NANOSCIENCE AND TECHNOLOGY (3170509), Sem- VII (CHEM)

Chapter Name: NANOMATERIALS CHARACTERIZATION

INTRODUCTION OF NANOMATERIALS CHARACTERIZATION

 Characterization refers to the study of material's features such as its composition, structure,&
various properties like physical, electrical, magnetic etc.
 Nanomaterials, dispersed in the form of colloids in solutions, particles (dry powders) or thin films,
are characterized by various techniques.
 Although the techniques to be used would depend upon the type of material and information one
needs to know, usually one is interested in first knowing the size, crystalline type, composition,
thermal, chemical state, and properties like optical or magnetic properties.

WHAT IS NANO CHARACTERIZATION?

 The characterization of nanoparticles is a branch of nanometrology that deals with the
characterization, or measurement, of the physical and chemical properties of nanoparticles.
 Nanoparticles measure less than 100 nanometers in at least one of their external dimensions, and
are often engineered for their unique properties. Nanoparticles are unlike conventional chemicals
in that their chemical composition and concentration are not sufficient metrics for a complete
description, because they vary in other physical properties such as size, shape, surface properties,
crystallinity, and dispersion state.
 Nanoparticles are characterized for various purposes, including nanotoxicology studies and
exposure assessment in workplaces to assess their health and safety hazards, as well as
manufacturing process control.
 There is a wide range of instrumentation to measure these properties, including microscopy and
spectroscopy methods as well as particle counters. Metrology standards and reference materials
for nanotechnology, while still a new discipline, are available from many organizations.

SCANNING ELECTRON MICROSCOPY

 scanning electron microscope (SEM), type of electron microscope, designed for directly studying
the surfaces of solid objects, that utilizes a focused beam of electrons of relatively low energy as an
electron probe that is scanned in a regular manner over the specimen.
 As the name suggests, an electron microscope utilizes a beam of accelerated electron to illuminate
the sample to be viewed.

PRINCIPAL
 The basic principle is that a beam of electrons is generated by a suitable source, typically a tungsten
filament or a field emission gun. The electron beam is accelerated through a high voltage (e.g.: 20
kV) and pass through a system of apertures and electromagnetic lenses to produce a thin beam of
electrons. Then the beam scans the surface of the specimen Electrons are emitted from the
specimen by the action of the scanning beam and collected by a suitably positioned detector.

CONSTRUCTION & WORKING

Scanning Electron Microscope's basic components are as following...
1. Electron gun (Filament):
 It generates a beam of energetic electrons. The most common from of an electron gun is a
thermionic emitter.

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2. Condenser lenses
 they aren't made of glass
 Instead, the Condenser lenses are made of magnets capable of bending the path of electrons.
 The Condenser lenses focus and control the electron beam, ensuring that the electrons end up
precisely where they need to go.

3. Objective Aperture
 The objective aperture arm fits above the objective lens in the SEM. It is a metal rod that holds a
thin plate of metal containing four holes.
 The aperture stops electrons that are off-axis or off energy from progressing down the column. It
can also narrow the beam below the aperture, depending on the size of the hole selected.

4. Scan coils
 The scanning coils consist of two solenoids oriented in such a way as to create two magnetic fields
perpendicular to each other.
 Varying the current in one solenoid causes the electrons to move left to right and another
solenoid move electrons upward and downwards. Means one each solenoid for the X and Y axes.

5. Objective lens:
 The objective lens gathers light from the specimen, magnifies the image of the specimen, and
projects the magnified image into the body tube.
 The condenser lens defines the size of the electron beam (which defines the resolution), while the
main role of the objective lens is to give more focused the beam onto the sample.

6. Vacuum chamber
 To avoid the oxidation of electron generator and contamination, vacuum chamber is there.
Usually vacuum about 10-5torr or better is necessary for a normal operation in SEM.

7. Detectors
 Detectors register secondary electrons, which are electrons dislodged from the outer surface of a
specimen. These detectors are capable of producing the most detailed images of an object's
surface.
 Other detectors, such as backscattered electron detectors and X-ray detectors, can tell researchers
about the composition of a substance.

APPLICATIONS
 To produced most detailed Nano and microscopic images.
 To detect and analyze surface fractures, provide information in microstructures, examine surface
contaminations, reveal spatial variations in chemical compositions, provide qualitative chemical analyses and
identify crystalline structures.
 for quality control (QC) and good-bad testing of pharmaceutical products, and it has proven useful for
detecting and identifying unknown contaminants in manufactured goods.

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 Surface structure morphology of polymer nano composites, fracture surfaces, nano fibers, nano coating can
be done effectively using SEM characterization.
 for viewing the dispersion of nanoparticles such as carbon nanotubes, nanoclay, and hybrid nanofillers in
bulk and on nano composite fibers.
 Heavy metal traces can be determined by EDX technique which requires SEM as an integral part of
technique.
 SEM applications specific for Nanotechnology : Nanowires for gas sensing, Examination of paint particles and
fibers, Investigation of gemstones and jewellery, Semiconductor inspections, Archaeological surveys,
Biological sciences, Solid and rock sampling, Forensic investigations

Figure: Scanning electron microscope.

Figure: Scanning electron microscope.

ADVANTAGES
 Detailed three-dimensional and topographical imaging with high resolution up to 1 nm and the
versatile information garnered from different detectors.

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 Easy to operate with training

 Works fast as compared to conventional EM. Analyses sample in less than five minutes.
 SEMs allow for the generation of data in digital form.
 Although all samples must be prepared before placed in the vacuum chamber, most SEM samples
require minimal preparation actions.

DISADVANTAGES
 The disadvantages of a Scanning Electron Microscope start with the size and cost.
 SEMS are expensive, large and must be housed in an area free of any possible electric, magnetic or
vibration interference.
 Maintenance involves keeping a steady voltage, currents to electromagnetic coils and circulation of
cool water.
 Special training is required to operate an SEM as well as prepare samples & to minimize chances of
sample contamination.
 limited to solid, inorganic samples small enough to fit inside the vacuum chamber that can handle
moderate vacuum pressure.
 Carries a small risk of radiation exposure associated with the electrons that scatter from beneath the sample
surface.

TRANSMISSION ELECTRON MICROSCOPY

 Transmission electron microscopes (TEM) are microscopes that use a particle beam of electrons to
visualize specimens and generate a highly-magnified image.
 TEMs can magnify objects up to 2 million times.
 TEM is also known as conventional transmission electron microscopy or CTEM.

PRINCIPLE:
 An extremely thin sample is required for scanning in TEM from which electron beam is passed
through rendering its interaction with the sample as a result of which image is produced. This
image can be magnified and focused on the device used for imaging, like a fluorescent screen, on a
photographic filmlayer, or to be identified by a sensor like a camera.

WORKING
 TEMs employ a high voltage electron beam in order to create an image. An electron gun at the top
of a TEM emits electrons that travel through the microscope’s vacuum tube.
 Rather than having a glass lens focusing the light (as in the case of light microscopes), the TEM
employs an electromagnetic lens which focuses the electrons into a very fine beam.
 This beam then passes through the specimen, which is very thin, and the electrons either scatter or
hit a fluorescent screen at the bottom of the microscope. An image of the specimen with its
assorted parts shown in different shades according to its density appears on the screen.
 This image can be then studied directly within the TEM or photographed. Figure 1 shows a diagram
of a TEM and its basic parts.

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Figure: Transmission electron microscope

PARTS OF A TRANSMISSION ELECTRON MICROSCOPE

 Parts of a transmission electron microscopeare
1. Electron Gun: This is the part that generates the electrons. The electron gun houses a tungsten
filament that is heated to produce electrons.
2. Electromagnetic Lens: The electromagnetic lens are a combination of three types of lenses which
aid in creating an image of the specimen:
3. Condenser lens: This lens focuses the electron beam on to the specimen. There is also a second
condenser lens that focuses the electrons into a thin beam
4. Objective lens: The electron beam that comes out of the specimen and passes through the second
set of magnetic lenses is usually termed as the objective lens and this lens produces the
intermediate magnified image of the specimen.
5. Ocular (projector) lens: The final image of the further magnified image is produced by the projector
lens which are another set of magnetic lenses that create higher magnification of images, all the
while maintaining excellent level of resolution and detail
6. Specimen Holder: This is very thin carbon film or a collodion that is held in place by a metal grid
7. Image viewer and recording mechanism: The final magnified image of the specimen appears on the
fluorescent screen. There is also a camera right below the screen that is used to record the image.

APPLICATIONS
 Scientific Research: The electron microscopes help researchers, students, and scientists to study
specimens, both biological and non-biological at a nanoscale

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 Natural Resources: oil and gas companies also use these microscopes in their exploration of oil and
gas reserves
 Industry: Electronics manufacturing industry, apparel, automotive, pharmaceutical, and
aeronautical industries among others use electron microscopes.
 Forensic Science: In the process of unravelling a criminal case, electron microscopes find application
as they help in analysis of the evidence at a nanoscale. For instance, analysis of samples like hair,
residue from gunshot, cloth fibres, biological evidence material like skin, tissue etc. using an
electron microscope yields better results.
 To view the shapes and sizes of microbial cell, bacteria, viruses, and fungi etc.
 To study and differentiate between plant and animal cells.
 It is used to detect and identify fractures, damaged micro-particles which further enable repair
mechanisms of the particles.
 To measure mechanical and electrical properties of individual nanowires and nanotubes, TEM can
help to understand relationship between structure and properties

ADVANTAGES
 High Magnification: Modern-day electron microscopes have an extremely high magnification power
and they can magnify a specimen up to 500,000 times. The higher magnitude of magnification
allows us to see the minute details.
 High Resolution: Apart from the magnification, electron microscopes have superior resolution
power and they can distinguish the features, details and properties of a specimen at a nanoscale
level thereby enabling better research and better outcomes.
 Give three dimensional imaging and real-time analysis of specimens
 Versatility: An electron microscope can be used to observe and analyse just about any kind of
specimen both inorganic and organic making them indispensable in the fields of industry,
biomedical research, forensics among others
 Images are high quality and detailed
 TEM can be used to acquire vast information on compounds and their structures.
 TEMs are able to yield information of surface features, shape, size and structure along with its
composition
 TEM can produce permanent images.
 Easy to operate with proper training

DISADVANTAGES
 No colour images: Electron microscopes only create black and white images and they need to be
colorized falsely
 Cost: TEM are very costly.
 TEM requires special housing and maintenance
 Carries a small risk of radiation exposure
 Can’t observe live specimens: The very fact that the specimens need to be observed in vacuum
renders the observation of live specimens useless as it is near impossible to stay alive in vacuum.
 Samples are limited to those that are electron transparent, able to tolerate the vacuum chamber
and small enough to fit in the chamber
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 Specialized knowledge required: The operation of an electron microscope is complex and requires
specialized knowledge and training to be able to operate it and maintain it.
 Larger in size creating limitation of area requirement
 Special training required before operation to minimize chances of sample contamination
 they are extremely sensitive to vibrations and electro-magnetic movements

SCANNING ELECTRON MICROSCOPY VS TRANSMISSION ELECTRON MICROSCOPY

 The below table summarizes the differences between SEMs and TEMs.
Scanning Electron Microscopes Transmission Electron Microscopes
(SEM) (TEM)
Electron stream Fine, focused beam Broad beam
Electron beam Scattered / Reflected Transmitted
Study Surface Ultrastructure
Image taken of for Analysis Topographical/surface Internal structure
Resolution Lower resolution (0.4nm) Higher resolution (0.4A)
Magnification Up to 2,000,000 times Up to 50,000,000 times
Image dimension 3-D 2-D
Sample thickness Thin and thick samples only Ultrathin samples only
Penetrates sample No Yes
Sample restriction Less restrictive More restrictive
Sample preparation Less preparation required/ easy More preparation
required/Ultrathin Sample
Cost Less expensive More expensive
Speed Faster Slower
Operation Easy to use More complicated
Specimen mounting Al Cu-grid
Field of View Large Small
Voltage needed SEM voltage ranges from Accelerating voltage much lower;
60-300,000 volts not necessary to penetrate the
specimen

FOURIER TRANSFORM INFRARED SPECTROSCOPY

 Fourier infrared spectroscopy is the study of interactions between matter and electromagnetic
fields in the IR region.
 Infrared spectrum is an important record which gives sufficient information about the functional
groups of a compound.

PRINCIPAL
 IR spectroscopy are work on principal of absorption. In IR spectroscopy when ir radiation we give
any compound it is excited and show vibration. Applied IR frequency should be equal to the natural
frequency of radiation.

COMPONENTS OF FTIR SPECTROSCOPY

1. Source: Infrared energy is emitted from a glowing black-body source.
2. Interferometer: The beam enters the interferometer where the "spectral encoding" takes place.The
resulting interferogram signal then exits the interferometer.
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3. Sample: The beam enters the sample compartment where it is transmitted through or reflected off
of the surface of the sample, depending on the type of analysis being accomplished. This is where
specific frequencies of energy, which are uniquely characteristic of the sample, are absorbed.
4. The Detector: The detectors used to measure the special interferogra m signal. Detectors transform
the input energy into an output then converted to a signal.
5. The Computer: The measured signal is digitized and sent to the computer where the Fourier
transformation takes place.
6. Moving mirror: It is the only moving part of the instrument.
7. Fixed mirror: It is a stationary mirror

Figure: Fourier Transform Infrared Spectroscope

WORKING
 Radiation from the broadband IR source is collimated and directed into the interferometer, and
impinges on the beam splitter.
 At the beam-splitter half of the IR beam is transmitted to the fixed mirror and the remaining half is
reflected to the moving mirror.
 After the divided beams are reflected from the two mirrors, they are recombined at the beam-
splitter.
 Due to changes in the relative position of the moving mirror, an interference pattern is generated.
The resulting beam then passes through the sample and is eventually focused on the detector.

APPLICATIONS
 Application of FT-IR in Textile:
 It can identify unknown materials
 It can determine the quality or consistency of a sample
 Identification of compounds by matching spectrum of unknown compound with reference
spectrum (fingerprinting)
 Identification of reaction components and Study of chemical reaction.
 Identification of molecular orientation in polymer films.
 Detection of impurities in a compound
 The same way it determines Percentage of trash particles or foreign matter present in fiber, yarn or
fabric.

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 Identification of polymers, plastics, and resins.

 Structure determination.

ADVANTAGES
 Sensitive, fast and easy.
 It is a non-destructive technique
 Internally calibrated (self-calibrating)
 Simpler mechanical design.
 Elimination of Stray light and emission contributions.
 Powerful data station.
 Majority of molecules in the universe absorb mid-infrared light, making it a highly useful tool.
 Universal technique.
 Relatively inexpensive and provides rich information.
 Low maintenance

DISADVANTAGES
 It cannot be used to detect all the vibration modes in a molecule.
 It is not possible to know molecular weight of substance
 It is not possible to know whether it is pure compound or a mixture of compound.
 Interferogram are difficult to interpret without first performing a Fourier transform to produce a
spectrum.
 Accuracy of FT-IR remains true if there is no change in atmospheric conditions throughout the
experiment.
 It can only scan a single nano sized image at a time of about 150 x 150nm
 Inaccurated at a may provide If not functionalized properly
 Skilled person needed
 Requires routine checkup with reference sample

ENERGY DISPERSIVE SPECTROSCOPY OR ENERGY-DISPERSIVE X-RAY SPECTROSCOPY (EDS)

 Interaction of an electron beam with a sample target produces a variety of emissions, including x-
rays. An energy-dispersive (EDS) detector is used to separate the characteristic x-rays of different
elements into an energy spectrum, and EDS system software is used to analyze the energy
spectrum in order to determine the abundance of specific elements. EDS can be used to find the
chemical composition of materials down to a spot size of a few microns, and to create element
composition maps over a much broader raster area. Together, these capabilities provide
fundamental compositional information for a wide variety of materials.

PRINCIPAL
 Energy Dispersive Spectroscopy (EDS) is a standard procedure for identifying and quantifying
elemental composition of sample areas of a micron or less. The characteristic X-rays are produced
when a material is bombarded with electrons in an electron beam instrument, such as a scanning
electron microscope (SEM). Detection of these x-rays can be accomplished by an energy dispersive
spectrometer, which is a solid state device that discriminates among X-ray energies.

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Figure: Energy-dispersive x-ray spectroscopy (EDS)

CONSTRUCTION & WORKING

 EDS systems are typically integrated into either an SEM or EPMA (Electron Probe Micro Analyzer)
instrument. EDS systems include a sensitive x-ray detector, a liquid nitrogen for cooling, and
software to collect and analyze energy spectra.
 The detector is mounted in the sample chamber of the main instrument at the end of a long arm,
which is itself cooled by liquid nitrogen. The most common detectors are made of Si(Li) crystals that
operate at low voltages to improve sensitivity, but recent advances in detector technology make
availabale so-called "silicon drift detectors" that operate at higher count rates without liquid
nitrogen cooling.
 An EDS detector contains a crystal that absorbs the energy of incoming x-rays by ionization, yielding
free electrons in the crystal that become conductive and produce an electrical charge bias. The x-
ray absorption thus converts the energy of individual x-rays into electrical voltages of proportional
size; the electrical pulses correspond to the characteristic x-rays of the element.

APPLICATIONS
 Elemental identification of material
 If the nanoparticles near or at the surface of a specimen are heavy metal ions, such as Au, Pd and
Ag nanoparticles, the composition and the amount can be estimated with EDX; however, elements
with low atomic numbers are difficult to detect. (below 11-Na)

ADVANTAGES
 Rapid elemental analysis of small features
 Two-dimensional elemental mapping
 When used in "spot" mode, a user can acquire a full elemental spectrum in only a few seconds.
Supporting software makes it possible to readily identify peaks, which makes EDS a great survey
tool to quickly identify unknown phases prior to quantitative analysis.
 EDS can be used in semi-quantitative mode to determine chemical composition by peak-height
ratio relative to a standard.
 low sensitive with molecules causing least change in composition, can analyze for all materials at
once, precision and accuracy are good with simple spectra, and surface sensitive. (penetration up to
100 μm)
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DISADVANTAGES
 There are energy peak overlaps among different elements, particularly those corresponding to x-
rays generated by emission from different energy-level shells (K, L and M) in different elements. For
example, there are close overlaps of Mn-Kα and Cr-Kβ, or Ti-Kα and various L lines in Ba.
Particularly at higher energies, individual peaks may correspond to several different elements; in
this case, the user can apply deconvolution methods to try peak separation, or simply consider
which elements make "most sense" given the known context of the sample.
 EDS cannot detect the lightest elements, typically below the atomic number of Na.
 Surface sensitive (not used for bulky materials) and modest limit of detection conventional
techniques.

ATOMIC FORCE MICROSCOPY

 It is a very high-resolution type of scanning probe microscope that can works more than 1000 times
better than the optical diffraction limit and can imaging, measuring, and manipulating matter at the
nano-scale. It can be called scanning force microscope (SFM)

PRINCIPAL
 The Atomic Force Microscope works on the principle measuring intermolecular forces and sees
atoms by using probed surfaces of the specimen in nanoscale.
 Its functioning is enabled by three of its major working principles that include Surface sensing,
Detection, and Imaging.
 The main principle behind of this microscopic technique is that In this method a laser beam is
focused on the back of a cantilever that moves up and down on the surface of a specimen and the
deflections of the beam are captured by a diode.

WORKING
 A laser beam is used to detect cantilever deflections towards or away from the surface. By
reflecting an incident beam off the flat top of the cantilever, any cantilever deflection will cause
slight changes in the direction of the reflected beam.
 A position-sensitive photo diode (PSPD) can be used to track these changes.
 Thus, if an AFM tip passes over a raised surface feature, the resulting cantilever deflection (and the
subsequent change in direction of reflected beam) is recorded by the PSPD.
 The microscope has a feedback loop that controls the length of the cantilever tip just above the
sample surface, therefore, it will maintain the laser position thus generating an accurate imaging
map of the surface of the image.

PARTS OF ATOMIC FORCE MICROSCOPE

1. Cantilever with a sharp tip.
 Cantilever with a sharp tip. The stiffness of the cantilever needs to be less the effective spring
constant holding atoms together, which is on the order of 1 10nN/nm.

2. tips
 Modified tips which are used to detect the sample surface and undergo deflections

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 The tip should have a radius of curvature less than 20-50 nm (smaller is better) a cone angle
between 10-20 degree

3. Piezo stage
 The movement of the tip or sample in the x, y, and z-directions is controlled by a piezo-electric tube
scanner, similar to those used in STM.

4. Feedback control unit

 Feedback control. The forces that are exerted between the tip and the sample are measured by the
amount of bending (or deflection) of the cantilever.
 By calculating the difference signal in the photodiode quadrants, the amount of deflection can be
correlated with a height.
 Because the cantilever obeys Hooke's Law for small displacements, the interaction force between
the tip and the sample can be determined.

AFM Tip

Figure: Atomic Force Microscope

DIFFERENT MODE OF OPERATION

Figure: Different Mode Of Operation

APPLICATIONS

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 This type of microscopy has been used in various disciplines in natural science such as solid-state
physics, semiconductor studies, molecular engineering, polymer chemistry, surface chemistry,
molecular biology, cell biology, medicine, and physics.
 Some of these applications include:
 Identifying atoms from samples
 Evaluating force interactions between atoms
 Studying the physical changing properties of atoms
 Studying the structural and mechanical properties of protein complexes and assembly, such as
microtubules.
 used to differentiate cancer cells and normal cells.
 Evaluating and differentiating neighboring cells and their shape and cell wall rigidity.

ADVANTAGES
 Easy to prepare samples for observation
 It can be used in vacuums, air, and liquids.
 Measurement of sample sizes is accurate
 It has a 3D imaging
 It can be used to study living and nonliving elements
 It can be used to quantify the roughness of surfaces
 It is used in dynamic environments.

DISADVANTAGES
 It can only scan a single nanosized image at a time of about 150x150nm.
 They have a low scanning time which might cause thermal drift on the sample.
 The tip and the sample can be damaged during detection.
 It has a limited magnification and vertical range.

X-RAY DIFFRACTION
 This method is used to get the knowledge of arrangements of atoms, particle shape and
morphology of nanomaterials.
 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.

PRINCIPAL
 X-ray diffraction is based on constructive interference of monochromatic x-rays and a crystalline
sample. Monochromatic X-rays are 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. This law relates the wavelength of
electromagnetic radiation to the diffraction angle and the lattice spacing in a crystalline sample.
Diffracted X-rays are then detected, processed and counted.
 A primary use of XRD analysis is the identification of materials based on their diffraction pattern.

WORKING
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 Crystals are regular arrays of atoms, whilst X-rays can be considered as waves of electromagnetic
radiation. Crystal atoms scatter incident X-rays, primarily through interaction with the atoms’
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:

 2dsinθ = nλ

 Where d is the spacing between diffracting planes, θ is the incident angle, n is an integer, and λ is
the beam wavelength. The specific directions appear as spots on the diffraction pattern called
reflections. Consequently, X-ray diffraction patterns result from electromagnetic 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 angstroms).

Figure: X-RAY DIFFRACTION

CONSTRUCTION
 X-ray diffractometer consists of three basic elements:
1. X-ray source tube (Cathode ray tube): X-rays are generated in a cathode ray tube by heating a
filament to produce electrons.
2. Entrance slit: It controls the electrons which are coming from the x-ray source.
3. Exit slit: It controls the electrons which are coming from the sample.
4. X-ray detector: It detects the electrons which are coming from the sample

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 X-rays are generated in a cathode ray tube by heating a filament to produce electrons, accelerating
the electrons toward a target by applying a voltage, and bombarding the target material with
electrons. When electrons have sufficient energy to dislodge inner shell electrons of the target
material, characteristic X-ray spectra are produced.

APPLICATIONS
 As a primary characterization tool for obtaining critical features such as crystal structures,
crystalline size, and strain, x-ray diffraction patterns have been widely used in nanoparticle
research. The randomly oriented crystals in nanocrystalline materials cause broadening of x-ray
diffraction peaks.

ADVANTAGES
 Powerful and Rapid technique≤ 20 min.
 Non-destructive method and minimum quantity of sample required
 XRD units are extensively available
 Data interpretation is relatively easier

DISADVANTAGES
 Homogeneous and single-phase material is best for identification of an unknown contents
 Peak overlay may occur and worsen for high angle ‘reflections’

DYNAMIC LIGHT SCATTERING

 Dynamic Light Scattering (DLS, also known as Photon Correlation Spectroscopy or Quasi-Elastic
Light Scattering) is one of the most popular light scattering techniques because it allows particle
sizing down to 1 nm diameter.

PRINCIPAL
 The sample is illuminated by a laser beam and the fluctuations of the scattered light are detected at
a known scattering angle θ by a fast photon detector.

CONSTRUCTION
1. The laser - A stable laser with low noise characteristics, as exemplified by certain Helium-Neon gas
lasers, is the most suitable.
2. The optical arrangement – Measuring only at 90o enables the production of a simple, cost-effective
system that provides an appropriate level of sensitivity for many applications as shown in Figure 1.
For experiments that require higher sensitivity levels or for more concentrated samples, measuring
at wider angles is preferable.
3. The detector(s)- Detectors are one of two types: cheaper, less sensitive photomultipliers or more
expensive, higher performance avalanche photodiode detectors (APD).

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Figure: Dynamic Light Scattering

WORKING
 Small particles in a dispersion or solution are subject to Brownian motion. DLS is driven by collisions
with the solvent molecules present, which are in constant movement due to their thermal energy.
 The speed of Brownian motion can be directly measured from the scattered light pattern produced
by the moving particles, a technique known as photon correlation spectroscopy (PCS) or quasi-
elastic light scattering (QELS) but presently referred to as DLS.
 The relationship between the speed of Brownian motion of a particle and that particle’s size is
defined by the Stokes-Einstein equation:

 Where D = Diffusion speed, k = Boltzmann’s constant, T = absolute temperature, η = viscosity, and

DH = hydrodynamic radius.
 This relationship shows how size can be determined from diffusion speed provided that the
temperature and continuous phase viscosity of the sample are known

APPLICATIONS
 Dynamic light scattering (DLS) is a valued sizing technique for emulsions, micelles, polymers,
nanoparticles, proteins, colloids and dispersions, which comfortably extends to the sub 1 nm
region.
 The observation of scattered light helps determine defining characteristics of a particle dispersion
or molecular solution such as particle size, molecular weight and zeta potential.
 Determination of the size of nano-particles and micro-beads
 Determination of Critical Micelle Concentration
 Monitoring Protein Complex Formation in Solution
 Vesicle Size Distribution Analysis
 Thermal Dissociation and Denaturation
 Protein Purification Confirmation
 Liposome Characterization

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ADVANTAGES
 Accurate, reliable and repeatable particle size analysis in one or two minutes.
 DLS is cheaper, and consists of simple experimental setup.
 Simple, material independent measurement of per peak particle concentration.
 Mean size only requires knowledge of the viscosity of the liquid.
 Simple or no sample preparation, high concentration, turbid samples can be measured directly.
 Size measurement of sizes from <1nm to >10um
 Low volume requirement (as little as 3µL).
 Operators can attain usable, detailed data without needing to have significant expertise.
 Sample volumes are small, down to just a few microliters, making this an appealing technique for
early stage research where valuable materials are involved.
 It is possible to measure both dilute and turbid systems with the concentration range for analysis
reaching down as low as 0.1ppm and up to 40% w/v.
 This size and concentration range, together with the high reproducibility of the technique makes
DLS suitable for a wide range of applications.

DISADVANTAGES
 Low resolution - When size populations are closely spaced, less than a factor of three difference in
size, DLS will not precisely characterize a polydisperse sample, making it advisable to apply
separation prior to measurement.
 Multiple light scattering - Multiple scattering is when the scattered light from one particle is
scattered by another before reaching the detector, and it compromises the accurate calculation of
particle size in more concentrated samples.
 Dispersant choice – Even though most dispersants are suitable, those with a viscosity greater than
100 mPa.s inhibit reliable measurements. Furthermore, light absorption by the dispersant can
interfere with detection
 Sedimentation - This is particularly likely with more dense particles. Increasing the density of the
dispersant by, for example, introducing sucrose is a helpful strategy but only for density matching
up to around 1.05 g/ml.
 DLS is Time consuming, especially for slow dynamics, Only transparent sample, Sensitive for
mechanical, Disturbances, Lack of selectivity and relatively low signal strength.

UV-VIS SPECTROPHOTOMETER
 Ultraviolet-visible (UV-Vis) spectrophotometers use a light source to illuminate a sample with light
across the UV to the visible wavelength range (typically 190 to 900 nm).

PRINCIPAL
 The instruments then measure the light absorbed, transmitted, or reflected by the sample at each
wavelength. Some spectrophotometers have an extended wavelength range, into the near-infrared
(NIR) (800 to 3200 nm).

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Figure: Uv-Vis Spectrophotometer

CONSTRUCTION
1. Light sources
 A light source that generates a broadband of electromagnetic radiation across the UV-visible
spectrum

2. Monochromators
 All the light sources produce a broad-spectrum white light. To narrow the light down to a selected
wavelength band, the light is passed through a monochromator, which consists of: An entrance slit
 A dispersion device, to spread the light into different wavelengths (like a rainbow) and allow the
selection of a nominated band of wavelengths
 An exit slit where the light of the nominated wavelengths passes through and onto the sample
 A single monochromator spectrophotometer is used for general-purpose spectroscopy and can be
integrated into a compact optical system. A double monochromator is typically found in high-
performance instruments.

3. Sample compartments
 The sample compartment of a UV-Vis spectrophotometer is typically a black-colored box with a
closing lid. The matt black inside the compartment helps to absorb stray light that may enter the
compartment. In the sample compartment, the sample is positioned to allow the beam from the
monochromator to pass through the sample. Glass, plastic, or quartz cuvettes are used for liquid
samples. Solid samples are held in position by a holder attached to the floor of the sample
compartment. The light can also be taken out of the sample compartment using fiber optics.

4. Detectors
 A detector converts the light from the sample into an electrical signal. Like the light source, it
should give a linear response over a wide wavelength range, with low noise and high sensitivity.
 Each detector has a different sensitivity and wavelength range.

WORKING

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 In the sample compartment, the sample is positioned to allow the beam from the monochromator
to pass through the sample. For the measurement of absorbance, liquid samples would typically be
held in a cuvette with a known, fixed pathlength.
 A cuvette is a rectangular liquid holder as shown below. They are made from glass, quartz, plastic,
or another material that transmits UV or visible light.
 Standard cuvettes have a 10 mm pathlength and are made from quartz, to ensure good
transmittance of UV wavelengths. Cheaper plastic cuvettes can also be used, but generally do not
transmit UV light so are only useful if measurements in the visible wavelength region are required.
 A multitude of cuvettes for special applications are available—from cuvettes that hold tiny volumes
of liquids through to those that have much longer pathlengths, for use with very dilute samples.

Figure: Cuvettes for measuring liquid samples.

 Solid samples can be held in place for simple transmission measurements. They can also be
measured at various angles of incidence. For more complex measurements, like diffuse reflectance
or transmission, other accessories may be installed into the sample compartment.
 The light can also be taken out of the sample compartment using fiber optics. Fiber optics are
useful when measuring very large, hot, cold, radioactive, or other dangerous samples.

APPLICATIONS
 UV-Vis spectroscopy is used in a wide variety of applications and a UV-Vis spectrophotometer can
be found in most laboratories. The Agilent handbook on the Basics of UV-Vis Spectrophotometry
contains detailed information on the main UV-Vis applications, or check out our UV-Vis and UV-Vis-
NIR application notes for a complete list, which include:
 Confirmation of identity—comparison of a measured spectrum with a reference spectrum
 Concentration measurements
 Reaction kinetics—see our video on kinetics measurement with the Agilent Cary 60 UV-Vis
spectrophotometer
 Color measurement
 Structural changes of compounds, including conformational studies of proteins
 Protein and nucleic acid melting temperature
 Multi-component analysis
 Multi-temperature analysis—the new Agilent Cary 3500 UV-Vis spectrophotometer measures up to
four temperature experiments, across eight cuvette positions, simultaneously

ADVANTAGES

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 The technique is non-destructive, allowing the sample to be reused or proceed to further

processing or analyses.
 Measurements can be made quickly, allowing easy integration into experimental protocols.
 Instruments are easy to use, requiring little user training prior to use.
 Data analysis generally requires minimal processing, again meaning little user training is required.
 The instrument is generally inexpensive to acquire and operate, making it accessible for many
laboratories.

DISADVANTAGES
 Stray light - In a real instrument, wavelength selectors are not perfect and a small amount of light
from a wide wavelength range may still be transmitted from the light source,1 possibly causing
serious measurement errors.9 Stray light may also come from the environment or a loosely fitted
compartment in the instrument.
 Light scattering - Light scattering is often caused by suspended solids in liquid samples, which may
cause serious measurement errors. The presence of bubbles in the cuvette or sample will scatter
light, resulting in irreproducible results.
 Interference from multiple absorbing species - A sample may, for example, have multiple types of
the green pigment chlorophyll. The different chlorophylls will have overlapping spectra when
examined together in the same sample. For a proper quantitative analysis, each chemical species
should be separated from the sample and examined individually.
 Geometrical considerations - Misaligned positioning of any one of the instrument's components,
especially the cuvette holding the sample, may yield irreproducible and inaccurate results.
Therefore, it is important that every component in the instrument is aligned in the same
orientation and is placed in the same position for every measurement. Some basic user training is
therefore generally recommended to avoid misuse.

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