BCHM 313 – Physical Biochemistry Spectroscopy Dr. Bruce Hill Email: hillb@queensu.ca Office: Rm. 628 Botterell Hall Lab: Rm. 633

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)

The Electromagnetic spectrum microwaves Wavelength λ (m) 10-13 10-10 10-6 10-2 102 106 x-rays γ-rays UV Radio frequencies v=c/λ Vis IR Frequency ν (Hz or s-1) 1022 1019 1015 1011 107 103 E=hv Energy per photon (J) 10-11 10-14 10-18 10-22 10-26 10-30

Useful spectroscopic regions for biomolecules Typical λ (m) Energy (kJ/mol) Spectroscopic region Application 10-13 109 γ-ray Mössbauer 10-10 106 X-ray Diffraction, scattering 10-7 103 Vacuum UV-UV Electronic spectra C-C bond energy 5 x10-6 5 x102 Visible Electronic spectra 10-5-10-4 1-10•••RT at 25oC•••IR Vibrational spectra 10-2 10-2 Microwaves EPR 1 10-3 Radiowaves NMR

The nature of electromagnetic radiation x 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)

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

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

When electromagnetic radiation and matter meet Scattering Absorption Emission Photochemistry

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

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

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

Ultraviolet – Visible absorption spectroscopy Transitions between different electronic energy states Spectral regions 200-400 nm (ultraviolet) 400-750 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 ~ 10-15 s 7) λmax and ε can be used for concentration measurements and for interactions with other molecules

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.

Chromophores of biological interest Chromophore λmax(nm) ε max(M-1cm-1) Peptide bond 190-200 7000 DNA bases ~ 260 10000 Aromatic amino acids TRP 280 5600 TYR 274 1400 PHE 257 200 NAD+ 260 18000 NADH 259 14400 340 6230 Flavin FMN 443 1000 FAD 460 1270 Heme 410 120000