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UV-Vis Spectroscopy – Principle, Instrumentation, Applications, Advantages, and Limitation

MN Editors

Spectroscopy is a technique that is used to study the interaction of matter and electromagnetic radiation. It involves the measurement of the energy absorbed or emitted by a sample at different wavelengths. Spectroscopy can be used to identify the composition of a sample, to study the physical and chemical properties of a sample, and to determine the structure of a molecule.

UV-Vis spectroscopy, also known as ultraviolet-visible spectroscopy, is a subtype of spectroscopy that specifically deals with the absorption of ultraviolet (UV) and visible light by a sample. It is a commonly used technique in analytical chemistry, biochemistry, and materials science. UV-Vis spectroscopy can be used to determine the concentration of a substance in a solution, to identify the functional groups present in a molecule, and to study the electronic and geometric structure of a molecule.

The significance of UV-Vis spectroscopy is the ability to measure the absorption of light by a sample and the resulting data can be used to identify and analyze the functional groups, concentration and electronic and geometric structure of a molecule. This technique has wide range of applications like in analytical chemistry, biochemistry, and materials science and also in industries like Pharmaceuticals, food, water and environment analysis, etc.

What is UV-Vis Spectroscopy?

  • UV-Vis spectroscopy, technically known as Ultra Violet-Visible spectrophotometry, is a method that falls under the category of absorption spectroscopy or reflectance spectroscopy. This technique specifically targets the ultraviolet and the full, adjacent visible regions of the electromagnetic spectrum.
  • Therefore, it is essential for the sample under examination to be a chromophore, meaning it should absorb in the UV-Vis region. Besides its ability to absorb, parameters such as absorbance (A), transmittance (%T), reflectance (%R), and their variation over time are also of significant interest.
  • An analytical tool known as the UV-vis spectrophotometer plays a pivotal role in this process. This instrument measures the amount of ultraviolet (UV) and visible light absorbed by a given sample. Then, it is widely recognized and employed in various fields, including chemistry and biochemistry, for the identification and quantification of compounds in diverse samples.
  • The operational mechanism of UV-vis spectrophotometers is relatively straightforward. They function by directing a beam of light through the sample. The instrument then quantifies the light absorbed at each specific wavelength. It is crucial to note that the light’s absorption level is directly proportional to the concentration of the absorbing compound present in the sample.
  • Delving deeper into the science of spectroscopy, it is the act of measuring and interpreting the electromagnetic radiation absorbed or emitted when a sample’s molecules, atoms, or ions transition between energy states.
  • UV spectroscopy is a subset of absorption spectroscopy. In this method, molecules absorb light from the ultra-violet region (200-400 nm), leading to the excitation of electrons from their ground state to a heightened energy state.
  • In the realm of UV-VIS spectroscopy, a graph plots the transition of electrons at various levels due to radiation absorption from the ultraviolet to the visible region. This graphical representation, showcasing various absorptivities at specific radiation levels, is attributed to the absorption capacities of compounds at certain levels. These specific levels are termed regions of absorption, and the compounds responsible for this absorption are called chromophores.
  • Furthermore, chromophores are ubiquitous and can be found in nearly every compound. This prevalence is evident in the fact that almost all compounds, especially organic ones, can be identified and quantified using UV-Vis spectroscopy. Therefore, UV-Vis spectroscopy stands as a vital tool in the scientific community, offering detailed and sequential insights into the nature and properties of various compounds.

What is the main purpose of UV spectroscopy?

The main purpose of UV spectroscopy is to determine the absorption or transmission characteristics of a sample in the ultraviolet (UV) region of the electromagnetic spectrum. By measuring the amount of light absorbed by a sample at different wavelengths, researchers can learn about its chemical composition, electronic structure, and other properties. UV spectroscopy is used in a wide range of applications, including:

  • Identifying and quantifying the concentration of a specific compound in a mixture
  • Characterizing the purity of a sample
  • Determining the electronic structure of a compound, such as the presence of conjugated double bonds or aromatic ring systems
  • Studying chemical reactions and reaction kinetics
  • Analysis of biological macromolecules like proteins, DNA, and RNA
  • Environmental analysis
  • Pharmaceuticals and drug analysis

Overall, the main purpose of UV spectroscopy is to provide structural and compositional information about the sample being analyzed.

Which solvent is used in UV spectroscopy?

The solvent used in UV spectroscopy depends on the nature of the sample being analyzed and the specific application. Some common solvents used in UV spectroscopy include:

  • Water: Water is often used as a solvent for UV spectroscopy of polar compounds, such as acids, bases, and biomolecules.
  • Alcohols: Alcohols like methanol and ethanol are commonly used as solvents for UV spectroscopy of organic compounds.
  • Acetonitrile: Acetonitrile is a polar, aprotic solvent that is often used for UV spectroscopy of polar compounds and for samples that are not soluble in water or alcohols.
  • Dichloromethane: Dichloromethane (DCM) is a non-polar solvent that is often used for UV spectroscopy of non-polar compounds.
  • Hexane: Hexane is also a non-polar solvent that is often used for UV spectroscopy of non-polar compounds.

It is important to note that some solvents absorb in the UV region, so the choice of solvent should be chosen carefully. In general, a solvent should be transparent in the region of the spectrum where the sample absorbs.

Types of UV Radiation Rays

There are different types of ultraviolet (UV) radiation rays, which are broadly classified into three main categories based on their wavelength:

  • UVA (320-400 nm): UVA rays are the longest wavelength and least energetic of the UV rays. They are mostly absorbed by the ozone layer and can penetrate the skin more deeply than UVB rays. They are associated with skin aging and wrinkling, and can also contribute to the development of skin cancer.
  • UVB (280-320 nm): UVB rays are shorter wavelength and more energetic than UVA rays. They are mostly absorbed by the atmosphere and are responsible for sunburns and suntans. They are also the primary cause of skin cancer, as well as cataracts and other eye damage.
  • UVC (100-280 nm): UVC rays are the shortest wavelength and most energetic of the UV rays. They are completely absorbed by the ozone layer and do not reach the Earth’s surface. However, they can be produced artificially by certain types of lamps, such as mercury vapor lamps, and are used for disinfection and sterilization.

It is worth noting that UV-A and UV-B are the harmful rays that can cause skin cancer, eye damage, and other health problems, while UV-C is less harmful and mostly used for disinfection.

UV Visible Spectroscopy Principle

UV-Vis Spectroscopy
UV-Vis Spectroscopy

Spectroscopy, at its core, delves into the intricate interaction between light and matter. When light, especially from the ultraviolet region, is absorbed by matter, it leads to a notable increase in the energy content of the atoms or molecules involved. This absorption process results in the excitation of electrons, propelling them from their ground state to a higher energy state.

Diving deeper into the molecular realm, molecules that possess π-electrons or nonbonding electrons (often denoted as n-electrons) have the capability to absorb energy in the form of ultraviolet light. This absorption facilitates the excitation of these electrons to higher anti-bonding molecular orbitals. Therefore, the ease with which these electrons are excited determines the wavelength of light they can absorb. To provide a clearer perspective, there are four potential types of transitions, namely π–π*, n–π*, σ–σ*, and n–σ*. These transitions can be hierarchically ordered based on their energy levels as σ–σ* > n–σ* > π–π* > n–π*.

Besides this, the absorption of ultraviolet light by a specific chemical compound generates a unique spectrum. This spectrum acts as a fingerprint, aiding in the precise identification of the compound in question.

Transitioning to the realm of ultraviolet-visible (UV-Vis) spectra, these are derived when the incident radiation interacts with the electron cloud in a chromophore. This interaction culminates in an electronic transition, where electrons from the ground state are promoted to a higher energy state.

It is essential to note that the UV and visible spectral bands of substances are generally broad. While they might not offer a high degree of compound recognition specificity, they are invaluable for quantitative analyses. Furthermore, they serve as an alternative detection method for various substances.

The principle of chance predicts the energy released at each specific wavelength, primarily based on the temperature of the solid. In contemporary practices, the tungsten-halogen lamp, which transmits radiation deep into the UV zone through its quartz envelope, has become a standard. For the UV region itself, the deuterium lamp stands as the most preferred source. A comprehensive UV-Visible spectrometer typically houses both types of lamps, ensuring coverage across the entire wavelength spectrum.

In conclusion, UV-Visible spectroscopy operates on the foundational principle of light-matter interaction, emphasizing the functions and transitions of electrons within molecules. This technique offers both qualitative and quantitative insights, making it a cornerstone in various scientific applications.

Ultraviolet-visible (UV-Vis) spectroscopy is a fundamental analytical technique that revolves around the interaction of light with matter. Delving deeper into its principles provides a comprehensive understanding of how this interaction results in the absorption of specific wavelengths, revealing the molecular and atomic composition of a sample.

  1. Basic Understanding: Spectroscopy, at its core, is the study of how light interacts with matter. When matter absorbs light, there’s a notable increase in its energy content, specifically at the molecular or atomic level.
  2. Absorption Spectrum: The absorption spectrum of a material is a direct reflection of its molecular and atomic composition. Each material has a unique spectrum, akin to a fingerprint, which arises due to the specific frequencies of light it absorbs. This frequency corresponds to the energy difference between two molecular energy states.
  3. Electron Excitation: Central to UV-Vis spectroscopy is the concept of electron excitation. When radiation is absorbed by a molecule, electrons are excited from a ground state to a higher energy state. This absorption results in an absorption line, and collectively, these lines form the absorption spectrum.
  4. Absorbance and Concentration: The absorbance of a compound is intrinsically linked to the number of electrons it excites from the ground state. This, in turn, is influenced by the concentration or number of molecules present in the sample. Therefore, the absorbance spectrum serves as a marker or identifier for the compound.
  5. Types of Transitions: There are four primary types of electronic transitions observed in UV-Vis spectroscopy: π–π*, n–π*, σ–σ*, and n–σ*. The energy required for these transitions varies, with the order being σ–σ* > n–σ* > π–π* > n–π*. Each transition type corresponds to specific molecular interactions and bond types.
  6. Spectrophotometer Function: A spectrophotometer is the instrument used in UV-Vis spectroscopy. It measures the amount of absorption by a sample across different wavelengths in the UV and visible spectrum range. The resulting plot, known as a spectrum, charts absorbance (A) against wavelength (ƛ).
  7. Molecular Orbitals and Excitation: At the heart of UV-Vis spectroscopy lies the concept of molecular orbitals. During excitation, electrons transition from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO), resulting in an excited or antibonding state.
  8. Specific Transitions: Certain transitions, like σ–σ*, require significant energy and are observed in compounds like methane. In contrast, n–σ* transitions, seen in compounds like water and ethanol, require less energy. Transitions such as π–π* and n–π* are associated with unsaturated functional groups, with alkenes and alkynes exemplifying π–π* transitions and carbonyl compounds showcasing n–π* transitions.

In conclusion, UV-Vis spectroscopy offers a detailed insight into the molecular and atomic structure of compounds by analyzing how they interact with light. Through a detailed understanding of the principles underlying this technique, researchers and scientists can derive valuable information about the composition and characteristics of various samples.

Electronic transitions

When a molecule absorbs electromagnetic radiation in the UV-visible region and becomes excited, its electrons are promoted from the ground state to the excited state or from the bonding orbital to the anti-bonding orbital.

  • 𝜎 − 𝜎* Transition: 𝜎 − 𝜎*  represents the transition of an electron from a bonding sigma orbital (𝜎) to an anti-bonding sigma orbital ( 𝜎 *). In alkanes, for instance, all the atoms are bound together by a sigma bond.
  • 𝑛 − 𝜎* & 𝑛 − 𝜋* Transition: Transitions from non-bonding molecular (𝑛) orbital to anti-bonding sigma orbital or anti-bonding pi orbital (𝜋 * ), are represented by 𝑛 − 𝜎 * or 𝑛 − 𝜋 * transition respectively. These transition required less energy than 𝜎 − 𝜎 * transition. For example, alkyl halide, aldehydes, ketones etc.
  • 𝜋 − 𝜋* Transition: This type of transition generally show in unsaturated molecules like alkenes, alkynes, aromatics, carbonyl compounds etc. This transition required less energy as compare to 𝑛 − 𝜎 * transition.
UV-Vis Spectroscopy
UV-Vis Spectroscopy
UV-Vis Spectroscopy
UV-Vis Spectroscopy

Absorption, intensity shift & UV spectrum

  • Bathochromic effect: An effect in which the absorption maximum is shifted to a longer wavelength due to the presence of an autochrome or a solvent change. Red shifts are also known as bathochromic shifts.
  • Hypsochromic shifts: This refers to the phenomenon in which the absorption maximum is shifted toward a shorter wavelength. It is also known as the blue shift. It may be caused by removing conjugation and altering the polarity of the solvent.
  • Hyperchromic effect: This is an effect in which the maximum absorption intensity, Emax, increases. B-band for pyridine at 275 mμ, Emax 2750 moved to 262 mμ, 2-methyl pyridine Emax 3560. Typically, the addition of autochrome increases the intensity of absorption.
  • Hypochromic effect: Hypochromic effect is described as the decrease in the maximum intensity of absorption, or the decrease in the extinction coefficient Emax. The hypochromic effect is caused by the insertion of the group which changes the geometry of the molecule.
Absorption, intensity shift & UV spectrum
Absorption, intensity shift & UV spectrum | Credit:

Types of absorption bands

There are several types of absorption bands that can be observed in the spectra of molecules:

  1. Rotational absorption bands: These occur when the rotation of a molecule causes a change in the dipole moment of the molecule. Rotational absorption bands are typically observed in the microwave and far-infrared regions of the spectrum.
  2. Vibrational absorption bands: These occur when the vibrational motion of a molecule causes a change in the dipole moment of the molecule. Vibrational absorption bands are typically observed in the infrared region of the spectrum.
  3. Electronic absorption bands: These occur when the electronic structure of a molecule changes as a result of absorption of light. Electronic absorption bands are typically observed in the ultraviolet and visible regions of the spectrum.
  4. Nuclear magnetic resonance (NMR) absorption bands: These occur when the nuclei of certain atoms (such as hydrogen, carbon, and phosphorus) in a molecule absorb energy in the radiofrequency region of the spectrum. NMR spectroscopy is widely used in analytical chemistry and biochemistry to study the structure and dynamics of molecules.
  5. Raman scattering bands: They are caused by inelastic scattering of light. It occurs when a photon is scattered by a molecule, but with a change in energy, which results in a shift in wavelength. Raman scattering is typically observed in the visible and near infrared regions and it is used to study the vibrational modes of a molecule.

Types of UV-Visible spectroscopy

Ultraviolet-Visible (UV-Vis) Spectrophotometry, a pivotal technique in analytical chemistry, employs various types of spectrometers to analyze samples. Delving into the details, there are primarily two types of UV-Vis spectrophotometers:

Single beam UV-Visible spectrophotometer
Single beam UV-Visible spectrophotometer
  1. Single Beam UV-Visible Spectrophotometer:
    • As the name suggests, a single beam UV-Vis spectrophotometer utilizes a singular beam of light. The process begins with the incident light emanating from the source, which is subsequently passed through a monochromator. This monochromatic light then traverses a slit and proceeds to pass through the sample solution. During this phase, a portion of the incident light is absorbed by the sample, while the remainder is transmitted.
    • The transmitted light is then detected by a specialized detector. Following detection, the light is amplified and recorded, culminating in its display on an appropriate readout device. A spectrum is then plotted, and the λmax (wavelength of maximum absorbance) is identified.
    • The integral components of a single beam UV-Vis spectrophotometer encompass:
      • Light source
      • Lens
      • Gratings
      • Wavelength selector
      • Sample container or cuvette
      • Detector
      • Digital meter or Recorder
  2. Double Beam UV-Visible Spectrophotometer:
    • The foundational instrumentation of both single and double beam spectrophotometers remains largely analogous. However, the distinguishing feature of a double beam UV-Vis spectrophotometer is its ability to simultaneously direct the beam of incident light towards both the reference and the sample cuvettes.
    • The incident light undergoes a division and is channeled towards the reference and sample cuvette in tandem. The beams that are refracted or transmitted are subsequently detected by detectors. It’s imperative to note that a double beam UV-Vis spectrophotometer necessitates two detectors. These detectors are pivotal in detecting the electron ratio, which is instrumental in measuring or calculating the absorbance in a test sample.
    • Additionally, a double beam UV-Vis spectrophotometer mandates a stabilized voltage supply to ensure accurate and consistent readings.
Double beam UV-Visible spectrophotometer
Double beam UV-Visible spectrophotometer

Instrumentation of UV Visible Spectrophotometer (Parts of UV Spectroscopy)

To acquire a better grasp of how a UVVis spectrophotometer operates, and despite the fact that there are numerous UVVis spectrophotometer variations, let’s explore the major components represented in Figure.

Instrumentation of UV Spectroscopy
A simplified schematic of the main components in a UV-Vis spectrophotometer. Credit: Dr. Justin Tom.

The primary components of U.V-Visible spectroscopy are:

  1. Light source
  2. Monochromator
  3. Chopper
  4. Sample container
  5. Detectors
  6. Amplifier
  7. Recorder

1. Light source

As a light-based approach, a constant source capable of emitting light over a broad spectrum of wavelengths is required. A single xenon lamp is frequently employed as a source of high-intensity UV and visible light. In contrast to tungsten and halogen lamps, xenon lamps are more expensive and less stable.

For equipment utilising two lamps, a tungsten or halogen lamp is typically utilised for visible light, whilst a deuterium lamp is typically employed as the source of ultraviolet light. As UV and visible wavelengths require two distinct light sources, the instrument’s light source must be switched during measurement. This transition often occurs during the scan between 300 and 350 nm, where the light emission from both light sources is comparable and the transition may be achieved more smoothly.

  • As they illuminate the entire UV spectrum, tungsten filament lamps and hydrogen-deuterium lamps are the most popular and suitable light sources.
  • The intensity of Hydrogen-Deuterium lights falls below 375 nm, whereas the intensity of Tungsten filament lamps exceeds 375 nm.

2. Wavelength selection

From the broad wavelengths radiated by the light source, specific wavelengths must be selected for sample inspection that correspond to the sample type and analyte to be detected. Methods for accomplishing this include:

a. Monochromators

A monochromator divides light into a limited wavelength band. It is often based on diffraction gratings that may be rotated to pick incoming and reflected angles in order to select the appropriate light wavelength. The groove frequency of a diffraction grating is frequently expressed as the number of grooves per millimetre. A greater groove frequency improves optical resolution but reduces the wavelength range that can be utilised. A lower groove frequency expands the wavelength range that can be utilised but diminishes the optical resolution. 300 to 2000 grooves per mm are acceptable for UV-Vis spectroscopy, with 1200 grooves per mm being the norm. Physical defects in the diffraction grating and optical setup can affect the precision of spectroscopic observations. Consequently, ruled diffraction gratings have a greater propensity for having faults than blazing holographic diffraction gratings. Typically, holographic diffraction gratings with a blazed surface yield much higher quality data.

b. Absorption filters

Absorption filters are usually constructed from colored glass or plastic that is designed to absorb certain frequencies of light.

c. Interference filters

These filters, which are often called dichroic filters, are constructed from many layers of dielectric material, with interference occurring between the thin layers. These filters can be used as a wavelength selector by blocking off frequencies of no use through destructive interference.

d. Cutoff filters

Light below (shortpass) or above (longpass) a specified wavelength is passed through by cutoff filters. Interference filters are frequently used to do this.

e. Bandpass filters

By combining shortpass and longpass filters, it is possible to create bandpass filters that permit a variety of wavelengths to pass through.

Due to their adaptability, monochromators are most typically employed for this procedure. Nevertheless, filters are frequently employed in conjunction with monochromators to further reduce the wavelengths of light selected for more accurate measurements and to increase the signal-to-noise ratio.

3. Sample analysis

Regardless of the wavelength choice is utilised in the spectrophotometer, the light passes through the sample. For all analyses, it is essential to measure a reference sample, sometimes known as a “blank sample,” such as a cuvette containing the same solvent used to create the sample. If an aqueous buffered solution containing the sample is utilised for measurements, the aqueous buffered solution without the component of interest serves as the standard. When analysing bacterial cultures, sterile culture media would serve as a standard. The equipment then uses the signal from the reference sample to determine the true absorbance values of the analytes.

In UV-Vis spectroscopy experiments, it is essential to be aware of the substances and circumstances employed. For instance, the majority of plastic cuvettes are unsuitable for UV absorption investigations due to the fact that plastic typically absorbs UV radiation. Glass can operate as a filter, often absorbing the bulk of UVC (100280 nm) and UVB (280315 nm), while permitting some UVA (315400 nm) to pass through. Due to the fact that quartz is transparent to the vast majority of UV light, quartz sample containers are necessary for UV analysis. Because molecular oxygen in air absorbs wavelengths of light shorter than around 200 nm, air can also be considered a filter. For observations with wavelengths less than 200 nm, a specialised and more expensive setup is required, often consisting of an optical system filled with pure argon gas. Also accessible are cuvette-free devices that enable the study of extremely small sample volumes, such as in DNA or RNA analysis.

4. Amplifier

The photocell-generated alternating current is passed to the amplifier. A tiny servometer is linked to the amplifier. Typically, the photocells generate a relatively weak current; therefore, the primary function of the amplifier is to amplify the signals so that we may obtain clear and recordable signals.

5. Detection or Detector

When exposed to light, a photoelectric coating emits negatively charged electrons. An electric current proportionate to the light intensity is generated when electrons are expelled. In UV-Vis spectroscopy, a photomultiplier tube (PMT) is one of the most used detectors. A PMT relies on the photoelectric effect to eject electrons upon exposure to light, followed by consecutive multiplication of the ejected electrons to generate a greater electric current. Four PMT detectors are particularly useful for detecting extremely low light levels.

When semiconductors are exposed to light, a current proportional to the intensity of the light can pass through. In particular, photodiodes and chargecoupled devices (CCDs) are two of the most used semiconductor-based detectors.

After the electric current has been generated by whichever detector was used, the signal is detected and sent to a computer or display. Below Figures depict simplified schematic examples of UV-vis spectrophotometer configurations.

Schematic diagram of a cuvette-based UV-Vis spectroscopy system.
Schematic diagram of a cuvette-based UV-Vis spectroscopy system. | Source:
Schematic diagram of a cuvette-free UV-Vis spectroscopy system.
Schematic diagram of a cuvette-free UV-Vis spectroscopy system. | Source:

6. Recording devices

The majority of the time, the amplifier is paired to a computer-connected pen recorder. The computer stores all generated data and generates the desired compound’s spectrum.

7. Sample and cells reference

One of the two separated beams is sent through the sample solution, while the other is passed through the standard solution. In the cells, both sample and reference solutions are enclosed. These cells are either composed of silica or quartz. Glass cannot be used for the cells because it also absorbs UV radiation.

Types of Detectors in UV-Visible Spectroscopy

In the domain of UV-Visible spectroscopy, detectors play an indispensable role. Their primary function is to convert light into proportional electrical signals, which subsequently determine the spectrophotometer’s response. Delving into the technicalities, there are four primary types of detectors utilized in UV-Visible spectroscopy. Therefore, for a comprehensive understanding, let’s explore each type in detail.

Types of Detectors in UV-Visible Spectroscopy
Types of Detectors in UV-Visible Spectroscopy
  1. Phototube: The phototube, also referred to as a photoelectric cell, is a detector filled with gas under low pressure. Within its evacuated quartz envelope, it houses a light-sensitive cathode and an anode. A potential difference of approximately 100 V is applied between these electrodes. When a photon enters the tube, it strikes the cathode, leading to the ejection of an electron. This electron, upon hitting the anode, results in the flow of current. However, the resultant current is of low intensity and necessitates amplification. The response of the phototube is contingent on the wavelength of the incident light. Phototubes operate based on the photoelectric effect, where light is absorbed by a metallic surface with a low work function. To prevent excessive current density on a part of the cathode, a larger spot on the photocathode is illuminated. However, due to thermionic emission at longer wavelengths, a dark current may be generated, necessitating cooling with liquid nitrogen.
  2. Photomultiplier Tube: The photomultiplier tube stands as a popular detector in contemporary UV-Vis spectrophotometers. Its structure comprises an anode, cathode, and multiple dynodes. When a photon enters the tube, it strikes the cathode, leading to the emission of electrons. These electrons are subsequently accelerated towards the first dynode, resulting in the production of several electrons. This process is reiterated across the dynodes, amplifying the number of electrons. The primary advantages of this detector include its ability to detect very low light levels, high wavelength resolution, and faster response time. However, it is susceptible to damage when exposed to high-intensity light.
  3. Diode Array Detector: A diode array detector is a multichannel photon detector capable of measuring all wavelengths of dispersed radiation simultaneously. It comprises an array of silicon photodiodes on a single silicon chip. Each diode in the array is reverse biased, and individual diode circuits are sequentially scanned. The primary advantage of this detector is its ability to record the complete spectrum in a short time frame. However, it may present challenges such as relevant dark current and high read noise.
  4. Charge Coupled Device (CCD): The CCD is renowned for its high sensitivity, making it ideal for detecting extremely low light intensity signals. Structurally similar to diode array detectors, CCDs utilize photo capacitors instead of diodes. Each photocapacitor in a CCD comprises millions of detector elements termed pixels. The silicon chip within the CCD converts light into an electric signal, with the buildup of charges corresponding to the pattern of the incident light. CCDs are known for their low dark count rate, high UV-Vis quantum efficiency, and low read noise.

In conclusion, the choice of detector in UV-Visible spectroscopy is pivotal, with each type offering unique advantages and functionalities. Whether one opts for the simplicity of a phototube or the precision of a CCD, the emphasis remains on accurate and efficient light-to-signal conversion, ensuring reliable and consistent results in spectroscopic analyses.

Factors affecting UV-Vis Spectroscopy

UV-Vis spectroscopy, a cornerstone technique in analytical chemistry, is influenced by a myriad of factors that can alter the results and spectra obtained. For a comprehensive understanding, let’s delve into a detailed and sequential explanation of each factor.

  1. Effect of Sample Temperature: Temperature variations in the sample can significantly influence the spectrum. As the temperature decreases, the sharpness of absorption bands intensifies. However, the total absorption intensity remains unaffected by temperature changes. Lower temperatures also reduce the rotational and vibrational energy states of molecules, producing finer absorption bands. Therefore, for precision, it’s imperative to maintain a consistent or specific temperature when obtaining the spectrum.
  2. Effect of Sample Concentration: The concentration of the sample directly correlates with the intensity of light absorption. High concentrations can lead to molecular interactions, altering the shape and position of absorption bands. The solvent’s nature also plays a pivotal role, with polar solvents producing broader bands compared to non-polar solvents. Thus, understanding the solvent-solute interactions is crucial for accurate spectral interpretation.
  3. Effect of Sample pH: The pH of the solution can markedly affect the absorption spectra of certain compounds. For instance, changes in pH can alter the spectra of aromatic compounds like amines and phenols. Acid-base indicators, due to their absorptions in the visible region, are particularly sensitive to pH changes. Maintaining a constant pH, often using buffer solutions, is essential for consistent spectral results.
  4. Effect of Solvent: The solvent in which the molecule is dissolved can shift the absorption peak. Polar solvents, for instance, can form hydrogen bonds with the substance, shifting the absorption bands of polar molecules. The spectrum recorded in a non-polar solvent can differ significantly from that in a polar solvent due to these interactions.
  5. Effect of Steric Hindrance: The molecular configuration, especially in terms of planarity and conjugation, can influence the spectrum. Steric hindrance, which prevents molecules from existing in a planar configuration, can shift the absorption peak. Geometric isomerism, particularly the difference between trans and cis isomers, also plays a role in determining the spectrum.
  6. Effect of Conjugation: Conjugation in molecules can shift the absorption peak. When chromophores are conjugated, the absorption peak shifts to a longer wavelength. An increase in the number of conjugated bonds can lead to the absorption of visible light, imparting color to compounds.

In conclusion, UV-Vis spectroscopy, while a powerful analytical tool, is influenced by various factors that can alter the results. Understanding these factors and their implications is pivotal for accurate spectral interpretation and analysis. Whether it’s the temperature, concentration, or the solvent used, each element plays a crucial role in shaping the final spectrum.

Types of Cuvette and Compatible Wavelengths for UV-Vis spectroscopy

In the realm of UV-Vis spectroscopy, the cuvette plays a pivotal role as it holds the sample to be analyzed. The material from which a cuvette is made determines its compatibility with specific wavelengths, thereby influencing the accuracy and range of measurements. Here’s a detailed exposition on the various cuvette materials and their respective wavelength compatibilities:

  1. Pyrex Glass or Optical Glass Cuvettes:
    • Material Composition: These cuvettes are primarily made from polystyrene, PMMA, and glass.
    • Wavelength Compatibility: They are suitable for the visible radiation range, boasting a transmission range from 340 nm to 2500 nm.
    • Applications: Given that a majority of spectroscopic applications fall within this range, these cuvettes are commonly used.
    • Cost Consideration: For those with budget constraints, optical glass cuvettes are a viable choice due to their affordability.
  2. UV Quartz Cuvettes:
    • Material Composition: UV quartz is a specialized type of plastic, distinct from regular optical glass.
    • Wavelength Compatibility: These cuvettes have an extended transmission range from 190 nm to 2500 nm, encompassing the UV spectroscopy range.
    • Applications: They are indispensable for UV spectra measurements, given their compatibility with ultraviolet wavelengths.
    • Cost Consideration: Being superior to optical glass in terms of transmission range, UV quartz cuvettes are priced higher.
  3. IR Quartz Cuvettes:
    • Material Composition: IR quartz is a refined material tailored for UV-Vis experiments.
    • Wavelength Compatibility: They offer a transmission range of 220 nm to 3500 nm, covering both the IR and some UV regions.
    • Applications: Ideal for experiments that require measurements in both the UV and IR spectra.
    • Cost Consideration: Their broader range and enhanced quality make them a pricier option compared to UV quartz and optical glass cuvettes.
  4. Sapphire Cuvettes:
    • Material Composition: Sapphire is renowned for its hardness and resistance to scratches.
    • Wavelength Compatibility: These cuvettes have an impressive transmission range of 250 nm to 5000 nm.
    • Applications: Given their broad optical range, they are suitable for diverse spectroscopic applications.
    • Unique Features: Besides their broad transmission range, sapphire cuvettes are resistant to electrical, thermal, and chemical damage.
    • Cost Consideration: Owing to their superior material quality and extensive transmission range, sapphire cuvettes are among the most expensive.

In conclusion, the choice of cuvette material is crucial in UV-Vis spectroscopy. It not only determines the range of wavelengths that can be measured but also influences the accuracy and reliability of the results. Therefore, when selecting a cuvette, it’s imperative to consider both the required wavelength range and the budgetary constraints.

Selection of Cuvette in Ultraviolet Visible Spectroscopy

The choice of cuvette material can significantly influence the accuracy and reliability of the results. Therefore, understanding the nuances of cuvette selection is paramount.

  1. UV-Vis Absorbance Cut-off:
    • Every cuvette material possesses a specific UV-Vis absorbance cut-off. Below this cut-off value, the solvent within the cuvette absorbs the light, potentially skewing results.
  2. Material Considerations:
    • For measurements in the UV spectrum, a quartz cuvette is imperative due to its transparency in this range.
    • In the visible spectrum, one has the flexibility to choose between glass, plastic, and quartz cuvettes.
    • For experiments demanding high purity, disposable plastic cuvettes are favored as they curtail the risk of contamination.
    • While aqueous solutions are amenable to any cuvette type, organic solvents necessitate the use of glass cuvettes due to their superior solvent resistance compared to their plastic counterparts.
  3. Durability and Reusability:
    • Plastic cuvettes, although cost-effective, have limited reusability.
    • In contrast, glass and quartz cuvettes, being reusable materials, can be meticulously cleaned and maintained for prolonged use. Their longevity makes them a cost-effective choice in the long run.
    • Quartz, with its enhanced light transmission capabilities and transparency, is the go-to choice for sensitive experiments. Its resilience to temperature fluctuations further underscores its utility.
  4. Polishing:
    • Cuvettes can be polished on either two or all four sides.
    • Given that light traverses the sample linearly, a two-sided polish suffices for most UV-Vis spectroscopy applications, ensuring optimal light transmittance.
    • However, for specialized experiments like fluorescence and scattering, where the signal is captured at a 90° angle to the incident light, a four-sided polish becomes essential.

Advantages of UV-Vis spectroscopy

  1. Non-Destructive Nature:
    • One of the primary advantages of UV-Vis Spectroscopy is its non-destructive nature.
    • This means that post-analysis, the sample remains intact and unaltered, allowing for its reuse or further processing in subsequent experiments or analyses.
  2. Speed and Efficiency:
    • UV-Vis Spectroscopy facilitates rapid measurements.
    • This swift nature ensures that it can be seamlessly integrated into various experimental protocols, enhancing the efficiency of the overall research process.
  3. User-Friendly Instruments:
    • Instruments used in UV-Vis Spectroscopy are notably user-friendly.
    • Their straightforward design and operation mean that users require minimal training before they can effectively utilize the equipment.
  4. Simplified Data Analysis:
    • The data procured from UV-Vis Spectroscopy typically requires minimal processing.
    • This simplicity in data analysis further reduces the need for extensive user training, ensuring that even novices can interpret the results with ease.
  5. Cost-Effectiveness:
    • From an economic standpoint, UV-Vis Spectroscopy instruments are generally affordable.
    • Not only is the initial acquisition cost reasonable, but the operational expenses are also relatively low. This cost-effectiveness makes the technique accessible to a wide range of laboratories, from academic settings to industrial research facilities.

Limitations of UV-Vis spectroscopy (DIsadvantages)

  1. Stray Light:
    • In practical scenarios, wavelength selectors in UV-Vis instruments are not flawless. They might inadvertently transmit a minor amount of light from a broader wavelength range originating from the light source.
    • Such stray light can introduce significant measurement errors.
    • Additionally, stray light can also emanate from external environments or due to improperly fitted compartments within the instrument.
  2. Light Scattering:
    • Light scattering is predominantly caused by suspended solids present in liquid samples. This phenomenon can lead to substantial measurement inaccuracies.
    • The presence of air bubbles within the cuvette or the sample can further exacerbate light scattering, leading to inconsistent results.
  3. Interference from Multiple Absorbing Species:
    • A single sample might contain multiple chemical species that absorb light. For instance, a sample could have various types of chlorophyll, each with its unique absorption spectrum.
    • When these species are analyzed together, their spectra might overlap, complicating the analysis.
    • For accurate quantitative analysis, it’s imperative to separate each chemical species from the sample and analyze them individually.
  4. Geometrical Considerations:
    • The precise alignment of the instrument’s components is paramount for accurate results. Any misalignment, especially concerning the cuvette holding the sample, can lead to inconsistent and erroneous outcomes.
    • Every component must maintain a consistent orientation and position for each measurement to ensure reproducibility.
    • Given these geometrical sensitivities, it’s recommended that users undergo basic training to prevent potential misuse and misalignment.

Applications of  UV-Vis spectroscopy

  1. Identification of Species:
    • UV-Vis spectroscopy plays a pivotal role in identifying both organic and inorganic species present in a solution.
  2. Concentration Determination:
    • This technique is adept at determining the concentration of unknown solutions, providing valuable insights into sample composition.
  3. Structural Elucidation:
    • UV-Vis spectroscopy aids in the determination of molecular structures. It provides data on bands and intensities associated with specific functional groups, facilitating a deeper understanding of molecular configurations.
    • It is instrumental in detecting the presence or absence of unsaturation and heteroatoms in organic molecules.
  4. Chemical Kinetics:
    • The study of chemical kinetics, specifically the appearance and disappearance of functional groups during reactions, is enhanced using UV-Vis spectroscopy. By passing UV radiation through a reaction cell, absorbance changes can be meticulously observed.
  5. Isomer Study:
    • In the realm of geometric isomerism, UV-Vis spectroscopy offers insights into species differentiation. For instance, trans-species, which absorb at higher wavelengths, exhibit larger molar absorptivity values compared to their cis counterparts.
  6. Detection of Conjugation:
    • The technique is proficient in detecting the presence of conjugation in molecules, further elucidating molecular behavior.
  7. Impurity Detection:
    • UV-Vis spectroscopy stands out as an optimal method for impurity determination in organic molecules. By comparing the sample’s absorption spectrum with that of a standard raw material, impurities can be identified.
  8. Functional Group Analysis:
    • The presence or absence of specific functional groups in compounds can be ascertained using this technique. The absence of a specific band at a designated wavelength serves as evidence of the absence of a particular group.
  9. Pharmaceutical Applications:
    • In the pharmaceutical domain, many drugs, whether in raw material form or as formulations, can be assayed. This is achieved by preparing a suitable solution of the drug and measuring its absorbance at specific wavelengths.
  10. Molecular Weight Determination:
    • By preparing suitable derivatives of compounds, their molecular weights can be determined spectrophotometrically.
  1. High-Performance Liquid Chromatography (HPLC):
    • UV spectrophotometers can be employed as detectors in HPLC, enhancing the precision of chromatographic analyses.
  1. Miscellaneous Applications:
    • UV-Vis spectroscopy finds its utility in clinics for drug analysis, in petrochemical industries, water quality control labs, forensic labs, and even in chemical and biological plants.


What are the Applications of UV-Visible Spectroscopy?

UV-Vis spectroscopy is a versatile and widely used analytical technique that has a wide range of applications in various fields, such as:
Analytical chemistry: Determining concentration and identifying unknown compounds
Biochemistry: Studying the structure and function of biomolecules
Environmental science: Monitoring water and air quality, detecting pollutants
Pharmaceuticals: Analysis of drugs, determining purity, monitoring synthesis
Food industry: Measuring concentration of food ingredients and monitoring product quality
Materials science: Studying the electronic and optical properties of materials
Organic chemistry: Identifying functional groups and studying reaction mechanisms
Medical research: Studying properties of blood, glucose level and photochemistry of biological systems
Forensics: Analyzing trace evidence, identifying sample source
Industrial process control: Monitoring chemical reactions, optimizing conditions and controlling final product quality.

What is UV-Visible spectroscopy?

UV-Vis spectroscopy, also known as ultraviolet-visible spectroscopy, is a technique that is used to study the interaction of matter and electromagnetic radiation. It specifically deals with the absorption of ultraviolet (UV) and visible light by a sample. The sample is exposed to a broad range of wavelengths of light and the absorption of light at different wavelengths is measured. The resulting data is plotted as a spectrum, from which the sample’s absorbance or transmittance can be determined. The absorbance or transmittance spectrum can be used to identify the presence of specific functional groups or to determine the concentration of a substance in a solution. UV-Vis spectroscopy is commonly used in analytical chemistry, biochemistry, and materials science, as well as in various industries like Pharmaceuticals, food, water and environment analysis, etc.

What are the limitations of UV Visible Spectroscopy?

The time required to prepare to utilise a UV-VIS spectrometer is the primary drawback of its application. UV-VIS spectrometers must be properly configured. Any external light, electrical noise, or other impurities that could interfere with the spectrometer’s reading must be eliminated.

Why is a glass cuvette not suitable for UV?

A glass cuvette is not suitable for UV spectroscopy because it absorbs UV light, which can interfere with the measurement being taken. Quartz cuvettes are typically used for UV spectroscopy because they are transparent to UV light and do not absorb it.

What is the main difference between UV and IR spectroscopy?

The main difference between UV and IR spectroscopy is the range of wavelengths they use to measure different compounds. UV spectroscopy typically uses wavelengths in the range of 190-400 nm, while IR spectroscopy uses wavelengths in the range of 4000-400 cm-1 (or roughly 750-2.5 µm). UV spectroscopy is useful for studying compounds that have conjugated double bonds or aromatic ring systems, while IR spectroscopy is useful for studying compounds that have vibrations of functional groups such as C-H, C-O, N-H and O-H. UV spectroscopy is more sensitive to changes in the electron density of a compound, while IR spectroscopy is more sensitive to changes in the vibrational energy of a compound.

What is the range of UV-Vis spectroscopy?

The range of UV-Vis (ultraviolet-visible) spectroscopy is typically from 190 nm to 800 nm, although some instruments can measure as low as 175 nm and as high as 2500 nm. This range covers both ultraviolet (UV) and visible regions of the electromagnetic spectrum. The UV region is typically divided into three regions, the vacuum ultraviolet (VUV, < 200 nm), the near ultraviolet (NUV, 200-380 nm) and the far ultraviolet (FUV, 380-200 nm) and the visible region is from 400 nm to 800 nm. The UV-Vis spectroscopy is sensitive to the absorption of light by a compound, which is directly related to its electron density, and can be used to identify and quantify a wide variety of compounds, including transition metal complexes, organic dyes, and biomolecules.

What is the unit of UV spectroscopy?

However, Beer–Lambert’s law is often applied to obtain the concentration of the sample (c) after measuring the absorbance (A) when the molar absorptivity (ε) and the path length (L) are known. Typically, ε is expressed with units of L mol‑1 cm‑1, L has units of cm, and c is expressed with units of mol L‑1.

Why we use HPLC instead of UV?

High-performance liquid chromatography (HPLC) and ultraviolet (UV) spectroscopy are both analytical techniques that can be used to identify and quantify compounds in a mixture, but they have different strengths and are often used in conjunction with each other.
One of the main reasons why HPLC is used instead of UV is that HPLC can separate and isolate individual components in a mixture, while UV spectroscopy typically provides a broad spectrum of all the compounds present in a sample. HPLC allows for the purification and isolation of specific compounds, which can then be further analyzed using UV spectroscopy or other techniques.
Another reason is that HPLC is a highly sensitive technique and can detect and quantify compounds at very low concentrations, while UV spectroscopy is relatively less sensitive. HPLC is also suitable for a wide variety of samples, such as liquid, gases, and even solid samples, while UV spectroscopy is mainly used for liquid samples.
Furthermore, HPLC can be used to identify and quantify a wide range of compounds, including polar, non-polar, ionized and non-ionized compounds, while UV spectroscopy is sensitive to the absorption of light by a compound, which is directly related to its electron density, and it is mainly used for organic compounds.
In summary, HPLC and UV spectroscopy are complementary techniques that are often used together for the analysis of complex samples. HPLC provides separation and isolation of individual components, while UV spectroscopy provides structural and compositional information about the compounds.

Why quartz cuvette is used in UV?

Quartz cuvettes are used in UV spectroscopy because they are transparent to UV light and do not absorb it. Quartz is a type of silica (SiO2) that has a high transmission in the UV-visible region of the spectrum, making it ideal for UV spectroscopy measurements. Quartz cuvettes are also relatively durable and easy to clean, which makes them suitable for use in analytical instruments.
Another reason is that quartz cuvettes have a low thermal expansion coefficient which makes them suitable for use in high-temperature measurements and also in thermal cycling.
Furthermore, quartz cuvettes are also chemically resistant to most of the solvents and samples that are used in UV spectroscopy, this makes them a suitable choice for many applications.
In contrast, glass cuvettes absorb UV light, which can interfere with the measurement being taken. They are not suitable for UV spectroscopy.
In summary, quartz cuvettes are used in UV spectroscopy because they are transparent to UV light, durable, easy to clean, chemically resistant, have low thermal expansion coefficient, and do not absorb UV light, which makes them ideal for use in analytical instruments.

What is the unit of absorbance?

Absorbance is directly proportional to concentration and length: A = εcl. ε is the wavelength-dependent molar absorbtivity coefficient and it is constant for a particular substance. ε has units of L mol – 1 cm – 1.

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