Separation of Organic Acids with Mixed-Mode LC Column and Mass Detector

Significance of the Topic

This technology brief addresses the rapid and reliable separation of organic acids in beverages, particularly fruit juices. Monitoring organic acids is essential for quality control, authenticity testing, and health diagnostics. Traditional anion-exchange methods often require long run times and non-selective detectors can suffer matrix interferences. A mixed-mode LC approach combined with mass detection offers a more efficient and selective solution.

Study Objectives and Overview

The goal of the study was to demonstrate the performance of the Waters Atlantis PREMIER BEH C18 AX mixed-mode column paired with the ACQUITY QDa mass detector for analyzing a panel of 15 organic acid standards and complex juice samples. Key objectives included reducing analysis time, improving retention and separation efficiency, and ensuring accurate quantitation in complex matrices.

Methodology

A series of 15 common organic acids were prepared at 20 ppm. Separation was performed on a 2.1 × 100 mm Atlantis PREMIER BEH C18 AX column with 1.7 µm particles at 30 °C. A three-component mobile phase system was used:

• Solvent A: 50 mM ammonium formate with 0.9% formic acid in water (pH 2.9)
• Solvent B: 0.9% formic acid in acetonitrile
• Solvent C: 0.9% formic acid in water

A gradient method delivered a total run time of 7 minutes, achieving baseline separation for most acids and resolving closely eluting pairs without requiring full baseline separation.

Instrumentation

• LC System: Waters ACQUITY UPLC H-Class
• Column: Atlantis PREMIER BEH C18 AX, 1.7 µm, 2.1 × 100 mm
• Mass Detector: Waters ACQUITY QDa
• Ionization Mode: Negative electrospray
• Capillary Voltage: 0.8 kV; Cone Voltage: 5 V; Probe Temperature: 600 °C
• Data Software: Empower 3 CDS

Main Results and Discussion

Using 11 selected ion recording (SIR) channels, all 15 organic acids were detected with high selectivity and minimal interference. The run time was reduced to 7 minutes—approximately one-sixth of typical anion-exchange methods. In orange juice samples diluted 100×, most target acids were observed, although some minor peaks remained unidentified. The mixed-mode column provided strong retention of polar analytes and high separation efficiency, while the QDa detector enhanced quantitation by eliminating matrix interferences.

Benefits and Practical Applications

• Significant reduction in analysis time for routine quality control of beverages.
• Improved retention and peak shape for polar organic acids.
• Highly selective mass detection reduces quantitation errors from complex matrices.
• Simplified method development by combining reversed-phase and weak anion-exchange interactions.

Future Trends and Applications

Continued advances in mixed-mode stationary phases and compact mass detectors will further streamline organic acid analysis workflows. Integration with autosamplers and automated dilution systems can enhance throughput for high-volume testing laboratories. Emerging applications may include clinical diagnostics of metabolic disorders and environmental monitoring of organic acid pollutants.

Conclusion

The combination of the Atlantis PREMIER BEH C18 AX column and ACQUITY QDa mass detector offers a fast, selective, and robust method for organic acid analysis in juices. This approach minimizes matrix interferences, accelerates run times, and delivers reliable quantitation, making it well suited for routine beverage quality control and broader analytical applications.

References

Yang J., Rainville P. Separation of Organic Acids with Mixed-Mode LC Column and Mass Detector. Waters Corporation, 2020.