Enhanced Performance for Basic Analytes at Low pH Using a CORTECS C18+ Column on an Agilent 1290 LC System

Importance of the Topic

The analysis of basic pharmaceutical compounds under low-pH conditions is critical for ensuring reliable purity assessment and robust method performance. At acidic pH, basic analytes become positively charged, leading to reduced retention in reversed-phase liquid chromatography (RPLC) and poor peak shapes due to secondary interactions with residual silanol groups on stationary phases. Improving peak shape and resolution for these analytes directly supports quality control, troubleshooting, and high-throughput workflows in pharmaceutical and bioanalytical laboratories.

Study Objectives and Overview

This application note compares the separation performance of a superficially porous CORTECS C18+ column with a conventional superficially porous C18 column from a competing vendor. Using an Agilent 1290 Infinity I LC System and the Waters Reversed-Phase QC Reference Material (RP-QCRM) containing seven neutral, acidic, and basic probes, the study evaluates peak shape, peak capacity, and retention behavior under identical low-pH conditions.

Methodology

A ready-to-use solution of the RP-QCRM standard (uracil, butylparaben, naphthalene, propranolol, dipropylphthalate, acenaphthene, amitriptyline) at pH 7 was injected (1 µL) onto both 2.1 × 50 mm, 2.7 µm columns. Mobile phases comprised 0.1% formic acid in water (A) and acetonitrile (B), with a linear gradient programmed on the Agilent 1290 system. UV detection at 254 nm and Empower 3 software managed data acquisition and processing. Peak capacity (Pc) was calculated using gradient time and average peak width.

Instrumentation

• LC system: Agilent 1290 Infinity I
• Detection: UV 254 nm
• Columns compared: CORTECS C18+ 90 Å, 2.7 µm vs. competitor superficially porous C18 100 Å, 2.7 µm
• Data system: Waters Empower 3 FR5

Main Results and Discussion

The CORTECS C18+ column, featuring a low-level positive surface charge, delivered notably sharper peaks for the basic analytes propranolol and amitriptyline compared to the unmodified competitor column. This improvement reduced average peak widths for basic probes and yielded a ~10% increase in overall peak capacity for the RP-QCRM separation. Neutral and weakly acidic compounds showed comparable performance between columns, indicating that basic analyte behavior drove the enhanced capacity. A slight decrease in retention for charged analytes on the CORTECS C18+ column was observed due to ionic repulsion, but this did not compromise resolution under the tested conditions.

Benefits and Practical Applications

• Enhanced peak shape for positively charged basic analytes without changing mobile phase additives.
• Up to 10% higher peak capacity supporting improved resolution in QC and method transfer.
• Simple column exchange approach to mitigate secondary interactions, reducing method development time.
• Compatibility with existing RPLC workflows and routine system performance monitoring using RP-QCRM.

Future Trends and Possibilities for Use

The incorporation of charged surface functionalities into superficially porous particles can be extended to mixed-mode and ion-exchange separations, enabling tailored selectivity for a wider range of analytes. Future work may explore optimization of surface charge density, particle pore structure, and coupling with mass spectrometry-friendly additives to further enhance sensitivity and throughput in bioanalytical and pharmaceutical testing.

Conclusion

Introducing a low-level positive charge on CORTECS C18+ superficially porous particles effectively reduces secondary interactions with basic analytes at low pH, yielding sharper peaks and higher peak capacity relative to unmodified C18 columns. This column swap strategy streamlines method optimization and enhances routine QC performance without altering mobile phase composition.

Reference

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