§ 436.344 - Thin layer chromatographic identity test for cefuroxime.

(a) Equipment—(1) Chromatography tank. Use a rectangular tank approximately 23×23×9 centimeters, with a glass solvent trough on the bottom and a tight-fitting cover. Line the inside walls of the tank with Whatman #3MM chromatographic paper or equivalent.

(2) Plates. Use 20 × 20 centimeter thin layer chromatography plates coated with Silica Gel F or equivalent to a thickness of 250 microns.

(b) Developing solvent. Mix chloroform, methanol, and formic acid in volumetric proportions of 90:16:4, respectively.

(c) Preparation of the spotting solutions. Dissolve approximately 200 milligrams each of the working standard and sample in 5 milliliters of a 50 percent aqueous acetone solution.

(d) Procedure. Pour the developing solvent into the glass trough at the bottom of the chromatography tank. Cover and seal the tank. Allow it to equilibrate for 1 hour. Prepare a plate as follows: On a line 2 centimeters from the base of the plate, and at intervals of 2 centimeters, spot 5 microliters each of the sample and working standard solutions. After all spots are thoroughly dry, place the plate directly into the glass trough of the chromatography tank. Cover and seal the tank tightly. Allow the solvent front to travel a minimum of 15 centimeters from the starting line. Remove the plate from the tank and allow it to air dry. Observe under ultraviolet light (254 nanometers).

(e) Evaluation. Measure the distance the solvent front traveled from the starting line and the distance the spots are from the starting line. Calculate the Rf value by dividing the latter by the former. The sample and standard should have spots of corresponding Rf values.