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Light scattering determines branching in polymers and other measures of sub-micron shape or structure, entirely in solution.

Conformation

Smaller than the wavelength of visible light, it is notoriously difficult to determine the shape, structure or conformation of macromolecules and nanoparticles. While imaging techniques such as AFM and cryoTEM can in some cases provide detailed information, they require significant alterations to native solution conditions with deposition on a surface, and measure a very small, often unrepresentative ensemble. Light scattering offers non-invasive determination of conformation, in solution, over large and representative ensembles.

The use of triple-detection GPC combining multi-angle static light scattering (MALS), differential viscometry (dVI) or dynamic light scattering (DLS), and differential refractometry (dRI), independently or in combination, provides quantitative analysis of:

  • Branching ratio in polymers and dendrites
  • The shape of protein aggregates or amyloid fibrils
  • Empty or filled carrier nanoparticles

across a broad size range.

Branching ratio

Conformation

Important properties of synthetic polymers such as stiffness, viscosity and solubility are governed by the branching ratio g. As outlined in the white paper 'Branching Revealed: Characterizing Molecular Structure in Synthetic and Natural Polymers by Multi-Angle Light Scattering' by Dr. Stepan Podzimek, the branching ratio is most reliably determined from the slope v of the log-log plot of the rms radius rg vs molar mass Mw. Since MALS determines both parameters - simultaneously and independently - downstream of a size-based separation technique such as SEC-MALS or FFF-MALS, it is the ideal technique for this purpose, as long as molecules are larger than about 10 nm in radius.

Branched Polymer

 

For smaller molecules, the hydrodynamic radius Rh as determined by differential viscometry or online DLS may be substituted for rg. The addition of a ViscoStar II differential viscometer to a MALS system introduces the parallel capability for dVI analysis to determine branching and the Mark-Houwink parameter.

Shape factor

For macromolecular assemblies besides branched polymers, the log-log plot of rg vs. Mw is indicative of shape. Increasing values of the slope v progress from spherical, to random coil, to linear conformations of polymers and aggregates. For example, amorphous protein aggregates are distinguished from amyloid fibrils by the value of v, which is in the range of spherical/random coil for amorphous aggregates but much closer to the value expected for a linear arrangement in the case of amyloid fibrils.

Similar information is obtained from the shape factor, ρ = rg/Rh, for each macromolecular size fraction. The theoretical shape factor of ellipsoids, often taken to represent macromolecules and their assemblies or aggregates, is well known as a function of semi-axis ratio. Hence MALS with simultaneous, integrated DLS by means of the WyattQELS module is an independent and orthogonal method to confirm the shape of an assembly or nanoparticle.

When the analyte is known to have a particular shape, the shape factor provides structural information. For example, since liposomes are always spherical, the shape factor ρ clearly differentiates between empty liposomes and those filled with a substance such as a drug compound.

Conformation of Liposomes

ρ = ~1.00

ρ = ~0.77

Selected references

  Ahmad, I. A. H.; Striegel, A. M. Influence of second virial coefficient and persistence length on dilute solution polymer conformation. Anal. Bioanal. Chem.  2011, 399, 1515-1521.

  Peng, Y.; Zhang, L. Characterization of a polysaccharide-protein complex from Ganoderma tsugae mycelium by size-exclusion chromatography combined with laser light scattering. J. Biochem. Bioph. Meth.  2003, 56, 243-252.

  Striegel, A. M. Architecture on the mechanochemical degradation of macromolecules. J. Biochem. Bioph. Meth.  2003, 56, 117-139.

  Tarazona, M. P; Saiz, E. Combination of SEC/MALS experimental procedures and theoretical analysis for studying the solution properties of macromolecules. J. Biochem. Bioph. Meth.  2003, 56, 95-116.

  Zhang, C.; Zhou, Y.; Liu, Q.; Li, S.; Perrier, S.; Zhao, Y. Facile synthesis of hyperbranched and star-shaped polymers by RAFT polymerization based on a polymerizable trithiocarbonate. Macromolecules  2011, 44, 2034-2049.

 BIBLIOGRAPHIC SEARCH

Application notes

Instrumentation for Conformation Analyses

  • MALS Detectors

    dawn-heleos-II

    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


    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.

  • Dynamic Light Scattering Detectors

    DynaPro NanoStar

    WyattQELS - A dynamic light scattering (DLS) module which integrates into a DAWN or miniDAWN MALS detector to provide simultaneous DLS measurements in the same scattering volume.

    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 which may be used for rapid assessment of conformation. The NanoStar does double duty as an online DLS detector by installing its optical collection fiber into a Wyatt MALS detector.

  • Field-Flow Fractionation Systems

    eclipse-dualtec

    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.

  • Refractive Index Detectors

    optilab-ut-rex

    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.

  • Accessories

    WyattCOMET Orbit, accessory

    Orbit - The Orbit helps conserve mobile phase by programmatically directing the solvent to either a waste bottle or back to the solvent reservoir.

    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.

    microCUVETTE - Allows static and/or dynamic light scattering measurements to be made in a quartz cuvette in a DAWN, miniDAWN or µDAWN, requiring only 10 µL.

    NanoFilter Kit - Makes it easy to filter, recover and refilter precious sample. This is especially useful when your total sample volumes are below 300 µL.

    RI Calibration Kit - Pre-made RI calibration standards are available to speed up RI calibration. 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.

  • Software

    ASTRA 6, software ASTRA - Our comprehensive software solution for MALS analysis in chromatography, AF4 or batch mode, included with each MALS, RI and viscosity 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 instruments, as well as molar mass and A2 in the NanoStar. 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).

   

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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