ELECTROPHORESIS / Principles 363
Hawcroft DM (1997) Electrophoresis the Basics. Oxford:
Oxford University Press.
Rickwood D and Hames BD (eds.) (1990) Gel Electro-
phoresis of Nucleic Acids: A Practical Approach, 2nd
edn. Oxford: Oxford University Press.
Righetti PG (1983) Isoelectric Focusing: Theory, Methodo-
logy and Applications. Amsterdam: Elsevier Science Pub-
lishers BV.
Principles
B Gas, Charles University, Prague,
Czech Republic
& 2005, Elsevier Ltd. All Rights Reserved.
Introduction
Electrophoresis is a separation method that is based
on the migration of charged species in a supporting
medium (a liquid or a hydrophilic gel) under the in-
fluence of an electric field. The ability of electro-
phoresis to separate charged species ranges from
small inorganic or organic ions to charged biopoly-
mers (like DNA or proteins), or even chromosomes,
microorganisms, or whole cells. Even though electro-
phoresis was established more than 100 years ago, it
is an important technique even today. In the form of
gel electrophoresis it is almost the only technique
used for solving complicated separation tasks in
genomics and proteomics, where thousands and
thousands of species must be separated. Further-
more, in the last two decades electrophoretic tech-
niques in capillaries or microchips have had a rapid
development. Due to the fast expansion of new
electrophoretic methods, many of the older termi-
nologies now have rather different meanings than
before. In this article, the basic principles are ex-
plained and discussed.
Basic Terms
The supporting medium where electrophoresis takes
place is called the electrophoretic system or the sepa-
ration system. The separation systems are solutions
of electrolytes that are filled into a separation column
or a separation channel (Figure 1). Both ends of the
column are connected with vessels, which are also
filled with the solution of electrolytes and serve as a
stock of the solution. The electric voltage (500–
30 000 V), which is called driving voltage, is pro-
vided by two electrodes in the two vessels and
Righetti PG (1990) Immobilized pH Gradients: Theory
and Methodology. Amsterdam: Elsevier Biomedical
Press.
Simpson CF and Whittaker M (eds.) (1983) Electrophore-
tic Techniques. London: Academic Press Inc.
Westermeier R and Naven T (2002) Proteomics in Practice:
A Laboratory Manual of Proteome Analysis. Weinheim:
Wiley-VCH.
rises the driving electric field. The analyzed species
(analytes) are introduced through a narrow sample
plug, which then migrate across the longitudinal axis
of the column in the liquid, the negatively charged
analytes (anions) in the direction of the anode and
the positively charged ones (cations) in the direction
of the cathode. As various species have different
velocities in the electric field, they are spatially sepa-
rated from each other and are detected by a suitable
detector, which is located at a certain position of the
column or, sometimes, at its end. The separation
column is often a very thin tube, with an inner di-
ameter of 10–500 mm, which is called the capillary;
the electrophoretic methods that utilize such a capil-
lary are called the capillary electrophoretic methods.
The separation column can also be formed by a
channel in various materials (glass, plastic, silica,
etc.), which is fabricated by means of technologies
normally used for the production of electronic micro-
chips. These approaches are called ‘lab-on-a-chip’
technologies and electrophoresis that uses this ap-
proach is called chip or microchip electrophoresis.
A hydrophilic cross-linked gel (mostly polyacryl-
amide or polysaccharide gel), which is soaked with a
solution of electrolytes, or a viscous solution of a
linear polymer with an electrolyte, can also serve as
the separation medium. Electrophoresis using such
gels or polymeric media is called gel electrophoresis.
The polymeric chains of the gel serve in most cases
not only as a supporting medium but also influence
to a great extent the movement of the separated spe-
cies. The gels or solutions of linear polymers can also
be filled into columns (channels), but as the structure
Driving voltage
Electrode Electrode
Solution of electrolytes
Column or channel
Solution of Solution of
electrolytes Separated Detector electrolytes
Vessel species Vessel
Figure 1 Schematic diagram of an electrophoretic instrument.
364 ELECTROPHORESIS / Principles

Driving voltage Table 1 Limiting ionic mobilities ui0 of some ions at 251C

Cations u0i , Anions u0i ,

Separated species 10
9 m2 V
1 s
1 10
9 m2 V
1 s
1

Cs þ 80.0a Br
80.9a
Kþ 76.2a Cl
79.1b
Na þ 51.9a F
57.0b
Li þ 40.1a SO24
82.9b
Ammediol þ 29.5b CH3COO
42.4a
b-Alanine þ 36.7b b-Alanine
30.8c
Hþ 362.3a OH
206.4a
Slab gel
a
Solution of electrolytes Atkins PW (1994) Physical Chemistry. Oxford: Oxford University
Press.
Figure 2 Electrophoresis in slab gel. b
Foret F, Křivánková L, and Boček P (1993) Capillary Zone
Electrophoresis. Weinheim: VCH.
c
of gels is cross-linked they are mechanically stable Hirokawa T, Gojo T, and Kiso Y (1987) Journal of Chro-
even without being filled into columns. Therefore, matography 390: 201–223. Ammediol, 2-amino-2-methyl-1,3-
propandiol.
they are used in the form of flat strips or layers that
are sticking to a supporting plastic or glass plate and
are called slab gels (Figure 2).
given temperature and solvent for many ions. By
convention, it is negative for negatively charged spe-
Mobility and Conductivity cies (anions) and positive for positively charged spe-
cies (cations). To a certain range it depends on the
When a charged particle of an ith species with charge
concentration of the electrolytes (or the ionic
number zi is placed in the electric field with an in-
strength) in solution. Therefore, the usual data for
tensity E (in V m
1), the Coulombic force Fc ¼ zi eE
tabulated electrophoretic mobilities of ions are the
acting on the particle causes its movement;
limiting or absolute ionic mobilities, u0i . They are the
e ¼ 1:602  10
19 C is the elementary charge. As
mobilities that are extrapolated for zero ionic
in electrophoresis the particles to be separated move
strength assuming further that the substance would
in liquid or gel form, they face obstacles during the
be completely in the given ionic form. Such condi-
movement – mostly molecules of the solvent or poly-
tions can be theoretically reached in infinitely diluted
mer chains of the gel. The collisions and/or interac-
solutions. Table 1 gives limiting ionic mobilities u0i
tions with them causes the friction force Ff, which is
for several ions. The ‘real’ ionic mobilities under a
proportional to the velocity of the moving particle,
given finite ionic strength and given conditions are
and which acts against the Coulombic force. After a
called actual mobilities.
very short time (10
13–10
10 s) both forces are
The electromigration of charged species in the
balanced, Fc ¼ Ff , and a mean velocity of the particle
separation column increases the electric current. The
attains a constant value. The net movement of
current density j (in A m–2) at a particular position of
charged particles in the electric field is called electro-
the column is given by
migration and the velocity of such movement is
X
n
called electrophoretic velocity. Various species have, j ¼ FE ci ui zi ½2
under given conditions, a different electrophoretic i¼1
velocity. When moving for a certain time, they are
spatially separated. This is a separation principle of where F¼ 96 487 C mol
1 is the Faraday constant, ci
the majority (but not all) electrophoretic separation is the concentration of the ith charged species (in
techniques. The electrophoretic velocity of the ith mol m–3), and n is the total number of charged spe-
species vi is almost perfectly proportional to the in- cies present. Notice that the product uizi is always
tensity of the driving electric field, vi BE, when other positive, so the movement of positive species in one
parameters (e.g., temperature) are constant. The co- direction and negative species in the opposite direc-
efficient of proportionality is a quantity called elect- tion contribute to the electric current density in the
rophoretic mobility, ui, and is defined as same way. According to Ohm’s law, the current den-
sity j is proportional to the intensity of the electric
vi ¼ ui E ½1 field E,
j ¼ kE ½3
The dimension of the mobility in SI units is
m2 V
1 s
1. The electrophoretic mobility is unique The proportionality constant is the specific con-
for a given species and is therefore tabulated for a ductivity, k, which is defined by the above relation.

The SI unit for specific conductivity is S m–1. Obviou-
sly, the specific conductivity can be calculated if con-
centrations, charge numbers, and mobilities of all the
charged species are known,
X
n substance that is added to the separation system in
k¼F ci ui zi ½4
i¼1
Mobility of Weak Electrolytes –
Dependence on pH
Most substances behaving as electrolytes, especially
organic acids, bases, and ampholytes, are weak elec-
trolytes. Under given conditions their dissociation or
protonation is not complete and reaches a chemical
equilibrium. Then, significant parts of the substance
can exist as ionic forms with various charge numbers
or in the neutral form; and all these forms have dif-
ferent velocities. In most cases the acid–base reactions
in solutions are very fast so the electromigration of the
substance in the electric field can be regarded as the
movement of one entity with all its ionic or neutral
species moving with a certain mean velocity. Such a
mean electrophoretic velocity vA of electromigration
of the weak electrolyte A is then
E X m
vA ¼ c u ½5
c%A k¼1 A;k A;k
P Enantioselective Electrophoresis
where c%A ¼ cA;0 þ m k¼1 cA;k is the total (analytical)
concentration, cA,k and uA,k are the concentration
and actual mobility of the kth ionic species, re-
spectively, cA,0 is the concentration of the neutral
form, and m is the total number of all ionic species of
the weak electrolyte A. The above equation allows a
definition of the effective mobility u%A of the weak
electrolyte A:
1 X m Xm
u%A ¼ cA;k uA;k ¼ xA;k uA;k ½6
c%A k¼1
k¼1
where xA,k is the molar fraction of the ionic species k.
The molar fractions of the ionic species are de-
pendent on pH, so the effective mobility of the weak
electrolyte is dependent on the pH of the solution as
well. This fact shows that changing pH is a very
powerful tool that allows one to ‘tune’ the effective
mobilities of analytes to obtain their best separation.
Other Ways to Influence
Electrophoretic Mobility
As the difference in electrophoretic mobilities of
analytes is of key importance for obtaining good
separation, additional ways are used to influence it.
ELECTROPHORESIS / Principles 365
Here, electrophoresis often employs principles used
in chromatography – interaction of the separands
with another phase, which is called the pseudo-
stationary phase. The pseudostationary phase is a
the column, and can interact with the species to be
separated. The substance can be neutral, then it does
not have its own electrophoretic movement; or it can
be charged, then it can move in the column with
certain mobility. In both cases, the analytes with their
own electrophoretic movements encounter on their
way the molecules of the pseudostationary phase and
interact with them by forming a temporary complex
or by association. During the time when the sepa-
rands are bound to the pseudostationary phase its
mobility is different; however, when it is free it moves
with its own mobility. As the rate constants of the
interaction are mostly very high, in analogy with
weak electrolytes, the analyte then moves with a cer-
tain mean mobility that lies somewhere between the
bound and free mobilities. The mean mobility is in
this way dependent on the interaction (complex-
ation) constant. Many substances can used for this
purpose, such as 2-hydroxyisobutyric acid, which
forms complexes with many ions, especially with
lanthanides, and enables their electrophoretic sepa-
ration when added to the separation systems.
An important task that can be solved by electro-
phoresis is the separation of enantiomers of chiral
compounds. It is performed by means of their dif-
ferent interactions with a chiral pseudostationary
phase – another chiral substance, called chiral selec-
tor, which is present in the separation system. The
enantiomers of a chiral analyte have the same phy-
sicochemical properties in the achiral environment,
so their mobilities are also the same and cannot be
separated when the separation system is achiral.
However, when a chiral substance, which is able to
interact (to form complexes) with the analyte, is
added to the system, the constants of complexity are
generally different and the mobilities of the com-
plexes with both enantiomers may also be different.
This enables their separation.
Micellar Electrokinetic Chromatography
In micellar electrokinetic chromatography (MEKC),
the added pseudostationary phase are micelles. The
micelles are spherical aggregates of several molecules
of a surface-active compound like sodium dodecyl
sulfate (SDS). The core of the SDS micelles is
composed of entangled aliphatic chains, while the
outer shell is formed by charged sulfate groups.
366 ELECTROPHORESIS / Principles
The micelles are highly charged and move in the
electric field. They can interact with molecules of
analytes, even when they are neutral, and form tran-
sient associations. In this way the analytes obtain an
electrophoretic movement and can be separated if
their interaction constants are different.
Mobility in Sieving Media:
Gel Electrophoresis
The hydrophilic gel soaked with an electrolyte solu-
tion, which is often used in electrophoresis as a sup-
porting medium has, additionally, an even more
important function: it serves as a sieving medium for
separation of high molecular mass biomolecules such
as DNA or proteins according their chain length or
molecular size. For example, in sequencing of DNA
by the Sanger method the resulting mixture contains
a series of single-stranded DNA fragments with the
chain lengths ranging from B20 to 600–1000 nuc-
leotides differing only by a single nucleotide. As
every nucleotide contains a phosphate group, which
has a negative charge, the total charge of any
fragment is exactly proportional to its length. Such
a mixture of chains cannot be separated by elect-
rophoresis in simple liquid solutions according to
their length. The reason is that the chains are only
slightly coiled and form the statistical free-draining
coils – that is, the electrolyte solution can flow
through the coils. Thus, both the Coulombic force
and the friction force are proportional to the chain
length in the same way, and hence the mobility of the
fragments in free solution m0 is not dependent on the
chain length land. The free solution mobility of DNA
is B37  10
9 m2 V
1 s
1 at 251C. The free solution
mobility was independent of DNA molecular mass,
from B400 bp to 48.5 kbp.
The diameter of the DNA coils can be characteri-
zed by the gyration diameter Rg, which is dependent
on the length of the chain. When the separation sys-
tem is formed by a neutral cross-linked hydrophilic
gel, e.g., polyacrylamide, with a certain mean size of
pores between polymer chains, the separated DNA
fragments being rather spherical coils mechanically
collide with the polymer chains and have to pass
through the pores in some way. When the polymer
chains are approximately regarded as straight long
rods with radius r, such a type of movement of the
DNA fragments is called the Ogston movement. Its
effective mobility u% is given by
u% ¼ u0 expð
KR FÞ ½7
where KR BðRg þ rÞ2 is the retardation coefficient
and F is the concentration of the gel. As the gyration
radius is dependent on the chain length, the effective
mobilities of the DNA fragments are also dependent
on the chain length. Thus, DNA fragments can be
separated by electrophoresis.
The DNA fragments and also other biopolymers
(proteins, carbohydrates) can be separated by electro-
phoresis in sieving media even if their chains are
much longer and their gyration radius is much greater
than the radius of pores. Here, other more complex
mechanisms of movement play a role: reptation
model (the separated molecule creeps in chains of the
sieving medium as a reptile in grass), biased reptation
model, biased reptation with fluctuation, model of
entropic barriers, and geometration. For very long
double-stranded DNA fragments (longer than
B10 kbp) pulsed field gel electrophoresis is used, a
technique that adopts alternating driving electric
field. Here, the mode of movement of the chains is
even more complicated.
Theory – Continuity Equation
A simple environment, in which the separation proc-
ess in electrophoresis takes place, allows easy for-
mulation of basic transport laws that describe
electromigration with good exactness and enables
the separation process to be understood well. For
example, the approximate continuity equations that
describe electromigration of n strong ions in free so-
lution are
@ci @ 2 ci @
ci ui 
¼ Di 2 þ j ; i ¼ 1; y; n ½8
@t @x @x k
where Di is the diffusion coefficient of the ith ion.
These equations are able to provide information
about the concentration of all n ions in the electric
field in any time t and at any position x of the sepa-
ration column, when the initial distribution of all
ions is known. The first term on the right-hand side
of eqn [8] describes diffusion and the second one
electromigration. The division in the electromigra-
tion term is a nonlinear mathematical operation.
This indicates that electromigration is intrinsically a
nonlinear process. The nonlinearity is responsible for
some phenomena in electrophoresis, such as self-
sharpening of boundaries between zones of analytes
or electromigration dispersion.
Modes of Electrophoresis
The configuration of electrolytes in the separation
system can substantially influence the electrophoretic
separation. Some of the configurations or modes
of electrophoresis showed to be well suited for

specific classes of analytical tasks and are now wide-
ly used. Here belong predominantly: zone elect-
rophoresis, isotachophoresis (ITP), and isoelectric
focusing (IEF).
Zone Electrophoresis
This mode is characterized by two features: (1) the
whole separation system together with electrode ves-
sels is filled with a homogeneous solution of electro-
lytes, which is called the background electrolyte
(BGE), (2) the mixture of analyzed species to be sepa-
rated is introduced or injected in a rather low
amount as a short plug at one end of the separation
column (Figure 3). The mode of zone electrophoresis
is often used in capillary columns, when it is called
capillary zone electrophoresis (CZE). The BGE is
mostly formed by a suitable buffer to maintain a
certain pH, which is optimal for the separation of a
given mixture. This mode is also almost exclusively
used for gel electrophoresis: here the whole separa-
tion system is formed by a buffer-soaked hydrophilic
slab gel and the mixture to be separated is introduced
in little amounts at one end. After applying the
driving electric field the analytes migrate in the
column or in the slab gel with mutually different
velocities so that they are spatially separated. After
some time they are detected with a suitable detector
located near the second end of the separation column;
in the case of the slab gel electrophoresis, the plate
with the gel is removed from electricity and the sepa-
rated analytes are visualized as spots by a suitable
reagent. The conductivity of the BGE in the separa-
tion system is only slightly influenced by the presence
of the separated analytes, if their amount is low
enough. Then, the k in the denominator of the sec-
ond term in eqn [8] is practically constant in time and
homogeneous across the column length so that the
Capillary
BGE
Optics
Injected Light Photodetector
sample
U Longitudinal coordinate x of the column (mm)
BGE BGE
Figure 3 Configuration of capillary zone electrophoresis. The
arrangement shown is close to a real instrument. The capillary is
often made of fused silica, whose typical length is 10–100 cm. It
has an inner diameter of 20–100 mm and its hydrodynamic re-
sistance is rather high. The electroosmosis is allowed to flow. A
photometric (UV) absorption detector is generally used.
ELECTROPHORESIS / Principles 367
equations can be regarded as linear. The individual
zones of analytes, although narrow at the beginning,
become broader when moving in the column as dif-
fusion causes their dispersion and their longitudinal
profile attains a Gaussian shape. If, however, the
amount of an analyte is rather high, it influences
conductivity in its own zone, so it is no longer con-
stant. Then, the nonlinearity arising in this way
causes the electromigration dispersion – another type
of broadening of the zones that further deforms
zones to the triangular shape (Figure 4). The quan-
titative determination of a particular analyte is per-
formed by integration of its detector signal.
Isotachophoresis
ITP is used for separation of smaller species like
inorganic or organic ions. The separation system,
which serves for separation of either cations or an-
ions, is composed of two different electrolyte solu-
tions. The main features of ITP are as follows: (1)
one electrode vessel and the separation column are
filled with the leading electrolyte, whose cation (an-
ion) has higher mobility (in absolute value) than any
cation (anion) of the separated mixture; (2) the sec-
ond electrode vessel is filled with the terminating
electrolyte, whose cation (anion) has lower mobility
than any cation (anion) of the separated mixture; (3)
a plug of mixture of cations (anions) to be separated
is introduced between the leading and terminating
electrolytes (Figure 5). After applying the driving
0.8 Na+
0.7
Li+ Cs+
Concentration (mmol dm−3)
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0 10 20 30 40 50
Figure 4 Longitudinal profiles of analytes in CZE as calculated
from eqn [8] by computer. Background electrolyte: 20 mmol l
1
acetic acid/10 mmol l
1 potassium. Sample introduction: 2 mm
plug of mixture of 1 mmol l
1 Cs þ , 1 mmol l
1 Na þ , 1 mmol l
1
Li þ ions, injected at position of 7 mm. Current density
j ¼ 200 A m
2. The picture shows resulting profiles at t ¼ 250 s,
when all ions are almost separated. A slight triangular deforma-
tion of analyte zones caused by electromigration dispersion is
obvious in the figure.
368 ELECTROPHORESIS / Principles
Terminating electrolyte
Injected sample
U
Leading electrolyte
Electrodes of conductivity detector
Conductivity detector
Semipermeable membrane
Leading electrolyte
Figure 5 Configuration of capillary isotachophoresis. The arrange-
ment shown is close to a real instrument. The capillary is often
made of plastic. The semipermeable membrane prevents electro-
osmotic and hydrodynamic flows of the electrolyte but allows
electromigration of ions. A conductivity detector is often used. It
has miniature electrodes placed inside the capillary.
electric field the ion of the leading electrolyte leaves
the separation column and ‘pulls’ the sample ions,
while the terminating ion starts to fill the column and
‘pushes’ the ions. The plug of the separated ions
moves ‘bracketed’ between the leading and termina-
ting electrolytes and every ion forms its own
individual zone after some time. This stage is called
the stationary state. All the zones of the ions are then
stacked one by one and move with the same velocity,
which is given by the velocity of the leading ion.
Unlike the mode of zone electrophoresis, here the
specific conductivities k of the zones of the separated
analytes are different and no longer constant. The
nonlinearity in ITP is responsible for a useful pheno-
menon: ‘sharpening’ of the boundaries between the
individual zones. The boundaries are very steep and
the longitudinal profile of the zones of the analytes is
almost rectangular (Figure 6). When the zones pass a
detector located near the second end of the separa-
tion column, the detector gives a rather stepwise
signal. The concentration of the analytes in their
zones is not the same, as it was in the original mix-
ture: it attains a certain value. For singly charged
univalent ions or strong multicharged ions it can be
derived from a simple relation, which is called the
Kohlrausch regulating function. As concentrations of
K+
10
Concentration (mmol dm−3)
Na+
8 Li+
Ammediol+
6
4
2
0
0 10 20 30 40 50
Longitudinal coordinate X of the column (mm)
Figure 6 Longitudinal profiles of analytes in capillary isotacho-
phoresis as calculated from eqn [8] by a computer. Leading
electrolyte: 20 mmol l
1 acetic acid/10 mmol l
1 potassium. Ter-
minating electrolyte: 20 mmol l
1 acetic acid/10 mmol l
1 am-
mediol. Sample introduction: 6 mm plug of mixture of 5 mmol l
1
Na þ , 5 mmol l
1 Li þ ions, injected at position of 7 mm. Current
density j ¼ 75 A m
2. The picture shows resulting profiles at
t ¼ 650 s, when sodium and lithium are separated. They form
isotachophoretic zones with virtually rectangular profiles stacked
one by one.
the analytes in the ITP zones are fixed, the longitu-
dinal length of the zone is directly proportional to
their amount. The proportionality enables a precise
quantitative determination of the analytes when mea-
suring the length of their zones.
Interestingly, the principle of ITP is applied in the
initial phase of disc electrophoresis, in the sample
and stacking gels, for the purpose of sorting and
concentrating (often by a factor of 10 000) the vari-
ous protein zones. The ITP process is disrupted as the
train of zones enters the running gel, where migra-
tion continues in the zonal mode.
Isoelectric Focusing
IEF is an electrophoretic technique for separation of
amphoteric species, mostly proteins. It is a technique
based on differences in isoelectric points (pI). The
species are separated in the pH gradient that is
formed in the separation system. The pH gradient is
generated in stabilizing matrices of special gels with
proteolytic groups, mostly in slabs. Alternatively, it
can be generated in a free solution without the gel by
passing the electric current through the mixture of a
series of ampholytes having isoelectric points in close
proximity to each other. The analyte migrates in the
separation system by electrophoretic movement ac-
cording to its net charge. When it reaches a position
where the pH is equal to its pI, the net charge be-
comes zero and the movement stops. The separated

Driving voltage
+ −
Molecules of protein
+ + − liquid from one electrode vessel to another. The EOF
+ − − − + −
+ + − in itself cannot separate the analytes but it is vec-
V=0 Vani
Vcat
pH
pI
Xf Position
Figure 7 Principle of isoelectric focusing. The protein has cat-
ionic or anionic movement at pH which is below or above its pI,
respectively. The velocity of the movement is zero, when protein
reaches the position xf, this is the site where it will be focused.
species are in this way focused at pH positions cor-
responding to their pI (Figure 7). When the slab gel is
used, they are visualized by a suitable reagent; when
focused in free solution in the column, the content of
the column is pushed out by pressure or by changing
electrolytes in the electrode vessels and detected by a
detector located at the end.
Electroosmotic Flow
The electroosmotic flow (EOF) is an electrokinetic
phenomenon accompanying almost all electrophore-
tic methods. It arises due to the existence of the
electric double layer at the inner surface of the sepa-
ration channel. The electric double layer is generally
formed at the interface between the liquid and solid
phases. A possible specific adsorption of some ions
from the liquid phase at the solid phase or dissoci-
ation of some ionogenic groups from the surface
layer of the solid phase causes a layer of a surface
charge, the Helmholtz layer, sticking to the channel
inner wall. This is compensated by the opposite
charge, which is distributed in the adjacent liquid
solution of the BGE and which ranges to some dis-
tance from the inner wall. This part of the double
layer is called the diffuse layer. The diffuse layer,
which has an excess of volume charge, can be
regarded as a very thin ‘sleeve’ of charged liquid ad-
jacent to the inner wall. When the driving electric
field is applied longitudinally to the channel axis, the
Coulombic force acting at the charged ‘sleeve’ causes
its movement. Due to friction forces the movement
is, in very short time (10
4–10
2 s), spread over the
whole volume of the channel and as a result the
whole volume of the liquid flows in the channel. This
ELECTROPHORESIS / Principles 369
is the EOF. Unlike the electrophoretic movement
where only charged species move in the separation
system and neutral molecules of the solvent have no
net movement, the EOF causes the liquid to move as
a whole. In fact, it acts as a pump transporting a
torially superposed to its electrophoretic movement.
The total electromigration velocity vEM;i of the ith
separated species is
vEM;i ¼ vi þ vEOF ½9
The EOF is especially important in CZE and MEKC.
Here, the columns are often made of fused silica, and
have negative charge at the inner wall due to disso-
ciation of the silanol groups, which causes the EOF
to flow in the direction of the cathode. Therefore, the
electrophoretic movement of cationic species is
speeded up, while the negative species move slower
as the EOF flows against them. Sometimes the latter
cannot reach the detector.
The distribution of the velocity of the EOF across
the radius of the channel or column is an important
issue. In the ‘normal’ laminar flow that is raised by a
difference in pressure, the radial profile of the velo-
city vector has a parabolic shape: it is zero at the
inner wall and maximum in the channel axis. The
radial profile of the EOF is quite different: it is also
zero at the wall but in a very short distance, which is
approximately equal to the thickness of the diffusion
layer, it attains a certain value, which is constant
across the rest of radius. The radial profile of the
EOF is ‘plug-like’. This is a very beneficial feature of
this type of flow, as it does not cause axial dispersion
of the separand.
The excess of the volume charge in the diffuse
layer causes the origin of the electric potential f(y) in
liquid solution. It is dependent on the distance y from
the Helmholtz layer (Figure 8). The potential f is
conventionally set zero at a big distance from the
wall. The value of the potential in the diffuse layer in
the closest vicinity to the Helmholtz layer ðy ¼ 0Þ is
called the zeta potential, fð0Þ ¼ z. When longi-
tudinal driving electric field E is applied, the velocity
vEOF of the plug-like EOF is related to the zeta
potential by the Helmholtz–Smoluchowski equation:
ez
vEOF ¼
E ½10
Z
where e is the permitivity and Z is the dynamic vis-
cosity, respectively, of the liquid solution. The zeta
potential and, consequently, also the velocity of the
EOF strongly depend on the quality of the surface.
Various materials have different zeta potentials.
370 ELECTROPHORESIS / Isotachophoresis
100

=100 mV
80
Electric potential  (mV)
60
40
20
0
0 5 10
Distance y from the Helmholtz layer (nm)
Figure 8 Course of electric potential in the diffuse layer of
10 mol dm
3 NaCl. The zeta potential is supposed to be 100 mV.
The curve is a solution of the Poisson–Boltzmann equation and is
approximately exponential.
As pointed out above, fused silica used as a material
for separation columns in capillary electrophoretic
methods has normally a negative charge. Various
ions, especially surfactants or big amphoteric ions,
can be, however, adsorbed on the surface, which
dramatically influences the zeta potential. For example,
the addition of a small concentration of a suitable
cationic surfactant like tetradecyltrimethylammonium
bromide to the BGE causes its adsorption at the inner
capillary wall and the reversal of the EOF in silica
capillaries to the anodic side. Such surfactants are
Isotachophoresis
L Křivánková and P Boček, Institute of Analytical
Chemistry, Brno, Czech Republic
& 2005, Elsevier Ltd. All Rights Reserved.
Introduction
Capillary isotachophoresis began to develop as a
modern instrumental analytical method at the end of
the 1960s and the beginning of the 1970s and is now
a well-established method with a good theoretical
background and available commercial instrumenta-
tion directed nowadays toward miniaturization. It is
often used in the separation of negatively charged
species as the EOF in the anodic direction speeds up
their movement. The EOF can be also utilized for
pumping liquids in electrophoretic methods, espe-
cially in electrophoresis in chips. Further, it is used
for pumping of the mobile phase in capillary electro-
chromatography. This method is a hybrid between
electrophoresis and chromatography. Instead of
using a mechanical pump for the transport of a liq-
uid phase through the column it utilizes the EOF
raised by the driving voltage.
See also: Capillary Electrophoresis: Overview. Micellar
Electrokinetic Chromatography.
15 20 Further Reading
Auroux PA, Iossifidis D, Reyes DR, and Manz A
(2002) Micro total analysis systems 1. Introduction,
theory, and technology. Analytical Chemistry 74:
2637–2652.
Chankvetadze B (1997) Capillary Electrophoresis in Chiral
Analysis. Chichester: Wiley.
Everaerts FM, Beckers JL, and Verheggen TPEM (1976)
Isotachoporesis. Theory, Instrumentation and Applica-
tions. Amsterdam: Elsevier.
Hames BD (1998) Gel Electrophoresis of Proteins. Oxford:
Oxford University Press.
Heller C (ed.) (1997) Analysis of Nucleic Acids by
Capillary Electrophoresis. Wiesbaden: Friedrich Vieweg.
Jones P and Rickwood D (1995) Gel Electrophoresis:
Nucleic Acids. New York: Wiley.
Righetti PG (1990) Immobilized pH Gradients, Theory
and Methodology. Amsterdam: Elsevier.
Righetti PG (ed.) (1996) Capillary Electrophoresis in An-
alytical Biotechnology. Boca Raton: CRC Press.
highly useful for analysis of complex mixtures in so-
lution, from simple inorganic ions up to proteins.
Isotachophoresis (ITP) can be performed, however,
also in any apparatus developed for capillary zone
electrophoresis (CZE), and the advantages of this
powerful analytical method can be employed in an
effective online hyphenation with CZE or transiently
within zone electrophoretic migration as well. Here,
the basic theory, instrumentation, principles of quali-
tative and quantitative analyses, and examples of
applications of single analytical ITP as well as com-
bined techniques are described. The article is orient-
ed to capillary ITP since capillaries provide high
separation efficiency and speed of analysis; however,