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 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: :A=\log_{10}(I_0/I)=\varepsilon c L, where
A is the measured
absorbance (formally dimensionless but generally reported in absorbance units (AU)), I_0 is the intensity of the incident light at a given
wavelength, I 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 1/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). 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 (n) and the extinction coefficient (k) 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==