NEEDLE WASH MECHANISM AND ITS IMPACT ON HILIC ANALYSIS

Significance of the Topic

Hydrophilic interaction liquid chromatography (HILIC) is increasingly used for polar analytes in regulated laboratories. When transferring HILIC methods between different HPLC systems, subtle differences in autosampler wash mechanisms and wash solvent composition can lead to chromatographic anomalies such as peak splitting. Understanding these effects is crucial for robust method migration, reliable system suitability, and compliance in quality control environments.

Study Objectives and Overview

This investigation evaluated how the needle wash mechanism and wash solvent composition influence the performance of a USP Cetirizine Hydrochloride organic impurities HILIC method on two HPLC platforms. The primary goals were to identify root causes of split peaks and to assess system suitability under varying wash conditions.

Methodology

A standardized isocratic HILIC method was applied using six wash solvent mixtures of acetonitrile and water (93:7, 80:20, 60:40, 40:60, 20:80, 7:93). System suitability criteria required a tailing factor ≤2.0 for cetirizine and resolution ≥2.0 between cetirizine and its related compound.

Instrumentation Used

• Alliance iS HPLC System with dual‐wavelength UV and variable‐wavelength detectors
• System X HPLC System (alternate autosampler design)
• XBridge BEH HILIC column (4.6 × 250 mm, 5 µm, 130 Å)
• Column temperature: 25 °C; sample compartment: 10 °C; flow rate: 1.0 mL/min; injection volume: 10 µL

Results and Discussion

The Alliance iS system achieved acceptable chromatograms and met system suitability requirements across all six wash compositions. In contrast, System X met criteria only with stronger organic washes (93:7 through 40:60); aqueous‐rich washes (20:80 and 7:93) led to pronounced peak splitting. Investigation of the autosampler mechanisms revealed that on Alliance iS, the needle is washed at the injection port both above and below, with excess solvent aspirated away before injection. On System X, the needle is dipped into a wash station and then sent directly to the injection port, carrying residual solvent into the sample stream. This residual water disrupts the HILIC retention mechanism, causing split peaks.

Benefits and Practical Applications of the Method

• Highlights critical role of wash solvent strength and autosampler design in HILIC method transfer
• Provides guidance for selecting suitable wash protocols to ensure system suitability
• Reduces troubleshooting time by identifying autosampler wash as a potential source of chromatographic artifacts

Future Trends and Applications

Advances may include adaptive wash protocols that adjust solvent composition in real time, sensor‐based detection of residual wash solvent, and improved autosampler designs that minimize carryover without impacting chromatographic integrity. Integration of predictive software tools and machine learning could optimize wash cycles based on analyte properties and system architecture.

Conclusion

Needle wash composition and mechanism are key factors affecting HILIC separations when migrating methods between HPLC systems. Detecting and controlling residual wash solvent through appropriate autosampler design and wash protocols is essential to achieving reliable, compliant analyses.

References

• USP‐NF Monograph: Cetirizine Hydrochloride, United States Pharmacopeia, Rockville MD; Sep 2021