Ch 10 Pages 530-538; 541-554

1. Lecture 24 – Introduction to Spectroscopy Ch 10 Pages 530-538; 541-554

2. Spectroscopy A central problem in quantum chemistry is to calculate the allowed energy states of atoms and molecules In the last two weeks, we learned how to calculate the energies of vibrational and electronic states of molecules through the quantum mechanical formalism The principal source of experimental information about the allowed energy states of atoms and molecules is spectroscopy.

3. Spectroscopy The electromagnetic spectrum is characterized in terms of wavelength, wavenumber, frequency or energy, which are related by the well-known relationships: Spectroscopy at different frequencies monitors different molecular properties. UV and visible spectroscopy monitor changes between electronic states, infrared spectroscopy monitors vibrational states of molecules. Nuclear magnetic resonance spectroscopy, perhaps the most important of all spectroscopic techniques nowadays, monitors the absorption of radiofrequency radiation by nuclei spins placed in a static magnetic field.

4. Spectroscopy In the broadest sense, spectroscopy is the study of the interaction of radiation with matter. When a beam of light impinges on a sample (see below) several things may happen:

5. Spectroscopy Electromagnetic radiation may have not interactions with the sample, in which case the light is said to be transmitted 2. Radiation may be absorbed into the sample; when this occurs, transitions between energy states take place and usually results in the production of heat when the energy is dissipated back into the sample. Absorbed light may also induce a chemical reaction.

6. Spectroscopy Light with different polarization can be absorbed with different efficiency; this is called linear dichroism (LD) or circular dichroism (CD) for linearly and circularly polarized light, respectively Light may be re-emitted at lower frequencies (e.g. fluorescence or phosphorescence) 5. Small amounts of light may be scattered at various angles relative to the angle of the incident/transmitted beams

7. Light scattering Light scattered at essentially the same frequency as the incident beam is called Rayleigh scattering. However, there is a distribution of frequencies in Rayleigh scattering which results from molecular motions and/or changes in the internal energy states of the molecules. Random molecular motions will cause a distribution of scattered frequencies. Brillouin scattering results when sound waves propagate through liquids and induce coherent molecular motions

8. Raman scattering A small fraction of light is scattered at frequencies very different from the frequency of the incident beam. The frequency is shifted because some of the energy of the scatterer is changed by DE=hDn. Raman scattering frequencies occur at nscat=n0±DE/h. Scattered light with increased frequencies nscat=n0+DE/h is called anti-Stokes scattering. Scattered light with increased frequencies nscat=n0-DE/h is called Stokes scattering.

9. Reflection Light scattered in the opposite direction of the incident beam leads to reflection. Reflected light re-combines with the incident beam to give rise to refraction, a physical effect which light appear as though it travels more slowly through the sample than if it traveled through a vacuum. The magnitude of this effect is measured by the index of refraction: Scattered beams can re-combine, giving rise to diffraction.

10. Absorption Experimentally, absorbance is measured in solution and the fraction of light absorbed depends on the thickness of the sample (and its concentration, as we shall see later) according to the equation: where dx is the thickness of a layer of absorbing medium, I is the intensity of the incident beam, and b is a constant called the absorption coefficient.

11. Absorption The differential form of the law of absorption, given above, means that the fraction of light absorbed per unit length is a constant. The integrated form of the absorption law is: where I0 is the intensity of the incident beam and I is the intensity of the beam a distance x from its point-of-entry into the absorbing medium. The log form of the absorption law is: T is the transmittance and a is the linear absorption coefficient.

12. Absorption In 1852, Beer showed that the linear absorption coefficient a is proportional to the concentration of the absorber: Where e is the molar absorption coefficient, and C is the concentration in moles/L. Beer’s law is often expression in terms of the absorbance A: Its units are M-1cm-1. The wavelength dependence of e is called absorption spectrum

13. Absorption of proteins and nucleic acids Both proteins and nucleic acids have strong absorption in the UV region of the spectrum, and these properties are used to measure their concentration as well as characterize their conformational properties. Proteins have absorption maxima at 280 nm and 200 nm due, respectively, to transitions of aromatic side chains and amide groups. Nucleic acids absorb much more strongly at 260 nm, and this is again due to transitions in the aromatic bases. For proteins, only Trp, Tyr and Phe absorb strongly at 280 nm, in fact Phe absorbs very weakly; the extinction coefficient of Tyr is: Changes in absorption spectra around 200 nm can be used to monitor conformational changes in proteins, although other methods (Circular Dichroism, CD) are far more effective.

14. Absorption of proteins and nucleic acids All bases in nucleic acids have similar absorption spectra with extinction coefficient per base Changes in absorption spectra are used very often to monitor conformational transition in DNA and RNA. The change is called hyperchromicity.