Module 6: Spectroscopic, Diffraction and Microscopic Techniques

a) Fundamental concepts in spectroscopic and instrumental

techniques
b) Principle and applications of UV-Visible Spectroscopy technique
c) Principle and applications of X-Ray Diffraction (XRD) technique
(including numerical)
d) Overview of various techniques:
i. Atomic Absorption Spectroscopy (AAS)
ii. Infrared (IR) Spectroscopy
iii. Nuclear Magnetic Resonance (NMR) Spectroscopy
iv. Scanning Electron Microscopy (SEM) &
v. Transmission Electron Microscopy (TEM)
a) Fundamental concepts in spectroscopic and instrumental techniques
Spectroscopy Basics:
• Spectroscopy is a branch of science that studies the interaction between
electromagnetic (EM) radiation and matter.
• Spectroscopy is used as a tool for studying the structures of atoms and molecules.
• The basic principle shared by all spectroscopic techniques is to shine a beam of EM
radiation onto a sample, and observe how it responds to such a stimulus. The
response is recorded as a function of radiation wavelength.
EM Radiation Basics:
• EM radiation is a form of energy that has both wave and particle properties as shown in the
classical sinusoidal wave model (Fig. shown below), and embodies parameters such as
Wavelength, λ (m), Frequency, υ (Hz), Velocity (m/s) and Amplitude (m).
• Compared to other wave phenomena such as sound, EM radiation requires no supporting
medium for its transmission, and thus passes readily in vacuum.
• EM radiation is represented as electric and magnetic fields that undergo in-phase, sinusoidal
oscillations at right angles to each other and to the direction of propagation.
Fig. Representation of a
single ray of plane-
polarized EM radiation.

Properties of EM radiation
• Wavelength (λ) is the spatial distance between two consecutive peaks (one cycle) in the
sinusoidal waveform and is measured in sub-multiples of meter, usually in nm.
• Maximum length of the vector is called Amplitude.
• Frequency (υ) of the EM radiation is the number of oscillations made by the wave within the
timeframe of one second. It therefore has the units of 1 s-1 = 1 Hz. Frequency is related to
the wavelength via the speed of light (c = 2.998x108 m.s-1), υ = c λ-1.
• Wavenumber (υ-1) describes the number of completed wave cycles per distance and is
typically measured in 1 cm-1.
Types of EM radiation interaction with matter:
• If matter is exposed to EM radiation e.g. IR light (Fig. shown below), the radiation can be
either absorbed, transmitted, reflected, scattered or undergo photoluminescence.
• Photoluminescence is a term used to designate a number of effects including fluorescence,
phosphorescence and Raman scattering.
• Complement of the absorbed light gets transmitted.
• Color of an object that we see is due to the wavelengths transmitted or reflected. Other
wavelengths are absorbed. The more absorbed, the darker the color (more concentrated
solution).

Fig. Schematic representation

of interaction of radiation and
matter

• Interaction of EM radiation with matter is a quantum phenomenon and is dependent on

both the properties of radiation and appropriate structural parts of the samples involved.
• Origin of EM radiation is due to energy changes within matter itself.
• In spectrochemical methods, we measure the absorbed radiation.
Frequency range of each transition during the spectroscopic analysis
 Type of radiation Frequency range (Hz) Wavelength range Type of transition
excitement of nucleus to a
Radio waves <3x1011 >1 mm
higher spin state
molecular rotations,
Microwaves 3x1011-1013 1 mm-25 mm
electron spin flips
Infrared 1013-1014 25 mm-2.5 mm molecular vibrations
Near-infrared 1~4x1014 2.5 mm-750 nm outer e- molecular vibrations
Visible 4~7.5x1014 750 nm-400 nm outer electron
Ultraviolet 1015-1017 400 nm-1 nm outer electron
X-rays 1017-1020 1 nm-1 pm inner electron
Gamma-rays 1020-1024 <10-12 m Nuclear

Spectrometric Instruments:
• Ultraviolet-Visible (UV-Vis), Atomic Absorption
Spectroscopy (AAS) and Atomic Emission Spectroscopy
(AES) are used for measurement of substances.
• IR, Raman, X-ray Fluorescence (XRF), Energy-dispersive
X-ray (EDX) and Nuclear Magnetic Resonance (NMR)
spectroscopy techniques are mainly used for
characterization of substances.
• Intensity of the radiation is mostly measured with a photoelectric transducer.
• The large number of wavelengths emitted by these systems makes it possible to
investigate their electron configurations of ground and various excited states.
 (b). Principle and applications of UV-Visible Spectroscopy technique
• In UV-Vis spectroscopy, energy is absorbed by a molecule in the UV region (1 nm-
400 nm) or visible region (400 nm-750 nm) resulting in electronic transition of valence
electrons.
• Different molecules absorb radiation of different wavelengths depending on their
structure. An absorption spectrum will show a number of absorption bands
corresponding to structural (functional) groups within the molecule.
• For ex. absorption by carbonyl group in acetone is of the same wavelength as the
absorption by carbonyl group in diethyl ketone.
Three types of electronic transitions involving:
(i). π, σ and n electrons; (ii). charge-transfer electrons and (iii). d and f electrons.
▪ Inorganic species show charge-transfer absorption and are called charge-transfer
complexes.
▪ For a complex to demonstrate charge-transfer behaviour, one of its components must
be able to donate electrons and other component must be able to accept electrons.
▪ Absorption of radiation then involves the transfer of an electron from the donor to an
orbital associated with the acceptor (ε will be very high > 10,000 dm3mol-1cm-1).
▪ Absorption of UV-Vis radiation in organic molecules is restricted to certain functional
groups (chromophores) that contain valence electrons of low excitation energy. The
spectrum of a molecule containing these chromophores is complex and broad.
Electronic excitations in UV- Visible spectroscopy

Empty
orbitals

σ to π* is forbidden transition

σ to σ* transitions: Electron in a bonding σ orbital is excited to the corresponding

antibonding σ* orbital. Energy required is large. For ex. methane having only C-H bonds
can undergo only σ to σ* transitions showing abs. maximum at 125 nm. Abs. maxima
due to σ to σ* transitions are not seen in typical UV-Vis. spectra (200-700 nm).
n to σ * transitions: Saturated compounds containing atoms with lone pairs (non-
bonding electrons) are capable of n to σ * transitions. These transitions usually need
lesser energy than σ to σ* transitions. They can be initiated by light whose wavelength
is in the range 150 - 250 nm. The number of organic functional groups with n to σ *
peaks in the UV region is small.
n to π* and π to π * transitions: need an unsaturated group in the molecule to provide
the π electrons. Most absorption spectroscopy of organic compounds is based on
these transitions, since their absorption peaks fall in the experimentally convenient
spectral region between 200 - 700 nm.
Chromophore: any isolated
covalently bonded group
that shows a characteristic
absorption in the UV-Vis.
region. The only molecular
moieties likely to absorb
light in the 200 to 800 nm
region are π-electron
functions and hetero atoms
having non-bonding
electron pairs.

~ λmax: 280 nm
Auxochrome: group of
atoms attached to a Auxochrome
chromophore which modifies ~ λmax: 255 nm
the ability of that
chromophore to absorb light.
Ex. COOH, -OH, -SO3H, -NH2, -
NH-R, -N-R2
~ λmax: 385 nm
~ λmax: 320 nm
Based on the functional group present and attached to chromophores…
Bathochromic shift: absorption maximum shifted

Absorbance
to longer wavelength (Blue to Red [Red shift]).
Hypsochromic shift: absorption maximum shifted
to shorter wavelength (Red to Blue [Blue shift]).
Hyperchromism: increase in molar absorptivity
Hypochromism: decrease in molar absorptivity
Components of a UV-Vis Spectrophotometer

Source Lamp Sample Holder Photometer/Detector

Monochromator
Signal Processor and Readout
Source lamp
▪ Tungsten filament incandescent lamp used in Visible and adjacent parts of UV and
IR regions.
▪ Hydrogen or deuterium discharge lamps are used in 160~360 nm (UV region).
▪ Deuterium lamps provide maximum intensity.
▪ Source used in UV- Vis spectroscopy should meet the following criteria: (i). Beam
produced should be in the detectable and measurable range, (ii). Should serve as a
continuous source of energy and (iii). Should be stable.
Monochromator
o Filter the energy source so that a limited portion is allowed to be incident on the
sample.
o Gratings are normally used as monochromators.
o A particular wavelength can be selected using monochromator.
Sample holder
• The selection of material used for constructing the cuvette is based on the selected
range of measurement.
• Cuvette thickness depends on the absorption intensity. Cuvettes with varied shapes
are used (rectangular, cylindrical or cylindrical with flat ends).
• Cell thickness: 1, 2 and 5 cm.
• Main factor is that the cuvette window should be normal to the beam direction.
• Requirement of cuvettes in terms of its make and thickness: UV region – quartz and
Visible region – glass or quartz cells.
Photometer/Detector
Mechanism behind the photoelectric devices is the conversion of radiant energy to
electrical signal. Basically, 3 types of photometers are used: (a). Photovoltaic cells, (b).
Phototubes and (c). Photoconductive cells.
Signal processing
Electrical signal generated by the transducer is sent to the signal processor, where it is
displayed in a more convenient form for the analyst.
Applications of UV-Vis spectrophotometer:
Qualitative analysis: Identification of chromophores by scanning the absorbance at each
wavelength.
Absorbance maximum
at this wavelength

Buta-1,3-diene
CH2=CH-CH=CH2

Quantitative analysis:
• Absorbance at a particular
λmax can be measured using
photometry mode.

• Determining the reaction rate and pKa values (dissociation

constants) of weak acids or bases.
▪ Determining the percentages of keto and enol forms.
▪ To analyze metals in waste water.
▪ Determining total serum protein, serum cholesterol, etc.
▪ Characterizing pharmaceuticals, food, paint, glass and metals.
(c). Principle and applications of X-Ray Diffraction (XRD) technique
• XRD is a versatile, non-destructive characterization technique widely used in materials
science and engineering for identifying unknown crystalline materials.
• XRD works by irradiating a material with incident X-rays, and then measuring the intensities
and scattering angles of the X-rays that leave the material.
• XRD is used to study the structure and function of many biological molecules, including
vitamins, drugs, proteins and nucleic acids such as DNA.
• XRD is also used to determine structural properties like lattice parameters, strain, grain size,
epitaxy, phase composition, preferred orientation, to measure thickness of thin films and
multi-layers and to determine atomic arrangement.
• XRD also yields information on how the actual structure deviates from the ideal one, owing
to internal stresses and defects.
Selected Nobel Prize Winners involving X-ray crystallography
Year Laureate(s) Prize Rationale
Discovery of diffraction of X-rays by crystals, an important
1914 Max von Laue Physics
step in the development of X-ray spectroscopy.
1915 William Henry Bragg Physics Analysis of crystal structure by means of X-rays.
Determination of the structures of important biochemical
1964 Dorothy Hodgkin Chemistry
substances.
Ada E. Yonath, T.A.
2009 Chemistry For studying the structure and function of the ribosome.
Steitz, R. Venkatraman
2012 Brian Kobilka Chemistry For studying G-protein-coupled receptors.
 Incident-beam optics • X-ray tube: source of X-rays
Receiving-side optics
• Incident-beam optics:
condition the X-ray beam before
it hits the sample.
• Goniometer: platform that
holds and moves the sample,
optics, detector, and/or tube.
• Sample holder
• Receiving-side optics: condition the X-ray
beam after it has encountered the sample.
• Detector: count the number of X-rays
scattered by the sample.
How XRD pattern produced?
• Crystalline atoms are a periodic array of
coherent scatterers and can diffract X-rays.
• The wavelength of X-rays are similar to the
distance between atoms.
(Interference

• Diffraction from different planes of atoms

pattern)

produces a diffraction pattern, which contains

information about the atomic arrangement
within the crystal.
• When X-ray hits the crystal planes at specific
angles and the diffracted waves are in the
same phase, then constructive interference (a
peak) will be produced.
• Based on structure of the crystal (and crystal planes), the angles at which diffraction occurs may vary.
What is diffraction?
• Diffraction refers to different phenomena which occur when a
wave encounters an obstacle.
• In classical physics, the diffraction phenomenon is described as
the apparent bending of waves around small obstacles and the
spreading out of waves past small openings.
Interference of diffracted waves
• Interaction between diffracted waves is called interference.
• Constructive Interference: Waves are in-phase when each of their crests and troughs
occur exactly at the same time. Those type of waves stack together to produce a resultant
wave that has a higher amplitude. For constructive interference, path difference should
be multiples of n*λ.
• Destructive Interference: If the waves are out of phase by multiples of (n/2)*λ, then
destructive interference occurs and the amplitude of the resultant wave will be reduced.

path difference = multiple of n*λ path difference = multiple of (n/2)*λ

XRD patterns for 3 different forms of SiO2

▪ These three phases of SiO2 are

chemically identical.
▪ The amorphous glass does not
have long-range atomic order and
therefore produces only broad
pattern.
▪ Cristobalite form polycrystalline
structure.
▪ Quartz form single crystal structure.
Bragg model of diffraction
• Crystals are regular arrays of atoms, whilst X-rays are waves of EM radiation. Crystal atoms
scatter incident X-rays, primarily through interaction with the atom’s electrons. This
phenomenon is known as elastic scattering; the electron is known as the scatterer.
• A regular array of scatterers produces a regular array of spherical waves. In the majority of
directions, these waves cancel each other out through destructive interference, however,
they add constructively in a few specific directions, as determined by Bragg’s law:
• nλ = 2dsinθ , where “n” is an integer, and “λ” is the beam wavelength, “d” is the spacing
between diffracting planes and “θ” is the incident angle.
• X-rays scattered from adjacent planes will
combine constructively (constructive
interference) when angle θ between plane and
X-ray results in path-length difference that is
integer multiple “n” of X-ray wavelength “λ”.

Constructive interference Destructive interference

• In this model, a given reflection is associated with a set of evenly spaced sheets running
through the crystal, usually passing through the centres of the atoms of the crystal lattice.
• Orientation of a particular set of sheets is identified by its three Miller indices (h, k, l), and let
their spacing be noted by “d”.
• Specific directions appear as spots on the diffraction pattern called reflections.
Consequently, XRD patterns result from EM waves impinging on a regular array of scatterers.
• X-rays are used to produce the diffraction pattern because their wavelength, λ, is often the
same order of magnitude as the spacing “d” between the crystal planes (1-100 Ǻ).

• The path difference between Ray 1

and 2: 2a = 2d sinθ
• For constructive interference
2a = nλ = 2d sinθ
• Because crystal structures contain planes of atoms, each plane will reflect incident X-rays
differently. For ex. let two monochromatic X-ray beams (of a specific wavelength) strike a
crystal structure at an incoming angle of θ.
• Ray 1 will reflect off of the top atomic plane while Ray 2 will reflect from the second atomic
plane. However, because Ray 2 has to cross deeper into the atomic plane, it travels a
distance “2a” farther than Ray 1.
• If the distance “2a” is equal to the integral number (n*λ) of wavelength of 2 waves, then Ray
1 and 2 will be in-phase (constructively interfere) when they both exit the crystal.
• If we know wavelength λ of X-rays going into crystal and also measure angle θ of diffracted
X-rays coming out of crystal, then we can determine d-spacing between atomic planes.
• If we now reorient the crystal to a different atomic plane, we can measure d-spacing in
other planes. By doing multiple x-ray diffractions at different crystal orientations, we can
determined crystal structure and size of crystal unit cell.

• Incident angle (ω) is defined between the X-ray

tube
X-ray source and sample.
Detect
• Diffracted angle (2θ) is defined between
or
the incident beam and the detector angle.
ω θ 2
• Incident angle (ω) is always ½ of the
θ
detector angle 2θ i.e. θ.
• In a typical XRD instrument, the X-ray tube
is fixed, the sample rotates at θ°/min and
detector rotates at 2θ°/min.
 11
11
11
11
11
11
11

11
11
11
11
11
11
11
11
11
11
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1
1111111 1
1111111 1
1111111 1
1111111 1
1111111 1
1111111 1
1111111 1
111111 1

1111111 1
1111111 1
1
Powder X-ray Diffraction

Single Crystal X-ray Diffraction

• Powder diffraction data consists of a record of photon intensity vs detector angle 2θ

• Diffraction data can be reduced to a list of peak positions and intensities.
• Diffraction patterns are best reported using dhkl and relative intensity rather than 2θ and
absolute intensity.
• Each dhkl corresponds to a family of atomic planes {hkl}
• Individual planes cannot be resolved. This is a limitation of p-XRD vs single crystal diffraction.
Calculation of Crystallite Size using p-XRD data
Crystallites smaller than ~120 nm create broadening of diffraction peaks. This peak broadening
can be used to quantify the average crystallite size of nanoparticles using the Scherrer equation
- used for nanoparticles characterization. Must know the contribution of peak width from the
instrument by using a calibration curve

Scherrer equation can be written as:

o τ is mean size of ordered (crystalline) domains, which may be smaller or equal to grain size;
o K is a dimensionless shape factor, with a value close to unity. The shape factor has a typical
value of about 0.9, but varies with the actual shape of the crystallite;
• β is the line broadening at half the maximum intensity (FWHM), after
subtracting the instrumental line broadening, given in radians
• λ is the X-ray wavelength
• θ is the Bragg angle.
• This quantity is also
denoted as Δ(2θ)
Fig. Simulated p-XRD
patterns for wurtzite CdS
spherical particles of different
sizes from 1 μm to 1 nm. The
inset shows the 1, 2, and 5
nm XRD patterns on an
expanded y-axis scale for
clarity.
 XRD Numericals

Q. 1. In a NaCl crystal, there is a family of planes 0.252 nm apart. If the first-order

maximum is observed at an incidence angle of 18.1°, what is the wavelength of the X-
ray scattering from this crystal?

Q. 2. Estimate the crystallite size of the given nanomaterial using p-XRD data:
Peak position 2θ = 21.61o, FWHM of sample = 2.51o, k = 0.9 and λ = 1.5406 Å (degree
to radian = Degree × π/180).
 XRD Numericals
Q. 1. In a NaCl crystal, there is a family of planes 0.252 nm apart. If the first-order
maximum is observed at an incidence angle of 18.1°, what is the wavelength of the X-
ray scattering from this crystal?
Ans.: Use the Bragg equation nλ = 2dsinθ to solve for θ.
For the first-order, n=1. Then λ = 2dsinθ/n = 2dsinθ/1
λ = 2(0.252×10−9 m)sin(18.1°)/1
= 0.504*0.31068 = 1.57×10−10m or 0.157 nm.

Q. 2. Estimate the crystallite size of the given nanomaterial using p-XRD data:
Peak position 2θ = 21.61o, FWHM of sample = 2.51o, k = 0.9 and λ = 1.5406 Å (degree
to radian = Degree × π/180).
Ans.: 2θ = 21.61o (θ = 10.805o) and FWHM = 2.51o (0.043825 radian)
Crystalline grain size calculation by Scherrer’s equation: k*λ/β*cosθ
k = 0.9, λ = 1.5406 Å (0.15406 nm), β = FWHM in radian and 2θ = Bragg’s angle in o obtained
from p-XRD data.
Crystallite size = (0.9*0.15406)/(0.043825*0.982257) nm = 3.22 nm
(d)-(i): Overview of Atomic Absorption Spectroscopy (AAS)
Basic Principle: In atomic spectrometry, the elements present in a sample are converted into
gaseous atoms by a process called atomization and their interaction with the specific radiation
is measured.
• The atomization is achieved by the thermal energy of the flame.
• Wavelength of the radiation absorbed and the extent of the absorption form the basis of
the qualitative and quantitative determinations.

Elements ( Pink colour)

detected by AAS

AAS components:
▪ Radiation source
▪ Atom reservoir
▪ Monochromator
▪ Detector
▪ Readout device
Sample Atomization process

Need to break
sample into atoms
to observe atomic
spectra
Basic steps:
(1). nebulization – solution sample, get
into fine droplets by spraying thru thin
nozzle
(2). desolvation - heat droplets to
evaporate off solvent just leaving analyte
and other matrix compounds
(3,4). volatilization – convert solid
analyte/matrix particles into gas phase
(5). dissociation – break-up molecules
in gas phase into atoms
(6). excitation – with light, heat, etc. for
spectra measurement.
(7). ionization – cause the atoms to
become charged
Working procedure

AAS consists of
(i). source delivering the characteristic radiation of the analyte
(ii). an atom reservoir into which the analyte is introduced and atomized
(iii). a monochromator
(iv). detector and
(v). readout device.
The radiation from a hollow cathode lamp is made to fall on the sample of the analyte
aspirated into the flame where a part of it is absorbed. The transmitted radiation is
then dispersed by a monochromator and sent to the detector.
Hollow Cathode Lamp
Coated with element
to be analyzed

Process: use element to detect element

(1). ionizes inert gas to high potential (300V): Ar 🡪 Ar+ + e-
(2). Ar+ go to “-ve” cathode and hit surfaces
(3). As Ar+ ions hit cathode, some of deposited element is excited and dislodged
into gas phase (sputtering)
(4). excited element relaxes to ground state and emits characteristic radiation
Advantages: sharp lines specific for element of interest
Disadvantages: can be expensive, need to use different lamp for each element
tested.
LIMITATIONS: Need for trace metal analyses at mg/L and even sub mg/L levels calls for
a more sensitive technique. To meet this trace analysis, one has to use GRAPHITE
FURNACE ATOMIC ABSORPTION.
(d)-(ii): Overview of Infrared (IR) Spectroscopy
• An IR spectrometer is an optical instrument used to measure properties of light
over a specific portion of the EM spectrum in the range of 5 to 20 µ.
• Fourier Transform Infrared (FTIR) spectrometer obtains IR spectra by first
collecting an interferogram of a sample signal using an interferometer and then
performs a Fourier Transform on the interferogram to obtain the spectrum.
• An interferometer is an instrument that uses the technique of superimposing
(interfering) two or more waves, to detect differences between them. FTIR
spectrometer uses a Michelson interferometer.
• Fourier transform defines a relationship between a signal in time domain and its
representation in frequency domain.
• Being a transform, no information is created or lost in the process, so the original
signal can be recovered from the Fourier transform and vice versa.
What is Finger Print region and Functional Group region in IR spectroscopy?
• It is convenient to split an IR spectrum into two approximate regions: 4000-1000
cm-1 known as the functional group region, and <1000 cm-1 known as the
fingerprint region.
• It usually contains a large number of peaks, making it difficult to identify individual
peaks.
Wavelength = 1/Wavenumber 5 µ corresponds to 2000 cm-1.
For the IR, wavelength is in µ. 20 µ corresponds to 500 cm-1.
Wavenumber is typically in 1/cm, or cm-1. 15 µ corresponds to 667 cm-1.
• The absorption of light, as it passes through a medium, varies linearly with the
distance the light travels and with concentration of the absorbing medium.
• Where, “a” is the absorbance, “ε” is a characteristic constant for each material at a
given wavelength (known as the extinction coefficient or absorption coefficient),
“c” is concentration, and “l” is the length of the light path, the absorption of light
may be expressed by the simple equation a= εcl
• Infrared spectroscopy is the measurement of the wavelength and intensity of the
absorption of mid-infrared light by a sample. Mid-infrared is energetic enough to
excite molecular vibrations to higher energy levels.
• The wavelength of infrared absorption bands is characteristic of specific types of
chemical bonds, and infrared spectroscopy finds its greatest utility for
identification of organic and organometallic molecules.
Theory of Infrared Absorption Spectroscopy
• For a molecule to absorb IR, the vibrations or rotations within a molecule must
cause a net change in the dipole moment of the molecule. The alternating
electrical field of the radiation (remember that EM radiation consists of an
oscillating electrical field and an oscillating magnetic field, perpendicular to each
other) interacts with fluctuations in the dipole moment of the molecule.
• If the frequency of the radiation matches the vibrational frequency of the molecule
then radiation will be absorbed, causing a change in the amplitude of molecular
vibration.
Beer-Lambert Law
• When IR light passes through a molecular material, absorption can occur.
• The extent of absorption is given by the Beer-Lambert Law (also known as Beer’s Law),
where A is the absorption, T is the transmittance, I0 is the incoming intensity of light, and I is
the light transmitted through the sample. On the right side, ε is the absorptivity, L is the path
length, and c is the concentration of the specific analyte.
• The absorptivity characterizes how much light is absorbed by a specific molecule at a specific
wavelength. The product Lc effectively represents how many molecules are in the beam.

• Rotational levels are quantized, and absorption of IR by gases yields line spectra.
• However, in liquids or solids, these lines broaden into a continuum due to molecular
collisions and other interactions.
• In general, a molecule which is an excited vibrational state will have rotational energy and
can lose energy in a transition which alters both the vibrational and rotational energy
content of the molecule.
• The total energy content of the molecule is given by the sum of the vibrational and
rotational energies.
• For a molecule in a specific vibrational and rotational state, denoted by the pair of quantum
numbers (v, J), we can write its energy as:
E(v, J)=Evib(v) + Erot(J)
 Bending
Vibrations

Stretching Vibrations
• An IR spectrophotometer is an instrument that passes
IR light through an organic molecule and produces a
spectrum that contains a plot of the amount of light
transmitted on the vertical axis against the
wavelength of infrared radiation on the horizontal
axis.

• The mirror moves at a fixed rate. Its position is

determined accurately by counting the interference
fringes of a collocated Helium-Neon laser.
• The Michelson interferometer splits a beam of
radiation into two paths having different lengths, and
then recombines them.
• Detector measures the intensity variations of the exit
beam as a function of path difference.
• Monochromatic source would show a simple sine
wave of intensity at detector due to constructive and
destructive interference as the path length changes.

• In IR spectra, the absorption peaks point downward

because the vertical axis is the % transmittance of the
radiation through the sample.
• Absorption of radiation lowers the % transmittance
value. Since all bonds in an organic molecule interact
with IR radiation, IR spectra provide a considerable
amount of structural data.
Absorbance of organic functional groups and bonds in the IR region
Examples:

Octane
Octane

Hydrocarbons
• Hydrocarbons compounds contain only C-H and C-C bonds, and more information can be
obtained from IR spectra arising from C-H stretching and C-H bending.
• In alkanes, which have very few bands, each band in the spectrum can be assigned:
• C–H stretch from 3000–2850 cm-1
• C–H bend or scissoring from 1470-1450 cm-1
• C–H rock, methyl from 1370-1350 cm-1
• C–H rock, methyl, seen only in long chain alkanes, from 725-720 cm-1
• Above IR spectrum is for Octane. Note the change in dipole moment with respect to distance
for the C-H stretching is greater than that for others shown, which is why the C-H stretch
band is the more intense.
 1-octene
1-octene

Above figure shows the IR spectrum of 1-octene. In alkenes compounds, each band in the spectrum can be
assigned:
• C=C stretch from 1680-1640 cm-1
• =C–H stretch from 3100-3000 cm-1
• =C–H bend from 1000-650 cm-1
• As alkanes compounds, these bands are not specific and are generally not noted because they are
present in almost all organic molecules.

1-hexyne

Spectrum of 1-hexyne, a terminal alkyne, is shown above. In alkynes, each band in the spectrum is assigned:
• –C≡C– stretch from 2260-2100 cm-1
• –C≡C–H: C–H stretch from 3330-3270 cm-1
• –C≡C–H: C–H bend from 700-610 cm-1
Above is the spectrum of toluene. In aromatic compounds, each band in spectrum is assigned :
• C–H stretch from 3100-3000 cm-1
• overtones, weak, from 2000-1665 cm-1
• C–C stretch (in-ring) from 1600-1585 cm-1
• C–C stretch (in-ring) from 1500-1400 cm-1
• C–H “oop” from 900-675 cm-1
• Note that this is at slightly higher frequency than is the –C–H stretch in alkanes. This is a very
useful tool for interpreting IR spectra.
• Only alkenes and aromatics show a C–H stretch slightly higher than 3000 cm-1.
What advantages does in-situ FTIR Spectroscopy bring to Reaction Analysis?
• The mid-IR energy region yields detailed “fingerprint” spectra of starting materials,
intermediates, products and by-products allowing continually tracking of these key species
as a function of time.
• Real-time measurement, performed every minute or less.
• In-situ, no extractive sampling required; measure chemistry without disturbing the reaction
• Non-destructive; preserving the integrity of the chemical reaction
• Measures reactions in batch, semi-batch or continuous flow operation
• Measures reactions run under pressure or at elevated or low temperature
• Measures reactions in aqueous or non-aqueous media and over a broad range of pH
• Spectral data can be converted into real-time concentration data, enabling the development
of key kinetic parameters and precise endpoint determination
• Provides a primary means of obtaining important kinetic data and factual evidence
supporting proposed mechanisms
• In-situ FTIR helps to identify and track transient intermediates that might affect product yield
and quality and is key to mechanistic understanding
• Reaction trends are followed in real time, allowing for the monitoring key reaction events
such as initiation, steady-state, endpoint and decomposition.
Where FTIR Spectroscopy used for?
Academic Research Pharmaceutical Industry Chemical Industry
Organocatalysis Chemical Synthesis Intermediates
Metal-Mediated Hydrogenation Reactions Surfactants
Chemistry Metal Catalyzed Reactions Flavors and Fragrances
Chemo- and Biocatalysis Biocatalysis/Enzymatic Catalysis Coatings/Pigments
C-H Activation Crystallization and Recrystallizatio Agrochemicals
Mechanistic Studies n (Supersaturation) Initiators
Reaction Kinetics/Reaction Halogenations/Lithiations/Fluorine Bulk Chemicals
Progress Kinetics Analysis and Fluorination Chemistry Isocyanate Reactions
Catalyst Cycles Suzuki and Other Cross-Coupling Ethylene Oxide and Propylene
Polymerization Kinetics Reactions Oxide (EO/PO)
Organometallic Chemistry Highly Oxidizing Reactions
Low Temperature Chemistry Hydroformylation
Quality by Design and Process Catalytic Reactions
Analytical Technology Phosgenations
Esterifications
Halogenations
(d)-(iii). Overview of Nuclear Magnetic Resonance (NMR) Spectroscopy
• NMR spectroscopy or magnetic resonance spectroscopy (MRS) is a spectroscopic
technique used to observe local magnetic fields around atomic nuclei.
• 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, in
modern organic chemistry practice, NMR spectroscopy is the definitive method to
identify monomolecular organic compounds.

• Similarly, biochemists use NMR to identify proteins

and other complex molecules. 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 13C-NMR spectroscopy, but it is
applicable to any kind of sample that contains
nuclei possessing spin.
Principle of NMR: usually involves 3 sequential steps:
• Alignment (polarization) of magnetic nuclear spins in an applied, constant magnetic field B0.
• Perturbation of this alignment of the nuclear spins by a weak oscillating magnetic field; usually
referred to as a radio-frequency (RF) pulse.
• Detection and analysis of EM waves emitted by nuclei of the sample as a result of this
perturbation.
The following features lead to the NMR phenomenon:
• A spinning charge generates a magnetic field. The resulting spin-magnet has a magnetic moment
(μ) proportional to the spin.
• In the presence of an external magnetic field (B0), two spin states exist [+1/2 and -1/2].
• Magnetic moment of the lower energy +1/2 state is aligned with the external field, but that of the
higher energy -1/2 spin state is opposed to the external field. Note that the arrow representing
the external field points North.
• Difference in energy between the two spin states is dependent on the external magnetic field
strength, and is always very small. The following diagram illustrates that the two spin states have
the same energy when the external field is zero, but diverge as the field increases. At a field equal
to Bx a formula for the energy difference is given (remember I = 1/2 and μ is the magnetic
moment of the nucleus in the field).
Chemical shift
• The precise resonant frequency of the energy transition is dependent on the
effective magnetic field at the nucleus. This field is affected by electron shielding
which is in turn dependent on the chemical environment. As a result, information
about the nucleus' chemical environment can be derived from its resonant
frequency.
• In general, the more electronegative the nucleus is, the higher the resonant
frequency. Other factors such as ring currents (anisotropy) and bond strain affect
the frequency shift.
• It is customary to adopt tetramethylsilane (TMS) as the proton reference
frequency. This is because the precise resonant frequency shift of each nucleus
depends on magnetic field used. Frequency is not easy to remember (for example,
the frequency of benzene might be 400.132869 MHz) so it was decided to define
chemical shift as follows to yield a more convenient number such as 7.17 ppm.
δ = (ν-ν0)/ν0
• The chemical shift, using this equation, is not dependent on the magnetic field and
it is convenient to express it in ppm where (for proton) TMS is set to ν0 thereby
giving it a chemical shift of zero. For other nuclei, ν0 is defined as Ξ νTMS where Ξ
(Greek letter Xi, uppercase Ξ, lowercase ξ) is the frequency ratio of the nucleus (e.
g., 25.145020% for 13C).
• In the case of 1H-NMR spectrum of ethyl
benzene, the methyl (CH3) group is the most
electron withdrawing (electronegative) and
therefore resonates at the lowest chemical
shift.
• The aromatic phenyl group is the most
electron donating (electropositive) so has the
highest chemical shift.
• The methylene (CH2) falls somewhere in the middle. However, if the chemical shift
of the aromatics were due to electro positivity alone, then they would resonate
between 4 and 5 ppm. The increased chemical shift is due to the delocalized ring
current of the phenyl group.
Uses of NMR spectroscopy
• NMR spectroscopy is used in quality control and research for determining the
content and purity of a sample as well as its molecular structure.
• For ex. NMR can quantitatively analyse mixtures containing known compounds. For
unknown compounds, NMR can either be used to match against spectral libraries
or to infer the basic structure directly. Once the basic structure is known, NMR can
be used to determine molecular conformation in solution as well as studying
physical properties at the molecular level such as conformational exchange, phase
changes, solubility and diffusion.
(d)-(iv). Overview of Scanning Electron Microscopy (SEM) &
(d)-(v). Transmission Electron Microscopy (TEM)

Scanning Electron Microscopy (SEM) Transmission Electron Microscopy (TEM)

Limitations of using Light Microscope (LM):
▪ LM has a magnification of 1000x with a resolution of 200 nm.
▪ Resolving power of LM is limited by the number and quality of the lenses but also by the
wavelength of the light used for illumination.
▪ Using light with a short wavelength (blue or ultraviolet) gave a small improvement.
▪ Immersing the specimen and the front of the objective lens in a medium with a high refractive
index (oil) gave another small improvement leading upto 100 nm.
SEM basics
o Imagine yourself alone in an unknown darkened room with only a fine beam torch. You
might start exploring the room by scanning the torch beam systematically from side to
side gradually moving down so that you could build up a picture of the objects in the
room in your memory.
o SEM uses an electron beam instead of a torch, an electron detector instead of eyes
and a fluorescent screen and camera as memory.
▪ Accelerated electrons behave in vacuum just like light. They travel in straight lines and
have a wavelength which is about 100 000 times smaller than that of light.
▪ Furthermore, electric and magnetic fields have the same effect on electrons as glass
lenses and mirrors have on visible light.
▪ The first electron microscope used two magnetic lenses and later added a third lens to
achieve a resolution of 100 nm, twice as good as that of the light microscope.
▪ Now, the electron microscope uses five magnetic lenses in the imaging system, a
resolving power of 0.1 nm at magnifications of over 1 million times.
Essential components of SEM
1. Electron gun as Source
2. Electron Lenses
3. Sample Stage
4. Detectors for all signals of interest
5. Display/Data output devices
6. Infrastructure Requirements: (a). Power Supply, (b). Vacuum System, (c). Cooling system,
(d). Vibration-free floor and (e). Room free of ambient magnetic and electric fields.
Vacuum
1. Electron gun as Source:
Electron gun consists of 3 important
components:
(i). Filament made from hair-pin shaped
Tungsten (or Lanthanum Hexaboride)
functioning as Cathode. Voltage applied
to the filament heats it up to 2700 oC;
(ii). Wehnelt cylinder (also known as
grid cap or cylinder) made from
platinum or tantalum foil. It is used for
focusing and controlling the electron
beam and has the shape of a hollow
cylinder. The bottom side of the
cylinder has an opening at its centre
with 200 to 1200 μm diameter; and
(iii). Anode, which is positive with
respect to the filament, forms powerful
attractive forces for electrons. This
causes electrons to accelerate (several
hundred thousand km/sec) toward the
anode. Some accelerate right by the
anode and down by the column onto These 3 components together form a triode electron
the sample. gun which is a very stable source of electrons.
2. Electron lenses - Magnification and resolution
o Magnification is entirely determined by electronic circuitry that scans the beam over the
specimen (and simultaneously over fluorescent screen of monitor where the image appears).
o Magnification can be as high as 3,00,000x. Increasing the magnification is achieved by
reducing the size of the area scanned on the specimen.
o Resolution of 1 nm can be attained depending on:
a) Diameter of electron beam on the specimen surface
b) Specimen properties
c) Specimen preparation technique
d) Instrumental parameters such as
i. beam intensity
ii. accelerating voltage
iii. scanning speed
iv. distance from the last lens to specimen (referred to as the working distance) and
v. angle of the specimen surface with respect to the detector.
3. Sample stage: What happens to the specimen during electron bombardment?
Electron beam scan the specimen in a rectangular spot less than 4 nm in diameter.
a) Specimen itself emits secondary electrons.
b) Some of the primary electrons are reflected (back-scattered electrons).
c) Electrons are absorbed by the specimen.
d) Specimen emits X-rays, and sometimes emits photons (light).
All these phenomena are interrelated and depends on the specimen topography, its chemical
state and atomic number. The number of backscattered electrons, secondary electrons and
absorbed electrons at each point of the specimen depends on the specimen’s topography to a
much greater extent than the other properties mentioned above.
Specimen orientation and manipulation
o SEM image quality depends on specimen orientation and distance from detectors and lens.
o Specimen stage allows the specimen to be moved in a horizontal plane (X and Y direction)
and up and down (Z direction) and rotated and tilted as required. These movements are
often motorized and controlled by the PC.
o SEM models differ in size of their specimen chambers allowing various sizes of specimens to
be introduced and manipulated. Maximum specimen size also determines the price because
larger the specimen chamber, larger the goniometer movement needed and larger the
pumping system needed to maintain a good vacuum.
o The simplest model accepts specimens of a few cm in diameter and can move them 50 mm
in the X and Y directions. Largest chamber accepts samples up to 200 mm in diameter and
can move them 150 mm in each direction.
o All models allow samples to be tilted up to high angles and rotated through 360o.
o There are special stages for heating, cooling and straining specimens.
4. Detectors for all signals of interest
▪ Detectors for backscattered electrons and secondary electrons are either a scintillation
detector or a solid state detector.
▪ In the case of scintillation detector, electrons strike a fluorescent screen, which then emits
light that is amplified and converted into an electrical signal by photomultiplier tube.
▪ In the case of solid state detector, it works by amplifying the minute signal produced by the
incoming electrons in a semiconductor device.
▪ Amplified signal of the electron beam is also impacted on a cathode ray tube. Both the beam
in the microscope and the one in the CRT are scanned at the same rate and the 1-to-1
5. Display/Data output devices
o SEM is usually equipped with two image monitors, one for observation by operator and
other a high resolution monitor, equipped with ordinary photo camera (or Polaroid)
o To facilitate the observation and correct choice of the parameters mentioned above, SEM
have an image store in which the image is built up scan by scan and displayed at TV speed so
that there is a steady, flicker-free image on the viewing monitor.
o Digital images are stored electronically for subsequent enhancement and analysis.
o Since the SEM image is electronically produced, it can be subjected to contrast
enhancement, inversion (black becomes white), mixing of images from various detectors,
6. subtraction
Importanceofofthe Vacuum
image infromSEM column:
one detector from that produced by a different detector,
• colour
In SEM,coding
the column through
and image which electron beam passes through must always be at vacuum.
analysis.
o• All
If the
thesesample is in may
techniques a gasbefilled environment,
applied if it suits thethe electron
primary aimbeam cannot the
of extracting be best
generated or
possible
maintained from
information because of a high instability in the beam. Also, gases could react with the
the specimen.
electron source, causing it to burn out, or cause electrons in the beam to ionize, which
produces random discharges and leads to instability in the beam.
• Transmission of beam through electron optic column would also be hindered by the
presence of other molecules from the sample or the microscope itself, and could form
compounds and condense on the sample. This would lower the contrast and obscure detail
in the image.
• Sufficiently low vacuum in SEM column is produced by either an oil diffusion pump or a
turbo-molecular pump backed by a rotary pre-vacuum pump.
• These combinations provide reasonable exchange times for specimen, filament and aperture
(less than 2 minutes) without the need to use vacuum airlocks.
Strengths of SEM
▪ Gold as conductive coating: A heavy element like gold is preferred for use as conductive
coating as it gives a good yield of secondary electrons and thereby a good quality image. In
addition, it gives a fine grain coating and is easily applied in a sputter coater. The layer
required to ensure a conducting layer is quite thin (about 10 nm).
▪ Operational features: Most SEM models are easier to operate, with user-friendly interfaces,
minimal sample preparation and rapid data acquisition (less than 5 min/image).
▪ SEM generate data in digital formats, which are highly portable.

Limitations of using SEM:

o Sample condition: Must be solid and stable in a vacuum to order of 10-5~10-6 torr. Samples
likely to outgas at low pressures (rock sample saturated with hydrocarbons, wet samples
such as coal, organic materials or swelling clays) are unsuitable for examination in
conventional SEM. However, they can be measured using "low vacuum" SEM.
o SEM cannot measure samples that fail to withstand the electron bombardment.
o If the specimen contains any volatile components such as water, it need to be removed using
a drying process (or can be frozen solid).
o Non-conducting specimens will charge up under electron bombardment, and hence an
electrically conductive coating must be applied to electrically insulating samples for study in
conventional SEM, unless the instrument is capable of operation in a low-vacuum mode.
o Detector type: Solid state x-ray detector (EDS) cannot detect elements with A.N. less than 11
(Na). While EDS is very fast and easy to utilize, they have relatively poor energy resolution
Transmission Electron Microscope (TEM)
o TEM can be compared with a slide projector. In projector case, light from a source is made
into a parallel beam by condenser lens, and passed through the slide (object), which is further
focused as an enlarged image onto the screen by the objective lens.
o In TEM case, the light source is replaced by an electron source (tungsten filament heated in
vacuum), glass lenses are replaced by magnetic lenses and the projection screen is replaced
by a fluorescent screen which emits light when struck by electrons.
o Electromagnetic lenses are variable, i.e. by varying the current through the lens coil, the
focal length which determines the magnification can be varied.
o Entire electron path from gun to screen has to be under vacuum (otherwise the electrons
would collide with air molecules and be absorbed). The final image is viewed through a
window in the projection chamber.
o Specimen (object) has to be very thin to allow the electrons to pass through it.
o TEM specimens are usually 0.5 µm or less
thick. Higher the electrons speed, higher the
accelerating voltage in the gun, and thicker
the specimen that can be studied.
Slide
projector

TEM
Layout of a basic TEM
What happens in the specimen during the electron bombardment in TEM?
When electrons impinge on the specimen, a number of things can happen:
a. Some of the electrons are absorbed as a function of thickness and composition of the
specimen, which causes amplitude contrast in the image.
b. Other electrons are scattered over small angles, depending on the composition of the
specimen, which causes phase contrast in the image.
c. In crystalline specimens, electrons are scattered in very distinct directions which are a
function of the crystal structure, which causes diffraction contrast in the image.
d. Some of the impinging electrons are reflected, and are called backscattered electrons.
e. The impinging electrons can cause the specimen itself to emit electrons (these are called
secondary electrons).
f. The impinging electrons cause the specimen to emit X-rays whose energy and wavelength
are related to the specimen’s elemental composition.
g. The impinging electrons cause the specimen to emit photons or light (called cathodo-
luminescence.
h. Finally, electrons which have lost an amount of energy because of interaction with the
specimen can be detected by an Energy Loss Spectrometer which is the equivalent of a
prism in light optics.
Essential components of TEM
1. Electromagnetic lenses
o When an electrical current is passed through the coils (C), an electromagnetic field is created
between the pole pieces (P) which form a gap in the magnetic circuit.
o By varying the current through the coils, the magnification of the lens can be varied. This is
the essential difference between the magnetic lens and the glass lens.
o Otherwise they behave in the same way and have the same types of aberration: spherical
aberration (the magnification in the centre of the lens differs from that at the edges),
chromatic aberration (the magnification of the lens varies with the wavelength of the
electrons in the beam) and astigmatism (a circle in the specimen becomes an ellipse in the
image).
o Spherical aberration is a very important characteristic which is largely determined by the lens
design and manufacture. Chromatic aberration is reduced by keeping the accelerating voltage
as stable as possible and using very thin specimens. Astigmatism can be corrected by using
variable electromagnetic compensation coils. The condenser lens system focuses the electron
beam onto the specimen under investigation as much as necessary to suit the purpose.
o The objective lens produces an image of the specimen which is then magnified by the
remaining imaging lenses and projected onto the fluorescent screen. If the specimen is
crystalline there will be a diffraction pattern at a different point in the lens known as the back
focal plane.
o By varying the strength of the lens immediately below the objective lens, it is possible to
enlarge the diffraction pattern and project this onto the fluorescent screen. In the Tecnai
series of TEMs, the objective lens is followed by four lenses: a diffraction lens, an
intermediate lens and two projector lenses.
o To guarantee a high stability and to achieve the highest possible magnification, the lenses
in a modern TEM are water cooled. On the way from the filament to the fluorescent
screen, the electron beam passes through a series of apertures with different diameters.
These apertures stop those electrons which are not required for image formation (e.g.
scattered electrons).
o Using a special holder carrying four different apertures, the diameter of the apertures in
the condenser lens, the objective lens and the diffraction lens can be selected from outside
the column as dictated by circumstances.
2. Observation and recording of the image
• Image on fluorescent screen can be observed through a large window in the projection
chamber. In order to examine fine detail or to assist correct focusing of image, a special
fine-grain focusing screen is inserted into beam and observed through high-quality 12x
binocular viewer.
• Electrons have same influence on photographic material as light. It’s only necessary to
replace the fluorescent screen with a photographic film in order to record the image.
• In practice the fluorescent screen hinges up to allow the image to be projected on the film
below. It is also possible to use a TV camera to record the image digitally, making it
suitable for subsequent enhancement and analysis. It can also be used for recording
dynamic phenomena using a video tape recorder or as the input signal for an image
analysis system.
3. Diffraction
• When a wave passes through a periodic structure whose periodicity is of the same order of
magnitude as the wavelength, the wave emerging is subject to interference which produces a
pattern beyond the object.
• This phenomenon can be observed when ocean waves pass through a regular line of posts or
when a street lamp is seen through an umbrella. The street lamp appears as a rectangular
pattern of spots of light, bright in the centre and then getting fainter. This is caused by
diffraction of light by the weave of the umbrella fabric and the size and form of the pattern
provide information about the structure (closeness of weave and orientation).
• In exactly the same way, electrons are diffracted by a crystal and the pattern of spots on the
screen of the microscope gives information about the crystal lattice (shape, orientation and
spacing of the lattice planes).

4. Vacuum
• Electrons behave like light only when they are manipulated in vacuum. The whole column
from gun to fluorescent screen and including the camera is evacuated. Various levels of
vacuum are necessary: the highest vacuum is around the specimen and in the gun; a lower
vacuum is found in the projection chamber and camera chamber.
• Different vacuum pumps are used to obtain and maintain these levels. The highest vacuum
attained is of the order of a ten millionth of mm Hg.
• To avoid having to evacuate the whole column every time a specimen or photographic
material or a filament is exchanged, a number of airlocks and separation valves are built in. In
modern TEMs the vacuum system is completely automated and the vacuum level is
continuously monitored and fully protected against faulty operation.
5. Electronics
• To obtain the very high resolution of which modern TEMs are capable, the accelerating
voltage and the current through the lenses must be extremely stable.
• The power supply cabinet contains a number of power supplies whose output voltage or
current does not deviate by more than one millionth of the value selected for a particular
purpose. Such stabilities require very sophisticated electronic circuits.
• Digital electronic techniques in general and microprocessor-based techniques in particular
play an important role in this respect. Modern electron microscopes employs a fast, powerful
PC (personal computer) to control, monitor and record the operating conditions of the
microscope.
• This results in a dramatic reduction in number of control knobs compared with earlier models
and a microscope which is very easy to use. Furthermore, it allows special techniques to be
embedded in the instrument so that the operator can carry them out using the same controls.
• PC can be attached to a network to allow automatic back-ups to be made and results to be
downloaded to other workstations. The microscope can always be brought up to date by
installing new software or by replacing the computer with the latest model.
6. Specimen orientation and manipulation
• With most specimens, it is not sufficient to move them only in the horizontal plane. Although
the specimen is thin, there is nevertheless information in the image coming from various
depths within the specimen.
• This can be seen by tilting the specimen and taking stereo photographs. In order to define the
axis of tilt, it is necessary to be able to rotate the specimen.
• Crystalline specimens need to have a second tilt axis perpendicular to the first tilt axis in order
to be able to orient a part of the specimen so as to obtain the required diffraction pattern.
These requirements can be fulfilled in a device called a goniometer.
• The goniometer is a specimen stage designed to provide, in addition to X and Y translation of
the specimen, tilt about one or two axes and rotation as well as Z movement (specimen height)
parallel to the beam axis.
• It is usual also to provide for heating, cooling and straining of the specimen for specialised
experiments in the microscope.
• The goniometer is mounted very close to the objective lens; the specimen is actually located in
the objective lens field between the pole pieces because it is there that the lens aberrations are
smallest and the resolution is highest.
• The goniometer itself provides motorised X, Y and Z movement and tilt about one axis. The
specimen is mounted near the tip of a rod-shaped holder which in turn is introduced into the
goniometer through an air lock.
• It is the specimen holder rod which provides the extra tilt axis or the rotation or heating,
cooling or straining, a special holder being needed for each purpose.
Specimen preparation
• TEM can be used in any branch of science and technology where it is desired to study the
internal structure of specimens down to the atomic level.
• It must be possible to make the specimen stable and small enough (~3 mm in dia.) to permit
its introduction into the evacuated microscope column and thin enough (less than about 0.5
µm) to permit the passage of electrons.

Every branch of research has its own specific methods of preparing the specimen for
electron microscopy.
1) In biology, tissues are treated as follows: at first, there is a chemical treatment to remove
water and preserve the tissue as much as possible in its original state; it is then embedded in a
hardening resin; after resin has hardened, slices (sections) with average thickness of 0.5 µm
are cut with an instrument called ultra-microtome equipped with a glass or diamond
knife.
2) The tiny sections thus obtained are placed on a specimen carrier – usually a 3 mm diameter
copper specimen grid which has been coated with a structure-less carbon film 0.1 µm thick.
3) In metallurgy, a 3 mm diameter disc of material (thickness ~0.3 mm) is chemically treated in
such a way that in the center of the disc the material is fully etched away. Around this hole,
there will usually be areas that are sufficiently thin (approximately 0.1 µm) to permit
electrons to pass through.
4) In a semiconductor, it is sometimes desired to cut out a section of material perpendicular to
the surface in order to investigate a defect. This is done by ion-beam etching.
Limitations of TEM:
Not all specimens can be made thin enough for the TEM. Only a very narrow region of the
specimen appears in focus in the image and there is considerable distortion. The technique has
not found wide application in the study of surfaces.

The most important differences between TEM and SEM are:

(1). the beam is not static as in the TEM: with the aid of an electromagnetic field, produced by
the scanning coils, the beam is scanned line by line over an extremely small area of the
specimen’s surface
(b). the accelerating voltages are much lower than in TEM because it is no longer necessary to
penetrate the specimen; in a SEM they range from 200 to 30.000 volts.
(c). specimens need no complex preparations.
TEM specimens have to be very thin in order to be imaged with electrons. Typically, the
specimen must be no thicker than a few hundred nm.
SEM TEM

Vacuum
SEM TEM