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Calibration of UV / Visible Spectrophotometer


Learn how to calibrate the UV - Visible spectrophotometer using Potassium dichromate Solution.

Ensure that the connections of the instrument are proper.

Control of Absorbance

Potassium dichromate Solution :
Dry a quantity of potassium dichromate by heating to constant weight at 130°C. Weigh & transfer accurately a quantity not less than 57.0 mg & not more than 63.0 mg to 1000 ml volumetric flask.   Dissolve & dilute in sufficient 0.005M H2SO4 to produce 1000 ml.
Measure the absorbance of potassium dichromate solution at the wavelengths given below.
Calculate value of A (1% 1cm) for each wavelength.
A (1% 1cm) = Absorbance X 10000 / Weight of Potassium dichromate in mg.
Acceptance criteria
Wavelengths (nm)
A [1% 1cm]
Limit
235.0
124.5
122.9 - 126.2
257.0
144.0
142.8 - 145.7
313.0
48.6
47.0 - 50.3
350.0
106.6
105.6 - 108.2
Stringent limits are adopted from IP & BP for acceptance criteria

Resolution power

Record the spectrum of a 0.02% v/v solution of toluene in hexane in the range of 260 nm to 420 nm (before use check the hexane for transmittance, using water as a blank between 260nm to 420nm & use only if transmittance is not less than 97%).
Acceptance criteria:
The ratio of the absorbance at the maximum at about 269nm to that at the minimum at about 266nm is not less than 1.5.

Limit of Stray Light

Prepare a 1.2 % w/v solution of Potassium chloride in water.
Measure absorbance of the above solution at  198.0, 199.0, 200.0, 201.0, 202.0 nm using water as blank.
Acceptance criteria:
Absorbance is greater than 2.

Wavelength accuracy, Resolution & Baseline flatness (inbuilt test)

Attach printer directly to the instrument (instead of computer).
Go to Þ MODE press F3 key i.e. Maintenance.
Press ‘1’.
Press Start/Stop key.
After screen changes, ensure that nothing is kept in the optical path & press the Start/Stop key again.
Prints will come after all the three tests are over.
Acceptance criteria:
For wave length accuracy : at 656.1 ± 0.3 nm & at 486.0 ± 0.3 nm.
Resolution: 1.0 nm or less.
Baseline flatness: ± 0.002Abs.
Frequency: Once in a month.

Maintenance / Repairs

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.
After repair / maintenance, calibrate the instrument.
REFERENCE
Indian Pharmacopoeia
British Pharmacopoeia
Instruction manual of UV-1700.

CALIBRATION REPORT OF UV / VISIBLE SPECTROPHOTOMETER

Page 1 of 2
SOP Reference –
Date -
Instrument No. 
Frequency
: Once in a month
Make –

             Model –

1.
Control of Absorbance:
Potassium dichromate solution :
Gross Wt. :  ………………….                                 Balance :
Tare Wt. :  ………………….
Net Wt.    : ………………….                                        ………..           ml with 0.005M sulphuric acid.
Dissolve and dilute 57.0 to 63.0 mg of potassium dichromate in sufficient 0.005M sulphuric acid to produce 1000.0 ml.
Wavelength (nm)
Absorbance
A (1%, 1cm)
Tolerance
235.0


122.9 to 126.2
257.0


142.8 to 145.7
313.0
47.0 to 50.3
350.0
105.6 to 108.2
Calculation :
             1%                    Absorbance  x 10000
          A1 cm    = -------------------------------------------------------
                          Weight of potassium dichromate in mg
Remark : Satisfactory / Not satisfactory.
Performed by :
Date :
Checked by :
Date :
2.
Resolution power :
0.02% v/v toluene in hexane : 
             ……….ml   → 100.0 ml with hexane  / ………  ml   → 100.0 ml with hexane
Absorbance at 269 nm = ……………
Absorbance at 266 nm = ……………
                          Absorbance at 269 nm
                Ratio = ---------------------------------- =    ---------------- =
                          Absorbance at 266 nm                             
  (Limit : Not less than 1.5)
Remark : Satisfactory / Not satisfactory.
Performed by :                                                                 Checked by:
Date :                                                                                Date           : 

 

CALIBRATION REPORT OF UV / VISIBLE SPECTROPHOTOMETER

Page 2 of 2
3.
Limit of stray light:
Potassium chloride solution
Gross Wt.                :  ………………….                                  Balance :                 
Tare Wt. :  ………………….
Net Wt.    : ………………….                          ……………. ml with water.
Dissolve and dilute accurately 1.2 g potassium chloride  in sufficient water to produce 100.0 ml.
Sr. No.
Wavelength (nm)
Absorbance
Tolerance
1
198.0
Absorbance is greater than 2
2
199.0
3
200.0
4
201.0
5
202.0
Remark : Satisfactory / Not satisfactory.
Performed by :
Date :
Checked by :
Date :
4.
 Wavelength accuracy (In built test)
Sr. No.
Wavelength Standard (nm)
Wavelength Observed (nm)
Difference
Limit (nm)
1
656.1
+ 0.3
2
486.0
+ 0.3
Remark : Satisfactory / Not satisfactory.
Performed by :                                                                 Checked by:
Date :                                                                                Date           : 
5.
Resolution (In built test)
Sr. No.
Limit
Observation
1
1.0 nm or less
nm
Remark : Satisfactory / Not satisfactory.
Performed by :                                                                 Checked by:
Date :                                                                                Date           : 
6.
Baseline flatness (In built test)
Sr. No.
Limit
Observation
1
± 0.002 Absorbance
Remark : Satisfactory / Not satisfactory.
Performed by :                                                                 Checked by:
Date :                                                                                Date           : 
Conclusion : Instrument working is Satisfactory / Not satisfactory.
Next calibration due on :

Performed by :
Date :
Checked by :
Date :




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