Vitamin D2 and D3 Separation by New Highly Hydrophobic UHPLC/HPLC Phase

Published on: 

The Application Notebook

The Application Notebook, The Application Notebook-09-01-2015, Issue 0
Pages: 12

Separations of structurally similar compounds such as vitamin D2 and vitamin D3 are very challenging.

Vitamin D2 (ergocalciferol) and D3 (cholecalciferol) can be found in different foods including fatty fishes, meat, egg, and some mushrooms. Both compounds are (indirectly) involved in a number of biological functions in the body, including bone metabolism and enhancement of intestinal absorption of calcium, iron, magnesium, phosphate, and zinc. A regular intake of vitamin D therefore is essential.



Standard C18 columns are not able to fully separate the two vitamins. A very hydrophobic phase with a higher carbon coverage and therefore a greater density of C18 chains is required. The highly hydrophobic phase YMC-Triart C18 ExRS (carbon load 25%!) is able to separate these two.




The isocratic high performance liquid chromatography (HPLC) method for vitamin D2 and D3 separation, using a 5 μm YMC-Triart C18 ExRS column, can easily be transferred to a ultrahigh-performance liquid chromatography (UHPLC) method using a 1.9 μm column, thereby reducing the analysis time by 50%. Furthermore, the resolution can be increased, resulting in a full baseline separation.

YMC-Triart C18 ExRS (5 µm, 8 nm), 150 × 3.0 mm

YMC-Triart C18 ExRS (1.9 µm, 8 nm),
        75 × 2.1 mm

Part No.:   TAR08S05-1503PTH/TAR08SP9-L5Q1PT
Eluent:THF/acetonitrile (10/90)
Flow rate:0.425 mL/min
Detection:UV at 265 nm
Temperature:30 °C



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