A spectrophotometer is a spectroscopic instrument that resolves polychromatic radiations into different wavelengths and then measured the light intensity at several wavelengths. It provides the measurement of absorbed light. Particularly, it is designed for the measurement of ultraviolet, visible, and infrared rays.

Spectrophotometric Instrumentation

This instrument consists of the following components:

  • Radiation source

It provides the continuous beam of EMR radiations in the required wavelength.

  • Monochromator

It is used to disperse the light into its components and then to choose a narrow range of wavelengths for analysis.

  • Sample holder

It depends upon the physical appearance of the sample, Such as in UV vis the dilute solution is taken in cuvettes

  • Detectors

It is a device, usually that converts the radiant energy into an electric signal.

  • Readout device

The electronic devices that will display the response of the detector

Spectrometer vs Spectrophotometer

A spectrometer is a component of a spectrophotometer, which detect the range of wavelengths whereas, a spectrophotometer is a device to analyze the sample analyte and qualifies the basis of absorption and emission of characteristic wavelengths.


The working principle of spectrophotometers is the interaction of light (electromagnetic radiations) EMR with matter. when a light source illuminates the sample it excites the energy levels of its components such as the nucleus, electrons, and bond pairs, based on provided energy. However, the interaction of light of different ranges has different consequences.

For example, when Low energy radio waves are used to illuminate the sample they resonated with nuclear energy and the UV-visible range excites the valence electrons of the sample. 

How it works

According to quantum chemistry, light is the form of energy. That energy is consist of small energy packets called quanta. In spectrophotometry, the light energy that is absorbed or emitted during excitation and de-excitation is fixed and can be used for quantification purposes. Because the amount of light absorbed depends on the amount of analyte and path length.

Types of spectrophotometer

Spectrophotometry is classified on the basis of the use of light sources and the type of interaction studied. Such as Nuclear magnetic resonance used radio waves while infrared rays are absorbed by chemical bonds in IR spectroscopy. Some of the commonly used spectrophotometers are discussed below.

Single beam spectrophotometer

It used a single beam from the light source. The incident light coming from the source is passed through a monochromator. Then that monochromatic light moves through a grating. Then it passes through the sample solution. Where some of the incident light is absorbed by the sample while other is transmitted. That transmitted light is detected by the detector. the commonly used detector is a photomultiplier tube. The spectrum is plotted and the λmax is located.

A single beam spectrophotometer comprises the following components;


single beam UV-vis spectrophotometer

The graph of amoxicillin obtained at the recorder is as under,

spectral analysis of amoxicillin in uv-vis spectrophotometer Spectronic 20TM

Since 1980, It is most commonly used UV visible spectrophotometer. the reason behind its application is its convenience ad used friendly operation. Firstly it is calibrated with the standards solutions. Then blank is performed to minimize the matrix effect. The blank cuvette is replaced by a sample’s cuvette and so on.

Double beam UV-Visible spectrophotometer

The double beam instrument is almost similar to a single beam but the only add-on is the light-beam divider. The significance of this instrument is that the analysis of a sample and reference or blank can be done simultaneously.

The incident light splits and is directed towards both the reference and sample cuvette. The refracted or transmitted beam is detected by the detectors. A double beam UV-vis spectrophotometer needs two detectors that detect electron ratio to measure or calculate absorbance in a test sample. It also requires a stabilized voltage supply.

double beam UV vis spectroscopy


Nanodrop spectrophotometers are highly accurate instruments. They can be used for the analysis of up to 2 microliters of sample. They are available under the brand names nanodrop 2000TM and nanodrop 8000TM.

iR spectrophotometer

Infrared spectrophotometers are used to detect the functional groups of organic compounds. It is considered as the best tool to fast detection and qualitative analysis.

Handheld spectrometer

Nowadays, some portable handheld devices are available in the market for the detection and estimation of samples in the working fields, environmentalists mostly use them. It greatly minimizes the freight during research work. some common examples, the x rite spectrophotometer, dr3900, and hach-dr3900.


spectrophotometers are utilized estimation of metals of chemistry biology and many others.

  • Determination of Iron
  • Determination of Iron in Vitamin Tablets
  • Determination of Nitrate Nitrogen in Water
  • Determination of Lead on Leaves Using Solvent Extraction
  • Determination of Inorganic Phosphorus in Serum
  • Reaction progress
  • estimation of blood profile

Concepts Berg

What Is a Spectrophotometer and How Does It Work?

It is an optical instrument used to detect the concentration of samples, both qualitatively and quantitatively.

What is the basic principle of a spectrophotometer?

It works on the principle of light interaction with matter. For example, a UV-Vis spectrophotometer work on the beer’s lambert law which states that the absorption of light, when passed through a sample solution, is directly proportional to the concentration and path length.

What are the three main components of a spectrophotometer?

The three major components of a general spectrophotometer are as follows,

  1. Light source e.g (Hollow cathode lamp)
  2. Sample holder e,g cuvettes
  3. Detectors

What is the unit of absorbance?

What are the types of spectrophotometers?

The common example of spectrophotometers are given below

  • UV- vis spectrophotometer
  • Nanodrop spectrophotometer
  • IR spectrophotometer
  • atomic absorption spectrophotometer
  • Fluorescence spectrophotometer
  • NMR spectrophotometer

Why do you blank the spectrophotometer?

The blank analysis is employed to nullify the possible absorption of light by the matrix such as solvent etc. The blank value is then subtracted from the sample reading.

Why is a spectrophotometer absorbance negative?

It can be negative if the reference value is greater than the sample results. This is because the sample is present in a dilute solution.

Why do we need to calibrate a spectrophotometer?

Calibration is done by preparing primary standard solutions of a known quantity. These samples are run in ascending order of concentration and the results are plotted on the graph. The best fit line is drawn through these points as a calibration curve. 

How does a spectrophotometer measure absorbance?

In the first step, the light source is used to illuminate the sample. The sample absorbed a characteristic wavelength. The amount of absorbed light is directly proportional to the concentration and path length. The light detect at the detector is the transmitted light. The absorbed light can be calculated from the formula,

A = log 1/T

What is the 1 cm path length cuvette in a spectrophotometer?

In UV visible spectrophotometer one cm cuvette is commonly used as its standard length. The  

Why is it bad to have bubbles in a cuvette when using a spectrophotometer?

According to Lambert’s law, the absorbance is directly proportional to the path length. therefore, if bubbles are present in the cuvette may deviate from accurate results.

Why do we use quartz cuvettes in the spectrophotometer?

Quartz materials are best to use as sample holders in UV vis spectrometer. This is due to the minimum absorbance of light and hence does not interfere with the results.

How does infrared spectroscopy differ from UV visible spectrophotometry?

IR spectrophotometry and UV spectrophotometry work on different principles of absorption of light. That is both absorbs light but of different ranges such as UV vis range is used to excite the valence electrons whereas, IR rays interfere with the bond energies.

Reference Books

  • Vogel’s Textbook of Quantitative Chemical Analysis sixth edition by  J Mendham, RC Denney, JD Barnes, M Thomas

Reference links