Wyatt Technology

Multi-Angle static Light Scattering (MALS) measures molar mass directly, in solution. Combined with a fractionation technique like SEC or FFF, MALS determines absolute molar mass distributions - independent of elution time.

Molar Mass

While there are many ways to determine the molar masses, or molecular weights, of macromolecules such as proteins, biopolymers and synthetic polymers, only one determines absolute molecular weights in solution, rapidly and effectively: Multi-Angle static Light Scattering (MALS). MALS combines the precise measurements of light scattering intensity and concentrations for a rigorous, first-principles calculation of solution-property molecular weights—making no assumptions of conformation or shape, and no perturbations to the sample. The theory of multi-angle light scattering for the determination of molecular masses is described in detail in Classical Light Scattering Theory.

BSA and Hemoglobin Molar Mass

While column calibration would suggest that hemoglobin is much smaller than BSA, MALS shows that they do in fact have similar molar masses. The polydispersity of hemoglobin, seen here as a trailing shoulder on the peak with decreasing molecular weight, is due to dynamic equilibrium between monomers, dimers and tetramers.

On-line (fractionated) MALS

MALS detectors are most commonly integrated with a separation technique such as size exclusion chromatography (SEC-MALS) or field-flow fractionation (FFF-MALS). Incorporation of independent, absolute MALS analysis eliminates reliance on unreliable column calibration, retention time and models of hydrodynamic size vs. molar mass in order to provide accurate molar mass distributions across the chromatogram or fractogram.

Important Applications of SEC-MALS and FFF-MALS

  • Molar mass distributions of heterogeneous samples such as carbohydrates or synthetic polymers
  • Accurate aggregation and oligomeric states of proteins, peptides and virus-like particles (VLPs)
  • Stoichiometry of tightly bound protein heterocomplexes

See the SEC-MALS and FFF-MALS pages to learn how these versatile techniques combine multiple detectors, including the DAWN HELEOS II or miniDAWN TREOS MALS detectors and the Optilab T-rEX refractive index concentration detector, with the ASTRA software in order to extract a wealth of information about your samples beyond molar mass.

Batch MALS

laser scattering from dextran in vials

Even if samples are not compatible with a separation technique, MALS can still be used to perform analysis of molar mass. Analysis of unfractionated samples yields the weight-average molecular weight Mw of the solution, as well as the second virial coefficient A2 (a.k.a. B22), a measure of solute-solute interactions.

These unfractionated, or 'batch', measurements are typically carried out via direct injection into a MALS flow cell. For maximum accuracy the measurement is carried out over several concentrations. Historically, batch measurements have been laborious and prone to operator error; the Calypso II automates batch MALS for accurate, reliable and rapid results.

When use of a flow cell is undesirable, Wyatt offers a microCUVETTE Adapter Kit which replaces the flow cell of a DAWN or miniDAWN with a high-quality, low-volume microCUVETTE, or a Batch Conversion Kit for disposable scintillation vials. Alternatively, the DynaPro NanoStar offers true static light scattering (SLS) analysis of molar mass for molecules < 10 nm in radius.

Batch MALS is also useful as an orthogonal technique to validate SEC-MALS results against potential loss of sample on the column as well as dissociation of aggregates due to shear or dilution.

Molar Mass via Dynamic Light Scattering (DLS)

Strictly speaking, DLS measures the diffusion constant of particles in solution and cannot determine molar mass directly and rigorously. DLS analysis estimates molar mass in the DYNAMICS software by converting the diffusion constant to an effective size and invoking models that assume relationships between size and mass. See DLS Theory for more details.

While less rigorous than MALS for precise molar masses, DLS is effective as a rapid, low-volume screen for aggregation and other shifts in solution properties. Sample volumes as small as 1.25 µL may be analyzed in the DynaPro NanoStar, while the DynaPro Plate Reader II offers high-throughput measurements in industry-standard microwell plates.

Molar Mass via Intrinsic Viscosity

Intrinsic viscosity and 'Universal Calibration' are commonly used to determine molecular weights of synthetic polymers. Though these techniques are less rigorous than MALS, they may be the optimal means of characterizing materials not amenable to MALS analysis:

  • When the solvent and solute have the same refractive indices (making dn/dc=0), the polymer does not scatter light
  • When the polymers fluoresce under excitation by the MALS laser, the fluorescence greatly increases the apparent scattering signal producing erroneous molecular weights. While a fluorescence blocking filter may be incorporated into the DAWN HELEOS II, this does not always provide a sufficient solution to strongly fluorescing polymers.

The ViscoStar III differential viscometer coupled with an Optilab T-rEX differential refractometer to any GPC system provides superior measurements of intrinsic viscosity for characterization of all types of polymers. Estimates of molar masses based on intrinsic viscosity and the Mark-Houwink equation η=KMα rely on empirical values of K and α established in the literature, while Universal Calibration is valid for all random coil polymers assuming no non-ideal column interactions.

The ASTRA software not only analyzes polymers based on the Mark-Houwink equation and Universal Calibration technique, but may also be used to establish Mark-Houwink coefficients using light scattering to determine molar masses ab initio.

Selected references

  Folta-Stogniew, E. J. Macromolecular Interactions: Light Scattering. In Encyclopedia of Life Sciences; Wiley: 2001.

  Lu, D.; Jimenez, X.; Zhang, H.; Atkins, A.; Brennan, L.; Balderes, P.; Bohlen, P.; Witte, L.; Zhu, Z. Di-diabody: a novel tetravalent bispecific antibody molecule by design. J. Immunol. Methods  2003, 279, 219-232.

  Mogridge, J. Using light scattering to determine the stoichiometry of protein complexes. In Protein-Protein Interactions: Methods in Molecular Biology; Fu, H., Ed.; Humana Press: New York, 2004; Vol. 261, p 113-118.

  Sondermann, H.; Zhao, C.; Bar-Sagi, D. Analysis of Ras:RasGEF interactions by phage display and static multi-angle light scattering. Methods  2005, 37, 197-202.

  Waters, J.; Leiske, D. Characterization of hyaluronic acid with on-line differential viscometry, multi-angle light scattering, and differential refractometry. LCGC  2005, 23, 302-310.

 BIBLIOGRAPHIC SEARCH

Application notes

Instrumentation for Molar Mass

  • MALS Detectors

    dawn-heleos-II, molar mass determination, molar mass measurement, molar mass analysis, molecular weight measurement, molar mass characterization

    DAWN HELEOS II - The most sensitive MALS detector available, anywhere. Incorporates detectors at 18 angles to determine molar masses from 200 Da to 1 GDa and radii from 10 – 500 nm.

    • Heated/cooled option -15°C to +150°C
    • High-temperature option to +210°C

    The HELEOS offers advanced options such as fluorescence or polarization filters and a 785 nm laser.


    miniDAWN TREOS - Second only to the DAWN in sensitivity. Incorporates detectors at 3 angles to determine molar masses from 200 Da to 20 MDa and radii from 10 – 50 nm. Ambient only.

    µDAWN - The first MALS detector for UHPLC, with interdetector dispersion as low as 2 µL, the µDAWN is also optimal for hollow fiber FFF. Incorporates detectors at 3 angles to determine molar masses from 200 Da to 20 MDa and radii from 10 – 50 nm. Ambient only.

  • Refractive Index Detectors

    optilab-t-rex, molar mass determination, molar mass measurement, molar mass analysis, molecular weight measurement, molar mass characterization

    Optilab T-rEX - A unique on-line differential refractometer for measuring concentration of any macromolecule, regardless of chromophores. The high-concentration Optilab accommodates protein concentration up to 180 mg/mL.

    Optilab UT-rEX - The first RI detector specifically designed for use with UHPLC systems. Temperature controlled from 4°C to 50°C, and is compatible with all UHPLC systems.


    Viscometer

    ViscoStar III - A highly sensitive, on-line differential viscometer used in conjunction with SEC-MALS to determine the size and conformation of all types of biopolymers and synthetic polymers. The ViscoStar III may also determine molar masses via Universal Calibration.

  • Field-Flow Fractionation Systems

    eclipse-dualtec, molar mass determination, molar mass measurement, molar mass analysis, molecular weight measurement, molar mass characterization

    Asymmetric Flow Field-Flow Fractionation (AF4) and Hollow-Fiber Flow Field-Flow Fractionation (HF5) perform versatile separation of macromolecules and nanoparticles ranging from 1 nm to > 1000 nm in radius. Combined with MALS and DLS instrumentation, FFF separation is the ideal means for obtaining distributions of molar mass and molecular size, in solution.

    Eclipse DualTec - The complete FFF fluidics control system that integrates with your HPLC pump and autosampler to provide all the advantages of FFF without losing SEC capabilities. Supports switching between any two of AF4, HF5 and SEC.

    Eclipse AF4 - The basic FFF fluidics control system, optimal when switching automatically between SEC and AF4 is not important. The AF4 is required for the fritless channel, and optimal for semi-preparative FFF.

    Eclipse Channels - The Eclipse system offers a wide selection of separation channels and membranes to accommodate a variety of analytical and even semi-preparative tasks.

  • Instruments for Batch Measurements

    DynaPro NanoStar, molar mass determination, molar mass measurement, molar mass analysis, molecular weight measurement, molar mass characterization

    DynaPro NanoStar - With sample volumes as small as 1.25 µL and temperature control spanning -15°C to +150°C, the NanoStar goes above and beyond traditional cuvette-based DLS instruments. It offers an optimized static light scattering detector in parallel to the DLS detection system in order to determine true molar mass.

    Calypso II - Preparative and sample delivery system for carrying out and analyzing automated batch (unfractionated) MALS in a DAWN or miniDAWN.

    microCUVETTE - Allows static and dynamic light scattering measurements to be made simultaneously in a MALS detector using a quartz cuvette, requiring only 10 µL.

    Microbatch Kit - Used to inject samples into the flow cell of a MALS detector, and in determining dn/dc.

  • Accessories

    Wyatt COMET, molar mass determination, molar mass measurement, molar mass analysis, molecular weight measurement, molar mass characterization

    COMET - An ultrasonic transducer that cleans MALS flow cells automatically by agitating the fluid and loosening particles from the glass surfaces. This module may be integrated into a DAWN, miniDAWN or µDAWN.

    Orbit - The Orbit helps conserve mobile phase by programmatically directing the solvent to either a waste bottle or back to the solvent reservoir. In a CG-MALS system, the Orbit is used to automatically choose wash solvents in the post-experiment cleaning cycle.

    SEC Columns - Wyatt offers silica-based columns specifically designed and tested for optimal SEC-MALS measurements of proteins and peptides.

    RI Calibration Kit - This set of refractive index calibration standards can be used to calibrate any Optilab or third-party differential RI instrument.

    Solaris - Prevents growth of bacteria, fungi or algae in solvents with high efficiency and protects your system from down-time due to contamination.

    WISH - An injector module that simplifies manual loading of SEC columns as well as "batch" determination of dn/dc values using an Optilab.

  • Software

    ASTRA 6, ASTRA software, molar mass measurement, molar mass analysis, molar mass characterization

    ASTRA - Our comprehensive software solution for MALS and DLS analysis in chromatography, FFF or batch mode, included with each detector. ASTRA is available in a 21CFR(11) compliant version and offers additional options such as particle analysis and a research database.

    DYNAMICS - Software for batch DLS measurements in the DynaPro and Möbiuζ instruments, as well as molar mass and A2 in the NanoStar and electrophoretic mobility in the Möbiuζ. Calculates size and size distributions, and derives parameters such as the melting temperature TM, the aggregation onset temperature Tonset and the concentration coefficient of diffusion (kD or D1).

    CALYPSO - The CALYPSO software controls the Calypso II composition gradient system and a MALS detector to measure solution weight-average molecular weight as a function of concentration or buffer conditions. These measurements are further analyzed by the CALYPSO software to determine binding constants, reaction rates and other parameters of macromolecular interactions.

    ECLIPSE - Controls the Eclipse DualTec and AF4 Field-Flow Fractionation (FFF) systems. Coupled with ASTRA for data collection and analysis, ECLIPSE provides users with a turn-key solution for macromolecular characterization.

   

For essential macromolecular and nanoparticle characterization—The Solution is Light™

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Wyatt Technology is the recognized leader in light scattering instrumentation and software for determining the absolute molar mass, size, charge and interactions of macromolecules and nanoparticles in solution.

Wyatt's line of multi-angle static light scattering products couple to size exclusion chromatography (SEC-MALS), field-flow fractionation (FFF-MALS), and stop-flow composition-gradient systems (CG-MALS). Our dynamic light scattering (DLS) products operate in traditional cuvette as well as on-line and automated, high-throughput modes. We also offer unique instruments for electrophoretic light scattering (MP-PALS), differential refractometry, and differential viscosity.



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