Shimadzi Nexera XS inert Ultra High Performance Liquid Chromatograph

Importance of the Topic

The analysis of biomolecules by ultra-high performance liquid chromatography (UHPLC) is challenged by adsorption onto metal surfaces and corrosion under high-salt or extreme pH mobile phases. Complete inertness of the sample flow path is essential for accurate quantitation of proteins, nucleic acids, and other labile analytes at low concentrations.

Objectives and Study Overview

This application note evaluates the performance of the Nexera XS inert UHPLC system compared to a conventional stainless steel-based UHPLC. Key aims include demonstrating improvements in recovery, sensitivity, chromatographic resolution, reproducibility, quantitative linearity, and automated operational reliability for biomolecular separations.

Methodology and Instrumentation

The Nexera XS inert employs a metal-free flow path designed to withstand pressures up to 105 MPa and resist corrosion. Comparative experiments used a metal-free column versus stainless steel column on a standard UHPLC platform. Test analytes included nucleotides (ATP, ADP, AMP), oligonucleic acids, and peptide/protein fragments. Additional components evaluated were TORAST-H Bio vials to minimize vial surface adsorption and Shim-pack™ series columns for varied separation modes. Optional accessories included a pH monitor (pHM-40) for real-time mobile phase monitoring and Analytical Intelligence features for auto diagnostics and method scouting.

Key Results and Discussion

• Unconstrained Recovery and Sensitivity – Nexera XS inert prevented adsorption of ATP/ADP/AMP at 2 ng injection levels, yielding sharp peaks and improved MS signal intensity for metal-sensitive oligonucleic acids compared to standard UHPLC.
• Clear Resolution – Chromatograms showed reduced tailing and superior peak shapes for nucleotides, demonstrating the benefit of inert surfaces.
• Reproducibility – RSD for ATP peak area over ten injections was 0.56% with Nexera XS inert versus 10.1% on the standard system, eliminating the need for pre-analysis passivation.
• Quantitative Linearity – Calibration curves for ATP achieved r2=0.9999 over 0–40 µg/mL on Nexera XS inert, compared to r2=0.9918 on the stainless steel system. QC samples at low (2 µg/mL), medium (20 µg/mL), and high (45 µg/mL) levels showed accuracy within 100–102% and precision <1% RSD for Nexera XS inert versus wider deviations on the standard system.
• Automated Reliability – Integrated diagnostics detected and corrected pump bubble events via auto-purge routines. Weight sensors on solvent trays alerted to low mobile phase volumes ahead of runs. Method scouting automated multi-column and gradient screening, reducing method development time.

Benefits and Practical Applications

The Nexera XS inert system delivers reliable, sensitive biomolecule quantitation without sample loss from surface adsorption. It streamlines operations by removing passivation steps, supports method development with automatic column/gradient switching, and ensures consistent data quality across routine QA/QC, proteomics, oligonucleotide analysis, and bio-pharmaceutical applications.

Future Trends and Opportunities

As analytical laboratories adopt digitalization and AI, UHPLC platforms will further integrate real-time diagnostics, remote monitoring, and automated decision-making. The inert UHPLC concept can expand into multi-dimensional separations, metabolomics, single-cell analysis, and in-line bioprocess monitoring, enabling faster turnaround and higher confidence in complex biomolecular assays.

Conclusion

The Nexera XS inert UHPLC system overcomes key limitations of biomolecule analysis by eliminating metal adsorption and corrosion effects, delivering superior sensitivity, resolution, reproducibility, and quantitative performance. Its advanced automation and inert flow path make it a robust solution for demanding biomolecular separations.

Used Instrumentation

• System Controller: SCL-40, CBM-40/40lite
• Detector: SPD-40/40V/M40 inert UV cells (5 mm path length)
• Solvent Delivery Unit: LC-40D XSi (corrosion-resistant, low pulsation, AI-enabled)
• Autosampler: SIL-40C XSi (non-metal wetted surfaces, direct sample rack access, dry-air cooling)
• Switching Valves: FCV-0206H2i (2-position/6-port), FCV-0607H2i (6-position/7-port) with inert materials
• pH Monitor: pHM-40 for real-time mobile phase pH measurement
• Vials: TORAST-H Bio vials for minimized biomolecule adsorption
• Columns: Metal-free UHPLC columns and Shim-pack™ series for reversed-phase, ion exchange, size-exclusion, HIC, and peptide separations