Nuclear magnetic resonance (NMR) spectroscopy exploits the magnetic properties of atomic nuclei. It can provide information about the physical and chemical properties, structure, dynamics, and kinetics of biochemical systems. NMR spectroscopy works by aligning atomic nuclei with an external magnetic field and then stimulating them with radiofrequency pulses. This causes the nuclei to absorb and emit radiofrequency radiation at characteristic frequencies. Analyzing these frequencies yields information about the molecule's structure. NMR is widely used to determine the structures of organic molecules and biomolecules like proteins and nucleic acids.
Nmr nuclear magnetic resonance spectroscopyJoel Cornelio
Basics of NMR. Suitable for UG and PG courses.
Includes principle, instrumentation, solvents. chemical shift and factors affecting it. Some problems. resolving agents, coupling constant and much more
NMR, principle and instrumentation by kk sahu sirKAUSHAL SAHU
Introduction
History
Principle
Assembly
Solvents
Chemical shift
Factors affecting chemical shift
2D NMR
NOE effect
NOESY
COSY
Application
Conclusion
References
NMR SPECTROSCOPY ,Relaxation,longitudinal / spin- spin relaxation,transverse / spin- spin relaxation,Shielding of proton ,Deshielding of proton,CHEMICAL SHIFT,Factors Influencing Chemical Shift,Inductive effect, Vander Waal’s deshielding,Anisotropic effect (space effect),Hydrogen bonding
,SPLITTING OF THE SIGNALS,COUPLING CONSTANT,NMR SIGNAL IN VARIOUS COMPOUND
Nmr nuclear magnetic resonance spectroscopyJoel Cornelio
Basics of NMR. Suitable for UG and PG courses.
Includes principle, instrumentation, solvents. chemical shift and factors affecting it. Some problems. resolving agents, coupling constant and much more
NMR, principle and instrumentation by kk sahu sirKAUSHAL SAHU
Introduction
History
Principle
Assembly
Solvents
Chemical shift
Factors affecting chemical shift
2D NMR
NOE effect
NOESY
COSY
Application
Conclusion
References
NMR SPECTROSCOPY ,Relaxation,longitudinal / spin- spin relaxation,transverse / spin- spin relaxation,Shielding of proton ,Deshielding of proton,CHEMICAL SHIFT,Factors Influencing Chemical Shift,Inductive effect, Vander Waal’s deshielding,Anisotropic effect (space effect),Hydrogen bonding
,SPLITTING OF THE SIGNALS,COUPLING CONSTANT,NMR SIGNAL IN VARIOUS COMPOUND
Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy or magnetic resonance spectroscopy, is a spectroscopic technique to observe local magnetic fields around atomic nuclei.
It would be use full to All Needy People. It involve information about NMR Spectroscopy ( a spectroscopic techniques), factors influencing , proton NMR and their applications of NMR as well as Nuclear magnetic imaging.
Nuclear magnetic resonance (NMR) spectroscopyVK VIKRAM VARMA
SPECTROSCOPY
NMR SPECTROSCOPY
HISTORY
THEORY
PRINCIPLE
INSTRUMENTATION
SOLVENTS USED IN NMR(PROTON NMR)
CHEMICAL SHIFT
FACTORS AFFECTING CHEMICAL SHIFT
RELAXATION PROCESS
SPIN-SPIN COUPLING
푛+1 RULE
NMR SIGNALS IN VARIOUS COMPOUNDS
COUPLING CONSTANT
NUCLEAR MAGNETIC DOUBLE RESONANCE/ SPIN DECOUPLING
FT-NMR
ADVANTAGES & DISADVANTAGES
APPLICATIONS
REFERENCE
NMR Spectroscopy is a powerful technique that can provide detailed information on the topology, dynamics and three-dimensional structure of molecules in solution and the solid state
Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy or magnetic resonance spectroscopy, is a spectroscopic technique to observe local magnetic fields around atomic nuclei.
It would be use full to All Needy People. It involve information about NMR Spectroscopy ( a spectroscopic techniques), factors influencing , proton NMR and their applications of NMR as well as Nuclear magnetic imaging.
Nuclear magnetic resonance (NMR) spectroscopyVK VIKRAM VARMA
SPECTROSCOPY
NMR SPECTROSCOPY
HISTORY
THEORY
PRINCIPLE
INSTRUMENTATION
SOLVENTS USED IN NMR(PROTON NMR)
CHEMICAL SHIFT
FACTORS AFFECTING CHEMICAL SHIFT
RELAXATION PROCESS
SPIN-SPIN COUPLING
푛+1 RULE
NMR SIGNALS IN VARIOUS COMPOUNDS
COUPLING CONSTANT
NUCLEAR MAGNETIC DOUBLE RESONANCE/ SPIN DECOUPLING
FT-NMR
ADVANTAGES & DISADVANTAGES
APPLICATIONS
REFERENCE
NMR Spectroscopy is a powerful technique that can provide detailed information on the topology, dynamics and three-dimensional structure of molecules in solution and the solid state
Applications of IR (Infrared) Spectroscopy in Pharmaceutical Industrywonderingsoul114
Various applications of IR (Infrared) Spectroscopy in Pharmaceutical industries related to drug discovery and structural elucidation is outlined in this presentation. Various qualitative and quantitative analysis of drug products are also outlined.
NMR - Nuclear magnetic resonance (NMR).pptxmuskaangandhi1
Nuclear magnetic resonance (NMR) spectroscopy is the study of molecules by recording the interaction of radiofrequency (Rf) electromagnetic radiations with the nuclei of molecules placed in a strong magnetic field.
It is concerned with absorption of certain amount of energy
by spinning nuclei in a magnetic field when irradiated with
radiofrequency radiation (RFR) of equivalent energy.
NMR gives the information about the number and configuration of
magnetically active atoms, like positions of different types
of Hydrogen over the C- skeleton of an organic molecule.
Absorption of RFR occurs when the nucleus undergoes
transition from one alignment in the applied magnetic field
to the opposite alignment, i.e. from parallel (ground state)
orientation to anti-parallel (excited state) orientation.
When the frequency of the oscillating electric field of the
incoming RFR just matches the frequency of the electric field
generated by the precising nucleus, then the 2 fields can
couple, and the energy can be transferred from the
incoming radiation to the nucleus, thus causing a spin change
(clock-wise to anti-clock-wise).
This condition is called "resonance", and the nucleus is said to
have resonance with the incoming electromagnetic wave
(RFR).
In NMR technique, the frequency of the RFR is kept constant
(60MHz) and the strength of magnetic field is varied.
At certain value of the applied field strength, depending
upon the nature of proton or nucleus, the energy required to
flip the proton matches the energy of radiation.
As a result, absorption takes place and a signal is observed
in the spectrum. Such a signal or peak is called an NMR
Spectrum.
NMR spectrum is graphical plot of relative intensity
(Y axis) and the δ value (x axis).
The nucleus of a hydrogen atom (proton) behaves as a spinning bar magnet because it possesses both electric and magnetic spin.
Like any other spinning charged body, the nucleus of hydrogen atom also generates a magnetic field.
Nuclear magnetic resonance Involves the interaction between an oscillating magnetic field of electromagnetic radiation and the magnetic energy of the hydrogen nucleus or some other type of nuclei when these are placed in an external static magnetic field.
The sample absorbs electromagnetic radiations in radio wave region at different frequencies since absorption depends upon the type of protons or certain nuclei contained in the sample)
Consider a spinning top. It also performs a slower waltz like motion,
in which the spinning axis of the top moves slowly around
the vertical.
This is processional motion & the top is said to be processing around the vertical axis of earth's gravitational field.
The precession arises from the interaction of spin with earth's gravity acting vertically downwards.
It is called Gyroscopic motion.
Proton will be showing processional motion due to interaction of Spin &
Gravitational force of Earth
Explaining all the difficult concepts with precise and accurate points, 3D models, animations and smart art graphics.
Principle
The NMR phenomenon
Theory
Precessional frequency (ν)
Chemical shift
Spin-spin interactions
Interpretation of NMR
Chemical shift (δ)
Multiplicity of the signal
Coupling constant
Instrumentation
Fourier NMR
Continuous wave NMR
Applications
Identification testing
Assay of drugs
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Comparing Evolved Extractive Text Summary Scores of Bidirectional Encoder Rep...University of Maribor
Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
2. • Nuclear magnetic resonance spectroscopy, most commonly known as
NMR spectroscopy, is a research technique that exploits the magnetic
properties of certain atomic nuclei.
• This type of spectroscopy determines the physical and chemical
properties of atoms or the molecules in which they are contained.
• Nuclear magnetic resonance (NMR) spectroscopy is a powerful
technique that can be used to investigate the structure, dynamics, and
chemical kinetics of a wide range of biochemical systems.
• The first NMR derived three dimensional solution structure of a
small protein was determined in 1985 means NMR is about 25
years earlier than X-ray crystallography
3. • NMR spectroscopy can provide information about conformational
dynamics and exchange processes of biomolecules at timescales
ranging from picoseconds to seconds
• Efficient in determining ligand binding and mapping interaction
surfaces of protein/ligand complexes.
• Nowadays, three-dimensional structures can be obtained for proteins
up to 50 kDa molecular weight, and NMR spectra can be recorded
for molecules well above 100 kDa.
4. • This technique relies on the ability of atomic nuclei to behave
like a small magnet and align themselves with an external
magnetic field.
• When irradiated with a radio frequency signal the nuclei in a
molecule can change from being aligned with the magnetic
field to being opposed to it.
• The instrument works on stimulating the “nuclei” of the atoms
to absorb radio waves. The energy frequency at which this
occurs can be measured and is displayed as an NMR spectrum.
• The most common atomic nuclei observed using this
technique are 1
H and 13
C, but also 31
P, 19
F, 29
Si and 77
Se NMR are
available.
5. History
• 1946 Bloch, Purcell introduced about nuclear magnetic
resonance
• 1955 Solomon gave concept about NOE (nuclear Overhauser
effect)
• 1966 Ernst, Anderson introduced Fourier transform NMR
• 1975 Jeener, Ernst gave concept about 2D NMR
• 1985 Wuthrich first solution structure of a small protein
(BPTI) from NOE derived distance restraints
• 1987 3D NMR 13
C, 15
N isotope labeling of recombinant
proteins
6. • Two common types of NMR spectroscopy are used to
characterize organic structure:
– 1
H NMR:- Used to determine the type and number of H
atoms in a molecule
– 13
C NMR:- Used to determine the type of carbon atoms in
the molecule
6
7. • The source of energy in NMR is radio waves which have long
wavelengths having more than 107
nm, and thus low energy
and frequency.
• When low-energy radio waves interact with a molecule, they
can change the nuclear spins of some elements, including 1
H
and 13
C.
7
8. In a magnetic field, there are two energy states for a proton: a lower
energy state with the nucleus aligned in the same direction as Bo, and
a higher energy state in which the nucleus aligned against Bo.
When an external energy source that matches the energy difference
between these two states is applied, energy is absorbed, causing the
nucleus to “spin flip” from one orientation to another.
The energy difference between these two nuclear spin states
corresponds to the low frequency RF region of the electromagnetic
spectrum.
When a charged particle such as a proton spins on its axis, it creates a
magnetic field. Thus, the nucleus can be considered to be a tiny bar
magnet.
Normally, these tiny bar magnets are randomly oriented in space.
However, in the presence of a magnetic field B0, they are oriented
with or against this applied field.
8
9. More nuclei are oriented with the applied field because this
arrangement is lower in energy.
The energy difference between these two states is very small (<0.1
cal).
9
10. • Thus, two variables characterize NMR: an applied magnetic
field B0, the strength of which is measured in tesla (T), and
the frequency n of radiation used for resonance, measured
in hertz (Hz), or megahertz (MHz).
The energy difference between two nuclear spin states (v)
needed for resonance and the applied magnetic field strength
(B0) are proportionally related:
The stronger the magnetic field, the larger energy difference
between two nuclear spin states (v) and higher the ν needed for
the resonance.10
A nucleus is in resonance when it absorbs RF radiation and
“spin flips” to a higher energy state.
A nucleus is in resonance when it absorbs RF radiation and
“spin flips” to a higher energy state.
ν α BO
ν α BO
11. • Both liquid and solid type of samples can be used in NMR
spectroscopy.
• For liquid sample, conventional solution-state NMR spectroscopy is
used for analysing where as for solid type sample, solid-state
spectroscopy NMR is used.
• In solid-phase media, samples like crystals, microcrystalline
powders, gels, anisotropic solutions, proteins, protein fibrils or all
kinds of polymers etc. can be used.
• In liquid phase, different types of liquid solutions, nucleic acid,
protein, carbohydrates etc. can be used.
11
13. 1.Sample holder :- Glass tube with 8.5 cm long,0.3 cm in diameter
2.Permanent magnet :- It provides homogeneous magnetic field at 60-100
MHZ
3.Magnetic coils :- These coils induce magnetic field when current flows
through them.
4.Sweep generator :- To produce the equal amount of magnetic field pass
through the sample
Radiofrequency
Transmitter
Sweep
Generator
Radiofrequency
Amplifier
Audio
Amplifier
Detector
Oscilloscop
and / or
Recorder
14. • Radio frequency transmitter:- A radio coil transmitter that
produces a short powerful pulse of radio waves
• Radiofrequency :- A radio receiver coil that detects Receiver
radio frequencies emitted as nuclei relax to a lower energy
level
• Readout system :- A computer that analyses and record the
data
15. • All subatomic particles (neutrons, protons, electrons) have the
fundamental property of spin. This spin corresponds to a small
magnetic moment.
• In the absence of a magnetic field the moments are randomly
aligned. When a static magnetic field, Bo is applied this field
acts as a turning force that aligns the nuclear spin axis of
magnetic nuclei with the direction of the applied field
• This equilibrium alignment can be changed to an excited state
by applying radio frequency (RF) pulses.
• When the nuclei revert to the equilibrium they emit RF
radiation that can be detected
Principles of nuclear magnetic
resonance
16. • The nuclei of many elemental isotopes have a characteristic spin
(I). Some nuclei have integral spins (e.g. I = 1, 2, 3 ....), some
have fractional spins (e.g. I = 1/2, 3/2, 5/2 ....), and a few have
no spin, I = 0 (e.g. 12
C, 16
O, 32
S, ....).
• Isotopes of particular interest and use are 1
H, 13
C, 19
F and 31
P, all of
which have I = 1/2.
• A spinning charge generates a
magnetic field and resulting spin-
magnet has a magnetic moment (μ)
proportional to the spin.
• In the presence of an external
magnetic field (B0), two spin states
exist, +1/2 and -1/2.
17. • The difference in energy between the two spin states is
dependent on the external magnetic field strength, and is
always very small.
• Strong magnetic fields are necessary for nmr spectroscopy.
• Modern nmr spectrometers use powerful magnets having
fields of 1 to 20. Even with these high fields, the energy
difference between the two spin states is less than 0.1
cal/mole.
18. • For nmr purposes, this small energy difference (ΔE) is usually
given as a frequency in units of MHz (106
Hz), ranging from 20
to 900 Mz, depending on the magnetic field strength and the
specific nucleus being studied.
• For spin 1/2 nuclei the energy difference between the two spin
states at a given magnetic field strength will be proportional to
their magnetic moments.
• In order to induce nmr, a oscillatory magnetic field has to be
applied at the frequency which corresponds to the separation
(ΔΕ) of the two spin energy levels.
19. How does it work
• To get the nuclei in a molecule to all align in the same
direction, a very strong magnetic field is generated using a
superconducting electromagnet, which requires very low
temperatures to function.
• The coils of the magnet are surrounded by liquid helium
(-269°C), which is prevented from boiling off too quickly by a
surrounding layer of liquid nitrogen (-196°C). These coolants
are all contained in double-layer steel with a vacuum between
the layers, to provide insulation just like a thermos.
• There is a narrow hole through the middle of the magnet, and
the sample tube and radio frequency coils ("probe”) are
located there.
20. • A solution of the sample in a uniform 5 mm glass tube is oriented between the
poles of a powerful magnet, and is spun to average any magnetic field variations.
• Radio frequency radiation of appropriate energy is broadcast into the sample from
an antenna coil (colored red). A receiver coil surrounds the sample tube, and
emission of absorbed rf energy is monitored by dedicated electronic devices and a
computer.
Radio Frequency
Transmitter
Magnet
Pole
Sweep Generator
Sweep
Coils
Sweep
Coils
Spinning
Sample
tube
Magnet
Pole
Radio Frequency
Receiver & Amplifier
Control
Console and
Recorder
21. • An nmr spectrum is acquired by varying the magnetic field
over a small range while observing the rf signal from the
sample. An equally effective technique is to vary the
frequency of the rf radiation while holding the external field
constant.
22. Chemical shifts
• The exact resonance frequency depends on the chemical
environment of each spin, such that for example the NMR
spectrum of a protein will show NMR signals with slightly
different frequencies. These differences are called chemical
shifts.
• The first step of a structure determination by NMR consists of
assigning the chemical shifts of all the atoms/spins of the
molecule which are observed in an NMR spectrum.
• The resonance frequencies are called chemical shifts and are
measured in parts per million (ppm) in order to have chemical
shift values independent of the static magnetic field strength.
23. • Backbone amide protons HN
in a protein resonate around 8
ppm, while Hα
spins have resonance frequencies between 3.5-
5.5 ppm.
24. Factors affecting chemical shift:
• Electronegative groups
• Magnetic anisotropy of π-systems
• Hydrogen bonding
• Electronegative groups:- Electronegative groups attached to the C-H
system decrease the electron density around the protons, and there is
less shielding (i.e.deshielding) and chemical shift increases
• Magnetic anisotropy of π-systems:- The word "anisotropic" means
"non-uniform". So magnetic anisotropy means that there is a "non-
uniform magnetic field".
• Electrons in π systems (e.g. aromatics, alkenes, alkynes, carbonyls etc.)
interact with the applied field which induces a magnetic field that
causes the anisotropy. It causes both shielding and deshielding of
protons. Example:-Benzene Hydrogen bonding:- Protons that are
involved in hydrogen bonding are typically change the chemical shift
values. The more hydrogen bonding, the more proton is deshielded and
chemical shift value is higher.
25. Proton NMR
• The most common form of NMR is based on the hydrogen-1 (1H), nucleus or
proton. It can give information about the structure of any molecule containing
hydrogen atoms.
26. Nuclear overhauser effect
• NOE is the transfer of nuclear spin polarization from one spin to
another spin via cross-talk between different spins (normally protons)
in a molecule and it depends on the through space distance between
these spins.
• The local field at one nucleus is affected by the presence of another
nucleus. The result is a mutual modulation of resonance frequencies.
• NOEs are typically only observed between protons which are
separated by less than 5-6 Å.
• NOE is related to the three-dimensional structure of a molecule. For
interproton distances > 5 Å, the NOE is too small and not observable.
27. J coupling constants
• Provide information about dihedral angles, and thus can define
the peptide backbone and side chain conformations.
• Mediated through chemical bonds connecting two spins. The
energy levels of each spin are slightly altered depending on
the spin state of a scalar coupled spin (α or β).
29. NMR spectrum
• An NMR spectrum appears as a series of vertical peaks/signals
distributed along the x-axis of the spectrum.
• Each of these signals corresponds to an atom within the
molecule being observed
• The position of each signal in the spectrum gives information
about the local structural environment of the atom producing
the signal.
• As we move towards bigger molecules with more and more
atoms, the 1D spectra become very complex, and 2D and 3D
spectroscopy becomes important in understanding the
relationships and interactions between different atoms in the
molecule.
30. • The information contained in 1D spectra can be expanded in a
second (frequency) dimension - 2D NMR
• In a 1D experiment a resonance (line) is identified by a single
frequency: NH(f1nh)
• In 2D spectra, a resonance (cross-peak) is identified by two
different frequencies: NH (f1nh, f2ha), NH (f1nh, f2ha)
• Usually, the second frequency depends on how the NMR
experiment is designed.
31. Resonance assignment
• The crosspeaks in NOE spectra cannot be interpreted without knowledge
of the frequencies of the different nuclei
• The frequencies can be obtained from information contained in COSY
(correlation spectroscopy) spectra
• The process of determining the frequencies of the nuclei in a molecule is
called resonance assignment (and can be lengthy...)
• Two-dimensional COSY NMR experiments give correlation signals that
correspond to pairs of hydrogen atoms which are connected through chemical
bonds.
• COSY spectra show frequency correlations between nuclei that are connected
by chemical bonds
• Since the different amino acids have a different chemical structure they give
rise to different patterns in COSY spectra. This information can be used to
determine the frequencies of all nuclei in the molecule. This process is called
resonance assignment
• Modern assignment techniques also use information from COSY experiments
with 13C and 15N nuclei
32. • NOE Spectroscopy experiments
give signals that correspond to
hydrogen atoms which are close
together in space (< 5A), even
though they may be far apart in the
amino acid sequence.
• Structures can be derived from a
collection of such signals which
define distance constraints between
a number of hydrogen atoms along
the polypeptide chain.
Example: short distance (< 5 A, NOE)
correlations between hydrogen atoms in a
helix
34. • NMR is used in biology to study the Biofluids, cells, organs and
macromolecules such as Nucleic acids (DNA, RNA), carbohydrates
Proteins and peptides and also Labeling studies in biochemistry.
• NMR is used in physics and physical chemistry to study High pressure
diffusion ,liquid crystals, Membranes.
• 3D structure determination of proteins, nucleic acids, protein/DNA
complexes, ...)
• NMR is used in pharmaceutical science to study Pharmaceuticals and
Drug metabolism.
• NMR is used in chemistry to determine the Enantiomeric purity.
Elucidate Chemical structure of organic and inorganic compounds.
• 1
H widely used for structure elucidation.
Inorganic solids- Inorganic compounds are investigated by solid state
1
H-NMR.eg CaSO4 H2O.⋅
Organic solids- Solid-state 1
H NMR constitutes a powerful approach to
investigate the hydrogen-bonding and ionization states of small organic
compounds.
Applications of NMR
35. • Direct correlation with hydrogen-bonding lengths could be
demonstrated, e.g. for amino acid carboxyl groups.
• Polymers and rubbers- Examine hydrogen bonding and acidity.
• In vivo NMR studies- concerned with 1H NMR of human brain. Many
studies are concerned with altered levels of metabolites in various brain
diseases.
• To determine the spatial distribution of any given metabolite detected
spectroscopically IS (image selected in vivo spectroscopy).
• MRI is specialist application of multi dimensional Fourier
transformation NMR for Anatomical imaging, for measuring
physiological functions, for flow measurements and angiography, for
tissue perfusion studies and also for tumors.
36. Limits
• Molecular weight limits for protein structure calculation (monomer):
5-15 kDa: routine
15-20 kDa: usually feasible
20-30 kDa: long term project
40-50 kDa: in the next future?
• Molecular weight limits for peptide/protein, protein/protein interactions
(MW of the AB complex, A < 10 kDa):
20-30 kDa: routine
30-50 kDa: feasible
50-100 kDa: in the next future
37. Advantages of NMR Limitation of NMR
Obtain angles, distances, coupling constants,
chemical shifts, rate constants etc. These are
really molecular parameters which could be
examined more with computers and molecular
modeling procedures.
This is good for the more accurate determination
of the structure, but not for the availability of
higher molecular masses
With a suitable computer apparatus we can
calculate the whole 3D structure
The resolving power of NMR is less than some
other type of experiments (e.g.: X-ray
crystallography) since the information got from
the same material is much more complex
There are lots of possibilities to collect different
data-sets from different types of experiments for
the ability to resolve the uncertanities of one
type of measurements
The highest molecular mass which was examined
successfully is just a 64kDa protein-complex
This method is capable to lead us for the
observation of the chemical kinetics
There are lots of cases when from a given data-
set - a given type of experiment – we may
predict two or more possible conformations, too
We can investigate the influence of the dielectric
constant, the polarity and any other properties of
the solvent or some added material
The cost of the experimental implementation is
increasing with the higher strength and the
complexity of the determination
38. Disadvantages
1) Sensitivity
• The greatest disadvantage of NMR spectroscopy and imaging compared with
other modalities is the intrinsic insensitivity of the methods. The signal that
can be generated in the
• NMR experiment is small and, for practical purposes, most strongly coupled
with the concentration of the nuclei in the sample.
• For example, the human body is composed of -70% water, and thus a
relatively large signal can be obtained from the 1
H nucleus in water that is
effectively at a concentration in the tens of molar range.
• Thus, it is possible to measure signals from cubes (voxels) of tissue as small as
= 0.3mm on a side from the human brain, generating the high-quality.
• The NMR signals from water will always be detectable at resolutions
approximately two orders of magnitude greater than those of other NMR-
sensitive nuclei.
• Thus, compounds present in submillimolar and certainly micromolar
concentrations cannot practically be detected directly in tissues.
• As a result, the sample size generally dictates the choice of magnet and field
strength; thus, the smaller the sample, the more sensitive the experiments.
39. 2) Working in a High-Magnetic-Field Environment
• An inevitable consequence of carrying out NMR investigations is the need to
work in a high-magnetic-field environment.
• No known intrinsic risks are associated with high magnetic fields; however, the
presence of the magnetic field can affect equipment routinely used in animal
research.
• For example, electronic monitors and computer-controlled devices may
function improperly or not at all.
• Due to the nature of the forces involved, the result can be a scalpel, a pair of
scissors, or even a gas cylinder becoming a flying object.
• Instruments that can be obtained that are non ferromagnetic, thus reducing the
potential difficulties associated with working in a high-magnetic-field
environment.
• Now a days, a steel passive shield or an active shield may be placed around the
magnet to reduce the magnet fringe fields and minimize the risks.
• This reduction can be particularly important when the space available to site
the instrument is limited.
40. 3) Motion Sensitivity
• Most MR techniques are motion sensitive. This sensitivity leads to
signal distortions that are visually most evident in artifacts on images
or more subtly in quantitative measurements.
• Some MR techniques such as functional MRI (fMRI1) are particularly
sensitive to motion artifact, thus great care must be taken not to
minimize the distorting effects of motion and thus minimize
misinterpretation of data.
• In animal studies, anesthesia is usually essential to avoid gross
movement of the animal during the study.
• Cardiac gating may be necessary even when imaging other organs
such as the brain because of motion caused by the pulsatile blood
flow. In some cases, such as lung imaging, it may also be necessary to
gate to respiratory motion or, alternatively, the subject may be
controlled via mechanical ventilation.
41. Examples/ uses of NMR Spectroscopy
• Several different NMR-sensitive nuclei can be used in the study of
biological systems, and the most common are 31
P, 1
H, 13
C, 23
Na, and 19
F.
• 31
P, 1
H, and 13
C-NMR spectroscopy are typically used to investigate
cellular metabolism and bioenergetics.
• Whereas 23
Na NMR studies usually focus on issues related to ion
transport and regulation of ion pumps. Fluorine does not occur naturally
in biological systems.
• However, it is a very sensitive NMR nucleus. Therefore, fluorine-
labeled compounds can be introduced into cells and used as an indicator
of a cellular process such as calcium concentration.
• 19
F-NMR spectroscopy has also been used to follow the metabolism of
drugs, such as 5-fluorouracil.
42. NMR and X-ray crystallography are
complementary
• Molecules are studied in solution.
• Protein folding studies can be done by monitoring NMR spectra
• Denatured states of a biomolecule, folding intermediates and even
transition states can be characterized
• Conformational or chemical exchange, internal mobility and
dynamics at timescales ranging from picoseconds to seconds
• Very efficient in mapping interactions with other molecules
• Upper weight limit for NMR is ~ 50 kDa
Editor's Notes
This important and well-established application of nuclear magnetic resonance will serve to illustrate some of the novel aspects of this method. To begin with, the nmr spectrometer must be tuned to a specific nucleus, in this case the proton. The actual procedure for obtaining the spectrum varies, but the simplest is referred to as the continuous wave (CW) method. A typical CW-spectrometer is shown in the following diagram. A solution of the sample in a uniform 5 mm glass tube is oriented between the poles of a powerful magnet, and is spun to average any magnetic field variations, as well as tube imperfections. Radio frequency radiation of appropriate energy is broadcast into the sample from an antenna coil (colored red). A receiver coil surrounds the sample tube, and emission of absorbed rf energy is monitored by dedicated electronic devices and a computer. An nmr spectrum is acquired by varying or sweeping the magnetic field over a small range while observing the rf signal from the sample. An equally effective technique is to vary the frequency of the rf radiation while holding the external field constant.