Polycyclic Aromatic Hydrocarbon (PAH) Separations Using ZORBAX Eclipse PAH Columns – Analyses from Six to 24 PAHs

Importance of Topic

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental and food contaminants formed by incomplete combustion of organic matter. Many PAHs are classified as probable carcinogens, prompting regulatory bodies such as OSHA, NIOSH, and the EU to set exposure limits. Robust analytical methods are essential to monitor PAHs across diverse sample types—including air, water, soil, and food—and ensure compliance with safety standards.

Study Objectives and Overview

This application note demonstrates the versatility of ZORBAX Eclipse PAH columns for separating PAH mixtures ranging from six to 24 compounds. The primary goals are to optimize resolution, analysis speed, and detector sensitivity by adjusting mobile-phase gradients, column dimensions, and temperature. Five representative PAH mixtures—covering food-screening protocols, regulatory lists, and environmental standards—are evaluated to illustrate method customization for various analytical needs.

Methodology and Instrumentation

• Chromatographic System: Agilent 1200 Series Rapid Resolution LC (RRLC) with degasser, binary pump, autosampler, column compartment, diode array (DAD) and fluorescence (FLD) detectors.
• Columns: ZORBAX Eclipse PAH in multiple configurations (2.1–4.6 mm I.D., lengths 30–250 mm, particle sizes 1.8–5 µm) to balance pressure, throughput, and resolution.
• Mobile Phase: Water (A) and acetonitrile (B) with programmed gradients optimized for each mixture.
• Detection: FLD for high sensitivity (picogram-level limits of detection for most PAHs) and UV at 220–230 nm for broader coverage.
• Temperature Control: Column temperatures from 15 °C to 35 °C to fine-tune selectivity, especially for critical PAH pairs.
• Sample Preparation: Ready-made standard mixtures or combinations of individual PAH standards dissolved in toluene or methylene chloride, diluted with ethanol.

Main Results and Discussion

• Six-Compound Food Screening: Rapid resolution of six priority PAHs with FLD sensitivity yielding signal-to-noise ratios above 10 at sub-nanogram levels.
• 20-Component Regulatory Mix: A gradient method successfully separated EPA 610 plus four additional PAHs (including methyl-naphthalenes and benzo[e]pyrene) on a 100 mm RRHT column.
• 24-Component Environmental Standard: The Quebec Ministry of Environment PAH mixture achieved baseline resolution on a 150 mm, 3.5 µm column in under 20 minutes. A shortened run on a 100 mm RRHT column delivered comparable separation in 14 minutes with higher pressure compatibility on RRLC systems.
• EU ‘‘15+1’’ Plus Additional Analytes: Eighteen PAHs, including JECFA’s benzo[c]fluorene additions, were separated in under 9 minutes on a 2.1 mm × 50 mm RRHT column. Lowering temperature from 25 °C to 15 °C improved resolution of critical peaks by up to 75%.
• Drinking-Water Screening: Isocratic separation of six priority PAHs in 2–4 minutes using short 50 mm columns (3.5 µm and 1.8 µm), supporting Directive 80/778/EEC requirements and high sample throughput.

Benefits and Practical Applications

• Scalability: A wide range of column dimensions allows laboratories to match throughput and resolution requirements to instrument pressure limits.
• Reproducibility and Robustness: Consistent selectivity and long column lifetimes support high-volume environmental and food-safety testing.
• Enhanced Sensitivity: FLD detection at picogram levels meets stringent regulatory limits, while UV detection offers cost-effective monitoring for less critical applications.
• Method Flexibility: Gradient, isocratic, and temperature adjustments provide rapid method development for new PAH lists or targeted compounds.

Future Trends and Potential Applications

• Integration with Mass Spectrometry: Coupling RRHT columns to high-resolution MS for confirmatory analysis and improved specificity.
• Green Chromatography: Exploration of alternative solvents or reduced solvent consumption via ultra-short columns and microflow systems.
• Automated Workflows: Implementation of online sample preparation and data processing to streamline high-throughput PAH analysis in QA/QC environments.
• Expanded Target Lists: Adapting method frameworks for emerging contaminants such as alkylated PAHs and heterocyclic analogues.

Conclusion

ZORBAX Eclipse PAH columns deliver reliable, high-resolution separations across a broad spectrum of PAH mixtures and sample matrices. By leveraging scalable column formats, controlled temperatures, and optimized gradients, analysts can achieve sensitive, reproducible results tailored to regulatory and research applications in environmental monitoring and food safety.

References

1. High-Throughput Gradient Optimization by Easily Minimizing Delay Volume, Agilent Technol. Pub. 5989-6665EN (2007).
2. Robustness of Eclipse PAH Columns, Agilent Technol. Pub. 5989-7828EN (2008).
3. Polynuclear Aromatic Hydrocarbons (PAHs) Analysis in Water with ZORBAX Eclipse PAH HPLC Column, Agilent Technol. Pub. 5989-7953EN (2008).
4. Analytical methods for polycyclic aromatic hydrocarbons in food and the environment needed for new food legislation in the European Union, Trends Anal. Chem., Vol. 25, No. 7 (2006).