Size Exclusion Chromatography in the Presence of an Anionic Surfactant

Significance of the Topic

Size exclusion chromatography is essential for non-denaturing analysis of protein size distributions. Introducing anionic surfactants such as SDS alters protein conformation, charge and micelle interactions, impacting retention behavior, mass measurement and aggregate stability. Understanding these effects enhances proteomic profiling, quality control and biopharmaceutical characterization.

Objectives and Study Overview

The study aimed to evaluate the impact of increasing SDS concentration on bovine serum albumin (BSA) monomer, dimer and higher-order aggregates. Key parameters included retention time, apparent molecular weight and hydrodynamic radius. Non-reduced BSA in phosphate buffered saline was analyzed across a gradient of SDS from 0 to 1.4% w/v.

Applied Methodology and Instrumentation

• Column: Agilent AdvanceBio SEC 300 Å, 2.7 µm, 7.8 × 300 mm
• LC system: Agilent 1260 Infinity Bio-inert Quaternary LC
• Detectors: UV absorbance at 280 nm; static light scattering at 15° and 90°; dynamic light scattering for hydrodynamic radius
• Mobile phases: PBS pH 7.4 (10 mM phosphate, 140 mM NaCl) with 0–2.0% w/v SDS
• Flow rate: 0.8 mL/min; stepwise increase of SDS-containing eluent

Main Results and Discussion

Retention times shortened with SDS up to 0.6% and then stabilized, indicating increased hydrodynamic size. Light scattering data showed proportional molecular weight increases due to SDS binding, reaching a steady SDS:BSA ratio of ~2:1 (g/g) above 0.6% SDS. Aggregate peaks persisted, confirming covalent disulfide linkages remain intact without reduction. Dynamic light scattering revealed monomer hydrodynamic radius growth from ~3.8 nm to ~5.7 nm and a background micelle signal around 2.5 nm.

Benefits and Practical Applications

• Quantifies SDS binding and its impact on protein size in SEC
• Assesses aggregate stability and surfactant saturation for biotherapeutic QC
• Identifies resolution limitations in surfactant-containing SEC, guiding method development

Future Trends and Potential Applications

• Extension to diverse surfactant and mixed micelle systems for protein interaction studies
• Coupling SEC with mass spectrometry for detailed proteoform mapping under denaturing gradients
• Integration of multi-detector platforms for comprehensive structural profiling combining SEC, light scattering and spectroscopy

Conclusion

SDS significantly alters protein retention and mass profiles in SEC by forming micelle–protein complexes without dissociating disulfide-linked oligomers. While the Agilent AdvanceBio SEC column tolerates low levels of SDS, surfactant use degrades separation resolution and should be minimized for high-precision analyses.

Reference

• Reynolds J A and Tanford C J Biol Chem 1970 245 5161-5165
• Pitt-Rivers R et al Biochem J 1968 109 825-830
• Aoki K et al Bull Inst Chem Res Kyoto Univ 1969 47 274-282
• Hagarová D et al Chem Papers 1991 45 341-348
• Shirahama K et al J Biochem 1974 75 309-319
• Wen J et al Anal Biochem 1996 240 155-166
• Lorber B et al Biochem Mol Biol Ed 2012 40 372-382
• Valstar A et al Langmuir 2000 16 922-927
• Tanner R E et al J Chem Phys 1982 76 3866-3872