New Technology for Environmental Assays – Improve Resolution, Run More Samples at Lower Cost

Significance of the Topic

Environmental monitoring often demands the simultaneous separation of many analytes with high resolution, minimal analysis time and reduced solvent consumption. Traditional HPLC columns with 5 µm particles can require long run times, high solvent volumes and even multiple columns to resolve complex mixtures such as polycyclic aromatic hydrocarbons (PAHs), explosives or pesticide panels. Sub-2 µm column technology offers a route to faster, more efficient assays that lower cost and increase laboratory throughput.

Objectives and Study Overview

The primary goal was to evaluate Agilent’s Rapid Resolution HT (RRHT) sub-2 µm HPLC columns and related column formats for environmental applications. Key targets included:

• Improving resolution and reducing run times for PAH priority pollutant mixtures (EPA-610, Florida-700, Quebec-24).
• Assessing solvent savings via narrow-bore and “solvent saver” column formats.
• Resolving complex explosives mixtures (EPA-8330) on a single column.
• Demonstrating rapid separations of multi-class pesticide panels with LC-MS/MS.

Methodology and Instrumentation

Columns and configurations:

• ZORBAX RRHT Eclipse PAH: 1.8 µm, 3.5 µm and 5 µm particles; 4.6 × 50–250 mm; 2.1 × 50–250 mm.
• Extend-C18 RRHT: 1.8 µm and 3.5 µm, 4.6 × 100 mm and 3.0 × 100 mm.

Detection and instrumentation:

• Agilent 1200 Series RRLC with 400 bar and 600 bar pumping capability.
• Diode Array Detection (DAD) at 220–254 nm, fluorescence detection (FLD) with multi-wavelength switching.
• AccuBOND C18 SPE for water sample preparation; 0.2 µm filtration.
• Agilent 6410 triple quadrupole MS (ESI+) for pesticide MRM screening.

Main Results and Discussion

PAH separations:

• Conventional 4.6×250 mm, 5 µm column required 25 min per run; 60 mL solvent.
• Reducing particle size to 3.5 µm on 4.6×100 mm cut time to 14 min and solvent by 45% while improving resolution (Rs > 2.4).
• RRHT 1.8 µm, 4.6×50 mm achieved a full 16-PAH separation in 6 min with Rs ≥ 2.0.
• Narrow-bore 2.1 × 50 mm, 1.8 µm columns saved 96% solvent (2.5 mL/run) and delivered 7 samples/hour throughput.
• Scale-up from 2.1 × 50 mm to 2.1 × 100 mm and 4.6 × 100 mm formats traded speed for maximum resolution (Rs up to 3.6).
• Columns demonstrated excellent lifetime stability over 5 000 injections with minimal loss of resolution.

Explosives analysis:

• EPA-8330 explosive standard resolved in 14 min with Extend-C18 RRHT 1.8 µm, 4.6×100 mm (no co-elutions, Rs > 1.6).
• 3.5 µm RR columns ran at lower pressure on any HPLC, with 280 bar max, maintaining full separation in 14 min.
• Solvent saver 3.0×100 mm, 3.5 µm columns achieved equivalent separation at 220 bar and reduced flow from 2.5 to 1.1 mL/min.
• Resolution of critical pairs was optimized by column temperature control (38–42 °C).

Pesticide screening:

• Rapid LC-MS/MS methods on Extend-C18 RRHT 1.8 µm, 2.1×100 mm provided full MRM separation of over 40 pesticides in fast (20 min) or slow (60 min) gradients, with limits of quantitation in low ng/mL.

Benefits and Practical Applications

• High-throughput PAH screening in environmental, drinking water and soil samples with minimal sample prep.
• Substantial solvent savings (up to 96%) and lower per-sample cost.
• Single-column confirmation for explosives, eliminating the need for multiple stationary phases.
• Flexible method scalability to address varying resolution, throughput and solvent use priorities.
• Simplified fluorescence detection via fewer wavelength changes using 1.8 µm columns.

Future Trends and Opportunities

• Extension of RRHT technology to higher-order PAH mixtures (> 24 congeners) and emerging contaminants.
• Integration with automated sample prep and online SPE for full laboratory workflow automation.
• Hybrid separations coupling sub-2 µm columns to high-resolution MS for non-target screening.
• Further miniaturization (capillary and microflow formats) for ultra-low solvent consumption.

Conclusion

Agilent’s RRHT sub-2 µm columns and related particle sizes deliver transformative improvements in environmental assay speed, resolution and cost efficiency. The flexibility in column geometry and particle size enables tailored workflows for PAHs, explosives and pesticides, meeting regulatory and high-throughput screening demands without compromise.