Spectrophotometer; Principles, Working, Types, and Applications


What is Spectrophotometer?

A spectrophotometer is a device that calculates how much light a sample has absorbed.

The Beckman DU spectrophotometer was developed in 1940 at the National Technologies Laboratory (NTL) by scientist Arnold J. Beckman and his coworkers.

A spectrophotometer measures the absorbance of a substance at a certain wavelength.

It uses a light source that can produce several wavelengths, each with a particular wavelength.

Part of the light source is absorbed after it passes through a test sample. The relationship between the sample’s absorbance and concentration is linear.

Light Absorption

When it hits a colored solution, light is either absorbed or transmitted. White light is made up of all colors, but a colored substance absorbs them all and only transmits one color. This color is what it is.

Before reading the test sample, the spectrophotometer must be calibrated by inserting a cuvette containing a control solution. Zeroing of the spectrophotometer refers to the procedure of device calibration.

Principle of a Spectrophotometer

The spectrophotometer method is used to calculate the wavelength-dependent intensity of light. This is accomplished by diffracting the light beam into a variety of wavelengths, using a charge-coupled device to measure the intensities, and visualizing the outcomes first on the detector and then on the display device.

When a beam of incident light with intensity I0 passes through a solution, some of the light is reflected (Ir), some of it is absorbed (Ia), and the remaining light is transmitted (photometric technique), according to the spectrophotometer.

Following the dispersive device and before the detector, the sample solution is put into the cuvette (glass tubes). The constant thickness and length of the optical path distinguish the cuvette from the test tubes. The cuvette is often constructed of quartz or glass. The spectrophotometer is founded on two basic equations of photometry that demonstrate the mathematical link between the amounts of light absorbed and the substance concentration.


The Chemistry behind Beer’s Law: A Closer Look

This was developed in 1852 by German scientist and mathematician August Beer, who stated that a substance’s absorptive ability is directly proportional to its concentration in a solution.

Beer’s Law can be expressed as follows:

                                              A = εcL


A= absorbance of light

ε = molar absorptivity

L = sample path length

c = solution concentration of the substance

The concentration of a specimen can be easily determined by making a graph using standard solutions, unlike many modern devices that calculate Beer’s Law calculations by simply comparing a blank cuvette with a sample. In the graphing approach, absorbance and concentration are assumed to have a linear relationship, which is ideal for diluted solutions. This was developed in 1852 by German scientist and mathematician August Beer, who stated that a substance’s absorptive ability is directly proportional to its concentration in a solution.

The Chemistry behind Beer’s Law: A Closer Look

According to Lambert’s law of absorption, similar parts inside the same absorbing material absorb equivalent amounts of light that penetrate them.

Lambert studied the transmission of monochromatic light (light with a single wavelength) by uniform solid materials. He noticed that each additional unit layer of the medium absorbed an equal amount of the light passing through it, demonstrating the significant influence the thickness of the solid substance had on how much light transmitted through it. The term “light path length” refers to the medium’s thickness.

In its simplest form,

Beer-law, Lambert’s which states that the amount of light absorbed by a color solution is directly proportional to the solution’s concentration and the length of a light path through the solution, serves as the foundation for the Spectrophotometer’s operation.


A = cL


            A = Absorbance / Optical density of the solution

            c = Concentration of solution

            L = Path length

             ∈ = molar absorptivity

Working of Spectrophotometers

  • A spectrophotometer must first be calibrated before it can be used, and this is done using standard solutions with the known concentration of the solute that has to be determined in the test solution. For this, the standard solutions are poured into the cuvettes and positioned in the spectrophotometer’s cuvette holder.
  • The solution is illuminated by a beam of light with a specified wavelength that is used in the test. The light beam travels through a sequence of mirrors, prisms, and diffraction gratings before arriving at the solution. The diffraction grating allows the required wavelength to pass through it and reach the cuvette containing the standard or test solutions.
  • These mirrors are used for light navigation in the spectrophotometer. The prism splits the beam of light into multiple wavelengths. It examines the reflected light and contrasts it with a known standard solution.
  • Monochromatic light enters the cuvette, where some of it is reflected, some of it is absorbed by the solution, and the remainder is transmitted through the solution and lands on the photosensitive detector. The photosensitive detector translates the electrical impulses that are sent to the galvanometer from the measured transmitted light intensity.
  • The galvanometer takes electrical signal readings and converts them to digital information. The solution under analysis has an absorbance or optical density, which is a digital representation of the electrical signals.
  • More light will be absorbed by the solution if the absorption is higher, and more light will be transmitted through the solution if the absorption is lower. This influences the galvanometer reading and reflects the solute concentration in the solution.
  • The beam splitters used in double-beam spectrophotometers divide the monochromatic light into two beams: one for the standard solution and the other for the test solution. This allows the absorbance of the standard and test solutions to be measured simultaneously and allows for the comparison of any number of test solutions to a single standard.

Instrumentation of Spectrophotometer


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Types of Spectrophotometer Technologies

Visible Spectrophotometer

The visible spectrophotometer is a device that measures absorbance and performs statistical analysis at visible light wavelengths (400–1000 nm). The visible light area of 400–1000 nm is where the visible spectrophotometer measures wavelengths. Single-beam spectrophotometer that measures light in the visible spectrum.

It is a tool used to analyze the substance in both a qualitative and quantitative way by measuring how much of a specific wavelength of light is absorbed. The equipment is utilized in the fields of medicine and health, clinical testing, environmental monitoring, food production, and others. At 600 nm, the density of bacterial cells can be measured.

UV VIS Spectrophotometer

The UV VIS spectrophotometer is an analytical tool that uses the absorption of photons in the ultraviolet-visible spectrum region by material molecules for analysis. It is based on the idea of ultraviolet-visible spectrophotometry. The use of this equipment allows for both qualitative and quantitative examination of the sample’s absorbance to visible or ultraviolet light.

There are three types of optical paths for the product: single beam, pseudo-double beam, and double beam. This equipment is currently one of the most widely utilized analytical tools available. It is extensively used in a variety of disciplines, including chemistry, chemical engineering, environmental science, agricultural science, biological science, and material science.

Infrared Spectrophotometer

The light released by the light source, which is split into two beams of equal energy and symmetry, is precisely what an infrared spectrophotometer refers to. The reference beam serves as the reference, while the first beam is the sample light that passes through the sample.

The fan-shaped mirror modulates the two light beams at a specific frequency after they reach the photometer through the sample chamber to create an alternating signal. The infrared spectrum longer than 760 nm is the general infrared spectrum.

Fluorescence Spectrophotometer

A fluorescence spectrophotometer is a device used to scan the fluorescence spectrum generated by fluorescent markers that resemble liquids. It can offer a variety of physical metrics, such as the excitation spectrum, emission spectrum, fluorescence intensity, quantum yield, fluorescence lifespan, fluorescence polarization, etc., that can reflect different aspects of the bonding and structure of molecules.

In order to better understand the connection between molecular structure and function, these factors can be measured in order to do general quantitative analysis as well as infer the conformational changes of the molecule in diverse settings.  A fluorescence spectrophotometer typically has an excitation wavelength scanning range of 190–650 nm and an emission wavelength scanning range of 200–800 nm.

Atomic Absorption Spectrophotometer

Another name for an atomic absorption spectrophotometer is an atomic absorption spectrometer.  This equipment evaluates the metal elements which is Based on how the ground state atomic vapor of the substance affects the characteristic radiation absorption. The distinctive spectrum radiation to be measured from the light source is emitted, and after passing through the atomizer, it is absorbed by the ground state atoms of the measuring elements in the sample vapor.

By measuring the amount of characteristic radiation absorbed, the content of the element being examined can be determined. It has developed into a potent tool for material analysis and quality control departments to examine major and trace metals because it can accurately and consistently identify trace elements (semi-metals).

Portable Spectrophotometers

You may take portable spectrophotometers with you wherever and at any time. It is easy to carry around in your pocket and pull out as needed.

Bench-top Spectrophotometer

This instrument is the best option for laboratory-based procedures, particularly when the highest level of precision and control is required.

Single beam spectrophotometer

It uses a single beam of light that ranges in wavelength from 325 nm to 1000 nm. Since the light only has one direction of travel, the test solution and blank are read in the same direction. suitable for detecting absorbance or light transmittance at a certain wavelength but not typically for full-band spectral scanning because this requires a very high light source and detector stability.


Double Beam Spectrophotometer

The performance of a double-beam spectrophotometer is superior to that of a single-beam instrument in the measurement of turbid samples as well as high concentrations and mixed multi-component samples.

In particular, for structural analysis, gets rid of light source instability, detector sensitivity variations, and other issues. Using two monochromators, the double beam UV–visible spectrophotometer can produce monochromatic light with two distinct wavelengths. At regular intervals, the same sample cell is irradiated alternately by the two light beams.


Applications of the Spectrophotometer

It is used in a variety of important applications, some of which are listed below:

  • Substance concentration detection
  • Finding of impurities
  • Tracking the amount of dissolved oxygen in freshwater and marine ecosystems
  • Biochemical analysis of proteins
  • Elucidation of chemical compound structures
  • Identification of functional groups
  • Hospitals analyze the respiratory gas flow
  • Calculating the molecular weight of chemicals
  • Classification of chemicals in both their pure form and in biological preparations can be done using the visible and UV spectrophotometer
  • The optical density or its absorbance can be measured using a spectrophotometer to determine the concentration of both colored and colorless chemicals
  • The rate of formation and absorption of the light-absorbing compound in the visible and ultraviolet (UV) regions of the electromagnetic spectrum can also be utilized to determine the reaction path

After reading this article, I hope that you will have a better understanding of the principles, working, types, and different applications of spectrophotometers.


How is a spectrophotometer used?

Spectrophotometers are used in a variety of applications, including analyzing the purity of chemicals, studying the properties of pigments, and determining the concentration of a chemical in a solution. They are also used to analyze biological samples, such as proteins, enzymes, and DNA.

What factors can affect the accuracy of spectrophotometer measurements?

There are several factors that can affect the accuracy of spectrophotometer measurements, including the purity of the reference solution, the quality of the light source, and the temperature of the sample. It is important to carefully control these variables in order to obtain accurate results.

What is the diferrence between spectrometer vs spectrophotometer

A spectrometer and a spectrophotometer are both instruments that are used to measure the properties of light and to analyze the composition of materials. However, there are some key differences between the two:
1. Range of wavelengths measured: Spectrometers are generally able to measure a wider range of wavelengths than spectrophotometers. Spectrometers are often used to measure the entire electromagnetic spectrum, while spectrophotometers are typically limited to the ultraviolet, visible, or near-infrared regions.
2. Type of measurement: Spectrometers measure the energy or frequency of the light, while spectrophotometers measure the intensity of the light.
3. Type of information obtained: Spectrometers are often used to identify the elements present in a sample, while spectrophotometers are used to determine the concentration of a specific substance in a sample.
Precision: Spectrometers are typically more precise than spectrophotometers, as they are able to measure the energy or frequency of light with higher accuracy.
Overall, spectrometers and spectrophotometers are both important tools in a variety of scientific fields, and they are often used in combination to analyze materials and understand their properties.

What are the three main components of a spectrophotometer

A spectrophotometer typically consists of three main components:
Light source: The light source generates a beam of light that is directed through the sample. The type of light source used depends on the type of spectrophotometer and the wavelengths of light being measured.
The monochromator is a device that filters the light from the light source and selects a specific wavelength. It is typically a prism or grating that is used to separate the light into its individual wavelengths.
Detector: The detector measures the intensity of the light after it passes through the sample. The detector is typically a photodetector, such as a photodiode or photomultiplier tube, which converts the light into an electrical current that can be measured.
There are other components that may be included in a spectrophotometer, such as a sample holder, a display screen, and controls for adjusting the wavelength and intensity of the light.



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