4.0 PROCEDURE:
4.1 Procedure For General Cleaning:
4.1.1 Ensure that the power supply to the instrument is switched OFF before cleaning.
4.1.2 Clean the equipment with a clean dry cloth every day. Occasionally wet cloth dipped in dilute soap solution may be used.
4.1.3 Precaution shall be taken to clean the equipment immediately with dry cloth to remove the moisture.
4.2 Operation:
4.2.1 Ensure that the instrument is properly connected to the stabilized power supply.
4.2.2 Switch ON the main power supply switch.
4.2.3 The Yellowish light flickers and the instrument starts INITIALISATION as under.
4.2.3.1 Lsi Initialize.
4.2.3.2 Rom Check.
4.2.3.3 Ram Check.
4.2.3.4 Filter Origin.
4.2.3.5 Light Source Org..
4.2.3.6 ג Org.(Coarse).
4.2.3.7 W Lamp Energy.
4.2.3.8 ג Org (Fine).
4.2.3.9 D2 Lamp Energy
4.2.3.10 Check
4.2.4 After initialization of the instrument push MODE key . The MAIN MENU appears on the screen as under:
4.2.4.1 Photometric
4.2.4.2 Spectrum
4.2.4.3 Quantitation
4.2.4.4 Kinetics
4.2.4.5 Time scan
4.2.4.6 Multiple component
4.2.4.7 Optional program pack
4.2.4.8 UTILITIES
4.3 Select Option:
4.3.1 For further operation on the instrument any option as listed in point no.4.2.4 is chosen.
4.4 Determination of Absorbance:
4.4.1 Set the cursor at PHOTOMETRY.
4.4.2 Press the “GO TO” on the keyboard.
4.4.3 “GO TO WL” [ 190.0-1100.0] appears on the screen.
4.4.4 Select the required wavelength from the keyboard [ e.g. 230nm-press key numbers 2,3,0 and ‘.’ then ‘0’ and press key ENTER].
4.4.5 On screen the wavelength drive will start moving to the selected wavelength [ i.e. 600.0nm-230.0nm].
4.4.6 Clean the quartz cells with distilled water and then with blank solution.
4.4.7 Fill both the quartz cells with blank solution. Clean cells with soft tissue paper.
4.4.8 Place the cuvettes into the reference and sample compartment of the instrument and close the shutter of the compartment.
4.4.9 Press Autozero on the keyboard “0.000” will appear on the screen.
4.4.10 Now the instrument is ready for taking absorption at chosen wavelength for the sample.
4.4.11 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.
4.4.12 Clean the cell with soft tissue paper and place it in the sample compartment of the instrument and close the shutter.
4.4.13 The absorbance of the sample will appear on the screen.
4.4.14 Print the absorbance of the sample by pressing PRINT DATA key on the key board.
4.5 Wave Length Scan:
4.5.1 Set cursor to wavelength scan, by pressing “ENTER” .
4.5.2 Now select options, i) ABS, ii) % T , iii) SINGLE BEAM.
4.5.3 Press ENTER again .TEST SET UP appears.
4.5.4 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.
4.5.5 ENTER the START WL by pressing numerical keys.
NOTE: START WL means the highest wavelength in the wavelength scanning range.
4.5.6 Then STOP WL appears ENTER VALUE [190.0-1100.0].
4.5.7 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.
4.5.8 Press ENTER UP - SCALE appears, select the maximum absorbance to be kept as UP -SCALE.
4.5.9 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.
4.5.10 Press ENTER TO ‘SCAN SPEED’ [nm/min], select the options out of 1 to 8 i.e. the options by pressing numerical keys.
4.5.11 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.
4.5.12 Press ENTER to go to ‘CYCLE TIME’ [sec], ENTER value from 0-9999 by pressing the numerical keys and then ENTER.
4.5.13 Press ENTER to DISPLAY FORMAT. Select options out of two i.e. i) SEQUENTIAL ii) OVERLAY.
4.5.14 Press ENTER to test name.
4.5.15 Press ENTER to “ENTER” to change string.
4.5.16 Feed the name of the test scanning by operating the CURSOR provided for SELECT –0-10 Charecters.
4.5.17 Enter to maximum 10 letters abbreviated or numericals to be coded for TEST NAME.
4.5.18 Enter to INSTR SET UP and fix parameters.
4.5.19 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).
4.5.20 ENTER to LAMP CHARGE WL (nm) ENTER VALUE from 325.0-370.0.
4.5.21 ENTER the following like this (one after another)
4.5.21.1 VISLAMP-ON (select options i. ON ii. OFF )
4.5.21.2 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.
4.5.22 GRAPH PRINT: -select option i.ON ii)OFF. Keep the text print –ON when simultaneous Printing of graph is required during scanning.
4.5.23 TEXT PRINT :-select option i) ON, ii)OFF. Keep the text print – ON when simultaneous printing of absorbances at particular WL is desired at particular intervals.
4.5.24 LIST INTERVAL:-Enter the value (0.1-100.0) by pressing the numerical keys.
4.6 Enter To User Base Line:
4.6.1 Keep the blank solution as described in point no. 6.1.7.
4.6.2 Again ENTER to START to measure USER BASE LINE. Make the USER BASE LINE ‘AUTO ZERO’ for the WL range opted for two times.
4.6.3 FORWARD to get the graph i.e. WL against Absorbance.
4.6.4 Fill the sample cell with test solution, wipe with tissue paper and fit in the sample compartment.
4.6.5 Start scanning and take prints if required. Trace peaks on the scan by operating numerical keys
4.6.6 Take out the quartz cells press ‘MAIN MENU’ on the keyboard to get back the instrument to its original position.
4.6.7 Switch OFF the instrument after operation and clean the quartz cells. For further operations, please refer manual of SHIMADZU UV/VIS SPECTROPHOTOMETER 1601.
4.7 Calibration Procedure:
4.7.1 Switch ON the instrument and allow it to warm for 10 minutes.
4.8 Preparation of Potassium dichromate solution-UV :
4.8.1 Dry a quantity of Potassium dichromate by heating to constant weigh at 130ºC. Weigh accurately 60.0 mg (57-63mg) and dissolve in 0.005M Sulphuric acid to produce 1000ml.
4.9 Control of absorbance:
4.9.1 Scan the user base line from 200nm-400nm with blank solution (0.005M Sulphuric acid) ands press Auto zero. Check the absorbance by using Potassium dichromate solution UV at various wavelengths indicated in the following table using 0.005M Sulphuric acid as blank.
……………………………………………………………………………………………
WAVELENGTH (nm) E1% (1% 1cm) MAXIMUM TOLERANCE
…….…..………………………………………………………………………………….
235 124.5 122.9-126.2
257 144 142.4-145.7
313 48.6 47.0-50.3
350 106.6 104.9-108.2
……………………………………………………………………………………………
4.9.2 The above table gives for each wavelength the exact value of E1% (1% 1cm) and permitted limits.
The E1% (1% 1cm) is calculated as follows:
E1% (1% 1cm) = Absorbance % 1000
Concentration % 100
4.9.3 Record the observation of absorbance calibration in calibration record as per the Annexure - 1.
4.10 Control of wave length:
4.10.1 Verify the wavelength scale using the absorption maxima of Holmium oxide filters.
4.10.2 Switch ON the instrument and allow it to warm up.
4.10.3 Bring the instrument in wavelength scanning position.
4.10.4 Set the Wavelength limit from 200-600nm.
4.10.5 Clean all the four windows of reference and sample compartment.
4.10.6 Make auto zero.
4.10.7 Put Holmium oxide film in the sample compartment of the instrument.
4.10.8 Start scanning and take the print of wavelength scan.
4.10.9 The obtained print should match with the reference spectra shown in Annexure - 1
4.10.10 For checking wavelength accuracy narrow absorption bands should only used.
4.10.11 The band at 360.8nm is particularly suitable.
4.10.12 Other suitable bands are at 279.3nm and 536.4nm.
4.10.13 Verify the wavelength scale using the absorption maxima.
4.10.14 The permitted tolerances are,
a. 1nm for the range 200-400nm.
b. 3nm for the range 400-600nm.
4.10.15 Record the observation of wavelength calibration in calibration record as per the Annexure - 1.
4.11 Limit Of Stray Light:
4.11.1 Preparation of Potassium chloride solution:
4.11.2 Weigh accurately 1.2 g of Potassium chloride (dried at 120°) in 100ml volumetric flask. Dissolve the content in water and make up the volume.
4.11.3 Messure the absorbance at 200 nm at path length 1.0 Cm.
4.11.4 Absorbance should be greater than 2.0 .
4.12 Ressolution :-Prepare a0.02% w/v solution of Toluene in Hexane .Record the spectrum
4.13 Frequency: Once In A Month
FORMAT FOR CALIBRATION RECORD OF UV VISIBLE SPECTROPHOTOMETER
INSTRUMENT CALIBRATION RECORD
Name of Instrument
|
: U.V. Visible Spectrophotometer
| ||
Instrument Ser. No.
|
:
| ||
Make & Model
|
:
| ||
Calibration date
|
:
| ||
Calibration due on
|
:
| ||
1. Control Of Wavelength: (Visible Region):
Maxima At Wavelength
(NM)
|
Observed Maxima Of Holmium Filter (NM)
|
Limit
(NM)
|
241.15
|
240.15 to 242.15
| |
287.15
|
286.15 to 288.15
| |
361.5
|
360.5 to 362.5
| |
536.3
|
533.3 to 539.3
|
2. Control Of Absorbance (UV Region)
Potassium dichromate (dried at 130°) ________ (about 0.60 mg) à 1000 ml with 0.005 N H2SO4
Wavelength NM
|
Observed absorbance
|
Obtained A1 %
|
Limit a1 %
|
235
|
122.9 – 126.2
| ||
257
|
142.4 – 145.7
| ||
313
|
47.0 – 50.3
| ||
350
|
104.9 – 108.2
|
Calculation:
Absorbance X 1000
A (1%, 1 CM) = -----------------------------------
Wt of Potassium Dichromate
Calibrated by : Checked by:
Date : Date :
FORMAT FOR CALIBRATION RECORD OF UV VISIBLE SPECTROPHOTOMETER
1. Limit of Stray Light:
Potassium chloride (dried at 120°) ________ (about 1.2g) à 100 ml water
Absorbance at 200 nm = (Limit: Not less than 2.0)
2. Resolution:
1 ml of toluene à 50 ml hexane 1 ml à 100 ml of hexane
Absorbance at 269 nm : 266 nm. :
Ratio of 269 nm and 266 nm : (Limit: Not less than 1.5)
Remarks: Satisfactory / Not Satisfactory
Calibrated by : Checked by:
Date : Date :
