Improved Aromatic Acids Analysis: Saving Significant Time, Solvent, Sample and Money with 1.9 μm Agilent InfinityLab Poroshell 120 Columns

Importance of the Topic

Aromatic acids such as protocatechuic acid, DOPAC, PABA, vanillic acid, syringic acid, and salicylic acid are critical biomarkers and quality indicators in agriculture, food testing, and small-molecule pharmaceutical analysis. Efficient, high-resolution liquid chromatography methods can reduce analysis time, solvent and sample consumption, and operational costs while maintaining or improving analytical performance.

Objectives and Study Overview

This study compared the separation performance of a traditional 5 µm Agilent ZORBAX Eclipse Plus C18 column (4.6 × 250 mm) to three superficially porous Agilent InfinityLab Poroshell 120 EC-C18 columns (2.1 × 50 mm) with 1.9 µm, 2.7 µm, and 4 µm particle sizes. The aim was to demonstrate method transferability, evaluate resolution, pressure requirements, runtime, and resource savings, and to push the limits of ultrafast separations using the 1.9 µm column.

Methodology and Instrumentation

The separation used a binary gradient of 0.2 % formic acid in water (A) and acetonitrile (B). Original and scaled gradients were applied to each column to maintain selectivity without further method development. Flow rates and injection volumes were geometrically scaled to column volume. Analyte concentration was 0.01 mg/mL per compound.

Instrumentation Used

• Agilent 1290 Infinity LC system with low-dispersion binary pump, high-performance autosampler, thermostatted column compartment, and diode array detector
• Columns evaluated:
  • ZORBAX Eclipse Plus C18, 4.6 × 250 mm, 5 µm
  • InfinityLab Poroshell 120 EC-C18, 2.1 × 50 mm, 1.9 µm, 2.7 µm, and 4 µm
• Mobile phases: 0.2 % formic acid in water and HPLC-grade acetonitrile

Main Results and Discussion

All columns provided similar selectivity, achieving baseline separation of six aromatic acids with minimum resolution ≥1.8. Key findings:

• 5 µm ZORBAX column: 36 min runtime, minimum resolution 2.4, system pressure 193 bar
• 1.9 µm Poroshell column (standard): 3 min runtime, minimum resolution 2.9, system pressure 472 bar, 12× faster, 96 % less mobile phase, 75 % less sample
• 2.7 µm Poroshell column: resolution 2.1, pressure 281 bar
• 4 µm Poroshell column: resolution 1.8, pressure 141 bar
• 1.9 µm Poroshell ultrafast mode: 0.35 min runtime, minimum resolution 2.3, pressure 1 150 bar, 103× faster, 98 % less mobile phase

Benefits and Practical Applications

• Significant time savings support high-throughput workflows
• Reduced solvent and sample usage lowers operating costs and environmental impact
• Easy method transfer between ZORBAX and Poroshell columns due to similar bonded phases
• Flexibility to choose particle size based on instrument pressure limits and performance goals

Future Trends and Opportunities

The move toward smaller superficially porous particles will continue to drive ultrafast, high-efficiency separations. Anticipated advances include integration with automated sample preparation and real-time QA/QC monitoring, further reduction of system dispersion, and development of novel stationary phases for enhanced selectivity in complex matrices.

Conclusion

The Agilent InfinityLab Poroshell 120 EC-C18 column with 1.9 µm particles significantly outperforms a traditional 5 µm column, offering faster analyses, improved resolution, and dramatic resource savings. When instrument pressure limits are reached, 2.7 µm and 4 µm Poroshell columns provide alternative options with minimal method reoptimization. Ultrafast separations at elevated temperature and flow rates demonstrate the full potential of superficially porous particles for modern analytical challenges.

References

1. Gratzfield-Hugsen A.; Naegele E. Maximizing efficiency using Agilent Poroshell 120 Columns. Agilent Technologies Application Note 5990-5602EN, 2016.
2. Meyer V. R. Practical High Performance Liquid Chromatography. Fourth Edition, Wiley, 2004, p. 34.
3. Agilent Technologies. Transfer of Methods between Poroshell 120 EC-C18 and ZORBAX Eclipse Plus C18 Columns. Application Note 5990-6588EN, 2011.