Date: 8-OCTOBER-2026 Last Updated: 13-OCTOBER-2026
Introduction
Fat-soluble vitamins D2 (Ergocalciferol), D3 (Cholecalciferol), E (α-Tocopherol), and K1 (Phylloquinone) are commonly found in dietary supplements and fortified products. Due to their hydrophobic nature and similar properties, their analysis can be challenging using conventional C18 columns. This application note demonstrates the simultaneous HPLC analysis of these vitamins using a C30 column, which provides enhanced selectivity and resolution.
Peaks:
1. α-Tocopherol (Vitamin E), CAS 59-02-9
2. Ergocalciferol (Vitamin D₂), CAS 50-14-6
3. Cholecalciferol (Vitamin D₃), CAS 67-97-0
4. Phylloquinone (Vitamin K₁), CAS 84-80-0
Method Conditions
Column:
Cogent C30™, 5 μm, 200 Å
Catalog No.
:
72530-25P
Dimensions
: 4.6 x 250 mm
Mobile Phase
: 80:20 Acetonitrile / Methanol (80:20) containing 0.1% formic acid
Injection vol.
: 4 μL
Flow rate
: 1.0 mL/minute
Detection
: UV @ 210 nm
Sample Preparation
: Phylloquinone (Vitamin K1), CAS 84-80-0, 1.7 mg/mL, α-Tocopherol, CAS 59-02-9, 2.0 mg/mL, Ergocalciferol (Vitamin D2), CAS 50-14-6, 2.0 mg/mL, Cholecalciferol (Vitamin D3), CAS 67-97-0, 2.15 mg/mL- all prepared in ethanol
Mixed Standard:
100 µL of each stock solution.
Notes: Vitamins D2 and D3 are structurally similar secosteroids, while Vitamin E (α-tocopherol) and Vitamin K1 (phylloquinone) are fat-soluble vitamins with distinct chromophores. Due to their hydrophobic nature, these compounds are well-suited for separation using C30 stationary phases.