Analysis of compounds in mobile phases with high pH

Significance of the Topic

The ability to perform liquid chromatography at high pH values is essential for the analysis of basic small molecules and peptides. Conventional stainless-steel systems suffer from corrosion and metal ion release when exposed to aggressive alkaline eluents, limiting their useful pH range. An iron-free flow path extends the pH window, improves column lifetime, and ensures consistent retention and resolution.

Objectives and Study Overview

This study evaluated the Agilent 1260 Infinity Bio-inert Quaternary LC system for generic high pH applications. Two sample sets were chosen: four β-blockers representing small drug molecules, and a tryptic bovine serum albumin (BSA) peptide digest as a model biomolecule. Retention time and resolution stability were monitored over extended run times at pH 10–11.

Methodology and Instrumentation

The bio-inert system configuration included:

• Agilent 1260 Infinity Bio-inert Quaternary Pump (G5611A)
• High-performance bio-inert autosampler (G5667A)
• DAD VL detector with bio-inert flow cell (G1315D)
• Thermostatted column compartment (G1316C)
• Agilent ZORBAX Extend-C18 column, 4.6×100 mm, 1.8 µm
• OpenLAB CDS ChemStation Edition software

Mobile phases:

• β-Blockers: 50 mM triethylamine, pH 11 (A) and methanol (B)
• Peptides: 15 mM ammonium hydroxide, pH 10 (A) and 90% acetonitrile with 15 mM ammonium hydroxide (B)

Chromatographic conditions such as gradient profiles, flow rate (1 mL/min), temperature (35 °C), detection wavelengths, and injection volumes (3 µL) were optimized for each sample set.

Main Results and Discussion

Small Molecules:

• Four β-blockers (acebutolol, pindolol, metoprolol, timolol) were separated at pH 11.
• Retention time RSD was below 0.2% over 20 hours (1200 min).
• Resolution remained stable throughout the extended run.

Peptides:

• A tryptic BSA digest was analyzed at pH 10.
• Four selected peptides exhibited retention time stability over 37.5 hours (2250 min).

These results confirm that high-pH mobile phases do not degrade chromatographic performance over long periods in a bio-inert system.

Benefits and Practical Applications

• Extended pH range (1–13, up to 14 short-term) for versatile method development.
• Reduced metal contamination enhances biomolecule recovery and reproducibility.
• Stable retention and resolution support high-throughput pharmaceutical QA/QC and bioanalysis.
• Compatibility with common buffers and salts used in proteomics and peptide mapping.

Future Trends and Opportunities

• Integration with mass spectrometry for advanced proteomic workflows at high pH.
• Development of new stationary phases optimized for alkaline conditions.
• Applications in metabolomics and basic compound screening.
• Further pH stability studies up to pH 14 for specialized separations.

Conclusion

The Agilent 1260 Infinity Bio-inert Quaternary LC system demonstrated robust performance for high pH separations of both small molecules and peptides. Retention times and resolution remained stable over many hours, validating the system’s suitability for demanding analytical applications in pharmaceutical and biochemical research.

Reference

• Proof of Performance ‑ Determination of low-metal release from the Agilent 1260 Infinity Bio-inert LC system with ICP-MS, Agilent publication 5990-9352EN, 2011.
• Peng I. and Farkas T. (2008) Analysis of basic compounds by reversed-phase liquid chromatography–electrospray mass spectrometry in high-pH mobile phases. Journal of Chromatography A, 1179, 131–144.
• Using the High-pH Stability of ZORBAX Poroshell 300Extend-C18 to increase Signal-to-Noise in LC/MS, Agilent publication 5989-0683, 2004.