Performance Evaluation of the Agilent 1290 Infinity II High Speed Pump using Phosphate Buffer as Mobile Phase

Significance of the Topic

The adjustment of mobile phase pH using phosphate buffers is crucial in reversed-phase HPLC to ensure optimal retention of ionizable compounds and prevent column damage. Consistent mobile phase composition and pump performance are essential for high reproducibility and longevity of HPLC systems.

Study Aims and Overview

This overview evaluates the Agilent 1290 Infinity II High Speed Pump performance when using a 25 mM phosphate buffer (pH 3.0) and acetonitrile in an antibacterial drug separation. The study assesses retention time precision, resolution, peak symmetry across 90 consecutive injections, and evaluates potential system pressure changes.

Methodology and Instrumentation

The analysis employed the following conditions:

• Column: ZORBAX Eclipse Plus C18, 3.0×100 mm, 1.8 µm.
• Mobile phase A: 25 mM KH2PO4, pH 3.0; B: acetonitrile.
• Gradient flow: 1.6 mL/min, 5 % B initial to 80 % B over 11 min, 15 min total runtime.
• Injection volume: 3 µL; sample temp: 8 °C; column temp: 40 °C.
• Detection: PDA at 204 nm (ref 360 nm).

Used Instrumentation

• Agilent 1290 Infinity II High Speed Pump (G7120A).
• Agilent 1290 Infinity II Multisampler with cooler (G7167B).
• Agilent 1290 Infinity II Multicolumn Thermostat (G7116B).
• Agilent 1290 Infinity II Diode Array Detector with Max-Light cell (G7117B).
• Data system: Agilent OpenLAB CDS ChemStation.

Main Results and Discussion

Retention time reproducibility was exceptional, with relative standard deviations below 0.05 % for most analytes over six runs and under 0.07 % over 90 runs. Resolutions remained high (e.g., >75 between key components), and peak symmetry stayed near unity. System backpressure increased by only 18 bar over 90 injections (0.02 % per run), indicating minimal column fouling.

Practical Benefits and Applications

The demonstrated robustness enables reliable analysis of ionizable and pH-sensitive compounds in pharmaceutical quality control, reducing downtime and maintenance costs. High throughput laboratories can leverage this performance for complex separations requiring buffer-based mobile phases.

Future Trends and Opportunities

Advancements in pump materials and seal technologies may further improve buffer compatibility and long-term stability. Integration with automated sample preparation and real-time monitoring by advanced detectors could expand applications to proteomics and small‐molecule drug discovery workflows.

Conclusion

The Agilent 1290 Infinity II High Speed Pump maintains exceptional performance with phosphate buffer mobile phases, delivering outstanding retention precision, resolution, and system integrity even over extended sequences. This capability supports rigorous analytical demands in pharmaceutical and research laboratories.