SOP for Flame Test Method Development in Analytical Method Development Laboratory

Department	Analytical Method Development
SOP No.	SOP/AMD/063/2025
Supersedes	SOP/AMD/063/2022
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 outlines the procedure for developing and validating flame test methods for the qualitative identification of metal ions in pharmaceutical materials by observing characteristic color emissions upon combustion.

2. Scope

This SOP applies to the Analytical Method Development (AMD) department and is intended for the identification of alkali and alkaline earth metals such as sodium, potassium, calcium, lithium, and barium in raw materials and finished drug substances.

3. Responsibilities

• Analytical Chemist: Prepares samples and standards, performs flame tests, and documents observations.
• Reviewer: Ensures accuracy of color observation records and proper documentation.
• QA Officer: Confirms method compliance with GMP and GLP guidelines.
• Head – AMD: Approves the developed method for routine use and regulatory support.

4. Accountability

The Head of AMD is accountable for the approval, integrity, and reliability of all flame test methods used in the identification of inorganic ions in pharmaceutical substances.

5. Procedure

5.1 Principle of Flame Test

1. Flame tests are based on the excitation of electrons in metal ions by heat energy, which release visible light of specific wavelengths upon returning to their ground state.
2. Each element emits a distinct flame color, allowing qualitative identification.
3. The intensity and clarity of the flame color are influenced by:
   • Purity of sample
   • Presence of masking agents or impurities
   • Type of flame used (oxidizing/reducing)

5.2 Apparatus and Reagents

1. Apparatus:
   • Bunsen burner or spirit lamp
   • Platinum or nichrome wire loop
   • Porcelain crucibles or watch glasses
   • UV-protective glasses for accurate flame color observation
2. Reagents:
   • Concentrated hydrochloric acid (for cleaning and preparing volatile salts)
   • Standard solutions of known metal ions (Na⁺, K⁺, Ca²⁺, etc.)
3. Record all materials and instruments in Annexure-1: Apparatus and Reagent Log.

5.3 Preparation of Standard and Sample

1. Prepare 1% (w/v) solutions of reference standards in deionized water or dilute HCl.
2. Dissolve test sample in minimal volume of water or HCl to form a clear solution.
3. Clean wire loop by dipping in HCl and holding in flame until no color is observed.
4. Document all solution details in Annexure-2: Sample and Standard Prep Log.

5.4 Performing the Flame Test

1. Dip cleaned loop into sample or standard solution.
2. Introduce the loop into the hottest part of a non-luminous Bunsen flame.
3. Observe the flame color and compare with known standards:
   • Sodium – Yellow
   • Potassium – Violet
   • Calcium – Brick red
   • Lithium – Crimson red
   • Barium – Pale green
4. For confirmation, use cobalt glass to filter overlapping emissions (e.g., Na in presence of K).
5. Document flame color, duration, and other observations in Annexure-3: Flame Test Observation Log.

5.5 Safety Precautions

1. Use flame-resistant gloves and UV-protective goggles during testing.
2. Handle concentrated HCl under fume hood.
3. Ensure open flame sources are used in flame-proof enclosures where applicable.

5.6 Method Optimization

1. Evaluate optimal sample concentration and solvent system.
2. Compare loop types (platinum vs nichrome) for clarity of emission.
3. Assess interference by excipients and remove using sample purification (e.g., ion-exchange or filtration).
4. Document improvements in Annexure-4: Optimization Report.

5.7 Method Validation

1. Specificity: Confirm that each metal ion produces a distinct color not reproducible by others.
2. Precision: Perform triplicate tests for each ion and evaluate observer agreement.
3. Robustness: Evaluate reproducibility under different flame intensities and loop materials.
4. Limit of Detection (LOD): Determine minimum detectable concentration visually.
5. Summarize findings in Annexure-5: Validation Summary.

6. Abbreviations

• SOP: Standard Operating Procedure
• AMD: Analytical Method Development
• LOD: Limit of Detection
• HCl: Hydrochloric Acid

7. Documents

1. Apparatus and Reagent Log – Annexure-1
2. Sample and Standard Prep Log – Annexure-2
3. Flame Test Observation Log – Annexure-3
4. Optimization Report – Annexure-4
5. Validation Summary – Annexure-5

8. References

• USP General Chapter <197> – Spectroscopic Identification
• IP Appendix – Identification of Ions by Flame Tests
• ICH Q2(R1) – Validation of Analytical Procedures

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Apparatus and Reagent Log

Item	ID/Batch No.	Preparation/Verification Date	Checked By
Bunsen Burner	BB-011	10/05/2025	Ajay Mehra

Annexure-2: Sample and Standard Prep Log

Material	Concentration	Solvent Used	Prepared By
Sodium Standard	1% w/v	Distilled Water	Sunita Reddy

Annexure-3: Flame Test Observation Log

Sample	Observed Flame Color	Reference Color	Status	Observer
Sample A	Yellow	Sodium	Confirmed	Rajesh Kumar

Annexure-4: Optimization Report

Parameter	Tested Variation	Observation	Conclusion
Loop Material	Nichrome vs Platinum	Brighter color with Platinum	Platinum preferred

Annexure-5: Validation Summary

Parameter	Acceptance Criteria	Result	Status
Specificity	Distinct flame color	Confirmed	Pass
Precision	Reproducibility in 3 trials	Consistent	Pass
LOD	0.01% solution	Detected	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Included optimization of loop material and validation section	Annual SOP Update