unidentified compound. The combination of the precision of the TLC method with the characteristic colors of Ihe spray reagent make it relatively simple to identify all the major components found. 

The TLC technique has also been applied to the study of reaction kinetics by Rogers and Smith (94). 

Stahl (95) described a pyrolysis procedure that also used TLC to identify 

The simplest and most universal method of reaction monitoring is thin layer chromatography (tic) and this will be discussed first of all, but it is not always the best or only method, and sometimes you may have to use a little ingenuity to find an appropriate reaction monitoring technique. 

Tic is a simple, but extremely powerful analytical tool. However it may take a little time before your expertise reaches a consistently high level, since a certain amount of intuition is always involved in choosing the appropriate solvent system, spotting the correct amount of sample, etc. Once you have gained experience and confidence in the use of tic, you will find it extremely useful for a variety of purposes. 

Tic is normally the simplest and quickest way to monitor a reaction and the reaction mixture should be chromatographed against starting materials (and a co-spot). This allows you to follow how the reaction is progressing, and to assess when is the best time to work it up. In all cases a record of the tic should be made in your lab book (see Chapter 2). 

Tic can be used to indicate the identity of a compound, by comparing the unknown sample with a known material. In general each substance is spotted separately and also together (co-spot). Caution should be applied as co-running on tic is not definitive proof of identity. Of course, substances that do not co-nm are definitely not the same. 

Tic usually gives a good indication of the purity of a substance. Diastereoisomers can usually (but not alivays), be separated. 

At some point in the near future you should watch the video entitled Thin-Layer Chromatography in the multimedia activity Practical techniques on the Experimental techniques CD-ROM that accompanies this book. This activity should take approximately 5 minutes to complete. 

A difficult choice is always that of which solvent to use to run or elute the TLC plate. There is no easy answer to this question. Chemists choose using a combination of experience and trial-and-error they would normally run several TLC plates, each using a different solvent or mixture of solvents, and find which gives the best separation. Table 2.2 lists common solvents in order of increasing polarity, as a guide to solvent selection. 

The main drawback with TLC, is that it can only be performed on a very small scale, and so is not useful for separating the entire reaction mixture into its various components — for this we need a large-scale version of TLC, which we discuss in the next section. Now try doing a TLC experiment for yourself in the next Computer Activity. 

COMPUTER ACTIVITY 2.5 Thin-layer chromatography In use an application from the food industry 

At some point in the near future you should watch the video entitled Thin-layer chromatography in use an application from the food industry in the multimedia activity Practical techniques on the Experimental techniques CD-ROM that accompanies this book. There you will see an experiment on the separation of food colourings. At various times you will be asked to take notes or make measurements from the screen, so you should make sure that you have an experiment notebook and pen to hand. This activity should take about 

This technique has been used extensively for the separation and determination of mixtures of compounds in water by migration on thin layers, usually, of silica or alumina. In the ceise of volatile compounds, such as aliphatic hydrocarbons, care is needed as volatiles may be lost during the 

Complementary to paper chromatography, but used more frequently because of the wide availabihty of stationary phases, is thin layer chromatography (TLC). It is simple, fast, reproducible and can achieve high resolution. It is usually performed on a square plate or on strips. A variation of this type of planar chromatography is carried out on a rotating circular plate using an instrument called a Chromatotron. 

A common, simple, inexpensive and relatively fast method for the separation of phenolic compounds from a mixture is thin layer chromatography (TLC). A small amount of the extract (40-100 pi) is applied approximately 2 cm from the bottom of a thin layer chromatography 

The TLC plate is then placed in a glass container with a solvent filled to approximately 1 cm from the bottom. The solvent will move to the top of the TLC plate as a result of capillary action. Since each compound in the mixture will have a unique way of interacting with the matrix and the solvent, some compounds will move faster towards the top of the TLC plate than others. The it /-value is the ratio of the distance of the compound has migrated divided by the distance the solvent has migrated, and has by definition a maximum value of 1. The -value tends to be constant for a given combination of compound, solvent, and matrix so that comparisons can be made between separations performed at different times. If a given compound is colored, it is easy to determine the / -value. For non-colored compounds staining methods are available (see section 1.3). 

The identification of phenolic compounds separated by TLC is somewhat challenging. The most common strategy is to include a set of reference compounds on the TLC plate. These compounds are applied individually, and if the mixture contains any of the reference compounds, they can be identified based on the i /-value. Note that this approach always leaves some room for uncertainty, because two different compounds can have the same / /-value. Further characterization is necessary to establish compound identity with more confidence. This can be achieved by scraping off the area on the TLC plate where the compound of interest has migrated to, followed by solvent extraction of the matrix, and more detailed chemical analyses, such as, for example gas chromatography-mass spectrometry or mass spectrometry (see Section 1.5 and Chapter 5). 

Below is a step-by-step protocol for TLC. In this example the goal is to separate anthocyanins isolated from flower petals. 

Pick the petals and place them in 1 ml methanol acidified with 0.1 or 1% (v/v) HC1. 

In spite of its indisputable simplicity and rapidity, this technique is now largely obsolete for analyzing such complex mixtures like essential oils, due to its low resolution. However, for the rapid investigation of the essential oil pattern of chemical races or the differentiation of individual plant species, this method can stiU be successfully applied (Gaedcke and Steinhoff, 2000). In addition, silver nitrate and silver perchlorate impregnated layers have been used for the separatiou of olefiuic compounds, especially sesquiterpene hydrocarbons (Prasad et al., 1947), and more recently for the isolation of individual sesquiterpenes (Saritas, 2000). 

Handbook of Essential Oils Science, Technology, and Applications 

FIGURE 2.1 Comparison of conventional and fast GC separation of lime oil. (From Mondello, L. et al., LC-GC Eur., 13, 495, 2000. With permission.) 

After successful application of enantioselective GC to the analysis of enantiomeric composition of monoterpenoids in many essential oUs (e.g., Werkhoff et al., 1993 Bicchi et al., 1995 and references cited therein), the studies have been extended to the sesquiterpene fraction. Standard mixtures of known enantiomeric composition were prepared by isolation of individual enantiomers from numerous essential oils by preparative GC and by preparative enantioselective GC. A gas chromatographic separation of a series of isolated or prepared sesquiterpene hydrocarbon enantiomers, showing the separation of 12 commonly occurring sesquiterpene hydrocarbons on a 2,6-methyl-3 pentyl-p-cyclodextrin capillary column has been presented by Kbnig et al. (1995). Further investigations on sesquiterpenes have been published by Kbnig et al. (1994). However, due to the complexity 

A large number of variously 2-, 4-, and 5-substituted thiazoles with alkyl, aryl, hydroxy, methylthio, mercapto, halo, and nitro groups have been analyzed by thin-layer chromatography on silica and alumina by the Stahl s technique (167, 170, 172). Among the many systems recommended for the elution of these compounds are the following  

Most of the thiazoles studied absorb in the ultraviolet above 254 nm, and the best detection for these compounds is an ultraviolet lamp (with plates containing a fluorescent indicator). Other indicator systems also exist, among which 5% phosphomolybdic acid in ethanol, diazotized sulfanilic acid or Pauly s reagent (Dragendorff s reagent for arylthiazoles), sulfuric anisaldehyde, and vanillin sulfuric acid followed by Dragendorff s reagent develop alkylthiazoles. Iodine vapor is also a useful wide-spectrum indicator. 

Linear relationships have been established on one hand between the Rf and pAa values of these azaaromatic bases (in the absence of steric hindrance of the ring nitrogen) and on the other hand, between the 

The steric effects of alkyl substituents (R= methyl, ethyl, i-propyl, f-butyl) on the nitrogen have been related to the steric factors of these same groups as measured in kinetic studies (152). 

Application of Snyder s theory of linear chromatographic adsorption (171) gives the variation in adsorption energy of the thiazole nitrogen atom as a function of this steric hindrance for silica and alumina (see Table III-22). These results show that alumina is more sensitive toward steric effects while silica shows a higher selectivity in the case of polar effects. 

Modem chromatographic methods which have caused such a tremendous development in chemical and biochemical analysis and in preparative separations, are unthinkable today without thin-layer chromatography (TLC), which is the subject of discussion of the present chapter. Its rapid development began in about 1958 mainly due to the work of Stahl who elaborated this method and standardized it in its present form. 

Included in this chapter is also a discussion of the modernised versions of liquid-solid column chromatography which have enabled this technique to hold its own against the competition offered by TLC, the latter having the advantage of being a convenient and rapid chromatographic technique. 

This technique may, in principle, be based on adsorption or partition, but usually its adsorption version is exmployed. Steps involved in TLC for separation and subsequent analysis (qualitative and quantitative) of the constituents of a given mixture are detailed below  

Like paper chromatography, thin-layer chromatography is a form of plane chromatography in that the stationary phase is held on a plane rather than in a column. Table 12.1 lists important stationary phases used in TLC along with the respective predominant sorption process operative with each of them. The solid phase is supported on to glass, metal or a plastic substance. (Microscope slides 

Alumina Silica gel Modified silica gel Kieselguhr Cellulose powder Modified cellulose, e.g. D E A E and C M Sephadex gels Adsorption or partition Adsorption or partition Adsorption, partition Partition Partition Ion-exchange Exclusion 

The first recorded works on TLC are those of Beyerinck in 1889 [1] and Wijsmann [2] in 1896 however it is generally accepted that it was Izmailov and Shraiber in 1938 [3] who enunciated the ideas and fundamental principles of using a chromatographic adsorbent in the form of a thin layer fixed on an inert rigid support. Meinhard and Hall [4] in 1949 developed this notion of an open column , and in 1951 Kirchner et al. [5] reported the separation of terpenes on a chromatostrip , prepared by coating a small glass strip with an adsorbent mixed with starch or plaster of Paris, which acted as a binder. 

Over the past decade there have been a number of further significant improvements in the technique. For instance, there are now a wide range of sorbents available in the form of pre-coated plates and the applicability of the technique has also been extended with the increasing range of bonded phase sorbents, e.g. reverse phases (Cg and Cig), those of medium-polarity (amino and cyano) and other specialised layers featuring chiral and mixed stationary phases. 

As well as being convenient these commercially produced plates give improved performance due to the narrower particle size range and reproducible physical properties such as surface area and pore size. For standard TLC, using silica gel, the particle size is between 5 and 17 pm, while typical pore size and layer thickness are 60 A and 0.25 mm, respectively. The use of a refined silica gel with a mean particle size of 5 pm and particle size range of 2-10 pm (c/. HPLC) has led to the development of high performance TLC (HPTLC) which uses thinner layers (100 pm) and requires smaller samples. 

Further advances have resulted from developments in instrumentation, particularly in the areas of scanning densitometry and automated sample application, which have now made fully instrumental quantitative TLC a reality far removed from the basic practice. TLC is now regarded as an indispensable tool in both quality control and research laboratories. The technique is easy to learn and is fast and versatile and in many instances may be preferred to the techniques of gas chromatography and high performance liquid chromatography. 

In the method, soil samples are extracted by shaking or vortexing with the solvent. Water samples are extracted by shaking in a separatory funnel. If there is a potential for the presence of compounds that interfere with the method and make the data suspect, silica gel can be added to clean the extract. Sample extract aliquots are placed close to the bottom of a glass plate coated with a stationary phase. The most widely used stationary phases are made of an organic hydrocarbon moiety bonded to a silica backbone. 

Eor the analysis of petroleum hydrocarbons, a moderately polar material stationary phase works well. The plate is placed in a sealed chamber with a solvent (mobile phase). The solvent travels up the plate, carrying compounds present in the sample. The distance a compound travels is a function of the affinity of the compound to the stationary phase relative to the mobile phase. Compounds with chemical structure and polarity similar to those of the solvent travel well in the mobile phase. For example, the saturated hydrocarbons seen in diesel fuel travel readily up a plate in a hexane mobile phase. Polar compounds such as ketones or alcohols travel a smaller distance in hexane than do saturated hydrocarbons. 

After a plate has been exposed to the mobile-phase solvent for the required time, the compounds present can be viewed by several methods. Polynuclear aromatic hydrocarbons, other compounds with conjugated systems, and compounds containing heteroatoms (nitrogen, oxygen, or sulfur) can be viewed with long-and short-wave ultraviolet light. The unaided eye can see other material, or the plates can be developed in iodine. Iodine has an affinity for most petroleum compounds, including the saturated hydrocarbons, and stains the compounds a reddish-brown color. 

The method is considered to be a qualitative and useful tool for rapid sample screening. Limitations of the method center on its moderate reproducibility, detection limits, and resolving capabilities. Variability between operators can be as high as 30%. Detection limits (without any concentration of the sample extract) 

Pour the appropriate developing solvent into a glass jar at least one hr before use. This is to saturate the jar with the running solvent vapors. 

Measure the distance of the solvent and the compound travelled to obtain the Rf values. 

Modern TLC can be conducted in both the normal-phase and the reversed-phase formats and can be extended to the separation of chiral compounds by modifying the stationary phase or mobile phase with chiral selectors. Using automated systems, performance equal to that achieved by HPLC is, in some cases, possible. Applications for quantitative analysis, including examples of stability-indicating and validated methods for pharmaceuticals, have been reviewed. 

To facilitate identification of separated bands, TLC/MS may be usedJ This approach may be as simple as removal of the band from the plate, extraction of the solvent into an appropriate solvent, and injection into the ion source of the MS. However, other approaches such as fast atom bombardment (FAB) and matrix- or surface-assisted laser desorption/ionization have also been used. 

frangulin A/E, glucofrangulin A/B, rhein, aloe-emodin and rhaponticoside (.stil-bene gliicoside) are applied a,s 0.1% meihanolic solutions. 

Chromatography is performed on silica gel 60F,j precoated plates (Merck, Germany). Adsorbent 

With the exception of Senna preparations, the solvent system is suitable for the 

UV 365 mil All anthracene derivatives give yellow or red-brown fluorescence 

After spraying with 5% or 10% ethanolic KOH, anthraqitinones appear red in the visible and show red fluorescence in UV-365 nm. 

TLC is still used in many pharmaceutical laboratories in either manual or semi-automatic operation on conventional, high performance or modified stationary phases [9, 10, 126]. TLC offers a quick, inexpensive, flexible and portable technique that has been the subject of some recent new developments. 

Despite these advancements, modern TLC has largely served as a complementary technique to other column-based liquid chromatographic methods such as HPLC. 

Advances in stationary phase technology have led to commercial availability of adsorbents such as high performance sihcas, aluminas, polyamides, celluloses and derivatised silicas [9,10], The development of automated method development (AMD) systems [127] now allow multi-step gradients of different elution strengths to be achieved in a relatively short time compared to earlier manual approaches. AMD systems are ideally suited for separation of complex mixtures with a wide range of polarities. Further improvements in sample resolution and reduced method development times in TLC include the use of two-dimensional development approaches [128] and forced-flow development by over-pressure liquid chromatography (OPLC) [129]. 

The main improvements in quahtative and quantitative detection systems in TLC are centred around the introduction of densitometry. These involve the use of slit scanning densitometers or video or CCD camera (image processing) [130]. 

Other method development approaches used in TLC include unidimensional multiple development [127] and multi-modal separation techniques [127], where TLC, in normal phase mode is used in conjunction with reversed-phase liquid column chromatography [131] or GC [131] to provide additional information in separations. This complementary strategy can prove very inportant even for well 

Polymer additives - mould release agents, plasticisers, antioxidants and UV absorbers, with molecular weights extending beyond 1000 - are generally unsuitable for GC or liquid chromatographic (LC) analysis because of their low volatility, lack of chromophore or thermal instability. 

The identification of mixtures of unknown additives in solvent extracts of polymers presents some difficult problems. The solvent extract is usually available in only fairly small quantities, often consists of a complex mixture requiring preliminary separation into pure components before identifications can be attempted, and is frequently a mixture of compounds of completely unknown type. 

Numerous works have been published on the experimental technique of TLC which will not be discussed further except in so far as is relevant to its application to additive identification. Dohmann [1] provides an excellent short review of techniques available. He discusses TLC in the normal sense of the word, i.e., with plate layers up to 250 pm thick and 20 cm x 20 cm or 20 cm x 8 cm in area and also discusses preparative layer chromatography which, with some loss in resolution, can separate considerably larger quantities of compounds on plate layers up to 2 mm thick and 100 cm x 20 cm in area. 

Halpaap [2] discusses in some detail the experimental techniqne of preparative layer chromatography with sample quantities between 0.1 and 100 mg, nsing plates up to 100 cm X 20 cm in area and adsorbent layers up to 2 mm thick. 

Quantity of substance mixture applied Several micrograms 0.1-50 g 1-250g 

TLC is the easiest and most economical technique of detecting limonoid aglycones. Unlike other methods, TLC can be used for a variety of tissue samples. TLC solvent systems were well developed and an appropriate system can be found for a particular type of tissue sample with just a few trials. The major disadvantage is the use of subjective visual comparison with standards for qualitation and quantitation. 

Maier and Grant (1970) extracted the fruit tissue-acid mixture, which was prepared as described in previous section, with chloroform. The chloroform extract was then evaporated to dryness and re-dissolved in acetonitrile. The acetonitrile sample was then spotted on silica gel TLC plates along with the appropriate standards, and developed in a benzene-ethanol-water-acetic acid solvent system (200 47 15 1, upper phase). 

Tatum and Berry (1973a) spotted 50-pl aliquots of commercial juice samples directly on a 20 X 20 cm silica gel G plate (Analtech Inc.) without extraction or other sample preparation. The plate was scribed into 1-cm channels prior to the spotting and standards of 0.1 through 0.5 p,g were spotted in the centre five channels of the plate. The plate was thoroughly dried with a heat gun and the dried plate was first developed with acetone to 3 cm mark from the applied spots. The acetone was used to extract limonin out from the dried juice solids. Then the plate was dried and developed in one of the 17 solvent systems listed in the paper. 

Since the solvent system which separates the limonoids in one sample may not be satisfactory for another, several solvent systems should be tried for a particular sample in order to find the one that has the best separation. For example, Fong et al. (1992) used solvent system (A) to quantify limonin and nomilin in the extract of Valencia orange flesh, and solvent system (C) to quantifjr deacetylnomilin in the same extract. For the analysis of the extract from Valencia orange peel, system (B) was used to quantify limonin, nomilin and deacetylnomilin. 

Final estimation of the limonoid spots in made by visual or spectrodensitometric comparison with the standards. 

To execute a TLC analysis, a small amount of the sample to be analyzed, or a solution of it, is first applied to a solid adsorbent bound to a rectangular glass or plastic plate (Fig. 6.2a). The adsorbent serves as the stationary phase. Next, the plate, with its spotted end down, is placed in a closed jar, called a developing chamber (Fig. 6.3). The chamber contains a saturated atmosphere of a suitable eluant or eluting solvent, which is the mobile phase and may be comprised of either a single solvent or mixture of two or more. A folded filter paper is often used to help maintain solvent equilibration in the chamber. It is important that the level of 

Thin-layer chromatography, (a) Original plate loaded with sample, (b) Developed chromatogram. 

TLC chamber, (a) Folded filter paper to be placed in developing chamber for solvent equilibration, (b) Developing chamber with filter paper and TLC plate. 

The solid adsorbent in TLC is usually alumina (AI2O3) or silica gel (silicic acid, Si02 X H2O), both of which are polar. Alumina is the more polar of the two and is commercially available in three forms neutral, acidic, and basic. Acidic and basic alumina are sometimes used to separate basic and acidic compounds, respectively, but neutral alumina is the most common form of this adsorbent for TLC. Silica gel, which is slightly acidic, is the adsorbent used in the experimental procedures described in this section. 

As you might imagine, consideration of the acidic or basic character of the solid adsorbent used for a TLC experiment can be particularly important if the substances to be analyzed contain functional groups that are sensitive to acids or bases. In a worst-case scenario, the adsorbent may function as a catalyst to destroy the functionality by chemical reaction during the course of the analysis this greatly complicates the interpretation of the TLC results. 

Szyfter and J. Chelkowski, Chem. Anal (Poland), 1976,21, 1267 (Chem. Abs., 1977,87,136 229k). 

Joseph C. Touchstone, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6080, United States 

The following constitutes a general guide to the use of thin layer chromatography (TLC). Pertinent reference sources on the subject include [1]-13]. The discussion here is focused on precoated plates, since these have largely supplanted homemade layers. For further information on the relationship between TLC and other types of chromatography - Basic Principles of Chromatography. 

The absolute configuration of numerous aliphatic lichen acids was determined by their CD spectra Huneck and Follmann (1967), Huneck et al. (1979), Huneck and Snatzke (1980b), Huneck and Hofle (1980), Huneck et al. (1983, 1986), and Huneck and Takeda (1992). 

The microcrystallization of lichen substances was developed mainly by Asahina (1936-1940) and Asahina and Shibata (1954). The microcrystal test can be performed on a microscope slide with a high degree of accuracy and speed. The substances are identified by their characteristic crystal formation in various reagents. Because of its simplicity, the method is rapidly accepted by lichenologists and has been used for chemo-taxonomic work on lichens. 

Micro spirit-lamp or micro Bunsen-burner 

GE acetic acid glycerol =1 3 An aniline glycerol ethanol = 1 2 2 oT o-toluidine glycerol ethanol =1 2 2 Py pyridine glycerol-.water =1 1 3 Q quinoline ethanol glycerol =1 2 2. 

Thin layer chromatography (TLC) is now the method of choice for the rapid determination and identification of lichen substances, and nu-merous papers have been published on this subject. Santesson (1967) reported the Rf values of about 80 lichen substances, and Yoshimura and Kurokawa (1977) investigated the sensitivity of TLC for several lichen substances lecanoric 

Preliminary ether extraction of dilaurylthiodipropionate (DLTDP) from the aqueous extractants. 

In this method the distilled water, sodium carbonate and 5% citric acid extractants (80 ml) are transferred to a separatory funnel. For the distilled water extractant only, solid sodium chloride (10%) is added to assist the extraction of DLTDP. The addition of sodium chloride to the distilled water extractant before ether extraction was essential in order to obtain good recoveries. Omission of the sodium chloride resulted in recoveries in the order of 5%. Addition of sodium chloride to citric acid solutions was deleterious the citric acid was salted out in the ether, and thus remained on evaporation of the ether. Some of this residue was dissolved along with the DLTDP and was present on the thin-layer chromatograph plate. 

Neutralisation of the citric acid before extraction resulted in low recoveries. The aqueous phase is extracted with several portions of diethyl ether which are subsequently combined and dried by shaking with anhydrous sodium sulfate. The ether extract is evaporated to dryness and the residue transferred with small volumes of ether to a 2 ml volumetric flask and made up to volume with ether. 

The ether alcohokwater extractant is evaporated to dryness on a water bath and transferred to a 2 ml volumetric flask as described previously. DLTDP is determined in the ether extracts as described next. 

The liquid paraffin extractant is diluted with petroleum ether 60 °C to 80 °C in a separatory funnel. The solution is then passed down a column of chromatographic grade silica gel which retains the DLTDP and allows liquid paraffin to percolate through the column. Petroleum ether is then passed through the column to remove the last traces of liquid paraffin. A mixture of chloroform and methanol is then poured down the silica column to desorb DLTDP which is quantitatively recovered in the column effluent. The total effluent is evaporated to dryness in a steam bath and the residue made up to a standard volume with diethyl ether. DLTDP is determined in the extracts as described next. 

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The reaction mixture in ethyl acetate is then transferred to a 100-ml reactor, purged under a nitrogen atmosphere, 340 mg of Lil is added, and the whole mass is then heated, with mechanical stirring, on an oil bath, up to ethyl acetate reflux temperature. The heating is continued for 5 hours, until the disappearance of the epoxide (II), as evidenced by the thin-layer chromatography. 

The infrared spectra of a set of 2-thiazolylthioureas are reported in Ref. 486. The ultraviolet spectra of l-aryl-3-(2-thiazolyl)thioureas are characterized by two bands of approximate equal intensity around 282 and 332 nm (492). For l-alkyl-3-(2-thiazolyl)thioureas these bands are shifted to 255 and 291 nm, respectively (492). The shape of the spectrum is modified further when l.l -dialkyl-3-(2-thiazolyl)thioureas are considered (491). Fragmentation patterns of various 2-thiazolylthioureas have been investigated (100, 493), some of which are shown in Scheme 158. Paper and thin-layer chromatography provide an effective tool for the analysis of these heterocyclic thioureas (494. 495). 

Most of the spectroscopic properties of 2-imino-4-thiazolines have been treated in Section II. Paper chromatography and thin-layer chromatography are particularly suitable for distinguishing 2-atnino-thiazoles from 2-imino-4-thiazolines their RfS and characteristic reactions are different (148, 494. 705). 

A-4-Thiazoline-2-thione and derivatives can be determined in mixtures as a result of sv Stematic studies on thin-layer-chromatography values... 

The nitration of the 2-anilino-4-phenylselenazole (103) is much more complicated. Even careful nitration using the nitrate-sulfuric acid method leads to the formation of a mixture of variously nitrated compounds in an almost violent reaction. By the use of column chromatography as well as thin-layer chromatography a separation could be made, and the compounds could be partly identified by an independent synthesis. Scheme 33 shows a general view of the substances prepared. Ring fission was not obser ed under mild conditions. 

V. Other physical determinations w. Thin-layer chromatography X. X-ray diffraction z. Optical properties... 

Thin-Layer Chromatography. Chiral stationary phases have been used less extensively in tic as in high performance Hquid chromatography (hplc). This may, in large part, be due to lack of avakabiHty. The cost of many chiral selectors, as well as the accessibiHty and success of chiral additives, may have inhibited widespread commerciali2ation. Usually, nondestmctive visuali2ation of the sample spots in tic is accompHshed using iodine vapor, uv or fluorescence. However, the presence of the chiral selector in the stationary phase can mask the analyte and interfere with detection (43). 

Antioxidants (qv) have a positive effect on oils when present in the proper concentration. Sterols and tocopherols, which are natural antioxidants, may be analy2ed by gas-Hquid chromatography (glc), high performance Hquid chromatography (hplc), or thin-layer chromatography (tic). Synthetic antioxidants maybe added by processors to improve the performance or shelf life of products. These compounds include butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), / fZ-butyUiydroquinone (TBHQ), and propyl gallate. These materials may likewise be analy2ed by glc, hplc, or tic. Citric acid (qv), which functions as a metal chelator, may also be deterrnined by glc. 

Thin-layer chromatography (tic) (16) is frequently used. The procedure allows for rapid screening for most dmgs of abuse using simple, inexpensive technology. A drawback to tic, however, is that the technique is not especially sensitive and low levels of dmgs may be missed. 

Refs. 293 and 294. Mobility ranking based on soil thin-layer chromatography (sdc). 

The total phosphoms content of the sample is determined by method AOCS Ja 5-55. Analysis of phosphoUpid in lecithin concentrates (AOCS Ja 7-86) is performed by fractionation with two-dimensional thin-layer chromatography (tic) followed by acid digestion and reaction with molybdate to measure total phosphorous for each fraction at 310 nm. It is a semiquantitative method for PC, PE, PI, PA, LPC, and LPE. Method AOCS Ja 7b-91 is for the direct deterrnination of single phosphoHpids PE, PA, PI, PC in lecithin by high performance Hquid chromatography (hplc). The method is appHcable to oil-containing lecithins, deoiled lecithins, lecithin fractions, but not appHcable to lyso-PC and lyso-PE. 

To determine the phosphoHpid and fatty acid compositions chromatographic methods (28) like gas chromatography (gc), thin-layer chromatography (tic), and high performance Hquid chromatography (hlpc) are used. Newer methods for quantitative deterrnination of different phosphoHpid classes include P-nmr (29). 

Finally, the techniques of nmr, infrared spectroscopy, and thin-layer chromatography also can be used to assay maleic anhydride (172). The individual anhydrides may be analyzed by gas chromatography (173,174). The isomeric acids can be determined by polarography (175), thermal analysis (176), paper and thin-layer chromatographies (177), and nonaqueous titrations with an alkaU (178). Maleic and fumaric acids may be separated by both gel filtration (179) and ion-exchange techniques (180). 

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