Biophysical and Nanoparticle Characterisation Using Light Scattering Techniques

Measurement Principles Dynamic Light Scattering Electrophoretic Light Scattering Static Light Scattering Combining DLS with Size Exclusion Chromatography Applications Conclusions... D

Biophysical and Nanoparticle Characterisation Using

Light Scattering Techniques

Measurement Principles

Dynamic Light Scattering

Electrophoretic Light Scattering

Static Light Scattering

Combining DLS with Size Exclusion Chromatography

Applications

Conclusions

-5 to 10,000-

1000 to 2 x 107

5 to 10,0000.6 to 6000

10,000 to 2 x 107

5 to 10,000

1 to 3000

10,000 to 2 x 107-

1 to 3000

1000 to 2 x 107-

0.6 to 6000

Molecular Weight Range

Zetasizer Nano Series:

Sample Specifications

Measurement Principles

Dynamic Light Scattering

Dynamic Light Scattering

Dynamic light scattering measures the time dependent fluctuations in the scattering intensity to determine the translational diffusion coefficient (D), and subsequently the

hydrodynamic diameter (DH) (from the Stokes-Einstein equation)

Where k = Boltzmann’s constant,

T = absolute temperature,

η = viscosity

kT 3πηDH

D =

Intensity Fluctuations, Correlation and Size Distributions

Correlate

Apply Algorithm

Intensity Size Distributions

Primary result obtained from

a DLS measurement

Based upon the intensity of

light scattered by particles

Sensitive to the presence of

large particles/aggregates

/dust

The only sample properties

required are the dispersant

viscosity and refractive index

Volume Size Distributions

Derived from the intensity

distribution using Mie theory

Equivalent to the mass or

weight distribution

The optical properties of the

particles are required to

make this transformation

Particle refractive index

Particle absorption

Number Size Distributions

Derived from the intensity

distribution using Mie theory

The optical properties of the

particles are required to

make this transformation

Particle refractive index

Particle absorption

Optical Configuration:

Non Invasive Back Scatter (NIBS)

Conventional DLS instruments detect the scattered light at an angle of 90o

An optical configuration that allows measurements of samples at lower concentrations of smaller sizes is

The NIBS design provides both a wide size and

concentration range due to high sensitivity,

selectable scattering volume and measurement

position

Non Invasive Back Scatter (NIBS)

High sensitivity enables the measurement of the size

of very small particles and molecules at low

concentrations

This arises from the use of patented fibre optics in combination with NIBS and ensures efficient signal collection from the selected scattering volume

Measurements of sizes less than 1nm have been

achieved with NIBS optics

Non Invasive Back Scatter (NIBS)

Concentrated Samples

Minimise path length Minimise multiple scattering

Focussing Lens Cuvette

Small Particles/

Dilute Samples

Maximise measurement volume

Minimise laser flare

Measurement Principles

Electrophoretic Light Scattering

The particles move with a characteristic velocity which

is dependent on:

 Field strength

 Dielectric constant of medium

 Viscosity of the medium

 Zeta potential

Electrophoretic Light Scattering

Electrophoresis is the movement of a charged

particle relative to the liquid it is suspended in under

the influence of an applied electric field

√√√√

√√√√

√√√√

√√√√

Electrophoretic Light Scattering

A laser beam is passed through the sample in the capillary cell undergoing electrophoresis and the

scattered light from the moving particles is frequency shifted

The frequency shift ∆f is equal to:

ν = the particle velocity

λ = laser wavelength

θ = scattering angle

∆

∆f = 2νννν sin(θθθθ/2)/λλλλ

Phase Analysis Light Scattering (PALS)

P hase A nalysis L ight S cattering - very accurate

determination of a frequency shift

PALS can give an increase in sensitivity of greater than 100 times than that associated with Fourier

Transformation techniques

Beneficial in applications where the particle mobility

is very low

Non-aqueous dispersants with low dielectric constants

(mobility is proportional to dielectric constant)

High viscosity samples (low mobilities)

High conductivity (must work at low voltages to avoid Joule heating)

Dispersion Stability

The stability of colloidal

dispersions is determined

by the sum of attractive

forces (Van der Waal)

and repulsive forces

(electrostatic) which

particles experience as

they approach one

another

The stability of a particle

dispersion can be lost

through various

mechanisms

Stable System Flocculation

Coagulation

Flocculation Sedimentation

Phase Separation

Maintaining Dispersion Stability

There are two fundamental mechanisms that affect

Easy to measure the controlling

parameter (zeta potential)

Reversible

May only require change in pH or

ion concentration

Origins of Surface Charge in

This surface charge results in an increased

concentration of counter ions close to the surface

Zeta Potential

The zeta potential is the electrical potential at the slipping plane (hydrodynamic plane of shear)

What is Zeta Potential?

Zeta potential is the charge a particle has in a particular medium

Depends on:

Chemistry of the particle surface

Chemistry of the dispersant

For electrostatically stabilised dispersions, the higher the value of zeta potential, the more stable the

dispersion is likely to be

Stability dividing line is generally considered to be

±30mV for aqueous systems

Factors Affecting Zeta Potential

Zeta potential can be affected by

 changes in pH,

 conductivity (concentration and/or type of salt)

 changes in the concentration of a formulation component (eg polymer, surfactant)

Zeta Potential and pH

Zeta Potential and

Non-Specific Ion Adsorption

Non-specific ion

adsorption has no effect

on the position of the IEP

Non-specific adsorption

can give rise to changes

in the zeta potential of the

particle dispersion

E.g Alumina and KNO3

Zeta Potential and

Specific Ion Adsorption

Specific ion adsorption

leads to a change in the

In some cases, specific

ion adsorption leads to

charge reversal

E.g Alumina and LiNO3

Measurement Principles

Static Light Scattering

Dynamic and Static Light Scattering

Dynamic light scattering measures

the time dependent fluctuations in

the scattering intensity to determine

the translational diffusion coefficient

(D), and subsequently the

hydrodynamic diameter (DH) (from the

Stokes-Einstein equation)

Static light scattering measures the

time averaged intensity of scattered

light, from which the weight-averaged

molecular weight (MW) and 2nd virial

coefficient (A2) can be determined

What Samples Are Suitable

Static Light Scattering (SLS)

The intensity of scattered light that a macromolecule produces is proportional to the product of the weight- average molecular weight and the concentration of

The Zetasizer Nano measures the intensity of

scattered light of various known concentrations of sample at one angle

This is called a Debye plot and allows for the

determination of

Weight-Averaged Molecular Weight

2nd Virial Coefficient

strength is equivalent to the molecule-molecule

interaction strength – the solvent is described as

being a theta solvent

When A2<0, the molecule will tend to crystallise or aggregate

measure using a differential or interferometric refractometer

Standard required with known Rayleigh ratio – this is

normally toluene (calibration)

Solvent

Zetasizer Nano SLS MW Specifications

For single angle MW measurements with Zetasizer Nano system:

Benefits of Sizing Proteins by DLS

Non-invasive

High sensitivity (< 0.1 mg/mL for typical proteins) Low volume (12 µL)

Scattering intensity is proportional to the square

of the protein molecular weight, making the

technique ideal for identifying the presence of trace amounts of aggregate

Molecular Weight From DLS

Using DLS for determining molecular weight has a number of advantages over SLS:

Simple model to convert size measured to MW

No material parameter input is required

No calibration

No second detector required

No alignment of second detector to get molecular weight

relationships, similar to GPC and other calibrated

techniques

Molecular Weight Estimator

Estimated Molecular Weight Example:

Lysozyme

Literature value for the molecular weight of lysozyme is 14.7kDa

This is less than 3% error in the molecular weight

Light Scattering Techniques Summary

Dynamic Light Scattering

Time dependent fluctuations in the scattering intensity

Diffusion coefficients, particle size and polydispersity (size distribution)

Electrophoretic Light Scattering

Frequency or Doppler shift of the scattered light

Electrophoretic mobility and zeta potential

Static Light Scattering

Concentration dependence of the scattering intensity

Weight-average molecular weight and 2 nd virial coefficient

Combining DLS with Size Exclusion Chromatography

Flow Mode

Flow System Overview

Flow Mode Requirements

SEC system

Zetasizer Nano S or ZS

Quartz Flow Cell ZEN0023

Two external detectors can be connected to the Zetasizer Nano (optional – requires modified

instrument backplate)

Data Analysis

There are two possible ways to analyse the data:

1 DLS: the z-average and scattered intensity data are

used to calculate PSDs and estimated molecular weights

No sample concentration knowledge required

2 SLS: using the concentration data from the UV or RI

detectors and the scattered intensity data to produce the Debye plot and obtain MW for each peak

This will be supported in the next version of Zetasizer Nano software (5.10) Research Only

Applications

Probing the Lower Size Limit

At sizes above 20nm, monodisperse, traceable latex standards are available

For sizes below 10 nm, certain proteins (e.g lysozyme and BSA) are often used, although preparation of a

sample of the monomer is not straightforward

For samples close to or below 1nm in size, something other than a protein must be considered

Suitable nanoparticle samples are difficult to obtain in a well dispersed state containing no aggregates

Crystalline, pure and readily soluble in water

M Kaszuba, D McKnight, M.T Connah, F McNeil-Watson and U Nobbmann (2007) J Nanoparticle Research (DOI 10.1007/s11051-007-9317-4)

Experimental Setup

Various sucrose concentrations prepared in ultra pure water: 5, 10, 15, 20, 25, 30 and 35% w/v respectively

Viscosities were determined by doping each sucrose

concentration with polystyrene latex standard of certified size and comparing the results (assuming the viscosity of water) with the size obtained for the latex in 10mM NaClSamples filtered through Whatman Anotop 20nm pore size syringe filters prior to DLS measurements

Intensity Particle Size Distributions

Volume Particle Size Distributions

Aluminium Polyoxocations

Aluminium ions can self assemble into aluminium polyoxocations such as the Al13-mer and Al30-mer,

(AlO4-Al12(OH)24(H2O)127+ ) (Al30 O8(OH)56(H2O)2418+

O Deschaume, K.L Shafran and C.C Perry (2006) Langmuir 22, 10078-10088

Aluminium Polyoxocations: Correlation Functions

Aluminium Polyoxocations:

Intensity Particle Size Distributions

Peak 1 Mode = 0.68nm

Aluminium Polyoxocations:

Volume Particle Size Distributions

Aluminium Polyoxocations:

Interactions With BSA

Zeta Potential of BSA = - 8.6mV

The concentration at which

micelles form is called the

critical micelle concentration Size and shape of surfactant micelles influenced by changes in

pH

ionic strength

temperature

Micelle Size Characterization

0.25 0.207

15.4 Nonidet P40

0.012 0.167

10.7 Tween 80

0.3 0.055

7.5 Triton X-100

CMC (mM)

PDI Mean Diameter

(nm) Surfactant

Micelle Size Characterization

0.25 0.207

15.4 Nonidet P40

0.012 0.167

10.7 Tween 80

0.3 0.055

7.5 Triton X-100

CMC (mM)

PDI Mean Diameter

(nm) Surfactant

Micelle Size Characterization

0.25 0.207

15.4 Nonidet P40

0.012 0.167

10.7 Tween 80

0.3 0.055

7.5 Triton X-100

CMC (mM)

PDI Mean Diameter

(nm) Surfactant

Micelle Size Characterization

0.25 0.207

15.4 Nonidet P40

0.012 0.167

10.7 Tween 80

0.3 0.055

7.5 Triton X-100

CMC (mM)

PDI Mean Diameter

(nm) Surfactant

Determining Critical Micelle Concentration

(CMC)

Correlation Functions

0.6mM Triton X-100

0.05mM Triton X-100

can influence the size

and shape of micelles

increase the diffusion

speed of the micelles

can influence the size

and shape of micelles

increase the diffusion

speed of the micelles

A co-surfactant (usually an alcohol)

When the concentrations of these components are favourable, they spontaneously emulsify to form a monodisperse, thermodynamically stable, transparent microemulsion

microemulsion prior to administration to the patient

Trend Measurements

Size/Intensity Versus Time

These measurements are useful for following

processes such as particle aggregation,

sedimentation, creaming or solubilisation

Information on the kinetics of such processes can be obtained from these measurements

seconds duration were

taken over a period of

over an hour

changes in the rate of

size and/or scattering

increases

Protein Melting Points

Protein Melting Points

Protein Melting Points

Size/Intensity Versus Temperature Trend Measurements

This next example

shows the temperature

dependent changes in

the conformation of

polymer particles

Size/Intensity Versus Temperature

This would also result in

an increase in the mean

count rate

However, in the results

obtained in this study,

the mean count rates

decrease upon heating

Temperature limits = 50 and 90 o C Temperature increment = 1 o C Step equilibration time = 5 minutes

Size/Intensity Versus Temperature

Trend Measurements

These results indicate

that the polymer

particles are swelling

with increasing

temperature

As the conformation of

these swollen particles

becomes more open

the mean count rate

Temperature limits = 50 and 90 o C Temperature increment = 1 o C Step equilibration time = 5 minutes

Pigment Dispersions

Pigment particle size is critical in determining

many product properties

Particle size reduction of pigments

High shear mixer running in batch operation

Continuous operation using in-line high shear mixers, mills or pumps

Most sizing techniques involve high sample

dilutions

May change the morphology of the sample

Measurement of the sample to original

concentration or as close as possible is very

desirable

Pigment Dispersions

Neat Conc n

(15% w/v)

Pigment Dispersions

Neat Conc n

(15% w/v)

Measured Conc n

(1.5% w/v)

Pigment Dispersions

Neat Conc n

(15% w/v)

Measured Conc n

(1.5% w/v)

90 o DLS (0.0015% w/v)

Pigment Dispersions (1.5% w/v)

Effect of Calcium on Emulsion Stability:

20% w/v

0 2.

Absolute Molecular Weight

Coupled DLS & SLS measurements of synapse polymer, with an SEC measured MW of 20 kDa.

Antibody Fragment Characterization

Mean diameter = 5.1nm

Mean zeta potential = -7.6mV

MW = 20.7KDa

A2 = - 0.0049 ml mol/g 2

Different Vaccine Formulations

Nanoparticles Used for Drug Delivery: Effect of Cations

Pharmaceutical Emulsions

Product A2 showed creaming after 6 months

storage at room temperature

Cationic Liposomes in Gene Therapy

Cationic liposomes (positively charged) are

complexed with DNA (plasmids)

The liposome:DNA ratio is seen to be essential for optimal transfection

Zeta potential measurements can be used to optimise the ratio required for particular

liposomes with various plasmids