Spectrophotometric Analysis

Abstract

Spectrophotometric techniques are used to measure the concentration of solutes in solution by measuring the amount of light that is absorbed by the solution in a cuvette placed in the spectrophotometer. Spectrophotometry takes advantage of the dual nature of light. Namely, light has: 1. a particle nature which gives rise to the photoelectric effect 2. a wave nature which gives rise to the visible spectrum of light A spectrophotometer measures the intensity of a light beam after it is directed through and emerges from a solution. An example with copper sulfate (CuS0 4) is shown on Figure 1. Figure 1. The red part of the spectrum has been almost complete absorbed by CuSO 4 and blue light has been transmitted. CuSO 4 absorbs little blue light and therefore appears blue. In spectrophotometry, greater sensitivity can be gained by directing red light through the solution because CuSO 4 absorbs strongest at the red end of the visible spectrum. For this, red wavelengths have to be isolated. In a spectrophotometer, a light source gives off white light which strikes a prism, separating the light into its component wavelengths (See Figure 2.). Thus, lightwaves can be separated by frequency. Figure 2. The spectrophotometer can measure the amount of light (of certain frequency) transmitted or adsorbed by the solution. This light that has not been absorbed by the solution in the cuvette, will strike the phototube. The photons of light that strike the phototube will be converted into electrical energy. This current that is produced is very small and must be amplified before it can be efficiently detected. The signal is proportional to the amount of light which originally struck the phototube and is thus an accurate measurement of the amount of light which has passed through (been transmitted by) the sample. Different compounds having dissimilar atomic and molecular interactions have characteristic absorption phenomena and absorption spectra. Concentration of every component may be found from the spectrophotometer measurements and calibration curve made using the samples of known concentration.