title: Preparative separation and identification of novel subsidiary colors of the color additive D&C Red No. 33 (Acid Red 33) using spiral high-speed counter-current chromatography.
abstract: Three low-level subsidiary color impurities (A, B, and C) often present in batches of the color additive D&C Red No. 33 (R33, Acid Red 33, Colour Index No. 17200) were separated from a portion of R33 by spiral high-speed counter-current chromatography (HSCCC). The separation involved use of a very polar solvent system, 1-BuOH/5mM aq. (NH4)2SO4. Addition of ammonium sulfate to the lower phase forced partition of the components into the upper phase, thereby eliminating the need to add a hydrophobic counterion as was previously required for separations of components from sulfonated dyes. The very polar solvent system used would not have been retained in a conventional multi-layer coil HSCCC instrument, but the spiral configuration enabled retention of the stationary phase, and thus, the separation was possible. A 1g portion of R33 enriched in A, B, and C was separated using the upper phase of the solvent system as the mobile phase. The retention of the stationary phase was 38.1%, and the separation resulted in 4.8 mg of A of >90% purity, 18.3mg of B of >85% purity, and 91 mg of C of 65-72% purity. A second separation of a portion of the C mixture resulted in 7 mg of C of >94% purity. The separated impurities were identified by high-resolution mass spectrometry and NMR spectroscopic techniques as follows: 5-amino-3-biphenyl-3-ylazo-4-hydroxy-naphthalene-2,7-disulfonic acid, A; 5-amino-4-hydroxy-6-phenyl-3-phenylazo-naphthalene-2,7-disulfonic acid, B; and 5-amino-4-hydroxy-3,6-bis-phenylazo-naphthalene-2,7-disulfonic acid, C. The isomers A and B are compounds reported for the first time. Application of the spiral HSCCC method resulted in the additional benefit of yielding 930 mg of the main component of R33, 5-amino-4-hydroxy-3-phenylazo-naphthalene-2,7-disulfonic acid, of >97% purity.
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 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: Bioseparation Technology Laboratory, 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; D003116:Color; D004396:Coloring Agents / Q000032:analysis*; D003377:Countercurrent Distribution; D057927:Hydrophobic and Hydrophilic Interactions; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009282:Naphthalenesulfonates / Q000032:analysis*
chemical_list: D004396:Coloring Agents; D009282:Naphthalenesulfonates; C044608:D.C. Red No. 33
keywords: 5-Amino-3-biphenyl-3-ylazo-4-hydroxy-naphthalene-2,7-disulfonic acid; 5-Amino-4-hydroxy-3,6-bis-phenylazo-naphthalene-2,7-disulfonic acid; 5-Amino-4-hydroxy-6-phenyl-3-phenylazo-naphthalene-2,7-disulfonic acid; D&C Red No. 33; NMR spectra of dye impurities; Spiral high-speed counter-current chromatography