UV Spectroscopy- Definition, Principle, Steps, Parts, Uses

Spectroscopy is the study of how matter interacts with electromagnetic radiation, such as light. Different types of spectroscopy use different regions of the electromagnetic spectrum to probe the structure, properties, and behavior of molecules. UV spectroscopy is a type of absorption spectroscopy that uses ultraviolet (UV) light, which has wavelengths between 200 and 400 nanometers (nm) .

Absorption spectroscopy measures how much light is absorbed by a sample at different wavelengths. When a molecule absorbs UV light, it gains energy and some of its electrons are promoted from lower to higher energy levels . The energy levels of the electrons depend on the molecular structure and the type of chemical bonds . Therefore, by analyzing the absorption spectrum of a molecule, we can learn about its structure and identify functional groups that absorb UV light .

UV spectroscopy is widely used in analytical chemistry for the qualitative and quantitative determination of various substances in a sample . For example, UV spectroscopy can be used to detect impurities, elucidate structures, measure concentrations, and study reaction kinetics of organic and inorganic compounds that have chromophores . Chromophores are parts of molecules that absorb light and give color to substances. Some common chromophores are double bonds, aromatic rings, carbonyl groups, and metal ions .

To perform UV spectroscopy, we need a device called a spectrophotometer . A spectrophotometer consists of several parts that work together to produce and measure the absorption spectrum of a sample . The main parts are:

• A light source that emits UV light
• A monochromator that selects a specific wavelength of light
• A sample cell that holds the sample solution
• A detector that measures the intensity of light after passing through the sample
• A recorder that displays the spectrum as a graph of absorbance versus wavelength

In this article, we will discuss each part in more detail and explain how they function. We will also explore some of the applications and uses of UV spectroscopy in various fields of science.

The principle of UV spectroscopy is based on the interaction of light with matter. When a beam of ultraviolet or visible light passes through a sample, some of the light is absorbed by the molecules or atoms in the sample. The amount of light absorbed depends on the wavelength of the light, the concentration of the sample, and the path length of the light through the sample.

The relationship between these factors is given by the Beer-Lambert law, which states that the absorbance (A) of a sample is proportional to its concentration (c) and the path length (b) of the light through the sample:

$$A = \epsilon b c$$

where $\epsilon$ is the molar absorptivity or extinction coefficient of the sample at a given wavelength. The molar absorptivity is a measure of how strongly a sample absorbs light at a given wavelength and is characteristic of the chemical structure and environment of the sample.

The absorbance of a sample can be measured by using a spectrophotometer, which is an instrument that measures the intensity of light before and after passing through a sample. The ratio of these intensities is called the transmittance (T) and is related to the absorbance by:

$$A = -\log_{10} T$$

By measuring the absorbance of a sample at different wavelengths, a spectrum can be obtained that shows how much light is absorbed by the sample at each wavelength. This spectrum can provide qualitative and quantitative information about the sample, such as its identity, purity, structure, and concentration.

The principle behind UV spectroscopy is that when molecules or atoms absorb ultraviolet or visible light, they undergo electronic transitions from lower to higher energy levels. These transitions involve electrons in different types of orbitals, such as $\sigma$, $\pi$, and $n$ orbitals. The energy required for these transitions depends on the type and arrangement of the orbitals involved and can be calculated by using molecular orbital theory.

There are four main types of electronic transitions that occur in UV spectroscopy: $\sigma$-$\sigma^$, $n$-$\sigma^$, $\pi$-$\pi^$, and $n$-$\pi^$. These transitions are ordered according to their energy requirements as follows:

$$\sigma-\sigma^ > n-\sigma^ > \pi-\pi^ > n-\pi^$$

The $\sigma$-$\sigma^$ transitions involve electrons in bonding or non-bonding $\sigma$ orbitals moving to anti-bonding $\sigma^$ orbitals. These transitions require very high energy and occur in the far-UV region (below 200 nm). They are usually not observed in organic compounds because they cause bond breaking and fragmentation.

The $n$-$\sigma^$ transitions involve electrons in non-bonding $n$ orbitals (such as lone pairs) moving to anti-bonding $\sigma^$ orbitals. These transitions also require high energy and occur in the near-UV region (200-300 nm). They are observed in compounds that have heteroatoms with lone pairs, such as oxygen, nitrogen, sulfur, etc.

The $\pi$-$\pi^$ transitions involve electrons in bonding or non-bonding $\pi$ orbitals moving to anti-bonding $\pi^$ orbitals. These transitions require moderate energy and occur in the UV-visible region (200-400 nm). They are observed in compounds that have conjugated $\pi$ systems, such as alkenes, aromatic rings, carbonyl groups, etc.

The $n$-$\pi^$ transitions involve electrons in non-bonding $n$ orbitals moving to anti-bonding $\pi^$ orbitals. These transitions require low energy and occur in the visible region (400-800 nm). They are observed in compounds that have heteroatoms with lone pairs adjacent to $\pi$ systems, such as aldehydes, ketones, amides, etc.

The absorption spectrum of a compound can be used to identify its functional groups and structure by comparing it with known spectra of similar compounds. The wavelength at which maximum absorption occurs is called the $\lambda_{max}$ and indicates the type of transition involved. The intensity of absorption is measured by the molar absorptivity ($\epsilon$) and indicates the extent of conjugation or delocalization of electrons in the compound. The shape and width of absorption bands can also provide information about the symmetry and environment of the compound.

UV spectroscopy is a technique that uses ultraviolet light to measure the absorption of molecules. The basic parts of a UV spectrophotometer are:

• Light source: This is the device that produces the ultraviolet radiation that is used to excite the electrons in the sample. The most common light sources are tungsten filament lamps and hydrogen-deuterium lamps, which cover the whole UV region from 200 to 400 nm. Tungsten filament lamps emit more red radiation, while hydrogen-deuterium lamps have higher intensity below 375 nm.

• Monochromator: This is the device that separates the light from the source into different wavelengths and selects a specific wavelength to pass through the sample. The monochromator consists of prisms and slits that disperse and filter the light. Most UV spectrophotometers are double-beam spectrophotometers, which means that the light from the monochromator is split into two beams: one for the sample and one for the reference.

• Sample and reference cells: These are the containers that hold the sample and reference solutions. The sample solution is the one that contains the molecule of interest, while the reference solution is usually a blank solvent or a standard solution. The cells are made of silica or quartz, because glass absorbs UV light and would interfere with the measurement. The cells are placed in the path of the light beams from the monochromator.

• Detector: This is the device that measures the intensity of the light beams after they pass through the cells. The detector converts the light into an electrical signal that can be amplified and recorded. The most common detectors are photocells, which generate a current proportional to the light intensity. The detector compares the intensity of the sample beam with that of the reference beam and calculates the absorbance of the sample.

• Amplifier: This is the device that increases the strength of the electrical signal from the detector. The amplifier is connected to a servometer, which is a device that controls the rotation of the prism in the monochromator. The amplifier adjusts the servometer so that the intensity of the reference beam remains constant, while scanning different wavelengths for the sample beam.

• Recording devices: These are the devices that display and store the data from the amplifier. The most common recording devices are pen recorders and computers. The pen recorder draws a graph of absorbance versus wavelength on a paper chart, while the computer displays a digital spectrum on a screen and saves it in a file.

The light source is the component that provides the electromagnetic radiation in the UV region for the spectroscopy. The light source should have the following characteristics:

• It should emit continuous and intense radiation in the UV range (200-400 nm).
• It should have a long and stable lifetime.
• It should be easy to operate and maintain.

There are two types of light sources commonly used in UV spectroscopy: tungsten filament lamps and hydrogen-deuterium lamps.

Tungsten filament lamps

Tungsten filament lamps are electric lamps that use a heated metal filament to produce light. They are rich in red radiations and emit radiation of about 375 nm and above. They are suitable for the visible and near-UV regions of the spectrum. They have a long lifetime and are relatively inexpensive. However, they have a low intensity and a broad emission spectrum, which may limit their resolution and sensitivity.

Hydrogen-deuterium lamps

Hydrogen-deuterium lamps are gas discharge lamps that use an electric current to ionize a mixture of hydrogen and deuterium gases. They emit radiation of about 160-375 nm, covering the entire UV region. They have a high intensity and a narrow emission spectrum, which improve their resolution and sensitivity. However, they have a short lifetime and are relatively expensive. They also require special precautions to handle the flammable and explosive gases.

The choice of the light source depends on the wavelength range and the analytical requirements of the UV spectroscopy. In some cases, both types of light sources may be used in combination to cover a wider range of wavelengths. The light source is usually placed in a housing that protects it from dust, moisture, and mechanical damage. The housing also has a window that allows the light to pass through to the monochromator.

Monochromator

A monochromator is a device that separates the light from the source into its different wavelengths and selects a narrow band of wavelengths to pass through the sample. The purpose of a monochromator is to ensure that only one wavelength (or a very small range of wavelengths) reaches the detector at a time. This way, the absorbance of the sample can be measured at each wavelength and a spectrum can be obtained.

There are different types of monochromators, but the most common ones are based on prisms or gratings. Prisms and gratings are optical elements that disperse light into its component colors by refraction or diffraction, respectively. A prism splits white light into a rainbow of colors, while a grating splits white light into a series of bright and dark lines called a spectrum.

A typical monochromator consists of an entrance slit, a collimating lens, a dispersing element (prism or grating), a focusing lens, and an exit slit. The entrance slit allows only a narrow beam of light to enter the monochromator. The collimating lens makes the light rays parallel before they reach the dispersing element. The dispersing element separates the light into its different wavelengths and angles them in different directions. The focusing lens collects the dispersed light and focuses it on the exit slit. The exit slit selects only a small range of wavelengths to pass through and blocks the rest.

The wavelength of the light that passes through the exit slit can be changed by rotating the dispersing element or moving the exit slit. This way, the monochromator can scan through the entire UV region and measure the absorbance of the sample at each wavelength.

The quality of a monochromator depends on several factors, such as the resolution, the bandwidth, and the stray light. The resolution is the ability of the monochromator to separate two closely spaced wavelengths. The bandwidth is the width of the wavelength range that passes through the exit slit. The stray light is the unwanted light that reaches the detector from other sources than the exit slit. These factors affect the accuracy and precision of the UV spectroscopy measurements.

After the monochromator, the beam of light is split into two paths: one that passes through the sample solution and one that passes through the reference solution. The sample solution contains the compound of interest dissolved in a suitable solvent, while the reference solution contains only the solvent. The purpose of using a reference solution is to eliminate the effect of the solvent`s absorption on the measurement of the sample`s absorbance.

Both sample and reference solutions are contained in transparent cells that are placed in the path of the light beams. These cells are usually rectangular in shape and have a fixed path length, typically 1 cm. The cells are made of either silica or quartz, which are transparent in the UV region. Glass cannot be used for the cells as it also absorbs light in the UV region .

The cells must be clean and free of scratches or fingerprints, as any impurities or defects can affect the accuracy and precision of the measurement. The cells are also filled carefully to avoid any bubbles or particles in the solution. The cells are usually capped or sealed to prevent evaporation or contamination of the solution.

The light beams that pass through the sample and reference cells are then detected by two photocells, which convert the light intensity into an electrical signal. The difference between the signals from the sample and reference cells is proportional to the absorbance of the sample at a given wavelength .

The detector is the part of the UV spectroscopy instrument that converts the light signal into an electrical signal. The detector should be sensitive to the wavelength range of interest, have a fast response time, and produce a low noise output. There are different types of detectors used in UV spectroscopy, such as:

• Photomultiplier tubes (PMTs): These are the most common detectors in UV spectroscopy. They consist of a photocathode that emits electrons when struck by photons, and a series of dynodes that multiply the electrons by secondary emission. The resulting current is proportional to the intensity of the light. PMTs have high sensitivity, wide dynamic range, and fast response time, but they are also expensive and bulky.
• Photodiodes: These are semiconductor devices that generate a current when exposed to light. They are cheaper, smaller, and more durable than PMTs, but they have lower sensitivity and dynamic range. Photodiodes are often used in array detectors, where multiple photodiodes are arranged in a linear or two-dimensional configuration to measure the spectrum simultaneously.
• Photovoltaic cells: These are similar to photodiodes, but they generate a voltage instead of a current when illuminated. They have low noise and high stability, but they also have low sensitivity and require a high light intensity to operate.
• Photoconductors: These are materials that change their electrical resistance when exposed to light. They have high sensitivity and low noise, but they also have slow response time and high dark current. Photoconductors are often used in infrared spectroscopy, where the light intensity is low.

The choice of detector depends on the application and the performance requirements of the UV spectroscopy instrument. The detector output is then amplified and recorded by a computer or a pen recorder.

The amplifier is a device that increases the intensity of the electrical signals generated by the detector. The amplifier is usually coupled to a small servometer that controls the rotation of the prism in the monochromator. The purpose of the amplifier is to amplify the signals to a level that can be recorded and displayed by the recording devices.

The amplifier also converts the alternating current (AC) signals from the detector into direct current (DC) signals that can be measured by a voltmeter or a potentiometer. The DC signals are proportional to the absorbance of the sample at each wavelength.

The amplifier can also perform some mathematical operations on the signals, such as taking the logarithm, subtracting the background noise, or calculating the ratio of sample and reference signals. These operations can improve the accuracy and precision of the measurements.

The amplifier is an essential part of UV spectroscopy as it enables the detection and quantification of very low concentrations of analytes in solution.

Recording devices are used to store and display the data obtained from the detector and amplifier. They can also be used to plot the spectrum of the sample by showing the absorbance or transmittance as a function of wavelength. There are different types of recording devices available for UV spectroscopy, such as:

• Pen recorder: This is a device that uses a moving pen to draw a graph on a paper chart. The pen is connected to the amplifier and moves according to the signal received from the detector. The paper chart is attached to a drum that rotates at a constant speed. The drum is synchronized with the monochromator so that the wavelength of the light corresponds to the position of the pen on the chart. Pen recorders are simple and inexpensive, but they have some limitations, such as low resolution, slow response, and paper consumption.

• Digital display: This is a device that shows the numerical values of absorbance or transmittance on a screen. The digital display is connected to an analog-to-digital converter (ADC) that converts the signal from the amplifier into digital data. The digital display can also show other parameters, such as wavelength, concentration, and calibration curve. Digital displays are fast and accurate, but they do not provide a visual representation of the spectrum.

• Computer: This is a device that can store, process, and display the data from the detector and amplifier using specialized software. The computer is connected to an ADC that converts the signal from the amplifier into digital data. The computer can also control the monochromator and other parts of the instrument. The computer can perform various functions, such as:

  • Displaying the spectrum of the sample on a monitor in different modes, such as absorbance vs wavelength, transmittance vs wavelength, or absorbance vs time.
  • Saving and retrieving the data in different formats, such as text files, spreadsheets, or images.
  • Performing mathematical operations on the data, such as baseline correction, smoothing, peak picking, integration, differentiation, or derivative spectroscopy.
  • Applying calibration methods to determine the concentration of unknown samples using standard solutions or regression analysis.
  • Comparing the spectrum of the sample with spectra of known compounds stored in a database or library.
  • Printing or exporting the data or spectrum to other devices or programs.

Computers are versatile and powerful devices that can enhance the performance and functionality of UV spectroscopy. However, they also require more maintenance and technical skills than other recording devices.

UV spectroscopy is a versatile technique that can be used for various purposes in different fields of science and industry. Some of the common applications of UV spectroscopy are:

• Detection of functional groups: UV spectroscopy can be used to identify the presence or absence of chromophores in a compound, such as unsaturated bonds, aromatic rings, carbonyl groups, nitro groups, etc. The absorption spectrum of a compound can be compared with the spectra of known compounds to determine its structure or confirm its identity.
• Quantitative analysis: UV spectroscopy can be used to measure the concentration of a compound in a solution by applying the Beer-Lambert law, which relates the absorbance to the concentration and path length. The molar absorptivity of the compound at a specific wavelength can be obtained from tables or calibration curves. UV spectroscopy can also be used to determine the purity of a compound by comparing its absorbance with that of a standard sample.
• Kinetic studies: UV spectroscopy can be used to monitor the progress of a chemical reaction by measuring the change in absorbance of the reactants or products over time. The rate constant and order of the reaction can be calculated from the kinetic data. UV spectroscopy can also be used to study the mechanism of a reaction by observing the formation or disappearance of intermediates.
• Biochemical analysis: UV spectroscopy can be used to analyze biological molecules such as proteins, nucleic acids, enzymes, hormones, vitamins, etc. The absorption spectrum of a biomolecule can provide information about its structure, function, interactions and modifications. UV spectroscopy can also be used to quantify biomolecules by using specific chromogenic or fluorogenic reagents that react with them to produce a color or fluorescence change.
• Quality control: UV spectroscopy can be used to check the quality of various products such as food, beverages, pharmaceuticals, cosmetics, textiles, etc. The absorption spectrum of a product can indicate its composition, freshness, stability, authenticity and safety. UV spectroscopy can also be used to detect impurities, adulterants or contaminants in a product by comparing its spectrum with that of a reference standard.
• Nanotechnology: UV spectroscopy can be used to characterize nanomaterials such as nanoparticles, nanotubes, nanowires, etc. The absorption spectrum of a nanomaterial can reveal its size, shape, surface properties and optical properties. UV spectroscopy can also be used to monitor the synthesis and modification of nanomaterials by measuring the change in absorbance during the process.

These are some of the applications of UV spectroscopy that demonstrate its usefulness and versatility in various domains. UV spectroscopy is a simple yet powerful technique that can provide valuable information about different substances and processes.

One of the main applications of UV spectroscopy is the detection of impurities in organic molecules. Impurities can affect the physical and chemical properties of the compounds, as well as their biological activity and safety. Therefore, it is important to identify and quantify the impurities in pharmaceuticals, food, cosmetics, and other products.

UV spectroscopy can detect impurities by measuring the absorbance of the sample at specific wavelengths and comparing it with that of the pure standard. Impurities can cause additional peaks or shifts in the absorption spectrum of the sample, indicating their presence and nature. For example, if a compound has a chromophore (a part of the molecule that absorbs UV light), such as a double bond or a benzene ring, its absorption spectrum will show peaks corresponding to the electronic transitions of the chromophore. If an impurity has a different chromophore or modifies the existing one, it will alter the absorption spectrum of the sample.

To detect impurities using UV spectroscopy, the following steps are usually followed :

• Prepare a solution of the sample in a suitable solvent that does not absorb UV light, such as water, ethanol, or methanol.
• Prepare a solution of the pure standard in the same solvent and concentration as the sample.
• Set up a UV spectrophotometer with a light source that covers the UV region (200-400 nm), such as a deuterium lamp or a tungsten filament lamp.
• Place a quartz or silica cell containing the solvent in the reference beam and adjust the baseline to zero absorbance.
• Place another cell containing the standard solution in the sample beam and record its absorption spectrum over the desired wavelength range.
• Replace the cell with the sample solution and record its absorption spectrum over the same wavelength range.
• Compare the spectra of the sample and the standard and look for any differences in peak positions, intensities, or shapes.

The differences in spectra can indicate the presence and type of impurities in the sample. For example, if an impurity has a higher or lower energy gap than the standard, it will cause a shift in peak position to shorter or longer wavelengths. If an impurity has a higher or lower molar absorptivity than the standard, it will cause a change in peak intensity. If an impurity has a different symmetry or polarity than the standard, it will cause a change in peak shape.

The amount of impurities can be estimated by measuring the absorbance at a specific wavelength where only the impurity absorbs and applying Beer-Lambert law:

$$A = \epsilon cl$$

where A is absorbance, $\epsilon$ is molar absorptivity, c is concentration, and l is path length.

The concentration of impurity can be calculated by rearranging this equation:

$$c = \frac{A}{\epsilon l}$$

The molar absorptivity of impurity can be obtained from literature values or from calibration curves using known concentrations of impurity.

UV spectroscopy is a simple, fast, and sensitive method for detecting impurities in organic molecules. However, it has some limitations, such as:

• It requires that the impurity has a different absorption spectrum than the standard.
• It may not be able to distinguish between different types of impurities that have similar absorption spectra.
• It may not be able to detect very low levels of impurities that are below the detection limit of the instrument.
• It may be affected by interference from other substances that absorb UV light, such as solvents or additives.

Therefore, UV spectroscopy should be used in combination with other analytical techniques, such as mass spectrometry or chromatography, to confirm and quantify impurities in organic molecules.

UV spectroscopy can be used to measure the concentration of compounds that absorb UV light by applying the Beer-Lambert law, which relates the absorbance of light to the properties of the material. The Beer-Lambert law is given by:

$$A = \epsilon b c$$

where A is the absorbance, $\epsilon$ is the molar absorptivity, b is the path length, and c is the concentration. The molar absorptivity is a constant for a given compound at a given wavelength, and it measures how strongly the compound absorbs light at that wavelength. The path length is the distance that the light travels through the sample, and it is usually 1 cm for standard cuvettes. The concentration is the amount of solute per unit volume of solution, and it has units of mol/L.

To use UV spectroscopy for quantitative analysis, one must first calibrate the instrument using a series of standard solutions with known concentrations of the analyte. The absorbance of each standard solution is measured at a selected wavelength, and a calibration curve is plotted with absorbance on the y-axis and concentration on the x-axis. The calibration curve should be linear according to the Beer-Lambert law, and its slope should be equal to $\epsilon b$. Then, the absorbance of an unknown sample is measured at the same wavelength, and its concentration is determined by interpolating from the calibration curve.

The choice of wavelength for quantitative analysis should be based on several factors, such as:

• The wavelength should be within the UV range (200-400 nm) where most organic compounds absorb light.
• The wavelength should correspond to a peak in the absorption spectrum of the analyte, where the molar absorptivity is high and the sensitivity is good.
• The wavelength should be selective for the analyte, meaning that it should not be absorbed by other components in the sample or by the solvent.
• The wavelength should be in a region where the absorption curve is relatively flat, meaning that small errors in setting or reading the wavelength will not affect the absorbance significantly.

Some examples of applications of UV spectroscopy for quantitative analysis are:

• Detection of impurities in organic molecules by comparing their absorption spectra with those of pure standards or by measuring their absorbance at specific wavelengths.
• Structure elucidation of organic compounds by identifying functional groups or chromophores based on their characteristic absorption bands or shifts due to solvent or pH effects.
• Determination of reaction kinetics or equilibrium constants by monitoring the absorbance changes of reactants or products over time or with varying conditions.
• Assay of drugs or pharmaceuticals by measuring their absorbance in suitable solvents and using appropriate calibration curves or molar absorptivities.
• Measurement of molecular weights or binding constants by using spectrophotometric titrations with absorbing reagents or indicators.

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