Unit 5 - Spectroscopy - Full

Unit-5
Advance Analytical Techniques: Spectroscopic,

Microscopic Techniques and their Applications
Course Objectives:
Acquire fundamental understanding of spectroscopic techniques to characterize novel
structures, materials and explore the applicability.
Course Outcomes:
Apply the knowledge of various advanced spectroscopic techniques and understand structure
property relationship to characterize novel materials for various engineering applications.
Dr. Ramya Chulliyil 1

Syllabus:
(1). Mass spectroscopy: Principle and instrumentation, Principles of spectroscopy and

selection rules.
(2). Nuclear Magnetic Resonance (NMR) Spectroscopy: Proton NMR
(3). UV-Visible absorption and emission (Fluorescence spectroscopy) and its
applications, Woodward-Fisher rule
(4). Surface characterization techniques: (a). Scanning Electron Microscopy (SEM), (b).
Transmission Electron Microscopy (TEM)
(5). Chromatography: Thin Layer Chromatography (TLC) and its applications

Spectrometric, Spectroscopic Techniques
and their Applications
Lectures
Background knowledge required to understand the chapter
Prepared by:
Dr. Ramya Chulliyil

MIT-ADT University, Pune
 Electromagnetic Radiation consists of photons which behave both as particles and
waves. Equation connecting energy, frequency, velocity and wavelength is
n =n h is Planck’s constant
 In an atom, the energy levels are quantised, ie, it can have only certain discrete
energy levels.
 The energy levels in an atom are Orbits
 The electrons will stay in the ground state of energy, if

no energy is absorbed or irradiated on the atom.
 If an energy which is the difference between any two

energy levels is given, electron will absorb that energy
and will jump to the higher energy state (excited state).

energy
4
Energy
2
(absorption spectra)
n=1
3
Energy
2
(Emission spectra)
n=1

 Similarly, the energy levels in a molecule are
 The energy levels are quantised.

 Each electronic level is associated with a number of vibrational energy levels. Each
vibrational energy level is usually associated with a number of rotational energy
levels.
 And the notations for these quantised energy levels are:
S1
J = 0, 1,
3 2..
2
S0
1
V=0
V = Vibrational Quantum Number

J= Rotational Quantum Number
Electromagnetic spectrum
 The range of wavelengths and frequencies in light is known as the electromagnetic
spectrum.
 This spectrum is divided into various regions extending from very short-wavelength,
high-energy radiation to very long-wavelength, low-energy radiation.

Interaction of Radiation with Matter (Molecules)
Ultraviolet (electron level

changes)
Visible
Energy
(molecular
Infrared Vibration)
Microwaves (molecular
Rotation)
Wavelength
 Absorption of energy causes a molecule to go from an initial energy state (ground

state) to a high-energy state (excited state) which could take the form of the
increased/changed rotation, vibration or electronic energy levels.
 Molecular rotations are excited/ changed by microwaves
 Vibrations of bonds are excited by infrared radiation

 Electrons are promoted to higher orbitals by the absorption of ultraviolet or visible
light
 Radio waves can cause nuclei in some atoms to change magnetic orientation and
this forms the basis of a technique called nuclear magnetic resonance (NMR)
spectroscopy.
 By studying the change in energy states, we can learn about the physical and
chemical properties of the molecules

Interaction of Radiation with Matter (Molecules)…..
Absorption & emission
Scattering
Elastic (~ 99 %)
Scattering
Inelastic (~1 %)

Spectroscopy- General Idea
 Spectroscopy is the general process of putting some energy into a system and
measure the characteristics of what comes out
Molecular Spectroscopy
 Spectroscopy is the use of the absorption, emission or scattering of
electromagnetic radiation by molecules (or molecular ions) to qualitatively or
quantitatively study the molecules, or to study physical processes.
 Molecular spectroscopy is the study of the interaction of electromagnetic (EM)

radiation with matter
 It is based on the analysis of EM radiation that is emitted, absorbed, or scattered
by molecules.

Electronic (UV-Visible)
Vibrational (IR)
Molecular Spectroscopy Rotational (Microwave)
NMR (Radio)
Raman (scattered)
 Mass spectrometry is another useful technique used by chemists to help them determine
the structure of molecules.
 Although sometimes referred to as mass spectroscopy it is, by definition, not a spectroscopic
technique as it does not make use of electromagnetic radiation.
• Instead, the molecules are ionised using high-energy electrons and these molecular ions
subsequently undergo fragmentation

Lectures
Portions covered from syllabus:
Mass Spectrometry- principle and instrumentation
Prepared by:
Dr. Ramya Chulliyil

Mass Spectrometry (MS)
 This is a very powerful analytical tool that can provide information on both molecular mass
and molecular structure. It is about 1000 times more sensitive than other analytical
techniques such as IR or NMR spectroscopy
 A mass spectrometer generates multiple ions from the sample under investigation, it then
separates them according to specific mass to charge ratio (m/z) and then records the relative
abundance of each type of ion.
Background knowledge required to understand this technique
1. Consider a wire carrying an electric current – a flow of negative electrons-. It has a magnetic
field associated with it.
2. If this current-carrying wire is placed in an
external magnetic field it would ‘jump’ as it is
deflected when the two magnetic fields interact.
3. An electromagnetic field is also

generated by a flow of positively charged
ions, such as those generated by a mass
spectrometer. It will be deflected by an
external magnetic field.
Working and Instrumentation
 In a mass spectrometer a stream of positively charged ions is produced along with an

associated magnetic field and their deflection in a controlled external magnetic field is
studied in detail.
 The processes involved in Mass spectrometry are
1. Vaporization
2. Ionisation and fragmentation
3. Acceleration
4. Deflection
5. Detection
(1). It is important that the atoms or the molecules of the substance being investigated are free
to move so if the sample is not a gas it must first be VAPORISED.
(2). Next, the sample must be IONISED. This is achieved by bombarding the sample with high-
energy electrons from an electron gun. These knock off an electron to produce a positive ion.
e.g. consider a helium atom

He(g) + e– He+(g) + 2e–
Mass spectrometer

• The high energy electron bombardment may also cause molecules to be broken into many
different fragments
e.g. methane molecules CH4 can be fragmented to produce CH3+ , CH2+ , CH+ and C+
CH4 CH3+ , CH2+ , CH+ and C+
NOTE: Because the positive ion formed has an unpaired electron it is sometimes shown with a
dot indicating that it is a free radical, e.g. CH3.+
(3). The positive ions are then ACCELERATED by an electric field and focused into a fine beam
by passing through a series of slits with increasing negative potential.
(4). The beam of fast-moving positive ions is DEFLECTED by a strong external magnetic field.
The magnitude of deflection depends upon two factors:
• The mass (m) of the ion – the lighter it is, the more it will be deflected.
• The charge (z) on the ion – ions with 2+ charges are deflected more than 1+.

• These two factors are combined into the mass to charge ratio (m/z). When m/z is small, the
deflection is large.
(5). Finally ions which make it right through the machine are DETECTED electronically. As the
positive ions arrive at the detector they pick up electrons to become neutral. This movement of
electrons is detected, amplified and recorded. The external magnetic field involved in deflection
can be adjusted so that ions with different m/z ratios can be detected.
As the molecule gets bigger the possibility of fragmentation increases and the mass spectra
become more complex. Final decisions about structure are made after combining evidence
from mass spectrometry with other analytical tools such as infrared, ultraviolet–visible and
nuclear magnetic resonance spectroscopy.
 On running the sample in Mass spectrometer, a graph is generated with
Relative Abundance vs m/z ratio of ions.
Abundance
Relative
(%)

m/z
Components in a mass spectra of simple molecules
1. Molecular ion: The ion obtained by the loss of an electron from the molecule. Molecular mass
can be determined from the molecular ion of mass spectra.
+
e. field
(Propane) (m/z = 44)

2. Fragment ions: Lighter cations formed by the decomposition of the molecular ion. These often
corresponds to stable carbocations.
+
+
(m/z = 29) (not detected by MS)
+
Dr. Ramya Chulliyil
(m/z = 15) 20
(not detected by MS)
3. Radical cation: Positively charged species with an odd number of electrons
+
- CH4 + (m/z = 28)
base peak
Abundance
Relative
(%)
4. Base Peak: The most intense (tallest)

peak in a mass spectrum due to the ion
with the greatest relative abundance. m/z
It is given the percentage 100 and the
% s of other ions are calculated
with respect to the intensity of the base peak.
Applications of Mass spectrometer
 The technique is used in industrial and academic fields for both routine and research
purposes.
(1). Academic: It is used to determine the molecular weight of compounds by separating

molecular ions on the basis of their mass and charge.
(2). Geological: Oil composition
(3). Biotechnology: the analysis of proteins and pesticides
(4). Environmental: Water quality and food contamination
(5). Clinical: haemoglobin analysis, drug testing

Mass spectrometer

Portion covered from syllabus:
NMR Spectroscopy
Prepared by:
Dr. Ramya Chulliyil

Nuclear Magnetic Resonance (NMR) Spectroscopy
 Nuclei of atoms are composed of protons and neutrons. Like electrons,

these particles also spin on their own axis- clockwise and anti-clockwise.
 NMR arises from the fact that certain atomic nuclei have a
property called “net spin”.
 The circulating nuclear charge generates a magnetic dipole

along the axis.
 The net spin number or spin quantum number I of a particular nucleus

can be obtained by adding spin numbers of individual protons and
neutrons of ½ each (+1/2 for clockwise spin and -1/2 for anti-clockwise spin)
(by considering their arrangement in separate SHELLS)
assuming that the spin number of neutrons cancel only neutrons and
protons cancel only protons.
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Dr. Ramya Chulliyil
 All nuclei are not associated with a magnetic dipole.
- Any atomic nucleus that has an odd mass number, an odd atomic number, or both has a spin
and a resulting nuclear magnetic moment.
- Such nuclei have “I” values as multiples of half integrals (1/2, 3/2, 5/2…) or integers (1,2,3..)
Inactive
Magnetic nuclei
Moving charges and magnetic dipole moments produce a magnetic
field.
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Dr. Ramya Chulliyil
 In the absence of an external magnetic field ,the direction of
the spin of the nuclei will be randomly oriented
 When a sample of these nuclei is placed in an external

magnetic field B0, the nuclear spins will adopt specific
orientations.
 For a nuclei with I value ½, (2I+1) = 2

spin orientations are possible,
-with the external field (i.e. parallel to and
in the same direction as the external field)
External Magnetic Field

-against the field (i.e. antiparallel to the
external field).
 The spins parallel to the field (α spin) has slightly

lower energy compared to anti-parallel spin states
(β spin).
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Dr. Ramya Chulliyil
 Orientations in presence of magnetic field

m = -1/2
m = +1/2
In the absence of
Magnetic Field
 The energy difference between the spin states is quite small and corresponds to the
radio frequency range of the EM spectrum
 On applying an external magnetic field, the spinning magnetic nuclei
(1). Adopt specific orientations- α and β spins
(2). Spin like a “top” around the external magnetic field axis. precess
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Dr. Ramya Chulliyil
 The angular frequency of the precessing motion of the
spinning nuclei is known as the Larmor Frequency.
 When Radiofrequency energy matching ( in

resonance), the Larmor Frequency is introduced at right
angle to the applied magnetic field, the precessing nucleus
will absorb the energy and magnetic moment will flip to its
I= -1/2 state (β state).
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Dr. Ramya Chulliyil
 When this occurs, the nucleus and radiofrequency beams are said to be in resonance,
hence the term Nuclear Magnetic Resonance.
 The energy required to cause the spin-flip, depends on the magnetic field
strength at the nucleus.
-With no applied field, there is no energy difference between the spin states,
-as the field increases, so does the separation of energies of the spin states and
therefore so does the frequency required to cause the spin-flip.
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Dr. Ramya Chulliyil
Proton Nuclear Magnetic Resonance (1H NMR) Spectroscopy
 While not all nuclei are NMR active (e.g. 12C and 16O are inactive), the most important nuclei
for organic chemists are 1H and 13C (both with nuclear spin = 1/2).
 1H 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.
 In this discussion, the word "proton" will be often used for "hydrogen atom",
because it is the proton in the nucleus of the 1H isotope that is observed in these
experiments
 If a nucleus/proton is bare, it “feels” the full magnitude of external magnetic field
applied and absorbs radiofrequency which is in resonance with this magnetic field.
 But in real systems, nuclei/protons are surrounded by valence electrons and in response to
applied magnetic field, the electron density generate a secondary magnetic field known
as Induced Magnetic field.
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Dr. Ramya Chulliyil
Shielding and de-shielding
 If the electronic effects in the molecule push the electron density into the region of the
nucleus, the induced magnetic field generated by the electron density opposes the applied
field, the nucleus is said to be shielded.
 If the electronic effects in the molecule (ie, hybridisation, presence of electronegative atoms
or resonance effect) pull the electron density away from the region of the nucleus, the
induced magnetic field generated by the electron density enhances the applied field, the
nucleus is said to be de-shielded.
 Shielded nuclei/protons do not “sense’ as large magnetic field as deshielded nuclei/protons

do. As a result, the nuclei/protons experiences a lower applied magnetic field
strength and absorbs more upfield (resonate at higher fields ).
 Deshielded protons experiences a higher applied magnetic field strength and absorbs more
downfield (resonate at a much lower frequency).
 The terms “upfield” and “downfield” describes the relative location of the peaks. Upfield
means to the left and downfield means to the left.
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Dr. Ramya Chulliyil
Chemical Shift
 Due to shielding/de-shielding, identical nuclei which have different chemical environment
resonate at different values of the applied field. These values being characteristic can be
used to identify various types of environment in which the nucleus is present.
 The shift in the position of resonance due to the difference in chemical environment is
called Chemical Shift.
 The chemical shift tells us the strength of the magnetic field that the nucleus feels.
Reference Standard - TMS

 Since we cannot measure the NMR of bare proton/nuclei, we have to use some reference
standard with respect to which we can measure the extent of shielding or de-shielding of the
external field in various chemical environment.
 The reference chosen is the molecule Tetra Methyl Silane (TMS).
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Dr. Ramya Chulliyil
The reasons for selecting TMS as standard:
1. The protons in TMS are highly shielded and hence resonance occurs at lower
magnetic fields compared to protons of most organic compounds.
2. TMS contains 12 chemically equivalent identical protons with high specific
proton concentration. So it give a single strong NMR in dilute solutions.
3. It is largely inert, and is not likely to react with most samples
4. It is relatively volatile and, thus, can be easily removed from the sample later
Unit of Chemical Shift

 Since the extent of shielding is proportional to the external magnetic field Bo, we use field
independent units for chemical shifts: δ values, whose units are ppm.
 The positions of shielded and de-shielded protons in an NMR spectrum is as shown
below:
TMS
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Dr. Ramya Chulliyil
Representation of Proton NMR spectrum
 NMR absorptions generally appear as sharp peaks.

 Increasing chemical shift is plotted from left to right.
 Most of the protons absorb between 0-10 ppm.

 NMR absorptions are measured relative to the position of the reference peak of TMS at 0
ppm on the scale.
H+ Bare Proton
 Thus protons in different chemical environment (shielded and de-shielded protons in

different extents) will show signals at different δ values.
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Dr. Ramya Chulliyil
 Hence by counting the number of signals, we can predict the number of chemically
different protons present in the sample.
eg: 1- methyl acetate

(methyl ethanoate)
Number of chemically different
protons present = 2
 Chemically equivalent protons give the same NMR signal.

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Dr. Ramya Chulliyil
TMS
Protons at X signal are more de-shielded due to adjacent electronegative O atom.
Other examples
The number of chemically different

protons in each of them = 1 hence they
give single peak in NMR spectrum
with the δ values mentioned.
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Dr. Ramya Chulliyil
NMR Integration
 Integration (or area under the peak) of a NMR signal is used to find out the number of
hydrogens bound to a given carbon atom.
 Integration is the measurement of peak areas on the NMR spectrum.
 The height of peak/ relative peak intensity or peak area is proportional to the number of
protons corresponds to the signal
 Integrator trace (line) is a computer generated line and it will appear over the NMR
spectrum
Relative peak area = number of protons
 eg: NMR spectrum of ethanol with red coloured integrator trace on top.
 The ratio of peak intensity of protons of OH, -CH2 and CH3 are 1: 2 : 3.
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Dr. Ramya Chulliyil
Spin-spin coupling (splitting)
 The interaction between the spins of neighbouring nuclei in a molecule may cause the
splitting of NMR signal into multiplets. This is known as spin-spin coupling or splitting.
 The splitting pattern is related to the number of equivalent H atoms at the nearby nuclei.
Rules of Spin-spin coupling (splitting)
1. Chemically equivalent protons which are on same carbon atoms or adjacent carbon atoms do
not show spin-spin splitting.
3 C-H protons are chemically

2 protons each on adjacent carbon atoms are
equivalent : no splitting.
chemically equivalent; they do not split the
signals each other.
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Dr. Ramya Chulliyil
2. Protons that are farther than two carbon atoms apart do not split each other.
Splitting not usually observed
3. Splitting is observed when protons are on chemically non-equivalent environment
X Y

n+1 rule
 The multiplicity of signals is calculated using n+1 rule where n is the number of protons
on adjacent carbon atom.
- No (Zero) H atom as neighbour--- n + 1 = 1 (singlet)
- One H atom as neighbour --- n + 1 = 1 + 1 = 2 (doublet)
- Two H atoms as neighbour-- n + 1 = 2 + 1 = 3 (triplet)
Pascal’s triangle
 The intensity ratio of multiplets can be predicted using Pascal’s triangle.

Number of different ways that the three hydrogen atoms can align themselves

Features required to draw an NMR pattern
1. Number of signals………. Indicates how many different kinds of protons are present.
2. Position of signal………... Indicates shielding or de-shielding of protons
3. Intensity of signals……… Proportional to the number of protons present (Integration)
4. Spin-spin coupling………. Indicates the number of chemically different nearby protons
5. Position of TMS…………… Show the position TMS at δ value zero.
NMR spectra of molecules

eg: 1- ethyl alcohol
Step 1: number of different kinds of protons = 3.

Step 2: Position of signals-
(a). Since Oxygen is an electronegative atom, proton on –OH group is highly deshielded hence
the peak appears at extreme left hand side.
(b). Since –CH3 group exhibit electron pumping effect (+I effect), the electron density on the 3
protons will be the highest and they are highly shielded from the applied magnetic field. Hence
the peak appears at the extreme right side.
(c). Since the protons on –CH2 groups are adjacent to electronegative Oxygen atom, it is
somewhat de-shielded and situates in between the above two signals.
Step 3: Intensity of signals

Number of protons on –CH3 group is the highest, hence it intensity is the maximum and the
lowest is for the proton attached to –OH group.
By considering the above 3 steps, the low resolution

NMR spectrum of ethanol is:
TMS
Step 4: spin-spin coupling
Proton NMR spectrum of impure ethanol

 The ordinary ethanol is slightly impure and the exchange of –OH protons among ethanol
molecules take place so rapid that the proton on Oxygen atom do not stay for sufficiently long
time for the coupling to be observed.
 Hence no spin-spin coupling is observed between -OH proton and adjacent –CH2 protons.
 Thus -CH2 protons splits only by coupling from –CH3 protons.
According to n+1 rule,

-CH2 signal will split into 4 signals by adjacent 3 protons (n=3) of –CH3 with intensity
ratio 1:3:3:1 (quartet)
-CH3 protons will split into 3 by adjacent 2 protons (n=2) of –CH2 group with intensity
ratio 1:2:1 (triplet)

Proton NMR spectrum of impure ethanol is
-CH3
-OH
Singlet -CH2-
TMS
eg: 2- Ethyl Bromide
TMS
eg: 3- propane
(2) (2) 1. Number of chemically distinct types of protons =2

therefore there are 2 peaks
2. Position of signals - -CH3 signal is at the right
(1) (1) hand side and –CH2 signal is the right hand side.
3. Relative Intensity of –CH3: -CH2 peak is 3:2
4. -CH3 proton peak is a triplet due to spin-spin coupling
from the 2 protons of –CH2
(1)  -CH2 peak is a quartet due to coupling by 3 protons
of –CH3 group.
(1) (1) (1)

pi electrons
2 types of electron
Densities
valence electron
on Hydrogen
Magnetic Fields Bind by pi electrons

induced
On application of Bo
Bind by H valence electron

 In alkenes and aromatics, pi bond is formed by the sideways overlap of p orbitals.
 The net orientation of pi electron cloud is above and below the plane.
 In alkynes, there are two pi bonds at right angles to one another.

 This generates a cylinder of electron density around the sigma bond.
NMR peak in aromatic compounds and alkenes
 On the application of external magnetic field (B0), an induced magnetic field Bind is
created by the (1). п Electrons and (2). valence electrons.
 Bind by п Electrons is in same direction of B0.
Bres = B0 + Bind
 Bind by valence electrons will be in different direction to Bres and it won’t be able
to shield the proton from feeling the externally (Bres) applied magnetic field.
 The protons feel a stronger magnetic field and require higher frequency for
resonance.
 Protons are deshielded and resonate at higher
Dr. Ramya Chulliyil frequency. 53
NMR peak in terminal alkynes
In terminal alkynes, circulating pi-electrons induce a

magnetic field which is in the opposite direction of
applied field
Acetyl hydrogen lies along the axis of applied field.

The induced field by the valence electrons is in the
opposite direction of applied field and hence shield
the nucleus from applied field.
 Bind is in the opposite direction of B0
Bres = B0 - Bind
 Acetyl protons are shielded and resonate at lower frequency.
Magnetic anisotropy
 The word “anisotropic” means “non-uniform”.
 On the application of external magnetic field, an induced magnetic field is created
by the п Electrons in п systems (eg: aromatics, alkenes, alkynes etc) and by the
valence electron in H atom.
 There is magnetic “anisotropy” as there are different induced fields created in the
same molecule which can be in the same direction or opposite direction to
externally applied magnetic field (directional).
 Depending on the position of the proton, it can be either shielded or deshielded.
In case of alkynes, shielding occurs but in case of alkenes and benzenes,

deshielding takes place.
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NMR Spectrum of Benzene
Since all protons are equivalent in benzene, the NMR

spectrum of Benzene shows only one signal which is
deshielded due to magnetic anisotropy.
TMS
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Dr. Ramya Chulliyil
NMR Spectrum of Toluene
-CH3
Using a high resolution NMR
TMS
instrument, a distinct multiplet
peak due to the 3 distinct protons
present on phenyl ring (ortho, meta
and para) is visible.
In a low-field instrument, those peaks would

overlap
Dr. Ramya Chulliyil to give you a broad multiplet 57
NMR Spectrum of ethylene and acetylene
(1) (1)
(1) (1)
(1) (1)
 All 4 protons in ethylene are  Both protons in acetylene are

equivalent and hence give a single equivalent and hence give a single
spectrum with δ value 5.3 since it is peak with δ value 1.8 since it is
deshielded due to magnetic shielded due to magnetic anisotropy.
anisotropy.
Dr. Ramya Chulliyil

58
Selection Rule of NMR Spectroscopy
 Selection rule explains which transition is quantum mechanically allowed in
spectroscopy. There are individual selection rules in all spectroscopic techniques.
 In the case of NMR, the selection rule is: only transitions in which changes in spin states m
changes by one (up or down) are allowed.
--Means when radiofrequency is absorbed and a proton from α spin flip to β spin state, the
change in m value is given by
∆m = +1/2 – (-1/2) = 1

m = -1/2
m = +1/2

1. Define the following:
a. Chemical shift b. Magnetic anisotropy c. Integration
d. shielding e. de-shielding
2. Write principle, selection rule of NMR spectroscopy
3. Write a short note on effect of shielding and de-shielding on chemical shift
4. Explain the NMR spectra of simple molecules like

a. Ethyl alcohol b. ethyl bromide c. benzene d. toluene e. ethylene f. acetylene

Lectures
UV-Visible Absorption spectroscopy- Transitions, Instrumentation (Double beam), terms-
(chromophores, auxochrome, λmax), Beer-Lambert’s Law, Applications, Principle.
Woodward-Fisher rule.
UV-Visible Emission Spectroscopy- Fluorescence, Jablonski Diagram, principle, Applications.
Prepared by:
Dr. Ramya Chulliyil

UV-Visible Absorption spectroscopy
Types of electrons present in organic molecule

1. Bonding electrons: Electrons that participate directly in bond formation like sigma
and pi bonds.
2. non-bonding or lone pairs of electrons
Sigma bond- single bond

pi bond- double and triple bond
n electrons- non-bonding electrons on atoms like oxygen, halogens etc.

The electronic energy levels in an organic molecule

Electronic Transitions
(1). σ-σ* transitions : These types of transitions are related to the promotion of an
electron from a σ bonding orbital to σ* antibonding orbital. This type of transitions is
common for saturated organic molecules without hetero-atoms. Usually this transition
occurs below 150 nm. These transitions cannot be observed in commercial
spectrometers (200 nm – 750 nm).
(2). π-π* transitions : This transition is available in compounds with unsaturated

centers, e.g., simple alkenes, aromatics, carbonyl compounds, etc.
These types of transitions are related to the promotion of an electron from a π
bonding orbital to π* anti-bonding orbital.
The unsaturated organic molecules like alkenes have π-bonding orbital and π* anti-
bonding orbital shows this type of transitions.
Ethylene molecule gives an absorption maximum at 169nm (which is not scanned by
commercial spectrometer). But conjugated molecules shows absorption in UV-Visible
range. Dr. Ramya Chulliyil 66
(3). n-π* transitions : These types of transitions are related to the promotion of an
electron from a nonbonding orbital to π* antibonding orbital. These transitions are
shown by unsaturated molecules which contain hetero atoms like N, O, Cl, Br, S etc.
These transitions are weak.
Example, in aliphatic aldehydes and ketones n-π* transitions generally occurs in the
wavelength range 270 to 300 nm.
(4). n-σ* transitions : These types of transitions are related to the promotion of an
electron from a nonbonding orbital to σ* antibonding orbital. These are forbidden
transitions and therefore are weak intense. The saturated organic molecules with
hetero atoms like N, O, S, Cl etc shows this type of transitions. Example ethers,
chloroform etc. Most of these absorptions takes place below 200nm, therefore
commercial spectrometers does not scan this.

In order to absorb light in the region from 200 to 800 nm (where spectra are
measured), the molecule must contain either π bonds or atoms with non-bonding
orbitals.

UV-Visible Spectrophotometer
Two types: single beam and double beam
Lens
Lens

Double Beam Spectrophotometer
 To measure the absorption in UV/Visible range by a sample

 Source: Combination of Deuterium lamp- (200-370 nm) and
Tungsten-Halogen lamp- (320 nm-800 nm)
 The output from the light source is focused onto the diffraction grating which splits
the incoming light into its component colours of different wavelengths, like a prism
(shown above) but more efficiently.
 Beam splitter: optical device that splits a beam of light in two
 Sample : For liquids the sample is held in an optically flat, transparent container
called a cell or cuvette.
 Reference cell contains the solvent in which the sample is dissolved and this is
commonly referred to as the blank.
 The detector (usually a photomultiplier tube) converts the incoming intensity of
light.
UV-Visible Spectrum: graph of Absorbance vs Wavelength
Absorbance (A), also known

as optical density (OD), is the
quantity of light absorbed by
chromophores in solution
UV-VIS absorption
spectrum for buta-1,3-diene
Terms:
(1). λmax: refers to the wavelength in the absorption spectrum where the absorbance
is maximum
(2). Blue shift: When absorption maximum (λmax )of a compound shifts towards lower
wavelength, (i.e. higher energy and higher frequency) it is called blue-
shift or hypsochromic shift.

Original
spectrum
Blue shifted Red shifted

spectrum spectrum
(3). Red shift : When the absorption maximum (λmax)of a compound shift towards
higher wavelengths (i.e. lower energy and lower frequency), it is called a red-shift or
a bathochromic shift.

(4). Chromophores: an atom or group whose presence is responsible for the colour of
a compound or absorption in UV-Visible region.
(5). Auxochromes: a saturated or unsaturated group with non-bonding electrons when

attached to a chromophore altering both wavelength as well as intensity of
absorption.
eg:-OH, -NH2, -OR, -NHR and –NR2

(6). hyperchromic shift: Increase in Intensity due to introduction of auxochrome.
(7). hypochromic shift: reduction in Intensity.

Beer-Lambert’s Law
Consider a cell (also called cuvette) of path length l, containing solution of an absorbing species
of concentration c. If an incident radiation having intensity Io enters the sample cell, its intensity
decreases as it penetrates the sample and it becomes I.
l
According to the Beer–Lambert Law, the absorbance is proportional to the concentration of the
substance in solution and the path length.
The Beer–Lambert Law can be expressed in the form of the following equation:
A= = εcl
where (1). A = absorbance, (2). l = optical path length in cm
(3). c = concentration of solution in mol/L,
(4). ε = molar absorptivity or molar extinction co-efficient which is constant for a particular
substance at a particular wavelength in L (mole-1)(cm-1).
 It is a measure of how strongly a chemical species or substance absorbs light at a particular
wavelength
A = εcl = =
 Transmittance (T) is the fraction of incident light which is transmitted
 It's the amount of light that “successfully” passes through the substance and comes out the
other side.
 Absorbance (A) is the flip-side of transmittance and states how much of the light the sample
absorbed

Problems:
1. The molar absorption coefficient of tyrosine in water is 1280 M-1cm-1 at 280
nm. Calculate the concentration of a tyrosine solution in water if the absorbance
of the solution is 0.34 in a 1 cm path length cell.
Ans: c = 250 µ Molar
2. Calculate the concentration of a tryptophan solution that gives an absorbance of 0.25 at 280
nm in a 1 mm path length cell (Given ε @ 280 nm = 5690 M-1cm-1)
Ans: c = 43.9 µ Molar

Principle of UV-Visible Absorption spectroscopy
 Ultraviolet- Visible spectroscopy (λ 200-800nm) studies the changes in electronic energy

levels within the molecule arising due to transfer of electrons from π or non-bonding orbitals.
It commonly provides the knowledge about π electron systems, conjugated systems,
aromatic compounds and conjugated non-bonding electron systems etc.
Since the absorption of ultra violet or visible radiation by a molecule leads to transition among
electronic energy levels of the molecule, it is also known as electronic spectroscopy.
From Beer-Lambert Law, it is clear that greater the number of molecules capable of absorbing
light of a given wavelength, the greater the extend of light absorption.

Applications of UV-Visible Absorption spectroscopy
 UV-VIS spectroscopy is used extensively in teaching, research and analytical laboratories for
the quantitative analysis of all molecules that absorb UV and visible electromagnetic radiation.
 In clinical chemistry UV-VIS spectroscopy is used extensively in the study of enzyme kinetics.
When cells are damaged by disease, enzymes leak into the bloodstream and the amount
present indicates the severity of the tissue damage. The relative proportions of different
enzymes can be used to diagnose disease, say of the liver, pancreas or other organs, which
otherwise exhibit similar symptoms.
 In environmental and agricultural fields the quantification of organic materials and heavy
metals in fresh water can be carried out using UV-VIS spectroscopy .
 Detection of impurities in organic molecules by the presence of extra peaks.

Woodward-Fisher rule
 Woodward Fisher rules are several sets of empirically derived rules which attempt to predict
the wavelength of the absorption maximum (λmax) in an UV-Visible spectrum of a given
compound: 1. dienes and 2. enones
 Each type of diene and enone (α,β unsaturated carbonyl compounds) system is having a
certain fixed value of λmax which is known as the Base value or Parent value.
 The base value depends upon whether the diene is a (1). cyclic diene- (a). homoannular or
(b). heteroannular diene or (2). Acyclic diene
Substituents such as (1). alkyl substituents or ring residue, (2). double bond extending
conjugation (3). Exocyclic double bond etc also contribute some numeral values which are
added to the base value to obtain λmax for a particular compound.
1. Dienes
Base value:
1. Cyclic systems: Homo and hetero annular conjugated dienes
(a). Homo-annular conjugated dienes: Both conjugated double bonds are in the same ring.
λmax is 253 nm

(b). Hetero-annular dienes: Conjugated double bonds are present in different rings.
λmax is 214 nm
(2). Acyclic conjugated dienes: λmax is 217 nm
Increments:
a) Alkyl substituent or Ring residue attached to the parent diene = 5 nm
Here, four alkyl substituents are there as shown as

1 ,2 ,3 and 4 attached to parent diene.

(b). Double bond extending conjugation = 30 nm
(c). Exo-cyclic double bonds = 5 nm

2. Enones:
They are α,β-unsaturated carbonyl compounds
Base value:
Acyclic and cyclic enones: λmax is 215 nm
Increments:
α- alkyl substituent: 10 nm
β-alkyl substituent : 12 nm
γ- alkyl substituent : 18 nm
δ-alkyl substituent : 34 nm
Important Notes:
1. While calculating λmax only those alkyl substituents or ring residues are counted which are
directly attached to the carbon atoms making conjugated system within the compound.
2. If a molecule has both homoannular diene and heteroannular diene, then always use the
homoannular diene as the core chromophore.
Examples:
acyclic diene: 217 nm

(1). 3 alkyl substituents: 15 nm
Total: 232 nm
acyclic diene: 217 nm

(2). 3 alkyl substituents: 15 nm
Extended conjugation: 30 nm
Total: 262 nm

(3).
Homo-cyclic diene: 253 nm
Extended conjugation: 30 nm
Exo-cyclic double bond : 05 nm
5 alkyl substituents: 25 nm
Total: 313 nm
Enones
(4).
Base value: 215 nm
β- alkyl substituent : 12 nm
Total : 227 nm

Base value: 215 nm
Extended conjugation : 30 nm
(5). γ- alkyl substituent : 18 nm
Exo-cyclic double bond: 5 nm
Total : 302 nm
Base value: 215 nm

Extended conjugation : 30 nm
(6).
Homo-annular system: 39 nm
γ- alkyl substituent : 18 nm
Total : 336 nm
The experimental values of λmax almost always comes nearer to the ones calculated using
Woodward Fisher rule.

UV-Visible Emission spectroscopy
In a UV/visible absorption experiment, the sample absorb light. After excitation, the molecules
somehow get rid of the excess energy and return back to the ground state. The electrons can
return back to the ground state in different ways such as releasing the excess energy through
collisions or through emitting a photon. Various electronic and molecular processes that occur
following excitation are usually represented on a Jablonski diagram
In order to understand this better, we need to understand what is meant by a singlet and triplet
state.
Singlet state: A singlet state refers to a system in which all the electrons are paired. The net
angular momentum of the particles in this type of system is zero. Therefore, we can say that
the overall spin quantum number, s is zero (s=0). Furthermore, if we take the spectrum of this
system, it shows one spectral line, and thus, got the name “singlet state”. Moreover, almost all
the organic molecules that we know exist in the singlet state.
Triplet state of a system describes that the system has two unpaired electrons. The net angular
momentum of the particles in this type of system is one. Therefore, the spin quantum number
is one. Moreover, this allows three values of the angular momentum. Hence, the spectral lines
that we obtain for this type of system split into three lines, and thus, got the name triplet state.

Spin Multiplicity: (2s+1)
It is the total number of possible orientations of the spin angular momentum
corresponding to a given total spin quantum number (s)
1. For singlet system: s=(+½ + - ½) =0

2s+1 = 1
So it is a singlet state
2. For triplet systems: s = (½ + ½ ) = 1
2s+1 = 3
So it is a triplet system
Spin selection Rule:
 Selection Rule determines whether a transition is allowed or forbidden during
absorption or emission of electromagnetic radiation.
Generally, organic molecules have no unpaired electrons. Their ground states are
singlet states. According to selection rules for electronic excitation, the excited state
must also have no unpaired electrons. In other words, the spin on the electron that
gets excited is the same after excitation as it was before excitation.
 Spin selection rule tells that the overall spin S of a complex must not change
during an electronic transition, hence, ΔS = 0.

Jablonski Diagram

Absorption: S0 state with 0th vibrational level is the state of lowest energy. Absorption of a
photon of resonant frequency usually results in the population of S1 or S2 electronic states; but
usually a higher vibrational state. Transition of electrons from low energy molecular orbital to a
high energy molecular orbital through absorption of light is a femtosecond (10-15 s)
phenomenon. The electronic transition, therefore, is too quick to allow any significant
displacement of the nuclei during transition.
 Excited states are short-lived, so there are two options for the release of excess energy. It
could happen through non-radiative transitions or through emission of light quanta.
 There are various types of Non-radiative transitions- vibrational relaxation, internal
conversion and intersystem crossing.
 Radiative transitions are Fluorescence and Phosphorescence
(1). Vibrational relaxation causes the electron to relax to the ground vibrational state from
higher vibrational levels of the same electronic energy level.
(2). Internal conversion: By absorption of energy, if the fluorophores are excited to higher
energy states (Sn), they rapidly relax to the lower vibrational state of S1 through non-radiative
processes. The timescale of internal conversion/vibrational relaxation is of the order of 10-12
seconds.
(3). Intersystem crossing: The electrons in the first excited state (S1) if goes to triplet state (T1)
by changing spin multiplicity, it happens in several orders of magnitude slower than
fluorescence. The low probability of intersystem crossing arises from the fact that the molecules
must first undergo spin conversion to produce unpaired electrons, which is a quantum
mechanically forbidden process. Dr. Ramya Chulliyil 94
Radiative Transitions
(1). Fluorescence: Fluorescence emission is the outcome of fluorophore returning back to the S0
state from S1 by emitting a photon. The molecules then return back to the lowest vibrational
state through vibrational relaxation. Fluorescence lifetimes are of the order of 10-8 seconds.
(2). Phosphorescence: The molecule in the triplet state, T1, can return back to the S0 state by
emitting a photon. This process is known as phosphorescence.
As this process involves an intersystem crossing and two flips in spin, it requires a much longer
time than fluorescence (from 10-4s up to few s). Therefore, the probability of phosphorescence,
and hence the intensity of the phosphorescence spectrum, is very low also due to high
possibility of radiationless deactivation.

Stokes shift
Stokes shift is the difference between

positions of the band maxima of the
Absorption Emission absorption and emission spectra in the same
electronic transition

Lectures
Surface characterization techniques: (a). Transmission Electron Microscopy (TEM)

(b). Scanning Electron Microscopy (SEM)
Prepared by:
Dr. Ramya Chulliyil

 Electron microscopy (EM) is a technique for obtaining high resolution images of biological
and non-biological specimens (like nanomaterials).
 An electron microscope uses an 'electron beam' to produce the image of the object and
magnification is obtained by 'electromagnetic fields'; unlike light or optical microscopes, in
which 'light waves' are used to produce the image and magnification is obtained by a system of
'optical lenses'.
 Electron microscope can resolve objects as small as 0.2 nm (as resolving power is inversely
proportional to excitation wavelength) compared to 0.2 microns for light microscopes.
 It produces useful magnification up to X 50L as compared to X 2000 in a light microscope.

Types of Electron Microscopes:
(1) Transmission Electron Microscope (TEM):
(a). Primary electrons generated by electron gun and focused by stages of condenser lenses into
bundles
(b). Electrons illuminate the sample:
─ at low magnification, a spread beam is used to illuminate a large area
─ at high magnification, a strongly condensed beam is used
(c). The pattern of electrons leaving the object, reaches the objective lens forms the image.
(d). The image is greatly enlarged by a projector lens.
(e). The traversing electrons (transmission) reach the scintillator plate at the base of the column
of the microscope.
(f). The scintillator contains phosphor compounds that can absorb the energy of the striking
electrons and convert it to light flashes, forming an image

 TEM offers two methods of specimen
observation,
(1). diffraction mode and (2). image
mode.
The objective lens forms a diffraction
pattern in the back focal plane with
electrons scattered by the sample.
 The diffraction pattern is entirely

equivalent to an X-ray diffraction pattern

 TEM uses transmitted electrons (electrons that are passing through the sample) to create
an image. As a result, TEM offers valuable information on the crystal structure of sample,
structural defects and impurities.
 TEM image spatial resolution of even less than 50 pm have been reported.
 TEM images are 2D projections of the sample.
 Due to the requirement of transmitted electrons, TEM samples must be very thin (generally
less than 150 nm) and in cases that high-resolution imaging is required, even below 30 nm.
 TEM users can magnify their samples by more than 5 million times
 TEM can be used to obtain (1). electron microscope images and (2). electron diffraction
patterns from same sample (SAED Patterns). (3). Chemical composition (4). Bonding (5).
Electronic structure and (6). Coordination number of the sample.
TEM images of nanoparticles

(2). Scanning Electron Microscope (SEM):
A scanning electron microscope (SEM) is a type of microscope which uses a focused beam of
electrons to scan a surface of a sample to create a high resolution image. SEM produces images
that can show information on a material’s surface composition and topography.

 The electrons emitted from the cathode are accelerated by an electric field up to the required
energy. The maximum electron energy in scanning electron microscopes is usually 30 keV which
can be adjusted towards the smallest energies.
 After acceleration, the electrons are focused and scanned by magnetic lenses. The electron
lenses work on the basis of the Lorentz force. The focal length of magnetic lenses and the size of
the beam spot can be changed by changing the current flowing through the coils. The minimum
diameter of the beam spot is ~1 nm.
 The beam scans the sample surface point by point and row by row.
 The image formation in a scanning electron microscope is by secondary electrons,
backscattered electrons and X-ray photons from sample.
 They are collected by dedicated detectors and utilizing their signals, an intensity map is
constructed on the screen; this is the microscopic image.
 Due to this image formation mechanism in SEM, there is no need to have a transparent to the
electrons specimen as in case of a transmission electron microscope (TEM).
 As the energies of emerging products are different, they provide information from different
depths.
The secondary electrons (SE) are specimen electrons, knocked out of the surface by inelastic
collisions with the incoming beam electrons and their majority are emitted with energies of a
few electron volts (3-5 eV) providing only near-surface information.
 In this case, the resolution is determined by the diameter of the focused electron beam.
The ultimate resolution in case of secondary electrons is ~1 nm.
SEM images of Nanoparticles
SEM images of Pollen grains
SEM images of insect


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Unit-5

Advance Analytical Techniques: Spectroscopic,

Dr. Ramya Chulliyil 1

(1). Mass spectroscopy: Principle and instrumentation, Principles of spectroscopy and

Dr. Ramya Chulliyil 2

Background knowledge required to understand the chapter

Dr. Ramya Chulliyil

 The energy levels in an atom are Orbits

 The electrons will stay in the ground state of energy, if

 If an energy which is the difference between any two

Dr. Ramya Chulliyil 4

Dr. Ramya Chulliyil 5

 The energy levels are quantised.

V = Vibrational Quantum Number

Dr. Ramya Chulliyil 8

Ultraviolet (electron level

 Absorption of energy causes a molecule to go from an initial energy state (ground

 Vibrations of bonds are excited by infrared radiation

Dr. Ramya Chulliyil 10

Absorption & emission

Dr. Ramya Chulliyil 11

 Molecular spectroscopy is the study of the interaction of electromagnetic (EM)

Dr. Ramya Chulliyil 12

Molecular Spectroscopy Rotational (Microwave)

Dr. Ramya Chulliyil 13

Mass Spectrometry- principle and instrumentation

Dr. Ramya Chulliyil

3. An electromagnetic field is also

 In a mass spectrometer a stream of positively charged ions is produced along with an

e.g. consider a helium atom

Dr. Ramya Chulliyil 17

CH4 CH3+ , CH2+ , CH+ and C+

Dr. Ramya Chulliyil 18

Dr. Ramya Chulliyil 19

(Propane) (m/z = 44)

4. Base Peak: The most intense (tallest)

(1). Academic: It is used to determine the molecular weight of compounds by separating

(3). Biotechnology: the analysis of proteins and pesticides

(4). Environmental: Water quality and food contamination

(5). Clinical: haemoglobin analysis, drug testing

Dr. Ramya Chulliyil 22

Dr. Ramya Chulliyil 23

Portion covered from syllabus:

Dr. Ramya Chulliyil

 Nuclei of atoms are composed of protons and neutrons. Like electrons,

 The circulating nuclear charge generates a magnetic dipole

 The net spin number or spin quantum number I of a particular nucleus

 When a sample of these nuclei is placed in an external

 For a nuclei with I value ½, (2I+1) = 2

External Magnetic Field

 The spins parallel to the field (α spin) has slightly

External Magnetic Field

 When Radiofrequency energy matching ( in

 Shielded nuclei/protons do not “sense’ as large magnetic field as deshielded nuclei/protons

Reference Standard - TMS

Unit of Chemical Shift

 NMR absorptions generally appear as sharp peaks.

 Most of the protons absorb between 0-10 ppm.

 Thus protons in different chemical environment (shielded and de-shielded protons in