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Determination of Protein Molecular Weight: 

SEC-MALS vs. Conventional Calibration

The physical characteristics and behavior of proteins in solution vary depending not only on various factors related to the protein’s purity and formulation but also on their inherent properties.

Size Exclusion Chromatography (SEC) is a powerful tool commonly used to examine factors such as protein recovery, molecular weight, and aggregation. 

The principle of SEC involves separating samples as they pass through a permeable and inert column matrix. Smaller molecules pass more deeply into the pores, while larger molecules are excluded, thus passing through the column more quickly. The result is a separation based on hydrodynamic volume, but the ultimate goal is usually to determine molecular weight.

 In the past, the molecular weight was estimated by comparing the elution time of unknown proteins to standard spherical proteins with known molecular weights, a method called ‘Conventional Calibration’. This method was performed using single concentration detectors such as UV.  

< Malvern Viscotek SEC-MALS 20 System > 

However, now it is possible to measure protein molecular weight using both a light scattering detector and a concentration detector (UV or refractive index, RI), regardless of retention time.  This is a very useful technique because most proteins are not spherical, and the measured molecular weight may not be accurate.  By adding additional detectors such as IV (Intrinsic Viscosity) and DLS (Dynamic Light Scattering), the amount of information available from a single SEC measurement significantly increases.

The Malvern Viscotek SEC-MALS 20 System is a light scattering device with 20 detection angles, capable of measuring protein molecular weight regardless of elution volume.  This application note guides you through the separation of various proteins using SEC. 

In addition, after measuring molecular weight using MALS (Multi Angle Light Scattering) or conventional calibration, the differences in the results are discussed.