——————————————————— as the path difference. This path difference

———————————————————

Fourier-Transform Infrared Radiation Spectroscopy (FTIR) is
a technique widely used to obtain an infrared spectrum or absorption of a
solid, liquid or gas. The first commercial production of an FTIR Spectrometer was
first exhibited in 1969, created by the company Digilab. This was made possible
by the invention of mini computers in 1965 with a greater computing capacity,
most notably the PDP-8. This was due to the fact they were needed to perform
the intense Fourier transform calculations necessary. This newly invented
product had come a long way from the first spectrophotometer with the ability
to record the infrared spectrum created by Perkin-Elmer in 1957.

 

The FTIR spectrometer consists of an interferometer, this is
most commonly the Michelson Interferometer. Infrared light is sent from a point
source through a beam splitter which separates the light into two beams, these
two separate beams reflect onto two different mirrors, where one is fixed and
one is movable. The light reflects and the two beams recombine at the beam
splitter to form one singular beam of light. The interferometer measures the
interference pattern produced for when two beams of light interfere with each
other.

 

When the two waves are in phase they have undergone
constructive interference and their intensity amplitude is increased. However,
when they are out of phase they undergo destructive interference and the
intensity is zero 1. The
difference in the path travelled by the two waves which have the same frequency
and velocity is known as the path difference. This path difference can be
adjusted by moving the mirror which is not fixed to different distances.

 

This light is then sent through a sample, the chemical bonds
within this sample absorb certain wavelengths of the incident infra-red light
unique to that of the sample. Not all wavelengths are absorbed and thus
transmit through the sample reaching the detector. A spectrum is therefore
created by the light incident on the detector. This spectrum has various peaks
and troughs, which are analysed by being compared to a database of known
measured elemental spectra which help to determine the samples chemical make-up.
In this spectrum there can often be carbon dioxide and water vapour included
which can cause a large room for error when analysing the spectrum.

 

If the optical path difference and the light intensity are
plotted against one another, an interferogram is produced. This is the foundation
of measurement that an FTIR obtains. The interferogram’s, which are produced
while scanning, are then Fourier Transformed by the FTIR software in order to
yield a spectrum onto a computer screen connected to the instrument. This is
where the name, FTIR, derives from as the mathematical concept known as the
Fourier Transform is used in the process to create a spectrum from the raw data
received 2. This concept is used
to calculate the superposition of sine and cosine waves for a specific
function.

 

When a sample is placed in the instrument that has an absorbance
(absorbing at a wavelength )
then the amplitude of that interferogram will decrease 3. When the number of scans is increased, this increases the
number of times the scanning (movable) mirror is moved forward and backwards. For
example if there were to be 10 scans then 10 interferograms would be produced
and an average of these would be calculated, this is known as signal averaging 4.

 

The instrument experiences background noise when conducting
measurements which can provide poor results. During the measurement, the signal
in the interferogram of the sample is always considered to be positive, however
the signal of the noise is arbitrary and fluctuates back and forth between a
positive and negative value. If one scan is taken the noise can have a very
large impact on the resulting spectrum causing poor results, however if 100
scans are taken and averaged, as previously mentioned, the noise cancels itself
out. The interferogram signal however is consistently positive and therefore
does not cancel itself out. Therefore, the greater the number of scans
performed the less background noise seen in the resulting spectrum. It is found
that the signal to noise ratio in a spectrum is proportional to the square root
of the number of scans added together 5.

 

The infrared detector has the ability to measure interferograms
as electrical signals, these have different frequencies and are displayed as
cosine waves which are presented as the spectrum of the sample. When an
interferogram is Fourier Transformed, a mathematical function is produced which
corresponds to it. This function being the spectrum of the infrared beam that
the detector picks up. As before mentioned, when the light passes through the
interferometer an interference pattern is produced due to the optical
transformation of the infrared beams spectrum. The interference pattern is then
measured by the detector and the Fourier Transform changes it back to the
spectrum. So, when a sample is placed in front of the infrared beam it alters
the interference pattern 6. When
an interferogram is Fourier transformed a single beam spectrum is produced. It
is noted that FTIR only uses a single beam of IR light as opposed to other
spectrometers which use two beams.

 

A background spectrum is obtained by taking measurements
with the FTIR spectrometer with no sample present. This is a spectrum which
includes contributions of the surrounding environment (e.g. the contributions
of water vapour and carbon dioxide) and from the instrument. This background
spectrum can be used to first determine the quantity of artifacts present, in
order to help analyse the data by subtracting these from the samples spectrum.

 

The data which FTIR collects is high-spectral-resolution
data which spans over a wide spectral range. This is a clear advantage FTIR has over a
common dispersive spectrometer which has a very narrow range of measurement for
intensity of wavelengths at a time. The range of infrared is
approximately 700nm ~ 1mm long 7. FTIR
is the third generation of Infrared spectroscopy, there are several reasons why
it has replaced the previous 2nd generation technique first
introduced in the 1960’s 8. Some
of the advantages include; high accuracy of wavenumber to an uncertainty of
±0.01,
a wide scan range (1000 ~ 10),
extremely high resolution (0.1 ~ 0.005)
and the scan time of all frequencies is short.

 

The FTIR has a major advantage over other infrared
spectrometers and that is due to the fact they can measure spectra with high
signal-to-noise ratios (SNR). The SNR is the measurement of the quality of
spectra, the more light that hits the detector the greater the amount of signal
in a spectrum. Many other instruments force light to pass through gratings and
prisms which reduces the amount of light hitting the detector and therefore
produce a weaker signal within a spectrum. However, in a FTIR spectrometer, the
beam of light does not pass through any such thing and thus has a greater
amount of light hitting the detector, giving it the ability to measure spectra
with high SNR 9.

 

It is also found that the SNR is directly proportional to
the number of scans taken added together to create a spectrum in FTIR
spectroscopy, therefore the greater number of scans taken, the greater the SNR.
This is known as the multiplex advantage. A high SNR is very advantageous as it
improves the instruments sensitivity making it easier to detect smaller peaks.
Another reason a high SNR is beneficial is quantitative accuracy, this is how
accurately peak areas and heights can be measured, as the absorbance spectra is
proportional to the concentration.

 

These SNR advantages of FTIR spectroscopy have provided an
abundance of applications that were never available using other types of
spectrometers. Different techniques of sample preparation, most notably
Attenuated Total Reflection (ATR), are now performing constructive analysis of
various samples with speed and ease. In a more medical frame, the spectra of
cancerous cells in the human body is now currently possible, due to these SNR
advantages which are being used to save lives by targeting cancer at an early
stage 10.

 

ATR is an accessory of FTIR spectrometry, it is used to
perform measurements of surface properties of thin or solid film samples directly
without further preparation, as opposed to their bulk properties. This process
tends to have a 1 – 2 micrometre penetration depth which can vary depending on
the conditions of the sample. ATR produces an evanescent wave using properties
of total internal reflection. IR light passes through a crystal and reflects
once, at the very least, off of the internal surface which is in contact with
the sample. An evanescent wave is produced in the process which penetrates the
sample. The value to which it penetrates is dependent on; the angle of
incidence, the wavelength of the light and the refraction of the ATR crystal
being used. Changing the angle of incidence can alter the amount of reflections
produced. Once the beam exits the crystal it is collected by a detector.

 

Evert instrument has imperfections and the FTIR Spectrometer’s
lie in the disadvantages of ‘artifacts’. These are features which are not from
the sample in question but are detected as part of the samples measurements.
Two very common artifacts are water vapour and carbon dioxide peaks. When using
FTIR spectroscopy the sample and background spectra must be measured at
different points in time, this means that if anything is to change within the
spectrometer between the two spectra measured, such as a change in H2O or
Carbon Dioxide concentration, then the peaks produced by this will contaminate
the spectrum of the sample 11.

 

It thus causes the need for one to use great caution in
avoiding making the mistake of interpreting atmospheric gas peaks as being from
the sample. The spectrum of the atmosphere needs to be well understood to avoid
this problem. There also must be great care when preparing the sample, all
scientists new to working in this field require time to perfect the art of the
preparation of a sample making sure no air bubbles are present to avoid spectra
contamination. Albeit this is a very common way to make mistakes and is a
disadvantage of FTIR spectroscopy, as a whole the advantages that have been
discussed far outweigh this inconvenience.

 

FTIR spectroscopy, as before mentioned, can produce information
about different samples in multiple states. This can help to identify an
unknown composition of elements within a material which can provide very
helpful in many areas. One of the most common applications of FTIR is in the
pharmaceutical industry. The strong regulatory nature of this industry and its
mass production scale require quick and effective analysis of the drugs or samples
in use, making FTIR spectroscopy an obvious choice and very beneficial. The applications
of the technique in this field ranges from drug formula characterisations to
clarification of kinetic processes in drug delivery. 12

A
paper written by H. Masmoudi titled “The evaluation of cosmetic and
pharmaceutical emulsions aging process using classical techniques and a new
method: FTIR” found that FTIR was the only technique found to be able to
characterise the evolution and chemical functions of emulsions during the
process of thermal aging. This paper also stated that whilst FTIR was
beneficial for the chemical modifications of emulsions, conductivity
measurements were necessary for the physical aspect of the emulsions and their
stability measurements. It was concluded the two techniques were complimentary
and should be used together to obtain the most accurate data 13.

Another field where FTIR plays an important role is in Forensics. Drug
enforcement agencies around the world must be able to effectively identify
drugs or illegal substances which are being brought into the country. Police
departments require the same speed of analysis when working with crime scene
evidence. One important application of FTIR within this field is in the fight
against endangered animal hunting and poaching for the reward of highly sought
after hair or fur. This can be beneficial by determining a specific animal’s
hair, which has been used for indigenous products and accessories to aid in the
attempt to save these endangered species and punish those responsible for doing
so.

A paper from 2008 titled ‘Forensic identification of elephant and giraffe hair
artifacts using HATR FTIR spectroscopy and discriminant analysis’ found success
by using horizontal-attenuated
total-reflection Fourier transform infrared spectroscopy to differentiate
between Elephant and Giraffe hair. This could prove very beneficial by using
FTIR to aid in uncovering illegal wildlife trade 14.

FTIR spectroscopy
has also proven very useful and is in fact a front running method which is
widely used in biophysics. This is a very broad field with seemingly limitless
applications for FTIR, however one of the most common is in the detection and
study of biological tissue, mainly proteins.

 

Proteins are a very
common sample used in FTIR. Whilst FTIR Spectroscopy may not provide the
same level of detail of a proteins structure as other techniques such as NMR or
X-Ray crystallography, it can be used readily to help our understanding of how these
biological components function. 15
This is due to the fact the data which
it collects has a high-spectral-resolution which spans over a wide spectral
range 16. The spectrum produced when exposing a sample to infrared
light in a FTIR spectrometer can give a large amount of information about the
protein. When exposed to Infrared Radiation, sample molecules absorb radiation
of certain wavelengths, this then changes the dipole moment of the molecule 17.
The absorption peak intensity is notably related to the change of dipole moment
and the possibility of the transition of energy levels.

 

The number of
vibrational freedoms of the molecule is directly related to the number of
absorption peaks recorded. Furthermore, the frequency of the absorption peaks
is determined by the vibrational energy gap 18. As can be seen, the
analysis of the infrared spectrum using FTIR spectroscopy can therefore provide
an abundance of structural information of the protein in question, which will
in turn provide information which can be applied to the enhancement of modern
day challenges in the medicinal industry such as drug binding.

 

A paper from 2007 titled “Fourier Transform Infrared Spectroscopic
Analysis of Protein Secondary Structures” written by J. Kong studied the
process of protein analysis by Infrared Spectroscopy and found that FTIR
spectroscopy is far more convenient to use in the process than other
techniques. This is because it can obtain IR spectra in a wide range of
environments with little sample volume. They also state the potential errors
that could arise from using the background subtraction procedure which is very
common when working with FTIR Spectroscopy. They concluded that it was
beneficial to use both CD and FTIR Spectroscopy together for more accurate
analysis 19.

 

Written by J. Zhang, “Solid-film sampling method for the
determination of protein secondary structure by Fourier transform infrared
spectroscopy” was published in 2017 and studies the ‘solid film method’ of
sample preparation which is used before the application of FTIR spectroscopy.
They found that this method renders the FTIR technique suitable for determining
the secondary structure of proteins in aqueous solutions at lower
concentrations, less than 0.5mg/mL, which is much lower than the traditional
method 20.

 

In 2016, A. C. S. Talari produced a paper named “Advances in Fourier
transform infrared (FTIR) spectroscopy of biological tissues”. In this
paper, there is a wide set of data collected of spectral peaks that are present
in FTIR Spectroscopy of biological tissues which were recorded and tabled to
aid in the study of spectral analysis to reduce time spent by scientists. The
data incorporates all varieties of biological tissue 21.

 

“Multivariate
Analysis for Fourier Transform Infrared Spectra of Complex Biological Systems
and Processes” which was
written by D. Ami and published in 2013 by InTechOpen confirmed FTIR
micro-spectroscopy to be a very useful tool as it is a quick and effective way
to provide a ‘chemical fingerprint’ of biological samples. However, the study
did highlight the problems that the method encounters such as overlapping of
main biomolecules which shows that multivariate analysis is needed if the data
is going to be successfully interpreted 22.

 

In conclusion FTIR
spectroscopy consistently remains as the main choice of spectroscopy for many
scientists when determining structural properties or elemental make up of a
solid in a gas, water or vapour state. There are many advantages to using this
technique when compared to other forms of IR spectroscopy, although there are
still issues with artifacts throughout the process, this can be disregarded as enough
of a reason to avoid using the technique once the large amount of advantages
have been taken into account, such as the high spectral resolution and the
ability attained to measure spectra with high SNR. We have also discussed
various applications of FTIR, however this only scratches the surface of fields
that it is currently being used for, not to mention the possible applications
of the future. We have already seen the advancement in recent years of FTIR, that
being the new ability to produce high resolution imaging which is imperative
for characterising tissue structures and cell types leading to accurate disease
diagnosis through ATR-FTIR. The future aim for FTIR would certainly be to
overcome the contamination of sample results by removing artifacts completely
from the instruments measurements. A modification to the instrument could
perhaps provide an elimination process for artifacts by running more intense
and thorough background checks before conducting the measurements. The future
looks bright for the application and development of FTIR regardless, it will
continue to progress and provide important information to make improvements in
many different fields.