Experiment 5: AAS, SEM and TEM Analysis

Abstract
The primary objective of this section is to familiarize ourselves with the basic operating principles, sample preparation,
and results interpretation of the AAS and SEM.

ATOMIC ABSORPTION SPECTROMETRY

Background
The optical methods of chemical analysis make up a large group of which optical spectroscopy is one of them. In optical
spectroscopy the light is resolved to components which have different wavelengths. The methods in this group can be
divided into two categories: atomic and molecular spectroscopy. Atomic methods are capable for the elemental analysis
of substances, while with methods belonging to the latter category molecular composition can be determined.

Development of Atomic Spectroscopy

Flame colouring was used for the identification of sodium and potassium as far back as 1758 by Margraff. The
investigation of the spectrum of the sun was a milestone in the development of atomic spectroscopy. Bunsen and Kirchhoff
in their common article in 1859 explained the structure of spectrum of coloured flames: when we transfer energy to the
atoms of certain gases, they emit a characteristic spectrum consisting of lines. Furthermore these atoms can absorb light
having the same wavelength as the wavelength of the emitted ones. These findings led to the discovery of new elements
such as Cs, Rb, He. The first flame spectrograph was built in 1928, which could be used for the quantitative determination
of elements. The real advance was the emergence of the atomic absorption spectrophotometer in 1957 equipped with
electric parts and proper light source. The advanced generations of this instrument nowadays still have a wide field of
applications (e.g. in research, industry, in medical-, water- and environmental protecting laboratories…).

Flame spectrometry includes three methods:

a) Flame emission spectroscopy (FES): We measure the intensity of molecular bands or atomic or ionic lines
emitted by excited molecules, excited atoms or even by excited ions.
b) Flame atomic absorption spectroscopy (FAAS): We let through the fire a light beam with such a wavelength that
can be absorbed by ground state atoms and thus we measure the decrease of light intensity.
c) Atomic fluorescent spectroscopy (AFS): We irradiate the ground state atoms with an external light source,
however we measure the intensity of emitted light at the characteristic wavelength instead of the decrease of
light intensity due to the absorption of light.

The greatest importance is credited to FAAS. FES can be applied at a much smaller application field. Although AFS is
rather sensitive, the potential of the method compared to FAAS only for a few elements is comparable, thus it is relatively
less popular. Recently flameless atomization (graphite furnace atomic spectroscopy, GFAAS) keeps getting more and
more significant since it has small sample volume demand, high sensitivity and good detection limits.

Sample Introduction
The aim of sample introduction is to introduce samples (mostly liquids) to the flame with good reproducibility and with
high efficiency so that the interfering effects should remain minimal (the liquid must be introduced as fine aerosol). The
sample introduction is most often spraying the liquid. The small droplets formed in the nebulization transform gradually
while going through the high temperature zones of the flame. It is an important aspect that the particle size of the droplets
(< 5 m) should be possibly in similar size ranges in order the particles to be desolvated and to be further transformed in
the same region of the flame.

For the nebulization of solutions the indirect pneumatic nebulization is the most frequently used method which has a
sample introduction efficiency about 10 %. In Figure 1(a), the structure of a typical pneumatic system used for premixed
flames can be found. The sample gets into the nebulizer through a plastic capillary. In the nebuliser shown in Figure 1(b),
the sample leaving the capillary is surrounded by the gas (air) feeding the flame. After this high speed gas comes out
through the tight emergent hole of the nozzle pressure decrease is established in the capillary which results the take up of
the liquid. By the way the high speed gas breaks the uptake liquid into small drops thus aerosol is formed. The latter
phenomenon is called Venturi effect (Figure 2).

(a) (b)
Figure 1. (a) Structure of concentric pneumatic nebulization system and (b) Venturi effect
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MSE 359/METE 357, Materials/Metallurgical Laboratory II, Materials Engineering Department, KNUST, Kumasi
The position of the nebuliser’s capillary can be adjusted by a screw and by this way the sample take up speed and the
aerosol size distribution can be optimized. The size distribution of the formed primer aerosol is further refined by a glass
impact bead placed in front of the nebuliser (seconder aerosol is formed). The larger drops of the aerosol hit the impact
bead, however, the smaller ones can go round of it without precipitating

Figure 2. Role of impact bead in the development of secondary aerosol

Processes Occurring in Flame

This process are shown in Figure 3. From the liquid drop the solvent evaporates first, and solid aerosol particles
(micronized crystals of salts) are formed, in the next step it loses its’ crystalline water (if possessed) then the crystals melt
and evaporate that is molecule vapour forms. In the higher regions of the flame the thermal dissociation of the molecules
occurs: ground state atoms are formed (for atomic absorption measurements only these particles are useful). Of course,
when the temperature of the flame makes it possible, the thermal processes go further, by the side of the ground state
atoms excited atoms, even ions will be present in the flame (these excited particles are useful only for emission
measurements).

Figure 3. Processes occurring in flame

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MSE 359/METE 357, Materials/Metallurgical Laboratory II, Materials Engineering Department, KNUST, Kumasi
Detectors
The light intensity passed through the detector can be measured with different measuring devices. In the beginning of
flame photometry photographic light measurements were used, but nowadays instead of them solely photocells or
photomultipliers are in use. The multiplying factor of photomultipliers might be as large as 10 10. The principle of
photomultipliers (Figure 4) is the following: Photons impacted to K photosensitive cathode produce electrons which flow
towards the P1 dinode due to the potential between P1 dinode and K cathode. Electrons impacting to P1 dinode creates a
seconder electron emission of which intensity is larger then the primer electron current. This process is repeated on the
following (P2-P11) dinodes until the anode. The number of electrons increases exponentially with the number of dinodes.

Figure 4. Schematics of photomultiplier

Evaluation Methods
In flame spectrometry comprehensive calibration and standard addition calibration are the two most often used evaluation
techniques.

Comprehensive technique can be applied in case of big number samples of the same type. The comprehensive solution
series (standards) contain increasing quantity of the examined elements. Moreover the standards may contain other
components present in the samples (cations, anions, organic compounds, solvents) in average concentration. Since the
composition of validation series made by this way is about the same with sample composition, the arising interfering
effects will be about the same. The parts of the calibration series are usually diluted from a stock solution with exactly
known concentration.

This method provides analytically reliable results only in case of samples belonging to the same type with relatively
steady composition. We would determine for example calcium with large error if we used the same composition calcium
standard for the analysis of blood serum, water sample and ore solution made from rock pieces. In the mentioned samples
not only the concentration of components present besides calcium differ significantly but also the type of arising
interfering effects may be different. A typical calibration diagram from comprehensive evaluation method is seen in
Figure 5.

Figure 5. Comparison evaluation method

Standard addition method is applied primarily in case of so called exponential interfering effects, when we do not know
the cause of interfering effect or we do not know the quality and concentration of interfering components respectively.
The point of the method is that the samples are divided into 3-5 equal parts. We give to each portion increasing amount
of examined metal ion in the same volume. To the first portion we do not add any metal ion solution thus it contains the
examined metal ion in the original concentration (this unknown is the “+0” member in addition series.), to the other
members known increasing amount of examined metal solution is given. The results should be plotted like in Figure 6.
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MSE 359/METE 357, Materials/Metallurgical Laboratory II, Materials Engineering Department, KNUST, Kumasi
The linear line fit to measure points (intensity of emission or absorbance) intersects the abscissa axis beyond 0 in negative
range. The scale is the same than in positive range. The absolute value of the distance between the intersect and the origin
gives the metal ion concentration of the sample.

The condition of the applicability of the method is that a linear line should be able to fit on measure points. To perform
standard addition on several samples however makes it much work demanding thus it is not capable for serial analysis. It
makes hard to use this method when the volume of sample solution is limited (1-2 mL). In case of complex samples
containing mostly organic substance matrixes, standard addition method is often used.

Figure 6. Standard addition method

SCANNING ELECTRON MICROSCOPY (SEM)

Background
SEM is one of the most versatile methods for analysis of solid materials. Since its inception in the middle of the 20th
century, the technique has evolved to allow for imaging of nanoscale features and integration with electron probe
microanalysis (EPMA) techniques. In SEM, an electron beam is moved in a raster pattern across the surface of a sample.
The beam interacts with the sample surface, producing a number of different signals, which can be analysed to provide
useful information about topography, composition, crystallography, etc. In many respects, the optical system in an SEM
is analogous to that in an optical microscope, however, instead of glass lenses used to focus light, electromagnetic lenses
are used to focus and deflect the electron beam. The primary motivation for using SEM instead of light microscopy is
related to the fact that electrons have a much shorter wavelength than light (higher resolution) and SEM uses a longer
focal length (greater depth of focus).

The main classification for different types of SEM instruments is related to the source of electron illumination. The most
common electron guns employ a tungsten filament which is resistively heated to nearly 3000 K until electrons have
sufficient energy to overcome the work-function (EW) energy barrier (thermionic emission). By choosing materials with
a lower Ew than tungsten, the brightness of the electron source can be increased by an order of magnitude.

The interaction of a high energy electron beam with the specimen will produce various effects resulting in a range of
signals being emitted. The incident electrons interact with specimen atoms and are significantly scattered by them (rather
than penetrating the sample in a line fashion). Most of the energy of the electron beam will eventually end up heating the
sample (phonon excitation of the atomic lattic); however, before the electrons come to rest, they undergo two types of
scattering: elastic and inelastic.

Figure 7. Electron beam specimen interaction

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MSE 359/METE 357, Materials/Metallurgical Laboratory II, Materials Engineering Department, KNUST, Kumasi
The electrons-specimen interaction can provide information on:
▪ Specimen composition
▪ Topography
▪ Crystallography
▪ Electrical Potential
▪ Local Magnetic Field

Secondary Electrons
The inelastic interaction is responsible for the production of secondary electrons and it takes place between the incident
electrons and the outer (not strongly bound) electrons of the atoms. These outer electrons can be ejected from the atom
with energies lower than 50eV. If these “secondary” electrons are produced near the surface, and its energy is higher than
the surface energy (~6eV) then, they can escape to the vacuum and reach the detector.
In elastic scattering, the electron trajectory changes, but its kinetic energy and velocity remain essentially constant (due
to large differences between the mass of the electron and nucleus). This process is known as electron backscattering
(although later we will confine the term "backscattered electrons" to those scatter out of the sample).

In inelastic scattering, the trajectory of the incident electron is only slightly perturbed, but energy is lost through
interactions with the orbital electrons of the atoms in the specimen. Inelastic interactions produce diverse effect including:
▪ Secondary electrons
▪ phonon excitation (heating)
▪ cathodoluminescence(visible light fluorescence)
▪ continuum radiation (bremsstrahlungor “braking”radiation)
▪ characteristic x-ray radiation
▪ plasmonproduction (secondary electrons)
▪ Auger electron production (ejection of outer shell electrons)

The Hitachi S4500 FESEM

The Hitachi S4500 is an advanced cold filed emission SEM that features upper and lower secondary electron SE) detectors
as well as an energy dispersive X-ray spectrometer (EDS). The 4500 SEM is capable of resolution less than 5 nm and
magnifications up to 500,000 X. Figure 7 is a diagram of the Hitachi S4500 SEM column.

The electrons emitted from the gun are accelerated toward the anode (just above the beam monitor aperture), which has
a positive bias (accelerating voltage) variable from 0.5 to 30 kV. The condenser lenses serve to demagnify the beam,
controlling the probe size and current. The objective aperture serves three main functions: minimizes the effects of lens
aberration, controls the final probe current and determines the convergence angle of the beam on the specimen. The
objective lens controls the final focusing of the beam onto the specimen and the deflector coils control the scanning of
the beam. Also, located between the two deflector coils (although not pictured above) are the stigmators, which allow for
correction of astigmatism.

Important Issues in SEM Imaging

1. Objective Aperture and Resolution
SE’s are weak, valence band electrons ejected from the sample surface by inelastic collisions with the incident
electron beam. Since SE’s are low energy, the bulk of the signal detected comes from the very surface of the sample
(top 1 – 10 nm). The resolution of SE image is therefore determined largely by the size of the probe at the sample
surface (also related to the efficiency of the SE detector). The diameter of the electron beam, d, is defined by
4𝑖𝑝 2
( 2 + (1.22𝜆) ) 𝐶𝑠 2 6 𝐶𝑐 Δ𝑉𝑖 2 2
0.62𝜋𝛽 𝑖
𝑑2 = + ( ) 𝛼 + ( ) 𝛼
𝛼2 2 𝑉𝑖
where ip is the beam current, βi is the brightness of the electron source,  is the wavelength of the incident electrons,
α is the convergence angle of the beam, Cs and Cc are the spherical and chromatic aberration coefficients and ∆Vi/Vi
is the fractional variation in the electron beam energy. By adjusting the objective aperture size, the resolution of an
image can be improved.
2. Minimizing Specimen Charging
When a sample is bombarded with the electron beam, some of the charge being introduced by the beam is released
as backscattered and secondary electrons that are collected for imaging. However, depending on the situation, a large
amount of the charge introduced by the beam does not escape the sample surface. Instead, the charge is captured by
the sample and if a path to ground is available, the charge can escape easily. With nonconductive samples, or
conductive samples that are isolated from ground, the charge accumulates in the surface of the sample. This
phenomenon is of major importance for SE imaging since the week SE’s are easily influenced by charging and can

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MSE 359/METE 357, Materials/Metallurgical Laboratory II, Materials Engineering Department, KNUST, Kumasi
 cause severe image distortion and artifacts. In addition to image degradation such as streaking and voltage contrast
effects, charging can cause damage to the sample by excessive heating and subsequent loss of material.

Two methods for reducing the effects of charging include (1) specimen coating and (2) low voltage microscopy.
One of the most common methods for dealing with charging involves sputter coating or the evaporation of a thin
conductive film on the sample’s surface. Materials commonly used for this include carbon and metals such as Au,
Aj/Pd, Pt, Ti, and Cr. The second method used to reduce sample charging is low voltage microscopy, for which a
cold cathode FESEM is ideally suited. The energy of the incident electrons (dictated by the accelerating voltage)
has a major impact on the nature and extent of charging. By decreasing the accelerating voltage, the total amount of
charge introduced into the sample surface is reduced.

Figure 7. Schematic of the Hitachi S4500 FESEM

Imaging from SEM

Images are generated in an SEM by the raster pattern scanning of the incident electron beam across the sample’s surface.
The emitted electrons are detected for each position in the scanned are by an electron detector. The intensity of the emitted
electron signal is displayed as brightness on a display monitor and/or in a digital image file. By sychromizing the position
in the image scan to that of the scan of the incident beam, the display represents the morphology of the sample surface
are. Magnification of the image is the ratio of the image display size to the sample area scanned by the electron beam.
Figures 8 – 10 shows typical micrographs.

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MSE 359/METE 357, Materials/Metallurgical Laboratory II, Materials Engineering Department, KNUST, Kumasi
Figure 8. Tensile testing specimen showing (a) cup and cone ductile fracture and (b) flat brittle fracture

Figure 9. Scanning electron fractograph showing dimples characteristic of ductile fracture (a) spherical dimples from
uniaxial loading and (b) elongated dimples from shear loading

Figure 10. Scanning electron fractograph showing brittle fracture (a) transgranular failure and (b) intergranular failure

Questions for Laboratory Write-Up

1. Give a general description about the most commonly used flames in flame spectrometry
2. What is the way of sample introduction to the spectrometers?
3. What is the way of the detection of light in spectrometers?
4. Describe the two most commonly used evaluation methods (comprehensive calibration, standard addition
calibration)
5. In this lab, the effects of objective aperture on resolution were discussed. Explain why decreasing the aperture size
improves resolution. How do you think the condenser lens strength would affect the resolution of an SE image?
How about accelerating voltage?
6. Describe each of the important signals produced by the interactions between the electron beam and a sample and
explain the information that can be derived from each.
7. The lab looked at two ways to reduce sample charging, namely low accelerating voltage and sample coating. Can
you think of any other ways to reduce charging without altering the beam conditions?
8. Since SEM is essentially a surface analysis technique, why doesn’t sputter coating obscure useful surface
information? Can you think of any cases where the coating might become a problem?
9. Low voltage microscopy is a useful method for minimizing charging effects. What are some of the trade-offs? Why
is cold field emission well suited for this technique?

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MSE 359/METE 357, Materials/Metallurgical Laboratory II, Materials Engineering Department, KNUST, Kumasi