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3.1 THIN-LAYER CHROMATOGRAPHY

APPENDIX 3 CHROMATOGRAPHY AND ELECTROPHORESIS

          CHROMATOGRAPHY Chromatography is defined as a procedure by which essential drug principles and other substances encountered in pharmaceutical products are separated by dynamic differential migration process in a system consisting of two or more phases, one of which moves continuously in a given direction and in which the individual substances exhibit different mobilities by reason of differences in adsorption, partition, solubility, vapour pressure, molecular size, or ionic charge density. The individual substances thus obtained can be identified or determined by analytical methods.

          The general chromatographic technique requires that a solute undergo distribution between two phases, one of them fixed (stationary phase), the other moving (mobile phase). It is the mobile phase that transfers the solute through the medium until it eventually emerges separated from other solutes that are eluted earlier or later. Generally, the solute is transported through the separation medium by means of a flowing stream of a liquid or a gaseous solvent known as the “eluant”. The stationary phase may act through adsorption, as in the case of adsorbants such as activated alumina, silica gel, and ion-exchange resins or it may act by dissolving the solute, thus partitioning the latter between the stationary and mobile phases. In the latter process, a liquid coating held on an inert support serves as the stationary phase. Partitioning is the predominant mechanism of separation in paper chromatography, forms of column chromatography designated as liquid-liquid chromatography, and gas chromatography. In practice, separations frequently result from a combination of adsorption and partitioning effects.

          The types of chromatography useful in qualitative and quantitative analyses that are employed in the assays and tests are Paper, Thin-layer, Column, Gas, and High-pressure Liquid Chromatography. Thin-layer chromatography and paper chromatography are ordinarily more useful for purposes of identification,because of their convenience and simplicity. Gas chromatography and high-pressure liquid chromatography require more elaborate apparatus and normally provide high-resolution methods that will identify and quantitate very small amounts of material.

          In paper and thin-layer chromatography, the ratio of the distance (this distance being measured to the point of maximum intensity of the spot) traveled on the medium by a given compound to the distance traveled by the front of the mobile phase, from the point of application of the test substance, is designated as the Rf value of the compound. The ratio between the distances traveled by a given compound and a reference substance is the RRf value.

          ELECTROPHORESIS Electrophoresis is a process in which charged species (ions or colloidal particles) are separated based upon differential migration rates in an electrolyte solution migrate in the direction of the electrode bearing the opposite polarity. In gel electrophoresis, the movements of the particles are retarded by interactions with the surrounding gel matrix, which acts as a molecular sieve. The opposing interactions of the electrical force and molecular sieving result in differential migration rates according to sizes, shapes and charges of particles. Because of their different physicochemical properties, different macromolecules of a mixture will migrate at different speeds during electrophoresis and will thus be separated into discrete fractions. Electrophoretic separations can be conducted in systems without support phases (e.g., free solution separation in capillary electrophoresis) and in stabilizing media such as thin-layer plates, films or gels.

3.1 THIN-LAYER CHROMATOGRAPHY

Conventional Thin-layer Chromatography

          Thin-layer chromatography is used for the rapid separation of compounds by means of a uniform layer of dry, finely powdered material applied to a glass, plastic, or metal sheet or plate, glass plates being most commonly employed. The coated plate can be considered as an “open chromatographic column” and the separations achieved may be based upon adsorption, partition, or a combination of both effects, depending on the particular type of adsorbant, its preparation, and its use with different solvents. Thin-layer chromatography on ion-exchange films can be used for the fractionation of polar compounds.

           As Rf 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 2 spots of identical Rf value and about equal magnitude. A visual comparison of the size of the spots may serve for semi-quantitative estimation. 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 The essential apparatus and materials for thin-layer chromatography are as follows:

          Flat glass plates of convenient size typically 20 cm × 20 cm.

          An aligning tray or a flat surface upon which to align and rest the plates during the application of the adsorbant.

          A storage rack to hold the prepared plates during drying and transportation. The rack holding the plates should be kept in a desiccator or be capable of being sealed in order to protect the plates from the environelectrical field. Under the influence of an electrical field, charged particles dissolved or dispersed in an ment after removal from the drying oven.

          The adsorbant consists of finely divided adsorbent material, normally 5 to 40 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 paste 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 adsorbant may contain fluorescing material to aid in the visualization of spots that absorb ultraviolet light.

          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 adjustable spreader would be particularly useful in such cases.

          A developing chamber of transparent material, usually glass, ground at the top to take a tightly fitting lid, of a size suitable for the plates used.

          A template (generally made of plastic) to aid in placing the test spots at definite intervals, to mark distances as needed, and to aid in labelling the plates.

          A graduated micropipette capable of delivering 10-μl quantities. Total quantities of sample and standard solutions are specified in the individual monograph.

          A reagent sprayer that will emit a fine spray and will not itself be attacked by the reagent.

          An ultraviolet light source suitable for observations with short (254 nm) and long (366 nm) ultraviolet wavelengths.

          PROCEDURE 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, then dry. It is important that the plates be completely free from lint and dust when the adsorbant 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 adsorbant with 2 parts of water (or in the ratio suggested by the supplier) by shaking vigorously for about 30 seconds in a glass-stoppered conical flask, and transfer the slurry to the spreader. Usually 30 g of adsorbant and 60 ml of water are sufficient for five 20-cm × 20-cm plates. Complete the application of adsorbants using plaster 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 undisturbed for 5 minutes, then transfer the square plates, layer side up, to the storage rack and dry at 105º 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 distribution 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.

          Place two filter-paper wicks, 18 cm in height and as wide as the length of the developing chamber, into the chamber, add about 100 ml of the solvent (sufficient to have a depth of 0.5 to 1 cm at the bottom of the chamber). Seal the cover to the top of the chamber, and allow the system to equilibrate; it is essential that the wicks become completely wet. Alternatively, the chamber may be completely lined with filter paper. In either case, assure that the filter paper dips into the solvent at the bottom of the chamber. Where vapour saturation of the chamber by these methods is undesirable, it is so indicated in the individual monograph.

          Apply the sample solution and the standard solution, as directed in the individual monograph, at points about 1.5 cm apart and about 2 cm from the lower edge of the plate (the lower edge is the first part over which the spreader moved in the application of the adsorbant layer), and allow to dry. Avoid physical disturbance 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 10-cm to 15-cm distance through which the solvent front should pass.

          Mark the sides of the plate 10 to 15 cm above the spot point. Place the plate in the developing chamber. Allow the solvent in the chamber to reach the lower edge of the adsorbant, but do not allow the spot points to be immersed. Put the cover in place, and maintain the system until the solvent ascends to a point 10 to 15 cm above the initial spots, this usually requiring about 15 minutes to 1 hour. Remove and air-dry the plates, and observe first under short-wavelengthultraviolet light (254 nm) and then under long-wavelength ultraviolet light (366 nm). Measure and record the distance of each spot from the point of origin, and indicate for each spot the wavelength under which it was observed. If further directed, spray the spots with the reagent specified, observe, and compare the sample with the standard chromatogram.

Continuous Thin-layer Chromatography

          In contrast to conventional thin-layer chromatography, which is carried out in a closed tank, the continuous development or continuous flow technique allows the upper end of the plate to project through a slot in the cover of the developing chamber. When the developing solvent reaches the slot, continuous evaporation occurs, producing a steady flow of solvent over the plate. In conventional thin-layer chromatography, spot migration ceases when the solvent reaches the top of the plate, after which the spots simply enlarge by diffusion.

In the continuous flow process, spot migration continues as long as the plate remains in the tank and the developing solvent is not exhausted.

          Development may be continued for several hours after the solvent reaches the top of the plate, to provide adequate migration of the spots. Usually spots of a standard solution, a test solution, and a mixture of equal amounts of test and standard solutions, are initially applied at a standard distance from the base of the plate. Identity of the standard and test substances is confirmed by their migrating equal distances from the origin and by the observation that the two substances applied as a mixture shows no tendency to separate.

          A major advantage of continuous development thinlayer chromatography stems from the greater solvent selectivity for solvents of low solvent strength. Solvent strength refers to the property of a developing solvent that causes solutes to migrate, and it is strongly influenced by the polarity of the solvent. Increasing the solvent strength by adding a more polar solvent causes the Rf value to increase. Solvent selectivity refers to the ability of a solvent system to produce different Rf values for closely related substances. In conventional thinlayer chromatography, a solvent system giving an Rf value in the range of 0.3 to 0.7, but with adequate selectivity to permit separation of the substances being examined is usually selected. It is much easier to find solvent systems producing adequate migration than to find those affording adequate selectivity. Solvent systems of lower strength generally exhibit higher selectivity, but are difficult to employ in conventional thin-layer chromatography because they result in very little migration before the solvent reaches the top of the plate. Migration may be increased, however, by repeated drying and redevelopment of the plate or, more conveniently, by providing means for evaporation of solvent at the top of the plate, which results in continuous development. An Rf value cannot be measured in continuous development thin-layer chromatography. Substances may be compared either by their migration distance over a fixed period of time or by comparison with the migration of a standard substance applied to the plate.

           APPARATUS Acceptable apparatus and materials for continuous development thin-layer chromatography are the same as those described under Conventional Thinlayer Chromatography, except as follows.

          A developing chamber being used consists of a rectangular tank, approximately 23-cm × 23-cm × 9-cm, equipped with a glass solvent trough and a platform about 3.75 cm high to elevate the solvent trough above the base of the tank. The chamber is fitted with a cover having a 21-cm × 6-cm slot in the front edge.

          PROCEDURE Apply the standard solution, the test solution and a mixture of equal amounts of the standard solution and the test solution to a line about 2 cm from the base of the plate. Place the plate in the elevated empty solvent trough with the adsorbent on the under side of the leaning plate. The adsorbent rests against a piece of heavy (about 1 mm thick) filter paper measuring 20 cm × 3 cm, folded lengthwise and placed over the front edge of the tank. Place the developing solvent in the trough, set the cover in place, and seal all openings except where the adsorbent contacts the paper wick. The plate extends about 1 cm beyond the top of the tank. After the solvent reaches the top of the tank, allow development to continue for an appropriate time. Then remove and dry the plate, and detect the spots by suitable means.

Identification of Steroids

          Use Method I unless otherwise specified.

METHOD I

          Carry out the test as described in the “Thin-layer Chromatography” (Appendix 3.1), using kieselguhr G as the coating substance. Impregnate the dry plate by placing it in a chromatographic chamber containing a shallow layer of the specified impregnating solvent, allowing the solvent to ascend to the top, removing the plate 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 (A) contains 2.5 mg per ml of the test substance. Solution (B) contains 2.5 mg per ml of the corresponding Reference Substance. Solution (C) is a mixture of equal volumes of solutions (A) and (B). Use the specified mobile phase. After removal of the plate, allow the solvent to evaporate, heat at 120º for 15 minutes and spray the hot plate with ethanolic sulfuric acid (20 per cent). Heat at 120º for a further 10 minutes, allow to cool and examine in daylight and under ultraviolet light (366 nm). The principal spot in the chromatogram obtained from solution (A) corresponds to that obtained from solution (B). The principal spot in the chromatogram obtained from solution (C) 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 propylene glycol and 9 volumes of acetone.

III. A mixture of 1 volume of liquid paraffin and 9 volumes of petroleum ether (boiling range, 40º to 60º).

          Mobile phases

A. Chloroform.

B. A mixture of 75 volumes of toluene and 25 volumes of chloroform.

C. Toluene.

D. A mixture of 80 volumes of cyclohexane and 20 volumes of toluene.

E. A mixture of equal volumes of cyclohexane and petroleum ether (boiling range, 40º to 60º).

F. A mixture of 60 volumes of water and 40 volumes of glacial acetic acid.

G. A mixture of 80 volumes of hexane and 20 volumes of dioxane.

H. A mixture of 115 volumes of cyclohexane, 56 volumes of chloroform and 29 volumes of toluene.

METHOD II

          Carry out the test as described in the “Thin-layer Chromatography” (Appendix 3.1), using silica gel G as the coating substance and a mixture of 85 volumes of ether, 10 volumes of toluene and 5 volumes of 1-butanol saturated with water as the mobile phase. Apply separately to the plate, 2 μl of each of four solutions in a mixture of 9 volumes of chloroform and 1 volume of methanol containing (A) 2.5 mg per ml of the test substance, (B) 2.5 mg per ml of the corresponding Reference Substance, (C) 1.25 mg per ml each of the test substance and the corresponding Reference Substance, and (D) 1.25 mg per ml each of the test substance and the specified Reference Substance. After removal of the plate, allow it to dry in air and spray with ethanolic sulfuric acid (20 per cent). Heat at 120º for 10 minutes or until spots are produced, allow to cool and examine in daylight and under ultraviolet light (366 nm). The principal spot in the chromatogram obtained from solution (A) is similar in colour in daylight, fluorescence under ultraviolet light (366 nm), position and size to the principal spot in the chromatogram obtained from solution (B) and the chromatogram obtained from solution (C) shows only one spot. The test is not valid unless the chromatogram obtained from solution (D) shows two principal spots that are close to, but separated from, one another.

          Related Impurities in Phenothiazines

METHOD

          Carry out in subdued light under an atmosphere of nitrogen the method for “Thin-layer Chromatography” (Appendix 3.1), using silica gel GF254 as the coating substance, 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 freshly prepared solutions of the test substance in a mixture of 95 volumes of methanol and 5 volumes of diethylamine containing (A) 20 mg per ml and (B) 100 μg per ml. Use the specified mobile phase. After removal of the plate, allow it to dry in air and examine under ultra-violet light (254 nm). Ignore any spot on the baseline. Unless otherwise specified any secondary spot obtained from solution (A) is not more intense than that obtained from solution (B).

          Mobile phase

A. A mixture of 80 volumes of cyclohexane, 10 volumes of acetone and 10 volumes of diethylamine.

B. A mixture of 85 volumes of hexane, 10 volumes of acetone and 5 volumes of diethylamine.

C. A mixture of 15 volumes of 1-butanol and 3 volumes of 1 M ammonia.

Related Foreign Steroids

METHOD I

          Carry out the test as described in the “Thin-layer Chromatography” (Appendix 3.1), using silica gel G as the coating substance and a mixture of 77 volumes of dichloromethane, 15 volumes of ether, 8 volumes of methanol, and 1.2 volumes of water as the mobile phase. Apply separately to the plate, 1 μl of each of three solutions in a mixture of 9 volumes of chloroform and 1 volume of methanol containing (A) 15 mg per ml of the test substance, (B) 15 mg per ml of the corresponding Reference Substance and (C) 300 μg per ml of each of Prednisolone RS, Prednisone RS and Cortisone Acetate RS. After removal of the plate, allow it to dry in air until the solvents have evaporated, heat at 105º for 10 minutes, cool, and spray with alkaline tetrazolium blue TS. The principal spot in the chromatogram obtained from solution (A) corresponds in position, colour and intensity to that obtained from solution (B). Any secondary spot obtained from solution(A) is not more intense than the proximate spot obtained from solution (C).

METHOD II

          Carry out the test as described in the “Thin-layer Chromatography” (Appendix 3.1), using silica gel G as the coating substance and a mixture of 95 volumes of 1,2-dichloroethane, 5 volumes of methanol and 0.2 volumes of water as the mobile phase. Apply separately to the plate, 1 μl of each of three solutions in a mixture of 9 volumes of chloroform and 1 volume of methanol containing (A) 15 mg per ml of the test substance, (B) 15 mg per ml of the corresponding Reference Substance and (C) 300 μg per ml of each of Prednisone RS, Prednisolone Acetate RS, Cortisone Acetate RS, and Deoxycortone Acetate RS. Complete the procedure described under Method I beginning with “After removal of the plate...”.

Related Substances in Sulfonamides

METHOD I

          Carry out the test as described in the “Thin-layer Chromatography” (Appendix 3.1), using silica gel H as the coating substance and a mixture of 15 volumes of 1-butanol and 3 volumes of 1 M ammonia as the mobile phase. Apply separately to the plate, 10 μl of each of two solutions, in a mixture of 9 volumes of ethanol and 1 volume of strong ammonia solution, containing (A) 10 mg per ml of the test substance and (B) 50 μg per ml of sulfanilamide. After removal of the plate, heat it at 105º for 10 minutes and spray with a 0.1 per cent w/v solution of 4-dimethylaminobenzaldehyde in ethanol containing 1 per cent v/v of hydrochloric acid. Any spot in the chromatogram obtained from solution (A), other than the principal spot, is not more intense than that obtained from solution (B).

METHOD II

          Carry out the test as described in the “Thin-layer Chromatography” (Appendix 3.1), using silica gel H as the coating substance and a mixture of 20 volumes of chloroform, 2 volumes of methanol and 1 volume of dimethylformamide as the mobile phase. Apply separately to the plate, 10 μl of each of two solutions, in a mixture of 9 volumes of ethanol and 1 volume of strong ammonia solution, containing (A) 2.5 mg per ml of the test substance and (B) 12.5 μg per ml of sulfanilamide. After removal of the plate, allow it to dry in air. Spray the plate with a 10 per cent v/v solution of sulfuric acid in ethanol, heat at 105º 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 7 M sulfuric acid dropwise to a solution containing 10 per cent w/v of sodium nitrite and 3 per cent w/v of potassium iodide). Place the plate in a current of warm air for 15 minutes and spray with a 0.5 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride in ethanol. If necessary, allow to dry and repeat the spraying. Any spot in the chromatogram obtained from solution (A), other than the principal spot, is not more intense than that obtained from solution (B).

METHOD III

          Carry out the test as described in the “Thin-layer Chromatography” (Appendix 3.1), using silica gel GF254 as the coating substance and a mixture of 50 volumes of dioxane, 40 volumes of nitromethane, 5 volumes of water, and 3 volumes of 6 M ammonia as the mobile phase. Apply separately to the plate, 5 μl of each of the following solutions. For solution (A), dissolve 100 mg of the test substance in 0.5 ml of strong ammonia solution and dilute to 5 ml with methanol; if the solution is not clear, heat gently until dissolution is complete. Solutions (B) to (D) are solutions in a mixture of 24 volumes of methanol and 1 volume of strong ammonia solution, containing (B) 4 mg per ml of the test substance, (C) 100 μg per ml of the test substance and (D) 4 mg per ml of the corresponding Reference Substance. After removal of the plate, dry it at 105º and examine under ultraviolet light (254 nm). Any secondary spot in the chromatogram obtained from solution (A) is not more intense than that obtained from solution (C).

APPENDICES • 3.1 THIN-LAYER CHROMATOGRAPHY
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