Transcript of "Practical capillary electrophoresis"
PREFACE TO THE FIRST EDITION
Capillary electrophoresis (CE) or high-performance CE (HPCE) is making the transition
from a laboratory curiosity to a maturing microseparations technique. Now used in
almost 1000 laboratories worldwide, CE is employed in an ever-widening scope of
applications covering both large and small molecules.
The inspiration for this book arose from my popular American Chemical
Society short course entitled, as is this text, "Practical Capillary Electrophore-
sis." During the first 18 months since its inception, nearly 500 students have
enrolled in public and private sessions in the United States and Europe.
I have been amazed at the diversity of the scientific backgrounds of my stu-
dents. Represented in these courses were molecular biologists, protein chemists,
analytical chemists, organic chemists, and analytical biochemists from indus-
trial, academic, and government laboratories. Interestingly enough, CE provides
the mechanism for members of this multidisciplinary group to actually talk with
each other, a rare event in most organizations.
But the diverse nature of the group provides teaching challenges as well. Most
of the students are well versed in the art and science of liquid chromatography.
However, CE is not chromatography (usually). It is electrophoresis, and it is gov-
erned by the art and science of electrophoresis. For those skilled in electrophore-
sis, CE offers additional separation opportunities that are not available in the
slab-gel format. Furthermore, the intellectual process of methods development
differs from that in either slab-gel electrophoresis or liquid chromatography.
The key to grasping the fundamentals of CE is to develop an understanding of
how ions move about in fluid solution under the influence of an applied electric
field. With this background, it becomes painless to wander through the elec-
trophoretic domain and explain the subdeties and permutations frequently illus-
trated on the electropherograms. Accordingly, a logical approach to methods
development evolves from this treatment. This is the goal of my course, and hope-
fully, I have translated this message into this text.
Since I work independently, without academic or industrial affiliations, the
writing of this text would have been impossible without the help of my friends
and colleagues. In particular, 1 am grateful to Professor Ira Krull and his grad-
uate student, Jeff Mazzeo, from Northeastern University for reviewing the entire
manuscript; Dr. Michael Albin from Applied Biosystems, Inc., for providing his
company's computerized bibliography on HPCE; and the Perkin-Elmer Corpo-
ration including Ralph Conlon, Franco Spoldi, and librarian Debra Kaufman
and her staff for invaluable assistance. I am also thankful to my associates
throughout the scientific instrumentation industry for providing information,
intellectual challenges, hints, electropherograms, comments, etc., many of
which are included in this text. Last, I thank my students for helping me con-
tinuously reshape this material to provide clear and concise explanations of elec-
Finally, many of the figures in this text were produced by scanning the illus-
tration in a journal article with subsequent graphic editing. While all efforts
were made to preserve the integrity of the original data, subtle differences may
appear in the figures produced in this book.
PREFACE TO THE SECOND EDITION
It is hard to believe that seven years have passed since I wrote the first edition of this
book. The time is ripe for a second edition. Not only has capillary electrophoresis
matured, but my ability to articulate the field has improved as well.
I have reorganized this book to better reflect usage in the field. There are now
ten chapters instead of twelve. The material on isotachophoresis has been com-
bined with the section on stacking, and the special topics chapter has been elimi-
nated. With the exception of the introduction and the chapter on basic concepts,
all of the other material has been extensively reorganized and rewritten. Empha-
sis has been placed on commercially available apparatus and reagents, although
gaps in the commercial offerings are discussed as well. Note that micellar electro-
kinetic capillary chromatography (MECC) is considered as a variant of capillary
zone electrophoresis (CZE) and is included in the chapter on secondary equilib-
rium. Cyclodextrins and chiral recognition are covered here as well.
Many thanks to Dr. Bruce McCord, Mr. Ira Lurie, and Professor Ira KruU for
reviewing some of the chapters in this second edition. The author gratefully
acknowledges the support of Hewlett-Packard and in particular Dr. David Heiger.
Much has been said about the ability of capillary electrophoresis (HPCE) to
replace liquid chromatography (HPLC). Clearly it has not. As the first high-
performance condensed phase technique, HPLC quickly replaced gas chro-
matography as the method of choice for separating polar molocules. As food for
thought, imagine if capillary electrophoresis had a 25-year head start over
HPLC. Then perhaps the chromatographers would be fighting the uphill battle
of displacing HPCE. As noted in this text, HPCE is clearly superseding the slab
gel, at least in the fields of DNA separations.
MASTER SYMBOL LIST
A Corrected peak area
A Raw peak area
a Fraction ionized
a Molar absorptivity
a Separation factor
h Detector optical pathlength
C, c Concentration
C Coefficient for resistance to mass transfer in the mobile phase
C^ Coefficient for resistance to mass transfer in the stationary phase
CLOD Concentration limit of detection
CMC Critical micelle concentration
%C Percentage of crosslinker in a gel
D Capillary diameter
D, D Diffusion coefficient
D^^ Solute diffusion in stagnant mobile phase
DR Dynamic reserve
d Particle diameter, chromatography
AH Height differential between capillary inlet and outlet
Ap^ Difference in mobility between two solutes
AP Pressure drop
6 Debye radius
^ Zeta potential
e Charge per unit area
E Field strength
E Acceptable increase in H
E Detector efficiency
8 Dielectric constant
8 Molar absorptivity
8o Permittivity of vacuum
XVi Master Symbol List
/ Frictional force (Stoke's law)
g Gravitational constant
Y Field enhancement factor
Y Obstructive factor for diffusion, Van Deemter equation
H Height equivalent of a theoretical plate
dH/dt Rate of heat production
If Fluorescence intensity
I Excitation source intensity
k' Capacity factor
k' Capacity factor in MECC
K, X Thermal conductivity
K Equilibrium constant
L Length of capillary
L^ Length of capillary to detector
I^ Length of the detector window
Lf Length of capillary from detector to fraction collector
L^ ^^ Length of the unpacked portion of a CEC capillary
L ^^^^^ Length of the packed portion of a CEC capillary
L^ Total length of capillary
l.^. Length of an injection plug
X Tortuosity factor, Van Deemter equation
M Actual mass
MLOD Mass limit of detection
N Number of segments in a polymer chain
N Number of theoretical plates
n Number of charges
P Partition coefficient between water and micelle
AP Pressure drop
O Polymer concentration, size separations
O Quantum yield
O Overlap threshold
Oj Fluorescence quantum yield
O* Entanglement threshold, size separations
Q Quantity of injected material
q Ionic net charge
R Peak ratio
R Displacement ratio
Master Symbol List XVll
r Ionic radius (Stokes' law)
r Capillary radius
S/N Signal to noise ratio
a Peak variance
a Peak variance due to capillary wall effects
cap ^ J
a^ Peak variance due to the detector
a, „ Peak variance due to diffusion
a^^ Peak variance due to electrodispersion
^heat P^ak variance due to Joule heating
a Peak variance due to injection
a^ Peak variance in units of length
o Peak variance from all sources
Absorption time to a stationary phase or wall
Desorption time from a stationary phase or wall
Migration time for a micellar aggregate
Migration time for a neutral "unretained" solute
TR Transfer ratio
%)T Percentage of monomer and crosslinker in a gel
|JL Ionic mobility
|i^ Apparent (measured) mobility
|Li^^ Electroosmotic mobility
|Li^ Electrophoretic mobility
V Partial molar volume of micelle
1) Ionic velocity
1) Mean linear velocity
D^ Electrophoretic velocity
1) Electroosmotic velocity
0)^ ^^ Solute velocity in the unpacked portion of a CEC capillary
) ^^^^^ Solute velocity in the packed portion of a CEC capillary
W.^ Width of an injection plug
W^ Spatial width of a sample zone
W^ Temporal width of a sample zone
X. Intital length of an injection plug
X^ Zone length after stacking
Z Number of valence electrons
1.2 Microchromatographic Separation Methods
1.3 Capillary Electrophoresis
1.4 Capillary Electrochromatography
1.5 Micromachined Electrophoretic Devices
1.6 Historical Perspective
1.7 Generic HPCE Systems
1.9 Sources of Information on HPCE
1.10 Capillary Electrophoresis: A Family of Techniques
Electrophoresis is a process for separating charged molecules based on their move-
ment through a fluid under the influence of an applied electric field. If two solutes
have differing electrophoretic mobilities, then separation v^U usually occur. The
separation is performed in a medium such as a semisolid slab-gel. Gels provide
physical support and mechanical stabiUty for the fluidic buffer system. In some
modes of electrophoresis, the gel participates in the mechanism of separation by
serving as a molecular sieve. Nongel media such as paper or cellulose acetate are
alternative supports. These media are less inert than gels, as they contain charged
surface groups that may interact with the sample or the run buffer.
A carrier electrolyte is also required for electrophoresis. Otherwise known as
the background electrolyte (BGE), the carrier electrolyte, or simply the run
buffer, this solution maintains the requisite pH and provides sufficient conduc-
tivity to allow the passage of current (ions), necessary for the separation. Fre-
quently, additional materials are added to the BGE to adjust the resolution of
the separation through the generation of secondary equilibria. Additives can
also serve to maintain solubility and prevent the interaction of solutes or excip-
ients with the gel matrix or, in the case of capillary electrophoresis, with the
^ Chapter 1 Introduction
capillary wall. The theory and practice of electrophoresis have been the subject
of many textbooks and conference proceedings (1-9).
Apparatus for conducting electrophoresis, such as that illustrated in Figure
1.1, is remarkably simple and low cost. The gel medium, which is supported on
glass plates, is inserted into a Plexiglass chamber. Two buffer reservoirs make
contact at each end of the gel. Electrodes immersed in the buffers complete the
electrical circuit between the gel and power supply. Many samples can be sepa-
rated simultaneously, since it is possible to use a multilane gel. One or two lanes
are frequently reserved for standard mixtures to calibrate the electropherogram.
Calibration is usually based on molecular size or, in isoelectric focusing, pi.
Gels such as polyacrylamide or agarose serve several important functions:
1. they may contribute to the mechanism of separation;
2. they reduce the dispersive effects of diffusion and convection; and
3. they serve to physically stabilize the separation matrix.
The gel composition is adjusted to define specific pore sizes, each for a nom-
inal range of molecular sizes. This forms the basis for separations of macro-
molecules based on size. By proper calibration, extrapolation to molecular
weight is straightforward.
Reduction of convection and diffusion is an important function of the gel
matrix. The production of heat by the applied field induces convective move-
ment of the electrolyte. This movement results in band broadening that reduces
the efficiency of the separation. The viscous gel media inhibits fluid movement
in the electric field. Such a material is termed anticonvective. Since the gel is of
high viscosity, molecular diffusion is reduced as well, further enhancing the effi-
ciency of the separation.
FIGURE 1.1 Drawing of an apparatus for slab-gel electrophoresis.
1.2 Microchromatographic Separation Methods 3
Finally, the gel must be sufficiently viscous to provide physical support. Low
viscosity solutions or gels would flow if the plate is not held level. Immersion
in detection reagents would be impossible, since handling or contact with fluid
solutions would destroy the matrix and separation. In the capillary format, the
gel is unnecessary since the walls of the capillary provide the mechanical sta-
bility for the separation.
The basic procedure for performing gel electrophoresis is as follows:
1. prepare, pour and polymerize the gel;
2. apply the sample;
3. run the separation;
4. immerse the gel in a detection reagent;^
5. destain the gel;
6. preserve the gel; and
7. photograph or scan the gel for a permanent record.^
These steps are extremely labor intensive. High performance capillary elec-
trophoresis (HPCE) is the automated and instrumental version of slab-gel elec-
trophoresis. In the DNA applications arena, the most important of which include
DNA sequencing, human identification, and genetic analysis, HPCE is rapidly
replacing the slab-gel as the separation method of choice.
The separation of some polymerase chain reaction (PCR) products is shown
in Figure 1.2. A restriction digest, used as a sizing standard, appears in the outer
lanes. The middle three lanes of the gel show a triplicate run of a 500-mer dou-
ble-stranded DNA PCR reaction. Quantitation for such a separation is difficult
and often imprecise, but such information can be obtained with the aid of a gel
scanner. Recoveries of material from the gel are performed using procedures
such as the Southern blot (10). Sufficient material is recoverable for sequenc-
ing or other bioassays.
Separations of the sizing standard and 500-mer PCR product by HPCE using
a size selective polymer network are shown in Figure 1.3. Quantitation is read-
ily performed using peak area comparison with the standard. However, fraction
collection is difficult relative to the slab-gel, particularly for trace impurities,
since only minuscule amounts of material are injected into the capillary.
The evolution of chromatographic methods over the last 40 years has produced
a systematic and rational trend toward miniaturization. This is particularly true
lOn-line detection is performed on an instrument such as an automated DNA sequencer.
^Automated gel scanners can be used in place of gel archiving or photography.
Chapter 1 Introduction
FIGURE 1.2 Slab-gel electrophoresis of a 500-mer double-stranded PCR reaction product in a
1.8% agarose ethidium bromide gel. Courtesy of Bio-Rad.
for gas chromatography, where the advantages of the open tubular capillary dis-
placed the use of packed columns for most applications.
Chromatographic separations all function via differential partitioning of a
solute between a stationary phase and a mobile phase. A packed column offers
solutes "a multiplicity of flow paths, some short, the majority of average length,
and some long (11)." Solute molecules select various paths through the chro-
matographic maze. The detected peak suggests this distribution and is broad-
ened. In the open tubular capillary, the choices for solute transport are limited,
so that the solute elutes as a narrow band.
In order for the open tubular capillary to function properly, its diameter must
be quite small. Larger diameter capillaries present a problem, since solutes away
from the walls do not sense the stationary phase in a timely fashion. However,
a major problem with narrow inner diameter (i.d.) capillaries is loading capac-
ity. Injection sizes must be kept small to avoid overloading the system. In gas
chromatography (GC) this problem is overcome in part, since sensitive detec-
tors such as the flame ionization detector (FID), electron capture detector
(ECD), and mass spectrometer are easily interfaced.
Improved efficiency is one of several advantages obtained through minia-
turization. The most important of those is improved mass limits of detection
1.2 Microchromatographic Separation Methods
FIGURE 1.3 Capillary gel electrophoresis of a 500-mer (top) double-stranded PCR reaction prod-
uct and a low molecular weight sizing standard (bottom). Capillary: 50 cm x 50 [im i.d. Bio-Rad
coated capillary; buffer: 100 mM tris-borate, pH 8.3, 2 mM EDTA with linear polymers; injection:
electrokinetic, 8 kV, 8 sec; detection UV, 260 nm. Courtesy of Bio-Rad.
(MLOD). Since dilution of the solute is minimized in the miniaturized system,
better MLODs are obtained than in large scale systems. This is particularly
important when the available sample size is small, as sometimes happens in bio-
Miniaturization of GC has been exquisitely successful. These triumphs could
not be directly transferred to liquid chromatography (LC) for several reasons.
The most important is the lack of good detectors. Interface to the FID and ECD
is not practical due to the incompatibility of the mobile phase with each detec-
tor. Pumping of the mobile phase at the low flow rates required by miniatur-
ization is also more complex, particularly when gradient elution is required.
Despite these problems, |I-LC systems are useful in sample-limited situations
and for mass spectrometry where the reduced liquid flow rate is advantageous.
Several books have been devoted to this important field (12-14).
6 Chapter 1 Introduction
Most of work with i-LC employs 250 |im i.d. packed columns, and so the
advantages enjoyed by open tubular GC are not realized in |Li-LC. The instru-
mental problems of injection and detection posed by open tubular LC have
inhibited most people from using this technology
1.3 CAPILLARY ELECTROPHORESIS
The arrival of HPCE solved many experimental problems of gels. Use of gels is
unnecessary since the capillary walls provide mechanical support for the car-
rier electrolyte.3 The daunting task of automation for the slab-gel format is
solved with HPCE. Sample introduction (injection) is performed in a repeatable
manner. Detection is on-line, and the instrumental output resembles a chro-
matogram. The use of narrow diameter capillaries allows efficient heat dissipa-
tion. This permits the use of high voltage to drive the separation. Since the speed
of electrophoresis is directly proportional to the field strength, separations by
HPCE are faster than those in slab-gels. On the other hand, the relative speed
of the slab-gel is enhanced, since multiple samples can be separated at once.
HPCE is a serial technique; one sample is followed by another. This limitation
has been overcome through the use of the capillary array for high throughput
applications such as DNA sequencing (15,16) and serum protein analysis (17).
Commercial instruments are now available for these applications.
HPCE represents a merging of technologies derived from traditional elec-
trophoresis and high performance liquid chromatography (HPLC). Both HPCE
and HPLC employ on-line detection. Developments in on-column micro-LC detec-
tion have directly transferred over to capillary electrophoresis. One of the modes
of HPCE, micellar electrokinetic capillary chromatography (Chapter 4), can be
considered a chromatographic technique. Electrically driven separations through
packed columns (Chapter 7) have been reported from many laboratories. While
there is much in common between chromatography and electrophoresis, the fun-
damentals of HPCE are based on electrophoresis, not chromatography.
Professor Richard Hartwick, formerly from the State University of New York
at Binghamton, started many of his lectures on capillary electrophoresis with a
discussion of transport processes in separations. While performing a separation,
there are two major transport processes occurring:
Separative transport arises from the free energy differences experienced by
molecules with their physicochemical environment. The separation mechanism
may be based on phase equilibria such as adsorption, extraction, or ion exchange.
Alternatively, kinetic processes such as electrophoresis or dialysis provide the
mechanism for separation. Whatever the mechanism for separation, each indi-
vidual solute must have unique transport properties for a separation to occur.
^Gels are occasionally used in HPCE for running size separations. Pumpable polymer networks
are preferred, since they can be changed for each run.
1.3 Capillary Electrophoresis 7
Dispersive transport, or band broadening, is the sum of processes of the dis-
persing zones about their center of gravities. Examples of dispersion processes
are diffusion, convection, and restricted mass transfer. Even under conditions
of excellent separative transport, dispersive transport, unless properly con-
trolled, can merge peaks together.
According to the late Professor Calvin Giddings as paraphrased by Hartwick,
"separation is the art and science of maximizing separative transport relative to
dispersive transport." In this regard, capillary electrophoresis is perhaps the
finest example of optimizing both transport mechanisms to yield highly effi-
Figures 1.4 and 1.5 illustrate this concept, using a series of barbiturate sep-
arations to compare HPCE and HPLC. The mode of electrophoresis used in Fig-
ure 1.5 is micellar electrokinetic capillary chromatography (MECC), an
electrophoretic technique that resembles reversed-phase LC. In the LC separa-
tion amobarbital and pentabarbital coelute, but they are resolved by HPCE.
With some optimization work, amobarbital and pentabarbital can be sepa-
rated by HPLC. But with HPCE, methods development often progresses rapidly
because of the enormous peak capacity of the technique. Peak capacity simply
describes the number of peaks can be separated per unit time. With a couple of
hundred thousand theoretical plates,"^ many separations occur without exten-
sive optimization efforts. In addition, peak symmetry is excellent using HPCE
unless wall effects (Section 3.3) occur. With the absence of a stationary phase,
many factors that contribute to peak broadening and tailing are minimized.
It would be misleading to state that all separations are superior by HPCE or
that methods development will always be straightforward. It is realistic, how-
ever, based on the experiences of many separation scientists skilled in the art of
both techniques, to predict that HPCE will provide the requisite speed and res-
olution in the shortest possible run time with the least amount of methods
development, under most circumstances.
These same two figures illustrate an important limitation of HPCE, the con-
centration limit of detection (CLOD). In Figure 1.4, the LC separation requires
a 1.25 |Lig/mL solution to give full scale peaks with 1-2% noise (the postcolumn
reagent merely alkalized the mobile phase, permitting sensitive detection at 240
nm). The CLOD is approximately 30-fold better by HPLC. The MECC separa-
tion shown in Figure 1.5 required a solute concentration of 100 |ag/mL for a
similar response, although the noise was lower (0.5%).^ On the other hand, the
MLOD by capillary electrophoresis exceeds HPLC by a factor of 100. The ideal
detector for HPCE will be mass sensitive and not depend on the narrow optical
pathlength defined by the capillary itself. Descriptions, advantages, and limita-
tions of many HPCE detectors can be found in Chapter 9.
^The theoretical plate (N) is a measure of the efficiency of a chromatographic of electrophoretic
peak; N = 5.5'(t^/Wiy, where t^ is the migration time and W is the peak width at half height.
5The CLOD can easily be improved through the use of stacking and/or extended pathlength
8 Chapter 1 Introduction
TIME (MIN.) 11
FIGURE 1.4 Reversed-phase liquid chromatography of barbiturates. Column: Econosphere Cis,
25 cm X 4.6 mm i.d.; mobile phase: acetonitrile : water, 55/45 (v/v); injection size: 20 jxL; flow rate:
1.2 mL/min; postcolumn reagent: borate buffer, pH 10, 0.2 mL/min; detection: UV, 240 nm; solutes:
(1) barbital, (2) butethel, (3) amobarbital and pentabarbital, (4) secobarbital; amount injected: 25
ng of each barbiturate from a 1.25 |Llg/mL solution.
The preceding comparison is significant since a |Li-separation technique is
compared with conventional HPLC using a 4.6 mm i.d. column. Would it be bet-
ter to compare HPCE with |i-LC? Perhaps so from an academic standpoint, but
this would not reflect the current usage and thinking in the real world. Chemists
are contemplating using HPCE to replace or augment conventional HPLC as well
as |i-LC. Table 1.1 provides a comparison of slab-gel electrophoresis, |I-LC,
HPLC, and HPCE. Two disadvantages of HPCE compared to conventional HPLC
are sensitivity of detection and precision of analysis. These have prevented the
most widespread use of HPCE. On the other hand, HPCE is replacing the slab-
gel for most high-throughput DNA applications. In this case, the ease of automa-
tion, precision and ruggedness of HPCE supercede the slab-gel.
1.3 Capillary Electrophoresis
TIME (MIN.) 10
FIGURE 1.5 Micellar electrokinetic capillary chromatography of barbiturates. Capillary: 50 cm
(length to detector) X 50 |lm i.d.; buffer: 110 mM SDS, 50 mM borate, pH 9.5; injection: 1 sec vac-
uum (5 nL); detection: UV, 240 nm; solutes: (1) phenobarbital, (2) butethel, (3) barbital, (4) amo-
barbital, (5) pentobarbital, (6) secobarbital; amount injected: 500 pg of each barbiturate from a 100
HPCE is a novel and alternative format for both liquid chromatography and
electrophoresis. The unique properties of this technique include the use of:
1. capillary tubing in the range of 25-100 jim;
2. high electric field strength;
3. on-line detection in real time;
4. only nanoliters of sample;
5. limited quantities of mostly aqueous reagents; and
6. inexpensive capillaries relative to HPLC columns.
The molecular weight range of analytes separable by HPCE is enormous. A
search of the literature reveals applications covering small ions, small molecules,
10 Chapter 1 Introduction
TABLE 1.1 Comparison of Slab-Gel Electrophoresis, p-LC, Conventional LC, and HPCE
peptides, proteins, DNA, viruses, bacteria, blood cells, and colloidal particles.
The molecular weight range of HPCE is easily from 3 for a lithium ion to
100,000,000 for a virus or particle.
1.4 CAPILLARY ELECTROCHROMATOGRAPHY
A hybrid of chromatography and electrophoresis, capillary electrochromatog-
raphy (CEC) employs the electrically driven electroosmotic flow (EOF) to
pump a mobile phase through a packed capillary. The use of the EOF to gener-
ate flow solves some of the instrumental problems of pumping at nL flow rates.
Capillary electrochromatography employs small diameter capillaries filled
with a stationary phase. Reversed-phase packings are most often used, although
an application with a cation-exchange material has been reported (18). An
amazing efficiency 8 million plates per meter was reported in that paper, though
the mechanism and reproducibility of the effect are still unclear.
1.6 Historical Perspective 11
Typically, 50 |im i.d. capillaries are used though larger diameter tubes can be
employed at the expense of efficiency. Particle diameters of 3-5 |im porus mate-
rial are most common, though it is possible to employ 1.5 |Lim pellicular pack-
ing. Since there is no pressure drop with an electrically pumped system,
relatively long capillaries can be employed to generate hundred of thousands of
theoretical plates. The reduction of eddy diffusion also contributes to the
enhanced efficiency (19).
The mobile phase is pumped using the EOF generated by both the wall of
the capillary and the chromatographic packing. Formulation of the mobile
phase is similar to conventional reversed-phase chromatography, except that a
dilute buffer—for example, 1-10 mM tris, borate, or phosphate—is added to
ensure sufficient electrical conductivity The capillary is usually pressurized to
a few atmospheres to suppress bubble formation.
The least mature of the electrically driven techniques, CEC capillaries and
second generation instruments are now available. One promise for this tech-
nique is the ability to employ the vast existing chromatographic database to
speed methods development.
1.5 MICROMACHINED ELECTROPHORETIC DEVICES
Employing technology used in the fabrication of integrated circuits, it is now
possible to create an electrophoretic apparatus on a chip (20-28). Designed for
dedicated applications such as clinical analysis, genetic analysis, or DNA
sequencing, chips can be manufactured at low cost in commercial quantities.
These devices can form the basis of an automated laboratory, where the dispos-
able chip serves as the separations device.
A diagram of a simple micromachined HPCE chip is shown in Figure 1.6. The
technological advantage of this device compared with a conventional capillary
is its ability to perform extremely small injections (29). As a result, a shorter
separation channel is required, again compared with the conventional capillary.
Detection problems resulting from the small injection are solved through the
use of laser-induced fluorescence (LIE). Micromachined electrophoretic devices
are expected to have a huge impact in the DNA applications area.
1.6 HISTORICAL PERSPECTIVE
A century of development in electrophoresis and instrumentation has provided
the foundation for HPCE. Reviews describing the history of electrophoresis were
published by Vesterberg (30) and Compton and Brownlee (31). The highlights
in the development of HPCE are given in Table 1.2.
1 4 Chapter 1 Introduction
(57, 58). One ofJorgenson's first papers in the field employed fluorescence (59).
Gassmann et al. (39) employed LIF, improving detectability to the attomole
range. Olivares et al (42) interfaced CZE to the mass spectrometer via the elec-
trospray. The use of on-line mass spectrometry is significant because of the dif-
ficulty of carrying out fraction collection. Wallingford and Ewing (43) developed
electrochemical detection, sensitive enough to measure catecholamines in a sin-
gle snail neuron. Kuhr and Yeung (44) employed indirect detection to measure
solutes that neither absorbed nor fluoresced. More exotic detection techniques
include electrochemical detection (43, 60) nuclear magnetic resonance (56),
Raman (45), chemiluminescence (55) and radioactivity (61).
The problem of protein adherence to the capillary wall was addressed from sev-
eral fronts. The use of treated capillaries was described by Hjerten (40) in 1985.
Around the same time, Lauer and McManigill (41) employed alkaline buffers above
the pi of the protein to effect solute repulsion from the anionic capillary wall. Based
on these and related developments, wall effects have been substantially reduced.
The relative instability of crosslinked polyacrylamide gel-filled capillaries for
protein and DNA separations was addressed by the first reports of polymer net-
works (47, 48). This led to the commercial introduction of kits for separations
of proteins, oligonucleotides, and DNA. DNA sequencing can now be performed
using various low viscosity polymer solutions (62, 63)
The first commercial instrument was introduced in 1988 by the late Bob
Brownlee's company, Microphoretics. The following year, new instruments from
Applied Biosystems, Beckman, and Bio-Rad were introduced. Later, Spectra-
Physics, Isco, Europhor, Dionex, Waters Associates, Hewlett-Packard, and Uni-
cam entered the fray. Modular systems from Lauer Labs, Groton Technologies,
Jasco, and Europhor became available over the next few years.
In the mid 1990s, the slow development of the HPCE generic marketplace
caused an industry shakeout as a number of instruments were withdrawn
from the marketplace. In 1990, the first report employing a multiple capil-
lary system was published (54). The mid to late 1990s provided the first
application specific instruments for performing serum protein analysis (Beck-
man) and DNA sequencing (Beckman, PE Biosystems, Molecular Dynamics).
Instruments from the latter two companies are sold with 96 capillary arrays.
These instruments are designed for high throughput DNA sequencing as
required by the Human Genome Project. It is expected that human identifi-
cation, another area that requires high throughput, will be implemented on
In 1998, Covergant Bioscience Limited of Ontario, Canada, reported on a
new dedicated instrument for capillary isoelectric focusing. The entire capillary
is imaged using a charged coupled device camera. The advantage of whole cap-
illary imaging is the elimination of the mobilization step. Electrochromatogra-
phy has attracted intense interest in the late 1990s. The present state of the
commercial offerings is given in Table 1.3.
16 Chapter 1 Introduction
1.7 GENERIC HPCE SYSTEMS
While application specific DNA systems are becoming wildly successful, generic
HPCE systems have not provided the returns expected by the scientific instru-
ment manufacturers. The generic HPCE system is designed for the user to
develop his or her own methods. In HPLC, this type of system forms the largest
segment of this multibillion dollar market. There are numerous reasons, beyond
the scientific, that this has not occurred with HPCE.
1. Liquid chromatography (HPLC) is the greatest analytical instrumenta-
tion success story in history. With a 23 year head start over HPCE, most
problems have been worked out. Methods development is straightfor-
ward, chemists are trained, and troubleshooting is usually simple.
2. HPLC scales up for preparative work and scales down to the capillary
format with relative ease. It is possible to have a single method for ana-
lytical, preparative, and commercial scale separations.
3. Many chromatographers consider HPCE to be the separations technique
of last resort.
4. It is far more difficult for an instrument company sales force to sell
HPCE. With quotas high and bonuses tied to performance, the sales-
person goes where the money is. Setting up a demo instrument in a users
lab is prone to failure, since the chemist is probably not trained in cap-
illary electrophoresis. Postsales customer support is also quite high.
5. Capillary electrophoresis is electrophoresis, not chromatography. Chro-
matographers must first master the principles of electrophoresis in order
to effectively develop and troubleshoot methods. The training require-
ments are not trivial. Methods development can seem overwhelmingly
complex to the new user.
6. Private industry is so downsized that scientists have no time to learn new
techniques. Many purchased instruments sit idle because of initial fail-
ures of methods development. Instrument companies are downsized as
well and have cut back on customer applications efforts. When faced with
a problem chemists retreat to the familiar, and that is frequently HPLC.
7. Capillary electrophoresis is not as rugged as HPLC. Changes in the cap-
illary surface chemistry lead to variable electroosmotic flow This in turn
causes changes in the solute migration time.
8. The sensitivity of HPCE is lower than HPLC. This has become less of an
issue as stacking techniques coupled with extended pathlength capillar-
ies come into play. The training issues prevail, as many chemists are
unaware of the variety of stacking techniques that exist.
9. There are few official methods of analysis employing HPCE. However,
much is now in the pipeline. A number of pharmaceutical companies
have submitted new drug applications to the Food and Drug Adminis-
tration citing HPCE methodology.
1.8 Instrumentation 17
10. Since a single HPCE instrument can replace as many as ten liquid chro-
matographs, the size of the market may become self-limiting.
The prospects for HPCE are not so bleak since once the learning curve is
scaled. Successful methods development and routine implementation has been
accomplished in many organizations.
The instrumental configuration for HPCE is relatively simple. Before 1988, all
work was done on simple homemade systems of a design similar to Jorgenson
and Lukacs's original work (35, 59, 64).
A schematic of a homemade system is shown in Figure 1.7. The system con-
sists of a high voltage power supply, buffer reservoirs, an HPLC ultraviolet detec-
tor, a capillary, and a Plexiglas cabinet. A safety interlock can be employed to
prevent activation of high voltage when the cabinet is open. The capillary can
be filled with buffer by a vacuum, generated using a syringe or handpump. Sam-
ples are injected either by siphoning (elevating the capillary for a defined time
at a specified height) or by electrokinetic injection. While these simple systems
provide good separations, precision may be poor due to the lack of temperature
control and system automation.
Another common problem in homemade systems is excessive detector noise. The
capillary is threaded through the detector and generally passes close to sensitive elec-
tronics, where the high electric field frequently causes electrical disturbances due
CATHOLYTE ELECTRODES ^ ^ Q ^ ^ T E
FIGURE 1.7 Basic schematic of an HPCE Instrument.
18 Chapter 1 Introduction
to inadequate grounding and shielding. This problem has been solved in com-
The advantage of homemade systems is primarily in the area of detection. It
is easy to interface HPCE to fluorescence detection and in particular laser-
induced fluorescence. With the introduction of commercial modular systems,
the advantages of homebuilt systems have all but disappeared excepting cost.
The arrival of commercial instruments has facilitated substantial growth in
the field. An illustration of the now obsolete Applied Biosystems 270A is shown
in Figure 1.8. This instrument provides the following basic features: a high volt-
age power supply that can provide up to 30 kV, an autosampler, electrodes, a
separation capillary, an air-cooled capillary temperature controller, a UV detec-
tor, a capillary filling apparatus, and microprocessor control.
Newer instruments have random access where any vial can be designated as
the inlet or outlet. Most of the newer instruments contain the capillary within
a cartridge for efficient cooling with either air or fluids. Pressure is used rather
than vacuum for filling the capillary; this is an advantage when using viscous
polymers or interfacing to the mass spectrometer. Many instruments can also
perform voltage programming and fraction collection, have alternative detec-
tors such as fluorescence, photodiode array, or conductivity, and possess cooled
Vacuum Buffer Reservoir Auto Sampler Carousel
FIGURE 1.8 Schematic of the Apphed Biosystems 270A. Courtesy of Apphed Biosystems.
1.9 Sources of Information on HPCE 1 9
autosamplers. Data systems that are specifically designed for HPCE are found
on most units. Computers now provide for system control on all fully auto-
1.9 SOURCES OF INFORMATION ON HPCE
Keeping up with the hterature in HPCE is no small task. Through 1998, about
7000 English language papers have appeared in the literature. The growth of
the literature in the field is illustrated in Figure 1.9. Note the large increase that
began in 1988, the year of commercial introduction of HPCE instrumentation.
The conference proceedings of the International Symposia on High Perfor-
mance Capillary Electrophoresis that have appeared in the Journal of Chro-
matography, Vols. 480, 516,559,608, 652,680, 717, 744, 745, 781,817, and 853,
contain an impressive concentration of state-of-the-art results. Other journals
containing numerous papers on HPCE are Analytical Chemistry,Journal ofMicro-
column Separations, Chromatographia,Journal ofHigh Resolution Chromatography,
Electrophoresis, Journal of Liquid Chromatography and Related Techniques, and
Journal of Capillary Electrophoresis. Many dedicated issues from some of these
journals covering HPCE, notably Electrophoresis and Journal of Liquid Chro-
matography, have been published as well.
For a comprehensive review of the literature, the biannual editions of
Analytical Chemistry entitled "Fundamental Reviews" should be consulted.
For general information on the theory of electromigration techniques, see
(65) for an excellent review. Two recent editions of Electrophoresis, Vol. 18
(1997) No. 12-13 and Vol. 19 (1998) No. 16-17, contain outstanding
1400 r -^ ^ - ^ ~ - - - ~- - ^ -^.-.--.- -;
HumbBf of P
1 6 4
il ^^ ^m
80 81 82 83 84 86 86 87 BB 89 90 91 92 93 94 95 96 97
FIGURE 1.9 The growth of the hterature of HPCE.
20 Chapter 1 Introduction
Table 1.4 Capillary Electrophoresis Books and Proceedings
Grossman, ED., Colbum,J.C., eds. Capillary Electrophoresis: Theory and Practice. 1992, Acadenic Press.
Vindevogel, J., Sandra, P Introduction to Micellar Electrokinetic Chromatography. 1992, Huthig.
Guzman, N., ed. Capillary Electrophoresis Technology. 1993, Marcel Dekker.
Weinberger, R. Practical Capillary Electrophoresis. 1993, Academic Press.
Camilleri, P., ed. Capillary Electrophoresis: Theory and Practice. 1993, CRC Press.
Foret, E, Krivankova, L., Bocek, P Capillary Zone Electrophoresis. 1993, VCH.
Jandik, P, Bonn, G. Capillary Electrophoresis of Small Molecules and Ions. 1993, VCH.
Baker, D. Capillary Electrophoresis. 1995, Wiley.
Righetti, P G., ed. Capillary Electrophoresis in Analytical Biotechnology. 1995, CRC.
Engelhardt, H., Beck, W, Schmitt, T. Capillary Electrophoresis: Methods and Potentials. 1996, Vieweg.
Cohen, A. S., Terabe, S., Deyl, Z., eds. Capillary Electrophoretic Separation of Drugs. 1996, Elsevier.
Altria, K.D., ed. Capillary Electrophoresis Guidebook: Principles, Operation, and Applications. 1996,
Jackim, E., ed. Capillary Electrophoresis Procedures Manual. 1996, Elsevier.
Lunte, S. M., Radzik, D. M., eds. Pharmaceutical and Biomedical Applications of Capillary Elec-
trophoresis. 1996, Pergamon.
Coleman, D., ed. Directory ofCapillary Electrophoresis: New Completely Revised Edition, 1996, Elsevier
Chankvetadze, B. Capillary Electrophoresis in Chiral Analysis. 1997, John Wiley 62: Sons.
Parvez, H., Caudy, P, Parvez, S., Roland-Gosselin, P, eds. Capillary Electrophoresis in Biotechnol-
ogy and Environmental Analysis. 1997, VSP
Shintani, H., Polonsky J., ed. Handbook of Capillary Electrophoresis Applications. 1997, Blackie.
Weston, A., Brown, PR., HPLC and CE: Principle and Practice. 1997, Academic Press.
Heller, C, ed. Analysis of Nucleic Acids by Capillary Electro phoresis. 1997, Vieweg.
Khaledi, M.G., ed. High Performance Capillary Electrophoresis. Theory, Techniques, and Appli-
cations. 1998, Wiley
reviews of most aspects of HPCE. There have been numerous textbooks and
conference proceedings in this field; a compilation is given in Table 1.4.
1.10 CAPILLARY ELECTROPHORESIS: A
FAMILY OF TECHNIQUES
Capillary electrophoresis comprises a family of related techniques with differ-
ing mechanisms of separation. These techniques, which are covered in the fol-
lowing chapters of this book, are:
capillary zone electrophoresis (CZE)
capillary isoelectric focusing (CIEF)
capillary gel electrophoresis (CGE)^
capillary isotachophoresis (CITP)-^
micellar electrokinetic capillary chromatography (MECC)^
capillary electroosmotic chromatography (CEC).
^CGE is now performed using replaceable polymer network reagents.
7CITP is considered here only for trace enrichment or sample stacking.
^MECC is the most significant application employing secondary equilibrium with CZE.
1. Westheimer, R., Electrophoresis in Practice: A Guide to Methods and Applications ofDNA and Pro-
tein Separations, 2nd Ed. 1997, Wiley.
l.Mosher, R. A., Saville, D. A., Thormann, W, The Dynamics of Electrophoresis. 1992, VCH.
3.Rickwood, D., Hames, B. D., Gel Electrophoresis of Nucleic Acids: A Practical Approach, 2nd Ed.
1990, IRL Press.
4. Rickwood, D., Hames, B. D., Gel Electrophoresis of Proteins: A Practical Approach, 2nd Ed. 1990,
5. Andrews, A. T., Electrophoresis: Theory, Techniques and Biochemical and Clinical Applications.
1981, Clarendon Press.
6. Righetti, P. G., Isoelectric Focusing: Theory, Methodology and Applications. Laboratory Techniques
in Biochemistry and Molecular Biology, ed. T. S. Work and R. H. Burdon. 1983, Elsevier Bio-
7. Chrambach, A., The Practice of Quantitative Gel Electrophoresis. 1985, VCH.
S.Dunn, M. J., ed. Gel Electrophoresis of Proteins. 1986, Wright.
9.Jorgenson, J. W, Phillips, M., eds. New Directions in Electrophoretic Methods ACS Symposium
Series 335. 1987. American Chemical Society.
10.Southern, E. M.J. Mol. Biol, 1975; 98:503.
1 I.Jennings, W, Analytical Gas Chromatography. 1987, Academic Press, p.5.
12.Novotny, M., Ishii, D., eds. Microcolumn Separations. 1985, Elsevier.
13.1shii, D., ed. Introduction to Microscale High Performance Liquid Chromatography. 1988, VCH.
14. Yang, E J., ed. Microbore Column Chromatography: A Unified Approach to Chromatography. 1989,
15.Carrilho, E., Miller, A. W, Ruiz-Martinez, M. C, Kotler, L., Kesilman, J., Karger, B. L. Factors
to Be Considered for Robust High-Throughput Automated DNA Sequencing Using a Multiple-
Capillary Array Instrument. Proc. SPIE-Int. Soc. Opt. Eng., 1997; 2985 (Ultrasensitive Biochem-
ical Diagnostics II):4.
16.Huang, X. C, Quesada, M. A., Mathies, R. A. Capillary Array Electrophoresis Using Laser-
Excited Confocal Fluorescence Detection. Anal. Chem., 1992; 64:967.
17.Bienvenu, J., Graziani, M. S., Rpin, E A., Bernon, H., Blessum, C, Marchetti, C, Righetti, G.,
Somenzini, M., Verga, G., Aguzzi, E Multicenter Evaluation of the Paragon CZE 2000 Capillary
Zone Electrophoresis for Serum Protein Electrophoresis and Monoclonal Component Typing.
Clin. Chem., 1998; 44:599.
18. Smith, N. W, Evans, M. B. The Efficient Analysis of Neutral and Highly Polar Pharmaceutical
Compounds Using Reversed-Phase and Ion-Exchange Electrochromatography. Chro-
matographifl, 1995; 41:197.
19.Dittman, M. M., Wienand, K., Bek, E, Rozing, G. P. Theory and Practice of Capillary Elec-
trochromatography LC-GC, 1995; 13:800.
22 Chapter 1 Introduction
20.Manz, A., Harrison, D. J., Verpoorte, E. M. J., Fettinger, J. C, Ludi, H., Widmer, H. M. Minia-
turization of Chemical Analysis Systems—A Look into Next Century's Technology or Just a
Fashionable Craze. Chimia, 1991; 45:103.
21.Manz, A., Harrison, D. J., Verpoorte, E. M. J., Fettinger, J. C, Paulus, A., Ludi, H., Widmer, H.
M. Planar Chips Technology for Miniaturization and Integration of Separation Techniques into
Monitoring Systems. Capillary Electrophoresis on a Chip. J. Chromatogr., 1992; 593:253.
22.Woolley, A. T, Mathies, R. A. Ultra-High-Speed DNA Sequencing Using Capillary Elec-
trophoresis Chips. Anal. Chem., 1995; 67:3676.
23. Chiem, N. H., Harrison, D. J. Microchip Systems for Immunoassay: An Integrated Immunoreactor
with Electrophoretic Separation for Serum Theophylline Determination. Clin. Chem., 1998; 44:591.
24. Colyer, C. L., Tang, T, Chiem, N., Harrison, D.J. Clinical Potential of Microchip Capillary Elec-
trophoresis. Electrophoresis, 1997; 18:1733.
25.Effenhausen, C. S., Manz, A. Miniaturizing a Whole Analytical Laboratory Down to Chip Size.
Am.Lah., 1994; 26:15.
26.Harrison, D. J., Manz, A., Fan, Z., Ludi, H., Widmar, H. M. Capillary Electrophoresis and Sam-
ple Injection Systems Integrated on a Planar Glass Chip. Anal. Chem., 1992; 64:1926.
27.Jacobson, S. C, Hergenroder, R., Koutny, L. B., Ramsey, M. J. High Speed Separations on a
Microchip. Anal. Chem., 1994; 66:1114.
28.Jacobson, S. C, Hergenroder, R., Koutny, L. B., Ramsey, M.J. Open Channel Electrochro-
matography on a Microchip. Anal. Chem., 1994; 66:2369.
29.Jacobson, S. C, Hergenroder, R., Koutny L. B., Warmack, R. J., Ramsey M. J. Effects of Injec-
tion Schemes and Column Geometry on the Performance of Microchip Electrophoresis Devices.
Anal. Chem., 1994; 66:1107.
30.Vesterberg, O. History of Electrophoretic Methods. J. Chromatogr, 1989; 480:3.
31. Compton, S. W, Brownlee, R. G. Capillary Electrophoresis. BioTechniques, 1988; 6:432.
32.Hjerten, S. Free Zone Electrophoresis. Chromatogr Rev., 1967; 9:122.
33.Pretorius, V, Hopkins, B. J., Schieke, J. D. A New Concept of High-Speed Liquid Chromatogra-
phy J. Chromatogr, 1974; 99:23.
34.Mikkers, F E. R, Everaerts, F M., Verheggen, T P. E. M. High Performance Zone Electrophore-
sis. J. Chromatogr, 1979; 169:11.
35.Jorgenson, J. W, Lukacs, K. D. Zone Electrophoresis in Open Tubular Glass Capillaries. Anal.
Chem., 1981; 53:1298.
36.Hjerten, S. High-Performance Electrophoresis: The Electrophoretic Counterpart of High Per-
formance Liquid Chromatography. J. Chromatogr, 1983; 270:1.
37.Terabe, S., Otsuka, K., Ichikawa, K., Tsuchiya, A., Ando, T. Electrokinetic Separations with
Micellar Solutions and Open-Tubular Capillaries. Anal. Chem., 1984; 56:111.
38.Hjerten, S., Zhu, M.-D. Adaptation of the Equipment for High-Performance Electrophoresis to
Isoelectric Focusing. J. Chromatogr, 1985; 346:265.
39.Gassmann, E., Kuo, J. E., Zare, R. N. Electrokinetic Separation of Chiral Compounds. Science,
40.Hjerten, S. High-Performance Electrophoresis: Elimination of Electroendosmosis and Solute
Adsorption. J. Chromatogr, 1985; 347:191.
41.Lauer, H. H., McManigill, D. Capillary Zone Electrophoresis of Proteins in Untreated Fused Sil-
ica Tubing. Anal. Chem., 1986; 58:166.
42.01ivares, J. A., Nguyen, N. T, Yonker, C. R., Smith, R. D. On-Line Mass Spectrometric Detec-
tion for Capillary Zone Electrophoresis. Anal. Chem., 1987; 59:1230.
43. Wallingford, R. A., Ewing, A. G. Capillary Zone Electrophoresis with Electrochemical Detec-
tion. Anal. Chem., 1987; 59:1762.
44.Kuhr, W G., Yeung, E. S. Indirect Fluorescence Detection of Native Amino Acids in Capillary
Zone Electrophoresis. Anal. Chem., 1988; 60:1832.
45. Chen, C. Y., Morris, M. D. Raman Spectroscopic Detection System for Capillary Zone Elec-
trophoresis. Appl. Spectrosc, 1988; 42:515.
References 2 3
46. Guttman, A., Paulus, A., Cohen, A. S., Grinberg, N., Karger, B. L. Use of Complexing Agents for
Selective Separation in High-Performance Capillary Electrophoresis: Chiral Resolution via
Cyclodextrins Incorporated Within Polyacrylamide Gel Columns. J. Chromatogr., 1988; 448:41.
47.Hjerten, S., Valtcheva, L., Elenbring, K., Eaker, D. High-Performance Electrophoresis of Acidic
and Basic Low-Molecular Weight Compounds and of Proteins in the Presence of Polymers and
Neutral Surfactants. J. Liq. Chromatogr., 1989; 12:2471.
48.Zhu, M., Hansen, D. L., Burd, S., Gannon, F. Factors Affecting Free Zone Electrophoresis and
Isoelectric Focusing in Capillary Electrophoresis. J. Chromatogr., 1989; 480:311.
49. Cohen, A. S., Najarian, D. R., Karger, B. L. Separation and Analysis of DNA Sequence Reaction
Products by Capillary Gel Electrophoresis. J. Chromatogr, 1990; 516:49.
50.Drossman, H., Luckey J. A., Kostichka, A. J., D'Cunha, J., Smith, L. M. High-Speed Separations
of DNA Sequencing Reactions by Capillary Electrophoresis. Anal. Chem., 1990; 62:900.
51.Luckey, J. A., Drossman, H., Kostichka, A. J., Mead, D. A., D'Cunha, J., Norris, T. B., Smith, L.
M. High Speed DNA Sequencing by Capillary Electrophoresis. Nud. Acids Res., 1990; 18:4417.
52.Swerdlow, H., Gesteland, R. Capillary Gel Electrophoresis for Rapid, High Resolution DNA
Sequencing. Nud. Adds Res., 1990; 18:1415.
53. Swerdlow, H., Wu, S., Harke, H., Dovichi, N. J. Capillary Gel Electrophoresis for DNA Sequencing:
Laser-Induced Fluorescence Detection with the Sheath Flow Cuvette.J. Chromatogr, 1990; 516:61.
54.Zagursky, R. J., McCormick, R. M. DNA Sequencing Separations in Capillary Gels on a Modi-
fied Commercial DNA Sequencing Instrument. BioTechniques, 1990; 9:74.
55.Dadoo, R., Colon, L. A., Zare, R. N. Chemiluminescence Detection in Capillary Electrophore-
sis. HRC & CC, 1992; 15:133.
56. Wu, N., Peck, T. L., Webb, A. G., Magin, R. L., Sweedler, J. V Nanoliter Volume Sample Cells
for ^H NMR: Application to Online Detection in Capillary Electrophoresis. J. Am. Chem. Soc,
57.Walbroehl, Y., Jorgenson, J. W On-Column UV Absorbance Detector for Open Tubular Capil-
lary Zone Electrophoresis. J. Chromatogr, 1984; 315:135.
58. Green, J. S., Jorgenson, J. W Design of a Variable Wavelength UV Absorption Detector for On-
Column Detection in Capillary Electrophoresis and Comparison of Its Performance to a Fixed
Wavelength UV Absorption Detector. J. Liq. Chromatogr, 1989; 12:2527.
59.Jorgenson,J. W, Lukacs, K. D. Free-Zone Electrophoresis in Glass Capillaries. Clin. Chem., 1981;
60. Wallingford, R. A., Ewing, A. G. Capillary Zone Electrophoresis with Electrochemical Detec-
tion in 12.7|Lim Diameter Columns. Anal. Chem., 1988; 60:1972.
61.Pentoney, S. L., Zare, R. N., Quint, J. F. On-Line Radioisotope Detection for Capillary Elec-
trophoresis. Anal. Chem., 1989; 61:1642.
62.Salas-Solano, O., Carrilho, E., Kolter, L., Miller, A. W, Goetzinger, W, Sosic, Z., Karger, B. L.
Routine DNA Sequencing of 1000 Bases in Less than One Hour by Capillary Electrophoresis
with Replaceable Linear Polyacrylamide Solutions. Anal. Chem., 1998; 70:3996.
63. Kim, Y., Yeung, E. S. Separation of DNA Sequencing Fragments up to 1000 Bases by Using
PolyCethylene Oxide)-Filled Capillaries. J. Chromatogr, A, 1997; 781:315.
64.Jorgenson, J. W, Lukacs, K. D. Zone Electrophoresis in Open-Tubular Glass Capillaries: Pre-
liminary Data on Performance. HRC & CC, 1981; 4:230.
65.Kleparnik, K., Bocek, P Theoretical Background for Clinical and Biomedical Applications of
Electromigration Techniques. J. Chromatogr, 1991; 569:3.
2.1 Electrical Conduction in Fluid Solution
2.2 The Language of Electrophoresis
2.6 Joule Heating
2.7 Optimizing the Voltage and Temperature
2.8 Capillary Diameter and Buffer Ionic Strength
2.9 Optimizing the Capillary Length
2.11 Temperature Effects
2.12 Buffer Additives
2.14 Sources of Band Broadening
2.1 ELECTRICAL CONDUCTION IN
Several simple concepts are important for understanding the physical processes
that occur upon passage of an electrical current through an ionic solution. ^
These processes are far more complex than the passage of current through a
metal. In metals, uniform and v^eightless electrons carry all the current. In fluid
^See any basic text on physical chemistry for a thorough description of electrical conduction in
26 Chapter 2 Capillary Zone Electrophoresis
solution, the current is carried by cations and anions. The molecular weight of
these charge bearing ions ranges from a simple proton to tens of thousands for
large complex ions such as proteins and polynucleotides.
Conduction in fluid solution is still described by Ohm's law,
E = IR, (2.1)
where E is the voltage or applied field, I is the current that passes through the
solution, and R is the resistance of the fluid medium.
The reciprocal of resistance is conductivity. Kohlrausch found that the con-
ductivity of a solution resulted from the independent migration of ions. As
illustrated in Figure 2.1, when a current passes through an ionic solution,
anions migrate toward the anode (positive electrode) while cations migrate
toward the cathode (negative electrode) in equal quantities. Despite the passage
of current, electroneutrality of the solution is always maintained because of
electrolysis at each electrode.
This is important because electrolysis produces protons at the anode and
hydroxide at the cathode (Figure 2.2). The resultant pH changes are due to the
process known as buffer depletion (1-3). Since pH is the single most important
experimental parameter in capillary electrophoresis, this effect must be mini-
1. Using the appropriate buffers
2. Having sufficiently large buffer reservoirs
3. Replacing buffers frequently
The introduction of a sample into the capillary changes the situation dra-
matically (Figure 2.3). The Ohm's law equation changes as well to that for a
E = IR^ + IRj . (2.2)
This process and the equation have important implications in HPCE. When low-
conductivity samples (relative to the BGE) are injected, a process known as
stacking (Section 8.6) occurs. This permits the use of large-volume injections
FIGURE 2.1 The independent migration of ions.
2 8 Chapter 2 Capillary Zone Electrophoresis
The mobility of ions in fluid solution is governed by their charge to size ratio.
The size of the molecule is based on the molecular weight, the three-dimensional
structure, and the degree of solvation (usually hydration). Data given in Table 2.1
(4) for alkali metals illustrate several of these important points:
1. The orders for the mobilities of the metal ions are the reverse of what is
expected based on the metal or crystal radii data. These smaller ions are
more hydrated than their larger counterparts.
2. The current generated by 100 mM solutions of various acetate salts is
proportional to the ionic mobility of the cation. This feature becomes
important when selecting the appropriate counterion for preparing
The forces governing this behavior are expressed by Stoke's law,
/ = 67rrirv , (2.3)
where 7] = viscosity, r = ionic radius, and v = ionic velocity. The competing
forces of mobility (velocity) and viscosity are illustrated in Figure 2.4 for an ion
of radius r. Ionic size modifies mobility because of a solute's exposure to fric-
tional drag as it migrates through the supporting electrolyte. The frictional drag
is directly proportional to viscosity, size, and electrophoretic velocity. An expres-
sion for mobility that contains these terms is
M—)=-5^^^^^ = ^ - , (2.4)
Vs E(V/cm) 6nrir
where q = the net charge and E = the electric field strength. Thus, mobility is
considered a charge-to-size ratio. Since the units for velocity are centimeters per
second and the field strength is expressed as volts per centimeter, the units of
mobility are cm^A^s.
2.2 THE LANGUAGE OF ELECTROPHORESIS
There are several distinguishing differences between the terminology of chro-
matography and that of capillary electrophoresis. For example, a fundamental para-
meter in chromatography is the retention time. In electrophoresis nothing should
ever be retained (except for CEC), so a more descriptive term is migration time:
the time it takes a solute to travel from the beginning of the capillary to the detec-
30 Chapter 2 Capillary Zone Electrophoresis
FIGURE 2.4 The competing forces of electrophoretic mobility and viscous drag.
The use of a detection window in HPCE (on-capillary detection) as opposed
to postcolumn detection must also be considered. In HPLC, the length of the
chromatographic column must be included in all methods. Figure 2.5 is a draw-
ing of a capillary. Both the total length of the capillary (L^ or L) and the length
to the detector (L^ or I) must be described. The segment of capillary that occurs
after the detector window is necessary to make electrical contact with the out-
let or detector-side electrolyte reservoir. Ideally, L^ - L^ should be as short as
practical. Otherwise, some system voltage (V) is wasted on maintaining field
strength (E) over part of the capillary that lies beyond the detector window and
hence does not participate in the separation.
FIGURE 2.5 Illustration of a capillary defining the total length (Lj) and the length to the detec-
2.3 Electroendoosmosis 31
Expressions for some other fundamental terms are given in the following
The preceding include the electrophoretic mobility (^ep^ cm^A^s), the elec-
trophoretic velocity (Vgp, cm/s), and the field strength (E, V/cm). These equa-
tions define some fundamental features of HPCE:
1. Velocities are measured experimentally (Eq. 2.5). They are determined
by dividing the length of capillary, from the injection side to the detec-
tor window (Ld), by the migration time t^.
2. MobiUties are calculated by dividing the electrophoretic velocity v^p by the
field strength (Eq. 2.6). The field strength is simply the voltage divided by
the total capillary length (L^). The field strength is the important parameter
governing electrophoretic migration. Field strength is changed when either
the voltage or the capillary length is altered. Mobility is the fundamental
parameter of capillary electrophoresis. This term is independent of voltage
and capillary length. Equations (2.6) and (2.7) define only the relative
mobility. To calculate the true mobility, a correction for a phenomenon
known as electroendoosmotic flow (Section 2.3) must first be made.
A. THE CAPILLARY SURFACE
One of the fundamental processes that accompanies electrophoresis is electroos-
mosis. One of the "pumping" mechanisms of HPCE, electroosmosis occurs
because of the surface charge, known as the zeta potential, on the wall of the cap-
illary. Fused silica is the most common material used to produce capillaries for
HPCE. Technology developed for manufacturing capillary columns for GC read-
ily transferred to HPCE. Fused silica is a highly crosslinked polymer of silicon
dioxide with tremendous tensile strength (5), although it is quite brittle. With its
polyimide coating, fused silica is quite durable, although some polyimide must
32 Chapter 2 Capillary Zone Electrophoresis
be removed to create a ultraviolet (UV) transparent optical window for detection.
Other materials such as Teflon and quartz have been used (6), but performance
and cost are less favorable.
Before use, capillaries are usually conditioned with 1 N sodium hydroxide. The
base ionizes free silanol groups and may cleave some silica epoxide linkages as well.
An anionic charge on the capillary surface results in the formation of an electrical
double layer. The resulting ionic distribution is shown in Figure 2.6 (7). Anions
are repelled from the negatively charged wall region, whereas cations are attracted
as counterions. Ions closest to the wall are tightly bound and immobile, even under
the influence of an electric field. Further from the wall is a compact and mobile
region with substantial cationic character. At a greater distance from the wall, the
solution becomes electrically neutral as the zeta potential of the wall is no longer
sensed. Expressions describing this phenomenon were derived by Gouy and
Chapman in 1910 and 1913, respectively This diffuse outer region is known as the
Gouy-Chapman layer. The rigid inner layer is called the Stem layer.
When a voltage is applied, the mobile positive charges migrate in the direc-
tion of the cathode or negative electrode. Since ions are solvated by water, the
fluid in the buffer is mobilized as well and dragged along by the migrating
charge. Although the double layer is perhaps 100 A thick, the electroendoos-
motic flow (EOF) is transmitted throughout the diameter of the capillary, pre-
sumably through hydrogen bonding of water molecules or van der Waals
interactions between buffer constituents.
The electroosmotic flow as defined by Smoluchowski in 1903 is given by
Veo = ^ E , (2.8)
where £ is the dielectric constant, 77 is the viscosity of the buffer, and f is the
zeta potential of the liquid-solid interface. The equation is only valid for capil-
laries sufficiently large that the double layers on opposite walls do not overlap
each other (8). Practical use of this equation is not forthcoming, as the zeta
potential is rarely measured and data for the dielectric constants of mixtures are
not readily available. Like electrophoretic mobility, the EOF is inversely pro-
portional to the viscosity of the BGE.
B. MEASURING THE ELECTROOSMOTIC FLOW
Since the migration time of a solute is influenced by the EOF, calculation of the
actual mobility requires measurement of the EOF:
Here jH^^ = the actual mobility, jU^pp = apparent (observed) mobility, and
jLl^o = electroosmotic mobility. The use of mobility as the "migration parameter"
34 Chapter 2 Capillary Zone Electrophoresis
The simplest method for measuring the EOF is to inject a dilute solution con-
taining a neutral solute and measure the time it takes to transit the detector (9-11).
Since the capillary length is known, the velocity in centimeters per second is eas-
ily calculated. Dividing that value by the field strength yields the electroosmotic
mobility in units of cmWs. Neutral solutes such as methanol, acetone, benzyl
alcohol, and mesityl oxide are frequently employed. When MECC is the mode of
separation (Chapter 4), a further requirement that the marker solute not partition
into the micelle is imposed.
When the EOF is slow, the migration time can be quite long. To reduce the
experimental time, it is useful to use the short end of the capillary to make the
measurement. The short end is the section of capillary normally found between
the detector window and capillary outlet. The injection can be made at the out-
let side and the system operated using reversed polarity Now the EOF is mea-
sured using a short capillary length of 6-10 cm depending on the brand of
instrument. As will be shown later, the short end of the capillary can be very
useful when performing screening runs during methods development.
When the EOF is very slow, as in the case with certain coated capillaries, spe-
cial techniques must be employed (12). It is seldom necessary to measure very
weak EOF, since it does not notably affect mobility or experimental precision.
C. EFFECT OF BUFFER P H
The impact of pH on the EOF and the mobility is illustrated in Figure 2.7. At
high pH the silanol groups are fully ionized, generating a strong zeta potential
and dense electrical double layer. As a result, the EOF increases as the buffer
pH is elevated (9, 13). A robust flow, typically around 2 mm/s at pH 9 in 20 mM
borate buffer at 30 kV, 30°C is realized. For a 50 |Lim capillary, this translates to
235 nL/min. Since the total volume of a 50 cm x 50 jim i.d. capillary is only
980 nL, a neutral compound would reach the detector in 4.2 min. At pH 3, the
EOF is much lower, about 30 nL/min.
The EOF must be controlled or even suppressed to run certain modes of HPCE.
On the other hand, the EOF makes possible the simultaneous separation of
cations, anions, and neutral species in a single run. For example, a zwitterion like
a peptide will be negatively charged at a pH above its pi. The solute will elec-
tromigrate toward the positive electrode. However, the EOF is sufficiently strong
that the solute's net migration is toward the negative electrode (Figure 2.7, top).
At low pH, the zwitterion has a positive charge and will migrate as well toward
the negative electrode (Figure 2.7, bottom). In untreated fused-silica capillar-
ies, most solutes migrate toward the negative electrode unless buffer additives
or capillary treatments are used to reduce or reverse the EOF (Section 3.3).
The EOF is exquisitely sensitive to pH (9,14,15). Hysteresis effects have been
reported (15) wherein the direction of approach to a particular pH value produces
a different pH (Figure 2.8). When approaching from the acid side, the measured
M,0 / * ep
4 . 4 . ^ 4 . 4 , 4 » 4.4> 4. 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 * 4 .
+ + + + + 4* + 4- + + +^+ + -f + -I" + -f +
FIGURE 2.7 Behavior of electroendoosmotic flow and electrophoretic migration of a zwitterion
(pi = 7) at high and low pH.
EOF is always lower, and vice versa. This means there is a kinetic parameter with
regard to the estabUshment of a stable charge on the capillary wall. Longer equili-
bration times would reduce hysteresis at the expense of increased total run time.
Since the EOF will affect migration time precision, it is important to design
experiments with these features in mind. The problems with EOF repro-
ducibility are often most severe in the pH range 4-6 (15).
D. EFFECT OF BUFFER CONCENTRATION
The expression for the zeta potential is (16)
where £ = the buffer's dielectric constant, e = total excess charge in solution
per unit area, and 5 is the double-layer thickness or Debye ionic radius. The
Debye radius is 5 = (3 x 10'')(Z)(Ci/^), where Z = number of valence electrons
and C = the buffer concentration.
As the ionic strength increases, the zeta potential and, similarly, the EOF
decreases in proportion to the square root of the buffer concentration. This was
36 Chapter 2 Capillary Zone Electrophoresis
H 1 1 1 1—I 1 1 1 1
2 3 4 5 6 7 8 9 10 11
FIGURE 2.8 Effect of experimental design on the EOF. Key: • , high pH titrated to low pH; •,
low pH titrated to high pH. Data from reference (15).
confirmed experimentally (17) for a series of buffers where the EOF was found
hnear to the natural logarithm^ of the buffer concentration. It was reported that
equivalent EOF is found for different buffer types as long as the ionic strength
is kept constant (17).
The effect of buffer concentration and field strength is shown in Figure 2.9 (18).
The electroosmotic mobility is plotted against field strength for phosphate buffer
at three different concentrations using a 50-|Lim-i.d. capillary. As expected, the
higher buffer concentrations showed lower EOF at all field strengths. Since
^The linear relationship of EOF with the buffer concentration is a square root relationship as
indicated by Eq. (2.10).
3 8 Chapter 2 Capillary Zone Electrophoresis
be carefully controlled. In this regard, wholly aqueous separations are often
F. CONTROLLING THE ELECTROOSMOTIC FLOW
The EOF is a double-edged sword. It allows the separation of cations, anions,
and neutral solutes in a single run. It is also the single most important contrib-
utor to migration time variability on a run-to-run, day-to-day, and capillary-to-
capillary basis. The EOF is affected by many parameters, including
In this list, the only factor not under direct experimental control is the cap-
illary surface. This single factor is often implicated as the cause for migration
time variation in HPCE. It is important to ensure that the capillary surface is
properly reconditioned after each run to maintain a reproducible surface. Coated
capillaries that suppress the EOF are useful here, as long as the coating is sta-
ble. Some new reagents^ that form a dynamic surface coating show great promise
for stabilizing the capillary surface (Section 3.3).
For some modes of HPCE, it is advantageous to suppress the FOE Capillary
isoelectric focusing (CIEF) and capillary isotachophoresis (CITP) separations
are usually performed under conditions of very low or carefully controlled EOF
Additives such as 0.5% hydroxypropylmethyl cellulose are effective in sup-
pressing the EOF, particularly in conjunction with a coated capillary (22).
Cationic surfactants such as cetyltrimethylammonium bromide can actually
reverse the direction of electroosmotic flow (14). This can be employed to pre-
vent proteins from sticking to the capillary wall (23, 24). While complete sup-
pression of the EOF is unnecessary for most applications, control is critical to
obtain reproducible migration times and resolution.
^CElixir, Scientific Resources, Inc., Eatontown, NJ.
2.4 Efficiency 3 9
The high efficiency of HPCE is a consequence of several unrelated factors:
1. A stationary phase is not required for HPCE. The primary cause of band
broadening in LC is resistance to mass transfer between the stationary and mobile
phases. This mass transfer problem is illustrated in Figure 2.10. When a solute
is in the mobile phase, its linear velocity is determined by the linear velocity of
the mobile phase. When attached to the stationary phase, the linear velocity
becomes zero. The solute is not of a single velocity as it moves down the chro-
matographic tube. Whenever differing velocities occur during a separation, band
broadening will occur. Minimizing the particle size of the packing improves but
does not eliminate this problem. Thus, the parameter that results in separation
also causes band broadening. The greater the retention, the greater the prob-
lem—as evidenced by broadened peaks as retention time increases. For most
modes of HPCE (except CEC), this dispersion mechanism does not operate.
Similarly, other HPLC dispersion mechanisms such as eddy diffusion and stag-
nant mobile phase are unimportant in HPCE.
2. In pressure-driven systems such as LC, the frictional forces of the mobile
phase interacting at the walls of the tubing result in radial velocity gradients
throughout the tube. As a result, the fluid velocity is greatest at the middle of
the tube and approaches zero near the walls (Figure 2.11). This is known as
laminar or parabolic flow. These frictional forces, together with the chromato-
graphic packing, result in a substantial pressure drop across the column.
In electrically driven systems, the EOF is generated uniformly down the
entire length of the capillary. There is no pressure drop in HPCE, and the radial
flow profile is uniform across the capillary except very close to the walls, where
the flow rate approaches zero (Figure 2.11).
FIGURE 2.10 The mass transport problem in HPLC.
40 Chapter 2 Capillary Zone Electrophoresis
Jorgenson and Lukacs derived the efficiency of the electrophoretic system
from basic principles (25-27) using the assumption that diffusion is the only
source of band broadening. Other sources of dispersion—including Joule heat-
ing (Section 2.6), capillary wall binding (Section 3.5), injection (Section 9.1),
detection (Section 9.5), and electromigration dispersion (Section 2.13)—lead
to fewer theoretical plates than the simple theory predicts.
The migration velocity for a solute is
V = luE =
where // = the mobility, E = field strength, V = voltage, and L = capillary
length. The time t for a solute to migrate the length L of the capillary is
FIGURE 2.11 Capillary flow profiles resulting from electroosmotic and hydrodynamic flow.
2.5 Resolution 41
Diffusion in liquids that leads to broadening of an initially sharp band is
described by the Einstein equation
(yl = 2Dt = =^^, (2.13)
where D = the diffusion coefficient of the individual solute. The number of the-
oretical plates N is given by
N = — . (2.14)
Substituting Eq. (2.11) into Eq. (2.12) gives an expression for the number
of theoretical plates:
N = ^ . (2.15)
Some important generalizations can be made from this expression:
1. The use of high voltage gives the greatest number of theoretical plates, since
the separation proceeds rapidly, minimizing the effect of diffusion. This
holds true up to the point where heat dissipation is inadequate (Section 2.6).
2. Highly mobile solutes produce high plate counts, because their rapid
velocity through the capillary minimizes the time for diffusion.
3. Solutes with low diffusion coefficients give high efficiency due to slow
diffusional band broadening.
Points 2 and 3 appear contradictory. This is clarified by Figure 2.12 and sup-
plemented with some calculations in Table 2.2. Because of the indirect but
inverse relationship between mobility and diffusion, high-efficiency separations
occur across a wide range of molecular weights.
HPCE can yield high-efficiency separations for both large and small mole-
cules. The greatest number of theoretical plates is found in capillary gel elec-
trophoresis (CGE). The use of an anticonvective gel matrix furthers the
advantages of HPCE. The combination of HPCE in the gel or polymer network
format (Chapter 6) can yield millions of theoretical plates.
While high efficiency is important, resolution is the key for all forms of sepa-
ration. In a high-efficiency system, inadequate resolution may result in a single
very sharp peak.
42 Chapter 2 Capillary Zone Electrophoresis
FIGURE 2.12 Diffusion and mobihty of small and large molecules.
The resolution (R) between two solutes is defined as
4 Mep + Meo
where A^ is the difference in mobility between two solutes, /i^^ is the average
mobility of the two solutes, and N is the number of theoretical plates. Substi-
tuting the plate count equation (Eq. (2.15) and V = EL) yields (25)
R, = O.UlAfl^
(/^ep + I^J^n
This expression suggests that increasing the voltage is not very effective
in improving resolution, since that parameter falls inside of the square root
of the resolution equation. A doubling of voltage results in only a 41%
improvement in resolution. The production of heat quickly limits this
Table 2.2 Calculated Theoretical Plates for a Small and Large Molecule
2.6 Joule Heating 43
approach. Another means of improving resolution as predicted by Eq. (2.17)
is to adjust the EOF. Akhough this also falls under the square root sign of the
resolution equation, this technique can be quite effective. There are three cat-
egories in this regard:
1. Both electrophoresis and electroosmosis are in the same direction. This
normally occurs when cations are being separated. In this case, decreas-
ing the EOF will enhance resolution at the expense of run time. Dou-
bling the run time produces a 41% improvement in resolution.
2. Electrophoresis and electroosmosis are in opposite directions. This occurs
on bare silica capillaries when anions are separated. Decreasing the EOF
will enhance run time at the expense of resolution, and vice versa.
3. Electrophoresis and electroosmosis are equal but in opposite directions.
Here the resolution is infinite, but so is the separation time. However, this
concept was used to generate ultrahigh theoretical plate numbers (28).
It is clear that improvements in resolution are best addressed by adjustments
to AjUgp, the difference in mobility between the two most closely eluting solutes
in a separation. Since A/i^p falls outside of the square root sign of the resolution
equation, the improvement in resolution is directly proportional to the change
in mobilities. This subject forms the basis for many of the chapters in this book.
2.6 JOULE HEATING
The conduction of electric current through an electrolytic solution generates
heat via frictional collisions between migrating ions and buffer molecules. Since
high field strengths are employed in HPCE, ohmic orJoule heating can be sub-
stantial. There are two problems that can result from Joule heating:
1. Temperature changes due to ineffective heat dissipation
2. Development of thermal gradients across the capillary
If heat is not dissipated at a rate equal to its production, the temperature
inside the capillary will rise and eventually the buffer solution will outgas. Even
a small bubble inside of the capillary disrupts the electrical circuit. At moder-
ate field strengths, outgassing is not usually a problem, even for capillaries that
are passively cooled.
The rate of heat production inside the capillary can be estimated by
^ = - ^ , (2.18)
where L = capillary length and A = the cross-sectional area. Rearranging this
equation using J = V/R, where the resistance R = L/kA and k = the conductivity.
44 Chapter 2 Capillary Zone Electrophoresis
The amount of heat that must be removed is proportional to the conductivity
of the buffer, as well as the square of the field strength.
Lacking catastrophic failure (bubble formation), the problem of thermal gra-
dients across the capillary can result in substantial band broadening (29-31).
This problem is illustrated in Figure 2.13. The second law of thermodynamics
states heat flows from warmer to cooler bodies. In HPCE, the center of the cap-
illary is hotter than the periphery. Since the viscosity of most fluids decreases
with increasing temperature, Eq. (2.4) and (2.8) predict that both mobility and
EOF increase as the temperature rises.
This situation becomes similar to laminar flow where the electrophoretic or
electroosmotic velocity at the center of the capillary is greater than the velocity
near the walls of the capillary. The temperature differential of the buffer between
the middle and the wall of the capillary can be estimated from
AT = 0.24
where W = power, r = capillary radius, and K = thermal conductivity of the buffer,
capillary wall, and polyimide cladding. A 2-mm-i.d. capillary filled with 20 mM
CAPS buffer draws 18 rtiA of current at 30 ky giving a AT of 75°C. A 50-|lm-i.d.
capillary filled with the same buffer draws only 12 |LIA of current, yielding a AT of
50 m°C. Since the thermal gradient is proportional to the square of the capillary
radius, the use of narrow capillaries facilitates high resolution. On the other hand,
the use of dilute buffers or isoelectric buffers (32) permits the use of wider bore
capillaries, but the loading capacity of the separation is reduced.
FIGURE 2.13 Impact of the radial temperature gradient on electrophoretic and electroosmotic flow.
5 0 Chapter 2 Capillary Zone Electrophoresis
generally more effective than air-cooled systems, since the heat capacity of most
fluids exceeds that of air.
B. CONSTANT VOLTAGE OR
Power can be applied to the system in one of two ways. The voltage can be fixed,
allowing the current to float based on the resistance of the buffer. Alternatively the
current can be fixed. Most published work in HPCE is in the constant-voltage
mode. There has been one report that found constant current more reproducible
than constant voltage (37). Until this is better understood, both modes should
be studied during methods development for CZE, MECC, and CGE separations.
CITP is typically performed in the constant-current mode, or else separation
time becomes long. CIEF may also benefit from the constant-current mode as
well, although there is no evidence published to that effect.
2.8 CAPILLARY DIAMETER AND
BUFFER IONIC STRENGTH
Some very subtle effects due to Joule heating can occur when comparing sepa-
rations run on capillaries with different inner diameters, or even the same cap-
illaries run on various instruments with different capillary cooling systems.
Some of these issues are illustrated in Figures 2.19 and 2.20.
The ionic strength of the buffer influences not only the EOF and jil^^, but
indirectly the viscosity of the medium. More concentrated buffers have greater
conductivity and generate more heat when the voltage is applied. The viscos-
ity depends on the temperature, and so there is also a dependence on the cap-
illary diameter. This is shown for a series of runs in 50- and 75-|Llm-i.d.
With the 50-|lm capillary, the migration times lengthen as the buffer con-
centration is increased. Ions in solution are always surrounded by a double
layer of ions of the opposite charge. The migration of these counterions is in
a direction opposite to that of the solute (Figure 2.21); hence, increasing the
concentration of the buffer reduces the mobility of the solute, due to increased
drag caused by countermigration of the more densely packed counterions.
With the 75-|Lim capillary, the solute migration times first increase as
expected, but then they decrease. This decrease is a consequence of the sig-
nificant effects of Joule heating at higher buffer concentrations. Note as well
the impact of buffer concentration on peak width. Sharper peak widths at
the higher buffer concentrations are due to stacking (Section 8.6). The