Topic%2001%20_%20Spectroscopy%20_%20F10

Topic%2001%20_%20Spectroscopy%20_%20F10 - Spectrophotometry...

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Spectrophotometry • Widely used in biochemistry o simple, sensitive and selective o usually non-destructive • Quantitation of substances that contain a chromophore o Chromophore: light-absorbing moiety • Light absorbed: UV, VIS or IR region
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Examples of chromophoric substances: Nucleotides/nucleoside and free bases: adenine, guanine, cytosine, thymine, uracil Cofactors: NADH and FADH 2 Aromatic amino acids: tyr, trp, phe Pigments: heme, chlorophyll Indicator dyes: bromphenol blue, p-nitrophenol
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Intensity of the incident light Intensity of the transmitted light I o I t Cuvette I t I o
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Beer’s Observation Concentration of light-absorbing solute I t I t 10 -c Transmission is not a linear function of concentration
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For example: 2 mg/ml 1 mg/ml I o = 1 I t = 0.8 I o = 1 I t = 0.64 (Not ½ of 0.8)
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Transmittance = T = = 10 - a l c I t I o I t I o -log T = -log = a l c Lambert-Beer Law a l c = A = absorbance
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Lambert-Beer Law Abs = a l c concentration Path length Absorption coefficient Absorbance (Extinction coefficient) ( ε ) Abs c, , and a Abs vs c results in a linear plot, slope = al l
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Measuring Abs in the lab: controls eg measuring a protein solution at 280 nm 1 mg/ml BSA I o I t 1 cm pathlength cuvette Abs = 0.671 at 280 n m The cuvette and solution may cause some diffraction and scattering of the light thus the transmitted light, I
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This note was uploaded on 11/21/2010 for the course MCB 120L 69059 taught by Professor Fairclough during the Fall '10 term at UC Davis.

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Topic%2001%20_%20Spectroscopy%20_%20F10 - Spectrophotometry...

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