Molar Mass Determination of Collagen Peptides

Significance of the Topic

Collagen peptides, produced by hydrolysis of natural proteins, are integral in dietary supplements and functional foods. Their performance depends on molar mass distribution, making reliable quality control essential for product consistency, safety, and efficacy.

Objectives and Study Overview

This study aimed to develop a cost effective, robust, high precision GPC/SEC method using only UV detection and widely available reference materials. The approach sought accurate molar mass distributions for collagen peptides without requiring advanced detectors, ensuring applicability across laboratories and enabling interlaboratory comparison.

Methodology and Instrumentation

The workflow involved three calibration steps: establishing a universal calibration via viscometry using pullulan and dextran standards; generating a base calibration using UV active CNBr collagen peptides; and applying a broad standard calibration to adjust parameters for true collagen peptide molar masses. A round robin test validated interlaboratory reproducibility.

Used Instrumentation

• Agilent GPC/SEC system with isocratic pump, degasser, column thermostat, autosampler
• Agilent PROTEEMA columns (5 µm, 100 Å, 8 mm × 300 mm) and precolumn
• Detectors: variable wavelength UV at 214 nm, online viscometer, refractive index
• Eluent: phosphate buffer (pH 5.30) with 0.2 M NaCl
• Software: Agilent WinGPC with guided broad calibration wizard

Main Results and Discussion

Universal calibration delivered a polynomial correlation between log([η]·M) and elution volume. Base calibration with CNBr peptides provided UV-based molar mass points. Broad calibration using five well-characterized collagen peptide references yielded a corrected calibration curve, enabling close-to-true molar masses. A four-laboratory round robin showed interlaboratory deviations below 4% from the mean and below 9% from absolute values.

Benefits and Practical Applications

• A cost effective QC method requiring only UV detection
• Robust interlaboratory reproducibility and comparability
• Avoids reliance on advanced detectors and rare standards
• Enables accurate molar mass distribution analysis in routine labs

Future Trends and Opportunities

Advances may include integration of automated calibration workflows, development of new peptide reference materials, extension to other protein hydrolysates, and potential incorporation of light scattering or mass-sensitive detectors for broadened applicability.

Conclusion

The presented broad standard calibration approach provides a simple, reliable, and precise GPC/SEC method for collagen peptide molar mass distribution analysis using only UV detection. It ensures accurate, reproducible results across laboratories without complex instrumentation.

References

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