The influence of silica pore size and particle size on insulin - a small molecule of protein separation

Importance of the Topic

The efficient and reproducible separation of insulin and its variants by reversed-phase HPLC is essential for pharmaceutical quality control, method development, and regulatory compliance. Understanding how silica pore size and particle size influence retention, resolution, and peak shape enables analysts to optimize methods for small proteins and peptides.

Objectives and Study Overview

This study evaluates the impact of silica pore diameters (80 Å, 95 Å, 120 Å, 170 Å, 300 Å) and particle morphologies (fully porous versus superficially porous) across particle sizes (5 µm, 3.5 µm, 1.8 µm, 2.7 µm) on the separation of porcine insulin and A21-desamido insulin. Multiple Agilent C18 columns meeting pharmacopeia specifications were compared under a standardized isocratic method.

Methodology and Instrumentation

A single isocratic mobile phase (74% aqueous sulfate buffer at pH 2.3, 26% acetonitrile) was used with a 1.0 mL/min flow rate and UV detection at 214 nm. Separation was performed on an Agilent 1200 SL LC system with thermostatted column compartment at 40 °C.

• Columns evaluated included ZORBAX SB-C18, Eclipse Plus C18, 300SB-C18, TC-C18(2) (fully porous) and Poroshell 120 SB-C18, Poroshell 120 EC-C18 (core-shell).
• Column dimensions ranged from 4.6×150 mm to 4.6×100 mm; particle sizes spanned 5 µm, 3.5 µm, 1.8 µm and 2.7 µm.

Results and Discussion

Columns with small pores (<100 Å) limited insulin access, yielding long retention times and broader peaks. A transition from 80 Å and 95 Å to 120 Å pores improved mass transfer and reduced tailing. Superficially porous Poroshell 120 columns delivered twice the efficiency of fully porous SB-C18 80 Å at comparable particle sizes. Decreasing particle size from 5 µm to 1.8 µm further enhanced resolution and peak shape. The Agilent TC-C18(2) column exhibited the highest surface area and strongest retention but required elevated organic content to optimize run times.

Benefits and Practical Applications

• Faster analysis with reduced retention times by adjusting pore size and particle diameter.
• Sharper peak shapes and higher theoretical plates meeting USP/Ph Eur requirements for insulin assays.
• Guidelines for selecting column formats in QC labs, improving throughput and consistency.

Instrumentation Used

• Agilent 1200 SL LC system with binary pump, high-performance autosampler, diode array detector.
• Columns: ZORBAX SB-C18, Eclipse Plus C18, ZORBAX 300SB-C18, TC-C18(2), Poroshell 120 SB-C18, Poroshell 120 EC-C18.

Future Trends and Applications

Emerging developments include ultrahigh-pressure UHPLC separations with sub-2 µm core-shell phases, advanced stationary phases for peptides and small proteins, and method transfer strategies from QC to research settings. Integration with mass spectrometry and automation will further accelerate workflow and data quality.

Conclusion

This investigation demonstrates that combining larger pore sizes (>100 Å) with reduced particle diameters, particularly in core-shell formats, significantly improves insulin separation performance. Analysts can leverage these insights to tailor HPLC methods for enhanced resolution, speed, and regulatory compliance.

References

1. China Pharmacopoeia (2010 edition), Insulin, pp. 845–846.
2. United States Pharmacopeia USP 31, Insulin, pp. 2403–2404.
3. Phu T. Duong, Analysis of Oxidized Insulin Chains Using Reversed Phase Agilent ZORBAX RRHD 300 SB-C18, Agilent Application Note 5990-7988EN.