Physical Biochemistry Vibrational Spectroscopy

In general, molecules can undergo three types of motion

 Translational – moving along a line
 Rotation
 Vibrational (bonds expand / contract)

All of these motions have energy associated with then, but only rotation and vibrational motion
have quantised energies (they have specific values)

Under certain circumstances, the vibrational and rotational motion energies can exchange EMR
 They can emit or absorb EMR
 EMR can interact with the vibrational or rotational motion of the molecule

Born-Oppenheimer approximation makes use of the fact that these phenomena take place at very
different timescales and energies.

Molecular rotation: 10-11s (360 rotation)

Molecular vibration: 10-13s (to expand and contract)
Electron transition: 10-15 (from ground state to excited state) - femtosecond scale

This is useful, for example if we are looking at vibrational motion then we can assume that the
molecule is ‘still’ in terms of rotation. The influence of these phenomena can be studied
separately.

Separation between energy levels:

Rotational < Vibrational < Electronic

A molecular energy state describes the electronic, vibrational and rotational states of the
molecule. It is the sum of these 3 energy components.

In this example, each electronic state has 3 vibrational states.

The total energy levels represent all three parameters.

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Molecular Rotation:

Can be probed with microwave radiation in molecules with electric dipole moments e.g.
Heteronuclear (non-identical masses) diatomic molecules. In order for EMR to interact with a
molecule, the molecule must have a permanent electric dipole moment (heteronuclear only).
Can be used to determine bond length in small molecules in rotational spectroscopy.

m = mass
c = centre of mass (the point at which the molecule is rotating)
r0 = bond length

Ramen spectroscopy is an easier indirect way to study rotation

Vibrational Rotation:

Simple Harmonic Oscillator (classical)

Represents a vibrating diatomic molecule
The bond is treated a spring, stretching and compression store equal amounts of energy

m = mass
req = equilibrium bond distance

Intermolecular distance (x) / Energy (y)

Parabolic curve

When bond is at equilibrium distance, there is no energy stored in the spring.

If you assume that the potential energy is changing

Simple Harmonic Oscillator (quantum)

v = vibrational energy level. Associated with quantum numbers (non-negative integers).

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The ΔE between consecutive vibrational energy levels is

always the same.
Even when v = 0, there is still energy in the system, the
molecule is always vibrating. If a molecule is not vibrating, its
location can be accurately determined; this breaks
fundamental quantum laws (Heisenberg uncertainty
principle). In order to measure something you need to disturb
it, if you disturb something you cannot measure it with
complete precision.

The potential energy has no defined limits

The graph shows wave functions, showing distribution of one

of the atoms e.g. In v = 0, the atom is likely to be at r eq. The wave function is not a physical
location, more a distribution of possible locations. ψ 2 gives the probability distribution of the atom.
In the higher vibrational states, curves2 show the atom is not likely to be found at the equilibrium
distance.

The max compression / stretch are ‘turning points’, the molecule is more likely to be found at these
points than at the equilibrium distance. Atoms are moving the slowest at the turning points as they
are changing direction (they need a velocity of 0 to change direction). Conversely, the atoms are
moving fastest at req.

It should be noted that the simple harmonic oscillator is not a fair description of molecular
vibration. The compression and stretching of a bond are not actually equivalent, however we
assume this in the simple harmonic oscillator model (quantum).

In reality, a bond behaves more like an anharmonic oscillator.

If a bond stretches beyond a certain point, the bond will break and the atoms will dissociate
(dissociation energy = D).
De = ΔE between 0 and dissociation energy
D0 = ΔE between v = 0 and dissociation energy
The spacing between energy levels decreases with increasing ν, it is no longer constant
Compression brings changes into the system – e- repulsion, nuclear repulsion etc.

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The Morse potential follows a different path to that of

the simple harmonic oscillator (left).
There is a lot of overlap between the two for v = 0 &
1. At room temperature, most molecules will be found
at vibrational state 0, making the simple harmonic
oscillator appropriate for biochemistry.

Normal H2O Modes:

Wavenumber [cm-1] = 1/λ (wavelength)

Proportional to frequency: Higher wavenumber corresponds to a higher energy
The EMR that can interact with vibrational energy transitions is usually in the IR region (2.5μ -
20μ). Vibrational spectroscopy is often called infrared spectroscopy.

A water molecule is vibrating. Three different types of vibration are shown:

Antisymmetric stretch – one compresses while the other bond stretches (3756
cm-1)

Symmetric stretch – both bonds are stretching and compressing at the same time (3651
cm-1)

Symmetric bend – the bond is undergoing angular movement (1595 cm -1)

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Physical Biochemistry Vibrational Spectroscopy

All molecules have a small group of vibrations, known as normal vibrational modes. Each mode
has an independent energy level. By using EMR of different wavelengths, each vibrational mode
can be studied independently.
The amount of energy put into the system will influence the amplitude of vibration.
Any molecular vibration can be described as a linear combination of the normal modes.
In order for a vibrational mode to interact with electromagnetic radiation the dipole of a molecule
needs to change as the molecule vibrates, but does not require a permanent dipole (rotational
spectroscopy requires a permanent electric dipole moment).

The symmetric stretch in CO2 will not produce a vibrational spectra using EMR (there is no varying
dipole moment), but the antisymmetric stretch and symmetric bend will.

Skeletal vibrations usually occur in the fingerprint region of a vibration spectrum (600 –
1500cm-1)
A complex region, can be used to identify compounds (forensics) as different molecules have
characteristic vibrational spectra.

Functional Group Vibrations:

Functional group vibrations (related to specific bonds) have typical absorbances in the region of
1000-4000cm-1

v = Vibrational frequency

Groups with light atoms have a higher frequency than groups with heavier atoms.
Stretching modes usually have higher frequencies than bending modes
The stronger the bond the higher the frequency (triple > double > single bond)

N–H: One or two sharper peaks at 3500-3300cm-1

O–H: Broad peak between 3650-3200cm-1
Analysis in this region can be difficult as peaks overlap.

C=O: Strong and sharp peak between 1820-1660cm -1

IR Spectrometer:

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Measurement is alternated between sample and n0-sample to minimize environmental effects and
background radiation. If the sample is contained within a solvent, a reference cell is used to
cancel out the solvent absorption spectrum.

The light is initially polychromatic (many different wavelengths)

The monochromator ensures only the desired light comes out (effectively a prism)
The beam splitter allows some light to pass through the sample, and some light to reach the
detector without passing through the sample (this would allow a reference cell to be placed).

Sample Handling:

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Solvents: Water and alcohol are rarely used as they absorb in IR and attack cell window
materials. CCl4 and chloroform are commonly used, however no solvents are completely invisible
in the IR region.
Liquid samples are often analysed in their pure form for this reason.
Cells: NaCl and KBr are often used as a transparent material to hold the sample. 0.1-1mm thick.
Glass and quartz are opaque in the IR region.

Attenuated Total Reflection (ATR) Infrared Spectroscopy:

Used to study samples

without using water. The
aqueous solution is
passed through the flow
cell. Uses total internal
reflection.

Total internal reflection

occurs when light crosses a
boundary of different refractive indices (n), an angle that is greater than the
critical angle.

With each internal reflection, the beam penetrates 1-10μm into the sample (evanescent wave),
and is attenuated depending on what is in this thin layer. Multiple reflections (~7 bounces) help to
amplify the signal.
Useful for studying thin layered samples such as membranes.

Fourier Transform Infrared (FTIR) Spectroscopy:

Does not use a monochromator, the sample is irradiated by the full IR spectrum of the source.
Studies using a monochromator are slow; it can take minutes to scan the entire spectrum.

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1. A beam of light passes through the beamsplitter, passes through the sample then is reflected
back through the sample and beamsplitter, then hits the detector.
2. The second beam of light passes straight through the beamsplitter, and is reflected back to the
detector, without passing through the sample.
The second mirror can be moved, for each position of this mirror a reading is taken using the
detector.
There will be both constructive and destructive interference at different wavelengths between
these beams. Movement of mirror 2 will suppress or accentuate other wavelengths.
The interference imposes a cosine modulation on the spectrum, which has a periodicity that is
dependant on path difference.

The detector will detect the Fourier transform of the spectrum, which is a type of encoding of the
spectrum. In order to reconstruct the absorption spectrum, the signal needs to be ‘decoded’ as a
function of path difference.
The ‘decoding’ can be carried out computationally in order to obtain the spectrum (Inverse FT).
This process is much faster than conventional means.

Interference:

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The frequency of the modulation imposed on the spectrum is increased with the path difference
(dx).

Advantages of FT
 Full spectrum is obtained in one go
 Less noise
 Faster (10s) – real time measurements much easier to carry out
 Uniform resolution over entire spectrum. When using a monochromator, resolution is not
constant at different wavelengths.

Isotope editing can be used to enhance results. Using a different isotope will specifically shift the
absorption frequency for a specific group. Spectra of edited and unedited molecules are
compared. Helpful analysing overlapping carbonyl groups.

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