This document details the procedure for the Operation and Calibration Of a visible spectrophotometer.
It is the policy of Xyz Limited that the written procedure shall be followed for the operation and calibration of a visible spectrophotometer and its use monitored to ensure that the instrument gives consistently accurate and reproducible results in compliance with regulatory requirements.
This
procedure is to be applied at the time of the Operation and Calibration of a visible spectrophotometer.
RESPONSIBILITY & ACCOUNTABILITY
Persons
along with their responsibilities & accountabilities are given below:
|
S. No |
Designation |
Responsibility |
|
01 |
Executive -
Corporate Quality Assurance |
To prepare SOP and follow the same |
|
02 |
Manager – Quality Assurance |
To ensure implementation of SOP. |
|
03 |
Section - in charge |
To ensure implementation of SOP. |
|
04 |
Manager
- Technical |
To ensure implementation of SOP. |
|
05 |
VP - Technical |
To ensures implementation of SOP. |
|
06 |
Assistant - General Manager |
To ensure implementation of SOP. |
5.0 PROCEDURE
5.1PRELIMINARY CHECK
5.1.1 Check and ensure that the instrument is clean and suitable for starting
the operation. If it is not, clean and ensures suitability.
5.1.2 Instrument should be located at the ambient temperature.
5.1.3Check calibration status of an instrument before use.
5.2 CLEANING OF CUVETTE
5.2.1 Never use a brush to clean the inside of the cuvette.
5.2.2 Clean the Cuvette using Purified water or any organic solvent (e.g. Acetone or
Methanol) & dry the cuvette
5.2.3 Add about 1ml of the solution to be measured. Tilt and turn the cuvette so that
the solution has contact with all the surfaces. Discard the solution and repeat
this rinse once more.
5.2.4 Fill the cuvette about ¾ full of the solution you wish to test.
5.2.5 Wipe the outside of the cuvette with a lint-free cloth, tissue paper to remove any
moisture or fingerprints from the outside surface.
5.3 PRECAUTIONS visible spectrophotometer
5.3.1 Do not touch the fine surfaces, always use the rough surface of the cells to hold.
5.3.2 Ensure cleanliness of the cells before and after use.
5.3.3 Use Purified water to rinse the cells.
5.3.4 Do not use directly Purified water to rinse cells after use of solutions
prepared with immiscible solvents, use ethanol or methanol first
and then water.
5.3.5 Use a lint-free cloth duster for wiping of the cells.
5.3.6 Use keeping cells in one direction in the cell holder throughout the test to
minimize the error in the blank and sample solutions.
5.3.7 Do not shake solutions of samples immediately before measurement.
5.3.8 The temperature of all solutions/reagents used in the test should not differ by
more than 0.5 ºC.
5.3.9 The solvent in the reference cell should be of the same batch as that used to
prepare the solutions
Operations of Spectrophotometer
Make sure the panel is closed and cuvette holders are empty
before starting.
Before
starting check power supply is On.
Turn the spectrophotometer "ON" by flipping the switch on the right side of
the instrument and allow 15-20 minutes for the instrument to warm up.
Set the appropriate wavelength with wavelength setting knob from the front
panel of the instrument.
Set the zero in the air with a zero setting knob. Fill the cuvettes with
blank / Solvent solution, wipe with lint-free cloth duster and place it in the
cell holders & set 100% Transmittance with 100 setting knob. And also
adjust the sensitivity with a sensitivity knob from the front panel of the
instrument.
Then remove the cuvette, discard the blank solution. Rinse the cuvette with the sample
solution. Then fill the cuvette with sample solution & replace it in the cell
holder. The monitor will display the Absorbance/Transmittance of the sample
solution at the wavelength displayed.
Switch “OFF” the instrument when not in use.
Maintain Logbook of Visible Spectrophotometer on XYZ/CQA/SOP-089/FR-01 Logbook for Visible Spectrophotometer (Appendix I)
6.0 CALIBRATION PROCEDURE Visible Spectrophotometer
6.0.1
CALIBRATION FREQUENCY: Half Yearly and After Every Maintenance Job
6.1 WAVELENGTH ACCURACY
6.2 LINEARITY OF ABSORBANCE
6.3 SPECIFICATION OF CELLS
6.1 WAVELENGTH ACCURACY
6.1.1 Preparation
of 0.15M CoCl2.6H2O:
Prepare
stock solution of 1.5M CoCl2.6H2O by dissolving 17.84 g of CoCl2.6H2O in 50ml
of water. Pipette out 10.0ml of stock solution to the 100ml volumetric flask
and make up the volume with water up to the mark (0.15M CoCl2.6H2O).
6.1.2 Set the wavelength 600nm and put the blank to set the 100% transmittance.
6.1.3 Clean, then rinse and fill the cuvette with 0.15M CoCl2.6H2O standard solution
and note the transmittance.
Repeat
the same for 650nm, 700nm, 750nm, 800nm, and 850nm.
|
Sr. No. |
Wavelength in nm |
Transmittance in % |
|
1.0 |
600 |
119.8 - 114.6 |
|
2.0 |
650 |
155.6 - 162.0 |
|
3.0 |
700 |
170.9 - 177.9 |
|
4.0 |
750 |
160.1 - 166.7 |
|
5.0 |
800 |
161.7 - 168.3 |
|
6.0 |
850 |
130.1 - 135.5 |
Acceptance
criteria:
Maximum Transmittance should be observed at 700 ±50nm.
6.1.5
Discard the solution after the calibration.
6.1.6
Record the calibration data as per XYZ/CQA/SOP-089/FR-02 Appendix II (Page 2 of
4).
6.2 LINEARITY OF ABSORBANCE:
6.2.1 Preparation of the stock solution:
Prepare a stock solution of 1.5M CoCl2.6H2O by dissolving 17.84 g of CoCl2.6H2O in 50ml of water. Working standard solution:
0.15M CoCl2.6H2O solution
Pipette
out 5.0ml of stock solution to the 50ml volumetric flask and make up the volume
with water up to the mark
0.12M
CoCl2.6H2O solution
Pipette
out 4.0ml of stock solution to the 50ml volumetric flask and make up the volume
with water up to the mark.
0.09M
CoCl2.6H2O solution
Pipette
out 3.0ml of stock solution to the 50ml volumetric flask and make up the volume
with water up to the mark.
0.06M
CoCl2.6H2O solution
Pipette
out 2.0ml of stock solution to the 50ml volumetric flask and make up the volume
with water up to the mark.
0.03M
CoCl2.6H2O solution
Pipette
out 1.0ml of stock solution to the 50ml volumetric flask and make up the volume
with water up to the mark
|
Sr.
No. |
concentration |
Transmittance
at 550 nm |
|
1 |
0.1500 |
44.1
to 45.9 |
|
2 |
0.1200 |
51.9
to 54.0 |
|
3 |
0.0900 |
60.8
to 63.2 |
|
4 |
0.0600 |
70.6
to 73.4 |
|
5 |
0.0300 |
83.3
to 86.7 |
Acceptance
criteria
Transmittance
should be ±2%.
The
linearity coefficient ‘r’ obtained from the linearity graph of CoCl2.6H2O for
different levels should not be less than 0.99.
6.2.3 Record the calibration data as per XYZ/CQA/SOP-089/FR-02 (Appendix II)
6.3 SPECIFICATION
OF CELL
In
`Photometry mode’ select transmittance mode. Set the wavelength at 400 nm. Then
set ‘zero’ to get 100 % transmittance with air blank in the cell holders. Then
place the cuvettes in the sample compartment filled with Milli Q water using
air blank and read the transmittance. Similarly repeat the operation for 450nm,
500nm, and 550nm. The transmittance values should meet the requirement as given
in the following table.
Acceptance
criteria
|
WAVELENGTH |
LIMIT OF % TRANSMITTANCE |
|
400nm |
Not Less Than 73% |
|
450nm |
Not Less Than 76% |
|
500nm |
Not Less Than 79% |
|
550nm |
Not Less Than 82% |
6.3.2
Record the calibration data as per XYZ/CQA/SOP-089/FR-02 (Appendix II) page 4
of 4.
6.3.3
Summarize all the data on XYZ/CQA/SPO-089/FR-03 Visible Spectrophotometer
Calibration Summary Sheet Appendix III
7.0 GENERATION OF INSTRUMENT CALIBRATION NUMBER
Generate
Instrument Calibration number on the calibration datasheet as INSCALXXYYZZZ
Where INS denotes Instrument, CAL denotes Calibration
XX
denotes the year, YY denotes Month ZZZ denotes sequence number.
8.0 ABBREVIATIONS
INS
CAL Instrument
Calibration
XYZ
Xyz Limited
SOP
Standard Operating Procedure
CQA
Corporate Quality Assurance
T
Transmittance
Appendix
& Logbook: - Download
Calibration of UV-vs Spectrophotometer | UV spectrometer
1.0 OBJECTIVE:- To lay down the procedure for UV-VIS SPECTROPHOTOMETER.
2.0 SCOPE
2.1 This procedure applies to the Quality Control Department of.
3.0 RESPONSIBILITIES
3.1 Chemists and Lab boy of QC to follow the SOP.
3.2 Head – QA-QC for complies of SOP.
4.0 PROCEDURE
4.1 CLEANING OF QUARTZ CELLS (Cuvettes)
4.1.1 Never use a brush to clean the inside of QUARTZ CELLS (Cuvettes).
4.1.2 Clean the QUARTZ CELLS (Cuvettes) using distilled water or any organic solvent (e.g. Acetone or Dried Methanol) & dry the QUARTZ CELLS (Cuvettes).
4.1.3 Add about 1ml of the solution to be measured. Tilt and turn the QUARTZ CELLS (Cuvettes) so that the solution has contact with all the surfaces. Discard the solution and repeat this rinse once more.
4.1.4 Fill the QUARTZ CELLS (Cuvettes) about ¾ full of the solution you wish to test.
4.1.5 Wipe the outside of QUARTZ CELLS (Cuvettes) with a lint-free, soft tissue to remove any moisture or fingerprints from the outside surface.
4.2 PRECAUTIONS:
4.2.1 Do use the unit for any purpose other than the analysis.
4.2.2 Do not touch the fine surface of the QUARTZ CELLS (Cuvettes), always use the rough surface of the cells to hold.
4.2.3 Ensure cleanliness of the cells before and after use.
4.2.4 Use Distilled water to rinse the cells.
4.2.5 Do not use directly Distilled water to rinse cells after use of solutions prepared with immiscible solvents, use ethanol or methanol first, and then use water.
4.2.6 Use keeping cells in one direction in the cell holder throughout the test to minimize the error in the blank and sample solutions.
4.2.7 Do not disassemble or modify the unit without the approval of authorized service personnel.
4.2.8 Ensure the lid of the UV compartment is closed properly.
4.2.9 The solvent in the reference cell should be of the same batch as that used to prepare the solutions.
Sodium Polysulfide Solution Preparation, Formula, and Uses
4.3 OPERATION PROCEDURE
4.3.1 Before starting check power supply is ON.
4.3.2 Switch on Instrument by using ON/OFF present on the rear side of UV-VIS Spectrophotometer.
4.3.3 Allow the UV lamp to glow for 10 minutes.
4.3.4 Now open the UV Probe Software icon (by double-clicking on the main window) an Instrument option will start.
4.3.5 Each time you start the instrument application (by double-clicking the instrument icon from the main window) an Instrument option will start.
4.3.6 Now go to any mode SPECTRUM or PHOTOMETRIC mode. Then press “CONNECT”
4.3.7 Now wait till initialization is completed and press “OK”. (Now the system is ready for analysis).
SPECTRUM MODE:
4.3.8 If all checks are all right, press analysis, it will show “SPECTRUM MODE”, after clicking it, it will show “Parameter”. Click on Parameter, it will further show “METHOD”, click it. Now type “RAW DATA”, then again press “NEXT”, it will further show “Method”, click it. Then set the parameter like wavelength range (from…….to…….) and then press “NEXT”.
4.3.9 Now put Reference Sample (BLANK) in both the QUARTZ CELLS (Cuvettes) and press“Baseline”.
4.3.10 Then remove front side QUARTZ CELLS (Cuvettes), discard the reference solution. Rinse the QUARTZ CELLS (Cuvettes) with the sample solution. Then fill the QUARTZ CELLS (Cuvettes) with sample solution & replace it in the cell holder and press “READ UNK”.
4.3.11 After completion, select the path where data is to be saved.
4.3.12 To see where ʎ max is press “PEAK PICK”.
4.3.13 Now for the print out click on “REPORT FORMAT”. Open the File, go to the “Spectrum report Format “, Print Preview, and Print.
PHOTOMETRIC MODE:
4.3.14 If all checks are all right, press analysis, it will show “PHOTOMETRIC MODE”, after clicking it, it will show “Parameter”. Click on Parameter, it will further show “METHOD”, click it. Now type “RAW DATA”, then again press “NEXT”, it will further show “Method”, click it. Then set the parameter like wavelength range (from…….to…….) and then press “NEXT”.
4.3.15 Now put Reference Sample (BLANK) in the QUARTZ CELLS (Cuvettes) and press “Auto Zero”.
4.3.16 Then remove front side QUARTZ CELLS (Cuvettes), discard the reference solution. Rinse the QUARTZ CELLS (Cuvettes) with the sample solution. Then fill the QUARTZ CELLS (Cuvettes) with sample solution & replace it in the cell holder and press “START”.
4.3.17 After completion, select the path where data is to be saved. now this window will appear
4.3.18 To see where ʎ max is press “PEAK PICK”.
4.3.19 Now for the print out click on “REPORT FORMAT”. Open the File, go to the “Photometric report Format “, Print Preview, and Print.
5.0 CALIBRATION:
5.1 WAVELENGTH CALIBRATION:
5.1.1 Go to mode ‘2’ and then select operation in ‘E’ mode by pressing ‘1’.
5.1.2 Press ‘2’ Enter scan range 660 nm ( 650 nm.
5.1.3 Select scan speed medium & press the ‘Start’ key. Check and note the peak indication.
5.1.4 Press ‘2’ Enter scan range 490 nm ( 480 nm.
5.1.5 Select scan speed medium & press the ‘Start’ key. Check and note the peak indication. Acceptance criteria: Peak indication should occur at
a) 656.1 ( 1.0 nm when scanning between 660 nm ~ 650 nm.
b) 486.0 ( 1.0 nm when scanning between 490 nm ~ 480 nm.
5.2 CONTROL OF ABSORBANCE:
5.2.1 Check the absorbance using potassium dichromate solutions at the wavelength of 235, 257
, 313 and 350 nm.
5.2.2 Potassium dichromate solution:
5.2.2.1 Add 5.4 ml of sulphuric acid in 500 ml purified water and dilute to 1000 ml with purified water (Solution A)
Observed O.D X 1000
E1 = -----------------------------------------------
Weight of Potassium dichromate in gm X 100
5.2.2.2 Further dilute 100 ml to 2000 ml with purified water to give a 0.005 M sulphuric acid solution. (Blank) [Alternatively, the ready-made solution can also be used.]
5.2.2.3 Dry Potassium dichromate (A.R. grade) at 130°C to constant weight and weigh a quantity accurately between 57.0 mg and 63.0 mg into a 1000.0 ml volumetric flask. Add 0.005 M Sulphuric acid to dissolve and dilute to the mark. (Solution B)
5.2.2.4 Also weigh 57.0-63.0 mg of Potassium dichromate UV into 100.0ml volumetric flask. Add
0.005M sulphuric acid to dissolve and dilute to the mark (Solution C)
5.2.2.5 The absorbance limits 144.0% (Tolerance 142.4% - 145.7%).
5.2.2.6 Check the absorbance of solution B at the following wavelengths (nm): 235, 257, 313, and 350.
5.2.2.7 Check the absorbance of solution C at wavelength 430.
5.2.2.8 Record the absorbance at each wavelength in the Calibration Data Sheet.
5.2.2.9Calculate the value A (1%, 1 cm) as per the following formula:
A (1 %, 1 cm) = Absorbance x 100 x 100
Weight of K2Cr2O7 in mg
5.2.2.10 Check the value of A at each wavelength against the acceptance criteria given below:
Acceptance criteria: Wavelength(nm)
5.3 PHOTOMETRIC ACCURACY
5.4 LIMIT OF STRAY LIGHT:
5.4.1 Note the absorbance of a 1.2 % w/v solution of Potassium chloride, at a path length of 1 cm the absorbance increases steeply between 220 nm and 200 nm and is greater than 2 at a wavelength between 198 nm and 202 nm, when compared with purified water as reference liquid.
Acceptance criteria: Absorbance should be greater than 2 and transmittance should be less than 1.0% T.
5.4.2 Note the absorbance of 0.025 % w/v solution of Potassium dichromate in 0.05 M KOH in
a path length of 1 cm at about 370 nm when compared with purified water as reference liquid.
Acceptance criteria: Absorbance should be greater than 2 and transmittance should be less than 1.0%
T.
6.0 ABBREVIATIONS
8.1.1 Instrument ID : ----------------------------
8.1.2 Frequency of Calibration : Half Yearly
8.1.3 Date of Calibration : ----------------------------
8.1.4 Next due date of Calibration : ----------------------------
8.1.5 PRELIMINARY CHECK
8.1.6 Cleanliness of the Instrument: Observation: Clean / Not Clean
Corrective action in the case of non-compliance:
8.1.7 Cleanliness of the QUARTZ CELLS (QUARTZ CELLS (Cuvettes) s): Observation: Ok / Not Ok Corrective action in the case of non compliance:
8.1.8 Humidity and temperature requirement: Observation: Ok / Not Ok Corrective action in the case
of non-compliance:
8.1.9 Condition of silica Gel: Corrective action in the case of non-compliance:
8.1.10 LCD Display: Observation: Ok / Not Corrective action in the case of non-compliance:
8.1.11 Instrument and printer are connected to a stabilized power supply Observation: Ok / Not Ok
8.1.12 Any other maintenance: Corrective action in the case of non-compliance:
