Optimization of HPLC Instrumentation for Use with Poroshell 120 Columns

Significance of the Topic

Superficially porous HPLC columns such as Poroshell 120 offer performance comparable to sub-2 µm materials while operating at substantially lower pressures. This advantage supports high-throughput separations, reduces solvent consumption and extends instrument lifetime in routine and advanced analytical laboratories.

Aims and Overview of the Study

This work explores how key HPLC system parameters—data acquisition rate, flow cell geometry and connecting tubing volume—can be optimized to fully realize the efficiency gains of Poroshell 120 columns. Comparative experiments under optimized and de-optimized conditions illustrate the impact of each parameter.

Methodology and Instrumentation

The study systematically varied detector scan rate and time constant, flow cell size and internal diameter of connecting tubing. Chromatographic performance was assessed on 2.1×100 mm, 3×100 mm and 4.6×100 mm Poroshell 120 EC-C18 columns using a multi-component QC mixture in 60/40 MeCN/water.

Instrumentation

• Agilent 1200 RRLC system
• G1312B Binary Pump
• G1367A Autosampler
• G1315C Diode Array Detector
• ChemStation software v. B.03.01

Main Results and Discussion

The key findings include:

• Data collection at 40 Hz with minimal detector time constants preserved signal-to-noise and maintained narrow peaks.
• Replacing the standard 13 µL flow cell with 5 µL or 2 µL micro-flow cells markedly improved column efficiency.
• Using 0.12 mm i.d. PEEK tubing (red) halved extra-column volume versus 0.17 mm i.d. tubing (green), reducing peak broadening.
• Properly installed low-dead-volume fittings eliminated tailing caused by voids and mis-seated ferrules.
• Optimized flow rates (2.0 mL/min for 4.6 mm, 0.85 mL/min for 3.0 mm, 0.42 mL/min for 2.1 mm columns) balanced efficiency and system pressure.

Benefits and Practical Applications

Poroshell 120 columns enabled:

• Transfer of traditional methods with minimal re-optimization.
• Lower solvent and energy usage due to reduced pressure.
• Seamless integration with LC–MS and bioanalytical workflows.
• Increased sample throughput in QC/QA and research laboratories.

Future Trends and Opportunities

Emerging developments may include:

• Next-generation detectors with higher sampling rates for ultrafast separations.
• Further miniaturization of flow cells and microfluidic interfaces to minimize volumes.
• Automated, AI-driven optimization of HPLC parameters.
• Expansion of superficially porous phases into novel chemistries and biomolecule separations.

Conclusion

Optimizing detector acquisition rate, minimizing extra-column volume and selecting appropriate flow cell dimensions are critical for exploiting the full potential of Poroshell 120 columns. Adherence to these guidelines delivers high efficiency, robust performance and streamlined method development.

References

1. Agilent 1200 Series DAD/MWD SL User Manual, G1315-90011 (2006).
2. Maintaining Your Agilent LC and LC/MS Systems, 5990-4957EN (2009).
3. The Influence of Sub-Two Micron Particles on HPLC Performance, 5988-9251EN.
4. Step-by-step Upgrade of Agilent 1100 to 1200 RRLC Systems, Part 1: 5989-6336EN; Part 2: 5989-6337EN.
5. Optimize Data Sampling Rate for RRHT Columns, 5989-5810EN.