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Practical Capillary Electrophoresis Textbook PDF Download

The document discusses the evolution and applications of capillary electrophoresis (CE), highlighting its transition from a laboratory curiosity to a widely used microseparation technique in over a thousand laboratories globally. It emphasizes the importance of understanding ion movement in fluid solutions under electric fields for effective methods development in CE, which differs from traditional chromatography. The text also acknowledges the diverse backgrounds of students in CE courses, indicating the multidisciplinary nature of the field.
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100% found this document useful (11 votes)
505 views14 pages

Practical Capillary Electrophoresis Textbook PDF Download

The document discusses the evolution and applications of capillary electrophoresis (CE), highlighting its transition from a laboratory curiosity to a widely used microseparation technique in over a thousand laboratories globally. It emphasizes the importance of understanding ion movement in fluid solutions under electric fields for effective methods development in CE, which differs from traditional chromatography. The text also acknowledges the diverse backgrounds of students in CE courses, indicating the multidisciplinary nature of the field.
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Practical Capillary Electrophoresis

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To Lisa, Julie and Jeremy
Preface

Capillary electrophoresis (CE) or high-performance C E (HPCE) is making the


transition from a laboratory curiosity to a maturing microseparations technique.
Now used in over one thousand laboratories worldwide, C E 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 Electrophoresis."
During the first eighteen months since its inception, nearly five hundred 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
students. Represented in these courses were molecular biologists, protein chemists,
analytical chemists, organic chemists, and analytical biochemists from industrial,
academic, and government laboratories. Interestingly enough, C E 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, C E is not chromatography (usually). It is electrophoresis, and it is
governed by the art and science of electrophoresis. For those skilled in electro­
phoresis, C E offers additional separation opportunities that are not available in the
slab-gel format. Furthermore, the intellectual process of methods development
differs from both slab-gel electrophoresis and liquid chromatography.
The key to grasping the fundamentals of C E 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 electro-
phoretic domain and explain the subtleties and permutations frequently illustrated
on the electropherograms. Accordingly, a logical approach to methods development
xi
xii Preface

evolves from this treatment. This is the goal of my course, and I am hopeful that I
have translated that 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, I am grateful to Professor Ira Krull and his graduate
student Jeff Mazzeo from Northeastern University for reviewing the entire manuscript;
Dr. Michael Albin from Applied Biosystems, Inc., for providing his company's
bibliography on HPCE; and the Perkin-Elmer Corporation, 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 instru­
mentation industry for providing information, intellectual challenges, hints,
electropherograms, comments, etc., many of which are included in this text.
Lastly, I thank my students for helping me continuously reshape this material to
provide clear and concise explanations of electrophoretic phenomena.
Finally, many of the figures in this text were produced by scanning the
illustration 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.

Robert Weinberger
Chappaqua, New York
Master Symbol List

4 Corrected peak area


^corr
4 Raw peak area
"raw
a Fraction ionized
a Molar absorptivity (Eq. (10.4))
a Separation factor
Β Viscosity dependence on temperature
b Detector optical pathlength
C,c Concentration
c c
Capacitance of the fused-silica wall
C ei Capacitance at the buffer-capillary wall interface
Cm Coefficient for resistance to mass transfer in the mobile phase
cs Coefficient for resistance to mass transfer in the stationary phase
%c Percent of crosslinker in a gel
D,Dm Diffusion coefficient
D Dynamic reserve (Eq. (10.5))
D Capillary diameter (Eq. (9.10))
δ Debye radius
ξ Zeta potential
e Charge per unit area
Ε Field strength
Ε Acceptable increase in Η (Eq. (9.8))
Ε Detector efficiency (Eq. (10.4))
ε Dielectric constant
/ Frictional force (Stokes' Law)
8 Gravitational constant
γ Field enhancement factor
Η Height equivalent to a theoretical plate
Ah Height differential between capillary ends
dHldt Rate of heat production

xiii
xiv Master Symbol List

/ Current
If Fluorescence intensity
h Excitation source intensity
k,K Conductivity
k' Capacity factor
k' Capacity factor in MECC
*c Desorption rate constant
Κ,λ Thermal conductivity
Κ Complex formation constant (Eq. (7.5))
Ld Length of capillary to detector
Length of the detector window
Li Length of capillary from detector to fraction collector
U Total length of capillary
Length of an injection plug
m Mass
Μ Actual mass (Eqs. (10.6), (10.8))
Ν Number of theoretical plates
Ν Number of segments in a polymer chain (Eq. (5.3))
η Number of charges
η Viscosity
AP Pressure drop
Φ Quantum yield
Ρ Density (Eq. (9.11))
Ρ Resistivity (Eq. (9.13))
Q Quantity of injected material
q Ionic net charge
Φ Overlap threshold
o Fluorescence quantum yield
f
R Resistance
R Peak ratio (Eq.(11.2))
R Displacement ratio (Eq. (10.5))
Rs Resolution
r Ionic radius (Stokes' Law)
r,rc Capillary radius
SIN Signal-to-noise ratio
σ Peak variance
σί Peak variance in units of length
Adsorption time to a stationary phase
Master Symbol List xv

id Βββοφίίοη time from a stationary phase


'L Lag time
'm Migration time
Migration time for a micellar aggregate
to Migration time for a neutral "unretained" solute
U "Retention" time in MECC
Τ Temperature
%T Percentage of monomer and crosslinker in a gel
μ Ionic mobility
Electrophoretic mobility
Mf Mobility of uncomplexed solute (Eq. (7.5))
V Voltage
υ Ionic velocity
υ Mean linear velocity (Eq. (8.2))
υ βρ Electrophoretic velocity
υ β0 Electroosmotic velocity
W Power
W i nj Width of an injection plug
ws Spatial width of a sample zone
Temporal width of a sample zone
Initial length of an injection plug
Zone length after stacking
Ζ Number of valence electrons
ζ Charge
"There are only three problems with capillary electrophoresis: injection, separation
1
and detection"

Chapter 1

Introduction

1.1 Electrophoresis

Electrophoresis is a process for separating charged molecules based on their


movement through a fluid under the influence of an applied electric field. The
separation is performed in a medium such as a semisolid slab-gel. Gels provide
physical support and mechanical stability for the fluidic buffer system. In some
modes of electrophoresis, the gel participates in the mechanism of separation as a
molecular sieve. Non-gel media such as paper or cellulose acetate are alternative
supports. These media are less inert than gels, since 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 or simply the run buffer, this solution maintains the
requisite pH and provides sufficient conductivity to allow the passage of current
(ions), necessary for the separation. Frequently, additional materials are added to
the buffer to adjust the selectivity of the separation. These reagents, known as
additives, can interact with a solute and modify its rate of electrophoretic migration.
The theory and practice of electrophoresis have been the subject of many textbooks
and conference proceedings (1-10).
Apparatus for conducting electrophoresis, such as that illustrated in Fig. 1.1, is
remarkably simple and low-cost. The gel medium, which is supported on glass
plates, is inserted into a Plexiglas 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 the power supply. Many samples can be separated
1
Comment from a conferee at HPCE '89, excerpted from a short course produced by Dr. Joseph
Olechno, Dionex Corporation.

1
2 1.1 Electrophoresis

BUFFER SOLUTION
Figure 1.1. Drawing of an apparatus for slab-gel electrophoresis.

simultaneously, since it is possible to use a multilane gel. One or two lanes are
frequently reserved for standard mixtures to calibrate the electropherogram.
2
Calibration is usually based on molecular size or in isoelectric focusing, pi.
Gels such as polyacrylamide or agarose serve several important functions:
molecular sieving, reduction of diffusion and convection, and physical stabiliza­
tion. The gel composition is adjusted to define specific pore sizes, each for a
nominal range of molecular sizes. This forms the basis for separations of macro­
molecules based on size. With proper calibration, extrapolation to molecular
weight is simple.
Reduction of convection and diffusion is another function of the gel matrix.
The production of heat by the applied field induces convective movement of the
electrolyte. This movement results in bandbroadening that reduces the efficiency of
the separation. The viscous gel medium inhibits fluid movement in the electric field.
Such a material is termed anticonvective. Since the gel is high-viscosity, molecular
diffusion is reduced as well, further enhancing the efficiency of the separation.
Finally, the gel must be sufficiently viscous to provide physical support.
Low-viscosity solutions or gels would flow if the plate were not held level.
Immersion in detection reagents would be impossible, since handling or contact
with fluid solutions would destroy the matrix and separation.
2
Calibration of capillaries for isoelectric focusing and size separations is discussed in Chapters
4 and 5, respectively.
Introduction 3

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.
4 1.1 Electrophoresis

The basic procedure for performing gel electrophoresis is

1. prepare and pour the gel


2. apply the sample
3. run the separation
3
4. immerse the gel in a detection reagent
4
5. photograph the gel for a permanent record

The separation of some polymerase chain reaction (PCR) products is shown in


Fig. 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 double-stranded
DNA PCR reaction. Quantitation for such a separation is difficult and often
imprecise, but it can be performed with the aid of a gel scanner. Recoveries of
material from the gel are performed using procedures such as the Southern blot
(11). Sufficient material is recoverable for sequencing or other bioassays.
Separations of the sizing standard and 500-mer PCR product by HPCE using
a size-selective polymer network (Chapter 5) are shown in Fig. 1.3. Quantitation
is readily performed using peak area comparison to the standard. However,

500

10 15 20
TIME (min.)

Figure 1.3. Capillary gel electrophoresis of a 500-mer (top) double-stranded PCR reaction product
and a low molecular weight sizing standard (bottom). Capillary: 50 cm χ 50 μπι i.d. Bio-Rad coated
capillary; buffer: 100 mM tris-borate, pH 2.3, 2 mM E D T A with linear polymers; injection:
electrokinetic, 8 kV, 8 s; detection UV, 260 nm. Courtesy of Bio-Rad.

3
On-line detection is performed on an instrument such as an automated DNA sequencer.
4
Automated gel scanners can be used in the place of gel archiving or photography.
Introduction 5

fraction collection is difficult, since only minuscule amounts of material are


injected into the capillary.

1.2 Microchromatographic Separation Methods

The evolution of chromatographic methods over the last 30 years has produced
a systematic and rational trend toward miniaturization. This is particularly true for
gas chromatography, where the advantages of the open-tubular capillary displaced
the use of packed columns for many applications.
Chromatographic separations all function based on 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" (12). Solute molecules select various paths through the chromato­
graphic maze. The detected peak suggests this distribution and is broadened. In the
open tubular capillary, the choices for solute transport are limited, so the solute
elutes as a narrow band.
In order for the open tubular capillary to properly function, 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 i.d. capillaries is loading capacity. Injection sizes
must be kept small to avoid overloading the system. In G C , this problem is
overcome in part because sensitive detectors 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
(MLOD). Since dilution of the solute is minimized in the miniaturized system,
improved MLODs compared with large-scale systems are obtained. This is partic­
ularly important when the available sample size is small, as often happens in
biomolecule separations.
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 because of the incompatibility of the mobile phase with each detector.
Pumping of the mobile phase at the low flow rates required by miniaturization is
also more complex, particularly when gradient elution is required. Despite these
problems, μ-LC systems are useful in sample limited situations. Several books have
been devoted to this important field (13-15).
Most work with μ-LC employs 250 μιη i.d. packed columns, so the advantages
enjoyed by open-tubular G C are not realized in μ-LC. The instrumental problems
posed by open-tubular L C have inhibited most people from using this technology.
6 1.3 Capillary Electrophoresis

1.3 Capillary Electrophoresis

The arrival of high-performance capillary electrophoresis (HPCE) solves many


experimental problems of gels. Use of gels is unnecessary, since the capillary walls
5
provide mechanical support for the carrier electrolyte. 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 chromatogram.
The use of narrow-diameter capillaries allows efficient heat dissipation.
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
may be overcome in the future if multicapillary instruments (16) are designed
and introduced.
HPCE represents a merging of technologies derived from traditional electro­
phoresis and high-performance liquid chromatography (HPLC). Both HPCE
and HPLC employ on-line detection. Developments in on-column micro-LC
detection have directly transferred over to capillary electrophoresis. One of the
modes of HPCE, micellar electrokinetic capillary chromatography (Chapter 7), is
a true chromatographic technique. Electrically driven separations through packed
columns (Chapter 8), while difficult in practice, have been reported from many
laboratories. While there is much in common between these two techniques, the
fundamentals of HPCE are based on electrophoresis, not chromatography.
Professor Richard Hartwick, from the State University of New York at
Binghamton, starts off many of his lectures on capillary electrophoresis with a
discussion of transport processes in separations (17). During a separation, there are
two major transport processes that are 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 individual
solute must have unique transport properties for a separation to occur.
Dispersive transport, or bandbroadening, is the sum of processes of the
dispersing 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 controlled,
can merge peaks together.
5
Gels are sometimes used in HPCE for running size separations.
Introduction 7

According to 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 efficient separations.
Fig. 1.4 and Fig. 1.5 illustrate this concept, using a series of barbiturate
separations to compare HPCE and HPLC. The mode of electrophoresis used in Fig.
1.5 is micellar electrokinetic capillary chromatography (MECC), an electropho-
retic technique that resembles reverse-phase L C . In the L C separation, amobarbital
and pentabarbital coelute, but both are resolved by HPCE.
With some optimization work, amobarbital and pentabarbital can be separated
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 that can be separated per unit time. With a couple of hundred
6
thousand theoretical plates, many separations occur without extensive optimiza­
tion efforts. In addition, peak symmetry is excellent using HPCE unless wall effects

,2

0 11
TIME (MIN.)
Figure 1.4. Reverse-phase liquid chromatography of barbiturates. Column: Econosphere C i 8 ,
25 cm χ 4.6 mm i.d.; mobile phase: acetonitrile:water, 55/45 (v/v); injection size: 20 μι; flow rate:
1.2 mL/min; postcolumn reagent: borate buffer, pH 10,0.2 mL/min; detection: UV, 240 nm; solutes:
1) barbital, 2) butethal, 3) amobarbital and pentabarbital, 4) secobarbital; amount injected: 25 ng
of each barbiturate from a 1.25 μ g / m L solution.

The theoretical plate (N) is a measure


2
of the efficiency of a chromatographic or
electrophoretic peak, Ν = 16 ( f m/ P W ) , where tm is the migration time and PW is the peak width at
the baseline.

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