Multi-Mode Water Soluble Vitamin Separation Using ZirChrom®-SAX

Dr. Bingwen Yan and Dr. Clayton McNeff, ZirChrom Separations, Inc.

Traditionally the analysis of water-soluble vitamins by reversed-phase HPLC has been complicated by the lack of retention for these compounds on conventional silica C18 columns. Here we demonstrate efficient, multi-mode, baseline resolution of six water-soluble vitamins in 6 min using a ZirChrom® -SAX column.

In this application note we focus on the HPLC analysis of Vitamin C and five B-complex vitamins; Vitamin B1 (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (nicotinic acid form), Vitamin B3 (nicotinamide form), and Vitamin B6 (pyridoxine). All of these vitamins are water-soluble.

Chromatographers oftentimes struggle in their attempts to analyze water-soluble vitamins by HPLC. Many water-soluble vitamins are very polar. Thiamin (Vitamin B1), pyridoxine (Vitamin B6), and ascorbic acid (Vitamin C), for example, show almost no retention on conventional C18 columns. Reversed-phase analytical methods employing ion pair reagents have been offered as a potential solution to this problem, but these methods tend to suffer from column-to-column reproducibility problems due to the somewhat unpredictable way ion-pairing reagents interact with the silica surface and the bonded phase.

In this technical bulletin we present a unique method for the analysis of water-soluble vitamins using a ZirChrom® -SAX HPLC column. ZirChrom® -SAX, an anion exchange material, is polyethleneimine-coated zirconia containing a substantial amount of hydrophobic cross-linker which imparts both ion-exchange and reversed-phase characteristics. The multi-mode retention characteristics of the ZirChrom® -SAX column create the unique selectivity ideal for this application (Figure 1).

Figure 1

For the analysis of water-soluble vitamins in serum, or other samples containing biological matrices, we recommend the addition of the ProTain® In-Line Protein Removal System; consisting of one guard holder (part# 850-00-2) and a set of three ProTain® inserts (part# PT01-0246). Please see technical bulletins #275 and #291, available at www.zirchrom.com, for further information on the use of ZirChrom's ProTain® In-Line Protein Removal System.

Experimental

Six water-soluble vitamin standards were prepared in an aqueous solution and injected on a ZirChrom® -SAX column. The separation conditions are as follows:

Column: ZirChrom® -SAX, 150 × 4.6 mm i.d., 3μm

(part number: ZR06-1546)

Mobile Phase: 50 mM Ammonium dihydrogenphosphate,

pH 4.5

Flow rate: 1.0 ml/min.

Temperature: 30°C

Injection Vol.: 5.0 μl

Detection: UV at 254 nm

The separation is shown in Figure 1. Under these conditions the separation of Vitamin C and the four B-complex vitamins is achieved, with good peak shape and baseline resolution, in 6 min.

This method can be tailored to your specific application needs. ZirChrom technical support can help to optimize and transfer this method to your site. Please contact ZirChrom technical support at 1-866-STABLE-1 or support@zirchrom.com for details.

ZirChrom phases offer unique selectivity, high efficiency, and excellent chemical and thermal stability.

ZirChrom Separations, Inc.

617 Pierce Street, Anoka, MN 55303

tel. 1-866-STABLE-1

Email: support@zirchrom.com

www.zirchrom.com