Precision SEC analysis of HSAs with BeSEC

The most abundant protein present in plasma is human serum albumin (HSA), which plays an important role in transporting a wide range of endogenous and exogenous substances and in maintaining colloid osmotic pressure.

HSA is used clinically to treat hypoalbuminemia in nephrotic syndrome or liver cirrhosis and restore blood volume in cases of burns or shock. It also serves as a drug carrier due to its strong binding capacity.

In research settings, HSA is often used as a standard due to its high stability and purity, and it supports cell growth as a key component of culture media.

Beyond biomedical applications, HSA is also incorporated into skincare formulations to strengthen the skin barrier and improve moisture retention. In specialized foods, it provides high-quality protein and contributes to stability and emulsification.

Experimental section

This study used a Size Exclusion Chromatography (SEC) system equipped with light-scattering (LS) and refractive index (RI) detectors.

The LS detector used was the BeSEC LS2 from Bettersize Instruments with detection angles of 90 ° and 7 °.

The BeSEC workstation integrates light scattering with UV or RI signals to calculate molecular weight distributions and averages, including Mn, Mw, and Mz.

System configuration:

• Detectors: Refractive Index (RI) and Light Scattering (LS)
• Column: Shodex PROTEIN LW-803
• Mobile phase: Phosphate-buffered saline (PBS)
• Flow rate: 0.7 mL/min
• Injection volume: 100 μL
• Column temperature: 40 °C
• dn/dc: 0.185 mL/g

Sample preparation:

Two HSA samples (A and B) were weighed and dissolved in PBS to a concentration of 2–5 mg/mL.

The solutions were stirred until transparent, then filtered through 0.22 μm PES syringe filters into vials and loaded into the autosampler.

Results and discussion

Figure 1. Elution profiles of the multi-detector signals for Sample A. Image Credit: Bettersize Instruments 

Figure 2. Elution profile of the molecular weight for Sample A. Image Credit: Bettersize Instruments 

Figure 1 demonstrates the elution profile of HSA after it passed through the SEC column. Many distinct peaks are observed, indicating that in PBS HSA exists in multiple aggregated states.

After baseline correction and integration, the RI and light-scattering signal ranges were defined, and the molecular weight of each peak was calculated. Table 1 summarizes these values.

Figure 2 gives the molecular weight profile derived from the full signal integration. As expected, when the elution volume increased, the molecular weight decreased gradually, consistent with the principle of SEC separation.

At each peak, the molecular weight remained constant, forming a flat plateau. This behavior reflects the narrow molecular-weight distribution of each oligomeric state of the protein.

Figure 3. Elution profiles of the multi-detector signals for Sample B. Image Credit: Bettersize Instruments 

Figure 4. Elution profile of the molecular weight for Sample B. Image Credit: Bettersize Instruments 

Table 1. Molecular weight results for peaks in Sample A and B. Source: Bettersize Instruments

Table 1 shows that Peak 4, which elutes last, has a molecular weight of approximately 68 kDa, consistent with the theoretical value for the HSA monomer. Peaks 1 through 3 scale as simple multiples of Peak 4, which is accordant with the dimer, trimer, and tetramer species.

Peak area analysis indicates that the monomer accounts for approximately 77 % of the total protein in both HSA samples, while a substantial portion is also present in aggregated forms.

Conclusion

This article demonstrates how the BeSEC LS2 with light scattering can be used to accurately determine the molecular weight of HSA samples. The results confirm that static light scattering can precisely provide molecular weight values for each elution peak across the entire chromatogram.

These measurements enable precise identification of aggregation states, distinguishing monomeric HSA from higher-order oligomers.

Acknowledgments

Produced using materials originally created by Zhibin Guo, from Bettersize Instruments 

About Bettersize Instruments

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