HPLC Method For Analysis of Ergocalciferol (Vitamin D2) and Cholecalciferol (Vitamin D3) on Primesep SB Column

Separation type: Liquid Chromatography Mixed-mode

High Performance Liquid Chromatography (HPLC) Method for Analysis of Ergocalciferol (Vitamin D2) and Cholecalciferol (Vitamin D3)
Ergocalciferol (Vitamin D2) and Cholecalciferol (Vitamin D3) are fat soluble vitamins that help your body absorb calcium and phosphorus. Your body can produce both when exposed to the sun, but you can also intake these essential vitamins through a daily multivitamin or a Vitamin D pill.
Vitamins D2 and D3 can be detected in the high UV regime. Using a Primesep SB reverse-phase column and a mobile phase consisting of water and acetonitrile with an acetic acid buffer, Vitamins D2 and D3 can be retained and separated. This analysis method can be UV detected at 265 nm.



Column Primesep SB, 4.6×150 mm, 5 µm, 100A
Mobile Phase MeCN/H2O – 80/20%
Buffer Acetic Acid – 0.1%
Flow Rate 1.0 ml/min
Detection UV 265 nm



Class of Compounds
Analyzing Compounds Ergocalciferol (Vitamin D2),  Cholecalciferol (Vitamin D3)


Application Column

Primesep SB

The Primesep family of mixed-mode columns offers a wide variety of stationary phases, boasting unprecedented selectivity in the separation of a broad array of chemical compounds across multiple applications. Corresponding Primesep guard columns, available with all stationary phases, do not require holders. SIELC provides a method development service available to all customers. Inquire about our specially-tailored custom LC-phases for specific separations.

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Application Analytes:
SIELC Technologies usually develops more than one method for each compound. Therefore, this particular method may not be the best available method from our portfolio for your specific application. Before you decide to implement this method in your research, please send us an email to research@sielc.com so we can ensure you get optimal results for your compound/s of interest.