An Electromagnetic Wave
An Electromagnetic Wave
An Electromagnetic Wave
Electromagnetic spectrum
Absorption Spectroscopy
If light with suitable energy n hits a molecule in the ground state 1, the
energy can be absorbed and the molecule is raised to an electronically
excited state 2.
The molecule can return to its ground state via spontaneous emission of
light (a photon).
E = h
The valence electrons are the only ones whose energies permit them to
be excited by near UV/visible radiation.
Singlet and triplet excited states
The origin of the absorptions
n to * and to * Transitions:
*
High energy required, vacuum UV range
CH4: = 125 nm
n *
Saturated compounds, CH3OH etc ( = 150 - 250 nm)
n * and *
Mostly used! = 200 - 700 nm
Chromophore:
Biological chromophores
A single-beam spectrophotometer
For UV:
- a common lamp is a deuterium arc lamp
- electric discharge causes D2 to dissociate and emit UV radiation (160 325 nm)
- other good sources are:
Xe (250 1000 nm) Hg (280 1400 nm)
Beer-Lambert Law
dl
Molar absorption
Absorbance Coefficient
Depends on wavelength
Molar absorption coefficient and cross sections
Then, the total opaque area on the slab due to the absorbers = N A d
Therefore, for a chromophore with cross sectional area 12, the maximal value (assuming
that the transition probability is 1) is 2.6 x 104 M-1cm-1.
Deviations from Beer-Lambert law
Low c
High c
Polychromatic light
Absorption Spectroscopy
Amino acids
160000
120000
80000 Series1
-carotene
Absorption due to *
40000 transitions
0
380 480 580 680
Biological chromophores
If conjugated
o 5 nm
Spectral nomenclature of shifts
Hyperchromic effect in unstacked bases
2
Sample purity
260:280 ratio has high sensitivity for nucleic acid contamination in protein
% protein % nucleic acid 260:280 ratio
100 0 0.57
95 5 1.06
90 10 1.32
70 30 1.73
Vibrational
relaxation
Jablonski diagram
Stokes shift
Kashas rule
Mirror-image rule
Characteristics of fluorescence emission
Solvent reorientation
Characteristics of fluorescence emission
Solvent reorientation
o Ample time for the solvent molecules to reorient around the excited-
state dipole, which lowers its energy.
Vibrational relaxation
Mirror image rule and Franck-Condon principle
pH sensitive fluorophore
Anthracene
Fluorescence emission spectra of aromatic amino acids
Fluorescence lifetimes and quantum yields
Rate of fluorescence
This can be seen by calculating the average time in the excited state
<t>.
F0/F is given by the ratio of the decay rate in the absence of quencher
() to the total decay rate in the presence of quencher ( + kq[Q]):
Therefore 2
Stern-Volmer equation
Fluorescence quenching
Fluorescence lifetime
Fluorescence lifetime, is the time it takes for intensity to decay to I0/e