Dls presentation edited

DYNAMIC LIGHT SCATTERING…..
BY: PRIYA KUMARI

ABSTRACT
 Dynamic light scattering(DLS) is an analytical tool for the
determination of diffusion coefficient and hydrodynamics
sizes of molecules and particles in solution.
 Dynamic light scattering are routinely used in biology
laboratories to detect aggregates in macromolecular solution
to determine the size of proteins,nucleic acid and complexes
or to monitor the binding of ligands.
 Widely used in biochemistry,biotechnology and
pharmaceutical developments,DLS is a popular wherever the
size and size distribution of macromolecules and
nanoparticles need to measured quickly and easily.

 Allows for the characterization in a size range from the nanometer to
lower micrometer scales,which makes it especially valuable in the
field of biotechnology.
 Review the basic concept of light scattering measurements and
addresses the critical aspects of the analysis and interpretation of
DLS results.
 To ensure reproducible quantitative data,attention should be paid to
control the preparation and handling of proteins or assemblies
because variation in the state of aggregation ,induced by minor
changes in experimental condition or techniques might compromise
DLS results and affect protein activity.
 Variables like temperature,solvent viscosity and interparticle
interaction may also influence particle size determination.

INTRODUCTION
 Dynamic light scattering or Photon Correlation
Spectroscopy is a well-known and well used
techniques for measuring particles in solution with
size ranging from a few monometers to a few
microns.
 In this process,a coherent monochromatic light
source is radiated upon a sample .
 The frequency spectrum of intensity of resulting
scatter is recorded and the sizes of particles are
determined.
 The shift in frequency is termed as Doppler Shift or
broading and it is related to the size of particles
causing shift.

 As a result of their higher average velocity ,small
particles cause a bigger shift in the light frequency
than the larger particles.
 It is this differences in the frequency of the scattered
among particles of various sizes that is used to
determine the sizes of particles present in the fluid.
 Compared with other methods,DLS is fast and
somewhat cheap process.
 It is mostly used to determine the characteristics of
bacteria as well as protein.
 Optometrists can use this method to detect the
development of cataract in the eyes.

 DLS is often used to analyze maromolecules like
protein.(Protein Crystallography and
nanotechnology application)
 The molecular mass and the concentration of the
protein in the solvent is directly proportional to
the scattered by it.

ASSUMPTION AND THEORY
 The theory behind this techniques is based on
two condition.
 The 1st condition is that the particles follow
Brownian motion in solution.This random motion
follows a mathematical formula in which the
probability function can be determined.
 The 2nd condition is that the particles are
relatively spherical and with a diameter of less
than a half of a wavelength of the incoming
radiation.

BROWNIAN MOVEMENT
EXPLANATION:
A suspended particles is constantly and
randomly bombarded from all sides by
molecules of the liquid.If the particles is very
small,the member of hits it takes from one sides
at a given time will be stronger than the bumps
from other sides.This makes the particle
jump.These small random jumps are what make
up Brownian motion.
D

STOKE-EINSTEIN EQUATION:
 D – diffusion constant
 -Boltzmann’s constant
 T - Absolute temperature
 - dynamic viscosity
 r -radius of sphere


BROWNIAN MOVEMENT:A zigzag motion of particles

APPLICATION OF DLS USING
STOKE-EINSTEIN EQUATION
 SIZE:Using Stoke-Einstein equation,DLS can be
used to easy,fast and accurate determination of
the hydrodynamic radius of particles.
(Typically range:1nm-1µm)
 SHAPE:Ellipsodial particles results in a small
fraction depolarized scattered light.Can be used
for estimation of ellipticity of the particles.

Some examples of sub-micron
systems………
 Micro-emulsion
 Peptides
 Micelles
 Macromolecules
 Polymers
 Paint pigments
 Bacteria,viruses

ESTIMATION OF ELLIPSODIAL
OBJECTS

RESEARCH USING DLS
 Determination of size on complex systems:
 “water-in-oil”
 Biomolecules
 Cellulose
 Glass transition dynamics
 Polymer dynamic

DLS INSTRUMENT………….
ZETASIZER NANO-ZS

DESCIPTION OF INSTRUMENT……
 The Zetasizer Nano-ZS uses dynamic light scattering
(DLS) to measure size,molecular weight and zeta
potential of dispersed particles and molecules in
solution.
 The temperature covered is 2 to 90ºC.
 DLS is a non-invasive(not having the tending to
spread very quickly and undesirably or harmfully)
techniques that measures a large population of
particles in a very short time period,with no
manipulation of the surrounding medium.

 This equipment can measure particles sizes as
small as 0.6nm and as large as 6 µm across a
wide range of sample concentrations.
 Because of the sensitivity to trace amounts of
aggregates and the ability to resolve multiple
particle sizes,DLS is ideally suited for colloidal
science and macromolecular and volume.

ADVANTAGES DISADVANTAGES
 Wide time range
 Cheap
 Simple experimental set up
 Only transparent sample
 Very clean sample need
 Sensitive for mechanical
disturbance

SCIENTIFIC INSTRUMENTS
 More laser power!
 Specially designed cryo-
furnaces
 Polarization options
 Vibration isolation table

WORKING OF DLS …………
 In DLS ,a detector is focused on a small volume that
is illuminated by a laser beam.
 The detector measures fluctuations in the intensity
of light scattered from this volume(in MALLS the
detector measures the time-averaged intensity from
the same volume).
 The fluctuations are the result of macromolecules
diffusing into and out of small volumes that the
detector is monitoring .
 From the data,the translational diffusion constant of
the macromolecules is determined which in turn can
be related to the radius that a sphere with the same
diffusion would have.

 This is the Stoke radius(also measured by sedimentation
velocity) contain contributions from the mass,shape and
band solvents components.
 A measure of the polydispersity of the sample is also
derived during the analysis with high polydispersity
suggesting aggregation in the case of protein.
 In some cases population of species of differing radii and
polydispersity can be resolved.
 Protein preparation exhibiting low polydispersity have
generally proven more suitable for crystallization trials than
those with high polydispersity.
 The method is well suited to the characterization of large
polymers and liposomes.

DLS SIGNAL:
Random motion of
particles lead to
random fluctuation
in signals(due to
changing/destructiv-
e interference of
scattered light.

FIG:speckle,pattern
in the far field.As
the particles move
in Brownian
motion,their
position changes,as
do the phases of
light that they
scatter and the
speckle pattern
fluctuates from one
random
configuration to
another.

TYPICAL INTENSITY FLUCTUATION FOR
LARGE AND SMALL PARTICLES

PROCEDURE……..
 There are different way to determine the dynamic
of a particles in Brownian motion.One such method
by using a laser as a light source.
 The laser passes through lens that would then hit
the particles .
 Then the light is scattered and passes through
another callimotor lens.
 The resultant of this diffraction light is “collected”
and read by photomultipler.
 The photomultiplier translates all the different
intensity in the form of voltage readings.
 It is necessary to note that two collimotor lens are
required:

 The first is to better focus the light to directly hit the cell
and to ensure the area on the cell that the light hits is far
enough away from the sides of the cell.
 The second lens is to get just the right amount of
scattered light to be collected by the photomultiplier.
 After the beam is measured by the photomultipler,the
signals gets amplified and all the information can be sent
to and analyzed by a computer.
 In order to ensure accurate measurements ,it is essential
to calibrate the instruments.
 It is important to make sure that the light beam is
shining at a consistent linear path.

 In other words,it needs to be at the same
height in the entire path.
 This is to ensure that the beam will pass right
through the first lens and straight into the
center of the cell.
 Another thing to note is that all other light
sources should be blocked out,other than the
scattered light from the laser source.
 This will allow for more accurate
measurement.

APPLICATION OF DLS….
 DLS technique is one of the most popular
method used to determine the size of particles
such as proteins,polymers,micelles,carbohydrate
and nanoparticles.
 It is applicable in range from about 0.001 to
several microns,which is difficult to achieve with
other techniques.
 Today,DLS is recorgnised as a standard
instrument widely used in industry such as
biopharmaceutical industry.
 DLS is mostly used in protein crystallization.

 Very fast detection of aggregates.
 Great dynamic range.
 Were suited to study kinetics of aggregates.
 DLS can be used to determine:
 Particle/molecule size
 Size distribution
 Relaxation in complex fluid
 Rapid,non-invasive techniques
 Stability studies can be done by using DLS.

LIMITATION
 Measures hydrodynamic radius which is
affected by shape.
 Cannot discriminate between shape effects
and changes in oligomeric states i.e non-
spherical shape mimics oligomerization.
 Need fractionation to resolve low number
oligomers when present in mixture.

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