This application note describes the method development options for separation of vitamin D2 and D3 by UHPLC. Partial separation of vitamin D2 and D3 was achieved by an isocratic LC method using a C18 column and methanol/acetonitrile mobile phase. Several optimization parameters were examined to increase resolution (Rs) including column selectivity and column length. A 2.1 mm × 150 mm Agilent ZORBAX StableBond-C18 column provided the best resolution. Further fine tuning included faster flow rates and mobile phase strength resulting in short analysis times of 3 to 1.5 minutes with Rs of 1.6 to 1.4. The performance of a 3.0 mm internal diameter (id) column was also compared to that of the 2.1 mm id column demonstrating higher sample loading before any loss of resolution in the closely spaced peaks.
This document describes the optimization of a Liquid Chromatography (LC) method for the separation of Vitamin D2 and Vitamin D3 in food samples. It evaluates various parameters to improve resolution and reduce analysis time, focusing on the transition from standard HPLC to UHPLC methods.
The study compares different column selectivities, specifically between Agilent ZORBAX Eclipse Plus C18 and ZORBAX StableBond-C18 (SB-C18), demonstrating that SB-C18 provides superior resolution for these vitamins. The document further investigates the impact of column length, flow rate, and column internal diameter (ID) on separation efficiency and solvent usage, recommending the use of Agilent RRHD (Robust High Pressure) columns operated at high pressures up to 1200 bar.
| System | Agilent 1290 Infinity with G4212A DAD |
|---|---|
| Detection Wavelength | 280 nm |
| Reference Wavelength | 360 nm |
| Column Temperature | 25°C |
| Column Dimensions | 2.1 mm x 150 mm |
A 2.1 mm x 150 mm Agilent ZORBAX StableBond-C18 provided the best resolution.
The RRHD columns are designed to operate up to 1200 bar.
Increasing the flow rate from 1 to 1.5 mL/min reduced peak retention by one-third.