Standard operating procedure for operation and calibration of U.V. Spectrophotometer U.V.1800.

Title: Standard operating procedure for operation and calibration of U.V. Spectrophotometer U.V.1800.

1.             Purpose:

To lay down the procedure for the operation and calibration of Spectrophotometer

 UV1800 ( Make : Shimadzu ) used       in the raw materials, intermediates and finished

 products analysis  in the Lab.  

3.         Scope:

This procedure shall be applicable for operation and calibration of Spectrophotometer UV1800 in Lab at Fermenta Biotech Limited.

 

4.         Responsibility: Research Associate/ Research Scientist .

5.0  Procedure :

  5.1    Start up and operation of instrument:

  5.1.1 Before starting the instrument Ensure, there should be no cuvette or the Sillica Bag in the   sample compartment.

    5.1.2 After initialization of Spectrophotometer is completed click O.K. and leave the instrument

             for 15-20 minutes to warm up.

     5.1.3 After warm up time, select the required mode i.e. photometric or spectrum by clicking the icon.

     5.1.4 Fill the Wavelength in the photometric mode and wavelength range in the spectrum mode.

     5.1.5 Click Baseline for Baseline correction in spectrum mode and click Auto zero in Photometric       method.

     5.1.6 Rinse and fill both the cuvettes with blank solution wipe the outer surface with the tissue paper   and place it in respective position in sample compartment.

     5.1.7 Now again click Baseline for Baseline correction in spectrum mode and click Auto Zero for        making the reading zero of Blank in photometric mode.

     5.1.8 Take out the front cuvette rinse and fill the cuvette with Test sample, place the cuvette in the        sample compartment.

     5.1.9 Click the start, in the spectrum mode spectrum will be displayed and in the photometric mode.    Absorbance will be displayed on the screen.

   5.2  Switch off the instrument:

    5.2.1  After Analysis take out cuvettes from sample compartment clean them and keep in cuvette          box.

            

                                    

        5.2.2 Click Return then click mode, a message will appear on screen 'current data not saved do you continue, if you want to exit the application, click O.K, UV probe file will close, and will return to mode menu. 

       5.2.3  Switch off the UV Spectrophotometer.

5.3  Calibration of the UV-Spectrophotometer

5.3.1 Instruments and apparatus: 

 Analytical weighing balance

Volumetric flask

Pipette

5.3.2  Reagents:

            Potassium dichromate (K₂Cr₂0₇).

            Sulphuric acid A.R Grade.

            Potassium chloride.

           Toluene.

            Hexane

           RO water

5.3.3 Calibration Procedure: Transfer accurately weighed about 60 mg K₂Cr₂0₇ previously dried at 120ºC in 1.0 liter volumetric flask. Add distilled water to dissolve, add 0.27 ml of Sulphuric acid and make up to 1.0 liter with RO water.                                                                                                               

5.3.4 Control of Absorbance: Switch on the instrument.

 5.3.4.1 Initialization starts.

5.3.4.2 After completion of initialization choose spectrum mode to check spectrum.

 

5.3.4.3 Go to method and select the wavelength range 400 nm to 230nm then enter.

5.3.3.4  Start base line correction.

 

 5.3.3.5 Fill the both cuvettes with the blank solution. Place the cuvettes in their respective position in      the instruments.

 5.3.3.6 Zero the reading by pressing start baseline correction.

 5.3.3.7 Take out front cuvette and fill with the 0.006 % K₂Cr₂0₇ after rinsing put into respective positions and click start.

  5.3.3.8 Then spectrum will come on the screen then click operation and point peak.

             Fill wavelength 350nm, 313nm, 257nm and 235nm.

  5.3.3.9 Then  absorbance  of  above  said  wavelength  will  come  on  screen  and  take  out the print      and calculate as under.

Calculation:                                  Absorbance value x1000

                                    A (1%, 1 cm)     = -----------------------------------------  

                                         Weight of K₂Cr₂0₇   in gm x100

 

     Wavelength                        A (%, 1cm)                              Maximum tolerance

               235                                124.5                                        122.9   to    126.2         

               257                               144.0                                        142.4   to    145.7        

               313                                48.6                                          47.0    to     50.3         

               350                                106.6                                          104.9   to    108.2

   5.3.5    Limit of stray light:

5.3.5.1  The absorbance of a 1.2% w/v solution of potassium chloride with a path length of 1.0 cm                           should be more than 2.0 at 200nm. When compared with water as reference liquid.

5.3.6     Resolution Power:

5..3.6.1Prepare 0.02% v/v solution of Toluene in Hexane and take the spectrum from 220nm to 300 nm    against water as a blank. Note the absorbance at 269 nm and 266nm and calculate the resolution           power as under.

                   Absorbance at 269nm

                  ------------------------------- = Theoretical ratio should be not less than 1.5

                         Absorbance at 266nm

5.3.6.2   After calibration is over, record the results.

 

6.0 Abbreviations:

gm	:	Grams
ml	:	milli liter
nm	:	nano meter
mg	:	milli gram
UV	:	Ultra Violet
cm	:	Centimeter
°C	:	degree Celsius
v/v	:	volume/volume

  

7 .Records:

Sr.No.	Name of the Document	Rev No.
1	UV Spectrophotometer Report	01

 

8.         References: : ICH Guidelines Q7 and spectrophotometer manual.