Safranin O
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This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition
Issued and maintained by the United States Pharmacopeial Convention (USP)
CAS RN®: 477-73-6.—Dark red powder consisting of a mixture of 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride, C20H19ClN4 —
350.85, and 3,7-diamino-2,8-dimethyl-5-o-tolylphenazinium chloride, C21H21ClN4 —364.88—Sparingly soluble in 70 percent alcohol yielding a
clear red solution with a yellowish-red fluorescence.
IDENTIFICATION
A. To 10 mL of a 0.5% w/v solution add 5 mL of hydrochloric acid: a bluish violet solution is produced.
B. To 10 mL of a 0.5% w/v solution add 5 mL of sodium hydroxide solution (1 in 5): a brownish-red precipitate is produced.
C. To 100 mg add 5 mL of sulfuric acid: a green solution is produced, which, on dilution, changes to blue and finally to red.
Absorption Characteristics: Dissolve 50 mg in 250 mL of 50 percent alcohol. Dilute 3 mL of this solution with 50 percent alcohol to 200 mL.
Determine the absorbance, in a 1-cm cell, with a suitable spectrophotometer. The absorbance maximum is in the range of 530 to 533 nm; the
ratio (P − 15)/(P + 15) is between 1.10 and 1.32, in which P is the wavelength of maximum absorbance.
