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Calibration of UV / Visible Spectrophotometer


Calibration of the UV spectrophotometer including control of absorbance using potassium dichromate solution, resolution power using toluene in hexane, limit of stray light and wavelength accuracy.
Ensure that the connections of the spectrometer are proper.

Control of Absorbance

Potassium Dichromate Solution :
Spectrophotometer Calibration in PharmaceuticalsDry 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 labelled “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 :

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
Email: .moc.enilediugamrahp@ofni Need Help: Ask Question


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