Basis of Spectrophotometer

A Spectrophotometer is a crucial tool used in various fields such as biotechnology, life science, chemical, forensics, agriculture, and more. It is used for quantitative and qualitative analysis of chemical and biological samples. It is interesting to know the basis of spectrophotometry and its configuration.

The spectrophotometer is the most common and inexpensive technique to measure light absorption and the quantity of chemicals present in the samples. The basis of a spectrophotometer is the light and the color. In simple words, it is said the color of the object is dependent on light.

Color and Light

Spectrophotometer color and light

The color of the object we see is the effect of the light produced on the object. When the light passes through the object, it gets either reflected, absorbed, or transmitted. For example, Glass transmits light radiation that comes in contact with it and so it is colorless. The snow reflects all the light that comes in contact with it and so it is white in color. 

A black color object absorbs all the light, a red color object reflects more red color than the other colors. What does this color of the object or solution mean? It resembles the chemical structure that absorbs, reflects, or transmits a certain wavelength of light.

We use white light as a source. A beam of white light constitutes many rays, when passed through a prism  (a triangular transparent object) the white light is dispersed into several bands of color. 

These dispersed seven bands of color that constitute red, orange, yellow, green, blue, indigo, and violet are called the spectrum. The band of spectrum always constitutes these colors and is organized in this order from left to right.

Here is the color variation chart, when the system undergoes a change in concentration of some substance,  the color of the substance also varies. It is the basis for colorimetric analysis.

Colorimetric analysis

For example, when the white light is passed through the yellow object. The light that is transmitted is mostly yellow and the complementary color blue is absorbed. This yellow color that is transmitted reaches our eyes and we see the object as yellow.

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Colorimetry, as the name mentions, is the measurement of color. It measures the relative absorption of light through which the concentration of the substance is determined. 

Generally, natural or artificial white light is used as a light source to determine the color and the instrument is termed a colorimeter. When the photoelectric cell is used as a detector, then it is termed a photoelectric colorimeter.

Colorimetric analysis works on the principle of the unique nature of light-absorbing characteristics of substances and their nature of forming colors. The color formed indicates the contraction of the substance. 

When the light radiation (I₀) is passed through a substance, the molecules of the substance absorb a portion of light and the radiation of intensity (I) is emitted. This difference in intensity of light radiation determines the colorimetric.

According to Lambert-Beer's law, the quantity of light radiation absorbed is given by, 

A = Ɛ · l · C

A    is absorbance

Ɛ   is the molar extinction coefficient [L/(mol·cm)]

l     is path length (cm)

C     is concentration (mol/liter)

Photometer vs Spectrophotometer

The photometer is used to measure light or electromagnetic radiation. The Photometer also separates the specific wavelength of electromagnetic radiation using filters. Band filters separate light into different color components and then match with the color components that we can see with our eyes. 

This provides the color value that is visible to the eye and does not provide any data that is invisible to the human eye. Thus spectral data requires a spectrophotometer to know the more detailed characteristics of the spectrum.

Spectrophotometers differ from photometers as we can measure the spectrum of all wavelengths of visible light and not the specific wavelength that is visible to our eyes. The spectrophotometer uses grating to separate the light spectrum. 

Then detector such as an array of silicon photodiodes or a photomultiplier tube is used to read the spectral data. This is ideal for the analysis of spectral emission of the light source and so it is widely used in scientific devices.

Different types of spectrophotometers based on optical configurations

Single Beam

In a single-beam spectrophotometer, the blank and the sample are measured consecutively, for a single wavelength to full-spectrum measurement with a conventional instrument. Over a long time, it may result in a significant error such as spectral drift.

Different types of spectrophotometers based on optical configurations

Double Beam

In a double-beam spectrophotometer, the sample and blank spectrum are measured simultaneously. In a double-beam spectrophotometer, the single light beam is split with a light chopper and produces two light beams of equal energy and optical path. 

One beam is passed through the reference sample and the other is passed through the sample for which the analysis is to be done. A Single beam spectrophotometer has high throughput and sensitivity as it involves fewer optical components compared to that of a double beam spectrophotometer.

Split Beam

The split beam spectrophotometer is similar to that of a double beam spectrophotometer only difference is it uses a beam splitter instead of a chopper to separate light beams of equal intensities to pass through the reference and the sample. 

This setup aslo enables the measurement of blank and the sample at a time. This setup is simpler than compared to that of the true double-beam spectrophotometer.

Applications of spectrophotometer 

The spectrophotometer is widely used in various industries and research fields. Some of the best uses of spectrophotometer include,

  • Concentration determination
  • Monitoring analyte concentration vs. time
  • Physico-chemical parameters
  • Elemental determination of water quality
  • Enzymatic analysis in wine 
  • To determine the purity of olive oil


The spectrophotometer works on the basis of light or electromagnetic radiation. It is crucial to understand the nature of light and the color formation of objects. The light absorbed by the particular object or solution defines the characteristics of the object or solution.

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