Ultraviolet–visible spectroscopy

thumb|right|270px|Beckman DU640 UV–Vis spectrophotometer

Ultraviolet–visible spectrophotometry (UV–Vis or UV-VIS) refers to absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet and the full, adjacent visible regions of the electromagnetic spectrum.

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

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. 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.

Applications

thumb|right|300px|An example of a UV–Vis readout

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

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. 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 λ<sub>max</sub>, 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:

:<math>A=\log_{10}(I_0/I)=\varepsilon c L</math>,

where A is the measured absorbance (formally dimensionless but generally reported in absorbance units (AU)), <math>I_0</math> is the intensity of the incident light at a given wavelength, <math>I</math> 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 <math>1/M*cm</math>.

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).

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 (<math>n</math>) and the extinction coefficient (<math>k</math>) of a given film across the measured spectral range.

Practical considerations

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. 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 and wavelength accuracy.

Spectral bandwidth

Spectral bandwidth of a spectrophotometer is the range of wavelengths that the instrument transmits through a sample at a given time. 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. 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. 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, which would mean that monochromatic measurements would deviate from the Beer–Lambert law.

Measurement uncertainty sources

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.

Ultraviolet–visible spectrophotometer

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

:<math>A=-\log(\%T/100\%)</math>

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 (<math>I</math>), and compares it to the intensity of light reflected from a reference material (<math>I_o</math>) (such as a white tile). The ratio <math>I/I_o</math> 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)

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, <math>L</math>, 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. 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. In addition, ultraviolet–visible spectrophotometry can be used to determine the thickness, along with the refractive index and extinction coefficient of thin films.

Additional applications

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.

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