Evaluating the Impact of LC System Dispersion on the Size-Exclusion Chromatography Analysis of Proteins

Importance of Topic

Size-exclusion chromatography (SEC) is a cornerstone technique for assessing noncovalent aggregation in biotherapeutic proteins. Advances toward higher throughput and smaller particle SEC columns have reduced peak volumes, making extra-column dispersion a critical factor that can compromise resolution, sensitivity, and reproducibility of protein separations.

Objectives and Study Overview

This study systematically evaluates how extra-column dispersion impacts SEC performance for protein analysis. By measuring dispersion volumes and correlating them with observed plate counts and resolution for various column geometries and particle sizes, practical guidelines are developed for column selection based on LC system characteristics and analytical requirements.

Methodology and Instrumentation

Extra-column dispersion (5σec) was quantified by injecting a low-volume caffeine standard, measuring peak width at 4.4% height, and comparing with second-moment variance calculations. A protein standard mix featuring thyroglobulin, IgG, BSA, and myoglobin was used to confirm dispersion effects on high molecular weight species. SEC separations were performed across columns differing in internal diameter (2.1, 4.6, 7.8 mm), length (150, 300 mm), and particle size (1.7, 2.5, 3.5 μm) under constant linear velocity, with injection volumes and flow rates scaled to column volume.

Used Instrumentation

• Waters ACQUITY UPLC H-Class Bio System
• Waters ACQUITY Arc Bio System
• Waters Alliance HPLC System
• ACQUITY UPLC Protein BEH SEC Columns (1.7 μm, 2.1–4.6 mm ID)
• XBridge Protein BEH SEC Columns (2.5–3.5 μm, 4.6–7.8 mm ID)
• ACQUITY UPLC TUV detector with 5 mm flow cell
• Empower 3 Chromatography Software

Key Findings and Discussion

• Extra-column dispersion volumes ranging from ~10 to ~60 μL were measured and found to correlate linearly between peak-width and second-moment methods, with peak-width offering better reproducibility.
• Small-ID (2.1 mm) columns exhibited significant losses in plate count (up to 36%) and resolution when dispersion volumes approached or exceeded analyte peak volumes.
• Medium-ID (4.6 mm, 1.7 μm) columns delivered superior resolution under low-dispersion conditions (5σec ≤ 25 μL) but performance degraded at higher dispersion levels.
• Large-ID (7.8 mm, 2.5 μm) columns provided robust resolution across a wider range of system dispersions, balancing efficiency, throughput, and operating pressure.

Advantages and Practical Applications

• Optimized column selection improves reproducibility and robustness of SEC methods across diverse LC platforms.
• Larger column IDs mitigate the deleterious impact of extra-column dispersion without sacrificing analysis time when flow rates are scaled appropriately.
• Tailored particle size and column geometry choices enable high-resolution separation of aggregates and low-molecular-weight fragments under realistic lab conditions.

Future Trends and Potential Uses

As protein analytical demands grow, future directions include integrating real-time dispersion monitoring, developing automated dispersion correction algorithms, and designing next-generation low-dispersion flow paths. Miniaturized and high-throughput SEC platforms will benefit from these advances, enhancing QC workflows and biopharmaceutical characterization.

Conclusion

Extra-column dispersion significantly influences SEC performance for protein analysis, especially with modern small-particle columns. By systematically measuring dispersion and understanding its effects on resolution and efficiency, practitioners can select column geometries and particle sizes that match their LC systems and analytical goals, ensuring reliable and high-quality separations.

References

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