3. Responsibilities

• Production Chemist/Operator: Prepare TLC samples and apply as per method instructions.
• QC Analyst: Prepare and develop TLC plates, interpret and document results.
• QA Officer: Review TLC documentation and ensure compliance with specifications and good documentation practices.

4. Accountability

QC Manager is accountable for ensuring accurate TLC procedure execution. QA Manager is responsible for ensuring documentation compliance.

5. Procedure

5.1 Preparation of TLC Plate

1. Use commercially available pre-coated silica gel TLC plates (e.g., silica gel 60 F254).
2. Cut the plate to required size (e.g., 5 x 10 cm) using a clean glass cutter or scissors.
3. Draw a baseline using pencil 1.5 cm from the bottom edge and mark spots for sample and standard.
4. Ensure plate is handled by the edges only to avoid contamination.

5.2 Sample and Standard Application

1. Prepare dilute solutions of test sample and reference standard in suitable solvent.
2. Apply 2–5 µL of sample/standard on the marked positions using a micropipette or capillary tube.
3. Allow the spots to dry before developing the plate.

5.3 Chamber Preparation and Development

1. Use a clean, dry TLC developing chamber (glass or stainless steel).
2. Add mobile phase solvent mixture as per the method and saturate the chamber with filter paper lining for 15 minutes.
3. Place the TLC plate inside upright without touching the sides and close the lid tightly.
4. Allow solvent to rise till ¾ of the plate height and remove the plate.
5. Mark the solvent front immediately using pencil and dry the plate in a fume hood.

5.4 Detection and Visualization

1. Visualize the plate using UV light (254 nm or 365 nm) or apply detection reagent (e.g., iodine, ninhydrin).
2. Compare spot positions and intensity between sample and standard.
3. Record Rf values (Retention factor) using the formula:
   Rf = Distance traveled by spot / Distance traveled by solvent front

5.5 Interpretation of Results

1. Acceptance criteria:
   • Matching Rf values within ±0.05 range between sample and standard.
   • Absence of unknown/extra spots indicates reaction completion or identity match.
2. Document observation and interpretation in the TLC Result Log (Annexure-1).
3. Affix TLC plate copy or image in the batch record if required.

5.6 Cleaning and Safety

1. Dispose solvents and used TLC plates in designated waste containers.
2. Clean chamber with suitable solvent and store it inverted to dry.
3. Wear gloves and safety goggles during entire procedure.

6. Abbreviations

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

7. Documents

1. TLC Result Log (Annexure-1)
2. Batch Manufacturing Record (if applicable)
3. TLC Method Sheet

8. References

• Pharmacopeia General Chapter on TLC (USP/EP/IP)
• ICH Q2 (R1) – Validation of Analytical Procedures
• 21 CFR Part 211 – cGMP

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 Result Log

Date	Batch No.	Sample Rf	Standard Rf	Observation	Result	Analyst
13/04/2025	API-20250413	0.72	0.73	Single spot, matched	Complies

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
01/01/2022	1.0	Initial SOP Release	Standard Procedure Introduction	QA Head
13/04/2025	2.0	Updated Rf Acceptance Range and Interpretation Criteria	Audit Observation	QA Head