LAB TECHNIQUE - UV-VIS SPECTROSCOPY
Spectroscopy is the study of the interaction of light and matter. A spectrophotometer is an instrument that uses electromagnetic radiation from a selected region of the electromagnetic spectrum, such as ultraviolet, visible, or infrared light, to analyze the absorption or transmission of radiation by a sample. The basic function of a spectrophotometer is shown below in figure 1.
The electromagnetic spectrum (see figure 2) is the entire range of possible wavelengths or frequencies of electromagnetic spectrum, from 380 nm to 750 nm. Typical light sources for visible spectrophotometers include xenon and tungsten lamps.
Figure 2. Electromagnetic Spectrum
Glass cuvettes or test tubes may be used as sample cells for visible spectrophotometers. More specialized spectrophotometers require quartz cells, which the are "invisible" to the detector and do not absorb ultraviolet radiation. In addition to the light source used in spectrophotometers, a diffraction grating called a monochromator is also built in. The monochromator separates light into its individual wavelengths. The desired wavelength is then focused onto the sample cell to detect any absorption or emission of light by a substance in the sample.
Spectrophotometry is an analytical procedure that uses electromagnetic radiation to measure the concentration of a substance. The success of a spectrophotometric technique requires that the absorption of light by the substance being analyzed must be distinct or different from that of other chemical species in solution. How do scientists select the desired wavelength for spectrophotometry?
The absorption of visible light by a substance result from electron transitions, that is, the promotion of an electron in its ground (lowest energy) state to a higher energy atomic or molecular orbital. Both light energy and electron energy levels are quantized, so that the specific wavelength of light absorbed by a substance depends on the energy difference between two electron energy levels. The optimum wavelength for spectrophotometric analysis of a substance is selected by measuring the visible spectrum of the substance, corresponding to a plot of absorbance (A) versus wavelength (λ, "lambda").
Just seven unique dyes are approved by the Food and Drug Administration for use in foods, drugs, and cosmetics. These seven FD&C dyes give rise to the entire palette of artificial food colors. Three FD&C dyes, FD&C Blue 1, FD&C Red 40, and FD&C Yellow 5, are provided in this lab for the analysis of sports drinks and other beverages. The structure of FD&C Blue 1 is shown in Figure 3. Notice the extensive series of alternating single and double bonds (also called conjugated double bonds) in the center of the structure. This feature is characteristic of intensely colored organic dyes and pigments. Every double bond added to the system reduces the energy difference between the bonding and nonbonding molecular orbitals so that the resulting energy gap corresponds to visible light
A solution containing FD&C Blue 1 appears blue under normal white light - this is the color of light transmitted by the solution. The colors or wavelengths of light that are absorbed by this solution are complementary to the transmitted color. A color wheel (see figure 4) provides a useful tool for identifying the colors or wavelengths of light absorbed by a substance. The blue solution absorbs yellow, orange, and red light and we would expect the visible spectrum of FD&C Blue 1 to contain a peak in the 580-650 nm region. The optimum wavelength for spectrophotometric analysis of a dye solution is generally determined from the wavelength of maximum absorbance (known as lambda max "λmax"
The wavelength of light absorbed by a substance is characteristic of its molecular or electronic structure. The intensity of light absorbed depends on the amount of the substance in solution. Generally, the more concentrated the solution, the more intense the color will be, and the greater the intensity of light the solution absorbs. A digital spectrophotometer measures both the percent transmittance of light and the absorbance. When light is absorbed, the radiant power (P) of the light beam decreases. Transmittance (T) is the fraction of incident light (P/Po) that passes through the sample (see Figure 5). The relationships between transmittance and percent transmittance (%T) and between transmittance and absorbance (A) are given in Equation 1 and 2, respectively. Figure 5
%T = T x 100 = P/Po x 100% Equation 1
A = absorbance = -logT Equation 2
The amount of light absorbed by a solution depends on its concentration (c) as well as the path length of the sample cell (b) through which the light must travel. See equation 3, which is know as Beer's law. The constant ε ("epsilon") in the equation is a characteristic of a substance and is known as the molar absorptivity coefficient
A = εbc Equation 3