The Separation of Seven Synthetic/Artificial Food Colors on Agilent HC(2)/TC(2) Reversed Phase Columns

Importance of the Topic

Objectives and Study Overview

Methodology and Instrumentation

• Instrumentation: Agilent 1200SL HPLC system with diode array detection at 254 nm.
• Column: Agilent TC-C18(2), 4.6 mm × 150 mm, 5 μm, 12 % carbon load; comparison with HC-C18(2) (17 % carbon load).
• Mobile phase: A – 20 mM phosphate buffer (pH 7.0); B – methanol; gradient from 10 % to 90 % B over 15 min.
• Flow rate: 1.0 mL/min; column temperature: 30 °C; injection volume: 5 µL (standard) and 50 µL (low-level samples).
• Method optimization: pH effect evaluated at pH 2.2, 4.5, and 7.0 to improve peak shape for acidic dyes.

Main Results and Discussion

• pH Optimization: pH 7.0 yielded symmetrical peaks, USP tailing factors near 1.0, and baseline resolution for all seven dyes. Lower pH values caused coelution of the most polar, acidic compounds.
• Column Comparison: TC-C18(2) provided stronger retention of polar colorants and better selectivity in low-organic gradients. HC-C18(2) retained nonpolar components more strongly, reversing elution order of some peaks.
• Reproducibility: Three different column lots showed negligible retention time drift and consistent peak shapes, demonstrating high manufacturing quality.
• Sample Analysis: In orange soda and beverage powder, three target colorants were fully resolved from unknown interferences. Large-volume (50 µL) injections allowed detection at low concentrations without loss of resolution.

Benefits and Practical Application of the Method

• High resolution and symmetrical peak shapes for a range of polar azo dyes.
• Excellent lot-to-lot consistency for routine QC/QA environments.
• Capacity for large-volume injections to meet low-level detection and quantitation needs.
• Flexibility in column selection based on analyte polarity when paired with HC-C18(2).

Future Trends and Application Opportunities

• Integration with mass spectrometry for enhanced sensitivity and structural confirmation.
• Adaptation to a wider range of food and beverage matrices, including complex formulations.
• Development of greener mobile phases and faster run times for high-throughput screening.
• Design of novel stationary phases to further tune selectivity for emerging additives and contaminants.

Conclusion

Reference

• Fang Yanyan. Separation of Seven Synthetic Food Colors on Agilent HC(2)/TC(2) Reversed Phase Columns. Agilent Technologies Application Note 5989-3639CHCN, 2008.