Skip to main content

Meet Our Expert: Tony Person

“At the forefront of our mission, we are harnessing the power of cutting-edge SEC-MALS (Size Exclusion Chromatography with Multi-Angle Light Scattering) analysis to propel us into a position of leadership within the protein product industry. Our unwavering commitment to excellence drives us to continuously refine and expand our protein product portfolio, ensuring that we not only meet but exceed the highest industry standards.”

-Anthony Person Ph.D.
Vice President, Business Unit Leader- Proteins & Enzymes

Tony Person Headshot


From clinical trials to basic science, recombinant proteins are critical for advancing your research. Bio-Techne understands that for researchers to maximize their success, our recombinant proteins must display rigorous quality control and consistency to provide you with a superior product you can trust. Beyond ensuring your recombinant proteins have high levels of biological activity across lots, we also utilize cutting-edge protein characterization techniques.

Protein characterization should have a minimal set of tests which include the following: purity, identity (molecular weight, MW), oligomeric state (monomer, dimer, trimer…) and homogeneity (aggregation state)1,2. SEC-MALS is an established method for precise and reliable quantification of protein molecular weight that can perform the above metrics and characterize proteins even further.

What is SEC-MALS?

SEC-MALS is a technique that is best described as the sum of its parts. The first part of the technique consists of Size Exclusion Chromatography – High Performance Liquid Chromatography (SEC-HPLC), which allows sample fractionation via a column. The second part of the technique involves a Multi-Angle Light Scattering (MALS) detector, which determines the MW of separated fractions by using well-defined equations3,5 and concentration detectors (UV/RI).

Graphic representation of a typical SEC-MALS set up included SEC column, UV detector, MALS detector, dRI detector and computer for analysis.

Figure 1: Typical SEC-MALS Set Up. A SEC-MALS configuration typically involves having a HPLC/FPLC system with a SEC column, a UV detector, a MALS detector and a dRI detector. A sample is injected into the system and flows through the attached SEC column where the sample is separated. Once eluted from the column, the sample enters the UV detector, through the MALS detector and ultimately through the dRI detector. By simultaneously measuring the light scattering and the concentration of the molecule(s) as they pass through the detectors, the molar mass of the molecule(s) can be determined.4

SEC-MALS journey of a monomer, dimer and trimer

Graphical explanation of how proteins are processed through SEC-MALS at 6 different stages.

Figure 2: 1. A protein sample is run through chromatography to separate monomers, dimers and trimers by their hydrodynamic volume. 2. The molecules then pass through the UV detector, which provides composition data based on the molecules absorbance level to UV light. 3. Traveling through the MALS detector, molecules are hit with a laser. There are at least three detectors positioned to measure the light scattering of the laser off the sample to determine molecular weight. 4. The sample passes through the differential refractive index (dRI) detector, which measures the change in refractive index of the sample solution to a blank solvent to determine concentration. 5. The output of these detectors is summarized in a chromatograph. 6. Analysis of the chromatograph can show the separation of the monomers, dimers and trimers over time, as well as their absorbance and molar mass concentration.

Principles Behind SEC-MALS

First, the principle of SEC-HPLC is based on the separation of molecules through a stationary phase (column) based on the molecules hydrodynamic volume (size) and shape – not MW.

Second, the principle of MALS involves a laser beam of polarized light that is focused onto the sample molecule and the scattered light is detected at multiple angles (at least x3 angles are required). By utilizing the MALS detector along with two concentration detectors, the molar mass and weight-fraction of a modifier can be determined.

Highlighting peaks from a typical SEC-MALS profile

Image A shows output of a graph from SEC-MALS analysis, including peaks for BSA protein and other aggregates. Image B shows a zoomed in view of Image A.

Figure 3: Understanding SEC-MALS analysis. (A) Example of a graph showing BSA run through SEC-MALS. (B) Zoom in of graph in A showing the separation of the monomer-dimer-trimer species of BSA using SEC-MALS.

There are a variety of methods available to characterize the molecular mass of proteins, which include the following: SEC-HPLC, SDS-PAGE, Native PAGE, Mass Spectrometry (MS), Analytical Ultracentrifugation (AUC), Capillary Electrophoresis (CE) and Light scattering (LS). Each of the above methods have pros and cons that go with them when determining molecular mass.5

SEC-MALS is a common method for characterizing the molecular mass of macromolecules; however, there are limitations that need to be considered (as described in the table below).

  Advantage Limitations

1. Easy, well established non-destructive method to measure masses of proteins based on hydrodynamic volumes and sizes.

2. Resolve different protein species: monomers/dimers/oligomers/fragments

1. Need to use known calibration standard(s).

2. Assumes sample of interest has similar conformation (globular) as calibration standards. Not fit to measure elongated proteins.

3. Assume sample does not interact with column. Not fit to measure “sticky” proteins.

Light Scattering SLS (MALS) DLS (QELS) 

1. Assessing homogeneity of sample using non-destructive measurements. Light scattering is able to detect trace amounts of aggregates.

2. Column-Free measurements possible.

1. Low resolution method that can’t separate molecules that are closely related (monomer/dimer).

2. Large aggregates, even a small amount, may affect measurement.


1. Provides absolute molar mass independent of retention time, calibration standards, protein conformation and column interactions.

2. Non-destructive measurement where no protein modification is needed.

3. Protein conjugate analysis: [Glycosylation, Antibody-Drug conjugate (ADC)…]

4. Determination of the stoichiometry of protein/protein complexes.

1. Identically sized molecules will not be separated.

2. Should only be used with highly pure samples containing well-resolved peaks to obtain accurate measurements.

3.  Large complexes that may be susceptible to dissociation by dilution and/or shear forces during chromatographic separation, SEC-MALS may underestimate MW.

4.  SEC-MALS typically requires extensive equilibration to obtain a baseline signal.

At Bio-Techne, we apply SEC-MALS to a variety of applications

Proteins Analyzed by SEC-MALS

SEC-MALS graph of recombinant human TNF-alpha protein determined to be a homotrimer
SEC-MALS Data Result
Retention Time 18.5-19.0 min
MW-Predicted (Monomer) 17.0 kDa
MW-MALS 53.1 kDa
Polydispersity 1.000
System Suitability: BSA Monomer 66.4 ± 3.32 kDa Pass

Recombinant Human TNF-alpha (Catalog # 210-TA) has a molecular weight (MW) of 53.1 kDa as analyzed by SEC-MALS, suggesting that this protein is a homotrimer.  MW may differ from predicted MW due to post-translational modifications (PTMs) present (i.e. Glycosylation).

SEC-MALS graph of recombinant human IFN-gamma protein determined to be a homodimer
SEC-MALS Data Result
Retention Time 18.3-18.8 min
MW-Predicted (Monomer) 16.9 kDa
MW-MALS 34.9 kDa
Polydispersity 1.001
System Suitability: BSA Monomer 66.4 ± 3.32 kDa Pass

Recombinant Human IFN-gamma (Catalog # 285-IF) has a molecular weight (MW) of 34.9 kDa as analyzed by SEC-MALS, suggesting that this protein is a homodimer.  MW may differ from predicted MW due to post-translational modifications (PTMs) present (i.e. Glycosylation).

SEC-MALS graph of recombinant human FGF-basic protein determined to be a monomer.
SEC-MALS Data Result
Retention Time 16.0-16.6 min
MW-Predicted (Monomer) 16.0 kDa
MW-MALS 17.2 kDa
Polydispersity 1.001
System Suitability: BSA Monomer 66.4 ± 3.32 kDa Pass

Recombinant human FGF basic/FGF2/bFGF, 145 aa TC Grade (Catalog # 4114-TC) has a molecular weight (MW) of 17.2 kDa as analyzed by SEC-MALS, suggesting that this protein is a monomer.  MW may differ from predicted MW due to post-translational modifications (PTMs) present (i.e. Glycosylation).