Experiment1-Spec

Experiment1-Spec - Experiment 1(Lab period 1 Spectrophotometry Absorption spectra and the use of light absorption to measure concentration

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Experiment 1 (Lab period 1) Spectrophotometry: Absorption spectra and the use of light absorption to measure concentration Spectrophotometry is a procedure that is frequently utilized in biological laboratories. Probably the most common application in biology of this technique is in the measurement of the concentration of a compound in solution. In this lab you will be introduced to the concepts of spectrophotometry as well as how it is used to measure the concentration of compunds in solution. Over the rest of the course you will apply spectrophotometry on several occasions. Spectrophotometry is the measurement of the interaction of light with matter. Many kinds of biological substances absorb visible light (400 to 700 nm) selectively and, as a result, appear colored. Substances that do absorb visible light are called pigments. Other substances do not absorb light of visible wavelengths and appear colorless. We can study colorless compounds in two different ways. One is that they can often react with other substances to form colored derivatives that we can see and measure (as you will see later on in this course). Another is that colorless compounds usually absorb light in the region of the spectrum that is not visible to the naked eye. Absorption of this light can be measured, even if we cannot observe it unaided. Using a spectrophotometer, which measures the absorption by a solution of light of specific wavelengths (visible or not), allows us to determine concentration as discussed below. A second application of spectrophotomerty is the determination of the absorption spectrum of a compound. (Both of these can be applied to colorless as well as colored solutions since a spectrophotometer can measure absorbance of light that we cannot see.) These two applications are discussed separately below. Absorption spectra and color Regardless of whether a solution is colorless or colored, the wavelength(s) absorbed are distinctive. A solution of a particular compound, such as hemoglobin, always absorbs light of specific wavelengths and reflects light of other wavelengths. Furthermore, absorption of light is not absolute. A compound will characteristically absorb a certain proportion, anywhere from 0% to 100%, of light of a specific wavelength. For every compound, if you measure the proportion of light absorbed, for any wavelength, you will always get the same answer. This is the basic idea of the absorption spectrum of a specific compound, which is the proportion of light absorbed for each wavelength of the spectrum. The wavelength(s) absorbed by a substance in the visible part of the spectrum is complementary to the color that we perceive. The "color" is a function of human perception, but absorption of specific wavelengths of light is a function of molecular interaction with light. If a substance absorbs blue and red light, but not green light, we will see it as green since that is the only light that reaches us from that substance. Therefore, when you look at the absorption
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This note was uploaded on 01/30/2012 for the course BIO 2004 taught by Professor Morton during the Spring '11 term at Columbia.

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Experiment1-Spec - Experiment 1(Lab period 1 Spectrophotometry Absorption spectra and the use of light absorption to measure concentration

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