ISO 13099-2:2012
(Main)Colloidal systems — Methods for zeta-potential determination — Part 2: Optical methods
Colloidal systems — Methods for zeta-potential determination — Part 2: Optical methods
This part of ISO 13099 specifies two methods of measurement of electrophoretic mobility of particles suspended in a liquid: video microscopy and electrophoretic light-scattering. Estimation of surface charge and determination of zeta-potential can be achieved from measured electrophoretic mobility using proper theoretical models, which are described in detail in ISO 13099-1.
Systèmes colloïdaux — Méthodes de détermination du potentiel zêta — Partie 2: Méthodes optiques
General Information
Standards Content (Sample)
INTERNATIONAL ISO
STANDARD 13099-2
First edition
2012-06-15
Colloidal systems — Methods for zeta-
potential determination —
Part 2:
Optical methods
Systèmes colloïdaux — Méthodes de détermination du potentiel zêta —
Partie 2: Méthodes optiques
Reference number
ISO 13099-2:2012(E)
©
ISO 2012
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ISO 13099-2:2012(E)
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ISO 13099-2:2012(E)
Contents Page
Foreword .iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms, definitions and symbols . 1
3.1 Terms and definitions . 1
3.2 Symbols . 2
4 Principles . 3
5 Microscopic methods . 4
6 Electrophoretic light-scattering (ELS) method. 5
6.1 General . 5
6.2 Cell design . 5
6.3 Reference beam optical arrangement . 6
6.4 Cross-beam optical arrangement . 6
6.5 Signal processing . 7
6.6 Determination of electrophoretic mobility . 9
7 Calculation of zeta-potential . 9
8 Operational procedures .10
8.1 Requirements .10
8.2 Verification .12
8.3 Sources of measurement error .13
8.4 Test report .15
Annex A (informative) Electroosmosis within capillary cells .16
Bibliography .19
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ISO 13099-2:2012(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
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Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 13099 was prepared by Technical Committee ISO/TC 24, Particle characterization including sieving,
Subcommittee SC 4, Particle characterization.
ISO 13099 consists of the following parts, under the general title Colloidal systems — Methods for zeta-
potential determination:
— Part 1: Electroacoustic and electrokinetic phenomena
— Part 2: Optical methods
The following part is under preparation
— Part 3: Acoustic methods
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ISO 13099-2:2012(E)
Introduction
Zeta-potential is a parameter that can be used to predict the long term stability of suspensions and emulsions
and to study surface morphology and adsorption on particles and other surfaces in contact with a liquid. Zeta-
potential is not a directly measurable parameter. It can be determined using appropriate theoretical models
from experimentally determined parameters, such as electrophoretic mobility. Optical methods, especially
electrophoretic light scattering, have been widely used to determine electrophoretic mobility of particles or
macromolecules in suspension or in solution. The purpose of this part of ISO 13099 is to provide methods for
measuring electrophoretic mobility using optical means and for calculating zeta-potential.
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INTERNATIONAL STANDARD ISO 13099-2:2012(E)
Colloidal systems — Methods for zeta-potential
determination —
Part 2:
Optical methods
IMPORTANT This part of ISO 13099 shall be read in conjunction with ISO 13099-1, which gives a
comprehensive overview of the theory.
1 Scope
This part of ISO 13099 specifies two methods of measurement of electrophoretic mobility of particles
suspended in a liquid: video microscopy and electrophoretic light-scattering. Estimation of surface charge
and determination of zeta-potential can be achieved from measured electrophoretic mobility using proper
theoretical models, which are described in detail in ISO 13099-1.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced document
(including any amendments) applies.
ISO 13099-1, Colloidal systems — Methods for zeta-potential determination — Part 1: Electroacoustic and
electrokinetic phenomena
ISO Guide 30: Terms and definitions used in connection with reference materials
3 Terms, definitions and symbols
3.1 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1.1
Brownian motion
random movement of particles suspended in a liquid cause by thermal movement of medium molecules
3.1.2
Doppler shift
change in frequency and wavelength of a wave for an observer moving relative to the source of the wave
3.1.3
electric surface potential
difference in electric potential between the surface and the bulk liquid
NOTE Electric surface potential is expressed in volts.
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ISO 13099-2:2012(E)
3.1.4
electrokinetic potential
zeta-potential
ζ-potential
ζ
difference in electric potential between that at the slipping plane and that of the bulk liquid
NOTE Electrokinetic potential is expressed in volts.
3.1.5
electroosmosis
motion of liquid through or past a charged surface, e.g. an immobilized set of particles, a porous plug, a
capillary or a membrane, in response to an applied electric field, which is the result of the force exerted by the
applied field on the countercharge ions in the liquid
3.1.6
electroosmotic velocity
υ
eo
uniform velocity of the liquid far from the charged interface
NOTE Electroosmotic velocity is expressed in metres per second.
3.1.7
electrophoretic mobility
µ
electrophoretic velocity per electric field strength
NOTE 1 Electrophoretic mobility is positive if the particles move toward lower potential (negative electrode) and
negative in the opposite case.
NOTE 2 Electrophoretic mobility is expressed in metres squared per volt second.
3.1.8
electrophoretic velocity
υ
e
particle velocity during electrophoresis
NOTE Electrophoretic velocity is expressed in metres per second.
3.1.9
slipping plane
shear plane
abstract plane in the vicinity of the liquid/solid interface where liquid starts to slide relative to the surface under
influence of a shear stress
3.2 Symbols
a particle radius
D diffusion coefficient
E electric field strength
k Boltzmann constant
B
I light intensity
N Avogadro’s number
A
n medium refractive index
R capillary radius
cap
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ISO 13099-2:2012(E)
S(ω) frequency power spectrum of scattering
Γ characteristic Lorentzian half peak width
ε medium permittivity
ζ electrokinetic potential (zeta-potential)
η medium viscosity
0
θ angle between incident light and scattered light
θ’ angle between two cross-beams
κ reciprocal Debye length
λ wavelength
µ electrophoretic mobility
µ electroosmotic mobility of liquid
eo
ν frequency
ξ angle between scattered light and electric field direction
τ delay time in autocorrelation function
ϕ volume fraction
ω rotational frequency (= 2πν)
4 Principles
A suspension of particles having a given electrokinetic charge is placed in a cell which has a pair of electrodes
placed some distance apart (Figure 1). This cell can be in the form of either a cylindrical or rectangular capillary
with electrodes at either end, or a pair of electrodes at a known fixed distance apart that are dipped into a
cuvette or other vessel. A potential is applied between the electrodes. Due to the process of electrophoresis,
particles carrying a net negative charge are drawn towards the electrode of opposite sign and vice versa. In
addition, if the capillary walls are charged, then an effect called electroosmosis causes the liquid to stream
along the capillary walls. The direction and velocity of this flow depends on the sign and magnitude of the wall
charge. The resulting velocity of the particle in the frame of references associated with the cell is superposition
of the electrophoretic velocity and the velocity of electroosmotic flow. Here it should be noted that the time
taken for the particle to reach the terminal electrophoretic velocity after the application of the electric field is
much shorter than the period of time needed to fully establish the electroosmosis flow throughout the whole
cell. This difference is exploited in some implementations. The velocity of the particles measured at a specific
position can be determined using either video microscope or electrophoretic light scattering through a laser
Doppler arrangement. Both the velocity and the direction of the moving particles in the frame of references
associated with the cell are determined. Provided that the distance between the electrodes is known together
with the applied electric potential, then the electrophoretic mobility can be established, from which a zeta-
potential can be calculated using established theories. Alternatively, calibration with particles having a known
zeta-potential can be used to eliminate the need to determine the unknown cell constant of a particular cell.
There are two distinctively different approaches to monitor particle motion in the electric field. Historically, the
first deals with particle images observed through a microscope. It is referred to as the “microscopic method”,
or alternatively as “microelectrophoresis”. The second relies on measuring light scattered by particles and
extracting information on electrophoretic mobility from the Doppler frequency shift of the scattered light.
This method is called the “electrophoretic light-scattering method”. For optical techniques, a cell constant for
many types of cells has to be determined, through either calculation or measurement of a solution of known
conductivity.
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ISO 13099-2:2012(E)
a
d
Key
d distance
a
Measurement zone.
Figure 1 — Schematic diagram of electrophoresis measurement
5 Microscopic methods
The main principles of these methods can be traced back over two centuries (Reference [1]) following the
development of microelectrophoresis. A light source illuminates particles migrating under the influence of a
d.c. or a.c. electric field. The illuminated particles can be observed due to scattering. This illumination can
be arranged either as a bright field or as a dark field or both (Reference [2]). The contrast afforded by the
bright field illumination is inadequate to illuminate particles with sizes smaller than about 0,2 µm. Dark field
illumination is suitable for capturing images of moving nano-particles with sizes down to nanometre scale.
There are several approaches to the treatment of microscopic images of the moving particles. Depending on
the degree of operator involvement, it can be classified as manual, semi-automatic and automatic. Manual
methods track the movement of one or several individual particles by eye and a stopwatch and therefore are
typically time consuming, tedious to employ and inaccurate.
In the semi-automatic methods, particles are tracked through a microscope manually while the apparatus
either scans the illuminating light or moves a prism reflecting the illuminated image of particles. When the light-
scanning velocity or prism-moving velocity is semi-automatically adjusted so that the particle image as viewed
in the microscope is static, such a velocity is the electrophoretic velocity of particles (References [3][4]). These
methods are only applicable to samples having a homogeneous electrophoretic mobility.
There are designs combining the manual microscopic observation with automatic electrophoretic light-
scattering signal analysis to measure samples of polydisperse electrophoretic mobility (References [5][6]).
The appearance of modern charge-coupled devices (CCD) and computers has made it possible to capture
images, transfer the images sequentially to a computer, and then using sophisticated image analysis to
reconstruct trajectories of particles moving under the influence of an electric field from the time-stamped
video frames. Only particles confined to video visibility can be measured. In order to record accurate moving
distances, from the time duration between frames and the distance each particle moved, the velocity of each
particle is calculated and combined with the applied field strength, and its electrophoretic mobility is obtained.
Dark field illumination extends this method to nano-particles. This method allows application of electric
field for very short periods of time, which resolves the problems of thermal convection and electrochemical
contamination. Concentration of particles shall be very low in order to track individual particles.
A 90° laser scattering device is a typical optical arrangement of modern instruments. The laser serves as the
illumination of the microscope focal plane. Both laser beam and microscope axis are perpendicular to the
electric field. In Figure 2, the field direction is perpendicular to the plane of the drawing. Laser illumination and
microscope require alignment with the stationary layer to avoid electroosmosis, which is explained in Annex
A. It is necessary precisely to locate this position in order to accurately measure the electrophoretic motion of
particles (Reference [7]).
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ISO 13099-2:2012(E)
1
3
2
4
Key
1 laser 3 microscopic objective
2 cell channel cross-section 4 video camera
Figure 2 — A typical electrophoresis video microscope
6 Electrophoretic light-scattering (ELS) method
6.1 General
Electrophoretic light scattering (ELS) is an indirect ensemble method for measuring electrophoretic mobility
via the Doppler shifts in scattered light. In an ELS experiment, coherent incident light illuminates dispersed
particles in a liquid that are subjected to an applied electric field. Charged particles move towards either the
anode or the cathode, depending on the sign of their net charge. Because of the motion, the frequency of
scattered light from particles is shifted due to the Doppler effect. From the frequency shift distribution, the
particle electrophoretic mobility distribution can be determined. ELS provides rapid, accurate, automatic, and
highly reproducible electrophoretograms of complex particulate samples suspended in either aqueous or non-
aqueous media without the need to use standard particles for calibration (Reference [8]).
6.2 Cell design
Many designs of measurement cell have been employed. All cells have at least three functions: holding the
sample containing the particles to be measured; supplying an electric field to the sample; and providing an
entrance and an exit for the incident light and scattered light, respectively. Some cells are designed with liquid
flow capability so that automatic titration can be performed with an additional device. In some implementations,
special cell designs, e.g. utilizing a transparent electrode and multiple refraction for both incident and scattered
light (Reference [9]), have been implemented to facilitate measurements of electrophoretic mobility at moderate
concentrations. The electric field at the place of measurement shall be stable, homogenous, and parallel. To
achieve that, either the two electrodes have to be placed very close to each other, in the case of cuvette cells,
or the field path has to be confined, in the case of capillary cells. The voltage applied to the electrodes induces
a current in the liquid if ions are present. This current can well be sufficiently high to cause Joule–Thompson
heating of the liquid and lead to electrolysis at the electrodes. Therefore, choosing an appropriate type of cell
and electrode material, sufficiently prompt temperature control, and properly applied field duration and field
strength are all important factors to ensure correct and reproducible results.
To reduce polarization on the electrodes and maintain homogenous distribution of particles in the sample,
the applied field direction is regularly reversed with an intervening off-time to minimize heating effects. In
capillary cells, because of electroosmosis of liquid caused by charges on the walls, particles do not move in
a static liquid. The liquid moves in a parabolic form across the closed capillary. Measurements are therefore
taken at the so-called stationary layer where there is no liquid movement or multiple measurements are taken
across the capillary to separate the liquid movement from the electrophoretic motion of the particles. Some
implementations offer disposable cells.
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ISO 13099-2:2012(E)
6.3 Reference beam optical arrangement
A typical example for a small angle light-scattering arrangement is shown in Figure 3.
2
1
4
3
10
5
6
7
8
9
Key
1 optical modulator 6 beam splitter
2 attenuator 7 reference beam
3 laser 8 scattered or reference light
4 sample cell with electrodes 9 processor
5 beam stop 10 photoelectric detector
Figure 3 — One configuration of the reference beam optics
A small angle scattering optical arrangement incorporating heterodyne detection is often employed. The
scattering angle, typically between 15° and 30°, exploits the advantage that the spectral broadening due
to Brownian motion is reduced. With non-spherical particles, rotational diffusion may increase the spectral
broadening. Means are provided in the measurement cell for the introduction of a pre-dispersed sample. The
cell may be temperature controlled; if it is not, the temperature shall be accurately known since viscosity,
permittivity, and refractive index of the liquid are all temperature dependent. A voltage is applied between the
electrodes of the cell, whose spacing is defined, to set up a potential gradient. In some implementations, extra
monitoring electrodes are employed at a defined spacing to provide a direct measure of the potential gradient.
Light from a coherent laser source of known wavelength is split into two beams, one called the incident beam
and the other the reference beam. The incident beam enters the cell directly or is refracted through a cell
window to illuminate particles in the sample. The reference beam, which may or may not go through the cell,
merges with the scattered light through conventional or fibre optics at the surface of the photoelectric detector,
which is either a photomultiplier tube or an avalanche photodiode. One or both of the laser beams pass through
a form of optical modulator to shift its frequency by a few hundred hertz from the original laser frequency so that
the two beams acquire a desired frequency difference. This moves the origin of the Doppler shift caused by
the velocity of the particles away from zero and enables the particle moving direction to be recognized and low
frequency environment interference to be minimized. The detector aperture may be variable so as to control
coherent detection and the scattering volume. The detected signal is passed to a signal processing unit that
can be a digital correlator, a spectrum analyser or an amplitude-weighted phase structure function system. The
voltage applied to the measuring cell can be reversed or pulsed and reversed as determined by the processor
which also synchronizes the data collection. The final control is often by a desktop computer which calculates
the zeta-potential.
6.4 Cross-beam optical arrangement
Another now less common optical arrangement is the cross-beam method. See Figure 4. In the cross-beam
method, the main beam is split into two beams of equal intensity, the frequency of one or both beams being
modulated. The two beams are made to cross symmetrically to enter one side of the cell. A detector is located
at the other side of the cell in between the two beams. The scattering from each particle is a product of the
illumination of both beams yet at different scattering angles. The Doppler shift resulting from scattered light is
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ISO 13099-2:2012(E)
independent of either scattering angle and depends only on the cross-beam angle. Another simplified way to
view the arrangement is to understand that since the two beams are coherent, they form an interference pattern
in the cell. The spacing between these fringes depends upon the wavelength and the angle between the two
beams. The detection of electrophoretic motion is through particle movement in the strip-like fringe pattern.
An optical modulator is used to impose a movement of the fringes across the particle at some known frequency
and in a defined direction. A particle in motion through the moving fringes resulting in a measured frequency
greater than the modulator frequency indicates that its movement is against the fringe moving direction. A
measured frequency lower than the imposed fringe frequency indicates that the particle is moving in the same
direction as the fringe motion. The fringe movement is always made greater than the anticipated maximum
particle velocity. By this means, both the velocity and the direction (charge sign) are determined.
4
1
a
3
2
Key
1 beam 1 3 photoelectric detector
2 beam 2 4 optical modulator
a
Scattered light.
Figure 4 — Configuration of cross-beam optics
6.5 Signal processing
6.5.1 Spectrum analysis
Consider a particle sample that is polydisperse both with respect to size and electrophoretic mobility. The
particles are undergoing Brownian motion together with electrophoretic motion under the influence of a d.c.
field of defined strength.
The spectrum, also called an electrophoretogram, for the reference beam optical arrangement can be written
as follows (Reference [8]):
ΔΔν
d
s,max
max
I Γ
s,ij i
2
SIωδ= 22π ω + I (1)
() ()
LL ∑ ∑
2
2
id= j=Δν
ωω +2πΔν +Γ
min s,min ()
Ms,ij i
where
I is the reference beam intensity;
L
I is the scattered intensity from particles of ith size and jth mobility;
s,ij
Γ is the characteristic Lorentzian half peak width at half height from particles of the ith size, which for
spherical particles is related to particle diameter;
ω is the rotational frequency;
ω is the modulator frequency;
M
Δν is the frequency shift caused by the electrophoretic motion of particles with ith size and jth mobility.
s,ij
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The symbol in the denominator denotes that there are two peaks in the spectrum. One is located in the
negative frequency region that cannot be observed and the other in the visible positive frequency region. If by
choosing a large modulator frequency, ω , so that the sum (ω + 2πΔν ) is always positive, a negative sign
M M s,ij
can be used instead.
According to Formula (1), the electrophoretic spectrum of any particulate sample has an intrinsic broadening
caused by Brownian motion of the particles in addition to any spectrum of electrophoretic mobility velocities.
The broadening due to Brownian motion becomes more pronounced as the particle size reduces or when the
scattering angle increases. One strategy of measuring the degree of broadening of the frequency spectrum as
a result of the Brownian motion is to conduct a measurement with the applied field switched off. By subtraction
of the Brownian motion only spectrum from the total spectrum a distribution of electrophoretic mobility can in
certain circumstances be established (Reference [10]).
6.5.2 Autocorrelation function
The autocorrelation function is a Fourier transform of the frequency power spectrum. Formula (2) shows the
intensity–intensity autocorrelation function in the reference beam optics as a function of delay time, τ:
Δν
d
s,max
max
()2 2
GIττ=+22II expc−ΓΔos ωπ+ ντ (2)
() ()
()
LL ∑ ∑ s,ij iiMs, j
id= j=Δν
min s,minn
Figure 5 shows a typical autocorrelation function together with an electrophoretic velocity spectrum. In the
autocorrelation function, the cosine wave is due to oriented electrophoretic motion and the decay is due to
random Brownian motion. In the spectrum, the peak location is related to optical modulator and electrophoretic
motion of the particles, and the peak shape is caused by Brownian motion of the particles, the mobility velocity
spectrum and any finite laser beam width restrictions.
1
τ
2
ω
Key
2
1 autocorrelation trace G (τ) autocorrelation function τ delay time
ω
2 spectrum S(ω) frequency power rotational frequency
Figure 5 — A typical autocorrelation function and spectrum from electrophoretic light scattering
6.5.3 Phase analysis light scattering (PALS)
The electrophoretic mobility of some particles in a non-polar solvent is very small, resulting in very small
differences between the modulator frequency and the Doppler frequency shifts from electrophoretic motion.
Such frequency differences can be less than 1 Hz. For particles suspended in a solution of high ion concentration,
only a very small field can be applied between the electrodes before the Joule–Thompson heating affects
measurement. Therefore, again, very small Doppler frequency shifts require detection.
In these instances, because of
...
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