Which reagent system contains sulfanilic acid, hydrochloric acid, and sodium nitrite?

• A. Jaffe
• B. Zimmerman
• C. Diazo
• D. Lowry

Answer: (C)

The main idea here is the diazo reaction used to colorize bilirubin. Sulfanilic acid is diazotized in strongly acidic solution by nitrous acid generated from sodium nitrite in hydrochloric acid, forming a diazonium ion. This diazonium compound then couples with bilirubin to produce a colored azo dye (azobilirubin) that can be measured spectrophotometrically. Because the reagent system described—the combination of sulfanilic acid, hydrochloric acid, and sodium nitrite—is the classic diazo reagent setup, it best fits the diazo method. The other options rely on different chemistries (for example, the Jaffe method uses a reaction of creatinine with picric acid in alkaline conditions, and Lowry involves protein-detection chemistry), so they do not match this diazotization/coupling mechanism.