Thin-Layer Chromatography (TLC Method and Apparatus)

Thin-layer chromatography is a technique in which a solute undergoes distribution between two phases, a stationary phase acting through adsorption and a mobile phase in the form of a liquid. 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 are most commonly used. Separation may also be achieved on the basis of partition or a combination of partition and adsorption, depending on the particular type of support, its preparation and its use with a different solvent.

Identification can be affected by observation of spots 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 and intensity of the spots usually serves for semiquantitative estimation.

Apparatus

(a) Flat glass plates of appropriate dimensions which allow the application at specified points of the necessary quantities of the solution being examined and appropriate reference solutions and which allow accommodation of the specified migration path-length. The plates are prepared as described below; alternatively, commercially available pre-coated plates may be used.
(b) An aligning tray or a flat surface on which the plates can be aligned and rested when the coating substance is applied.
(c) The adsorbent or coating substance consisting of finely divided adsorbent materials, normally 5 microns to 40 microns 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 Sulphate) or with any other suitable binders. The adsorbent may contain fluorescing material to help in visualizing spots that absorb ultraviolet light.
(d) A spreader which, when moved over the glass plate, will apply a uniform layer of the adsorbent of desired thickness over the entire surface of the plate.
(e) A storage rack to support the plates during drying and transportation.
(f) A developing chamber that can accommodate one or more plates and can be properly closed and sealed. The chamber is fitted with a plate support rack that supports the plates, back to back, with the lid of the chamber in place.
(g) Graduated micro-pipettes capable of delivering microliter quantities say 10 ml and less.
(h) A reagent sprayer that will emit a fine spray and will not itself be attacked by the reagent.
(i) An ultraviolet light, suitable for observation at short (254 nm) and long (365 nm) ultraviolet wavelengths.
Related: Principle and Working of HPLC

Preparation of plates

Unless otherwise specified in the monograph, the plates are prepared in the following manner. Prepare a suspension of the coating substance in accordance with the instructions of the supplier and, using the spreading device designed for the purpose, spread a uniform layer of the suspension, 0.25 to 0.30 mm thick, on a flat glass plate 20 cm long. Allow the coated plates to dry in air, heat at 100° to 105° for at least 1 hour (except in the case of plates prepared with cellulose when heating for 10 minutes is normally sufficient) and allow to cool, protected from moisture. Store the plates protected from moisture and use within 3 days of preparation. At the time of use, dry the plates again, if necessary, as prescribed in the monograph.

Method

Unless unsaturated conditions are prescribed, prepare the tank by lining the walls with sheets of filter paper; pour into the tank, saturating the filter paper in the process, sufficient of the mobile phase to form a layer of solvent 5 to 10mm deep, close the tank and allow standing for 1 hour at room temperature. Remove a narrow strip of the coating substance, about 5 mm wide, from the vertical sides of the plate. Apply the solutions being examined in the form of circular spots about 2 to 6 mm in diameter, or in the form of bands (10 to 20 mm x 2 to 6 mm unless otherwise specified) on a line parallel with, and 20 mm from, one end of the plate, and not nearer than 20 mm to the sides; the spots should be 15 mm apart.

If necessary, the solutions may be applied in portions, drying between applications. Mark the sides of the plate 15 cm, or the distance specified in the monograph, from the starting line. Allow the solvent to evaporate and place the plate in the tank, ensuring that it is as nearly vertical as possible and that the spots or bands are above the level of the mobile phase. Close the tank and allow standing at room temperature, until the mobile phase has ascended to the marked line. Remove the plate and dry it.

For two-dimensional chromatography dry the plate after the first development and carry out the second development in a direction perpendicular to the first.

When the method prescribed in the monograph specifies 'protected from light' or 'in subdued light' it is intended that the entire procedure is carried out under these conditions.

Adjustment of chromatographic conditions

Minor adjustments to the parameters of the test may be made in order to satisfy the system suitability criteria. These may be:
Mobile phase: Minor solvent component of a mixture: ± 30 percent relative or ± 2 percent absolute, whichever is the larger; no other component altered by more than 10 percent absolute;
Concentration of salts: In the buffer component of the mobile phase; ± 10 percent; pH of the aqueous component of the mobile phase. ± 0.2 pH, unless otherwise stated in the monograph, or ± 0.1 pH, when neutral substances are to be examined;
Volume of solutions applied: 10-20 percent of the prescribed volume if plates have fine particles (2-10 micron).

Visualisation

After development, the plate should be examined under an ultraviolet light having a maximum output at about 254 nm or at about 365 nm, as the case may be. Alternatively, it may be visualised as directed in the monograph; where a spraying technique is prescribed it is essential that the reagent be evenly applied as a fine spray. The term secondary spot means any spot other than the principal spot. Similarly, a secondary band is any band other than the principal band.

Semi-quantitative 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 in respect of the color, the size and the Rf of the spots.

Test for Related Substances. 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.

Quantitative measurement

The substances that have been separated after the development of the plate and that respond to UV-Vis irradiation can be estimated directly on the plate with suitable instrumentation. Measurement is of the reflectance of the incident light from the spots by moving the plate or the measuring device. Likewise, fluorescence may be measured using an appropriate optical system.

Apparatus

The apparatus for direct measurement consists of a device for exact positioning and reproducible application of the number of solutions onto the plate, a mechanical device for moving the plate or the measuring device along the x-axis or the y-axis, a recorder and a suitable integrator or a computer, and a photometer with a source of light, an optical device for generating monochromatic light and a photo cell of adequate sensitivity; for measurement of fluorescence, a suitable filter to prevent light used for excitation from reaching the detector while permitting emitted light or a specific portion thereof to pass.

Method

Prepare the test solution and reference solutions as prescribed in the individual monograph. Use the same solvent for all the solutions and apply the same volume of each and develop the plate. Prepare and apply not fewer than 3 reference solutions of the substance under examination, the concentrations of which span the expected value in the test solution (about 80 percent, 100 percent and 120 percent).

Treat with the prescribed reagent, if necessary, and record the reflectance, the transmittance or fluorescence in the chromatograms obtained with all the solutions. Use the measured results to calculate the amount of substance in the test solution. The requirement for resolution and separation are prescribed in the individual monograph.

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