Updating Old Methods:Is the Gain Worth the Pain? - Becoming a Better Chromatographer HPLC educational webinar

Importance of the Topic

Laboratories today require high sample throughput, reduced solvent consumption and improved separation performance. Updating traditional HPLC methods by adopting superficially porous particle columns can meet these demands without extensive system upgrades.

Study Objectives and Overview

This study reviews allowable method adjustments under USP General Chapter 621 and demonstrates the transfer and optimization of isocratic and gradient HPLC procedures onto Agilent InfinityLab Poroshell 120 superficially porous columns to enhance efficiency, resolution and sensitivity.

Methodology and Used Instrumentation

Methods were adjusted within USP 621 limits for particle size, column dimensions, flow rate, temperature and mobile phase composition. Comparative separations employed Agilent InfinityLab Poroshell 120 columns (1.9, 2.7 and 4 µm) and traditional totally porous columns on Agilent 1260 Infinity systems. Detection included UV-DAD at 240 nm, UV at 220 nm and MS in ESI mode for HILIC separations. High-pH stability was tested on Poroshell HPH-C18 phases.

Key Results and Discussion

• Decreasing particle size from 4 µm to 1.9 µm increased plate counts up to 120% and resolution (Rs) from 0.7 to over 2.2 under similar pressure conditions.
• Catechin and ibuprofen analyses were accelerated by 40–60% while maintaining system suitability criteria (N, Rs).
• Naproxen method transfer onto 2.7 µm columns achieved equivalent performance with up to 38% shorter column length and maintained N > 4000 and Rs > 11.5.
• Gradient method scaling preserved retention factors by adjusting gradient time, flow rate and column volume proportionally.
• Notoginsenoside baseline separation improved from Rs=1.51 to Rs=2.31 using Poroshell 120 at 4 µm.
• Amino acid separation at pH 8.3 remained stable after over 2000 injections on HPH-C18.

Benefits and Practical Applications

• Throughput gains of 30–50% on existing HPLC instruments.
• Solvent savings of 50–80%, reducing operating costs and waste.
• Extended column lifetime and compatibility with high-pH mobile phases.
• Rapid method transfers across isocratic, gradient, HILIC and chiral analyses.

Future Trends and Potential Applications

Advancements in superficially porous particles will support peptide and protein separations, multidimensional chromatography and high-resolution MS workflows. Emerging column chemistries tailored to challenging analytes and AI-driven method optimization promise further gains.

Conclusion

Transitioning legacy HPLC methods to superficially porous columns delivers substantial improvements in speed, efficiency and sustainability with minimal revalidation. These updates justify the effort by unlocking new analytical capabilities on standard instrumentation.

References

1. Pittcon 2012, W. Long and A. Brooks, Transfer and Optimization of HPLC Methods to Superficially Porous UHPLC (Oral Presentation 420-3)