Peak Shape (Why it matters and how to get good peak shape)

Importance of Peak Shape

Good peak shapes are symmetrical (Gaussian) peaks defined by a tailing factor near 1.0, high column efficiency, and narrow peak width.

• Enhanced resolution, sensitivity, and precision
• More accurate quantitation
• Longer column lifetime based on system suitability criteria

Study Objectives and Overview

This application note examines how to define and measure peak shape, identifies common peak distortions, explores factors influencing peak symmetry, and offers guidelines for optimizing chromatographic performance.

Methodology and Instrumentation

Peak shape metrics: USP tailing factor at 5% height, asymmetry factor at 10% height, column efficiency via plate number (N) and Van Deemter analysis to establish optimal flow rates.

Used Instrumentation

• Agilent 1260/1290 Infinity II LC systems
• Agilent InfinityLab Poroshell 120, ZORBAX Eclipse Plus, StableBond, Bonus-RP, Extend-C18 columns
• Diode array detectors with multiple flow cell volumes and Max-Light lamp cartridges
• PEEK-lined bio-inert flow paths, InfinityLab Quick Connect/Quick Turn fittings, InfinityLab deactivator additive

Main Results and Discussion

Key factors affecting peak shape:

• Column chemistry: silica acidity, metal content, bonding, endcapping, pore and particle size, packing quality
• Mobile phase: pH, buffer type and concentration, temperature, organic modifier choice (MeOH vs. ACN), additives (TEA, TFA, deactivator)
• Extracolumn effects: capillary volume, fitting alignment, detector flow cell volume, data acquisition rate
• Sample: injection solvent strength, load, cleanliness, metal-complexing analytes and system passivation

Case studies demonstrated improved tailing and efficiency using high-purity, double-endcapped or hybrid-particle columns, optimized buffers, system passivation with phosphoric acid, and bio-inert configurations.

Benefits and Practical Applications

Optimized peak shapes enable robust quantitative assays in pharmaceutical QC, environmental analysis, and proteomics. Poroshell superficially porous particles and high-pH-stable bonded phases increase throughput and resolution. Bio-inert LC systems and additives expand capabilities for metal-sensitive compounds.

Future Trends and Opportunities

Emerging hybrid particle designs and small-pore superficially porous columns will further boost efficiency. Integration of RFID-enabled consumables, predictive maintenance, and automated system passivation promises improved uptime. Advanced mobile phase additives will address ever more challenging analytes.

Conclusion

A comprehensive strategy—incorporating column selection, mobile phase design, system configuration, and sample preparation—is essential for achieving ideal peak shape, enhancing sensitivity, resolution, and reproducibility in modern HPLC analyses.