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Nuclear magnetic resonance spectroscopy

Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful analytical technique used in chemistry,
physics, and biochemistry to study the properties of atomic nuclei. It exploits the magnetic properties of
certain atomic nuclei to provide valuable information about the structure, dynamics, and composition of
molecules. NMR is widely used in research and various industries, including pharmaceuticals, food
science, materials science, and environmental analysis.

Principle of NMR Spectroscopy: The basis of NMR spectroscopy lies in the behavior of atomic nuclei in a
magnetic field. Nuclei with an odd number of protons or neutrons possess a magnetic moment and act like
tiny magnets. When placed in an external magnetic field, these nuclei align with the field or against it,
resulting in two possible energy states. The energy difference between these two states depends on the
strength of the magnetic field and the properties of the nucleus.

How NMR Spectroscopy Works:

1. Sample Preparation: The sample, usually in liquid or solid form, is placed in a strong and uniform
magnetic field.
2. Resonance Absorption: A radiofrequency (RF) pulse is applied to the sample, causing the nuclei
to temporarily deviate from their equilibrium positions. When the RF pulse is removed, the nuclei
relax back to their equilibrium states, emitting radiofrequency signals in the process. The
frequency of these signals corresponds to the energy difference between the nuclear spin states.
3. Signal Detection: A sensitive radiofrequency detector picks up the emitted signals. These signals
are then mathematically processed to obtain an NMR spectrum.
4. Data Analysis: The NMR spectrum is analyzed to determine the chemical shifts, which provide
information about the local environment of specific nuclei in the molecule. It can also yield details
about molecular connectivity, dynamics, and interactions.

Chemical Shift and Peak Assignment: The chemical shift in NMR spectroscopy refers to the variation in
the resonance frequency of nuclei due to their chemical environment. It is expressed in parts per million
(ppm) and is referenced to a standard compound (usually tetramethylsilane, TMS) with a defined chemical
shift of 0 ppm. By analyzing the chemical shifts in an NMR spectrum, chemists can identify different types
of nuclei and gain insights into the molecular structure.

Applications of NMR Spectroscopy:

1. Structural Elucidation: NMR is extensively used to determine the structures of small organic
molecules, complex natural products, and biomolecules like proteins and nucleic acids.
2. Quantitative Analysis: NMR can be used for quantitative analysis, such as determining the
concentration of a specific compound in a mixture.
3. Drug Discovery: NMR plays a vital role in drug discovery, helping researchers understand
interactions between small molecules and biological targets.
4. Material Characterization: NMR can be used to investigate the properties of materials like
polymers, catalysts, and nanomaterials.
5. Quality Control: NMR spectroscopy is employed in industries like pharmaceuticals and food
science to assess the quality and purity of products.

NMR spectroscopy provides a non-destructive, highly versatile, and valuable tool for researchers and
scientists to gain valuable insights into the structure and properties of a wide range of molecules.
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Principles of H- NMR and C- NMR

Hydrogen Nuclear Magnetic Resonance (H-NMR) and Carbon Nuclear Magnetic Resonance
(C-NMR) are two specific applications of NMR spectroscopy, focusing on the analysis of
hydrogen and carbon nuclei, respectively, in organic molecules. While both techniques share
some common principles with NMR spectroscopy in general, they have specific features due
to the differences in the properties of hydrogen and carbon nuclei.

Principles of H-NMR:

1. Sensitivity to Hydrogen Nuclei: H-NMR spectroscopy is concerned with the behavior of

hydrogen nuclei (protons) in a magnetic field. Hydrogen is the most abundant nucleus in
organic compounds, making it a crucial target for NMR analysis.
2. Chemical Shift: The chemical environment around a hydrogen nucleus influences its
resonance frequency. The chemical shift in H-NMR represents the difference between the
resonance frequency of a particular hydrogen nucleus and a reference standard (TMS) and is
measured in parts per million (ppm). It provides valuable information about the local
electronic environment of hydrogen atoms in a molecule.
3. Integration: The area under each NMR peak in an H-NMR spectrum is proportional to the
number of equivalent protons responsible for that peak. Integration allows for quantitative
analysis and helps determine the relative abundance of different hydrogen environments in a
molecule.
4. Coupling: Hydrogen nuclei can interact with each other through a phenomenon called
coupling or spin-spin splitting. This results in a characteristic splitting pattern for each set of
coupled protons, providing valuable information about the neighboring hydrogen atoms.

Principles of C-NMR:

1. Sensitivity to Carbon Nuclei: C-NMR spectroscopy focuses on the behavior of carbon nuclei
in a magnetic field. Carbon-13 (13C) is the isotope of carbon usually studied because it has a
nuclear spin (I = 1/2) and is, therefore, NMR active.
2. Lower Sensitivity: Compared to hydrogen, carbon-13 has a lower natural abundance (about
1.1%), resulting in lower sensitivity in C-NMR. As a consequence, C-NMR spectra generally
require more time to acquire and may require larger sample amounts.
3. Chemical Shift: Similar to H-NMR, C-NMR also experiences chemical shifts due to the
influence of the local electronic environment on the resonance frequency of carbon nuclei.
The chemical shift values are measured in ppm with respect to a reference standard.
4. Lack of Coupling: In contrast to H-NMR, carbon nuclei have a low gyromagnetic ratio, and
their natural abundance is relatively low. As a result, they do not typically exhibit significant
spin-spin coupling with neighbouring carbon nuclei in organic molecules. This simplifies the
C-NMR spectra, and each carbon environment gives rise to a single peak.

Both H-NMR and C-NMR are essential tools for structural determination and analysis of organic
molecules. H-NMR provides detailed information about the hydrogen atoms and their local
environment, while C-NMR gives insights into the carbon atom connectivity and the overall
molecular framework. By combining the data from both techniques, chemists can elucidate the
complete structure of organic compounds.

Chemical shift, factors affecting chemical shift

Chemical shift is a fundamental concept in nuclear magnetic resonance (NMR) spectroscopy,
referring to the variation in resonance frequency of a nucleus due to its specific electronic
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environment within a molecule. It is measured in parts per million (ppm) and is referenced to a
standard compound, usually tetramethylsilane (TMS), which is assigned a chemical shift of 0 ppm.
The chemical shift is a critical parameter in NMR spectroscopy as it provides valuable information
about the local chemical environment of the NMR-active nucleus.

Factors Affecting Chemical Shift:

1. Electronegativity: The electronegativity of neighboring atoms has a significant impact on the

chemical shift. Electronegative atoms (e.g., oxygen, nitrogen, and halogens) pull electron
density away from the NMR-active nucleus, resulting in deshielding (higher chemical shift)
of the nucleus. In contrast, electron-donating groups tend to shield the nucleus, leading to a
lower chemical shift.
2. Hybridization: The hybridization state of the carbon or other NMR-active nuclei influences
the chemical shift. As the s-character in the hybridization increases, the electron density is
pulled closer to the nucleus, resulting in deshielding. For example, sp hybridized carbon
atoms generally have higher chemical shifts than sp3 hybridized carbon atoms.
3. Pi Bonding: In molecules with pi (π) bonds, such as alkenes, alkynes, and aromatic
compounds, the π electrons are above and below the molecular plane. These π electrons can
influence the magnetic field experienced by the NMR-active nucleus, causing deshielding and
resulting in higher chemical shifts.
4. Ring Currents: Aromatic compounds with delocalized π electrons generate ring currents,
which create an additional magnetic field. This can lead to either upfield (shielding) or
downfield (deshielding) shifts, depending on the orientation of the nucleus relative to the ring
current.
5. Hydrogen Bonding: Hydrogen bonding can influence the chemical shift of hydrogen nuclei
involved in the hydrogen bonding interactions. Hydrogen bonds can cause significant
deshielding, leading to higher chemical shifts for these protons.
6. Steric Effects: Steric hindrance around the NMR-active nucleus can affect the chemical shift.
Bulky substituents can shield the nucleus by blocking the access of external magnetic fields,
resulting in lower chemical shifts.
7. Magnetic Anisotropy: In molecules with asymmetric substituents, such as acetylenic or
alkynyl groups, the anisotropic distribution of electrons affects the chemical shift. This is
especially evident in solid-state NMR spectroscopy.
8. Solvent Effects: The choice of solvent can influence the chemical shift, particularly for
protons. Solvent molecules can interact with the solute, leading to changes in local electronic
environments and affecting the chemical shifts of certain nuclei.

Overall, chemical shift is a powerful tool for the structural elucidation of organic and inorganic
compounds. By analyzing the chemical shifts in NMR spectra, researchers can deduce valuable
information about molecular structure, connectivity, and electronic environments.

Coupling constant, Spin -spin coupling, relaxation , instrumentation and

application
Coupling Constant:

In nuclear magnetic resonance (NMR) spectroscopy, the coupling constant, often denoted as "J,"
refers to the interaction between two neighboring nuclei in a molecule. This interaction arises due to
the coupling of the nuclear magnetic moments of these nuclei. The coupling constant is a key
parameter that provides valuable information about the connectivity and relative arrangements of
atoms in a molecule.
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Spin-Spin Coupling:

Spin-spin coupling, also known as scalar coupling or J-coupling, is the specific interaction between
two nearby NMR-active nuclei with non-zero spins (e.g., protons, carbon-13). When two nuclei are
coupled, the magnetic field experienced by one nucleus is influenced by the spin state of the other
nucleus, resulting in splitting of NMR signals.

For example, in proton-proton coupling (H-H coupling), when one proton is in a magnetic field, its
spin can align with or against the field, creating two distinct energy levels. The presence of a
neighboring proton with a different spin state can cause the energy levels to split further, resulting in a
characteristic splitting pattern in the NMR spectrum. The number of lines in the splitting pattern (n+1
rule) depends on the number of equivalent neighboring nuclei (n) interacting with the target nucleus.

Relaxation:

Relaxation in NMR spectroscopy refers to the process by which the nuclear spins return to their
equilibrium positions after being perturbed by a radiofrequency pulse. There are two types of
relaxation processes:

1. T1 Relaxation (Longitudinal or Spin-Lattice Relaxation): T1 relaxation refers to the time it

takes for the nuclear spins to return to their equilibrium positions parallel to the magnetic field
(z-axis) after being tilted by a radiofrequency pulse. T1 relaxation is influenced by the
interactions of the nuclear spins with their surroundings (e.g., molecular motions, chemical
environment), and it provides information about the dynamics of the system.
2. T2 Relaxation (Transverse or Spin-Spin Relaxation): T2 relaxation refers to the decay of the
transverse magnetization after the radiofrequency pulse is applied. It is caused by interactions
between nuclear spins and each other, leading to dephasing of the spins. T2 relaxation
provides insights into the homogeneity and molecular motion in the sample.

Instrumentation:

NMR spectrometers consist of several essential components, including:

1. Magnet: A strong and uniform magnetic field is crucial for NMR spectroscopy.
Superconducting magnets are commonly used to provide a constant and stable magnetic field.
2. Radiofrequency (RF) System: The RF system generates the pulses used to excite and
manipulate the nuclear spins.
3. Probe: The NMR probe contains the sample and the RF coil for transmitting and receiving
NMR signals.
4. Data Acquisition System: This system captures the NMR signals, which are then processed
and transformed into an NMR spectrum.

Applications:

NMR spectroscopy has a wide range of applications in various fields:

1. Structural Elucidation: NMR is extensively used to determine the structures of organic

compounds, natural products, and biomolecules like proteins and nucleic acids.
2. Drug Discovery: NMR is employed in drug discovery to study ligand-receptor interactions
and assess the binding affinities of potential drug candidates.
3. Metabolomics: NMR is used in metabolomics studies to identify and quantify metabolites in
biological samples.
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4. Material Science: NMR spectroscopy is applied to study the structure and dynamics of
materials like polymers, catalysts, and nanomaterials.
5. Quality Control: NMR is used in industries like pharmaceuticals and food science to assess
the quality and purity of products.

These are just a few examples of the numerous applications of NMR spectroscopy, demonstrating its
versatility and importance as an analytical tool in various scientific disciplines.

mass spectroscopy - principles , fragmentation, ionisation techniques- electron impact,

chemical ionization, MALDI , FAB, Analysers - time of flight and quadrupole ,
instrumentation and application
Mass Spectrometry (MS) is an analytical technique used to determine the mass-to-charge ratio (m/z)
of ions, providing information about the molecular composition and structure of a sample. MS has
wide-ranging applications in various fields, including chemistry, biochemistry, pharmaceuticals,
environmental science, and forensic analysis.

Principles of Mass Spectrometry:

1. Ionization: The first step in MS involves ionizing the sample, converting neutral molecules
into charged ions. Various ionization techniques are used to achieve this.
2. Acceleration: The ions are accelerated by an electric field, which imparts kinetic energy to
them.
3. Deflection: The accelerated ions pass through a magnetic or electric field, which causes them
to deflect based on their m/z ratio. Lighter ions are deflected more than heavier ions.
4. Detection: The deflected ions are detected, and their m/z values are recorded, creating a mass
spectrum.

Fragmentation in Mass Spectrometry:

Fragmentation occurs when ions are subjected to collision-induced dissociation (CID) or other
processes that break the ions into smaller fragments. The resulting fragments provide information
about the structure and composition of the original molecule. Fragmentation patterns are unique to
different compounds and are crucial in identifying unknown substances.

Ionization Techniques:

1. Electron Impact (EI): In EI, a high-energy electron beam collides with the sample, leading to
the removal of an electron and the formation of positively charged molecular ions (M+•). EI
is commonly used in gas-phase samples and produces abundant fragmentation.
2. Chemical Ionization (CI): In CI, the sample is ionized through collisions with reagent ions,
often generated from a reagent gas such as methane or isobutane. CI typically produces
molecular ions (M+H or M+•) and less extensive fragmentation compared to EI.
3. Matrix-Assisted Laser Desorption/Ionization (MALDI): MALDI is a soft ionization technique
used primarily for large biomolecules like proteins and peptides. The sample is mixed with a
matrix, and a laser is used to ionize the analyte molecules from the solid matrix. MALDI
produces molecular ions and minimal fragmentation.
4. Fast Atom Bombardment (FAB): FAB is another soft ionization technique used for
biomolecules. High-energy atoms, such as xenon or argon, bombard the sample, leading to
desorption and ionization of the molecules.

Analyzers:
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1. Time-of-Flight (TOF) Analyzer: TOF analyzers measure the time taken for ions to travel a
fixed distance from the ionization source to the detector. The m/z ratio is calculated based on
the time of flight, and TOF MS offers high mass accuracy and sensitivity.
2. Quadrupole Analyzer: Quadrupole analyzers use oscillating electric fields to selectively filter
ions based on their m/z ratio. By adjusting the electric field parameters, specific ions can be
allowed to pass through the quadrupole and reach the detector, while others are excluded.

Instrumentation:

Modern mass spectrometers are typically equipped with ionization sources, analyzers (e.g., TOF,
quadrupole), ion detectors, and data analysis systems. Hybrid instruments, combining multiple
analyzers, have become common to provide enhanced capabilities for various applications.

Applications:

Mass spectrometry has diverse applications, including:

1. Identification of Unknown Compounds: MS is used to identify unknown substances by

comparing their mass spectra to databases of known compounds.
2. Proteomics and Protein Characterization: MS is a valuable tool in proteomics for identifying
and characterizing proteins in complex biological samples.
3. Drug Discovery: MS is employed to study drug metabolism, pharmacokinetics, and
interactions between drugs and receptors.
4. Environmental Analysis: MS is used to detect and quantify pollutants and environmental
contaminants.
5. Forensic Analysis: MS is utilized in forensic investigations for identifying illegal drugs,
explosives, and other trace evidence.

Overall, mass spectrometry is a versatile analytical technique with broad applications in various
scientific fields, enabling researchers to gain valuable insights into the structure and composition of
diverse substances.