Electronic Spectroscopy – Emission ( ) Fluorescence is the emission of light by a molecule in the excited state Fluorescence – Decay occurs between states of the same spin – Decay occurs from vibrational ground state of excited electronic state Phosphorescence occurs when the excited state is converted to another excited state of different spin (intersystem crossing) Phosphorescence – Decay from an excited state to a ground state of different spin occurs very slowly – Intersystem crossing often occurs due to overlap of vibrational wavefunctions between different excited states Intersystem crossing Emission spectroscopy can be useful, but it has its problems – Quenching of the excited state by another species in solution can cause emission to be very low (non-radiative transition) – Other species may absorb emitted photons…but this can be used to our advantage

Fluorescence Resonance Energy Transfer (FRET, 19.13) Many monomers in a biopolymer have chromophores that absorb and emit photons at different energies – It is possible that one chromophore emits energy similar to that needed to excite another chromophore – The donor chromophore loses energy to the acceptor chromophore (this does not occur through emission and absorption of light and is called FRET)FRET The rate at which FRET occurs is dependent on how long the donor stays in its excited state and how far the donor and acceptor are from one another – R 0 is the distance at which k FRET is equal to the rate of radiative decay (i.e., fluorescence) FRET can be used to determine how far apart chromophores are from one another in a biopolymer – Luminescent dyes can be placed in biopolymers to help determine tertiary structure, since efficiency of FRET depends on distance between chromophores Luminescent dyes

Radiative Transitions in Molecules

Intersystem Crossing and Phosphorescence

Donor-Acceptor Photon Arrangement

FRET Efficiency as a Function of Distance