PRINCIPLES OF SPECTROPHOTOMETRY

Introduction In physics, spectrophotometry is the quantifiable study of electromagnetic spectra. It is more specific than the general term electromagnetic spectroscopy in that spectrophotometry deals with visible light, near-ultraviolet, and near-infrared. Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the color (or more specifically the wavelength) of light. Important features of spectrophotometers are spectral bandwidth and linear range of absorption measurement. Perhaps the most common application of spectrophotometers is the measurement of light absorption, but they can be designed to measure diffuse or specular reflectance. Strictly, even the emission half of a luminescence instrument is a kind of spectrophotometer. The use of spectrophotometers is not limited to studies in physics. They are also commonly used in other scientific fields such as chemistry, biochemistry, and molecular biology. [1] They are widely used in many industries including printing and forensic examination. What is color?

Without light there is no color. Light falls on an object and is partially absorbed. The light that is not absorbed is reflected and falled on the color receptions in the eyes. The receptors convert the incident light into stimuli, which are then conducted via nerves to the brain. Here the stimuli are interpreted and a color perceptions is formed.

As with all sensory impression, each person "experiences" color differently. This is not just because no two eyes are identical, but because color interpretation also varies from person can even perceive colors differently at difference times and according to the mood he or she is in. Major Classes Double beam spectrophotometer Single beam spectrophotometer

Double beam spectrophotometer

A double beam spectrophotometer compares the light intensity between two light paths, one path containing a reference sample and the other the test sample.

Single beam spectrophotometer

A single beam spectrophotometer measures the relative light intensity of the beam before and after a test sample is inserted. Although comparison measurements from double beam instruments are easier and more stable, single beam instruments can have a larger dynamic range and are optically simpler and more compact.

Design aspect

The spectrophotometer quantitatively compares the fraction of light that passes through a reference solution and a test solution. Light from the source lamp is passed through a monochromator, which difracts the light into a "rainbow" of wavelengths and outputs narrow bandwidths of this diffracted spectrum. Discrete frequencies are transmitted through the test sample. Then the intensity of the transmitted light is measured with a photodiode or other light sensor, and the transmittance value for this wavelength is then compared with the transmission through a reference sample. In short, the sequence of events in a spectrophotometer is as follows: 1. The light source shines into a monochromator. 2. A particular output wavelength is selected and beamed at the sample. 3. The sample absorbs light. Principles A spectrophotometer consists of two instruments, namely a spectrometer for producing light of any selected color (wavelength), and a photometer for measuring the intensity of light. The instruments are arranged so that liquid in a cuvette can be placed between the spectrometer beam and the photometer. The amount of light passing through the tube is measured by the photometer. The photometer delivers a voltage signal to a display device, normally a galvanometer. The signal changes as the amount of light absorbed by the liquid changes.

If development of color is linked to the concentration of a substance in solution then that concentration can be measured by determining the extent of absorption of light at the appropriate wavelength. For example hemoglobin appears red because the hemoglobin absorbs blue and green light rays much more effectively than red. The degree of absorbance of blue or green light is proportional to the concentration of hemoglobin. When monochromatic light (light of a specific wavelength) passes through a solution there is usually a quantitative relationship (Beer's law) between the solute concentration and the intensity of the transmitted light, that is,
I = I 0 *10 KCL
Where,

I= intensity of the transmitted light when the colored compound is added I0 = intensity of transmitted light using the pure solvent l = distance the light passes through the solution, and k = constant.

If the light path l is a constant, as is the case with a spectrophotometer, Beer's law may be written,
I I 0 = 10 KC = T

where k is a new constant and T is the transmittance of the solution. There is a logarithmic relationship between transmittance and the concentration of the colored compound. Thus,
log T = log 1 / T = kc = opticaldensity

The O.D. is directly proportional to the concentration of the colored compound. Most spectrophotometers have a scale that reads both in O.D. (absorbance) units, which is a logarithmic scale, and in % transmittance, which is an arithmetic scale. Various types UV spectrophotometers Visible region 400700 nm spectrophotometry is used extensively in colorimetry science. Ink manufacturers, printing companies, textiles vendors, and many more, need the data provided through colorimetry. They take readings in the region of every 1020 nanometers along the visible region, and produce a spectral reflectance curve or a data stream for alternative presentations. These curves can be used to test a new batch of colorant to check if it makes a match to specifications e.g., iso printing standards. IR spectrophotometers Spectrophotometers designed for the main infrared region are quite different because of the technical requirements of measurement in that region. One major factor is the type of photosensors that are available for different spectral regions, but infrared measurement is also challenging because virtually everything emits IR light as thermal radiation, especially at wavelengths beyond about 5 m.