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Capillary Electrophoresis Methods and Scope.

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Volume 32 . Number 5
May 1993
Pages 629 - 766
International Edition in English
Capillary Electrophoresis: Methods and Scope
New Analytical
Methods
(44)
By Heinz Engelhardt," Wolfgang Beck, Jorg Kohr, and Thomas Schmitt
Capillary electrophoresis is a new analytical technique that has seen a great upswing in recent
years, because this adaptation of electrophoresis may be automated and simple, direct quantification is possible. As in conventional electrophoresis, samples with a large range of molecular weights, from inorganic ions to biopolymers such as DNA and proteins, can be separated.
Uncharged molecules can also be separated when micelle-forming detergents are added to the
buffer; the distribution mechanism that then comes into play increases the separation efficiency of this micellar electrokinetic chromatography. Chiral additives such as cyclodextrins make
the separation of enantiomers possible. The rapid increase in the popularity of capillary
electrophoresis is reflected in instrument sales and the growing number of scientific publications dealing with the method, and it seems that the future of this technique is assured.
1. Introduction'""]
Analytical separation methods such as chromatography
and electrophoresis have played an important role in the
determination of the composition of complex biological, environmentally relevant, and industrial samples. Gas-liquid
chromatography (GLC) and high-performance liquid chromatography (HPLC) allow the separation, identification,
and quantification of the components of complex mixtures in
a very short time. The combination of the high-performance
separation systems with sensitive, selective, and specific detectors, such as UVjVIS diode array detectors (DAD), mass
[*] Prof. Dr. H. Engelhardt, DipLChem. W. Beck, DipILChem. T. Schmitt
Institut fur angewandte physikalische Chemie der Universitiit
D-W-6600 Saarbriicken 11 (FRG)
Dr. J. Kohr
Lonza AG. CH-3930 Visp (Switzerland)
[**I
Frequently used abbreviations: CE = capillary electrophoresis. CGE =
capillary gel electrophoresis, EC = electrochromatography, EOF = electroosmotic flow, GLC = gas-liquid chromatography, HPLC = highperformance liquid chromatography, IEF = isoelectric focusing, ITP =
isotachophoresis. MEC = micellar electrokinetic chromatography,
RSD = relative standard deviation, SDS = sodium dodecylsulfate.
Angel$.. Chem h i . Ed. Engl. 1993, 32. 629-649
spectrometers, and Fourier transform infrared (FTIR) spectrometers makes it possible to identify the separated substances unequivocally. Development in instrumentation is so
far advanced that chromatographic analysis is now almost
fully automated.
The areas of application of GLC and HPLC are clearly
defined by the demands made by the analytical method on
the sample. In view of the vast number of nonvolatile compounds it is not surprising that HPLC is today the most
widely used separation technique with a continuing high
growth rate. (The market for HPLC instruments is at present
growing at over 10% per year.) Problems always arise with
HPLC when polar and ionogenic (and in particular basic)
samples or biopolymers are to be analyzed rapidly and effciently. This is an inherent problem of chromatography with
stationary phases based on silica gel. Although phases are
now available for which such problems are hardly noticeable
(polymer-based stationary phases have not been applied
widely because of their swelling capacity, lower efficiency,
and limited pressure stability), the development of a technique for the separation of ionogenic substances still requires
great skill and a detailed knowledge of the multifarious sorption and exchange processes involved.
Q VCH VEriufs~eseIlschafifi mhH, W-6940 Weinheim, 1993
0570-0833l93lOSo5-06293 l0.00i .25!0
629
Charged particles migrate at different rates in solution
under the influence of an electric field. In the first half of the
century the terms “electrophoresis” o r “electrical transportation” were introduced for this behavior.“ -41 Differences in
the rate of migration can have two causes: either the molecules have different charges and are thus accelerated to different extents by the applied field, or the migrations of the
molecules are slowed down to differing degrees as a result of
their individual frictional resistances. In the simplest case the
separation of a solution of an electrolyte can be carried out
in a tube, since here no interactions with a stationary phase
can occur. In practice, however, distortions of the substance
zones occur on account of differing densities of the electrolyte and convection currents originating from the transport of Joule heat to the capillary wall. In classical electrophoresis, as introduced by T i ~ e l i u s ,gels
~ ~ ~or strips of
paper impregnated with solutions of electrolytes are used to
reduce the distortions arising from convection. In addition,
frictional resistance introduced for macromolecules with low
charge differences increases the separation efficiency. Polyacrylamide gel electrophoresis (PAGE) makes possible the
efficient separation of D N A molecules and proteins. The
separation performance can be optimized by varying the
degree of cross-linking of the gels. Gel electrophoresis can be
used for direct determination of the molecular weight of
proteins denatured by sodium dodecyl sulfate (SDS). In this
case the separation is due solely to the hindrance of the
migration by the gel (in the absence of a gel all proteins
denatured by SDS would migrate at almost equal rates).
Classical electrophoresis (with gels or paper) unfortunately
has two significant disadvantages. Firstly, quantitative analysis is possible only with reflection measurements; proteins
must first be dyed, and the results are subject to considerable
errors. Secondly, the voltage gradient that can be applied
across the gel bed is limited. Since the heat evolved increases
with the square of the voltage, efficient cooling is necessary
to prevent the gel from drying out. The time required for an
analysis using a gel bed 10 cm in length can thus be as much
as several hours. The advantage with respect to modern automated separation processes is that several separations can
be carried out simultaneously in one gel by using separate
tracks; the sample throughput is thus greatly increased. In
Heinz Engelhardt
Jorg Kohr
Wolfgang Beck
Thomas Schmitt
Heinz Engelhardt, born in Nuremberg in 1936, studied chemistry at the University of Erlangen-Nuremberg and gained his doctorate
in 196.5. He was a postdoctoral fellow at Northeastern University in Boston in 1969-f970. In 1971 he obtained his “Habilitation”
in analvtical organic chemistry, and in that year accepted a chair at the University of the Saarland, where he is currently Professor
of Applied Physical Chemistry. Engelhurdt has published more than 130 papers on chromatography and is the author ofthe book
“Hochdruck-Fliissigkeits-Chromatographie”,
which was also published in English, Russian, and Chinese. He is editor of several
,journals, chairman ofthe Arbeitskreis Chromatographie of the Gesellschaft Deutscher Chemiker (GDCh), and a member of the
board ofthe Subdivision of Analytical Chemistr-v ofthe GDCh. In 1992 he obtained the Dal Nogare Award and in 1993, the A . J. P.
Martin Award of the Chromatogruphic Society of Great Britain.
Wolfgang Beck was born in 1964 in FiirthlBavaria and studied chemistry at the University of the Saarland,fiom 1985 to 1990. In
his diploma thesis with H. Engelhardt he synthesized and characterized HPLC stationary phases for the separation of chiral
compounds. Since 1990 he has been working towards his doctorate. His work deals with the improvement ofdetection methods and
the optimization of UV detection, diode arruy detection, and indirect UV detection.
Jorg Kohr, born in 1963 in Saarlouis/Saarland, studied chemistry at the University qf the Saarland (diplorpa 1989) and received
his doctorate in 1992 for his research with H . Engelhardt on the application qf polymer-coated quartz capillaries in capillary
electrophoresis.
Thomas Schmitt was born in 1964 in QuierschiedlSaar andstudied chemistry from f985 to 1991 at the University of the Saarland.
His diploma thesis dealt w’ith the separation of D N A restriction,fragments by capillary gel electrophoresis. Since 1991 he has been
working towards his doctorate in Saarbriicken on the separation of chirul compounds by capillary electrophoresis.
630
Angrn. Chem. I n t . Ed. Engl. 1993, 32, 629-649
addition, the flatbed method readily allows two-dimensional
(2D) operation in which different separation mechanisms are
employed. The excellent separation performance of 2D gel
electrophoresis in protein analysis will be mentioned here
only briefly.
2. Principles of Capillary Electrophoresis
Various attempts have been made to reduce convection
currents in free electrolytes when open tubes are used, for
example, by rotating the tube about its axis.[61The use of thin
capillaries, either of glasst6- or Teflon,I8]with an internal
diameter between 200 and 500 pm was found to be successful. Quartz capillaries[’- 1 2 1 with internal diameters of 25200 pin made it possible to carry out highly efficient separations of proteins and dansyl amino acids. The negative influence of thermally induced convection was greatly reduced
with these capillaries because of the relatively large surface to
volume ratio. Quartz capillaries of the type used in GLC
make it possible to use detectors for the on-line detection of
the separated substances within the capillary. In the second
half of the 1980s the simplicity of the apparatus required led
to an increased interest in capillary electrophoresis (CE),
often referred to as capillary zone electrophoresis (CZE).
Since in all separations the substances are injected as discrete
zones at the beginning of the capillary and the components
of the samples migrate as discrete zones, we shall refer to this
simple case simply as capillary electrophoresis (CE).
The basic construction of a CE apparatus is shown schematically in Figure 1. The thin quartz capillary (25- 100 pm
of the sample zones but not to their separation. This EOF
depends strongly on the pH value of the buffer and the
surface properties of the capillary. It can be so large that not
only neutral molecules but even negative ions are transported
to the detector, that is, in the opposite direction to that of
their electrophoretic migration. Since in most buffers dissociation of silanol groups leads to the presence of negative
charges at the surface of the quartz capillaries, in turn inducing positive charges in the vicinity of the surface, the EOF is
directed towards the cathode; thus the detector is usually
positioned near the cathode. The EOF helps in the transport
of the sample zones to the detector, so that when the EOF is
sufficiently large anions can also be transported to the
cathode. The result of a separation of cationic, anionic, and
neutral substances by means of CE is shown in Figure 2.
Under these conditions all uncharged molecules migrate
with the same velocity, that of the EOF, and cannot be separated, whereas the separation of the ions is possible because
of their differing electrophoretic mobilities.
0.015
I
A
rc“]
acquisition
capillary
inlet
capillary
outlet
@q
_-_ _
-
buffer
reservoir
I
high-voltage
power supply
--
in diameter), 20 to 100 cm in length, bridges the two buffer
vessels, between which a voltage of up to about 30000 V is
applied. A relatively small “slug” of the sample (a few nL!)
is introduced at the anodic end of the capillary. This is done
by replacing the buffer vessel by a sample vial; in other
words, the end of the capillary is dipped into the sample
solution. The advantages and disadvantages of the various
methods of sample introduction are discussed in detail in
Section 3 “Apparatus”.
The migration of the sample is induced by the applied
voltage. The electrophoretic migration is always accompanied by an electroosmotic flow (EOF) of a certain magnitude, which contributes in a passive manner to the transport
AnXm
Chvm. I t i t . Ed Ennl. 1993, 32, 629-649
n
‘L
-
~
t Iminl
‘
‘
Fig. 2. Demonstration of a separation of positive and negative ions using capillary electrophoresis. Conditions capillary: L (length) = 30/37 cm. I D (inner
diameter) =75 pn; buffer: 33 mM borate, pH 9.5; field: E = 350 Vcm-’, UV
detection at 214 nm. Sample: 1 = trimethylphenylammonium bromide, 2 =
histamine, 3 = 4-aminopyridine, 4 = benzyl alcohol. 5 = phenol. 6 = syrinyaldehyde, 7 = 2-@-hydroxypheny1)acetic acid, 8 = benzoic acid. 9 = vanillic
acid, 10 = p-hydroxybenzoic acid.
Apart from this simplest form of CZE in which the separation depends only on mobility differences, numerous variations have been introduced : in capillary gel electrophoresis
(CGE) the capillary is filled with a gel; the electrophoretic
migration of macromolecules is influenced by the gel matrix,
leading to a separation according to molecular size. Uncharged molecules can be separated by means of micellar
electrokinetic chromatography (MEC). When detergents are
added to the buffer, the neutral molecules are distributed
between the buffer and the micelles according to their hydrophobic properties. Since a distribution process is involved,
this is a chromatographic technique. The separation is due to
the mobility of the micelles, which are generally negatively
charged. In the case of isoelectric focusing (IEF) the separa631
5
tion is carried out in a p H gradient, which is formed in the
electric field when ampholytes are added to the buffer. Of
less importance is electrochromatography (EC), in which
HPLC stationary phases are used and the transport of the
samples is brought about only by the electroosmotic flow.
Finally we should mention the oldest capillary separation
technique, isotachophoresis (ITP), which has recently become more important because of its use in CE for sample
concentration.
2.1. Electrophoretic Migration
An increase in voltage and consequently an increase in the
field strength E leads to an increase in the electrophoretic
migration velocity u of the ions and thus to a faster analysis.
The applied voltage decreases over the total length L,,, of the
capillary. The velocity and hence the electrophoretic mobility p is determined from the time t the sample stays in the
capillary between sampling (usually at the anodic end of the
capillary) and detection, and the effective capillary length
L,,, . With commercial equipment the capillary length between detection window and capillary end (usually the cathodic end) can be between 5 and 20cm. This additional
length must be considered when determining mobilities. The
correlation between these values is given by Equation (1 j . A
single ion experiences an accelerating force KB [Eq. (2)],
their size (provided that they have similar comp~sitions);['~J
this makes the electrophoretic separation of large molecules
more difficult, as the migration rate of D N A molecules and
proteins denatured by SDS is identical in pure electrolytes.
Separation can only be achieved when the migration is influenced by sieve effects (e.g. in gels) or exclusion effects.
2.2. Electroosmotic Flow
While electrophoresis is responsible for the separation of
molecules with differing mobilities, electroosmosis causes a
flow of the buffer solution in the electric field. In most cases
the electrophoretic migration of the ions in CE is reinforced
by the electroosmotic flow (EOF), which depends on the
distribution of the charges in the vicinity of the capillary
surface. Almost every surface carries charges; in the case of
quartz capillaries these are negative and induced by dissociation of the silanol groups. The surface charges are balanced
by corresponding opposite charges in the buffer. In a quartz
capillary this double layer, shown schematically in Figure 3,
is dominated by positive ions arranged in a static and a
diffuse layer at the surface. If a field is applied parallel to the
electric field
b
direction of the electroosrnotic flow
electrolyte
KB = z F E
(2)
F is the Faraday constant (96500 Cmol-') and z the effective charge of the ion. This force is opposed by the frictional
force K,, which is approximated by Stokes' Law [Eq. (3)]; 1
is the dynamic viscosity [Pa s] and r the Stokes radius [cm] .
diffuse
layer
mobile
ions
static
layer
adsorbed
ions
capillary wall
Fig. 3. Schematic diagram of the charge distribution at the capillary surface.
The electrophoretic migration velocity u is thus defined by
Equation (4). Electrophoretic separations are possible only
when the ions have different mobilities. The effective charge
equals the formal charge minus the charge contribution of
the surrounding oppositely charged rigid double layer.['41
During the migration the ion takes this fraction of the double
layer with it and thus migrates more slowly than expected on
the basis of its formal charge. This is known as the electrophoretic effect and is greatest for a thin diffuse double
layer surrounding the ion. This characteristic double layer
can be calculated by Debye-Huckel theory, and its thickness
is inversely proportional to the square root of the electrolyte
concentration. It can be shown that the effective ionic
charge, and thus the migration velocity, decreases with increasing ionic strength.
In the case of large molecules with radii greater than the
thickness of the double layer, their mobility is independent of
632
surface, the counterions in the mobile layer are pulled parallel to its axis along with the liquid in the capillary. Thus for
quartz capillaries the induced EOF is in the direction of the
cathode. An extremely flat piston-shaped flow profile results. This leads to considerably less band broadening than
obtained with hydrodynamic flow with applied pressure, for
which a parabolic Hagen-Poisseuille flow profile is observed, which depends strongly on the radius of the capillary
and the flow velocity. In capillaries packed with glass beads
or silica gel particles the E O F should be independent of the
particle diameter of the packing material and should lie in
the same direction as in the empty capillary. It is thus theoretically possible to use either very small particles (1 pm or
less in diameter) o r long columns in a chromatographic separation. The procedure known as electrochromatography
(EC) is thus a subject of increasing interest,["] since it should
combine the selectivity of HPLC with the separation efficiency of CE. By using nonporous particles it is possible to eliminate the contribution to the band broadening caused by
diffusion within the pores.
Angew. Chem. In!. Ed. Engl. 1993, 32, 629-649
The magnitude of the E O F can be described simply by the
Helmholtz equation [Eq. ( 5 ) ] . It is inversely proportional to
the viscosity
of the electrolyte and proportional to its
dielectric constant E , to the applied field strength E, and to
the number of charges at the capillary wall (and thus to (, the
“zeta potential” they induce). In the case of quartz capillaries the E O F decreases with the concentration of the electrolyte and the amount of organic components added, and
increases with the degree of dissociation of the silanol groups
at the surface; in other words, it increases with increasing
pH. The pH dependence of the E O F for quartz capillaries
and the corresponding reproducibility of the mobility is
shown in Figure 4. The largest variations occur at intermediate pH values in the vicinity of the pK value of silicic acid. The
deviations can be reduced, however, by increasing the conditioning time following a change in the pH value of the buffer;
the hysteresis effects are then decreased. For reproducible
analyses conducted with untreated capillaries the rinsing and
conditioning times following a change in the buffer must be
standardized; this can reduce hysteresis effects in the
EOF.[ 6l
10.
a6-
121
2
3
~
5
PH
6
7
8
2.3. Band Broadening
The main contribution to band broadening in capillary
chromatography is that due to the Hagen-Poiseuille flow profile. This contribution increases with the square of the diameter of the capillary and is inversely proportional to the diffusion coefficient of the sample in the electrolyte (Aris-Taylor
equation or mass transfer term of the Golay equation[‘91).
Because of the slow radial diffusion in liquids the flow profile
is not balanced, and capillary liquid chromatography is thus
not possible with capillaries > 50 pm in diameter. The diffusion coefficients of samples in gases are larger by a factor of
I 04, however, and the parabolic flow profile is rapidly balanced OUT by radial diffusion. Capillary gas chromatography
is thus a highly efficient separation technique. Since in CE
the flow profile is piston-shaped as a result of the electroosmotic flow, the contribution of the flow profile to band
broadening can be neglected, and only the longitudinal diffusion term must be taken into account.
The band broadening H can be calculated from Equation (6), where D is the diffusion coefficient of the sample in
9
Fig. 4. pH Dependence of the electroosmotic flow p. Conditions: capillary:
L = 40’47 cm. ID = 7 S pm; buffer: l O m phosphate;
~
neutral marker: benzyl
alcohol: E = 425 Vcm-’
Electroosmotic flow is observed for all electrophoretic
separation techniques, since surface charges can never be
completely eliminated. On the one hand it can lead to a
convective mixing of the electrophoretic zones, while on the
other it plays an important role in the transport of the zones
through the capillary. In capillary electrophoresis the detector is generally situated near the cathode because of the
omnipresent EOF. Since anions are also transported to the
cathode when the velocity of their electrophoretic migration
is less than the EOF, it is possible to separate cations and
anions in one single.analysis. In other CE techniques (e.g. in
MEC) the E O F is used solely for the transport of the (in part
uncharged) samples to the detector. The capillary surface
can be chemically modified to control, eliminate, and even
reverse the EOF. Measuring the magnitude of the EOF is the
single means of determining changes a t the capillary surface,
for example, the irreversible adsorption of components of
the sample. All other methods for the characterization of the
Angcn Clieni. Inf. Ed. EngI. 1993, 32, 629-649
surface fail because of the very low surface area ( < 10 cm2).
Surface-modified capillaries show hardly any hysteresis effects when the buffer is changed, and because of the reduced
adsorption they are extremely well suited for the analysis of
proteins (see Section 4.2.2 “Chemically Modified Capillaries”).
The addition of long-chain cationic detergents such as
cetyltrimethylammonium salts, which are adsorbed at the
silanol groups of the surface, makes a reversal of the EOF
possible.”71 A detergent double layer is formed in which the
positive charges are arranged in the direction of the electrolyte. Capillaries thus coated can be used for the separation of rapidly migrating inorganic ions.“ 81
the electrolyte. Using the relation H = LjN we can calculate
the number of theoretical plates from Equation (7). U is the
(7)
voltage, which decreases along the length L of the column. It
should be mentioned that the mobility p of the ions is a
combination of the electrophoretic mobility pepand the electroosmotic mobility pet,. The number of plates increases with
increasing voltage and decreasing diffusion coefficients of
the samples (in contrast to HPLC, where the number of
plates decreases rapidly with decreasing diffusion coefficients). Giddings[l4]showed that at room temperature and
across a wide range this relationship can be reduced to Equation (8). When voltages between 100 and 50000 V are em-
N=20zU
(8)
ployed for the separation of ions with effective charges z
between 1 and 10, it is possible to achieve up to 10 million
633
theoretical plates per meter. In this respect CE is clearly superior to HPLC. The mathematical expressions derived for
chromatography have been adapted for the description of
the band broadening in CE and can as such also be used to
describe transport phenomena in the capillary. It should be
mentioned, however, that in chromatography samples are
always transported through the detector (after elution from
the column and the consequent dilution) with the same velocity. In CE with on-column detection the migration rate of
the samples at the detector window can vary. A comparison
of the number of theoretical plates is thus not completely
valid.
The high numbers of theoretical plates predicted have indeed been realized for the analysis of DNA molecules in gelfilled capillaries.[2o1However, DNA molecules are a special
case, since they d o not interact with the capillary surface
because of their negative charges. Such high numbers of
plates have not yet been achieved for separations of proteins,
although up to 500000 plates per meter have been obtained
with coated capillaries. A slight degree of adsorption at the
surface leads to local changes in the EOF and thus to a
distortion of the piston-shaped profile. In addition, any adsorption leads to a decrease in mass transfer. The mass transfer term of the Golay equation can no longer be neglected,
and the band broadening increases.
As mobility and the EOF increase with increasing voltage,
the analysis time can be decreased. Because the sample spends
less time in the column, band broadening decreases and the
number of plates increases. When the voltage is increased,
however, the buffer solution warms due to Joule heating.
Heat transport occurs solely across the capillary wall, and a
radial temperature (and thus viscosity) gradient is built up at
right angles to the electrophoretic migration.1z'.221 Cooling
the capillary increases the temperature gradient, but this is
necessary in order to avoid degassing and local overheating.
Viscosity differences between the center of the capillary and
the capillary wall lead to migration differences, and thus to
band broadening and loss of resolution.
The negative influence of this radial temperature gradient
can only be reduced by reducing the inner diameter of the
capillary. In a cylindrical tube the temperature difference
between the center and the wall of the capillary increases
with the square of the capillary diameter. This is the reason
for the use of extremely narrow capillaries (diameter 25100 pm) in CE. However, a decrease in the capillary diameter
implies a reduction in the length of the light path and thus in
the detection sensitivity (Lambert-Beer Law). Alternatively,
Joule heating can also be reduced by reducing the buffer
concentration and/or using buffers with lower ionic conductivity.
The reduction in the conductivity of the buffer is subject
to limits. If there is a large difference between the conductivity in the buffer and sample zones, the induced local variations in the electrical field will lead to distortion of the zones
and thus to a decrease in the separation efficiency. If the
conductivity in the sample zone is greater than that in the
carrier electrolyte, the diminished resistance will lead to a
reduction in the field strength. The molecules at the maximum sample concentration will thus migrate more slowly
than at the flanks of the peak. This leads to an extreme distortion of the zones, with a slow increase and a rapid falloff
634
(leading). When conductivity in the buffer is greater than in
the sample zone, peaks with a high degree of tailing are
observed. Symmetrical peaks are obtained only when the
conductivities are identical. The buffer concentration must
thus be adjusted for the given separation problem (dissociation and mobility of the samples). In addition, mobility differences between sample ions and buffer ions can lead to isotachophoretic effects, which generally result in triangular peak
forms that may cause integration problems.
The buffer concentration can be varied only within narrow limits. An increase in the buffer concentration leads to
more Joule heating, which can be compensated for by lowering the voltage. This, however, increases the time required
for the analysis and thus the time the sample spends in the
capillary. As a result the increase in the longitudinal diffusion leads to a greater band broadening. In the optimization
of the separation efficiency in CE it is thus necessary to take
into account the influence of Joule heating, the conductivity
differences between samples zones and electrolytes, and the
detection sensitivity that can be achieved, in order to obtain
high resolution with short analysis times.
3. Apparatus
Commercial capillary electrophoresis instruments have
been available since 1988, and the number of suppliers is
increasing. The various instruments are quite similar, since
the actual separation system is very simple. Slight differences
are found in the way in which the sample is introduced and
in the number and type of detectors offered. We have no
intention of surveying the market but intend to emphasize
the typical requirements made on the various components of
the apparatus.
3.1. Voltage Source
The voltage should be adjustable within the range from
30 to + 30 kV and should remain constant at the value
required. In addition it is useful to be able to adjust the
voltage and current separately. When the voltage, or better
the current, is monitored during the analysis it may be possible to localize any problems that may occur. In commercial
instruments the high-voltage source shuts off automatically
when the analysis compartment is opened to prevent accidents due to high voltage. Similar precautions must be taken
when an apparatus is constructed in the laboratory.
-
3.2. Sample Introduction
The most difficult problem in capillary electrophoresis is
reproducible sample introduction. The sample zone must be
kept small to avoid band broadening; this requires the injection of very small sample volumes in the range 550 nL.123- 51 Excessively large sample volumes rapidly lead to
peak distortions and loss of resolution.[26. "I To meet these
extreme requirements and for the manipulation of such small
sample volumes, miniaturized"*, 301 and automated sample
introductionrz9 311 is indispensable. Reproducible introduc~
Angeic. Chem. In[. Ed.
Engl. 1993, 32. 629-649
Table 1. Types of injection used in capillary electrophoresis.
Electrokinetic
injection
Hydrostatic
injection
Hydrodynamic
injection
Injection by sample splitting
Injection by
electric field
siphoning effect
pressure or
vaccum
electrical sample splitter and
split-flow systems
Automatization
yes
Yes
yes
no
Smallest sample volume
<10wL
(multiple injection)
<10 WL
(multiple injection)
10 pL
(multiple injection)
> 10 pL by
dosing capillary or HPLC syringe
(no multiple injection)
Discrimination
effects with sample
introduction
Yes
no
no
yes (for electrical splitters)
no (for flow split systems)
RSD values
4.1 %
(from ref. [30])
< 2.9 Y o
2-3%
(experimentally
found)
-
(from refs. (30, 331)
tion of the small sample volumes is an important requirement for quantitative analysis with acceptable standard deviations. The most important types of sample injection used in
various commercial instruments are summarized in Table 1.
can thus be neglected. The amount of sample introduced can
be calculated by using Equation (10). The amount injected
p g x r4 Ah tic
'=
3.2.1. Electrokinetic Injection
In this injection method the sample vial into which the
capillary dips is connected to the voltage source; a voltage is
applied for a short interval causing the components of the
sample to migrate into the separation capillary. The amount
Q of the sample depends on the magnitude of the applied
voltage U i , the time t i during which the voltage is applied,
and the mobility of the components of the sample according
to Equation (9),1301where c is the concentration of the sample
(9)
in the solution. This relationship makes clear the problem of
this method of injection-the discrimination between sample
components of differing mobilities. The electrical resistance
of the sample solution (the ionic strength) relative to that of
the electrolyte solution also affects the reproducibility of the
procedure.[32' A relative standard deviation (RSD) of 4.1 %
for peak area measurements has been determined for automatic electrokinetic sample i n t r o d ~ c t i o n . ' ~
In~general
'
i t is
possible to improve the reproducibility of quantitative analysis greatly by using an internal standard, as is the case for
all procedures which are accompanied by problems with
sample introduction (e.g. capillary gas chromatography).
< 3 % (ideal case for electrical
splitter) (from ref. [34])
- 2 % (for split flow systems)
(from ref. [ 3 5 ] )
8qL
thus depends only on the difference in levels and the injection
time. Injection times of several seconds are normally used,
and the level difference must be adjusted accordingly. Manual injection leads to poor reproducibility in quantification
(RSD 10YO),
while with automatic, computer-aided injection
RSDs of 2%[301 or
can be achieved.
3.2.3. Pressure Injection
In this method the sample is injected by applying a pressure difference between the sample vial and the end of the
capillary; to achieve this the pressure is either increased in
the sample vial or reduced at the end of the capillary. These
two possibilities also make it easy to rinse the capillary with
fresh buffer solutions. The amount of sample introduced is
calculated from Equation (1 I ) and depends only on the pressure difference and the injection time. This is the most com-
Q=
Ap K r4 ti c
8VL
monly used technique in commercial instruments. According
to our experience the RSD lies between 2 and 3 Yo;it can be
reduced to below 1 YOwhen an internal standard is used.
3.2.4. Sample Splitters
3.2.2. Hydrostatic injection
This method takes advantage of a difference in level between the buffer reservoir and the sample vial for the introduction of small sample volumes; the sample solution is introduced into the capillary by siphoning. The sample volume
introduced depends on the difference in levels (generally 510 cm), the time, and the hydrodynamic properties (viscosity
q, density p ) of the electrolyte solution. Density and viscosity
differences between the sample and the electrolyte solution
Angru U w m .h i t Ed. Engl 1993. 32. 629-649
Splitting systems analogous to those used in capillary gas
chromatography have also been described for CE sample
introduction; we can distinguish between electrical and hydrodynamic splitting systems. In the case of the electrical
sample splitter (Fig. 5 ) the junction with the separation capillary is situated at the center of the dosage capillary. Since
the field strengths applied to the two capillaries are unequal,
the sample moves within two different current circuits; the
splitting ratio is determined by the ratio of the two currents
635
dosage
capillary
I
i I
separation
capillary
Fig. 5. Schematic diagram of an electric sample splitter. i,, i,, and i, denote the
currents in the corresponding sections of the capillary: n , is the amount of
sample prior to sample splitting, n1 the amount introduced, and n3 the remainder. Left: during sample splitting. Right: after the sample is split.
in the capillaries. The RSD for this method has been reported to be < 3
In the hydrodynamic splitting system an
HPLC syringe is used to introduce the sample volume into a
T-piece. The splitting ratio is determined by the ratios of
diameter and length of the separation capillary and overflow
capillary.[351An R S D of approximately 2 % has been reported for this technique, which requires, however, relatively
large sample volumes. Other splitting systems, some of
have been described, but they
which use HPLC
have not provided better reproducibility.
3.2.5. Reproducibility of Sample Introduction
The reproducibility of sample introduction is influenced
by a number of factors. Conductivity differences between the
separation electrolyte and the sample s0lution,[~’1 large differences in the concentrations of the various components of
the sample[331and their electrophoretic mobilities,[38] and
differences in the sample matrixL3’] all affect the reproducibility. CE is the method of choice, however, when only very
small sample volumes are available, for example, in the analysis of ions in drops of rainwater.[391In commercial, automated instruments miniaturization of sample introduction is so
advanced that a sample volume of 3 pL suffices for several
injections.
The problems with the reproducibility of sample introduction in CE are due to the small pressure differences and short
injection times required. When larger volumes are injected,
the efficiency of the separation rapidly decreases. Attempts
have therefore been made to introduce larger volumes and to
sharpen the zones prior to the actual separation. This can be
done by making use of isotachophoresis effects (ITP) prior
to the actual CE separation, as will be described in Section 4.6.
Samples may also be concentrated by means of transient
increases in the field strength. This can be carried out most
easily when a water ‘‘plug’’ is injected into the capillary prior
to the actual sample introduction (“sample stacking”).[“’I
The voltage decrease across the “nonconductor” water is so
great that the following sample zone is concentrated in the
much larger field present there. Enrichment factors of up to
100 have been achieved.
3.3. Thermostatting
The main purpose of thermostatting is to remove Joule
heat. Both air and liquid thermostats are used in commercial
636
instruments and allow the temperature to be controlled in
the range from 15 to roughly 60 “C. For most applications
intensive air cooling suffices.[411The influence of temperature on efficiency and selectivity is a t present still a subject of
discussion.One reason for this is that in commercial instruments only the capillary (or sections thereof) is thermostatted, while the buffer reservoir is not always kept at the same
temperature as the capillary. It has been shown, however,
that for the separation of D N A fragments in gel-filled capillaries the actual separation performance decreases with increasing temperature, although the relative migrations, in
other words the selectivity of the separations, can be increased.[421
3.4. Capillaries
Polyimide-coated quartz capillaries 25 to 100 pm in diameter are generally used in CE. In principle glass o r plastic
capillaries could be used, though these are not sufficiently
transparent in the short-wave UV range. The polyimide layer of the quartz capillaries must be removed at the site of the
detector either mechanically or by burning off before the capillary is coated or used.“ 31 Capillaries with UV-transparent
coatings have recently become available. Untreated and unmodified capillaries are used for most applications. The
quartz capillaries obtained from different suppliers differ
with respect to the precision and constancy of their internal
diameter as well as to the treatment of the inner surface and
the optical transmission in the lower wavelength region. It is
thus advisable to treat new capillaries with 1 M NaOH prior
to their initial use in order to obtain complete hydroxylation
of the surface.
Surface modification of the capillaries can be carried out
with the same methods described for the modification of
silica gels for the preparation of stationary phases for HPLC
or for coating capillary columns for gas chromatography. As
already mentioned, changes in the E O F can be used to characterize modified capillary surfaces; characterization by gaschromatographic techniques has also been described.[431The
advantages and disadvantages of modified capillaries and
gel-filled capillaries will be discussed in combination with the
various separation techniques for which they are used.
3.5. Detection
Detection is still the greatest technical problem in CE. Because of the short cell length (mean capillary diameter) the
demands made on the detectors with respect to sensitivity,
noise, stray light effects, etc. are extremely high. Nevertheless, modified UV detectors designed for HPLC are most
frequently used. Detection is carried out directly in the capillary in order to avoid efficiency loss due to peak broadening
outside the capillary. Typical bandwidths of the zone in the
capillary are around 5 mm ( N = 500000), which corresponds
to a volume of 10 nL (for a 50 Fm capillary). These extremely
small volumes are also the reason for the mass sensitivity,
which has often been referred to as astounding. The world
record at present is a reported detection limit of 300 molecules with a signal-to-noise ratio of 3:1[““] for derivatized
Angen. Chem. I n l . Ed. Engl. 1993, 32, 629-649
amino acids detected by laser-induced fluorescence. Although
concentration sensitivity is more important for routine use,
it is restricted by the short absorption path (mean capillary
diameter!). UV Detection in CE gives concentration limits
that are 30 to 100 times lower than those in HPLC.[451In the
case of irradiation at right angles to the capillary axis the
limit depends on the detector noise and the effective cell
length, which is lower than the nominal value (the inner
diameter of the capillary). Stray light effects due to inefficient focusing (light from the capillary wall!) and nonideal
cylindrical capillary geometry[461increase the background
noise; these effects can be largely eliminated by optimization
of the optics (slit, lens, etc.). The detector most often used is
a UV detector with a fixed or variable wavelength. In spite of
the short path length it is also possible to record UV spectra
with sensitive diode array d e t e c t o r ~ [ ~(see
' * ~Fig.
~ ~ 14).
Samples that do not absorb in the UV can be detected with
commercial instruments by means of indirect UV detection,[16.49-531 An electrolyte that absorbs in the UV whose
mobility is similar to that of the sample to be separated is
added to the buffer or is used as a buffer component. Because of the necessity for electroneutrality the amount of
added electrolyte must be less where the sample migrates
(displacement mechanism). As a result the buffer has a
higher transmission, and a negative peak, as shown schematically in Figure 6, is recorded. Examples are given in Sections
4.1.4 and 4.2.1 in the description of ion analysis (Figs. 12 and
17). The detection sensitivity in indirect UV detection depends on the molar extinction coefficient of the added UVabsorbent background electrolyte and corresponds to that of
normal UV absorption.[54'
Fig. 6. Principle of indirect UV detection. 0 = UV-absorbing buffer ions;
o = ionic sample components that are not UV absorbing.
In addition to UV detectors, fluorescence detectors have
also recently become commercially available. The main difference between the two is the light source. Besides the usual
deuterium and xenon flashlamps, much more complex laser
systems are on the market. Since in the latter the excitation
lies in visible range, samples must be derivatized accordingly.
Indirect fluorescence detection is also possible and should
become universally applicable when suitable fluorophores
without quenching effects become available.[55- 571
Conductivity detection and other electrochemically based
techniques appear promising but are as yet not commercially
available. As a highlight we should mention the trace analysis of alkali metal and alkaline earth ions directly in the
capillary by using micro electrode^.^^^^ The problem with
Angen.. Chem. Int. Ed. EngI. 1993, 32. 629-649
conductivity detection is that the conductivity in the zone
containing the substance to be analyzed is only slightly hgher
than that of the electrolyte. Suppression techniques for the
reduction of the buffer conductivity such as those used in
HPLC cannot be applied here. Conductivity detection in CE
has already been described by a number of worker^.[^^-^^] It
has, for example, been possible by using amperometric detection to analyze neurotransmitters in nerve cells directly;
the separation was carried out in 5 pm c a p i l l a r i e ~ . [ ~ ~ - ~ ~ l
Because of the low flow rates (100 nLmin-'), coupling
CE with mass spectrometry also appears feasible. The main
problem is that the transition from the capillary to the ion
source, where high vacuum is necessarily present, must be
constructed in such a way that the eluent is not sucked out
of the capillary. A pressure drop of 1 bar leads to a linear
flow velocity of 1 cm s - ' when applied to a capillary 1 m in
length and with an internal diameter of 50 pm. The laminar
parabolic flow profile thus induced would lead to a noticeable loss of efficiency; a "protective flow" must thus be
applied around the capillary prior to ionization. Electrospray MS permits mass spectrometric detection of biopolymers by multiple ionization.f6'- 7 2 1
Other detection methods (Raman s p e c t r o ~ c o p y ~and
'~~
measurements of on-line radioacti~ity,['~]circular dichro-
Table 2. Detection methods in capillary electrophoresis
Detection
method
Detection limits
absolute
concenamount
tration
[moll
Imoli~l
Typical
applications
Remarks
10-'-10-4
aromatic compounds, standard detection
proteins, nucleic
in CE (available in
all commercial
acids, . . .
instruments)
Indirect UV lO-"-lO-"
absorption
10-8-10-4
metal ions, amines,
possible with
organic and inorganic commercial
ions, sugars
instruments
Fluorescence 10- "-1 0-
10- 9-1 0 - 4
derivatized amino
derivatization
acids, DNA, peptides, necessary for many
proteins
samples
Laser10-21-]0-'7
induced
fluorescence
lO-"-lO-'
DNA fragments,
derivatized amino
acids
lasers are still very
expensive (use in
VIS and near-UV)
Indirect
10-'6-10-14
fluorescence
10-7-10-5
alcohols, amines,
anions, cations,
sugars
only a few
applications
described
Amperometry
10- I6-1O- l 4
10-8-10-6
reducible or oxidizable capillaries with inner
substances, e.g.,
diameters up to
2 pm can be used
neurotransmitters
Conductometry
10- la-] 0-
10-7-10-5
ionic samples. e.g.,
metal ions, amines,
carboxylic acids
changing capillaries
is difficult
Potentiometry
10-8
alkali metal and
alkaline earth ions;
selective detection by
ion-selective
microelectrodes
use and preparation
of microelectrodes
difficult
10-l7
Mass
spectrometry
10-8
proteins, peptides,
drug monitoring
interfaces
commercially
available, coupling
with automated
systems possible
10- "-1o-"j
On-line
radioactivity
measurements
10-'o-10-8
32Pand l4C in biochemically relevant
compounds
good detection
sensitivity using
stop-flow systems
l6
637
ism,1751
refractive index,[76]and capillary
have
been described in the literature. It is not possible at present
to estimate their suitability for routine use.
The limits of detection for the most frequently used detection systems are listed in Table 2. It is clear that excellent
mass sensitivities can be achieved because of the small volumes involved. The concentration sensitivities lie in the
range of those found for HPLC.
Chromophores can naturally be introduced into non-UVactive and nonfluorescing samples prior to the separation by
using the standard derivatization reagents. Special reagents
for electrophoresis that permit the simultaneous introduction of charges are a topic of d i ~ c u s s i o n . [ ~ * - ~ ~ ~
detector
0.006
10
0
I
II
I
A
Iji,
4. Separation Techniques in Capillary
Electrophoresis
I , . .
.., .....,,, ,
,
, ,
,
1 AMP
2 dAMP
3 CMP
L dCMP
5 dTMP
6 GMP
7 dGMP + UMP
8 dADP
9 ADP
10 dGDP
11 COP
12 GDP
13 dCDP
14 dTDP
, 1 5 ~ 0 ~
4.1. Capillary Electrophoresis in Uncoated Capillaries
4.1.1. The Influence of the p H Value
The influence of the pH value on the transport of samples
to the detector has two causes. Firstly, as already mentioned,
a separation based on electrophoretic migration is dominated
more or less by the EOF,whose magnitude is determined by
the dissociation of the surface silanol groups. Secondly, the
mobility of the ions is determined by their degree of dissociation in the carrier electrolyte and thus by its pH value. The
separation can therefore be optimized by changing the pH
value and the buffer. In the case of weak electrolytes the
greatest migration differences, in other words the highest
selectivities, are obtained when the pH value of the buffer lies
between the pKs values of the sample components, as is already known from classical electrophoresis. Nondissociated
samples migrate through the capillary at the velocity of the
EOF. As has been shown, the EOF also depends on the pH
value and the other properties of the electrolyte, in particular
on its ionic strength. The dependence of the EOF on the pH
value in quartz capillaries is shown in Figure 4.
The possibilities provided by the combination of electrophoretic migration and EOF can be demonstrated on nucleotides. The EOF is directed towards the cathode, and the
nucleotides migrate to the anode; triphosphates are the
fastest because of their high charge. Even at high pH values
(pH > 10) the EOF does not suffice to transport the triphosphates to the detector at the cathode. If the electrophoretic
mobilities of the mono- and diphosphates are lower than the
EOF, they will be transported to the detector and will reach
it at different times. The separation of this type of sample is
shown in Figure 7. Under these experimental conditions the
vector component of the EOF is insufficient to transport the
triphosphates, which migrate to the anode compartment. The
triphosphates can be analyzed by reversing the polarity,
while the di- and monophosphates are driven by the EOF in
the opposite direction. The mobility can be varied by the
addition of ion pair-forming substances in such a manner that
all the nucleotides can be separated in one
The separation of anions with greatly different mobilities in one single
analysis is also possible when the EOF is reversed by surface
modification and simultaneous field reversal (see Fig. 16).
638
4.1.2. The Influence of Ionic Strength
The lower the ionic strength, the lower the Joule heating of
the electrolyte. On the other hand, the differences in the conductivity between the sample zone and the carrier electrolyte
should be negligible. The larger the ionic strength of the electrolyte, the lower the EOF.
The influence of the ionic strength on the EOF and the
mobility of the ions is shown in Figure 8a. In all cases the
current is held constant. The neutral marker (benzyl alcohol)
is eluted after about 15 minutes at a concentration of 100 mM,
a1
I
1 1
lOOmM 5.5 k V
5 0 m M 1NpA
r
t Iminl-
30
0
t [min]
-
Fig. 8. Dependence of the EOF on the ionic strength of the buffer. Conditions:
capillary: L = 44 cm. ID =75 pm; buffer: borate solutions of various concentrations, pH 9.5; a) constant current (100 bA); b) constant field (230 Vcm-’).
Sample: 1 = benzyl alcohol, 2 = benzoic acid. 3 = benzene-1,2-dicarboxylic
acid, 4 = benzene-l.3,5-tricarboxylicacid.
Angew. Chem. Int. Ed. Engl. 1993,32, 629-649
30
while the carboxylic acids which migrate to the anode are not
eluted. If the ion concentration is reduced to 20 mM the separation is complete after about 20 minutes, at 10 mM after
5 minutes. The analysis time is determined here by the size of
the EOF, since the effective sample migration is almost independent of the buffer concentration. Because of their lower
mobility with respect to the borate ions of the buffer, the diand tricarboxylic acids migrate as asymmetrical peaks with
tailing. Such triangular peaks are always observed when the
mobility difference between the carrier electrolyte and the
sample is too great.[*]Samples with mobilities greater than
that of the buffer ion exhibit leading. Symmetrical peaks,
and thus high numbers of theoretical plates, are obtained
only when the mobilities of the sample and carrier electrolyte
are equal. Analogous effects are observed when a constant
voltage is used, as shown in Figure 8 b. Comparison of Figures 8 a and 8 b clearly demonstrates the advantages of low
buffer concentrations and high possible voltages: the analysis time can be reduced tremendously (Fig. 8a). Thus low
bufferconcentrations and high field strength are oprimal.
Figure 9 shows the influence of the buffer concentration
on the band broadening H of samples with different mobilities. Low band-broadening and high separation perfor-
concentration of the ions in the sample solution; this also
leads to sharply defined, symmetrical zones. However, high
ionic strength implies a high current density for a given
voltage gradient, and thus an increase in Joule heating.
These effects can be simply measured in the form of deviations from Ohm's law, as shown in Figure 10 for two buffer
systems and capillaries with different inner diameters. The
200,
1
150
I IpAI
100
50
0
0
I
300
5
10
20
25
30
100pm
2100 (
0
P
/c
5
10
15
U [kV]
20
25
30
Fig. 10. Current-voltage dependence as a function of the capillary diameter
and the buffer concentration. Top: buffer: 20 mM borate, pH 9.5. Bottom:
buffer: 25 m M CAPS, pH 11.0.
" o ~ : - ~ 7 *
0
60
-
I
1
LO
15
U [kV]
LOO;
Hlpml
200
75 prn
100 pm
80
100
t [mrnoll
120
34
140
160
Fig. 9. Influence of the buffer concentration on the band width H . Conditions:
capillary: L = 50j54 cm. ID = 50 pm; buffer: borate solutions of various concentrations. pH 8.5; field: E = 560 Vcm-'; UV detection at 214 nm. Sample:
1 = benzene-1.3.5-tricarboxylicacid, 2 = benzene-l,2-dicarboxylicacid, 3 = p hydroxybenzoic acid. 4 = benzyl alcohol.
mance are observed for the neutral marker benzyl alcohol
(breakthrough of the zone with EOF) and for the only weakly dissociated p-hydroxybenzoic acid even at a low buffer
concentration (50 mM). At this buffer concentration the
band broadening for the di- and tricarboxylic acids is intolerably high. Even at higher buffer concentrations the band
broadening is not as low as that obtained for nondissociated
or weakly dissociated samples.
4.1.3. Choice of the Buffer
Several factors must be taken into consideration when
choosing the buffer. As shown above, the mobilities must be
equal in order to obtain a high separation performance. The
concentrations of the buffer ions should be greater than the
Angew. Clwm. Inr. Ed. Engl. 1993, 32, 629-649
advantage of organic buffers, which have low conductivities,
can clearly be seen. Even for the thicker capillary Ohm's law
is obeyed throughout the whole range for the buffer 3-(cyciohexy1amino)-1-propanesulfonic acid (CAPS), while with the
same capillary and a borate buffer the range of linearity only
extends up to 10 kV. The advantages of the thinner capillaries for heat transport can also be seen for the borate
buffer; it is still possible to work at 25 kV with the 50 pm
capillary. It is also possible to achieve shorter analysis times,
since these are inversely proportional to the applied field
strength.
4.1.4. Areas of Application
Electrophoresis in uncoated capillaries is the standard
technique particularly suitable for the separation of small
molecules with permanent charges. Thus it is possible to
separate aliphatic and aromatic carboxylic acids, sulfonic
acids, amino acids, phenols, nucleotides, and amines without
great difficulty. A good survey of recent applicatons is available in the two articIes written by K ~ h r . ~841
' ~An
, example of
a separation of sugars and sugar acids is shown in Fig639
ure 11 ;1s51 Figure 12 shows the separation of cations and
lower aliphatic amines with indirect UV detection. Imidazole
is used here as both an electrolyte component and a UV
Figure 13 shows the CE of a natural tannin
mixture. The separation of charged molecules with relatively
large hydrophobic residues can be improved by the addition
of SDS, as is demonstrated in Figure 14a with aromatic
sulfonic acids. The resolution of the fast migrating components is only possible in the presence of SDS; the migration
of the letter acids is, as expected, not influenced by the SDS.
The scope for the optimization of CE separations by adding
detergents will be discussed in detail in Section 4.5, which
deals with micellar electrokinetic chromatography. The per-
11
10 I
I
I
0
8
4
12
t Iminl
16
20
Fig. 11. Separation ofcarbohydrates by CZE. Conditions: capillary: L =loo/
122 cm, ID = 50pm; buffer: 6 mM sorbate, pH 12.1; field: E = 203 Vcm-'.
I = 13 pA; T = 30°C; UV detection at 256 nm; CE instrument: ABI model
270A. Samp1e:l = raffinose, 2 = 2-deoxy-~-ribose, 3 = galactose, 4 =
glucose, 5 = rhamnose, 6 = mannose, 7 = N-acetylneuraminic acid, 8 =
gluconic acid, 9 = galacturonic acid, 10 = glucuronic acid, 11 = mannuronic
acid (from ref. [ S S ] ) .
8
P
2.541
1
I
2.53
2.52
I
A 2.51
0
-
20
10
tlminl
2.50
0.12,
2.491
1.5
2.0
-
2.5
3.0
t Iminl
3.5
4.0
Fig. 12. Separation of metal ions, amines, and amino alcohols (indirect UV
detection). Conditions: capillary: L = 50/58 cm, ID =75 pm; buffer: 5 mM
imidazole, pH4.5; field: E = 430Vcm-', I = 9pA. Sample:l= N H f , K',
2 = Na', 3 = dimethylamine, 4 = C a t , 5 = trimethylamine, 6= M g Z + ,7 =
Lit, 8 = diethylamine, 9 = triethylamine. 10 = diethanolamine, 11 = triethanolamine. Sample concentrations: 1-3 ppm (from ref. [Sl]).
-
0 190
-
190
AInml
430
190
0.10 7
AInml
430
190
3-0
-
430
6
TKL
oo6190
0
Alnml-
L30
190
XInml
-
I
-
430
' 1".\;
A
0
190
5
t lminl-
Alnrnl-
130
o'rr!!!L
0190
,
A lnml
130
Fig. 13. Separation of tannic acids and their characterization (diode array detection). Spectra are shown for the marked peaks. Conditions: capillary: t =
50/80cm, ID =75 pm; buffer: 50 mM borate, pH 9.5; field: E = 375 Vcm-',
Z = 32 pA. Detection: Perkin Elmer LC-480.
640
190
AInml-
Fig. 14. Separation of aromatic sulfonic acids and their characterization (diode
array detection) a) Conditions: capillary: L = 50/80 cm, ID =75 pm; buffer:
59 mM borate, 50 mM SDS, pH 9.5; field: E = 310 Vcm-', I = 39 pA. Sample.
1 = 2-naphthylamine-6-sulfonic acid, 2 = sulfanilic acid, 3 = benzenesulfonic
acid, 4 = anthracene-2-sulfonic acid, 5 = 1-naphthylamine and 2-methylnaphthylene, 6 = l-hydroxymethyI-2-naphthol-6-sulfonic
acid, 7 = 2-naphthol-6sulfonic acid, 8 = R-acid. 9 = G-acid, 10 = H-acid. b) Spectra for the given
components. Detection was carried out using a Perkin Elmer LC-480 adapted
for capillary electrophoresis.
Angew. Chem. I n [ . Ed. Engl. 1993, 32, 629-649
formance of diode array detectors (DAD) in capillary electrophoresis is also demonstrated in Figures 13 and 14 b. Purity determination by means of UV spectra recorded at the
peak maxima and (for overlapping peaks) at the points of
inflection is possible even for the sharp, narrow peaks obtained in CE; Figure 14b shows the overlap observed for the
on-line UV spectra of the acids.
n u m l CE
tsst anionr
CE mm
lwumd
4.2. Capillary Electrophoresis in Surface-Modified
Capillaries
The EOF can be controlled by chemical modification or
dynamic coating of the capillary surface. The possibility of
the absorption of sample components at the capillary surface
is simultaneously reduced and the reproducibility of the
analysis improved. Surface-modified capillaries are, for example. absolutely necessary for the analysis of proteins.
4.2.1. Dynamically Modified Capiliaries
The simplest method of modifying the surface of quartz
capillaries is to add to the buffer solution a component which
will be preferentially adsorbed by the surface silanol groups.
The resulting layer influences the EOF and also reduces the
absorption due to hydrophobic or eiectrostatic repulsion.
As shown in Figure 15, the use of cationic detergents leads
to the formation of a double layer in which the positive
charges are directed towards the interior of the capillary; the
EOF is reversed as a result. This technique also permits the
J-1
EOF
/
0b1-
ncm
+
i
in&
*anRrn
tsstanium
)i
I
electroosmotic
mobility
@
detectw
L
4
electrophoretic
mobility
overall
mobility
Fig. 16. Migration of ions with normal and with reversed EOF
detection. Chromate is used as the UV absorber,["] and the
capillary is coated with cetyl trimethylammonium bromide,
for example. Care must be taken that the critical micellar
concentration is not exceeded when the detergent is present.
1
2.4
1
2.2
A
2.0
0
0
electric field
1.8
direction of the dedrwsmoticflow
1.6
t Imin 1
double layer of
cationic tensides
capillary wall
Fig. 15 Schematic representation of EOF reversal induced by cationic detergents.
separation of rapidly migrating anions when the field is also
reversed,['71 as is shown schematically in Figure 16; this
should be compared with the case demonstrated in Figure 7,
in which the EOF is directed towards the cathode and the
rapidly migrating triphosphates migrate to the anode. It was
possible to detect the latter by means of polarity reversal,
though the slowly migrating anions were no longer observed.
If the EOF is also reversed, both rapidly and slowly migrating anions can be detected and determined in one single
analysis. Figure 17 demonstrates this for the analysis of inorganic and organic anions in the ppm range with indirect UV
A n g e w Chem. Inr. Ed. Ennl. 1993,32, 629-649
2.A
2.0
2.8
Fig. 17. Separation of inorganic and organic anions. Conditions: capillary:
L = 42/50 cm, ID = 75 pm; buffer: 5 mM chromate, 0.5 mM hexadecyltrimethylammonium bromide (dynamic coating), pH 8.1 ; field: E = 600 Vcm-'.
Sample: 1 = bromide, 2 = chloride, 3 = sulfate, 4 = nitrite, 5 = nitrate, 6 =
azide, 7 = fluoride, 8 = phosphate, 9 = carbonate, 10 = acetate, I 1 = propionate, 12 = butyrate, 13 = valeriate, 14 = D-gluconate.
Positive surface charges are also used in the separation of
proteins. The adsorption of cationic proteins is hindered by
the electrostatic repulsion achieved either by adding detergentsLg2]or by coating the capillary surface with ethylene
imine, which can also be cross-linked at the surface.['081A
high number of theoretical plates is achieved, and symmetrical peaks are observed for basic proteins. When lower
polyamines are added to the buffer[90] the adsorption of
proteins at the capillary surface can be reduced. The efficiencies are high, though the field strength must be kept low
because of the high intrinsic conductivity of the buffer. Since
the EOF is also decreased, the analysis times are relatively
high. Figure 18 shows, as an example, the separation of standard proteins. Table 3 lists the separation systems generally
used for the separation of proteins in uncoated capillaries.
641
Fable 4. Survey of capillary coating methods.
2
Functionality
pH range
Conventional Coatings
trimethylsilyl7
trimethylsilyl9
RP-C8 [a]
9
RP-Cl8 [b]
9
polyethylenglycol3-5
diol3-5
[3-(2,3-epoxypropo~y)propyl]-5
trimethoxysilane [c]
maltose
3-7
pentafluoroaryl7
a-lactalbumin
8
Poljmer coatings
linear polyacrylamide
0
20.0
50.0
t fminl-
Applications
Ref.
small molecules
MEC
proteins
proteins
proteins
proteins
proteins
proteins
proteins
proteins
2-8
proteins
(to 10) [d] IEF
DNA fragments
3-11
proteins
2-6
proteins
7
proteins
7
MEC
7
MEC
7
chiral separations
Fig. 18. Separation of basic proteins in a n untreated capillary. Conditions:
capillary: Polymicro, L = 55/78 cm, ID = 50 pm; buffer:80 mM phosphate,
4 0 m ~1,3-diaminopropane, 2 0 m ~K,SO,. pH 3.5; field: € = 200 Vcm-'
(from refs. [YO, 1131). Sample: 1 = cytochrome C, 2 = lysozyme. 3 = ribonuclease, 4 = chymotrypsinogen.
polyethylenimine
1 -vinyl-2-pyrrolidine
poly(methylglutamate)
polymethyisiloxane (OV1)
polyethyleneglycol
polysiloxanelB-cyclodextrin
Table 3. Protein separations with buffer additives in an uncoated capillary.
[a] RP-CX = n-octylsilyl. [b] RP-C18 = n-octadecylsilyl. [c] Also commercially
available as "glycidoxypropylsilane". [dl Stability range given in ref. [I051 or highest
pH used in measurements.
Buffer [a]
c ImMl
pH
tricine/KCI
CHES/K,SO,/EDTA
phosphate/betaine/K,SO,
phosphate/sultone
1.3-diaminopropane/K,S04
phosphate/BRIJ 35
phosphate/FC 134
borate/SDS
10120
8.3
100/250/1
9.0
40/2000/100 6.7
7.0
100/1000
3.5
40120
7.0
lOjl0 ppm
S0/100 ppm 7.0
10010.5%
10.0
PI
x
Ref.
[x1O3rn-']
____
5-7
5-11
9-11
5-11
9-11
3-11
7-11
4-6
~
500
100
300
300
300
100
300
?
is possible to work at normal field strengths, so that the
separation is complete after about 5 minutes. A further advantage of coated capillaries lies in the greater constancy and
reproducibility of the EOF. A disadvantage is their reduced
stability at high pH values (pH > 7). The use of ionic coatings1'08."31 can in addition lead to a pH-independent flow
or to a reversal of the EOF.
~
[a] Abbreviations: tricine = N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine, CHES = 2-(cyclohexylamino)ethanesulfonic acid, BRIJ 35 = polyethyleneglycol lauryl ether, FC 134 = Fluorad (fluorine-containing cationic
detergent; 3M Company, St. Paul, MN (USA)).
4
I
4.2.2. Chemically Modijied Capillaries
The techniques developed for gas chromatography (polymer coating) and methods used for the modification of silica
gel surfaces (silylation reactions, surface polymerization)
have been applied to the modification of capillaries for use in
electrophoresis. Table 4 contains a list of the most important
coating methods, and their main areas of application, and
the stability ranges of the coatings. The greatest limitation of
the coatings prepared by silanization is their limited stability
at higher pH values, a fact which is painfully well known
from HPLC. The disadvantage of polymer coatings is their
extreme hydrophobicity, which has led to the widespread use
of such phases for protein separation in the presence of nonionic or zwitteri~nic[~']
detergents. As already mentioned,
the coating and its stability can be most easily characterized
by the reduction or constancy of the EOF.
In analogy to classical electrophoresis, acrylamide coatings have often been used for the separation of proteins by
CE;IL6-1 0 3 - 1 0 5 1 these coatings are often described as "linear
acrylamide". Figure 19 shows the separation of the same
standard proteins as in Figure 18 with a polyacrylamidecoated capillary. In spite of the considerably reduced EOF it
642
0
5.0
tlminl
-
10.0
Fig. 19. Separation of basic srdndard proteins i n a capillary coated with linear
polyacrylamide. Conditions: capillary: L = 31/48 cm; buffer: 30 mM citrate,
pH 3.0; field: € = 410 Vcm-'. Sample: 1= cytochrome C. 2 = Iysoryme.
3 = ribonuclease A, 4 = chymotrypsinogen [ c = 0.2 mgmL-'1.
As shown in Figures 18 and 19, the separation of proteins
can be carried out successfully with both dynamically and
permanently coated capillaries. Apart from the general remarks made here, no conclusion as to the advantages and
disadvantages of the two separation techniques is possible at
present.
Apart from protein separation, the main area of application of coated capillaries is in micellar electrokinetic chromatography (MEC), where the EOF must be reduced to
Angew. Chem. Int. Ed. Engl. 1993, 32, 629-649
2 3
obtain long residence times in the capillary.[100. ''I When
gel-filled capillaries are used, the EOF must be eliminated by
capillary coatings in order to keep the gel stationary. Enantiomers could be separated with a capillary coated with a
polysiloxane containing cyclodextrin groups," 'I although
interactions with the capillary surface otherwise always lead
to extreme losses in efficiency.
1
4.3. Isoelectric Focusing (IEF)
In classical electrophoresis, IEF is a highly efficient separation process, and it can also be used for the determination
of the isoelectric points of proteins. A pH gradient is established along the separation pathway in an ampholyte-stabilized ge1.['141In this pH gradient a protein migrates only as
long as it is charged and stops at the pH value corresponding
to its isoelectric point (PI). The ampholytes are either added
to the buffer or covalently bound to the gel.["5-"S1 This is
necessary for the stabilization of the pH gradient because of
the relatively long analysis times. The pH gradient is established before the sample is introduced by applying a voltage.
Stabilizing gels are not required for the transfer of IEF in the
capillary; however. the EOF must be reduced or completely
eliminated in order to allow formation of the pH gradient,[11y-'221Th'is can be done either by coating the capillary
surface or by increasing the viscosity of the buffer solution
by adding highly viscous polymers (e.g. hydroxymethylcellulose).[' 231 The latter is particularly useful, since IEF requires
the use of very high pH values.
In contrast to flatbed IEF, in CE the formation of the pH
gradient in the capillary and the focusing of the proteins is
carried out in one step. The capillary is filled with the solution of the samples in the ampholyte mixture; dilute sodium
hydroxide is introduced into the capillary at the anode side
and phosphoric acid at the cathode side. A high current flows
when the voltage is first applied. The end of the focusing is
evident by the reduction in the current to a constant low
value. After the sample has been focused (and thus separated)
in the capillary, it must be transported to the detector. This
is generally carried out by exchanging the cathode electrolyte
for a NaOH-NaCI solution, which destroys the pH gradient,
mobilizes the focused proteins, and allows their transport to
the detector.['241Since the focusing occurs throughout the
whole capillary, also between the detector and the cathode,
proteins with extremely high PI values cannot be detected by
this method. This can be prevented by filling the capillary
with NaOH beyond the detection site. An example demonstrating the scope of IEF in capillaries is shown in Figure 20.
4.4. Capillary Gel Electrophoresis (CGE)
The enormous growth in popularity of capillary electrophoresis and the introduction of commercial instruments were
induced by the American Human Genome Project. DNA
fragments from sequencing reactions, restriction fragments,
etc. have been separated almost solely by CGE.[", lZ5,
Of the various types of gels used in classical flatbed gel electrophoresis, a~rylarnide,['~']agarose,['28. I3O1 and celluare generally used as the matrix in CGE. The preAngiw Cheni. Int. Ed. Engl. 1993, 32. 629-649
3
0
6
t tminl
-
9
12
Fig. 20. lsoelectric focusing of a protein standard mixture (PI 5.1 -7.5). Conditions: capillary: L = 1 2 cm, ID = 25 mm, coated capillary; buffer: Bio-Lyte
ampholyte, p H 3- 10 (catholyte: 0.01 M phosphoric acid. anolyte: 0.02 M
NaOH); mobilization with 0.08 M NaCl and 0.02 M NaOH; voltage for focuslng
and mobilization: 7 kV; UV detection at 280 nm (0.1 AUFS) (Application
Note 31, BioRad). Sample: 1 = human hemoglobin C, 2 = human hemoglobin A. 3 = equine myoglobin, 4 = equine myoglobin (minor band), 5 =
human carbonic anhydrase. 6 = bovine carbonic anhydrase. 7 = b-lactoglobuIin B.
requisite for the stability of the gel in the capillary is the
complete elimination of the EOF. This can be achieved by
coating or chemically modifying the capillary wall.[1o3,
Initially the gel was often cross-linked with the surface coating during the polymerization in the capillary.[20*13'] The
selectivity of the gels was varied by changing the ratio of the
acrylamide monomer concentration (YOT) to the concentration of the cross-linker (YOC). The remarkable separation
performance of these capillaries is demonstrated in Figure 21.1132.1331 The number of theoretical plates amounts to
several million and increases with increasing migration
This apparent contrast to the situation in chromatography shows that apparently only the longitudinal diffusion contributes to the band broadening, and that at least
for CGE Equation (6) correctly describes the dependence of
the number of plates on the diffusion coefficient.
In practice these fixed gels had some disadvantages. They
dried out very rapidly at the ends of the capillary (bubble
formation) and were thus of no further use. An exchange of
the buffer in the capillaries was either impossible or required
a great deal of time. The thermal stability of such gels was
also poor, and commercial applications could not be realized. The preparation of these capillaries requires a great deal
of skill in the modification of the surface and, in particular,
in carrying out the polymerization in the capillary.[132.1341
These problems can be avoided when the capillary is filled
with a solution of a high molecular weight, water-soluble polymer instead of polymerizing cross-linked gels directly in the
capillary.[' 1361 "Linear polyacrylamide" (0 YOC) is also
used for this purpose.[421The polymers are introduced into
the buffer reservoir, and the capillaries are filled by the application of pressure. The EOF must, of course, also be eliminated, so that the gel remains stationary in the capillary during
the separation; any flow reduces the efficiency of the separation. The selectivity of very high molecular weight "linear
gels" (e.g. 1 2 % T) is similar to that of cross-linked gels
359
643
LO
0
30
30
60
weight fragments, and the separation improves although the
number of plates decreases with increasing temperature. Theory and practice agree here, too. Apart from linear polyacrylamides, hydroxyethylcellulose has also been used suc1391
cessfully for the separation of restriction
UL
120
90
LOO
A
130
\,
120
t [minl
-
I
5
6
thin]
7
Fig. 22. Separation of DNA restriction fragments with "liquid gels". Conditions: capillary: L = 20j27 cm: buffer. 100 r n M Tris-borate buffer. pIf 8.3. 3 %
(w!v) linear polyacrylamide, capillary coated with linear polyacrylamide; field:
E - 300 Vom ' : UV detection at 2.54 nm. T - 20 'C. Sample: pBR 322 flae 111
restriction fragments. Apparatus: Pace System. Beckmnn Instruments.
644
1
tlminl
5
110
polymerized in the capillary. Figure 22 shows the separation
of restriction fragments (PBR 322) in a capillary filled with
"linear gel" ( 3 % T, 0 % C).[1371
Here the number of theoretical plates is greater than 600000 plates per meter. At such
high plate numbers the capillaries should not be coiled, since
this reduces the
As already mentioned. the
separation of DNA fragments in gels is temperature dependent. As a comparison between Figures 22 and 23 (the same
separation carried out at 20 and 50 " C )shows, the selectivity
increases with temperature, particularly for higher molecular
L
- L A
Fig. 3 .I'emperature inlluence o n the separalior. of DNA restrichon fragments. Conditions as in Figure 22: 7 = 5 0 ' T
131
Fig. 21. Separation o f poly(uridine-5'-phosphate). Conditions: capillary:
LE,<
= 45 om. I D = 100 pm: gel- polyacrylamide 6 % T. 5 % C ; buffer. 0.1 M
Tris10.25 M boric acid;7 M urea; field: E = 300 Vcm-l. I = 12 PA: injection:
5000 V. 2 s . UV detection at 260 nm (taken from ref [I-iZ]).
-1
,
3
~
CGE has been used almost solely for the separation of
DNA fragments. The sensitive detection of proteins in gelfilled capillaries at low wavelengths ( < 230 nm) in the UV is
not possible. as acrylamides are not optically transparent
below 250 nm; cellulose derivatives should be useful here.
Interactions between the proteins and the gels may also play
a role, since until now only the successful separation of
proteins denatured with SDS in gel-filled capillaries has been
140. l 4 I 1 Only laser-induced fluorescence has
been used for detection in the DNA sequencing. because the
amounts of sample available are very small; the limit of
detection lies at I O - " M.
4.5. Micellar Electrokinetic Chromatography (MEC)
In CE any uncharged molecules present are eluted between
the cations and the anions and are not separated. The addition
of anionic detergents to the buffer (SDS is generally used)
leads to the formation of micelles around the uncharged molecules; since the micelles carry a negative charge, they migrate to the anode.1142,
1431If the capillaries are not coated,
and the EOF is larger than the rate of migration of the
micelles, they will also be transported to the detector at the
cathode. In this case the effective rate of migration of the
sample components is equal to the vector sum of the electrophoretic migration to the anode and the electroosmotic
velocity. Since micelles always migrate with almost the same
rate, the separation is due to the distribution of the uncharged molecules between the buffer solution and the interior of the micelles; this is shown schematically in Figure 24.
Chromatography is indeed the correct term here, as the
mechanism leading to the separation of the uncharged molecules is based on a distribution equilibrium. In the case of
ions this distribution process is superimposed on the actual
electrophoretic migration.
6,
+--
(3---
direction of
cathode
a6,-
Q--/
Fig. 24. Schematic representation of micellar electrokinetic chromatography
(MEC). S = sample components. p., = electroosmotic mobility, pmc= micelle
mobility; p,,, = net mobility
Optimization strategies for the MEC method will be
demonstrated below with the example of the separation of
fluorenylmethyloxycarbonyl (Fmoc) derivatives of amino
acids. The separation of eleven Fmoc amino acids is shown
in Figure 25. Because of the relatively large contribution
5
0
f [mini
-
10
Fig. 26. Separation of 16 Fmoc amino acids by MEC. The peaks are identified
by the one-letter-code for amino acids; * = system peak. Conditions: buffer:
50 mM borate, pH 9.5, 50 mM SDS; otherwise as in Figure 25.
Besides anionic detergents and organic solvents, neutral or
charged components such as urea, zwitterions, and other
Sources of ion Pairs may be added to the buffer. As the
separation is based on distribution equilibria, it is naturally
also temperature-dependent. Of course high concentrations
of organic components cannot be used, since hardly any
micelles will be formed. However, separation is still possible
I
TG
0
2
1
tlminl
6
I
5
15
10
tlrninl
20
Fig. 25. Separation of eleven Fmoc amino acids by CZE. Conditions: capillary: Chrompack. L = 50/75 cm, ID = 50 pm; buffer: 50 mM borate, pH 9.5;
field: E = 330 Vcm- ': UV detection a t 200 nm. Sample: Fmoc amino acids.
Fig. 27. Separation of 16 Fmoc amino acids by MEC with methanol as buffer
additive. For peak identification see Figure 26. Conditions: buffer. 50 mM borate, pH 9.5, 50 mM SDS, 10% methanol (vjv); otherwise as in Figure 25.
made by the neutral derivatizing reagent, which is the same
for all the samples, the electrophoretic mobility of the derivatives is very similar and almost no separation is observed.
The addition of SDS to the buffer leads to a much better
separation of the Fmoc amino acids (Fig. 26) under otherwise identical conditions. The addition of an organic component to this buffer can lead to a further improvement in the
separation. The separation shown in Figure 27 was carried
out under conditions identical to those in Figure 26 but with
methanol added to the buffer. This additive has an influence
on the distribution equilibrium of the sample between the
buffer and the micelles, changes the EOF, and affects the
solubility of the sample in the
when the concentration of the detergent is in the submicellar
range. It was possible to separate chiral compounds by adding
chiral components such as alkylated amino acid derivatives
and cyclodextrins to the SDS-containing b ~ f f e r . [ ' ~ ~ - ' ~ ' ~
Such a system was also used to separate polycyclic aromatic
hydrocarbons by MEC.r'481High concentrations of urea
and cyclodextrins were used to improve the solubility of the
aromatic compounds in the aqueous buffer. A typical example of the application of MEC for the separation of neutral molecules is shown in Figure 28. Since the variations
with respect to both the samples to be separated and the
organic additives to the aqueous buffer system appear to be
unlimited, MEC is an extremely versatile technique. A very
Angew. Chem. In!. Ed Ennl. 1993,32, 629-649
645
Isotachophoresis
start
steadv state
r i
I
0
5
10
f lrninl
-
I
15
20
Capillary Electrophoresis
Fig. 28. Separation of phenols by MEC. Conditions: capillary: L = 65crn.
1D = 50 p;buffer: 1 mmol SDS in 20 mM borate-phosphate buffer, pH =7.0;
field: E = 300Vcm-', I = 28 pA;UVdetectionat270 nm, T - 25°C. Sample:
1 = water. 2 = acetylacetone, 3 = phenol, 4 = u-cresol, 5 = m-cresol, 6 = p cresol, 7 = o-chlorophenols 8 = m-chlorophenol, 9 = p-chlorophenol, 10 =
2,6-xylenol, 11 = 2,3-xylenol, 12 = 2,5-xylenol, 13 = 3,4-xylenol, 14 = 3,5xylenol. 15 = 2,4-xyIenol, 16 = p-ethylphenol (from ref. [142]). a.u. = absorption units fall scale.
I3
@---I
1
i.
F O
@A
electroosmotic flow
detector
recent monograph['491 provides an excellent survey of its
wide range of applications.
4.6. Isotachophoresis (ITP)
While the electrophoretic separation techniques with capillaries that we have described so far correspond to elution
chromatography (discontinuous sample introduction, constant eluent composition, different rates of migration for the
sample components), ITP" 501 corresponds to displacement
chromatography. In both cases all the components of the
sample migrate with the same velocity. ITP was described
many years ago and was a t that time carried out in Teflon
tubes. The method did not find wide use as an analytical
technique because of the problems associated with the selection of suitable electrolytes. In ITP the sample is introduced
between two electrolytes of different ionic mobility; these
must be chosen in such a way that they limit the mobility
range of the components of the sample. In general the lead
electrolyte has the highest and the terminating electrolyte the
lowest mobility of all the migrating ions. All ions with the
same charge migrate at an equal rate when the stationary
state is achieved.['51* 5 2 1 This is shown schematically in Figure 29 and compared with the potential variation in CE. For
ITP the field strength is different in each zone but constant
within each zone; the field jumps suddenly at the zone
boundary. Thus in ITP only ions with the same charge are
separated. The concentration dependence of the sample zones
can be described by a square-wave function. As in displacement chromatography, the zones follow one another directly. A step-shaped signal is obtained when a conductivity
detector is used.
ITP has been used mainly for the separation of inorganic
ions and of organic carboxylic acids. The method is not
widely used because of detection problems and the difficulty
of finding suitable electrolytes for samples of unknown composition. One specific problem is that the separation of
proteins and other complex mixtures requires suitable spacers, electrolytes with migration rates between those of the
646
+@
&
injection
Fig. 29. Schematic comparison of ITP and CZE.
sample components, for better separation of the zones. Because suitable spacers are not available, ITP has hardly been
used in bioanalytical work. Like displacement chromatography, ITP causes dilute samples to be concentrated and is thus
useful as a concentration process prior to a CE separat i ~ n . [ '1561
~ ~Problems arising from the dosage of relatively
large volumes of dilute samples can thus be overcome. The
concentration step can be carried out directly in the separation capillary used for CE. A short zone of the lead electrolyte is introduced into the capillary filled with the separation electrolyte; then the sample is injected. Sample volumes
~
T
1
1
0
I
I
I
I
0
1
6
8
10
12
11
16
18
20
t IrninlFig. 30. isotachophoresis for sample enrichment in CZE. Lower electropherogram: fluoresceinisothiocyanate (Fitc)-derivatized amino acids (without enrichment by ITP), injection: 2 sj5 kV, separation voltage 25 kV. Upper electropherogram: coupling of an ITP capillary to the CZE capillary. Voltage in ITP:
10 kV. in CZE. 25 kV, injection and sample as above. detection: laser-induced
fluorescence at 488j514 nm (from ref. [153]).
Angew. Chrm. Int. Ed. Engl. 1993, 32, 629-649
in the pL range can be applied. The normal electrolyte is then
introduced as a terminating electrolyte. The enrichment
takes place in the direction of the lead electrolyte with enrichment factors of up to
It has been shown that
with proteins the addition of ammonium acetate to the
sample
can have the same effect; the acetate ion
acts as the lead electrolyte. The coupling of two instruments,
ITP for enrichment and CE for separation, each with a UV
detector. has also been described.“ 5 3 1 When the enriched
ITP zone reaches the first detector, the system is switched
over to CE separation. The enrichment and subsequent CE
separation achieved by such an arrangement is demonstrated
in Figure 30.
4.7. Electrochromatography (EC)
Band broadening in HPLC results primarily from the
parabolic flow profile and diffusion into and out of the pores
of the stationary phase. Attempts have been made to reduce
this contribution to band broadening by reducing the particle
diameter; this reduction is opposed by the high pressure drop
required. In the ideal case the EOF is independent of the
particle diameter of the material used to pack the capillaries.
In addition the piston-shaped flow profile of the EOF leads
us to expect high separation performance. It thus appears
possible in principle to combine the high selectivity of HPLC
with the efficiency of CE by using packed columns.[2’. 1581
The separation principles and stationary phases used are those
of HPLC, but the transport of ionic components is due to
electric migration. Extremely small nonporous particles (1 pm
in diameter) are used in order to reduce the contribution of
the sorption process to band broadening. The favorable theoretical predictions for this technique have not yet been verified experimentally. The reduced plate heights achieved are
not much better than those attained in HPLC.[”] One reason
for this is the detection problem: in CE detection is carried out
directly through the separation capillary ; this is no longer
possible when packed columns are used. Thus the problems
known from micro-HPLC resulting from the coupling
of the detector to the separation capillary also occur
here.
5. Outlook
The separation systems discussed above based on electric
migration are summarized in Table 5. The technique most
often used is certainly CE in uncoated or surface-modified
quartz capillaries (open tubes). Neutral molecules can also
be separated in this system when a micelle-forming agent is
added.
CE has several advantages over classical electrophoresis:
- extremely high separation efficiency,
- simple on-line detection and quantification,
- simple operation,
- short analysis times,
- simple automation,
- low consumption of buffers.
The advantages compared with HPLC are
- the high separation efficiency,
- the rapid establishment of equilibrium when the analytical
conditions are changed,
- short analysis times,
- low consumption of buffers,
- minimal sample preparation (suspensions can be injected
directly).
The disadvantages of CE lie in
- the use of UV detectors because of their low concentration
sensitivity,
- the poor reproducibility and control of the electroosmotic flow,
- the adsorption of the analytes at the surface of the capillary, which is always accompanied by a loss of efficiency.
Commercial instruments, some already second-generation,
are presently available for the routine CE analysis. This is also
reflected in the nature of publications appearing, where practice-oriented papers are slowly outnumbering those describing theory and methods. Whereas in 1990 only 225 papers
were listed under the heading “capillary electrophoresis” in
the review volume of Analytical
(more than
half of these dating from 1988/89), the literature survey for
the two following years[841contains 523 publications. It was
not our intention when compiling this article to provide a
complete literature survey, but to give an objective overview
(although personal preferences have certainly played a part)
Table 5. Separation systems driven by an electric field in a capillary.
Capillary zone
electrophoresis
Capillary gel
electrophoresis
modified/
unmodified capillaries
Electrochromatography
Isotachophoresis in
capillaries
Isoelectric focusing in
capillaries
modified and gel-filled unmodified
capillaries
capillaries
packed
capillaries
unmodified
capillaries
modified/
unmodified capillaries
EOF
controlled/
uncontrolled
EOF
suppressed
with EOF
with EOF
with EOF
EOF
suppressed/
very low
separation by electrophoretic migration;
influence of EOF.
separation by electrophoretic migration
with sieve effect;
no EOF
separation by electrophoretic migration and distribution of the sample between micelle and buffer;
influence of EOF
separation by interseparation by electroaction with a stationary phoretic migration;
phase (LC separation
influence of EOF
system);
EOF functions as pump
wide application range
for small, neutral, and
charged molecules
application range as
limited application range separation of zwitterionic
for HPLC (no practical sample preconcentration samples according to their PI
use so far)
technique for CE
values
many applications for separation according
small and large charged to molecular size of
molecules
DNA molecules
and SDS-denatured
proteins
A n g w . Chetn. In!. Ed. EngI. 1993. 32, 629-649
Micellar electrokinetic
chromatography
separation according to the isoelectric point of the sample;
mobilization of the sample zone
towards detector not necessary
when small EOF present
647
of the current scope and limiations of CE. CE complements
the established separation techniques, HPLC and GC; its
advantages are found in situations where the latter reach
their limits, that is, in the separation of polar and ionogenic,
low molecular weight materials and in the analysis of
proteins and other biopolymers.
As the history of the development of the established separation techniques has shown, method-oriented publications
are typically followed by those dealing with applications in
various areas. The tendency of the analytical chemist to test
as many separations as possible with the new technique can
also be observed here. Even polycyclic aromatic hydrocarbons have been separated with CE; however, we make no
comment as to whether this is a worthwhile application (see
Section 4.5). Further developments are possible by coupling
CE with other separation techniques and with spectroscopic
techniques, in particular mass spectrometry.
The first monographs and collected editions dealing with
I6O1
CE have appeared or been
Our own work in this area has been supported by the
Deutsche Forschungsgemeinschaft, the Fonds der Chemischen
Induslrie, and by graduate scholarships provided by the Saarland to J. Kohr and 7: Schrnitt. We thank friends and colleagues for an intensive and fruitful exchange of ideas. 7:
Schmitt thanks Prof. B. L. Karger, Northeastern University,
Boston, for allowing him to carry out experimental work in his
laboratories. We also thank Beckman and Millipore for
providing us with instruments.
Received: September 16, 1992 [A 904 IE]
German version: Angew. Chem. 1993, 105, 659
Translated by Prof. Dr. T. N. Mitchell, Dortmund (FRG)
[I]P. Konig, Actas Trab. Congr. Sud. Am. Chim. III Congr. Rio de Janeiro,
1937, 2, 334. See also Arch. Pathol. 1939, 192, 271.
[2] G. Berraz, An. Asoc. Quim. Argent. 1943, 31, 96.
[3] T. Wieland, Angew. Chem. 1950, 62, 31.
141 E. L. Durrum, J. Am. Chem. Soc. 1950, 72,2943.
[ 5 ] A. Tiselius, Trans. Faraday SOC.1937, 33, 527.
[6] S. Hjerten, Chromatogr. Rev. 1967, 9, 122-219.
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