STANDARD OPERATING PROCEDURE Title: Spectrophotometer Calibration

1. PURPOSE

• It is established to assure the accuracy of results.
2. SCOPE
• It is applicable to Quality Control Department.

3. RESPONSIBILITY
• Quality Control Manager
• Assistant Manager Quality Control
4. PROCEDURE

WAVELENGTH CALIBRATION:

1. Go to Calibration mode by pressing “8” on the keyboard and “Enter”. Press “1” “Enter”, for wavelength calibration then the “Start”.
2. SAMPLE ZERO: Insert Shutter (solid Black Cuvette) in Sample Holder (closest to the front of the instrument) and close sample door. Press “9” “Enter”, then “Start” key.
3. REFERENCE ZERO: Insert Shutter (solid Black Cuvette) in Reference Holder (towards back of the instrument) and close the sample door. Press “10” “Enter”, then “Start” key. Remove Shutter.
4. Set CLOCK Press “3” “Enter” (Cursor will be on right menu). Press “1” “Enter”, to turn CLOCK ON (Cursor will move to HOUR). Enter “Hour” on numeric keypad, then “Enter Key” (this is a 24 hour clock,.i.e.2 P.M. should be keyed as “14”) Enter Minute,Month,Day,Year,and date format.
5. System Baseline Press “2” “Enter”, then the Green” Start” key.]
6. SET UP IS NOW COMPLETE.Press blue “Main Menu” key.
CONTROL OF ABSORBANCES:
Check the absorbance using a solution of potassium Dichromate at the wavelength indicated in the following table, which gives for each wavelength the exact value of the specific absorbance (A 1%,1 cm) and the permitted limits. The tolerance for the absorbance is ± 0.01.
Wavelength A (1%, 1cm) Maximum tolerence
nm
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 109.2
Use a solution of potassium dichromate prepared in the following manner. Dissolve 57.0 to 63.0 mg of potassium dichromate, previously dried to constant weight at 130oC, in sufficient 0.005M sulphuric acid to produce 1000 ml.
The absorbance of a potassium dichromate solution containing exactly 60.06 mg of k2Cr207 1000 ml of 0.005M of sulphuric acid were used as the basis for the table above. Measured at a path length of 1 cm, the absorbances are as shown in the table below.
_______________________________________________________________________
Wave length_(nm)_____________________Absorbance_____________________________
235 0.748
257 0.865
313 0.292
350 0.640
Limit of stray light Stray light may be detected at a given wavelength with suitable filters or solutions. For example, the absorbance of a 1.2% w/v solution of potassium chloride with a path length of 1 cm should be more than 2 at 200nm when compared with water as reference liquid.
Resolution When prescribed in a monograph record the spectrum of a 0.02 % v/v solution of toluene in hexane. The ratio of the absorbance at the maximum 269 nm to that at the minimum at 266 nm is stated in the monograph.
Spectral slit width When measuring the absorbance at an absorption maximum and when using an instrument the slit-width should be small compared with the half width of the absorption band.However,it should be as large as possible to obtain a high values of I and should be such that further reduction does not result in an increased absorbance reading.