§ 436.308 - Paper chromatography identity test for tetracyclines.

(a) Equipment—(1) Sheet (chromatographic). Whatman No. 1 filter paper for chromatography, 20 × 20 centimeters.

(2) Chamber (chromatographic). Cylindrical glass chromatographic jar, 25 centimeters high by 12 centimeters in diameter, with a ground-glass lid.

(3) Preparation of solutions—(i) pH3.5 buffer. Mix 13.93 volumes of 0.1M citric acid with 6.07 volumes of 0.2M of disodium phosphate.

(ii) Solvent (organic phase). Mix chloroform, nitromethane, and pyridine in volumetric proportions of 10:20:3, respectively.

(b) Preparation of spotting solutions. Prepare solutions of the working standard and sample as follows: Accurately weigh a portion of the working standard and sample and dilute with methanol to obtain a concentration of 1 milligram per milliliter of antibiotic to be tested.

(c) Procedure. Fill the chamber to a depth of 0.6 centimeter with freshly prepared solvent. Draw a starting line about 2.5 centimeters from and parallel to the bottom of the sheet. Wet the sheet thoroughly with the pH 3.5 buffer and blot it firmly between sheets of absorbent paper. Starting about 5 centimeters from the edge of the sheet and at 1.5-centimeter intervals, apply to the starting line 2 microliters each of standard solution, sample solution, and a 1:1 mixture of the standard and sample solutions. Allow a few minutes for the sheet to dry partially, and while still damp place it in the chamber with the bottom edge touching the solvent. When the solvent front has risen about 10 centimeters, remove the sheet from the chamber. Expose the paper to ammonia vapor. Examine the dried sheet under a strong source of ultraviolet light and record the position of any fluorescent spots. Measure the distance the solvent front traveled from the starting line and the distance that the fluorescent spots are from the starting line. Calculate the Rf value by dividing the latter by the former.