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.
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.
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.
Application notes
Automated Electrophoretic Mobility Measurement of High Salt Solutions
Automated Measurements of Electrophoretic Mobility
Computation of Protein Net Charge from Electrophoretic Mobility
Discriminating Heparin from Chondroitin Sulfate by Charge:Mass Ratio
Charge and Interaction Analysis for Predicting Antibody Formulation Stability
Selected references
Heida, R. et al. Assessing the Immunomodulatory Effect of Size on the Uptake and Immunogenicity of Influenza- and Hepatitis B Subunit Vaccines In Vitro. Pharmaceuticals 2022, 15(7).
Qu, H.; Tong, S.; Song, K.; Ma, H.; Bao, G.; Pincus, S.; Zhou, W.; O'Connor, C. Controllable in situ synthesis of magnetite coated silica-core water-dispersible hybrid nanomaterials. Langmuir 2013, 29, 10573-10578.
Mendivil-Alvarado, H. et al. Extracellular Vesicles and Their Zeta Potential as Future Markers Associated with Nutrition and Molecular Biomarkers in Breast Cancer. Int. J. Mol. Sci. 2023, 24(7).
Pisani, A.; et al. Chemokine-Decorated Nanoparticles Target Specific Subpopulations of Primary Blood Mononuclear Leukocytes. Nanomaterials 2022, 12(20).
Roberts, D.; Keeling, R.; Tracka, M.; van der Walle, C. F.; Uddin, S.; Warwicker, J.; Curtis, R. The role of electrostatics in protein-protein interactions of a monoclonal antibody. Mol. Pharm. 2014, 11, 2475-2489.
Saito, S.; Hasegawa, J.; Kobayashi, N.; Tomitsuka, T.; Uchiyama, S.; Fukui, K. Effects of ionic strength and sugars on the aggregation propensity of monoclonal antibodies: influence of colloidal and conformational stabilities. Pharm. Res. 2013, 30, 1263-1280.
Seidel, L. et al. Composition, ζ Potential, and Molar Mass Distribution of 20 Must and Wine Colloids from Five Different Cultivars Obtained during Four Consecutive Vintages. J. Agric. Food Chem. 2022.
Instrumentation for Charge & Zeta Potential
ELS Detector
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
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.
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.
