Spectrophotometer

Introduction

Spectrophotometer is an optical instrument that measures and shows how much light energy is transmitted by a substance in solution at different wavelengths of radiant energy. Light passes into a monochromator where only a very narrow range of wavelengths can pass through. From there, light is transmitted through a sample solution, and on to a phototube where the light energy is converted to an electric current that is registered on a meter. To measure the amount of light absorbed by the solutes in sample solution, comparison to the initial intensity of light reaching the solution is needed. Each solution with a different solute has its own characteristic absorption property or spectrum. Spectrophotometer can also be used to find out the concentration of compounds in solution because the amount of light absorbed is directly proportional to the concentration of absorbing compounds in that sample. When a solution like bromophenol blue absorbs certain wavelengths of light in the visible spectrum and transmits or reflects others, it appears coloured. However, water is a colourless solution because it absorbs only wavelength outside of the visible light spectrum. By analysing the pattern of wavelength absorbed by a given sample, spectrophotometer can be used to distinguish compounds as different compounds absorb light at different wavelength. Figure 1 shows the schematic diagram of the components in a spectrophotometer.

Figure 1. A schematic diagram of the components in a spectrophotometer. The arrows indicate the pathway of light.

The spectrophotometer consists of a light source, a prism or diffraction grating that separates the light into different wavelengths. A narrow beam of the desired wavelength passes through a slit (the incident light, I0) which is then passing through the sample solution in the cuvette. The photosensitive tube measures the transmittance or absorbance value for the sample by detecting the light that passes through the...