NMR spectroscopy is a powerful analytical technique used to characterize organic molecules. It exploits the magnetic properties of atomic nuclei. When placed in a strong magnetic field, atomic nuclei absorb and emit radio frequency radiation. The frequency depends on the magnetic field strength and chemical environment of the nucleus. NMR spectroscopy is used for a variety of applications including analysis of mixtures, elemental analysis, structure determination, and pharmaceutical analysis such as drug identification, quantification, and quality control. It provides both static structural information and dynamic information about molecular motion.

1. Laraib Tariq
Pharm D
Nuclear Magnetic Resonance (NMR):
Definition:
It is the phenomenon where magnetic nuclei emit and absorbs energy in the presence of
magnetic field. This energy is at specific resonance frequency which depends upon strength
of magnetic field and magnetic properties of isotopes of the atom.
 Introduction:
NMR is a powerful analytical technique used to characterize organic molecules by
identifying carbon hydrogen frameworks within the molecules. It is a research technique
that exploits the magnetic properties of certain atomic molecule. It determines the physical
and chemical properties of atoms and molecules in which they are contained.
NMR spectroscopy just like IR and UV is regarded as a process whereby energy from an
external source is absorbed and brings about a change or resonance to an ‘excited’ or high
energy state. The energy required for NMR lies in the low energy or long wavelength radio-
frequency end of the electromagnetic spectrum. Hence, NMR is also known as Nuclear Spin
Resonance (NSR) spectroscopy. NMR spectroscopy has broadened the scope and absolute
possibility for performing more extensive as well as intensive studies with regard to
recording the spectrum of isolated and synthesized organic molecules in addition to their
mechanistic and stereochemical details.
 Principle of NMR:

2. The sample is dissolved in a solvent, usually deuteron-chloroform and placed in a magnetic
field.
A radio frequency generator then irradiates the sample with a short pulse of radiation, causing
resonance.
When the nuclei fall back to their lower energy state, the detector measures the energy
released and a spectrum is recorded.
Proton in different environments absorb at slightly different frequencies, so they are
distinguishable by NMR.
The frequency at which a particular proton absorbs is determined by its electronic
environment.
The size of magnetic field generated by electrons around a proton determines where it absorbs.
Modern NMR spectrometers use a constant magnetic field strength and then a narrow range of
frequencies is applied to achieve the resonance of all protons.
Only nuclei that contain odd mass numbers or odd atomic numbers give rise to NMR signals.

3.  Types:
 Continuous Wave spectroscopy:
Continuous wave spectrometer consists of a control console, magnet, and two
orthogonal coils of wire that serve as antennas for radiofrequency radiation. One coil is
attached to an rf generator and serves as a transmitter. The other coil is the rf pick up
coil and is attached to the detection electronics.
The first CW-NMR spectrum of ethanol demonstrated the ability of NMR to distinguish
chemical groups of methyl, methylene and hydroxyl by their chemical shift and relative
areas. In this type of spectroscopy experiment a CW excitation is swept across the
frequency range, slow enough that the transverse response of the spin-systemcan be
modelled by the steady-state solution to the Bloch equations .A limitation of these early
methods was a slow spectral sweep rate to avoid artifacts from previously excited spins.
These efficiency issues were addressed by later improvements to CW spectroscopy
 Fourier Transform NMR Spectroscopy:
It uses a pulse of rf radiation which causes nuclei in a magnetic field to flip into higher
energy alignment. Due to Heisenberg uncertainty principle, the frequency width of the
rf pulse is wide enough to simultaneously excite nuclei in environment. All nuclei will re-
emit rf radiation at their respective resonance frequencies, creating an interference
pattern in the resulting rf emission vs time known as free induction decay (FID).

4. Fourier transform NMR (FT-NMR) using pulsed excitation (Ernst and Anderson, 1966)
provided a large efficiency and sensitivity improvement on the CW-NMR methods. The
spectral range is excited with a single short duration hard RF pulse and the following FID
is Fourier transformed to reconstruct the spectrum as shown in. This method of
acquiring NMR spectra is equivalent to slow passage CW-NMR spectra
 Applications of NMR:
 Analysis of multi-component mixture: Hollis has described a method for
the determination of aspirin, phenacetin and caffeine in commercial analgesic
preparation.
 Elemental analysis: The total concentration of a given kind of magnetic nucleus in
sample can also be determined by NMR.
 Identification of compound: The structure of unknown compound from its
NMR can be easily decided by certain principles, some of them are mention here,
The no of main NMR signal should be equal to the no of equivalent protons in interested
compound.
The type of methylene hydrogen atom, methyl group hydrogen, ether hydrogen etc is
indicated by chemical shift.
The possible arrangement of group in the molecule is indicated by spin spin splitting.
The area under NMR is directly proportional to the no of nuclei present in each group.
 Hydrogen bonding: it causes a decreasing the electron shielding on the proton.
Breaking of intermolecular hydrogen bond is indicated by up field shift of signal. The
downfield shift depends upon the strength of hydrogen bonding
 Ketoenol Tautomerism: The Ketoenol Tautomerism has also been studied by
NMR spectroscopy.
 Structural determination: NMR spectroscopy is very helpful is studying and
establishing the structure of complexes, organic and inorganic compounds. For example;
o Structure of SOF4: only one resolution field signal is obtained while 19F
spectrum of SOF4 is recorded indicating that all the four fluorine in molecule of
SOF4 are equivalent.

5. o Structure of HF2: if 19F magnetic resonance spectrum of HF2 is recorded,
only one signal is recorded showing that HF2 has linear structure.
 Non destructive Testing: Nuclear magnetic resonance is extremely useful for
analyzing samples non-destructively. Radio-frequency magnetic fields easily penetrate
many types of matter and anything that is not highly conductive or
inherently ferromagnetic. For example, various expensive biological samples, such
as nucleic acids, including RNA and DNA, or proteins, can be studied using nuclear
magnetic resonance for weeks or months before using destructive biochemical
experiments. This also makes nuclear magnetic resonance a good choice for analyzing
dangerous samples
 Segmental and molecular motion: In addition to providing static
information on molecules by determining their 3D structures, one of the remarkable
advantages of NMR over X-ray crystallography is that it can be used to obtain important
dynamic information. This is due to the orientation dependence of the chemical-shift,
dipole-coupling, or electric-quadrupole-coupling contributions to the instantaneous
NMR frequency in an anisotropic molecular environment. When the molecule or
segment containing the NMR-observed nucleus changes its orientation relative to the
external field, the NMR frequency changes, which can result in changes in one- or two-
dimensional spectra or in the relaxation times, depending on the correlation time and
amplitude of the motion.
 Process control: NMR has now entered the arena of real-time process
control and process optimization in oil refineries and petrochemical plants. Two
different types of NMR analysis are utilized to provide real time analysis of feeds and
products in order to control and optimize unit operations. Time-domain NMR (TD-NMR)
spectrometers operating at low field (2–20 MHz for 1H) yield free induction decay data
that can be used to determine absolute hydrogen content
values, rheological information, and component composition. These spectrometers are
used in mining, polymer production, cosmetics and food manufacturing as well
as coal analysis. High resolution FT-NMR spectrometers operating in the 60 MHz range
with shielded permanent magnet systems yield high resolution 1H NMR spectra
of refinery and petrochemical streams. The variation observed in these spectra with
changing physical and chemical properties is modeled using chemometrics to yield

6. predictions on unknown samples. The prediction results are provided to control
systems via analogue or digital outputs from the spectrometer.
 Magnetometers: Various magnetometers use NMR effects to measure magnetic
fields, including proton precession magnetometers (PPM) (also known as proton
magnetometers), and Overhauser magnetometers.
 SNMR: Surface magnetic resonance (or magnetic resonance sounding) is based on the
principle of Nuclear magnetic resonance (NMR) and measurements can be used to
indirectly estimate the water content of saturated and unsaturated zones in the earth's
subsurface. SNMR is used to estimate aquifer properties, including quantity of water
container in the aquifer, Porosity, and Hydraulic conductivity.
 Pharmaceutical application of NMR:
1. Quantitative analysis: The concentration of species can be determined directly
by making use of signal area per proton and the area of that identifiable peak of one of
the constituent for e.g if solvent present in known amount were benzene, cyclohexane
and water the area of single proton peak for these compound could be used in order to
set the required information.
2. Medicine: It is also widely used in biochemical studies, notably in NMR spectroscopy
such as proton NMR, carbon-13 NMR, deuterium NMR and
Phosphorus-31 NMR. Biochemical information can also be obtained from living tissue
(e.g. human brain tumor) with the technique known as in vivo magnetic resonance
spectroscopy or chemical shift NMR microscopy.
These spectroscopic studies are possible because nuclei are surrounded by orbiting
electrons, which are charged particles that generate small, local magnetic fields that add
to or subtract from the external magnetic field, and so will partially shield the nuclei.
The amount of shielding depends on the exact local environment. For example, a
hydrogen bonded to an oxygen will be shielded differently from a hydrogen bonded to a
carbon atom. In addition, two hydrogen nuclei can interact via a process known as spin-
spin coupling, if they are on the same molecule, which will split the lines of the spectra
in a recognizable way.

7. 3. Identification Testing: The versatility and ability of NMR to distinctly
differentiate nuclei in various intramolecular environments has placed it as the most
reliable and dependable technique for carrying out the identification testing of a host of
pure drugs. Hence, any apparent deviations of the spectrum of a sample under
investigation vis-a-vis the spectrum of the pure and the authentic pharmaceutical
substance usually give rise to an enormous information not only confined to the true
identity of the substance but also the probable nature of the impurities it possesses.
The survey of literature provides ample evidence of the NMR spectra of a good number
of medicinal compounds belonging to various categories, namely: sulphonamides,
barbiturates, amphetamines, steroids, antihistamines, penicillin’s and cephalosporin’s to
name a few.
4. Assay of Drugs: A plethora of pure drugs, their respective combinations and their
dosage forms have been assayed by NMR-spectroscopy quantitatively by various
researchers and the result thus obtained were duly verified and compared with the
standard methods prescribed in various official compendia. A few typical examples of
such drugs shall be described briefly here;
 Quinidine in Mixtures and Hydroquinidine:
A given sample containing a mixture of quinidine (I) and hydroquinidine (II) is dissolved
in requisite quantity of deutrochloroform (CDCl3) along with 2, 3, 5-triboromothiophene
as the internal standard.The quantitative determination is carried out by comparing the
peak area attributed by ethylene of (I) at 5.16 ppm to the internal standard peak at 6.93
ppm. The coefficient of variation was found to be 1%.
 Assay Methsuximide and Phensuximide Capsules:

8. The analysis of methsuximide (I) is performed in carbon tetrachloride and of phensuximide (II)
in 10% v/v dichloromethane in carbon tetrachloride. In this particular analysis
hexamethylcyclotrisiloxane (III) is employed as an internal standard for (I) and (II); whereas the
frequencies are referenced to usual tetra-methylsilane (TNS).
 Assay of Meprobamate and Mebutamate
The assay of meprobamate (1) and mebutamate (II) have been accomplished* by using malonic
acid as the internal standard and acetone as the solvent. The results obtained were fairly
comparable to the lengthy official procedures.
 Assay of Meclizine and Methaqualone
NMR-assay of meclizine (I) and methaqualone (II), besides a number of other potent
hypnotics and their corresponding mixtures have been successfully carried out using an
external standardization procedure reported. It is, however, interesting to observe that

9. additional sources of variability are usually incorporated into an assay employing external
standardization, and the same has been duly shown in the results thus obtained i.e., a large
coefficient of variation to the extent of 4% achieved.
 Analysis of multi-component mixture:
Hollis has described a method for the determination of aspirin, phenacetin and caffeine
in commercial analgesic preparation.