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APPENDIX 3 CHROMATOGRAPHY
3.1 THIN-LAYER CHROMATOGRAPHY

          Thin-layer chromatography (TLC) is used for the rapid separation of compounds by mean of a uniform layer of dry, finely powdered material applied to a glass, plastic, or metal sheet or plate. The coated plate can be considered as an “open chromatographic column”. 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).

          The retardation factor (R_f) 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. As R_f values may vary significantly with the experimental conditions, it is always necessary to prepare chromatograms of authentic specimens or reference substances; preferably in varied quantities, alongside the chromatogram of the sample. Positive identification may be effected by observation of two spots of identocal R_f value and about equal magnitude. A visual comparison of the size of the spots may serve for semi-quantitative es timation. More accurate quantitative measurements can be made by densitometry, fluorescence, and fluorescence quenching, or careful removal of the spots from the plate, followed by elution with a suitable solvent and spectrophotometric measurement. For two-dimensional thin-layer chromatography, the chromatographed plate is turned at a right angle and again chromatographed, usually in another chamber equilibrated with a different solvent system.

Apparatus

          Plate The chromatography is carried out using the TLC plate (typically 20 cm × 20 cm) of which the stationary phase has an average particle size of 10 to 15 μm, and that of highperformance thin-layer chromatography (HPTLC) plates (typically 10 cm × 10 cm) has an average particle size of 5 μm. Commercial plates with a pre-adsorbent zone can be used if they are specified in a monograph.

          Spreader A spreader, which, when moved over the plate, will apply a uniform layer of adsorbant, 250 to 300 μm thick, over the entire surface of the plate. Other thicknesses might be desirable in some procedures, and an adjus table spreader would be particularly useful in such cases.

          Preparation of the TLC plate Use flat glass plates of convenient size typically 20 cm × 20 cm.

         The adsorbent consists of finely divided solid material,normally 10 to 15 μm in diameter, suitable for chromatography. It can be applied directly to the plate or can be bonded to the plate by means of plaster of Paris (hydrated calcium sulfate) at a ratio of 5 to 15 per cent, or with starch pas te or other binders. The former will not yield as hard a surface as will the starch,but it is not affected by strongly oxidizing spray reagents. The adsorbent may contain fluorescing material to aid in the visualization of spots that absorb uitraviolet light. Clean the plates scrupulously, as by immersion in chromic acid cleansing mixture, rinsing them with copious quantities of water until the water runs off the plates without leaving any visible water or oily spots, and then dry. It is important that the plates be completely free from lint and dust when the adsorbent is applied.

          Arrange the plate or plates on the aligning tray, place a 5-cm × 20-cm plate adjacent to the front edge of the first square plate and another 5-cm × 20-cm plate adjacent to the rear edge of the last square, and secure all of the plates so that they will not slip during the application of the adsorbant. Position the spreader on the end plate opposite to the raised end of the aligning tray. Mix 1 part of adsorbent with 2 parts of water (or in the ratio sugges ted by the supplier) by shaking vigorously for about 30 seconds in a glass-stoppered conical flask,and transfer the siurry to the spreader. Usually 30 g of adsorbent and 60 ml of water are sufficient for five 20-cm x 20 cm plates. Complete the application of adsorbents using plas ter of Paris binder within 2 minutes of addition of the water, since thereafter the mixture begins to harden. Draw the spreader smoothly over the plates towards the raised end of the aligning tray, and remove the spreader when it is on the end plate next to the raised end of the aligning tray. (Wash away all traces of adsorbant from the spreader immediately after use). Allow the plates to remain undis turbed for 5 minutes, then transfer the square plates, layer side up, to the s torage rack and dry at 105° for 30 minutes. Preferably place the rack at an angle in the drying oven to prevent the condensation of moisture on the back side of the plates in the rack. When the plates are dry, allow them to cool to room temperature, and inspect the uniformity of the dis tribution and the texture of the adsorbant layer; transmitted light will show uniformity of texture. Store the satisfactory plates over self-indicating silica gel in a suitable chamber

          Pre-treatment of the plate It may be necessary to wash the plates 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° for 20 minutes.

          Developing chamber A developing chamber with a flat bottom or twin trough, of inert, transparent material, of a size suitable for the plates is used and provided with a tightly fitting lid. For horizontal development, the chamber is provided with a trough for the mobile phase and it additionally contains a device for directing the mobile phase to the stationary phase.

         Micropipette, microsyringe, calibrated disposable capillary A micropipette, microsyringe, calibrated disposable capillary or other application devices suitable for the proper application of the solutions are used.

         Template A template (generally made of plastic) is used to aid in placing the tes t spots at definite intervals, to mark distances as needed, and to aid in labelling the plate.

         Detection/Visualization device An ultraviolet (UV) light source suitable for observations under short- (254 nm) and long- (366 nm) wavelength UV light and a variety of other spray reagents to make spots visible are used.

         A device may be used to provide documentation of the visualized chromatogram, for
example a photograph or a computer file.

Procedure

          Sample application Apply the prescribed volume of the solutions at a dis tance of at leas t 15 mm (5 mm on HPTLC plates) 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 leas t 10 mm (5 mm on HPTLC plates) between the centres of circular spots and 5 mm (2 mm on HPTLC plates) between the edges of bands.

          Apply the solution in sufficiently small portions to obtain circular spots of 2 to 5 mm in diameter (1 to 2 mm on HPTLC plates) or bands of 10 to 20 mm (5 to 10 mm on HPTLC plates) by 1 to 2 mm (0.5 to 1 mm on HPTLC plates) and allow to dry. Avoid physical dis turbance of the adsorbant during the spotting procedure (by the pipette or other applicator) or when handling the plates. The template will aid in determining the spot points and the specified distance through which the solvent front should pass.

          Development Line the walls of the developing chamber with filter paper. Pour into the developing chamber a sufficient of the mobile phase for the size of the chamber 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 developing chamber, replace the lid and allow to s tand for 1 hour. Unless otherwise indicated in the monograph, the chromatographic separation is performed in a saturated chamber. Apply the prescribed volume of solutions as described above. When the solvent has evaporated from the applied solutions, place the plate in the developing chamber, 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 developing chamber. Remove the plate when the mobile phase has moved over 15 cm, or over three-quarters of the length of the plate, above the initial spots or bands, unless otherwise indicated in the monograph. Dry the plate and visualize the chromatograms as prescribed.

         Horizontal development can be used in place of vertical development, if specified in the monograph. For two-dimensional chromatography,dry the plates after the first develop- ment and carry out a second development in a direction perpendicular to that of the first development.

          Detection/Visualization Observe the principal spot or band in the chromatogram first under short-wavelength ultraviolet light (254 nm) and then under long-wavelength ultraviolet light (366 nm). Measure and record the dis tance of each spot or band from the point of origin, and indicate for each spot or band the wavelength under which it was observed. If further directed, spray the spots or bands with the wavelength under which it was observed. If the sample with the standard chromatogram.