Investigation of system performance – of the KNAUER AZURA® UHPLC system

Significance of the topic

The efficiency of chromatographic methods is crucial in pharmaceutical analysis, where high sample throughput, cost savings, and reliable resolution of active ingredients are required. Fast UHPLC separations reduce run times and operational costs while maintaining data quality in routine quality control of anti-epileptic drugs.

Objectives and overview of the study

This work evaluates the performance of the KNAUER AZURA UHPLC system equipped with a binary P 8.1L pump, focusing on method acceleration, system dispersion, reproducibility, efficiency and resolution using a model mixture of anti-epileptic drug compounds.

Methodology and instrumentation

Sample preparation:

• Single standards at 1 mg/ml in acetonitrile:water 15:85 (v/v), mixed to 0.01 mg/ml each.
• Injection volume of 2 µl for the mixed standard.

Chromatographic conditions:

• Column: Agilent Zorbax SB-C18 RRHD, 2.1×50 mm, 1.8 µm.
• Eluents: water (A) and acetonitrile (B), gradient at 60 °C.
• Flow rates tested: 0.2 to 1.5 ml/min.
• Detection: DAD at 204 nm, data rate 100 Hz.
• Gradient program adjusted to each flow rate to maintain consistent percentage changes.

Instrumentation used:

• KNAUER AZURA P 8.1L binary pump, up to 1240 bar.
• AZURA AS 6.1L autosampler with 10 µl loop.
• AZURA DAD 6.1L diode array detector with 2 µl flow cell.
• AZURA CT 2.1 column thermostat.

Main results and discussion

The extra-column volume was measured at 9 µl, reflecting low dispersion. Ten replicate injections at each flow rate showed retention time variation below 0.6 s. Peak symmetry remained stable or improved slightly with higher flow rates. Van Deemter plots identified 0.6 ml/min as the optimal flow for minimal plate height. Although doubling the flow to 1.2 ml/min reduced efficiency by 10%, it cut analysis time by 43%. Resolution between critical peak pairs remained acceptable even at 1.5 ml/min, with phenytoin–carbamazepine resolution decreasing from 1.71 to 0.91.

Benefits and practical applications

The system enables a 77% reduction in run time (from 7.0 to 1.6 min) and over 400% productivity gain without compromising separation quality. Its high-pressure capacity and adaptive pulsation compensation support robust high-throughput analysis in pharmaceutical QC laboratories.

Future trends and potential applications

Integration of UHPLC with automated method optimization, multi-column setups, and AI-driven data analysis will further enhance throughput and flexibility. Applications may extend to complex matrices and real-time process monitoring.

Conclusion

The KNAUER AZURA UHPLC system delivers low dispersion, precise delivery up to 1240 bar, and consistent performance across a wide flow range, meeting the demands of high-throughput, high-resolution pharmaceutical analyses.

References

• ACQUITY UPLC I-Class: Optimized System Dispersion for Ultimate UPLC Performance; Waters Corporation (2011).
• Optimized System Dispersion for UPLC Performance in a Versatile LC Design; Waters Corporation (2011).
• Ahmad et al., A 14-Parameter Study of UHPLC for Method Development Transfer and Troubleshooting; J. Chromatogr. A (2017).