BIO5061B Bioanalytical technqiues

Introduction
NMR Spectroscopy is abbreviated as Nuclear Magnetic
Resonance spectroscopy.
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.
Zeeman first observed the strange behavior of certain
nuclei when subjected to a strong magnetic field at the
end of the nineteenth century, but the practical use of the
so-called “Zeeman effect” was only made in the 1950s
when NMR spectrometers became commercially
available.
The NMR spectroscopy determines the physical and
chemical properties of atoms or molecules.
 NMR Spectroscopy
Where is it?

1nm 10 102 103 104 105 106 107

(the wave) X-ray UV/VIS Infrared Microwave Radio

Frequency
(the transition) electronic Vibration Rotation Nuclear
(spectrometer) X-ray UV/VIS Infrared/Raman NMR
Fluorescence
 The problem the we want to
solve by NMR

What we want to “see”

What we “really” see

NMR
 Before using NMR
What are N, M, and R ?
Properties of the Nucleus
Nuclear spin
Nuclear magnetic moments

The Nucleus in a Magnetic Field

Precession and the Larmor frequency
Nuclear Zeeman effect & Boltzmann distribution

When the Nucleus Meet the right Magnet and radio wave
Nuclear Magnetic Resonance
 Solvents used in NMR
• Solvent used in NMR spectroscopy should have the following properties:
• ✓ Non viscous.
• ✓ Should dissolve analyte.
• ✓ Should not absorb within spectral range of analysis.
• ✓ All solvents used in NMR must be aprotic that is they should not possess
proton.
• • Chloroform-d (CDC|3) is the most common solvent for NMR measurements
• • Other deuterium labeled compounds, such as deuterium oxide (D20), benzene-
d6 (C6D6), acetone-d6 (CD3COCD3) and DMSO-d6 (hexaduetereo
dimethylsulfoxide) are also available for use as NMR solvents.
• • DMF (dimethylformide), DMSO, cyclopropane, dimethyl ether can also be used.
 Properties of the Nucleus​
Nuclear spin ​
• Nuclear spin is the total nuclear angular momentum quantum
number. This is characterized by a quantum number I, which may be
integral, half-integral or 0.​
• Only nuclei with spin number I 0 can absorb/emit electromagnetic
radiation. The magnetic quantum number mI has values of –I, -I+1, …..+I
.
( e.g. for I=3/2, mI=-3/2, -1/2, 1/2,3/2)
​1. A nucleus with an even mass A and even charge Z nuclear spin I is zero​
Example: 12C, 16O, 32S No NMR signal​
2. A nucleus with an even mass A and odd charge Z integer value I ​
Example: 2H, 10B, 14N NMR detectable​
3. A nucleus with odd mass A I=n/2, where n is an odd integer​
Example: 1H, 13C, 15N, 31P NMR detectable
 Nuclear magnetic moments​

Magnetic moment μ is another important parameter for a nuclei ​

μ =  I (h/2π) ​
I: spin number; h: Plank constant; ​
 : gyromagnetic ratio (property of a nuclei)​
1H: I=1/2 ,  = 267.512 *106 rad T-1S-1​
13C: I=1/2 ,  = 67.264*106 ​
15N: I=1/2 ,  = 27.107*106​
 Nuclear Zeeman effect​​

Zeeman effect: when an atom is placed in an external magnetic field, the

energy levels of the atom are split into several states. ​
• The energy of a given spin sate (Ei) is directly proportional to the
value of mI and the magnetic field strength B0​
Spin State Energy EI=- . B0 =-mIB0 r(h/2p)​
• Notice that, the difference in energy will always be an integer multiple of
B0r(h/2p). For a nucleus with I=1/2, the energy difference between two
states is​
ΔE=E-1/2-E+1/2 = B0 r(h/2p)​
The Zeeman splitting is proportional to the strength of the magnetic field​
 Boltzmann distribution​​
•Quantum mechanics tells us that, for net absorption of radiation to
occur, there must be more particles in the lower-energy state than in the
higher one. If no net absorption is possible, a condition called saturation.​
• When it’s saturated, Boltzmann distribution comes to rescue:​
•Pm=-1/2 / Pm=+1/2 = e -DE/kT ​
where P is the fraction of the particle population in each state, ​
T is the absolute temperature, ​
k is Boltzmann constant 1.381*10-28 JK-1​
​
• Example: At 298K, what fraction of 1H nuclei in 2.35 T field are in the
upper and lower states? (m=-1/2 : 0.4999959 ; m=1/2 :
0.5000041 )​
• The difference in populations of the two states is only on the order
of few parts per million. However, this difference is sufficient to
generate NMR signal. ​
• Anything that increases the population difference will give rise to a more
intense NMR signal.
 When the Nucleus Meet the Magnet​
Nuclear Magnetic Resonance​
​
•For a particle to absorb a photon of electromagnetic radiation, the particle must first be
in some sort of uniform periodic motion​
​
• If the particle “uniformly periodic moves” (i.e. precession)​
at vprecession, and absorb energy. The energy is E=hvprecession​
​
•For I=1/2 nuclei in B0 field, the energy gap between two spin states:​
DE=rhB0/2p
• The radiation frequency must exactly match the precession frequency​
E =
photon hv hv
precession= photon=DE=rhB0/2p ​

This is the so called “ Nuclear Magnetic RESONANCE”!!!!!!!!! ​

Instrumentation,
working and
principle
 1. The principle behind NMR is that many nuclei have spin and all nuclei are
electrically charged.
Principle of Nuclear 2. If an external magnetic field is applied, an energy transfer is possible between the
base energy to a higher energy level (generally a single energy gap).
Magnetic Resonance 3. The energy transfer takes place at a wavelength that corresponds to radio
(NMR) Spectroscopy frequencies.
4. and when the spin returns to its base level, energy is emitted at the same
frequency. The signal that matches this transfer is measured in many ways and
processed in order to yield an NMR spectrum for the nucleus concerned.
Instrumentation
1.Sample holder - Glass tube with 8.5 cm long, 0.3 cm
in diameter.​
2.Permanent magnet - It provides a
homogeneous magnetic field at 60-100 MHz (or 1.4
Tesla) ​
3.Sweep coils - These coils induce a magnetic
field when current flows through them​
4.Sweep generator - To produce an equal amount of
magnetic field pass through the sample​
5.Radio frequency transmitter/Generator - A
radio transmitter produces a short powerful pulse
of radio waves​
6.Radio frequency receiver/detector - A radio receiver
coil that detects radio frequencies emitted as nuclei relax
to a lower energy level ​
7.Recorder - A computer that analyses and records the
data​
 Working of Nuclear Magnetic
Resonance (NMR) Spectroscopy
• The sample is placed in a magnetic field and the NMR
signal is produced by excitation of the nuclei sample
with radio waves into nuclear magnetic resonance, which
is detected with sensitive radio receivers.
• The intramolecular magnetic field around an atom in a
molecule changes the resonance frequency, thus giving
access to details of the electronic structure of a molecule
and its individual functional groups.
• As the fields are unique or highly characteristic to
individual compounds, NMR spectroscopy is the
definitive method to identify monomolecular organic
compounds.
• Besides identification, NMR spectroscopy provides
detailed information about the structure, dynamics,
reaction state, and chemical environment of molecules.
• The most common types of NMR are proton and carbon-
13 NMR spectroscopy, but it is applicable to any kind of
sample that contains nuclei possessing spin.
Interpreting
NMR spectra
 NMR spectroscopy graph
A plot of the frequencies of the absorption peaks versus peak
intensities constitutes an NMR spectrum. The signals on the left side of
the spectrum(downfield) are high-frequency signals. Signals on the
right side of the spectrum(up field) are low frequencies signals because
they result from shielded protons that absorb lower frequencies of
radiation
 Chemical shift in
NMR spectroscopy
Chemical shift is defined as the difference in parts per million(ppm)
between the resonance frequency of the observed proton and that of
the reference compound, tetramethyl silane(TMS).

Why tms is used as reference

compound in NMR spectroscopy?
Tetramethyl silane is a universally accepted reference compound because of the
following facts.
1.TMS is chemically inert, symmetrical, volatile and soluble in most organic
solvent.
2.TMS gives a signal, intense, sharp NMR peak and its protons are more shielded
than almost all other protons in the organic compounds.
Factors affecting chemical shift
Chemical shifts is influenced by the following factors;

ELECTRONEGATIVE GROUPS:
• More electronegative atoms Deshields more and give larger shift values.
• Effect decreases with distance.
• Additional electronegative atoms cause increase in chemical shift.
• Attachment of electronegative atom cause a downward shift.

Anisotropic effect:
• The word anisotropic means “non uniform”. So magnetic anisotropy
means that there is a non uniform magnetic field. • Electron in a system
(aromatics, alkenes, alkynes, carbonyl etc.) interact with the applied field
that cause the anisotropy.
• It cause both shielding and deshielding.
Hydrogen bonding:
Protons that are involved in chemical bonding are typically change
the chemical shift values.
The more hydrogen bonding is , the more proton is deshielded and
chemical shift value is higher.
 Coupling Constants
The distance between the peaks in a given multiplet is a measure of the
magnitude of splitting effect. , it is referred to as coupling constant and is denoted
by the symbol J. Numerical value of J is expressed in Hz or cps.
Unlike the chemical shifts, the value of J is independent of the applied field
strength and depends only upon the molecular structure.
For a pair of mutually coupled protons, the coupling constant due to splitting by
one proton has the same value as the coupling constant due to the splitting by the
second proton. In other words, mutually coupled protons show the same
magnitude of the splitting of each other signals.
In general, the size of coupling constant is determined by the number and kind of
intervening chemical bonds and the spatial relations between the protons as
illustrated below as few cases.
(a) For protons attached to the same carbons atom (i.e., germinal protons), the
values of J varies from 0-20 Hz depending upon the bond angle and overall
structure of the molecule.
(b) For protons attached to adjacent carbon atoms (i.e., vicinal protons), J varies
from 2-18 Hz depending upon the spatial positions and the structure of the
molecules as a whole.
 Ring Current
The ring current is induced from the delocalized π electron in a
magnetic field and generates an additional magnetic field. In the
center of the arene ring this induced field is in the opposite direction t
is the external magnetic field.
 Spin-Spin Coupling
The interaction between the spin magnetic moments of the different
sets of H atoms in the molecule under study, is known as spin-spin
coupling. It is imperative that a minimum of 2 sets of protons are
present in adjacent positions.
The magnetic spins of these resonating nuclei interact with each other
and affect each other’s precession frequencies.
The effective magnetic field (Beff) experienced by neighboring protons
as a result of magnetic spins thereby affect the chemical shift
values. [5] In addition to the chemical shifts, the nature of the peaks in
the NMR spectrum is also affected.
 NMR relaxation​
The term relaxation indicates the process by which the magnetic atomic nuclei
reach thermal equilibrium with the chaotic molecular environment. In NMR, this
process can be very slow, requiring between a fraction of a second to many
minutes, depending on the sample and the environmental conditions. To NMR
spectroscopists, relaxation is a friend, since it generates the nuclear
magnetisation from which all NMR signals are derived; at the same time,
relaxation can be a foe, since it limits how long any different forms of spin order
last. For many years, it was generally thought that relaxation was intrinsic to the
sample, and could not readily be manipulated or overcome. However, recent
experiments have shown that in some cases the nuclear spin systems may be
held in special configurations called long-lived states that are to some extent
protected against relaxation.
 Spin-Lattice(T1) Relaxation
Spin-lattice relaxation occurs by transfer of energy to the surroundings (heat); dipolar coupling to
other spins.
Since molecule contains magnetic nuclei, the random nuclear motion will cause these nuclei to
give rise to fluctuating magnetic fields. When this field is properly phased (to match processional
frequency) and properly oriented, a nucleus which is in an upper spin state can be relaxed to the
ground state by imparting its excess energy to the lattice as rotational and translational energy.
This mechanism for spin-relaxation is referred as nuclear-dipolar interaction. The total energy of
the system is unchanged by this process, and the efficiency of the relaxation mechanism depends
upon (a) magnitude of the local fields (b) the rate of fluctuation of the local fields. A quantity
referred to as the spin lattice relaxation time, T1 , can be defined to indicate the rate of this
proves. A large value for T1 , indicates an inefficient relaxation process, and a long lifetime for the
excited state. In the absence of other effects, a sharp line results as predicted by equation: W α
1/t, when T1 is large.
 Spin-Lattice(T1) Relaxation

Gain and loss of magnetization in the z-direction.

NMR lines are at least as wide as specified by the Heisenberg Uncertainty
Principle broadening due to inherent lifetime of spin states (the actual width is
governed by T2 ).
For most spin 1/2 nuclei T2 is between 0.2 and 50 seconds.
 Spin-Spin(T2) Relaxation
T2 relaxation is caused by transient magnetic fields (usually due to molecular
motion) at any frequency. Thus T2 keeps getting shorter as molecular
reorientation rates slow down.
T2 relaxation is also caused by swapping of chemical shifts or coupling constants
due to chemical exchange or interconversion of conformations. Line broadening
due to chemical exchange provides an important tool for measurement of the
rates of a variety of molecular processes.
A third source of T2 relaxation is interconversion of spin states of a quadrupolar
nucleus coupled to it.
 Conclusion
• T2 ≤ T1
• T1 and T2 are routinely equivalent for most NMR
experiments
• NMR Linewidths ~1/ T2 for spin ½ nuclei
• NMR spectra of solids are very broad because T2 is short.
• Because of the very broad lines usually observed when
solid spectra are examined, most inorganic applications
are carried out on gases, liquids, or solutions.
Nuclear Overhauser Effect(NOE)​
The nuclear Overhauser effect (NOE) is the transfer of nuclear spin polarization from one
population of spin-active nuclei (e.g. 1H, 13C, 15N etc.) to another via cross-relaxation. A
phenomenological definition of the NOE in nuclear magnetic resonance spectroscopy (NMR) is the
change in the integrated intensity (positive or negative) of one NMR resonance that occurs when
another is saturated by irradiation with an RF field. The change in resonance intensity of a nucleus
is a consequence of the nucleus being close in space to those directly affected by the RF
perturbation.
The NOE is particularly important in the assignment of NMR resonances, and the elucidation and
confirmation of the structures or configurations of organic and biological molecules. The 1H two-
dimensional NOE Spectroscopy (NOESY) experiment and its extensions are important tools to
identify stereochemistry of proteins and other biomolecules in solution, whereas in solid form
crystal x-ray diffraction typically used to identify stereochemistry. The heteronuclear NOE is
particularly important in 13C NMR spectroscopy to identify carbons bonded to protons, to provide
polarization enhancements to such carbons to increase signal-to-noise, and to ascertain the extent
the relaxation of these carbons is controlled by the dipole-dipole relaxation mechanism.
Chemical Exchange​
Chemical exchange refers to any process in which a nucleus
exchanges between two or more environments and results in a
change in chemical shift, scalar coupling, and/or NMR
relaxation rate. This change of nuclear environment can occur
as part of a chemical reaction (whether bond-breaking or bond-
making) or by a conformational change (bond rotation). The
effect that this change has on the NMR spectrum depends on
the rate of the exchange process relative to the NMR time
scale, which is of the order of milliseconds and represents the
time taken to record one NMR spectrum
Types of NMR
Adv.& Disadv
Applications
 Types of NMR Spectroscopy
•Two common types of NMR Spectroscopy which
are used to characterize organic structure:​
1. H NMR​
2. C NMR​
​

​
 1H NMR Spectroscopy
• Used to determine the type and number of H atoms in a molecule.​
•H NMR is the go-to technique to help identify or confirm the structure of organic
compounds or those that contain protons. A solution-state proton spectrum is
relatively fast to acquire, compared with other nuclei, and a lot of information about
the structure of a compound can be deduced from it.​
•H NMR spectroscopy is used more often than 13C NMR, partly because proton
spectra are much easier to obtain than carbon spectra. ​
•The 13C isotope is only present in about 1% of carbon atoms, and that makes it
difficult to detect.​
•The 1H isotope is almost 99% abundant, which helps make it easier to observe​
 ¹³C NMR Spectroscopy
​
•Carbon-13 nuclear magnetic resonance is the application of
nuclear magnetic resonance spectroscopy to carbon. ​
•It is analogous to proton NMR and allows the identification of
carbon atoms in an organic molecule just as proton NMR identifies
hydrogen atoms. ​
•¹³C NMR detects only the ¹³ C isotope​

​
 Advantages
1. Ability to analyse intact algae​
2. Structural Details can be obtained​
3. Non Destructive analysis​
4. Lower maintenance Cost​
5. Lower analysis time​
6. Easy sample preparation​
7. Ability to distinguish between polar and neutral lipids when combined with "P NMR​
8. Allows for continuous monitoring of oil content in growing cells​
9. Automated Technique​
10. Require very small sample volumes​
11. Ability to measure TAG content across all oleaginous micro algae species
producing triacylglycerides. ​
12. TD-NMR is an extremely fast technique ​
13. High reproducibility​
14. Can be used as an online technique for continuous lipid analysis, under different stages
and cultivation conditions, as live cells can be analyzed directly.​
15. Can be used to study oxidation of lipids and biodiesel.​
 Disadvantages
1. Expensive instrument​
2. Relatively new technique​
3. Use of C NMR requires large sample volume,
because of its lower natural abundance​
4. Although more accurate, compared to TD and 'H
NMR, C NMR is time taking.​
5. TD-NMR provides limited qualitative results​
 Applications
Spectroscopy is the study of the interaction of electromagnetic radiation
with matter. NMR spectroscopy is the use of the NMR phenomenon to
study the physical, chemical, and biological properties of matter.
1.It is an analytical chemistry technique used in quality control.
2.It is used in research for determining the content and purity of a
sample as well as its molecular structure. For example, NMR can
quantitatively analyze mixtures containing known compounds.
3.NMR spectroscopy is routinely used by chemists to study chemical
structure using simple one-dimensional techniques. Two-dimensional
techniques are used to determine the structure of more complicated
molecules.
4.These techniques are replacing x-ray crystallography for the
determination of protein structure.​
5.Time domain NMR spectroscopy techniques are used to probe
molecular dynamics in solution.​
6.Solid state NMR spectroscopy is used to determine the molecular
structure of solids.​
7.Other scientists have developed NMR methods-of measuring
diffusion coefficients.​
​
​
8.Identification Of Substances​
•Like infrared spectrum, the PMR spectrum of a substance also serves as the
'fingerprint' of the substance.​
•If the PMR spectrum of a given sample is exactly the same as that of a
known substance, the given substance must be identical with the known
substance. In this way PMR helps in establishing the identities of substances.​
​9.Distinction between geometrical isomers.​
•Due to the difference in the chemical shifts as well as coupling constants,
the cis- and trans- isomers of a compound can be easily distinguished from
each other.​
​
​

10. Detection of hydrogen bonding.​

•If a hydrogen atom is involved in hydrogen bonding it will get deshielded due to the
strongly electronegative atom attached to it. As such absorption is shifted downfield.​
•The extent of inter-molecular hydrogen bonding varies with the nature of solvent,
concentration of solution and the temperature.​
•For example, in concentrated solution hydroxyl protons absorb around 4-5 8 while in
dilute solution (where there is much less hydrogen bonding) the absorption takes place
at higher field around 1.0 8.​
•The intramolecular hydrogen bonding also shifts the absorption downfield but the
shift does not depend upon concentration. This observation can be used for
distinguishing between inter and intra molecular hydrogen bonding.​
11. Also helps in proton exchange process.​
12. It is used for quantitative analysis of mixtures of compound.​
13. It is used for quality control.​
14. Also used in food science.​
15.beside this used in stud of drugs, biofluids, cells, nucleic acids.​
 NMR Spectroscopy - Key takeaways
•NMR spectroscopy is an analytical technique used to determine molecule shape and
structure.
•To carry out NMR spectroscopy, you dissolve your sample in a suitable solvent, add a
small amount of the reference molecule TMS, place it in an external magnetic field and fire
radio waves at the sample. A detector produces a graph of energy absorbed against a
property called chemical shift.
•A nucleus’s environment is all the different atoms and groups of atoms surrounding it.
Different nuclei have different chemical shift values depending on their environment.
•The two most common types of NMR spectroscopy are carbon-13 NMR and hydrogen-1
NMR. Hydrogen-1 NMR spectroscopy provides more information about the environments
of the nuclei it detects through integration traces and spin-spin coupling.
•NMR spectroscopy produces unique, well-resolved spectra. It can be used to distinguish
between different functional groups and environments in a molecule. However, it is
expensive and slow.
 Group Members:

Seema Hedaoo 1132220144

Sonal Gurav 1132220463
Isha Chourey 1132220476
Harvi Ghedia 1132220825