absorbance versus concentration should be linear with a y-intercept of zero. Of course, small deviations from this are to be expected. That is why knowing the R or R2 value is helpful…it indicates how closely the substance adheres (follows) Beer’s Law. EXPERIMENT: SPECTROPHOTOMETRIC ANALYSIS OF FOOD DYES INTRODUCTION Spectrophotometry is a very important and useful tool, which involves the interaction of matter with electromagnetic radiation (EM). The spectrophotometer that you will use in this experiment measures the visible portion of the EM spectrum, from 400-800 nm (1 nm = 10-9 m). The spectrophotometer will be used to find the absorption of a food dye at several different concentrations and then used to determine the unknown concentration of the same food dye. THEORY White light (the light we observe) contains all wavelengths of light in the visible region of the EM. A substance appears colored because it absorbs light at one or more wavelengths in the visible region and transmits the remaining wavelengths. The human eye, a type of EM radiation detector, sees the light transmitted, not absorbed. The color of the light transmitted is complementary to the color of light absorbed. The idea of a complementary color can be understood with the use of a color wheel. A linear relationship exists between absorbance and concentration for dilute solutions at a specific wavelength. This relationship is known as Beer's Law and is given by the equation: A = εbc, where A = the absorbance (measured) ε = the absorptivity constant (unique for each substance) b = the thickness of the sample (determined by the width of the sample cuvet and usually 1 cm) C = the concentration of the absorbing substance. In analytical analyses, a wavelength of light that is strongly absorbed is selected. It is determined by finding the maximum absorbance that occurs in the EM spectrum being analyzed. Since absorbance is directly proportional to the concentration for dilute solutions, a plot of absorbance versus concentration can be used to construct a linear plot. After the plot is constructed, the concentration of an unknown substance can be determined using the absorbance of the unknown substance and the graph.
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