§ 436.310 - Thin layer chromatography identity test for mitomycin.

(a) Equipment—(1) Chromatography tank. A rectangular tank, approximately 9 × 9 × 3.5 inches, lined with filter paper and with a solvent trough on the bottom.

(2) Plates. Use 20 by 20 centimeter thin layer chromatography plates coated with silica gel G or equivalent, to a thickness of 250 microns.

(b) Reagents—(1) Developing solvent. Mix n-butanol, glacial acetic acid, and water in volumetric proportions of 4:2:1, respectively.

(2) Spray solution. Prepare a one-percent solution of ninhydrin in ethanol.

(c) Preparation of spotting solutions. Prepare solutions of the sample and working standard, each containing 1 milligram of mitomycin per milliliter, in water.

(d) Procedure. Pour the developing solvent into the solvent trough on the bottom of the tank and onto the paper lining the walls of the tank. Cover and seal the tank. Allow it to equilibrate for 30 minutes. Prepare a plate as follows: Apply spotting solutions on a line 2.5 centimeters from the base of the silica gel plate and at points 2.0 centimeters apart. Apply approximately 2 microliters of the working standard solution to points 1 and 3. When these spots are dry, apply approximately 2 microliters of sample solution to points 2 and 3. After all spots are thoroughly dry, place the silica gel plate into the trough in the chromatography tank. Cover and seal the tank tightly. Allow the solvent front to travel about 10 centimeters from the starting line. Remove the plate and allow it to air dry. After the plate is dry, spray lightly with the spray solution. Heat the plate in an oven at 110° C. for 10-15 minutes. Mitomycin appears as a pink spot.

(e) Evaluation. The sample and standard should have spots of corresponding Rf value (approximately 0.51), and standard and sample combined should appear as a single spot of corresponding Rf value.