Spectrophotometry is an important branch of spectroscopy that focuses on the technique of measurement. In this technique, the amount of light that a sample absorbs at a particular wavelength is measured and used to determine the concentration of the sample by comparison with appropriate standards or reference data.
Ultraviolet and visible spectrometers have been in general use for the last 35 years and over this period have become the most important analytical instrument in the modern day laboratory. In many applications other techniques could be employed but none rival UV-Visible spectrometry for its simplicity, versatility, speed, accuracy and cost-effectiveness.
The basic parts of a spectrophotometer are a light source, a holder for the sample, a diffraction grating or monochromator to separate the different wavelengths of light, and a detector. The radiation source is often a Tungsten filament (300-2500 nm), a deuterium arc lamp, which is continuous over the ultraviolet region (190-400 nm) or more recently, light emitting diodes (LED) and Xenon arc lamps for the visible wavelengths.
A spectrophotometer can be either single beam or doble beam. In a single beam instrument, all of the light passes through the sample cell. P 0 must be measured by removing the sample This was the earliest design, but is still in common use in both teaching and industrial labs. In a double-beam instrument, the light is split into two beams before it reaches the sample. One beam is used as the reference; the other beam passes through the sample. The reference beam intensity is taken as 100%
Transmission (or 0 Absorbance), and the measurement displayed is the ratio of the two beam intensities. Some double-beam instruments have two detectors (photodiodes), and the sample and reference beam are measured at the same time. In other instruments, the two beams pass through a beam chopper, which blocks one beam at a time. The detector alternates between measuring the sample beam and the reference beam in synchronism with the chopper.
Samples are typically placed in a transparent cell, known as a cuvette. Cuvettes are typically rectangular in shape. The most widely applicable cuvettes are made of high quality fused silica or quartz glass because these are transparent throughout the UV, visible and near infrared regions. Glass and plastic cuvettes are also common, although glass and most plastics absorb in the UV, which limits their usefulness to visible wavelengths. For convenience of reference, definitions of the various spectral regions have been set by the Joint Committee on Nomenclature in Applied Spectroscopy (Table 1).