1. BCHM 313 – Physical Biochemistry
Spectroscopy
Dr. Bruce Hill
Office: Rm. 628 Botterell Hall
Lab: Rm. 633

2. BCHM 313 – Spectroscopy
Spectroscopy refers to the study of the interaction of electromagnetic radiation with matter. Spectroscopy is useful in both qualitative (what does the matter look like) and quantitative analysis (how much).
We are going to consider aspects of,
1) UV-visible absorption
2) Fluorescence
3) Circular dichroism
4) Electron Paramagnetic Resonance (very briefly)

3. The Electromagnetic spectrum
microwaves
Wavelength λ (m)
x-rays
γ-rays UV
Radio
frequencies
v=c/λ
Vis IR
Frequency ν (Hz or s-1)
E=hv
Energy per photon (J)

4. Useful spectroscopic regions for biomolecules
Typical λ (m) Energy (kJ/mol) Spectroscopic region Application
γ-ray Mössbauer
X-ray Diffraction, scattering
Vacuum UV-UV Electronic spectra
C-C bond energy
5 x x Visible Electronic spectra
•••RT at 25oC•••IR Vibrational spectra
Microwaves EPR
Radiowaves NMR

5. The nature of electromagnetic radiationx y z
Wave of electric and magnetic vectors
Photons with discrete energy, E = hν
Speed of propagation (c) is related to frequency (ν) and wavelength (λ)
c = ν λ
(cm/s) (cm) (s-1)

6. Stuff, what stuff- what is matter?
Properties of matter are dependent on,
Energetic state- energy levels are quantized
Shape- e.g., bond angle between H and O atoms in H2O-
minimal energy
- the well-defined shape of folded protein-energetic minimum
Dynamics- molecules in solution have kinetic energy
rotate, translate, vibrate
These properties are interrelated

7. Energy levels Ground state- state of lowest energy
Excited state – states of energy higher than ground state
States of equal energy are referred to as degenerate
Energy classes
translation
rotation
vibration
electronic
electron spin orientation
nuclear spin orientation

8. When electromagnetic radiation and matter meet
Scattering
Absorption
Emission
Photochemistry

9. The absorption process
Absorption occurs when there
is a match between the energy
of the impinging radiation and the
gap between the two states
Excited state
Ground state hv
E2-E1= ΔE = hν, h= Planck’constant
ν= frequency (s-1)
For there to be net absorption there must be a population
difference between the two states, favouring the ground state

10. Boltzmann distribution
Governs the distribution of molecules across the available energy
levels
Excited state E2
nE2
nE1
= exp(-ΔE/RT) , for 1 mole
If ΔE<<RT, exp(-ΔE/RT) ~e0→1
ΔE>>RT, nE2<<nE1
#of molecules in E2
#of molecules in E1 =
Ground state E1

11. Boltzmann distribution (cont.)
At T= 300 K,
For ΔE = 11.9 J/mol,
(rotational transition)
nE2
nE1
=0.9952
For an electronic transition,
ΔE = 119 kJ/mol,
=1.86 x 10-21

12. Ultraviolet – Visible absorption spectroscopy
Transitions between different electronic energy states
Spectral regions nm (ultraviolet)
nm (visible)
Chromophore-group giving rise to electronic transition
Characterized by position of maximum (λmax)
and the extinction coefficient (ε)
5) Electronic energy levels are described by molecular orbitals,
π, π*, n, and d, charge transfer
6) Timescale- electronic transitions occur in ~ s
7) λmax and ε can be used for concentration measurements
and for interactions with other molecules

13. The Beer-Lambert Law What about scattering ?
Relates absorbance to the concentration of a chromophore.
Electromagnetic radiation
i.e., light of intensity Io
Some is transmitted, intensity I
%T= I/Io
A= log (1/%T)= log(Io/I) = ε c l
A – absorbance
ε – extinction coefficient (M-1cm-1)
c – concentration (M)
l – pathlength (cm)
What about scattering ?
For a chromophore with ε of
10,000 M-1cm-1, at a concen-
tration of 1 mM in a pathlength
of 1 cm - A would be 10.

14. Chromophores of biological interest
Chromophore λmax(nm) ε max(M-1cm-1)
Peptide bond
DNA bases ~
Aromatic amino acids
TRP
TYR
PHE
NAD
NADH
Flavin
FMN
FAD
Heme