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Ultraviolet–visible spectroscopy
Ultraviolet–visible spectroscopy
Ultraviolet–visible spectroscopy
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Beckman DU640 UV–Vis spectrophotometer

Ultraviolet–visible spectrophotometry (UV–Vis or UV-VIS)[1][2][3] refers to absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet and the full, adjacent visible regions of the electromagnetic spectrum.[2] Being relatively inexpensive and easily implemented, this methodology is widely used in diverse applied and fundamental applications. The only requirement is that the sample absorb in the UV–Vis region, i.e. be a chromophore. Absorption spectroscopy is complementary to fluorescence spectroscopy. Parameters of interest, besides the wavelength of measurement, are absorbance (A) or transmittance (%T) or reflectance (%R), and its change with time.[4][5]

A UV–Vis spectrophotometer is an analytical instrument that measures the amount of ultraviolet (UV) and visible light that is absorbed by a sample. It is a widely used technique in chemistry, biochemistry, and other fields, to identify and quantify compounds in a variety of samples.[6]

UV–Vis spectrophotometers work by passing a beam of light through the sample and measuring the amount of light that is absorbed at each wavelength. The amount of light absorbed is proportional to the concentration of the absorbing compound in the sample.

Optical transitions

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Most molecules and ions absorb energy in the ultraviolet or visible range, i.e., they are chromophores. The absorbed photon excites an electron in the chromophore to higher energy molecular orbitals, giving rise to an excited state.[7] For organic chromophores, four possible types of transitions are assumed: π–π*, n–π*, σ–σ*, and n–σ*. Transition metal complexes are often colored (i.e., absorb visible light) owing to the presence of multiple electronic states associated with incompletely filled d orbitals.[5]

Applications

[edit]
An example of a UV–Vis readout

UV–Vis can be used to monitor structural changes in DNA.[8]

UV–Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination of diverse analytes or sample, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules. Spectroscopic analysis is commonly carried out in solutions but solids and gases may also be studied.

  • Organic compounds, especially those with a high degree of conjugation, also absorb light in the UV or visible regions of the electromagnetic spectrum. The solvents for these determinations are often water for water-soluble compounds, or ethanol for organic-soluble compounds. (Organic solvents may have significant UV absorption; not all solvents are suitable for use in UV spectroscopy. Ethanol absorbs very weakly at most wavelengths.) Solvent polarity and pH can affect the absorption spectrum of an organic compound. Tyrosine, for example, increases in absorption maxima and molar extinction coefficient when pH increases from 6 to 13 or when solvent polarity decreases.
  • While charge transfer complexes also give rise to colors, the colors are often too intense to be used for quantitative measurement.

The Beer–Lambert law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length.[9] Thus, for a fixed path length, UV–Vis spectroscopy can be used to determine the concentration of the absorber in a solution. It is necessary to know how quickly the absorbance changes with concentration. This can be taken from references (tables of molar extinction coefficients), or more accurately, determined from a calibration curve.

A UV–Vis spectrophotometer may be used as a detector for HPLC. The presence of an analyte gives a response assumed to be proportional to the concentration. For accurate results, the instrument's response to the analyte in the unknown should be compared with the response to a standard; this is very similar to the use of calibration curves. The response (e.g., peak height) for a particular concentration is known as the response factor.

The wavelengths of absorption peaks can be correlated with the types of bonds in a given molecule and are valuable in determining the functional groups within a molecule. The Woodward–Fieser rules, for instance, are a set of empirical observations used to predict λmax, the wavelength of the most intense UV–Vis absorption, for conjugated organic compounds such as dienes and ketones. The spectrum alone is not, however, a specific test for any given sample. The nature of the solvent, the pH of the solution, temperature, high electrolyte concentrations, and the presence of interfering substances can influence the absorption spectrum. Experimental variations such as the slit width (effective bandwidth) of the spectrophotometer will also alter the spectrum. To apply UV–Vis spectroscopy to analysis, these variables must be controlled or accounted for in order to identify the substances present.[10]

The method is most often used in a quantitative way to determine concentrations of an absorbing species in solution, using the Beer–Lambert law:[11]

,

where A is the measured absorbance (formally dimensionless but generally reported in absorbance units (AU)[12]), is the intensity of the incident light at a given wavelength, is the transmitted intensity, L the path length through the sample, and c the concentration of the absorbing species. For each species and wavelength, ε is a constant known as the molar absorptivity or extinction coefficient. This constant is a fundamental molecular property in a given solvent, at a particular temperature and pressure, and has units of .

The absorbance and extinction ε are sometimes defined in terms of the natural logarithm instead of the base-10 logarithm.

The Beer–Lambert law is useful for characterizing many compounds but does not hold as a universal relationship for the concentration and absorption of all substances. A 2nd order polynomial relationship between absorption and concentration is sometimes encountered[13] for very large, complex molecules such as organic dyes (xylenol orange or neutral red, for example).[14][15]

UV–Vis spectroscopy is also used in the semiconductor industry to measure the thickness and optical properties of thin films on a wafer. UV–Vis spectrometers are used to measure the reflectance of light, and can be analyzed via the Forouhi–Bloomer dispersion equations to determine the index of refraction () and the extinction coefficient () of a given film across the measured spectral range.[16]

Practical considerations

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The Beer–Lambert law has implicit assumptions that must be met experimentally for it to apply; otherwise there is a possibility of deviations from the law.[14] For instance, the chemical makeup and physical environment of the sample can alter its extinction coefficient. The chemical and physical conditions of a test sample therefore must match reference measurements for conclusions to be valid. Worldwide, pharmacopoeias such as the American (USP) and European (Ph. Eur.) pharmacopeias demand that spectrophotometers perform according to strict regulatory requirements encompassing factors such as stray light[17] and wavelength accuracy.[18]

Spectral bandwidth

[edit]

Spectral bandwidth of a spectrophotometer is the range of wavelengths that the instrument transmits through a sample at a given time.[19] It is determined by the light source, the monochromator, its physical slit-width and optical dispersion and the detector of the spectrophotometer. The spectral bandwidth affects the resolution and accuracy of the measurement. A narrower spectral bandwidth provides higher resolution and accuracy, but also requires more time and energy to scan the entire spectrum. A wider spectral bandwidth allows for faster and easier scanning, but may result in lower resolution and accuracy, especially for samples with overlapping absorption peaks. Therefore, choosing an appropriate spectral bandwidth is important for obtaining reliable and precise results.

It is important to have a monochromatic source of radiation for the light incident on the sample cell to enhance the linearity of the response.[14] The closer the bandwidth is to be monochromatic (transmitting unit of wavelength) the more linear will be the response. The spectral bandwidth is measured as the number of wavelengths transmitted at half the maximum intensity of the light leaving the monochromator.

The best spectral bandwidth achievable is a specification of the UV spectrophotometer, and it characterizes how monochromatic the incident light can be. If this bandwidth is comparable to (or more than) the width of the absorption peak of the sample component, then the measured extinction coefficient will not be accurate. In reference measurements, the instrument bandwidth (bandwidth of the incident light) is kept below the width of the spectral peaks. When a test material is being measured, the bandwidth of the incident light should also be sufficiently narrow. Reducing the spectral bandwidth reduces the energy passed to the detector and will, therefore, require a longer measurement time to achieve the same signal to noise ratio.

Wavelength error

[edit]

The extinction coefficient of an analyte in solution changes gradually with wavelength. A peak (a wavelength where the absorbance reaches a maximum) in the absorbance curve vs wavelength, i.e. the UV–VIS spectrum, is where the rate of change of absorbance with wavelength is the lowest.[14] Therefore, quantitative measurements of a solute are usually conducted, using a wavelength around the absorbance peak, to minimize inaccuracies produced by errors in wavelength, due to the change of extinction coefficient with wavelength.

Stray light

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See also: Stray light

Stray light[20] in a UV spectrophotometer is any light that reaches its detector that is not of the wavelength selected by the monochromator. This can be caused, for instance, by scattering of light within the instrument, or by reflections from optical surfaces.

Stray light can cause significant errors in absorbance measurements, especially at high absorbances, because the stray light will be added to the signal detected by the detector, even though it is not part of the actually selected wavelength. The result is that the measured and reported absorbance will be lower than the actual absorbance of the sample.

The stray light is an important factor, as it determines the purity of the light used for the analysis. The most important factor affecting it is the stray light level of the monochromator.[14]

Typically a detector used in a UV–VIS spectrophotometer is broadband; it responds to all the light that reaches it. If a significant amount of the light passed through the sample contains wavelengths that have much lower extinction coefficients than the nominal one, the instrument will report an incorrectly low absorbance. Any instrument will reach a point where an increase in sample concentration will not result in an increase in the reported absorbance, because the detector is simply responding to the stray light. In practice the concentration of the sample or the optical path length must be adjusted to place the unknown absorbance within a range that is valid for the instrument. Sometimes an empirical calibration function is developed, using known concentrations of the sample, to allow measurements into the region where the instrument is becoming non-linear.

As a rough guide, an instrument with a single monochromator would typically have a stray light level corresponding to about 3 Absorbance Units (AU), which would make measurements above about 2 AU problematic. A more complex instrument with a double monochromator would have a stray light level corresponding to about 6 AU, which would therefore allow measuring a much wider absorbance range.

Deviations from the Beer–Lambert law

[edit]

At sufficiently high concentrations, the absorption bands will saturate and show absorption flattening. The absorption peak appears to flatten because close to 100% of the light is already being absorbed. The concentration at which this occurs depends on the particular compound being measured. One test that can be used to test for this effect is to vary the path length of the measurement. In the Beer–Lambert law, varying concentration and path length has an equivalent effect—diluting a solution by a factor of 10 has the same effect as shortening the path length by a factor of 10. If cells of different path lengths are available, testing if this relationship holds true is one way to judge if absorption flattening is occurring.

Solutions that are not homogeneous can show deviations from the Beer–Lambert law because of the phenomenon of absorption flattening. This can happen, for instance, where the absorbing substance is located within suspended particles.[21][22] The deviations will be most noticeable under conditions of low concentration and high absorbance. The last reference describes a way to correct for this deviation.

Some solutions, like copper(II) chloride in water, change visually at a certain concentration because of changed conditions around the colored ion (the divalent copper ion). For copper(II) chloride it means a shift from blue to green,[23] which would mean that monochromatic measurements would deviate from the Beer–Lambert law.

Measurement uncertainty sources

[edit]

The above factors contribute to the measurement uncertainty of the results obtained with UV–Vis spectrophotometry. If UV–Vis spectrophotometry is used in quantitative chemical analysis then the results are additionally affected by uncertainty sources arising from the nature of the compounds and/or solutions that are measured. These include spectral interferences caused by absorption band overlap, fading of the color of the absorbing species (caused by decomposition or reaction) and possible composition mismatch between the sample and the calibration solution.[24]

Ultraviolet–visible spectrophotometer

[edit]
See also: Spectrophotometry

The instrument used in ultraviolet–visible spectroscopy is called a UV–Vis spectrophotometer. It measures the intensity of light after passing through a sample (), and compares it to the intensity of light before it passes through the sample (). The ratio is called the transmittance, and is usually expressed as a percentage (%T). The absorbance, , is based on the transmittance:

The UV–visible spectrophotometer can also be configured to measure reflectance. In this case, the spectrophotometer measures the intensity of light reflected from a sample (), and compares it to the intensity of light reflected from a reference material () (such as a white tile). The ratio is called the reflectance, and is usually expressed as a percentage (%R).

The basic parts of a spectrophotometer are a light source, a holder for the sample, a diffraction grating or a prism as a monochromator to separate the different wavelengths of light, and a detector. The radiation source is often a tungsten filament (300–2500 nm), a deuterium arc lamp, which is continuous over the ultraviolet region (190–400 nm), a xenon arc lamp, which is continuous from 160 to 2,000 nm; or more recently, light emitting diodes (LED)[4] for the visible wavelengths. The detector is typically a photomultiplier tube, a photodiode, a photodiode array or a charge-coupled device (CCD). Single photodiode detectors and photomultiplier tubes are used with scanning monochromators, which filter the light so that only light of a single wavelength reaches the detector at one time. The scanning monochromator moves the diffraction grating to "step-through" each wavelength so that its intensity may be measured as a function of wavelength. Fixed monochromators are used with CCDs and photodiode arrays. As both of these devices consist of many detectors grouped into one or two dimensional arrays, they are able to collect light of different wavelengths on different pixels or groups of pixels simultaneously.

Simplified schematic of a double beam UV–visible spectrophotometer

A spectrophotometer can be either single beam or double beam. In a single beam instrument (such as the Spectronic 20), all of the light passes through the sample cell. must be measured by removing the sample. This was the earliest design and is still in common use in both teaching and industrial labs.

In a double-beam instrument, the light is split into two beams before it reaches the sample. One beam is used as the reference; the other beam passes through the sample. The reference beam intensity is taken as 100% Transmission (or 0 Absorbance), and the measurement displayed is the ratio of the two beam intensities. Some double-beam instruments have two detectors (photodiodes), and the sample and reference beam are measured at the same time. In other instruments, the two beams pass through a beam chopper, which blocks one beam at a time. The detector alternates between measuring the sample beam and the reference beam in synchronism with the chopper. There may also be one or more dark intervals in the chopper cycle. In this case, the measured beam intensities may be corrected by subtracting the intensity measured in the dark interval before the ratio is taken.

In a single-beam instrument, the cuvette containing only a solvent has to be measured first. Mettler Toledo developed a single beam array spectrophotometer that allows fast and accurate measurements over the UV–Vis range. The light source consists of a Xenon flash lamp for the ultraviolet (UV) as well as for the visible (VIS) and near-infrared wavelength regions covering a spectral range from 190 up to 1100 nm. The lamp flashes are focused on a glass fiber which drives the beam of light onto a cuvette containing the sample solution. The beam passes through the sample and specific wavelengths are absorbed by the sample components. The remaining light is collected after the cuvette by a glass fiber and driven into a spectrograph. The spectrograph consists of a diffraction grating that separates the light into the different wavelengths, and a CCD sensor to record the data, respectively. The whole spectrum is thus simultaneously measured, allowing for fast recording.[25]

Samples for UV–Vis spectrophotometry are most often liquids, although the absorbance of gases and even of solids can also be measured. Samples are typically placed in a transparent cell, known as a cuvette. Cuvettes are typically rectangular in shape, commonly with an internal width of 1 cm. (This width becomes the path length, , in the Beer–Lambert law.) Test tubes can also be used as cuvettes in some instruments. The type of sample container used must allow radiation to pass over the spectral region of interest. The most widely applicable cuvettes are made of high-quality fused silica or quartz glass because these are transparent throughout the UV, visible and near infrared regions. Glass and plastic cuvettes are also common, although glass and most plastics absorb in the UV, which limits their usefulness to visible wavelengths.[4]

Specialized instruments have also been made. These include attaching spectrophotometers to telescopes to measure the spectra of astronomical features. UV–visible microspectrophotometers consist of a UV–visible microscope integrated with a UV–visible spectrophotometer.

A complete spectrum of the absorption at all wavelengths of interest can often be produced directly by a more sophisticated spectrophotometer. In simpler instruments the absorption is determined one wavelength at a time and then compiled into a spectrum by the operator. By removing the concentration dependence, the extinction coefficient (ε) can be determined as a function of wavelength.

Microspectrophotometry

[edit]

UV–visible spectroscopy of microscopic samples is done by integrating an optical microscope with UV–visible optics, white light sources, a monochromator, and a sensitive detector such as a charge-coupled device (CCD) or photomultiplier tube (PMT). As only a single optical path is available, these are single beam instruments. Modern instruments are capable of measuring UV–visible spectra in both reflectance and transmission of micron-scale sampling areas. The advantages of using such instruments is that they are able to measure microscopic samples but are also able to measure the spectra of larger samples with high spatial resolution. As such, they are used in the forensic laboratory to analyze the dyes and pigments in individual textile fibers,[26] microscopic paint chips[27] and the color of glass fragments. They are also used in materials science and biological research and for determining the energy content of coal and petroleum source rock by measuring the vitrinite reflectance. Microspectrophotometers are used in the semiconductor and micro-optics industries for monitoring the thickness of thin films after they have been deposited. In the semiconductor industry, they are used because the critical dimensions of circuitry is microscopic. A typical test of a semiconductor wafer would entail the acquisition of spectra from many points on a patterned or unpatterned wafer. The thickness of the deposited films may be calculated from the interference pattern of the spectra. In addition, ultraviolet–visible spectrophotometry can be used to determine the thickness, along with the refractive index and extinction coefficient of thin films.[16] A map of the film thickness across the entire wafer can then be generated and used for quality control purposes.[28]

Additional applications

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UV–Vis can be applied to characterize the rate of a chemical reaction. Illustrative is the conversion of the yellow-orange and blue isomers of mercury dithizonate. This method of analysis relies on the fact that concentration is linearly proportional to concentration. In the same approach allows determination of equilibria between chromophores.[29][30]

From the spectrum of burning gases, it is possible to determine a chemical composition of a fuel, temperature of gases, and air-fuel ratio.[31]

See also

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References

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Revisions and contributorsEdit on WikipediaRead on Wikipedia

Ultraviolet–visible spectroscopy

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Ultraviolet–visible spectroscopy (UV-Vis spectroscopy) is an analytical technique used to measure the absorption or transmission of (typically 200–400 nm) and visible (400–800 nm) by a sample relative to a reference, providing insights into the electronic structure and concentration of substances. The method relies on the principle that molecules or ions absorb photons at specific wavelengths corresponding to electronic transitions, where electrons are excited from ground-state orbitals (such as σ, n, or π) to higher-energy antibonding orbitals (σ*, π*), with the most common transitions being n → π* and π → π* in organic compounds featuring conjugated systems. This absorption follows the Beer-Lambert law, which relates to concentration, path length, and the molar absorptivity of the , enabling both qualitative identification and quantitative determination. The instrumentation for UV-Vis spectroscopy typically consists of a light source (such as a or tungsten-halogen lamp for UV and visible ranges, respectively), a wavelength selector like a or to isolate specific s, a sample compartment (often a to transmit UV light), and a detector such as a or array to measure transmitted intensity. Spectra are plotted as versus , revealing characteristic peaks that indicate the presence of chromophores—structural features responsible for light absorption, such as conjugated double bonds or aromatic rings. Solvents used must be spectrally pure to avoid interference, as even trace impurities can absorb in the UV region and obscure analyte signals. UV-Vis spectroscopy finds broad applications across disciplines, including quantitative analysis of biomolecules like nucleic acids and proteins in biochemistry, monitoring reaction kinetics and product purity in organic synthesis, determining metal ion concentrations in environmental samples, and quality control in pharmaceuticals and food industries. For instance, it is routinely employed to quantify DNA concentration at 260 nm due to the strong absorption by nucleotide bases and to assess bacterial growth by measuring optical density at 600 nm. Despite its simplicity and cost-effectiveness, the technique has limitations, such as sensitivity to scattering in turbid samples and inability to provide detailed structural information without complementary methods like NMR or IR spectroscopy.

Principles

Optical transitions

Ultraviolet–visible (UV-Vis) spectroscopy probes electronic transitions in molecules, where absorption of in the 200–800 nm range promotes electrons from the to higher energy excited states. These transitions primarily involve valence electrons in organic and inorganic molecules, occurring between molecular orbitals such as bonding (σ, π) and antibonding (σ*, π*) orbitals, as well as non-bonding () orbitals. The main types of electronic transitions observed in UV-Vis spectra include π → π* transitions, where an electron is excited from a π bonding orbital to a π* antibonding orbital, typically in conjugated systems; n → π* transitions, involving promotion of a non-bonding (e.g., from oxygen or lone pairs) to a π* orbital; n → σ* transitions, from non-bonding to σ* antibonding orbitals, which require higher ; and charge-transfer transitions, where an electron moves between different atoms or ligands, often in coordination complexes or dyes. π → π* and n → π* transitions are most common in organic molecules due to their lower energy requirements compared to σ → σ* transitions, which often fall outside the typical UV-Vis range. These transitions are depicted in a , which illustrates the electronic states of a with the ground (S₀) at the lowest and excited s (S₁, S₂, etc.) at higher levels, separated by energy gaps corresponding to UV-Vis photon energies. Vertical arrows represent absorption from S₀ to S₁ or S₂, while horizontal lines within each state account for vibrational sublevels; rapid vibrational relaxation follows excitation, but intersystem crossing to triplet states is less relevant for direct UV-Vis absorption. Several factors influence the energy and intensity of these transitions. The length of conjugation in chromophores lowers the energy gap between π and π* orbitals, shifting absorption to longer wavelengths (bathochromic shift). arise from stabilization of ground or excited states; polar solvents can blue-shift n → π* transitions by solvating the n orbital more strongly, while stabilizing π* orbitals in π → π* transitions. Selection rules, governed by , dictate allowed transitions (e.g., ΔS = 0 for singlets, strong dipole moment change) versus forbidden ones (e.g., n → π* often weak due to poor overlap), though vibronic coupling can relax these rules. Representative examples include , which exhibits a π → π* transition at approximately 255 nm due to its aromatic ring, and carbonyl compounds like acetone, showing an n → π* transition around 280 nm from the oxygen to the π* orbital of the C=O bond. These transitions provide the qualitative basis for UV-Vis spectra, with quantified via the .

Beer–Lambert law

The Beer–Lambert law, also known as Beer's law or the Bouguer–Beer–Lambert law, provides the fundamental quantitative relationship in ultraviolet–visible (UV-Vis) spectroscopy between the absorption of light by a sample and its concentration. It states that the absorbance AA, defined as A=log⁡10(I0/I)A = \log_{10}(I_0 / I) where I0I_0 is the incident light intensity and II is the transmitted intensity, is directly proportional to the molar concentration cc of the absorbing species, the path length ll of the sample, and the molar absorptivity ϵ\epsilon (a wavelength-specific constant for the species): A=ϵlcA = \epsilon l c This logarithmic form is the standard in modern spectroscopy, enabling the determination of unknown concentrations from measured absorbance values./Spectroscopy/Electronic_Spectroscopy/Electronic_Spectroscopy_Basics/The_Beer-Lambert_Law) The law's development spans centuries, beginning with Pierre Bouguer's 1729 observation of light attenuation proportional to the thickness of a transmitting medium (). Johann Heinrich Lambert formalized the exponential decay in 1760 using natural logarithms, establishing I=I0e−κlI = I_0 e^{-\kappa l} where κ\kappa is an absorption coefficient. August Beer extended this in 1852 by incorporating solute concentration, showing that κ\kappa is proportional to cc, thus yielding the full form A=ϵlcA = \epsilon l c. The derivation starts from the differential equation for light attenuation: the fractional decrease in intensity dI/IdI / I through an infinitesimal path length dldl is proportional to the concentration cc and a proportionality constant related to ϵ\epsilon, giving dI/I=−ϵc dldI / I = -\epsilon c \, dl. Integrating from l=0l = 0 (where I=I0I = I_0) to ll (where II is the transmitted intensity) yields ln⁡(I0/I)=ϵcl\ln(I_0 / I) = \epsilon c l. Converting to base-10 logarithm for absorbance provides the standard equation, with ϵ\epsilon defined in units of L mol⁻¹ cm⁻¹ to ensure dimensional consistency (assuming ll in cm and cc in mol L⁻¹)./Spectroscopy/Electronic_Spectroscopy/Electronic_Spectroscopy_Basics/The_Beer-Lambert_Law) The molar absorptivity ϵ\epsilon quantifies the intrinsic absorption strength and typically ranges from 10310^3 to 10510^5 L mol⁻¹ cm⁻¹ for allowed electronic transitions in UV-Vis spectroscopy, such as π→π∗\pi \to \pi^* in conjugated systems, reflecting high transition probabilities. The law assumes monochromatic illumination to avoid wavelength-dependent variations in ϵ\epsilon, negligible scattering or emission (e.g., no fluorescence), and dilute solutions where solute-solute interactions do not alter absorption properties./Spectroscopy/Electronic_Spectroscopy/Electronic_Spectroscopy_Basics/The_Beer-Lambert_Law) Deviations from linearity can arise at high concentrations (e.g., above 0.01 M), where the assumptions break down, leading to nonlinear responses.

Instrumentation

Spectrophotometer components

A UV-Vis spectrophotometer consists of several essential hardware components that work together to generate, select, and detect light in the and visible regions, enabling the measurement of sample . These components include the light source, , sample holder, detector, and associated , arranged in a linear to ensure accurate spectral analysis. Light sources in UV-Vis spectrophotometers are chosen to provide stable, continuous radiation across the required wavelength range. For the ultraviolet region (typically 190–400 nm), a is commonly used, emitting a continuum spectrum due to the excitation and recombination of gas molecules. In the visible region (320–1100 nm), a tungsten-halogen lamp serves as the , offering high intensity and stability through incandescent emission from a filament in a halogen-filled envelope. Many instruments incorporate both lamps, with an optical switch or beam combiner to seamlessly transition between UV and visible spectra, ensuring broad coverage without interruptions. The disperses the polychromatic from the source into its spectral components, allowing selection of a specific for analysis. Traditional designs use either prisms, which rely on material-dependent to separate , or diffraction gratings, which achieve dispersion through constructive interference of reflected from ruled surfaces. Grating-based are preferred in modern instruments for their higher efficiency and flatter dispersion across the UV-Vis range, though prisms offer simplicity in compact setups. Slit widths at the entrance and exit of the control the spectral bandpass, with narrower slits improving resolution (down to 0.1–1 nm) but reducing throughput and signal-to-noise ratio. Sample holders, typically in the form of cuvettes, position the in the for interaction with the monochromatic light. Quartz cuvettes are standard for UV-Vis measurements due to their high transparency below 350 nm, where absorbs strongly; these cells have parallel optical faces to minimize and refractive errors. The conventional path length is 1 cm (10 mm internal), providing a balance between sensitivity and sample volume requirements, though variations from 0.1 cm to 5 cm are available for specialized applications. Detectors convert the transmitted light intensity into an electrical signal proportional to , following the for quantitative output. tubes (PMTs) are widely used for their high sensitivity and low noise, amplifying photocurrent through a series of dynodes to detect weak UV-Vis signals. Photodiodes, often silicon-based, offer simplicity, speed, and cost-effectiveness in array configurations for simultaneous multi-wavelength detection. Instruments operate in either single-beam or double-beam configurations to handle reference and sample measurements. In single-beam setups, light passes sequentially through the reference and sample, providing high sensitivity but requiring manual baseline correction for source fluctuations. Double-beam designs split the beam with a chopper or beamsplitter, directing one path through the sample and the other through a reference cuvette, enabling real-time ratioing to compensate for drift and enhance accuracy. The overall layout follows a where the light source feeds into the for selection, followed by the sample holder and then the detector, with collimating mirrors or lenses ensuring efficient transfer throughout the system. This linear arrangement minimizes losses and , supporting reliable spectra from 190 nm to 1100 nm.

Operational principles

Ultraviolet–visible (UV-Vis) spectrophotometers operate by directing a beam of through a sample and measuring the intensity of transmitted, absorbed, or reflected, typically across wavelengths from 190 to 800 nm. In mode, the instrument quantifies the amount of absorbed by the sample, calculated as A=−log⁡10(T)A = -\log_{10}(T), where TT is the transmittance ratio of sample to beams; transmittance mode directly reports the fraction of passing through the sample, while reflectance mode measures scattered back from opaque or solid samples using an . These modes enable the of electronic transitions in molecules, with being the most common for solution-based analyses due to its sensitivity to concentration changes. Many modern UV-Vis instruments employ double-beam configurations to enhance measurement accuracy by simultaneously comparing the sample and reference beams, minimizing temporal fluctuations from the light source or detector. In these systems, a divides the monochromatic light into two paths—one through the sample and the other through a reference containing solvent or blank—while a mechanical chopper or optical switch alternates the beams to a single detector, or separate detectors are used for each path; this real-time ratioing reduces baseline drift and improves signal stability over long scans. Double-beam designs are particularly advantageous for kinetic studies or samples prone to , as they allow rapid, drift-corrected . Wavelength selection in UV-Vis spectrophotometers occurs via a , often a , which disperses the polychromatic from the source into its components for sequential analysis. In scanning instruments, the grating rotates stepwise to isolate wavelengths in increments of 1–5 nm, with the detector measuring intensity at each step; , defined as the minimum resolvable wavelength difference, is typically 1 nm, limited by slit width and grating quality to balance throughput and detail. Alternatively, detection systems, such as photodiode , capture the entire simultaneously by dispersing across multiple detector elements, enabling faster acquisition without mechanical scanning and supporting high-throughput applications. Photodiode detectors contribute to the high sensitivity of these instruments, often achieving detection limits in the micromolar range for chromophores. Following measurement, UV-Vis spectrophotometers process raw intensity data into spectra, plotting or against to visualize absorption bands. Baseline correction is applied by subtracting a reference spectrum—typically obtained from the blank or —to eliminate instrumental offsets, contributions, or slow drifts, often using automated software algorithms that fit and remove linear or baselines. The resulting spectra provide quantitative insights into peak positions and intensities, with outputs exportable as digital files for further analysis. The operational principles of UV-Vis spectrophotometers have evolved significantly since the mid-20th century, transitioning from manual single-beam instruments requiring sequential wavelength adjustments by hand to automated systems in the 1970s. Early manual spectrophotometers, like the Beckman DU introduced in 1941, relied on operator-controlled prism or grating adjustments for wavelength selection, limiting speed and precision. The advent of automated scanning in the 1960s improved reproducibility, but it was the introduction of diode-array detectors in the late 1970s—exemplified by the 8450A in 1979—that revolutionized operation by enabling simultaneous multi-wavelength detection and computer-controlled data processing. This shift to array-based instruments reduced measurement times from minutes to seconds per and facilitated integration with microcomputers for real-time baseline corrections and spectral manipulation. Subsequent developments from the 1980s onward include the widespread adoption of (CCD) detectors for improved sensitivity and resolution, advanced software for chemometric analysis and artificial intelligence-driven spectral interpretation, and the emergence of portable and miniaturized spectrophotometers for field applications, enhancing accessibility and integration with other analytical techniques as of 2025.

Microspectrophotometry

Microspectrophotometry adapts for the of microscopic samples by integrating a spectrophotometer with an , achieving spatial resolutions down to sub-micron scales. This configuration employs high-quality microscope objectives for both sample illumination and light collection, focusing UV-Vis light onto areas as small as 1–100 μm. Such setups scale down conventional spectrophotometer principles to enable localized spectral measurements without extracting the sample from its context. Key advantages include examination of minute samples, preserving their native state, and the potential to map spatial variations in concentration gradients across heterogeneous materials. This non-destructive approach facilitates detailed where bulk analysis would average out important heterogeneities. Common techniques involve transmission microspectroscopy for thin, translucent sections, where light passes through the sample, and microspectroscopy for opaque specimens, capturing backscattered light to derive absorption or properties. In material science, the method supports the investigation of dyes embedded in polymers, such as those in thin films or synthetic fibers, revealing molecular distributions and interactions. Biological applications focus on within cellular structures, exemplified by the spectral analysis of visual in retinal photoreceptors to determine maxima and pigment types. Despite these benefits, limitations persist, including diminished signal-to-noise ratios from restricted light throughput in the microscopic optical path, which demands specialized UV-transmissive optics to mitigate losses and enhance sensitivity.

Applications

Quantitative analysis

Quantitative analysis in ultraviolet–visible (UV-Vis) spectroscopy relies on the Beer–Lambert law, which relates the absorbance of a sample to its concentration, enabling precise determination of analyte amounts in solution. A fundamental approach involves constructing curves by measuring (A) at a selected against known concentrations (c) of the , typically using least-squares to fit the data. These curves exhibit linearity within a specific range, often up to an absorbance of 1–2, beyond which deviations occur due to factors like or non-monochromatic radiation; for optimal accuracy, at least five standard concentrations spanning the expected sample range are recommended. For pure compounds, direct methods employ single-wavelength measurements where the analyte's maximum occurs, allowing straightforward concentration calculation from the . In mixtures, indirect methods such as multi-wavelength or spectroscopy are used to resolve overlapping spectra; for instance, first- spectra enhance resolution by measuring the rate of change in , enabling quantification at zero-crossing points without physical separation. Representative examples include the Bradford assay for protein quantification, where the G-250 dye binds to proteins, shifting absorbance to 595 nm; the resulting intensity is proportional to protein concentration in the range of 0.1–1.4 mg/mL. In pharmaceuticals, UV-Vis is routinely applied for active pharmaceutical ingredient () purity assessment, such as determining acetylsalicylic acid content at 277 nm in tablet extracts, ensuring compliance with specified limits. Sensitivity in UV-Vis quantitative analysis typically achieves detection limits of 10⁻⁵ to 10⁻⁶ M for analytes with molar absorptivities (ε) around 10⁴–10⁵ L mol⁻¹ cm⁻¹ in a 1 cm pathlength cell, as lower ε values reduce sensitivity while instrument and background interference set the practical floor. Real-world protocols emphasize meticulous , including accurate weighing, dissolution in suitable solvents (e.g., or ), and serial dilutions to fall within the linear range, often followed by to remove particulates. Validation adheres to pharmacopeia standards like USP <857>, which specify procedures for linearity, accuracy, precision, and limit of detection through replicate measurements and statistical evaluation.

Qualitative analysis

In ultraviolet–visible (UV-Vis) spectroscopy, qualitative analysis involves interpreting spectral features to identify compounds and functional groups based on absorption patterns arising from electronic transitions, such as π→π* and n→π* excitations. Key indicators include the position of absorption maxima (λ_max) and relative intensities, which provide clues about molecular structure without relying on concentration measurements. Spectral features such as shifts in λ_max are particularly diagnostic for conjugation extent; extended conjugation lowers the energy gap between ground and excited states, causing bathochromic (red) shifts to longer wavelengths. For instance, in α,β-unsaturated ketones, each additional in conjugation can shift λ_max by approximately 30–40 nm, as established by empirical rules for predicting absorption in conjugated systems. Intensity ratios (ε values) also reveal substitution patterns; in derivatives, ortho and para disubstitutions often enhance absorption intensity due to better orbital overlap compared to meta, where electronic interaction is weaker, leading to distinct ε ratios for the B-band around 250 nm. Both electron-donating and electron-withdrawing groups often induce bathochromic shifts in conjugated systems; for example, auxochromes like -OH extend conjugation through , lowering the transition energy. Fingerprinting compares observed spectra to reference libraries for compound identification, leveraging unique bathochromic or hypsochromic effects from structural motifs. The NIST Chemistry WebBook UV-Vis database, containing spectra for thousands of compounds, enables matching of λ_max and band shapes to confirm identities, such as distinguishing conjugated dienes from isolated ones. Examples include differentiating ortho- and para-substituted benzenes, where para isomers exhibit greater bathochromic shifts (e.g., ~20 nm more than ortho for nitro-substituted toluenes due to enhanced conjugation) and higher intensities in the 250–300 nm region. UV-Vis also monitors reaction progress qualitatively, such as conjugation buildup in Diels-Alder adducts, where a new λ_max emerges at longer wavelengths as the product forms. Solvent effects further refine identification, as polarity influences solvatochromism; protic solvents stabilize excited states via hydrogen bonding, often causing bathochromic shifts of 20–50 nm relative to aprotic media for charge-transfer complexes. For example, in nitroaromatics, (protic) induces larger red shifts than (nonpolar), aiding in confirming presence. UV-Vis serves as a rapid screening tool, complemented by IR for vibrational details or NMR for atomic connectivity, to validate identifications in complex mixtures.

Specialized techniques

Derivative spectroscopy enhances the resolution of (UV-Vis) absorption spectra by computing the first or second s of the signal with respect to , allowing separation of overlapping peaks that are indistinguishable in standard spectra. This technique is particularly useful for analyzing complex mixtures where band broadening or proximity hinders direct peak identification, as the amplifies subtle changes in slope while suppressing baseline drift. However, it introduces amplification, necessitating algorithms like Savitzky-Golay filtering to maintain ; for instance, second- spectra reveal peak maxima as extrema (peaks or troughs), improving quantification in pharmaceutical assays. Stopped-flow spectroscopy is a rapid-mixing technique adapted for UV-Vis detection, enabling kinetic studies of fast chemical reactions on millisecond timescales by abruptly halting reactant flow after mixing in a observation cell. It is widely employed in , such as monitoring substrate binding or catalysis in real time, where transient intermediates absorb in the UV-Vis range; typical setups achieve mixing times under 1 ms, with detection volumes as low as 100 μL for . In biochemical research, it has been instrumental in elucidating mechanisms like the Michaelis-Menten kinetics of oxidoreductases. Fiber-optic probes extend UV-Vis spectroscopy to remote or in situ measurements by transmitting light through flexible silica fibers to a sample site, reflecting or absorbing spectra back to a detector for analysis without direct instrument contact. These probes facilitate real-time process control in industrial settings, such as monitoring reaction progress in chemical reactors, and environmental sensing, like detecting pollutants in flows via changes. Their small size (often <1 mm diameter) and immunity to electromagnetic interference make them ideal for harsh environments, with examples including multipoint arrays for spatial profiling. In the food industry, UV-Vis-based colorimetry using specialized setups assesses product ripeness or quality, such as quantifying anthocyanin pigments in fruits through absorbance at 520 nm to determine harvest readiness non-destructively. Clinically, it measures bilirubin levels in blood via diazo reaction products absorbing at 540 nm, aiding neonatal jaundice diagnosis with portable analyzers for point-of-care testing. Emerging developments since 2000 include portable UV-Vis devices, often battery-powered and smartphone-integrated, for field applications like on-site water quality assessment or agricultural nutrient analysis, achieving detection limits comparable to benchtop systems through micro-optics and LED sources. Hyphenation with chromatography, particularly UV detection in , provides selective monitoring of eluents at multiple wavelengths, essential for separating and quantifying compounds in complex samples like pharmaceuticals or metabolites. Microspectrophotometry complements these by enabling spatial resolution in heterogeneous samples.

Practical considerations

Error sources

In ultraviolet–visible (UV-Vis) spectroscopy, several instrumental factors can introduce errors in spectral measurements. Spectral bandwidth, determined by the monochromator slit width, affects resolution by broadening absorption peaks when the instrument's bandwidth exceeds about one-tenth of the analyte's natural bandwidth; for example, this leads to an overestimation of the molar absorptivity (ε) at the half-width of narrow peaks, such as those in aromatic compounds. Wavelength errors arise from calibration drifts in the monochromator or detector alignment, typically on the order of ±1 nm, which shift the observed λ_max and compromise accurate peak identification, particularly for multicomponent mixtures. Stray light, defined as unintended radiation outside the selected wavelength band reaching the detector due to optical imperfections or leaks in the spectrophotometer components, reduces measured absorbance values, especially at high absorbances (A > 2), where it compresses the and causes nonlinear deviations from expected signals. Chemical factors also contribute to inaccuracies by violating the assumptions of the , which requires monochromatic light, dilute solutions, and stable analytes. At higher concentrations, molecular aggregation alters the effective path length and scattering properties, leading to positive or negative deviations in proportional to concentration. occurs when UV exposure breaks down light-sensitive samples, such as certain dyes or pharmaceuticals, reducing over time during and introducing temporal errors. Changes in can shift absorption maxima or alter molecular , causing spectral shifts that do not follow linear Beer–Lambert behavior if not controlled. To mitigate these errors, routine verification using standards is essential; for instance, holmium oxide solutions or glass filters provide sharp, certified peaks (e.g., at 241.0 nm, 287.2 nm) for checking wavelength accuracy within ±1 nm across the UV-Vis range.

Uncertainty and calibration

Uncertainty in ultraviolet–visible (UV-Vis) spectroscopy arises primarily from variations in the molar absorptivity (ε), path length (l), and instrument noise, which propagate through Beer's law to affect concentration determinations. The relative uncertainty in concentration (u_c/c) can be estimated as the quadratic sum of the relative uncertainties in (u_A/A), ε (u_ε/ε), and l (u_l/l), where instrument noise typically contributes 0.001–0.005 absorbance units (AU) to u_A, depending on the detector and . According to ISO 11352:2025, for such physicochemical methods, including , is evaluated using validation data and results from single laboratories, incorporating both Type A (statistical) and Type B (systematic) evaluations to achieve combined standard uncertainties with coverage factors for 95% confidence intervals. Calibration procedures in UV-Vis spectroscopy involve preparing multi-point standard curves with , such as solutions at concentrations yielding absorbances from 0.1 to 1.5 AU, to verify and photometric accuracy. Blank corrections are performed by measuring the or matrix alone to set the zero baseline, subtracting any background signal to minimize systematic offsets. Inter-laboratory comparisons, often through proficiency testing schemes, ensure by aligning results against values from networks like those coordinated by the , with discrepancies typically below 2% for routine analytes. Validation metrics for UV-Vis methods emphasize , quantified by the (CV) of readings, which should be less than 1% across multiple replicates under identical conditions, and testing via of standard curves up to an absorbance of 1.5 AU, ensuring R² > 0.999 with residuals below 0.005 AU. These metrics confirm method robustness, with often assessed using least-squares fitting to detect deviations from Beer's law at higher absorbances. Software tools facilitate uncertainty reduction through automated baseline subtraction, which fits or asymmetric least-squares algorithms to remove drift and offset spectra, improving signal clarity by up to 20% in noisy datasets, and outlier detection via statistical thresholds like Grubbs' test or to identify and exclude spikes from cosmic rays or electrical interference. Examples include StellarPro for integrated preprocessing with spike removal and LabSolutions UV-Vis for correction checks during acquisition. Regulatory compliance in pharmaceutical applications follows USP <857>, mandating periodic performance verification for accuracy (±1 nm), photometric accuracy (±0.01 AU at 1 AU), (deviation <0.5% up to 2 AU), and (<0.01% T), using oxide for and for absorbance tests to ensure suitability for quantitative assays. In environmental testing, standards such as ISO 7887 for the determination of color in employ similar with multi-point standards and uncertainty estimation via ISO 11352 to meet reporting limits with expanded uncertainties below 15–20%.

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