Thin-layer chromatography is a separation technique in which a stationary phase consisting of an appropriate material is spread in a uniform thin layer on a support (plate) of glass, metal or plastic. Solutions of analytes are deposited on the plate prior to development. The separation is based on adsorption, partition, ion-exchange or on combinations of these mechanisms and is carried out by migration (development) of solutes (solutions of analytes) in a solvent or a suitable mixture of solvents (mobile phase) through the thin-layer (stationary phase). Apparatus Plates The chromatography is carried out using pre-coated plates as described under Reagents.
Preconditioning of the plates It may be necessary to wash theplates prior to separation. This can be done by migration of an appropriate solvent. The plates may also be impregnated by procedures such as development, immersion or spraying. At the time of use, the plates may be activated, if necessary, by heating in an oven at 120 °C for 20 min. Chromatographic tank A chromatographic tank with a flat bottom or twin trough, of inert, transparent material, of a size suitable for the plates used and provided with a tightly fitting lid is required. For horizontal development the tank is provided with a trough for the mobile phase and it additionally contains a device for directing the mobile phase to the stationary phase. Micropipettes, microsyringes, calibrated disposable capillaries Or other application devices suitable for the proper application of the solutions. Fluorescence detection device To measure direct fluorescence or the inhibition of fluorescence. Visualisation reagents Suitable devices are used for derivatisation to transfer to the plate reagents by spraying, immersion or exposure to vapour and, where applicable, to facilitate heating for visualisation of separated components. Documentation A device may be used to provide documentation of the visualised chromatogram, for example a photograph or a computer file. Method Sample application Apply the prescribed volume of the solutions at a suitable distance from the lower edge and from the sides of the plate and on a line parallel to the lower edge; allow an interval of at least 10 mm (5 mm on high-performance plates) between the centres of circular spots and 5 mm (2 mm on high-performance plates) between the edges of bands. Apply the solutions in sufficiently small portions to obtain circular spots 2-5 mm in diameter (1-2 mm on high-performance plates) or bands 10-20 mm (5-10 mm on high-performance plates) by 1-2 mm. In a monograph, where both normal and high-performance plates may be used, the working conditions for high-performance plates are given in the brackets [ ] after those for normal plates. Vertical development Line the walls of the chromatographic tank with filter paper. Pour into the chromatographic tank a sufficient quantity of the mobile phase for the size of the tank to give after impregnation of the filter paper a layer of appropriate depth related to the dimension of the plate to be used. For saturation of the chromatographic tank, replace the lid and allow to stand at 20-25 °C for 1 h. Unless otherwise indicated in the monograph, the chromatographic separation is performed in a saturated tank. Apply the prescribed volume of solutions as described above. When the solvent has evaporated from the applied solutions, place the plate in the chromatographic tank, ensuring that the plate is as vertical as possible and that the spots or bands are above the surface of the mobile phase. Close the chromatographic tank, maintain it at 20-25 °C and protect from sunlight. Remove the plate when the mobile phase has moved over the prescribed distance, measured between the points of application and the solvent front. Dry the plate and visualise the chromatograms as prescribed. For two-dimensional chromatography, dry the plates after the first development and carry out a second development in a direction perpendicular to that of the first development. Horizontal development Apply the prescribed volume of the solutions as described above. When the solvent has evaporated from the applied solutions, introduce a sufficient quantity of the mobile phase into the trough of the chamber using a syringe or pipette, place the plate in the chamber after verifying that the latter is horizontal and connect the mobile phase direction device according to the manufacturer’s instructions. If prescribed, develop the plate starting simultaneously at both ends. Close the chamber and maintain it at 20-25 °C. Remove the plate when the mobile phase has moved over the distance prescribed in the monograph. Dry the plate and visualise the chromatograms as prescribed. For two-dimensional chromatography, dry the plates after the first development and carry out a second development in a direction perpendicular to that of the first development. Visual estimation Identification The principal spot in the chromatogram obtained with the test solution is visually compared to the corresponding spot in the chromatogram obtained with the reference solution by comparing the colour, the size and the retention factor (Rf) of both spots. The retardation factor (RF) is defined as the ratio of the distance from the point of application to the centre of the spot and the distance travelled by the solvent front from the point of application. Verification of the separating power for identification Normally the performance given by the suitability test described in Reagents (4.1.1) is sufficient. Only in special cases an additional performance criterion is prescribed in the monograph. Related substances test The secondary spot(s) in the chromatogram obtained with the test solution is (are) visually compared to either the corresponding spot(s) in the chromatogram obtained with the reference solution containing the impurity(ies) or the spot in the chromatogram obtained with the reference solution prepared from a dilution of the test solution. Verification of the separating power The requirements for the verification of the separating power are prescribed in the monographs concerned. Verification of the detecting power The detecting power is satisfactory if a spot or band is clearly visible in the chromatogram obtained with the most dilute reference solution. Quantitative measurement The requirements for resolution and separation are prescribed in the monographs concerned. Substances separated by thin-layer chromatography and responding to UV-Vis irradiation can be determined directly on the plate, using appropriate instrumentation. While moving the plate or the measuring device, examine the plate by measuring the reflectance of the incident light. Similarly, fluorescence may be measured using an appropriate optical system. Substances containing radionuclides can be quantified in 3 ways: either directly by moving the plate alongside a suitable counter or vice versa (see Radiopharmaceutical preparations (0125)), by cutting the plates into strips and measuring the radioactivity on each individual strip using a suitable counter or by scraping off the stationary phase, dissolving it in a suitable scintillation cocktail and measuring the radioactivity using a liquid scintillation counter. Apparatus The apparatus for direct measurement on the plate consists of: —a device for exact positioning and reproducible dispensing of the amount of substances onto the plate; —a mechanical device to move the plate or the measuring device along the x-axis or the y-axis; —a recorder and a suitable integrator or a computer; —for substances responding to UV-Vis irradiation: a photometer with a source of light, an optical device able to generate monochromatic light and a photo cell of adequate sensitivity are used for the measurement of reflectance or transmittance; if fluorescence is measured, a suitable filter is required to prevent light used for excitation from reaching the detector while permitting emitted light or a specific portion thereof to pass; —for substances containing radionuclides: a suitable counter for radioactivity. The linearity range of the counting device is to be verified. Method Prepare the solution of the substance to be examined (test solution) as prescribed in the monograph and, if necessary, prepare the reference solutions of the substance to be determined using the same solvent as in the test solution. Apply the same volume of each solution to the plate and develop. Substances responding to UV-Vis irradiation Prepare and apply not fewer than 3 reference solutions of the substance to be examined, the concentrations of which span the expected value in the test solution (about 80 per cent, 100 per cent and 120 per cent). Treat with the prescribed reagent, if necessary, and record the reflectance, the transmittance or fluorescence in the chromatograms obtained with the test and reference solutions. Use the measured results for the calculation of the amount of substance in the test solution. Substances containing radionuclides Prepare and apply a test solution containing about 100 per cent of the expected value. Determine the radioactivity as a function of the path length and report the radioactivity in each resulting peak as a percentage of the total amount of radioactivity. Criteria for assessing the suitability of the system are described in the chapter on Chromatographic separation techniques (2.2.46). The extent to which adjustments of parameters of the chromatographic system can be made to satisfy the criteria of system suitability are also given in this chapter. Additional points for monographs other than those from the European Pharmacopoeia When the method prescribed in a monograph carries the instructions ‘protected from light’ or ‘in subdued light’ it is intended that the entire procedure is carried out under these conditions. Unless otherwise indicated in the monograph, the mobile phase should be allowed to ascend 15 cm above the line of application. The phrase ultraviolet light (254 nm) indicates that the plate should be examined under an ultraviolet lamp having a maximum output at 254 nm (see below); other wavelength maxima may be specified. The term secondary spot means any spot other than the principal spot. Similarly, a secondary band is any band other than the principal band. Where a spraying technique is prescribed it is essential that the reagent is evenly applied as a fine spray. The following method of visualisation is used when directed in the monograph. Method I Spray the dried plate with ethanolic sulphuric acid (20%), heat at 105° for 30 minutes and immediately expose to nitrous fumes in a closed glass tank for 15 minutes (the nitrous fumes may be generated by adding 7M sulphuric acid dropwise to a solution containing 10% w/v of sodium nitrite and 3% w/v of potassium iodide). Place the plate in a current of warm air for 15 minutes and spray with a 0.5% w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride in ethanol (96%). If necessary, allow to dry and repeat the spraying. Materials The coating substances and precoated plates are described in Appendix I A: General Reagents. Prepare suspensions of the coating substances as recommended by the manufacturer unless otherwise prescribed. Commercial pre-coated plates may be used for pharmacopoeial tests where a coating substances is prescribed provided that they comply with the test for chromatographic separation described for the corresponding coating substance and with any additional test for verification of separating power required in the monograph test. Ultraviolet Ray Lamps for Analytical Purposes (Ph. Eur. text 2.1.3) Mercury vapour in quartz lamps is used as the source of ultraviolet light. A suitable filter may be fitted to eliminate the visible part of the spectrum emitted by the lamp. When the Pharmacopoeia prescribes in a test the use of ultraviolet light of wavelength 254 nm or 365 nm, an instrument consisting of a mercury vapour lamp and a filter which gives an emission band with maximum intensity at about 254 nm or 365 nm is used. The lamp used should be capable of revealing without doubt a standard spot of sodium salicylate with a diameter of about 5 mm on a support of silica gel G R, the spot being examined while in a position normal to the radiation. For this purpose apply 5 µl of a 0.4 g/l solution of sodium salicylate R in alcohol R 1 for lamps of maximum output at 254 nm and 5 µl of a 2 g/l solution in alcohol R1 for lamps of maximum output at 365 nm. The distance between the lamp and the chromatographic plate under examination used in a pharmacopoeial test should never exceed the distance used to carry out the above test. Identification of phenothiazines (Ph. Eur. method 2.3.3) Carry out the method for thin-layer chromatography protected from light using kieselguhr G as the coating substance. Impregnate the dry plate by placing it in a tank containing a shallow layer of a solution containing 10% v/v of 2-phenoxyethanol and 5.0% w/v of polyethylene glycol 300 in acetone so that the plate dips about 5 mm beneath the surface of the liquid and allowing the impregnating solvent to ascend at least 17 cm above the line of application. Remove the plate from the tank and use it immediately. Carry out the chromatography in the same direction as the impregnation. For the mobile phase shake a mixture of 100 volumes of petroleum spirit (boiling range, 50° to 70°) and 2 volumes of diethylamine with 6 to 8 volumes of 2-phenoxyethanol until a persistent cloudiness is obtained, decant and use the supernatent layer even if cloudy. Apply separately to the plate 2 l of each of two solutions in chloroform containing (1) 0.2% w/v of the substance being examined and (2) 0.2% w/v of the corresponding European Pharmacopoeia Chemical Reference Substance. After removal of the plate, examine under ultraviolet light (365 nm) and observe the fluorescence produced after a few minutes. The spot in the chromatogram obtained with solution (1) is similar in position, colour, fluorescence and size to that in the chromatogram obtained with solution (2). Spray the plate with ethanolic sulphuric acid (10%) and observe the colour produced. The colour of the spot in the chromatogram obtained with solution (1) is the same as that in the chromatogram obtained with solution (2) and has a similar stability over a period of at least 20 minutes after spraying. Related Substances in Phenothiazines Carry out the method for thin-layer chromatography protected from light using silica gel GF254 as the coating substance and the mobile phase prescribed in the monograph, but allowing the solvent front to ascend 12 cm above the line of application. Unless otherwise specified, apply separately to the plate 10 µl of each of two solutions of the substance being examined prepared immediately before use in a mixture of 95 volumes of methanol and 5 volumes of diethylamine containing (1) 2.0% w/v and (2) 0.010% w/v. After removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm). Disregard any spot remaining on the line of application. Unless otherwise specified any secondary spot in the chromatogram obtained with solution (1) is not more intense than the spot in the chromatogram obtained with solution (2) (0.5%). Mobile phases A. A mixture of 10 volumes of acetone, 10 volumes of diethylamine and 80 volumes of cyclohexane. B. A mixture of 5 volumes of diethylamine, 10 volumes of acetone and 85 volumes of hexane. C. A mixture of 18 volumes of 1M ammonia and 90 volumes of butan-1-ol . Identification of steroids Carry out the method for thin-layer chromatography using kieselguhr G as the coating substance. Impregnate the dry plate by placing it in a tank containing a shallow layer of the specified impregnating solvent, allowing the solvent to ascend to the top, removing the plate from the tank and allowing the solvent to evaporate; use within 2 hours, with the flow of the mobile phase in the direction in which impregnation was carried out. Unless otherwise specified, apply separately to the plate 2 µl of each of the following three solutions in a mixture of 9 volumes of chloroform and 1 volume of methanol . Solution (1) contains 0.25% w/v of the substance being examined. Solution (2) contains 0.25% w/v of the corresponding British Pharmacopoeia Chemical Reference Substance or European Pharmacopoeia Chemical Reference Substance. Solution (3) is a mixture of equal volumes of solutions (1) and (2). Use the specified mobile phase. After removal of the plate, allow the solvent to evaporate, heat at 120° for 15 minutes and spray the hot plate with ethanolic sulphuric acid (20%). Heat at 120° for a further 10 minutes, allow to cool and examine in daylight and under ultraviolet light (365 nm). The principal spot in the chromatogram obtained with solution (1) is similar in position, colour in daylight, fluorescence in ultraviolet light (365 nm) and size to that in the chromatogram obtained with solution (2). The principal spot in the chromatogram obtained with solution (3) appears as a single, compact spot. Impregnating solvents I. A mixture of 1 volume of formamide and 9 volumes of acetone. II. A mixture of 1 volume of propane-1,2-diol and 9 volumes of acetone. III. A mixture of 1 volume of liquid paraffin and 9 volumes of petroleum spirit (boiling range, 40° to 60° or 50° to 70°). Mobile phases A. Chloroform. B. A mixture of 25 volumes of chloroform and 75 volumes of toluene. C. Toluene. D. A mixture of 20 volumes of toluene and 80 volumes of cyclohexane. E. A mixture of equal volumes of cyclohexane and petroleum spirit (boiling range, 40° to 60° or 50° to 70°). F. A mixture of 40 volumes of glacial acetic acid and 60 volumes of water . G. A mixture of 20 volumes of 1,4-dioxan and 80 volumes of hexane. H. A mixture of 29 volumes of toluene, 56 volumes of chloroform and 115 volumes of cyclohexane. The alcohol R used must be free from fluorescence. (Appendix IIIA The British Pharmacopoeia 2007)