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Bioanalytical Application of UV/ vis Spectroscopy

Bioanalytical Application of UV/ vis Spectroscopy. Week 4 Lab S117 Fall 2011. Overview. Post-lab Discussion Historical Perspective on Balmer , Rydberg, Bohr How science builds Pre-lab discussion Molecular Spectroscopy UV- vis Spectrometer Quantitative use of spectroscopy

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Bioanalytical Application of UV/ vis Spectroscopy

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  1. BioanalyticalApplication of UV/vis Spectroscopy Week 4 Lab S117 Fall 2011

  2. Overview • Post-lab Discussion • Historical Perspective on Balmer, Rydberg, Bohr • How science builds • Pre-lab discussion • Molecular Spectroscopy • UV-vis Spectrometer • Quantitative use of spectroscopy • Standard curves

  3. Balmer’s Contribution • Experimental • Empirical observation of a series • Mathematical description of series n2 Wavelength = constant * n2 - 22

  4. Rydberg’s Contribution • Generalization • Recognizing use of inverse wavelength

  5. Bohr’s Contribution • Theoretical • Assume classic interpretation • BUT…only if angular momentum restricted by quantum rule • From classical treatment mixed with quantum rule:

  6. Why is Bohr Accepted? • Direct match to experimental results • Explains Rydberg in terms of more fundamental constants • Philosophical question: How right is a “right theory” and how wrong is a “wrong theory?”

  7. Pre-lab: UV/vis Spectroscopy • Investigating a Dow Chemical Resin • Look at this UV-vis spectrum • How is it different than atomic spectrum? • Full spectrum and lmax

  8. Molecule for UV/vis Conjugation leads to molecules absorbing in UV/vis range 463nm, 494 nm

  9. Spectrometer

  10. Beer’s Law • Absorbance depends on • Characteristics of molecule under experimental conditions (molar absorptivity) • Size of cuvette (pathlength) • Concentration of molecules A = e b c

  11. Standard Curve • Determine molar absorptivity • Only good for one wavelength and one set of conditions • Use the data to determine concentrations of unknowns

  12. This Week • Goal: Determine [protein] using intrinsic absorbance of protein, then with added dye. Which is better? • For intrinsic absorption data • Determine lmax for protein unknowns and standard • Use this data to make a standard curve • Use standard curve to determine concentration of two unknown protein sample • For dye test, lmaxis given as 595nm • Need to be able to determine concentrations of dilutions and convert between concentration units

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