1.0
OBJECTIVE:
To lay down procedure for operation &
calibration of U.V Visible
spectrophotometer.
2.0
SCOPE:
This SOP covers procedure for operation &
calibration of U.V Visible spectrophotometer.
3.0
RESPONSIBILITY:
QC Officer: Responsible
for operation, calibration of the instrument as per procedure.
QC Manager/QC Executive: Review the records and
governing the document.
4.0
PROCEDURE:
4.1
CLEANING:
4.1.1
After completion of testing remove cuvette
from sample compartment.
4.1.2
Remove the solution from the cuvette and
wash with water then with methanol and dry with tissue paper. Preserve it in
cuvette box.
4.1.3
Clean the outer surface of instrument with
Isopropyl alcohol and dry it with tissue
paper.
4.2
OPERATION:
4.2.1
Ensure that the instrument is clean and free from dust.
4.2.2
Connect the plug pins of UV Visible spectrophotometer and computer to
electric line on the electric switch board and make then ‘ON’.
4.2.3
Ensure that UV spectrophotometer switch is ‘ON’. Click ‘UV Probe 2.21’
from desktop of computer.
4.2.4
Allow the instrument for 15 to 20 min after
switching on to stabilize.
4.2.5
Select a
particular mode for specific operation.(e.g. photometric, spectrum, kinetic,).
If absorbance is to be measured at a particular wavelength select kinetic or
photometric mode. If scanning is to be done within wavelength range, then
select spectrum mode.
4.2.6
Auto zero – Baseline – Go to WL – Connect –
Disconnect parameters will apepear in UV-Probe software. Click on “connect”.
4.2.7
Automatically the instrument
is checked and initialized. This initialization requires approximately 15-20
minutes. When initialization is complete, circles appearing in software
indicating different parameters will become green. If it become red means that
parameter is fail.
4.2.8
In kinetic mode, Select method à measurement à Enter
wavelength.
4.2.9
Click on Instrument parameters tab, select
Absorbance in measuring mode and click ‘OK’.
4.2.10
Press ‘AUTO
ZERO’, keep blank solution in both cuvettes in the sample compartment and again
press ‘ AUTO ZERO’ key.
4.2.11
Remove
front side cuvette and replace it with sample solution and click on ‘START’.
4.2.12
Measure the
absorbance. Save the file and
take print out of graph by pressing ‘PRINT’.
4.2.13
After
measuring the absorbance clean the cuvettes with purified water and wipe with
tissue paper. Preserve the cuvettes in the cuvette box.
4.2.14
Switch off
the instrument after use.
4.3
CALIBRATION:
4.3.1
Consider the
following parameters for calibration:
1. Control
of Absorbance
2. Limit
of stray light
3. Resolution Power
4.3.2
CONTROL OF ABSORBANCES:
4.2.2.1 Check the
absorbance at 235nm, 257nm, 313nm & 350nm using 0.006 % w/v potassium dichcromate (previously
dried at 130 ° C to constant weight) in 0.005 M Sulphuric acid and at 430nm using
0.06% w/v potassium dichcromate (previously dried at 130 ° C to constant
weight) in 0.005 M Sulphuric acid.
4.2.2.2 Preferably weigh 57.0 mg to 63.0 mg of potassium dichcromate in a 1000 ml
volumetric flask for 0.006% solution and 57.0 mg to 63.0 mg of potassium
dichcromate in a 100 ml volumetric flask for 0.06% solution. Dissolve and
dilute to volume with 0.005 M Sulphuric acid.
4.2.2.3
Measure the
absorbance of the resulting solution against 0.005 M Sulphuric acid at the
following wavelengths. Then calculate
the A 1%, 1 cm as follows:
Calculation: Abs
.
Conc. in %
Sr. No.
|
Wavelength
|
A % 1 cm as per IP
|
Limits as per IP
|
1
2
3
4
5
|
235 nm
257 nm
313 nm
350 nm
430 nm
|
124.5
144.5
48.6
107.3
15.9
|
122.9 to 126.2
142.8 to 146.2
47.0 to 50.3
105.6 to 109 .0
15.7 to 16.1
|
4.3 LIMIT STRAY LIGHT:
4.2.3.1 Stray light shall be detected
at a given wavelength (200nm) using 1.2% W/V of potassium chloride in water.
4.2.3.2 Weigh accurately about 1.2 g of
potassium chloride in 100 ml volumetric flask, dissolve and dilute with water
to 100 ml.
4.2.3.3 Check the absorbance of resulting solution at
200 nm against water as blank. Record the absorbance. (Limit: Not less than 2.0).
4.3.4
RESOLUTION
POWER:
4.2.4.1 Dilute 1 ml of toluene to 100 ml with hexane.
4.2.4.2 Dilute 5 ml of this solution to 50 ml with hexane. Further dilute 10 ml
to 50 ml with hexane.
4.2.4.3 Measure the absorbance at 266 nm and 269 nm
and record the spectrum. Calculate the
ratio of the absorbencies at the maximum of 269 nm to the minimum at 266 nm.
Limit :
Not less than 1.5 .
4.2.4.4 Record calibration detail in format (Refer Annexure –I) and also record
the usage of instrument in respective log book of instrument. (Refer Annexure
–II).
4.4
CALIBRATION
FREQUENCY:
Internal calibration : Quarterly
External calibration : Yearly
4.5
PRECAUTIONS:
4.5.1
Do not
carry out operations involving corrosive liquids near to the instrument.
4.5.2
Also
ensure & avoid spillage of solvent & water inside the cuvettes chamber.
4.5.3
Ensure
that the instrument is located and maintained at a temperature below 25° C.
4.5.4
Avoid direct exposure to air-blow from air-conditioners. Avoid exposure
to direct sun light and vibrations.
5.0 TRAINING:
Trainer : Manager / Executive - Quality Control
Trainees : Quality Control Officers
6.0 ANNEXURE(S):
Annexure – I Calibration record
Annexure – II - Instrument Usage log book
Annexure – 1
|
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CALIBRATION
DATA OF ULTRA-VIOLET AND VISIBLE
SPECTROPHOTOMETER
|
Page
No. :
01 of
02
|
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Calibration Date : Next
Due date for Calibration :
|
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(1) CONTROL OF
ABSORBANCES :
Potassium
dichromate (Grade) & B. No. : Balance
No. :
Make
:
Weight
of potassium dichromate taken:
diluted 1000 ml with 0.005 M H2SO4.
Diluted
100 ml with 0.005 M H2SO4. (for 430nm)
Calculation: Abs.
Conc. in %
|
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Remarks :
|
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(2) CONTROL OF WAVELENGTH
Holmium Perchlorate Solution: 4% w/v
solution of Holmium Oxide in 1.4M perchloric acid.
B.
No. :
Make
:
|
|||||||||||||
Remarks : Satisfactory / Not Satisfactory
Calibrated by : Checked by :
|
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Annexure – 2
|
|
CALIBRATION
DATA OF ULTRA-VIOLET AND VISIBLE
SPECTROPHOTOMETER
|
Page
No. :
02 of
02
|
Calibration Date : Next
Due date for Calibration :
|
|
(3) LIMITS OF STRAY LIGHT
Potassium
chloride Grade & B. No. :
Balance No. :
Make :
Weight of
potassium chloride taken: g diluted 100 ml with Purified
Water.
Absorbance
at 200 nm against water as blank is ---> (
Limit : Not less than 2.0 )
|
|
Remarks : Satisfactory / Not Satisfactory
|
|
(4) RESOLUTION POWER
Toluene (Grade)
& B. No. :
Make :
Hexane (Grade)
& B. No. :
Make :
0.02
% V/V toluene in Hexane
Absorbance
at 269 nm is (Maximum) --->
Absorbance at 266 nm is (Minimum) --->
* Ratio
= at
269 nm =
at
266 nm (
Limit : Not less than 1.5 )
|
|
Remarks
: Satisfactory / Not Satisfactory
|
|
Calibrated
by : Checked by :
|
|
SOP No. : XXX/SOP/000-00 Format No. : XXX/FRM/000-00
|
|
Annexure – 1I
|
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Date
|
Name of Sample
|
A. R. No.
|
B. No.
|
Used by
|
Remark
|
|
SOP No. : XXX/SOP/000-00 Format No. : XXX/FRM/000-00
|
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