Optical Spectroscopy UVVis Biophysical Tools 02 UVVis spectroscopy

Optical Spectroscopy: UV/Vis Biophysical Tools '02 - UV/Vis spectroscopy 1/13/2022 1

Principles of Spectroscopy Electromagnetic radiation plane and circularly polarized Interaction of electromagnetic wave with matter • Scattering • Absorption • Emmision Biophysical Tools '02 - UV/Vis spectroscopy 1/13/2022 2

Scattering oscillating E-field electrons oscillate radiation Elastic – same frequency Inelastic – lower frequency (Raman) Small and large molecules • Small: Rayleigh M/ 4 • Large: interference Dynamic Scattering P( ) RG (Guinier plots) ln A( ) D Biophysical Tools '02 - UV/Vis spectroscopy 1/13/2022 3

Absorption Lineshape • • • Position Intensity Width Boltzmann Biophysical Tools '02 - UV/Vis spectroscopy 1/13/2022 4

Electronic absorption (UV/Vis) Spectrophotometers Beer-Lambert Law: d. I ~ I*dl*sample Biophysical Tools '02 - UV/Vis spectroscopy 1/13/2022 • Single beam • Double beam • Diode array 5

Absorption bands Solvent effects – blue/red shifts peptides p p* @ 190 nm (e ~ 10, 000) n p* @ 210 -220 nm (e ~ 100) Sidechains Trp, Phe, Tyr (p p* @ 280 nm ) Biophysical Tools '02 - UV/Vis spectroscopy Hypochromism and hyperchromism Ionization state 1/13/2022 6

Polarized light: Different reffractive index for L &R = rotation (ORD) Different light absorption = ellipticity (CD) Circular Dichroism dichroism/ORD – secondary structure Absorption Instruments Biophysical Tools '02 - UV/Vis spectroscopy 1/13/2022 7

Vibrational Spectroscopy Infrared Raman Spectroscopy Biophysical Tools '02 - UV/Vis spectroscopy 1/13/2022 8