Exceptional Selectivity of Agilent ZORBAX Eclipse Plus Phenyl-Hexyl Columns to Separate Estrogens

Significance of the Topic

Accurate separation of estrogenic steroids is critical in pharmaceutical, environmental and clinical analyses due to their biological activity and regulatory requirements. High selectivity is required to resolve structurally related phenolic compounds with overlapping retention times.

Objectives and Study Overview

This study evaluates the selectivity of the Agilent ZORBAX Eclipse Plus Phenyl-Hexyl stationary phase compared to C18 and other phenyl-type columns for the isocratic separation of five estrogen steroids under identical conditions. The goal is to demonstrate how column chemistry impacts resolution and to guide method development strategies.

Methodology and Instrumentation

The separation used an Agilent 1200 Rapid Resolution LC system with a binary pump (1 mL/min, 60% methanol/40% water), an ALS with 3 µL injections, a column compartment at 25 °C, and a diode array detector at 220 nm.
Columns tested included:

• ZORBAX Eclipse Plus Phenyl-Hexyl (4.6×100 mm, 5 µm; 3.5 µm; 1.8 µm)
• ZORBAX Eclipse Plus C18 (4.6×100 mm, 3.5 µm)
• Various commercial phenyl-bonded phases (4.6×100 mm, 5 µm)

Samples comprised a composite mixture of five estrogens (estriol, estradiol, ethynylestradiol, diethylstilbestrol, dienestrol) at ~0.12 mg/mL each prepared in methanol/water.

Main Results and Discussion

The Phenyl-Hexyl phase delivered complete baseline separation of all five estrogens, outperforming the C18 column despite its lower theoretical plate count. Selectivity differences among phenyl columns were pronounced, driven by π-π interactions and ligand linkage length. Efficiency gains alone (smaller particles) could not compensate for suboptimal selectivity on other phases.

Benefits and Practical Applications of the Method

• Rapid optimization via column substitution to achieve ideal selectivity
• Enhanced resolution of aromatic steroids relevant for pharmaceutical QC and environmental assays
• Compatibility with standard HPLC setups and straightforward scale-up using sub-2 µm particles

Future Trends and Applications

• Adoption of UHPLC with sub-2 µm Phenyl-Hexyl columns for higher throughput
• Automated column screening and multivariate method development software
• Extension to other aromatic analytes and complex sample matrices
• Integration with mass spectrometry for enhanced detection sensitivity

Conclusion

Experimental evaluation of stationary phases is essential for optimal chromatographic selectivity. The Eclipse Plus Phenyl-Hexyl column offers distinct π-π interaction capabilities that yield superior separation of estrogenic steroids, underscoring the value of a diverse column portfolio for robust HPLC method development.

References

• Automated Method Development Using Agilent 1100 Series HPLC Systems, ChemStation, ChromSword Software, Agilent Technologies, publication 5988-8927EN.
• V. R. Meyer, Practical High-Performance Liquid Chromatography, 4th Ed., Wiley, 2004.
• M. Yang et al., Impact of Methanol and Acetonitrile on Separations Based on π-π Interactions with Reversed Phase Phenyl Column, Journal of Chromatography, 1097, 124–129, 2005.