Page 70 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

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Encyclopedia of Physical Science and Technology EN002E-79 May 17, 2001 20:28

356 Capillary Zone Electrophoresis

conventional electrophoretic techniques have been dem- with an electrolyte. A schematic of a CE system is pre-
onstrated: zone electrophoresis, isotachophoresis, isoelec- sented in Fig. 1. The capillary ends are immersed in
tric focusing, and sieving separations. Unlike conven- electrolyte-filled reservoirs containing electrodes con-
tional electrophoresis, however, the separations are nected to a high-voltage power supply. A sample is in-
usually performed in free solution without the require- troduced at one end of the capillary (the inlet) and ana-
ment for a casting a gel. As in HPLC, detection is lytes are separated as they migrate through the capillary
accomplished as the separation progresses, with resolved towards the outlet end. As separated components migrate
zones producing an electronic signal as they migrate past through a section at the far end of the capillary, they are
the monitor point of a concentration-sensitive (e.g., ul- sensed by a detector, and an electronic signal is sent to a
traviolet absorbance or fluorescence) detector; therefore, recording device.
the need for staining and destaining is eliminated. Data As in conventional gel electrophoresis, the basis of sep-
presentation and interpretation is also similar to HPLC; arations in capillary electrophoresis is differential migra-
the output (peaks on a baseline) can be displayed as an tion of analytes in an applied electric field. The elec-
electropherogram and integrated to produce quantitative trophoretic migration velocity ν EP (in units of cm/sec)
information in the form of peak area or height. In most will depend upon the magnitude of the electric field, E,
commercial CE instruments, a single sample is injected and electrophoretic mobility, µ EP , of the analyte:
at the inlet of the capillary and multiple samples are
ν EP = µ EP E (1)
analyzed in serial fashion. This contrasts to conventional
electrophoresis in which multiple samples are frequently In a medium of a given pH, mobility of an analyte is given
run in parallel as lanes on the same gel. This limitation by the expression:
of CE in sample throughput is mitigated by the ability
µ EP = q/6πηr (2)
to process samples automatically using an autosampler.
Several multicapillary systems have been introduced to where q is the net charge of the analyte, η is the vis-
increase sample throughput; most of these are designed cosity of the medium, and r is the Stoke’s radius of the
specifically for high-throughput DNA sequencing. analyte. Since the Stoke’s radius is related to molecular
Compared to its elder cousins, CE is characterized mass and therefore to frictional drag, mobility will in-
by high resolving power, sometimes higher than elec- crease inversely with molecular weight and directly with
2
trophoresis or HPLC. The use of narrow-bore capillar- increasing charge. Mobility in units of cm sec −1 V −1 can
ies with excellent heat-dissipation properties enables the be determined from the migration time and field strength
use of very high field strengths (sometimes in excess by:
of 1000 volts/centimeter), which decreases analysis time
µ EP = (I/t)(L/V ) (3)
and minimizes band diffusion. When separations are per-
formed in the presence of electroosmotic flow (EOF), the where I is the distance from the inlet to the detection
plug-flow characteristics of EOF also contribute to high point (termed the effective length of the capillary), t is the
efficiency. In contrast, the laminar flow properties of liq- time required for the analyte to reach the detection point
uid chromatography increase resistance to mass transfer, (migration time), L is the total length of the capillary, and
reducing separation efficiency. V is the applied voltage.
Because of its many advantages, CE is finding increas- In contrast to most forms of gel electrophoresis, the ve-
ing use as a tool in analytical chemistry. In some cases it locity of an analyte in capillary electrophoresis will also
may replace HPLC and electrophoresis, but more often it depend upon the rate of electroosmotic flow. This phe-
is used in conjunction with existing techniques, providing nomenon is observed when an electric field is applied to
a different separation selectivity, improved quantitation, a solution contained in a capillary with fixed charges on
or automated analysis. It was the adaptation of CE tech- the capillary wall. Typically, charged sites are created by
nology in the design of 96-channel DNA sequencers that ionization of silanol (SiOH) groups on the inner surface
enabled a draft of the human genome sequence to be com- of the fused silica. Silanols are weakly acidic and ionize
pleted in less than two years. at pH values above about 3. Hydrated cations in solution
associate with ionized SiO groups to form an electrical
−
double layer: a static inner Stern layer close to the surface
I. PRINCIPLES OF CAPILLARY and a mobile outer layer known as the Helmholtz plane.
ELECTROPHORESIS Upon application of the field, hydrated cations in the outer
layer move toward the cathode, creating a net flow of the
As the name implies, capillary electrophoresis separates bulk liquid in the capillary in the same direction (Fig. 2).
species within the lumen of a small-bore capillary filled The rate of movement is dependent upon the field strength