UV/VIS spectrophotometer Calibration SOP


     
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 Ϸ 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.
Sr.#
Maxima wavelength (nm)
Tolerance (nm)
Observed wavelength (nm)
1.
241.15nm
240.15nm to 242.15nm

2.
287.15nm
286.15nm to 288.15nm

3.
361.5nm
360.50nm to 362.50nm

4.
536.3nm
533.30nm to 539.30nm


Remarks: satisfactory/ not satisfactory.
Performed by/date
Checked by/ date


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.
Sr.#
Wavelength (nm)
Absorbance
Specific absorbance
A (1%,1cm)
Maximum tolerance (nm)
1.
235.0


122.9 to 126.2
2.
257.0


142.8 to 146.2
3.
313.0


47.0 to 50.3
4.
350.0


105.6 to 109.0
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.
Sr.#
Wavelength (nm)
Absorbance
Specific absorbance
A (1%,1cm)
Maximum tolerance (nm)
1.
430.0nm


15.7 to 16.1
Calculation: 
                                     1cmA1% =                  absorbance x 10000______
Wt. of potassium dichromate in mg














Remarks: satisfactory/ not satisfactory.
Performed by/date
Checked by/ date



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.

Sr.#
Wavelength (nm)
Absorbance
Tolerance
1.
198.0nm


2.
199.0nm


3.
200.0nm


4.
201.0nm


5.
202.0nm









Remarks: satisfactory/ not satisfactory.
Performed by/date
Checked by/ date



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.
Performed by/date
Checked by/ date



5.      WAVELENGTH ACCURACY, RESOLUTION & BASELINE FLATNESS (INBUILT TEST):
Wavelength accuracy (inbuilt test):
Sr.#
Wavelength standard (nm)
Wavelength observed
(nm)
Difference
Limit (nm)
1.
656.1nm


±0.3
2.
486.0nm


±0.3
Remarks: satisfactory/ not satisfactory.
Performed by/date
Checked by/ date



Resolution (inbuilt test):
Sr.#
Limit
Observation
1.
1.0nm or less

Remarks: satisfactory/ not satisfactory.
Performed by/date
Checked by/ date



Baseline flatness (inbuilt test):
Sr.#
Limit
Observation
1.
±0.002 Absorbance

Remarks: satisfactory/ not satisfactory.
Performed by/date
Checked by/ date


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
Wt.
Weight
B.P
British Pharmacopoeia


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