that are colorless or dissolved in aqueous or organic solvents.

2. Scope

This procedure applies to the Analytical Method Development (AMD) department and covers RI-based techniques for raw material qualification, purity profiling, and concentration measurement in liquid formulations, extracts, and solvents used in pharmaceutical analysis.

3. Responsibilities

• Analytical Chemist: Conducts method development and validation trials using RI instrumentation, records observations, and ensures instrument calibration.
• Reviewer: Reviews data for consistency, verifies calibration records, and ensures traceability.
• QA Officer: Ensures regulatory compliance, proper documentation, and audit readiness.
• Head – AMD: Approves the developed and validated method for routine or regulatory application.

4. Accountability

The Head of Analytical Method Development is accountable for the approval, implementation, and maintenance of accurate RI methods in line with regulatory and pharmacopoeial standards.

5. Procedure

5.1 Principle of Refractive Index

1. Refractive index (n) is the ratio of the speed of light in vacuum to its speed in a given substance.
2. RI varies with concentration, temperature, and wavelength of light; RI measurements can help determine identity or concentration of solutes in solvents.
3. Measurement is performed using an Abbe refractometer or digital refractometer at standard temperature (usually 20°C or 25°C).

5.2 Instrumentation and Calibration

1. Use calibrated digital or analog refractometers suitable for pharmaceutical applications.
2. Calibrate instrument using certified reference standard such as:
   • Distilled water (RI = 1.3330 at 20°C)
   • Sodium chloride or sucrose solution (known RI)
3. Clean prism surfaces thoroughly before and after each use.
4. Document instrument ID and calibration in Annexure-1: Instrument Calibration Log.

5.3 Sample Preparation

1. Use transparent, colorless, particle-free liquids for accurate RI reading.
2. Filter solutions using 0.45 µm membrane if particulate matter is present.
3. Maintain temperature control during sample preparation and analysis.
4. Document sample batch, concentration, solvent system, and preparation method in Annexure-2: Sample Preparation Log.

5.4 RI Measurement Procedure

1. Switch on the refractometer and allow for stabilization as per manufacturer’s instructions.
2. Verify instrument calibration using reference standard.
3. Place 2–3 drops of test sample onto the prism using a clean glass rod or dropper.
4. Close the prism lid gently and allow temperature equilibrium.
5. Record RI value displayed by the instrument. For analog, read from scale or via compensation wheel.
6. Clean the prism using distilled water followed by ethanol and wipe with lint-free tissue.
7. Perform triplicate measurements for each sample and calculate average.
8. Document all readings in Annexure-3: RI Observation Log.

5.5 Calculation

Average RI = (RI₁ + RI₂ + RI₃) / 3

Where RI₁, RI₂, and RI₃ are individual replicate readings.

5.6 Method Optimization

1. Evaluate:
   • Effect of sample temperature (e.g., 20°C vs 25°C)
   • Concentration-dependence for quantitative interpretation
   • Matrix interference (e.g., solvent effect)
2. Develop calibration curve for quantitative analysis using standard solutions (e.g., sucrose, API in solvent).
3. Determine linearity range and optimum concentration window.
4. Document in Annexure-4: Method Optimization Report.

5.7 Method Validation

1. Specificity: Confirm that the sample matrix does not interfere with RI reading.
2. Linearity: Plot RI vs. known concentrations. Acceptable R² ≥ 0.999.
3. Accuracy: Perform recovery study with known standard solution and compare theoretical RI.
4. Precision: Evaluate repeatability across 6 replicates (RSD ≤ 1.0%).
5. Robustness: Check reproducibility under ±2°C temperature variation.
6. Document validation results in Annexure-5: Validation Summary Report.

6. Abbreviations

• SOP: Standard Operating Procedure
• RI: Refractive Index
• RSD: Relative Standard Deviation
• AMD: Analytical Method Development

7. Documents

1. Instrument Calibration Log – Annexure-1
2. Sample Preparation Log – Annexure-2
3. RI Observation Log – Annexure-3
4. Method Optimization Report – Annexure-4
5. Validation Summary Report – Annexure-5

8. References

• USP General Chapter <831> – Refractive Index
• EP 2.2.6 – Refractive Index
• ICH Q2(R1) – Validation of Analytical Procedures
• Pharmacopoeia of India – Refractive Index Sections

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

Instrument ID	Standard Used	Observed RI	Acceptance Range	Status	Analyst
RI-102	Distilled Water	1.3331	1.3329–1.3331	Pass	Sunita Reddy

Annexure-2: Sample Preparation Log

Sample Code	Solvent	Concentration	Temperature	Prepared By
API-SOL-A	Water	1.0% w/v	25°C	Rajesh Kumar

Annexure-3: RI Observation Log

Trial	RI Value	Average RI	Status
1	1.3625	1.3624	Pass
2	1.3623
3	1.3624

Annexure-4: Method Optimization Report

Parameter	Variation	Observation	Conclusion
Temperature	20°C vs 25°C	RI increased slightly	Use 25°C consistently

Annexure-5: Validation Summary Report

Parameter	Criteria	Result	Status
Accuracy	±0.001 RI units	±0.0004	Pass
Precision	RSD ≤ 1.0%	0.38%	Pass
Linearity	R² ≥ 0.999	0.9992	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Expanded method validation and included digital RI instrumentation steps	Annual SOP Review