Waters UPLC, UHPLC, and HPLC Column Selection and Mobile-Phase Guide

Importance of Mobile-Phase and Column Selection in Liquid Chromatography

Choosing the right column chemistry and mobile-phase composition is central to achieving reliable and reproducible separations in UPLC, UHPLC, and HPLC workflows. Proper selection influences retention, peak shape, method robustness, and compatibility with detectors such as MS.

Study Objectives and Overview

This guide from Waters Corporation aims to help chromatographers match column types and mobile-phase buffers to target analytes. It surveys a broad range of bonded phases—C18, C8, phenyl, HILIC, HSS, CSH, CORTECS—and mobile-phase additives covering acidic, neutral, and basic pH zones. The goals are:

1. Clarify buffer selection for pH control and MS compatibility.
2. Highlight column chemistries suited for different analyte classes.
3. Provide practical guidance on instrument bandspread and system dispersion.

Methodology and Used Instrumentation

Buffers and pH ranges were tabulated for common additives (formic acid, acetic acid, ammonium salts, phosphates, trimethylamines) noting pKa, required concentration, and pH adjustment strategies. Chromatographic performance illustrations compare retention profiles for acidic, basic, and neutral compounds across multiple stationary phases. Key instrumentation includes Waters Acquity UPLC systems equipped with PDA detectors, and reference QC materials to monitor system suitability.

• Systems: ACQUITY UPLC, ACQUITY Arc, Alliance 2695 HPLC, Agilent 1290 UHPLC
• Columns: BEH C18/C8/Shield/Phenyl, HSS C18/T3/PFP/CN, CSH C18/Phenyl-Hexyl/Fluoro-Phenyl, CORTECS C18+/C8/Shield
• Detectors: MS, UV, ELSD, fluorescence
• QC Reference: EPA 8330 nitroaromatic standard

Main Findings and Discussion

Retention and selectivity are strongly modulated by mobile-phase pH relative to analyte pKa. Low-pH conditions enhance retention of bases in CSH and HSS phases, while high-pH buffers on BEH phases improve peak shape for acidic analytes. HILIC and amide phases excel at polar compound separations under high organic content. Solid-core particle chemistries deliver sharper peaks and lower system dispersion.

Instrument bandspread considerations guide column particle-size choices: smaller particles (<2 µm) suit high-pressure UPLC, while larger phases (3–5 µm) remain effective on conventional HPLC.

Benefits and Practical Applications

This comprehensive pairing of buffer systems and column technologies enables:

• Optimized method development across diverse analyte classes.
• Seamless scale-up from UPLC to HPLC with matched selectivity.
• Improved MS sensitivity through volatile buffer use.
• Extended column lifetime using guard and VanGuard cartridges.

Future Trends and Potential Applications

Anticipated developments include advanced hybrid particle surfaces for greater reproducibility, integrated column formats with high-performance surface treatments, and AI-driven selection tools that predict optimal buffer-stationary phase combinations. Enhanced MS-compatible buffer chemistries and microflow columns will further improve sensitivity in proteomics and metabolomics.

Conclusion

A strategic approach to mobile-phase and column selection is vital for robust, high-efficiency separations. By understanding the interplay between pH, buffer composition, and particle technology, analysts can tailor methods to their specific application requirements, ensuring peak performance and reliability.

Reference

Waters Corporation. Waters UPLC, UHPLC, and HPLC Column Selection and Mobile-Phase Guide. April 2021.