Maximizing Resolution and Selectivity: Superficially Porous Column Chromatography Options

Importance of the Topic

High resolution and selectivity are essential for accurate, rapid, and robust chromatographic analyses in pharmaceutical, environmental, and food laboratories. Superficially porous particle (SPP) columns have emerged as a powerful solution, combining the efficiency of sub‐2 µm fully porous phases with the lower backpressure of larger particles, making them compatible with most HPLC and UHPLC systems.

Objectives and Overview of the Study

This whitepaper examines the specifications and advantages of Agilent Poroshell 120 SPP columns, outlines key challenges in method development, and demonstrates how selectivity can be tuned via bonded phase and mobile phase pH. The goal is to guide analysts toward maximizing resolution and throughput while maintaining method robustness.

Methodology and Instrumental Setup

Analyses were conducted on Agilent HPLC and UHPLC instruments with pressure limits up to 600 bar. Poroshell 120 columns (2.7 µm and 4 µm) feature a 1.7 µm solid core and 0.5 µm porous shell. Multiple bonded chemistries (EC-C18, C8, CN, phenyl-hexyl, Bonus-RP, SB-Aq, PFP, HILIC) support a wide pH range (2–12). Typical mobile phases included 0.1% formic or acetic acid buffer with acetonitrile or methanol gradients. Detection was by UV/DAD at 210–260 nm using microflow cells.

Main Results and Discussion

• Efficiency and Pressure: 2.7 µm SPP columns achieved ≈12 000 plates with < 200 bar, matching sub-2 µm performance at half the pressure and double the efficiency of 3.5 µm fully porous columns.
• Resolution Improvements: Naproxen tablet assays met USP criteria (> 4000 plates, Rs > 11.5) with Poroshell 120 EC-C18 in shorter columns and reduced run times (2–4x faster).
• Selectivity Control: Bonded phase screening highlighted phenyl-hexyl and Bonus-RP phases for aromatic and polar analytes. Mobile phase pH shifts altered retention of ionizable compounds, enabling orthogonal separations.
• Applications: Steroid and NSAID separations demonstrated optimized resolution across chemistries. Quinolones in milk achieved high peak capacity and reduced pressure with 4 µm columns on low-pressure systems.
• Column Lifetime: Over 1800 injections of undiluted green tea at 550 bar showed no loss of performance. Guard columns effectively protected analytical columns in complex matrices.
• Operational Tips: Use spring-loaded Agilent A-Line fittings to minimize dead volume and ensure reproducible connections. Adjust data collection rates to preserve theoretical plates at high flow.

Benefits and Practical Applications

• Drop-in Replacement: SPP columns integrate seamlessly into existing HPLC/UHPLC setups, reducing development time.
• High Throughput: Lower backpressure and shorter columns deliver faster run times without sacrificing efficiency.
• Method Robustness: Broad pH stability and reproducible guard column protection extend column life and ensure consistent results.
• Flexible Selectivity: A diverse bonded phase portfolio allows fine-tuning of separation for polar, non-polar, and ionizable compounds.

Future Trends and Applications

• Expanded Phase Chemistries: Development of new core-shell phases tailored for chiral, ion-exchange, and mixed-mode separations.
• AI-Driven Method Development: Integration of cheminformatics and machine learning tools for automated phase and condition selection.
• Miniaturization: Adoption of micro- and nano-flow SPP columns to further reduce solvent consumption and increase sensitivity.
• High-pH Workflows: Wider availability of stable-pH phases to improve separations of basic compounds and reduce silanol interactions.

Conclusion

Poroshell 120 superficially porous columns provide an optimal balance of efficiency, selectivity, and pressure for modern chromatographic challenges. Their broad chemistry range, pH tolerance, and low backpressure enable rapid method development, high throughput, and robust performance across diverse applications.