Absolute Molecular Weight Determination of HSAs using the BeSEC

Abstract: Human serum albumin (HSA) is widely used in biomedical, pharmaceutical, and industrial applications, where accurate molecular weight determination is essential. In this study, size-exclusion chromatography coupled with static light scattering and refractive index detection was applied to determine the absolute molecular weight and aggregation states of HSA, enabling reliable identification and quantification of monomeric and oligomeric species.

Keywords: Human serum albumin (HSA), Absolute molecular weight, Protein aggregation, Size-exclusion chromatography (SEC), Static Light Scattering, Biopolymer characterization

 Introduction 

Human serum albumin (HSA) is the most abundant protein in plasma, playing a critical role in maintaining colloid osmotic pressure and transporting a wide range of endogenous and exogenous substances. Clinically, HSA is used to restore blood volume in cases of shock or burns, treat hypoalbuminemia in liver cirrhosis or nephrotic syndrome, and serves as a drug carrier due to its strong binding capacity.

In research, HSA is widely employed as a standard due to its high purity and stability, and it supports cell growth as a key component in culture media. Beyond biomedical applications, HSA is incorporated into skincare formulations to enhance moisture retention and strengthen the skin barrier. In specialized foods, it provides high quality protein and contributes to emulsification and stability.

 Experimental Section 

Figure 1. Elution profiles of the multi-detector signals for Sample A

Figure 2. Elution profile of the molecular weight for Sample A

Figure 1 illustrates the elution profile of HSA after passing through the SEC column. Multiple distinct peaks are observed, indicating that HSA exists in several aggregated states in PBS. After baseline correction and defining integration ranges for both RI and light scattering signals, the molecular weight of each peak was calculated. These values are summarized in Table 1.

 

Figure 2 shows the molecular weight profile derived from the full signal integration. As expected, molecular weight decreases gradually with increasing elution volume, consistent with the SEC separation principle. Within each peak, the molecular weight remains 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

 

 

Figure 4. Elution profile of the molecular weight for Sample B

 

Table 1. Molecular weight results for peaks in Sample A and B

Peak	Sample A		Sample B
	Mw (Da)	Area (%)	Mw (Da)	Area (%)
Peak 1	274,459	1.5	271,545	1.7
Peak 2	199,148	4.6	201,826	3.8
Peak 3	138,694	16.5	141,070	14.4
Preak 4	68,516	76.1	68,403	77.3

 

From Table 1, Peak 4, which elutes last, has a molecular weight of approximately 68 kDa, matching the theoretical value of HSA monomer. Peaks 1 through 3 scale as simple multiples of Peak 4, corresponding to the dimer, trimer and tetramer species. Peak area analysis indicates that the monomer accounts for roughly 77% of the total protein in both HSA samples, while a significant fraction exists in aggregated forms.

 

 Conclusion 

 

This application note demonstrates the use of the BeSEC LS2 with light scattering for accurate molecular weight determination of HSA samples. The results confirm that static light scattering provides precise molecular weight values for each elution peak across the entire chromatogram. These measurements enable precise identification of aggregation states, clearly distinguishing monomeric HSA from higher-order oligomers.

 

 

 

About the Authors

Zhibin Guo Application Research Lab, Bettersize Instruments Ltd.

 

BeSEC Advanced Light Scattering Detector   Proteins: Determine molecular weight, oligomer state, and aggregates Polymers & Polysaccharides: Analyze molecular weight distribution and size No column calibration required since Mw is independent of elution volume Low-angle detection enables accurate molecular weight Compatible with any GPC or SEC system   Learn more