Update: 30 September, 2023
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.
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MD5 Checksum: 6052C147EBCBBEDF90FC35CE7E69E37A
Publication date: 02 July, 2012
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PDF Link: AGILENT UHPLC Method Development Options for a Vitamin D2 D3 Separation Application Note PDF