Page 76 - 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

362 Capillary Zone Electrophoresis

3. Mass Spectrometry IV. CAPILLARY ELECTROPHORESIS
SEPARATION MODES
On-line coupling of a capillary electrophoresis system to a
mass spectrometer (CE–MS) enables mass and structural
One of the major advantages of CE as a separation tech-
information to be obtained for separated components. The
nique is the wide variety of separation modes available.
most common configuration is introduction of the capil-
Analytes can be separated on the basis of charge, molecu-
lary outlet into an electrospray ionization (ESI) interface.
lar size or shape, isoelectric point, or hydrophobicity. The
In this configuration, the outlet electrode of the CE is
same CE instrument can be used for zone electrophoresis,
eliminated and the MS becomes the ground. Two types
isoelectric focusing, sieving separations, isotachophore-
of CE–ESI interfaces have been employed. For conven-
sis, and chromatographic techniques such as micellar
tional capillaries of 50- to 100-µm i.d., a makeup flow
electrokinetic chromatography and capillary electroki-
is introduced via a coaxial sheath flow or liquid junction
netic chromatography. The following sections provide a
coupling to transport the capillary eluant into the ioniza-
brief description of each separation mode and typical
tion source and produce a stable electrospray. In these de-
applications.
signs, the concentric stainless steel capillary serves as the
electrical connection for the electrospray voltage. An al-
ternative design, termed microspray or nanospray, permits V. CAPILLARY ZONE ELECTROPHORESIS
direct introduction of the CE eluant without makeup flow.
Small diameter capillaries are used, with the outlet tip ta-
Incapillaryzoneelectrophoresis(CZE),thecapillary,inlet
pered and metal-coated to establish the ESI high-voltage
reservoir, and outlet reservoir are filled with the same elec-
connection. The major limitation of CE–ESI/MS is the re-
trolyte solution. This solution is variously termed “back-
quirement for volatile buffers, which narrows the choice of
ground electrolyte,”“analysis buffer,” or “run buffer.” In
CE separation modes and resolving power. However, this
CZE, the sample is injected as a plug at the inlet end of
constraint may be relaxed to a degree by the recent intro-
the capillary, and sample components are resolved into
duction of orthogonal or “Z-spray” ESI designs. In some
separated zones according to their mass-to-charge ratio as
studies, an in-line, hollow-fiber dialysis tube has been used
they migrate towards the detection point. It is the simplest
between the separation capillary and the ESI interface.
form of CE and the most widely used.
This technique has been used to remove ampholytes in the
performance of capillary isoelectric focusing–ESI–MS of
proteins.
A. Separation Conditions for Capillary
Zone Electrophoresis
E. Preparative Capillary Electrophoresis
Capillary zone electrophoresis may be performed in the
Because of its high resolving power, CE is often con- presence or absence of EOF. Separations carried out in
sidered for micropreparative isolation. Many of the com- the presence of EOF permit the analysis of cations and
mercially available CE systems have the capability for anions in the same analysis, and the resolution of analytes
automatic fraction collection; however, the desirability of moving counter to EOF can be enhanced. Uncoated fused
using CE as a preparative tool has to be carefully weighed silica capillaries exhibit electroosmotic flow toward the
against the problems encountered in fraction collection. cathode, and the magnitude of EOF increases with pH.
When using narrow-bore capillaries, the volume injected Capillaries coated with an adsorbed or covalent positively
into the capillary is quite small (typically a few nanoliters). charged coating exhibit reversed electroosmotic flow to-
Unless the analyte is in very high concentration, recov- ward the anode. Reversed-charge CZE can be used for
ery of sufficient material requires repetitive injections of analysis of cationic analytes which would adsorb to bare
the same sample. In this case, the run-to-run migration silica capillaries, and for analysis of high-mobility anions
times must be highly reproducible to ensure accurate col- which would not be resolved with normal EOF flow. A dis-
lection of the analyte peak. Also, the recovered analyte advantage of performing CZE in the presence of EOF is
must be stable under the collection conditions for the time variation in the magnitude of EOF, which can compromise
required to collect the desired amount of material (of- migration time and peak area reproducibility. Electroos-
ten several hours). An alternative strategy is the use of motic flow can be controlled or eliminated by coating the
larger diameter capillaries (≥75 µm). However, thermal capillary with an adsorbed or covalent neutral material,
effects may compromise resolution, and low voltages or usually a hydrophilic polymer. Coated capillaries are of-
low-conductivity buffers may be necessary to prevent ex- ten used when analyzing species that have strong affinities
cessive heating. for silica, such as proteins.