Course: PG Pathshala-Biophysics

Paper: Biophysics paper 10

Module:31 Dynamic Light Scattering

Introduction:

Dynamic light scattering (DLS), also known as photon correlation spectroscopy or quasi-

elastic light scattering, is a technique used to measure the flow anddiffusive properties of

scattering particles in suspension or polymersin solution. DLS is a technique used for

measuring the size of particles/biomolecules typically in the sub-micron region. Usually DLS

is concerned with measurement of size of particles suspended in a liquid medium. In DLS

when a monochromatic light is passed onto a solution with particles in Brownian motion,

Doppler Shift arises as the light strike the moving particle, thus altering the wavelength of the

incoming light and the motion established determines the size (or Dτ) of the particles. Motion

of particles is influenced by their size, solvent viscosity and temperature. Since, viscosity of

solvent related to the temperature therefore the information of accurate temperature is needed

for DLS measurements. While the motion of particles over a particular range of time is

examined, information based on the size of macromolecules can be achieved, as particles of

large size will diffuse slowly, attaining similar positions at different points of time, as

compared to the particles of small size which move faster and consequently do not

accomplish a specific position. DLS is a non-invasive technique, most commonly used for

measuring the size of the particles usually in the submicron region, size distribution,

hydrodynamic radius and diffusion coefficient. The diffusion coefficient can be measured by

using correlation function. Advantages associated with DLS are accuracy, reliable and

repeatable analysis, low sample volume requirement (200µl), particle size measurement

below 1 nm, short duration of experiment, low development cost, does not require sample

preparation, simple set up and fully automatic/computerized measurement.

Objective: In this module we discuss following things in detail,

1. Principle of DLS 2. DLS Spectra

3. DLS instrumentation

4. Factors affecting DLS measurements

5. Application of DLS

6. Limitations

7. Experimental conditions and general maintenance

8. Summary

1. PRINCIPLE OF DYNAMIC LIGHT SCATTERING

DLS measures the speed at which the particles are diffusing due Brownian motion of the

macromolecules in the suspension which is relates to size of the particles. Brownian motion

defines random thermal motion of the particles that occur due to the bombardment by the

solvent molecules surrounding them. The larger the particle size, the slower the Brownian

motion will be, smaller particles are hit out further by the solvent molecules and move faster

than the larger one. The intensity of scattered light fluctuates defines the diffusion rate of the

particles. DLS measures alteration in scattered light intensity with time at a fixed scattering

angle (typically 90º), whereas static light scattering measures scattered light intensity as a

function of angle. The velocity of the Brownian motion is defined by translational diffusion

coefficient (usually given the symbol, D). The particle size can be estimated by using the

Stokes- Einstein equation;

d(H) = hydrodynamic diameter

D= translational diffusion coefficient

k=Boltzmann’s constant

η = viscosity of the medium

T= absolute temperature

With the help of Stroke-Einstein equation, DLS can be used for the resolution of

hydrodynamic diameter and ellipticity of the particles in the suspension. The diameter

attained by this technique is the diameter of the sphere that has the identical translational

diffusion coefficient as that of the particle. Variables influencing the determination of particle

size include viscosity, temperature and interparticle interaction in the medium.

2. Spectra of DLS

A typical DLS spectrum is shown in the Figure 1.

Figure 1: Spectra for DLS: A. Hypothetical DLS spectra of two samples: upper panel

represents the spectra for large particles whereas lower one is for smaller particles. B.

Representative dynamic light scattering spectra.

3. Instrumentation

A DLS system consists of six components as shown in the Figure 2.

i. A laser which offer a light source to irradiate the sample holding in a cell.

ii. In dilute concentration, most of the monochromatic light passes through the sample,

however some of them get dispersed within the sample at different angles.

iii. A detector is employed for measuring the dispersed light. The intensity of scattered

light must be within a certain range for effective detection by detector. Saturation

may occur during the increased intensity. To overcome this problem, an attenuator is

employed so as to decrease incident light therefore decreasing the intensity of

scattered light.

iv. From the detector the scattered light intensity proceeds to the correlator.

v. The correlator measures the scattered light intensity at consecutive time intervals to

derive the variations in intensities.

vi. The data from correlator then passed onto the computer where data will be evaluated

by Nano software and size of the particle will be determined.

A. B.

Figure 2: Showing components of DLS system.

In DLS, the speed at which the particles are moving under the Brownian motion is recorded.

It is done by measuring the rate at which the intensity of the scattered light fluctuates when

detected using a suitable optical arrangement. How do these variations in the intensity of

scattered light arise?

If we consider a cuvette, having stationary particles, is illuminated by a laser beam and glass

screen is used to see the sample cell. A particular type of speckle pattern will be observed

DLS-

Machine

(Figure 3). The observed pattern will be stationary both in size and position asthe whole

system is stationary. The dark spaces areobserved when the phases of scattered light

areconjointly destructive and cancel out each other (Figure 4A). Whereas the bright spots are

seen in the speckle pattern when the scattered light from the particles reaches with the same

phase and constructive interference taking place (Figure 4B).

Figure 3: Representative speckle pattern observed from scattering of particles.

Figure 4: The signal observed from the particle scattering mainly depends upon the phase

addition. A. When two phases shows destructive interference, cancel out each

other and intensity of detected light is decreased. B. If interference is

constructive, the intensity of detected light increases.

In case of a system where particles undergoing Brownian motion continuously, a

specific speckle pattern have been observed in which the location of each speckle is seems to

be in constant and continuous motion. It happens as the phaseaddition from the moving

particles is persistently changing and forms new patterns. The rates at which these

fluctuations of intensity occur depend on the size of the particles. In Figure 5 representative

intensity fluctuations that has been arising from a dispersion of large and small particles.

Generally, smaller particles induce the intensity fluctuations at a higher rate as compared to

large ones.

Figure 5:Typical fluctuations in the intensity of larger and small particles.

Correlation function: While employing with DLS instrument, when the monochromatic (i.e.

laser) light encounters macromolecules in the solution, the incident light gets disperse in all

directions (Rayleigh scattering) and scattering intensity of light is recorded by a detector.

Doppler broadening/shift will be observed by the incident light as thesemacromolecules are

in continuous Brownian motion in solution.The dispersed light will either lead to mutually

constructive phases to exhibit a detectable signal and mutually destructive phases

whichnullify each other out.Sample Preparation of sample either by centrifugation or

filtration is required so to eliminate dust particles and artefacts from the solution.

The dynamic information of the macromolecules can be generated from digital

autocorrelator which associates intensity fluctuations of dispersed light with respect to time

(ns-μs) so as to define fluctuation intensity of light, which is directly proportional to the

diffusion behaviour of macromolecules. In a DLS experiment, the second order

autocorrelation curve is derived from the intensity trace and can be standardised as:

g2(q;τ) = ⟨(t)I ( t+ τ)⟩

[ ⟨I (t)⟩]𝟸

Here, g2(q;τ) represents the autocorrelation function that defines the motion of

macromolecules in the solution under examination and can be illustrated as an integral over

the product of intensities at time (t) and delayed time (t + τ). Moreover, the angular brackets

(<>) symbolize the expected value operator.

For mono-dispersed particles the decay i.e., the autocorrelation function defines a single

exponential. The Siegert relation associate the second-order autocorrelation function 2(q;τ)

with the first-order autocorrelation function g1(q;τ) which is based on an estimation thatthe

scattering of incident light is homodyne (i.e photo detector identify scattered light only) and

that the photon counting is based on random Gaussian method, which is normalised as :

g2(q; τ) = 1 + β[g1(q; τ)]2

Here, β is a coherence factor that relies on the optical alignment in the light scattering setup

and scattering behaviour of macromolecules.

4. Factors Affecting DLS Measurements

Ionic properties of medium: The ionic strength of the medium will influence the

diffusion rate of the particles by altering the thickness of the electric double layer

known as Debye length (K-1). High conductivity medium will repress the electric

double layer resulting in smaller the hydrodynamic diameter whereas, conversely low

conductivity medium produce double layer resulting in larger hydrodynamic diameter.

Temperature:An accurately known temperature is necessary for DLS experiments

because any change in temperature directly effects viscosity of medium used for the

measurements. The temperature also needs to be stable; or else convection currents

developed due to the change in temperatureof sample will induce non-random

movements that will effects the accuracy ofsize interpretation.

Viscosity: DLS measurement of particle size is significantly influenced by the

viscosity of medium/sample, viscosity accounts for both in the data interpretation and

in behaviour of sample. Care should be taken to mentionprecise values for viscosity

and the sample should be prepared in such a way that the viscosity does not

excessivelyaffect the results of experiments. It can be easily observed from the

Stokes- Einstein equation that viscosity have inverse relation with size measurements.

There is a requirement for DLS based measurements that the particles move freely

and independently devoid of any impact on their respective velocity. As the viscosity

of the medium increases, the velocity of particle decreases correspondingly.

Different types of viscosity representations have been used to define the non-

flowability of pure liquids and suspensions. In case of DLS, dynamic (absolute)

viscosity has been considered. Dynamic viscosity is the value of viscosity measured

for Newtonian fluids likemilk, water, organic solvents, gasoline, many oils, etc.

Newtonian fluids are those whose viscosity does not change significantly while

stirring or agitation. In practical terms, a Newtonian fluid showssteadiness of the ratio

of shearing stress to shear rate.

Another type of viscosity is termed as Apparent Viscosity. It is also

determined by using the same experimental methods and equations as is used for

dynamic viscosity. However, this has been used for Non-Newtonian fluids which are

complex systems of emulsions or suspensions like peanut butter, paint, blood,

shampoo, toothpaste, etc. In chemical sense, these materials are not pure fluids as

required by the Stokes-Einstein Equation. Apparent viscosity can alter the size of

particle nearly by 30%.

The effect of an error induced due to the change in viscosity or change in entry

of the viscosity value in theStokes-Einstein equation can be reasonably significant.

The example shown below represents the results of an analysis of a molecule

dispersed in water. The data was analyzed using a viscosity value of 1.0 (blue graph)

and 2.0 mPa·s (red). It can be observed that the data value shift solely due to changes

in the viscosity. Thus, accuracyof measurements requiresthe accurate information of

viscosity.

Figure 6: Effect of viscosity on the measurement of size.

Concentration of particles: Measurements based on the DLS principle considered an

ideal system in which there are no other substancespresent around a particle that

will hamper its motion. Taking this into the account, theuse of the pure dispersant

solutionscan give accurate results. In reality, nevertheless, the probability of

collisionbetween the particles increasesas theconcentration of the sample increases. It

hasbeen estimated empirically that inmost of the cases, when the concentration

of a solution reaches a particular value, the effect of interparticle

bombardmentbecomes no longer be negligible. So, whenever this type of particle

crowding takes place, it results in the decrease of the rate of movement of the

particleand finally increases in the viscosity of thedispersion.

Figure 7: Effect of high concentration on the movement of particles

Surface properties/structure of particles: Change in the surface structure will affect

the size of the particle. An adsorbed polymer in the suspension will decrease the

diffusion speed of the particle as compared to the polymer lying flat on the

surface.Any change to the surface of aparticle that affects the diffusion speed will

correspondingly change the apparent size of the particle. The nature of the surface and

the polymer, as well as the ionic concentration of the medium can affect the polymer

conformation, which in turn can change the apparent size by several nanometres.

Shape of/non-spherical particles: All techniques used for particle-sizing have a

characteristic problem in defining the size of non-spherical particles. Sphere is the

only body whose sizecan be unambiguously described by asingle figure. Alteration in

the shape of a particle will influence the diffusion rate resulting in change in

hydrodynamic size. Minor changes in the diameter of the rod particle affect the

diffusion speed, however alteration in the length of the rod shaped particle will

influence the size of the particle.

Generally, the hydrodynamic diameter of a nonspherical particle is equal to the

diameter of a sphere having same translational diffusion speed as the particle. So, any

change in the shape of a particle that affects the diffusion speed, ultimately changes

the hydrodynamic size. For example, even small changes in the length of a rod-shaped

particle will affect the size, while changes in the diameter ofrods, will hardly affect

the diffusion speed and will be difficult to notice. Similarly, the conformation of

proteins and other biomacromolecules are usually depends on the nature of the

dispersing medium/buffer/solvent. As discussed above that any conformational

changes will directly affect the diffusion speed, so DLS is a very sensitive technique

for detecting these changes.

5. Application of DLS:

DLS is an analytic tool which has wide variety of applications as follows:

DLS is used to measure the hydrodynamic size of the particles which includes

proteins, polymers, carbohydrates, micelles and nanoparticles (like quantum dots).

For example, it can be used to distinguish between a monomer and a dimer and can

also determine particles size that is below 1nm. This technique can be employed for

the estimation of molecular weight of the particles and to detect different molecules

in the same solution.

It used for the diffusional studies of macromolecules like that of proteins, lipids etc.

Widely used biopharmaceutical industry. For example: DLS aids in determining the

size of liposomes, which is crucial for effective drug delivery systems.

They are also used in protein crystallization. DLS is suitable for the evaluation of

protein homogeneity under multiple conditions and at adequate concentrations using

crystallization conditions.

Figure 8: DLS determines size distributions without fractionation, providing polydispersity

estimates as well as hydrodynamic radii.

DLS is used to check the quality of molecules/biomolecules prior to start expensive

analyses such as SPR or SANS.

DLS was also used to analyse thermal stability and chemical denaturation of protein,

differentiating pure unfolding from aggregation. It is also used to characterize the

folding intermediates of proteins.

It is used to determine size of metal nanoparticles or quantum dots.

Self-assembly progressions of polypeptides or DNA were also quantified with the

help of DLS.

Used to assess the colloidal stability as a precursor of aggregation or precipitation.

DLS is used to measure the viscosity of concentrated biotherapeutics formulations

with just a few microliters of solution.

It is widely used for the detection of population ofaggregates for small molecules,

proteins or liposomes. They are used for screening the unwanted aggregates in drugs

that may reduce the efficacy of the drugs and can lead to produce an immune reaction

or toxicity. Also DLS have been used for the investigation of protein aggregation

diseases such as Alzheimer’s disease and Huntington’s disease.

DLS is particularly more effective if it is employed in combination with other

techniques commonly used for characterizing protein behaviour, which may

includesizeexclusion chromatography (SEC) and native polyacrylamide gel

electrophoresis (PAGE).

Study the homogeneity of in vitro transcribed RNA.

DLS can be used in combination with static light scattering for the assumption shape

of the particles.

DLS can be used as a probe of complex fluids such as concentrated solutions.

6. Limitations of DLS

Despite a good technique, DLS has following limitations:

i. It does not measure the particle size above 5000nm.

ii. It measures the hydrodynamic radius of the particles but unable to measure the actual

particle size.

iii. Since, DLS is sensitive to solvent viscosity and temperature, therefore temperature

must be kept constant and solvent viscosity must be documented for reliable DLS

experiment.

iv. DLS known for low-resolution technique that cannot separate closely related

molecules.

v. DLS is also limited for transparent sample preparation.

vi. Since, DLS rely on the size and concentration of macromolecules, therefore for

reliable measurements requirement of optimization of range concentration is essential.

vii. It is time consuming technique exclusivelyfor slow dynamics.

viii. They lack selectivity and possess low signal strength.

7. Experimental Conditions and General Maintenance

DLS is a valuable and widely used technique for the study of different parameters like

estimation of protein conformation, size of particle/biomolecules, etc. DLS is also a widely

used technique in the field of nanotechnology, crystallography and chemical synthesis.

Because it is a very sensitive method, a minor error or experimental mismatch may create a

big problem in the targeted study. That is why some guidelines will be useful to get a reliable

data.

Cuvettes: DLS measurements are carried out in high transparency quartz cuvettes (cells).

Cuvettes should be handled, used and storedcarefully.Quartz cells should be washed and

dried between runs.The typical procedure we have used involves a 1 minwash with specific

detergent solutions, followedby thorough washing with distilled water, thenethanol, followed

by drying with a vacuum pump.

Preparation of Samples: Buffers/solvents used for DLS measurements must not contain any

component that interferes with the free movements of particles. All buffers should be

properly prepared, filtered and degassed under vacuum. In case of temperature dependent

studies, the viscosity factor of dispersion medium must not be ignored. To further improve

the quality of signal, each sample must be homogenously dispersed.

Calibration of DLS Instrument: The process of DLS calibration is very delicate. The first

procedure is to verify ifthe laser beam is at the same high in its entire path, then the focus the

first lensin the middle of the cell and the second lens into the photomultiplier. A usefulcontrol

is the one to check if there is incoming light other than the scattered one,reaching the

photomultiplier. Once that the laser beam has been set, the secondstep is controlling the

response of the photomultiplier. With a pulsing LED it waspossible to observe that in the

power spectrum of both the peaks and thebackground was rolling-off. This was confirmed by

the power spectrum of a constant LED lightwhere it is possible to observe a roll-off point at

high frequencies. An increasing of gain will increase the noise of the signal.

8. Summary

DLS is a widely used technique in numerousareas of biology, physics, chemistry and

medicine. Fundamental understanding of basics of DLS likephysical properties of dispersant,

viscosity, temperature, concentrations and instrumental knowledge is an important

prerequisite for the study of a widerange of biological and physical systems.DLS is most

commonly used to analyze size of particles. Examples comprise determining of nanoparticle

size, latex size, colloid size and protein size. In general, the technique is best used for

submicron particles and can be used to measure particle with sizes less than a nm. DLS can

also be used as a probe for complex solutionslike concentrated solutions but with some

limitations. DLS is preferred due to its peculiar sensitivity, specificity and low cost as

compared to other size determining analytical methods.Thus it is abroadly used technique for

a variety of environmental, pharmacological,engineering and biological purposes.

End of Module

Thank you

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