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Electrophoresis 2
Electrophoresis 2
Hawcroft DM (1997) Electrophoresis the Basics. Oxford: Righetti PG (1990) Immobilized pH Gradients: Theory
Oxford University Press. and Methodology. Amsterdam: Elsevier Biomedical
Rickwood D and Hames BD (eds.) (1990) Gel Electro- Press.
phoresis of Nucleic Acids: A Practical Approach, 2nd Simpson CF and Whittaker M (eds.) (1983) Electrophore-
edn. Oxford: Oxford University Press. tic Techniques. London: Academic Press Inc.
Righetti PG (1983) Isoelectric Focusing: Theory, Methodo- Westermeier R and Naven T (2002) Proteomics in Practice:
logy and Applications. Amsterdam: Elsevier Science Pub- A Laboratory Manual of Proteome Analysis. Weinheim:
lishers BV. Wiley-VCH.
Principles
B Gas, Charles University, Prague, rises the driving electric field. The analyzed species
Czech Republic (analytes) are introduced through a narrow sample
& 2005, Elsevier Ltd. All Rights Reserved. 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
Introduction of the cathode. As various species have different
velocities in the electric field, they are spatially sepa-
Electrophoresis is a separation method that is based
rated from each other and are detected by a suitable
on the migration of charged species in a supporting
detector, which is located at a certain position of the
medium (a liquid or a hydrophilic gel) under the in-
column or, sometimes, at its end. The separation
fluence of an electric field. The ability of electro-
column is often a very thin tube, with an inner di-
phoresis to separate charged species ranges from
ameter of 10–500 mm, which is called the capillary;
small inorganic or organic ions to charged biopoly-
the electrophoretic methods that utilize such a capil-
mers (like DNA or proteins), or even chromosomes,
lary are called the capillary electrophoretic methods.
microorganisms, or whole cells. Even though electro-
The separation column can also be formed by a
phoresis was established more than 100 years ago, it
channel in various materials (glass, plastic, silica,
is an important technique even today. In the form of
etc.), which is fabricated by means of technologies
gel electrophoresis it is almost the only technique
normally used for the production of electronic micro-
used for solving complicated separation tasks in
chips. These approaches are called ‘lab-on-a-chip’
genomics and proteomics, where thousands and
technologies and electrophoresis that uses this ap-
thousands of species must be separated. Further-
proach is called chip or microchip electrophoresis.
more, in the last two decades electrophoretic tech-
A hydrophilic cross-linked gel (mostly polyacryl-
niques in capillaries or microchips have had a rapid
amide or polysaccharide gel), which is soaked with a
development. Due to the fast expansion of new
solution of electrolytes, or a viscous solution of a
electrophoretic methods, many of the older termi-
linear polymer with an electrolyte, can also serve as
nologies now have rather different meanings than
the separation medium. Electrophoresis using such
before. In this article, the basic principles are ex-
gels or polymeric media is called gel electrophoresis.
plained and discussed.
The polymeric chains of the gel serve in most cases
not only as a supporting medium but also influence
Basic Terms to a great extent the movement of the separated spe-
cies. The gels or solutions of linear polymers can also
The supporting medium where electrophoresis takes be filled into columns (channels), but as the structure
place is called the electrophoretic system or the sepa-
ration system. The separation systems are solutions
Driving voltage
of electrolytes that are filled into a separation column Electrode Electrode
Solution of electrolytes
or a separation channel (Figure 1). Both ends of the Column or channel
column are connected with vessels, which are also
filled with the solution of electrolytes and serve as a Solution of Solution of
stock of the solution. The electric voltage (500– electrolytes Separated Detector electrolytes
Vessel species Vessel
30 000 V), which is called driving voltage, is pro-
vided by two electrodes in the two vessels and Figure 1 Schematic diagram of an electrophoretic instrument.
364 ELECTROPHORESIS / Principles
Driving voltage Table 1 Limiting ionic mobilities ui0 of some ions at 251C
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 1019 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 mol1 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.
ELECTROPHORESIS / Principles 365
The SI unit for specific conductivity is S m–1. Obviou- Here, electrophoresis often employs principles used
sly, the specific conductivity can be calculated if con- in chromatography – interaction of the separands
centrations, charge numbers, and mobilities of all the with another phase, which is called the pseudo-
charged species are known, stationary phase. The pseudostationary phase is a
X
n substance that is added to the separation system in
k¼F ci ui zi ½4 the column, and can interact with the species to be
i¼1 separated. The substance can be neutral, then it does
not have its own electrophoretic movement; or it can
Mobility of Weak Electrolytes – be charged, then it can move in the column with
certain mobility. In both cases, the analytes with their
Dependence on pH own electrophoretic movements encounter on their
Most substances behaving as electrolytes, especially way the molecules of the pseudostationary phase and
organic acids, bases, and ampholytes, are weak elec- interact with them by forming a temporary complex
trolytes. Under given conditions their dissociation or or by association. During the time when the sepa-
protonation is not complete and reaches a chemical rands are bound to the pseudostationary phase its
equilibrium. Then, significant parts of the substance mobility is different; however, when it is free it moves
can exist as ionic forms with various charge numbers with its own mobility. As the rate constants of the
or in the neutral form; and all these forms have dif- interaction are mostly very high, in analogy with
ferent velocities. In most cases the acid–base reactions weak electrolytes, the analyte then moves with a cer-
in solutions are very fast so the electromigration of the tain mean mobility that lies somewhere between the
substance in the electric field can be regarded as the bound and free mobilities. The mean mobility is in
movement of one entity with all its ionic or neutral this way dependent on the interaction (complex-
species moving with a certain mean velocity. Such a ation) constant. Many substances can used for this
mean electrophoretic velocity vA of electromigration purpose, such as 2-hydroxyisobutyric acid, which
of the weak electrolyte A is then forms complexes with many ions, especially with
lanthanides, and enables their electrophoretic sepa-
E X m
ration when added to the separation systems.
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 An important task that can be solved by electro-
and actual mobility of the kth ionic species, re- phoresis is the separation of enantiomers of chiral
spectively, cA,0 is the concentration of the neutral compounds. It is performed by means of their dif-
form, and m is the total number of all ionic species of ferent interactions with a chiral pseudostationary
the weak electrolyte A. The above equation allows a phase – another chiral substance, called chiral selec-
definition of the effective mobility u%A of the weak tor, which is present in the separation system. The
electrolyte A: enantiomers of a chiral analyte have the same phy-
sicochemical properties in the achiral environment,
1 X m Xm
so their mobilities are also the same and cannot be
u%A ¼ cA;k uA;k ¼ xA;k uA;k ½6
c%A k¼1 separated when the separation system is achiral.
k¼1
However, when a chiral substance, which is able to
where xA,k is the molar fraction of the ionic species k. interact (to form complexes) with the analyte, is
The molar fractions of the ionic species are de- added to the system, the constants of complexity are
pendent on pH, so the effective mobility of the weak generally different and the mobilities of the com-
electrolyte is dependent on the pH of the solution as plexes with both enantiomers may also be different.
well. This fact shows that changing pH is a very This enables their separation.
powerful tool that allows one to ‘tune’ the effective
mobilities of analytes to obtain their best separation. Micellar Electrokinetic Chromatography
In micellar electrokinetic chromatography (MEKC),
Other Ways to Influence the added pseudostationary phase are micelles. The
micelles are spherical aggregates of several molecules
Electrophoretic Mobility
of a surface-active compound like sodium dodecyl
As the difference in electrophoretic mobilities of sulfate (SDS). The core of the SDS micelles is
analytes is of key importance for obtaining good composed of entangled aliphatic chains, while the
separation, additional ways are used to influence it. outer shell is formed by charged sulfate groups.
366 ELECTROPHORESIS / Principles
The micelles are highly charged and move in the radius is dependent on the chain length, the effective
electric field. They can interact with molecules of mobilities of the DNA fragments are also dependent
analytes, even when they are neutral, and form tran- on the chain length. Thus, DNA fragments can be
sient associations. In this way the analytes obtain an separated by electrophoresis.
electrophoretic movement and can be separated if The DNA fragments and also other biopolymers
their interaction constants are different. (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
Mobility in Sieving Media: than the radius of pores. Here, other more complex
Gel Electrophoresis mechanisms of movement play a role: reptation
The hydrophilic gel soaked with an electrolyte solu- model (the separated molecule creeps in chains of the
tion, which is often used in electrophoresis as a sup- sieving medium as a reptile in grass), biased reptation
porting medium has, additionally, an even more model, biased reptation with fluctuation, model of
important function: it serves as a sieving medium for entropic barriers, and geometration. For very long
separation of high molecular mass biomolecules such double-stranded DNA fragments (longer than
as DNA or proteins according their chain length or B10 kbp) pulsed field gel electrophoresis is used, a
molecular size. For example, in sequencing of DNA technique that adopts alternating driving electric
by the Sanger method the resulting mixture contains field. Here, the mode of movement of the chains is
a series of single-stranded DNA fragments with the even more complicated.
chain lengths ranging from B20 to 600–1000 nuc-
leotides differing only by a single nucleotide. As Theory – Continuity Equation
every nucleotide contains a phosphate group, which
has a negative charge, the total charge of any A simple environment, in which the separation proc-
fragment is exactly proportional to its length. Such ess in electrophoresis takes place, allows easy for-
a mixture of chains cannot be separated by elect- mulation of basic transport laws that describe
rophoresis in simple liquid solutions according to electromigration with good exactness and enables
their length. The reason is that the chains are only the separation process to be understood well. For
slightly coiled and form the statistical free-draining example, the approximate continuity equations that
coils – that is, the electrolyte solution can flow describe electromigration of n strong ions in free so-
through the coils. Thus, both the Coulombic force lution are
and the friction force are proportional to the chain
length in the same way, and hence the mobility of the @ci @ 2 ci @ ci ui
¼ Di 2 þ j ; i ¼ 1; y; n ½8
fragments in free solution m0 is not dependent on the @t @x @x k
chain length land. The free solution mobility of DNA
where Di is the diffusion coefficient of the ith ion.
is B37 109 m2 V1 s1 at 251C. The free solution
These equations are able to provide information
mobility was independent of DNA molecular mass,
about the concentration of all n ions in the electric
from B400 bp to 48.5 kbp.
field in any time t and at any position x of the sepa-
The diameter of the DNA coils can be characteri-
ration column, when the initial distribution of all
zed by the gyration diameter Rg, which is dependent
ions is known. The first term on the right-hand side
on the length of the chain. When the separation sys-
of eqn [8] describes diffusion and the second one
tem is formed by a neutral cross-linked hydrophilic
electromigration. The division in the electromigra-
gel, e.g., polyacrylamide, with a certain mean size of
tion term is a nonlinear mathematical operation.
pores between polymer chains, the separated DNA
This indicates that electromigration is intrinsically a
fragments being rather spherical coils mechanically
nonlinear process. The nonlinearity is responsible for
collide with the polymer chains and have to pass
some phenomena in electrophoresis, such as self-
through the pores in some way. When the polymer
sharpening of boundaries between zones of analytes
chains are approximately regarded as straight long
or electromigration dispersion.
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 Modes of Electrophoresis
u% ¼ u0 expðKR FÞ ½7 The configuration of electrolytes in the separation
system can substantially influence the electrophoretic
where KR BðRg þ rÞ2 is the retardation coefficient separation. Some of the configurations or modes
and F is the concentration of the gel. As the gyration of electrophoresis showed to be well suited for
ELECTROPHORESIS / Principles 367
specific classes of analytical tasks and are now wide- equations can be regarded as linear. The individual
ly used. Here belong predominantly: zone elect- zones of analytes, although narrow at the beginning,
rophoresis, isotachophoresis (ITP), and isoelectric become broader when moving in the column as dif-
focusing (IEF). fusion causes their dispersion and their longitudinal
profile attains a Gaussian shape. If, however, the
Zone Electrophoresis amount of an analyte is rather high, it influences
This mode is characterized by two features: (1) the conductivity in its own zone, so it is no longer con-
whole separation system together with electrode ves- stant. Then, the nonlinearity arising in this way
sels is filled with a homogeneous solution of electro- causes the electromigration dispersion – another type
lytes, which is called the background electrolyte of broadening of the zones that further deforms
(BGE), (2) the mixture of analyzed species to be sepa- zones to the triangular shape (Figure 4). The quan-
rated is introduced or injected in a rather low titative determination of a particular analyte is per-
amount as a short plug at one end of the separation formed by integration of its detector signal.
column (Figure 3). The mode of zone electrophoresis
is often used in capillary columns, when it is called Isotachophoresis
capillary zone electrophoresis (CZE). The BGE is ITP is used for separation of smaller species like
mostly formed by a suitable buffer to maintain a inorganic or organic ions. The separation system,
certain pH, which is optimal for the separation of a which serves for separation of either cations or an-
given mixture. This mode is also almost exclusively ions, is composed of two different electrolyte solu-
used for gel electrophoresis: here the whole separa- tions. The main features of ITP are as follows: (1)
tion system is formed by a buffer-soaked hydrophilic one electrode vessel and the separation column are
slab gel and the mixture to be separated is introduced filled with the leading electrolyte, whose cation (an-
in little amounts at one end. After applying the ion) has higher mobility (in absolute value) than any
driving electric field the analytes migrate in the cation (anion) of the separated mixture; (2) the sec-
column or in the slab gel with mutually different ond electrode vessel is filled with the terminating
velocities so that they are spatially separated. After electrolyte, whose cation (anion) has lower mobility
some time they are detected with a suitable detector than any cation (anion) of the separated mixture; (3)
located near the second end of the separation column; a plug of mixture of cations (anions) to be separated
in the case of the slab gel electrophoresis, the plate is introduced between the leading and terminating
with the gel is removed from electricity and the sepa- electrolytes (Figure 5). After applying the driving
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
0.8 Na+
of the separated analytes, if their amount is low
enough. Then, the k in the denominator of the sec- 0.7
Li+ Cs+
Concentration (mmol dm−3)
0.4
Capillary
0.3
BGE 0.2
Optics 0.1
Injected Light Photodetector 0.0
sample 0 10 20 30 40 50
U Longitudinal coordinate x of the column (mm)
Figure 4 Longitudinal profiles of analytes in CZE as calculated
BGE BGE from eqn [8] by computer. Background electrolyte: 20 mmol l 1
acetic acid/10 mmol l 1 potassium. Sample introduction: 2 mm
Figure 3 Configuration of capillary zone electrophoresis. The plug of mixture of 1 mmol l 1 Cs þ , 1 mmol l 1 Na þ , 1 mmol l 1
arrangement shown is close to a real instrument. The capillary is Li þ ions, injected at position of 7 mm. Current density
often made of fused silica, whose typical length is 10–100 cm. It j ¼ 200 A m 2. The picture shows resulting profiles at t ¼ 250 s,
has an inner diameter of 20–100 mm and its hydrodynamic re- when all ions are almost separated. A slight triangular deforma-
sistance is rather high. The electroosmosis is allowed to flow. A tion of analyte zones caused by electromigration dispersion is
photometric (UV) absorption detector is generally used. obvious in the figure.
368 ELECTROPHORESIS / Principles
K+
10
6
Injected sample
4
U
Leading electrolyte
2
0
Electrodes of conductivity detector 0 10 20 30 40 50
Longitudinal coordinate X of the column (mm)
Conductivity detector 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-
Semipermeable membrane 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
Leading electrolyte
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
Figure 5 Configuration of capillary isotachophoresis. The arrange- one by one.
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 the analytes in the ITP zones are fixed, the longitu-
electromigration of ions. A conductivity detector is often used. It dinal length of the zone is directly proportional to
has miniature electrodes placed inside the capillary.
their amount. The proportionality enables a precise
quantitative determination of the analytes when mea-
electric field the ion of the leading electrolyte leaves suring the length of their zones.
the separation column and ‘pulls’ the sample ions, Interestingly, the principle of ITP is applied in the
while the terminating ion starts to fill the column and initial phase of disc electrophoresis, in the sample
‘pushes’ the ions. The plug of the separated ions and stacking gels, for the purpose of sorting and
moves ‘bracketed’ between the leading and termina- concentrating (often by a factor of 10 000) the vari-
ting electrolytes and every ion forms its own ous protein zones. The ITP process is disrupted as the
individual zone after some time. This stage is called train of zones enters the running gel, where migra-
the stationary state. All the zones of the ions are then tion continues in the zonal mode.
stacked one by one and move with the same velocity,
which is given by the velocity of the leading ion.
Isoelectric Focusing
Unlike the mode of zone electrophoresis, here the
specific conductivities k of the zones of the separated IEF is an electrophoretic technique for separation of
analytes are different and no longer constant. The amphoteric species, mostly proteins. It is a technique
nonlinearity in ITP is responsible for a useful pheno- based on differences in isoelectric points (pI). The
menon: ‘sharpening’ of the boundaries between the species are separated in the pH gradient that is
individual zones. The boundaries are very steep and formed in the separation system. The pH gradient is
the longitudinal profile of the zones of the analytes is generated in stabilizing matrices of special gels with
almost rectangular (Figure 6). When the zones pass a proteolytic groups, mostly in slabs. Alternatively, it
detector located near the second end of the separa- can be generated in a free solution without the gel by
tion column, the detector gives a rather stepwise passing the electric current through the mixture of a
signal. The concentration of the analytes in their series of ampholytes having isoelectric points in close
zones is not the same, as it was in the original mix- proximity to each other. The analyte migrates in the
ture: it attains a certain value. For singly charged separation system by electrophoretic movement ac-
univalent ions or strong multicharged ions it can be cording to its net charge. When it reaches a position
derived from a simple relation, which is called the where the pH is equal to its pI, the net charge be-
Kohlrausch regulating function. As concentrations of comes zero and the movement stops. The separated
ELECTROPHORESIS / Principles 369
0
0 5 10 15 20 Further Reading
Distance y from the Helmholtz layer (nm)
Auroux PA, Iossifidis D, Reyes DR, and Manz A
Figure 8 Course of electric potential in the diffuse layer of
(2002) Micro total analysis systems 1. Introduction,
10 mol dm 3 NaCl. The zeta potential is supposed to be 100 mV.
theory, and technology. Analytical Chemistry 74:
The curve is a solution of the Poisson–Boltzmann equation and is
approximately exponential. 2637–2652.
Chankvetadze B (1997) Capillary Electrophoresis in Chiral
Analysis. Chichester: Wiley.
Everaerts FM, Beckers JL, and Verheggen TPEM (1976)
As pointed out above, fused silica used as a material Isotachoporesis. Theory, Instrumentation and Applica-
for separation columns in capillary electrophoretic tions. Amsterdam: Elsevier.
methods has normally a negative charge. Various Hames BD (1998) Gel Electrophoresis of Proteins. Oxford:
ions, especially surfactants or big amphoteric ions, Oxford University Press.
Heller C (ed.) (1997) Analysis of Nucleic Acids by
can be, however, adsorbed on the surface, which
Capillary Electrophoresis. Wiesbaden: Friedrich Vieweg.
dramatically influences the zeta potential. For example,
Jones P and Rickwood D (1995) Gel Electrophoresis:
the addition of a small concentration of a suitable Nucleic Acids. New York: Wiley.
cationic surfactant like tetradecyltrimethylammonium Righetti PG (1990) Immobilized pH Gradients, Theory
bromide to the BGE causes its adsorption at the inner and Methodology. Amsterdam: Elsevier.
capillary wall and the reversal of the EOF in silica Righetti PG (ed.) (1996) Capillary Electrophoresis in An-
capillaries to the anodic side. Such surfactants are alytical Biotechnology. Boca Raton: CRC Press.
Isotachophoresis
L Křivánková and P Boček, Institute of Analytical highly useful for analysis of complex mixtures in so-
Chemistry, Brno, Czech Republic lution, from simple inorganic ions up to proteins.
& 2005, Elsevier Ltd. All Rights Reserved. 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
Introduction within zone electrophoretic migration as well. Here,
Capillary isotachophoresis began to develop as a the basic theory, instrumentation, principles of quali-
modern instrumental analytical method at the end of tative and quantitative analyses, and examples of
the 1960s and the beginning of the 1970s and is now applications of single analytical ITP as well as com-
a well-established method with a good theoretical bined techniques are described. The article is orient-
background and available commercial instrumenta- ed to capillary ITP since capillaries provide high
tion directed nowadays toward miniaturization. It is separation efficiency and speed of analysis; however,