Increasing Resolution and Speed by Operating UHPLC Columns up to 1200 Bar

Importance of the Topic

Ultra-high-pressure liquid chromatography (UHPLC) with sub-2 µm particles has become a cornerstone in modern analytical chemistry, offering unmatched speed and resolution. By operating columns at pressures up to 1200 bar, laboratories can further shorten run times, improve separation of complex mixtures, and increase sample throughput—critical factors for pharmaceutical development, quality control, and advanced research.

Objectives and Overview of the Study

This technical note evaluates how Agilent ZORBAX Rapid Resolution High Definition (RRHD) columns, designed for 1.8 µm particles, leverage the full 1200 bar capability of the Agilent 1290 Infinity LC System. Three representative applications demonstrate gains in analysis speed, chromatographic resolution, and peak capacity for complex samples.

Used Instrumentation

• Agilent 1290 Infinity UHPLC System (operating range up to 1200 bar)
• ZORBAX RRHD columns (1.8 µm) in various formats: 2.1×50 mm, 2.1×100 mm, 2.1×150 mm, and serial configurations

Methodology

Analyses employed gradient elution with aqueous acidic mobile phases (phosphoric or formic acid) and organic modifiers (acetonitrile, methanol). Flow rates spanned 0.4–1.6 mL/min. The study presents three workflows:

• High-speed separation of a 10-antioxidant mixture on a 2.1×50 mm column at 1.6 mL/min.
• Enhanced resolution of alkylphenones using tandem columns (150 mm + 100 mm) at 0.5 mL/min.
• Increased peak capacity for a licorice root extract using 100 mm and 150 mm columns with a 30-min gradient at 0.4 mL/min.

Main Results and Discussion

• Speed: A sub-two-minute gradient separation of 10 antioxidants was achieved at 1070 bar, demonstrating a threefold runtime reduction compared to conventional UHPLC limits.
• Resolution: Serial coupling of 150 mm and 100 mm RRHD columns raised resolution by 36% and theoretical plate count by 60%, operating near 1100 bar.
• Peak Capacity: Extending column length from 100 mm (nc ≈ 485) to 150 mm (nc ≈ 589) improved separation performance for a complex natural extract at pressures up to 768 bar.

Benefits and Practical Applications

High-pressure UHPLC unlocks:

• Rapid analyses for high-throughput screening and routine QA/QC.
• Superior separation of trace components in complex matrices.
• Modular method design through selectable column lengths or series assemblies.

Future Trends and Potential Applications

Ongoing developments may include:

• Integration with high-resolution mass spectrometry for comprehensive profiling.
• Design of sub-1.8 µm particles and advanced stationary phases.
• Further hardware enhancements to exceed current backpressure limits.
• Implementation in multidimensional LC for in-depth characterization.

Conclusion

Agilent ZORBAX RRHD columns, in combination with the 1290 Infinity UHPLC system, exploit a 1200 bar pressure range to deliver unparalleled speed and resolution. This capability empowers analytical laboratories to address increasingly complex separation challenges with efficiency and robustness.