Detectors UVVisible Detection

The relationship between the intensity of UV light transmitted through a cell (IT) and the concentration of solute in it (c) is given by Beer's law:

where I0 is the intensity of the light entering the cell, l is the path length of the cell, and s is the molar extinction coefficient of the solute for the specific wavelength of the UV light.

The sensitivity of the detector is directly proportional to the value of the absorption coefficient and the path length of the cell. Thus, the sensitivity of the detector can be increased by increasing the path length. However, the volume of the cell must also be constrained to avoid more than a small fraction of a peak existing in the cell at any one time (this can cause peak dispersion and, at the extreme, when two peaks exist in the tube, peak merging). Consequently, as the length is increased, the radius must be reduced, which will increase the detector noise and cause a reduction in sensitivity. Thus, increasing the detector sensitivity by increasing the path length has limitations, and a well-designed cell involves a careful compromise between cell radius and length to provide the maximum sensitivity. Most modern UV detector sensors have path lengths that range between 1 and 10 mm and internal radii that range from about 0.25 to 1 mm.

where A is termed the absorbance.

The term AA is often employed to define the detector sensitivity where the value of AA is the change in absorbance that provides a signal-to-noise ratio of 2, i.e.:

where (Ac) is the detector concentration sensitivity or minimum detectable concentration. Thus:


It is clear that two detectors having the same sensitivity, defined as AA, will not necessarily have the same sensitivity with respect to solute concentration.

Two detectors having the same minimum detectable change in absorbance will only exhibit the same concentration sensitivity if the path lengths of the two sensors are identical.

UV detectors can be used with gradient elution providing the solvents do not absorb significantly over the wavelength range that is being used for detection. The solvents employed in reversed-phase chromatography are usually water, methanol, aceto-nitrile and tetrahydrofuran (THF), all of which are transparent to UV light over the wavelength range commonly used. In normal-phase operation, however, more care must be taken in solvent selection, as many solvents that would be appropriate for the separation absorb UV light very strongly. The n-paraffins, dichloromethane, and n-paraffins containing small quantities of aliphatic alcohols or THF are useful solvents that are transparent in UV, and can be used with normal distribution systems (e.g. with a polar stationary phase such as silica gel).

There are two types of UV detector: fixed-wavelength detector and multiple-wavelength detector.

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