- Ultraviolet–visible spectroscopy
Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible 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, molecules undergo electronic transitions. This technique is complementary to fluorescence spectroscopy, in that fluorescence deals with transitions from the excited state to the ground state, while absorption measures transitions from the ground state to the excited state.
UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds, 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 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 ligands. For instance, the colour of a dilute solution of copper sulfate is a very light blue; adding ammonia intensifies the colour and changes the wavelength of maximum absorption (λmax).
- Organic compounds, especially those with a high degree of conjugation (e.g. DNA, RNA, protein), 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 colours, the colours 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. 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.
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, I0 is the intensity of the incident light at a given wavelength, I is the transmitted intensity, L the pathlength 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 1 / M * cm or often AU / M * cm.
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 for very large, complex molecules such as organic dyes (Xylenol Orange or Neutral Red, for example).
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.
A given spectrometer has a spectral bandwidth that characterizes how monochromatic the light is. If this bandwidth is comparable to the width 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.
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.
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.
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 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.
The instrument used in ultraviolet-visible spectroscopy is called a UV/Vis spectrophotometer. It measures the intensity of light passing through a sample (I), and compares it to the intensity of light before it passes through the sample (Io). The ratio I / Io is called the transmittance, and is usually expressed as a percentage (%T). The absorbance, A, is based on the transmittance:
- A = − log(%T / 100%)
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 (I), and compares it to the intensity of light reflected from a reference material (Io)(such as a white tile). The ratio I / Io 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 in a monochromator or a prism 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), Xenon arc lamps, 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, 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.
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. Io 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, 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. Cuvettes are typically rectangular in shape, commonly with an internal width of 1 cm. (This width becomes the path length, L, 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.
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 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.
Multiple biological applications (e.g. array CGH, qPCR, FISH, nucleic acid labelling) require quantitative as well as qualitative analysis of nucleic acids (DNA, RNA) 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.
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  , 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 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. A map of the film thickness across the entire wafer can then be generated and used for quality control purposes.
- Ultraviolet-visible spectroscopy of stereoisomers
- Infrared spectroscopy and Raman spectroscopy 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
- Near-infrared spectroscopy
- Vibrational spectroscopy
- Rotational spectroscopy
- Applied spectroscopy
- Slope spectroscopy
- Benesi-Hildebrand method
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