Capillary Electrophoresis and Capillary Electrochromatography.
Chapter 30 An Overview of Electrophoresis.
Electrophoresis is a separation method based on the differential rate of
migration of charged species in a buffer solution across which has been
applied a dc electric field. Positively charged ions migrate towards a
negative electrode and negatively charged ions migrate towards a positive
electrode. For safety reasons one electrode is usually at ground and the other
is biased positively or negatively. Ions have different migration rates
depending on their total charge, size, and shape, and can therefore be
separated. This separation technique was first developed by the Swedish
chemist Arne Tiselius in the 1930s for the study of serum proteins; he was
awarded the 1948 Nobel Prize for this work.
Electrophoresis has been applied to a variety of difficult analytical
separation problems: inorganic anions and cations, amino acids,
catecholamines, drugs, nucleic acids, nucleotides, polynucleotides, and
numerous other. A particular strength of electrophoresis is its unique ability
to separate charged macromolecules of interest in the biotechnology industry
and in biochemical and biological research. For many years, electrophoresis
has been the powerhouse method of separation of proteins (enzymes,
hormones, antibodies) and nucleic acids (DNA, RNA) for which it offers
unparalleled resolution.
An electrophoretic separation is performed by injecting a small band
of the sample into an aqueous buffer solution that is contained in a narrow
tube on a flat porous support medium such as paper or a semisolid gel. A
high de potential is applied across the length of the buffer by means of a pair
of electrodes located at either end of the buffer. This potential causes ions of
the sample to migrate towards one or the other of the electrodes.
30A-1 Types of Electrophoresis
Electrophoretic separations are currently performed in two quite
different formats: one is called slab electrophoresis and the other capillary
electrophoresis. Slab separations are carried out on a thin flat layer or slab of
a porous semisolid gel containing an aqueous buffer solution within its
pores. Ordinarily this slab has dimensions of a few centimeters on a side
and, like a chromatographic thin layer plate, is capable of separating several
samples simultaneously.
Slab electrophoresis is currently the most widely used separation tool
of the biochemist and the biologist. Capillary electrophoresis, which is an
instrumental version of electrophoresis, has been developed and used only in
the last decade and one half and has become an important separation tool
used by chemists and life scientists. In many cases, this new method of
performing electrophoretic separations appears to be a satisfactory substitute
for slab electrophoresis with several important advantages, which will be
described subsequently.
30A-2 The Basis for Electrophoretic Separations
The migration velocity v of an ion in centimeters per second in an
electric field is equal to the product of the field strength E (Vcm –1) and the
electrophoretic mobility ue(cm2V-1s-1). That is
v = ue E
The electrophoretic mobility is in turn proportional to the ionic charge on the
analyte and inversely proportional to frictional retarding factors. The electric
field acts only on ions.
30B) Capillary Electrophoresis.
As useful as conventional slab electrophoresis has been, and continues
to be, this type of electrophoretic separation is typically slow, labor
intensive, difficult to automate, and does not yield very precise quantitative
information. Capillary electrophoresis yields high-speed, high-resolution
separations on exceptionally small sample volumes (0.1 to 10nL in contrast
to slab electrophoresis, which requires samples in the uL range).
30B-1 Migration Rates and Plate Heights in Capillary Electrophoresis.
As we have already seen in the previous equation, an ion’s migration
velocity v depends upon the electric field strength. The electric field in turn
is determined by the magnitude of the applied potential (V, in volts) and the
length L over which it is applied. Thus,
v = ue (V/L)
This relationship indicates that high-applied potentials are desirable to
achieve rapid ionic migration and a rapid separation. It is desirable to have
rapid separations, but it is even more important to achieve high-resolution
separation. So we need to examine the factors that determine the resolution
in electrophoresis.
30B-2 Plate Heights in Capillary Electrophoresis.
In chromatography, both longitudinal diffusion and mass-transfer resistance
contribute to band broadening. However, since only a single phase is
involved for electrophoresis, only longitudinal diffusion need be considered.
It has been shown for electrophoresis, that the plate count (N) is given by
N = (ue*V/2D)
Where D is the diffusion coefficient of the solute in cm2s-1. Because
resolution increases as the plate count increases, it is desirable to use highapplied potentials in order to achieve high-resolution separation.
30B-3 Electroosmotic Flow
When a high potential is applied across a capillary tube containing a
buffer solution, electroosmotic flow usually occurs, in which the solvent
migrates toward the cathode or the anode. The rate of migration can be
appreciable. For example, a 50-mM pH 8 buffer has been found to flow
through a 50cm capillary toward the cathode at approximately 5cm/min with
an applied potential of 25KV. As shown in Figure 30-2 above, the cause of
electroosmotic flow is the electric double layer that develops at the
silica/solution interface.
The rate of electroosmotic flow is generally greater than the
electrophoretic migration velocities of the individual ions and effectively
becomes the mobile-phase pump of capillary zone electrophoresis. Even
though analytes migrate according to their charges within the capillary, the
electroosmostic flow rate is usually sufficient to sweep all positive, neutral,
and even negative species toward the same end of the capillary, so that all
can be detected as they pass by a common point. The resulting
electropherogram looks just like a chromatogram.
The electroosmostic flow velocity v is given by the- equation
V = ueoE
In the presence of electroosmosis, an ion’s velocity is the sum of its
migration velocity and the velocity of electroosmostic flow. Thus,
V = (ue + ueo ) E
Note that ue for an anion will carry a negative sign.
30B-4 Instrumentation for Capillary Electrophoresis.
As shown below, the instrumentation for capillary electrophoresis is simple.
A buffer-filled fused-silica capillary, that is typically 10 to 100 um in
internal diameter and 40 to 100 cm long, extends between two buffer
reservoirs that also hold platinum electrodes. Sample introduction is
performed at one end, and detection at the other. The polarity of the highvoltage power supply can be as indicated, or can be reversed to allow rapid
separation of anions.
Although the instrumentation is conceptually simple, there are
significant experimental difficulties in sample introduction and detection due
to the very small volumes involved. Since the volume of a normal capillary
is 4 to 5 uL, injection and detection volumes must be on the order of a few
nanoliters or less.
Sample Introduction:
The most common introduction methods are electokinetic injection and
pressure injection. With electrokinetic injection, one end of the capillary and
its electrode are removed from their buffer compartment and placed in a
small cup containing the sample. A potential is then applied for a measured
time, causing the sample to enter the capillary by a combination of ionic
migration and electroosmotic flow.
Detection.
Because the separated analytes move past a common point in most
types of capillary electrophoresis, detectors are similar in design and
function to those described for HPLC. One difference in behavior of
detectors is encountered, however, because in capillary electrophoresis each
ion migrates at a rate determines by its electrophoretic mobility. Thus,
analyte bands passes through the detector at different rates, which results in
peak areas that are somewhat dependent upon retention times. In contrast, in
HPLC all species pass through the detector at the velocity of the mobile
phase, and peak areas are independent of retention times. Ordinarily this
time dependence is of no concern.
Table 30-1 lists the detection methods that have been reported through 1988
for capillary electrophoresis. The second column of the table lists
representative detection limits for these detectors.
Absorbance Methods.
Both fluorescence and absorbance detectors are widely used in capillary
electrophoresis, although the latter are more common because they are more
generally applicable. In order to improve the sensitivity of absorbance
measurements, several techniques have been suggested for increasing the
path length of the measurements.
Indirect Detection.
Indirect absorbance detection has been used for detection of species that are
difficult to detect because of low molar absorptivities without derivatizaton.
An ionic chromophore is placed in the electrophoresis buffer. The detector
then receives a constant signal due to the presence of this substance.
Fluorescence Detection.
Just as in HPLC, fluorescence detection yields increased sensitivity and
selectivity for fluorescent analytes or fluorescent derivatives. Laser-based
instrumentation is preferred in order to focus the excitation radiation on the
small capillary and to achieve the low detection limits available from intense
source. Laser fluorescence detection has allowed detection of only 10
zeptomoles or 6000 molecules.
Electrochemical Detection.
Two types of electrochemical detection have been used with capillary
electrophoresis: conductivity and amperometry. One of the problems with
electrochemical detection has been that of isolation the detector electrodes
from the high potential required for the separation. One method for isolation
involves inserting of a porous glass or graphite joint between the end of the
capillary and second capillary containing the detector electrodes.
Mass Spectrometric Detection.
The very small volumetric flow rates of under 1 uL .min from
electrophoresis capillaries makes it feasible to couple the effluent from the
capillary of an electrophoretic device directly to the ionization source of a
mass spectrometer. Currently the most common sample
introduction/ionization interface used for this purpose is electrospray,
although fast atom bombardment has also been applied. Capillary
electrophoresis with mass spectrometric detection is only a decade and a half
old but has become of major interest to biologists and biochemists for the
determination of large molecules that occur in nature, such as proteins, DNA
fragments, and peptides.
30C APPLICATION OF CAPILLARY ELECTROPHORESIS.
Capillary electrophoretic separations are performed in several ways called
modes. It is noteworthy that these modes were first employed in slab
electrophoresis and were subsequently adapted for capillary electrophoretic
separations. These modes include capillary zone electrophoresis (CZE),
capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF),
and capillary isotachophoresis (CITP).
30C-1 Capillary Zone Electrophoresis.
In capillary zone electrophoresis (CZE), the buffer composition is constant
throughout the region of the separation.
30C-2 Capillary Gel Electrophoresis.
Capillary gel electrophoresis (CGE) is generally performed in a porous gel
polymer matrix, the pores of which contain a buffer mixture in which the
separation is carried out.
30C-3 Capillary Isotachophoresis.
In capillary isotachophoresis (CITP) all analyte bands ultimately migrate at
the same velocity; hence, the name from iso for same and tach for speed.
30D CAPILLARY ELECTROCHROMATOGRAPHY.
Electrochromatography is a hybrid of capillary electrophoresis and HPLC
that offers some of the best features of the two techniques. Two types of
capillary electrochromatography have been developed since the early 1980s:
packed column and micellar electrokinetic capillary.
Capillary electrochromatography CEC appears to offer several advantages
over either of the parent techniques. First, like HPLC, it is applicable to the
separation of uncharged species. Second, like capillary electrophoresis, it
provides highly efficient separations on microvolumes of sample solution
without the need for a high-pressure pumping system. In
electrochromatography, a mobile phase is transported through a stationary
phase by electroosmostic-flow pumping rather than by mechanical pumping,
thus simplifying the system significantly.
References:
http://www.acs.org
http://www.cas.org
http://www.chemcenter/org
http://www.sciencemag.org
http://www.kerouac.pharm.uky.edu/asrg/wave/wavehp.html
http://www.scimedia.com/chem-ed/sep/electrop/electrop.htm
http://www.scimedia.com/chem-ed/sep/electrop/cap-el.htm