UV/VIS spectrophotometer Operational SOP



1.0  OBJECTIVE:
To lay down the operating procedure of UV/VIS Spectrophotometer.
2.0  SCOPE:
This shall be applicable to the UV/VIS Spectrophotometer of Quality Control Instrument Laboratory.
3.0  RESPONSIBILITY:
3.1  Technical Assistant/ Helper.
3.2  Q.C Analyst.
4.0  ACCOUNTABILITY:
4.1  Manager-Quality Control.
5.0  MATERIAL AND EQUIPMENT:
5.1  UV/VIS Spectrophotometer.
6.0  PROCEDURE:
6.1   Ensure that the instrument is properly connected to the stabilized power supply.
6.2  Switch ON the main power supply switch.
6.3  The yellowish light flickers and the instrument start INITIALISATION as under.
6.3.1        Lsi initialize.
6.3.2        Rom check.
6.3.3        Ram check.
6.3.4        Filter origin.
6.3.5        Light source org.
6.3.6        λ org. (coarse).
6.3.7        W lamp energy.
6.3.8        λ org (fine).
6.3.9        D2 lamp energy.
6.3.10    Check.
6.4  After initialization of the instrument push MODE key. The MAIN MENU appears on the screen as under:
6.4.1        Photometric.
6.4.2        Spectrum.
6.4.3        Quantitation.
6.4.4        Kinetics.
6.4.5        Time scan.
6.4.6        Multiple components.
6.4.7        Optional program pack.
6.4.8        UTILITIES.
6.5  Select option:
6.5.1        For further operation on the instrument any option as listed in point no. 6.4 is chosen.
6.6  Determination of absorbance:
6.6.1        Set the cursor at photometry.
6.6.2        Press the “GO TO” on the keyboard.
6.6.3        “GO TO WL” [190.0-1100.0] appears on the screen.
6.6.4        Select the required wavelength from the keyboard [e.g. 230nm- press key number 2, 3, 0 and ‘.’ Then ‘0’ and press key ENTER].
6.6.5        On screen the wavelength drive will start moving to the selected wavelength [i.e. 600.0nm-230.0nm].
6.6.6        Clean the quartz cells with purified water and then with blank solution.
6.6.7        Fill both the quartz cells with blank solution. Clean cells with soft tissue paper.
6.6.8        Place the cuvettes into the reference and sample compartment of the instrument and close the shutter of the compartment.
6.6.9        Press Auto zero on the keyboard “0.000” will appear on the screen.
6.6.10    Now the instrument is ready for taking absorption at chosen wavelength for the sample.
6.6.11    Take out the cell from the sample compartment of the instrument and rinse it with the sample solution, and fill it to ¾ the height.
6.6.12    Clean the cell with soft tissue paper and place it in the sample compartment of the instrument and close the shutter.
6.6.13    The absorbance of the sample will appear on the screen.
6.6.14    Print the absorbance of the sample by pressing PRINT DATA key on the key board.
6.7  Wavelength scan:
6.7.1        Set cursor to wavelength scan, by pressing “ENTER”.
6.7.2        Now select options,
6.7.2.1  ABS,
6.7.2.2  %T ,
6.7.2.3  SINGLE BEAM.
6.7.3        Press ENTER again .TEST SET UP appears.
6.7.4        Press ENTER .One more cursor will appear on the screen i.e. on START WL [nm] simultaneously ENTER VALUE [190.0-1100.0] appears below the PARAM table.
6.7.5        ENTER the START WL by pressing numerical keys.
NOTE: START WL means the highest wavelength in the wavelength scanning range.
6.7.6        Then STOP WL appears ENTER VALUE [190.0-1100.0].
6.7.7        Select the lowest wavelength to be scanned by pressing the numerical keys and ENTER.
NOTE: STOP WL [nm] is the lowest wavelength in the wavelength scanning range.
6.7.8        Press ENTER UP - SCALE appears, select the maximum absorbance to be kept as UP -SCALE.
6.7.9        Press ENTER to arrive ‘LO SCALE’. Select the lowest absorbance on the or below the base line and feed by pressing numerical keys [e.g. 0.000 the base line absorbance], normally it is kept – 0.100 i.e. below the base line.
6.7.10    Press ENTER TO ‘SCAN SPEED’ [nm/min], select the options out of 1 to 8 i.e. the options by pressing numerical keys.
6.7.11    Press ENTER to arrive to ‘INIT-DELAY’ [sec] i.e. the time delay to start the scan, ENTER VALUE [0-9999] to feed the initial delay.
6.7.12    Press ENTER to go to ‘CYCLE TIME’ [sec], ENTER value from 0-9999 by pressing the numerical keys and then ENTER.
6.7.13    Press ENTER to DISPLAY FORMAT. Select options out of two i.e.
6.7.13.1     SEQUENTIAL.
6.7.13.2    OVERLAY.
6.7.14    Press ENTER to test name.
6.7.15    Press ENTER to “ENTER” to change string.
6.7.16    Feed the name of the test scanning by operating the CURSOR provided for SELECT – 0-10 Characters.
6.7.17    Enter to maximum 10 letters abbreviated or numerical to be coded for TEST NAME.
6.7.18    Enter to INSTR SET UP and fix parameters.
6.7.19    BASELINE: select option i) system ii) user press ENTER to RESPONSE: select option i) Fast, ii) Medium iii) Slow. (MEDIUM response for WL SCAN is desirable).
6.7.20    ENTER to LAMP CHARGE WL (nm) ENTER VALUE from 325.0-370.0.
6.7.21    ENTER the following like this (one after another).
6.7.21.1    VISLAMP-ON (select options i. ON ii. OFF).
6.7.21.2    UV LAMP-ON (select options i.  ON ii. OFF).
NOTE: When we are doing any scanning in the UV region or VISIBLE region, we need not keep the other lamp ON.
6.7.22    GRAPH PRINT: select option i. ON ii. OFF. Keep the text print –ON when simultaneous Printing of graph is required during scanning.
6.7.23    TEXT PRINT: select option i) ON, ii) OFF. Keep the text print- ON when simultaneous printing of absorbance at particular WL is desired at particular intervals.
6.7.24    LIST INTERVAL: Enter the value (0.1-100.0) by pressing the numerical keys.
6.8  Enter to user baseline:
6.8.1        Keep the blank solution.
6.8.2        Again ENTER to START to measure USER BASE LINE. Make the USER BASE LINE ‘AUTO ZERO’ for the WL range opted for two times.
6.8.3        FORWARD to get the graph i.e. WL against Absorbance.
6.8.4        Fill the sample cell with test solution, wipe with tissue paper and fit in the sample compartment.
6.8.5        Start scanning and take prints if required. Trace peaks on the scan by operating numerical keys.
6.8.6        Take out the quartz cells press ‘MAIN MENU’ on the keyboard to get back the instrument to its original position.
6.8.7        Switch OFF the instrument after operation and clean the quartz cells. For further operations, please refer manual.
7.0  REVISION LOG:
Revision No.
Effective Date
Reason
00

New SOP

8.0  REFERENCES:
8.1  Not Applicable.
9.0  ANNEXURES:
9.1  Not Applicable.
10.0    ABBREVIATIONS:
Abbreviation
Expanded Form
SOP
Standard operating procedure
&
And
No.
Number  
Ltd.
Limited
Q.C
Quality control
QCEO
Quality control equipment operation
F
Format
UV/VIS
Ultraviolet/ visible
λ
Lambda
nm
Nanometer
D2
Deuterium-2
ABS
Absorbance
%
Percentage
T
Transmittance
e.g.
For example


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