Separation of Vitamin K Isomers with Enhanced Selectivity

September 1, 2011

The Application Notebook

The Application Notebook, The Application Notebook-09-01-2011, Volume 0, Issue 0

Vitamin K is a group of structurally similar, fat-soluble vitamins that are needed for the post-translational modification of certain proteins, mostly required for blood coagulation but also involved in metabolic pathways in bone and other tissue.

Vitamin K is a group of structurally similar, fat-soluble vitamins that are needed for the post-translational modification of certain proteins, mostly required for blood coagulation but also involved in metabolic pathways in bone and other tissue. The primary members of the group are Vitamins K1 and K2 (Figure 1). Plants synthesize Vitamin K1 while bacteria can produce a range of Vitamin K2 forms, including the conversion of K1 to K2 by bacteria in the small intestines. Exposure to light is known to isomerize vitamin K in the isoprenoid chain.

Figure 1: Chemical structure of Vitamin K1 and Vitamin K2.

Strongly hydrophobic molecules, such as triglycerides, carotenoids, and fat-soluble vitamins may be analyzed using reversed-phase LC with low-aqueous or even non-aqueous mobile phases. While standard C18 silica columns fail to provide adequate shape selectivity to resolve structural isomers of these long chain hydrophobic compounds, the C30 phase is ideal for this application due to its excellent shape selectivity.

By choice of surface area and pore size in the silica substrate, the Thermo Scientific Acclaim™ C30 stationary phase has been designed to offer enhanced selectivity without excessive retention times. In many ways, it can be used as a general-purpose RP column same as the Acclaim C18 column, but with improved performance. For example, the Acclaim C30 column can separate highly hydrophilic analytes with high-aqueous mobile phases, or strongly hydrophobic analytes with low-aqueous mobile phases.

Results and Discussion

Figure 2a shows the resolution of the photo-isomers of Vitamins K1 and K2 using the Acclaim C30 column with a 98% methanol mobile phase at 20 °C. Under the same conditions, the Acclaim C18 column fails to resolve these structural isomers (Figure 2b). We also evaluated the effect of temperature and found that as temperature decreased, selectivity improved but the peak width also increased. The temperature of 20 °C was determined to be optimal for the separation. Acetonitrile can also be used instead of methanol, with slightly different selectivity, and somewhat longer retention times.

Figure 2: Columns: (a) Acclaim C30 and (b) Acclaim C18, 3 μm, 3.0 ×150 mm. Mobile phase: 98% methanol, 2% D.I. water; temperature: 20 °C; flow rate: 0.65 mL/min. Injection volume: 2.5 μL. Detection: Diode Array (UV at 250 nm shown). Sample: 500 μg/mL Vitamins K1 and K2 in acetonitrile, (exposed to UV light for 20 min). Peaks: (1) Vitamin K2; (2) Isomer of Vitamin K2; (3) Vitamin K1; (4) Isomer of Vitamin K1.

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