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A prism splits the incoming light into its components by refraction. Refraction occurs

because radiation of different wavelengths travels along different paths in medium of

higher density. In order to maintain the principle of velocity conservation, light of shorter

wavelength (higher speed) must travel a longer distance (i.e. blue sky effect). At a grating,

the splitting of wavelengths is achieved by diffraction. Diffraction is a reflection phenom-

enon that occurs at a grid surface, in this case a series of engraved fine lines. The distance

between the lines has to be of the same order of magnitude as the wavelength of the

diffracted radiation. By varying the distance between the lines, different wavelengths

are selected. This is achieved by rotating the grating perpendicular to the optical axis.

The resolution achieved by gratings is much higher than the one available by prisms.

Nowadays instruments almost exclusively contain gratings as monochromators as

they can be reproducibly made in high quality by photoreproduction.

The bandwidth of a colorimeter is determined by the filter used as monochromator.

A filter that appears red to the human eye is transmitting red light and absorbs almost

any other (visual) wavelength. This filter would be used to examine blue solutions, as

these would absorb red light. The filter used for a specific colorimetric assay is thus made

of a colour complementary to that of the solution being tested. Theoretically, a single

wavelength is selected by the monochromator in spectrophotometers, and the emergent

light is a parallel beam. Here, the bandwidth is defined as twice the half-intensity

bandwidth. The bandwidth is a function of the optical slit width. The narrower the slit

width the more reproducible are measured absorbance values. In contrast, the sensitivity

becomes less as the slit narrows, because less radiation passes through to the detector.

In a dual-beam instrument, the incoming light beam is split into two parts by a half-

mirror. One beam passes through the sample, the other through a control (blank,

reference). This approach obviates any problems of variation in light intensity, as

both reference and sample would be affected equally. The measured absorbance is

the difference between the two transmitted beams of light recorded. Depending on

the instrument, a second detector measures the intensity of the incoming beam,

although some instruments use an arrangement where one detector measures the

incoming and the transmitted intensity alternately. The latter design is better from

an analytical point of view as it eliminates potential variations between the two

Vis light

UV light

Half-mirrors

Mirror Mirror

Computer

Detector

Beam

selector

Slit

Grating

Prism

Slit

Mirrors

Mirror

Sample

cuvette

Reference

cuvette

Fig. 12.6Optical arrangements in a dual-beam spectrophotometer. Either a prism or a grating constitutes

the monochromator of the instrument. Optical paths are shown as green lines.

489 12.2 Ultraviolet and visible light spectroscopy