Module 6: Spectroscopic, Diffraction and Microscopic Techniques

Fundamental concepts in spectroscopic and instrumental techniques

1. Principle and applications of UV-Visible Spectroscopy technique
2. Principle and applications of X-Ray Diffraction (XRD) technique (including
numerical)
3. Scanning Electron Microscopy (SEM)
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.
1. 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 an 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 UV-vis spectrum of Formaldehyde
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
orbital
s

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 chromophore Auxochrome
which modifies the ability of ~ λmax: 255
that chromophore to absorb nm
light. Ex. COOH, -OH, -SO3H, -
NH2, -NH-R, -N-R2

~ λmax: 385
~ λmax: 320
Based on the functional group present and attached to chromophores…
Bathochromic shift: absorption maximum shifted to longer

Absorbance
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 n to σ * transitions:
Saturated compounds
at this wavelength containing atoms with
lone pairs (non-
bonding electrons) are
capable of n to σ *
transitions. These
Buta-1,3-diene transitions usually
CH2=CH-CH=CH2 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
Quantitative analysis: the π electrons. Most
absorption
• Absorbance at a particular spectroscopy of organic
compounds is based on
λmax can be measured using these transitions, since

• photometry mode. their absorption peaks

Determining the reaction rate and pKa values (dissociation fall in the
constants) of weak acids or bases. experimentally
convenient spectral
▪ Determining the percentages of keto and enol forms. region between 200 -
▪ To analyze metals in waste water. 700 nm.

▪ Determining total serum protein, serum cholesterol, etc.

Principle and applications of X-Ray Diffraction (XRD) technique
• XRD is a non-destructive (sample will not consumed) 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 step in the
1914 Max von Laue Physics
development of X-ray spectroscopy.
1915 William Henry Bragg Physics Analysis of crystal structure by means of X-rays.
1964 Dorothy Hodgkin Chemistry Determination of the structures of important biochemical substances.
Ada E. Yonath, T.A. Steitz, R.
2009 Chemistry For studying the structure and function of the ribosome.
Venkatraman
2012 Brian Kobilka Chemistry For studying G-protein-coupled receptors.
X-ray diffraction- Basics
When X-rays with a definite wave length (CuKα = 1.54Å) is passed in to the
powder, the atomic planes in the crystals causes an incident beam of X-ray to
interfere with one another as they leave the crystal. This phenomenon is called
X-ray diffraction

Bragg’s Law, nλ =
2dSinθ
Crystalline or powder structures are deduced by the
diffraction of known radiation incident from solid materials.
The three-dimensional regularity of unit cells in crystalline
materials results in coherent scattering of the radiation.
The directions of the scattered beams are a function of the
wavelength of the radiation and the specific inter-atomic
spacing (dhkl ) of the plane from which the radiation scatters.
 The intensity of the scattered beam depends on the position
of each atom in the unit cell and on the orientation of the crystal
relative to the direction of the incident X-ray beam.
Powder XRD – Instrumentation

The simplest of all modern X-ray analyses is powder

analysis using an X-ray diffractometer.
 The technique can be used to characterize powders as well
as polycrystalline materials.
The material of interest is ground to produce a fine,
randomly orientated powder, with each particle in the
powder consisting of small single crystals or an aggregate of
crystals 10 μm or less.
The powder is placed into a recess of a plastic sample
holder and leveled flat in the sample holder with a straight
edge
XRD - Instrumentation

1. X-ray Tube: The source of X Rays

2. Incident-beam optics: Condition the X-ray beam before
it hits the sample
3. The goniometer: The platform that holds and moves the
sample, optics, detector, and/or tube
4. Sample holder
5. Receiving-side optics: Condition the X-ray beam after it
has encountered the sample
6. Detector: Count the number of X Rays scattered by the
sample
XRD - Instrumentation

X-ray
Source
Detector

θ θ
2θ

Sample
Typical XRD Instrument
XRD - Instrumentation
1. Sealed X-ray tubes tend to operate at 1.8 to 3 kW. Rotating anode X-ray
tubes produce much more flux because they operate at 9 to 18 kW. Both
sources generate X rays by striking the anode target with an electron beam
from a tungsten filament. The target must be water cooled. The target and
filament must be contained in a vacuum.
2. Goniometer: The mechanical assembly that makes up the sample holder,
detector arm and associated gearing is referred to as goniometer.
3. Detectors:
 Point detectors : observe one point of space at a time, it is very slow, but
compatible with most/all optics,
 Scintillation and gas proportional detectors: count all photons, within an energy
window, that hit them
 Si(Li) detectors : Can electronically analyze or filter wavelengths
 Position sensitive detectors: Linear PSDs observe all photons scattered along a
line from 2 to 10°Long, 2D area detectors observe all photons scattered along a
conic section, gas proportional (gas on wire; microgap anodes), limited
resolution,
Applications of XRD

1. Phase identification (e.g TiO2, SiO2, TiN etc)

2. Identification of crystalline phases and orientation (hlkl
(100, 111)
3. Structural determination by calculating lattice
parameters
4. Grain size (D) using Scherrer’s equation (D = kλ/Bcosθ)
5. Phase composition
6. Preferred orientation of a materials (100, 111 etc.,)
based on the intensity of peak
7. Thermal expansion of a materials
Applications of XRD

Identification :
The most common use of powder (polycrystalline) diffraction is chemical analysis. This can
include phase identification, investigation of high/low temperature phases, and
determinations of unit cell parameters of new materials (hkl).
Polymer crystallinity :
A polymer can be considered partly crystalline and partly amorphous. The crystallinity
parts give sharp narrow diffraction peaks and the amorphous component gives a very broad
peak (halo). The ratio between these intensities can be used to calculate the amount of
crystallinity in the material.
Residual stress :
Residual stress is the stress that remains in the material after the external force that caused
the stress have been removed. Stress is defined as force per unit area. Positive values
indicate tensile (expansion) stress, negative values indicate a compressive state. The
deformation per unit length is called strain. The residual stress can be introduced by any
mechanical, chemical or thermal process. The principals of stress analysis by the X-ray
diffraction is based on measuring angular lattice strain distributions.
That is, we choose a reflection at high 2-Theta and measure the change in the d-spacing
with different orientations of the sample.
Using Hooke’s law the stress can be calculated from the strain distribution. F = KX, K is
constant, X is the distance moved.
Applications of XRD

Texture analysis : The determination of the preferred orientation of the

crystallites in polycrystalline aggregates is referred to as texture analysis, and
the term texture is used as a broad synonym for preferred crystallographic
orientation in the polycrystalline material, normally a single phase.

The preferred orientation is usually described in terms of polefigures. A

polefigure is scanned be measuring the diffraction intensity of a given
reflection (2-Theta is constant) at a large number of different angular
orientations of the sample.

A contour map of the intensity is then plotted as a function of angular

orientation of the specimen. The most common representation of the
polefigures are sterographic or equal area projections.

The intensity of a given reflection (h, k , l) is proportional to the number of h,

k , l planes in reflecting condition (Bragg’s law). Hence, the polefigure gives the
probability of finding a given crystal-plane-normal as function of the specimen
orientation. If the crystallites in the sample have a random orientation the
recorded intensity will be uniform.
Calculation of Grain size (D) using Scherrer’s equation (D = kλ/Bcosθ)

B= FWHM = Imax/2
(D = kλ/Bcosθ)
Peak position (2θ)

Imax Can be fit with Gaussian

software to calculate the
grain size
Intensity

Imax/2

Bragg angle (2θ)

Peak width (B) is inversely proportional to crystallite size, D

 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:

D = 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

Advantages of XRD
1. Non-destructive technique, fast and easy sample preparation
2. High accuracy for d-spacing calculations
3. In-situ measurement is possible
4. Single crystal, polycrystalline, amorphous powder and thin films can be analyzed
5. JCPDS (Joint committee on powder diffraction standards ) till 1969 and replaced by
ICDD (International centre for Diffraction Data) in 1978 available for several
compounds

JCPDS
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

 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(2 θ)

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.
Overview of Scanning Electron Microscopy (SEM) & 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.

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.
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 360 o.
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 relationship between each point on the CRT screen
and the corresponding point on the specimen is used to build the SEM image.
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, subtraction of the image from one detector from that produced by a
different detector, colour coding and image analysis.
o All these techniques may be applied if it suits the primary aim of extracting the best possible information from the
specimen.

6. Importance of Vacuum in SEM column:

• In SEM, the column through which electron beam passes through must always be at vacuum.
• If the sample is in a gas filled environment, the electron beam cannot be generated or maintained because of a high instability in the beam. Also, gases
could react with the 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.
• SEM vacuum system is fully automatically controlled and protected against operating failures.
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 and sensitivity to elements present in low abundances when
compared to wavelength dispersive x-ray detectors (WDS) on most electron probe micro-analyzers.
o Power supply: Voltages and currents required for the electron gun and condenser lenses should be sufficiently stable to attain
the best resolution. Similarly, stability of electronic circuitry associated with the detectors should be extremely well-controlled
Electron Microscopy - Basics

Microscopy is the characterization of objects smaller than what can be seen with
the naked human eye.

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100µm 10µm 1µm 100nm 10nm 1nm 1Å
Optical Microscopy
Near Field Scanning Optical Microscopy
Scanning Electron Microscopy
Transmission Electron Microscopy

Fig: Comparison of the characterization size regimes for optical and electron microscopy
 Types of Electron Microscope

Scanning Electron Microscope (SEM) uses focused beam of electrons scanning over
the surface of thick or thin specimens. Images are produced by focusing at one spot at a
time in a grid-like raster pattern.

Transmission Electron Microscope (TEM) uses a wide beam of electrons passing

through a thin sliced specimen to form an image. The magnification and resolution is
better than SEM

Scanning Transmission Electron Microscope (STEM) uses a focused beam of

electrons scanning through a thin sliced specimen to form an image. The STEM looks
like a TEM but produces images as does an SEM (one spot at a time). It is most
commonly used for elemental analysis of samples.
Scanning Electron Microscopy
A SEM typically has several orders of magnitude
SEM better depth of focus than a optical microscope
making SEM suitable for studying rough surfaces.
The higher magnification, the lower depth of focus

EDAX

SEM and EDAX of Ni thin

film
SEM - Instrumentation

288 electrons!

Electron gun
156 electrons!

Small portion
of sample is Detector
bombarded with
electrons

Image
SEM components SEM - Instrumentation

I. Electron optical column:

Contains electron source to produce electrons, magnetic lenses to de-magnify the beam, magnetic
coils to control and modify the beam and an aperture to define the beam to prevent electron spray
> The electron gun may of the following
1. Heated tungsten : A heated filament made from the metal tungsten causes the flow
of electrons from the filament when a high voltage is applied in between filaments
2. LanthHexaboride (LaB6) : The LaB6 filament is a thermal filament. It is more efficient
since the work function is lower than tungsten filament.
3. Tungsten field emission gun (FEG) : It emits electrons when a very powerful electric
field is applied close to the filament tip.
II. Vacuum System:
Chamber which contains vacuum pumps to produce vacuum, valves and control units
III. Signal detection and display:
Collects the signal and produce the image from the signal.
Electron – Specimen SEM - Instrumentation
interaction

When a sample is irradiated

with electron beam several
information is obtained
depending on the
characteristics of out
coming beams
1. Secondary electrons gives
image
2. X-ray gives composition of
the material
SEM and Energy Dispersive X-ray Analysis (EDAX)- Instrumentation

SEM provides information regarding the species

present at varying depths of the sample
(i) Elastic scattering of electrons by atomic nuclei
of the sample results in BSE – useful for generating
images based on Z-contrast.
(ii) Inelastic scattering of electrons by sample
atoms results in low-energy secondary
electrons – useful for providing topographic
information regarding the sample surface.
(iii) Inelastic scattering of electrons by sample
atoms results in X-ray generation
from lower sample depths – useful for chemical
analysis of the bulk sample. https://link.springer.com/article/10.1007/s1085
(iv) Inelastic scattering results in Auger electrons 4-022-08280-z
emitted from sample atoms near
the sample surface – useful for surface chemical S. Arockiasamy et al., (2022)
analysis.