Calibration of UV Visible Spectrophotometer

1.0    PURPOSE

1.1    To lay down the procedure for Calibration of UV/Visible Spectrophotometer. 

2.0    SCOPE

2.1    This procedure is applicable for Calibration of   UV/Visible Spectro- photometer in Quality control lab.

3.0    RESPONSIBILITY

3.1    Assistant-QC.

4.0    ACCOUNTABILITY

4.1        Executive-QC.

5.0    PROCEDURE

5.1    Operate the instrument as per the respective SOP and calibrate the UV/Visible Spectrophotometer as follows;
5.2    Perform the following tests for calibration of instrument.
5.2.1    CONTROL OF WAVELENGTH
5.2.2    CONTROL OF ABSORBANCE
5.2.3    LIMIT OF STRAY LIGHT
5.2.4    RESOLUTION
5.3    CONTROL OF WAVELENGTH

5.3.1    Preparation of solutions:
5.3.1.1    Perchloric acid solution(1.41%): Dilute 8.5mL of Perchloric acid (70-73%) to 100mL with water.
5.3.1.2    Holmium perchlorate solution: Transfer accurately weighed 0.4 grams of Holmium oxide into a 10mL volumetric flask. Add about 5mL of 1.41% Perchloric acid solution and heat gently on water bath until solution becomes clear. Cool and make up to the volume with the same solution.

5.3.2    Scan the above-prepared Holmium perchlorate solution in the range of 200 nm to 600 nm using the Perchloric acid solution as blank.

5.3.3    Verify the wavelength scale using the absorption maxima of holmium perchlorate solution, the line of a hydrogen or deuterium discharge lamp or the lines of a mercury vapour as shown below. 

        
5.3.4    Record the results in the format No.xxxxx

5.4    CONTROL OF ABSORBANCE

5.4.1    Preparation of potassium dichromate solution:
5.4.1.1    Weigh 57 to 63 mg of dried Potassium dichromate into a 100ml volumetric flask.
5.4.1.2    Add 50ml of 0.005 M Sulphuric acid, dissolve and make up to the mark with the same.
5.4.1.3    Transfer 10ml of the above solution to another 100ml volumetric flask.
5.4.1.4    Make up to the mark with 0.005 M Sulphuric acid.
5.4.1.5    Measure the absorbance of the solution at 235nm, 257nm, 313nm and 350nm after performing blank with 0.005 M Sulphuric acid.
5.4.2    Preparation of 0.005 M Sulphuric acid.

5.4.2.1    Dilute 2.8 mL of Conc. Sulphuric acid with 100 ml of water.  Further dilute 5mL of the above solution to 500 ml with water.

5.3.3    calculate the % A
                   E
Absorbance (1%, 1 cm)    =  ------
                                      CL
Where, 
E is absorbance of sample at particular wavelength.
C is concentration of sample in percentage (w/v).
L is thickness of the cell.

5.4.3    Record the results in the format No. xxxxx
Wavelength
(nm)    Specific absorbance
A (1%, 1cm)    Acceptance criteria 
235    124.5    122.9 to 126.2
257    144.5    142.8 to 146.2
313    48.6    47.0 to 50.3
350    107.3    105.6 to 109.0

5.5    LIMIT OF STRAY LIGHT

5.5.1    Weigh 1.2 grams of previously dried Potassium Chloride into a 100ml volumetric flask.
5.5.2    Add 50ml of water dissolve and make up to the mark with the same.
5.5.3    Measure the absorbance at 200nm using water as blank.
Record the results in the format No. xxxxx

Acceptance criteria: Absorbance at 200 nm is not less than 2.0.

5.6    RESOLUTION 

5.6.1    Transfer 2 ml of Toluene into 100 ml into a volumetric flask.
5.6.2    Make up to the mark with Hexane.
5.6.3    Transfer 1 ml of the resulting solution into another 100 ml volumetric flask.
5.6.4    Make up to the mark with hexane. (0.02% v/v solution of Toluene in hexane)
5.6.5    Scan the solution at the range of 255 nm to 275 nm.
5.6.6    Record the ratio of Absorbance at the maximum at 269 nm to that at the minima at 266 nm. 
5.6.7    Record the results in the format No. xxxxx

Acceptance criteria: Ratio shall not be less than 1.5

5.7    FREQUENCY

5.7.1    Once in three months.

uv visible, spectrophotometer, spectro photometer, volumetric flask