Ionic Additives and LC/MS Conditions for Polypeptide Analysis

Significance of the Topic

Reversed-phase high performance liquid chromatography (RP-HPLC) of polypeptides relies on ionic additives to control mobile phase pH, suppress silanol interactions and enhance retention. However, commonly used additives such as trifluoroacetic acid (TFA) significantly reduce mass spectrometric sensitivity. Achieving a balance between chromatographic performance and MS detection is crucial in proteomics, pharmaceutical analysis and peptide characterization.

Objectives and Study Overview

This work investigates the effects of TFA concentration on peptide retention, peak shape and MS response. It compares alternative volatile acids like formic acid and ammonium acetate buffers in conjunction with highly inert wide pore stationary phases. Quantitative measurements of retention shifts, ionization suppression and column performance are presented.

Methodology and Instrumentation

• Columns: Discovery BIO Wide Pore C18 (300Å, 3µm, 15cm × 2.1mm ID) and capillary formats (3µm, 10cm × 0.32mm ID).
• Mobile Phases: 25mM formic acid or 10mM ammonium acetate (pH 7.0) versus 0.005–0.1% TFA in water and 50:50 water:acetonitrile gradients.
• Samples: RP peptide performance mixture and angiotensin I–III standard peptides.
• Detection: Positive electrospray ionization MS, UV at 215nm; flow rates of 0.208mL/min (analytical) or 6µL/min (capillary); injection volumes from 0.2 to 50µL.
• Column temperature maintained at ambient or 35°C.

Main Results and Discussion

• TFA markedly increases retention and improves peak shape but suppresses MS signal by up to 80% at 0.1% concentration compared to formic acid conditions.
• Retention times decrease linearly with reduced TFA levels, altering selectivity of closely related peptides.
• Formic acid preserves ionization efficiency yet may lead to peak tailing unless paired with an inert wide pore stationary phase.
• Discovery BIO Wide Pore C18 demonstrates superior resolution of basic peptides under low or no TFA conditions, minimizing silanol interactions.
• Capillary LC/MS using ammonium acetate buffer successfully resolved and identified angiotensin peptides with minimal sample consumption via peptide mass fingerprinting.

Benefits and Practical Applications

• Avoiding or minimizing TFA enhances MS sensitivity for trace level peptide detection.
• Inert wide pore C18 phases enable good peak shape under volatile acid mobile phases.
• Capillary and microbore columns reduce sample and solvent consumption in proteomics and peptide mapping.
• Approach is applicable to pharmaceutical QA/QC, biomarker screening and complex sample analysis.

Future Trends and Applications

• Design of novel volatile ion-pairing agents that balance weak pairing with MS compatibility.
• Development of stationary phases with ultralow silanol activity and enhanced chemical stability.
• Integration of ultra-high resolution LC columns with accurate mass MS for advanced peptide fingerprinting.
• Expansion of capillary and nanoLC/MS workflows for single cell proteomics and low abundance biomarker discovery.

Conclusion

For optimal peptide analysis by LC/MS, volatile acids such as formic or acetic acid combined with highly inert wide pore C18 columns provide an effective compromise. This strategy maintains chromatographic resolution while maximizing MS sensitivity by avoiding the detrimental effects of TFA.

References

• Apffel A et al Journal of Chromatography 712 1995 177