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

Size exclusion chromatography with multi-angle static light scattering (SEC-MALS) is used to measure masses of macromolecules.  Size-exclusion chromatography (SEC) separates molecules based on hydrodynamic volume, but is dependent on similarity to a set of reference standards for accurate mass determination and fails for elongated or sticky proteins.  MALS uses the intensity and the angular dependence of the scattered light to measure absolute molar mass and size of the molecules (root mean square radius, rg) in solution.  Combining SEC and MALS in an SEC-MALS experiment allows for more accurate mass measurements that either method alone.  An inline Quasi-Elastic Light Scattering (QELS), also called a Dynamic Light Scattering (DLS) detector, enables measurement of a hydrodynamic radius.

 

For information on access fees, policies and getting started at the CMI, see the CMI Access Page.

SEC-MALS System Components

MALS

Chromatography

  • Agilent 1260 Infinity Isocratic Liquid Chromatography System
  • Agilent 1260 Infinity Autosampler
  • inline solvent degasser
  • variable wavelength UV detector

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

SEC-MALS data - BSA, Agilent AdvanceBio 300 column

Column: Agilent AdvanceBio 300Å, 7.8 mm x 300 mm

  • Resolution: 5KDa – 1.25 MDa (17 KDa - 667 KDa for MALS)
  • pH range: 2-8.5
  • Max pressure: 150 bar

Experimental conditions

  • Buffer:  25 mM HEPES pH 7.5, 150 mM NaCl, 0.02% NaN3
  • Sample: 100 µl BSA, 2mg/ml (Pierce 23209)
  • Flowrate: 0.5 ml/min
  • Runtime: 35 min
  • Pressure: 67 bar

SEC-MALS Data -BSA

Trained users who have not used ASTRA 7, should talk to Kelly before using the SEC-MALS system.  We are demoing a new module available in the ASTRA 7 that incorporates HPLC control.  Users will need to be trained on the new software before collecting data.

CMI SEC-MALS Getting Started Guidelines and Standard Protocol with ASTRA 7 and HPLC control 

CMI SEC-MALS Getting Started Guidelines and Standard Protocol (old software without HPLC control)

SEC-MALS Technology page from Wyatt Technologies.

  • purified protein samples (must be filtered before loading)
  • running buffer (1L)
  • analytical SEC column (the CMI usually has 1-2 working SEC-MALS columns that users may borrow, if their samples are purified by SEC and in an appropriate buffer)

Samples

maximum injection volume: 100 µl

minimum injection volume: 5 µl

 

typical protein amount: 50-500 µg

typical protein volumes: 20-120 µl

(add 10+ µl for autosampler dead volume)

  • The limiting factor in SEC-MALS analysis the light scattering of the molecules being tested.  The amount of sample needed depends on the size of the molecule, as large molecules scatter more light than small molecules.
    • 100 µg of BSA (67 KDa) gives a very good light scattering signal
  • samples should be prepared in running buffer to minimize the RI peak due to sample solvent (this is particularly important for samples running near the solvent peak)
    • it's good practice to run a standard size-exclusion chromatography purification just before analytical SEC-MALS to buffer exchange and ensure protein is well-behaved.  This is a requirement if you are using a shared CMI column (don't put junk on the column!)
  • samples must be filtered or centrifuged prior to injection
  • Know the concentration (in mg/ml) of your protein
  • Know the UV extinction coefficient in mL/mg•cm

 

Running Buffers

  • most standard aqueous SEC running buffers should be fine
    • some buffer components (eg. glycerol) will require customization of the solvent profile (especially if measuring dynamic light scattering)
    • buffer should be chosen to ensure that the sample is well-behaved
  • Make sure you know the buffer compatibility of the SEC column you are using
    • recommended: 25 mM HEPES pH 7-7.5, 150 mM NaCl, 0.02% NaN3 (filtered)
    • The CMI Tosoh G4SWxl and G2SWxl columns are stable at pH 2.5-7.5 (and MAY NOT be run at pH above 7.5)
    • The CMI Agilent AdvanceBio 300 column is stable at pH 2.5-8.5