Analysis of Water-Soluble Vitamins on an Agilent InfinityLab Poroshell 120 HILIC-OH5 Column

Significance of the Topic

Hydrophilic interaction chromatography is a powerful approach for the analysis of highly polar compounds such as water soluble vitamins. The combination of superficially porous particle columns and HILIC mode delivers high efficiency, rapid analysis, and improved resolution. Fast and reliable vitamin profiling is critical in food quality control, nutraceutical development, and regulatory compliance.

Objectives and Overview

This study aims to demonstrate the performance of an Agilent InfinityLab Poroshell 120 HILIC OH5 column for the baseline separation of eight water soluble vitamins in under four minutes. The work assesses chromatographic conditions, compares alternative HILIC phases, and evaluates key performance metrics such as resolution and peak shape.

Methodology

The separation employed a 2.1 × 100 mm superficially porous column with 2.7 micrometer particles. A binary mobile phase gradient combined an ammonium acetate buffer with acetonitrile using the following parameters.

• Flow rate 0.5 milliliter per minute
• Initial composition 87 percent acetonitrile
• Gradient to 50 percent acetonitrile over 3.5 minutes
• Re equilibration time 3 minutes
• Column temperature 40 degrees Celsius
• Detection by diode array at 260 nanometers

Calibration solutions were prepared from individual vitamin standards dissolved in appropriate solvent mixtures such as acetonitrile water or acetonitrile DMSO blends.

Applied Instrumentation

• Agilent 1290 Infinity binary pump with low dispersion solvent mixing
• Agilent 1290 Infinity autosampler and thermostatted injector
• Agilent 1290 Infinity column compartment
• Agilent 1290 Infinity diode array detector
• OpenLAB CDS software for data acquisition
• Agilent InfinityLab Poroshell 120 HILIC OH5 column, 2.1 × 100 millimeter

Main Results and Discussion

Baseline separation of eight vitamins including B1 B2 B3 B6 B9 B10 B12 and vitamin C was achieved within a four minute run time. The critical resolution between riboflavin and PABA exceeded 2.7 under the optimized conditions. Alternative HILIC phases were evaluated but showed either coelution of key vitamins or unacceptable peak shapes, highlighting the distinct selectivity and performance advantage of the OH5 stationary phase.

Benefits and Practical Applications

• High throughput screening of vitamin profiles in food and supplement matrices
• Reduced analysis time enhances laboratory productivity
• Reproducible retention and peak shape support stringent quality control
• Minimal sample preparation enables workflow simplicity

Future Trends and Opportunities

The integration of superficially porous HILIC phases with mass spectrometry may expand sensitivity and specificity. Further method development could optimize green solvent usage and broaden compound coverage. Automation and multiplexed analysis of vitamin complexes represent emerging directions for rapid nutritional profiling.

Conclusion

The Agilent InfinityLab Poroshell 120 HILIC OH5 column provides a robust fast separation of water soluble vitamins with excellent resolution and peak quality. This method supports efficient routine analysis in food testing and related fields.

References

1. Gratzfield Hugsen A Naegele E Maximizing efficiency using Agilent InfinityLab Poroshell 120 Columns Agilent Technologies Application Note publication number 5990 5602EN 2016