Standard Operating Procedure for Zeta Potential Determination in Analytical Method Development

Department	Analytical Method Development
SOP No.	SOP/AMD/117/2025
Supersedes	SOP/AMD/117/2022
Page No.	Page 1 of 14
Issue Date	19/05/2025
Effective Date	20/05/2025
Review Date	19/05/2026

1. Purpose

This SOP defines the method for measuring zeta potential using electrophoretic light scattering (ELS) for pharmaceutical dispersions, nanoparticles, and colloidal systems in order to assess their physical stability and surface charge properties.

2. Scope

This procedure applies to the Analytical Method Development (AMD) laboratory for the characterization of formulations, APIs, and nanoformulations requiring evaluation of zeta potential in both research and product development stages.

3. Responsibilities

• Analytical Scientist: Prepares samples, conducts measurements, and analyzes data.
• Instrument Operator: Calibrates equipment and ensures maintenance logs are updated.
• QA Officer: Reviews and verifies the records for GMP compliance.
• Head – AMD: Approves method development and interpretation reports.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring method integrity, accuracy, and traceability in zeta potential determination processes.

5. Procedure

5.1 Instrument Preparation

1. Power on the zeta potential analyzer (e.g., Malvern Zetasizer Nano ZS) and allow full system initialization.
2. Use appropriate cuvette type (disposable or reusable capillary cell).
3. Verify calibration using standard solutions (e.g., 0.1 mM KCl or certified latex particles).
4. Log instrument verification in Annexure-1: Instrument Calibration Log.

5.2 Sample Preparation

1. Disperse 1–10 mg of sample in 1–5 mL of electrolyte solution (1 mM KCl preferred).
2. Use bath sonication (3–5 minutes) to ensure complete dispersion.
3. Avoid air bubbles during transfer into the cuvette.
4. Ensure pH measurement is recorded if pH-dependence is evaluated.
5. Document all sample details in Annexure-2: Sample Preparation Log.

5.3 Measurement Conditions

1. Enter method parameters in software:
   • Temperature: 25 ± 2°C
   • Voltage: 30–150 V (auto-adjust or manual)
   • Dispersant properties: viscosity and refractive index
2. Run triplicate measurements and record average and standard deviation.
3. Accept measurement if:
   • Polydispersity index (PDI) ≤ 0.3 (if DLS measured concurrently)
   • %RSD between replicates ≤ 10%
4. Log results in Annexure-3: Zeta Potential Measurement Record.

5.4 Data Interpretation

1. Evaluate zeta potential results for indication of physical stability:
   • |Zeta Potential| > 30 mV: High stability
   • ±20 to 30 mV: Moderate stability
   • ±10 to 20 mV: Low stability
   • < ±10 mV: Likely aggregation
2. Document interpretation and observations in Annexure-4: Analysis Summary.

5.5 Cleaning and Maintenance

1. Flush cells with water or ethanol depending on solvent used.
2. Inspect for particle residues and clean with lint-free tissue.
3. Log cleaning activity in Annexure-5: Post-Use Cleaning Record.

6. Abbreviations

• ELS: Electrophoretic Light Scattering
• PDI: Polydispersity Index
• RSD: Relative Standard Deviation
• pH: Potential of Hydrogen
• SOP: Standard Operating Procedure

7. Documents

1. Instrument Calibration Log – Annexure-1
2. Sample Preparation Log – Annexure-2
3. Zeta Potential Measurement Record – Annexure-3
4. Analysis Summary – Annexure-4
5. Post-Use Cleaning Record – Annexure-5

8. References

• USP <729> – Globule Size Distribution in Lipid Injectable Emulsions
• ICH Q6A – Specifications for New Drug Substances
• Manufacturer’s User Manual – Malvern Zetasizer

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Instrument Calibration Log

Date	Standard Used	Result	Performed By
18/05/2025	0.1 mM KCl	Within range	Rajesh Kumar

Annexure-2: Sample Preparation Log

Sample ID	Electrolyte	Concentration	pH	Prepared By
ZETA117-A	1 mM KCl	2 mg/mL	6.5	Sunita Reddy

Annexure-3: Zeta Potential Measurement Record

Run	Zeta Potential (mV)	RSD (%)	Comments
1	-35.2	2.5	Stable

Annexure-4: Analysis Summary

The sample showed a zeta potential of -35.2 mV, indicating high colloidal stability and surface charge repulsion. The formulation is considered electrostatically stable.

Annexure-5: Post-Use Cleaning Record

Date	Cuvette Type	Solvent Used	Cleaned By
18/05/2025	Disposable DTS1070	DI Water	Sunita Reddy

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added Annexures and clarified pH and PDI recording requirements	Annual Review