Column choice based on Tanaka characterization - not all C18 columns are the same

Significance of the Topic

Reversed-phase liquid chromatography (RPLC) with C18-modified silica dominates over 90% of HPLC applications. Despite sharing the same USP code (L1), C18 phases differ in surface chemistry and performance. Tanaka characterization provides a practical framework to distinguish these subtle differences, guiding analysts toward optimal column selection for specific separation challenges.

Objectives and Overview

This study applies the Tanaka test to six KNAUER C18 phases—Eurospher II C18, C18 A, C18 H, C18 P, Eurospher I C18, and Eurosil Bioselect C18—to evaluate hydrophobic retention (HR), hydrophobic selectivity (HS), and shape selectivity (SS). Ion exchange and hydrogen-bonding capacities showed minimal variation and were not included in the comparative analysis.

Methodology and Instrumentation

Tanaka parameters were measured under isocratic conditions using methanol/water (80:20, v/v) at 30 °C, 254 nm detection, 1 mL/min flow, and 10 µL injections. All analytical columns were 150 × 4 mm ID; pore sizes were 100 Å for Eurospher phases and 300 Å for Bioselect.

• HPLC system: KNAUER AZURA® Educational System (pump, UV detector, manual injector)
• Column thermostat: AZURA® CT 2.1
• Detector: UV at 254 nm, data rate 20 Hz
• Columns evaluated: ES II C18, ES II C18 A, ES II C18 H, ES II C18 P, ES I C18 (100 Å), EB C18 (300 Å)

Main Results and Discussion

Hexagonal Tanaka plots highlighted significant differences in hydrophobic retention, with ascending HR ranking: EB C18 (most polar) < ES II C18 A < ES I C18 < ES II C18 < ES II C18 H < ES II C18 P (most hydrophobic). Hydrophobic selectivity values remained in a narrow range (1.45–1.64), indicating similar ligand coverage patterns. Shape selectivity was consistent except for Eurospher I C18, which exhibited a higher SS value attributed to incomplete endcapping. Both ion exchange and hydrogen-bonding parameters showed negligible variation across phases.

Benefits and Practical Applications

Detailed Tanaka profiling empowers analysts to:

• Select C18 A for robust performance in highly aqueous mobile phases without phase collapse.
• Use high-carbon phases (C18 H, C18 P) for improved pH stability across wide ranges.
• Choose large-pore Bioselect C18 for biomolecules or analytes above 2 000 Da.

Future Trends and Potential Applications

Advancements in stationary-phase design will focus on enhanced selectivity for polar and high-molecular-weight compounds, sustainable solvent compatibility, and data-driven column recommendations. Hybrid materials and novel ligand architectures may further refine separation efficiency.

Conclusion

Tanaka characterization offers a systematic method to distinguish C18 phases beyond generic USP codes. Recognizing variations in hydrophobic retention and shape selectivity enables tailored column choices, optimizing analytical performance across diverse applications.

References

1. Chromatography Online. Column selection for reversed-phase HPLC. Available at: http://www.chromatographyonline.com/column-selection-reversed-phase-hplc