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Calibration of UV-Visible spectrophotometer

In Pharmaceutical UV-Visible spectrophotometer is widely used for analytical purposes in the Quality control department. So to maintain its accuracy and working conditions, calibration of UV-Visible spectrophotometer need to be performed on a timely basis.

Calibration of UV-Visible spectrophotometer by parts

Check the following During the calibration of UV-Visible spectrophotometer

Calibration of UV-Visible spectrophotometer
  1. Absorption cells
  2. Control of absorbance
    • For UV region
    • For visible region
  3. Photo metric linearity
  4. Limit of stray light
  5. Resolution power
  6. Control of Wavelengths

Absorption cells

  • Fill the cuvette with milli-Q water at 240 nm, and absorption is not greater than 0.093 of the individual cuvette.
  • Rotate both cuvettes (180°) one by one.

Acceptance criteria: On rotation of the cuvette should give an absorbance difference not more than 0.005.

Control of absorbance:

For UV region

Solution A: Prepared 0.005M sulfuric acid:

Weigh accurately 60.0 mg of potassium dichromate, which previously dried to constant weight at 130°C in 1000 ml and dissolved in 0.005 M sulphuric acid.

Measure the absorbance at 235nm, 257 nm, 313nm, and 350 nm using 0.005 M H2SO4 as blank.

For Visible region

Solution B: Weight about 60.0 mg of potassium dichromate, which previously dried to constant weight at 130°C and transferred to 100 ml volumetric flask, dissolved in 0.005 M sulphuric acid.

Measure the absorbance at 430 nm using 0.005M sulphuric acid as blank.

Acceptance criteria

Soln. type/ wavelengths (nm)Specific abs.Max.Tolerance
Solution A: 235 124.5122.9 to 126.2
Solution A: 257 144.5142.8 to 145.7
Solution A: 313 48.647.0 to 50.3
Solution A: 350 107.3104.9 to 108.2
Solution B: 430 15.915.7 to 16.1

Photometric Linearity

Weight 50 mg Potassium dichromate and dissolved in 0.005 M Sulphuric acid in a 50 ml volumetric flask. Further, dilute the above solution in the following way

1 ml ≥ 100 ml

2 ml ≥ 100 ml

3 ml ≥ 100 ml

4 ml ≥ 100 ml

5 ml ≥ 100 ml

Take consecutive three readings of each dilution at wavelengths 235, 257, 313, and 350 nm.

Acceptance criteria:

At each wavelength mean value ≥of 0.999

Limit of stray light

Prepares 1.2 % v/v solution by using Potassium chloride in 50 ml Distilled water and make up the volume up to 100 ml by using distilled water. Run the solution at 200 nm using water as a blank.

Acceptance criteria: Absorbance at 200 nm should be greater than 2.0

Resolution power

Prepared 0.02% v/v solution by using toluene in hexane and scanned at 250 nm to 300 nm using hexane as a reference.

Record the absorption at 269 nm and 266 nm.

Calculate the absorption ratio by dividing the absorbance at 269 nm (Maxima) and 266 nm (minima).

Acceptance criteria: The absorbance maxima at 269 nm to the minima at 266 nm should not be less than 1.5.

Control of Wavelengths

This is a very important test we can verify that instrument wavelength and the detector is properly working, by using the Holmium perchlorate method.

Dissolved 1.0 g of holmium oxide in 1.4 M perchloric acid by heating on a water bath, keep aside for cool and then dilute to 25 ml by using the same solvent to prepare a 4.0% w/v solution of holmium oxide.

Record the spectrum of holmium perchlorate solution from 200 nm to 600 nm using 1.4 M perchloric acid as a reference solution.

Acceptance criteria

Maxima Wavelength (nm)Maxi. Tolerance (nm)
241.15 240.15 to 242.15
287.15 286.15 to 288.15
361.50 360.50 to 362.50
536.30 533.30 to 539.30

By using Discharge Lamp Methods:

Take 100 mg potassium dichromate in 0.05 N KOH (use 20 ml KOH), then further makeup volume up to 500 ml with the same solution. Use the blank 0.05 N KOH to take the reading by using UV. Take reading after autozero by using the above solution and scan the wavelength from 340 to 400 nm.

Acceptance criteria: maxima length shall be observed at 370 nm to pass the test.

Out of Calibration

Suppose the instrument or any parts of it are found out of calibration. Immediately Stopped any activity and Labeled the instruments “Out of Calibration.” Follow the “Handling of out of calibration” SOP.

Frequently Asked Questions (FAQs):
Why do we use Potassium Dichromate for the Linearity test?

Because It has more solubility and stability as well as that it may be the primary standard so we used pot. di chromate in the linerity of calibration steps.

Why there is a need for calibration?

When the addition of a new instrument, time elapsed, and variations.

Differernce between UV region and Visible region

The main differrence is a range between them, UV ranges is from 200 to 400 nm, and Range for visible is 400 to 800 nm.

Types of Cuvettes used in UV-Spectrophotometers is?

Four types; UV qaurtz, IR qaurtz, Saphire qaurtz, Pirex glass and Optical glass

Can we determine qualitative and quanitative analysis by using UV-Spectrophotometers?

Yes, We can use UV-Spectrophotometers for both qualitative and quanitative analysis.

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