Detailed description of UV-vis-AD

Ultraviolet-visible (UV-vis) light absorption detectors typically contain a deuterium lamp to generate UV-light (i.e., 200 - 400 nm) and a tungsten filament lamp to generate visible to near infrared light (400 - 1600 nm). The light emitted from the lamps is passed through a monochromator − either a monochromatic filter or a diffraction grating − to obtain light of only one desired wavelength, so-called monochromatic light. Its wavelength is dependent on the position of the monochromator relative to the light source.

The beam of monochromatic light is then split into a measurement and a reference beam. The intensity of the measurement beam I() is determined after it passed through a flow cell that is connected to the outlet of the HPLC column. Contrarily, the intensity of the reference beam I⁰() is directly measured (i.e., the reference beam does not pass through the flow cell). If an organic analyte elutes from the column and if that analyte absorbs light at the wavelength of the monochromatic light, I() is attenuated while I⁰() is not affected.

For a compound i and a measurement wavelength , absorbance A_i () (unitless) is defined as:

where ε_i() (L mol ^-1 cm^-1) is the molar absorptivity (or molar extinction coefficient) of compound i at the wavelength , b (cm) is the length over which the measurement beam passes through the sample solution, and c_i (mol L^-1) is the concentration of compound i.

In other words, UV-vis detectors measure the fraction of monochromatic light that passes through a sample. Changes in the intensity of the measurement beam relative to the reference beam are detected and reported as a chromatographic peak.

UV-vis absorption detectors are the predominant type of detector used in combination with HPLC. The universal use of this detector is due to its high sensitivity, its capability to detect a variety of organic compounds, and its wide linear response range.