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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5647_Библиотеки_им_академика_М_И_Перельмана.pdf
Comprehensive Insights into Molecular Emission Spectroscopy
Abstract
Molecular emission spectroscopy (MES) is an analytical technique that exploits
the phenomenon of molecular luminescence, offering valuable insights into the
composition and properties of various substances. MES operates on the principle
of energy absorption and subsequent emission of light at specific wavelengths by
molecules when exposed to excitati on energy, typically in the form of electro-
magnetic radiation. This technique encompasses a wide range of applications in
fields such as analytical chemistry, environmental science, biomedical research,
materials science, and more. The versatility of MES is evident in its applications,
including trace analysis, environmental monitoring, pharmaceutical and food
analysis, and quality control. In biological and medical research, MES enables
cellular imaging, biom arker discovery, and the study of molecular interactions.
Moreover, it plays a pivotal role in materials science, nanotechnology, and
environmental monitoring. Advancements in MES instrumentation, miniaturiza-
tion, and multichannel detection have further expanded its capabilities, making it
an indispensable tool for scientific research and practical applications.
8
Keywords
Molecular emission spectroscopy · Fluorescence spectroscopy · Instrumentation ·
Drug analysis · Environmental monitoring · Biomedical applications
8.1 Introduction
Molecular emission spectroscopy is a powerful analytical technique used to measure
the emission of radiation from excited atoms or molecules present in a sample. The
process begins with the excitation of molecules from their ground state (low energy
level) to an excited state (high energy level) by absorbing radiation from an external
321
322 8 Comprehensive Insights into Molecular Emission Spectroscopy
Fig. 8.1 Energy-level diagram depicting molecular emission. The diagram illustrates the emission
of light by a molecule or atom. The process begins with the excitation of an electron from a lower
energy state (E₁) to a higher energy state (E₂). After a brief period, the electron relaxes back to its
ground state (E₁), releasing energy in the form of a photon (hν). This emitted light corresponds to the
difference in energy between the two levels, represented as hν, where “h” is Planck’s constant and
“ν” is the frequency of the emitted light
source, typically a UV, visible, or laser light source (Fig. 8.1). The excitation occurs
at a specific wavelength, known as the excitation wavelength, which is unique to
each element or molecule depending on its electronic structure. The amount of
energy absorbed during this excitation is what raises the molecule to a higher energy
state.
Once the molecules absorb energy, they become unstable and rapidly return to
their ground state by releasing the absorbed energy. This release occurs in the form
of emitted radiation, characteristic of the specific molecule or atom. The radiation
emitted by these molecules can be detected at a longer wavelength than the absorbed
energy, referred to as the emission wavelength. In certain cases, if the emission is
nearly immedi ate, it is classified as fluorescence; however, if there is a delayed return
to the ground state due to intersystem crossing, the emission is called phosphorescence. The intensity, wavelength, and duration of the emitted light provide valuable
quantitative and qualitative information about the molecules present in the sample.
The excitation process in molecular emission spectroscopy can be achieved
through a variety of techniques, each suited for different applications and levels of
sensitivity:
• Flame excitation: In this method, the sample is atomized in a flame, which
provides sufficient thermal energy to excite the atoms or molecules. Flame
excitation is commonly used in atomic emission spectroscopy for metal analysis
due to its simplicity and cost-effectiveness.
• Plasma excitation: In this highly efficient technique, a plasma source (such as
inductively coupled plasma, or ICP) is used to excite the sample. The plasma
provides extremely high temperatures (up to 10,000 K), which ensures complete
atomization and excitation of a wide range of elements. Plasma excitation is
particularly beneficial for analyzing multiple elements simultaneously and for
complex matrices such as environmental or biological samples.
• Arc and spark
excitation: These methods involve electrical discharges, either in
the form of an arc or spark, to excite atoms in solid samples. The y are widely used
in metallurgical applications to analyze trace elements in metals.
8.1 Introduction 323
• Laser-induced excitation: This technique uses a focused laser beam to excite
specific atoms or molecules in the sample. The laser can be tuned to deliver
energy at specific wavelengths, enabling selective excitation of target molecules.
Laser-induced breakdown spectroscopy (LIBS) and laser-induced fluorescence
(LIF) are examples of this highly precise method. Laser excitation is known for its
high sensitivity, making it suitable for detecting low-concentration species.
• Electrothermal vaporization
(ETV): In
ETV, the sample is heated to extremely
high temperatures, allowing it to vaporize and enter the excitation source. This
technique, often coupled with ICP, enhances detection limits for trace elements in
solid and liquid samples.
Each of these excitation methods provides distinct advantages in terms of sensi-
tivity, precision, and selectivity, making molecular emission spectroscopy a versatile
tool in chemical analysis. The emitted radiation, once measured and analyzed,
allows for the accurate ident ification and quantification of various elements or
molecules in the sample, making it invaluable for applications in environmental
monitoring, biomedical analysis, industrial processes, and research.
What happens after a molecule has absorbed light (Fig. 8.2)? Many substances
are capable of absorbing UV or visible (VIS) light energy and then dissipating the
excess energy as heat through collisions with neighboring atoms or molecules.
However, some substances only release a portion of this excess energy as heat,
Fig. 8.2 Schematic representation of light absorption and emission by molecules. The diagram
illustrates the process of light absorption and emission by a molecule. Upon exposure to exciting
light, a normal molecule absorbs energy and transitions to an excited state. Approximately 80% of
the absorbed energy is dissipated as heat, while the remaining energy is either emitted as light (20%,
photoluminescence) or leads to photobleaching if excessive energy is applied. Photobleaching
should be minimized to preserve the molecule’s fluorescence properties. This cycle of excitation
and emission is fundamental in fluorescence-based studies
324 8 Comprehensive Insights into Molecular Emission Spectroscopy
emitting the remaining energy as electromagnetic radiation (ER) at a wavelength
longer than that which was absorbed. This process of emitting radiation is known as
luminescence or photoluminescence, and substances that exhibit this characteristic
are referred to as luminescent.
8.2 Electronic Spectra
Electronic spectra refer to the study of the interaction of electromagnetic radiation
with molecules, leading to electronic transitions between different energy levels.
This phenomenon occurs when molecules absorb or emit energy in the form of light,
causing electrons to jump from one electronic state to another. Electronic spectra are
a cornerstone in molecular spectroscopy, helping us understand the structure,
properties, and dynamics of molecules.
Electronic spectra play a crucial role in molecular emission spectroscopy (MES)
by providing insights into the electronic transitions of molecules. When molecules
absorb energy, their electrons transition from lower energy ground states to higher
energy excited states, resulting in an absorption spectrum that reflects these specific
energy levels. As the excited molecules return to their ground states, they emit
energy in the form of light, leading to an emission spectrum. The correlation between
electronic spectra and molecular emission spectroscopy lies in their shared focus on
electronic transitions. The wavelengths at which absorption occurs correspond to the
wavelengths of emitted light, allowing for the identification and characterization of
molecular species. By analyzing electronic spectra, researchers can gain valuable
information about the molecular structure, bonding, and energy levels, which are
essential for understanding the mechanisms of light emission and enhancing the
sensitivity and selectivity of molecular emission techniques. This interrelationship is
fundamental for interpreting spectral data and applying it to various fields, such as
chemistry, biology, and environmental science.
8.2.1 Basic Principles of Electronic Spectra
• Electronic transitions: Molecules consist of electrons arranged in various energy
levels, or orbitals, based on their quantum states. In their lowest energy state,
molecules exist in the ground state (S₀). Upon absorbing energy, typically from
ultraviolet (UV) or visible light, electrons in a molecule can be excited from the
ground state to a higher energy excited state. The absorbed energy must match the
energy gap between these two states for the transition to occur.
– Excitation: This is the process where a photon of appropriate energy excites an
electron from the ground state (S₀) to an excited singlet state (S₁, S₂, etc.).
– Relaxation:
radiation in the process.
• Absorption and emissi
when a sample absorbs photons at specific wavelengths corresponding to the
The excited
molecule can relax back to the ground state, emitting
on: In electronic spectra, absorption spectra are generated
8.2 Electronic Spectra 325
energy required for electronic transitions. This absorption process excites the
molecules to higher energy states. On the other hand, emission spectra occur
when the excited molecules return to the ground state and release energy in the
form of emitted photons.
– Fluorescence: If the emission occurs quickly after excitation (within
nanoseconds), it
is termed fl
uorescence. Fluorescence usually involves a
transition from the first excited singlet state (S₁) back to the ground state
(S₀), resulting in the emission of light at longer wavelengths than the absorbed
radiation (since energy is lost through non-radiative processes such as vibrational relaxation).
– Phosphorescence: If the molecule undergoes intersystem crossing to a tri
state (T
₁), and the return to the ground state happens more slowly
plet
(microseconds to seconds), this process is called phosphorescence. Phosphorescence typically occurs at even longer wavelengths due to the forbidden
nature of the triplet–singlet transition, making it slower and less probable.
• Franck–Condon principle: Electronic transitions often occur very rapidly com-
pared to the motion of nuclei in a molecule. According to the Franck–Condon
principle, electronic transitions are vertical, meaning they occur so quickly that
the nuclei do not have time to move, and the transition happens between vibrational states of the two electronic states. This principle explains why absorption
bands are broad and have distinct shapes, as multiple vibrational levels are
involved in the transition.
• Types of Transitions:
– π → π* transitions: Common in molecules with
conjugated
systems or double
bonds. The transition involves an electron moving from a bonding π-orbital to
an antibonding π*-orbital. This type of transition typically occurs in the UV or
visible region.
– n → π* transitions: Involve an electron in
a non-bon
ding orbital (n) moving to
an antibonding π*-orbital. These transitions occur at lower energy compared
to π → π* transitions and often appear in organic molecules containing lone
pairs (e.g., carbony l compounds).
– n → σ * transitions: Occur when an electron in a non-bonding orbital moves to
an antibonding σ -orbital. These transitions typically require higher energy and
occur in the UV region.
8.2.2 Excitation Techniques in Electronic Spectroscopy
Several methods are employed to excite molecules, and the choice of technique
depends on the type of sample, the required sensitivity, and the information needed:
• Absorption spectroscop
sample absorbs light at specific wavelengths. A UV– visible spectrophotometer is
y: This is the most straightforward technique, where the
326 8 Comprehensive Insights into Molecular Emission Spectroscopy
commonly used to measure the absorption spectrum. The resulting data reveal
absorption bands corresponding to electronic transitions.
• Fluorescence spectroscopy: In this technique, the sample is irradiated with light at
the excit
ation wave
length, and the resulting emitted light (fluorescence) is
detected at longer wavelengths. Fluorescence spectroscopy is highly sensitive
and widely used in biochemical applications, where it allows for the detection of
very small quantiti es of analytes.
• Phosphorescence spectroscopy: This method measures the longer-lived emission
of light as the molecule transitions from a triplet excited state to the ground state.
Since phosphorescence occurs on a longer timescale, special detectors are
required, and it is typically measured in low temperatures or using delayed
detection methods to distinguish it from fluorescence.
• Laser-induced fluorescence (LIF): This technique uses a laser to excite
molecules
within a sample, leading to highly selective and sensitive detection of
specific
fluorescence. LIF is particularly useful for gas-phase molecules or trace detection
in analytical chemistry.
• Raman spectroscopy: Although Raman scattering involves a different process
than electronic absorption, it provides complementary information about the
vibrational states of a molecule. Raman transitions involve the inelastic scattering
of light, which can provide data on molecular vibrations and electroni c
transitions.
8.2.3 Spectral Analysis
• Wavelength: The specific wavelengths at which absorption or emission occurs
correspond to the energy levels of the electronic transitions, providing insight into
the electronic structure of the molecule.
• Band shape and intensity: The shape and intensity of absorption/emission bands
can be influenced by factors such as molecular structure, solvent effects, and
intermolecular interactions. Broad bands often indicate transitions involving
multiple vibrational or rotational states, while sharp lines may be seen in
gas-phase molecules with little molecular interaction.
• Chemical analysis: Electronic spectra provide detailed information about the
molecular structure, composition, and concentration of samples. They are commonly used in environmental monitoring, food safety, and chemical
manufacturing.
• Biochemical analysis:
biological molecules such as proteins, nucleic acids, and small molecules. Fluorescent labels (e.g., GFP) allow for the visualization and quantification of
biomolecules in living systems.
• Material science:
properties of materials such as semiconductors, dyes, and polymers. It aids in
characterizing electronic transitions, band gaps, and material stability.
Fluorescence
spectroscopy is widely used in studying
Electronic spectroscopy is crucial for studying the optical
8.3 Types of Luminescence 327
• Pharmaceuticals: UV–visible and fluorescence spectroscopy are used to quantify
drugs, study drug interactions, and monitor stability and degradation in
formulations .
8.3 Types of Luminescence
There are several types of luminescence, each characterized by the way in which
light is emitted from a substance. The main types of luminescence are provided in the
following sections.
8.3.1 Fluorescence
Fluorescence is a common type of luminescence where a substance absorbs photons
of a specific energy, often in the ultraviolet (UV) or visible range, and then quickly
reemits the energy as lower-energy photons (visible light). This emission happens
almost immediately after excitation and stops when the excitation source is removed.
Fluorescence is widely used in various applications, such as in fluorescent dyes and
markers.
8.3.2 Phosphorescence
Phosphorescence is similar to fluorescence but involves a delayed emission of light
after the excitation source is removed. Phosphorescent materials can continue to emit
light for some time even in the absence of an excitation source. This is why
phosphorescent materials are often used in glow-in-the-dark products.
8.3.3 Electroluminescence
Electroluminescence is the emission of light resulting from the passage of an electric
current through a material. It is commonly seen in devices like light-emitting diodes
(LEDs) and organi c light-emitting diodes (OLEDs) used in displays and lighting.
8.3.4 Radioluminescence
Radioluminescence is the emission of light due to the interaction of ionizing radiation with certain materials, often in the form of phosphors. This is used in
applications such as glow-in-the-dark watch dials, where a radioactive material
(usually tritium) interacts with phosphors to produce visible light.
328 8 Comprehensive Insights into Molecular Emission Spectroscopy
8.4 Types of Molecular Emission Spectroscopy
Molecular emission spectroscopy encompasses various techniques and methods
used to study the emission of light from molecules. The following sections provide
some of the main types of molecular emission spectroscopy.
8.4.1 Fluorescence Spectroscopy
This is one of the most common and widely used techniques within molecular
emission spectroscopy. It involves the excitation of molecules by absorbing photons
at a specific wavelength and the subsequent emission of light at longer wavelengths,
typically in the visible or ultraviolet range.
8.4.2 Phosphorescence Spectroscopy
Phosphorescence is similar to fluorescence but involves a longer-lived excited state,
leading to the delayed emission of light. This technique is often used in the study of
materials with longer-lasting excited states.
8.4.3 Photoluminescence Spectroscopy
Photoluminescence encompasses the study of ligh t emission resulting from the
absorption of photons or other forms of electromagnetic radia tion. This includes
both fluorescence and phosphorescence, as well as other types of luminescence.
8.4.4 Raman Spectroscopy
Raman spectroscopy is a unique form of molecular emission spectroscopy that
involves the scattering of photons by molecules, resulting in shifts in wavelength
that provide information about molecular vibrations and rotational states.
8.4.5 Laser-Induced Breakdown Spectroscopy (LIBS)
LIBS involves the use of high-energy lasers to induce the emission of light from
samples. It is often used for elemental analysis in a variety of fields.
8.4 Types of Molecular Emission Spectroscopy 329
8.4.6 Cathodoluminescence Spectroscopy
Cathodoluminescence spectroscopy studies the light emission from materials when
they are irradiated with electrons, typically in electron microscopy. It is valuable for
materials characterization.
8.4.7 Plasma Emission Spectroscopy
This technique involves the generation of a high-temperature plasma to excite and
emit light from samples. It is commonly used for elemental analysis, especially in
analytical chemistry.
8.4.8 Chemiluminescence Spectroscopy
Chemiluminescence involves the emission of light because of a chemical reaction,
rather than external excitation by photon s. It is commonly observed in processes
such as bioluminescence in fireflies and the chemiluminescent glow of chemical
reactions used in assays and analytical chemistry.
8.4.9 Bioluminescence Spectroscopy
Bioluminescence is a type of chemiluminescence exhibited by living organisms,
such as fireflies and certain marine species. It involves the emission of light due to a
biochemical reaction, and it is widely used in biological research and medical
diagnostics.
8.4.10 Difference Between Chemiluminescence
and Bioluminescence
Chemiluminescence and bioluminescence are two processes involving the emission
of light, but they differ significantly in their mechanisms and contexts. Chemiluminescence refers to the emission of light resulting from a chemical reaction that does
not require external radiation. This phenomenon can occur in various chemical
systems, including synthetic and natural environments, and is not limited to
biological contexts. In contrast, bioluminescence is a specific type of chemiluminescence that occurs exclusively in living organisms. It involves biochemical reactions
typically between luciferin, a light-emitting molecule, and luciferase, an enzyme,
resulting in light production. While chemilum inescence can utilize a variety of
reactants, bioluminescence relies primarily on luciferin and luciferase, with the
emitted light color determined by the structure of luciferin. The applications of
these two processes also differ; chemiluminescence is commonly employed in
330 8 Comprehensive Insights into Molecular Emission Spectroscopy
laboratory assays, detection methods, and industrial applications, whereas bioluminescence is studied in ecology and biology for understanding organism behavior and
ecological interactions. Ultimately, while both processes involve light emission,
bioluminescence serves distinct biological functions, such as attracting mates or
deterring predators, making it crucial for the survival of the organisms that
produce it.
8.5 Theory
The theory of molecular emission spectroscopy is based on the principles of atomic
and molecular spectroscopy and involves the study of how molecules emit light
when transitioning from higher energy states to lower energy states. Here is an
overview of the key theoretical aspects: Following steps (Fig.
molecular emission spectroscopy.
8.3) are involved in
8.5.1 Vibrational Relaxation
Surplus energy from vibrationally excited species is transferred to solvent molecules.
This process occurs within a very short time, typically on the order of 10
causing solvent molecules to return to a lower vibrational energy state from an
electronically excited state. Molecules in a singlet excited state lose energy more
readily through c ollisions with surrounding solvent molecules.
-15
8.5.2 Internal Conversion
When the lower and upper electronic states of the excited singlet have the same
multiplicity, this phenomenon is known as internal conversion.
8.5.3 Photon Emission
Molecules in the singlet excited state return to the ground state, resulting in the
emission of photons, which is referred to as fluorescence.
8.5.4 Energy Transfer
Energy is transferred from the singlet state to the triplet state as the molecule returns
to the ground state. This process is also known as intersystem crossing.
Figure 8.4 illustrates
concept in fluorescence spectroscopy. Molecules absorb excitation energy, often
from specific wavelengths of light or electromagnetic radiation, promoting electrons
the fluorescence excitation–emission cycle, a fundamental
s,