LAB 7: SPECTROPHOTOMETRYObjectives: After studying this material, you will be able to:1) Understand the general idea of electromagnetic radiation and especially visible and ultraviolet light.2) Know what a spectrophotometer is, how it works, and for what it is used.3) Understand the concept of absorbance, and how it can be used to characterize compounds and determine their concentrations.4) Determine the concentration of a colored solution, the concentration of which is unknown.5) Understand how Breathalyzers can be used to determine the blood alcohol concentration (BAC).The purpose of this experiment is to understand how to determine the concentrations of solutionsusing spectrophotometry. We will specifically use spectrophotometry to detect the unknownconcentration of alcohol using breathalyzers as an example.BACKGROUNDWHAT IS SPECTROPHOTOMETRY?Spectrophotometry is a commonly used tool in biochemical research and clinical science. Physicians rely heavily on data obtained from spectrophotometric assays for both diagnosis and management of many disorders. Spectrophotometry involves measurement of the amount of light of a given wavelength absorbed by a colored solution. Because the amount of light absorbed by a colored solution depends on the number of colored molecules absorbing the light, spectrophotometry is often used to quantitate the concentration of material in a solution. In some cases, the concentration of a compoundcan be determined directly (for example, concentrations of the colored solutions we will use in today’s experiments). More often, however, the compound being investigated is colorless and hence must first be subjected to a chemical reaction that yields a colored product. The concentration of the original compound is then quantitated by spectrophotometrically measuring the amount of colored product formed and relating it to the amount of colorless product originally present. Such colorimetric assays, which measure the concentrations of various compounds in blood and other body fluids, are common in clinical chemistry labs. In addition, various molecules or groups of molecules often have characteristic absorption spectra and examination of these spectra is often useful in biochemical research. A brief discussion of the principles of spectrophotometry follows.Many molecules absorb specific types of radiant energy in a predictable fashion. When a beam of whitelight passes through a prism, it emerges as a band of colors known as the visible spectrum, which rangesfrom violet to red. A rainbow is formed because, when sunlight, a form of white light, passes throughtiny water droplets in the atmosphere, each droplet acts as a prism. Each color in this spectrumcorresponds to a wavelength of light, with red having the longest wavelength (about 800 nanometers; 8x 10-7 meters) and violet having the shortest (about 400 nm). When white light illuminates an object,one or more of the colors from the source of white light are absorbed. The remaining colors arereflected (or transmitted), and these determine the color of the object we see. An object or solutionthat appears blue actually reflects or transmits blue light, while absorbing the other colors present inwhite light.Biochem 107L7-2Visible light is a form of electromagnetic energy that travels in waves. The visible spectrum(electromagnetic energy waves we can see) is only a small part of the entire electromagnetic spectrum.We encounter radiation from other portions of the electromagnetic spectrum every day, although wemay not always be aware of this fact. This radiation includes cosmic rays from space, X-rays (for findingcavities and broken bones), ultraviolet radiation (for tans and burns), infrared radiation (those big lampsthat keep biscuits warm at the take out), microwaves (for warming up our croissants), and radio waves(remember radio?). The wavelength (distance between consecutive peaks) of a wave of electromagnetic radiation is afunction of its energy, with the shorter wavelengths having the highest energy and vice versa, and allthat really distinguishes these different parts of the electromagnetic spectrum from one another arethese differences in energies and wavelengths. Wavelengths and energies of various types ofelectromagnetic radiation are shown in the figure below. Note that the energy of the radiationdecreases (going from left to right in the figure) as the wavelength () increases.The colors of the visible spectrum and their approximate wavelengths are shown in the expandedportion of the figure. As the wavelength changes, the colors of light absorbed and reflected also changegradually from one color to the next (a rainbow), with no clear line of demarcation. The sum of thecolors of the reflected or transmitted light forms the apparent color of an object or solution as perceivedby the viewer.Spectrophotometry is the quantitative measurement of the amount and wavelengths of light absorbedby molecules in solution. It takes advantage of the property of colored solutions to absorb light ofspecific wavelengths. This ability of a colored solution to selectively and differentially absorb certainwavelengths of light determines the absorption spectrum of that solution. The absorbance spectrum fora given compound in solution is a unique pattern of absorption maxima and minima at differentwavelengths, and these spectra are often used to identify and/or characterize biochemical compounds.Examples of the usefulness of absorbance spectra are shown in the two figures below.Biochem 107L7-3The figure on the right contrasts the absorbancespectra of oxidized and reduced NAD+. NAD+ isinvolved in cellular energy metabolism (you willhear more about NAD in lecture). Transfer ofenergy derived from oxidation of foodstuffsinvolves passing electrons through NAD+, which isreduced to NADH and then re-oxidized during thiscycle. The reduction of NAD+ to NADH can befollowed by measuring the absorbance at 340 nm.Spectrophotometry was instrumental in determining just how this transfer of electrons results in theproduction of useful metabolic energy. The figure below compares the absorbance spectra forchlorophylls a and b with the photosynthetic rate. The