Use of Methanol Solvent for Separation

Significance of Methanol Solvent in Reverse Phase HPLC

Reversed-phase liquid chromatography often relies on acetonitrile for its elution strength and low viscosity. However, when enhanced selectivity for closely related compounds is required, methanol–water mixtures offer distinct chromatographic advantages. High-pressure UHPLC systems such as Shimadzu’s Nexera can accommodate the increased back pressure of viscous solvent blends, widening the scope of mobile phase choices.

Objectives and Study Overview

This study compares the separation of cortisone and cortisol in spiked urine samples using acetonitrile- versus methanol-based mobile phases. The goal is to assess resolution, analysis time, and system performance under each condition.

Methodology and Instrumentation

• UHPLC System: Shimadzu Nexera with a pressure limit up to 130 MPa
• Column: ODS (2.1 × 100 mm, 1.8 μm particle size)
• Mobile Phase A: 0.1% formic acid in water; B: acetonitrile or methanol
• Gradient Programs: Acetonitrile 10→60% B over 3.5 min (system pressure ~69 MPa); Methanol 30→90% B over 3.0 min (system pressure ~108 MPa)
• Flow Rate: 0.6 mL/min; Column Temperature: 40 °C
• Detection: UV at 245 nm

Main Results and Discussion

The methanol-based method delivered superior peak resolution between cortisone and cortisol compared to acetonitrile. Despite a higher operating pressure, the Nexera system maintained stable performance. Additionally, the steeper methanol gradient shortened the run time by 0.5 min, increasing sample throughput.

Benefits and Practical Applications

• Improved selectivity for steroid isomers
• Reduced analysis time with optimized gradients
• Greater flexibility in mobile phase design for complex matrices

Future Trends and Opportunities

Advancements in UHPLC pressure capabilities will enable broader use of high-viscosity solvents. Future developments may include tailored solvent blends, integration with mass spectrometric detection for enhanced sensitivity, and extension to other analyte classes requiring high selectivity.

Conclusion

Methanol as a mobile phase in UHPLC significantly improves the separation of cortisol and cortisone, demonstrating that high-pressure systems can leverage viscous solvents to achieve enhanced chromatographic performance.

Reference

• Shimadzu Corporation. Application Data Sheet No. 6: Use of Methanol Solvent for Separation; issued October 2010.