Page 72 - 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

358 Capillary Zone Electrophoresis

the capillary wall remain constant from run to run. Com-
pounds such as polypeptides and organic amines are no-
torious for interacting with silica surfaces and changing
the level of EOF. A great variety of capillary surface treat-
ments and buffer additives have been developed for reduc-
ing adsorption and controlling EOF, and this continues to
be an active area of research in capillary electrophoresis.
Separation efficiency N in capillary electrophoresis is
given by the following expression:
N = µ APP V/2D (8)
where D is the diffusion coeffcient of the analyte in the
separation medium. This predicts that efficiency is only
diffusion limited and increases directly with field strength.
Note that V = E/L, so increasing capillary length will in-
crease the diffusion time, decreasing efficiency. Capillary
FIGURE 3 Magnitude of electroosmotic flow as a function of pH.
electrophoresis separations are usually characterized by
very high effiency, often as high as several hundred thou-
and charge density of the capillary wall. The population of sand plates. Efficiency in CE is much higher than in HPLC
charged silanols is a function of the pH of the medium, so because there is no requirement for mass transfer between
the magnitude of EOF increases directly with pH until all phases and because the flow profile in EOF-driven systems
available silanols are fully ionized (Fig. 3). The velocity is flat (approximating plug flow) in contrast to the laminar-
ν EOF , of electroosmotic flow can be expressed as: flow profiles characteristic of pressure-driven flow in chro-
matography columns. Resolution, R,inCEisdefined as:
ν EOF = µ EOF E (4)
1/2
R = 1 4
µ EP N µ EP + µ EOF (9)
where µ EOF is the electroosmotic mobility, defined as:
This implies that resolution will be greatest when µ EP
µ EOF = εξ w /η (5) and µ EOF are of similar magnitude but of opposite sign;
however, high resolution will be at the expense of analysis
where ε is the dielectric constant, ξ w is the zeta poten-
time. It is also evident from Eq. (9) that, although Eq. (8)
tial of the capillary wall, and η is the viscosity of the
suggests that use of higher field strengths is the most direct
medium. Electroosmotic mobility can be determined ex-
route to high efficiency, a doubling of voltage yields only
perimentally by injecting a neutral species and measuring
a 1.4-fold increase in resolution, at the expense of Joule
the time, t EOF , when it appears at the detection point:
heat. The most direct route to optimize resolution is to
µ EOF = (I/t EOF )(L/V) (6) increase
µ.
The apparent velocity, ν APP , of an analyte in an elec-
tric field will therefore be the combination of its elec- II. BAND BROADENING IN CAPILLARY
trophoretic velocity and its movement in response to EOF:
ELECTROPHORESIS
ν APP = ν EP + ν EOF = (µ EP + µ EOF )E (7)
Band broadening and the resultant reduction in resolution
Inthepresenceofelectroosmoticflow,analyteswhichpos-
can arisefromseveral contributingfactors. If the total band
sess net positive charge will migrate faster than the rate of
broadening is expressed as plate height H, the contribu-
EOF, analytes which have zero net charge will be carried
tions to band broadening due to initial zone width, diffu-
toward the cathode at the rate of EOF, and anionic analytes
sion and electrodispersion, Joule heating, and adsorption
will migrate towards the cathode at a rate which is the dif-
can be expressed qualitatively as:
ference between their electrophoretic velocity and ν EOF .
If the magnitude of EOF is sufficiently great, all analytes H = h inj + h diff+cond + h joule + h ads (10)
regardless of their charge state will migrate past the de-
tection point. In this regard, performing separations in the
A. Initial Zone Width (h inj )
presence of EOF is highly desirable. However, achieving
reproducible separations requires that EOF be constant, Best resolution will always be obtained by keeping the
and this in turn requires that the surface characteristics of initial sample zone as small as possible. The starting zone