Principles of Spectrophotometry - Molecules either absorb or transmit energy in the form of electromagnetic radiation. White light (normal daylight) is made up of all the wavelengths of electromagnetic radiation in the visible spectrum. How objects or chemical substances absorb and transmit the light that strikes them determines their color.
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| Principles of Spectrophotometry |
What we see as the color of an object, or a solution, is determined by what wavelengths of light are “left over” to be transmitted or reflected by the object after certain wavelengths are absorbed by its constituent molecules. For example, the pigment chlorophyll, present in the leaves of plants, absorbs a high percentage of the wavelengths of light in the red and violet to blue ranges. Green light, not absorbed by chlorophyll molecules, is reflected from the surface of the leaf—thus most plants appear to be green. A solution of chlorophyll extracted from a leaf would also be green. The spectrophotometer can be used to measure the amount of light absorbed or transmitted by molecules in a solution. The spectrophotometer operates on the following principle. When a specific wavelength of light is transmitted through a solution, the radiant light energy absorbed, absorbance (A), is directly proportional to (1) the absorptivity of the solution—the ability of the solute molecules to absorb light of that wavelength; (2) the concentration of the solute; and (3) the length of the path of light (usually 1 cm) from its source, through the solution, to the point where the percentage of light energy transmitted or absorbed is measured by a phototube.
Spectrophotometers that employ ultraviolet or visible light are the types most often used to study
biological structures and reactions. The investigator selects a wavelength of light that will be maximally absorbed by a particular solute in solution. (If visible light is used and the molecule of interest does not absorb light, it is often possible to set up a chemical reaction that will yield a colored product.) After passing through the solution, the light energy received at the phototube is expressed as the ratio of transmitted light IT (the light that passes through the sample) to incident light I0 (the intensity of light at the source before it enters the sample). The light received at the phototube is measured as percent transmittance (T), or as the log of its inverse, absorbance (A):
By measuring the absorbance (or transmittance) it is possible to determine the concentration of the absorber (molecule) in solution. Concentration can be calculated directly if the molar absorptivity of the molecule (the amount of light at a specific wavelength absorbed by a specified concentration of solute in moles per liter) is known. Usually, however, molar absorptivity is not known and absorbance readings indicate only relative concentrations—a higher absorbance (A) resulting from a higher concentration. In such cases, the concentration can be found by locating the absorbance reading of the unknown concentration on a graph of the absorbances of known concentrations (standard curve).
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