wavelengths in spectrophotometer even at every 5 or 10 nm ranges. It gives a data
spectrum instead of giving a specific absorbance reading.
The spectrophotometers are of two types–single beam and double beam. In
single beam spectrophotometers, there is single beam from light source. The refer-
ence sample also called blank sample isfirst kept in sample holder, and wavelength
absorption is measured. Then it is removed, and sample under consideration is
placed in sample holder, and absorption of light is measured again. The absorbance
value of reference sample is subtracted from the absorbance value of samples under
consideration. This process removes the additive effects from the solvent and the
cell. In double beam spectrophotometer, the light from source is split in two beams.
One beam illuminates the reference or blank sample, and other beam passes through
sample under consideration. These beams may be recombining before they reach
monochromator. In some cases, two monochromator may be used. The display
shows measurement of reading as difference of reference and test sample. In these
instruments, 2–6 samples may be read in single run (Figs.5.2 and 5.3).
Fig. 5.2Spectrophotometer
Fig. 5.3Working principle of double beam spectrophotometer
5.1 Colorimeter and Spectrophotometer 19