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In thin-layer chromatography, the adsorbent is a relatively thin, 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 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 stationary phase, its preparation, and its use with different solvents. Thin-layer chromatography on ion-exchange layers can be used for the fractionation of polar compounds. Presumptive identification can be effected by observation of spots or zones of identical RF value and about equal magnitude obtained, respectively, with an unknown and a reference sample chromatographed on the same plate. A visual comparison of the size or intensity of the spots or zones may serve for semiquantitative estimation. Quantitative measurements are possible by means of densitometry (absorbance or fluorescence measurements), or the spots may be carefully removed 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— Acceptable apparatus and materials for thin-layer chromatography consist of the following.
A TLC or HPTLC plate. The chromatography is generally carried out using precoated plates or sheets (on glass, aluminum, or polyester support) of suitable size. It may be necessary to clean the plates prior to separation. This can be done by migration of, or immersion in, 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. The stationary phase of TLC plates has an average particle size of 10–15 µm, and that of HPTLC plates an average particle size of 5 µm. Commercial plates with a preadsorbant zone can be used if they are specified in a monograph. Sample applied to the preabsorbant region develops into sharp, narrow bands at the preabsorbant-sorbent interface. Alternatively, flat glass plates of convenient size, typically 20 cm × 20 cm can be coated as described under Preparation of Chromatographic Plates.
A suitable manual, semiautomatic, or automatic application device can be used to ensure proper positioning of the plate and proper transfer of the sample, with respect to volume and position, onto the plate. Alternatively, a template can be used to guide in manually placing the test spots at definite intervals, to mark distances as needed, and to aid in labeling the plates. For the proper application of the solutions, micropipets, microsyringes, or calibrated disposable capillaries are recommended.
For ascending development, a chromatographic chamber made of inert, transparent material and having the following specifications is used: a flat bottom or twin trough, a tightly fitted lid, and a size suitable for the plates. For horizontal development, the chamber is provided with a reservoir for the mobile phase, and it also contains a device for directing the mobile phase to the stationary phase.
Devices for transfer of reagents onto the plate by spraying, immersion, or exposure to vapor and devices to facilitate any necessary heating for visualization of the separated spots or zones.
A UV light source suitable for observations under short (254 nm) and long (365 nm) wavelength UV light.
A suitable device for documentation of the visualized chromatographic result.
Procedure— Apply the prescribed volume of the test solution and the standard 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 by 1 to 2 mm (5 to 10 mm by 0.5 to 1 mm on HPTLC plates) at an appropriate distance from the lower edge—during chromatography the application position must be 3 mm (HPTLC) to 5 mm (TLC) above the level of the developing solvent—and from the sides of the plate. Apply the solutions on a line parallel to the lower edge of the plate with an interval of at least 10 mm (5 mm on HPTLC plates) between the centers of spots or 4 mm (2 mm on HPTLC plates) between the edges of bands, and allow to dry.
Ascending Development— Line at least one wall of the chromatographic chamber with filter paper. Pour into the chromatographic chamber a quantity of the mobile phase sufficient for the size of the chamber to give, after impregnation of the filter paper, a level of depth appropriate to the dimension of the plate used. For saturation of the chromatographic chamber, close the lid, and allow the system to equilibrate. Unless otherwise indicated, the chromatographic separation is performed in a saturated chamber.
Place the plate in the chamber, ensuring that the plate is as vertical as possible and that the spots or bands are above the surface of the mobile phase, and close the chamber. The stationary phase faces the inside of the chamber. Remove the plate when the mobile phase has moved over the prescribed distance. Dry the plate, and visualize 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— Introduce a sufficient quantity of the developing solvent into the reservoir of the chamber using a syringe or pipet. Place the plate horizontally in the chamber, connect the mobile phase direction device according to the manufacturer's instructions, and close the chamber. If prescribed, develop the plate starting simultaneously at both ends. Remove the plate when the mobile phase has moved over the distance prescribed in the monograph. Dry the plate, and visualize 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.
Detection— Observe the dry plate first under short-wavelength UV light (254 nm) and then under long-wavelength UV light (365 nm) or as stated in the monograph. If further directed, spray, immerse, or expose the plate to vapors of the specified reagent, heat the plate when required, observe, and compare the test chromatogram with the standard chromatogram. Document the plate after each observation. Measure and record the distance of each spot or zone from the point of origin, and indicate for each spot or zone the wavelength under which it was observed. Determine the RF values for the principal spots or zones (see Glossary of Symbols).
Quantitative Measurement— Using appropriate instrumentation, substances separated by TLC and responding to ultraviolet-visible (UV-Vis) irradiation prior to or after derivatization can be determined directly on the plate. While moving the plate or the measuring device, the plate is examined 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 three ways: (1) directly by moving the plate alongside a suitable counter or vice versa; (2) by cutting the plates into strips and measuring the radioactivity on each individual strip using a suitable counter; or (3) by scraping off the stationary phase, dissolving it in a suitable scintillation cocktail, and measuring the radioactivity using a liquid scintillation counter (see Radioactivity 821).
The apparatus for direct quantitative measurement on the plate is a densitometer that is composed of a mechanical device to move the plate or the measuring device along the x-axis and the y-axis, a recorder, a suitable integrator or a computer; and, for substances responding to UV-Vis irradiation, a photometer with a source of light, an optical device capable of generating monochromatic light, and a photo cell of adequate sensitivity, all of which are used for the measurement of reflectance. In the case where fluorescence is measured, a suitable filter is also required to prevent the light used for excitation from reaching the photo cell while permitting the emitted light or specific portions thereof to pass. The linearity range of the counting device must be verified.
For quantitative tests, it is necessary to apply to the plate not fewer than three standard solutions of the substance to be examined, the concentrations of which span the expected value in the test solution (e.g., 80%, 100%, and 120%). Derivatize with the prescribed reagent, if necessary, and record the reflectance or fluorescence in the chromatograms obtained. Use the measured results for the calculation of the amount of substance in the test solution.
Preparation of Chromatographic Plates—
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 adsorbent.
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 environment after removal from the drying oven.
The adsorbent consists of finely divided adsorbent materials, normally 5 to 40 µm in diameter, suitable for chromatography. It can be applied directly to the glass plate or can be bonded to the plate by means of plaster of Paris [calcium sulfate hemihydrate (at a ratio of 5% to 15%)] or with starch paste or other binders. The plaster of Paris 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 UV light.
A spreader, which, when moved over the glass plate, will apply a uniform layer of adsorbent of desired thickness over the entire surface of the plate.
Procedure— [NOTE—In this procedure, use Purified Water that is obtained by distillation.] Clean the glass plates scrupulously, using an appropriate cleaning solution (see Cleaning Glass Apparatus 1051), 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 adsorbent is applied.
Arrange the plate or plates on the aligning tray, place a 5- × 20-cm plate adjacent to the front edge of the first square plate and another 5- × 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 adsorbent. Position the spreader on the end plate opposite the raised end of the aligning tray. Mix 1 part of adsorbent with 2 parts of water (or in the ratio suggested by the supplier) by shaking vigorously for 30 seconds in a glass-stoppered conical flask, and transfer the slurry to the spreader. Usually 30 g of adsorbent and 60 mL of water are sufficient for five 20- × 20-cm plates. Complete the application of adsorbents using plaster of Paris binder within 2 minutes of the addition of the water, because thereafter the mixture begins to harden. Draw the spreader smoothly over the plates toward 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 adsorbent 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 105 for 30 minutes. Preferably place the rack at an angle in the drying oven to prevent the condensation of moisture on the back sides of 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 adsorbent layer; transmitted light will show uniformity of distribution, and reflected light will show uniformity of texture. Store the satisfactory plates over silica gel in a suitable chamber.