Vitamin K1 (phylloquinone) is an essential human nutrient produced in plants, especially green leafy vegetables. The vitamin K1 in natural products exists mainly as the trans form, while the vitamin K1 used in food supplementation is often synthetic K1, which may contain appreciable amounts of the cis form. The trans-vitamin K1 is bioactive, while the cis-K1 is not. It is highly desirable to separate the trans- and the cis-vitamin K1 isomers to truly evaluate the nutritional value of the supplement ingredient. Available HPLC methods for the separation of vitamin K1 isomers require C30 columns. Their typical run time is about 20 minutes, and chlorinated solvents are used in some of the methods. UltraPerformance Convergence Chromatography (UPC2) is a separation technique that leverages the unique properties (i.e., low viscosity and high diffusivity) of compressed CO2 at or near its supercritical state, as well as sub-2 micron particle packed columns to significantly improve the separation efficiency, speed, and selectivity. This application note demonstrates a fast separation of vitamin K1 trans and cis isomers and menatetrenone (MK-4), a common form of vitamin K2, by UPC2in less than three minutes on an ACQUITY UPC2 HSS C18 SB Column. Compared to current LC-based vitamin K1 trans and cis isomers analysis methods, this UPC2 method is faster, simpler (no need to use a C30 column), and it uses less organic solvent.