CERM: CONSIDERATIONS FOR DEVELOPING A GREENER CHROMATOGRAPHIC METHOD

Significance of the Topic

Reducing the environmental footprint of high-performance liquid chromatography is critical in modern analytical laboratories. By minimizing use of toxic or energy-intensive solvents and lowering solvent consumption through column miniaturization, greener methods help meet regulatory expectations and sustainability goals without compromising analytical performance.

Objectives and Study Overview

This work explores two strategies to develop more eco-friendly reversed-phase chromatography methods:

• Study 1: Reduction of acetonitrile consumption by scaling column dimensions while maintaining separation efficiency using a model NSAID (naproxen sodium).
• Study 2: Replacement of acetonitrile with less toxic solvents (methanol or isopropyl alcohol) in intact monoclonal antibody (mAb) mass analysis.

Methodology

Study 1 employed three column formats (4.6×150 mm, 3.0×75 mm, 2.1×50 mm) packed with C8 particles of decreasing size to preserve the length-to-particle diameter ratio. The mobile phase was acetonitrile : water : acetic acid (45 : 54 : 1 v/v/v) and flow rates were scaled to achieve comparable analysis times.
Study 2 used reversed-phase LC – MS to analyze a NIST reference mAb with three mobile phases (95% acetonitrile, methanol or isopropyl alcohol, each with 0.1% formic acid), assessing raw and deconvoluted mass spectra quality.

Used Instrumentation

• Alliance HPLC with UV/Vis detector
• ACQUITY Arc and H-Class UPLC systems with PDA detectors
• BioAccord UPLC I-Class PLUS with ACQUITY RDa mass detector
• Columns: XBridge BEH C8 (5 µm), XBridge BEH C8 XP (2.5 µm), CORTECS UPLC C8 (1.6 µm), BioResolve RP mAb Polyphenyl (2.7 µm)

Main Results and Discussion

Column miniaturization led to a four-fold reduction in acetonitrile usage by switching from 4.6×150 mm to 3.0×75 mm format, and up to thirteen-fold reduction with a 2.1×50 mm column, while preserving chromatographic performance.
In mAb analyses, isopropyl alcohol produced comparable or slightly improved raw mass spectra compared with acetonitrile, showing better desolvation and charge-state envelopes. Methanol performed acceptably but gave slightly higher valley intensities. System pressure increased with IPA but remained within safe operational limits.

Benefits and Practical Applications

• Substantial reduction in hazardous solvent consumption lowers cost and waste disposal burden.
• Maintained or improved analytical quality in small-molecule and biotherapeutic separations.
• Compatibility with existing high-pressure systems enables rapid adoption.

Future Trends and Applications

Advances may include wider adoption of sub-2 µm and superficially porous particles in ultra-high-pressure systems, exploration of alternative green solvents and solvent mixtures, and use of AI-driven method optimization to further reduce solvent usage and energy demands. Continuous flow and microfluidic approaches could drive next-generation sustainable chromatography.

Conclusion

Method development strategies that combine column dimension scaling and solvent selection deliver greener reversed-phase HPLC and LC – MS methods. By reducing acetonitrile consumption and validating isopropyl alcohol for mAb analysis, laboratories can achieve robust separations while meeting sustainability targets.

References

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