Automated Measurements of Zeta Potential and Size

Significance of the topic

The electrophoretic mobility-derived zeta potential and dynamic light scattering (DLS)–derived hydrodynamic radius are fundamental, rapid indicators of colloidal stability for nanoparticles, polymers, and biotherapeutics. Routine use of these measurements supports formulation development, stability screening, and quality control. However, manual electrophoretic light scattering (ELS) and DLS workflows are often low-throughput, labor-intensive, and susceptible to operator-dependent variability and carryover. Automating sample delivery and measurement acquisition addresses these limitations, enabling standardized, high-throughput characterization without compromising data quality.

Objectives and study overview

This application note demonstrates an automated workflow that combines a DynaPro ZetaStar instrument with a Waters Arc HPLC System to perform unattended, repeated measurements of zeta potential (ζ) and hydrodynamic radius (Rh). The primary objectives were to (1) validate automation for simultaneous DLS and ELS measurements, (2) quantify reproducibility across many injections and (3) assess carryover following repeated analyses of polystyrene latex standards with different sizes and surface chemistries.

Methodology and instrumentation

Samples: four polystyrene latex (PSL) sphere standards with different nominal radii and surface coatings were diluted 1000× in 10 mM phosphate buffer (4 mM Na2HPO4, 6 mM NaH2PO4). For each standard, 24 identical 2 mL vials were prepared and arranged across two ANSI-48 vial plate holders.

Autosampler and flow conditions: 900 µL of sample were injected per run. After each injection the system flushed the flow cell and sample loop with 10 mL of wash buffer at 2 mL·min−1 and then executed seven additional wash measurements to confirm removal of residual sample.

Data acquisition: for each injection, seven replicate measurement cycles were collected. Each zeta potential (ELS) measurement used thirty 1-second acquisitions; each DLS measurement used ten 1-second acquisitions; ELS and DLS were acquired simultaneously. Adaptive collection mode was used for ELS with a medium electric field setting. Electrophoretic mobility was converted to zeta potential using the Henry model with the known ionic strength.

Data handling: the DYNAMICS software controlled ZetaStar experiment execution while HPLC CONNECT coordinated pump and autosampler actions, enabling full automation of injection, measurement, and wash sequences.

Used instrumentation

• DynaPro ZetaStar instrument (DLS + ELS detectors; back-scatter DLS and ELS detector)
• Waters Arc HPLC System: Quaternary Solvent Manager and Sample Manager with two ANSI-48 2 mL vial holders
• Autosampler plumbing with standard PEEK tubing and a 500-psi backpressure regulator downstream of the flow cell
• Software: DYNAMICS (instrument control) and HPLC CONNECT (pump/autosampler orchestration)

Main results and discussion

The automated workflow produced highly reproducible size and zeta potential results across many injections with negligible carryover. Typical repeatability within a single injection (seven replicate measurements) showed relative standard deviations (RSD) <2% for most measurements and never exceeding 5%. Across 24 sequential injections per sample (168 measurements per sample overall), RSD values for hydrodynamic radius remained within 4%.

Example—Sample A: first injection Rh = 51.1 ± 0.5 nm (1% RSD) and ζ = −58.1 ± 1.2 mV (2% RSD). After 93 subsequent injections, the final measured values were Rh = 51.5 ± 1.7 nm (3% RSD) and ζ = −57.5 ± 1.7 mV (3% RSD), demonstrating stability of measured parameters and minimal drift or contamination.

These precision metrics exceed the ISO guideline for zeta-potential methods that specifies a coefficient of variation for mean electrophoretic mobility below 10%. The combined automation and wash strategy effectively prevented carryover, as evidenced by stable baselines and consistent replicate statistics across the full injection sequence.

Measured differences among standards reflected expected influences of surface chemistry and particle size: sulfate-coated PSL samples (A, B) exhibited similar negative zeta potentials, while carboxylate-coated samples (C, D) displayed more distinct ζ values likely attributable to differing particle sizes and surface group densities. These subtleties were resolvable thanks to the high precision afforded by automation.

Benefits and practical applications

• Hands-free, high-throughput combined DLS and ELS measurements reduce operator time and user-to-user variability.
• Simultaneous size and zeta potential acquisition in a single injection improves efficiency and data co-registration.
• Standardized procedures and automated washing increase reproducibility and minimize carryover, supporting compliance with ISO standards.
• Suitable for formulation screening, biopharmaceutical development, QC workflows, and routine stability testing where many conditions or samples must be evaluated.

Future trends and opportunities

Automation of light-scattering characterization opens several avenues for expanded capability: integration with high-throughput formulation libraries and robotic sample prep; inline buffer exchange or dilution modules to extend measurable concentration ranges; automated decision logic to adjust acquisition parameters in real time; coupling results into LIMS for traceability and regulatory workflows; and applying multivariate or machine learning analysis across large datasets to identify formulation-dependent stability trends. Expanding validation across broader particle types, complex matrices, and regulatory contexts will further increase adoption in industry.

Conclusion

Coupling a DynaPro ZetaStar instrument with a Waters Arc HPLC System and coordinating software enables robust, unattended measurements of zeta potential and hydrodynamic radius. The automated workflow demonstrated excellent precision, negligible carryover across many injections, and preserved sensitivity to differences in surface chemistry and particle size. This approach streamlines screening and QC workflows, accelerates formulation development, and supports consistent compliance with relevant ISO standards.

References

1. International Organization for Standardization. Particle size analysis — Dynamic light scattering (DLS). ISO Standard No. 22412:2025.
2. International Organization for Standardization. Colloidal systems — Methods for zeta-potential determination. ISO Standard No. 13099-2:2025.