Selectivity nad Particle Size Consideration for High Resolution and High Speed LC

Significance of the Topic

High resolution and speed in liquid chromatography are essential for modern analytical workflows. Selectivity remains the primary factor that drives separation efficiency, and the introduction of sub2um particle columns has enabled unprecedented resolution in both short and traditional column formats. This development offers laboratories the flexibility to optimize separations while reducing analysis times and solvent consumption.

Objectives and Study Overview

The study aimed to compare multiple phenyl bonded phases against conventional C18 columns to explore differences in selectivity and retention behavior. A broad set of analytes, including aliphatic compounds, nitro substituted aromatics, nucleobases, polyaromatic hydrocarbons and natural product extracts, was evaluated to demonstrate orthogonality and method development strategies.

Methodology and Instrumentation

Columns tested included various phenyl phases such as phenylhexyl, phenylethyl, phenylpropyl and C18 phases in both Eclipse and stablebond families. Mobile phases were composed of acetonitrile or methanol mixed with water or buffer at controlled pH. Key chromatographic parameters included a flow rate of 1 to 1.85 mL per minute, detection by UV at 205 or 254 nm and gradient elution profiles tailored for each analyte class.

Used Instrumentation

• Agilent 1200 series LC system with 600 bar pump
• RRHT columns (4.6 x 50 mm, 1.8 um and 4.6 x 100 mm, 5 um)
• DAD detector operating at 220 or 254 nm
• Single quad ESI mass spectrometer in positive mode

Main Results and Discussion

Phenyl bonded phases exhibited distinct selectivity profiles compared to C18, with nitro substituted aromatics showing the greatest retention differences. Scatter plot analysis of log k revealed that methanol enhanced pi pi interactions on phenyl phases more than acetonitrile. Orthogonality studies with NSAIDS and estrogens confirmed that phenyl columns can resolve peak order and improve separation of structurally similar compounds.

Benefits and Practical Applications

• Enhanced method development with multiple selectivity options
• Reduced analysis time using sub2um particles without sacrificing resolution
• Improved separation of challenging compound classes such as aromatic and polyaromatic analytes
• Potential for streamlined QA QC workflows and high throughput screening

Future Trends and Opportunities

Anticipated developments include expansion of high pH stable phenyl phases, integration with smaller particle superficially porous columns, and deeper exploitation of aromatic interactions. Coupling these chemistries with advanced mass spectrometry and environmentally friendly solvents may further increase analytical performance and sustainability.

Conclusion

Introducing a variety of phenyl bonded phases alongside conventional C18 extends the selectivity toolkit for chromatographers. Combined with sub2um technology, this approach delivers fast, high resolution separations and supports robust method development across diverse sample types.

References

• William Long Maureen Joseph James D Martosella John W Henderson Jr Agilent Application Note 5989-4506EN Pittcon 2008