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Admission cell 2024-2025

Thin-Layer Chromatography (TLC)

Standard Operating Procedure (SOP) for Thin-Layer Chromatography (TLC)

1. Purpose:

To provide a standardized method for performing Thin-Layer Chromatography (TLC) to separate and analyze components of a mixture.

2. Scope:

This SOP applies to all personnel involved in performing TLC in the laboratory.

3. Responsibility:

  • Laboratory Technicians: Perform TLC according to the SOP.
  • Lab Manager: Ensure the SOP is followed and updated as needed.

4. Equipment and Materials:

  • TLC plates (silica gel, alumina, or other adsorbents)
  • TLC developing chamber
  • Mobile phase (solvent system)
  • Spotting equipment (capillary tubes or micropipettes)
  • Visualizing reagents (if needed)
  • Ruler
  • Pencil
  • UV lamp (if required)
  • Personal Protective Equipment (PPE): Lab coat, gloves, safety goggles

5. Procedure:

5.1 Pre-Operation:

  1. Ensure the TLC plates, developing chamber, and other equipment are clean and dry.
  2. Prepare the mobile phase according to the procedure or protocol.
  3. Wear appropriate PPE before starting the procedure.

5.2 Preparing the TLC Plate:

  1. Draw a light pencil line about 1 cm from the bottom edge of the TLC plate. This will serve as the baseline.
  2. Mark evenly spaced spots along the baseline for sample application.
  3. If needed, activate or pre-treat the TLC plate according to the procedure (e.g., pre-wet the plate or activate it in an oven).

5.3 Spotting the Samples:

  1. Dissolve the sample(s) in an appropriate solvent if not already in solution.
  2. Use a capillary tube or micropipette to apply a small amount of the sample solution to the baseline at the marked spots.
  3. Allow the spots to dry completely before proceeding to the next step.

5.4 Developing the TLC Plate:

  1. Pour the prepared mobile phase into the developing chamber to a depth of about 0.5 cm. Ensure the chamber is well-sealed.
  2. Place the TLC plate into the chamber with the baseline above the solvent level.
  3. Cover the chamber and allow the solvent to rise up the plate by capillary action until it is near the top edge.
  4. Remove the plate from the chamber once the solvent front has reached the desired height.

5.5 Visualizing the Results:

  1. Remove the TLC plate from the developing chamber and immediately mark the solvent front with a pencil.
  2. Allow the plate to dry completely if necessary.
  3. Visualize the spots using a UV lamp if the compounds are UV-active or use other visualization methods (e.g., staining with a reagent) as required.
  4. Measure the distance traveled by each spot from the baseline and the distance traveled by the solvent front.

5.6 Calculating Rf Values:

  1. Calculate the Rf value for each spot using the formula:

 Rf = Distance traveled by the compound / Distance traveled by the solvent front

                       Record the Rf values and compare them to known standards if available.

5.7 Post-Operation:

  1. Clean up the workspace and dispose of any waste according to laboratory protocols.
  2. Store the TLC plates and any remaining solvents or reagents appropriately.
  3. Document the results, including Rf values, visualization observations, and any other relevant information in the lab notebook.

5.8 Maintenance:

  1. Regularly clean the TLC developing chamber and other equipment used in the procedure.
  2. Inspect the TLC plates for any signs of degradation and replace them as needed.
  3. Record maintenance activities in the equipment logbook.

6. Safety Measures:

  1. Always wear appropriate PPE when handling solvents, reagents, and TLC plates.
  2. Handle chemicals and solvents with care, following all safety guidelines.
  3. Work in a well-ventilated area to avoid inhalation of fumes.
  4. Dispose of used solvents and TLC plates according to the laboratory’s waste disposal guidelines.

7. Documentation:

  1. Record the date, time, and purpose of each TLC run in the lab notebook.
  2. Note any deviations from the SOP and report them to the lab manager.
  3. Document the results, including Rf values and visual observations.

8. References:

  • Laboratory Manuals and Protocols for TLC
  • Manufacturer’s Instructions for TLC Plates and Developing Chambers
  • Laboratory Safety Guidelines
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