The principle and performance of capillary electrophoresis

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The principle and performance of capillary electrophoresis
Ljubica Glavaš-Obrovac
Introduction
Capillary electrophoresis (CE) is a relatively new and a very versatile technique (also
known as high-performance capillary electrophoresis) for the separation and analysis of a variety
of molecules – from small inorganic ions to huge biopolymers. Compared to the highperformance
liquid chromatography, and gas chromatography CE offers several advantages, including highly
efficient and fast separation, relatively inexpensive and long lasting capillary columns, small
sample size requirements and low reagent consumption. It can be used for analysis of polar ionic,
polar non-ionic and non-polar non-ionic compounds, as well as high molecular weight
biomolecules and chiral compounds. Further, CE separations are highly efficient (N>105 to 106),
sensitive in terms of mass and concentration, fast (usually less than 30 min), simple, and require
extremely small amounts of solvents and other consumables. This highly sophisticated, user
friendly and very effective CE has become commercially available at affordable costs (Slide 2).
Capillary electrophoresis theory
When a sample is injected into a capillary tube and an electric field is applied across the
capillary tube, sample’s components migrate as the result of two types of action: electrophoretic
mobility and electroosmotic mobility. Electrophoretic mobility is the solute’s response to the
applied electrical field. Cations move toward the negatively charged cathode, anions move toward
the positively charged anode, and neutral species remain stationary. The other contribution to a
solute’s migration is electroosmotic flow, which occurs when the buffer moves through the
capillary in response to the applied electrical field. Under normal conditions the buffer moves
toward the cathode, sweeping most solutes, including the anions and neutral species, toward the
negatively charged cathode1 (Slide 3).
Electrophoretic mobility
The velocity with which a solute moves in response to the applied electric field (Slide 4) is called
its electrophoretic velocity, νep; it is defined as
vep = μepE

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where μep is the solute’s electrophoretic mobility, and E is the magnitude of the applied electrical
field. A solute’s electrophoretic mobility is defined as
μep = q6πηr
where q is the solute’s charge, η is the buffer viscosity, and r is the solute’s radius.
Electrophoretic mobility and, therefore, electrophoretic velocity, increases for more highly charged
solutes and for solutes of smaller size. Because q is positive for a cation and negative for an anion,
these species migrate in opposite directions. Neutral species, for which q is zero, have an
electrophoretic velocity of zero.
Electroosmotic flow (EOF)
The EOF is caused by applying high-voltage to an electrolyte-filled capillary (Slide 5). This
flow occurs when the buffer running through the silica capillary has a pH greater than 3 and the
silanol (SiOH) groups lose a proton to become silanate (SiO-) ions. The capillary wall then has a
negative charge, which develops a double layer of cations attracted to it. The inner cation layer is
stationary, while the outer layer is free to move along the capillary. The applied electric field causes
the free cations to move toward the cathode creating a powerful bulk flow. The rate at which the
buffer moves through the capillary, its electroosmotic flow velocity, νeof,is a function of the applied
electric field, E, and the buffer’s electroosmotic mobility, μeof.
νeof =μeofE
Electroosmotic mobility is defined as
Μeof = εζ4πη
Electroosmotic flow is defined as
μEOF=ϵ4πηEζ
where ε is the dielectric constant of the solution, η is the viscosity of the solution, E is the field
strength, and ζ is the zeta potential.
Since the electrophoretic mobility is greater than the EOF, negatively charged particles,
which are naturally attracted to the positively charged anode, will separate out as well. The EOF

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works best with a large zeta potential between the cation layers, a large diffuse layer of cations to
drag more molecules towards the cathode, low resistance from the surrounding solution, and buffer
with pH of 9 so that all the SiOH groups are ionized. The main benefit of the EOF is a separation
in a single run, of both anions and cations, and also the separation of ions with quite different
charge-to-size ratios in reasonable length of time as well.
Capillary electrophoresis instrumentation
The capillary electrophoresis instrument1, 2 (Slide 6) consists of:
1. Capillary
2. Injection system
3. High voltage source
4. Electrodes and electrode jars
5. Detector
1. Capillary
In CE, the separation occurs in the capillary and this is also the compartment where injection
and detection takes place. Ideally a capillary should be chemically and physically resistant,
precisely machined with narrow internal diameters (d = 20-100 µm; length of 20-100 cm), does not
adsorb solutes, transparent to UV-visible radiation, with a high thermal conductivity, and low cost.
Fused silica capillaries with an external protective polyimide coating meet almost all these
requirements and are hereof a standard choice in CE. Capillaries can be internally ‘uncoated’,
having the inner silica surface in direct contact with buffers and solutes, but, under some
experimental conditions, these may adsorb analytes. Alternatively, the capillaries can be internally
coated with a thin layer of polymers (adsorbed or chemically bound) shielding the silica surface
from interactions with solutes. Capillaries can be also filled with gels, mimicking slab gel
electrophoresis, or with particles carrying a stationary phase, reproducing a chromatographic
system. The most common coatings are polyacrylamide, cellulose, polyvinyl alcohol (PVA), amino
acids, amines, surfactants, and poly(vinylpyrrolidinone) or polyethyleneimine. Polymers well
known as liquid chromatographic (C2, Cg, C1g) or gas chromatographic stationary phases
(polyethylene glycol (PEG), phenyl-methyl silicone) can be used for this purpose too (Slide 7).

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2. Injection system
Modern instrumentation uses two injection principles: hydrodynamic or electrokinetic. In the
first case, a pressure difference is generated between the two ends of the capillary, while the
injection end is dipped in the specimen vial. Alternatively, a voltage difference is established
between the samples and the opposite vial, while samples are being injected. Although
hydrodynamic injection is nonselective, electrokinetic injection (potential driven) is selective. This
is because the sample components enter the capillary according to their electrophoretic mobility,
the mobility and concentration of total ions in the sample and the EOF. Electrokinetic injection
allows field-amplified sample stacking, i.e. a highly efficient method for increasing analytical
sensitivity (in terms of concentration), to be accomplished (Slide 8).
3. High voltage source
The high-voltage power supplies used for CE are generally able to give voltages up to 20-30
kV and currents up to 200-300 µA. Separations are generally carried out under constant potential,
but voltage gradients or steps; and constant current separation is sometimes used. Because EOF in
fused silica capillaries is usually directed towards the cathode, the common polarity in a capillary
electropherograph is with the anode at the injection end of the capillary and the cathode at the
opposite end, close to the detector; however, under specific analytical conditions, the polarity is
reversed (Slide 8).
4. Electrodes and electrode jars
Each side of the high voltage power supply is connected to an electrode. These electrodes help
to induce an electric field to initiate the migration of the sample from the anode to the cathode
through the capillary tube (Slide 8).
5. Detector
In order to gain useful information from the separation technique, it is necessary to detect and
measure analytes. Detection may be qualitative and/or quantitative. While the capillary itself is the
detection cell it is linked to the detection device. It is also possible to couple to detectors that are
outside of the separation capillary although this does require a specialized interface. The most
frequently used method of detection involves absorbance of energy as the analytes move through a
focused beam of light. The CE detection, most often based on UV-visible absorption, is carried out
in-capillary, in order to avoid any possible post-separation added volumes. This can cause
unacceptable band spreading (Slide 9). Indeed, the wall of fused silica capillaries, after removing
the polyimide coating, is highly transparent. Thus, picogram amounts of analytes can easily be

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detected even by UV absorption, but, due to the limitations in the sample volume (nL) which can
be injected, the concentration sensitivity with UV detectors is limited to 10-5 M. However, this limit
in sensitivity can be overcome by using other detection techniques, e.g. laser-induced fluorescence
(sensitivity up to 10-12 M) or electrochemical detection (sensitivity up to 10-8 M) and/or by adopting
high efficiency sample stacking methods, which can assure improvements in sensitivity of 2-3
orders of magnitude. By implementation of diode-array or fast-scanning UV detectors allows on-
line recording of the UV spectra of the peaks, improving the information content of the CEanalysis.
Laser induced fluorescence, conductometric detection, and mass spectrometric with electrospray
ionization detection modes have recently become commercially available.
Indirect detection is based on the addition into the running buffer of an ionic additive which is
detectable at trace levels by the detector. This ionic compound, having the same charge and similar
mobility to the analyte(s) of interest, is displaced from the zones where the analyte(s) migrate to
preserve electroneutrality, and this displacement gives rise to ‘reversed peaks’, the area of which
is proportional to the concentration of the given analyte(s). Drawbacks of this detection mode are
sensitivity, which is usually lower than with the corresponding ‘direct’ mode, and the narrower
range of linearity
Capillary electroseparationtechniques
The most common CE separation techniques1, 2,3, 4 (Slide 10, Slide 11) are:
1. Capillary zone electrophoresis (CZE)
2. Micellar electrokinetic capillary chromatography (MECC or MEKC)
3. Capillary electrochromatography (CEC)
4. Capillary isoelectric focusing (CIEF)
5. Capillary gel electrophoresis (CGE)
6. Capillary isotachophoresis (CITP)
7. Capillary electrophoretic immunoassay (CEIA)
1. Capillary zone electrophoresis (CZE)
The CZE, also known as free solution capillary electrophoresis, is very quickly, easy and the
most commonly used mode of the CE (Slide 12). The separation technique is based on the
differences in electrophoretic mobility, which is directed proportional to the charge on the

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molecule, and inversely proportional to the viscosity of the solvent and radius of the atom. Usually
the end of the capillary containing the sample is the anode and solutes migrate toward the cathode
at a velocity determined by their electrophoretic mobility and the EOF. In the CZE, in addition to
the solutes, the buffer solution usually also moves through the capillary under the influence of an
electric field. Coating the capillary’s walls with a nonionic reagent eliminates the EOF. In this form
of the CZE cations migrate from the anode to the cathode. Anions elute into the source reservoir
and neutral species remain stationary. The CZE provides effective separations of charged species,
including inorganic anions and cations, organic acids and amines, and large biomolecules such as
proteins.
2. Capillary gel electrophoresis (CGE)
In the CGE a capillary is filled with a polymeric gel like cross-linked polyacrylamide, agarose,
or solutions of linear polymers (Slide 13). While the gel is porous, a solute migrates through the
gel with a velocity determined both by its electrophoretic mobility and by its size. The ability to
effect a separation using size is helpful when the solutes have similar electrophoretic mobilities.
For example, fragments of DNA of varying length have similar charge-to-size ratios, making their
separation by CZE difficult. Because the DNA fragments are of different size they can be separated
by the CGE. The capillary used for the CGE is usually treated to eliminate the EOF, preventing the
gel’s extrusion from the capillary tubing. Samples are injected electrokinetically because the gel
provides too much resistance for hydrodynamic sampling. The primary application of CGE is the
separation of large biomolecules, including DNA fragments, proteins, and oligonucleotides. The
main advantages over slab-gel electrophoresis are a wider range of gel matrix and composition,
online detection, improved quantification and automation.
3. Capillary isoelectric focusing (CIEF)
The CIEF is used to separate biological molecules mainly proteins, based on differences
between the isoelectric points (pI). This technique is performed by filling the capillary with a
mixture of ampholytes and the sample, and then forming a pH gradient (Slide 14). By applying an
electric field across the capillary with a basic solution at the cathode and an acidic solution at the
anode, the ampholytes and solutes migrate until they reach a region where their overall charge is
neutral (pH = pI). The ampholyte and solute zones remain extremely narrow because diffusion to
a zone of different pH results in the generation of charge and subsequent migration back to the
proper zone.

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4. Micellar electrokinetic chromatography (MEKC)
By the MEKC is possible to separate neutral and charged solutes as well. Sodium
dodecylsulfate, or SDS, has a long-chain hydrophobic tail and a negatively charged ionic functional
group at its head (Slide 15). When the concentration of SDS is sufficiently large micelles, with the
hydrophobic moieties of the surfactant in the interior and the charged moieties at the exterior, are
formed. Micelles have. The separation of neutral molecules is based on the hydrophobic interaction
of solutes with the micelles. The MEKC selectivity can be controlled by the choice of surfactant
and by the addition of modifiers to the buffer. Because micelles have a negative charge, they
migrate toward the cathode with a velocity less than the EOF velocity. Neutral species partition
themselves between the micelles and the buffer solution in a manner similar to the partitioning of
solutes between the two liquid phases in the HPLC. Hydrophilic neutrals are insoluble in the
micelle’s hydrophobic inner environment and elute as a single band, as they would in the CZE.
Neutral solutes that are extremely hydrophobic are completely soluble in the micelle and elute with
the micelles as a single band. Those neutral species that exist in the partition equilibrium between
the buffer solution and the micelles elute between the completely hydrophilic and completely
hydrophobic neutral species. The MECK is used to separate a wide variety of samples, including
mixtures of pharmaceutical compounds, vitamins, and explosives.
5. Isotachophoresis (ITP)
The ITP uses two different buffer systems where solutes are sandwiched between leading and
trailing electrolytes, creating a steady state in which the solute zones migrate in order of decreasing
mobility. Two unique aspects of the ITP are that all solute zones migrate at the same velocity and
that they all adopt the concentration of the leading electrolyte. The latter aspect makes ITP a very
useful technique for the analysis of dilute solutions. Samples can be concentrated to many orders
of magnitude.
6. Capillary electrochromatography (CEC)
In the CEC the capillary is packed with a stationary phase and when an electric field is applied
the EOF moves the mobile phase through the packed column (slide 16). The separation selectivity
depends on partition between the stationary and mobile phases. This allows high efficiency
separation of neutral compounds within very short analysis times. By the CEC is enabled separation
of closely related compounds, shorter run times, and increased sample transience. As in liquid
chromatography, the CEC separations are carried out by the partition of a solute between the mobile

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and stationary phases. The additional separation by motilities resolves charged and neutral
components.
7. Capillary electrophoretic immunoassay (CEIA)
Coupling competitive immunoassays with CE-driven bound/free fraction separation has
recently gained considerable attention, particularly whenever fluorescent tracers are used. Both, the
CZE or the MECC, can be used to separate the bound from the free fraction of the tracer in a
competitive immunoassay, allowing indirect but highly sensitive quantitation of several analytes in
a variety of biological samples. Additionally, since different tracers can be easily spotted in one
electropherogram, multicomponent immunoassays can be simultaneously carried out in the same
experiment. Many commercial immunoassay reagents can be adapted to CEIA.
Applications of the capillary electrophoresis
Capillary electrophoresis has had extensive applications in a variety of scientific domains,
including chemistry, biochemistry, pharmacology, toxicology, forensic toxicology, forensic
biology/biochemistry (DNA profiling, analysis of proteins and other biological compounds), and
biomedicine as well5, 6, 7, 8.
References
1 Whatley H. Basic principles of capillary electrophoresis. In Clinical and Forensic Applications of
Capillary Electrophoresis. Petersen, J., Mohammad, A.A. (Eds.), Humana Press Inc. pp 21-58
(2001).
2 Harstad, R.K., Johnson, A.C.,Weisenberger, M.M.,Bowser, M.T. Capillary Electrophoresis.
Anal. Chem., 88, 299–319 (2016) DOI: 10.1021/acs.analchem.5b04125
3 Karger, B.L. Guttman, A. DNA Sequencing by Capillary Electrophoresis. Electrophoresis. 30,
S196–S202. (2009) doi: 10.1002/elps.200900218
4 Wuethrich, A., Quirino J.P. Derivatisation for separation and detection in capillary electrophoresis
(2015-2017). Electrophoresis. 39, 82-96 (2018), doi: 10.1002/elps.201700252.
5 Tavares, M. F. M., Jager A., da Silva, C.L.; Morales E.P. Applications of capillary electrophoresis
to the analysis of compounds of clinical, forensic, cosmetological, environmental, nutritional and
pharmaceutical importance. J. Braz. Chem. Soc. 14, 281-290 (2003). 6 Thormann, W. et al.

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Clinical and Forensic Drug Toxicology. In Clinical and Forensic Applications of Capillary
Electrophoresis. Petersen, J., Mohammad, A. (Eds.), Humana Press Inc. 397-422 (2001).
7 Sarr, S.O., et al. (2016) Validation of a Capillary Electrophoresis method for analyzing
chlorphena- mine maleate-based drugs using the accuracy profile approach. Am. J Anal. Chem.,
7, 452-459 (2016).
8 Ticova A., Prikryl J. Reproducible preparation of nanospray tips for capillary
electrophoresis coupled to mass spectrometry using 3D printed grinding device, Electrophoresis,
37, 924-930 (2016).