1. OBJECTIVE
To lay down the procedure for operation, Cleaning and Calibration of UV-Vis Spectrometer
2. SCOPE
This procedure is applicable to Quality Control Department
3. RESPONSIBILITY
• Officer QC
• Manager QC
4. SAFETY INSTRUCTIONS
• Do not open sample compartment during initialization.
• Do not open sample compartment during baseline correction, auto zero and sampling measuring mode.
• Before placing the cuvettes in the sample holder clean the outer surface of cuvettes with tissue paper.
• Do not hold the cuvettes from its transparent optical surface.
• Operate the instrument after proper initialization.
5. PROCEDURE
5.1 OPERATION
5.1.1 Switch on the main power.
5.1.2 Switch on the instrument.
5.1.3 Wait for 15 minutes for proper stabilization.
5.1.4 After each parameter checking by system selects the program you want.
5.1.5 There are two method for measurements
Photometric mode: It is used to measure the absorbance transmittance (T %) at affixed wavelength.
Spectrum mode: It is used to measure the absorbance of sample in a wide range of selected wavelength. It will scan the sample in selected range and produce the spectrum
5.1.6 After that select the wavelength.
5.1.7 For blank correction, take two clean cuvettes, after proper rinsing keep the blank solution prior to the measurement in the both cuvette and wipe them clean from the outside using tissue paper.
5.1.8 Place one cuvette in the reference cell holder and the other in the sample cell holder. Close the sample compartment.
5.1.9 Press ‘AUTO ZERO’ key. In case of multi wavelength press ‘Baseline key.
5.1.10 After Blank correction, keep the sample in sample compartment
5.1.11 Click on ‘Start’ to measure the absorbance
5.1.12 The reading shown in the data position is the absorbance.
5.1.13 Record the result and make the entry in instrument log book.
5.1.14 Remove the cuvette and wash it with water and methanol as required.
5.2 CLEANING
5.2.1 After completion of testing, remove cuvette from the sample compartment.
5.2.2 Remove the solution from the cuvette and wash with a respective solvent in which solution is prepared.
5.2.3 Rinse with water and then with methanol and wipe with tissue papers.
5.2.4 Air dry and keep it at proper place.
5.2.5 Clean the outer surface of instrument with isopropyl alcohol and dry it with tissue paper
5.3 CALIBRATION
Ensure that all connections of spectrometer are proper.
5.3.1 Control of Absorbance
Potassium Dichromate Solution
5.3.1.1 Dry a quantity of potassium dichromate by heating to constant weight at 130 ̊C.
5.3.1.2 Weigh and transfer accurately a quantity not less than 57.0mg and not more than 63.0mg to 1000ml of volumetric flask.
5.3.1.3 Dissolve and dilute in sufficient 0.005M H2SO4 to produce 100ml.
5.3.1.4 Measure the absorbance of potassium dichromate solution at wavelength given below.
5.3.1.5 Calculate the value of A (1% 1cm) for each wavelength
A (1% 1cm) = Absorbance X 10000/ Weight of potassium dichromate in mg
5.3.1.6 Acceptance Criteria
Wavelength (nm) A (1% 1cm) Limit
235 124.5 122.9 – 126.2
257 144.0 142.8 – 145.7
313 048.6 47.0 – 50.3
350 106.6 105.3 – 108.2
5.3.2 Control of Absorbance
5.3.2.1 Record the spectrum of a 0.02% v/v solution of toluene in hexane in the range of 260nm to 420nm.
5.3.2.2 Acceptance Criteria: The ratio of absorbance at the maximum at about 269nm to that at the minimum at about 266nm is not less than 1.5.
5.3.3 Limit of Stray Light
5.3.3.1 Prepare a 1.2% w/v solution of potassium chloride in water.
5.3.3.2 Measure absorbance of above solution at 198, 199, 200, 201, 202 nm using water as blank.
5.3.3.3 Acceptance Criteria: Absorbance is greater than 2.
5.3.4 Maintenance/ Repair
5.3.4.1 When instrument does not complies with requirement/ tolerance range specified above, the instrument should be labeled “OUT OF CALIBRATION” and should be repair/ serviced.
5.3.4.2 After repair/ Maintenance calibrate the instrument.
6. RELATED DOCUMENTS
Operational Log Book Format – Annex. I
Cleaning Log Book Format – Annex. II
Calibration Report Format – Annex. III
7. DISTRIBUTION INDEX
Original Copy will be retained by Quality Assurance Department.
DISTRIBUTION INDEX
Distributed To Copy No. Received By/Date
Quality Control Department 01
8. ABBREVIATIONS
Q.C Quality Control Department
UV-VIS Ultra violet- Visible
9. REVISION HISTORY
Revision No Issue Date Supersedes
Reason of Change/ Description
Revision No. Dated
00 25-12-2015 NA NA First Version