Impact of Autosampler Design on Carryover Performance During Method Migration

Importance of the topic

Carryover in liquid chromatography can lead to inaccurate quantitation, failed system suitability tests and increased downtime when methods are migrated between instruments. Understanding how autosampler design and needle wash protocols influence both volumetric and adsorptive carryover is essential for robust method transfer and reliable results in regulated and research laboratories.

Objectives and study overview

This study evaluates the impact of autosampler architecture and needle washing settings on carryover performance during method migration. Two representative analytes—caffeine (to assess volumetric carryover) and chlorhexidine (to probe adsorptive carryover)—were analyzed on an ACQUITY Arc System and a comparator system (System X). The goal was to identify optimal wash modes and solvents that minimize residual analyte on successive injections.

Methodology and instrumentation

Two test protocols were developed: one based on a 4-minute isocratic caffeine assay (90:10 water:acetonitrile) using a CORTECS™ C18 column, and a second following the USP monograph for chlorhexidine HCl employing an HSS C18 SB column with an isocratic 67:33 water:acetonitrile + TFA mobile phase. Carryover was calculated after high-concentration sample injections followed by blank injections. Pre- and post-injection or pre- and post-aspiration wash modes were varied in duration and solvent composition to assess their effect on residual analyte levels.

Used Instrumentation

• Waters ACQUITY Arc System with flow-through needle and high-pressure seal
• Comparator autosampler (“System X”) with pre-injection/post-aspiration wash only
• CorteCS™ C18, 2.7 μm, 3 mm × 100 mm column for caffeine assay
• XSelect™ HSS C18 SB, 3.5 μm, 250 × 4.6 mm column for chlorhexidine assay
• PDA detection at 273 nm (caffeine) and 257 nm (chlorhexidine)

Main results and discussion

Volumetric carryover of caffeine was highest when no needle wash was applied. Introducing both pre- and post-injection washes on the ACQUITY Arc System reduced carryover to well below 0.002%. Solvent composition had minimal influence on volumetric carryover, indicating that wash duration and sequence are the primary control factors. In contrast, adsorptive carryover of chlorhexidine was strongly affected by wash solvent strength: a 50:50 water:acetonitrile wash delivered the lowest residual levels. The ACQUITY Arc System eliminated detectable carryover after a single blank injection, whereas System X exhibited persistent carryover across multiple blanks under its default wash settings.

Benefits and practical applications

• Enhanced confidence in method transfers across different LC platforms
• Optimized needle wash protocols to meet stringent carryover specifications
• Reduced sample re-runs and improved laboratory throughput
• Applicable to routine QC assays and compliance with pharmacopeial monographs

Future trends and possibilities

Advances in autosampler designs—such as flow-through needles with dynamic sealing—will further lower both volumetric and adsorptive carryover. Development of novel wash solvents tailored to specific analyte binding characteristics and integration of automated wash optimization algorithms are expected to streamline method migration and reduce manual troubleshooting.

Conclusion

This investigation demonstrates that both autosampler architecture and wash regimen critically determine carryover performance. Employing combined pre- and post-injection washes and selecting appropriate wash solvents can virtually eliminate residual analyte, facilitating reliable method migration and compliance with stringent analytical requirements.

References

• Waters Corporation. ACQUITY Arc System Guide, Document 715004747 Version 04.
• United States Pharmacopeia. USP Monographs, Chlorhexidine Hydrochloride, USP–NF, 2021.
• Agilent Technologies. 1260 Infinity II Multisampler Data Sheet.