Spectrophotometry

Spectrophotometry is a quantitative method of analysis involving the principles associated with how visible light interacts with atoms. Visible light is a small portion of the electromagnetic spectrum and includes the colors we observe (red, orange, yellow, green, blue and violet). It consists of electromagnetic radiation whose wavelengths range from 400-700 nm.When white light is observed what is actually being seen is all the colors of light combined. When this light passes through a substance certain energies (or colors) of light are absorbed while the other color(s) are allowed to pass through or are reflected. This is why some substances appear colored.

The color we see is the combination of the energies of visible light which are not absorbed by the sample. If the substance does not absorb any light, it appears white or colorless. A solution appears a certain color due to the absorbance and transmittance of visible light.

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For example, an orange solution appears orange because it is absorbing all of the colors except orange. A sample may also appear orange if all colors of light except blue are transmitted. This is because blue and orange are complimentary colors. (see figure below)The wavelength associated with the complimentary color is known as the wavelength of maximum absorbance.

The wavelength of maximum absorbance is used when determining the concentration of a colored solution since at this wavelength a slight change in concentration allows for a significant change in the absorbance of light.Many compounds involving transition elements are colored. This is because the transition metals include “d” electrons in its atomic structure. The spacing of these “d-orbitals” allow for electronic transitions within the energy range of the visible region of the electromagnetic spectrum. Compounds containing the alkali and alkaline earth metals are white due to having only “s” electron transitions. More energy is required to cause this type of transition and, thus, light of shorter wavelength is involved.The amount of light absorbed by a solution is dependent on the ability of the compound to absorb light (molar absorptivity), the distance through which the light must pass through the sample (path length) and the molar concentration of the compound in the solution.

This relationship is known as Beer’s Law and is represented by the equation:A = * l Cwhere A is the absorbance, * is the molar absorptivity, l is the path length and C is the molar concentration. If the same compound is being used and the path length is kept constant, then the absorbance is directly proportional to the concentration of the sample.A spectrophotometer is used to provide light of certain energy (wavelength) and to measure the absorbance of that light. The basic operation of the spectrophotometer includes a white light radiation source which passes through a monochromator. The monochromator is either a prism or a diffraction grating which separates the light into the colored components and allows only light of a particular wavelength to strike the sample. The sample is poured into a cuvette which is similar to a small test tube. It is marked so that it can be positioned in the light beam the same way each time to avoid variations due to the differences in the composition of the glass.

The light passes through the sample and the unabsorbed portion strikes a photodetector which produces an electrical signal proportional to the intensity of light. The signal is converted to a readable output which is used in the analysis of the sample.OBJECTIVES• To introduce the use of spectrophotometry for qualitative (what is it) and quantitative (how much is there of it) analysis of biological samples and molecules.• To apply laboratory skills in the lab• To determine absorbance spectrum of unknown compounds (qualitative analysis).• To prepare and graph standard or calibration curves for quantitative analysis using colorimetry.