Radio-waves

Electromagnetic waves having longer wave length and low energy and frequency.
 Nuclear Magnetic Resonance (NMR) spectroscopy
Introduction
 NMR spectroscopy is the name given to the technique which exploits the
magnetic properties of certain nuclei.
 It is a spectroscopic technique which deals with the study of molecules on the
basis of interaction of radiofrequency of electromagnetic radiations with the
nuclei of molecule in a strong magnetic field.
 NMR spectroscopy involves absorption of electromagnetic radiation in the
radio frequency region (4 to 900 MHz). Absorption of radio waves in the
presence of magnetic field is accompanied by a special type of nuclear
transition and for this reason, this type of spectroscopy is called nuclear
magnetic resonance (NMR) spectroscopy.
 NMR is a most powerful tool available for organic structure determination.
 NMR spectroscopy is exhibited by Nuclei Which is composed of an odd
number of protons (1H and its isotopes, 14N, 19F, and 31P) or an odd number of
neutrons (13 C) because they show magnetic behavior. If both the proton and
neutron counts are even (12C or 16O) the nuclei are non-magnetic.
 It is used to study a wide variety of nuclei like 1H , 13C , 15N , 11F , 31P etc.
 1H NMR spectroscopy is one of the most powerful tools for elucidating the no.
of H or proton in the compound.
NMR active and NMR inactive nuclei
 NMR active criteria
 To be NMR-active, a nucleus must have a non-zero nuclear spin (I ≠ 0). It is this
non-zero spin that enables nuclei to interact with external magnetic fields and
show signals in NMR.
 Atoms with both an odd number of protons and an odd number of neutrons, or
an odd sum of protons and neutrons, exhibit half-integer values for the nuclear
spin quantum number (I = 1/2, 3/2, 5/2, and so on). These atoms are NMR-active
because they possess non-zero nuclear spin.
 Conversely, atoms with an even number of both protons and neutrons, or an
even sum of protons and neutrons, have a nuclear spin quantum number of zero
(I = 0).
 These nuclei do not exhibit active spin and are therefore not NMR-active.
 NMR-active nuclei, particularly those with a spin quantum number of 1/2, are of
great significance in NMR spectroscopy. Examples include 1H, 13C, 15N, and 31P.
 Principle of NMR
 NMR theory is based on the spin of the nucleus. Like
electrons, nucleus of an atom is also spin on its own axis. As
a result, generates its own magnetic field (magnetic
moment). Hence, nuclei of an atom act as a tiny bar magnet.
Spinning proton

 The nuclei are spinning randomly without magnetic field. Without magnetic
field, the nuclear spins are random in direction
 When an external magnetic field (Bo or Ho) is applied, the nuclear magnetic field
(magnetic moment) can be aligned with or opposed to the external magnetic
field . Now, there is two energy states: higher energy state (ant-parallel spin, β
position) and lower energy state (parallel spin) α position.
E2 – E1 = ΔE – h𝜈
 The difference between those two orientations is denoted as ΔE. The difference in
energy is however small and lies within the range of radiofrequency (region of
electromagnetic radiation).
 A nucleus (proton) in an external field is exposed to radio wave radiation with
energy. When the energy of the applied radiation (radio-frequency or RF)
matches with the energy gap ΔE between two energy states, the proton absorbed
the RF radiation, and it will flip the proton (transition of proton from lower spin
state to opposite spin state). the nuclei are said to be in resonance with the
electromagnetic radiation. This condition is said to be resonance.
 When the spin returns to its ground state level, the absorbed radio frequency
energy is emitted at the same frequency level. The emitted radio frequency signal
that give the NMR spectrum of the concerned nucleus.
 The NMR spectrum is a plot of intensity of NMR signal Vs. magnetic field
(frequency) in reference to TMS (TMS is such a common standard that its 1H
chemical shift (δ) is defined as zero).
 Based on the spin of the nucleus
Anti-parallel spin

E2

(β-position)

Energy gap
No applied magnetic field ΔE
(Randomly oriented spin)

E1

Parallel spin
External magnetic field (α -position)
E2

Energy gap
ΔE hν

E1
 High energy state

Resonance
Spectra

Low energy state

If the radio-wave (equivalent to ΔE), is supplied to nucleus it flips from lower energy
(α –position) state to higher energy state (β-position).
 Circulating π electrons
Chemical shift (δ) create a ring current
 In this process, nuclei not only interact with the
magnetic field but also with the surrounding
nuclei and their electrons. Induced magnetic
field reinforces the
 When nucleus of an atom is placed in a external magnetic
magnetic field, the circulation of the electrons field (de-shielded)
around nucleus produces secondary magnetic
field, called induced magnetic field.
 If the induced magnetic field and the applied magnetic field, both are in same
direction, then the field felt by the proton is augmented and the proton is said to be
de-shielded. Shielding effect shifts the absorption down-field in NMR spectra
 If the induced magnetic field and applied magnetic field are in opposed direction, it
reduces the field experienced by the nucleus. Then the proton is said to be shielded.
and Shielding effect shifts the absorption up-field.
 The shift in the position of the NMR region resulting from shielding and de-shielding by
electrons is called chemical shift.
Downfield Up-field
 1H NMR spectra
o Plot of the intensity of a peak against chemical shift (ppm)
o Increasing chemical shift is plotted from left to right = x axis
o Most proton absorbs between 0-10 ppm. Sample CH3OC(CH3)3
o NMR absorption generally appear as sharp peaks
o The term “upfield” and “downfield describe the location of peaks.
Upfield means to the right. Downfield means to the left.

Downfield direction Up field direction

(TMS is such
a common standard
Intensity

that its 1H chemical

shift (δ) is defined as
zero).

Increasing chemical shift

 Equivalent & Non-equivalent proton
 Equivalent proton: The protons which are bonded to similar atoms or the proton
within the molecule which posses same magnetic field.
eg methane ethylene acetylene
 Non-equivalent proton: The protons which are bonded to dis-similar atoms. The
proton which are in different magnetic environment. eg methyl alcohol, ethyl
alcohol, di-ethylether.
 Protons with identical environments have the same chemical shift and, therefore,
give only one 1H NMR signal. If different give more signals.

Equivalent protons (one signal)

CH3-OH CH3 – CH2-OH CH3 -CH2 -O –CH2 -CH3
(a) (b) (a) (b) (c) (b) (a) (a) (b)
Two signal Three signal Two signal
 Spin-spin splitting of signals
 The absorption signal due to particular proton may split into double (two peaks),
triple (3 peaks) and so on. This phenomenon is commonly known as spin-spin
splitting. It results from the interaction or coupling of the nuclear spin of the
hydrogen atoms on an adjacent carbon (neighboring proton) atoms. It is caused
only non-equivalent neighboring protons.
 The spinning of the nucleus creates a magnetic field that produces changes in
the magnetic field in the neighboring nuclei. This change in the magnetic field
results in the splitting of chemical shifts.
Splitting of the signal into
 It depends on the protons on the adjacent atom (n) . Themultiplets
splitting of signals can
be calculated using (n+1) rule.
n = number of neighboring protons.
 Spin-spin splitting of signals

n=2 n=1
 Spin-spin splitting of signals

No. of signal = n+1 = 1+1

= 2 (Doublet)
No. of signal = n+1 = 2+1
= 3 (Triplet)
 NMR spectra of ethanol (Ethyl alcohol)
CH3-CH2-O-H
a) Tetrametyhl silane (TMS) is taken as standard reference and chemical shift of
its protons are arbitrarily taken as zero.
b) Hydrogen of ethanol has three different environments. Therefore, low
resolution of H-NMR spectra show 3 peaks.
c) CH3- protons are split by –CH2-protons into 2+1 splitting. Therefore , it shows
triplet peaks. The δ value at 1.26
d) -CH2- protons are spilt by CH3-protons into 3+1 splitting. Therefore , it shows
quartet peaks. The δ value is at 3.69
e) The –O-H proton resonance is not split by adjacent protons. Therefore, there
is no splitting. Hence, there is one peak. The δ value is at 2.61.
 Spectrum of ethanol in low and high resolution

Low resolution spectrum:

Three peaks are observed
arising from absorption by
the CH3, CH2 and OH
protons.

High resolution spectrum:

Two of the three peaks can
be resolved into additional
peaks.
(TMS is such
a common
standard that
its 1H
chemical shift
(δ) is defined
as zero).
 NMR spectra of ethanal (Acetaldehyde)
CH3-CHO
a) There are two types of protons (protons of CH3- and protons of CHO).
Therefore, there are two NMR signals under low resolution.
b) Proton signals of CH3- are split by –CH=. Therefore, two signal under high
resolution. The δ value is 2.21 ppm. This gives a doublet.
c) - CH = proton is split by – CH3 – protons. Therefore, there are 4 signals
under high resolution. it shows triplet peaks. The δ value is at 9.79 ppm.
Resonance splitting gives quartet.
 Application of NMR spectroscopy
1. Structure elucidation: Structure of the unknown compounds can be explained from its
NMR spectrum.( No of signals = no. of protons, Chemical shift = types of hydrogen Spin-
spin splitting = arrangement of hydrogen)
2. Determination of optical purity: Since dia-sterioisomers differs in ther NMR
characteristics, So it is used for determination of enantiomeric purity.
3. Moisture analysis: Water absorbed in food products appear in NMR spectra as relative
sharp band.
4. Medicine: MRI, a multidimensional NMR imaging technique, is used for diagnostic
purposes. It is useful for diagnostic imaging of soft tissues, including the heart, brain, and
muscles, to discover tumors of the body.
5. Structural analysis: To study the mol. structure of compounds (chemical identity and
position of atoms, orientation of chemical bonds of molecules eg nucleic acids and
proteins.
6. Quality control: used for quality control in the food, and chemical industries to ensure the
purity and composition of products.
7. Pharmaceuticals: To study the drug metabolism.
 Mass spectrometry
 Mass spectroscopy is the most accurate method for determining the
molecular mass of the compound and its elemental composition.
 Valuable technique in which the molecules in a test sample are
converted to gaseous ions that are subsequently separated according
to their mass-to-charge ratio.
 Used for measuring the mass-to-charge ratio (m/z) of
one or more molecules present in a sample.
 It is used to determine i) Relative atomic mass ii)
Relative abundance of isotopes iii) Relative molecular Mass/charge ratio
mass iv) Molecular structure of compound
 Principle of mass spectrometry
 A sample is introduced into the spectrometer and vaporized where charge ions are
produced by bombarding the sample with high energy electrons. The collision
between an electron and the molecule causes an electron to be ejected from the
sample molecule, leaving a positively charged ion:
M + e- M+ + 2e-
 These ions possess so much energy that they often fragment through various bond
cleavages to produce new positively charged ions. Each kind of ion has a particular
ratio of mass to charge i.e. m/e or m/z ratio (value). For most of the ions, the
charge is one and thus m/z ratio is simply the molecular mass of the ion.
 The ions passes through the magnetic and electric fields in the spectrometer. Then
the ions are separated according to their mass-to-charge ratio and reach detector
where they are detected.
 Signals are recorded to give mass spectra. A mass spectrum of the molecule is thus
produced.
• Forms Ions from Molecules (Molecular Ion)
• Analyzes Ions by mass to charge (m/z)
• Detects the separated Ions
• Collects the Data as spectrum
Mass Spectrometer
 Fragmentation process
 Bombardment of molecules by an electron beam with energy between (10-15 ev)
usually results in the ionization of molecules by the removal of one electron
(Molecular ion formation)
2
When the energy of electron beam is
Cation
increased between (50-70 ev) these
molecular ions acquire a high
excitation resulting in their break
down into various fragments. This
process is called as fragmentation.

Each kind of ion has a particular ratio of mass to charge.

The ions passes through the magnetic and electric fields to reach detector
Fragmentation of methane molecule

CH4 + e- CH4+ + 2 e-

Molecular ion (CH4+) Mol. Mass 16

Fragmentation

CH3+ CH2+ CH+ C+

15 14 13 12
 Mass spectrum
 The mass spectrum is a plot of the relative abundance of the ions at each m/z ratio.
 The largest peak found in a mass spectrum (that of highest intensity) is called the
base peak, and is given the numerical value of 100. The intensities of all other peaks
are expressed relative to the height of the base peak.
Mass spectrum of pentane:

Base peak

m/e ratio
 CH3+
CH4+

CH2+

CH2+

C+
CH4+
 Interpretation of mass spectrum of methane

The spectrum of methane displays a strong molecular ion at m/z = 16,

corresponding to the formula 12CH4+. It is assigned an abundance of 100, and it is
referred to as the base peak.
The peaks at m/Z of 15, 14, 13 and 12 are due to these lower molecular weight
fragments.
The mass spectrum of CH4 contains one additional peak. Though most C atoms
have an atomic mass of 1.11% having mass of 13. Thus 13CH4+ is responsible for
the peak at m/z = 17. This is called the M+1 peak.
Provides the information on:
 Molecular weight
 Fragmentation information
 Nature and structure of their precursor molecule

Mass spectrum of octane

 Major Applications of Mass Spectrometry

1. For determining or confirming the structure or the identity of drugs.

2. MS in conjunction with either gas chromatography (GC-MS) or liquid
chromatography (LC-MS) provides a method for characterizing the impurities.
3. GC-MS and LC-MS provides a highly sensitive and specific method for
determining drugs and their metabolites in biological fluids and tissues.
4. MS has become an important tool in proteomics ( study on proteins and their
cellular activities), which is currently the major tool in drug discovery
5. It is the best method for getting rapid identification of trace impurities.