Molecular charge and nanoparticle zeta potential are core properties of a solute and a solvent, relevant to colloidal stability, chemical modifications and other key qualities.
 

Charge & Zeta Potential

Zeta potential, ζ, is defined as the electric potential arising from a particle’s surface charge and attached counterions. Measurements of ζ are combined with additional information to calculate net molecular charge, Q*.

Commonly associated with the stability of suspensions, zeta potential or net molecular charge are also useful in assessing:

  • the N/P ratio of LNPs or engineered nanoparticles for drug or gene delivery
  • chemical modifications to biomolecules such as polysaccharides or IgG
  • confirming functionalization of theranostic nanoparticles
  • pKa of complex polymers

In addition, zeta potential of extracellular vesicles has been identified as a biophysical marker for cancer (see Selected references).

Both molecular charge and zeta potential are calculated from two primary measurements: electrophoretic mobility µE and hydrodynamic radius Rh. Analysis of molecular charge further requires knowledge of or assumptions regarding the Debye screening length in the solvent.

Particle motion under an applied electric field leads to a Doppler shift in scatter light, detected in an interferometer. Wyatt’s unique FIDELIS technology, implemented in the ZetaStar, utilizes fiber-coupled lasers, acousto-optic modulators and detectors to achieve kHz frequency modulation, overcoming mechanical noise that can affect conventional free-space interferometers.

Zeta potential by ELS

Light scattering determines electrophoretic mobility via electrophoretic light scattering (ELS), and hydrodynamic radius via dynamic light scattering (DLS). Please see Electrophoretic Light Scattering Theory for more details.

Wyatt’s DynaPro ZetaStar ELS/DLS/SLS detector offers simultaneous measurement of µ and Rh, reducing overall measurement time.

In order to be relevant, the charge must be measured in the solvent or formulation buffer of interest. The ZetaStar’s flow cell can be pressurized to suppress bubble formation, enabling measurements in buffers with high ionic strength.

Wyatt’s unique fiber-interferometer Doppler-shift electrophoretic light scattering (FIDELIS) technology, implemented in the ZetaStar, utilizes fiber-coupled lasers, acousto-optic modulators and detectors to overcome mechanical noise that can affect conventional free-space interferometers.

ELS

The isolelectric point of a protein – in this case IgG - can be determined in native formulation or physiological saline using ELS and the ZetaStar. In cases of high salt concentration, the flow cell is pressurized to suppress bubble formation.


Zeta potential by EAF4

Electrical/asymmetric-flow field-flow fractionation is a variant of field-flow fractionation that can determine zeta potential of each component in a mixture, using Wyatt’s Eclipse FFF system with the Mobility EAF4 module. Based on standard asymmetrical-flow field-flow fractionation, EAF4 incorporates an additional electrical field, applied perpendicular to the membrane. Charged particles exhibit a shift in height above the membrane, and a corresponding shift in retention time, that varies with applied field strength, from which the electrophoretic mobility µE may be calculated.

The Mobility module incorporates a conductivity sensor, while hydrodynamic radius may be determined directly from on-line dynamic light scattering, or indirectly based on FFF retention time. See Understanding Field-Flow Fractionation for additional details.

ELS

EAF4 analysis of a mixture of polystyrene latex particles. Hydrodynamic radius is determined from retention time at zero applied field, while electrophoretic mobility, charge and zeta potential are determined from the shift in retention time relative the applied field.


Selected references

Instrumentation for Charge & Zeta Potential

ELS Detector

ZetaStar

DynaPro ZetaStar - The ZetaStar provides walk-up or automated measurements of particle size and concentration, as well as zeta potential, molar mass and turbidity, with just microliters of sample. Parallel dynamic, static and electrophoretic light scattering measurements take place in distinct detection channels that are optimized for each type of measurement.

Measurements can be performed manually using single- or multi-use microcuvettes, the zeta disposable cell or Wyatt’s zeta potential flow cell. Automated measurements are enabled by an autosampler and pump that provide sample to the flow cell.

Conventional ELS detectors have difficulty measuring zeta potential in high salt solutions due to bubble generation during electrolysis. ZetaStar overcomes bubbles by pressurizing the flow cell, enabling relevant zeta potential measurements under physiological conditions or in high-salt formulation buffers.

EAF4 System

ZetaStar

Mobility - The Mobility module, which is an add-on to an Eclipse FFF system, combines an innovative EAF4 channel design with outstanding software control and analysis. The Mobility module incorporates pH and conductivity sensors which are required to analyze electrophoretic mobility and related properties.

Use Mobility to:

  • Understand if all components in the sample have similar surface chemistry
  • Evaluate N/P ratio of drug and gene delivery nanoparticles
  • Assess biopharmaceuticals for chemical and physical degradation

Software

DYNAMICS - Software for electrophoretic, dynamic and static light scattering measurements in the ZetaStar. Calculates

  • size and size distributions
  • electrophoretic mobility, zeta potential, Debye-Hückel-Henry and effective charge
  • molecular weight
  • particle concentration
  • turbidity/opalescence
  • colloidal stability via second virial coefficient (A2 or B22) or diffusion interaction parameter (kD)
  • thermal stability via thermal ramps as temperature of protein melting or aggregation onset (Tm, Tonset, Tagg)
  • kinetics from the evolution of size and molar mass with time

DYNAMICS enables comparison of multiple samples and measurements, even if taken on different days or with different instruments. It supports Wyatt’s DynaPro NanoStar and DynaPro Plate Reader DLS/SLS instruments as well as the ZetaStar and legacy DLS and ELS instruments. DYNAMICS also controls autosamplers and pumps for hands-off analyses of dozens of samples per day.

DYNAMICS is offered with a 21 CFR Part 11 compliant Security Pack option, enabling the use of the ZetaStar, NanoStar and DynaPro Plate Reader in a GMP environment.

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DYNAMICS Touch - measure and analyze DLS, ELS, and SLS data directly on the ZetaStar touch screen with the on-board DYNAMICS Touch app. Presenting a user-friendly and minimalistic interface, DYNAMICS Touch enables walk-up measurements by non-expert users, guiding them through the measurement process and assessing data quality with actionable feedback. Reports can easily be sent to a network location, USB flash drive or cell phone. Naturally, you can import your DYNAMICS Touch data into DYNAMICS for advanced data analysis.

DYNAMICS Touch is also provided with NanoStar, Wyatt’s cuvette-based DLS/SLS instrument.

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