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

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 spectroscopy, also known as UV-visible spectrum (UV-Vis also known as UV/Vis). UV-Vis spectroscopy is a technique used to measure the absorption of ultraviolet (UV) and visible light by a sample. It is commonly used to determine the concentration of a substance in a solution or to identify the functional groups present in a molecule. The absorption of light by a sample is measured at different wavelengths, and the resulting data is plotted as a spectrum. The absorption 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 an analytical technique that measures the amount of discrete wavelengths of UV or visible light that are absorbed by or transmitted through a sample in comparison to a reference or blank sample. This property is influenced by the sample composition, potentially providing information on what is in the sample and at what concentration. Since this spectroscopy technique relies on the use of light, let’s first consider the properties of light.

Light has a certain amount of energy which is inversely proportional to its wavelength. Thus, shorter wavelengths of light carry more energy and longer wavelengths carry less energy. A specific amount of energy is needed to promote electrons in a substance to a higher energy state which we can detect as absorption. Electrons in different bonding environments in a substance require a different specific amount of energy to promote the electrons to a higher energy state. This is why the absorption of light occurs for different wavelengths in different substances. Humans are able to see a spectrum of visible light, from approximately 380 nm, which we see as violet, to 780 nm, which we see as red. UV light has wavelengths shorter than that of visible light to approximately 100 nm. Therefore, light can be described by its wavelength, which can be useful in UV-Vis spectroscopy to analyze or identify different substances by locating the specific wavelengths corresponding to maximum absorbance (see the Applications of UV-Vis spectroscopy section).

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

Spectroscopy is fundamentally concerned with the interplay between light and matter.
When light is absorbed by matter, the outcome is an increase in the atomic or molecular energy content. Absorption of UV radiation results in the excitation of electrons from their ground state to a higher energy state. Molecules with -electrons or nonbonding electrons (n-electrons) can absorb ultraviolet light energy to excite these electrons to higher anti-bonding molecular orbitals. When electrons are more easily excited, they can absorb longer wavelengths of light. There are four sorts of possible transitions (π–π, n–π, σ–σ, and n–σ), which can be arranged as follows: σ–σ* > n–σ* > π–π* > n–π*. Absorption of UV light by a chemical substance will generate a spectrum that can be used to identify the component.

Utilizing ultraviolet and visible spectroscopy, also known as electronic spectroscopy, one may determine the quantity of double bond conjugation and aromatic conjugation within a molecule. This entails the transition of electrons from HOMO to LUMO (HOMO means Highest Occupied Molecular Orbital whereas LUMO means Lowest Unoccupied Molecular Orbital). As conjugation increases, the HOMO-LUMO gap decreases.

Visible zone corresponds to 800-400 nm while ultraviolet region corresponds to 400-200 nm. UV-visible spectroscopy based on the law of Beer and Lambert

log I_o/I = ε. 𝑙. 𝑐 or ϵ = 𝐴/𝑐 𝑙

Where,

• I_o = intensity of incident light
• I = intensity of transmitted light
• ε = molar absorptivity
• l = path length of sample
• c = concentration of sample
• A = Absorbance

Absorbance (A): It is reciprocal of Transmittance

Absobance (A) = Optical density (D) = log 1/T or log I_o/I

Transmittance (T): The fraction of incident light transmitted is known as Transmittance.

Transmittance (T) = I/I_o

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.

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.

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

There are present two types of U.V-Visible spectroscopy

1. Single beam spectroscopy

In the single beam configuration of a single beam spectroscopic monochromator, the sample and detector are placed in series. Here, Io-intensity monochromator light is transmitted through the sample to excite electrons from a lower energy state to a higher energy one.

2. Double beam spectroscopy

In double beam spectroscopy, the splitter or chopper divides monochromatic light into two beams, one of which goes through the sample and the other through the standard.

How does a UV-Vis spectrophotometer work? / 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.

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.

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.

Advantages of UV-Vis spectroscopy

UV-Vis spectroscopy, like any other technology, has its flaws. Popularity can be attributed to the technique’s notable advantages, which are outlined below.

• Versatility: UV-Vis spectroscopy can be used to study a wide range of samples, including liquids, gases, and solids. It can also be used to study samples in different states, such as solutions, suspensions, and emulsions.
• Non‑destructive: The procedure is nondestructive, allowing the sample to be reused or processed or analysed further.
• Rapid: Rapid measurements allow for simple integration into experimental methods.
• Sensitivity: UV-Vis spectroscopy is a highly sensitive technique that can detect even small concentrations of a substance in a sample.
• Easy to use: The instruments are simple to operate and require minimal training prior to use.
• Minimal processing: In general, data analysis needs minimum processing, hence requiring minimal user training.
• Quantitative analysis: UV-Vis spectroscopy can be used to determine the concentration of a substance in a sample, which makes it useful for quantitative analysis.
• Non-destructive: UV-Vis spectroscopy does not damage or alter the sample being studied, making it a non-destructive technique.
• High-throughput: UV-Vis spectroscopy can be used to analyze multiple samples simultaneously, which makes it useful for high-throughput applications.
• Low cost: UV-Vis spectroscopy equipment is relatively low cost and easy to operate and maintain.
• Widely available: UV-Vis spectroscopy is widely available in research and industrial laboratories, making it easily accessible for a wide range of applications.
• High-resolution: UV-Vis spectroscopy can be used to study samples at high-resolution, which allows for detailed analysis of the sample.
• Wide range of applications: UV-Vis spectroscopy has a wide range of applications, including in analytical chemistry, biochemistry, materials science, and in industries such as Pharmaceuticals, food, water and environment analysis, etc.

Limitations of UV-Vis spectroscopy (DIsadvantages)

While the benefits of this method seem to be to be overwhelming, they are certain flaws:

• Stray light: In a practical instrument, wavelength selectors are imperfect, and a small amount of light from a broad range of wavelengths may still be transmitted from the light source, potentially resulting in significant measurement errors. Additionally, stray light may originate from the environment or a loosely-fitting chamber within the device.
• Light scattering: Suspended particulates in liquid samples frequently produce light scattering, which can lead to significant measurement errors. The presence of bubbles in the cuvette or sample will scatter light, producing results that cannot be replicated.
• Interference from several absorbing species: A sample may contain various forms of the green pigment chlorophyll, for instance. When many chlorophylls are observed in the same sample, their spectra will overlap. For an accurate quantitative analysis, each chemical species must be extracted from the sample and analysed separately.
• Geometrical considerations: Inaccurate and unreproducible results may come from the misalignment of any of the instrument’s components, especially the cuvette containing the sample. For each measurement, it is crucial that each component of the instrument is aligned in the same orientation and positioned in the same location. Therefore, basic user training is often necessary to prevent misuse.
• Interference: UV-Vis spectroscopy can be affected by interferences such as scattered light and fluorescence, which can make it difficult to obtain accurate results.
• Sample preparation: Some samples require special preparation or handling before they can be analyzed using UV-Vis spectroscopy, which can be time-consuming and labor-intensive.
• Limited to surface analysis: UV-Vis spectroscopy is limited to analyzing the surface of a sample, so it is not suitable for studying the bulk properties of a material.
• Limited to specific functional groups: UV-Vis spectroscopy is sensitive to specific functional groups, such as conjugated double bonds and aromatic rings, which limits its ability to detect other functional groups.
• Complex samples: UV-Vis spectroscopy can be complicated when analyzing complex samples, such as biological samples that contain multiple components that absorb light at the same wavelength.
• Limited to transparent samples: UV-Vis spectroscopy requires that the sample is transparent at the wavelengths of interest, so it is not suitable for samples that are opaque or highly colored.
• High cost: some advanced UV-Vis spectroscopy instruments are quite expensive and may not be accessible to small laboratories or research groups.
• Requires a high degree of skill: UV-Vis spectroscopy requires a high degree of skill to operate and interpret the results, which can be a limitation for some users.

Applications of UV-Vis spectroscopy

UV-Vis has been adapted to numerous situations and purposes such as but not limited:

1. DNA and RNA analysis

• The rapid verification of the purity and concentration of RNA and DNA is a ubiquitous application. Table 1 provides a summary of the wavelengths employed in their analysis and what they represent. When preparing DNA or RNA samples for downstream applications such as sequencing, it is sometimes crucial to ensure that neither sample is contaminated with the other or with protein or chemicals derived from the isolation procedure.

Wavelength used in absorbance analysisin nanometers	What does UV absorbance at this wavelength indicate the presence of?	What causes UV absorbance at this wavelength?
230	Protein	Protein shape
260	DNA and RNA	Adenine, guanine, cytosine, thymine, uracil
280	Protein	Mostly tryptophan and tyrosine

• The ratio of absorbance at 260 to 280 nanometers (260/280) is beneficial for detecting probable contamination in nucleic acid samples, as detailed in Table 2. Pure DNA often has a 260/280 ratio of 1.8, whereas pure RNA typically has a ratio of 2.0. Because thymine, which is replaced by uracil in RNA, has a lower 260/280 ratio than uracil, pure DNA has a lower 260/280 ratio than RNA. Protein-contaminated samples will have a lower 260/280 ratio due to increased absorbance at 280 nm.

Absorbance ratio	Typical values
260/280	1.8 absorbance ratio typical for pure DNA2.0 absorbance ratio typical for pure RNA
260/230	Absorbance ratio varies; 2.15 to 2.50 typical for RNA and DNA

• The 260 nm/230 nm absorbance ratio (260/230) is also helpful for determining the cleanliness of DNA and RNA samples, as it can indicate protein or chemical contamination. Proteins can absorb light at 230 nm, reducing the 260/230 ratio and indicating contamination of DNA and RNA samples with proteins. 10 Guanidinium thiocyanate and guanidinium isothiocyanate, two popular chemicals employed in the purification of nucleic acids, absorb substantially at 230 nm, lowering the 260/230 absorbance ratio.

2. Pharmaceutical analysis

• In the pharmaceutical sector, UV-Vis spectroscopy is one of the most prevalent applications. Specifically, analysing UV-Vis spectra with mathematical derivatives enables the resolution of overlapping absorbance peaks in the original spectra to identify individual pharmaceutical drugs.
• By applying the first mathematical derivative to the absorbance spectra, benzocaine, a local anaesthetic, and chlortetracycline, an antibiotic, can be recognised simultaneously in commercial veterinary powder formulations.
• By constructing a calibration function for each component, simultaneous quantification of both chemicals was feasible over a concentration range of micrograms per millilitre.

3. Bacterial culture

• UV-Vis spectroscopy is utilised frequently in bacterial cultivation. To assess cell concentration and monitor growth, 600 nm OD readings are routinely and rapidly acquired at a wavelength of 600 nm.
• 600 nm is widely employed and recommended because to the optical properties of bacterial growth conditions in which they are cultivated and to avoid injuring the cells when they are needed for further investigation.

4. Beverage analysis

• Another common application of UV-Vis spectroscopy is the determination of specific chemicals in beverages. Caffeine content must be under specified legal limits which can be measured with UV light.
• Certain types of colourful compounds, such as the anthocyanin present in blueberries, raspberries, blackberries, and cherries, can be easily identified using UV-Vis absorbance by matching their known peak absorbance wavelengths in wine.

5. Other applications

• Analytical chemistry: UV-Vis spectroscopy is used to determine the concentration of a substance in a sample, to identify unknown compounds, and to monitor chemical reactions.
• Biochemistry: UV-Vis spectroscopy is used to study the structure and function of biomolecules, such as proteins, nucleic acids, and pigments.
• Environmental science: UV-Vis spectroscopy is used to monitor the quality of water and air, to detect pollutants and to study the photochemistry of atmospheric gases.
• Food industry: UV-Vis spectroscopy is used to measure the concentration of food ingredients, to monitor the quality of food products, and to detect contaminants.
• Materials science: UV-Vis spectroscopy is used to study the electronic and optical properties of materials, such as semiconductors, dyes, and pigments.
• Organic chemistry: UV-Vis spectroscopy is used to study the electronic structure of organic molecules, to identify functional groups and to study the mechanism of chemical reactions.
• Medical research: UV-Vis spectroscopy is used to study the properties of blood, to monitor the level of glucose in blood, and to study the photochemistry of biological systems.
• Forensics: UV-Vis spectroscopy is used to analyze trace evidence, such as fibers and paint, to identify the source of a sample.
• Industrial process control: UV-Vis spectroscopy is used to monitor the progress of chemical reactions in industrial processes, to optimize conditions and to control the quality of the final product.

Terms used in UV Spectroscopy

• Chromophores: A chromophore is a part of a molecule that is responsible for its color. Chromophores absorb specific wavelengths of light, and it is this absorption that causes the molecule to appear a certain color. They are found in many natural pigments, such as those found in plants and animals, as well as in synthetic dyes and pigments. Chromophores can be formed from various types of chemical groups, such as conjugated double bonds, aromatic rings, or specific types of atoms or ions.
• Auxochromes: An auxochrome is a functional group that when present in a molecule, it increases the molecule’s ability to absorb light. This can enhance the color of the chromophore, making it appear more intense. Auxochromes are typically groups that contain atoms such as oxygen, nitrogen, or sulfur, and they can interact with the chromophore by forming hydrogen bonds or through electronic interactions. The presence of an auxochrome can also shift the absorption maxima of a chromophore, causing the color of the compound to change. In dyes and pigments, auxochromes can be used to fine-tune the color and make it more resistant to fading.
• Fluorescence: Fluorescence is a phenomenon in which a molecule absorbs light at a specific wavelength (the excitation wavelength) and then emits light at a longer wavelength (the emission wavelength). The light emitted is usually in the visible region of the spectrum, making the process responsible for the fluorescence a common way to visualize and detect molecules in various fields such as chemistry, biology and medicine. Fluorescence occurs when an electron in a molecule is excited from a lower energy state to a higher energy state by absorbing a photon of light. The excited electron then relaxes back to its ground state, emitting a photon of light in the process. Fluorescence is different from phosphorescence which is a similar process, but the relaxation of the excited state happens over a longer period of time. Fluorescence is also different from the absorption of light, which causes a change in the color of a substance, but not the emission of light.
• Phosphorescence: Phosphorescence is a type of luminescence that occurs when a molecule absorbs light and then emits light at a longer wavelength over an extended period of time. Unlike fluorescence, in which the excited state relaxes back to the ground state very quickly, the excited state in phosphorescence relaxes back to the ground state slowly, over milliseconds or even minutes. This allows the emitted light to be detected long after the excitation source has been removed. Phosphorescence is used in a variety of applications, including in glow-in-the-dark materials, night-vision goggles, and biological imaging. The phosphors that are used in these applications are typically made of inorganic compounds such as zinc sulfide or europium-doped compounds, but can also be found in organic compounds such as certain species of fish and sea creatures.

FAQ

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