Wyatt Technology

Mobility

Eclipse AF4

 

Tunable, multi-dimensional separation of macromolecules and particles

The Mobility™, in conjunction with an Eclipse® DualTec® or Eclipse AF4 asymmetrical-flow field-flow fractionation (AF4) system, separates macromolecules and particles from 1 nm to 10,000 nm by size and charge.

Multi-dimensional separation using EAF4 and the Mobility provide two unique benefits beyond standard AF4:

  • enhanced separations of particles with the same size but different charge
  • true size / zeta potential distributions

The Mobility is invaluable for studying viral vectors, liposomal or polymeric drug delivery vehicles, colloidal stability of proteins and sub-micron particles, and a host of other macromolecules and colloids.

  • Principal of EAF4 separation

    A new dimension in FFF

    Standard field-flow fractionation using AF4 is a versatile, size-based separation technique that can be tuned to adjust resolution and range. With no packed stationary phase, AF4 is free of shear, and adverse surface interactions are minimal, making it an ideal technology for complex and difficult analytical fractionation tasks.

    The Mobility adds the ability to modulate separation by charge. Depending on the particle’s charge and polarity, its motion under the electric field combines with AF4 cross flow and parabolic channel flow to produce later or earlier elution. Hence analytes with the same size but different charge leave the EAF4 channel and reach the detectors at different times. Each fraction is characterized separately by the detectors.

    Combining electrical fractionation with AF4 in a single separation channel in EAF4, the Mobility is the most powerful and versatile separation technology available for macromolecules and nanoparticles from 1 nm to 10,000 nm.

    What's inside?

     

    EAF4 combines conventional asymmetric-flow field-flow fractionation with an applied electric field to separate particles by size and charge.

     

    • an EAF4 channel incorporating two electrodes with a standard AF4 frit and membrane
    • a conductivity cell and a pH cell
    • an electrode control unit.

    These are used with a standard Eclipse FFF controller and HPLC components from Agilent, Shimadzu or Thermo. Temperature control of the EAF4 channel is provided by a Thermos[Pro] chamber.

    The electrodes, placed above and below the AF4 channel, create a programmable electric field but are never actually touched by sample. These platinum-coated steel electrodes are compatible with a large range of salt concentrations: they do not corrode, and the pressure present in the channel prevents bubble formation under electrolysis.

    Rounding out the Mobility package, the VOYAGER CDS® chromatography data software and SCOUT DPS® data processing and simulation software perform optimization, control and analysis of EAF4 methods, to maximize size and zeta potential characterization.

    Analysis of zeta 
          potential by EAF4

    Exploded view of the Mobility channel

     

  • Multi-dimensional separation

  • Determining zeta potential

    Click to enlarge

    Zeta potential of macromolecules and particles can be determined with EAF4 in two ways:

    • Online detection - A Möbiuζ zeta potential/DLS detector is placed in line with the EAF4 eluent. This method is suitable for homogeneous as well as heterogeneous samples and will produce continuous or discrete size / zeta potential distributions.
    • SCOUT DPS - By running the sample at a series of applied electric fields and measuring the shift of elution time with field, the zeta potential and hydrodynamic radii of particles of discrete sizes (that do not overlap in the fractogram) are determined from EAF4 theory.

     

    Learn more about EAF4 and how it determines zeta potential in:
    C. Johann, S. Elsenberg, H. Schuch, U. Rosch., "Instrument and Method to Determine the Electrophoretic Mobility of Nanoparticles and Proteins by Combining Electrical and Flow Field-Flow Fractionation," Analytical Chemistry 87(8) , 4292-4298 (2015) [DOI: 10.1021%2Fac504712n.

  • Detectors for online analysis

    EAF4 is combined with one or more downstream, online detectors for particle characterization:

    • DAWN® HELEOS® II multi-angle light scattering (MALS) detector with WyattQELS™ embedded dynamic light scattering (DLS) for molar mass, size and shape
    • Optilab® T-rEX™ refractive index detector for universal concentration measurement
    • Möbiuζ® zeta potential and DLS detector for size, charge and zeta potential
    • UV/Vis or fluorescence detectors for concentration and spectral information
    • ICP-MS for elemental composition
    DAWN HELEOS II

    DAWN HELEOS II MALS detector

    Mobius zeta potential detector

    Mobius zeta potential detector

     

  • ECLIPSE softwareVOYAGER CDS® and SCOUT DPS®

    • Simulation and method design of EAF4 experiments
    • Eclipse fluidics control
    • Mobility field control & data acquisition of pH and conductivity
    • Agilent HPLC module control and data acquisition
    • MALS, DLS, UV & RI data acquisition and analysis of molar mass, size, shape and concentration via ASTRA®
    • distributions of hydrodynamic radius and zeta potential based on FFF and EAF4 theory

    ASTRA, VOYAGER CDS and SCOUT DPS are launched and integrated via the VISION CSH® Chromatography Software Hub

   

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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