The Beer-Lambert law states that the absorbance of a solution is directly proportional to the solution's concentration. Thus UV/VIS spectroscopy can be used to determine the concentration of 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 which can be 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 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, 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 or often .
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 basic parts of a spectrophotometer are a light source (often an incandescent bulb for the visible wavelengths, or a deuterium arc lamp in the ultraviolet), a holder for the sample, a diffraction grating or monochromator to separate the different wavelengths of light, and a detector. The detector is typically a photodiode or a CCD. Photodiodes are used with monochromators, which filter the light so that only light of a single wavelength reaches the detector. Diffraction gratings are used with CCDs, which collects light of different wavelengths on different pixels.
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, 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. 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.
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 best cuvettes are made of high quality quartz, although glass or plastic cuvettes are common. (Glass and most plastics absorb in the UV, which limits their usefulness to visible wavelengths.)
An ultraviolet-visible spectrum is essentially a graph of light absorbance versus wavelength in a range of ultraviolet or visible regions. Such a spectrum can often be produced directly by a more sophisticated spectrophotometer, or the data can be collected one wavelength at a time by simpler instruments. Wavelength is often represented by the symbol λ. Similarly, for a given substance, a standard graph of the extinction coefficient (ε) vs. wavelength (λ) may be made or used if one is already available. Such a standard graph would be effectively "concentration-corrected" and thus independent of concentration. For the given substance, the wavelength at which maximum absorbance in the spectrum occurs is called λmax, pronounced "Lambda-max".
The Woodward-Fieser rules rules are a set of empirical observations which can be used to predict λmax, the wavelength of the most intense UV/Vis absorption, for conjugated organic compounds such as dienes and ketones.
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. UV/Vis absorption is not, however, a specific test for any given compound. The nature of the solvent, the pH of the solution, temperature, high electrolyte concentrations, and the presence of interfering substances can influence the absorption spectra of compounds, as can variations in slit width (effective bandwidth) in the spectrophotometer.