Investigation of carryover under consideration of different washing solvents and volumes

Significance of the Topic

A thorough understanding of sample carryover in liquid chromatography is essential to avoid cross-contamination, false positives, and ensure data reliability in trace analysis, especially when using highly sensitive detectors such as MS/MS or electrochemical sensors.

Objectives and Study Overview

• Evaluate how different washing solvents and volumes affect carryover in the KNAUER AZURA AS 6.1L autosampler
• Compare carryover in three injection modes: microliter pickup, partial loop, and full loop
• Optimize a multi-step washing protocol using caffeine and chlorhexidine as model analytes

Methodology and Instrumentation

The study employed an HPLC configuration with an AZURA P6.1L pump, AZURA AS 6.1L autosampler, and AZURA DAD 6.1L detector operating at 272 nm for caffeine and 257 nm for chlorhexidine. Carryover was assessed following one to three wash steps of 250 µL each, and an extended wash function combining a strong organic solvent (isopropanol) followed by the weakest eluent (mobile phase) with varied volumes up to 1500 µL each.

Main Results and Discussion

For caffeine, single wash steps eliminated carryover in microliter pickup and full loop modes, while partial loop required at least two washes (< 0.005%). Chlorhexidine exhibited residual adsorption; two standard 750 µL washes lowered carryover below 0.005% across all modes. Implementing the extended wash with 1500 µL isopropanol followed by 1500 µL mobile phase reduced carryover to 0.0003% in partial loop mode, an order of magnitude improvement, highlighting the importance of solvent strength and volume in removing adsorbed residues.

Benefits and Practical Applications

• Enhances accuracy and reproducibility in trace-level analyses
• Prevents false positives in high-sensitivity detection methods
• Offers a standardized approach for reducing system contamination

Future Trends and Potential Applications

Further developments may include automated optimization of wash protocols, exploration of alternative solvents for diverse analytes, and integration of real-time carryover monitoring within lab automation platforms. Adapting these findings to microfluidic and high-throughput systems could broaden applicability.

Conclusion

Effective multi-step washing, particularly with an extended organic-to-aqueous sequence, can reduce autosampler carryover to negligible levels. Such rigorous cleaning procedures should be integral to method development to ensure reliable chromatographic results.

References

1. Zeng W., Musson D.G., Fisher A.L., Wang A.Q. A new approach for evaluating carryover and its influence on quantitation in high-performance liquid chromatography and tandem mass spectrometry assay. Rapid Commun. Mass Spectrom. 20:635-640 (2006).