Separation of Water-Soluble Vitamins on the Agilent InfinityLab Poroshell 120 Aq-C18 Column

Significance of the Topic

Water-soluble vitamins play essential roles in health and nutrition. Their high polarity challenges conventional reversed-phase separations, demanding advanced stationary phases capable of retaining and resolving these analytes.

Objectives and Study Overview

This application note evaluates the performance of the Agilent InfinityLab Poroshell 120 Aq-C18 column for simultaneous separation of ten B vitamins, ascorbic acid, and caffeine. The goals include achieving baseline separation, symmetrical peak shapes, and robust reproducibility under highly aqueous conditions.

Methodology and Instrumentation

An Agilent 1290 Infinity LC system with a binary pump, autosampler, column compartment, and diode array detector was employed. Key parameters included:

• Column: Poroshell 120 Aq-C18, 4.6 × 100 mm, 2.7 µm
• Mobile phase A: 10 mM phosphate buffer, pH 2.5; B: acetonitrile
• Gradient: 0–2 min, 0–6% B; 2–6 min, 6–25% B; 6–8 min, 25–70% B; hold to 10 min
• Flow rate: 1.5 mL/min; temperature: 30 °C; detection at 210 and 260 nm

Main Results and Discussion

Baseline separation of 12 compounds was achieved with symmetrical peaks. Highly polar analytes, including thiamine and ascorbic acid, showed strong retention under 100% aqueous start conditions. Pantothenic acid and biotin, undetectable at 260 nm, were monitored at 210 nm. Reproducibility testing with six consecutive injections demonstrated minimal variation in retention time and peak area, highlighting column stability and resistance to dewetting in polar mobile phases.

Benefits and Practical Applications

• Enhanced retention and resolution of polar nutrients in a single run
• Reduced analysis time and improved peak shapes for routine QC
• Compatibility with highly aqueous mobile phases without dewetting

Future Trends and Opportunities

Continued development of superficially porous phases may extend analysis to highly polar biomolecules and complex vitamin matrices. Coupling with mass spectrometry and automated sample preparation could streamline workflows in pharmaceutical, food, and clinical laboratories.

Conclusion

The Agilent InfinityLab Poroshell 120 Aq-C18 column delivers reliable, high-resolution separations of water-soluble vitamins and caffeine under highly aqueous gradients, with excellent reproducibility and peak performance.

Reference

Fu RJ, Wei TC. Analysis of Polar Compounds Using an Agilent InfinityLab Poroshell 120 Aq-C18 Column with Improved and Reliable Performance. Agilent Technologies application note 5994-5555EN. 2022.