#  Size-Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS) 

 



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[**CMI SEC-MALS resources and user guide**](/node/1655855#secmals-resources)**⬇︎**

[Size exclusion chromatography with multi-angle static light scattering (SEC-MALS](https://www.wyatt.com/solutions/techniques/sec-mals-molar-mass-size-multi-angle-light-scattering.html)) is used to accurately measure weight-averaged masses (Mw) of macromolecules in solution by measure the intensity of scattered light of a sample as it elutes from an SEC column. SEC-MALS can determine oligomeric state and sample polydispersity.

Size-exclusion chromatography (SEC) separates molecules based on hydrodynamic volume, but is dependent on similarity to a set of reference standards for accurate estimates of mass and fails for elongated or sticky proteins. Multi-Angle Light static Scattering (MALS) is used to measure light scattering intensity accurately, which is proportional to the weight-averaged mass in solution. Combining SEC, MALS and concentration detectors in an SEC-MALS experiment allows for more accurate mass measurements that SEC or MALS alone.

 ![SEC-MALS Sample Data and Mw illustration](/sites/g/files/omnuum4016/files/2025-02/SEC-MALS_data_Mw.jpg)

 

By using two concentration detectors (RI and UV), the molar mass and weight-fraction of a modifier can be determined. This can be used to measure protein and modifier masses of integral membrane proteins in detergent micelles and of glycosylated proteins.

The CMI has a SEC-MALS system with a [Wyatt](https://www.wyatt.com/solutions/techniques/sec-mals-molar-mass-size-multi-angle-light-scattering.html) Dawn Heleos Multi-Angle Light Scattering (MALS, 18-angle) Detector, with in-line DLS detector and Optilab TrEX refractive index detector; [Agilent](https://www.agilent.com) 1260 Infinity Isocratic Liquid Chromatography System with autosampler (5-100 μl) and variable wavelength UV detector.

\*\* There is no fraction collector on this system, as it is intended for analytical and not preparative chromatography.

   ![wyatt helleos image](/sites/g/files/omnuum4016/files/styles/hwp_1_1__360x360_scale/public/cmi/files/wyatt_sec_malsm1581283482itokrjndvwre.jpg?itok=v4OP7VNZ) 

 

**Key Features**

- Measure molar mass and oligomeric state for wide range of particle sizes
    - proteins ~5MDa – 5kDa
- Deconvolute mass contribution from two components
    - Protein and detergent micelle
    - Protein and glycan
    - Protein and DNA/RNA
- Determine hydrodynamic radius (Rh) from DLS data
- RMS radius (Rg) for large particles



 



###    SEC-MALS Column Data  expand\_more  

[Sepax SRT SEC-300 (pH 2-7.5, 5kDa-1.25MDa)](/sites/g/files/omnuum4016/files/cmi/files/sepax_srt-300_new_column_performance_20180612.pdf "sepax_srt-300_new_column_performance_20180612.pdf")

[Sepax SRT SEC-150 (pH 2-7.5, 1kDa-150kDa)](/sites/g/files/omnuum4016/files/cmi/files/sepax_srt-150_new_column_performance_20180613.pdf "sepax_srt-150_new_column_performance_20180613.pdf")

 

 



 

 

 

 

 SEC-MALS Resources SEC-MALS Supplies SEC-MALS Sample Preparation 

## SEC-MALS Resources

Generally, SEC-MALS data collection is done in ASTRA 7 with HPLC control.

[CMI SEC-MALS Getting Started Guide](/sites/g/files/omnuum4016/files/2025-03/CMI%20SEC-MALS%20Getting%20Started%20Guide.pdf "CMI SEC-MALS Getting Started Guide")

[CMI SEC-MALS Guide to Protein Conjugate Analysis](/sites/g/files/omnuum4016/files/2025-03/CMI%20SEC-MALS%20Guide%20to%20Protein%20Conjugate%20Analysis.pdf "CMI SEC-MALS guide to protein conjugate analysis"), guide to measuring dn/dc values and performing protein conjugate data analysis.

[CMI SEC-MALS Guide to Custom Solvents](/sites/g/files/omnuum4016/files/2025-03/CMI%20SEC-MALS%20Guide%20to%20Custom%20Solvents.pdf "CMI SEC-MALS Guide to Custom Solvents"), for solvents that cannot use PBS or water as the solvent model.

[SEC-MALS Technology](http://www.wyatt.com/solutions/techniques/sec-mals-molar-mass-size-multi-angle-light-scattering.html) page from Wyatt Technologies.

 

 

 

## SEC-MALS Supplies

- Sample filters, 0.02-0.2 μm filters (recommended)
- Autosampler vials (provided by the CMI)
- Analytical SEC Column:
    - A range of analytical SEC columns can be used including silica and agarose columns
    - silica columns (e.g. SEPAX SRT SEC-300, 7.8 mm x 300 mm)
        - generally excellent performance
        - low shedding
        - maximum pH of 7.5
    - agarose columns (e.g. Superdex 200 Increase 3.2 x 300mm)
        - more shedding, requires longer equilibration times
        - wide pH range
    - The CMI usually has 2-3 working SEC-MALS columns that users may borrow, if their samples are purified by SEC and in an appropriate buffer (pH ≤ 7.5, salt > 100 mM). No guarantees on performance are offered.
    - Make sure that the column is compatible with your sample. Some detergents and membrane proteins will require special columns designed for hydrophobic samples and buffers.

 

 

 

## SEC-MALS Sample Preparation

**Assay Buffers**

- Running buffer should always be chosen to be compatible with both the SEC column and the protein sample.
- Recommended Buffer: 25 mM HEPES pH 7-7.5, 150 mM NaCl (filtered).
- **Make sure you know the buffer compatibility of the SEC column you are using.**
    - Most ***silica columns will not tolerate pH above 7.5***.
- Some buffer components (e.g. glycerol) will require customization of the solvent profile. Take note of the absolute refractive index of the solvent after equilibration and before data collection.

**Samples**

- The sample should be prepared in running buffer to minimize RI peak due to sample solvent.
    - This is particularly important for samples that run near the solvent peak.
    - It is good practice to run a size-exclusion chromatography purification prior to analytical SEC-MALS to buffer exchange and clear aggregates. **This is a requirement if you are using a shared CMI column.**
- Samples must be filtered or centrifuged prior to injection.
- Concentration should be accurately measured to assess column recovery.
    - Know the concentration (in mg/ml) of your protein.
    - Know the UV extinction coefficient (in ml/mg•cm) of your protein
- Protein aggregates can damage the column.
    - Filter or centrifuge samples before use.
    - Assess protein heterogeneity via dynamic light scattering (in the DynaPro plate reader).
    - Purify protein samples with soluble aggregates by size-exclusion chromatography.
- Recommended protein concentration varies depending on protein mass:
    - Scattering is proportional to mass
        - Larger proteins require less sample than smaller proteins.
    - Typical range 5 – 500 µg/injection
        - BSA (67 KDa) 100 µl at 2 mg/ml always gives a good light scattering signal
        - **mg ~ 10/mass(KDa)**
- Sample volume:
    - Maximum injection volume: 100 µl
    - Minimum injection volume: 5 µl
    - Glass autosampler vial with low volume insert has 10 µl dead volume.
        - Fill vial with at least 110 µl for a 100 µl injection.

 

 

 

 

 

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 See also:- [ Light Scattering ](/cmi-terms/light-scattering)
- [ Static Light Scattering ](/cmi-terms/static-light-scattering)
- [ Multi-Angle Light Scattering ](/cmi-terms/multi-angle-light-scattering)