Improved Chromatographic Performance with a PREMIER Peptide C18 Column Versus a Titanium-Lined C18 Column Technology

Importance of the Topic

The analysis of peptides bearing acidic modifications such as phosphorylation is critical in proteomics and quality control of synthetic peptides. Adsorption of these analytes on metal surfaces can lead to sample loss, compromised sensitivity, and inaccurate quantitation.

Objectives and Study Overview

This study compares the chromatographic performance and stability of the ACQUITY PREMIER Peptide C18 Column featuring MaxPeak High Performance Surface (HPS) Technology against a commercially available titanium-lined C18 column. Key metrics include peptide recovery, peak shape, peak capacity, and column robustness under stress conditions.

Used Methodology and Instrumentation

Instrumentation and conditions:

• Columns: ACQUITY PREMIER Peptide CSH C18 (1.7 µm, 2.1×50 mm) and titanium-lined C18 (1.6 µm, 2.1×50 mm)
• System: ACQUITY UPLC H-Class Bio with UV detection at 220 nm
• Mobile phase: 0.1% formic acid in water/acetonitrile gradient (0.7–25% ACN over 5 min)
• Sample: MassPREP 4-component phosphopeptide standard (including doubly phosphorylated T43pp)
• Procedures: triplicate injections for initial performance, peak area and tailing assessment, and packed bed stability tests at high pressure using acenaphthene

Main Results and Discussion

The PREMIER column consistently outperformed the titanium-lined variant:

• Recovery: T43pp recovery was ~5% on first injection with titanium-lined vs baseline performance of PREMIER; titanium-lined required multiple injections to approach stable recovery still 65% lower than PREMIER.
• Peak capacity: ~20% higher on PREMIER column, enabling resolution of low-abundance sequence variants.
• Peak shape: Initial tailing factors were 63% higher for titanium-lined, remaining 25% higher after 10 injections; PREMIER demonstrated superior symmetry without extensive conditioning.
• Stability: PREMIER endured >1000 high-pressure cycles (>11,000 psi) without loss in efficiency or increased tailing, whereas titanium-lined degraded after <200 cycles.

Benefits and Practical Applications

Key advantages of the PREMIER column include:

• Enhanced recovery of metal-sensitive peptides
• Improved peak shape and higher resolution
• Minimal pre-use conditioning
• Greater mechanical stability at ultrahigh pressures
• Compatibility with high-throughput proteomics and impurity profiling

Future Trends and Potential Applications

MaxPeak HPS technology paves the way for broader adoption in phosphoproteomics, therapeutic peptide impurity analysis, and other challenging biomolecule separations. Integration with mass spectrometry workflows and further miniaturization may expand its utility in single-cell and microflow applications.

Conclusion

The ACQUITY PREMIER Peptide C18 Column with MaxPeak HPS provides a robust solution for reproducible, high-fidelity separation of phosphorylated and acidic peptides. Its inert hybrid surface and mechanical resilience deliver reliable performance with minimal sample preparation.

References

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2. Lough J et al. Analyte Adsorption in Liquid Chromatography Valve Injectors. J. Chromatogr. A. 1996;726:67–75.
3. De Pra M et al. Effects of Titanium Contamination on Peak Shape. J. Chromatogr. A. 2020;1611:460619.
4. Tuytten R et al. Stainless Steel Electrospray Probe: A Dead End for Phosphorylated Organic Compounds. J. Chromatogr. A. 2006;1104:209–21.