title: Preparative separation of two subsidiary colors of FD&C Yellow No. 5 (Tartrazine) using spiral high-speed counter-current chromatography.
abstract: Specifications in the U.S. Code of Federal Regulations for the color additive FD&C Yellow No. 5 (Color Index No. 19140) limit the level of the tetrasodium salt of 4-[(4',5-disulfo[1,1'-biphenyl]-2-yl)hydrazono]-4,5-dihydro-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylic acid and that of the trisodium salt of 4,4'-[4,5-dihydro-5-oxo-4-[(4-sulfophenyl)hydrazono]-1H-pyrazol-1,3-diyl]bis[benzenesulfonic acid], which are subsidiary colors abbreviated as Pk5 and Pk7, respectively. Small amounts of Pk5 and Pk7 are needed by the U.S. Food and Drug Administration for confirmatory analyses and for development of analytical methods. The present study describes the use of spiral high-speed counter-current chromatography (HSCCC) to separate the closely related minor components Pk5 and Pk7 from a sample of FD&C Yellow No. 5 containing ∼3.5% Pk5 and ∼0.7% Pk7. The separations were performed with highly polar organic/high-ionic strength aqueous two-phase solvent systems that were chosen by applying the recently introduced method known as graphic optimization of partition coefficients (Zeng et al., 2013). Multiple ∼1.0g portions of FD&C Yellow No. 5 (totaling 6.4g dye) were separated, using the upper phase of the solvent system 1-butanol/abs. ethanol/saturated ammonium sulfate/water, 1.7:0.3:1:1, v/v/v/v, as the mobile phase. After removing the ammonium sulfate from the HSCCC-collected fractions, these separations resulted in an enriched dye mixture (∼160mg) of which Pk5 represented ∼46% and Pk7, ∼21%. Separation of the enriched mixture, this time using the lower phase of that solvent system as the mobile phase, resulted in ∼61mg of Pk5 collected in fractions whose purity ranged from 88.0% to 92.7%. Pk7 (20.7mg, ∼83% purity) was recovered from the upper phase of the column contents. Application of this procedure also resulted in purifying the major component of FD&C Yellow No. 5 to >99% purity. The separated compounds were characterized by high-resolution mass spectrometry and several (1)H and (13)C nuclear magnetic resonance spectroscopic techniques.
authors.affiliation: Office of Cosmetics and Colors, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD 20740, USA. Electronic address: adrian.weisz@fda.hhs.gov.
authors.forename: Adrian
authors.initials: A
authors.lastname: Weisz
authors.affiliation: Office of Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD 20740, USA.
authors.forename: Clark D
authors.initials: CD
authors.lastname: Ridge
authors.affiliation: Office of Cosmetics and Colors, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD 20740, USA.
authors.forename: Jose A
authors.initials: JA
authors.lastname: Roque
authors.affiliation: Office of Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD 20740, USA.
authors.forename: Eugene P
authors.initials: EP
authors.lastname: Mazzola
authors.affiliation: Laboratory of Bioseparation Technology, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Ito
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004396:Coloring Agents / Q000737:chemistry / Q000302:isolation & purification*; D003377:Countercurrent Distribution / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D009994:Osmolar Concentration; D012997:Solvents / Q000737:chemistry; D013645:Tartrazine / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D004396:Coloring Agents; D012997:Solvents; D013645:Tartrazine
keywords: FD&C Yellow No. 5; Highly polar organic/high-ionic strength aqueous two-phase solvent systems; NMR; Spiral high-speed counter-current chromatography; Subsidiary colors; Tartrazine
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