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Chapter 23
Electrophoresis
Chapter Outline
23.1 Introduction: The Human Genome Project
23.1A What Is Electrophoresis?
23.1B How Is Electrophoresis Performed?
23.2 General Principles of Electrophoresis
23.2A Factors Affecting Analyte Migration
23.2B Factors Affecting Band-Broadening
23.3 Gel Electrophoresis
23.3A What Is Gel Electrophoresis?
23.3B How Is Gel Electrophoresis Performed?
23.3C What Are Some Special Types of Gel Electrophoresis?
23.4 Capillary Electrophoresis
23.4A What Is Capillary Electrophoresis?
23.4B How Is Capillary Electrophoresis Performed?
23.4C What Are Some Special Types of Capillary Electrophoresis?
23.1 INTRODUCTION: THE HUMAN The Sanger method was originally developed as a
GENOME PROJECT manual technique that took long periods of time to per-
form. Thus, one thing that had to be addressed early in
February 2001 saw one of the greatest achievements of the Human Genome Project was the creation of faster,
modern science. It was at this time that two scientific automated systems for sequencing DNA.5,6 Both tradi-
papers appeared, one in the journal Science and the other tional and newer systems for accomplishing this
in Nature, reporting the sequence of human DNA (or the sequencing utilize a separation method known as
“human genome”).1,2 These papers were the result of a electrophoresis. In this chapter we learn about elec-
major research effort known as the Human Genome trophoresis, look at its applications, and see how
Project, which was formally begun in 1990 under the improvements in this technique made the Human
sponsorship of the U.S. Department of Energy and the Genome Project possible.
National Institutes of Health.3
Although it was anticipated to take 15 years to fin-
23.1A What Is Electrophoresis?
ish, this project was “completed” in about a decade. This
early completion was made possible by several advances Electrophoresis is a technique in which solutes are sepa-
that occurred in techniques for sequencing DNA. One rated by their different rates of migration in an electric
common approach for sequencing DNA is the Sanger field (see Figure 23.2).7–10 To carry out this method, a
method (see Figure 23.1). In the Sanger method, the sec- sample is first placed in a container or support that also
tion of DNA to be examined (known as the “template”) is contains a background electrolyte (or “running buffer”).
mixed with a segment of DNA that binds to part of this When an electric field is later applied to this system, the
sequence (the “primer”). This mixture is placed into four ions in the running buffer will flow from one electrode to
containers that have the nucleotides and enzymes the other and provide the current needed to maintain the
needed to build on the template. These containers also applied voltage. At the same time, positively charged
have special labeled nucleotides that will stop the elonga- ions in the sample will move toward the negative elec-
tion of DNA after the addition of a C, G, A, or T to its trode (the cathode), while negatively charged ions will
sequence. The DNA strands formed in each container are move toward the positive electrode (the anode). The
later separated according to their size. By comparing the result is a separation of these ions based on their charge
length of these strands and by knowing which labeled and size. Because many biological compounds have
nucleotides are at the end of each strand, the sequence of charges or ionizable groups (e.g., DNA and proteins),
the DNA can be determined.4 electrophoresis is frequently utilized in biochemical and
571
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DNA replication
Add DNA and primer to
four reaction mixtures
Primer
for replication,
each mixture containing
Sample of DNA
a different elongation-
stopping nucleotide
Stops Stops Stops Stops
at C at T at A at G
FIGURE 23.1 Sequencing of DNA by the Sanger method. This method is named after F. Sanger, one of the
scientists who originally reported this technique.4 The final DNA sequence is determined in this method by
looking at the sequence of the primer strands and using the complementary nucleotides (C for G, A for T, G
for C, and T for A) to describe the sequence of the original DNA.
medical research. This approach can also be adapted for of moving boundaries between regions that contained dif-
work with small ions (like Cl - or NO3 - ) or for large ferent mixtures of proteins, as shown in Figure 23.3.10,16
charged particles (such as cells and viruses). Today it is more common to use small samples to allow
Even though it has been known for one hundred years analytes to be separated into narrow bands or zones, giving
that substances like proteins and enzymes have a character- a method known as zone electrophoresis.8–10,16 An example of
istic rate of travel in an electric field,11–13 electrophoresis did zone electrophoresis is shown in Figure 23.1, where DNA is
not become a routine separation method until around the sequenced by separating its strands of various lengths into
1930s. One notable advance occurred in 1937 when a scien- narrow bands on a gel.
tist named Arne Tiselius (Figure 23.3) used electrophoresis There are many ways in which electrophoresis is
for the separation of serum proteins.3,14 Tiselius conducted used for chemical analysis. These include the sequencing
this separation by employing a U-shaped tube in which he of DNA, as well as the purification of proteins, peptides,
placed his sample and running buffer. When he applied an and other biomolecules. In clinical chemistry, elec-
electric field, proteins in the sample began to separate as trophoresis is an important tool for examining the pat-
they migrated toward the electrodes of opposite charge. terns of amino acids, serum proteins, enzymes, and
However, the use of a large sample volume gave a series of lipoproteins in the body. Electrophoresis is also used in
broad and only partially resolved regions that contained the analysis of organic and inorganic ions in foods, com-
different mixtures of the original proteins.15 mercial products, and environmental samples. In addi-
The method employed by Tiselius is now known as tion, electrophoresis is an essential component of medical
moving boundary electrophoresis, because it produced a series and pharmaceutical research for the characterization of
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Protein 1
Protein 1 ⫹ 2
Protein 3
Protein 2 ⫹ 3
Proteins 1–3
Sample with a mixture
of proteins (1–3)
FIGURE 23.3 Arne W. K. Tiselius (1902–1971), and an example of a protein separation performed by
moving boundary electrophoresis. Tiselius was a Swedish scientist who won the 1948 Nobel Prize in chemistry
for his early work in the field of electrophoresis. Tiselius began this research while working as a graduate
student at the University of Uppsala in Sweden. He received his doctorate degree in 1930 and later returned
in 1937 to the University of Uppsala as a professor of biochemistry. It is here that he explored the use of
moving boundary electrophoresis to separate chemically similar proteins in blood.3,15 Electrophoresis is still
used today by clinical chemists when they examine the pattern of major and minor proteins in blood, urine,
and other samples from the body.
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Electrophoresis gel/support
(⫺) Electropherogram
Detector
Sample Standards
(⫺) (⫹)
Migration distance
FIGURE 23.4 Examples of the results produced by (a) gel electrophoresis, and (b) capillary electrophoresis.
electropherogram. The migration times in this plot can of a charged solute toward the electrode of opposite
be used to help in analyte identification, while the peak charge. This force depends on the strength of the applied
heights or areas are used to determine the amount of electric field (E, units of volts per distance) and the charge
each analyte. An internal standard is usually injected on the solute (z). The second force acting on the solute is
along with the sample to correct for variations during resistance to its movement, as created by the surrounding
injection or small fluctuations in the experimental con- medium. The force of this resistance (F–) depends on the
ditions during the separation. “size” of the solute (as described by its solvated radius r),
the viscosity of the medium (h), and the solute’s velocity
23.2 GENERAL PRINCIPLES of migration (v, in units of distance per time).
OF ELECTROPHORESIS When an electric field is applied, a solute will accel-
erate toward the electrode of opposite charge until the
The separation of analytes by electrophoresis has two key forces F+ and F– become equal in size (although opposite
requirements. The first requirement is there must be a dif- in direction).10,21 At this point a steady-state situation is
ference in how analytes will interact with the separation produced in which the solute begins to move at a con-
system. In electrophoresis this requirement means the stant velocity. This velocity can be found by setting the
analytes must have different migration times or migra- expressions for F+ and F– equal to each other and rear-
tion distances. The second requirement is that the bands ranging the resulting equation in terms of v.
or peaks for the analytes must be sufficiently narrow to
allow them to be resolved. Ez
6prhv = E z or v = (23.1)
6prh
23.2A Factors Affecting Analyte Migration
Electrophoretic Mobility. Electrophoresis is similar to
chromatography in that both involve the separation of com- Attraction of solute to
pounds by differential migration. Chromatography brings electrode
(F⫹ ⫽ E z)
about differential migration through chemical interactions
between analytes with the stationary phase and mobile (⫹) ⴙ (⫺)
phase. In electrophoresis, differential migration is produced Resistance to solute
by the movement of analytes in an electric field, where their movement
(F⫺ ⫽ 6rv)
rate of migration will depend on their size and charge.
The overall rate of travel of a charged solute in elec-
trophoresis will depend on two opposing forces (see FIGURE 23.5 Forces that determine
Figure 23.5). The first of these forces (F+) is the attraction electrophoretic mobility.
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To see how this velocity will be affected by only the If we lower the applied voltage from 20 kV to 10 kV
strength of the electric field, we can combine the other (a twofold change), the migration times will increase and
terms in Equation 23.1 to give a single constant (m), the migration velocities for these proteins will decrease
(also by twofold), but their electrophoretic mobilities will
v = mE (23.2) remain exactly the same. This situation occurs because
the electrophoretic mobility is independent of voltage
where m = z>(6 p r h). This new combination of terms is and electric field strength, while migration times and
known as the electrophoretic mobility, which is repre- velocities are not. Thus, if there is a decrease in V and E,
sented by the symbol m .7,9 The value of m is often Equation 23.3 indicates there must be a proportional
expressed in units of m2>V # s or cm2>kV # min and is con- decrease in v and tm to keep m constant.
stant for a given analyte under a particular set of temper-
ature and solvent conditions. The value of m also
Secondary Interactions. To obtain good separations
depends on the apparent size and charge of the solute, as
in electrophoresis it is often necessary to adjust the con-
represented by the ratio z/r in Equation 23.1. This last fea-
ditions of this method to change the electrophoretic
ture means that any two solutes with different charge-to-
mobility of a solute. We can accomplish this goal by
size ratios can, in theory, be separated by electrophoresis.
using secondary reactions that alter the charge or appar-
ent size of the solute. If an analyte is a weak acid or
weak base, for example, its net charge can be varied by
EXERCISE 23.1 Determining the Electrophoretic changing the pH. In the case of a weak monoprotic acid,
Mobility for an Analyte the main species at a pH well below the pKa will be the
The apparent electrophoretic mobility for an analyte in neutral form of the acid (HA), while the dominant
capillary electrophoresis can be found by rewriting species at a pH much greater than the pKa will be the
Equation 23.2 in the form shown. negatively charged conjugate base (A - ). At an interme-
diate pH, we will have a mixture of these two forms and
v (Ld>tm) the average charge for all of these species will be some-
m = = (23.3)
E (V>L) where between “0” and “–1.” As a result, the overall
observed electrophoretic mobility for such a compound
In this equation, V is the voltage applied to the elec- (as well as for other weak acids and weak bases) can be
trophoretic system over a length L, and Ld is the distance adjusted by varying the pH.
traveled from the point of application to the detector by It is also possible to use side reactions to change the
the analyte in migration time tm. effective size or charge of the analyte. This effect occurs
A sample of several proteins is applied to a neutral- in a method known as sodium dodecyl sulfate polyacry-
coated capillary with a total length of 25.0 cm and a distance lamide gel electrophoresis (SDS-PAGE), which is a tech-
to the detector of 22.0 cm. Two of the proteins in the sample nique for separating proteins according to their size (see
give migration times of 15.3 min and 16.2 min when using Section 23.4C). This analysis begins by first denaturing
an applied voltage of 20.0 kV. What are the migration veloc- the proteins and coating them with sodium dodecyl sul-
ities and electrophoretic mobilities of these proteins under fate, a negatively charged surfactant. The coating
these conditions? What will their electrophoretic mobilities process can be thought of as a type of complexation reac-
and migration times be at an applied voltage of 10.0 kV? tion. The negative coating not only alters the overall
charge but helps convert a protein into a rod-shaped
SOLUTION structure, which alters its size and shape.18,19
The electrophoretic mobility of the first protein can be Another approach for altering the apparent elec-
found by substituting the known values for Ld (22.0 cm), tm trophoretic mobility of an analyte is to use a solubility
(15.3 min), V (20.0 kV), and L (25.0 cm) into Equation 23.3. equilibrium. As an example, we could include a second
phase within the running buffer into which the analyte
(22.0 cm>15.3 min) can partition as it moves through the system (such as
Protein 1: m = = 1.80 cm2>kV # min through the use of micelles, a method we will examine
(20.0 kV>25.0 cm)
in Section 23.4C). Because the analyte in such a system
A similar calculation for the second protein gives an elec- will usually have different mobilities when it is present
trophoretic mobility of 1.70 cm2>kV # min . The lower in the running buffer or in the second phase, the parti-
electrophoretic mobility of the second protein makes tioning of an analyte between these regions leads to a
sense because it takes longer for this protein to migrate change in the analyte’s rate of travel through the elec-
through the system. The migration velocities for these trophoretic system. Physical interactions can also affect
proteins can be found by simply dividing their distance analyte migration. For instance, DNA sequencing by gel
of travel by their migration times (v = Ld>tm) , which electrophoresis uses a porous support to separate DNA
gives (22.0 cm/15.3 min) = 1.44 cm/min and (22 cm/ strands of different lengths. The same strategy is used in
16.2 min) = 1.36 cm/min for proteins 1 and 2. SDS-PAGE for protein separations.
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Electroosmosis. Up until now we have examined overall observed electrophoretic mobility (mNet) for an
only the direct movement of an analyte in an electric analyte will be equal to the sum of its own electrophoretic
field. It is also possible for the running buffer to move mobility (m) and the mobility of the running buffer due to
in such a field. This phenomenon can occur if there are electrosmotic flow (meo).
any fixed charges present in the system, such as on the
mNet = m + meo (23.5)
interior surface of an electrophoretic system or on a
support within this system (see Figure 23.6). The pres- In gel electrophoresis, electroosmotic flow is often small
ence of these fixed charges attracts ions of opposite compared to the inherent rate of analyte migration. This
charge from the running buffer and creates an electrical is not usually true in capillary electrophoresis, where the
double layer at the surface of the support. In the pres- support has a relatively large charge and high surface
ence of an electric field, this double layer acts like a pis- area compared to the volume of running buffer (see
ton that causes a net movement of the buffer toward the Section 23.3).
electrode of opposite charge versus the fixed ionic
groups. This process is known as electroosmosis and 23.2B Factors Affecting Band-Broadening
results in a net flow of the buffer and its contents
The same terms used to describe efficiency in chromatogra-
through the system.7
phy (e.g., the number of theoretical plates N and the height
The extent to which electroosmosis affects the buffer
equivalent of a theoretical plate H) can be used to describe
and analytes in electrophoresis is described by using a
band-broadening in electrophoresis. Two particularly
term known as the electroosmotic mobility (or meo).7 This
important band-broadening processes in electrophoresis
term has the same units as the electrophoretic mobility m.
are (1) longitudinal diffusion and (2) Joule heating.
The value of meo depends on such factors as the size of the
electric field, the type of running buffer that is being
Longitudinal Diffusion. You may recall from Chapter 20
employed, and the type of charge that is present on the
that longitudinal diffusion occurs when a solute diffuses
support. This relationship is described by Equation 23.4,
away from the center of its band along the direction of
meo = A e zE B >h (23.4) travel, causing this band to broaden over time and to
become less concentrated. One factor that affects the extent
where E is the electric field, e and h are the dielectric con- of this band-broadening is the “size” of the diffusing solute,
stant and viscosity of the running buffer, and z is the zeta or its solvated radius. Because larger analytes have slower
potential (which represents the charge on the support). diffusion, they will be less affected by longitudinal diffusion
Depending on the direction of buffer flow, electroos- than smaller substances. The rate of this diffusion will also
mosis can work either with or against the inherent migra- decrease as we increase the viscosity of the running buffer
tion of an analyte through the electrophoretic system. The or lower the temperature of the system.
Fixed charges
on support wall
Ions in ⴚ ⴚ ⴚ ⴚ ⴚ ⴚ ⴚ ⴚ
double layer ⴙ ⴙ ⴙ ⴙ ⴙ ⴙ ⴙ
ⴚ ⴚ
ⴚ ⴚ
Other ions in ⴙ ⴙ
ⴚ ⴚ
running buffer ⴙ ⴚ
ⴙ ⴚ
ⴚ ⴙ
ⴙ
ⴚ
(⫹) ⴙ (⫺)
ⴚ
ⴙ
ⴚ
Electroosmosis
FIGURE 23.6 The production and effects of electroosmosis. This particular example
shows a support that has a negatively charged interior. Such a situation is often
encountered when working with a support that is an uncoated silica capillary. The
interior wall of this capillary has silanol groups at its surface, which can act as weak
acids and form a conjugate base with a negative charge. The extent of electroosmosis
in this case will depend on the pH of the running buffer, because this will affect the
relative amount of the silanol groups that are present in their neutral acid form or
charged conjugate base form.
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The extent of longitudinal diffusion will depend on use more efficient cooling for the system, which would
the amount of time that is allowed for this process to allow higher voltages to be used and provide shorter sepa-
occur.10 This time, in turn, will be affected in elec- ration times. Another possibility is to add a support to the
trophoresis by the size of the electric field, because lower electrophoretic system that minimizes the effects of Joule
electric fields result in smaller migration velocities and heating due to uneven heat distribution and density gradi-
longer migration times.22 Electroosmosis will also affect ents in the running buffer. Examples of these approaches
the time needed for an electrophoretic separation and dif- will be given later when we examine the methods of gel
fusion. If electroosmosis moves in a direction opposite to electrophoresis and capillary electrophoresis.
that desired for the separation of analytes, the effective Another factor that affects Joule heating is the ionic
rate of travel for these analytes is decreased and the time strength of the running buffer. A lower ionic strength for
allowed for longitudinal diffusion is increased. If elec- this buffer will lower heat production, because at low
troosmosis instead occurs in the same direction as analyte ionic strengths there are fewer ions in this buffer. This
migration, longitudinal diffusion is decreased. lower ionic strength creates a greater resistance R to cur-
One way we can minimize the effects of longitudi- rent flow at any given voltage because fewer ions are
nal diffusion in electrophoresis is to have an analyte available to carry the current. We can see from Ohm’s law
move through a porous support. If the pores of this sup- in Equation 23.6 that as R increases a smaller current is
port are sufficiently small, they will inhibit the move- needed at voltage V. This smaller current, in turn, will
ment of analytes due to diffusion and help provide create lower heat production, as shown by Equation 23.7.
narrower bands. If the pore size becomes too small, a
size-based separation will also be created. Although this Other Factors. Eddy diffusion (a process we discussed
last feature is not always desirable, in some cases it can in Chapter 20 for chromatography) is another factor that
be an advantage, such as in the sequencing of DNA by can sometimes lead to band-broadening in electrophore-
gel electrophoresis. sis. This type of band-broadening can occur if a support
is used to minimize the effects of Joule heating, a situa-
Joule Heating. The most important band-broadening tion that creates multiple flow paths for analytes through
process in electrophoresis is often Joule heating.21-23 This the support. If the support interacts with analytes, band-
process is caused by heating that occurs whenever an broadening due to these secondary interactions will be
electric field is applied to the system. According to Ohm’s introduced as well; this extra band-broadening also
law (see Chapter 14), placing a voltage V across a medium occurs when secondary interactions are used to adjust
with a resistance of R requires that a current of I be pres- analyte mobility, such as complexation reactions or parti-
ent to maintain this voltage across the medium.10 tioning into a micelle. These latter effects are similar to
those described in Chapter 20 for stationary phase mass
Ohm’s law: V = I#R (23.6)
transfer in chromatography. Broadening of the peaks
As current flows through the system, heat is gener- before or after separation can be another issue when deal-
ated. This heat production depends on the voltage, cur- ing with highly efficient systems, such as those used in
rent, and time t the current passes through the system, as capillary electrophoresis.
shown below. Wick flow is another source of band-broadening that
occurs in gel electrophoresis.19 In such a system, the gel is
Heat = V # I # t (23.7)
kept in contact with the electrodes and buffer reservoirs
As heat is produced, the temperature of the elec- through the use of wicks. Because this support is often
trophoretic system will begin to rise. This rise in tempera- open to air, the presence of any Joule heating will lead to
ture will increase longitudinal diffusion and lead to some evaporation of solvent in the running buffer from
increased band-broadening. In addition, if the heat is not the support. As this solvent is lost, it is replenished by the
distributed uniformly throughout the electrophoretic sys- flow of more solvent through the wicks and from the
tem, the temperature will not be the same throughout the buffer reservoirs. This flow leads to a net movement of
system. An uneven temperature will lead to regions with buffer from each reservoir towards the center of the sup-
different densities (causing mixing) and different rates of port. The rate of this flow depends on the rate of solvent
diffusion, which results in even more band-broadening. evaporation, so it will increase with the use of a high volt-
Other problems created by an increase in temperature age or high current. The extent of this flow varies across
include possible degradation of the analytes or compo- the support, with the fastest rates occurring furthest from
nents of the system and the evaporation of solvent from the center of the support.
the running buffer, the latter of which can alter the pH
and composition of the buffer. All of these factors lead to a 23.3 GEL ELECTROPHORESIS
loss of reproducibility and efficiency in the system. 23.3A What Is Gel Electrophoresis?
One way Joule heating can be decreased is by using a
lower voltage for the separation. A lower voltage, how- One of the most common types of electrophoresis is the
ever, will lower the migration velocities of analytes and method of gel electrophoresis. This technique is an elec-
give longer separation times. An alternative approach is to trophoretic method that is performed by applying a sample
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to a gel support that is then placed into an electric field.17–20 be carried out on agarose, a support that we discussed in
Typical separations obtained by gel electrophoresis were Chapter 22. The resulting approach is known as “agarose
shown previously in Figures 23.1 and 23.4. In this type of electrophoresis.” In addition to its low nonspecific binding
system, several samples are usually applied to the gel and for many biological compounds, agarose has a low inher-
allowed to migrate along the length of the support in the ent charge. Agarose also has relatively wide pores that
presence of an applied electric field. The separation is allow it to be employed in work with large molecules, such
stopped before analytes have left the end of the gel, with as during the sequencing of DNA.
the location and intensities then being determined. The most common support used in gel electrophoresis
It is important to remember in gel electrophoresis is polyacrylamide. This combination is often referred
that the velocity of an analyte’s movement will be related to as polyacrylamide gel electrophoresis, or PAGE.17–19
to the distance it has traveled in the given separation time Polyacrylamide is a synthetic, transparent polymer that is
(as represented by the migration distance). The farther prepared as shown in Figure 23.8. It can be made with a
this distance is from the point of sample application, the variety of pore sizes that are smaller than those in agarose
higher the migration velocity is for the analyte and the and of a size more suitable for the separation of proteins and
larger its electrophoretic mobility. This migration dis- peptide mixtures. Like agarose, polyacrylamide has low
tance will, in turn, be related to the size and charge of the nonspecific binding for many biological compounds and
analyte and can be used in identifying such a substance. does not have any inherent charged groups in its structure.
Sample Application. The samples in gel electrophore-
23.3B How Is Gel Electrophoresis Performed? sis are applied to small “wells” that are made in the gel
Equipment and Supports. Some typical systems for during its preparation (see Figures 23.4 and 23.7). A sam-
carrying out gel electrophoresis are shown in Figure 23.7. ple volume of 10–100 µL is then placed into one of these
These systems may have a support that is held in either a wells by using a micropipette. These sample volumes
vertical or horizontal position. This support contains a help provide a sufficient amount of analyte for later
running buffer with ions that carry a current through the detection and collection, but they also create a danger of
support when an electric field is applied. To replenish this introducing band-broadening by creating a large sample
buffer and its components as they move through the sup- band at the beginning of the separation.
port or evaporate, the ends of the support are placed in A common approach to create narrow sample bands
contact with two reservoirs that contain the same buffer is to employ two types of gels in the system: a “stacking
solution and the electrodes. Once samples have been gel” and a “running gel.”19 The running gel is the support
placed on the support, the electrodes are connected to a used for the electrophoretic separation of substances in the
power supply and used to apply a voltage across the sup- sample. In a vertical gel electrophoresis system, this gel is
port. This electric field is passed through the system for a formed first and is located throughout the middle and
given amount of time, causing the sample components to lower section of the system (see right-hand portion of
migrate. After the electric field has been turned off, the gel Figure 23.7). The stacking gel has a lower degree of cross-
is removed and examined to locate the analyte bands. linking (giving it larger pores) and is located on top of the
The type of support we use in such a system will running gel. The stacking gel is also the section of the sup-
depend on our analytes and samples.17,19 Cellulose port in which the sample wells are located. After a sample
acetate, filter paper, and starch are useful supports for has been placed in the wells and an electric field has been
work with relatively small molecules, like amino acids and applied, analytes will travel quickly through the stacking
nucleotides. Electrophoresis involving large molecules can gel until they reach its boundary with the running gel.
BOX 23.1
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Matrix-assisted laser desorption/ionization time-of-flight mass absorbs some of this light, it transfers its energy to molecules in
spectrometry (MALDI-TOF MS) is a type of mass spectrometry in the sample, causing these to form ions. These ions are then
which a special matrix capable of absorbing light from a laser is passed through an electric field into a time-of-flight mass ana-
used for chemical ionization. The term “MALDI” was first used in lyzer, where ions of different mass-to-charge ratios will travel at
1985 to describe the use of a laser to cause ionization of the different velocities. The number of ions arriving at the other
amino acid alanine in the presence of tryptophan (the “matrix” in end is measured at various times, allowing a mass spectrum to
this case).28 In 1988 it was shown almost simultaneously by two be obtained for analytes in the sample.31
research groups, one in Germany and one in Japan, that MALDI- MALDI-TOF MS is a soft ionization approach that results
TOF MS could also be employed in work with large biomolecules, in a large amount of molecular ions and few, if any, fragment
such as proteins.29,30 The value of this method was recognized in ions for most analytes. This method also has a low background
2002 when members of both these groups shared the Nobel signal, a high mass accuracy, and can be used over a wide
Prize in chemistry for the development of this technique. range of masses. These properties make MALDI-TOF MS valu-
Figure 23.9 shows the typical way in which a sample is able in the study and identification of proteins after they have
analyzed by MALDI-TOF MS. First, the sample is mixed with a been separated by techniques like SDS-PAGE or 2-dimensional
matrix that can readily absorb UV light. This mixture is then (2-D) electrophoresis (see Section 23.3). MALDI-TOF MS can
placed on a holder in the MALDI-TOF instrument, where pulses also be used to look at peptides, polysaccharides, nucleic acids,
of a UV laser are aimed at the sample and matrix. As the matrix and some synthetic polymers.31,32
Pulsed N2
laser beam Sample ions
(337 nm) ⴙ (to mass spectrometer)
ⴙ ⴙ
ⴙ Sample in matrix that
absorbs UV light
Laser
Drift tube
Detector
Ionization ⴙ ⴙ
chamber ⴙ
ⴙ
Electric field
In SDS-PAGE, the proteins in a sample are first dena- At the end of an SDS-PAGE run, the positions of
tured and their disulfide bonds broken through the use of a protein bands from a sample are compared to those
reducing agent. This pretreatment converts the proteins obtained for known protein standards applied to the
into a set of single-stranded polypeptides. These polypep- same gel. This comparison is made either qualitatively or
tides are then treated with sodium dodecyl sulfate (SDS), a by preparing a calibration curve. The calibration curve is
surfactant with a nonpolar tail and a negatively charged typically prepared by plotting the log of the molecular
sulfate group. The nonpolar end of this surfactant coats weight (MW) for the protein standards versus their
each protein, forming roughly linear rods that have an exte- migration distance (dm) or retardation factor (Rf). The retar-
rior layer of negative charge. The result for a mixture of dation factor for an analyte band in SDS-PAGE is calcu-
proteins is a series of rods with different lengths but similar lated by using the ratio of a protein’s migration distance
charge-to-mass ratios. Next, these protein rods are passed over the migration distance for a small marker com-
through a porous polyacrylamide gel in the presence of an pound (ds), where Rf = dm>ds. The resulting plot of
electric field. The negative charges on these rods (from the log(MW) versus dm or Rf gives a curved response with an
SDS coating) cause them to all move toward the positive intermediate linear region for proteins with sizes that are
electrode, while the pores of the gel allow small rods to neither totally excluded from the pores nor able to access
travel more quickly to this electrode than large rods. all pores in the support.
96943_23_ch23_p571-596 1/8/10 2:54 PM Page 581
Sample pretreatment
ⴚ ⴚ
Denature proteins and ⴚⴚ ⴚ ⴚⴚ ⴚ ⴚ
reduce disulfide bonds
Coat proteins with SDS ⴚ ⴚ ⴚ
ⴚⴚ ⴚ
ⴚ ⴚ ⴚ
(a)
Protein separation
(⫺)
Standard Sample 1 Sample 2
High MW
Protein migration
Low MW
(⫹)
(b)
High
ⴚ pH ⬎ pI
Isoelectric Focusing. Another type of electrophore-
sis that often employs supports is isoelectric focusing
(IEF). 10 IEF is a method used to separate zwitterions ⴙ ⴚ
(substances with both acidic and basic groups, as dis- pH ⴙ ⴚⴙ ⴚ pH ⫽ pI
cussed in Chapter 8). Zwitterions are separated in IEF
based on their isoelectric points by having these com-
ⴙ pH ⬍ pI
pounds migrate in an electric field across a pH gradi-
ent. In this pH gradient, each zwitterion will migrate Low
until it reaches a region where the pH is equal to its (⫹) (⫹)
isoelectric point. At this point, the zwitterion will no
longer have any net charge and its electrophoretic FIGURE 23.11 Isoelectric focusing.
96943_23_ch23_p571-596 1/8/10 2:54 PM Page 582
D SOLUTION
We can use Equation 23.8 along with the conditions given
in Exercise 23.1 and the electrophoretic mobility calcu-
A,B lated earlier for protein 1 to get the expected value for N
E K at 20.0 kV.
L
A 1.80 cm2>kV # min B # 20.0 kV # 22.0 cm
Response
between these electrodes and the solution within the direction. The analysis of these ions in a silica capillary is
capillary, an on-line detector, and a means for injecting performed by injecting them at the end by the negative
samples onto the capillary. Because these instruments electrode and allowing them to migrate toward the posi-
can use voltages of up to 25–30 kV, they include safety tive electrode and against electroosmotic flow. This
features that protect the user from the high-voltage method is known as the “reversed polarity mode” of CE.8
region and that can turn off this voltage when the system In addition, electroosmotic flow can be altered by chang-
is opened for maintenance or the insertion of samples ing the pH (which changes the degree of deprotonation
and reagents. and charge on silica), or by placing a coating on the sur-
The capillary in a CE system is typically made of fused face of the support. In this second case, a neutral coating
silica. This capillary can be used directly or it can be modi- helps to reduce electroosmosis while a positively charged
fied to place various coatings on its interior surface. An coating will reverse the direction of this flow toward the
uncoated silica capillary can lead to a significant amount of positive rather than negative electrode.
flow due to electroosmosis when working at a neutral or
basic pH, due to deprotonation of the silica’s surface silanol Injection Techniques. There are two features of capillary
groups. One useful feature of this electroosmosis is it tends electrophoresis that place special demands on how samples
to cause all analytes, regardless of their charge, to travel in can be injected. First, the small volume of a CE capillary
the same direction through the CE capillary. This effect must be considered. A typical 50 µm I.D. * 25 cm long cap-
means that a sample containing many types of ions can be illary for CE will contain only 0.5 mL of running buffer.
injected at one end of the capillary (at the positive elec- Another factor to consider is the high efficiency of capillary
trode), with electroosmosis then carrying these through to electrophoresis. Both of these factors restrict the sample vol-
the other end (to the negative electrode) and past an on-line umes that can be injected without introducing significant
detector. This format is called the “normal polarity mode” of band-broadening (< 10 nL for a 0.5 mL volume capillary).8
CE.8 It is important to remember in this situation that a sep- There are two techniques that make it possible to
aration of ions will still occur, but that the observed mobility inject these small sample volumes onto a CE system. The
will now be equal to the sum of an analyte’s inherent elec- first technique is hydrodynamic injection, which uses a dif-
trophoretic mobility plus the mobility created by electroos- ference in pressure to deliver a sample to the capillary.
mosis (see Equation 23.5). This effect on observed mobility This method can be carried out by placing one end of the
will, in turn, affect the observed migration time and the effi- capillary into the sample in an enclosed chamber and
ciency and resolution obtained for the separation. applying a pressure to this chamber for a fixed period of
Although many analytes will travel in the same time, where the amount of injected sample will depend on
direction as electroosmotic flow through a CE system, it the size of the pressure difference and the amount of time
is possible for some to have migration rates faster than that this pressure is applied. Once the sample has entered
electroosmosis, which will carry them in the opposite the capillary, the separation is begun after the capillary
Data acquisition
(and control)
Detector
Net migration
Capillary
Inlet Outlet
reservior reservior
or sample
High voltage
power supply
FIGURE 23.14 General design of a capillary electrophoresis system, and a commercial instrument for
capillary electrophoresis. (The picture on the right is courtesy of Beckman Instruments.)
96943_23_ch23_p571-596 1/8/10 2:54 PM Page 585
has been put back into contact with the running buffer Initial conditions
and electrodes. A second technique that allows the injec-
tion of small sample volumes is electrokinetic injection. This Sample Separation buffer
(low ionic strength (higher ionic strength
method again begins by placing the capillary into the and conductivity) and conductivity)
sample, but an electrode is also now in contact with the
sample. When an electric field is applied across the capil-
ⴙ ⴙ
lary, electroosmostic flow and the electrophoretic mobility
of the analytes cause them to enter the capillary. The (⫹) (⫺)
ⴙ Sample and buffer
amount of each analyte that is injected in this method will
depend on the analyte’s electrophoretic mobility, the elec- ⴙ interface
tric field, and the time over which this field is applied.8
There are various methods for concentrating sam-
ples and providing narrow analyte bands in CE. One Conditions after sample stacking
such method is sample stacking (see Figure 23.15).21
Sample stacking occurs when the ionic strength (and
therefore the conductivity) of the sample is less than that ⴙ
of the running buffer. When an electric field is applied to ⴙ
(⫹) (⫺)
such a system, analytes will migrate quickly through the ⴙ
sample matrix until they come to the boundary between
ⴙ
the sample and running buffer. Because the running
buffer has a higher ionic strength than the sample, the
rate of analyte migration decreases at this boundary. This FIGURE 23.15 Principle of sample self-stacking.
decrease in migration rate causes the analytes to concen-
trate into a narrower band as they enter the running Another difference between detection in LC and
buffer. The overall effect is similar to what occurs when CE concerns how their signals vary with analyte reten-
using stacking gels in traditional electrophoresis. tion or migration. In LC, all analytes pass at the same
flow rate (that of the mobile phase) through the detec-
Detection Methods. Examples of detection methods tor and spend the same amount of time in this device.
that are used for capillary electrophoresis are shown in This effect makes it possible to directly compare the
Table 23.1. Many of these methods are also used in liquid peak areas of two analytes with different retention
chromatography (see Chapter 22).8,21 An important dif- times. However, in capillary electrophoresis analytes
ference between detection in LC and CE is the need in with different migration times also spend different
CE for methods that can work with very small sample amounts of time in the detector. A correction must be
sizes. This need is a result of the small injection volumes made for this difference if we wish to compare the
that are required in capillary electrophoresis to avoid areas of two analytes in the same CE run. We can make
excessive band-broadening. Selective monitoring meth- this adjustment by using a corrected peak area (A c ),
ods that work well for this purpose are electrochemical which is equal to the ratio of the measured peak area
and fluorescence detection. Ultraviolet-visible (UV-vis) (A) for an analyte divided by its migration time.
absorbance, conductance, and mass spectrometry detec-
tion are also often employed in CE. Ac = A>tm (23.10)
This correction allows areas for different analytes to be Along with the various detection methods we dis-
compared, as well as areas that are obtained for the same cussed for liquid chromatography in Chapter 22, another
analyte under different electrophoretic conditions. detection approach that is used in capillary electrophoresis
is laser-induced fluorescence (LIF).6,8,21 This method
employs a laser to excite a fluorescent compound, allowing
EXERCISE 23.4 Correcting Peak Areas for the detection of this agent through its subsequent emission
Analyte Migration Times of light. There are several advantages to using a laser as the
Proteins 1 and 2 in Exercise 23.1 have measured areas of excitation source. First, the laser is monochromatic and has
1290 and 1360 units at 20.0 kV when examined by an a high intensity, which allows for the selective and strong
absorbance detector. If it is known that these two proteins excitation of a compound with an excitation spectrum that
have a similar response to the detector, what is the cor- overlaps with the emission wavelength of the laser. Also,
rected area and relative amount of each protein in the the laser beam can be focused as a very narrow beam. This
given sample? feature is extremely valuable in work with the small-bore
capillaries found in capillary electrophoresis. One limita-
SOLUTION
tion of LIF detection is it does require an analyte that is nat-
The migration times for these proteins (given in urally fluorescent or that can be converted into a fluorescent
Exercise 23.1) are 15.3 min and 16.2 min. Placing these derivative. This second option makes use of a fluorescent
data into Equation 23.10 along with the measured peak tag like fluorescein or rhodamine (see Chapter 18).
areas gives the following results. LIF detection with CE was used in the automated
Protein 1: Ac,1 = A 1290 B > A 15.3 min B = 84.3 DNA sequencing systems that made early completion of
the Human Genome Project possible. This detection
Ac,2 = A 1360 B > A 16.2 min B = 84.0
involved the use of several fluorescent dyes, one for each of
Protein 2:
the four terminating nucleotides present during the Sanger
If these proteins have a similar response to the detector, reaction. These labeled DNA strands were then separated
then we can say from these corrected areas that there is based on their lengths by capillary electrophoresis (see
approximately the same amount of both proteins in the Figure 23.16). It was possible to further increase the speed
sample. If we had used the uncorrected areas for this cal- of this analysis by using a single laser beam to simultane-
culation, we would have incorrectly concluded that pro- ously examine a whole array of capillaries, each sequencing
tein 2 was present at a greater level. a different segment of DNA. The utilization of multiple
Detected Sequence
CA T G C T GCAGG T C G A C T C T A G A G G A T CCC C G G G T A C GA G C T C G AA TT C G T
10 20 30 40 50
Relative signal
FIGURE 23.16 DNA sequencing by a capillary electrophoresis system. The top panel shows the original electrophoretic data, and the
bottom panel shows the same data after they have been processed to determine the nucleotide sequence of the DNA segment being
examined (as given by the symbols A, C, T, and G). (Based on data from J. Bashkin, in Capillary Electrophoresis of Nucleic Acids, K.R.
Mitchelson and J. Cheng, Eds., Humana Press, Totowa, NJ, 2001, Chapter 7.)
96943_23_ch23_p571-596 1/8/10 2:54 PM Page 587
capillaries in a single CE system is known as capillary array system to give additional types of separations. Examples
electrophoresis (CAE).6,7,35 Such a system can examine many include CE methods that separate analytes based on their
DNA sequences at the same time, which increases sample size, isoelectric points, or interactions with additives in the
throughput and lowers the cost per analysis. running buffer. The use of CE in microanalytical systems
has also been a topic of great interest (see Box 23.2).
23.4C What Are Some Special Types of Capillary
Capillary Sieving Electrophoresis. One useful fea-
Electrophoresis?
ture of gel electrophoresis is the ability of some sup-
The main capillary electrophoresis method that has been ports to separate analytes based on size, as occurs for
discussed up to this point is zone electrophoresis, in which proteins in SDS-PAGE. The same effect can be obtained
differences in the charge/size ratio of analytes is the only in capillary electrophoresis by including an agent in
means employed for their separation. It is possible to the CE system that “sieves” the analytes, or
include other chemical and physical interactions in a CE separates them based on size. This approach is known
BOX 23.2
Analytical Chemistry on a Chip
The development of silicon microchips created a revolution in addition, the elimination of Joule heating allows CE to work
the computer and electronic industries. The result over the past with short separation channels and high electric fields, as is
few decades has been a continuous decrease in the size of elec- used in Figure 23.17. The creation of an electric field to sepa-
tronic devices and an increase in their capabilities. A similar rate analytes and to generate electroosmotic flow for CE is rel-
change is occurring in analytical chemistry. This change began atively easy to obtain with a microchip by including electrodes
in 1979, when methods developed for the creation of as part of this device. The availability of detection schemes like
microchips where used to make a gas chromatographic system LIF that are capable of working with small detection volumes is
on a silicon wafer.36 In 1990 it was proposed that all of the also valuable when placing a CE system on a microchip.38–41
components of a chemical analysis could even be placed onto a CE is not the only analytical method that has been carried
miniaturized system. The resulting device is now known as a out on microchips. Other methods have included liquid chro-
“lab-on-a-chip” or a micro total-analysis system (mTAS).37 matography, gel electrophoresis, biosensing, water analysis, flow
Capillary electrophoresis was one of the first analytical injection analysis, and solid-phase extraction. There are several
methods that was adapted for use on a microchip.38 An exam- potential advantages of using microchips with these techniques.
ple of such a device is given in Figure 23.17. There are now One is the small sample requirements of these devices. The abil-
many reports that have used microchips for CE.39–41 One fea- ity to make these systems fully portable or disposable are addi-
ture that makes CE and microchips a good match is the need in tional advantages. The possibility of making microchips on a
CE for narrow channels to avoid the effects of Joule heating. In large scale and at a low cost are other attractive features.39–41
Buffer
DCF
Sample
Sample waste
Response
RB
Injection
2 mm valve
Inject
Separation
channel
FIGURE 23.17 Design of a microchip-based system for performing electrophoresis and the use of this
device in the fast separation of the dyes rhodamine B (RB) and dichlorofluorescein (DCF). (Reproduced
with permission from S.C. Jacobson, C.T. Culbertson, J.E. Daler, and J.M. Ramsey, Analytical Chemistry, 70
(1998) 3476–3480.)
96943_23_ch23_p571-596 1/8/10 2:54 PM Page 588
as capillary sieving electrophoresis (CSE).7 A compar- dodecyl sulfate (SDS). We saw earlier that SDS has a long
ison of the results of CSE and a size separation by gel nonpolar tail attached to a negatively charged sulfate
electrophoresis (e.g., by SDS-PAGE) is given in group. When the concentration of a surfactant like SDS
Figure 23.18. reaches a certain threshold level (known as the “critical
There are several ways we can perform capillary micelle concentration”), some of the surfactant molecules
sieving electrophoresis. The first way is to place a porous come together to form micelles. If these micelles form in a
gel in the capillary, like the polyacrylamide gels polar solvent like water, the nonpolar tails of the surfactant
employed in SDS-PAGE. This method is called capillary will be on the inside of the aggregate (giving a nonpolar
gel electrophoresis (CGE).7 One problem with these gels is interior), while the charged groups at the other end will be
they are not always stable in the high electric fields used on the outside by the solvent (see Figure 23.19).
in capillary electrophoresis and must frequently be When micelles based on SDS are placed into the running
replaced. A second approach, and the one now used in buffer of a CE system, they will be attracted toward the posi-
DNA sequencing by CE, is to add to the running buffer a tive electrode. If a sample with several neutral compounds is
large polymer that can entangle with analytes and alter now injected into this system, some of these neutral sub-
their rate of migration. This approach provides a system stances may enter the micelles and interact with their nonpo-
with better reproducibility and stability than those using lar interior. This interaction involves a partitioning process
gels, because the polymer is continuously renewed as similar to that found in liquid–liquid extractions and some
the running buffer passes through the capillary. types of liquid chromatography (see Chapters 20 and 22), in
which the micelles act as the “stationary phase.” Although
Electrokinetic Chromatography. Ordinary capillary these neutral compounds normally travel with the electroos-
electrophoresis works well for separating cations and motic flow through the capillary, while they are in the micelles
anions, but it cannot be used to separate neutral sub- they migrate with the micelles in the opposite direction. The
stances from each other. Instead, these substances migrate result is a separation of neutral compounds based on the
as a single peak that travels with the electroosmotic flow. It degree to which they enter the micelles. Micelles can also alter
is possible to employ CE with such compounds if we place the migration times for charged substances through partition-
in the running buffer a charged agent that can interact with ing and charge interactions between the analytes and micelles.
these substances. This approach is called electrokinetic
chromatography. One common way of carrying out this Other Methods. There are many other types of CE that
method is to employ micelles as additives, giving a subset have been explored for use in chemical analysis and sepa-
of electrokinetic chromatography known as micellar rations. For example, isoelectric focusing can be carried
electrokinetic chromatography (MEKC).7,21,42,43 out in a capillary, creating the method of capillary isoelectric
A micelle is a particle formed by the aggregation of a focusing (CIEF).7,21 This technique involves the production
large number of surfactant molecules, such as sodium of a pH gradient across the capillary for the separation of
200 kDa
116 kDa 5.40
97 kDa
66 kDa
45 kDa 5.14
31 kDa
log(MW)
21 kDa 4.88
14 kDa
59,217
4.62
4.36 25,669
⫺4.10
1.60 1.89 2.18 2.47 2.76 3.05
Normalized migration time (min)
2 4 6 8 10
Time (min)
FIGURE 23.18 Comparison of capillary electrophoresis and traditional SDS-PAGE in the separation and analysis
of proteins. The figure on the left shows an electropherogram obtained for a series of proteins with molecular
masses ranging from 14 to 200 kDa. The inset shows an SDS-PAGE gel for the same proteins. The calibration
curve on the right shows how the migration times for these proteins in CE are related to their molecular masses.
(Adapted with permission from Bio-Rad Laboratories.)8
96943_23_ch23_p571-596 1/8/10 2:54 PM Page 589
ⴚ ⴚ
ⴚ ⴚ ⴚⴚ
ⴚ ⴚ ⴚ ⴚ ⴚ
ⴚ ⴚ ⴚ
ⴚ ⴚ ⴚ
ⴚ ⴚ ⴚ
(⫹) ⴚ ⴚ ⴚ (⫺)
ⴚ ⴚ
ⴚ ⴚ ⴚ ⴚ ⴚ ⴚⴚ
zwitterions. One way CIEF can be conducted is shown in pushed through the capillary and past the detector by
Figure 23.20. In this example, the electrodes are in contact applying pressure to the system.
with two different electrolyte solutions: (1) the Another type of capillary electrophoresis occurs
“catholyte,” which is a basic solution located by the cath- when biologically related agents are placed as additives
ode, and (2) the “anolyte,” which is an acidic solution in the running buffer. As analytes travel through this
located by the anode. The capillary contains a mixture of buffer, their overall mobility will be affected by their
ampholytes that will create a pH gradient when an elec- binding to these agents (see Figure 23.21). The result is a
tric field is applied between these electrodes. A coated method known as affinity capillary electrophoresis
capillary is also used in this case to minimize or eliminate (ACE).7,21,44,45 One common use of ACE is in the separa-
electroosmotic flow. When a sample (generally a mixture tion of chiral analytes through the use of binding agents
of proteins) is injected onto this system, its zwitterions like cyclodextrins or proteins (see Chapters 8 and 22).
will migrate until they reach a region where the pH is This method can also be used in clinical and pharmaceu-
equal to their pI. Once these bands have formed, they are tical assays and for the study of biological interactions.
Sample with
Detector
protein mixture
(⫹) Electrode (⫺) Electrode
Solution with
mixture of
amphyloytes
Anolyte Catholyte
solution solution
(a)
Detector
(⫹) Electrode Pressure (⫺) Electrode
Focused
protein bands
(b)
FIGURE 23.20 Capillary isoelectric focusing. The diagram in (a) shows the initial
configuration of this system before an electric field is applied. The diagram in (b)
shows the separation of proteins from the original sample after the electric field has
been applied. These protein bands are later passed through the capillary and past the
detector by applying pressure to the system.
96943_23_ch23_p571-596 1/8/10 2:54 PM Page 590
(⫹) (⫺)
Key Words
Capillary electrophoresis 582 Electrophoretic mobility 575 Micellar electrokinetic Southern blot 579
Capillary sieving Gel electrophoresis 577 chromatography 588 Two-dimensional
electrophoresis 588 Isoelectric focusing 581 Migration distance 573 electrophoresis 582
Electroosmosis 576 Joule heating 577 Migration time 573 Western blot 579
Electropherogram 574 Laser-induced Northern blot 579
Electrophoresis 571 fluorescence 586 SDS-PAGE 579
Other Terms
Affinity capillary Densitometer 579 Micro total-analysis Silver staining 579
electrophoresis 589 Electrokinetic injection 585 system 587 Sodium dodecyl sulfate 580
Ampholytes 582 Electrokinetic Moving boundary Wick flow 577
Capillary array chromatography 588 electrophoresis 572 Zone electrophoresis 572
electrophoresis 587 Electroosmotic mobility 576 PAGE 578
Capillary gel Hydrodynamic injection 584 Polyacrylamide 578
electrophoresis 588 MALDI-TOF MS 579 Retardation factor 580
Capillary isoelectric Micelle 588 Sample stacking 585
focusing 588
Questions
WHAT IS ELECTROPHORESIS? is the migration velocity of this protein? If an applied voltage
1. Define “electrophoresis” and explain how this method is of 200 V is used instead, how long will it take the same protein
used to separate chemicals. to migrate a distance the entire length of the 10 cm gel?
2. What is “zone electrophoresis”? How does this technique FACTORS AFFECTING ANALYTE MIGRATION
differ from “moving boundary electrophoresis”? Which of
these methods is more common in modern laboratories? 6. Explain why a charged substance will tend to move at a
constant velocity through an electric field. What forces are
HOW IS ELECTROPHORESIS PERFORMED? involved in this process?
3. Define each of the following terms and explain how they are 7. What is “electrophoretic mobility”? How is this term related
used in electrophoresis. to the movement of a substance in an electric field? What
(a) Migration distance are some general factors that affect the size of the elec-
(b) Migration time trophoretic mobility for an analyte?
(c) Electropherogram 8. A peptide is found to have a migration time of 8.31 min at
4. Chloride is found to migrate a distance of 35 cm in a capillary an applied voltage of 10.0 kV and on a capillary elec-
electrophoresis system with a migration time of 5.63 min. trophoresis system with a total length of 25.0 cm. The detec-
What is the migration velocity of chloride under these condi- tor is located at 21.5 cm from the point of sample injection
tions? At the same velocity, what distance would chloride and conditions are used so that electroosmotic flow is negli-
have traveled in 2.5 min? gible. What is the migration velocity of the peptide? What is
5. A protein is found to migrate a distance of 3.2 cm in 30 min its electrophoretic mobility?
when 100 V is applied to a 10 cm long polyacrymide gel. What 9. How would the migration velocity and migration time for
the peptide in the previous problem change if the voltage
96943_23_ch23_p571-596 1/8/10 2:54 PM Page 591
• Questions 591
was changed to 15.0 kV? What change, if any, would occur 19. How does longitudinal diffusion affect band-broadening in
in the electrophoretic mobility? electrophoresis? How is this type of band-broadening related
10. What are some examples of secondary interactions that can to the time of the separation and the size of the analytes? Why
affect analyte migration in electrophoresis? can a porous support help minimize the effects of this process?
11. Dicamba is a herbicide commonly used on broadleaf weeds. 20. What is “Joule heating”? What causes this heating? Why does
Its major metabolite is dichlorosalicylic acid (DCSA). Both Joule heating result in band-broadening in electrophoresis?
compounds are weak acids. Dicamba has a single weak-acid 21. What are some approaches that can be used to minimize the
group, which is a carboxylic acid group with a pKa of 1.94. effects of Joule heating in electrophoresis?
DCSA has two weak-acid groups: a carboxylic acid group 22. One useful tool in optimizing a separation for capillary elec-
with a pKa of 2.08 and a phenol group with a pKa of 8.60. A trophoresis is an “Ohm’s law plot.” This graph is prepared
separation of these compounds at pH 7.4 by capillary elec- by plotting the measured current of the electrophoretic sys-
trophoresis (CE) gives a migration time of 2.05 min for tem at various applied voltages.21
dicamba and 2.35 min for DSCA. The same compounds had (a) Based on Equation 23.6, what information will be pro-
migration times of 2.06 min and 4.1 min at pH 10.0.46 vided by the slope of this plot?
Explain why there is a large shift in the migration time for (b) Deviations from linearity are often seen at high voltages
DCSA over this pH range but no significant shift in the in an Ohm’s law plot. What do you think is usually the
migration time for dicamba. source of these deviations?
12. Two chiral forms of a drug have identical electrophoretic 23. Under what conditions will eddy diffusion be present dur-
mobilities in a CE system. However, it is possible to sepa- ing electrophoresis?
rate these forms when b -cyclodextrin (a complexing agent 24. What is “wick flow”? How does wick flow create band-
we discussed in Chapter 7) is placed as an additive into the broadening? In what types of electrophoresis can wick flow
running buffer. Based on your knowledge of chemical reac- be important?
tions, explain why the presence of b -cyclodextrin might
lead to such a separation. WHAT IS GEL ELECTROPHORESIS?
13. What is “electroosmosis”? What causes electroosmosis to 25. What is “gel electrophoresis”? How is this technique used
occur? How does electroosmosis affect the movement of for analyte identification and measurement?
analytes in an electrophoresis system? 26. A biochemist looking for a particular protein in a cell sam-
14. What is the “electroosmotic mobility”? What factors affect ple obtains the following results when using gel elec-
this mobility? trophoresis and a Western blot to compare this sample with
15. A neutral compound injected onto a capillary elec- standards containing the same protein. What is the approxi-
trophoresis system has a migration time of 1.52 min mate amount of this protein in the unknown cell sample?
through a 50 cm long capillary at 30.0 kV, with the detector
being located 30.0 cm from the point of injection. If it is
assumed that the observed electrophoretic mobility for Amount of Protein (ng) Relative Band Area
this compound is equal to the electroosmotic mobility, 0.0 50
what is the value of µeo under these conditions?
5.0 560
16. The electroosmotic mobility for a particular separation is
found to be 8.3 * 10 - 10m2>V # s. A 25 cm long capillary is 10.0 1120
used for this separation at an applied voltage of 20.0 kV, 20.0 2040
with the detector being located 20 cm from the point of
Unknown sample 980
injection. What is the expected migration time for a neutral
analyte in this system (i.e., an analyte that travels through
the system only due to electroosmotic flow)?
27. Figure 23.22 shows a typical result that is obtained for the
FACTORS AFFECTING BAND-BROADENING analysis of a human serum sample by gel electrophoresis.
Explain how this information might be used by a physician
17. Use the same equations as given in Chapter 20 for chro-
to detect both qualitative and quantitative changes in serum
matography to calculate the following values. (Note: You
proteins for their patients.
can assume Gaussian peaks are present.)
(a) The plate number of a peak in capillary electrophoresis
HOW IS GEL ELECTROPHORESIS PERFORMED?
that has a migration time of 7.30 min and baseline width
of 0.12 min 28. Draw a diagram of a typical gel electrophoresis system and
(b) The plate height of the system in Part (a) for a capillary label its main components. Explain the difference between
with a total length of 35.0 cm horizontal and vertical gel electrophoresis systems.
(c) The plate number of a band in gel electrophoresis with a 29. List some supports that can be used in gel electrophoresis.
migration distance of 4.2 cm and baseline width of 2.1 mm What type of support is often used with DNA? What type is
18. Use the equations given in Chapter 20 to calculate the fol- often used with proteins?
lowing values. 30. Describe how samples are usually applied to a support in
(a) The resolution of two peaks in capillary electrophoresis gel electrophoresis. Explain the purpose of a “stacking gel”
with migration times of 10.1 min and 10.4 min with versus a “running gel.”
baseline widths of 0.15 min and 0.16 min, respectively 31. Describe how each of the following items can be used for
(b) The resolution of two bands in gel electrophoresis with detection in gel electrophoresis.
migration distances of 2.3 cm and 2.6 cm with an aver- (a) Densitometer
age baseline width of 1.5 mm (b) Coomassie Brilliant Blue
96943_23_ch23_p571-596 1/8/10 2:54 PM Page 592
(c) Silver staining 37. What is “isoelectric focusing”? Describe how this method
(d) Blotting separates analytes.
32. What is the difference between a Southern blot and a 38. What is an “ampholyte”? How is an ampholyte used in iso-
Northern blot? What is the difference between a Southern blot electric focusing?
and a Western blot? How is each of these methods performed? 39. What is “2-D electrophoresis”? What types of electrophore-
33. What is MALDI-TOF MS (matrix-assisted time-of-flight sis are often used in this method? What advantages are
mass spectrometry)? Explain how this method can be used there in the use of 2-D electrophoresis for complex samples?
for identifying the contents of bands in gel electrophoresis.
WHAT IS CAPILLARY ELECTROPHORESIS?
WHAT ARE SOME SPECIAL TYPES OF GEL ELECTROPHORESIS? 40. What is meant by the term “capillary electrophoresis”?
34. Explain how SDS-PAGE (sodium dodecyl sulfate polyacry- How does this method differ from gel electrophoresis?
lamide gel electrophoresis), is performed. Describe why 41. The amount of a nitrate in an unknown sample is to be
SDS-PAGE can provide information on the molecular mass quantitated by capillary electrophoresis. Another anion is
of a protein. added to all samples and standards as an internal standard
35. The molecular mass of a protein is to be estimated by SDS- (IS) prior to injection. The following results are obtained.
PAGE. The following migration distances are obtained for pro- What is the amount of the nitrate in the unknown sample?
teins of known mass on the gel: 200 kDa, 0.33 cm; 116.3 kDa,
0.57 cm; 66.3 kDa, 0.91 cm; 36.5 kDa, 1.63 cm; 21.5 kDa, 1.96 cm;
14.4 kDa, 2.24 cm. The unknown protein has a migration dis- Concentration of
tance of 1.25 cm on the same gel. What is the approximate Concentration of Peak Internal Peak
molecular weight of this protein? Nitrate (mg/L) Height Standard (mg/L) Height
36. A biochemist uses the same conditions as in the last problem 0.0 0.2 2.5 9.8
to look for proteins with approximate masses of 18.5 kDa,
40.2 kDa, and 91.8 kDa. What are the expected migration dis- 5.0 18.8 2.5 10.2
tances for these proteins in this gel? 10.0 43.1 2.5 11.5
15.0 55.2 2.5 10.1
Unknown sample 15.1 2.5 9.7
Densitometer scan of stained gel
• Questions 593
45. A chemist wishes to use the plate number measured for a 59. What is “capillary isoelectric focusing”? Describe one way
CE to estimate the diffusion coefficient for a new drug. This this method can be performed.
drug is injected onto a 42.5 cm long neutral coated capillary, 60. What is “affinity capillary electrophoresis”? What are some
which has no binding for the drug and produces negligible applications of this method?
electroosmotic flow. The detector is located 38.0 cm from the
point of injection. The drug has a measured migration time CHALLENGE PROBLEMS
of 14.8 min and a baseline width of 26 s, when a voltage of 61. Compare the capillary electrophoresis system in Figure 23.14
15.0 kV is applied across the capillary. with the electrochemical cells that are discussed in Chapter 10.
(a) What is the number of theoretical plates for the peak (a) What similarities can you find in these two types of sys-
due to the drug, if it is known that this peak has a tems? Based on this comparison, which part of the elec-
Gaussian shape? trochemical cell would be equivalent to the capillary in
(b) What is the diffusion coefficient for the drug (in units of a CE system? Which part of an electrochemical cell do
cm2/s)? What assumptions did you make in reaching you think would be equivalent to the gel or support in a
your answer? gel electrophoresis system?
(b) Describe how current is carried from the power supply
HOW IS CAPILLARY ELECTROPHORESIS PERFORMED? and throughout an electrophoresis system. What is the
46. What are the main components of a capillary electrophore- role of the running buffer in this regard? What are the
sis system? How does this system differ from the equipment roles of the electrodes?
used in gel electrophoresis? (c) One tool we used in Chapter 6 for solving chemical
47. Why does the use of an uncoated silica capillary lead to problems was to use the method of charge balance,
electroosmotic flow in capillary electrophoresis? What is the which says that the number of positive and negative
direction of this flow? How does electroosmosis affect the charges in a system must be equal. And yet, in elec-
apparent migration of analytes through the CE system? trophoresis we use an electric field to separate analytes
48. What is the “normal polarity” mode of CE? What is the with positive and negative charges. Why do you think
“reversed polarity” mode? In what general situations are this separation is possible? (Hint: Consider your
these two modes utilized? answers to Parts (a) and (b).)
49. Explain why it is necessary in capillary electrophoresis to use 62. The effect of electroosmotic flow on the overall observed elec-
small injection volumes. What are some difficulties in work- trophoretic mobility (mNet) and migration velocity (tm) for an
ing with these small volumes? What are some advantages? analyte in electrophoresis is given by the following equations,
50. What is “hydrodynamic injection”? What is “electrokinetic
(m+mosm)V
injection”? How does each of these methods work? v = mNet E = (23.11)
51. What is “sample stacking”? Describe one way sample stack- L
ing can be accomplished in capillary electrophoresis. LdL Ld
52. List some general and some selective detectors that are used tm = = (23.12)
(m+mosm)V mNet E
in capillary electrophoresis. How does this list compare to
that given in Chapter 22 for liquid chromatography? where all terms are the same as described earlier in this
53. Two isoforms of a protein are found to elute with migration chapter.21,23
times of 20.3 min and 24.5 min from a capillary elec- (a) A cation has an electrophoretic mobility of
trophoresis system. These protein peaks have measured 2.50 cm2>kV # min on a CE system containing a 30.0 cm
areas of 3430 and 1235 units, respectively. What is the rela- long coated, neutral capillary with a detector located
tive amount of each protein in the sample? 25.0 cm from the point of injection. What migration time
54. A drug is found to have a peak area of 11,250 units and it and migration velocity would be expected for this
migrates through a 50.0 cm long capillary in the presence of cation when using an applied voltage of 15.0 kV?
an applied voltage of 15.0 kV. The detector is located at a (b) What migration time and velocity would be obtained
distance of 45.0 cm from the point of injection. What will the for the same cation as in Part (a) if a switch was made
expected area be for this sample if it is injected onto the from the neutral capillary to a negatively charged capil-
same system, but now using an applied voltage of 20.0 kV? lary of an identical size, but that gives an electroosmotic
55. What is “laser-induced fluorescence”? Explain why this mobility of 4.10 cm2>kV # min?
technique is useful in capillary electrophoresis. (c) Repeat the calculations in Parts (a) and (b) now using an
anion that has an electrophoretic mobility of
WHAT ARE SOME SPECIAL TYPES OF CAPILLARY -2.50 cm2>kV # min . Compare your results with those
ELECTROPHORESIS? obtained for the cation. What does this comparison tell
56. What is “capillary sieving electrophoresis”? What are two you about the role electroosmotic flow plays in the
ways in which this method can be performed? analysis of cations and anions in CE?
57. A protein is injected onto the same system used in 63. The effect of electroosmotic flow on the efficiency and reso-
Figure 23.18. What is the molecular weight of this protein if lution of a separation in electrophoresis is given by the
it has a normalized migration time of 2.65? equations shown,23
58. Define each of the following terms. (m + mosm)V
(a) Electrokinetic chromatography N = (23.13)
2D
(b) MEKC
(c) Micelle V
Rs = 0.177 (m1 - m2) (23.14)
(d) Critical micelle concentration A D(mavg + mosm)
96943_23_ch23_p571-596 1/8/10 2:54 PM Page 594
where m1 and m2 are the electrophoretic mobilities of the first TOPICS FOR REPORTS AND DISCUSSION
and second eluting solutes, mavg is the average electrophoretic 67. Obtain more information on the Human Genome Project.
mobility of solutes 1 and 2, and D is their diffusion coefficient. Discuss the challenges this project presented to analytical
(a) The same protein as in Exercise 23.1 is examined on a CE chemists. What changes in DNA sequencing methods were
system with a 25.0 cm long negatively charged capillary made to make this project possible?
(22.0 cm to the detector) at 20.0 kV. This new capillary
has an electroosmotic mobility of 3.0 cm2>kV # min . If
68. Now that human DNA has been sequenced, scientists have
begun to examine the vast number of proteins that are
the protein still has an inherent electrophoretic mobility
of 1.70 cm2>kV # min and a diffusion coefficient of
encoded by this DNA. This research has lead to an area
2.0 * 10 - 7 cm2>s, how many theoretical plates are pos-
known as “proteomics.” Obtain more information on pro-
teomics and the challenges that are presented by this field to
sible for this system? How does this result compare with chemical analysis. Describe some analytical methods that
that in Exercise 23.1? are being used in this field.
(b) Make a plot showing what resolution would be expected 69. Contact or visit a local hospital or biochemical laboratory.
at various values for µosm in the case where the applied Report on how electrophoresis is used in these laboratories.
voltage is 10.0 kV and two analyte peaks have elec- 70. Compare and contrast the advantages and disadvantages
trophoretic mobilities of 1.70 and 1.72 cm2>kV # min, for each of the following pairs of methods
with an average diffusion coefficient of 2.0 * 10 - 7 cm2>s (a) Gel electrophoresis versus capillary electrophoresis
. At what values of mosm versus mavg will the largest reso- (b) Gel electrophoresis versus HPLC
lutions be obtained? What values of mosm will give the (c) Capillary electrophoresis versus HPLC
smallest resolutions? 71. Use the Internet to obtain material safety data sheet (MSDS)
64. The amount of sample applied to a capillary by hydrody- information for the various chemicals that are shown in
namic injection can be determined by the following form of Figure 23.8 for the preparation of a polyacrylamide gel.
the Hagen–Poiseuille equation, Identify any chemical or physical hazards that are associ-
ated with these reagents.
¢P d4pt
Sample volume = (23.15) 72. Work with Northern and Southern blots in gel electrophore-
128h L sis often involves the use of phosphorus-32 (32P) as a radiola-
where ¢ P is the pressure applied across the capillary during bel. Obtain further information on any special requirements,
injection, d is the inner diameter of the capillary, t is the time training or facilities that are needed for dealing with this
over which the pressure is applied, h is the viscosity of the agent. In addition, find out why phosphorus-32 is used as a
applied solution, and L is the total length of the capillary.8 label for these applications. Write a report discussing your
(a) What volume of sample would be applied to a findings.
50 mm ID * 20 cm long capillary if a pressure of 0.5 psi is 73. There are several additional types of electrophoresis
applied for 1 s to a solution with a viscosity of 0.01 poise? besides those that were discussed in this chapter. A few
(b) How much sample would be applied under the same examples are listed below. 10,17 Write a report on one of
conditions but using a 1 s pulse on a 25 mm ID * these methods. Include of description of how the method
20 cm long capillary? separates analytes, its applications, and its advantages
65. The amount of an analyte that is applied to a capillary by and disadvantages.
electrokinetic injection is described by Equation 23.16, (a) Isotachophoresis
(b) Pulsed-field electrophoresis
(m + mosm)VACt
Q = (23.16) (c) Dielectrophoresis
L (d) Immunoelectrophoresis
where Q is the quantity of analyte injected, m is the elec- 74. The need for small sample sizes originally created several
trophoretic mobility of the analyte, mosm is the mobility due challenges in the design of CE instruments. This same fea-
to electroosmosis, V is the applied voltage, A is the cross- ture has made capillary electrophoresis attractive for the
sectional area of the capillary, C is the analyte concentration analysis of samples for which only small volumes are avail-
in the original sample, t is the time over which the electric able. For instance, it has been shown that CE can be used to
field is applied for injection, and L is the distance over analyze the content of single cells. Obtain a recent research
which the voltage is applied.8 article or review on this topic. Write a report that discusses
(a) Based on Equation 23.16, which types of analytes will have how CE was used to analyze single cells in the subject paper.
the largest injected quantities in electrokinetic injection: 75. Some early examples of capillary electrophoresis can be found
those that move with electroosmotic flow or against it? in Referemces 23 and 34. Look up these articles and examine
(b) How does the value of Q change with the size of elec- how electrophoresis was performed in them. How do the
troosmotic flow? Are small values or large values for methods described in these papers compare to those that are
µosm desirable in this method? now commonly used in CE, as described in this chapter?
66. Compare and contrast the following analytical methods in 76. Capillary electrochromatography is a method in which the
terms of the way in which they separate analytes. movement of an analyte through a stationary phase is
(a) Electrokinetic chromatography and reversed-phase achieved through the use of electroosmotic flow rather than
chromatography by only a difference in pressure.7,47,48 Obtain more informa-
(b) Capillary electrophoresis and ion-exchange tion on this method. Report on how this method works and
chromatography on some of its recent applications. Discuss how this method
(c) SDS-PAGE and capillary gel electrophoresis is related to both traditional liquid chromatography and
(d) Affinity capillary electrophoresis and affinity capillary electrophoresis, even though it is usually classified
chromatography as a chromatographic method.
96943_23_ch23_p571-596 1/8/10 2:54 PM Page 595
• References 595
77. Four chemicals that can be used as matrices in MALDI- 78. Find a recent research article that describes or uses a “lab-
TOF MS are nicotinic acid, sinapinic acid, a -cyano-4- on-a-chip” or mTAS device. Describe this device and the
hydroxycinnamic acid, and 2,5-dihydroxybenzoic acid. application for which it was employed. What advantages or
Obtain more information on one or more of these chemi- disadvantages were reported for this device versus more
cals and learn about how they are used in MALDI-TOF traditional methods for chemical analysis?
MS. What chemical or physical properties make these
chemicals used in this method? What are some analytes
that can be examined with these matrices?
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