Spectrophotometer Principle

Spectrophotometer is an instrument that measures the amount of intensity of light absorbed by the sample solution as a function of wavelength. This technique of measuring the amount of absorbed light through sample solution is known as spectrophotometry. Uses of spectrophotometer includes quantitative analysis of various known compounds in a mixture. It is used in various fields such chemistry, biochemistry, chemical engineering etc. This instrument is used by scientists also for various purposes. 

The spectrophotometer was invented by Arnold J. Beckman in 1940. The instrumentation of spectrophotometer is given below – 

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Working of spectrophotometer - In spectrophotometer, commonly used UV radiation sources are hydrogen and deuterium lamps and for visible radiation tungsten filament lamp is used. While for IR radiation Nernst Glower is used. A monochromator is used to resolve polychromatic radiation into individual wavelengths and differentiate them into very narrow bands. A monochromator includes collimator, prism or grating and a slit. Sample solution is taken into cuvettes or sample containers. One detector is used to detect the current. Generally, photocells are used as detector. So, it works on photoelectric effect. The current which detector detects is proportional to the light intensity so indirectly it measures the light intensity. Digital meter displays the reading. 

What is spectrometer? 

It is used as a part of spectrophotometer. It is a device for detecting and analyzing wavelengths of electromagnetic radiation, generally used for molecular spectroscopy. In spectrophotometer, it is used to produce desired range of wavelength of light. Thus, spectrometer uses electromagnetic radiation for spectroscopic analysis of sample materials. 

Photometer is also used in spectrophotometer. Photometer detects the amount of intensity of light. It includes detector or digital display. 

Spectrophotometer principle 

Spectrophotometer can be operated in UV region, Visible spectrum and IR spectrum as well. This instrument is based on photometric technique. According to photometric technique when a beam of incident light of intensity I0 , passes through a solution, a part of the incident light is reflected, a part is absorbed and rest of the light is transmitted. If part of incident light which got reflected is Ir , part which got absorbed is Ia and part which got transmitted is It . Then we can write – 

I0 = Ir + Ia + It

In spectrophotometer Ir is eliminated because measurement of It and I0 is enough for the measurement of Ia. So, Ir is kept constant. Relationship between the amount of light absorbed and the concentration of the substance can be established by following two laws- 

Beer’s law 

According to this law the amount of light absorbed is directly proportional to the concentration of solute in the solution under analysis. 


Where Ia = light absorbed 

c = concentration of solute in the solution. 

Lambert’s law 

According to this law the amount of light absorbed is directly proportional to the length or thickness of the solution under analysis. 

Ia l

Where Ia = light absorbed

l = length or thickness of the solution 

Thus, in simple words spectrophotometer is based on the Beer-Lambert Law which states that the amount of light absorbed is directly proportional to the concentration of the solute in the solution and thickness of the solution under analysis. 

A cl

Where A = absorbance 

c = concentration 

l = path length 

A = cl

Where = absorption coefficient 

Difference between spectrometer and spectrophotometer 

By spectrometer, we can measure the wavelength of absorbed light and reflected light while by using spectrophotometer, we can measure the relative intensity of light absorbed and reflected. 

Applications of spectrophotometer 

  • Spectrophotometer is used to know the concentration of solutes colourless or coloured in a solution. 

  • It is used for determination of rate of reaction by measuring the rate of formation and disappearance. 

  • It is used in forensic sciences. 

  • It is used in molecular biology. We can measure the growth of microorganisms like bacteria by spectrophotometer. 

  • UV – spectrophotometer is used in pharmaceutical industry to determine the composition of the drugs. 

  • It is used in foods and paints industry. 

  • It is used in water quality check. 

  • Blood is analysed by spectrophotometer. 

  • It is used in diagnosis of diseases. 

  • It is used in detection of impurities in organic compounds. 

This topic is the first detailed introduction of spectroscopy with students. This an important topic for students to make their base stronger for higher studies in spectroscopy or chemical sciences.