SOP For Calibration of UV-Visible Spectrophotometer

OBJECTIVE:
To lay down a procedure for Operation & Calibration of UV-Visible Spectrophotometer.
SCOPE:
This procedure is applicable for procedure for Operation & Calibration of UV-Visible Spectrophotometer.
RESPONSIBILITY:      
QC Officer/ QC Executive
ACCOUNTABILITY:
QC Manager.
PROCEDURE:
Operating Procedure:
Ensure the instrument is connected to a stabilized power supply.
Switch ON the mains.
Ensure that the sample compartment is empty and is completely closed.
Ensure that the printer is connected and is ON.
Switch ON the instrument.
The instrument initializes it self through various parameters and return to the MODE menu.
If any parameter is detected as not correct during initialization then the initialization process is interrupted at that item without the star being highlighted.
Check the error message displayed and refers the manual for Initialization and error display.
The initialization will take place for about five minutes.
The contrast of the screen may be adjusted using an adjustment knob given on the right rear side of the instrument.
Keep the instrument in ON position unless if the next reading will not be taken for long time. A warm up period of 30 minutes is desirable to get better results. This will reduce the drift.
Set the D2 lamp off time as 10 minutes. (To be set once during installation).
Ensure the calibration status is OK and is current for the instrument.
Calibration:
Control of wavelength:
Prepare the holmium perchlorate solution (Take about 400 mg of holmium oxide in 1.4M Perchloric acid).Take the graph of the solution from 200 to 600 nm against the diluents. Check the wavelength for (Ho) 241.15 nm, 287.15, 361.5, 536.3, and 486nm. The permitted tolerance is ± 1 nm for the range 200 to 400 nm, ± 3 nm for the range 400 to 600nm. Record the results in calibration record. Attach the graphs to the calibration record.
Control of absorbance for UV Range:
For the control of absorbance at 235 nm, 257 nm, 313 nm and 350 nm, dissolve 57.0- 63.0 mg of previously dried at 130 ºC potassium dichromate in 1000 ml dried and clean volumetric flask and for the control of absorbance at 430 nm, dissolve the same quantity in 100 ml volumetric flask.
Make up the volume with 0.005 M Sulphuric acid and mix.
Take the spectrum of the solution against 0.005 M Sulphuric acid as blank.
Record the absorbance of the solution at about 235nm, 257nm.313nm and 350nm, 430 nm using peak pick option.
Attach the print out of peak and valley and the graph with the calibration record.

Wavelength – A(1% 1cm) – Maximum Tolerance       
235 nm – 124.5 – 122.9 to 126.2

257 nm – 144.5 – 142.8 to 146.2

313 nm – 48.6 – 47.8 to 50.3

350 nm – 107.3 – 105.6 to 109.0

430 nm – 15.9 – 15.7 to 16.1

Calculation:

For UV, take 57.0
to 63.0 K₂Cr₂O₇ 1000 ml of 0.005 M H₂SO₄   
Calculate using formula Eq. (for wavelength 235, 257, 313, & 330)
Absorbance × 1000 × 100 /Weight of sample / 100  
For Visible, take 57.0 to 63.0 K₂Cr₂O₇ 100 ml of 0.005 M H₂SO₄
Calculate using formula Eq (for wavelength 430)
Absorbance × 100 × 100 Weight of sample / 100  
Remark:
Potassium Dichromate (K₂Cr₂O₇) has been previously dried at 105ºC and cooled in desiccators.
Calculate the specific absorbance (A1%1cm) and compare the results with the giventable.
Limit of stray light:
Weigh accurately about 1.2 gm of potassium chloride in 100 ml dried and clean volumetric flask, and make up the volume with water.
Take the absorbance of the solution against water. Absorbance of the above solution should be greater than 2.0 at 200 nm with l cm cells.
Resolution power:
Prepare a 0.02 % v/v solution of toluene in hexane UV.
Using hexane UV in the reference cell carry out the baseline correction.
The ratio of the absorbance at the maximum at about 269 nm to that the minimum at about 266 nm is not less than 1.5.
Record the results in calibration record.
Solvents:
Take water, ethanol, ethanol (95%), methanol and cyclohexane.
Take the absorbance at 254 nm with respect water as a blank.
Acceptance criteria: Not more than 0.100 reference to water.
TRAINING:
Trainer   : Manager – Quality Control
Trainees: Staff of the QC departments  
DISTRIBUTION:
Master Copy                  :           QA Department
Controlled Copy            :       QC Department
Display Copy                 :           QC Department (If Required)
REFERENCES:
In-House