Scaled Separation of Nine Water‑Soluble Vitamins on the Agilent InfinityLab Poroshell 120 SB‑Aq Column

Significance of the topic

Efficient separation of water-soluble B vitamins is essential in food analysis, nutritional studies, and quality control workflows. Superficially porous particle columns such as Agilent InfinityLab Poroshell 120 SB-Aq offer high chromatographic efficiency at moderate pressures, enabling rapid, high-resolution analysis. Scaling the separation method across different particle sizes enhances method transferability and instrument compatibility, supporting diverse laboratory needs.

Objectives and Study Overview

This study aimed to develop and geometrically scale a reversed-phase LC method for nine water-soluble B vitamins using Agilent Poroshell 120 SB-Aq columns with 1.9, 2.7, and 4.0 µm particles. The goals were to preserve selectivity and resolution, adjust flow rates and gradient times to maintain linear velocity, and demonstrate consistent performance across column formats.

Methodology and Instrumentation

The separation employed an Agilent 1290 Infinity II LC system with reduced system volume and low-dispersion flow paths. Key components included:

• Flexible binary pump with 35 µL mixer and seal wash
• Vialsampler autosampler with 20 µL loop and capillary heater
• Integrated column compartment for precise temperature control
• Diode array detector operating at 260 nm, 80 Hz acquisition

Three Poroshell 120 SB-Aq columns (2.1 × 50 mm, 1.9 µm; 3.0 × 100 mm, 2.7 µm; 4.6 × 150 mm, 4.0 µm) were packed with superficially porous silica. The mobile phase consisted of a phosphate buffer (pH ~2.0) and acetonitrile, using a gradient from 0 to 30% organic. Sample concentrations ranged from 0.1 to 0.8 mg/mL in water.

Main Results and Discussion

All three column formats achieved comparable retention patterns and peak selectivity for the nine B vitamins. Scaling the gradient duration (3, 6, and 12 minutes) and flow rates (0.5, 1.0, and 1.5 mL/min) maintained similar L/dp ratios and separation profiles. Maximum column pressures were within acceptable limits (220, 280, and 245 bar).

Key observations:

• The 1.9 µm column provided the fastest analysis, reducing runtime and solvent use but required a low-volume LC system to avoid extra-column dispersion and was more sensitive to sample particulates due to smaller frit sizes.
• The 2.7 µm and 4.0 µm columns offered greater robustness against clogging and broader LC system compatibility, with only moderate increases in runtime.

Benefits and Practical Applications

Method scaling across particle sizes allows laboratories to:

• Optimize throughput and solvent consumption by choosing smaller particles when instrument capabilities permit.
• Extend method applicability to standard HPLC systems using larger particles for clogging resistance and flexibility.
• Maintain consistent selectivity and resolution for quality control, nutritional profiling, and regulatory compliance.

Future Trends and Opportunities

Advances may include further reductions in superficial particle dimensions to push efficiency limits, integration of mass spectrometry detectors for multi-vitamin confirmation, and automated method scaling software. Green solvent systems and high-throughput UHPLC platforms will expand applicability in industry and research.

Conclusion

The study demonstrates that geometric scaling of gradient time and flow rate preserves chromatographic performance for nine water-soluble B vitamins across Agilent Poroshell 120 SB-Aq columns with 1.9, 2.7, and 4.0 µm particles. Users can select column formats to balance analysis speed, solvent usage, system compatibility, and robustness.

References

1. Gratzfield-Huesgen A, Naegele E. Maximizing Efficiency Using Agilent Poroshell 120 Columns; Agilent Technologies Application Note 5990-5602EN, June 2016.
2. Meyer VR. Practical High-Performance Liquid Chromatography, 4th Edition; Wiley, 2004, p. 34.