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 MOVEMENTEXPLANATION: 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”BiomoleculesCellulose 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 sizeSize distributionRelaxation 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.