Selectivity Choices in Reversed-Phase Fast LC

Importance of the Topic

Reversed-phase fast liquid chromatography (LC) is a cornerstone technique in analytical chemistry for separating complex mixtures rapidly and with high resolution. Selecting the appropriate stationary phase chemistry is critical to optimize separation performance, minimize analysis time, and improve peak shapes for a wide range of analytes in pharmaceutical, environmental, food, and biochemical applications.

Objectives and Study Overview

This study aims to characterize and compare the surface interactions and selectivity profiles of three Thermo Scientific reversed-phase column families: Accucore (2.6 µm solid core), Hypersil GOLD (1.9 µm fully porous), and Syncronis (1.7 µm high-surface area). Key interaction modes—hydrophobicity, steric selectivity, hydrogen bonding, ion exchange, base activity, acid interactions, and metal chelation—were quantified using standard diagnostic probes.

Methodology and Instrumentation

Diagnostic chromatographic tests adapted from Tanaka et al. were performed on a high-pressure UHPLC system equipped with UV detection at 254 nm. Test conditions included:

• Mobile phases: Water/methanol or phosphate buffers at pH 2.7 and 7.6 mixed with methanol
• Flow rate: 0.5–0.6 mL/min; temperature: 40 °C; injection volumes: 5–10 µL
• Columns: Accucore C18, C8, aQ, phenyl-hexyl, PFP; Hypersil GOLD C18, C8, C4, aQ, phenyl, PFP; Syncronis C18, C8, aQ, phenyl

Main Results and Discussion

Hydrophobic retention (HR) was highest on Syncronis C18 and aQ due to the elevated surface area, followed by Hypersil GOLD and Accucore RP-MS. Hydrophobic selectivity (HS) profiles were comparable among C18, RP-MS, and aQ phases. Steric selectivity (SS) peaked on pentafluorophenyl (PFP) phases, driven by fluorine interactions, while phenyl-hexyl demonstrated mixed hydrophobic and π–π interactions. Base activity and metal chelation (secondary interactions) were minimal for Hypersil GOLD and Syncronis due to high purity silica and advanced endcapping. Accucore solid core columns offered reduced backpressure and efficient peak shapes with low tailing factors.

Benefits and Practical Applications

• Wide selectivity range: Users can tailor method development by choosing phases with distinct hydrophobic and steric profiles.
• Fast separations: Solid core technology reduces analysis time without sacrificing resolution, making it ideal for high-throughput QA/QC.
• Enhanced peak shape: Low secondary interactions on high-purity silica improve detection sensitivity for basic and chelating analytes.
• Scalability: Phases cover polar to highly nonpolar analytes, supporting metabolite profiling, impurity screening, and isomer differentiation.

Future Trends and Applications

Advances in stationary phase design will focus on hybrid chemistries to further reduce nonideal interactions, bespoke ligands for chiral or ultra-polar analytes, and integration with multidimensional LC. Emerging applications include ultra-fast omics workflows, inline sample cleanup, and real-time process monitoring. Combining chemometric selection tools with predictive modelling will streamline column choice and method optimization.

Conclusion

The comparative characterization highlights that no single phase is universally optimal; instead, practitioners should leverage differences in hydrophobicity, steric selectivity, and secondary interaction profiles to meet specific analytical goals. The availability of Accucore, Hypersil GOLD, and Syncronis phases provides a versatile toolkit for fast, high-resolution reversed-phase separations.

Reference

1. Tanaka N. et al., Journal of Chromatographic Science 27 (1989) 721–728.