Standard Operating Procedure for Testing of Reaction Completion by TLC in API Manufacturing

Department	API Manufacturing
SOP No.	SOP/API/094/2025
Supersedes	SOP/API/094/2022
Page No.	Page 1 of 10
Issue Date	13/04/2025
Effective Date	15/04/2025
Review Date	13/04/2026

1. Purpose

To establish a standardized procedure for the use of Thin Layer Chromatography (TLC) in determining the completion of chemical reactions during the manufacturing of Active Pharmaceutical Ingredients (APIs).

2. Scope

This SOP applies to all production and quality control personnel performing TLC as an in-process control method to evaluate the endpoint of chemical reactions during API synthesis.

3. Responsibilities

• Production Chemist: Collect reaction samples and perform TLC as per method.
• QC Analyst: Confirm TLC results when required and provide training/support for method development.
• QA Officer: Review TLC data and ensure compliance with documentation practices.

4. Accountability

Production Head is accountable for proper sampling and testing during the reaction. QC Head is responsible for method compliance and training. QA Head ensures overall adherence to GMP standards.

5. Procedure

5.1 Preparation

1. Use pre-coated silica gel TLC plates (e.g., silica gel 60 F254).
2. Ensure proper availability of standard/reference compound and suitable solvent system (mobile phase) as defined in the method.
3. Use capillaries or micropipettes for sample spotting.

5.2 Sampling for TLC

1. Draw ~1 mL of the reaction mixture using a clean pipette or glass rod.
2. Quench if necessary (e.g., by adding ice or diluent) to stop the reaction before spotting.
3. Prepare two spots:
   • Standard/reference compound (starting material or desired product)
   • Reaction mixture

5.3 TLC Procedure

1. Mark a baseline (~1 cm from bottom edge of the plate).
2. Spot ~1–2 μL of sample and standard using a capillary tube.
3. Develop the plate in a TLC chamber pre-saturated with mobile phase.
4. Allow solvent front to migrate ~2/3 of the plate.
5. Remove the plate and mark the solvent front immediately.
6. Dry the plate and visualize under UV light (254 nm) or by spraying with iodine, ninhydrin, or appropriate visualizing agent.

5.4 Interpretation

1. Compare the R_f values of the sample and reference spots.
2. Absence of starting material or presence of single spot corresponding to the desired product indicates reaction completion.
3. If starting material is still visible, continue reaction and repeat test after a defined interval (e.g., 30 minutes).

5.5 Documentation

1. Paste the developed TLC plate in the Batch Manufacturing Record (BMR) or retain a photo copy if the plate is used for multiple batches.
2. Record the following in the TLC Observation Log (Annexure-1):
   • Date and Time
   • Batch No.
   • Reaction Stage
   • Solvent System
   • Result (Completed/Incomplete)
   • Initials

5.6 Precautions

1. Use fresh solvents and standard materials.
2. Avoid touching the silica surface with fingers.
3. Ensure uniform spot size for reproducibility.

6. Abbreviations

• TLC: Thin Layer Chromatography
• SOP: Standard Operating Procedure
• QA: Quality Assurance
• QC: Quality Control
• BMR: Batch Manufacturing Record
• Rf: Retention Factor

7. Documents

1. TLC Observation Log (Annexure-1)
2. Batch Manufacturing Record
3. Reaction Monitoring Checklist

8. References

• ICH Q7 – GMP for APIs
• USP General Chapter <1058> Analytical Instrument Qualification
• 21 CFR Part 211 – US FDA GMP

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: TLC Observation Log

Date	Batch No.	Reaction Stage	Solvent System	Result	Performed By	Remarks
13/04/2025	API-20250413	Post-heating	Toluene:Ethyl Acetate (8:2)	Reaction Complete		Confirmed

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
01/01/2022	1.0	Initial Release	New Reaction Monitoring SOP	QA Head
13/04/2025	2.0	Added Visualization Techniques and Logbook Format	Audit Observation	QA Head