Procedure—
Prepare the necessary number of chromatographic sheets (Whatman No. 1 filter paper, or equivalent), about 20 × 20 cm in size, by drawing a pencil line parallel to and 2.5 cm from one edge of the paper. Mark the line at points 2.5 and 5 cm from each edge of the paper. Impregnate the paper by dipping it in the immobile solvent (prepared fresh by dissolving 30 mL of redistilled formamide in 70 mL of acetone) for 30 seconds. Remove the paper, drain for 10 seconds, and blot between filter paper. Place the impregnated paper on dry filter paper, and air-dry for 3 to 5 minutes. With a micropipet, and with repeated applications, streak 100 µL of the
Assay Preparation along the starting line, applying the volume in five streaks of about 20 µL each and evaporating the solvent with a gentle stream of nitrogen between applications.
[NOTE—Make the streak as narrow as possible along the starting line, and keep within the 5-cm border.
] Rinse the tip of the pipet with a drop of methanol-ammonia TS mixture (9:1), and then streak the rinse along the starting line between the 5- and 2.5-cm points at the right edge. Repeat the rinsing with two additional drops, and then blow out the pipet.
Apply 10 µL of the Mixed Standard Preparation at the mark 2.5 cm from the left edge.
Place 50 mL of methylene chloride (mobile solvent) in a tray in a 23- × 23- × 7.5-cm chromatographic chamber arranged for ascending chromatography (see
Chromatography 
621
), and allow the chamber to equilibrate for about 15 minutes. Remove the cover, place from 7 to 10 mL of water in a second tray, and without delay, suspend the prepared chromatographic paper sheet so that it dips into the mobile solvent. Cover and seal the chamber, and allow the chromatogram to develop for 1 hour. Remove the paper from the chamber, and allow to air-dry for 5 minutes. Place the chromatogram on a dry sheet of filter paper, and view it under short-wavelength UV light.
[NOTE—Conduct the following identification and marking without delay to avoid excessive exposure of the sulfonamide spots to UV irradiation.
] Identify and mark the respective spots by matching
RF values with those of the spots produced by the
Mixed Standard Preparation. [NOTE—Sulfadiazine and sulfamerazine are chromatographed with increasing
RF, respectively.
]
Cut the marked zones from the paper, cut each zone into five or six pieces, and place the pieces from each spot in separate, glass-stoppered, 50-mL flasks. Add 20.0 mL of dilute hydrochloric acid (1 in 100) to each flask, and allow to stand for about 30 minutes, swirling each flask at least five times during this period. Filter the solutions through dry glass wool into separate test tubes, discarding the first 5 mL of the filtrate. Transfer 5.0 mL of the subsequent filtrate from each solution into separate 10-mL volumetric flasks. Transfer 3.0 mL of each required Standard Preparation into separate, 10-mL volumetric flasks. To each flask, and to a blank flask containing 5 mL of dilute hydrochloric acid (1 in 100), add 1.0 mL of sodium nitrite solution (1 in 1000) and 0.10 mL of hydrochloric acid, and allow to stand for 5 minutes with frequent swirling. To each flask add 1.0 mL of ammonium sulfamate solution (1 in 200), and allow to stand for 5 minutes, swirling frequently. Finally, to each flask add 1.0 mL of freshly prepared N-(1-naphthyl)ethylenediamine dihydrochloride solution (1 in 1000), mix, dilute with water to volume, and mix. Allow each solution to stand between 15 and 60 minutes, and then concomitantly determine the absorbances of the solutions, in 1-cm cells, recording the spectra from 440 to 700 nm, with a suitable spectrophotometer, using the blank to set the instrument. Draw a baseline, and determine the corrected absorbance for each solution at the wavelength of maximum absorbance at about 545 nm.
Calculate the concentration, in mg per mL, of each sulfonamide in the Assay Preparation by the formula:
0.12C(AU / AS),
in which C is the concentration, in µg per mL, of the pertinent USP Reference Standard in the Standard Preparation; AU is the corrected absorbance of the Assay Preparation; and AS is the corrected absorbance of the pertinent Standard Preparation. From the concentration of the Assay Preparation thus determined, and applying appropriate dilution factors, calculate the percentage of sulfonamide in the specimen taken.
for 5 minutes or until bright yellow spots become visible. The RF values of the yellow spots obtained from each Test Preparation correspond to those obtained from the mixed Standard Preparations on the respective plates. The individual sulfonamides may be identified by comparison of the RF values of the yellow spots obtained from the Test Preparations and individual Standard Preparations on the respective plates.