IR Spectroscopy and FTIR Spectroscopy:

How an FTIR Spectrometer Works and

FTIR Analysis
Article Published: August 16, 2022| Simon Thain PhD

Credit: iStock

Fourier transform infrared (FTIR) spectroscopy is a hugely popular technique

today, due to its unique combination of sensitivity, flexibility, specificity and
robustness. Able to cope with solid, liquid and gaseous analytes, it has become one
of the most widely practiced analytical instrumental techniques in science.
Although there are a number of known limitations to FTIR, such as its relative
intolerance of water and its sensitivity to the physical properties of the analysis
matrix, it is nevertheless hugely popular, and commonly used right across
industries as diverse as food and beverage,1 chemical, engineering,
environmental,2 pharmaceutical3 and biomass4 and in clinical settings.5 Suitable
instrumentation forms now include both benchtop, handheld and online real-time
devices.

Contents

What is IR spectroscopy?

What is FTIR spectroscopy and what’s the difference between FTIR vs IR

spectroscopy?

How does FTIR work?

FTIR analysis and collecting FTIR data

How to interpret an IR spectrum and an FTIR spectrum

IR spectrum chart

Advantages, disadvantages and uses of mid vs near-IR/FTIR spectroscopy

FTIR applications – present and future

What is IR spectroscopy?

The human eye can only see a small part of the much broader spectrum of
electromagnetic radiation (Figure 1). On the high-energy side of the visible
spectrum lies the ultraviolet (UV) region, while on the lower energy side is the
infrared (IR). IR regions most useful for the analysis of organic compounds tend to
be 2,500 to 16,000 nm in wavelength. Far-, mid- and near-IR (NIR) are included
under the umbrella of “molecular spectroscopy”.

Figure 1: The electromagnetic spectrum, with inset indicating the sub-region

typical for infrared spectroscopy types.

IR spectroscopy is the study of the interaction of IR light with matter, where IR light
is characterized by the wavenumber range spanning 12,800 to 10 cm-1. Historically,
by convention, IR tends to be described in “wavenumber”, where any wavenumber
is inversely proportional to its wavelength. Thus, a shorter wavelength will have a
larger wavenumber, referring to the fact that more waves would be able to fit into
a given distance. Far-IR is typically defined as radiation between 500 and 20 cm-1,
mid-IR between 4,000 and 500 cm-1 and NIR as typically between ~ 10,000 and
4,000 cm-1.

Continue reading below...

Listicle

Applications of FTIR Spectroscopy

Read More

IR light is absorbed by molecules at specific frequencies based on the molecular

bonds between atoms and the types of atoms present at the end of the bonds.
Photon energies in the IR region induce vibrational excitation of covalently bonded
atoms. These covalent bonds are often considered to act like stiff springs that can
stretch, bend, rotate and scissor (Figure 2). The higher-energy mid-IR radiation
excites fundamental vibrations when energy is absorbed by molecules, raising
them from ground state to the first vibrational state. In contrast, NIR spectroscopy
is comprised of combination bands of “overtones” produced from those
fundamental vibrations. The reader is also directed to useful additional
introductory material available from the Royal Society of Chemistry.

Figure 2: Animation showing the 3-dimensional movements that can occur for
molecular atomic bonds when excited by IR light. These movements cause the IR
spectral absorbance bands we observe. Credit: Taken from YouTube
https://www.youtube.com/watch?v=0S_bt3JI150?utm_source=363938&utm_medium=pd
f&utm_campaign=pdf_lead_conversion

What is FTIR spectroscopy and what’s the difference between

FTIR vs IR spectroscopy?

The difference between IR and FTIR is that the latter is constructed from an
interferogram as the raw signal. This represents the light intensity as a function of
the position of a mirror inside the interferometer, not as a function of wavelength
(as occurs in dispersive instruments). This is the “FT“. The signal must first be
Fourier-transformed (FT) to produce the intensity as a function of wavenumber.

By convention when we speak of FTIR, we think of it as operating in the mid-IR

region. However, FT instrumentation is available for both UV and NIR spectral
forms. FTIR and FT-NIR are potentially complementary techniques, but usually the
analyst must make a choice of which to use for a particular application, so it is
worth considering their relative strengths and weaknesses.

The acquisition of FTIR spectra is much faster than by conventional dispersive

instruments. The FT method produces spectra that show a much better signal-to-
noise ratio and, as the wavelength scale is calibrated with a very precise reference
laser, provides higher wavelength accuracy than IR.

How does FTIR work?

Infrared spectrophotometers were developed in the mid-1940s. Initially, their

applications were confined mostly to research work on organic compounds, and
mainly in the petrochemical field. These first instruments were dispersive scanning
spectrophotometers (Figure 3) and slow. Dispersive instruments are still around
and have found a new lease of life in novel applications, as they can be more
readily miniaturized and manufactured much more cheaply, to produce small,
palm-top packages with simple operating systems run on mobiles phones.

Figure 3: Schematic diagram showing the layout of a dispersive IR

spectrophotometer.

Today, most research and development grade mid-IR instruments are of the FT
type. Their development can be traced back to the 1890s and the work of Albert
Michelson, who, while studying the speed of light, invented the “interferometer”,
for which he received the Nobel Prize. An FTIR instrument uses an interferometer
(Figure 4), which consists of a source, beam splitter, two mirrors, a laser and a
detector. The energy goes from the source to the beam splitter which splits the
beam into two parts. One part is transmitted to a moving mirror, the other is
reflected to a fixed mirror. The moving mirror moves back and forth at a constant
velocity, controlled by the calibrating laser’s response. The two beams are reflected
from the mirrors and recombined back at the beam splitter, generating an
interference pattern, which is transmitted though the sample compartment (and if
present the sample where absorbance occurs) to the detector. This signal is then
subjected to the FT function to generate a spectrum.

The resulting interference waveform, called an “interferogram”, (discussed later)

produced by the FT instrument encodes all the information across all the
wavelengths measured. However, to generate an interpretable spectrum, the
signal must first be subjected to a computationally intensive Fourier transform
mathematical function. In 1966 the development of the Coey-Tukey algorithm6, 7
provided a short-cut calculation, the “fast Fourier transform” or FFT. This, along
with the advent of the first commercial computing systems, allowed the launch of
the first commercial FTIR, the FTS-14, in 19698 (Figure 5).

Figure 4: Schematic diagram showing the working principle of an interferometer.

Figure 5: Schematic diagram showing the working principle of an FTIR
spectrometer. Numbers indicate the steps of FTIR analysis discussed below.

Analysis using an FTIR proceeds as follows.

1. The source: IR energy is emitted from a glowing black-body source and the
beam passes through an aperture, which controls the amount of energy.

2. The interferometer: The IR beam enters the interferometer where “spectral

encoding” takes place as already described. The interferometer uses a
reference laser for precise wavelength calibration.

3. The sample: The IR beam enters the sample compartment where it is

transmitted through or reflected off the surface of the sample; specific
frequencies of energy that are uniquely characteristic of the sample are
absorbed by the sample.

4. The detector: The beam finally passes to the detector for final measurement.
 5. The computer: The signal is digitized, the FFT calculation takes place and final
infrared spectrum is presented to the user.

FTIR analysis and collecting FTIR data

There are now a whole range of FTIR instruments and versatile interchangeable
accessories that allow gaseous, liquid and solid samples of different sizes and
forms to be analyzed by the same basic instrument. It is worth noting that normal
glass is not mid-IR transmissible, so all instrument optics and sampling accessories
have to be constructed from other suitable IR optical materials. Early techniques
developed for solid samples required the analyte to be ground and mixed with IR-
transmissible substrates, often potassium bromide (KBr), under high pressure into
a small solid clear disk. These were then mounted in a holder for transmission
measurements. Liquid (non-water containing) samples were often formed as thin
films between two such IR-transmissible discs with a small spacer. Time and
reproducibility were both issues with this method.

Over the last 30 years, there has been increasing adoption of alternatives, notably
the now ubiquitous techniques of “ATR” (attenuated total reflectance). This device
can accommodate small amounts of liquid or solid sample placed onto a crystal
window with no real sample preparation required, allowing a spectrum to be
gathered in a few seconds. When solids are analyzed, they are firmly pressed onto
the crystal window by a top fixing clamp (Figure 6). Most published applications for
solid samples now use this form of device. Other types of devices, such as a
reflective hemisphere for diffuse reflectance or gas sample sealed cells are also
available for specific applications. There are even 96-position microtiter format
plates made from gold and other IR-compatible materials, allowing for high-
throughput screening using specially adapted FTIR accessory units.9
Figure 6: Use of an ATR sampling accessory.

A typical mode of operation (Figure 7) first requires a background “blank” spectrum

to be collected. This will contain absorbance values from the entire light beam path
(optics and atmospheric). The sample is then analyzed, and the blank spectrum
subtracted from it to yield the spectral responses unique to the sample alone. Both
wavenumber resolution (typically 4 to 16 cm-1) and co-added scans (typically 8 to
64) require application-specific optimization to achieve an acceptable balance of
signal-to-noise. Individual scans are rapid, typically less than 1 second on modern
instruments, so with co-adding scans and background spectral subtraction,
analysis workflows for a single sample on an ATR device can be accomplished in
less than 2 minutes. This makes FTIR-ATR in particular ideal for measuring 100’s or
1000’s of samples in manufacturing or screening applications, including
metabolomic fingerprinting.10
Figure 7: Workflow for producing a typical spectrum.

How to interpret an IR spectrum and an FTIR spectrum

FTIR spectra are information rich, but this very fact can make using and
interpreting them challenging. A useful primer for interpreting FTIR can be found
here. Even a simple, pure, single compound sample, like vanillin (Figure 8), has a
multi-peak spectrum. In such cases, library matching approaches to an
authenticated standard may identify it in a single component mixture, but this
would be impossible in complex mixtures of other compounds. The problem is
that most organics contain combinations of carbon (C), hydrogen (H), nitrogen (N)
or oxygen (O) atoms on single or double bonds, and so the same multiple
absorption peaks overlap from compound to compound. Simply trying to “eyeball”
lots of spectra to try to judge if or how the samples they come from are different in
some way then rapidly becomes an overwhelming challenge, even for an
experienced analyst. To address this problem, FTIR data is frequently used in
combination with statistical modeling approaches, such as multivariate
analysis (MVA), which in a chemistry context is commonly termed “chemometrics”.

Figure 8: FTIR spectrum of vanillin, with major peak wavenumbers indicated.

Wavenumber (cm-1) is shown on the X axis against absorbance on the Y axis. Credit:
Author.

FTIR spectral data is very amenable to MVA techniques, which at their heart simply
need multiple spectra to be collected from each sample and constructed into a
single data matrix. Here, each table row is an individual sample’s complete
spectrum, and each column is the aligned absorbance for particular consecutive
wavenumbers across all the samples. In this form, techniques like principal
components analysis (PCA) can be applied to explore and visualize possible class-
based relationships between different sample group’s spectral responses
efficiently via their scores plots. This gives an immediate “interpretability” of
sample differences, which is very difficult to gauge from simply over-laying spectra
from different sample classes. An example of a PCA scores plot is shown in Figure
9 for raw vs processed bio-oil sample differentiations.
Figure 9: Bio oil samples showing raw (red) compared to processed (green)
material. The FTIR spectrum (left) and the derived PCA scores plot (right) are
shown. Credit: Author.

When quantitation is the goal (e.g., concentration values), MVA approaches, such
as partial least-squares regression, can be used to build quantitative value-based
calibration predictions for properties such as chemical concentrations, using data
previously gathered on each sample’s composition from other assay techniques.
These known values are then fed into the algorithmic calculation to identify
spectral features that best correlate with the external value of interest. This latter
approach is very popular as, when properly planned and validated, it can allow
FTIR to effectively replace wet chemistry assays for new unknown samples (of the
same types), saving time and money. One useful consequence of constructing MVA
models is that the statistical output table produced, in addition to the sample
plots, allows the most important wavelength used in building the model to be
identified. From this information it is often possible to interpret some direct
chemical insights.

FTIR is virtually always assumed to be "mid"-FTIR. Non-FT mid-IR dispersive

instruments can't produce this broad spectrum as the scan rate is slow and its
power (signal-to-noise) is so much poorer. However, NIR has so much more energy
that a non-FT dispersive instrument can produce similar spectra to mid-FTIR
instruments. But it would take longer and so the resolution (number of actual
wavenumbers measured) tends to be lower.

IR spectrum chart

The chart below (Figure 10) shows the bands produced by the major functional
groups (1500 cm-1 and above). The region 500-1500 cm-1, which is in the mid-IR
region, is called the fingerprint region and provides molecular fingerprints unique
to specific compounds that cannot be faked.

Figure 10: Chart showing the IR bands produced by the major functional groups
and the fingerprint region specific to each compound.
Advantages, disadvantages and uses of mid vs near-IR/FTIR
spectroscopy

The shape and structure of mid-IR vs NIR spectra are quite different, with mid-IR
spectra containing sharper and more defined spectral absorption bands (Figure
11) for organic species, which thus lend the technique to structural elucidation and
compound identification. Also, detailed data tables of characteristic molecular
function group wavenumber regions have been collated and published over the
years, many within specific application areas. Organic molecules strongly absorb
mid-IR radiation, so good spectra can be obtained from relatively little sample
material (e.g., a few powder grains). Disadvantages include its intolerance of water
(which quenches the IR signal even when present at just a few percent). In
addition, the very fact that organics generally absorb mid-IR so well means that the
resulting spectrum will only be from a few microns of sample penetration and
represent limited homogeny. Therefore, considerably more careful sample
preparation or analytical replication is needed.

NIR’s advantages include a strong response to both chemical and physical

attributes of the sample (e.g., making it useful for overall sample grading
applications). NIR radiation achieves much more sample penetration, as it is only
weakly absorbed, so increased sampling volume may increase sensitivity, gain
better homogeny and require far less sample preparation for a measurement to
be performed. The major disadvantages of NIR are associated with chemical
specificity. Most NIR molecular responses are first order (or higher) overtones,
which exhibit degrees of signal overlap, potentially limiting discriminatory power.
However, what are considered advantage vs disadvantage is entirely application
specific, so both mid-IR and NIR instruments are widely used for different
applications.
Figure 11: Example of typical NIR (left) vs mid-IR (right) spectra. Note that
additional spectral features are resolved in mid-IR. Samples were dried mosquitos,
aiming to identify vertebrate blood meals in the malaria vector, Anopheles
arabiensis. Credit: Reproduced from Mwanga, E.P., Mapua, S.A., Siria, D.J. et al. 2019
under the Creative Commons Attribution 4.0 International License.

FTIR applications – present and future

FTIR sits in quite a unique “sweet spot” in terms of equipment costs, ease of use
and the information it can produce. Its flexibility has seen it applied to more areas
than there is room to discuss in this article. A range of pharmaceutical and medical
applications are becoming increasingly common.11

In addition, a particularly interesting technical development in the last 20 years has

been the appearance and evolution of chemical imaging based in FTIR video chips
(focal place array, “FPA”) for microscopy use. While FTIR microscopes have been
around since the 1970’s in standard format, they just use a single point IR detector,
and any imaging can only be achieved by stitching together large numbers of the
single spatial measurements. Such images, even of something small like a cut
tissue piece of a cm-2, would take hours to collect. Modern mid-infrared imaging
chips, such as a typically sized 128 x 128 pixel arrays (but can be higher resolution),
have now become standard. A 128x128 array produces 16,000 spatially resolved
spectra in one scan. Images can be acquired in under 30-60 second, and have
become used widely This makes spatially resolved applications such, forensics,
archeological artifacts, physical contaminants such as microplastics,
pharmaceutical pressed tablet testing12 and tissue biopsy screening for disease
state and diagnostic prediction possible.13, 14 FTIR-FPA can be usefully applied to
situations where chemical signals need to interpreted within a broad spatial
context.15, 16

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