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. Switch on the zeta potential analyzer (e.g., Malvern Zetasizer Nano ZS) and allow it to stabilize.
2. Verify the cuvette type:
   • Disposable folded capillary cells (DTS1070)
   • Glass cuvettes (for non-aqueous systems)
3. Calibrate instrument as per manufacturer’s instructions with 0.1 mM KCl or latex standard solution.
4. Record in Annexure-1: Instrument Calibration Log.

5.2 Sample Preparation

1. Prepare the formulation/sample at a concentration ensuring obscuration between 5–10%.
2. Use freshly prepared or filtered dispersions to avoid particulate interference.
3. If necessary, dilute with 1 mM electrolyte (e.g., KCl, NaCl) to maintain ionic strength.
4. Avoid bubbles or precipitates; mix gently to ensure uniformity.
5. Document preparation in Annexure-2: Sample Preparation Log.

5.3 Measurement Settings

1. Set parameters:
   • Measurement type: Zeta Potential
   • Medium: Water or selected solvent
   • Temperature: 25°C (unless otherwise justified)
   • Voltage: As per instrument auto-adjust or manual setting (30–150 V)
   • Run duration: 10–20 cycles per sample
2. Load the sample into the cuvette and place in the holder.
3. Start measurement and monitor electrophoretic mobility values.
4. Software will auto-calculate zeta potential via Henry’s equation.
5. Repeat triplicate readings and report average with standard deviation.
6. Record results in Annexure-3: Zeta Potential Data Sheet.

5.4 Data Analysis and Interpretation

1. Evaluate mean zeta potential and standard deviation across triplicates.
2. Acceptable RSD: ≤ 10% across replicates.
3. Typical interpretation:
   • > +30 mV or < -30 mV: High stability
   • ±20–30 mV: Moderate stability
   • ±10–20 mV: Low stability
   • < ±10 mV: Likely to aggregate
4. Include summary in Annexure-4: Interpretation and Observation Sheet.

5.5 Cleaning and Post-Use Protocol

1. Flush cuvettes with deionized water or ethanol (non-aqueous samples) after use.
2. Store reusable cuvettes in dust-free containers.
3. Log cleaning activities in Annexure-5: Post-Use Maintenance Record.

6. Abbreviations

• ELS: Electrophoretic Light Scattering
• RSD: Relative Standard Deviation
• mV: Millivolts
• SOP: Standard Operating Procedure

7. Documents

1. Instrument Calibration Log – Annexure-1
2. Sample Preparation Log – Annexure-2
3. Zeta Potential Data Sheet – Annexure-3
4. Interpretation and Observation Sheet – Annexure-4
5. Post-Use Maintenance Record – Annexure-5

8. References

• USP General Chapter <790>: Zeta Potential Determination
• ICH Q6A – Specifications for New Drug Substances
• Instrument User Manual – Zetasizer Nano Series

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	Expected Value	Measured Value	Status
18/05/2025	Latex standard	-42 mV	-41.8 mV	Pass

Annexure-2: Sample Preparation Log

Sample ID	Formulation	Diluent	Concentration	Prepared By
ZP-117-A	API-Nano Dispersion	1 mM KCl	0.5 mg/mL	Rajesh Kumar

Annexure-3: Zeta Potential Data Sheet

Trial	Zeta Potential (mV)	Remarks
1	-31.2	Stable
2	-30.7	Stable
3	-31.5	Stable

Annexure-4: Interpretation and Observation Sheet

Average Zeta Potential: -31.1 mV; RSD: 1.3%. Indicates good colloidal stability and electrostatic repulsion adequate to prevent aggregation.

Annexure-5: Post-Use Maintenance Record

Date	Cuvette Type	Cleaning Agent	Cleaned By	Verified By
18/05/2025	Disposable (DTS1070)	DI Water	Sunita Reddy	QA

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Expanded interpretation ranges and added robustness in cleaning protocol	Annual Review