1.0 OBJECTIVE:
To
lay down the calibration procedure for reliable and accurate results 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.
5.2
Analytical
weighing balance.
5.3
Desiccator.
5.4
Glassware (according
to requirement beakers, stirrer, spatula, pipette).
5.5
Printer.
5.6
Black marker.
5.7
1.0g of Holmium
oxide.
5.8
1.4M Perchloric
acid solution.
5.9
Potassium
dichromate 60mg (taken twice separately).
5.10 0.005M
sulphuric acid solution.
5.11 1.20g
of dried potassium chloride.
5.12 0.02ml of toluene.
5.13 Hexane (q.s).
5.14 Purified
water (q.s).
6.0 PROCEDURE:
6.1
Operate the
instrument as per SOP.
6.2
Calibration of UV/VIS Spectrophotometer is done in
five steps.
6.2.1
Control of wavelength.
6.2.2
Control of absorbance.
6.2.3
Limit of stray light.
6.2.4
Resolution power.
6.2.5
Wavelength accuracy, resolution & baseline flatness
(inbuilt test).
6.3
Control of wavelength:
6.3.1
Weigh accurately
1.0g of Holmium oxide and dissolve it in 1.4M Perchloric acid solution. Make up
to 25.0ml with the same solvent.
6.3.2
Select the method
file of CONTROL OF WAVELENGTH in the instrument.
6.3.3
After selecting
the file press reference button for baseline correction.
6.3.4
Then fill the
cuvette with 1.4M Perchloric acid and put in the sample cubicle and press
reference to zero.
6.3.5
After auto zero
put the Holmium perchlorate solution in sample cubicle then press start key.
6.3.6
Scan it and verify
the wavelength using absorption maxima of holmium perchlorate solution. The
permitted tolerance is given in annexure-1.
6.3.7
The permitted
tolerance is ±1nm for UV range and ±3nm for visible range.
6.3.8
Observed the
wavelength; tolerance is given in annexure-1.
6.4
Control of absorbance:
6.4.1
Dry a quantity of
the potassium dichromate by heating to constant weight at 130oC.
6.4.2
Solution
A:
6.4.2.1
Weigh accurately
about 57.0-63.0mg of dried potassium dichromate, suppose 60.0mg will be taken.
6.4.2.2
Dissolve it in
0.005M sulphuric acid solution.
6.4.2.3
Make up to
1000.0ml with the same solvent.
6.4.2.4
Mark the solution
as A, by using black marker.
6.4.3
Solution
B:
6.4.3.1
Weigh accurately
about 57.0-63.0mg of dried potassium dichromate, suppose 60.0mg will be taken.
6.4.3.2
Dissolve it in
0.005M sulphuric acid solution.
6.4.3.3
Make up to 100.0ml
with the same solvent.
6.4.3.4
Mark the solution
as B, by using black marker.
6.4.4
Select the method
file of CONTROL OF ABSORBANCE in the instrument.
6.4.5
After selecting
the file press reference button for baseline correction.
6.4.6
Then fill the
cuvette with 0.005M sulphuric acid for blank and put in both sample cubicle and
press reference to zero.
6.4.7
After auto zero
put the potassium dichromate solution labeled as solution ‘A’ in sample
cubicle, then press start key taking absorbance individually for first four λ
mentioned in annexure-2.
6.4.8
Now take
absorbance at 430.0nm for solution ‘B’.
6.4.9
Note the
absorption maxima of potassium dichromate solution at different wavelength.
6.4.10
Calculate the
absorbance; tolerance is given in annexure-2.
6.5
Limit of stray light:
6.5.1
Dry a quantity of
the potassium chloride by heating to constant weight at 130oC.
6.5.2
Weigh accurately
1.20g of dried potassium chloride and dissolve it in 50.0ml of purified water.
6.5.3
Make up to 100.0ml
with the same solvent.
6.5.4
Select the method
file of LIMIT OF STRAY LIGHT in the instrument.
6.5.5
After selecting
the file press reference button for baseline correction.
6.5.6
Measure absorbance
of the above solution at 198.0, 199.0, 200.0, 201.0 & 202.0nm using water
as blank.
6.5.7
Acceptance
criteria:
6.5.7.1
Absorbance is
greater than 2.0.
6.6
Resolution power:
6.6.1
Prepare 0.02% v/v solution of toluene in hexane.
6.6.2
Dissolve 0.02ml of toluene in sufficient hexane and make
up to 100.0ml by using same solvent.
6.6.3
Record the spectrum in the range of 260.0nm to 420.0nm.
(Before use of above solution, check hexane for transmittance using water as a
blank between 260.0nm to 420.0nm & use only if transmittance is not less
than 97%).
6.6.4
Select the method
file of RESOLUTION POWER in the instrument.
6.6.5
After selecting
the file press reference button for baseline correction.
6.6.6
Measure absorbance
of the above solution at 266nm and 269nm using hexane as blank solution.
6.6.7
The ratio of
absorbance maxima at 269nm to that of 266nm minima can be calculated as:
Ratio=
Absorbance at 269nm
Absorbance at 266nm
6.6.8
Acceptance
criteria:
6.6.8.1
The ratio of
absorbance maxima at 269nm to that of 266nm minima should be more than 1.5.
6.7 Wavelength accuracy, resolution &
baseline flatness (inbuilt test):
6.7.1
Attach printer
directly to the instrument (instead of computer).
6.7.2
Go to Ï· MODE press
F3 key i.e. Maintenance.
6.7.3
Press ‘1’.
6.7.4
Press start/stop
key.
6.7.5
After screen
changes, ensure that nothing is kept in the optical path & press start/stop
key again.
6.7.6
Print will come
after all the three tests are over.
6.7.7
Acceptance
criteria:
6.7.7.1
For wavelength
accuracy:
6.7.7.1.1
At 656.1±0.3nm.
6.7.7.1.2
At 486.0±0.3nm.
6.7.7.2
Resolution:
6.7.7.2.1
1.0nm or less.
6.7.7.3
Baseline flatness:
6.7.7.3.1
±0.002 Abs.
6.7.8
Frequency:
6.7.8.1
Once in a month.
6.7.9
Maintenance/
repairs:
6.7.9.1
When the
instrument does not comply with the requirement/ tolerance range specified
above, the instrument should be labeled “OUT OF CALIBRATION” and should be
repaired/ serviced.
6.7.9.2
After repair/
maintenance, calibrate the instrument.
6.8
Note down the
report in the internal calibration certificate and in instrument logbook.
7.0 REVISION LOG:
|
Revision No.
|
Effective Date
|
Reason
|
|
00
|
|
New
SOP
|
8.0 REFERENCES:
8.1 The British Pharmacopoeia. Vol V., Official Monograph /
Ultraviolet and visible Absorption Spectrophotometry: 2015, pp. 169-170.
9.0 ANNEXURES:
Annexure 1: Calibration
Report of UV/VIS Spectrophotometer.
Annexure:
1
Calibration
Report of UV/VIS Spectrophotometer
|
CALIBRATION
REPORT OF UV/VIS SPECTROPHOTOMETER
SOP ref.:
______________________
Date: __________________
Instrument No.:
_________________
Frequency: once in a month.
Make:
_________________________
Model: _________________
1.
CONTROL
OF WAVELENGTH:
Holmium
perchlorate solution:
Holmium oxide:
Gross
wt.: ____________g.
Balance: ___________
Tare
wt.: _____________g.
Net
wt.: ______________g.
1.4M Perchloric acid:
Quantity:
_____________ml.
Dissolve
1.0g of Holmium oxide in sufficient 1.4M Perchloric acid solution. Make up to
25.0ml with the same solvent.
Absorption
maxima of Holmium perchlorate solution for control of wavelength.
Remarks: satisfactory/
not satisfactory.
2.
CONTROL
OF ABSORBANCE:
Solution
A:
Potassium dichromate:
Gross
wt.: ____________g.
Balance: ___________
Tare
wt.: _____________g.
Net
wt.: ______________g.
0.005M sulphuric acid:
Quantity:
_____________ml.
Dissolve
60.0mg of potassium dichromate in sufficient 0.005M sulphuric acid solution.
Make up to 1000.0ml with the same solvent.
Calculation:
1cmA1% = absorbance x 10000______
Wt. of potassium dichromate in mg
Solution
B:
Potassium dichromate:
Gross
wt.: ____________g.
Balance: ___________
Tare
wt.: _____________g.
Net
wt.: ______________g.
0.005M sulphuric acid:
Quantity:
_____________ml.
Dissolve
60.0mg of potassium dichromate in sufficient 0.005M sulphuric acid solution.
Make up to 100.0ml with the same solvent.
Calculation:
1cmA1% = absorbance x 10000______
Wt. of potassium dichromate in mg
Remarks: satisfactory/
not satisfactory.
3.
LIMIT OF STRAY LIGHT:
Potassium
chloride solution:
Potassium chloride:
Gross
wt.: ____________g.
Balance: ___________
Tare
wt.: _____________g.
Net
wt.: ______________g.
Water:
Quantity:
_____________ml.
Dissolve
and dilute 1.2g of potassium chloride in sufficient water to produce 100.0ml.
Remarks: satisfactory/
not satisfactory.
4.
RESOLUTION POWER:
0.02%
v/v toluene in hexane:
Toluene:
Quantity:
_____________ml.
Hexane:
Quantity:
_____________ml.
Dissolve
and dilute 0.02ml of toluene in sufficient hexane to produce 100.0ml with
hexane.
Absorbance
at 269nm= __________________
Absorbance
at 266nm= __________________
Ratio= Absorbance at 269nm
Absorbance at 266nm
Limit: (not
less than 1.5).
Result: _______________
Remarks: satisfactory/
not satisfactory.
5.
WAVELENGTH ACCURACY, RESOLUTION &
BASELINE FLATNESS (INBUILT TEST):
Wavelength accuracy (inbuilt test):
Remarks: satisfactory/
not satisfactory.
Resolution (inbuilt test):
Remarks: satisfactory/
not satisfactory.
Baseline flatness (inbuilt test):
Remarks: satisfactory/
not satisfactory.
Conclusion: Instrument
working is satisfactory/ not satisfactory.
Next calibration due on: _______________________________
|
10.0
ABBREVIATIONS:
|
Abbreviation
|
Expanded Form
|
|
SOP
|
Standard operating
procedure
|
|
&
|
And
|
|
No.
|
Number
|
|
Ltd.
|
Limited
|
|
Q.C
|
Quality control
|
|
Q.A
|
Quality assurance
|
|
QCEB
|
Quality control
equipment calibration
|
|
F
|
Format
|
|
UV/VIS
|
Ultraviolet/ visible
|
|
g
|
Grams
|
|
mg
|
Milligram
|
|
M
|
Molar
|
|
ml
|
Milliliter
|
|
q.s
|
Quantity sufficient
|
|
nm
|
Nanometer
|
|
oC
|
Degree centigrade
|
|
λ
|
Lambda
|
|
%
|
Percentage
|
|
v/v
|
Volume by volume
|
|
Abs
|
Absorbance
|
|
Vol
|
Volume
|
|
Ref.
|
Reference
|
|
B.P
|
British Pharmacopoeia
|