Module 6

SPECTROSCOPIC, DIFFRACTION AND

MICROSCOPIC TECHNIQUES
UV TECHNIQUE
UV-Vis spectroscopy is an analytical technique that measures the amount of discrete
wavelengths of UV or visible light that are absorbed by or transmitted through a
sample in comparison to a reference or blank sample. This property is influenced by
the sample composition, potentially providing information on what is in the sample
and at what concentration. Since this spectroscopy technique relies on the use of
light, let’s first consider the properties of light.

Light has a certain amount of energy which is inversely proportional to its

wavelength. Thus, shorter wavelengths of light carry more energy and longer
wavelengths carry less energy. A specific amount of energy is needed to promote
electrons in a substance to a higher energy state which we can detect as absorption.
Electrons in different bonding environments in a substance require a different
specific amount of energy to promote the electrons to a higher energy state. This is
why the absorption of light occurs for different wavelengths in different substances.
Humans are able to see a spectrum of visible light, from approximately 380 nm,
which we see as violet, to 780 nm, which we see as red.1 UV light has wavelengths
shorter than that of visible light to approximately 100 nm. Therefore, light can be
described by its wavelength, which can be useful in UV-Vis spectroscopy to analyze
or identify different substances by locating the specific wavelengths corresponding to
maximum absorbance (see the Applications of UV-Vis spectroscopy section).
Whilst there are many variations on the UV-Vis spectrophotometer, to gain a better
understanding of how an UV-Vis spectrophotometer works, let us consider the main
components, depicted in Figure 1.

Figure 1: A simplified schematic of the main components in a UV-Vis

spectrophotometer. Credit: Dr. Justin Tom.
Light source

As a light-based technique, a steady source able to emit light across a wide range of
wavelengths is essential. A single xenon lamp is commonly used as a high intensity
light source for both UV and visible ranges. Xenon lamps are, however, associated
with higher costs and are less stable in comparison to tungsten and halogen lamps.

For instruments employing two lamps, a tungsten or halogen lamp is commonly used
for visible light,2 whilst a deuterium lamp is the common source of UV light.2 As two
different light sources are needed to scan both the UV and visible wavelengths, the
light source in the instrument must switch during measurement. In practice, this
switchover typically occurs during the scan between 300 and 350 nm where the light
emission is similar from both light sources and the transition can be made more
smoothly.

Wavelength selection

In the next step, certain wavelengths of light suited to the sample type and analyte
for detection must be selected for sample examination from the broad wavelengths
emitted by the light source. Available methods for this include:
- Monochromators
A monochromator separates light into a narrow band of wavelengths. It is most often
based on diffraction gratings that can be rotated to choose incoming and reflected
angles to select the desired wavelength of light.1,2 The diffraction grating's groove
frequency is often measured as the number of grooves per mm. A higher groove
frequency provides a better optical resolution but a narrower usable wavelength
range. A lower groove frequency provides a larger usable wavelength range but a
worse optical resolution. 300 to 2000 grooves per mm is usable for UV Vis
spectroscopy purposes but a minimum of 1200 grooves per mm is typical. The
quality of the spectroscopic measurements is sensitive to physical imperfections in
the diffraction grating and in the optical setup. As a consequence, ruled diffraction
gratings tend to have more defects than blazed holographic diffraction gratings.3
Blazed holographic diffraction gratings tend to provide significantly better quality
measurements.3

- Absorption filters
Absorption filters are commonly made of colored glass or plastic designed to absorb
particular wavelengths of light.2

- Interference filters
Also called dichroic filters, these commonly used filters are made of many layers of
dielectric material where interference occurs between the thin layers of materials.
These filters can be used to eliminate undesirable wavelengths by destructive
interference, thus acting as a wavelength selector.1,2
- Cutoff filters
Cutoff filters allow light either below (shortpass) or above (longpass) a certain
wavelength to pass through. These are commonly implemented using interference
filters.

- Bandpass filters
Bandpass filters allow a range of wavelengths to pass through that can be
implemented by combining shortpass and longpass filters together.

Monochromators are most commonly used for this process due to their versatility.
However, filters are often used together with monochromators to narrow the
wavelengths of light selected further for more precise measurements and to improve
the signal-to-noise ratio.

Sample analysis

Whichever wavelength selector is used in the spectrophotometer, the light then

passes through a sample. For all analyses, measuring a reference sample, often
referred to as the "blank sample", such as a cuvette filled with a similar solvent used
to prepare the sample, is imperative. If an aqueous buffered solution containing the
sample is used for measurements, then the aqueous buffered solution without the
substance of interest is used as the reference. When examining bacterial cultures,
the sterile culture media would be used as the reference. The reference sample
signal is then later used automatically by the instrument to help obtain the true
absorbance values of the analytes.

It is important to be aware of the materials and conditions used in UV-Vis

spectroscopy experiments. For example, the majority of plastic cuvettes are
inappropriate for UV absorption studies because plastic generally absorbs UV light.
Glass can act as a filter, often absorbing the majority of UVC (100-280 nm)2 and
UVB (280-315 nm)2 but allowing some UVA (315-400 nm)2 to pass through.
Therefore, quartz sample holders are required for UV examination because quartz is
transparent to the majority of UV light. Air may also be thought of as a filter because
wavelengths of light shorter than about 200 nm are absorbed by molecular oxygen in
the air. A special and more expensive setup is required for measurements with
wavelengths shorter than 200 nm, usually involving an optical system filled with pure
argon gas. Cuvette-free systems are also available that enable the analysis of very
small sample volumes, for example in DNA or RNA analyses.

XRD TECHNIQUE
Max von Laue, in 1912, discovered that crystalline substances act as three-
dimensional diffraction gratings for X-ray wavelengths similar to the spacing of
planes in a crystal lattice. X-ray diffraction is now a common technique for the study
of crystal structures and atomic spacing.
X-ray diffraction is based on constructive interference of monochromatic X-rays and
a crystalline sample. These X-rays are generated by a cathode ray tube, filtered to
produce monochromatic radiation, collimated to concentrate, and directed toward the
sample. The interaction of the incident rays with the sample produces constructive
interference (and a diffracted ray) when conditions satisfy Bragg's Law (nλ=2d sin θ).
This law relates the wavelength of electromagnetic radiation to the diffraction angle
and the lattice spacing in a crystalline sample. These diffracted X-rays are then
detected, processed and counted. By scanning the sample through a range of
2θangles, all possible diffraction directions of the lattice should be attained due to the
random orientation of the powdered material. Conversion of the diffraction peaks to
d-spacings allows identification of the mineral because each mineral has a set of
unique d-spacings. Typically, this is achieved by comparison of d-spacings with
standard reference patterns.

Numerical: https://www.youtube.com/watch?v=6IzrOWIw3zQ

AAS
Atomic absorption spectrometry (AAS) detects elements in either liquid or solid
samples through the application of characteristic wavelengths of electromagnetic
radiation from a light source. Individual elements will absorb wavelengths differently,
and these absorbances are measured against standards. In effect, AAS takes
advantage of the different radiation wavelengths that are absorbed by different
atoms.

In AAS, analytes are first atomized so that their characteristic wavelengths are
emitted and recorded. Then, during excitation, electrons move up one energy level in
their respective atoms (figure 1) when those atoms absorb a specific energy. This
energy corresponds to a specific wavelength that is characteristic of the element.
Depending on the light wavelength and its intensity, specific elements can be
detected and their concentrations measured.

As electrons return to their original energy state, they emit energy in the form of light

(figure 2).
AAS has an unlimited number of applications and is still a popular choice for
uncomplicated trace elemental analysis. Flame atomic absorption spectrometry
(FAAS) is widely accepted in many industries, which continue to utilize the unique
and specific benefits of this technology. Graphite furnace atomic absorption
spectrometry (GFAAS) is an established technology for measuring elements at parts
per billion (ppb or µg/l) concentrations with incredibly low sample volumes.

Refer to: https://www.youtube.com/watch?v=maKYfe2cVSs

IR
Infrared spectroscopy exploits the fact that molecules absorb specific frequencies that are
characteristic of their structure. These absorptions are resonant frequencies, i.e. the frequency
of the absorbed radiation matches the frequency of the bond or group that vibrates. The
energies are determined by the shape of the molecular potential energy surfaces, the masses
of the atoms, and the associated vibronic coupling.

In particular, in the Born-Oppenheimer and harmonic approximations, i.e. when the

molecular Hamiltonian corresponding to the electronic ground state can be approximated by a
harmonic oscillator in the neighborhood of the equilibrium molecular geometry, the resonant
frequencies are determined by the normal modes corresponding to the molecular electronic
ground state potential energy surface. Nevertheless, the resonant frequencies can be in a first
approach related to the strength of the bond, and the mass of the atoms at either end of it.
Thus, the frequency of the vibrations can be associated with a particular bond type.

In order for a vibrational mode in a molecule to be "IR active," it must be associated with
changes in the permanent dipole.

A molecule can vibrate in many ways, and each way is called a vibrational mode. Linear
molecules have 3N - 5 degrees of vibrational modes whereas nonlinear molecules have
3N - 6 degrees of vibrational modes (also called vibrational degrees of freedom). As an
example H2O, a non-linear molecule, will have 3 × 3 - 6 = 3 degrees of vibrational freedom,
or modes.

Simple diatomic molecules have only one bond and only one vibrational band. If the
molecule is symmetrical, e.g. N2, the band is not observed in the IR spectrum, but only in the
Raman spectrum. Unsymmetrical diatomic molecules, e.g. CO, absorb in the IR spectrum.
More complex molecules have many bonds, and their vibrational spectra are correspondingly
more complex, i.e. big molecules have many peaks in their IR spectra.
The atoms in a CH2 group, commonly found in organic compounds, can vibrate in six
different ways: symmetric and antisymmetric stretching, scissoring, rocking, wagging and
twisting.

Refer to playlist: https://www.khanacademy.org/science/organic-chemistry/spectroscopy-

jay/infrared-spectroscopy-theory/v/introduction-to-infrared-spectroscopy

NMR

Refer to: https://www.youtube.com/watch?v=SBir5wUS3Bo

SEM

A scanning electron microscope (SEM) scans a focused electron beam over a

surface to create an image. The electrons in the beam interact with the sample,
producing various signals that can be used to obtain information about the surface
topography and composition.
Given sufficient light, the human eye can distinguish two points 0.2 mm apart,
without the aid of any additional lenses. This distance is called the resolving power
or resolution of the eye. A lens or an assembly of lenses (a microscope) can be used
to magnify this distance and enable the eye to see points even closer together than
0.2 mm.
The main SEM components include:
▪ Source of electrons
▪ Column down which electrons travel with electromagnetic lenses
▪ Electron detector
▪ Sample chamber
▪ Computer and display to view the images
Electrons are produced at the top of the column, accelerated down and passed
through a combination of lenses and apertures to produce a focused beam of
electrons which hits the surface of the sample. The sample is mounted on a stage in
the chamber area and, unless the microscope is designed to operate at low
vacuums, both the column and the chamber are evacuated by a combination of
pumps. The level of the vacuum will depend on the design of the microscope.
Schematic of a Scanning Electron Microscope
The position of the electron beam on the sample is controlled by scan coils situated
above the objective lens. These coils allow the beam to be scanned over the surface
of the sample. This beam rastering or scanning, as the name of the microscope
suggests, enables information about a defined area on the sample to be collected.
As a result of the electron-sample interaction, a number of signals are produced.
These signals are then detected by appropriate detectors.

Refer to: https://www.youtube.com/watch?v=KfQ4VNpWN4M

TEM

Refer to: https://www.youtube.com/watch?v=qsrwpx3K8AI