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Biological Spectroscopy

Dr. W. Richard Thilagaraj


SRM UNIVERSITY
Infrared spectroscopy
Principles and practice
Infrared spectroscopy
Principles and practice
Infrared interpretation
Step 1
Look first for the carbonyl C=O band.
Look for a strong band at 1820-1660 cm
-1
. This
band is usually the most intense absorption
band in a spectrum. It will have a medium
width. If you see the carbonyl band, look for
other bands associated with functional groups
that contain the carbonyl by going to step 2.
If no C=O band is present, check for alcohols
and go to step 3.
Infrared interpretation
Step 2
If a C=O is present you want to determine if it
is part of an acid, an ester, or an aldehyde or
ketone. At this time you may not be able to
distinguish aldehyde from ketone.
Infrared interpretation

ACID
Look for indications that an O-H is also present.
It has a broad absorption near 3300-2500 cm
-1
.
This actually will overlap the C-H stretch. There
will also be a C-O single bond band near 1100-
1300 cm
-1
. Look for the carbonyl band near
1725-1700 cm
-1
.
ESTER
Look for C-O absorption of medium intensity
near 1300-1000 cm
-1
. There will be no O-H
band.
Infrared interpretation
ALDEHYDE
Look for aldehyde type C-H absorption bands.
These are two weak absorptions to the right of
the C-H stretch near 2850 cm
-1
and 2750 cm
-1

and are caused by the C-H bond that is part of
the CHO aldehyde functional group. Look for
the carbonyl band around 1740-1720 cm
-1
.
KETONE
The weak aldehyde CH absorption bands will
be absent. Look for the carbonyl CO band
around 1725-1705 cm
-1
.
Infrared interpretation
Step 3
If no carbonyl band appears in the spectrum,
look for an alcohol O-H band.
ALCOHOL
Look for the broad OH band near 3600-3300
cm
-1
and a C-O absorption band near 1300-
1000 cm
-1
.
Infrared interpretation
Step 4
If no carbonyl bands and no O-H bands are in the
spectrum, check for double bonds, C=C, from an
aromatic or an alkene.
ALKENE
Look for weak absorption near 1650 cm
-1
for a double
bond. There will be a CH stretch band near 3000 cm
-1
.
AROMATIC
Look for the benzene, C=C, double bonds which appear
as medium to strong absorptions in the region 1650-
1450 cm
-1
. The CH stretch band is much weaker than in
alkenes.
Infrared interpretation
Step 5
If none of the previous groups can be
identified, you may have an alkane.
ALKANE
The main absorption will be the C-H stretch
near 3000 cm
-1
. The spectrum will be simple
with another band near 1450 cm
-1
.
Infrared interpretation
Step 6
If the spectrum still cannot be assigned you
may have an alkyl halide.
ALKYL BROMIDE
Look for the C-H stretch and a relatively simple
spectrum with an absorption to the right of
667 cm-1.
Biological applications
Biological Applications of Infrared Spectroscopy
Barbara H. Stuart (Editor), David J. Ando (Editor)

ISBN: 0471974145
Publisher: John Wiley & Sons
Published: 01 July 1997
Paperback
Infrared spectroscopy
Use in determining protein
secondary structure
Secondary structure of proteins
Changes in the group frequencies may be
used to derive information regarding the
secondary structure of biological
molecules.
The carbonyl group of the amide bond in
proteins is particularly useful for the
determination of secondary structure.
Protein Structure
Primary
Secondary
Tertiary
Quaternary
Protein secondary structure
Protein secondary structure
The stretching, normal mode of the
carbonyl has been shown to have a
specific frequency associated with -
helices, -sheets and other characteristic
structures.
Approximate wave numbers
corresponding to the three most common
features found in proteins are: -
-helix (1650 cm
-1
)
-sheet (1632 cm
-1
and 1685cm
-1
)
Random coil (1658cm
-1
)

Protein secondary structure
Known structures are often used to
calibrate the vibrational frequency
measurements.
The vibrational spectrum of the amide
bond of a protein is often complex
because of the many amide bonds in
multiple environments.
The spectrum can be deconvoluted to
provide information about the amount and
types of secondary structures present.
Protein secondary structure
The assumption is usually made that the
observed vibrational frequency is a linear
combination of the frequencies associated with
the various secondary structures that are
present.
Each frequency is weighted by the percent of a
given structure present.
Results are compared with known structures and
with circular dichroism measurements.
Raman spectroscopy has proved particularly
useful since water solutions can be used.
Near infrared
spectroscopy
Biological applications
Near-Infrared spectroscopy
Near-IR spectrometry is characterized by
low molar absorptivities and scattering,
which permit nearly effortless evaluation of
pure materials, and broad overlapping
bands, which diminish the demand for a
large number of wavelengths in calibration
and analysis.
The near-IR region of the electromagnetic
spectrum, once regarded as having little
potential for biological work, has become
one of the most promising for molecular
spectrometry.
Near-Infrared spectroscopy
The advent of inexpensive and
powerful computers has contributed
to the surge of near-IR spectrometric
applications.
The near-IR region is usually
estimated to include wavelengths
between 700 nm (near the red end of
the visible spectrum) and 3000 nm
(near the beginning of infrared
stretches of organic compounds).

Near-Infrared spectroscopy
Absorbance peaks in the near-IR region
originate from overtones and combinations
of the fundamental (mid-IR) bands and from
electronic transitions in the heaviest atoms.
For example, C-H, N-H, and O-H bonds are
responsible for most major absorbances
observed in the near-IR, and near-IR
spectrometry is used chiefly for identifying
or quantifying molecules including unique
hydrogen atoms.
Near-Infrared spectroscopy
Near-IR spectrometry is thus in
routine service for quantitative
analyses of water, alcohols, amines,
and any compounds comprising C-H,
N-H, and/or O-H groups. Numerous
other elementary bond combinations
are also likely to generate near-IR
absorbance peaks.


Advantages of near-infrared
spectroscopy
Analysis times under 1 second are
possible
Simultaneous multicomponent analysis
is the norm
No sample preparation is usually
required for liquids, solids, or gases.
Non-invasive and non-destructive
analysis is possible
Cost per analysis is very low (no
reagents are used)

Advantages of near-infrared
spectroscopy
Physical properties and biological effects
can be calculated from spectra of samples.
Automated correction of background and
interferences is performed in instruments
using computer algorithms
Detection limits can be very low
A wide range of sample sizes can be
analyzed.
Molecular structural information can be
derived from spectra.

A visible-light image of a human carotid
bifurcation exposed at the beginning of
endarterectomy
Near-IR images at 6 selected wavelengths (from top to
bottom and left to right, 1678, 1944, 2098, 2180, 2230,
and 2312 nm) of the carotid
Raman Spectroscopy
Biological applications
Raman Spectroscopy
This is an alternative approach to studying
transitions between vibrational energy
levels.
In addition to absorbing light samples also
scatter light.
The amount of scattered light is at a
maximum at 90 to the beam.
Most of the scattered light is at the same
frequency as that of the incident light
(Rayleigh scattering)
Raman Spectroscopy
At the molecular level the electric field of
the light perturbs the electron distribution
but no transitions between energy levels
occurs.
Scattering is inversely proportional to the
forth power of the wavelength.
This is why the sky is blue (Shorter
wavelengths (blue) are scattered more
than longer wavelengths (red).
Raman Spectroscopy
Rayleigh scattering is observed at all
wavelengths.
The intensity of the scattered light is
related to the polarisability of the
molecules.
A small number of molecules return to a
different vibrational energy level after
scattering.

Raman Spectroscopy
The vibrational energy level can be either
higher or lower than the initial state.
As a result of this change some of the
scattered light will be at a slightly higher
or lower level frequency than the incident
light.
This is called Raman scattering.
Raman spectroscopy
Raman spectroscopy
The Raman effect occurs when light impinges
upon a molecule and interacts with the electron
cloud of the bonds of that molecule.
A molecular polarizability change, or amount of
deformation of the electron cloud, with respect to
the vibrational coordinate is required for the
molecule to exhibit the Raman effect.
The amount of the polarizability change will
determine the intensity, whereas the Raman shift
is equal to the vibrational level that is involved.
Raman spectroscopy
The incident photon (light quantum), excites one
of the electrons into a virtual state.
For the spontaneous Raman effect, the molecule
will be excited from the ground state to a virtual
energy state, and relax into a vibrational excited
state, and which generates Stokes Raman
scattering.
If the molecule was already in an elevated
vibrational energy state, the Raman scattering is
then called anti-Stokes Raman scattering.

Raman Spectroscopy
A very intense light source is required to
observe Raman scattering because only a
very small amount of the scattered light
displays a change in frequency.
The advent of lasers has permitted this to
be done routinely.
Higher concentrations are often required.
Raman spectroscopy is a scattering
phenomenon as compared with absorption
spectroscopy.
Advantages of Raman Spectroscopy
A permanent dipole moment is not
required.
It is only necessary for the polarisability of
the molecule to change between different
vibrational energy levels.
Visible light may be used rather than
infrared light so than Raman spectra may
be readily obtained in water.
Crystals and films may also be studied.
Raman Spectroscopy
Due to the fact that the intensity of the Raman
lines is weak, intense light sources and higher
concentrations than those needed for infrared
spectroscopy are often needed.
Raman and infrared are often regarded as
complimentary techniques.
Since infrared depends on a permanent dipole
moment and Raman on the polarisability a
vibrational transition is usually observed in either
the infrared or Raman spectrum (but not both).
Raman spectra of sucrose
Raman spectrum of amylose
Raman microscopy
Raman spectroscopy offers several
advantages for microscopic analysis. Since
it is a scattering technique, specimens do
not need to be fixed or sectioned.
Raman spectra can be collected from a
very small volume (< 1m in diameter);
these spectra allow the identification of
species present in that volume.
Raman microscopy
Water does not interfere very strongly. Thus,
Raman spectroscopy is suitable for the
microscopic examination of minerals, materials
such as polymers and ceramics, cells and
proteins.
A Raman microscope begins with a standard
optical microscope, and adds an excitation laser,
a monochromator, and a sensitive detector (such
as a charge-coupled device (CCD) or
photomultiplier tube (PMT)). FT-Raman has also
been used with microscopes.

Raman microscopy
In direct imaging, the whole field of view
is examined for scattering over a small
range of wavenumbers (Raman shifts).
For instance, a wavenumber characteristic
for cholesterol could be used to record the
distribution of cholesterol within a cell
culture.
Raman microscopy
Another approach is hyperspectral imaging, in
which thousands of Raman spectra are acquired
from all over the field of view. The data can then
be used to generate images showing the location
and amount of different components. Taking a
cell culture example, a hyperspectral image could
show the distribution of cholesterol, as well as
proteins, nucleic acids, and fatty acids.
Sophisticated signal- and image-processing
techniques can be used to ignore the presence of
water, culture media, buffers, and other
interferences.

Raman microscopy
Raman microscopy, and in particular confocal
microscopy, has very high spatial resolution. For
example, the lateral and depth resolutions are
250 nm and 1.7 m, respectively, using a
confocal Raman microspectrometer with the
632.8 nm line from a He-Ne laser with a pinhole
of 100 m diameter.
Raman microscopy for biological and medical
specimens generally uses near-infrared (NIR)
lasers (785 nm diodes and 1064 nm Nd:YAG are
especially common). This reduces the risk of
damaging the specimen by applying high power.