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170 4 Comprehensive Insights into Infrared Spectroscopy
Fig. 4.4 IR spectra of primary, secondary, and tertiary amines. The IR spectra for primary
(aniline), secondary (diethylamine), and tertiary (triethylamine) amines exhibit distinct peaks that
help differentiate between these amine types. In primary amines (top spectrum), two N-H stretching
bands are observed at 3442 cm
stretching, along with a scissoring (N-H bend) vibration around 1619 cm
(middle spectrum) show a single N-H stretching band near 3288 cm
spectrum) lack N-H stretching bands due to the absence of N-H bonds. C-N stretching vibrations are
observed for all amines in the range of 1200–1350 cm
nature of the compound. (Adapted from [
-1
and 3360 cm
-1
, corresponding to symmetric and asymmetric
-1
, depending on the aliphatic or aromatic
-1
-1
. Secondary amines
, and tertiary amines (bottom
https://www.orgchemboulder.com/Spectroscopy/irtutor/
aminesir.shtml])
4.7 Differentiating Between Amide I, Amide II, and Amide III Bands 171
Fig. 4.5 IR spectral representation of amide I, II, and III bands: The IR absorption spectrum
demonstrates the characteristic Amide I, II, and III bands corresponding to the vibrational modes in
peptide bonds. The Amide I band (1600–1700 cm
stretching vibration and is highly sensitive to the protein’s secondary structure, such as α-helices
or β-sheets. The Amide II band (1500–1600 cm
bending and C-N stretching vibrations, providing further insight into the peptide backbone structure. The Amide III band (1300–1400 cm
involving C-N stretching and N-H in-plane bending, typically used to probe structural details in
proteins. The inset highlights the molecular structure of peptide bonds and the vibrational modes
contributing to each respective amide band. (Adopted from [Mallamace F, Corsaro C,
Mallamace D, Vasi S, Vasi C, Dugo G. The role of water in protein’s behavior: The two dynamical
crossovers studied by NMR and FTIR techniques. Comput Struct Biotechnol J. 2014;13:33–7
(License: CC BY 4.0)])
-1
-1
, red peak) is primarily due to the C=O
-1
, blue peak) arises from a combination of N-H
, green peak) is associated with complex vibrations
1600–1700 cm
-1
(Fig. 4.10). Its precise position depends on factors such as
hydrogen bonding, protein secondary structure, and the molecular environment.
This band is crucial for studying protein secondary structures, with α-helices,
β-sheets, and random coils displaying slightly different amide I peaks, making it
an important tool for protein structural analysis.
• Amide II band:
The amide II band results mainly from a combination of N-H
bending and C-N stretching vibrations, typically found in the 1500–1600 cm
region (Fig. 4.10). The amide II band provides complementary information about
the peptide backbone and can be influenced by the protein’s secondary structure
and its hydrogen bonding environment. This band is also useful for probing the
peptide bond interactions in complex biological molecules.
-1
172 4 Comprehensive Insights into Infrared Spectroscopy
• Amide III band: The amide III band arises from a combination of C-N stretching
and N-H bending, typically observed in the 1200–1350 cm
-1
range (Fig. 4.10).
Although weaker compared to amide I and II, this band provides additional
insights into the conformational aspects of proteins, particularly in relation to
secondary structures. It is often used in conjunction with Amide I and II bands for
a more detailed analysis of protein folding and interactions.
Together, these three bands (amide I, II, and III) serve as key mark ers in IR
spectroscopy for analyzing the structure, dynamics, and interactions of proteins and
peptides. Their unique positions and sensitivities to molecular environments make
them invaluable tools in the study of biological systems.
4.8 Components of IR Spectrophotometer
An IR spectrophotometer is a specialized analytical instrument used to measure the
absorption, transmission, and reflection of infrared light as it interacts with a sample.
The key components of an IR spectrophotometer typically include:
Radiation Source IR spectroscopy employs various types of radiation sources to
generate the infrared radiation necessary for the analysis of chemical compounds.
The choice of radiation source depends on the specific needs of the analysis and the
range of wavelengths required. Radiation source should have the following
properties:
1. Intensity of radiation should be continuous over the λ range and covers a wide λ
range.
2. The intensity of radiation should be constant over long periods of time.
3. Source should have the normal operating temperatures, that is, between 1100 and
1500 K.
4. Sources should have maximum intensity between 4000 and 400 cm
-1
.
5. Sources should be enclosed in an insulator to reduce noise.
Common radiation sources used in IR spectroscopy include:
Mid-IR Sources Mid-IR sources are essential components in IR spectroscopy
instruments, particularly for analyzing molecules in the mid-infrared region, which
ranges from approximately 2.5 to 25 micrometers (μm) or 4000 to 400 cm
-1
. Mid-IR
sources generate radiation in this specific range. Here are some common Mid-IR
sources used in IR spectroscopy:
• Globar source: A
Globar is a silicon carbide (SiC) rod that serves as a versatile
and widely used mid-IR source. It emits a broadband spectrum of IR radiation
when heated. Globar sources cover a broad range of Mid-IR wavelengths, making
them suitable for many routine spectroscopy applications.
4.8 Components of IR Spectrophotometer 173
• Nernst glower: The Nernst glower is a ceramic rod typically made of rare-earth
oxides, which emits mid-IR radiation when h eated. It offers stability and reproducibility and is often used in high-temperature applications.
• Blackbody source: A blackb ody source is a heated cavity that emits
radiation across
a wide range of wavelengths. The temperature of the blackbody
thermal
source determines the specific Mid-IR wavelengths it emits. They are versatile
sources, and their emission can be tailored for specific applications.
• Quantum cascade lasers (QCLs): QCLs are semiconductor lasers designed to
emit mid-IR radiation at specific, tunable wavelengths. These lasers are used in
applications requiring high sensitivity and precision, such as trace gas analysis
and environmental monitoring.
• Synchrotron light sources: Synchrotron facilities produce intense, tunable mid-IR
radiation by accelerating electrons to emit synchrotron light. These sources are
suitable for advanced research and materials science applications, where highintensity, narrow bandwidth, and tunable radiation are required.
• Interband cascade lasers (ICLs): ICLs are another type of semiconductor laser
that emits mid-IR radiation. They are particularly useful for high-performance gas
sensing and spectroscopy applications.
The choice of mid-IR source depends on the specific requirements of the analysis,
including the desired wavelength range, sensitivity, and application demands.
Globar and Nernst glower sources are commonl y used in routine IR spectroscopy
applications due to their versatility and stability. However, QCLs and synchrotron
sources are employed when higher precision, narrow bandwidth, and specific wavelength coverage are necessary.
Near-IR Sources NIR sources are crucial components in near-infrared spectroscopy instruments, which analyze molecules and materials in the near-infrared region
of the electromagnetic spectrum. The NIR region typically covers the wavelength
range from about 780 to 2500 nm. Here are some common Near-IR sources used in
NIR spectroscopy:
• Tungsten-halogen lamp
: Tungsten
-halogen lamps are widely used Near-IR
sources. These lamps produce a continuous spectrum of radiation in the NIR
region and are relatively inexpensive. They are suitable for a wide range of
routine NIR spectroscopy applications.
• Deuterium-haloge
p: A deuterium-halogen lamp combines a deuterium lamp
n lam
and a tungsten-halogen lamp. The deuterium lamp emits light in the UV region,
while the tungsten-halogen lamp provides NIR radiation. This dual-source setup
is common in UV-visible-NIR spectrophotometers.
• Super-luminescent diodes (SLEDs): SLEDs are semiconductor light sources that
emit broad-spectrum, white-light-like radiation. They can be tailored to emit in
the NIR range and are often used in optical coherence tomography (OCT) and
other high-resolution NIR spectroscopy applications.
174 4 Comprehensive Insights into Infrared Spectroscopy
• Light emitting diodes (LEDs): LEDs can be designed to emit specific wavelengths
in the NIR region. While they may not provide a broad spectrum, they are useful
for targeted applications, such as remote sensing, medical diagnostics, and optical
communications.
• Diode lasers: Diode lasers can be engineered to emit in the NIR range. They offer
high coherence and wavelength precision, making them suitable for applications
like fiber optic communications, environmental monitoring, and gas sensing.
• Synchrotron light sources: Synchrotrons can produce intense NIR radiation by
accelerating electrons to emit synchrotron light. These sources are valuable for
advanced research and materials science applications that require high-intensity,
tunable, and narrow bandwidth radiation.
The choice of a near-IR source depends on the specific requirements of the
analysis, including the desired wavelength range, spectral characteristics, and application demands. Tungsten-halogen lamps are common in routine NIR spectroscopy,
but other sources, such as SLEDs and diode lasers, are used in specialized
applications requiring greater precision and specific wavelengths.
Far-IR Sources Far-IR sources are essential components in far-IR spectroscopy
instruments, which analyze materials in the far-IR region of the electromagnetic
spectrum. The far-IR region typically covers wavelengths greater than 25 μm. Far-IR
spectroscopy is particularly useful for studying low-energy molecular vibrations and
intermolecular interactions. Common sources used in far-IR spectroscopy include:
• Globar source: Globar can also serve as a
source for
far-IR radiation. When
heated, it emits broadband radiation that includes the far-IR region. Globar
sources are versatile and can be used in various spectroscopy applications.
• Nernst glower: Similar to the mid-IR, Nernst glowers can be used as a source of
far-IR radiation when heated. They are known for their stability and are employed
in high-temperature applications.
• Free electron lasers (FELs): FELs are powerful sources of coherent and tunable
far-IR radiation. They produce intense radiation over a wide range of
wavelengths, making them valuable for research in physics, chemistry, and
materials science.
• Synchrotron light sources: Synchrotron facilities are capable of producing intense
and tunable far-IR radiation by accelerating electrons to emit synchrotron light.
These sources are often used in advanced research and materials science
applications.
• Gas l
asers: C
ertain gas lasers, such as the CO
the far-IR region. CO
lasers are often employed in spectroscopy for their
2
laser, can be configured to emit in
2
wavelength range and tunability.
The choice
of far-IR source depends on the specific requirements of the analysis,
including the desired wavelength range, sensitivity, and application demands. While
Globar and Nernst glower sources are commonly used in many far-IR spectroscopy
4.8 Components of IR Spectrophotometer 175
applications due to their stability, FELs, synchrotron sources, and specialized gas
lasers are chosen for advanced research and precise control over the radiation
characteristics.
Laser Sources for IR Radiation Lasers are powerful sources of IR radiation used
in various IR spectroscopy techniques. They offer advantages such as high coherence, tunability, and intense radiation, making them well-suited for specific spectroscopic applications. Here are some common types of laser sources for IR radiation in
IR spectroscopy:
• CO
lasers: CO2 lasers are widely used for their strong emission in the mid-IR
2
region, particularly in the 9.4–10.8 μm range. They are employed in many IR
spectroscopy applications, including FT-IR (Fourier-transform infrared)
spectroscopy.
• Quantum cascade lasers (QCLs): QCLs are semiconductor lasers designed for
precise tunability in the mid-IR and far-IR regions. They offer high resolution and
are suitable for applications that require the analysis of specific chemical
compounds or trace gases.
• Diode lasers: Diode lasers are compact and can be engineered to emit at specific
IR wavelengths, including those in the near-IR and mid-IR ranges. They are used
in applications such as gas sensing and fiber optic spectroscopy.
• Dye lasers: Dye lasers can be tuned to specific wavelengths within the near-IR
and mid-IR regions. They are often employed in resear ch and specialized spectroscopy experiments where tunability is critical.
• Free electron lasers (FELs): FELs are powerful sources that provide coherent and
intense radiation across a wide range of IR wavelengths, from the near-IR to the
far-IR regions. The y are employed in advanced research applications.
• Solid-state lasers: Some solid-state lasers, such as erbium-doped fiber lasers, can
be used to generate IR radiation. These lasers are employed in
telecommunications and materials processing.
• Optical parametric oscillator (OPO): OPOs are nonlinear optical devices that can
produce tunable IR radiation in the near-IR and mid-IR regions. They are used for
high-resolution spectroscopy and imaging.
The choice of IR laser source depends on the specific wavelength range required
for the analysis and the characteristics of the sample. Lasers are particularly valuable
for applications where precise wavelength control and high intensity are essential,
such as in trace gas analysis, chemical identification, and materials characterization.
4.8.1 Sample Cell
It holds the sample and allows monochromatic light to pass through it for the
measurement of IR spectra. The samples to be analyzed may be in solid, liquid, or
gaseous forms. It is typically made of alkali halides, such as NaCl or KBr. These
176 4 Comprehensive Insights into Infrared Spectroscopy
halides are soluble in water; therefore, aqueous solvents cannot be used for analysis
because they dissolve the sample cell if it is composed of alkali halides. Only organic
solvents, such as CS
and CCl4, are used for analytical purposes.
2
4.8.2 Monochromator
The monochromator is used to separate the polychromatic radiation into its component wavelengths, either by using prisms (such as metal halide or NaCl prisms),
gratings, or both. The quality of mirrors and the width of the slit determine the
resolution of the IR spectrum. A rock salt prism, made of metal with a polished front
surface, is typically used in the range of 650–4000 cm
Beam Splitter The beam splitter divides the incoming IR beam into two beams:
one that interacts with the sample and another that serves as a reference. Common
types of beam splitters include KBr or CaF₂ plates.
Detectors IR spectroscopy utilizes various types of detectors to measure the intensity of IR radiation after it interacts with a sample. The choice of detector depends on
the specific wavelength range, sensitivity, and application requirements. Here are
some common types of detectors used in IR spectroscopy:
-1
.
• Thermocouples: These detectors work based on the principle that
the absorp
tion
of IR radiation causes a change in temperature in the detector material, leading to
a change in electrical voltage. Thermocouples are suitable for the mid-IR range.
• Bolometers: Bolometers are highly sensitive detectors that measure changes in
electrical resistance due to temperature variations caused by IR radiation absorption. They are often used in the far-IR and terahertz regions.
• Photovoltaic (PV) detectors: PV detectors, such as photodiodes and
phototransistors, convert absorbed IR radiation directly into an electrical current.
They are commonly used in the near-IR and mid-IR regions and offer high
sensitivity and speed.
• Pyroelectric detectors: Pyroelectric detectors measure changes in polarization in
a crystal due to temperature changes caused by IR radiation absorption. They are
often used in the mid-IR region and offer good sensitivity.
• Deuterated triglycine sulfate (DTGS) detectors: DTGS detectors are widely used
in mid-IR spectrophotometers. They offer good sensitivity and cover a broad
range of IR wavelengths.
• Mercury cadmium telluride (MCT) detectors: MCT detectors are highly sensitive
and cover a wide range of IR wavelengths, including the mid-IR and far-IR
regions. They are commonly used in high-performance IR spectrometers.
• Infrared arrays: Moder
n IR spectrophotometers may use infrared arrays, which
are solid-state detectors composed of multiple detector elements. Infrared arrays
provide rapid data acquisition and are suitable for various IR regions.
4.9 Sampling Techniques for IR Spectroscopy 177
• Fourier-transform infrared (FT-IR) detectors: FT-IR spectrometers use detectors
such as photodiodes, liquid nitrogen-cooled MCT detectors, or specialized
detectors like helium-cooled mercury-cadmium-telluride (HgCdTe) detectors.
These detectors are used for FT-IR measurements.
The choice of detector depends on the specific application and the wavelength
range of interest. MCT detectors are often used for high-performance and researchgrade instruments, while photovoltaic detectors, pyroelectric detectors, and DTGS
detectors are suitable for routine applications in the mid-IR and near-IR regions.
Bolometers and thermocouples are used in the far-IR and terahertz regions, where
extreme sensitivity is required.
Data Acquisition System The data acquisition system collects, processes, and
converts the detector’s signal into a digital format that can be used for spectral
analysis. This system includes analog-to-digital converters and computer interfaces.
Display/Output Device The results of the analysis, typically displayed as an IR
spectrum, are shown on a screen or output to a computer for further analysis and
interpretation.
Software Specialized software is used for controlling the instrument, collecting and
processing data, and generating IR spectra.
Alignment Optics Precise alignment optics are crucial to ensure that the IR beam is
properly directed through the sample and to the detector.
4.9 Sampling Techniques for IR Spectroscopy
In IR spectroscopy, samples for analysis can be in the form of solids, liquids, or
gases. The choice of the appropriate sample and sampling technique is crucial, as an
unsuitable sample can significantly impact the accuracy of the analysis. Therefore,
various sampling techniques are employed, depending on the nature of the sample.
These techniques include:
4.9.1 Solid Samples
When dealing with solid samples, various techniques can be employed to prepare
them for IR spectroscopy:
4.9.1.1 Mulling
In this technique, the solid sample is ground to a fine powder (Fig. 4.6a). A few drops
of a viscous liquid, such as Nujol, are added to create a thick slurry of the sample.
This slurry is then pressed between salt plates to form a thin film.
178 4 Comprehensive Insights into Infrared Spectroscopy
Fig. 4.6 Techniques for solid sample preparation in IR spectroscopy: This illustration presents two
common methods for preparing solid samples for IR spectroscopy. In (a), the mulling technique is
depicted, where the solid sample is ground into a fine powder and mixed with a mulling agent,
typically an oil, to form a homogeneous paste. The mull is spread between two salt plates, which are
transparent to IR radiation, allowing for accurate spectral analysis. In (b), the pelleting technique is
shown, where the solid sample is mixed with a transparent matrix material such as potassium
bromide (KBr) and pressed into a thin, transparent pellet using a hydraulic press. The pellet is then
placed in the IR spectrometer for analysis. Both techniques are essential for ensuring the solid
sample is in a suitable state for optimal IR absorption and accurate spectral data collection
4.9.1.2 Pelleting
Pelleting is a vital technique in IR spectroscopy for solid sample analysis. It involves
compressing finely ground samples into pellets or disks, ensuring uniform thickness.
This method is ideal for non-soluble solid samples and guarantees sample homogeneity. The process includes grinding the solid to fine particles, mixing with a binder
like KBr, pressing under high pressure, and optional drying. The resulting pellet
offers consistency, making it ideal for materials like polymers, minerals, and
pharmaceuticals in mid-IR analysis. The choice of binder and press ure can vary
based on specific analysis requirements and sample characteristics, enhancing the
reproducibility of results (Fig.
4.6b).
Optimal KBr-to-Sample Ratios for Pellet Formation
In transmission IR spectroscopy, samples are often prepared as pellets by mixing the
sample with KBr, a transparent salt in the infrared range. The KBr pellet technique
provides a simple way to study solid samples, but the ratio of KBr to the sample is
critical for obtaining high-quality spectra.
The typi
Br-to-sampl
e ratio.
cal ratio for preparing KBr pellets is 100:1 by weight (KBr). This means
• Ideal K
that for every gram of sample, 100 gm of KBr are used. This dilution helps to
ensure that the sample is spread uniformly in the matrix and that it does not absorb
too much infrared radiation. A higher sample concentration could result in
overabsorption, leading to broad and poorly resolved peaks.
4.9 Sampling Techniques for IR Spectroscopy 179
• Sample concentration.
The sample concentration within the KBr matrix generally falls between 0.2%
and 1% by weight. At this concentration, the sample absorbs enough IR radiation
to generate clear peaks without overwhelming the spectrum. If the sample
concentration is too high, it may result in saturated absorbance bands, making it
difficult to interpret the spectrum.
• Pellet formation.
For optimal results, the KBr and sample mixture is finely ground to a homogenous powder and then pressed into a transparent pellet under high pressure (about
10 tons). This pressure ensures that the pellet is clear and free of air pockets,
which could scatter light and reduce spectral quality.
• KBr quality.
It is also essential to use high-purity, infrared-grade KBr, which does not absorb
in the IR region. Impurities in KBr can introduce unwanted background signals,
reducing the accuracy and clarity of the resulting spectrum.
4.9.1.3 Thin Film Formation
Thin film formation is a valuable technique in IR spectroscopy for solid sample
analysis. It involves preparing a solid sample as a thin, uniform film on a transparent
substrate, such as KBr or NaCl, facilitating efficient IR analysis. This method is
particularly advantageous for samples challenging to grind into fine powders or
when uniform sample thickness is critical. In the process, the solid samp le is finely
ground, applied as a thin film onto the substrate, optionally dried to remove
interfering substances, and then subjected to IR analysis. It’s commonly used for
mid-IR solid sample analysis, offering advantages like sample uniformity and the
ability to analyze materials difficult to pelletize. The choice of substrate and deposition technique can vary depending on the specific analysis requirements. Thin film
formation finds applications in materials science and chemistry, especially for
studying polymers, coatings, and thin layers.
4.9.2 Liquid Samples
In IR spectroscopy, liquid samples are analyzed using specialized cells with transparent windows like NaCl or KBr to allow IR radiation transmission. Preventing air
or moisture ingress is essential to avoid interference. Liquid cells come in various
types, including demou ntable, sealed, and ATR cells, enhancing sensitivity. IR
spectra of liquids provide valuable insights into functional groups and molecular
vibrations, supporting qualitative and quantitative analysis. This technique finds
wide application in chemistry, aiding substance identification, reaction monitoring,
and concentration determination. It is also crucial in the pharmaceutical industry for
evaluating drug formulations and in environmental science for detecting water
pollutants. To prevent spectral interference, the choice of a suitable, non-absorbing
solvent like CCl
, CS2, or CH3Cl may be necessary when diluting the sample.
4