Calibration of UV by Control of Absorbance with Demo image

Calibration of UV-VIS spectrophotometer by Control  of  Absorbance

Control  of  Absorbance

Potassium Dichromate Solution:-

Weigh & transfer a quantity about 30 mg of previously dried Potassium Dichromate ( NIST ) at least 4 hour at 130 C ) to 500 ml volumetric flask. Dissolve & dilute in sufficient 0.005M H2SO4 (136 μL) to produce 500 ml. 

Blank 0.005M H2SO4 solution :-

Add 136 μL of concentrated sulfuric acid ( 98% H2SO4   specific gravity g 1.84) into a 500 ml volumetric flask containing 250 ml of purified water. Mix, cool and dilute to volume.

Go to Photometric mode

  • press Ctr+M
  • Add wavelength 235,257,313 & 350 nm {Select RAW DATA}
  • Make sure that Reference and sample cell holder is empty and press button for baseline correction(230 to 350 nm). 
  • Then keep the Potassium dichromate Solution in the sample cell holder & using 0.005 M H2SO4 solution as a blank 
  • Close the cell compartment 
  • Click Start
  • Calculate value of A (1% 1 cm) for each wavelength and record it.
  • Specific Absorbance = Observed Absorbance/ 0.006

Acceptance criteria:-

Wavelengths (nm)Limit
235122.9 - 126.2
257142.8 - 145.7
31347.0 - 50.3
350105.6 - 108.2

Control  of  Absorbance demo image

If calibration is satisfactory then affix the status label on it with the next due calibration date.


Once in a 3 month


Indian Pharmacopoeia/ British Pharmacopoeia/ US Pharmacopoeia / TGA Appendix D SOP : Operation Of Spectro Photometer

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