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SOP for Calibration of High Performance Thin Layer Chromatography (HPTLC)

To check the performance of High performance thin layer chromatography.

This procedure is applicable to calibrate high performance thin layer chromatography.

Doing : Technical Assistant
Checking : Executive /Manager

Head of the Department

  • Check the performance of the instrument in terms of
  1. Linearity of spotting (Annexure-I)
  2. Reproducibility of spotting (Annexure-II)
  • Prepare the mobile phase as per the method given in Annexure-I.
  • Prepare the solution as mentioned in Annexure-I
  • Use 10 X 10 cm HPTLC plate (Kieselgel 60F254 or Identical)

For Linearity of Spotting
  1. Apply 2 μl, 4μl, 6μl, 8μl and 10 μl of solution on HPTLC plate with spotter.
  2. Allow the plate to run in mobile phase.
  3. Dry the plate with drier.
  4. Scan the plate with scanner.
  5. Check the linearity and corelation coefficient.
  6. Fill in the Data as mentioned in Annexure-I

Reproducibility of Spotting
  • Apply 10μl solution on HPTLC plate for five times in sequence
  • Allow the plate to run in mobile phase.
  • Dry the plate with drier.
  • Scan the plate with scanner.
  • Calculate the RSD for five track ( RSD Limit : N.M.T 3.0%)
  • Fill in the data mentioned in Annexure-II

%= Percentage
RSD= Relative standard deviation
N.M.T = Not more than
N.L.T = Not less than
μl = Microlitter
HPTLC = High performance thin layer chromatography
API= Active pharmaceuticals ingredient


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