| Thin Layer chromatography full Analysis |
| 1. Procedure 1.1.Apparatus: >TLC chamber >TLC plate >Injector >Scale 1.2 Method: 1.2.1Unless 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 forms a layer of solvents 5 to 10 mm deep. 1.2.2 Close the tank and allow standing for 1 hour at room temperature. 1.2.3 Remove a narrow strip of the coating substance, about 5 mm wide, from the vertical sides of the plate. 1.2.4 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 & allow the solvent to evaporate from the plate after application of the samples. 1.2.5Mark the sides of the plate 15 cm, or the distance specified in the monograph, from the starting line. 1.2.6Place 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 stands at room temperature, until the mobile phase has ascended to the marked line. 1.2.7Remove the plate and dry it. 1.2.8For two-dimensional chromatography dry the plate after the first development and carry out the second development in a direction perpendicular to the first 1.2.9When 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. 1.3 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: 1.3.1Mobile phase: Minor solvent component of a mixture: ± 30 per cent relative or ± 2 per cent absolute, whichever is the larger; no other component altered by more than 10 per cent absolute. 1.3.2Concentration of salts: In the buffer component of the mobile phase; ± 10 per cent. 1.3.3pH 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. 1.3.4 Volume of solutions applied: 10-20 per cent of the prescribed volume if plates have fine particles (2-10 μm). 1.4 Visualization: 1.4.1After 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 visualized as directed in the monograph. 1.4.2Where a spraying technique is prescribed it is essential that the reagent be evenly applied as a fine spray. 1.4.3The term secondary spot means any spot other than the principal spot. Similarly, a secondary bands is any band other than the principal band. 1.5Semi-quantitative estimation: 1.5.1Identification. 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 colour, the size and the Rf of the spots. 1.5.2Test for Related substances. The secondary spot(s) in the chromatogram obtained with the test solution are visually compared to either the corresponding spot(s) in the chromatogram obtained with the reference solution containing the impurities or the spots in the chromatogram obtained with the reference sol. prepared from a dilution of the test solution. |