Lecture 4.

Electrokinetics and Electrohydrodynamics

Electrophoresis
Electroosmosis
Capillary Electrophoresis (CE)
DEP preconcentrator

Dielectrophoresis (DEP)
AC Electroosmosis
 Electrophoresis
• An ion with charge q in an electric field E moves toward opposite
electrode due to Coulombic force. A steady-state speed is reached
when the accelerating force equals the frictional force generated by
the medium.
VDC

- -
Medium
Anode + + Cathode

FE = qE FFriction = f ⋅ u E = 6πη r ⋅ u E

q uE q
uE = ⋅E µE = =
6πη r E 6πη r
Electrophoretic mobility is a function of viscosity and charge to radius ratio.
 Applications of Electrophoresis
• Many important biological molecules such as amino acids, peptides,
proteins, nucleotides, and nucleic acids, possess ionisable groups
(COOH, NH2, phosphates) and, therefore, at any given pH, exist in
solution as electically charged species either as cations (+) or
anions (-). DNA is negatively charged because the phosphates that
form the sugar-phosphate backbone of a DNA molecule have a
negative charge.
• Depending on the nature of the net charge, the charged particles
will migrate either to the cathode or to the anode at different rates.
Electrophoresis has been applied to a variety of analytical
separation problems.
– Amino acids
– Peptides, proteins (enzymes, hormones, antibodies)
– Nucleic acids (DNA, RNA), nucleotides
– Drugs, vitamins, carbohydrates
– Inorganic cations and anions
 Gel Electrophoresis
• Gel electrophoresis is a separation technique widely used for the
separation of nucleic acids and proteins. The separation depends
upon electrophoresis and filtering effect by gel (molecular sieve).
Under electric field, charged macromolecules are forced to move
through the gel with pores.
• Their rates of migration depend on the field strength, size and shape
of the molecules, hydrophobicity of the samples, and on the ionic
strength, and pH, temperature of the buffer in which the molecules
are moving. After staining, the separated molecules can be seen in a
series of bands spread from one end of the gel to the other.
 Electric Double Layer
• In general, a surface carries a net charge which comes about either
through dissociation of the chemical groups on the surface or by
adsorption of ions or molecules from the solution onto the surface.

• Glass surface is covered with silanol groups (Si-OH) that carry a

negative charge (Si-O-) at pH>2. When the surface is immersed in an
electrolyte, positive ions will be attracted toward the surface forming
diffuse layer.
 Electric Double Layer
• In a thin region between the surface and
the diffuse layer, there is a layer of bound
positive ions, referred to as Stern layer.
In this region, it is assumed that the potential
falls linearly. The potential decays exponentially
in the diffuse layer.
• zeta potential, ζ represents the value of
electrostatic potential at the interface between
stern and diffuse layers. The thickness δ of the
diffuse layer is defined as the distance from the
stern layer to a point at which the electrostatic
potential has dropped to 37% of zeta potential.
• The zeta potential of an aqueous solution in
contact with glass can have a magnitude as high
as 100 mV. pH and amount of counter ions affect
the zeta potential.
 Electro-Osmotic Flow (EOF)
• In a tangential electric field, excess cations in the diffuse layer are
attracted to the cathode, and this imparts a pumping action onto the
whole fluid column which moves toward the cathode.
• pH, dielectric constant, and temperature of the electrolyte are important
parameters for the electroosmotic mobility.

εζ
uEOF = µ EOF E µ EOF =
4πη
 EOF vs. Pressure-Driven Flow

• Uniform flow over more than 99.9% of the cross section of a

capillary column. The speed of the flow falls off immediately
adjacent to the capillary wall.

Electroosmosis driven flow Pressure-driven flow

uEOF = µ EOF E u( y) =
( Po − Pi )
2 µL
⋅ ( y2 − h2 )

εζ h 2 ( Po − Pi ) 3
µ EOF = u=
3µ
⋅
L
= ⋅ u max
2
4πη
2h 3 dP 2h 3 ( Po − Pi )
Q= (− ) = ⋅
Q = u EOF ⋅ A 3µ dx 3µ L
 Capillary Electrophoresis (CE)

• Electrophoresis performed in glass capillaries

• Electroosmotic mobility > electrophoretic mobility
Working Principle of CE

Ld
ua =
t
ua Ld Lt
µa = = ⋅
E t V

Ld : distance from injection to detector

Lt : Capillary total length
t : migration time from injection to detector

q εζ
µa = µ E + µ EOF = +
6πη r 4πη

ζ can be obtained from μa

 Some Issues about CE

• Unlike chromatography, analytes pass through the detector at

different rates. This results in peak areas that are somewhat
dependent on retention time.

• Band broadening occurs due to longitudinal diffusion, Joule

heating, injection length, sample adsorption, etc.

• Electrokinetic injection leads to sampling bias, because a

disproportionately large quantity of the species with higher
electrophoretic mobility migrates into the tube and can cause
problems for quantitative analysis.

• Electroosmotic flow rates tend to change over time because the

zeta potential changes over time, thus leading to changes in
mobility.
 Microfabricated CE

• In electrophoresis, the separation efficiency (number of theoretical

plates) and analysis speed depend on capillary length and diameter
(N ~ L/d, t ~ Lxd). This means, the only way of increasing the speed
of an analysis and keeping the efficiency constant is to reduce the
diameter and the length of a capillary by the same factor.

• High speed and efficiency, however, usually

require small injection and detection volumes.
These features can easily be arranged in
microfabricated CE systems.

• Using planar micromachined channels instead of

glass capillaries offers the potential for mass
production, the choice of many materials
(e.g., plastics), and integration of many processing
step on one chip. DNA/Protein Chip
(Caliper Technologies)
 Microfabricated CE
Low-cost Glass or PMMA MicroCE CE + PCR + Optical sensor

Parylene MicroCE
UV gel
 Microfabricated CE
(A) (B) (C)

Small channel Æ High E field

Æ High speed separation in Geometrically defined Smallest possible sample
seconds or even milliseconds injected volume plug + longer channel
ÆHigh separation efficiency
 AC Electrokinetics: Dielectrophoresis (DEP)

• A dielectric particle placed in an electric field becomes electrically

polarized as a result of partial charge separation, which leads to
an induced dipole moment. The dipole moment is a consequence
of the generation of equal and opposite charges at the boundary of
the particle.
• The induced surface charge is only about 0.1% of the net surface
charge normally carried by biological cells and microorganisms
and is generated within about a microsecond.
 Dielectrophoresis (DEP)
• In a nonuniform electric field, the particle experience a net dielectro-
phoretic force. The magnitude of the induced dipole depends on the
polarizability of the particle with respect to that of the medium.
• If a suspended particle has polarizability higher than the medium, the
DEP force will push the particle toward regions of higher electric field
(positive DEP). If the medium has a higher polarizability than the
suspended particle, the particle is driven toward regions of low field
strength (negative DEP).

What if the bias voltage is reversed?

 Dielectrophoresis (DEP)

Electrophoresis Dielectrophoresis
Motion of a particle is determined by Motion is determined by the
a net intrinsic electrical charge arried magnitude and polarity of charges
by that particle. induced in the particle by an applied
field.
DC field, usually homogeneous AC field of a wide range of ω.
Must be inhomogeneous
 Effective Dipole Moment
• For a spherical particle of radius r and complex permittivity εp*,
suspended in a fluid (εm*), the effective dipole moment is derived as

r ⎛ ε *
− ε *
⎞ 3r σ ε p* − ε m *
p = 4πε m ⎜ *
p m ⎟r E ε =ε − j
*
K (ω ) =
⎜ ε + 2ε ⎟ * ω ε p* + 2ε m *
⎝ p m ⎠
(Simplified)
• The effective dipole moment of the particle is frequency dependent.
This dependence is described by the Clausius-Mossotti factor, which
indicates the relative polarizability of the particle with respect to its
suspending medium.
• The electrokinetic force on a particle subjected under an E field is
r r r r r r
F = FEP + FDEP = qE + ( p ⋅ ∇) E
• The time-averaged dielectrophoretic force is
r 1 r r* ε *
− ε *
r 2
< F DEP >= Re[( p ⋅ ∇ ) E ] = πε m r Re[ * ]∇ E
3 p m

2 ε p + 2ε m *

r* r
Assume there is no spatially varying phase ( E = −(∇φ R + i∇φ I ) = −∇φ R = E )
 Clausius-Mossotti Factor
• The real part of CM factor defines the frequency dependence and
direction of the force.
ε p* − ε m * σ r 1 r ε p * − ε m* r2
K (ω ) = ε =ε − j
*
< FDEP >= Re[( p ⋅ ∇) E ] = πε m r Re[ *
* 3
]∇ E
ε p* + 2ε m * ω ε p + 2ε m *
2

CM factor as a function of frequency

Cm εm
Gm σm
σm σcyto
εm εcyto
σp
εp

(A and B) εp = 2.4 εm = 81 Theoretical K(ω) values for bioparticles

σp = 2e-4 S/m, σm = 0 to 0.05 S/m (Huang Y. et al, Anal Chem 2001)
 Dielectrophoretic Force

r ε *
− ε *
r2
< FDEP >= πε m r Re[ * ]∇ E
3 p m

ε p + 2ε m *

• The DEP force is zero if the electric field is uniform.

• The DEP force scales with the square of the voltage (or electric field).
Reversing the bias does not reverse the force. Spatial dependence of
the DEP force arise from electric field component.
• The DEP force scales inversely with the cube of the electrode gap.
Decreasing the characteristic dimensions of the electrode by one
order of magnitude can lead to a three orders of magnitude increase
in the DEP force.
• The DEP force is proportional to the particle volume. Strong electric
fields (104-105 V/m) are required to manipulate micron-scale particles.
• Microfabricated electrodes can provide the required high E field only
with several volts. Joule heating effect can also be minimized with
microfluidic channels with high surface/volume ratio.
 Dielectrophoretic Mobility
• If a particle moves under the influence of a DEP force, the equation of
motion is
dv
m = FDEP − Fη (Other forces ignored)
dt
Steady-state

FDEP = Fη = 6πηrv
r ε *
− ε *
r2
< FDEP >= πε m r Re[ * ]∇ E
3 p m

ε p + 2ε m *

DEP mobility
ε *
− ε *

ε m r 2 Re[ p* m
]
v ε p + 2ε m *

µ DEP = r2 =
∇E 6η
 DEP with a Spatially Dependent Phase
• For a AC field, such as that generated by the application of multiple
potentials of different phase, the derivation of the dielectrophoretic
force is more involved and the DEP force can be expressed as.
r*
E = −(∇φR + i∇φI )
r ε *
− ε *
r 2 ε *
− ε *
r* r*
< FDEP >= πε m r Re[ * ]∇ E + 2πε m r Im[ * ](∇ × (Re[ E ] × Im[E ]))
3 p m 3 p m

ε p + 2ε m *
ε p + 2ε m *

Electrorotation Travelling wave DEP

 Gravity and Brownian Motion
• Gravity and Buoyant forces
The effective mass of a spherical particle suspended in a medium
4
∆m = πr 3 ( ρ p − ρ m )
3
Sedimentational force Fgrav − Fbuoy = ∆m ⋅ g
Sedimentation rate in a steady-state condition
∆m ⋅ g 2 r ( ρ p − ρ m ) g
2

v= =
6πηr 9 η
(What if there is a non-uniform heating in the system?)

• Brownian motion
Particles in solution experience a random force due to the thermal
energy of the system, causing them to move in a random manner.
The rms velocity of the particle is
3kT
< v >1/ 2 =
2

m
 Competition of Forces

• Submicron particle manipulation requires very high field strength to

overcome Brownian motion. The DEP potential must exceed the
thermal energy. ε p * − ε m* r 2
πε m r Re[ *
3
] E ≥ kT
ε p + 2ε m *

• Side effects: AC electroosmosis and possible cell damage or

electroporation.
 Particle-Particle Interaction
• The particles are not isolated entities and particle-particle interaction
must be considered.

• Two or more particles with the same sign of charges suspended in an

insulating medium will repel each other.

• However, in electrolyte the available free charges screens the

particle’s charge and the field produced by the particle’s charge
rapidly decays with distance. Therefore any long-range electrostatic
interaction with other particles does not occur. But when the particles
are very close, both electrostatic interactions and van der Waals may
become visible, resulting in the formation of long chains.
 Microfabricated DEP

Au electrode

Spacer

Electrode substrate
Without fluid flow With fluid flow

Flow

Separation, trapping, movement, levitation, rotation, identification,

and other manipulations of cells and microorganisms
DEP: Electrode Design
 DEP Applications

• The characterization of blood cell subpopulations on normal blood

and the detection of perturbations of those subpopulations resulting
from diseases.

• Sample collection (enrichment) for biological warfare agents or

pathogens (bacteria, viruses) detection.

• Integrated isolation and molecular analysis of tumor cells.

• Bioparticle fractionation based on DEP-FFF or DEP chromatography.

• Cell patterning for in-vitro cell culture

• Particle focusing for flow cytometry

• Single cell manipulation

• Detection of molecular binding

Example: Single Cell Cage
Example: Endothelial Cell Patterning
Example: 3D Particle Focusing for Microcytometry
Example: Molecular Separation Using DEP
 AC Electroosmosis

• In the planar microelectrode arrays used for AC electrokinetics,

divergent electric fields are generated, and as a result a component
of the electric field lies tangential to the electrical double layer
which is induced on the electrode surface. Therefore the ions in the
diffuse double layer experience a force similar to DC
electroosmosis.
Local fluid flow by
E E AC electroosmosis
–Fq Et Et Fq
+++++++ ––––––– Surface charge
–V +V movement
Electrodes