Ultraviolet-visible spectroscopy
Overview
 
Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) refers to absorption spectroscopy
Absorption spectroscopy
Absorption spectroscopy refers to spectroscopic techniques that measure the absorption of radiation, as a function of frequency or wavelength, due to its interaction with a sample. The sample absorbs energy, i.e., photons, from the radiating field. The intensity of the absorption varies as a...

 or reflectance spectroscopy in the ultraviolet
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...

-visible
Visible spectrum
The visible spectrum is the portion of the electromagnetic spectrum that is visible to the human eye. Electromagnetic radiation in this range of wavelengths is called visible light or simply light. A typical human eye will respond to wavelengths from about 390 to 750 nm. In terms of...

 spectral region. This means it uses light in the visible and adjacent (near-UV and near-infrared (NIR)) ranges. The absorption or reflectance in the visible range directly affects the perceived color of the chemicals involved.
Encyclopedia
Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) refers to absorption spectroscopy
Absorption spectroscopy
Absorption spectroscopy refers to spectroscopic techniques that measure the absorption of radiation, as a function of frequency or wavelength, due to its interaction with a sample. The sample absorbs energy, i.e., photons, from the radiating field. The intensity of the absorption varies as a...

 or reflectance spectroscopy in the ultraviolet
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...

-visible
Visible spectrum
The visible spectrum is the portion of the electromagnetic spectrum that is visible to the human eye. Electromagnetic radiation in this range of wavelengths is called visible light or simply light. A typical human eye will respond to wavelengths from about 390 to 750 nm. In terms of...

 spectral region. This means it uses light in the visible and adjacent (near-UV and near-infrared (NIR)) ranges. The absorption or reflectance in the visible range directly affects the perceived color of the chemicals involved. In this region of the electromagnetic spectrum
Electromagnetic spectrum
The electromagnetic spectrum is the range of all possible frequencies of electromagnetic radiation. The "electromagnetic spectrum" of an object is the characteristic distribution of electromagnetic radiation emitted or absorbed by that particular object....

, molecule
Molecule
A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...

s undergo electronic transitions
Molecular electronic transition
Molecular electronic transitions take place when electrons in a molecule are excited from one energy level to a higher energy level. The energy change associated with this transition provides information on the structure of a molecule and determines many molecular properties such as color...

. This technique is complementary to fluorescence spectroscopy
Fluorescence spectroscopy
Fluorescence spectroscopy aka fluorometry or spectrofluorometry, is a type of electromagnetic spectroscopy which analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit...

, in that fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...

 deals with transitions from the excited state
Excited state
Excitation is an elevation in energy level above an arbitrary baseline energy state. In physics there is a specific technical definition for energy level which is often associated with an atom being excited to an excited state....

 to the ground state
Ground state
The ground state of a quantum mechanical system is its lowest-energy state; the energy of the ground state is known as the zero-point energy of the system. An excited state is any state with energy greater than the ground state...

, while absorption measures transitions from the ground state to the excited state.

Applications

UV/Vis spectroscopy is routinely used in analytical chemistry
Analytical chemistry
Analytical chemistry is the study of the separation, identification, and quantification of the chemical components of natural and artificial materials. Qualitative analysis gives an indication of the identity of the chemical species in the sample and quantitative analysis determines the amount of...

 for the quantitative
Quantitative analysis (chemistry)
In chemistry, quantitative analysis is the determination of the absolute or relative abundance of one, several or all particular substance present in a sample....

 determination of different analytes, such as transition metal
Transition metal
The term transition metal has two possible meanings:*The IUPAC definition states that a transition metal is "an element whose atom has an incomplete d sub-shell, or which can give rise to cations with an incomplete d sub-shell." Group 12 elements are not transition metals in this definition.*Some...

 ions, highly conjugated
Conjugated system
In chemistry, a conjugated system is a system of connected p-orbitals with delocalized electrons in compounds with alternating single and multiple bonds, which in general may lower the overall energy of the molecule and increase stability. Lone pairs, radicals or carbenium ions may be part of the...

 organic compound
Organic compound
An organic compound is any member of a large class of gaseous, liquid, or solid chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of carbon-containing compounds such as carbides, carbonates, simple oxides of carbon, and cyanides, as well as the...

s, and biological macromolecules. Determination is usually carried out in solutions.
  • Solutions of transition metal ions can be colored (i.e., absorb visible light) because d electrons
    Electron configuration
    In atomic physics and quantum chemistry, electron configuration is the arrangement of electrons of an atom, a molecule, or other physical structure...

     within the metal atoms can be excited from one electronic state to another. The colour of metal ion solutions is strongly affected by the presence of other species, such as certain anions or ligand
    Ligand
    In coordination chemistry, a ligand is an ion or molecule that binds to a central metal atom to form a coordination complex. The bonding between metal and ligand generally involves formal donation of one or more of the ligand's electron pairs. The nature of metal-ligand bonding can range from...

    s. For instance, the colour of a dilute solution of copper sulfate is a very light blue; adding ammonia
    Ammonia
    Ammonia is a compound of nitrogen and hydrogen with the formula . It is a colourless gas with a characteristic pungent odour. Ammonia contributes significantly to the nutritional needs of terrestrial organisms by serving as a precursor to food and fertilizers. Ammonia, either directly or...

     intensifies the colour and changes the wavelength of maximum absorption ().
  • Organic compound
    Organic compound
    An organic compound is any member of a large class of gaseous, liquid, or solid chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of carbon-containing compounds such as carbides, carbonates, simple oxides of carbon, and cyanides, as well as the...

    s, especially those with a high degree of conjugation
    Conjugated system
    In chemistry, a conjugated system is a system of connected p-orbitals with delocalized electrons in compounds with alternating single and multiple bonds, which in general may lower the overall energy of the molecule and increase stability. Lone pairs, radicals or carbenium ions may be part of the...

     (e.g. DNA, RNA, protein), also absorb light in the UV or visible regions of the electromagnetic spectrum
    Electromagnetic spectrum
    The electromagnetic spectrum is the range of all possible frequencies of electromagnetic radiation. The "electromagnetic spectrum" of an object is the characteristic distribution of electromagnetic radiation emitted or absorbed by that particular object....

    . The solvents for these determinations are often water for water-soluble compounds, or ethanol
    Ethanol
    Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...

     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 colours, the colours are often too intense to be used for quantitative measurement.


The Beer-Lambert law
Beer-Lambert law
In optics, the Beer–Lambert law, also known as Beer's law or the Lambert–Beer law or the Beer–Lambert–Bouguer law relates the absorption of light to the properties of the material through which the light is travelling.-Equations:The law states that there is a logarithmic dependence between the...

 states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length. 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
Calibration curve
In analytical chemistry, a calibration curve is a general method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard samples of known concentration...

.

A UV/Vis spectrophotometer may be used as a detector for HPLC
High-performance liquid chromatography
High-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...

. 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
Response factor
Response factor, usually in chromatography and spectroscopy, is the ratio between a signal produced by an analyte, and the quantity of analyte which produces the signal. Ideally, and for easy computation, this ratio is unity...

.

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 diene
Diene
In organic chemistry a diene or diolefin is a hydrocarbon that contains two carbon double bonds.Conjugated dienes are functional groups, with a general formula of CnH2n-2. Dienes and alkynes are functional isomers...

s and ketone
Ketone
In organic chemistry, a ketone is an organic compound with the structure RCR', where R and R' can be a variety of atoms and groups of atoms. It features a carbonyl group bonded to two other carbon atoms. Many ketones are known and many are of great importance in industry and in biology...

s. 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.

Beer-Lambert law

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

where A is the measured absorbance, is the intensity of the incident light at a given wavelength
Wavelength
In physics, the wavelength of a sinusoidal wave is the spatial period of the wave—the distance over which the wave's shape repeats.It is usually determined by considering the distance between consecutive corresponding points of the same phase, such as crests, troughs, or zero crossings, and is a...

, is the transmitted intensity, L the pathlength through the sample, and c the concentration
Concentration
In chemistry, concentration is defined as the abundance of a constituent divided by the total volume of a mixture. Four types can be distinguished: mass concentration, molar concentration, number concentration, and volume concentration...

 of the absorbing species. For each species and wavelength, ε is a constant known as the molar absorptivity
Molar absorptivity
The molar absorption coefficient, molar extinction coefficient, or molar absorptivity, is a measurement of how strongly a chemical species absorbs light at a given wavelength...

 or extinction coefficient. This constant is a fundamental molecular property in a given solvent, at a particular temperature and pressure, and has units of or often .

The absorbance and extinction ε are sometimes defined in terms of the natural logarithm
Natural logarithm
The natural logarithm is the logarithm to the base e, where e is an irrational and transcendental constant approximately equal to 2.718281828...

 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 for very large, complex molecules such as organic dyes (Xylenol Orange
Xylenol orange
Xylenol orange is an organic reagent, most commonly used as a tetrasodium salt as an indicator for metal titrations. When used for metal titrations it will appear red in the titrand and yellow once it reaches its endpoint...

 or Neutral Red
Neutral red
Neutral Red is a eurhodin dye used for staining in histology. It stains lysosomes red. It is used as a general stain in histology, as a counterstain in combination with other dyes, and for many staining methods. Together with Janus Green B, it is used to stain embryonal tissues and supravital...

, for example).

Practical considerations

The Beer-Lambert law has implicit assumptions that must be met experimentally for it to apply. 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.

Spectral bandwidth

A given spectrometer has a spectral bandwidth that characterizes how monochromatic the light is. If this bandwidth is comparable to the width
Spectral linewidth
The spectral linewidth characterizes the width of a spectral line, such as in the electromagnetic emission spectrum of an atom, or the frequency spectrum of an acoustic or electronic system...

 of the absorption features, then the measured extinction coefficient will be altered. In most reference measurements, the instrument bandwidth is kept below the width of the spectral lines. When a new material is being measured, it may be necessary to test and verify if the bandwidth is sufficiently narrow. Reducing the spectral bandwidth will reduce the energy passed to the detector and will, therefore, require a longer measurement time to achieve the same signal to noise ratio.

Wavelength error

In liquids, the extinction coefficient usually changes slowly with wavelength. A peak of the absorbance curve (a wavelength where the absorbance reaches a maximum) is where the rate of change in absorbance with wavelength is smallest. Measurements are usually made at a peak to minimize errors produced by errors in wavelength in the instrument, that is errors due to having a different extinction coefficient than assumed.

Stray light

Another important factor is the purity of the light used. The most important factor affecting this is the stray light level of the monochromator 
. The detector used 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 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.

Absorption flattening

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.
The deviations will be most noticeable under conditions of low concentration and high absorbance. The reference describes a way to correct for this deviation.

Measurement uncertainty sources

The above factor contribute to the measurement uncertainty
Measurement uncertainty
In metrology, measurement uncertainty is a non-negative parameter characterizing the dispersion of the values attributed to a measured quantity. The uncertainty has a probabilistic basis and reflects incomplete knowledge of the quantity. All measurements are subject to uncertainty and a measured...

 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.

Ultraviolet-visible spectrophotometer

The instrument
Measuring instrument
In the physical sciences, quality assurance, and engineering, measurement is the activity of obtaining and comparing physical quantities of real-world objects and events. Established standard objects and events are used as units, and the process of measurement gives a number relating the item...

 used in ultraviolet-visible spectroscopy is called a UV/Vis spectrophotometer. It measures the intensity of light 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
Diffraction grating
In optics, a diffraction grating is an optical component with a periodic structure, which splits and diffracts light into several beams travelling in different directions. The directions of these beams depend on the spacing of the grating and the wavelength of the light so that the grating acts as...

 in a monochromator
Monochromator
A monochromator is an optical device that transmits a mechanically selectable narrow band of wavelengths of light or other radiation chosen from a wider range of wavelengths available at the input...

 or a prism
Prism (optics)
In optics, a prism is a transparent optical element with flat, polished surfaces that refract light. The exact angles between the surfaces depend on the application. The traditional geometrical shape is that of a triangular prism with a triangular base and rectangular sides, and in colloquial use...

 to separate the different wavelengths of light, and a detector. The radiation source is often a Tungsten
Halogen lamp
A halogen lamp, also known as a tungsten halogen lamp, is an incandescent lamp with a tungsten filament contained within an inert gas and a small amount of a halogen such as iodine or bromine. The chemical halogen cycle redeposits evaporated tungsten back on to the filament, extending the life of...

 filament (300-2500 nm), a deuterium arc lamp
Deuterium arc lamp
A deuterium arc lamp is a low-pressure gas-discharge light source often used in spectroscopy when a continuous spectrum in the ultraviolet region is needed.-Principle of operation:...

, which is continuous over the ultraviolet region (190-400 nm), Xenon arc lamp
Xenon arc lamp
A xenon arc lamp is a specialized type of gas discharge lamp, an electric light that produces light by passing electricity through ionized xenon gas at high pressure to produce a bright white light that closely mimics natural sunlight...

s, which is continuous from 160-2,000 nm; or more recently, light emitting diodes (LED) for the visible wavelengths. The detector is typically a photomultiplier tube, a photodiode
Photodiode
A photodiode is a type of photodetector capable of converting light into either current or voltage, depending upon the mode of operation.The common, traditional solar cell used to generateelectric solar power is a large area photodiode....

, a photodiode array or a charge-coupled device
Charge-coupled device
A charge-coupled device is a device for the movement of electrical charge, usually from within the device to an area where the charge can be manipulated, for example conversion into a digital value. This is achieved by "shifting" the signals between stages within the device one at a time...

(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.
A spectrophotometer can be either single beam or double beam. In a single beam instrument (such as the Spectronic 20
Spectronic 20
The Spectronic 20 is a spectrophotometer developed by Bausch & Lomb in 1954. While of simple design, requiring manual setting of the wavelength and making readings from a moving-needle analog display, the unit is rugged, accurate, and easy to use...

), all of the light passes through the sample cell. must be measured by removing the sample. This was the earliest design, but 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
Optical chopper
An optical chopper is a mechanical device which periodically interrupts a light beam. Three types are available: variable frequency rotating disc choppers, fixed frequency tuning fork choppers, and optical shutters...

, which blocks one beam at a time. The detector alternates measuring between 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.

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
Cuvette
A cuvette is a small tube of circular or square cross section, sealed at one end, made of plastic, glass, or fused quartz and designed to hold samples for spectroscopic experiments. The best cuvettes are as clear as possible, without impurities that might affect a spectroscopic reading...

. 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 tube
Test tube
A test tube, also known as a culture tube or sample tube, is a common piece of laboratory glassware consisting of a finger-like length of glass or clear plastic tubing, open at the top, usually with a rounded U-shaped bottom....

s 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.

Specialized instruments have also been made. These include attaching spectrophotometers to telescopes to measure the spectra of astronomical features. UV-visible nanophotometers allow cuvetteless measurement of very small sample volumes (starting with 0.3 µl). UV-visible microspectrophotometers consist of a UV-visible microscope
Optical microscope
The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest design of microscope and were possibly designed in their present compound form in the...

 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.

Nanophotometry

Multiple biological applications (e.g. array CGH, qPCR
Real-time polymerase chain reaction
In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction or kinetic polymerase chain reaction , is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule...

, FISH
Fluorescent in situ hybridization
FISH is a cytogenetic technique developed by biomedical researchers in the early 1980s that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high...

, nucleic acid labelling) require quantitative as well as qualitative analysis of nucleic acids (DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

, RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....

) and Proteins with sample volumes in a submicroliter range. This becomes possible with a specialized NanoPhotometer. A drop of sample (0.03 µl to 5 µl) is pipetted directly onto the measuring window of the instrument. In the measuring chamber the sample is squeezed to exactly defined path lengths ranging from 0.04 mm up to 2 mm. This reduction of the path length compared to a measurement with standard cuvettes (path length 1 cm) offers an automatic sample dilution between 1/250 and 1/5 in comparison to a standard cuvette measurement. Due to this virtual sample dilution measurements can be processed with undiluted samples. The reproducibility of the results is extremely high and samples can be retrieved after the measurement for further processing.

Microspectrophotometry

UV-visible spectroscopy of microscopic samples is done by integrating an optical microscope with UV-visible optics, white light sources, a monochromator
Monochromator
A monochromator is an optical device that transmits a mechanically selectable narrow band of wavelengths of light or other radiation chosen from a wider range of wavelengths available at the input...

, and a sensitive detector such as a charge-coupled device
Charge-coupled device
A charge-coupled device is a device for the movement of electrical charge, usually from within the device to an area where the charge can be manipulated, for example conversion into a digital value. This is achieved by "shifting" the signals between stages within the device one at a time...

 (CCD) or photomultiplier
Photomultiplier
Photomultiplier tubes , members of the class of vacuum tubes, and more specifically phototubes, are extremely sensitive detectors of light in the ultraviolet, visible, and near-infrared ranges of the electromagnetic spectrum...

 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 , microscopic paint chips 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
Vitrinite
Vitrinite is one of the primary components of coals and most sedimentary kerogens. Vitrinite is a type of maceral, where "macerals" are organic components of coal analogous to the "minerals" of rocks. Vitrinite has a shiny appearance resembling glass . It is derived from the cell-wall material or...

 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
Thin-film interference
Thin-film interference is the phenomenon that occurs when incident light waves reflected by the upper and lower boundaries of a thin film interfere with one another to form a new wave. Studying this new wave can reveal information about the surfaces from which its components reflected, including...

 of the spectra. A map of the film thickness across the entire wafer can then be generated and used for quality control purposes.

See also

  • Ultraviolet-visible spectroscopy of stereoisomers
    Ultraviolet-visible spectroscopy of stereoisomers
    Ultraviolet-visible spectroscopy can distinguish between enantiomers by showing a distinct Cotton effect for each isomer. UV-vis spectroscopy sees only chromophores, so other molecules must be prepared for analysis by chemical addition of a chromophore such as anthracene...

  • Infrared spectroscopy
    Infrared spectroscopy
    Infrared spectroscopy is the spectroscopy that deals with the infrared region of the electromagnetic spectrum, that is light with a longer wavelength and lower frequency than visible light. It covers a range of techniques, mostly based on absorption spectroscopy. As with all spectroscopic...

     and Raman spectroscopy
    Raman spectroscopy
    Raman spectroscopy is a spectroscopic technique used to study vibrational, rotational, and other low-frequency modes in a system.It relies on inelastic scattering, or Raman scattering, of monochromatic light, usually from a laser in the visible, near infrared, or near ultraviolet range...

     are other common spectroscopic techniques, usually used to obtain information about the structure of compounds or to identify compounds. Both are forms of Vibrational spectroscopy.
  • Fourier transform spectroscopy
    Fourier transform spectroscopy
    Fourier transform spectroscopy is a measurement technique whereby spectra are collected based on measurements of the coherence of a radiative source, using time-domain or space-domain measurements of the electromagnetic radiation or other type of radiation....

  • Near-infrared spectroscopy
  • Vibrational spectroscopy
  • Rotational spectroscopy
    Rotational spectroscopy
    Rotational spectroscopy or microwave spectroscopy studies the absorption and emission of electromagnetic radiation by molecules associated with a corresponding change in the rotational quantum number of the molecule...

  • Applied spectroscopy
    Applied spectroscopy
    Applied spectroscopy is the application of various spectroscopic methods for detection and identification of different elements/compounds in solving problems in the fields of forensics, medicine, oil industry, atmospheric chemistry, pharmacology, etc....

  • Slope spectroscopy
    Slope Spectroscopy
    Slope spectroscopy is a spectroscopic technique that is used to quantify concentrations of various compounds, proteins and antibodies in which the operating path length is varied and the absorbance is measured.- Equations :...

  • Benesi-Hildebrand method
    Benesi-Hildebrand method
    The Benesi-Hildebrand method is a mathematical approach used in physical chemistry for the determination of the equilibrium constant K and stoichiometry of non-bonding interactions...

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