Increasing Peak Capacity in Reversed-Phase Peptide Separations with Charged Surface Hybrid (CSH) C18 Columns

Importance of the Topic

Reversed-phase peptide separations are fundamental to bottom-up proteomics workflows and biopharmaceutical peptide mapping. Achieving high peak capacity and sharp peak shapes under mass spectrometry–compatible conditions is essential for comprehensive peptide analysis in complex mixtures.

Objectives and Study Overview

This study evaluates the performance of a charged surface hybrid C18 stationary phase (CSH130 C18) for peptide separations. Benchmarks include peak capacity, selectivity, and compatibility with formic acid (FA)–only mobile phases, comparing CSH130 C18 to traditional fully porous and superficially porous C18 columns.

Methodology

• Columns tested: Waters ACQUITY UPLC CSH130 C18 (1.7 µm and 2.5 µm XP), BEH130 C18 (1.7 µm), superficially porous C18 (1.7 µm), and conventional silica C18 (5 µm).
• Mobile phases: water and acetonitrile with variable ratios of 0.1 % formic acid and 0.1 % trifluoroacetic acid.
• Gradient: initial 2 % ACN for 1 min ramped to 50 % ACN over 60 min at 0.3 mL/min.
• Sample: MassPREP nine-peptide mixture at ~0.6 mg/mL in 0.1 % FA.
• Detection: UV at 214 nm and Xevo G2 QTof MS in ESI+ mode.
• Peak capacity calculated from UV peak widths at half-height (peptides 2–7) and converted to 4σ values.

Instrumentation

• Waters ACQUITY UPLC H-Class Bio System with column heaters for 20 cm and 30 cm columns.
• ACQUITY UPLC TUV detector and Xevo G2 QTof mass spectrometer.
• Qsert glass vials and MassPREP peptide mixture standard.

Main Results and Discussion

• CSH130 C18 produced the best peak shapes under both TFA and FA mobile phases, outperforming other C18 materials.
• Under FA-only conditions, CSH130 C18 resolved peptides 8 and 9 with over 3 min separation, a selectivity not achieved by other columns.
• Retention on CSH130 C18 was slightly reduced due to its low-level positive surface charge repelling positively charged peptides.
• With 0.1 % TFA, CSH130 C18 peak capacity increased by ~20 %, and with 0.1 % FA by ~90 %, compared to other 1.7 µm C18 columns.
• TFA caused a 12-fold suppression of MS signal; CSH130 C18’s reduced dependence on TFA enhances LC–MS sensitivity.
• Method transfer from 1.7 µm UPLC to 2.5 µm XP HPLC (1.5× longer gradient, reduced flow) maintained selectivity and cut back pressure from ~8000 to ~3000 psi.

Benefits and Practical Applications

• Enhanced peak capacity and sharper peak shapes under acidic, MS-friendly conditions.
• Minimized use of strong ion-pairing reagents improves electrospray efficiency and detection limits.
• Scalable protocols for both UPLC and conventional HPLC platforms support diverse laboratory requirements.

Future Trends and Applications

Further exploration of charged surface hybrid technologies may extend to larger polypeptides and intact protein separations. Integration with high-resolution mass spectrometry and automated high-throughput workflows will deepen proteome coverage. Continued development of surface chemistries could optimize retention of highly hydrophilic peptides and novel analytes.

Conclusion

The CSH130 C18 stationary phase delivers significant improvements in reversed-phase peptide separations, offering higher peak capacity, unique selectivity, and reduced reliance on signal-suppressing additives. Its compatibility with both UHPLC and HPLC instruments makes it a versatile solution for advanced proteomic and biopharmaceutical analyses.

References

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