Separation of Peptides Using a Core Enhanced Technology Accucore HPLC Column

Importance of the Topic

Peptide analysis is central to proteomics, pharmaceutical development and quality control of biologics. Rapid and high-resolution separation of peptides enables efficient characterization, impurity profiling and method development in research and industrial laboratories. Core enhanced HPLC columns address challenges of high backpressure and limited resolution associated with fully porous particles, making them valuable tools for peptide separations.

Objectives and Study Overview

This application note evaluates the performance of a Thermo Scientific Accucore C18 column with core enhanced technology for the rapid separation of a mixture of peptides ranging from small dipeptides to large proteins. The goal was to achieve baseline resolution of six model peptides in under four minutes using an optimized reversed-phase gradient on an HPLC/UHPLC system.

Methodology and Instrumentation

Sample Preparation:

• A standard mixture of six peptides at 50 µg/mL was prepared in water.

Chromatographic Conditions:

• Column: Accucore C18, 2.6 µm, 100 × 2.1 mm
• Instrumentation: Thermo Scientific Accela HPLC/UHPLC with PDA/UV detection at 230 nm
• Column temperature: 40 °C; Flow rate: 0.50 mL/min; Injection volume: 2 µL
• Mobile phase A: 0.1% TFA in water; B: 0.1% TFA in acetonitrile
• Gradient: 90% A to 30% A over 6 min

Main Results and Discussion

The Accucore C18 column achieved baseline separation of six peptides with molecular weights from 238 (Gly-L-Tyr) to 13 700 Da (ribonuclease) in under 3.5 minutes. Retention times ranged from 0.6 min for the smallest peptide to 3.23 min for insulin (5 733 Da). The superficially porous 2.6 µm particles provided high efficiency and acceptable backpressure (242 bar), demonstrating their suitability for rapid peptide profiling.

The solid core and porous shell design reduced diffusion path length and allowed high linear velocities without sacrificing resolution. Robust C18 bonding ensured consistent retention and selectivity for hydrophobic peptide analytes.

Benefits and Practical Applications

This method delivers:

• High-throughput peptide separations in less than four minutes
• Baseline resolution across a broad molecular weight range
• Lower backpressure compared to sub-2 µm fully porous columns
• Robust performance suitable for routine QC and research workflows

Applications include peptide mapping, purity testing, impurity profiling and method development in proteomics and bioanalysis laboratories.

Future Trends and Potential Applications

Advances in superficially porous particle technology are expected to continue, with trends toward smaller particle sizes and higher pressure systems to further reduce analysis times. Integration with mass spectrometry will enhance peptide identification and quantitation. Emerging stationary phases with mixed-mode chemistries may expand selectivity for post-translational modifications and peptide isomers.

Conclusion

The Accucore C18 column with core enhanced technology provides a rapid, high-resolution solution for peptide separations with low backpressure and robust performance. This approach streamlines peptide analysis workflows, supporting high-throughput applications in research, QA/QC and pharmaceutical development.

References

1. Khan A. Separation of Peptides Using a Core Enhanced Technology Accucore HPLC Column. Thermo Fisher Scientific Application Note ANCCSCETPEPTIDE, 2011.