CSH130 C18 Columns for Peptide Separations

Importance of the Topic

Charged Surface Hybrid (CSH) C18 stationary phases represent a significant advance for reversed-phase peptide separations, enabling robust performance with both trifluoroacetic acid (TFA) and formic acid (FA) eluents. This flexibility addresses a common trade-off between chromatographic peak quality and MS sensitivity.

Objectives and Study Overview

The study evaluates Waters CSH130 C18 particles for peptide and small protein separations up to ~10 kDa. Key aims include comparing peak capacity, mass loading tolerance, and scalability across different particle sizes and acid modifiers, and validating column reproducibility with a tryptic digest standard.

Methodology and Instrumentation

Columns tested: CSH130 C18 (1.7 µm UPLC, 2.5 µm HPLC XP, and preparative formats). Mobile phases: 0.1% FA, 0.1% TFA, and mixtures. Sample: Waters MassPREP Peptide Standard; QC digest: cytochrome c tryptic mixture. Systems: Waters ACQUITY UPLC and eXtended Performance HPLC instruments with low dispersion flow paths.

Main Results and Discussion

• Peak capacity with CSH130 C18 exceeded competing superficially porous and BEH-based C18 phases by 15–30% under both FA and TFA conditions.
• MS signal suppression by TFA was substantially reduced compared to traditional TFA-only columns.
• CSH130 C18 tolerated higher peptide loads without loss of peak shape; performance differences narrowed at low loads.
• Scaled methods from 1.7 µm to 2.5 µm columns maintained resolution by adjusting flow rate and gradient time.
• Batch QC using cytochrome c digest confirmed reproducible performance across lots.

Benefits and Practical Applications

• Eliminates need to choose between chromatographic resolution and MS sensitivity.
• Supports high-throughput proteomics, peptide mapping, and lab-scale peptide purification.
• Scalable from nano-UPLC to preparative chromatography.
• Reliable column-to-column consistency for validated workflows.

Future Trends and Opportunities

Continued development of hybrid particle chemistries may further enhance load capacity and selectivity. Integration with advanced low-dispersion instrumentation and automation will expand applications in high-throughput proteomics. Exploration of mixed-acid modifiers or ion-pairing alternatives could optimize MS compatibility.

Conclusion

Waters CSH130 C18 columns deliver superior peptide separation performance, combining high peak capacity, versatile eluent compatibility, and robust load tolerance. They offer a flexible, reproducible solution for modern LC and LC-MS proteomic analyses.

References

1. Lauber MA, Koza SM, Fountain KJ. Waters Application Note 720004568EN (2013).
2. Lauber MA, Koza SM, Fountain KJ. Waters Application Note 720004571EN (2013).
3. Iraneta PC et al. Waters White Paper 720003929EN (2011).
4. Wyndham KD et al. Anal Chem. 2003;75(24):6781–6788.