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.
This application note details a high-performance liquid chromatographic (HPLC) method developed to separate a mixture of six B vitamins and vitamin C. The study utilizes Agilent ZORBAX Eclipse Plus C-18 stationary phases in three different particle sizes (5 µm, 3.5 µm, and 1.8 µm) and column dimensions. The method employs a simple phosphate buffer-methanol mobile phase gradient, designed to supplement United States Pharmacopeia (USP) methods which often require complex ion-pair reagents.
| Mobile Phase A | 25 mM NaH2PO4 pH = 2.5 |
|---|---|
| Mobile Phase B | Methanol |
| Flow Rate | 1.00 mL/min |
| Column Temperature | 35°C |
| Detection Wavelength | 220 nm |
All compounds could be separated on the 1.8 µm column in 3.5 minutes.
All compounds could be separated on the 1.8-um column in 3.5 minutes.
The flow rate was set to 1.0 mL/min.
The column compartment must be kept at a temperature of 35°C.