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_5647_Библиотеки_им_академика_М_И_Перельмана.pdf
10.11 Types of NMR Spectroscopy 471
10.11.4.2 Interactions in SSNMR
• CSA: In solids, the chemical shift varies depending on the orientation of the
molecule relative to the magnetic field, leading to anisotropic shifts. MAS helps
average out these shifts.
• Dipolar coupling: In solids, nuclei interact strongly with each other through
dipole–dipole interactions, which are not fully averaged out as they are in liquids.
• Quadrupol ar interactions: Nuclei with a spin greater than ½ (such as
14
N) experience quadrupolar interactions, which are strong in solids and lead to
2
11
H,
B, or
broader lines.
10.11.4.3 Applications of SSNMR
• Structure of biomolecules: Solid-state NMR is used to study membrane proteins,
amyloid fibrils, and other biomolecules that are difficult to analyze in solution.
These studies provide insights into protein folding, dynamics, and interactions
within solid envir onments such as membranes or fibrils.
• Pharmaceuticals: SSNMR is applied in the analysis of drug formulations to
examine the polymorphism, crystallini
ty, and molecular
structure of active phar-
maceutical ingredients in their solid state.
• Polymers and materials science : Solid-state NMR is widely used in
characterizing the structure
and proper
ties of polymers, providing detailed infor-
mation on molecular orientation, crystallinity, and phase transitions.
• Inorganic and catalytic materials: SSNMR is valuable in the study of zeolites,
catalysts, and other inorganic materials, helping to elucidate their structural and
dynamic properties.
10.11.4.4 Techniques in SSNMR
13
•
C CPMAS (cross-polarization magic angle spinning): This is one of the most
common techniques in SSNMR, used to improve the signal intensity of
13
C by
cross-polarizing from abundant protons while spinning the sample at the magic
angle.
2
H NMR: Deuterium NMR is used to study molecular dynamics and order in
•
solids, as
2
H are sensitive to quadrupolar interactions.
• Direct polarization: In some cases, direct polarization techniques are used for
nuclei where cross -polarization is less effective, especially in rigid solids.
10.11.4.5 Advantages of SSNMR
• Nondestructive: SSNMR can be applied without altering or destroying the sam-
ple, making it ideal for studying materials in their native state.
• Applicable to a wide range of materials: From crystalline to amorphous materials,
SSNMR can provide structural insights into a variety of solids.
• Detailed structural
information: SSNMR can resolve atomic-level details about
molecular structure, intermolecular interactions, and molecular motions in solid
systems.
472 10 Comprehensive Insights into Nuclear Magnetic Resonance Spectroscopy
10.11.4.6 Limitations of SSNMR
• Lower sensitivity: Solid-state NMR generally has lower sensitivity compared to
solution-state NMR due to the broader line shapes caused by restricted molecular
motions.
• Longer acquisition times: The experiments often require longer acquisition times
to collect high-quality spectra, especially for low-abundance nuclei such as
15
N.
13
C or
• Complex spectral interpretation: Due to the various interactions present in solids,
interpreting SSNMR spectra can be more challenging than in solutionstate NMR.
10.11.5 High-Resolution NMR
High-resolution NMR (HR-NMR) spectroscopy refers to NMR techniques that yield
highly resolved spectra, allowing detailed analysis of chemical structures, dynamics,
and molecular interactions, especially in solution-state samples. The term “highresolution” indicates the ability of the NMR instrument to clearly separate and
distinguish individual resonance signals, which is crucial for studying complex
molecules, such as organic compounds, proteins, and nucleic acids.
10.11.5.1 Principle of HR-NMR
In HR-NMR, well-defined, sharp resonance peaks are produced because the molecular motions in liquids or solutions effectively average out many interactions that
would otherwise broaden the signals, such as dipolar couplings and anisotropic
effects. The technique focuses on detecting and analyzing nuclei such as
15
N, and
31
P under conditions that maximize spectral clarity and resolution.
1
13
H,
C,
10.11.5.2 Key Features of HR-NMR
• Chemical shifts: Each nucleus in a molecule experiences a unique electronic
environment, leading to variations in the resonance frequency, which is observed
as chemical shifts in the NMR spectrum. HR-NMR can accurately measure these
shifts, revealing the chemical environment and connectivity of atoms.
• Spin–spin coupling (J-coupling): Interactions between nuclei that are chemically
bonded or close in space result in splitting patterns in the NMR spectrum. Highresolution NMR can resolve fine coupling constants (J values) that provide
structural information about bonding relationships.
• Sharp, narrow peaks:
HR-NMR, peaks appear narrow due to the fast tumbling
In
of molecules in solution, which averages out anisotropic interactions such as
dipolar couplings and chemical shift anisotropy. This makes it possible to distinguish closely spaced resonance signals.
• NOE: NOE is
observed when there is spatial proximity between nuclei, which
leads to changes in signal intensities. HR-NMR can utilize NOE to determine the
three-dimensional structure of molecules in solution.
10.11 Types of NMR Spectroscopy 473
10.11.5.3 Types of HR-NMR
•1 H NMR: Proton NMR is the most common type of HR-NMR, used to study
hydrogen atoms in organic and biological molecules. It provides information on
the number of protons, their chemical environment, and connectivity to other
atoms.
13
C NMR: Carbon-13 NMR is used to study carbon atoms in molecules,
•
providing complementary structural information to proton NMR. HR
is valuable in determining the framework of organic molecules.
• Heteronu clear NMR: HR-NMR can also be applied to other nuclei such as
31
P. Heteronuclear experiments enhance the resolution and sensitivity of these
13
C NMR
15
N or
nuclei through techniques such as decoupling and cross-polarizat ion.
10.11.5.4 Applications of HR-NMR
• Structura l elucidation of organic molecules: HR-NMR is widely used to deter-
mine the stru cture of small organic molecules by providing detailed information
on the number of atoms, their connectivity, and spatial arrangement.
• Protein structure determination: In combination with 2D and 3D NMR
techniques, HR-NMR is used to study proteins and other biomolecules in solution. It provid es information on the folding, dynamics, and interactions of
proteins at the atomic level.
• Metabolomics and quantitative analysis: HR-NMR is employed in metabolomics
to identify and quantify metabolites in biological samples, such as urine, plasma,
and tissues. The nondestructive nature of NMR makes it ideal for analyzing
complex mixtures.
• Drug discovery and pharmacology: HR-NMR is used in drug discovery to study
drug–receptor interactions, determine the structures of drug candidates, and
analyze the purity and composition of pharmaceutical compounds.
• Chemical kinetics and reaction mechanisms: HR-NMR is useful in studying
chemical reactions and kinetics by monitoring the changes in the NMR spectra
over time. This provides insights into reaction intermediates and mechanisms.
10.11.5.5 Techniques Enhancing HR-NMR
• Decoupling: In heteronuclear NMR (e.g.,
13
C NMR), proton decoupling
techniques remove the splitting caused by proton–carbon coupling, resulting in
simplified spectra with sharper lines and clearer chemical shifts.
• Pulsed field gradients: These are used to suppress unwanted signals and improve
the quality of NMR spectra by enhancing the resolution of small peaks and
reducing spectral artifacts.
• Shimming: Shimming refers to adjusting the homogeneity of the magnetic field,
ensuring that the magnetic field is uniform across the sample, which is critical for
achieving high-resolution spectra.
10.11.5.6 Advantages of HR-NMR
• Nondestructive: NMR is a nondestructive technique, allowing for the study of
materials without altering or destroying the sample.
474 10 Comprehensive Insights into Nuclear Magnetic Resonance Spectroscopy
• Detailed molecular information: HR-NMR provides comprehensive structural
information, including the identification of functional groups, bonding patterns,
and spatial arrangements.
• Versatile: It can be applied to a wide range of compounds, from small organic
molecules to large biomolecules such as proteins and nucleic acids.
• Dynamic studies: NMR can study molecular dynamics, providing insights into
conformational changes, interactions, and reaction mechanisms.
10.11.5.7 Limitations of HR-NMR
• Low sensitivity for certain nuclei: Nuclei such as
1
H NMR, requiring longer acquisition times and higher sample
than
13
C and
15
N are less sensitive
concentrations.
• Sample size: High-resolution NMR typically requires milligram quantities
samples, making
it less suitable for very small sample amounts.
of
• Overlapp ing peaks: In complex mixtures or large molecules, signals may overlap,
making it difficult to assign individual resonances.
10.11.6 Multinuclear NMR Spectroscopy
Multinuclear NMR spectroscopy refers to NMR techniques that study a variety of
nuclei beyond the commonly analyzed
used for structural analysis, multinuclear NMR expands the range of nuclei that can
be studied, including isotopes such as
approach allows researchers to gain more comprehensive insights into molecular
structure, dynamics, and interactions across different chemical environments.
1
H nucleus. While proton NMR is widely
13
15
31
C,
N,
19
P,
F, and many others. This
10.11.6.1 Principle of Multinuclear NMR Spectroscopy
Each nucleus has a charact eristic gyromagnetic ratio and resonance frequency,
which dictates its behavior in an external magnetic field. When different nuclei are
exposed to a magnetic field and excited by RF pulses, they resonate at specific
frequencies based on their magnetic properties. Multinuclear NMR spectroscopy
involves adjusting the spectrometer to detect the resonance signals of these less
abundant or less commonly analyzed nuclei.
10.11.6.2 Common Nuclei Studied in Multinuclear NMR
13
•
C: One of the most commonly studied nuclei after protons. It provides detailed
information about the carbon skeleton in organic compounds, including the types
of carbon atoms (e.g., sp
15
N: Used for studying nitrogen-containing compounds, particularly in proteins
•
and nucleic acids.
backbone structures and interactions in biological macromolecules.
31
P: Phosphorus is found in many biological molecules, such as DNA, RNA, and
•
phospholipids.
31
2
, sp3 , carbonyl carbons) and their connectivity.
15
N NMR is often used in biomolecular NMR to investigate
P NMR spectroscopy is commonly used to study phosphate
groups and their role in chemical and biological processes.
10.11 Types of NMR Spectroscopy 475
19
•
F: Fluorine is often used in pharmaceuticals, agrochemicals, and materials.
19
NMR provides information about the environment of fluorine atoms in these
compounds, especially useful for drug development and material science.
29
Si: Silicon-containing materials, such as silicates, ceramics, and organosilicon
•
compounds, can be analyzed using
29
Si NMR, which is essential in material
sciences and geochemistry.
10.11.6.3 Features of Multinuclear NMR
1. Broad range of nuclei: Multinuclear NMR enables the study of numerous
elements, offering insights into molecules that contain diverse chemical elements
beyond hydrogen and carbon. It allows for the investigation of non-protonbearing elements such as metals, halogens, and metalloids.
2. Different gyromagnetic ratios: Each nucleus resonates at a different frequency
based on its gyromagnetic ratio. For example,
1
H for a given magnetic field strength. NMR spectrometers can be tuned to
than
13
C resonates at a lower frequency
detect specific frequencies, making mul tinuclear studies possible.
3. Chemical shift and coupling information: Just like in proton NMR, each nucleus
has a unique chemical shift that provides information about its chemical environment. Additionally, spin–spin coupling (J-coupling) can occur between different
nuclei, revealing connectivity and spatial relationships.
4. Decoupling techniques: Heteronuclear decoupling is often used in multinuclear
NMR to simplify spectra by removing splitting patterns from proton coupling,
enhancing the clarity of the signal from the nucleus of interest (e.g.,
13
C or
15
N).
F
10.11.6.4 Applications of Multinuclear NMR
1. Structural elucidation: Multinuclear NMR is essential for detailed structural
analysis of molecules containing elements such as nitrogen, phosphorus, fluorine,
and silicon. It provides complementary information to proton and carbon NMR,
helping to build a complete picture of molecular structure.
2. Protein and nucleic acid studies: In biomolecular NMR,
15
N and
31
P NMR are
used alongside proton and carbon NMR to study the backbone and side chains of
proteins, as well as the phosphate groups in DNA and RNA. This is critical for
understanding the structure, dynamics, and interactions of biological
macromolecules.
3. Pharmaceutical and chemical industry:
study fluorinated compounds, which are common in pharmaceuticals and
agrochemicals.
31
P NMR is used to analyze phosphorus-containing drugs,
metabolites, and catalysts.
4. Material science: Multinuclear NMR, including
19
F NMR is used in drug development to
29
Si and
27
Al, is widely used to
investigate the structure and properties of inorganic materials such as glasses,
ceramics, and zeolites. These studies provide insights into the atomic structure
and composition of materials, which are essential for the development of new
materials with specific properties.
5. Metabolomics
and metabolic pathways:
31
P NMR is used in metabolic studies to
analyze phosphorus-containing metabolites, such as ATP, in living cells. This
476 10 Comprehensive Insights into Nuclear Magnetic Resonance Spectroscopy
technique helps to understand energy metabolism and phosphate-based biochemical pathways.
10.11.6.5 Challenges in Multinuclear NMR
• Low natural abundance: Many nuclei studied in multinuclear NMR (e.g.,
15
29
N,
Si) have low natural abundance, leading to weaker signals. This often
13
C,
requires longer acquisition times or the use of isotopically enriched samples.
• Lower sensitivity: Some nuclei have lower gyromagnetic ratios and lower sensi-
tivity compared to
1
H, making their detection more challenging. This often
necessitates high concentrations of the sample or advanced signal enhancement
techniques.
• Instrument calibration: Each nucleus requires the NMR
instrument
to be
recalibrated for its specific resonance frequency. This involves adjustments to
the radiofrequency channels, coils, and decoupling settings.
10.11.6.6 Advantages of Multinuclear NMR
• Versatility: Multinuclear NMR enables the study of a wide range of elements,
providing comprehensive molecular information beyond what is possible with
proton and carbon NMR alone.
• Detailed structural information: By analyzing multiple nuclei, researchers can
gather detailed information on different parts of a molecule, including non-carbon
atoms that play critical roles in molecular function.
• Nondestructive analysis: Like other forms of NMR, multinuclear NMR is nonde-
structive, allowing the study of materials without altering or consuming the
sample.
10.11.7 Time-Domain NMR (TD-NMR)
Time-domain NMR (TD-NMR) refers to a type of NMR spectroscopy where the
primary data collected is in the time domain, as opposed to the frequency domain
used in traditional NMR. Instead of immediately transforming the signal into the
frequency spectrum, TD-NMR directly measures the respon se of nuclei to
radiofrequency pulses in the time domain. This technique focuses on the relaxation
and dynamic properties of materials, rather than on obtaining high-resolution spectra
for structural analysis. TD-NMR is commonly used for applications such as studying
molecular motion, relaxation times, and sample compositions, and it is particularly
well-suited for analyzing solid and semisolid materials.
10.11.7.1 Principle of TD-NMR
In TD-NMR, a sample is placed in a magnetic field, and radiofrequency pulses are
applied to excite the nuclear spins. After the excitation, the system’s return to
equilibrium (relaxation) is monitored. The relaxation of the nuclear magnetization
is recorded as a function of time, creating what is called the free induction decay
(FID). This FID signal contains valuable information about the molecular
10.11 Types of NMR Spectroscopy 477
environment and dynamics of the sample. Two primary relaxation times are
measured in TD-NMR:
• T1 relaxation time: This measures how quickly the nuclear spins transfer energy
to their surroundings (lattice) and return to thermal equilibrium.
• T2 relaxation time: This measures the dephasing of nuclear spins as they interact
with each other and lose coherence, causing the signal to decay.
10.11.7.2 Features of TD-NMR
• Relaxation (T1 and T2) time measurements: Unlike high-resolution NMR that
focuses on chemical shifts and fine spectral details, TD-NMR primarily measures
relaxation times. These relaxation parameters provide insights into molecular
motion, viscosity, and interactions between molecules.
• No Fourier transformation needed for basic applications: In TD-NMR, many
applications do not require the transformation of data into the frequency domain
using a Fourier transform (FT). Instead, the time-domain data itself provides
valuable information about the material’s properties.
• Nondestructive analysis: Like other NMR techniques, TD-NMR is nondestruc-
tive, allowing materials to be analyzed without being altered or consumed.
• Rapid measurements: TD-NMR offers fast and robust measurements, often in the
range of milliseconds to seconds, making it ideal for industrial applications where
high throughput is required.
10.11.7.3 Applications of TD-NMR
TD-NMR is widely used for practical and industrial applications, especially where
rapid and noninvasive analysis of bulk material properties is needed.
• Food industry: TD-NMR is used to measure moisture content, fat content, and
water distribution in foods such as meat, cheese, and bakery products. It is
particularly useful for quality control and determining the texture and freshness
of products.
• Polymers and plastics: TD-NMR is applied in the study of polymers to determine
crystallinity, phase composition, and cross-link density. These parameters help
assess the mechanical and thermal properties of materials such as rubber and
plastics.
• Pharmaceuticals:
TD-NMR
can be used to analyze the moisture content and
distribution in solid dosage forms, as well as to study the molecular dynamics of
active pharmaceutical ingredients and excipients. This information is critical for
optimizing drug stability and performance.
• Oil and petro
leum industry: In the oil industry, TD-NMR is used to measure
hydrogen content in crude oil and its derivatives. It helps determine properties
such as oil viscosity, porosity in rock samples, and the quantity of fluid in
geological formations.
478 10 Comprehensive Insights into Nuclear Magnetic Resonance Spectroscopy
• Porosity and pore size distribution: TD-NMR is used to study porosity in
materials such as porous rocks, soil, and construction materials. By measuring
relaxation times, it can reveal the size and distribution of pores in these materials.
• Moistur e and fat content: TD-NMR is used in industries to quantify the moisture
and fat content in various products, from agricultural products to foodstuffs. This
is important for both quality control and product formulation.
10.11.7.4 Advantages of TD-NMR
• Noninvas ive and nondestructive: TD-NMR allows the analysis of materials
without destroying or altering them, making it suitable for sensitive or valuable
samples.
• Fast and efficient: TD-NMR provides quick measurements, making it ideal for
routine industrial applications where speed is critical.
• Versatile: It can be used to analyze solids, semisolids, liquids, and gels, offering
versatility across multiple industries.
• Simple operation: TD-NMR systems are often designed for easy operation and
are used in industrial environments by nonspecialists. This ease of use makes
them well-suited for routine quality control tasks.
10.11.7.5 Limitations of TD-NMR
• Limited chemical information: TD-NMR does not provide detailed chemical shift
information, which is essential for structural analysis. It focuses more on physical
and dynamic properties such as relaxation times.
• Lower resolution: Compared to traditional high-resolution NMR, TD-NMR
offers less detailed spectral information. It is not suitable for resolving complex
chemical structures or small differences in chemical environments.
• Sensitivity: Some nuclei, such as
TD-NMR due to their lower natural abundance and sensitivity compared to
1
H NMR .
13
C and
15
N, may not provide strong signals in
10.11.8 In Vivo NMR Spectroscopy
In vivo NMR spectroscopy is a specialized application of NMR that allows for the
noninvasive study of biological processes, metabolites, and tissue composition
within living organisms. This technique is often used to study the molecular and
metabolic changes in tissues and organs in real-time, without needing to remove
samples or disrupt the natur al physiological environment. It is especially useful in
medical research and diagnostics, providing detailed insights into the biochemical
and metabolic state of tissues, particularly the brain, liver, and muscles.
10.11.8.1 Principle of In Vivo NMR Spectroscopy
In vivo NMR operates on the same principles as conventional NMR spectroscopy,
where atomic nuclei (commonly hydrogen, phosphorus, or carbon) resonate when
placed in a strong magnetic field and exposed to specific RF pulses. The nuclei’s
10.11 Types of NMR Spectroscopy 479
resonant frequencies provide information about their chemical environment, which
can be used to identify different molecules and their concentrations within the tissue.
However, in vivo NMR spectroscopy faces unique challenges, such as:
• Motion artifacts: Movements of the living organism (such as breathing or heart-
beat) can introduce noise into the data.
• Lower signal-to-noise ratio (SNR): Compared to in vitro NMR, the signal
strength is weaker due to the complexity and heterogeneity of biological tissues.
10.11.8.2 Common Nuclei Studied in In Vivo NMR
•1 H NMR: Proton NMR is the most commonly used form of in vivo NMR due to
the high natural abundance of hydrogen in water and organic molecules, making
it sensitive and capable of detecting a wide variety of metabolites.
31
P NMR: Phosphorus NMR is used to study energy metabolism in vivo by
•
monitoring the concentrations of phosphates, such as ATP, ADP, and phosphocreatine, which are essential for cellular energy transfer.
13
C NMR: Carbon NMR provides information about glucose metabolism, lipid
•
profiles, and other metabolic processes involving carbon-containing compounds,
though its low natural abundance and sensitivity require isotope labeling for
sufficient detection.
19
F NMR: Fluorine NMR is occasionally used for in vivo studies, especially in
•
cases involving fluorinated drugs, as fluorine is not naturally abundant in
biological systems, providing a clean background for drug tracking.
10.11.8.3 Features of In Vivo NMR Spectroscopy
• Noninvas ive and nondestructive: One of the biggest advantages of in vivo NMR
is its ability to observe metabolic and molecular changes in real-time within living
organisms, without damaging tissues or requiring invasive procedures.
• Metabolite monitoring: In vivo NMR spectroscopy can track the concentrations
and changes of metabolites such as lactate, glucose, and neurotransmitters (such
as glutamate and GABA), providing insights into the metabolic health of the
tissues being studied.
• Spatial localization: In vivo NMR often incorporates MRI to localize the region
of interest (ROI). This combination is called MRS and allows researchers to focus
on specific tissues or organs, combining metabolic and spatial data.
• Dynamic processes: It enables the real-time monitoring of biochemical changes
over time, allowing the study of dynamic physiological processes such as glucose
metabolism, energy production, and oxygen utilization.
10.11.8.4 Applications of In Vivo NMR Spectroscopy
In vivo NMR spectroscopy has a wide range of applications, especially in the
medical and biological sciences:
• Brain metabolism
and neurological disorders: In vivo NMR, particularly proton
and phosphorus NMR, is used to study brain metabolites such as lactate, N-acetyl
480 10 Comprehensive Insights into Nuclear Magnetic Resonance Spectroscopy
aspartate (NAA), and neurotransmitters. It is instrumental in understanding neurological disorders such as Alzheimer’s disease, Parkinson’s disease, epilepsy,
and brain tumors. Monitoring changes in these metabolites helps in assessing
disease progression and response to treatment.
• Cancer research: In vivo NMR spectroscopy is used to study tumor metabolism,
detect the presence of specific metabolites, and monitor the effects of anticancer
therapies. Abnormal metabolic profiles can reveal tumor growth and malignancy.
• Cardiovascular health: By examining metabolites in cardiac muscle, in vivo
NMR can track changes in energy metabolism (e.g., ATP, phosphocreatine)
during heart failure, ischemia, and other cardiovascular diseases. This helps in
the early detection of heart diseases and in evaluating the ef ficacy of therapeutic
interventions.
• Muscle metabolism: It is used to investigate muscle metabolism in both healthy
and diseased states, particularly in understanding conditions such as muscular
dystrophy and other metabolic myopathies. It can track changes in energy
metabolism during exercise or muscle recovery.
• Liver and kidney function: In vivo NMR can help monitor liver and kidney
metabolism, providing insights into diseases such as fatty liver disease, cirrhosis,
and kidney dysfunction by measuring metabolite concentrations and assessing
metabolic function in real-time.
• Pharmacokin etics and drug monitoring: Fluorine NMR can track fluorin
drugs in
vivo, providing detailed information on drug distribution, metabolism,
ated
and excretion within the body. This is particularly useful in preclinical studies and
drug development.
• Diabete s and metabolic disorders: In vivo NMR spectroscopy can be used to
study glucose metabolism and insulin sensitivity in tissues, which is important for
understanding diabetes and other metabolic disorders.
10.11.8.5 Advantages of In Vivo NMR Spectroscopy
• Real-time monitoring: Provides the ability to observe physiological and metabolic
changes as they happen, offering invaluable insight into dynamic processes.
• Noninvas ive: The nondestructive nature of the technique allows for longitudinal
studies, where the same subject can be studied multiple times over a period
of time.
• Versatile: In vivo NMR can be applied to a wide range of tissues and organs,
providing metabolic information that complements structural data from other
imaging techniques such as MRI.
• Combining with MRI:
can be combined with MRI to provide both spatial
MRS
and biochemical information, offering a complete picture of tissue health and
function.
10.11.8.6 Limitations of In Vivo NMR Spectroscopy
• Sensitivity: The sensitivity of in vivo NMR is lower compared to in vitro
techniques, making it harder to detect low-concentration metabolites or smaller
regions of interest.