Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5925_Библиотеки_им_академика_М_И_Перельмана.pdf
8.5 Theory 331
Fig. 8.3 Schematic representation of electronic energy levels and transitions in fluorescence and
phosphorescence. This diagram illustrates the electronic transitions involved in fluorescence and
phosphorescence. Upon absorption of light, the molecule transitions from the ground state (S₀) to
higher singlet excited states (S₁, S₂). The molecule undergoes vibrational relaxation and internal
conversion to the lowest excited singlet state (S₁), from where it can return to the ground state (S₀)
by emitting light through fluorescence (10
occur, where the molecule transitions to a triplet excited state (T₁), followed by phosphorescence
emission (10
state, and the timescales of absorption, fluorescence, and phosphorescence are indicated. The
diagram also highlights the differences in energy and timescales between these radiative transitions
-4
–10 s) as it returns to the ground state. Vibrational relaxation occurs in each excited
-9
-6
–10
s). Alternatively, intersystem crossing may
to higher energy states. Subsequently, these excited molecules emit light as they
return to their ground state, generating a unique emission spectrum. This cycle is
vital in molecular emission spectroscopy, enabling qualitative and quantitative
analysis across various fields, such as analytical chemistry, biochemistry, and
environmental science, by identifying and measuring specific molecules in samples.
The emitted light’s intensity and wavelength character
standing
the investigated molecules, making fluorescence spectroscopy an invalu-
istics are crucial for under-
able analytical technique.
332 8 Comprehensive Insights into Molecular Emission Spectroscopy
Fig. 8.4 Schematic representation of fluorescence excitation–emission cycle. This figure illustrates
the fluorescence excitation–emission cycle, showcasing the transitions between electronic states.
Upon excitation by absorbing photons (hν
first singlet excited state (S₁). After rapid relaxation to the lowest vibrational level of S₁, the molecule
emits photons (hν
as relaxation and intersystem crossing to the triplet state (T₁) may also occur, leading to delayed
emission as phosphorescence (hν
indicated with solid and dotted lines, respectively. The formation of photoproducts through
degradation pathways is also highlighted
) as it returns to the ground state via fluorescence. Non-radiative transitions such
x1
ph
), the molecule moves from the ground state (S₀) to the
ex
). The transitions between radiative and non-radiative states are
8.6 Principle
Molecular emission spectroscopy is based on molecular luminescence, where
molecules absorb energy (e.g., light or electromagnetic radiation) and emit light at
specific wavelengths. Key aspects include energy absorption, unique excitation
wavelengths, temporary molecular instability in the excited state, emission of electromagnetic radiation (often visible or UV light), identification of longer emission
wavelengths, spectral analysis for molecular identification, quantitative concentration determination, and diverse applications in fields such as chemistry, environmental monitoring, and materials science. In essence, it centers on energy absorption
and light emission, serving as a versatile analytical technique. When one electron
from an elect ron pair within a molecule gets excited (Fig.
level, it can lead to the production of either a singlet or triplet state. In the excited
singlet state of the molecule, the spin of the excited electron remains opposite to that
8.5) to a higher energy
8.7 Types of Fluorescence 333
Fig. 8.5 Schematic representation of electronic states in molecules: ground and excited states. This
figure illustrates the different electronic states of a molecule, including the ground state and two
types of excited states. In the ground singlet state (a), all electrons are paired, and their spins are
opposite, representing the molecule’s lowest energy configuration. When the molecule absorbs
energy, one of the electrons is excited to a higher energy level while maintaining its opposite spin,
resulting in the excited singlet state (b). This state still has paired spins but with one electron in a
higher energy orbital. In contrast, the excited triplet state (c) occurs when an electron is excited, but
its spin is no longer paired with the ground-state electron, resulting in unpaired, parallel spins. The
triplet state is often lower in energy than the excited singlet state and has distinct implications in
photophysical processes such as phosphorescence. These states are crucial for understanding
molecular behavior in light absorption and emission phenomena
of the remaining ground state electron. However, in the triplet state, the spins of both
electrons become parallel and unpaired. The excited singlet state is relatively higher
in energy compared to its corresponding unpaired singlet state.
8.7 Types of Fluorescence
There are several types of fluorescence, each characterized by specific mechanisms
and applications. The following are the main types of fluorescence:
• Steady-state fluorescence: Common; molecules absorb
and emit
microscopy, flow cytometry, and spectroscopy.
• Time-resolved fluorescence: Focus on emissio
n time;
measures fluorescence
lifetimes; used in microscopy (FLIM) and molecular dynamics studies.
• Fluorescence anisotropy: It measures the polarization, assesses fluorophore
mobility, useful in binding studies and structural analysis.
• Fluorescence resona
nce
energy transfer (FRET): Energy transfer between nearby
donor and acceptor fluorophores, applied in molecular interactions and conformation studies.
• Fluorescence polar
ization: Measures molecule alignment; useful in binding
studies and biological dynamics analysis.
light, used in
334 8 Comprehensive Insights into Molecular Emission Spectroscopy
• Fluorescence quenching: Reduces fluorophore fluorescence due to interactions;
applied in assays for molecular interactions and concentration analysis.
• Two-photon fluorescence: Uses two
low-energy
photons for deep-tissue imaging,
minimizes photodamage, and improves spatial resolution.
• Fluorescence correlation spectroscopy (FCS): Analyzes intensity fluctuations in
small volumes; useful for studying diffusion, interactions, and concentrations at a
small scale.
8.8 Components of MES
MES is a sophisticated analytical technique that requires a well-designed instrumentation setup to effectively excite molecules, capture their emitted light, and
analyze the resulting emission spectra. The key components of the instrumentation
involved in molecular emission spectroscopy are outlined in the following sections
(Fig.
8.6).
Fig. 8.6 Schematic representation of atomic emission spectrophotometer components. This figure
illustrates the key components of an atomic emission spectrophotometer. A mercury vapor lamp
serves as the light source, emitting a spectrum of light. The light passes through a primary filter and
a condensing lens, where it is focused and directed to an excitation monochromator, which selects
the desired excitation wavelength. After passing through a secondary filter and another condensing
lens, the monochromatic light illuminates the sample contained in a sample cuvette, causing the
atoms within the sample to emit light. The emitted light from the sample passes through an emission
monochromator, where the various emission wavelengths are separated and focused onto a detector.
The detector captures the intensity of the emitted light, and the signal is amplified by an amplifier
before being sent to a readout device for analysis. This setup enables the detection and quantification of elements based on their characteristic emission spectra
8.8 Components of MES 335
8.8.1 Light Source
The light source is critical for providing the excitation energy needed for the sample
analysis. Several types of light sources are commonly employed:
• Lamps: Mercury vapor lamps and xenon lamps are often utilized for generating
specific wavelengths of light necessary for excitation. These sources provide
broad spectra that can effectively excite various molecular species.
• Laser systems: Lasers are increasingly used in advanced spectroscopy setups due
to their ability to produce monochromatic and intense light. This precision in
wavelength selection allows for highly specific excitation of target molecules,
enhancing sensitivity and resolution.
8.8.2 Sample Compartment
The sample compartment houses the sample during analysis. It may include various
types of holders such as cuvettes or microplates. The design of the sample compartment is crucial, as it should minimize stray light and ensure uniform illumination of
the sample, thereby enhancing the accuracy and reproducibility of measurem ents.
8.8.3 Monochromator
A monochromator is an essential component that isolates specific excitation and
emission wavelengths. By allowing the selection of a narrow range of wavelengths,
it facilitates precise excitation of the sample and accurate detection of the emitted
light, which is critical for obtaining reliable spectral data.
8.8.4 Sample Excitation and Emission Pathways
The optical components, including lenses and mirrors, direct the excitation light
toward the sample and collect the emitted light. The proper alignment and configuration of these optical elements are essential to ensure efficient light transmission and
to optimize the collection of emitted signals.
8.8.5 Detector
Detectors are essential components in MES, responsible for capturing and
converting emitted light from excited atoms and molecules into electrical signals
for analysis. The choice of detector significantly influences the sensitivity, speed,
and resolution of the measurement. The following sections provide an overview of
336 8 Comprehensive Insights into Molecular Emission Spectroscopy
the common types of detectors used in MES, their working principles, and their
applications.
8.8.5.1 Photomultiplier Tubes (PMTs)
Photomultiplier tubes are highly sensitive light detectors that amplify weak light
signals through a series of dynodes.
Working principle:
• PMTs consist of a photocathode, which emits electrons when exposed to light.
• These electrons are then accelerated toward a series of dynodes, each of which
emits more electrons upon impact.
• This cascade effect results in a significant amplification of the initial light signal,
enabling the detect ion of very low light levels.
Characteristics:
• High sensitivity and fast response times make PMTs suitable for detecting faint
emissions.
• Wide wavelength range typically from UV to visible light.
• Good signal-to-noise ratio.
Applications: Commonly used in fluorescence spectrometry, especially for mea-
suring low concent rations of analytes.
8.8.5.2 Charge-Coupled Device (CCD) Cameras
CCD detectors are imaging devices that capture light across an array of pixels,
allowing for simultaneous detection of multiple wavelengths.
Working principle:
• Photons strike the individual pixels in a CCD chip, generating electron–hole
pairs.
• The accumulated charge in each pixel is then read out sequentially, creating a
digital representation of the light intensity across the detected spectrum.
Characteristics:
• High spatial resolution and sensitivity.
• Capability to capture entire spectra or images in a single acquisition.
• Wide dynamic range, suitable for varying light intensities.
Applications:
• Used in imaging-based fluorescence spectroscopy, where capturing a full spec-
trum of emitted light is essential.
• Useful in applicati
ons such as bioimaging, where spatial distribution of emissions
is important.
8.8 Components of MES 337
8.8.5.3 Avalanche Photodiodes (APDs)
Avalanche photodiodes are semiconductor devices that offer high sensitivity and fast
response times.
Working principle:
• Similar to standard photodiodes, APDs operate by generating electron–hole pairs
when exposed to light.
• They utilize a process called avalanche multiplication, where the generated
electrons are accelerated by a reverse bias, causing additional electron–hole
pairs to be produced, thereby amplifying the signal.
Characteristics:
• High gain and sensitivity, allowing for the detection of weak light signals.
• Fast response time, making them suitable for time-resolved measurements.
• Operate effectively in the visible to near-infrared wavelength range.
Applications: Used in applications requiring rapid response times, such as fluores-
cence lifetime measurements and time-resolved spectroscopy.
8.8.5.4 Silicon Photodiodes
Silicon photodiodes are simple semiconductor detectors that convert light into
electrical current.
Working principle: When light hits the silicon material, it generates electron–hole
pairs, leading to a flow of current proportional to the intensity of the incident light.
Characteristics:
• Cost-effective and easy to use.
• Suitable for measuring light in the UV to visible range, although less sensitive
than PMTs or APDs.
Applications: Often used in general-purpose fluorescence spectroscopy and
applications where cost is a significant factor.
8.8.5.5 Photon Counting Modules (PCMs)
Photon counting modules combine a photomultiplier or avalanche photodiode with
electronics designed to count individual photons.
Working principle:
• PCMs detect single photons and generate an electrical pulse for each detected
photon.
• The counting
capability allows for precise measurements of low-intensity light.
338 8 Comprehensive Insights into Molecular Emission Spectroscopy
Characteristics:
• High sensitivity and ability to measure very low light levels.
• Excellent timing resolution for time-resolved measurements.
Applications: Used in low-light applications, such as single-molecule fluorescence
detection and
quantum
optics experiments.
8.8.5.6 Microchannel Plate (MCP) Detectors
Microchannel plates are two-dimensional electron multipliers used for detecting
photons with high spatial and temporal resolution.
Working principle: When a photon strikes the MCP, it generates secondary
electrons that are multiplied as they travel through the microchannels, resulting in
an amplified signal.
Characteristics:
• High gain and fast response time, allowing for detection of fast phenomena.
• Suitable for detecting very weak light emissions.
Applications: Often used in applications requiring high spatial resolution, such as
imaging in fluorescence microscopy.
8.8.6 Data Acquisition System
This system plays a crucial role in processing and recording the signals generated by
the detector. It typically consists of analog-to-digital converters (ADCs), amplifiers,
and dedicated data acquisition software, which collectively enable efficient data
collection and manag ement.
8.8.7 Spectrometer
A spectrometer is a critical instrument in MES, designed to separate and analyze the
emitted light from excited atoms and molecules. It plays a vital role in the measurement and characterization of the emission spectra, which provide qualitative and
quantitative information about the sample. The following sections provide an overview of the components, types, and functionalities of spectrometers used in MES.
8.8.8 Components of a Spectrometer
• Entrance slit: The entrance slit allows light emitted from the sample to enter the
spectrometer. It is designed to control the amount of light entering the system,
ensuring optimal signal intensity.
8.8 Components of MES 339
• Dispersive element: This is a key component that separates light into its constitu-
ent wavelengths. Common dispersive elements include:
– Prisms: Use refraction to disperse light based on wavelength. Prisms are often
employed in visible light spectrometers
for broad
spectral ranges.
– Diffraction gratings: Use interference patterns to separate light into various
wavelengths. They are preferred for their high resolution and efficiency across
a wide range of wavelengths, making them suitable for UV, visible, and nearinfrared spectroscopy.
• Exit slit: After the light is dispersed, the exit slit selects a specific wavelength or
range
of wavelengt
hs to be directed to the detector.
• Detector: The detector converts the optical signal into an electrical signal for
analysis. Common detectors in MES include:
– Photomultiplier tubes (PMTs): Highly sensitive and capable of detecting low
light levels, PMTs are often used for their rapid response and wide dynamic
range.
– Charge-coupled device (CCD): CCD detectors are commonly employed in
imaging applications, allowing for simultaneous detection of multiple
wavelengths and providing high-resolution spectral information.
• Optical components: Lenses and mirrors direct light throughout the spectrometer,
focusing and aligning it to optimize performance.
• Data acquisition system: This system records and processes the electrical signals
from the detector, often including analog-to-digital converte rs (ADCs) and data
processing software to analyze the spectra.
8.8.9 Types of Spectrometers Used in MES
• Fluorescence spectrometers: Designed specifically to measure fluorescence emis-
sion spectra, these spectrometers utilize excitation light sources and appropriate
optical filters to isolate the emitted light from background signals.
• Atomic emission spectrometers: These are used for analyzing the emission spectra
from atomic species. They are often coupled with plasma or flame excitation
sources to enhance sensitivity for elemental analysis.
• Multiwavelength spectrometers: These systems can capture spectra at multiple
wavelengths simultaneously, facilitating the analysis of complex mixtures or
rapid measurements in various applications.
• Time-resolved spect
sient signals and are often used in studies of dynamic processes, such as energy
transfer and reaction kinetics.
rometers: These spectrometers are capable of capturing tran-
340 8 Comprehensive Insights into Molecular Emission Spectroscopy
8.8.10 Functionalities of Spectrometers in MES
• Spectral resolution: Spectrometers in MES are designed to provide high spectral
resolution, allowing for the distinction between closely spaced emission lines,
which is crucial for accurate identification and quantification of analytes.
• Quantitative analysis: By measuring the intensity of the emitted light at specific
wavelengths, spectrometers enable the quantification of the concentration of
substances in the sample, following calibration with known standards.
• Qualitative analysis: Spectrometers provide spectral fingerprints of substances,
allowing for the identification of compounds based on their characteristic emission spectra.
• Sensitivity and selectivity: Advanced spectrometers equipped wi
performanc
trace amounts of analytes while minimizing interference from background
signals.
e detectors and optics enhance sensitivity, enabling the detection of
th high-
8.8.11 Computer and Software
A computer system is essential for controlling the instrumentation, collecting and
analyzing data, and generating spectral outputs. Specialized software is often used
for data processing, spectral analys is, and deconvolution, allowing for advanced
interpretation of the collected data.
8.8.12 Accessories
Various accessories can augment the capabilities of the instrumentation. These may
include temperature-controlled sample holders to maintain consistent conditions,
flow cells for continuous measurements, and automated sample changers that facilitate high-throughput analysis for efficient sample processing.
8.8.13 Optical Filters
Optical filters are crucial components in MES systems, as they enhance the quality of
the detected signals by selectively allowing certain wavelengths of light to pass
while blocking others. This selective filtration is essential for improving the signalto-noise ratio and ensuring accurate measurements. The following sections provide
the types, functions, and applications of optical filters used in MES:
8.8.13.1 Types of Optical Filters
• Band-pass filters: Band-pass filters transmit light within a specific wavelength
range while blocking wavelengths outside this range. They are used to isolate the
desired emission wavelengths from the sample. In MES, band-pass filters are