Dynamic Light Scattering
Basic Theory & Practical Application
Introduction
Dynamic light scattering (DLS), also
known as photon correlation
spectroscopy (PCS) and quasi-elastic
light scattering (QELS) provides many
advantages as a particle size analysis
method. DLS is a non-invasive
technique that measures a large
population of particles in a very short
time period, with no manipulation of
the surrounding medium. Modern
DLS instruments, notably the Malvern
Zetasizer Nano system, can measure
particle sizes as small as 0.6 nm and
as large as 6 um, 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 macromolecular
applications necessitating low sample
concentration and volume, such as
the development of stable food, drug,
and surfactant formulations and in the
screening of protein samples for
crystallization trials.
Particles and macromolecules in
solution undergo Brownian motion.
Brownian motion arises from
collisions between the particles and
the solvent molecules. As a
consequence of this particle motion,
light scattered from the particle
ensemble will fluctuate with time. In
DLS, theses fluctuations are
measured across very short time
intervals to produce a correlation
curve, from which the particle
diffusion coefficient (and subsequently
the particle size) is extracted.
In contrast to separation techniques,
where particles are separated and
then counted, in the dynamic light
scattering technique, all of the size
Zetasizer Nano application note
information for the ensemble of
particles is contained within a single
correlation curve. As such, particle
size resolution requires a de-
convolution of the data contained in
the measured correlation curve.
While standard algorithms exist for
transforming the correlation curve to a
particle size distribution, an
understanding of the precision and
accuracy of the distribution
necessitates a solid understanding of
the underlying principles behind the
dynamic light scattering technique
itself. In this application note, we
present a brief overview of the DLS
technique, along with common
algorithms used to deconvolute the
size distribution from the measured
correlation curve.
Light Scattering
Light scattering is a consequence of
the interaction of light with the electric
field of a particle or small molecule.
This interaction induces a dipole in
the particle electric field, that
oscillates with the same frequency as
that of the incident light. Inherent to
the oscillating dipole is the
acceleration of charge, which leads to
the release of energy in the form of
scattered light.
For a collection of solution particles,
illuminated by a monochromatic light
source such as a laser, the scattering
intensity measured by a detector
located at some point in space will be
dependent upon the relative positions
of the particles within the scattering
volume. The scattering volume is
defined by the crossover section of
the light source and the detector
optics. The position dependence of
the scattering intensity arises from
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constructive and destruction
interference of the scattered light
waves. If the particles are static, or
frozen in space, then one would
expect to observe a scattering
intensity that is constant with time, as
described in Figure 1. In practice
however, the particles are diffusing
according to Brownian motion, and
the scattering intensity fluctuates
about an average value equivalent to
the static intensity. As noted in Figure
1, these fluctuations are known as the
dynamic intensity.
Figure 1: Intensity time trace
showing the lack of discontinuity
expected for a random signal, when
viewed across a short time interval.
Across a long time interval, the
dynamic signal appears to be
representative of random fluctuations
about a mean value. When viewed
on a much smaller time scale
however (Figure 1 inset), it’s evident
that the intensity trace is in fact not
random, but rather composed of a
series of continuous data points. This
absence of discontinuity is a
consequence of the physical
confinement of the particles to be in a
position very near to the position
occupied a very short time earlier. In
other words, on short time scales, the
particles have had insufficient time to
move very far from their initial
positions, and as such, the intensity
signals are very similar. The net
result is an intensity trace that is
smooth, rather than discontinuous.
Correlation Statistics
Correlation is a second order
statistical technique for measuring the
degree of non-randomness in an
apparently random data set. When
applied to a time dependent intensity
trace, as measured with a dynamic
light scattering instrument, the
correlation coefficients are calculated
as shown below, where τ is the delay
time.
∞
G (τ ) = ∫ I(t ) I(t + τ )dt
0 Z Average Size
Typically, the correlation coefficients
are normalized, such that G(∞) = 1.
For monochromatic laser light, this
normalization imposes an upper
correlation curve limit of 2 for G(to)
and a lower baseline limit of 1 for
G(∞). In practice, experimental upper
limits for a DLS correlogram is
typically around 1.8 to 1.9.
In dynamic light scattering
instrumentation, the correlation
summations are performed using an
integrated digital correlator, which is a
logic board composed of operational
amplifiers that continually add and
multiply short time scale fluctuations
in the measured scattering intensity,
to generate the correlation curve for
the sample. Examples of correlation
curves measured for two sub-micron
particles are given in Figure 2. For
the smaller and hence faster diffusing
protein, the measured correlation
curve has decayed to baseline within
100 us, while the larger and slower
diffusing silicon dioxide particle
requires nearly 1000 us before
correlation in the signal is lost.
Zetasizer Nano application note
Figure 2: Intensity correlograms for ovalbumin and silicon dioxide, measured
with a Malvern Zetasizer Nano ZS static, dynamic, and electrophoretic light
scattering instrument.
In dynamic light scattering, all of the
information regarding the motion or
diffusion of the particles in the solution
is embodied within the measured
correlation curve. For monodisperse
samples, consisting of a single
particle size group, the correlation
curve can be fit to a single
exponential form as given in the
following expression, where B is the
baseline, A is the amplitude, and D is
the diffusion coefficient. The
scattering vector (q) is defined by the
second expression below, where ñ is
the solvent refractive index, λo is the
vacuum wavelength of the laser, and
θ is the scattering angle.
∫ I(t ) I(t + τ)dt = B + A e
-2q2 Dτ
4πñ ⎛ θ ⎞
q= sin ⎜ ⎟
λo ⎝ 2⎠
The hydrodynamic radius is defined
as the radius of a hard sphere that
diffuses at the same rate as the
particle under examination. The
hydrodynamic radius is calculated
using the particle diffusion coefficient
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and the Stokes-Einstein equation
given below, where k is the
Boltzmann constant, T is the
temperature, and η is the dispersant
viscosity.
kT
RH =
6πηD
A single exponential or Cumulant fit of
the correlation curve is the fitting
procedure recommended by the
International Standards Organization
(ISO). The hydrodynamic size
extracted using this method is an
average value, weighted by the
particle scattering intensity. Because
of the intensity weighting, the
Cumulant size is defined as the Z or
intensity average.
Intensity Size Distribution
While the Cumulant algorithm and the
Z average are useful for describing
general solution characteristics, for
multimodal solutions consisting of
multiple particle size groups, the Z
average can be misleading. For
multimodal solutions, it is more
appropriate to fit the correlation curve
to a multiple exponential form, using
common algorithms such as CONTIN
or Non Negative Lease Squares
(NNLS). Consider for example, the
correlation curve shown in Figure 3.
This correlation curve, measured for a
10 mg/mL lysozyme sample in 100
o
mM NaCl at 69 C, clearly exhibits two
exponential decays, one for the fast
moving monomer at 3.5 nm and one
for the slow moving aggregate at 388
nm. The size distribution shown in
Figure 3 was derived by fitting the
measured correlogram to a multi-
exponential using the CONTIN
algorithm. When the single
exponential Cumulant algorithm is
used, a Z average of 12.4 nm is
indicated. As evident in Figure 3, the
Z average, while beneficial for the
purposes of citing a single average
value, is clearly inadequate for giving Figure 3: Correlation curve and CONTIN distribution for 10 mg/mL lysozyme
a complete description of the in 100 mM NaCl at 69 oC, measured with a Malvern Zetasizer Nano ZS static,
distribution results. dynamic, and electrophoretic light scattering system. The Z average of 12.4
nm is indicated by the solid line in the distribution results.
Volume/Mass Distribution
For many applications, the first 2
The area under each peak in the DLS
assumptions are reasonable. The
measured intensity particle size
third assumption however, will always
distribution is proportional to the
fail, due to the ill-posed nature of the
relative scattering intensity of each
correlogram fitting in the DLS
particle family. Since the scattering
technique. In other words, regardless
intensity is proportional to the square
of how monodisperse the sample is,
of the molecular weight (or R6), the
the DLS measured distribution will
intensity distribution will tend to be
always have a small degree of
skewed towards larger particle sizes.
inherent polydispersity, i.e. you’ll
While this behavior is expected, it can
never be able to achieve a single
lead to some confusion with new
band distribution as one might
users of DLS instrumentation.
achieve using TEM measurements.
A transformation of the intensity to a As such, the volume transform should
volume or mass distribution can be not be used to report particle size, but
accomplished using Mie theory, rather to report mass composition.
wherein the optical properties of the Consider for example Figure 4, which
analyte are used to normalize the shows both the intensity and volume
effects of the R6 dependence of the distributions for an ovalbumin sample
o
scattering intensity. The assumptions in PBS buffer at 79 C. Two
required for the transformation are: populations are evident – one at 6.0
nm and one at 46 nm. By intensity, Figure 4: DLS measured intensity
1) The particles can be modeled as the larger particle size family
spheres. and mass distributions for an
dominates the distribution, even ovalbumin sample in PBS buffer at 79
though it represents only 6% of the o
2) All particles have an equivalent C, along with the resultant intensity
and homogeneous density. total mass of the sample. and mass compositions.
3) There is no error in the intensity
particle size distribution.

Zetasizer Nano application note MRK000-00

High Concentration DLS
The issues that should be considered
when attempting dynamic light
scattering based particle size
measurements on concentrated
samples can be loosely grouped into
five categories.
Multiple Scattering
Multiple scattering describes the
phenomenon wherein light scattered
from diffusing particles is re-scattered
by other particles prior to being
detected. Symptoms of multiple
scattering include:
• a reduction in the correlation curve
intercept
• a decrease in the apparent size
• an increase in the polydispersity
with increasing concentration.
The effects of multiple scattering can
be minimized by performing
measurements near the edge of the
cell (or sample surface) using the
patented NIBS backscatter optics in
the Zetasizer Nano system. The
effects of multiple scattering on the
resultant DLS measured size
distribution can be seen in Figure 5.
As seen in this figure, the dilute and
concentrated distributions are
indistinguishable when the NIBS
technology is used.
Restricted Diffusion
Restricted diffusion describes the
phenomenon wherein the presence
of other particles hinders free
particle diffusion. Symptoms of
restricted diffusion effects include:
• a shifting of the size distribution,
with no change in the modality or
polydispersity, to larger sizes
when the solvent viscosity values
are used for size calculations at
high sample concentrations.
• a concentration dependence of
the Z average which parallels that
of the bulk viscosity of the sample.
Zetasizer Nano application note
Figure 5: A comparison of DSL
measured size distributions of dilute
and concentrated latex samples,
collected with both classical 90o and
NIBS optical configurations.
As a general rule of thumb, restricted
diffusion effects can be minimized by
using the bulk, rather than the solvent,
viscosity for the size distribution
calculations. An example of this
correction can be seen in Figure 6,
which shows a comparison of the
concentration dependence of the DLS
measured size distributions,
calculated using the diluent viscosity
and the bulk sample viscosity.
Aggregation Equilibrium
Aggregation equilibrium describes the
phenomenon wherein an increase in
sample concentration leads to an
increase in the aggregation of primary
particles. Symptoms of aggregation
Figure 6: A comparison of the concentration dependence of DLS measured size
distributions for an emulsion, calculated using both the diluent viscosity and the
bulk viscosity of the sample.
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equilibrium include:
• an increase in both the modality
and the polydispersity of the sample
as the concentration is increased.
An example of DLS measured size
distributions for a sample exhibiting
aggregation equilibrium effects is
given in Figure 7, which shows a
distinct increase in both the modality
and the polydispersity as the sample
concentration is increased.
Electrostatic Interactions
As high sample concentrations,
interacting electric fields can lead to
“soft particle” interactions that can
influence the translational diffusion.
While it’s difficult to predict the effect
on the size results, the influence of
electrostatic interactions can be
minimized by reducing the Debye
length, and hence the size of the
electric field, via the use of shielding
electrolytes, e.g. 50-100 mM NaCl.
Hard Sphere Interactions
The phrase hard sphere interactions
refers to sticky type, e.g. hydrophobic,
interactions that can lead to mutual
diffusion or collections of particles
diffusing together at high sample
concentration. These types of
interactions will typically manifest
themselves as large polydisperse size
peaks, that decrease in amplitude and

Figure 7: Concentration effect on
the DLS measured size distribution
for a TiO2 sample exhibiting
aggregation equilibrium effects.
apparent size as the sample is
diluted, eventually vanishing in the
limit of infinite dilution. DLS size
measurements of samples exhibiting
hard sphere interactions will also tend
to be much less reproducible, due to
the random nature of the “group” size.
High Concentration Summary
As a consequence of multiple
scattering and particle interaction
effects at high sample concentration,
classical DLS measurements have
been generally restricted to the dilute
solution regime. With the introduction
of the Zetasizer Nano system
however, high concentration DLS
measurements can now be collected.
But, while the Nano system will
accommodate high concentration
measurements by minimizing optical
configuration dependent multiple
scattering effects, it cannot eliminate
sample dependent particle interaction
effects. As discussed above
however, the types of particle
interactions present in the sample can
be evaluated, if a dilution experiment
is performed. Since the stability of a
formulation is inherently dependent
upon the types of particle interactions
present at high concentration, the
importance of examining the
concentration dependence of the DLS
size distribution cannot be over
stated.
Zetasizer Nano application note
Figure 8: Concentration & ionic
strength dependence of the DLS
measured Z average diameter for
lysozyme at pH 7.
MW Estimates
Similar to the size vs. elution time
calibration curve approach used to
estimate particle size in size exclusion
chromatography and GPC, size vs.
molecular weight calibration curves
can be coupled with dynamic light
scattering measurements to estimate
sample molecular weight. As with
GPC techniques, the correlation
between the specific volume (1/ρ) of a
particle and its tertiary conformation
complicates the development of a
universal calibration curve for DLS.
However, empirical mass vs. size
1000
„
S
100 „ z
„„ S
„‹ z
„
S z
„ ‹z
10
z
‹ S Branched Polymers
‹
1
1 10
Hydrodynamic Radius (nm)
Figure 9: Molecular family dependent empirical mass vs. size calibration
curves for use in DLS applications for estimating molecular weight from
measured hydrodynamic size.
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calibration curves, each particular to a
specific ‘family’ of macromolecules,
are available for use in DLS
applications. The calibration curves
used for estimating molecular weight
from DLS data in the DTS software for
the Malvern Zetasizer Nano system
are shown in Figure 9. For reference
purposes, it is noted that all of the
proteins in the protein family are
globular; the linear polymers are
linear polysaccharides; the branched
polymers are densely branched
polysaccharides; and the spherical
polymers are starburst type
dendrimers, described as spherical,
with a density that increases with
radial distance from the core.
Additional Reading
Brown, R. G. W. “Miniature Laser
Light Scattering Instrumentation for
Particle Size Analysis”, Applied Optics
1990, 29(28), 1.
D’Arcy, Allan “Crystallizing proteins –
a rational approach”, Acta Cryst.
1994, D50, 467-471.
Hutchinson, Fiona J.; Francis, Sheila
E.; Lyle, Ian G.; Jones, Malcolm N.
“The characterization of liposomes
z
„ S
S
S
z
„ Globular Proteins
z Linear Polymers
‹ Spherical Polymers
100
with covalently attached proteins”, consequence of these features, the
Biochim. Biophys. Acta 1989, 978(1), Zetasizer Nano specifications for
17-24. sample size and concentration
exceed those for any other
Moradian-Oldak J.; Leung W.; commercially available dynamic light
Fincham A. G. “Temperature and pH- scattering instrument, with a size
dependent supramolecular self- range of 0.6 nm to 6 um, and a
assembly of amelogenin molecules: a concentration range of 0.1 ppm to
dynamic light-scattering analysis”, 40% w/v.
Journal Of Structural Biology 1998,
122(3) 320-327. Complementary to the patented and
award winning hardware design, is
Phillies, G. D. “Quasielastic Light the Malvern DTS software, providing
Scattering”, Analytical Chemistry instrument control and data analysis
1990, 62(20), 1049A-1057A. for the Zetasizer Nano System. The
Pecora, R. “Dynamic Light DTS software utilizes self analyzing Figure 10: The Zetasizer Nano &
Scattering: Applications of Photon algorithms to insure that the optical MPT2 Automatic Titrator combined
Correlation Spectroscopy”, Plenum setup is optimized for each set of static, dynamic, and electrophoretic
Press, 1985. experimental conditions, and includes light scattering system.
a unique "one click" measure,
Piekenbrock, T.; Sackmann, E. analyze, and report feature designed
“Quasielastic light scattering study of to minimize the new user learning
thermal excitations of F-actin curve.
solutions and of growth kinetics of
actin filaments”, Biopolymers 1992, The MPT-2 Titrator is a Zetasizer
32(11), 1471-1489. accessory designed to facilitate
measurement automation. When
coupled with the Zetasizer Nano
Zetasizer Nano System system, the MPT-2 Titrator brings full
The Zetasizer Nano system from automation to the researcher’s
Malvern Instruments is the first fingertips. The system incorporates
commercial instrument to include the micro-syringes for the delivery of up to
hardware and software for combined three titrants of user defined
static, dynamic, and electrophoretic composition and concentration,
light scattering measurements. The facilitating the automation of pH,
wide range of sample properties dilution, additive, and conductivity
available for measurement with the type titrations, requiring sample
Nano system include, particle size, volumes as small as 3 mL.
molecular weight, and zeta potential.
The Zetasizer Nano system was
specifically designed to meet the low
concentration and sample volume
requirements typically
associated with
pharmaceutical and Malvern Instruments Ltd
biomolecular applications, Enigma Business Park • Grovewood Road • Malvern • Worcestershire • UK • WR14 1XZ
along with the high Tel: +44 (0)1684 892456 • Fax: +44 (0)1684 892789
concentration requirements for
colloidal applications.
Malvern Instruments Worldwide
Satisfying this unique mix of
requirements was Sales and service centers in over 50 countries for details visit www.malvern.co.uk/contact
accomplished via the
integration of a backscatter
optical system and the design
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more information at www.malvern.co.uk

Zetasizer Nano application note MRK000-00