Alliance Carryover Performance Part 1: Carryover Improvement Achieved Through Instrument Design Changes for the Alliance HPLC System

Importance of the topic

High sample carryover in HPLC workflows can compromise data quality, leading to out-of-specification results, failed batches and reduced reproducibility. Optimizing injector design and wash protocols is crucial to minimize residual analyte transfer between injections and ensure reliable routine analyses across varied compound chemistries.

Study objectives and overview

This application note compares carryover performance between the original Alliance HPLC System and the enhanced 2018 Alliance configuration. Four analytes with diverse properties—caffeine, chlorhexidine, coumarin and quetiapine fumarate—were selected to evaluate improvements achieved by redesigning the injector seal pack and optimizing needle wash flow.

Instrumentation

• Waters Alliance e2695 Separations Module (firmware 3.03) and 2018 Alliance Module with e2695 Enhancement Kit (firmware 3.04)
• 100 μL syringe and 2998 PDA Detector
• CH-30 passive column preheater
• XBridge BEH C18 (3.5 μm, 4.6 × 50 mm) and CORTECS C18 (2.7 μm, 3 × 100 mm) columns
• Empower 3 Chromatography Data Software

Methodology

Two carryover quantification approaches were employed. For caffeine and coumarin, a high-concentration challenge sample was injected to saturate the detector, followed by a low-level standard and a post-challenge blank. Carryover was calculated as post-blank response relative to standard response scaled by 0.01. For chlorhexidine and quetiapine fumarate, challenge concentrations fell within the detector linear range and carryover was expressed as blank peak area divided by standard peak area multiplied by 100. Standard HPLC conditions—mobile phases, flow rates, temperatures and wash solvents—were held constant, with only the injector seal pack design differing between systems.

Main results and discussion

The enhanced 2018 Alliance configuration demonstrated markedly lower carryover for all test compounds. Caffeine carryover decreased from 0.0011% to 0.00016% (6.9× reduction); chlorhexidine from 0.010% to 0.0005% (20×); coumarin from 0.0002% to 0.00006% (3.3×); and quetiapine fumarate from 0.028% to 0.019% (1.5×). These reductions reflect improved exterior needle washing achieved by the redesigned seal pack, without altering standard wash durations.

Benefits and practical applications of the method

By integrating the new seal pack design, laboratories can achieve lower baseline carryover across a range of chemistries, enhancing method robustness and reducing re-analysis rates. This improves throughput, data integrity and confidence in trace-level quantitation for QA/QC, pharmaceutical assays and environmental testing.

Future trends and opportunities

Further advances may focus on adaptive wash cycles based on analyte property prediction, automated seal pack diagnostics and integration of real-time carryover monitoring. Expansion to additional column chemistries and higher pressure systems can extend the benefits to ultra-high-performance liquid chromatography platforms.

Conclusion

Redesigning the injector seal pack on the 2018 Alliance HPLC System significantly lowers sample carryover for diverse analytes. This enhancement delivers more reliable chromatographic performance and reduces the risk of false positives or inaccurate quantitation due to residual sample contamination.

References

1. United States Pharmacopeia and National Formulary Quetiapine Fumarate USP 40-NF35 S1 United Book Press Inc 2017 p 5939
2. Jenkins T Waite M Low Sample Carryover with Key Performance Indicators on the Alliance HPLC System Waters Technology Brief 720004534EN
3. Jenkins T Waite M Screening of Commercial Vanilla Extracts for Authenticity using the Breeze 2 Modular HPLC System Waters Application Note 720002877EN
4. Thurmond M Hodgin JC A Carryover-Elimination Method for a Broad Range of Analytical Sample The Application Notebook September 2003
5. Waters Corporation Waters e2695 Separations Module Instrument Specifications 720002552EN September 2008
6. Waters Corporation Waters e2695 Separations Module Instrument Specifications 720004547EN April 2018