Ultra-fast liquid chromatography using the Agilent 1100 Series HPLC system and 1.8-μm ZORBAX SB C18 Rapid Resolution HT columns

Importance of the topic

High-throughput liquid chromatography is critical for timely assessment of purity, chemical and metabolic stability, and quality control in chemical and pharmaceutical settings. Advances in sub-2 µm column technology and high-pressure instrumentation make ultra-fast separations possible without sacrificing resolution or robustness.

Study Objectives and Overview

This technical overview presents a recommended configuration of the Agilent 1100 Series HPLC system equipped with 1.8 µm ZORBAX SB C18 Rapid Resolution HT columns. The goals are to achieve baseline separation of nine alkylphenones in under 30 seconds, evaluate performance across flow rates (2.3–4.35 mL/min) and temperatures (32–60 °C), and demonstrate long-term system stability over thousands of injections, with and without alternating column regeneration.

Methodology

• Analytes: Nine alkylphenones (acetanilide to octanophenone) at 100 ng/µL in acetonitrile.
• Column: ZORBAX StableBond C18, 4.6 × 50 mm, 1.8 µm particles.
• Mobile phase: Gradient from 50% to 100% acetonitrile (0.1% formic acid) in 0.65 min at 32 °C.
• Flow rates: 2.3–4.35 mL/min producing backpressures of 259–390 bar.
• Injection: 1 µL via well-plate autosampler with Automatic Delay Volume Reduction (ADVR) and Overlapped Injections (OI).
• Detection: Diode-array detector at 245 nm, 500 nL flow cell, 20 Hz acquisition.
• Alternating column regeneration: 2-position/10-port valve and second pump to overlap regeneration with analysis.

Instrumentation

• Agilent 1100 Series binary pump (G1312A) with Upchurch 80 µL filter and vacuum degasser (G1322A).
• Well-plate autosampler (G1367A) with ADVR and OI capabilities.
• Thermostatted column compartment (G1316A) with integrated 2-position/10-port valve.
• DAD UV detector (G1315B) with 500 nL flow cell (G1315-60014) and 0.125 mm capillaries.
• ZORBAX SB C18 RRHT column 4.6 × 50 mm, 1.8 µm (822975-902).

Results and Discussion

• Baseline separation of nine alkylphenones in 29 s at 32 °C and 2.9 mL/min, with 0.5 s peak widths and resolution >2.65 between critical peaks.
• Elevating flow to 4.35 mL/min at 60 °C reduced analysis to 0.65 min while maintaining sharp peaks and backpressure <400 bar.
• Alternating column regeneration shortened cycle time from 1.5 to 1.3 min without significant loss of resolution or precision.
• Minimized carry-over achieved by combining ADVR, OI, and a high flush-out factor (×20), eliminating residual peaks.
• Comparison of 500 nL vs. standard (0.17 mm) flow cells showed sharper peaks and higher resolution with 500 nL, while the standard cell reduced baseline noise by ~3.75×.
• Long-term testing over 8000 injections (4000 per column) demonstrated stable retention times, peak widths, and resolution, confirming column and system robustness.

Practical Benefits and Applications

The optimized configuration supports ultra-fast, high-throughput LC-UV analyses for quality control, stability testing, and process monitoring, reducing solvent consumption and instrument downtime. Automated ADVR, OI, and alternating column regeneration enable continuous, unattended operation.

Future Trends and Potential Applications

• Integration with mass spectrometry and other detectors for enhanced specificity and sensitivity.
• Development of microfluidic and capillary LC systems to further lower solvent use.
• Advanced software for real-time method optimization, predictive maintenance, and data analytics.
• Expansion of ultra-fast LC to high-throughput screening, metabolomics, and process analytical technology (PAT) in biopharmaceutical and chemical manufacturing.

Conclusion

The Agilent 1100 Series combined with 1.8 µm ZORBAX RRHT columns delivers ultra-fast separations with high resolution and long-term reliability. Through optimized flow rates, temperatures, and automation features, nine-component mixtures can be resolved in under one minute, significantly enhancing laboratory productivity.

References

1. Application Note 5988-9997EN, “Increasing Sample Throughput Using ZORBAX Eclipse XDB C18 Rapid Resolution HT Columns.”
2. Application Note 5989-0540EN, “High efficiency, high throughput LC and LC/MS applications using ZORBAX Rapid Resolution HT columns.”
3. Application Note 5989-0599EN, “ZORBAX Rapid Resolution HT Columns – A Breakthrough in High-Throughput HPLC Column Technology.”
4. Application Note 5988-7831EN, “High throughput HPLC – Alternating column regeneration with the Agilent 1100 Series valve solutions.”
5. Waters Brochure 720000880EN, “Ultra Performance LC by Design.”