Lecture 32: Spectroscopy (continued)
Review
o Two basic rules of spectroscopy
o Transition probability
o Lifetime broadening
Today
UV visible spectroscopy
o Fundamentals
o Beer-Lambert’s law
o Examples
o Proteins
 Secondary structure
o DNA
Fluorescence
o Fundamentals
o Examples
o Quantitative aspects
o Fluorescence lifetime and quantum yield
o Excitation transfer
o Quenching
Rules of spectroscopy
In spectroscopy we deal with interaction of radiation with
molecules. Given that quantum mechanics gives us the
energy levels and the associated wavefunctions, we can
summarize two fundamental aspects of the spectroscopy.
Whether a given transition of the molecular systems
between the energy levels 1 to 2 will take place or not
depends on whether the following integral is nonzero or
not.
    2*  i 1d
In this formula,  is the dipole moment, i=x,y,z. If this
integral is nonzero we call the transition from energy level
1 to 2 is “dipole” allowed. In UV-visible and IR
spectroscopies, the selection rules are governed by the
dipole interaction. However, the selection rule is of general
validity. For example, a given NMR transition is allowed or
not depends on the angular momentum operator Ix.
Similarly there are quadrupole allowed transitions.
Second important rule concerns the
linewidth of a spectral line. The
uncertainty principle can be restated
as follows:
E.t  h / 2 or  t  1 / 4
Longer the lifetime of the
excited state,
sharper/narrower is the
linewidth.
UV-visible spectroscopy
Here we are concerned mainly with the electronic
transitions, as shown below.
Within each electronic level,
there are the vibrational
energy levels approximated
as harmonic oscillator.
During the excitation of
molecule, it may end up in
one of the vibrationally
excited state. This is not
very pronounced for the
spectra taken from solutions
due to broadening caused by
collisions with the solvent
molecule.
Note in the diminished linewidth broadening in vapor phase
UV-visible spectroscopy of mixtures: Beer-Lambert law
Example of the isobestic points
Since isobestic points occur if and only if there are only two
absorbing species, it can be a very useful diagnostic technique, as
shown below.
The reason for this unique property of the isobestic point is that the
chances of having the same extinction coefficient for three
compounds are extremely low.
Typical Absorption spectra of proteins and DNA
In proteins, two maxima (280 and 200nms) have been ascribed to
pi-pi* transitions in aromatic rings and the amide bonds
respectively. Unlike proteins, nucleic acids generally show one
maximum at 260nm, whose intensity decreases in organized
helical structure. This is the so-called hypo-chromic effect. The
ratio of A260/A280 has been used to determine nucleic acid to
protein concentration ratio. Additionally, the UV-visible
spectroscopy is sensitive to the secondary structure of protein:
Fluorescence
We will use the kinetics of the first order process to describe
terminology of fluorescence.
Kinetics of Fluorescence
However, the kd contains many contributions.
Contributions to Kd and the quantum yield
Fluorescence quenching
Many molecules that having energy level separation
comparable to the excited state energy of the photo-excited
molecules. They can quench the fluorescence. Molecular
oxygen is one prime example of fluorescence quencher. In
this case the kinetic scheme is described as:
Excitation transfer
Using
We can easily show that
Thus one tries to detect the fluorescence of the acceptor.
The key to excitation transfer process is the distance
between the donor and the acceptor. Forrester pointed out
that it is due to dipolar interactions and has r6 dependence.
Molecular Ruler
The basic idea that the excitation transfer depends on the
distance of separation between the donor and the acceptor
species can be used to design molecular ruler. Beautiful
studies of Stryer et al demonstrated the potential of this
approach in the studies of nucleic acids. These researchers
labeled two termini of the oligo-proline strands and
observed the excitation transfer efficiency as the
fluorescent acceptor to donor distance was systematically
varied. Shown below are their results.
Note the excellent fit to R6 model.
The key criteria for this method to work is that absorption
spectrum of the acceptor must overlap the emission
spectrum of the donor. It is only then the resonance energy
transfer can take place.
Single molecule fluorescence
One of the difficulties in performing the fluorescent energy
transfers, and hence the studies of inter-molecular
interaction, is that one needs fluorescent labeled materials.
Covalent attachment of these marker molecules is time
consuming and requires special synthetic techniques.
However the beauty of the fluorescent-labeled studies is
that concentration of the material needed is small. As
matter of fact, single molecule fluorescence can be
observed owing to the extraordinary sensitivity of the
photon counting instruments. An example of application of
this technique to study ATP synthesase is shown below.
Single molecule fluorescence
In their experiment Noji et al attached the “motor “ portion
of the protein to the glass slide by chemical bonding. And a
fluorescently labeled actin molecule replaced the
transmembrane part of the protein. When the slide was
placed in solution containing ATP they observed rotation of
actin filament by the monitoring emission of light of the
probe under a fluorescent microscope. It is hindered
rotation occurring in jumps of 120 degrees.
Modern DNA and Protein chips also make use of this
extraordinary sensitivity. A complementary strands on
DNA are synthesized on the chip surface as shown below
Fluorescently labeled complementary strand is then
introduced. Binding event only occurs when the sequence
introduced is complementary to the structure present on the
chip. In this way, today genes are detected.