Agilent Reversed-Phase HPLC Separation of Water-Soluble Vitamins on Agilent ZORBAX Eclipse Plus Columns Manual

Update: 30 September, 2023

This file introduces a method for separating a mixture of six B vitamins and vitamin C on the same Agilent ZORBAX Eclipse Plus C-18 stationary phase in three different particle sizes and column dimensions: 5 µm (4.6 × by 150 mm); Rapid Resolution (RR), 3.5 µm (4.6 × 100 mm), and RR High Throughput (RRHT), 1.8-µm (4.6 × 50 mm). A simple phosphate buffer- methanol mobile phase gradient was used. The method was designed to supplement United States Pharmacopeia (USP) methods in which these vitamins are separated singly on different columns by different methods, some involving complex ion-pair reagents. The shorter columns provided more rapid separations with little change in res- olution. All compounds could be separated on the 1.8-µm column in 3.5 minutes. A gain in sensitivity was noted when the column length and particle size were reduced. The HPLC method was applied to the analysis of vitamins in a variety of commercial products, including multivita- mins and various soft drinks. The columns chosen allowed for good separation, but the RRHT column also allowed quick method development without giving up the resolution and sensitivity.


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Publication date: 12 June, 2012

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