Preparative separation of 1,3,6-pyrenetrisulfonic acid trisodium salt from the color additive D&C Green No. 8 (pyranine) by pH-zone-refining counter-current chromatography

In developing analytical methods for batch certification of the color additive D&C Green No. 8 (G8), the U.S. Food and Drug Administration needed the trisodium salt of 1,3,6-pyrenetrisulfonic acid (P3S) for use as a reference material. Since P3S was not commercially available, preparative quantities of it were separated from portions of a sample of G8 that contained ∼3.5% P3S. The separations were performed by pH-zone-refining counter-current chromatography using dodecylamine (DA) as the hydrophobic counterion. The added DA enabled partitioning of the polysulfonated components into the organic stationary phase of the two-phase solvent system used, 1-butanol–water (1:1). Thus, a typical separation that involved 20.3 g of G8, using sulfuric acid as the retainer acid and 20% DA in the stationary phase and 0.1 M sodium hydroxide as the mobile phase, resulted in ∼0.58 g of P3S of greater than 99% purity. The identification and characterization of the separated P3S were performed by elemental analyses, proton nuclear magnetic resonance, high-resolution mass spectrometry, ultra-violet spectra, and high-performance liquid chromatography.

► Minor component 1,3,6-pyrenetrisulfonate (P3S) of D&C Green No. 8 (G8) separated.
► pH-zone-refining CCC separation of 20.3 g of G8 using dodecylamine as ion-exchanger.
► Major component 8-hydroxy-1,3,6-pyrenetrisulfonate (HP3S) also separated.
► Obtained 0.58 g P3S and 12.1 g HP3S of ∼99% purity.
► Identification and characterization by 1H NMR, HR-MS, UV–vis, and HPLC.