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Introduction to Spectroscopy
Purpose of Course To provide an introduction to a number of physical techniques that are generally
acknowledged to be of value in biological research.
As biochemists and biophysicists the focus of our interest is broadly defined as "How do
living cells carry out their functions ?" These functions are usually specific and detailed; some
-How does cytochrome c oxidase couple electron transfer to proton transport?
-How does the interaction of actin and myosin lead to muscle contraction?
-What controls the various conformational states of DNA?
-What are the consequences of a photon impacting a rod or cone in the retina?
-What are the structural consequences of adrenaline binding to its receptor?
The answers to these and similar questions invariably require a knowledge of the structure of
the responsible molecules (or supramolecular structures or organelles) and thus much of
Biochemistry can be seen as an investigation of bio-molecular structure and the relationship of this
structure to biological function. This is usually called the structure-function paradigm.
Unfortunately biosystems have two properties that complicate this undertaking:
a) Limited stability restricts the techniques that can be used. We need
i) a pH near neutrality
ii) a temperature close to ambient
iii) and the solvent is limited to water
b) Most systems of interest are not readily crystallized. X-ray analysis, the method of choice
for structure determination, requires high quality crystals (and the availability of heavy atom
derivatives if the structure is of a new protein). Thus this technique may not be applicable.
Modern nmr has the potential to obtain data on protein backbone folding comparable to X-ray
crystallography, i.e. to about 0.2 nm resolution, particularly if 13C and 15N substituted proteins can
be prepared. However nmr is currently restricted to proteins of mass < 20 kDa. Moreover to collect
the necessary data requires about two weeks of uninterrupted data acquisition; many pure proteins
denature when maintained at room temperature or above for this length of time.
It is thus apparent that we need methods that allow us to circumvent the intrinsic limitations
of the biological systems, methods that can complement x-ray crystallography and nmr.
Why Spectroscopy?
Experience has shown that the most reliable structural and dynamic information on molecules
of biological interest, on sub-cellular organelles and on cells is obtained by probing these systems
with "light" i.e. with electromagnetic radiation of a wavelength selected because it reveals the
property to be studied. Thus "light" provides a window into the properties of matter, in this case
molecules such as proteins, nucleic acids and metabolites, and of more complex structures such as
membranes. In particular this probe is usually able to provide both static (e.g. as from X-ray) and
dynamic insights into bio-structures.


In general a particular method provides "limited information" and will either provide
extremely detailed information about a small region of the protein or macromolecule or lowresolution data about the molecule as a whole.
Of particular importance is that these methods allow us to study changes in structures as
molecules implement their biological function. They often provide the high time resolution necessary
for functional studies (i.e. for mechanism of action). In favorable cases processes that occur within
picoseconds (psec, 10-12 sec) can be studied.
Finally these spectroscopic techniques are non-destructive i.e. the sample is re-usable.
(Traditional i.r. measurements were not applied to biological samples because the material to be
studied was first subjected to procedures which denature proteins-rendering the measurement
invalid. Thus vibrational spectroscopy of biomolecules has only recently become routine using either
the Raman or FTIR approaches (See Ch. VI) which overcome this limitation.)
It should be appreciated that the most desirable circumstance is to have detailed structural
information such as that provided by x-ray crystallography AND the specific information provided
by spectroscopy. These two areas of research are by no means mutually exclusive and the most
rewarding progress is made when both kinds of information are to hand.
Which Spectroscopy?
There is some bias in the selection of techniques for this course, as will be apparent from the
table of contents. Other methods could easily be included were more time available.
Because the emphasis in these lectures will be on the interaction of light with matter we begin
with a brief
Review of Electromagnetic Radiation
Electro-magnetic radiation is conventionally described by the wave model in which the
radiation is represented as oscillating electric and magnetic fields (Fig I-1).
These waves vary in both space and time as depicted in the figure. By convention the wave
advances from left-to-right (z-direction in this figure). An observer at +z looking at the wave
approaching will see the electric component (E) oscillating in a single plane (conventionally assigned
to the vertical, xz plane) and the magnetic component (H, not shown) oscillating, in phase, in the
plane perpendicular to E (in this case, the horizontal yz plane). [Remember that this is only one such
wave; normally a light source consists of many waves each with its E (and H) oriented in an unique
direction in the xy plane-the beam is unpolarized. Note also that the energy content of the E and H
components is the same.]
The wave is characterized by several quantities. The most familiar is the wavelength (), the
separation (in meters) between two equivalent points (e.g. between 2 peaks). Because many
wavelengths of interest are of order 10-9 meters a common unit is the nanometer (nm); visible light
falls in the range 400-700 nm. An older unit still very much in use is the angstrom (); 1 = 0.1 nm
= 10-8 cm; an atom is roughly 1 in diameter.






Fig. I-1
Quantities related to are
(i) The wavenumber ('), the number of waves/cm. = 1/(cm); this is called the kaiser and
has the unit cm-1. 1000 cm-1 = 1 kK is the kilokaiser. The symbol ' is often written as with a bar
over it. It is very common to interchange nm and cm-1; a simple aid for this is the rule (No. of
nm)*(No. of cm-1) = 107 . So 500 nm = 20,000 cm-1.
(ii) the frequency (), the number of oscillations per sec. = c (cm sec-1)/(cm), where c is
the velocity of light (3 x 1010 cm/sec). 500 nm = 500 x 10-9 m = 5 x 10-5 cm = 6 x 1014 Hz.
(iii) the energy of the light E = h = hc/ = hc' (h = Planck's constant = 6.623 x 10-27 erg
sec (per photon) or 6.623 x 10-34 J s). This energy is the energy per photon; the amplitude of the
wave is specified by the number of photons/sec in the beam. Thus the amplitude of the beam does
not change smoothly but in increments; these increments are of course very small and are not
apparent on a macroscopic scale.
A mole of photons is called an einstein. Usually when we talk about the energy in a wave of
some we implicitly mean the energy in an einstein of that . Thus the energy in a given
monochromatic beam is given by (No. of photons)*(Energy per photon (a.k.a. wavelength)).
Useful numeric relationships are 1000 nm = 10,000 cm-1 = 28.6 kcal/einstein = 1.24 eV =
1.97 x 10 -12 erg/ photon.
Radiation that consists of a single wavelength is called monochromatic; if it is oriented in
only a single plane (as in Fig. I-1) it is called linearly or plane polarized. A linearly polarized beam is
composed of two counter-rotating circularly polarized beams (see Fig 3 of Appendix I). If all the
monochromatic waves pass through their maxima at the same moment (i.e. they have the same
phase) they are called coherent. For simplicity we usually use sketches in which the waves are


monochromatic, linearly polarized and coherent. Waves from most real light sources are only
approximately monochromatic, usually unpolarized and almost never coherent.

An overview of the electromagnetic spectrum.

7.1 x 1014 Hz




9 10 11 12 13 14 15 16 17 18 19 20 21


30 um

300 nm

0.3 nm



400 nm
25000 cm - 1








Fig. I-2


The interaction of light with matter.

The response to the electromagnetic radiation impinging on a molecule can be divided into 3
common categories; scattering, absorption and emission. This is illustrated for the particular case of
the absorption of ultraviolet or visible light (Uv-Vis) where the various processes are conveniently
summarized in a Jablonski diagram (Fig. I-3).


Fig. I-3: Jablonski Diagram
We define G, the Ground-state: this is the equilibrium state in the absence of any radiation.
Sn are the n excited s inglet states of the molecule (n=1,2,3...); these represent states in which a
loosely bound (valence) electron has adopted a new "shape" in response to the light beam without
any change in the orientation of its spin. The ground and excited states are called stationary states as
they are either stable (G) or metastable (S). For example in the hydrogen atom the sole electron is in
a 1s orbital and it can be excited into one of the 2p orbitals; the change in shape is obvious. In
molecules the ground state is usually the highest occupied molecular orbital (HOMO) and the first
excited state (S1 ) is the lowest unoccupied molecular orbital (LUMO).
When exposed to an oscillating electric field the electron cloud of the molecule distorts
rhythmically in response to the oscillations. When the energy in the beam is smaller than that needed
to drive the molecule from G to one of the Sn (i.e. into an excited state) the distortion yields a
"Virtual State"- a non-stationary state that is not described by a single wavefunction but can be


described as a linear combination (a mixture) of initial and final wavefunctions (e.g. G + S1) with
the relative proportion of S1 increasing as the distortion gets increasingly large. When the energy in a
photon is comparable to the energy difference (S1 minus G) the molecule may be driven into the
metastable state represented in this instance by S1. This occurs when allowed by a selection rule, a
"spectroscopy specific" rule that controls whether-or-not this transition has a high probability. The
excited states are called singlet states because the electron that has been promoted maintains its
original spin orientation; with low probability an electron in S can reverse its spin and pass into a
Triplet state.
In Uv-Vis, electronic transitions from G to the various sub-states of S1 occur in a time
comparable to 10-15 sec, the reciprocal of the frequency of visible light responsible for the
transition. Immediately following promotion the excited molecule is in the equilibrium nuclear
configuration of the ground state (Frank-Condon principle, electron motions are very much more
rapid than nuclear motions) and over the next picosecond the molecule's nuclei change to the
equilibrium geometry of the excited state.
In general we can identify four fates to the excited molecule (Fig. I-3):
1) The excited molecule leaves the virtual state or Sn and returns immediately to precisely that
state from which it originated. The photon that is emitted has exactly the same energy as that which
was absorbed though its direction of propagation may well have changed. This is elastic or Rayleigh
scattering. (Immediately means synchronous with the frequency of the exciting radiation; this is a
time shorter than 1 vibrational oscillation). If the material is ordered e.g. a crystal, and the
wavelength is comparable with the size of an atom, then x-ray crystallography can be conducted as
is described in the other half of this course.
2) During the 10-15 sec. in either the virtual state or Sn the excited molecule may undergo a
vibration (this has a low probability, e.g. 0.01% ) before or during its return to the ground state so
that the emitted photon has an energy that differs slightly, either greater or smaller, from that which
was absorbed, the difference being the energy associated with the vibrational transition. Again this is
an "immediate" event. It is the inelastic or Raman or Stokes scattering. Typically the emitted light
falls within 3000 cm-1 of the energy of the exciting light. This shift can be measured by Raman
spectroscopy and allows the vibrational spectrum of the molecule to be studied.
3) The excited molecule stays in the excited state for a time of the order 1 nsec; during this
time many vibrational events occur and, regardless of the initial excited level (S1, S 2....) the
molecule finds itself in the lowest vibrational level of S1 before emission occurs. Emission is to any
vibrational level of G. The emitted photon has a significantly smaller energy than the photon that was
absorbed originally and light of substantially longer wavelength is emitted. This delayed emission is
called fluorescence.
4) The excited molecule stays in the excited state for 1 nsec or longer as in (3) but returns to
the ground state by processes that do not involve the emission of light. These processes are called
internal conversion (typically arising from collisions with the solvent), or quenching (from collisions
with some solute) or intersystem crossing (singlet triplet conversion) and are the principal
processes responsible for the Uv-Vis phenomenon. The triplet state is relatively long-lived (msec.sec.) and can itself emit light, called phosphorescence, or also return to the ground state by internal


When the incident light beam is reduced in intensity without any change in direction then we
have absorption. The frequency dependence of the magnitude of this absorption (i.e. the spectrum) is
an fundamental experimental quantity. Most types of spectroscopy provide data of this form.
5) An additional possibility is that the light causes a chemical reaction. This is called
photochemistry; a prime example is photosynthesis. Typically light of wavelength < 1000 nm is
needed for photochemistry i.e. light of energy comparable with the strength of chemical bonds.
Some people classify all of the first 4 processes just listed as spectroscopy. However my preference
is to omit Rayleigh scattering from this category though I admit that this is a personal prejudice (i.e. I
do not consider X-ray crystallography to be spectroscopy although there are versions of the
crystallographic technique (e.g. MAD) that involve absorption of the radiation).
In principle the shape of an absorption spectrum should be described by the Lorentz
formula: the reason for this can be found in the optional background material present in appendix II.
The Lorentz lineshape (L()) is given by
L() =
1 + ((-o)/)2


where L() is the spectral amplitude at frequency , L o is the amplitude at the absorption maximum
which occurs at frequency o, and is the half-width of the curve when the amplitude has fallen to
one-half the maximum value (at v either greater and smaller than o; is called the Half Width at
Half Height (HWHH)). As the spectrum is symmetric about o the HWHH can be measured on
either side of the curve.
Frequently the observed absorption curve consists of a number of overlapping, Lorentzian
curves that can not be separated, either for fundamental reasons or for limitations in the instrument.
Then the observed absorption curve approximates a Gaussian lineshape:
G() = G o exp{-ln(2)((-o)/)2}


One often sees the ln(2) omitted from the Gaussian formula whereupon is replaced by 1.2
(symbolized as ) the width when G() = 0.37Go (= G o/e). If the Go is replaced by (2) 1/2 then the
area under the Gaussian curve is 1.
Fig. I-4 shows the relative shapes of the Lorentz and Gaussian curves; small (but
occasionally important) differences will be apparent. The curves were adjusted to have the same
height-consequently they have different areas.
Why the emphasis on Magnetic Resonance in this part of the course ?
1. Many people believe that this is the single most valuable spectroscopic technique available
today for determining structures and that its importance can only increase in the future.
2. The connection between result and theory is easy to make and hence structural and
dynamic information is readily and reliably obtained without extensive training in quantum


Comparison of Gaussian and Lorentzian Lineshape


Solid line is gaussian

Broken line is Lorentzian














Fig I-4
Generic Uses.
Many experiments are conceptually independent of the spectroscopic method and can be
performed with any of them. Thus many experiments that are carried out using spectroscopic
methods do not depend upon a detailed knowledge of the physical principles of the selected method.
For example: an important area of biochemical research involves a characterization of the interaction
of two or more molecules, e.g. the binding of enzyme + substrate, antigen + antibody, repressor +
nucleic acid, hormone + receptor etc. To characterize any one of these interactions all one needs is a
spectral property of one or other species that changes upon binding of the second species. Frequently
several methods can be used to monitor these changes and one's choice of which to use may often be
made for relatively trivial reasons. It is, of course, prudent that such measurements be performed
under the guidance of someone who is familiar with the principles of the selected method. Even
apparently simple methods might have limitations or potential artifacts that can undermine the
significance of your experiment.
The CcO example!
However the emphasis this semester will be the use of spectroscopy to provide more
illuminating insights into biomolecules. In particular the focus will be on how these methods can
provide specialized structural information in the absence of an X-ray structure.
General Texts: (i) Campbell and Dwek: Biological Spectroscopy (Out of print. Selected material
has been put on reserve). (ii) Cantor and Schimmel: Biophysical Chemistry (On reserve).
Appendices I and II are more extensive discussions on Waves and the Forced Damped Harmonic
Oscillator. Any material in these appendices that is not covered in class will not be tested.