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Ultraviolet–visible spectroscopy or 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 spectral regions. This means it uses light in the visible and adjacent 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, atoms and molecules undergo electronic transitions. Absorption spectroscopy 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.^[1]

Molecules containing bonding and non-bonding electrons (n-electrons) can absorb energy in the form of ultraviolet or visible light to excite these electrons to higher anti-bonding molecular orbitals.^[2] The more easily excited the electrons (i.e. lower energy gap between the HOMO and the LUMO), the longer the wavelength of light it can absorb. There are four possible types of transitions (π–π*, n–π*, σ–σ*, and n–σ*), and they can be ordered as follows :σ–σ* > n–σ* > π–π* > n–π*.

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

${\displaystyle A=-\log(\%T/100\%)}$

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 (${\displaystyle I}$), and compares it to the intensity of light reflected from a reference material (${\displaystyle I_{o}}$) (such as a white tile). The ratio ${\displaystyle I/I_{o}}$ 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 lamp, which is continuous from 160 to 2,000 nm; or more recently, light emitting diodes (LED)^[1] 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.

Simplified schematic of a double beam UV–visible spectrophotometer

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. ${\displaystyle I_{o}}$must be measured by removing the sample. This was the earliest design and 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 between measuring 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.

In a single-beam instrument, the cuvette containing only a solvent has to be measured first. Mettler Toledo developed a single beam array spectrophotometer that allows fast and accurate measurements over the UV/VIS range. The light source consists of a Xenon flash lamp for the ultraviolet (UV) as well as for the visible (VIS) and near-infrared wavelength regions covering a spectral range from 190 up to 1100 nm. The lamp flashes are focused on a glass fiber which drives the beam of light onto a cuvette containing the sample solution. The beam passes through the sample and specific wavelengths are absorbed by the sample components. The remaining light is collected after the cuvette by a glass fiber and driven into a spectrograph. The spectrograph consists of a diffraction grating that separates the light into the different wavelengths, and a CCD sensor to record the data, respectively. The whole spectrum is thus simultaneously measured, allowing for fast recording^[10].

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, ${\displaystyle L}$, 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.^[1]

Specialized instruments have also been made. These include attaching spectrophotometers to telescopes to measure the spectra of astronomical features. 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.