Calibration of the UV spectrophotometer

Learn the UV/VIS spectrophotometer calibration process, including wavelength check with holmium oxide, resolving power with toluene in hexane, absorbance check with a potassium dichromate solution and limit scattered light with potassium chloride solution.

UV-Vis (ultraviolet-visible) spectrophotometry is a technique for measuring the absorption of light in the ultraviolet and visible wavelength range of the electromagnetic spectrum. Incident light can be absorbed, transmitted, or reflected when it strikes objects. Atomic excitation is due to the absorption of UV-Vis light, which refers to the transition of molecules from a low-energy ground state to an excited state.


  1. Purpose of calibration
  2. Preparation of the solvent
  3. Compartment classification
  4. Wavelength control
  5. Absorbance control
  6. Stray light limit
  7. resolving power
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Purpose of UV/VIS Spectrophotometer Calibration:

The purpose of calibration is to ensure the performance, the accuracy of the result and to establish the reliability of the spectrometer. It is used in qualitative and quantitative analysis of analytes based on Lamberts law of beer, which is why we often need to verify instrument performance either in-house or by a service engineer.

UV calibration is one of the basic requirements to ensure compliance with Indian Pharmacopoeia (IP) and British Pharmacopoeia (BP) standards. The instrument qualification test parameter includes wavelength control, absorbance control, stray light limit and resolving power.

Wavelength control: It is a process by which accuracy is determined by scanning a known solution for the maximum lambda.

Absorbance control: It is a process by which precision is determined by manually scanning a chromophore solution and recording the absorbance.

Stray Light Limit: Stray light is electromagnetic radiation that is not needed for spectrophotometric analysis and only interferes with the process.

Resolving power: It is the ability to resolve spectral features and bands into their separate components.

Calibration of the UV/VIS spectrophotometer

Preparation of solvents required for calibration:

Preparation of the perchloric acid solution (1.4 M):

Take 11.50 ml of AR grade perchloric acid (70%) with a pipette, dilute with 100 ml of distilled water in a clean, dry volumetric flask and mix well.

Preparation of the holmium oxide solution (4% w/v):

Take 01.00 g of AR grade holmium oxide and dissolve it in a 1.4 M perchloric acid solution using a heater. Once completely dissolved, bring the volume to 25 ml.

Preparation of the sulfuric acid solution (0.005 M):

Take 0.6 ml of sulfuric acid (H2SO4) with a pipette, dilute 2000 ml of distilled water in a volumetric flask and mix well.

Preparation of potassium dichromate solution:

Take 57-63 mg of pre-dried AR grade potassium dichromate (K2Cr2O7) and dissolve in 1000 ml of 0.005 M sulfuric acid.

Preparation of potassium chloride (1.2% w/v):

Take 01.20 g of previously dried potassium chloride and dissolve in 50 ml of distilled water. When completely dissolved, make up the volume to 100ml with distilled water.

Preparation of the toluene solution in hexane (0.02%):

Pipette 01.00 mL of HPLC grade toluene, dilute with 50 mL of HPLC grade hexane in a volumetric flask and mix well. Take 0.5 ml of this solution and further dilute to 50 ml with hexane.

Calibration procedure:

Here are the UV Visible Spectrophotometer calibration parameters and their strict limits are adopted from IP, USP and BP Pharmacopoeia for the acceptance criteria. For operation of the instrument, follow the standard operating procedure (SOP) and laboratory operating guidelines.

Classification of tanks:

Take the transmittance of the clean, pristine cuvette at 200 nm. Acceptance criteria at 200 nm are T% > 80% and must agree within 1.5%.

Fill the cuvette with Millipore water, take the absorbance at 240 nm, record the reading. The individual cuvette absorbance acceptance criterion should not exceed 0.093.

Wavelength control:

Take the UV spectrum from the holmium oxide filter or prepare a 4% w/v holmium oxide solution in the range of 200 nm to 600 nm using 1.4 M perchloric acid as a blank .

Acceptance criteria :

Wave length



240.15 to 242.15


286.15 to 288.15


360.50 to 362.50


533.30 to 539.30

Absorbance control:

Take the absorbance of 1.2% potassium dichromate at the wavelength shown below and calculate the value of A (1% 1 cm) for each wavelength. A (1% 1cm) = absorbance x 1000/weight of K2Cr2O7.

Acceptance criteria :




122.9 to 126.2


142.8 to 146.2


47.0 to 50.3


105.6 to 109.0

Stray Light Limit:

Using water as a blank, measure the absorbance of the prepared 1.2% potassium chloride solution using a 1 cm path length at 200 nm. Acceptance criteria for absorbance at 200 nm must be greater than 2.0.

Resolving power:

Take the UV spectrum of the prepared 0.02% v/v toluene solution in the range of 250 nm to 300 nm using hexane as a blank. Record absorbance at 269 nm for maxima and 266 nm for minima. The acceptance criteria for the calculated resolution must not be less than 1.2.

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