§ 436.327 - Thin layer chromatographic identity test for cyclacillin.

(a) Equipment—(1) Chromatography tank. Use a rectangular tank approximately 23 x 23 x 9 centimeters, with a glass solvent trough on the bottom and a tight-fitting cover.

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

(b) Reagents—(1) Developing solvent. One percent ammonium formate aqueous solution.

(2) Spray solution. Dilute starch iodide paste TS (U.S.P. XIX) with an equal volume of water. Mix diluted starch iodide paste, glacial acetic acid, and 0.1N iodine in volumetric proportions of 50:3:1, respectively.

(c) Assay solutions—(1) Preparation of working standard solution. Accurately weigh an amount of cyclacillin working standard and dissolve the material with sufficient 0.1N sodium hydroxide to obtain a solution containing 1 milligram per milliliter. Allow the solution to stand for 15 minutes before using.

(2) Preparation of sample solution. Using the sample solution prepared as described in the section for the antibiotic to be tested, proceed as described in paragraphs (d) and (e) of this section.

(d) Procedure. Pour the developing solvent into the glass trough on the bottom of the tank. Cover and seal the tank. Allow it to equilibrate. Prepare a plate as follows: On a line 2 centimeters from the base of the thin layer chromatography plate and at intervals of 2 centimeters, spot 5 microliters each of the working standard solution and sample solution. Dry the spots thoroughly with a stream of dry air. Place the plate in the trough in the chromatography tank. Cover and seal the tank. Allow the solvent front to travel about 15 centimeters from the starting line and then remove the plate from the tank. Dry the plate by heating for 30 minutes at 80° C in a circulating air oven. Visualize the spots by applying the spray solution.

(e) Evaluation. Measure the distance the solvent front traveled from the starting line, and the distance the spots are from the starting line. Divide the latter by the former to calculate the Rf value. The sample and standard should appear as white spots against a blue background at an Rf of approximately 0.6. The test is satisfactory if the Rf value of the sample compares with that of the working standard.