INCREASE SPEED, SENSITIVITY, AND RESOLUTION WITH UPLC COLUMN TECHNOLOGY

Significance of the Topic

UltraPerformance Liquid Chromatography (UPLC) represents a major advancement in liquid chromatography by combining sub-2 µm particle technology with low-dispersion instrumentation to deliver unparalleled increases in resolution, speed, and sensitivity. This capability is essential for modern analytical challenges ranging from complex small-molecule separations to bioseparations of peptides, proteins, glycans, and oligonucleotides. By optimizing both stationary phase design and instrument hardware, UPLC enables high-throughput analysis, robust method development, and reproducible performance in research, quality control, and industrial laboratories.

Study Objectives and Overview

The primary goal of this white paper is to present Waters’ holistic UPLC column platform, detailing key particle technologies (BEH, CSH, HSS, and CORTECS) and complementary components (pre-columns, vials, standards) that together maximize chromatographic performance. The document reviews:

• The range of UPLC column chemistries for small molecules and biomolecules
• Innovations in particle synthesis and column manufacture
• Instrumentation requirements for achieving theoretical efficiency gains
• Practical examples illustrating speed, resolution, and sensitivity improvements

Methodology and Instrumentation

Waters’ UPLC approach integrates several critical elements:

• Sub-2 µm fully porous particles (BEH, CSH, HSS) and 1.6 µm solid-core particles (CORTECS) engineered for high chemical and mechanical stability across wide pH and temperature ranges
• Low-dispersion ACQUITY UPLC instrument hardware and software to preserve column efficiency and peak shape
• VanGuard pre-columns for guard protection without compromising chromatographic performance at pressures up to 18 000 psi
• Certified TruView vials tested for dimensional accuracy, UV/MS cleanliness, and low analyte adsorption
• Quality control reference materials and standards for method validation and instrument qualification

Main Results and Discussion

Comparative experiments demonstrate:

• Efficiency gains: Solid-core CORTECS columns achieve sharper, narrower peaks and higher peak capacities (e.g., Pc≈182 vs 179) at doubled flow rate compared to fully porous counterparts.
• pH stability: BEH columns operate reliably from pH 1 to 12, allowing retention tuning for acidic and basic analytes without sacrificing peak shape.
• Peak shape improvement: CSH columns with a controlled positive surface charge dramatically enhance basic analyte peak shapes under low-ionic-strength acidic conditions, outperforming both fully porous and competitive solid-core phases.
• Selectivity range: HSS columns offering five bonded phases (C18, T3, C18 SB, cyano, PFP) extend the selectivity space for complex mixtures such as nitroaromatic explosives.
• Guard column performance: VanGuard pre-columns maintain analyte recovery and shape while protecting analytical columns from matrix contamination.

Benefits and Practical Applications

Key advantages of the integrated UPLC column system include:

• High throughput analysis for routine small-molecule screening and complex bioseparation workflows
• Flexible method development through broad pH, temperature, and selectivity options
• Robustness and reproducibility for regulated QC environments
• Compatibility with UHPLC and HPLC platforms, enabling seamless transfer of methods
• Improved MS performance via reduced dispersion and minimized secondary interactions

Future Trends and Potential Applications

Emerging directions in UPLC column technology may include:

• Tailored surface chemistries for improved enantiomeric and chiral separations
• Nano-bore and microfluidic integration for single-cell and metabolomics studies
• Advanced machine-learning algorithms for automated method scouting and predictive retention modeling
• Next-generation particle architectures combining solid-core, hybrid, and monolithic elements for ultra-high efficiency
• Increased synergy with high-resolution mass spectrometry and ion mobility separations

Conclusion

Waters’ UPLC column platform exemplifies the convergence of advanced particle engineering and precision instrumentation to address modern analytical demands. By offering a comprehensive portfolio of column chemistries, robust hardware, and supporting consumables, this technology empowers laboratories to achieve faster run times, higher resolution, and greater sensitivity, while maintaining method robustness and reproducibility. Continued innovation in materials, software, and instrument integration promises to further expand the capabilities and applications of UPLC in research and industry.

References

• Waters Corporation. UPLC Column Technology White Paper, 2018.
• US Patent 6,686,035; US Patent 7,223,473; US Patent 7,250,214; US Patent 8,449,769; US Patent 9,724,621.