Fourier transform infrared

spectroscopy (FTIR)

 Fourier transform infrared spectroscopy (FTIR) is a

technique which is used to obtain
an infrared spectrum of absorption, emission, photoco
nductivity or Raman scattering of a solid, liquid or gas.
An FTIR spectrometer simultaneously collects spectral
data in a wide spectral range. This confers a significant
advantage over a dispersive spectrometer which
measures intensity over a narrow range of wavelengths
at a time.
The term Fourier transform infrared
spectroscopy originates from the fact that a Fourier
transform (a mathematical process) is required to
convert the raw data into the actual spectrum

 The goal of any absorption

spectroscopy (FTIR, ultraviolet-visible ("UV-Vis")
spectroscopy, etc.) is to measure how well a sample
absorbs light at each wavelength.
The most straightforward way to do this, the
"dispersive spectroscopy" technique, is to shine
a monochromatic light beam at a sample, measure
how much of the light is absorbed, and repeat for
each different wavelength.
This is how UV-Vis spectrometers work, for example.

 Fourier transform spectroscopy is a less spontaneous

way to obtain the same information. Rather than
shining a monochromatic beam of light at the
sample, this technique shines a beam containing
many frequencies of light at once, and measures how
much of that beam is absorbed by the sample.
Next, the beam is modified to contain a different
combination of frequencies, giving a second data
point. This process is repeated many times.
Afterwards, a computer takes all these data and
works backwards to infer what the absorption is at
each wavelength.

 The beam described above is generated by starting

with a broadband light sourceone containing the
full spectrum of wavelengths to be measured.
The light shines into a Michelson interferometera
certain configuration of mirrors, one of which is
moved by a motor. As this mirror moves, each
wavelength of light in the beam is periodically
blocked, transmitted, blocked, transmitted, by the
interferometer, due to wave interference. Different
wavelengths are modulated at different rates, so that
at each moment, the beam coming out of the
interferometer has a different spectrum.

 As mentioned, computer processing is

required to turn the raw data (light absorption
for each mirror position) into the desired
result (light absorption for each wavelength).
The processing required turns out to be a
common algorithm called the Fourier
transform (hence the name, "Fourier
transform spectroscopy").
The raw data is sometimes called an
"interferogram"

 Fourier Transform Infrared (FT-IR) spectrometry was

developed in order to overcome the limitations
encountered with dispersive instruments.
The main difficulty was the slow scanning process. A
method for measuring all of the infrared frequencies
simultaneously, rather than individually, was needed.
A solution was developed which employed a very
simple optical device called an interferometer.
The interferometer produces a unique type of signal
which has all of the infrared frequencies encoded
into it.
The signal can be measured very quickly, usually on the
order of one second or so. Thus, the time element per
sample is reduced to a matter of a few seconds rather
than several minutes.

 Most interferometers employ a beam splitter which

takes the incoming infrared beam and divides it into
two optical beams.
One beam reflects off of a flat mirror which is fixed in
place. The other beam reflects off of a flat mirror
which is on a mechanism which allows this mirror to
move a very short distance (typically a few millimeters)
away from the beamsplitter.
The two beams reflect off of their respective mirrors
and are recombined when they meet back at the
beamsplitter. Because the path that one beam travels is
a fixed length and the other is constantly changing as
its mirror moves, the signal which exits the
interferometer is the result of these two beams
interfering with each other.

 The resulting signal is called an interferogram

which has the unique property that every
data point (a function of the moving mirror
position) which makes up the signal has
information about every infrared frequency
which comes from the source
This means that as the interferogram is
measured, all frequencies are being measured
simultaneously. Thus, the use of the
interferometer results in extremely fast
measurements

 Because the analyst requires a frequency

spectrum (a plot of the intensity at each
individual frequency) in order to make an
identification, the measured interferogram signal
can not be interpreted directly.
A means of decoding the individual frequencies
is required. This can be accomplished via a wellknown mathematical technique called the
Fourier transformation.
This transformation is performed by the
computer which then presents the user with the
desired spectral information for analysis

The Sample Analysis Process

1. The Source: Infrared energy is emitted from a
glowing black-body source. This beam passes through
an aperture which controls the amount of energy
presented to the sample (and, ultimately, to the
detector).
2. The Interferometer: The beam enters the
interferometer where the spectral encoding takes
place. The resulting interferogram signal then exits the
interferometer.
3. The Sample: The beam enters the sample
compartment where it is transmitted through or
reflected off of the surface of the sample, depending
on the type of analysis being accomplished. This is
where specific frequencies of energy, which are
uniquely characteristic of the sample, are absorbed

The Sample Analysis Process

4. The Detector: The beam finally passes to
the detector for final measurement. The
detectors used are specially designed to
measure the special interferogram signal.
5. The Computer: The measured signal is
digitized and sent to the computer where the
Fourier transformation takes place. The final
infrared spectrum is then presented to the
user for interpretation and any further
manipulation.

 Because there needs to be a relative scale for the

absorption intensity, a background spectrum
must also be measured. This is normally a
measurement with no sample in the beam. This
can be compared to the measurement with the
sample in the beam to determine the percent
transmittance.
This technique results in a spectrum which has all
of the instrumental characteristics removed.
Thus, all spectral features which are present are
strictly due to the sample. A single background
measurement can be used for many sample
measurements because this spectrum is
characteristic of the instrument itself.

Why does absorption occur?

When can absorption occur ?

 As the name suggests,

dispersive spectrometers generate spectra by
optically dispersing the incoming radiation
into its frequency or spectral components.
Common dispersive elements include prisms
and gratings
An FTIR spectrometer simultaneously collects
spectral data in a wide spectral range. This
confers a significant advantage over
a dispersive spectrometer which measures
intensity over a narrow range of wavelengths
at a time

Attenuated Total Reflectance (ATR) cell -

ATR products successfully replace constant

path transmission cells and salt plates used for
analysis of liquid and semi-liquid materials.
ATR is well suited for both qualitative and
quantitative applications.
Several heating options are available

mercury cadmium telluride (MCT)

Most mid-infrared (mid-IR) analyses are performed
with the standard deuterated L-alanine doped
triglycine sulfate (DLaTGS) detector due to its ease of
use, high sensitivity, and excellent linearity.
When sample measurements must be made at high
speed or when IR throughput is low, the highly
sensitive mercury cadmium telluride (MCT) detector
provides the ability to scan faster than an DLaTGS
detector while maintaining a constant IR response.
The response of the DLaTGS detector is reduced by
half for every two-fold multiple of the scanning
velocity. Scanning faster with an MCT detector
reduces sampling time without affecting sensitivity.

This is a spectrometer

To begin using the spectrometer, turn the PC on.

The Spectrometer is NEVER turned off. You should see a green LED,
and a blinking yellow LED, indicating that the spectrometer is scanning.
The spectrometer contains moisture-sensitive components,
that are kept warm by the infrared source, so it is always left on.

Advantages of FT-IR
Some of the major advantages of FT-IR over the
dispersive technique include:
Speed: Because all of the frequencies are measured
simultaneously, most measurements by FT-IR are made
in a matter of seconds rather than several minutes.
This is sometimes referred to as the Felgett Advantage.
Sensitivity: Sensitivity is dramatically improved with
FT-IR for many reasons. The detectors employed are
much more sensitive, the optical throughput is much
higher (referred to as the Jacquinot Advantage) which
results in much lower noise levels, and the fast scans
enable the coaddition of several scans in order to
reduce the random measurement noise to any desired
level (referred to as signal averaging).

Advantages of FT-IR
Mechanical Simplicity: The moving mirror in
the interferometer is the only continuously
moving part in the instrument. Thus, there is
very little possibility of mechanical
breakdown.
Internally Calibrated: These instruments
employ a HeNe laser as an internal
wavelength calibration standard (referred to
as the Connes Advantage). These instruments
are self-calibrating and never need to be
calibrated by the user.

SUMMARY

 These advantages, along with several others, make measurements

made by FT-IR extremely accurate and reproducible.
Thus, it is a very reliable technique for positive identification of
virtually any sample. The sensitivity benefits enable identification
of even the smallest of contaminants.
This makes FT-IR an invaluable tool for quality control or quality
assurance applications whether it be batch-to-batch comparisons
to quality standards or analysis of an unknown contaminant.
In addition, the sensitivity and accuracy of FT-IR detectors, along
with a wide variety of software algorithms, have dramatically
increased the practical use of infrared for quantitative analysis.
Quantitative methods can be easily developed and calibrated and
can be incorporated into simple procedures for routine analysis

 Thus, the Fourier Transform Infrared (FT-IR)

technique has brought significant practical
advantages to infrared spectroscopy.
It has made possible the development of
many new sampling techniques which were
designed to tackle challenging problems which
were impossible by older technology.
It has made the use of infrared analysis
virtually limitless

 The use of Fourier Transform Infrared spectroscopy (FTIR) to

determine the structure of biological macromolecules has
dramatically expanded.
FTIR spectroscopy requires only small amounts of proteins
(1mM) in a variety of environments.
Therefore, high quality spectra can be obtained relatively
easy without problems of background fluorescence, light
scattering and problems related to the size of the proteins.
The omnipresent water absorption can be subtracted by
mathematical approaches.
Methods are now available that can separate
subcomponents that overlap in the spectra of proteins.
These facts have made practical biological
systems amenable to studies by FTIR spectroscopy.

Basic principles of infrared (IR) absorption

IR spectroscopy is the measurement of

the wavelength and intensity of the
absorption of infrared light by a sample.
Infrared light is energetic enough to
excite molecular vibrations to higher
energy levels.

Fourier Transform Infrared (FTIR) spectroscopy

To use the Fourier Transform Infrared
Spectroscopy, a continuum source of light
(such as a Nernst Globar) is used to produce
light over a broad range of infrared
wavelengths.
Light coming from this continuum source is
split into two paths using a half-silvered
mirror; this light is then reflected from two
mirrors back onto the beam splitter, where it
is recombined.

Fourier Transform Infrared (FTIR) spectroscopy

One of these mirrors is fixed, and the second
is movable.
If the distance from the beamsplitter to the
fixed mirror is not exactly the same as the
distance from the beamsplitter to the second
mirror, then when the two beams are
recombined, there will be a small difference in
the phase of the light between these two
paths.

Fourier Transform Infrared (FTIR) spectroscopy

Because of the "superposition principle"
constructive and destructive interference exist
for different wavelengths depending on the
relative distances of the two mirrors from the
beamsplitter.
It can be shown that if the intensity of light is
measured and plotted as a function of the
position of the movable mirror, the resultant
graph is the Fourier Transform of the intensity
of light as a function of wavenumber

Fourier Transform Infrared (FTIR) spectroscopy

In FTIR spectroscopy , the light is directed
onto the sample of interest, and the intensity
is measured using an infrared detector.
The intensity of light striking the detector is
measured as a function of the mirror position,
and this is then Fourier-transformed to
produce a plot of intensity vs. wavenumber.
As radiation source a Michelson
Interferometer is used (see the drawing
below).

 It is necessary to increase the sensitivity

somehow, because the absorption due to one
monolayer of molecules typically results in a
change in intensity of only about one part in 105.
For semiconductors, one way of increasing the
sensitivity is to use multiple internal reflection.
In this technique, the edges of the sample are
polished, and the light is sent in at an angle. The
light bounces around inside the sample, making
about 30-50 bounces. This increases the
sensitivity by about a factor of 30-50, making it
possible to measure the absorption of less than
one monolayer of molecules on a surface

Band assignments
Amide vibrations
The peptide group, the structural repeat unit of proteins, gives up
to 9 characteristic bands named amide A, B, I, II ... VII.
The amide A band (about 3500 cm-1) and amide B (about 3100 cm1) originate from a Fermi resonance between the first overtone of
amide II and and the N-H stretching vibration.
Amide I and amide II bands are two major bands of the protein
infrared spectrum.
The amide I band (between 1600 and 1700 cm-1) is mainly
associated with the C=O stretching vibration (70-85%) and is
directly related to the backbone conformation.
Amide II results from the N-H bending vibration (40-60%) and from
the C-N stretching vibration (18-40%). This band is conformationally
sensitive.
Amide III and IV are very complex bands resulting from a mixture of
several coordinate displacements.
The out-of-plane motions are found in amide V, VI andVII

 Amide A is with more than 95% due to the the N-H stretching
vibration. This mode of vibration does not depend on the backbone
conformation but is very sensitive to the strength of a hydrogen bond.
It has wavenumbers between 3225 and 3280 cm-1 for hydrogen bond
lengths between 2.69 to 2.85
Amide I is the most intense absorption band in proteins. It is primilary
governed by the stretching vibrations of the C=O (70-85%) and C-N
groups (10-20%). Its frequency is found in the range between 1600 and
1700 cm-1. The exact band position is determined by the backbone
conformation and the hydrogen bonding pattern.
Amide II is found in the 1510 and 1580 cm-1 region and it is more
complex than amide I. Amide II derives mainly from in-plane N-H
bending (40-60% of the potential energy). The rest of the potential
energy arises from the C-N (18-40%) and the C-C (about 10%)
stretching vibrations.
Amide III, V are very complex bands dependent on the details of the
force field, the nature of side chains and hydrogen bonding. Therefore
these bands are only of limited use for the extraction of structural
information.

CONCLUSION
FTIR is a chemically-specific
analysis technique
It can be used to identify
chemical compounds,
and substituent groups