Chromatographic Resolution of 7 of 8 Stereoisomers of Vitamin K1 on an Amylose Stationary Phase Using Supercritical Fluid Chromatography

摘要

In a previous report, it was shown that the trans-isomers of the 2′–3′ double bond on the side chain of vitamin K1 (phylloquinone) represented 88.65 % of a specific sample, while the cis-isomer represented 11.35 %. However, the side chain contains 2 chiral centers, indicating there are 8 possible stereoisomers. Seven of these 8 stereoisomers were partially resolved in 20 min, on a RegisPack 4.6 × 250 mm, 5 μm column, using 5 % methanol in CO2, at 2 mL/min, 30 °C, 150 bar. This is the first reported separation of these enantiomers by supercritical fluid chromatography, and, apparently, the first separation, by any technique, on a widely available commercial chiral stationary phase. The chiral separation produced one dominant peak, accounting for approximately 58 % of the total area, most likely due to 2′,3′-trans-4R,11R-philloquinone. Three other peaks each accounted for 9.7, 13.4, and 10.3 % of the total area, for a total of an additional ±33.4 %. These 4 peaks account for ≈91.4 % of the total area, in near agreement with the achiral findings for the separation of the trans-isomers. Three smaller, broad, poorly quantified peaks, collectively accounted for 8.1 % of the total area. Mass balance suggests these 3 small peaks are all cis-enantiomers, leaving approximately 3.2 % of the total area, representing the 4th cis-enantiomer, unaccounted for, and likely a co-elution with one of the 4 larger peaks.