pg instruments
Fig 1: PG Instruments T80+ UV-Visible Spectrophotometer.

A spectrophotometer is an instrument that measures the amount of light absorbed by a sample. The use of spectrophotometer is based on Beer-Lambert’s law which relates the amount of light transmitted through a solution to be a function of the length of light path and the concentration of the absorbing media.
Spectrophotometer techniques are used to:
1. Measure the concentration of a solution by measuring the amount of light absorbed by the solution.
2. Measure light intensity as a function of wavelength. It does this by diffracting the light beam into a spectrum of wavelengths, detecting the intensities with a charge –coupled device, and displaying the result as a graph on the detector and then the display device.
3. Identify organic compounds by determining the absorption maxima.
4. Color determination within the spectral range of 380 to 700nm.

A Spectrophotometer is made up basically of:

1. Light Source: This is to provide a sufficient amount of light which is suitable for making measurement. The light source typically yields a high output of polychromatic light over a wide range of the spectrum, and light sources are usually tungsten and deuterium lamps.

2. Monochromator: This is an optical device that transmits a mechanical selectable narrow band of wavelength of light or other radiation chosen from a wider range of wavelength available at the input. It consists of three parts: entrance slit, exit slit, and dispersion device. Types of dispersion devices are prisms, filters, and diffraction gratings.

3. Sample Cell: These are small rectangular glass or quartz containers. They are designed such that light beam travels a distance of 1cm through the content. The sample cell functions as the sample holder containing the solution of substance to be analyzed.

4. Detector: This is a photosensitive device that detects how much light is transmitted through the sample and convert optical signal to electrical signal. Example of a detector is silicon photodiodes or photomultiplier tube.

Spectrophotometers are basically of two types – single beam and double beam.

Fig 2: Single Beam and Double Beam.

Single beam
In single beam spectrophotometers, a single beam of light passes through the sample holder. The instrument is standardized by placing a reference in the sample holder, and the resulting value is subtracted from subsequent sample measurements to remove effects from the solvent and the cell. This was the earliest design, but still in common use in both teaching and industrial labs.

The advantages of the single beam design are, low cost, high sensitivity, because the optical system is simple while the disadvantage is that an appreciable amount of time elapses between taking the reference (I) and making the sample measurement(Io) such that there can be problems with drift

Double beam
In a double beam spectrophotometer 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.

The advantage of double beam is high stability because reference and sample are measured virtually at the same time while the disadvantages are higher cost, lower sensitivity because throughput of light is poorer due to complex optics and lower reliability.

mr sewen
Authored by: Oluwaseun Adeogun; a Business Support Executive at Applied Analytical Systems Litd., 8, Atunwa Street, Ikeja, Lagos, Nigeria. Phone (234) 8037396888, email: [email protected] website www.aasnig.com