A Novel HILIC Column for High Speed N-linked Glycan Analysis

Importance of Topic

Reversed-phase HPLC method development often struggles to achieve both high resolution and selectivity, especially for mixtures containing acidic, basic and neutral analytes. Adjusting buffer pH offers a powerful way to modify analyte charge and hence retention without changing organic modifier or stationary phase, enabling rapid screening of chromatographic conditions. The advent of high-pH–stable superficially porous columns broadens the scope of pH-dependent separations, improves peak shapes for basic compounds, and supports robust LC–MS workflows.

Objectives and Study Overview

This study evaluates Agilent Poroshell HPH-C18 columns under alkaline conditions as a tool for HPLC method development. Key aims include:

• Investigating the impact of mobile phase pH on selectivity and elution order of acid, base and neutral probe compounds.
• Comparing retention time correlations between low pH (3.0, 4.8) and high pH (10.0) using methanol and acetonitrile modifiers.
• Assessing ESI+ LC–MS sensitivity for basic analytes at high vs. low pH.
• Testing column lifetime under 2,000 injections at pH 10 to demonstrate stability.

Used Instrumentation

The work was performed on an Agilent 1260 Infinity LC with binary pump, thermostatted column compartment and diode array detector. Samples were injected with an Agilent ALS autosampler. In some experiments an Agilent 6140D single-quadrupole LC/MS in positive electrospray mode was used. Data acquisition and processing were managed by OpenLab C.01.05 software.

Methodology

A generic linear gradient (5–95% organic over 4 min) at 0.42–2 mL/min was applied to mixtures of acidic (e.g., acetylsalicylic acid, diflunisal), basic (e.g., procainamide, diltiazem) and neutral (e.g., hexanophenone, caffeine) compounds. Mobile phases consisted of 10 mM ammonium formate (pH 3.0), ammonium acetate (pH 4.8) or ammonium bicarbonate (pH 10.0), with methanol or acetonitrile as organic modifiers. Retention times were compared across pH values to calculate correlation coefficients (R2) as a measure of orthogonality. High-pH vs. low-pH LC–MS experiments evaluated peak shape, retention and ESI signal intensity for bases.

Main Results and Discussion

Altering pH from acidic to alkaline dramatically reordered elution of ionizable analytes while neutrals remained unchanged. At pH 3.0, acids were neutral and strongly retained; at pH 10.0 they were ionized and eluted earlier, while bases showed the opposite trend. Retention time correlations between pH 3.0 and 10.0 yielded R2≈0.49 with methanol and R2≈0.40 with acetonitrile, confirming enhanced selectivity differences under alkaline conditions. LC–MS of bases such as lidocaine, procainamide and diltiazem at pH 10 displayed sharper peaks, higher retention and up to two-fold intensity gains compared with low-pH formic acid buffers. Column stability tests with 2,000 injections at pH 10 showed negligible changes in retention for most probes on HPH-C18, whereas a conventional C18 suffered significant retention drift for ionizable analytes.

Advantages and Practical Applications

Using elevated pH on a high-pH–stable superficially porous column enables:

• Rapid adjustment of selectivity via pH without changing bonded phase or gradient profile.
• Improved peak shape and detection sensitivity for basic analytes in ESI+ LC–MS.
• Extended column lifetime under alkaline conditions, reducing method redevelopment frequency.
• Orthogonal separation strategies that enhance resolution in complex mixtures.

Future Trends and Applications

High-pH RPLC is poised to integrate with automated method development platforms and retention modeling tools within a Quality by Design framework. Emerging stationary phases with wider pH tolerance will further expand analyte coverage. Coupling high-pH separations with advanced mass spectrometers and software for orthogonal screening will streamline biopharmaceutical and environmental analyses.

Conclusion

Agilent Poroshell HPH-C18 columns demonstrate robust performance at pH 10, enabling flexible pH-based selectivity control while maintaining column integrity and enhancing LC–MS detection of basic compounds. This strategy accelerates method development and broadens applicability in pharmaceutical and chemical analysis.

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