Electrophoresis

C H A P T E R
12
Electrophoresis
James M. Karlinsey
Department of Chemistry, Penn State Berks, Reading, Pennsylvania 19610, USA
O U T L I N E
12.1. Introduction 375 12.2.6. Capillary Isoelectric Focusing 388

12.1.1. Theory 376
12.3. Detection Methods for CE 389
12.1.2. Formats 377
12.3.1. UV Absorbance 389
12.2. CE Methods 381 12.3.2. Laser-Induced Fluorescence 390
12.2.1. Capillary Zone Electrophoresis 381 12.3.3. Other Detection Methods 391
12.2.2. Micellar Electrokinetic
12.4. Applications of CE for Food Analysis 392
Chromatography 383
12.2.3. Capillary 12.5. Conclusions 400
Electrochromatography 385
12.2.4. Capillary Gel Electrophoresis 386
12.2.5. Capillary Isotachophoresis 387
12.1. INTRODUCTION strength (7.25 V cm1), chosen to reduce

the band-broadening effects of Joule heating.
Electrophoresis is the movement of charged The research that followed involved the
particles under the influence of an applied elec- development of anticonvective media for elec-
tric field, and it has found widespread use as trophoresis, including silica gel (Consden
a separation technique for biological molecules. et al., 1946), paper (Kunkel and Tiselius,
The first application of electrophoresis to sepa- 1951), starch gel (Smithies, 1955), acrylamide
rate samples in a mixture is widely attributed gel (Raymond and Weintraub, 1959), and
to Tiselius in 1937 for the separation of albumin agarose gel (Hjertén, 1961), reported in that
and globulin fractions in blood serum (Tiselius, order over a 15-year period (1946e1961). These
1937). This initial work was limited by large methods were widely applicable to peptides,
sample volumes and a low electrical field proteins, and oligonucleotides and preceded
Chemical Analysis of Food: Techniques and Applications

DOI: 10.1016/B978-0-12-384862-8.00012-1 375 Copyright Ó 2012 Elsevier Inc. All rights reserved.
376 12. ELECTROPHORESIS
further developments by Hjertén (1967) using During electrophoresis, a steady state is

capillary electrophoresis (CE) techniques for achieved where the electrical and frictional
the separation of diverse analytes ranging forces are equal and opposite, producing a rela-
from small molecules to whole cells. Benefits tionship that can be solved for the particle’s
of using capillaries for electrophoresis include velocity:
smaller volumes and the ability to apply higher
electric fields due to better heat dissipation vp ¼ qE$ð6phrÞ1 (12.3)
[e.g., human serum proteins were separated at The velocity may also be described as the
222 V cm1 (Caslavska et al., 1994)]. The further product of the electrophoretic mobility of
progression of electrophoresis from capillary a particle mp (with units of cm2 V1 s1) and
to microchips was due, in part, to advances in the applied field:
the semiconductor industry, where methods
were developed to pattern microscale features vp ¼ mp E (12.4)
in silicon substrates. This allowed capillary
channels to be patterned in glass so that many When Eqns (12.3) and (12.4) are combined, it is
of the electrophoresis techniques described possible to solve for mp, the preferred variable
below could be adapted from the capillary to when considering key electrophoretic parame-
the microchip. ters (e.g., amount of sample injected and
resolution):
12.1.1. Theory mp ¼ q$ð6phrÞ1 (12.5)

The following discussion summarizes the
This result indicates that the mobility of
fundamental theoretical concepts that must be
a particle is dependent on both its charge and
accounted for in an electrophoresis experiment.
size, such that a small, highly charged particle
A more thorough and detailed discussion,
will exhibit high mobility.
including calculated examples, can be found in
When a sample is loaded into a lane or
a recent text focused on capillary and microchip
channel for electrophoresis, the initial
electrophoresis (Landers, 2008). In its simplest
boundary between the sample and the buffer
form, electrophoresis deals with the relationship
solution is sharp and orthogonal to the direc-
between a charged particle in a homogeneous
tion of the applied electric field (and subse-
electric field. When an electric field E is applied
quent electrophoretic migration). During
across a conductive medium (i.e., buffer solu-
electrophoresis, as in any liquid phase separa-
tion in a capillary), a particle in the solution
tion, the sharp boundary deteriorates as the
will experience an electrical force Fel that is
concentrated sample diffuses across the
dependent on its charge q:
boundary. For a discrete sample zone, with
Fel ¼ qE (12.1) the buffer solution flanking both ends, the
sharp boundaries will deteriorate as the
While moving through the buffer, the particle is sample migrates through the electric field.
also subject to an opposing frictional force Ffr Assuming a Gaussian profile, the peak
that takes into account the velocity np and the maximum is dependent on the initial concen-
Stokes radius r of the spherical particle, as well tration of the sample and the peak width is
as the viscosity h of the buffer: dependent on both time t spent in the system
(until the sample passes a detector) and the
Ffr ¼ 6phrvp (12.2) diffusion constant for the particle Dp. The
I. ANALYTICAL TECHNIQUES
12.1. INTRODUCTION 377
resulting width of the curve, described by the The two factors affecting resolution are selec-
standard deviation s, is shown below, along tivity, which depends on the mobilities, and effi-
with the variance (s2): ciency, indicated by the theoretical plates. Both
of these factors can be related by combining
s ¼ ð2Dp tÞ1=2 (12.6) Eqns (12.8) and (12.9) to determine R:
s2 ¼ 2Dp t (12.7)
R ¼ 1=4 ðDmapp =mavg Þ$N 1=2 (12.10)
Increased variance in electrophoresis is
observed as a broadening of the sample zone
(i.e., band broadening). In addition to diffusion, 12.1.2. Formats
factors affecting band broadening include
temperature, the volume of sample introduced, As a separation technique, electrophoresis
and interactions between particles and the was first performed using a moving boundary
channel wall (Huang et al., 1989; Jones et al., method (Tiselius, 1937). This involved the use
1990). of a U-shaped tube filled with a buffer solution
When separating components of a mixture, it that contained the sample to be separated,
is desirable to maintain narrow sample zones so with no defined boundary between the sample
that components may be more easily resolved. and the buffer solutions. This required a signifi-
The resolution R of two components in an elec- cant amount of sample and, more importantly,
trophoresis separation is determined by their allowed complete isolation of only part of the
respective electrophoretic mobilities and diffu- fastest and slowest migrating components. The
sion constants. The apparent mobility mapp desire to perform electrophoresis using
refers to the observed mobility of an analyte a defined sample zone in order to isolate indi-
and takes into account any effects due to the vidual component zones led to the concept of
separation method used: zone electrophoresis. Early efforts focused on
the incorporation of anticonvective media in
R ¼ 1=4Dmapp Et$ð2Davg tÞ1=2 (12.8) the separation channel to reduce band broad-
ening and preserve sample zones. Smithies
In chromatography, where a partition equi- (1955) was the first to implement a gel matrix
librium between two phases is the physical by preparing potato starches in buffer for the
basis of separation, it is customary to use theo- separation of serum proteins. Shortly after,
retical plates N to measure the efficiency of the Raymond and Weintraub (1959) pioneered the
separation. There is no such partition in elec- use of acrylamide as a stable, flexible, and trans-
trophoresis, since the separation of two parent gel for zone electrophoresis. To date,
components is determined by their relative polyacrylamide gels (as well as agarose gels)
mobilities in an electric field, but the concept continue to be used for protein and DNA sepa-
of theoretical plates can still be used to rations. They are typically referred to as slab
describe peak shape and assess factors that gels because the gels are cast as thin slabs.
affect separation. Because N refers to the Figure 12.1 shows a schematic of a slab gel,
behavior of a single component during a sepa- along with a separation of proteins from several
ration, it is calculated using the average elec- fish extracts that was performed on a polyacryl-
trophoretic mobility mavg of the components amide gel.
to be separated: In the experiments described above, low elec-
tric field strengths (<10 V cm1) were used to
N ¼ ðmavg EtÞ2 $ð2Davg tÞ1 (12.9) minimize heating effects. It became obvious
(a) (b) or resistive heating), a phenomenon where

–
current passing through a conductive medium
ST
L 1 2 3 + –
10
11
1
2
3
4
5
6
7
8
9
kDa
produces a temperature increase. From Eqns
94 (12.8) and (12.9), it is evident that increased elec-
migration
67 tric field strengths should produce greater reso-
43
lution and efficiency, while also increasing the
30
overall speed of the separation [Eqn (12.4)]. In
20
slab gel systems, however, where the entire
14
substrate is composed of the separation
+
medium, the poor heat dissipation limits the
FIGURE 12.1 Slab gel electrophoresis. (a) Schematic of separation to low field strengths and long run
electrophoresis on a slab gel. The gel is cast to provide times.
wells where the sample is loaded, and the wells define The benefits of using slab gels include the
separation lanes that travel the length of the gel. Voltage is ability to multiplex a single electrophoresis
applied from the sample wells to the opposite end of the
run by loading the sample into multiple lanes
gel to mobilize negatively charged species toward the
positively charged anode. A typical experiment might (Fig. 12.1) and the potential for performing
include a ladder (L) for evaluating electrophoretic perfor- two-dimensional separations on the same
mance, unknown samples (1,2,3), and both positive (þ) slab gel (by performing a second electropho-
and negative (e) controls. (b) Representative data from resis separation orthogonal to the first).
a polyacrylamide gel electrophoresis experiment. Protein
Component zones are detected once the sepa-
profiles from 11 different raw fish extracts (1e11) are
shown, along with a protein sizing standard (ST). The ration is completed, usually following a stain-
separation was performed with an applied field of ing step where dye binds the analytes, and it
12 V cm1 with sample migrating from the bottom of the is possible to collect isolated zones by cutting
gel to the top (as shown). Detection was performed using out the bands or “picking spots.” Drawbacks
a commercial silver stain. Source: Reprinted from Piñeiro
to working with slab gels include time-
et al. (1999).
consuming preparation (e.g., casting the gel,
loading the sample, and staining the gel)
during early electrophoresis experiments that and poor reproducibility (especially with
an increase in temperature due to a combination two-dimensional separations). This makes it
of electric field and buffer selection would difficult to automate the procedure, which,
negatively affect the separation performance. coupled with the poor heat dissipation and
Tiselius noted during the moving boundary long run times, led to the development of
experiments that, although the highest possible capillary-based systems.
electric field strength is preferred for resolving CE differs from gels in that separations are
the components of a mixture, the resulting performed in capillaries instead of gel lanes.
generation of heat causes thermal convection A capillary is a thin-walled narrow-bore tube
currents and hinders the separation (Tiselius, that defines a single electrophoresis channel
1937). According to Smithies (1955), the temper- (i.e., lane). Capillaries in CE separations are
ature increase during an electrophoresis separa- typically made of glass (e.g., fused silica and
tion could be kept under 5 C for a starch gel quartz) with inner diameters ranging from 10
in a field strength of 6 V cm1 if the plastic to 200 mm, and a thin layer of polymer is coated
containment tray was kept thin and allowed to onto the capillary to impart flexibility without
rest on a copper plate with free air circulation. fracturing the glass. Because of the small cross-
The observed increases in temperature were sections, even longer separation lengths (e.g.,
due to Joule heating (also referred to as ohmic 100 cm) feature volumes less than 50 mL, which
12.1. INTRODUCTION 379
results in a significant decrease in sample and along with a separation of riboflavin in wine
buffer volume requirements compared to slab samples performed on a capillary.
gels. To apply an electric field across a buffer- CE experiments can be readily automated,
filled capillary, the ends of the capillary are owing, in large part, to offline (isolated) sample
immersed in vials containing additional buffer vials and the straightforward fluidic control
solution. Electrodes from a high voltage power associated with pumping solution through
supply are then placed in the buffer vials, and a single capillary channel. The introduction of
voltages up to 120 kV can be applied to the sample solutions into a capillary can be per-
system (Hutterer and Jorgenson, 1999), though formed by using either hydrostatic or electroki-
typical values in the literature range from 10 to netic methods (Ermakov et al., 1994). Injection
30 kV. The effects of Joule heating are countered volumes on capillary are typically in the low
by efficient dissipation due to the good thermal nanoliter range, and calculations can be per-
conductivity of glass, high surface-to-volume formed to determine the quantity of sample
ratio of capillaries, and the ability to thermostat injected depending on the dimensions of the
the external capillary environment at ambient capillary and the nature of the sample and
temperature. Figure 12.2 shows a cross-section buffer solutions. A hydrostatic injection is per-
of a capillary and a conventional CE setup, formed by immersing the capillary inlet in the
(a) polymer coating (b)

channel (12 - 20 m thickness)
(10 - 200 m I.D.)
substrate
(150 -360 m O.D.)
migration
detection
point
high voltage
power supply
+ –
sample
buffer inlet buffer outlet
FIGURE 12.2 Capillary electrophoresis. (a) Schematic of a CE setup. Both ends of the capillary are placed in buffer
vials so a high voltage power supply can deliver a voltage across the length of the capillary. The positions of the positive
(þ) and negative (e) electrodes refer to normal polarity. The resulting field strength mobilizes the sample toward the
buffer outlet using electroosmosis. In order to introduce the sample, the capillary inlet must be transferred from the buffer
vial to the sample vial. Exploded cross-sectional view of the capillary includes ranges for inner (I.D.) and outer (O.D.)
diameters as well as thicknesses of the polymer coating used to impart flexibility to the capillary. Values were taken from
a major capillary supplier. (b) Representative data from a CE experiment. Wine samples were diluted in water and
analyzed for riboflavin (RF) content. Separations were performed at 357 V cm1, and the naturally fluorescent RF
molecules were excited at 442 nm and detected at 515 nm. Data are shown for Italian samples of (a) white, (b) rosé, and
(c) red wines. Source: Reprinted from Cataldi et al. (2002).
sample solution and either pressurizing the inlet increased surface area-to-volume ratio associ-
vial to force solution into the capillary or pulling ated with small diameter capillaries.
vacuum on the outlet vial to pull the solution in. Initial reports of flow due to electroosmosis,
Electrokinetic injections are performed by or electroosmotic flow (EOF), have been attrib-
immersing the capillary inlet in the sample solu- uted to Helmholtz in the late 1800s after he
tion and the outlet in the separation buffer. A observed net flow of an aqueous salt solution
voltage is then applied for some predetermined in a glass tube when an electric field was
time to mobilize the sample into the capillary. applied (Landers, 2008). Nearly a century later,
Pioneering separations using a 3-mm quartz EOF was used to pump solvent in both thin
tube were performed by Hjertén (1967) with layer and liquid chromatography (Pretorius
the goal of reducing band broadening in free et al., 1974), with the principal benefit being
zone electrophoresis (i.e., electrophoresis with the plug-like flow profile obtained (Fig. 12.3a).
no anticonvective media) by rotating the tube When using laminar flow (driven, for example,
about its longitudinal axis during the separa- by pressure), friction between the liquid and
tion. The method was shown to be successful the solid surface results in a parabolic flow
in the separation of low-molecular-weight profile since there is less resistance to flow at
substances (e.g., inorganic ions and amino the center of a circular cross-section and more
acids) and larger structures (e.g., proteins, resistance at the boundaries. With EOF, the
nucleic acids, and cells) using small amounts flow is initiated at the interface, as counter
of sample (e.g., 20 mg of protein). Though the ions in solution stack up at the charged surface
method was successful, Hjertén reported to form a double layer of static and mobile
several technical problems that needed to be ions (Fig. 12.3b). The mobile layer of ions is
addressed before the method could be fully real- free to migrate toward the appropriate electrode
ized. These included the need for uniform cross- (e.g., cations migrate toward the cathode), and
sections, narrow sample zones, and control of the ions migrate with waters of hydration
electroosmosis. Electroosmosis is the flow of when an electric field is applied. The resulting
liquid that is in contact with a charged solid bulk flow of solution is uniform and plug-like
surface when an electric field is applied, and it throughout the circular cross-section. The most
becomes an important consideration with the fundamental CE experiments take advantage
FIGURE 12.3 Electroosmotic flow. (a)

(a) Flow profile diagrams in a capil-
(b)
channel buffer
lary. EOF produces a plug-like flow
due to bulk solution flowing through wall
a charged capillary surface, while + + +
pressure-driven flow produces a para-
EOF
+ +
bolic profile. (b) Schematic of the +
+
double ionic layer. The double ionic + + +
layer that forms within the capillary +
+ +
channel is shown after the channel
wall assumes a negative charge. The + + + +
static layer of cations refers to the Stern
+ +
layer, while the mobile layer contain- +
ing predominantly cations refers to the + +
outer Helmholtz plane. pressure
+ + +
static mobile
12.2. CE METHODS 381
of this behavior in order to mobilize all analytes the shape of the injected sample plug by taking
past the detector, regardless of charge. In the into account the distribution of electric fields
absence of EOF, either the positively charged in each channel segment on the microchip
or negatively charged ions could be analyzed (Effenhauser et al., 1993; Jacobson et al.,
in a single separation. In either case, neutral 1994a,b). Compared to capillary, microchip
species would not be detected because they electrophoresis offers additional benefits in
lack the necessary charge character for elec- terms of the ability to integrate multiple analyt-
trophoresis. The ability to perform CE with ical steps and the portability associated with
EOF thus represents a significant advantage reducing instrumentation size. Figure 12.4
over the previous capabilities of slab gel shows a schematic of injection and separation
electrophoresis. steps on a microchip, along with a separation
Microchip electrophoresis, described in of DNA fragments from maize samples per-
more depth in Chapter 7 (Microfluidic devices, formed on a microchip.
biosensors), was initially described in 1992
when Harrison and coworkers transferred the
fundamental principles of capillary electropho- 12.2. CE METHODS
resis onto a planar glass chip (Harrison et al.,
1992). Borrowing from fabrication technologies Of the three formats presented above, CE has
in the semiconductor industry, a glass slide was played the largest role in food analysis and will
etched to create capillary-like microchannels be the focus of the following separation modes.
with a depth of 10 mm. Despite a difference in While the behavior of an analyte in an electric
the geometry of the channel cross-sections field depends primarily on its charge and size
due to an isotropic etch (i.e., hemispherical [Eqn (12.5)], it can be modified by chemical or
instead of circular), there is little difference physical interactions between the analyte and
regarding the principles of electrophoretic the separation media (e.g., additives and poly-
migration. Bulk flow due to EOF remains mer networks). Different separation modes
possible because of the high surface area-to- have been established to take advantage of these
volume relationship, even in the absence of modified behaviors, resulting in a variety of CE
the radial symmetry common to glass capil- modes that include capillary zone electrophoresis
laries. Depending upon the microchip (CZE), micellar electrokinetic chromatography
substrate, however, there can be a significant (MEKC), capillary electrochromatography
difference in the magnitude of EOF using the (CEC), capillary gel electrophoresis (CGE), capil-
same electrophoresis conditions (Ocvirk et al., lary isotachophoresis (CITP), and capillary
2000; Bilitewski et al., 2003). The most impor- isoelectric focusing (CIEF).
tant difference when transitioning from
capillary to microchip is the integration of
12.2.1. Capillary Zone Electrophoresis
multiple intersecting microchannels, since this
is necessary to introduce the sample into the CZE has also been referred to as free solution
separation channel on-chip and allows the capillary electrophoresis because the separation
possibility of incorporating additional analyt- is entirely dependent upon the ratio of charge to
ical steps (e.g., sample pretreatment) onto the size and does not include anticonvective media
microchip. For sample introduction, the region in the capillary. Jorgenson and Lukacs (1981b)
defined by the channel intersection serves as were the first to use EOF for zone electropho-
the injection volume, and several different resis in narrow-bore capillaries (75 mm I.D.).
techniques have been developed to control Just 2 years earlier, Mikkers et al. had reported
(a) migration (b) (c) 175

+ Unit: bp 2652
Electric field (V/cm, dotted line)

buffer inlet 150
1.0 RFU
125
sample waste
175
500
25
(i) DNA
75
reservoir reservoir ladder 125
+ – 101 151 100
RFU
(ii) GM-maize
75
migration
151
50
(iii) Non GM-maize 25

detection point
(iv) Negative control
buffer outlet 0
35 75 115 155 195
– Migration time (s)
FIGURE 12.4 Microchip electrophoresis. (aeb) A schematic showing electrophoresis on a microchip. In order to intro-
duce the sample into the separation channel, an injection step (a) is required to mobilize the sample from the sample
reservoir to the waste reservoir into the cross-tee intersection. Following the injection, a separation is performed (b) as
voltage applied between the buffer inlet and outlet reservoirs mobilizes the sample out of the tee intersection toward the
cathode. (c) Representative data from a microchip electrophoresis experiment. Genetically modified (GM) and nongeneti-
cally modified (non-GM) maize samples were injected (58.8 V cm1) and electrophoresed (117.6 V cm1) through a polymer
sieving matrix to discriminate DNA fragments by length (using the DNA ladder with fragments of known lengths).
Fragments were detected by fluorescence (RFU) after associating with ethidium bromide in the buffer. Source: Reprinted from
Kumar and Kang (2007).
zone electrophoresis in polytetrafluoroethy- is static due to the strong interaction with

lene (PTFE) capillaries (200 mm I.D.), where the oppositely charged wall, the strength of
the selection of the chemically and electrically which is dependent upon the pH and ionic
inert PTFE was based, in part, on the elimina- strength of the buffer. The positive charge
tion of EOF (Mikkers et al., 1979) following the density at the wall decreases exponentially
earlier observations by Hjertén (1967). Jorgen- with distance, producing a more diffuse
son and Lukacs used glass, however, and concentration of positive charge with weaker
took advantage of EOF to separate various attraction that is free to migrate toward the
mixtures (e.g., amino acids, dipeptides, and cathode under an applied electric field. The
amines) as well as a human urine sample con- cations carry with them waters of hydration
taining both charged and neutral analytes. and, due to hydrogen bonding of these water
EOF was possible due to the negatively molecules to those in the bulk solution, the
charged glass surface that attracted counter entire buffer solution is mobilized toward the
ions from the buffer. Specifically, silanol cathode.
groups on the glass surface (Si-OH, pKa 4e6) EOF is always present when an electric field
will become ionized in basic buffer (Si-O), is applied to an aqueous buffer in contact with
and cationic species from the buffer solution a charged surface (e.g., glass), but the effect is
will be attracted to the oppositely charged magnified with the reduced scale of the capil-
wall. The double layer that results consists of lary and the high electric field strengths used
the static Stern layer (i.e., inner Helmholtz in CE. The magnitude of EOF is expressed as
plane) and the diffuse layer (i.e., outer a mobility term, mEOF, and can be determined
Helmholtz plane) (Fig. 12.3b). The Stern layer from the velocity of the bulk flow in an applied
electric field [Eqn (12.4)]. It is important to magnitude of EOF is greater. It should be noted
account for EOF when determining the that CZE can be operated in reverse polarity,
apparent electrophoretic mobility (mapp) of where EOF is driven in the opposite direction
a particle [referred to in Eqns (12.8) and by introducing a positive charge on the capillary
(12.10)]. When EOF is present, it is the combina- wall to mobilize an anionic double layer toward
tion of both mobility terms that produces the the anode. Figure 12.5 shows a schematic of CZE
observed flow of analytes: with normal polarity, along with a separation of
sugars in orange juice.
mapp ¼ mEOF þ mp (12.11)
12.2.2. Micellar Electrokinetic

In normal polarity, cations and neutral
species migrate with EOF and anions migrate
Chromatography
against it. It is the cumulative effect of both CZE enables neutral analytes to be separated
mobilities that results in the flow of anions from both cationic and anionic species, but
toward the cathode, provided that the neutral species cannot be resolved because
(a) (d)
Si O Si O Si O Si O Si O Si O Si O Si O Si O Si O
O– O– O– O– O– O– O– O– O– O–
0.008
O
– –
O O
– –
O O
– –
O O
– –
O O
– O
–
Si O Si O Si O Si O Si O Si O Si O Si O Si O Si O 0.006
A
(b) u
Sucrose Glucose Fructose
– – – – – – – – – –
0.004
– – – – – – – – – –
(c) 0.002
migration
– – – – – – – – – – Eof Unknown
+ EOF – 0.000
– – – – – – – – – – 15 20 25 30
Minutes
FIGURE 12.5 Capillary zone electrophoresis. (aec) A stepwise schematic of CZE. (a) A negative charge on the
channel wall is obtained by deprotonating the silanol groups (Si-OH) on the native glass surface using basic buffer.
(b) A mixed sample containing some combination of positive (circles), negative (squares), and neutral (triangles) species
is introduced into the separation channel. (c) When a voltage is applied across the channel, EOF mobilizes all species
toward the cathode. (d) Representative data from a CZE experiment. Orange juice was diluted 1:50 (v/v) in water, and
neutral sugar molecules (sucrose, glucose, and fructose) in the sample were mobilized with EOF (labeled Eof) using an
applied field of 267 V cm1. Sugar molecules were detected by UV absorbance (Au) at 270 nm. Source: Reprinted from
Rovio et al. (2007).
Dmapp is equal to zero [Eqn (12.10)]. In 1984, distribution of analyte between the micelle and
Terabe and coworkers demonstrated that the surrounding buffer.
a neutral analyte could gain an apparent electro- Sodium dodecyl sulfate (SDS) is a common
phoretic mobility when incorporated into an surfactant, producing an anionic micelle that
ionic micelle (Terabe et al., 1984). The addition migrates toward the anode in the absence of
of ionic surfactant to the separation buffer at EOF. In this example, an MEKC separation
a concentration above its critical micellar would produce a micelle peak that lags behind
concentration was sufficient to allow the separa- both the leading EOF marker and the incorpo-
tion of neutral species, and no modification of rated analyte. In the presence of a cationic
the instrumentation was necessary. Basically, surfactant such as cetyltrimethylammonium
MEKC incorporated chromatography into bromide (CTAB), resolution would be achieved
a CZE separation. The resulting micelles are by reversing the polarity. The use of electroki-
considered a pseudostationary phase because netic chromatography (EKC) on capillary has
they function as a stationary phase in liquide also been demonstrated without the use of
liquid partition chromatography, while at the micelles, using other pseudostationary phases
same time migrating electrophoretically within such as charged cyclodextrins (CDs), which
the capillary. The micelles themselves are pseu- can be used to separate chiral species, and
dophase because they exist only in equilibrium microemulsions. In general, MEKC is better
with surfactant molecules in solution. As suited to the separation of small molecules and
neutral analytes partition into the micelle, the peptides, and, while originally employed to
mapp of the micelle increases. Overall, the migra- separate neutral analytes, it has also been used
tion time is dependent upon the micelle to improve the selectivity of charged analytes.
mobility, the velocity of EOF, and the Figure 12.6 shows a schematic of EKC, along
EGC
(a) (c) (+)–C
– – – – – – – – – –
– – – – – – – – (–)–C
– – – – – – –
– – – – – – –
– – –
EGCG
CF
– – – – – – – – – –
4.0 4.2 4.4 4.6 4.8 5.0 5.2
EC
migration
(b) ECG
– – – – – – – – – – 25 mAU
– – – – – – – – IS (–)–C
+ EOF –
–
–
–
–
–
–
–
–
–
–
–
–
– –
– (+)–C (+)–GC (–)–GC
– – – – – – – – – –
0 2 4 6 8
time, min
FIGURE 12.6 Micellar electrokinetic chromatography. (aeb) A stepwise schematic of MEKC. (a) Anionic surfactant
(e.g., SDS) is added to the separation buffer, with some molecules forming micelles while others are free in solution.
(b) During a separation, some neutral molecules will migrate with EOF, while others will become trapped in a micelle and
exhibit a delayed migration. (c) Representative data from an EKC experiment. A green tea extract was separated using an
applied field of 500 V cm1 and neutral CDs as the pseudostationary phase. The extract included catechins (EC, ECG, EGC,
EGCG, C, and GC) and the methylxanthine CF. The internal standard (IS, syringic acid) serves as an EOF marker, and the (þ)
and (e) designations indicate chirality. UV absorbance detection (mAU) was performed using a DAD. Source: Reprinted from
Gotti et al. (2009).
with a separation of catechins in a green tea a stationary phase. Previous discussion of the
extract. electrical double layer required for EOF
focused on the charge on the capillary wall,
12.2.3. Capillary but it is necessary in CEC to account for the
total surface area contributed by the packed
Electrochromatography
stationary phase. Because the latter makes up
Just as MEKC brought together the advan- the greater percentage of the surface area, the
tages of chromatography with electrophoresis, total surface charge density is assumed to be
CEC is the result of efforts to combine CZE that of the stationary phase. In many cases,
with high-performance liquid chromatography a fused silica capillary is packed with silica
(HPLC). In place of the pressure gradient particles so that all surfaces are negatively
used to flow mobile phase through a packed charged due to the dissociation of the silanol
chromatography bed, the plug-like flow of groups. To determine the magnitude of EOF
EOF is used in CEC. By avoiding the parabolic in CEC, a neutral, unretained marker like
flow profile characterized by a pressure-driven acetone is used. In general, CEC is used to
separation, the efficiency of the separation can separate small molecules, peptides, proteins,
be improved. The first example of CEC was and carbohydrates. Figure 12.7 shows a sche-
performed in 1974 using EOF to flow mobile matic of CEC, along with a separation of mela-
phase through an LC column packed with tonin in red wine.
particles ranging from 75 to 125 mm (Pretorius CEC capillaries have traditionally been
et al., 1974). Jorgensen and Lukacs (1981a), after prepared by sintering silica particles at one
their initial description of CZE, demonstrated end of the capillary to serve as a retaining frit.
CEC in 1981 using a capillary packed with The capillary is then packed with a slurry of
10 mm particles, and current methods use the desired stationary phase before a second
even smaller particles (<5 mm) to create retaining frit is sintered at the opposite end of
(a) (c)
– – – – – – – – – – 800
– – – – – – – – –
– – – – – – – – –
Correct area
600
– – – – – – – – – – MT
400
migration
(b) – – – – – – – – – – 200
– – – – – – –
+ EOF
– – – – – – –
–
– 0
– – – – – – – – – – 0 2 4 6 8 10 12
Time (min)
FIGURE 12.7 Capillary electrochromatography. (aeb) A stepwise schematic of CEC. (a) The surfaces of the solid phase in
the capillary (open circles) have a negative charge like the channel walls and also produce an electrical double layer. (b) The
resulting EOF drives the sample through the solid phase during electrophoresis. (c) Representative data from a CEC
experiment. Melatonin (MT) was resolved from other compounds in red wine by mobilizing the sample through an
immobilized carboxylic multiwalled carbon nanotube stationary phase. UV absorbance was detected at 254 nm.
Source: Reprinted from Stege et al. (2010).
the capillary. This method is labor intensive and fragments are well sieved. As the fragment
introduces reproducibility issues due to bubble becomes significantly larger than the pores, it
formation at the frit interface, which has led to migrates through the polymer network with
the formation of monolithic columns as an alter- a snakeline motion (i.e., reptation). In this
native solid phase. In order to simplify the prep- regime, the mobility of the fragment is closely
aration and provide more flexibility in the related to its size. The remaining regimes include
localization of the stationary phase within the intermediate and large molecules, where frag-
capillary, in situ polymerization of monomers ments are likely to get trapped in a U-shaped
can be used to form a continuous, rod-like conformation or orient in the field direction,
porous bed. A wide variety of monomers is respectively, so that no separation is possible.
available to obtain the desired surface chem- In the Ogston and reptation regimes, DNA
istry, and the polymerization process is well fragments up to 1000 bases are well separated
controlled either thermally or by ultraviolet by CGE based on size (Viovy and Duke, 1993).
(UV) radiation. Because of the development of With the ability to amplify fragments in this
HPLC methodologies, there is a wide range of range from much larger DNA strands using
stationary phases to choose from depending the polymerase chain reaction (PCR), it is
upon the nature of the analytes to be separated. possible to generate relatively large quantities
of sample to be analyzed by CGE. PCR offers
the benefit of specificity, with the ability to
12.2.4. Capillary Gel Electrophoresis
design primers for target DNA fragments,
As opposed to the packing of stationary phase and sensitivity, with the ability to exponen-
in a CEC separation, CGE separations feature tially increase the quantity of the target
a capillary filled with a viscous buffer solution DNA. In food analysis, separations of PCR-
containing entangled polymer strands. This amplified DNA fragments have only been
separation mode is reserved almost entirely for reported in the last decade, starting in 2002
nucleic acid separations (i.e., DNA and RNA). with reports of genetically modified maize
The presence of the entangled polymer network samples (Garcia-Canas et al., 2002a,b). Further
is necessary because the electrophoretic mobility applications have included genetically modi-
of nucleic acids is independent of fragment fied organisms (Garcia-Canas et al., 2004a,b;
length. Specifically, the ratio of charge to size is Nadal et al., 2006; Sanchez et al., 2007), food-
maintained throughout the length of the nucleo- borne pathogens (Alarcon et al., 2004), bacteria
tide chain because of the charge on the sugare (Garcia-Canas et al., 2004), species identifica-
phosphate DNA backbone. As a result, a sieving tion (Sun and Lin, 2003), and food authenticity
matrix or “gel” is required for size resolution, (Archak et al., 2007; Spaniolas et al., 2008;
where longer fragments spend more time associ- Spaniolas et al., 2008). Although CGE applica-
ating with the matrix and smaller fragments pass tions to DNA samples will likely have the
more easily though pores within the matrix. The greatest impact, it should be noted that
relationship between mobility and fragment size proteins, too, have been successfully separated
has been characterized by different regimes using CGE (Braddock et al., 2001; Gomis et al.,
(Viovy and Duke, 1993). When the DNA frag- 2003). In these applications, SDSeprotein
ment size is small, it assumes a rod-like shape complexes provided uniform charge to size
and migrates as if in free solution (i.e., no sieving ratios for the sample components. Figure 12.8
matrix). In the Ogston regime, the fragment shows a schematic of CGE, along with a sepa-
behaves as an unperturbed spherical object ration of DNA fragments from transgenic
with a size comparable to the gel pores and the maize samples.
10
(a) (d)
T25
Zein
Relative Fluorescence Units

Btl76
(b) 6
T25’
(c) migration 2 Mon810
GA21
Btl76’
– + Bt11
0
14 18 22 26 30
Minutes
FIGURE 12.8 Capillary gel electrophoresis. (aec) A stepwise schematic of CGE run in reverse polarity. (a) The capillary
is initially filled with a sieving matrix (shown as a gray grid). (b) The sample is loaded using either pressure- or voltage-
driven methods. (c) When the voltage is applied across the capillary, smaller fragments pass more easily through the sieving
matrix while larger fragments take longer to migrate through the capillary. (d) Representative data from a CGE experiment.
A sample containing DNA from transgenic maize (Bt11, T25, Mon810, GA21, and Bt176) and a reference fragment (indi-
cating the Zein gene) was electrophoresed (217 V cm1) through a polymer sieving matrix to discriminate DNA fragments
by length. Fragments were detected by fluorescence after associating with an intercalating dye (YOPRO1) in the buffer.
Source: Reprinted from Garcia-Canas et al. (2004b).
12.2.5. Capillary Isotachophoresis (e.g., mA and mB) should be between that of the
LE and TE:
The previous methods feature sample
components migrating with different mobilities mLE > mA > mB > mTE (12.12)
under the influence of a homogeneous electric
field. In CITP, however, the components migrate CITP is a technique for separating similarly
with the same mobility (after forming distinct charged particles, and the counter ion is the
zones with different conductivity) under the same in each solution (e.g., Naþ or Hþ for a sepa-
influence of a heterogeneous electric field. ration of anions). EOF does not play a role in
Because the field strength is not the same CITP, since the magnitude would be affected
throughout the capillary, CITP is considered by the nature of the electrolytes in the different
a discontinuous system with different conduc- zones, and so it is common to see electrically
tivity zones. These zones typically include inert capillaries (e.g., Teflon) utilized.
a leading electrolyte (LE) zone, with the highest If a constant voltage source was used to
electrophoretic mobility (mLE) in the system, generate the electric field, as in CZE, the field
a terminating electrolyte (TE) zone, with the would be distributed evenly throughout the capil-
lowest mobility (mTE), and a sample zone. The lary. By using a constant current source, however,
mobility of the components in the sample zone the ions in the different buffer solutions will
migrate at different speeds until a steady state is determination of organic acids (Jezek and Suhaj,
reached at the boundaries and conductivity zones 2001; Masar et al., 2001; Sadecka and Polonsky,
are stabilized. The stabilized zones exhibit 2003). Figure 12.9 shows a schematic of CITP,
different conductivity values, and the electric along with a separation of organic acids in apple
field becomes discontinuous in that the LE exists juice.
in a region of low conductivity and the TE exists
in a region of high conductivity. As the name 12.2.6. Capillary Isoelectric Focusing
implies (i.e., iso e same, tacho e speed), the zones
migrate through the capillary with the same Contrary to the other CE methods that mobi-
speed (though they experience different electric lize the sample toward the detector under an
field strengths), maintaining sharp boundaries applied electric field, CIEF uses the field to focus
between the different conductivity zones. The the components of a mixture within a pH
technique was originally called displacement gradient. The focused sample zones are then
electrophoresis when it was demonstrated in mobilized toward the detector using low-pres-
1967 (Martin and Everaerts, 1967) since it was sure flow. A stable pH gradient is obtained by
considered to be analogous to displacement chro- loading the capillary with carrier ampholytes
matography, where sample components are (CAs), placing the ends of the capillary in ano-
resolved into concentrated zones with no solvent lyte (e.g., phosphoric acid) and catholyte (e.g.,
in between. This concentration effect in CITP has sodium hydroxide) solutions, and applying
proven to be particularly useful as a preliminary a voltage. Components of a sample mixture,
separation step coupled to CZE in food analysis loaded onto the capillary along with the CAs,
(Kvasnicka, 2003; Kvasnicka et al., 2006, 2007; reach equilibrium within the pH gradient where
Kvasnicka and Voldrich, 2000), though it has their isoelectric point (pI) equals zero. From
also been used as a stand-alone method in the Eqn (12.5), it is clear that if the particle has no
(a) (d)
TE A+B LE
citrate (T)
(b) fumarate
– TE B A+B A LE + R
phosphate
migration
(c)
sulphate
– TE B A LE + chloride (L)
E 450 455 460 465 470 475 480

time (s)
FIGURE 12.9 Capillary isotachophoresis. (aec) A stepwise schematic of CITP. (a) The capillary is filled with leading
electrolyte (LE), the sample (containing A and B components), and terminating electrolyte (TE) solutions. (b) Under constant
current, buffer ions move at different velocities to create different zones of conductivity. (c) Once the zones have been
established, all of the solutions move at the same velocity. The relative electric field strengths in the zones are provided to
indicate the difference in conductivity due to the buffer composition. (d) Representative data from a CITP experiment.
Components of apple juice (10 times diluted) were separated (250 mA) to isolate the fumarate component. Data plotted as
response (R) of conductimeter. Source: Reprinted from Kvasnicka and Voldrich (2000).
12.3. DETECTION METHODS FOR CE 389
(a) (d)
400
C. zeylanoides
300 C. lusitaniae
G. candidum
C. kefyr
(b) pH gradient (low to high)
Y. lipolytica
T. asahii
200
4.7 C. tropicalis
+ – S. cerevisiae
100 1.8
(c) pH gradient (low to high) 0
0 2 4 6 8 10 12
+ low pressure – t[min.]
FIGURE 12.10 Capillary isoelectric focusing. (aec) A stepwise schematic of CIEF. (a) The sample is initially loaded onto
the capillary along with carrier ampholytes. (b) Upon application of an electric field, the sample migrates to a pH zone
where its pI is equal to zero. Samples having the lowest pI are shown with open triangles, and the dark triangles represent
samples with the highest pI. (c) Pressure is then applied to mobilize the sample past the detector, while it remains focused
under the applied field. (d) Representative data from a CIEF experiment. Eleven clinically significant yeasts were stained
with PBePEG to provide a fluorescence signal and separated according to pI values. Standards were included in the run to
indicate pH 1.8 and 4.7. One of the samples, S. cerevisiae, is used in baking and commonly referred to as Baker’s yeast.
Fluorescence emission was collected at 480 nm. Source: Reprinted from Horká et al. (2007).
charge, then it will have no mobility at its pI. IEF commonly features a UV detector with the
separations are primarily used for peptides and option of adding LIF detection capabilities
proteins to take advantage of their amphoteric (with the additional cost of a laser source).
character, but this technique has found limited These methods will be considered below, along
application in food analysis. Figure 12.10 shows with less common methods such as chemilumi-
a schematic of CIEF, along with a separation of nescence (CL) and electrochemical (EC) detec-
yeast samples that includes Baker’s yeast. tion. Mass spectrometry (MS) is a sensitive
detection technique that will be considered
briefly here, although a more in-depth discus-
12.3. DETECTION METHODS sion on MS instrumentation and its various
FOR CE modes of detection can be found in Chapter 9
(Mass spectrometry).
The small volumes used in CE, especially
considering the narrow sample zones passing
12.3.1. UV Absorbance
the detector, require detection with high sensi-
tivity and specificity. Early CE analysis Absorbance is determined by measuring the
primarily involved UV absorbance or laser- transmission of light through a sample, where
induced fluorescence (LIF) detection, with LIF molecules in the sample may absorb a photon
requiring the addition of a fluorophore but to assume an excited state. The transmission is
providing better sensitivity for low analyte easily determined by observing the ratio of light
concentrations. Commercial instrumentation intensity before and after passing through the
sample, and the absorbance A is directly related a photodiode or a photomultiplier tube. In

to the transmission T: some applications, a diode array detector is
used along with a prism or spectrograph to
A ¼ log T (12.13) provide absorbance data over a range of wave-
lengths from which single (Gel-Moreto et al.,
The absorbance can then be related to the
2001; Rodriguez et al., 2001) or multiple wave-
sample using the BeereLambert law, taking
lengths (Cifuentes et al., 2001; del Castillo et al.,
into account the molar absorptivity ε of an ana-
2002; Garcia-Villar et al., 2006) can be reported.
lyte, its concentration c in the sample, and the
Absorbance detection is sufficient for many
path length l through which the light passes:
biological analytes, but in some cases the analyte
A ¼ εcl (12.14) has a weak chromophore and does not appre-
ciably absorb the UV wavelengths. Carbohy-
Because the path length is fixed by the capil- drates, for example, are important in the
lary channel and the absorptivity will be analysis of foods and beverages but do not
constant for a set of given experimental condi- exhibit strong UV absorbance. In order to take
tions (e.g., pH and wavelength), the measured advantage of the automated CE systems with
absorbance is linearly related to the concentra- absorbance detection for carbohydrate analysis,
tion of analyte within a sample zone. On the researchers have used both indirect UV
capillary, there is a trade-off in sensitivity due detection and UV derivatization techniques.
to the effect of the reduced path length, though Indirect detection uses a wavelength-absorbing
this is necessary to ensure high-resolution substance in the separation medium that serves
separations with efficient heat dissipation as a continuous background signal. As the carbo-
(due to the high surface area-to-volume ratio). hydrate samples are electrophoresed through
Approaches to increase the path length include the medium, they displace the absorbing species
increasing the diameter in a short segment of and produce a decrease in the detected absor-
the capillary to produce a bubble cell (Heiger bance (Soga and Serwe, 2000; Vorndran et al.,
et al., 1994) or introducing bends in the capil- 1992). The benefit of indirect detection is that it
lary so that the light enters and propagates in does not require any derivatization of the carbo-
the direction of flow before exiting (Bruin hydrates but is not as sensitive (104 to 106 M)
et al., 1991). as direct UV detection (105 to 107 M) (Scanlan
Light sources for UV detection include lamps, et al., 2008). In order to take advantage of
lasers, and light-emitting diodes (LEDs). While the increased sensitivity of direct detection,
some sources are limited to specific wave- samples can be derivatized with a strong chro-
lengths, such as atomic emission lamps or lasers, mophore. Several different groups have used
it is more common to use lamps that provide this approach for carbohydrate detection
broadband emission. A deuterium arc lamp, (Cortacero-Ramı́rez et al., 2004; Doner et al.,
for example, has stable output from 160 to 2001; Zhang et al., 2001), though it should be
400 nm. Laser sources are coherent and can be noted that the derivatization usually adds an
focused rather easily within the capillary, additional processing step.
whereas the incoherent lamp and LED sources
require both collection and focusing optics
12.3.2. Laser-Induced Fluorescence
(Bruin et al., 1991). After interacting with the
sample, the beam is spectrally filtered to eval- LIF detection involves the use of a laser
uate absorption at the desired wavelength(s). source to excite electrons in a fluorescent mole-
Detection can be performed using either cule. When the excited molecule relaxes to its
12.3. DETECTION METHODS FOR CE 391
ground state, a photon is emitted at a slightly re-excited following fluorescence (i.e., after
higher wavelength. This difference between relaxing to its ground electronic state), and the
excitation and emission maxima is known as Stokes shift allows filters to be selected such
the Stokes shift and accounts for any energy that the signal can remain high while keeping
lost. Fluorescence excitation and emission the noise low. Despite the increased sensitivity
spectra are relatively broad due to the various attainable with LIF, a limited number of mole-
energy states that exist in a molecule, and optics cules contain a natural fluorophore. Because of
are chosen so that a fluorophore is excited near this, almost all reports of CEeLIF involve deriv-
its excitation maximum and then filtered to atization either on- or off-capillary. Carbohy-
obtain signal near the emission maximum. Fluo- drates, for example, have been derivatized
rescence emission is similar to absorbance in with different fluorescent dyes to take advan-
that its intensity IF is related to path length l, tage of common laser excitation sources (e.g.,
concentration c, and molar absorptivity ε. the 488-nm line from an argon ion laser) (Liu
However, it also accounts for the intensity of et al., 1991; Guttman, 1996).
the laser IL and the fluorescence quantum yield
QF of the fluorophore:
12.3.3. Other Detection Methods
IF ¼ ðlog εÞ$cl$IL $QF (12.15) Relatively few light-based methods exist
apart from UV and LIF detection, though CL
Though the equation is somewhat similar to has been reported for the analysis of derivatized
Eqn (12.14), it takes into account the laser source carbohydrates (Wang et al., 2003) and amino
and the behavior of the molecule once it is excited. acids (Lin et al., 2006). CL detection is based
Regarding the former, it should be noted that the on the production of an electronically excited
laser intensity has an optimal value despite its species following a chemical reaction. The
linear relationship with fluorescence emission excited species can then luminesce, releasing
and should not simply be run at full power light energy as it relaxes to its ground state, or
(Mathies et al., 1990). Regarding the latter, the donate energy to another molecule that lumi-
emission spectrum of a fluorescent molecule is nesces. The principal benefit of CL detection is
dependent on environmental factors such as pH the minimal instrumentation requirements
and intermolecular forces that exist with the since no external source is necessary for excita-
solvent or sample (e.g., DNA intercalating dyes). tion. The lack of source also ensures that there
Several variations of LIF detection have is no background signal due to light scatter.
been implemented, but all feature a laser source, Common CL systems in food analysis include
focusing optics to direct light into the capillary, the luminol reaction, the peroxyoxalate reaction,
additional optics to collect the fluorescence, and the tris (2,20 -bipyridine) ruthenium (II)
2þ
appropriate filters for the wavelengths system ½RuðbpyÞ3 , each of which has been
involved, and a sensitive photodetector applied to pesticide analysis (Gámiz-Gracia
(Johnson and Landers, 2004). Compared to UV et al., 2005).
detection, direct fluorescence detection is signif- As an alternative to spectroscopy, electroana-
icantly more sensitive. A general range of detec- lytical methods have been applied to food
tion limits is 109 to 1012 M (Scanlan et al., samples in order to perform EC detection. In vol-
2008), with single molecule detection first tammeric or amperometric detection (which
demonstrated on a capillary in 1996 (Chen and measures voltage or current differences, respec-
Dovichi, 1996). This sensitivity is aided by the tively), signal is based on an EC reaction (i.e.,
ability of the fluorescent molecule to become oxidation or reduction) when an analyte interacts
with a working electrode at the capillary outlet. field. Quantitative detection is based on the
This type of detection is dependent upon the ana- distance travelled by the ions in the field. In the
lyte contacting the electrode, and so the method is coupling of CE and MS techniques, electrospray
suited to the reduced volume of the capillary. injection (ESI) is the method most often used to
Because it is not dependent on path length, EC introduce a sample in solution into the mass
detection does not lose sensitivity as the capillary spectrometer. ESI, described in 1989 by John
diameter is reduced and is generally more sensi- Fenn (who later received a Nobel Prize in chem-
tive than UV detection and comparable to LIF istry for his work) (Fenn et al., 1989), features
detection. In one report, the separation and a high voltage applied between the capillary
amperometric detection of 19 chlorophenols outlet and the walls of an electrospray chamber
(and phenol) were optimized, and it was found such that the solution exiting the capillary is
that EC was more sensitive than UV by one to dispersed into a fine spray of charged droplets.
three orders of magnitude (van Bruijnsvoort The solvent is evaporated, and the analyte is
et al., 1997). A more recent EC technique is desorbed as ions that are introduced into the
contactless conductivity detection, where the mass spectrometer. Although there is an inherent
difference in conductivity of a sample zone challenge in decoupling the high separation
compared to the separation buffer is used to detect voltage from the electrospray voltage, several
various analytes. This has been shown for carbo- approaches have been reported since the initial
hydrates (Carvalho et al., 2003) and amino acids report of CEeMS in 1987 (Olivares et al., 1987).
(Tanyanyiwa et al., 2003; Coufal et al., 2003). The combination of the resolving power of CE
Although EC detection offers the benefit of not with the sensitivity of MS has been implemented
requiring a native chromophore or derivatization in numerous applications with a great deal of
to detect the sample, it has not been as widely success, as evidenced by a thorough tabulated
reported on the capillary. This is, in part, due to review covering the 15 years that followed
the difficulty in aligning the capillary with the (Schmitt-Kopplin and Frommberger, 2003), as
electrodes and the need to decouple the high elec- well as reviews focused on quantitation (Ohne-
tric field strengths used to separate analytes from sorge et al., 2005) and the different CE modes
the detection zone which is necessarily sensitive to coupled to MS for the analysis of biomolecules
current or voltage, though methods have been (Hernández-Borges et al., 2004). More germane
designed for both end-column and off-column reviews have focused on applications of CEeMS
detection to account for this (Voegel and to various food types (Campa et al., 2006; Simó
Baldwin, 1997). The different types of EC detec- et al., 2005a,b; Stutz, 2005) and food quality
tion, including examples and considerations, (e.g., contaminants and residues) (Font et al.,
have been reviewed for both capillary (Holland 2008; Kumar et al., 2010; Ravelo-Pérez et al.,
and Leigh, 2002; Tanyanyiwa et al., 2002) and 2009; Robledo and Smyth, 2009).
microchip (Vandaveer et al., 2004), with a signifi-
cant improvement in the alignment of electrodes
with the separation channel possible on 12.4. APPLICATIONS OF CE FOR
microchip. FOOD ANALYSIS
MS has been referred to as a universal detector
because of its application to a large variety of CE has clearly found a place in food analysis
samples. Briefly, samples in the gas phase are due to its high resolving capability, reduced
ionized using an ion source, and the ionized sample consumption, and ease of automation.
samples then have a characteristic mass-to- The first review of CE applied to food analysis
charge (m/z) ratio that is subjected to an electric was in 1992 (Zeece, 1992) and featured only five
12.4. APPLICATIONS OF CE FOR FOOD ANALYSIS 393
applications from the literature. By 1996, this Carbohydrates range from simple sugars to
number had increased 10-fold according to complex polysaccharides, with more specific
a review by Lindeberg (1996b), and it should categories referring to structure and function.
be noted that an additional 25 references were CE is an attractive technique for carbohydrate
added in an addendum (Lindeberg, 1996a) to analysis because it does not require different
include work that was published during the columns when analyzing different types or sizes
interval between submission and publication. of carbohydrates, and it allows for the simulta-
The number of reports of CE methods to analyze neous analysis of other noncarbohydrate species
foods and food components has continued to (Campa et al., 2006). Carbohydrates have already
grow significantly, as evidenced by a series been mentioned because of the need for derivati-
of well-organized reviews on the subject (Frazier zation in order to perform UV or LIF detection
et al., 1999; Frazier, 2001; Frazier and Papadopou- since they lack a strong chromophore. Regarding
lou, 2003; Cifuentes, 2006; Garcia-Canas and CE, carbohydrates also lack a charge at neutral
Cifuentes, 2008; Herrero et al., 2010). These pH, so conditions for CZE usually feature high
reviews span the last decade, averaging more pH (e.g., pH 10) and/or complex formation
than 150 references each. In addition, there have with borate ions in the buffer. Applications are
been several recent reviews focused on CE appli- most common in evaluating food composition,
cations for food analysis, including composition, especially for nutritional labeling. CZE is the
quality, safety, and authenticity (Table 12.1). The most common separation mode, with detection
majority of the referenced reviews are focused performed by UV, LIF, or MS. Figure 12.11 shows
solely on CE in food analysis, but some of the a CZE separation with UV absorbance detection
more expansive reviews provide additional and a CZE separation with ESIeMS detection.
applications of other separation methods (e.g., Proteins are another main component of food,
HPLC) or samples (i.e., not food related). with major protein groups separated into meat,
Considering the broad array of reviews that dairy, and cereal proteins. There is a high degree
already provide thorough descriptions of of variability in terms of protein structure and
methods employed and results obtained, it is function due to their heterogeneity, which makes
evident that a singular, collective review would them both interesting and challenging from an
be beyond the scope of this work. Issues to analysis standpoint. Proteins are important in
consider when evaluating a food analysis tech- nutrition (e.g., growth), health (e.g., allergies),
nique include the separation mode, which will and food composition (e.g., structure and flavor),
depend upon the target analyte(s) as well as the and analytical methods are needed to determine
sample matrix, and the detection mode, which protein composition and elucidate protein func-
depends upon the analyte(s). Because CE is tion (Bean and Lookhart, 2001). CE offers the
strictly a separation mode, quantitative analysis benefit of being able to separate protein samples
is primarily dependent upon the detection using several different separation modes on capil-
mode, though it should be noted that the sensi- laries, including CEC and CIEF, although CZE is
tivity of a CE-based assay can be aided with the most common. This is despite the tendency
a concentrating effect within the capillary (e.g., of proteins to adsorb to the capillary wall,
CITP). In light of the possible combinations although this can be remedied through polymer
available when performing CE for food analysis, wall coatings or careful buffer selection (i.e.,
the following discussion will present the most high ionic strength, extreme pH, or addition of
common separation and detection modes of an additive). Detection is usually performed
some of the more commonly analyzed groups with UV, owing to the absorption character of
of compounds. the ring-containing amino acid residues that
TABLE 12.1 Summary of Reviews for CE Applications to Food Analysis Since 2005
Year Subject References
2005 CEeMS in food analysis Simó et al. (2005b)
CEeMS of amino acids in foods Simó et al. (2005a)

CEeMS for proteins and peptides Stutz (2005)
Analysis of natural antioxidants by CE Herrero et al. (2005)
Analysis of plant pigments by CE Sun et al. (2005)
Analysis of carboxylic acids by CE Baena et al. (2005)
Analysis of pesticides by CE Hernández-Borges et al. (2005)
CE in routine food analysis Castaneda et al. (2005)

CE in food authenticity Kvasnicka (2005)
Determination of organic contaminants in food by CE Juan-Garcia et al. (2005)
2006 CEeMS for carbohydrates Campa et al. (2006)
Analysis of antibiotics by CE Garcı́a-Ruiz and Marina (2006)
Amino acid analysis by CE Poinsot et al. (2006)
Analysis of biologically active amines by CE Chiu et al. (2006)

CE of proteins Dolnı́k (2006)
CZE for bioactive phytochemical compounds Li et al. (2006)
Pesticide analysis by CE Ravelo-Pérez and Hernández-Borges
(2006)
2007 Determination of additives and organic contaminants in Boyce (2007)
food by CE and CEC
Microchip CE for food analysis Escarpa et al. (2007)
CE methods for the determination of nonprotein amino Castro-Puyana et al. (2007)
acids in foods
CE methods for the analysis of micronutrients in foods Cheung et al. (2007)
Analysis of bioactive compounds in food by CE Kvasnicka (2007)
Detection of microbial food contaminants and their Garcı́a-Cañas and Cifuentes (2007)
products by CE techniques
CE for the characterization of Maillard reaction products Vallejo-Cordoba and
in foods González-Córdova (2007)
Determination of organic acids by CE and CEC Klampfl (2007)
Analytical methods for the determination of biogenic Önal (2007)
amines in foods
TABLE 12.1 Summary of Reviews for CE Applications to Food Analysis Since 2005 (cont’d)
Year Subject References
2008 CEeMS for food contaminants and residues Font et al. (2008)
Analysis of antibiotics by CE and CEC Castro-Puyana et al. (2008)
Microchip CE for food analysis Escarpa et al. (2008)
CE in bioanalysis Kostal et al. (2008)

CE of complex natural polysaccharides Volpi et al. (2008)
CE of proteins Dolnı́k (2008)
Determination of amino acids by MEKC Iadarola et al. (2008)
Methods for determining polyphenol compounds Kartsova and Alekseeva (2008)
CE applications for food analysis Vallejo-Cordoba and Martinez (2008)
2009 Food safety and food quality applications of CEeMS Ravelo-Pérez et al. (2009)
CEeMS for trace analysis of environmental pollutants and Robledo and Smyth (2009)
food contaminants
Analysis of sulfonamides by CE Hoff and Kist (2009)

Analytical methods for the determination of mycotoxins Turner et al. (2009)
2010 Determination of pesticides in foods using CEeUV/MS Kumar et al. (2010)
Analysis of antibiotics by CE and CEC Castro-Puyana et al. (2010)
CE for the analysis of contaminants for food safety and Vallejo-Cordoba and
food traceability González-Córdova (2010)
CE and CEC of proteins El Rassi (2010)
Developments in coupled SPEeCE Ramautar et al. (2010)
Analytical applications of CEeCL Lara et al. (2010)

CE and CEC of nucleosides and nucleotides in food Chen et al. (2010)
materials
make up the protein (or smaller peptide chain). groups allow amino acids to be well separated by
Figure 12.12 shows a CZE separation with LIF CZE, and the first report of CZE in 1981 actually
detection and a CZE separation with UV absor- featured a mixture of neutral and charged amino
bance detection. acids (Jorgenson and Lukacs, 1981b). The chirality
Amino acids are important in nutrition and are associated with amino acids, where two different
primarily obtained from protein, though they can forms of the same molecule exist as mirror images
also be obtained in free form. Like carbohydrates, of one another, can be used to evaluate food
amino acids require derivatization for UV or quality (Simó et al., 2005a), but the chiral mole-
LIF detection, though the presence of amine and cules would exhibit the same mobility in CZE.
carboxylic acid functional groups creates oppor- In order to resolve the chiral species, either
tunities for efficient labeling. The same functional MEKC with SDS micelles or EKC separations
(a) 7
(b) 25000
TIC
Non-alcohol
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

Light alcohol
Absorbance (280 nm)
minutes
2000 1
m/z 133
2914
m/z 149 2 3 4
Special
4 10000 11
2
7 8 10
65 3 * 1 Special Black m/z 179
5000
12
5 6 7 8 9 10 11 m/z 181
Migration time (min)
FIGURE 12.11 Sample data for carbohydrate analysis. (a) Representative data from a CZE separation with UV detection.
Beer samples were diluted 1:20 in water and derivatized with p-aminobenzoic acid (PABA). The separation was performed at
251 V cm1, and the sample was detected by monitoring UV absorbance at 280 nm. The four beer samples (nonalcoholic, light
alcohol, special, and special black) were run separately along with other samples (not shown). Labeled peaks were determined
from a standard mixture of 7 PABAecarbohydrate derivatives and include (1) glucose, (2) maltose, (3) maltotriose, (4) mal-
totetraose, (5) maltpentaose, (6) maltohexaose, (7) maltheptaose, and (R) unreacted PABA. Source: Reprinted from Cortacero-
Ramı́rez et al. (2004). (b) Representative data from a CZE separation with electrospray ionization mass spectrometry detection. A
red wine sample was diluted 1:5 in water and analyzed for the determination of underivatized carbohydrates. The separation
was performed at 286 V cm1, and the sample was introduced into a quadrupole mass spectrometer using electrospray
ionization. (Top) The total ion current (TIC) provides an electropherogram of separated carbohydrates. (Bottom) The selected
ion monitoring (SIM) data indicate ion count at selected mass-to-charge (m/z) ratios. Labeled peaks were determined from
a standard mixture of 17 carbohydrates and include (1) deoxyribose, (2) arabinose, (3) ribose, (4) xylose, (7) galactose,
(8) glucose, (10) fructose, (11) inositol, and (12) mannitol. Source: Reprinted from Klampfl and Buchberger (2001).
(a) (b)
0.25 β-LGB
Absorbance (214 nm)
β-LGA
0.20 p-κ-CN
0.15
F.I. (a.u.)
0.03
0.10
0.05 BSA β-Lg
α-LA
0.00
0
–0.05
0 10 20 30 40 10 20 30 40
Time (min) Time (min)
FIGURE 12.12 Sample data for protein analysis. (a) Representative data from a CZE separation with LIF detection. Bovine
whey was obtained from fresh raw milk samples and derivatized on-capillary with a fluorescent dye (2,6-TNS). The separation
was performed at 115 V cm1, and LIF was detected at 450 nm. Labeled peaks were determined from a standard mixture and
include a-lactalbumin (a-LA), b-lactoglobulin B (b-LGB), b-lactoglobuline (b-LGA), and bovine serum albumin (BSA).
Source: Reprinted from Benito et al. (1999). (b) Representative data from a CZE separation with UV detection. Processed cheese was
analyzed for the degradation products of its casein fraction. The separation was performed at 417 V cm1, and UV absorbance
was recorded at 214 nm. Acidic conditions were used to separate the previously unresolved p-k-casein (p-k-CN), a degradation
product of casein, from b-lactoglobulin (b-Lg), the major whey protein in milk. Additional peaks represent various whey
proteins, caseins, and their degradation products. Source: Reprinted from Miralles et al. (2001).
(a) (c)
18
a 1 2 3 4 5 40 pA
16 6 7 8
14
12 3
10 15 20 25 30 35
RFU
10 1
2 5
8 b 4 6 7
6
6 7 1 40 pA
4 8 5
2 4 8
0 3
5 6 7 8 9 10 11 12 13 14 15 2
Minutes
(b) 15 20 25 30 35 40
0.05
Arg His+Ile+Leu+Phe
Try
Met+Gln+Tyr
Abs (A.U.)
20 pA
0.000 Val+Thr
8
7
Orn
5 6 Ser
1 4 Lys Ala
Gly Cys Glu Asp
3
2
0 10 15 20 25 30 35 40
Time (min) t (min)
FIGURE 12.13 Sample data for amino acid analysis. (a) Representative data from an MEKC separation with LIF
detection. A beer sample was diluted 1:20 in water and derivatized with a fluorescent dye (SAMF). The separation was
performed at 395 V cm1, and the sample was excited at 488 nm and detected at 520 nm (RFU). Data are shown for (a) a beer
sample and (b) the same sample spiked with 250 nmol L1 standard amines. Labeled peaks correspond to (1) unreacted
SAMF, (2) ethanolamine, (3) methylamine, (4) ethylamine, (5) n-propylamine, (6) n-butylamine, (7) n-pentylamine, and
(8) n-hexylamine. Source: Reprinted from Cao et al. (2005). (b) Representative data from a CZE separation with indirect UV
detection. Purchased salmon was crushed and homogenized prior to pervaporation (to leach and collect volatile analytes).
The separation was performed at 286 V cm1, and UV absorbance was monitored at 214 nm. Labeled peaks were determined
from a standard mixture of eight biogenic amines and include (1) trimethylamine, (2) putrescine, (3) cadaverine, (4) sper-
mine, (5) tryptamine, (6) spermidine, (7) phenylethylamine, and (8) tyramine. (Ruiz-Jiménez and Luque de Castro, 2006).
(c) Representative data from a CZE separation with EC detection. A beer sample was diluted 1:10 in water and derivatized
with an electroactive tag (NDA) that reacts with primary amines in the presence of cyanide. The separation was performed
at 225 V cm1, and EC detection was carried out with a three-electrode system (working electrode, auxiliary electrode, and
reference electrode). Data are shown for a beer sample (top) before and (middle) after derivatization, as well as (bottom)
a standard solution of 19 amino acids (labeled) after derivatization. Source: Reprinted from Dong et al. (2002).
with CDs are usually performed. The bacterial biogenic amines being of particular interest
degradation products of amino acids are called regarding contamination and toxicity. Separation
biogenic amines. These are found in all foods is typically performed by either CZE or MEKC,
and are a part of the normal composition of fer- with detection performed by UV, LIF, or MS.
mented foods (e.g., beer, wine, and cheese). Figure 12.13 shows an MEKC separation with
Conditions for the separation and detection of LIF detection, a CZE separation with indirect
biogenic amines are similar to those of amino UV absorbance detection, and a CZE separation
acids. Both are analyzed for food quality, with with EC detection.
Phenolic compounds are naturally occurring antioxidants, Herrero et al. include several
metabolites in plants that feature a phenol applications (including limits of detection) to
group (i.e., an aromatic hydrocarbon with flavonoids and catechins, a subgroup of flavo-
a hydroxyl group), and they are of primary noids that has been reported to offer health
interest in food analysis due to their contribu- benefits (Herrero et al., 2005). The presence of
tion to taste. Polyphenols, which contain at one or more phenol groups explains the antiox-
least one phenol group, can be broadly catego- idant behavior of these compounds, as well as
rized as either flavonoids or nonflavonoids, their strong UV absorption. CZE and MEKC
though the latter can be further split into nine separation modes are most often reported,
different types based on structure (e.g., couma- with detection performed by either UV absor-
rins, phenolic acids, and lignins). Flavonoids bance or EC detection. Figure 12.14 shows an
make up more than half of the phenolic MEKC separation and a CITPeCZE separation,
compounds and are typically found in both with UV absorbance detection.
fruits, seeds, and leaves. In their analysis of Food additives are substances that are
CE methods for the analysis of natural purposely added to food to improve flavor,
(a) (b) T
0.020 6
0.018
R Interfering compounds
0.016 Analytes
4 7
L
0.014
t[min]
AU
0.012 6 7 8 9 10 11 12
10
2;3
0.010
9
1 5
0.008
5 A254nm 4 7
3 11
1 6 8
0.006
t[min]
1.5 2.0 2.5 3.0 3.5 4.0 4.5 25 30 35 40
MINUTES
FIGURE 12.14 Sample data for phenolic compound analysis. (a) Representative data from an MEKC separation with UV
detection. A sample of bottled green tea was analyzed for catechin and xanthine composition. The separation was performed
at 638 V cm1, and UV absorbance was recorded at 200 nm. Labeled peaks were determined from a standard mixture and
include (1) theobromine, (3) gallocatechin, (4) caffeine, (5) catechin, (6) epigallocatechin, (7) epigallocatechin-3-gallate, (9)
epicatechingallate, (10) epcatechin, and (11) gallic acid. Source: Reprinted from Bonoli et al. (2003). (b) Representative data from
a CITPeCZE separation with UV detection. A sample of Cabernet Sauvignon was diluted 1:10 in water. CITP was performed
at 200 mA in a 9-cm polymer capillary as a preconcentrating step, and the signal was recorded with a conductimeter (R). CZE
was subsequently performed at 169 V cm1, and UV absorbance was recorded at 254 nm. (top) Conductimeter data indicate
the presence of (L) leading ion, interfering compounds, desired analytes, and (T) trailing ion. (Bottom) Labeled peaks were
determined from a standard mixture and include (1) protocatechuic acid, (2,3) gallic and caffeic acids, (4) vanillic acid,
(5) syringic acid, (6) ferulic acid, (7) p-foumaric acid, and (8) quercitrin. Source: Reprinted from Hamoudová et al. (2004).
appearance, or shelf life. Several different types in the Boyce review is the presence of unde-
of additives have been analyzed by CE, sired food contaminants. This has become
including dyes, preservatives, and sweeteners, a greater focus of regulatory bodies in recent
and Boyce (2007) has provided a recent review years. Contaminants of interest range from
of the different separation and detection pesticides in plant crops to antibiotics in
methods (including limits of detection) meat. Several reviews have been published
applied. In the review, Boyce points out that since 2008 alone dealing with issues of contam-
although all of the principal additives had inants, toxins, environmental pollutants, food
previously been analyzed by CE, it was not quality, and food safety (Font et al., 2008;
until 2001 when the focus switched to the anal- Kumar et al., 2010; Ravelo-Pérez et al., 2009;
ysis of real food samples. Separations of food Robledo and Smyth, 2009; Castro-Puyana
additives are typically performed using CZE et al., 2008, 2010; Hoff and Kist, 2009; Turner
or MEKC using UV absorbance detection. et al., 2009; Vallejo-Cordoba and González-
Figure 12.15 shows a reverse polarity CZE Córdova, 2010). Further discussion regarding
separation and an MEKC separation, both contaminants and safety issues can be found
with UV absorbance detection. Also reported in Volume II of this text.
FIGURE 12.15 Sample data for food additive analysis. (a) Representative data from a reverse polarity CZE separation
with UV detection. Nitrite and nitrate preservatives were evaluated in meat products and vegetables using a capillary
coated with a cationic polymer to suppress EOF. The separation was performed at 373 V cm1, and UV absorbance was
recorded at 210 nm. Labeled peaks for (top) spinach and (bottom) salami samples were determined from a standard mixture
of (1) nitrite, (2) nitrate, and (3) thiocyanate (used as an internal standard). Source: Reprinted from Öztekin et al. (2002).
(b) Representative data from an MEKC separation with UV detection. Various soft drink samples were analyzed to identify
artificial sweeteners, preservatives, and food colors. The separation was performed at 412 V cm1, and UV absorbance was
recorded at 200 nm. Labeled peaks for (top) a mixed-flavor soft drink, (middle) sugar-free bubblegum-flavor sparkling
drink, and (bottom) low-sugar soda were determined from a standard mixture of (1) aspartame, (2) benzoic acid, (3)
saccharin, (4) acesulfame K, (5) sunset yellow FCF, (6) Class IV caramel, (7) brilliant blue FCF, and (8) ponceau 4R.
Source: Reprinted from Frazier et al. (2000).
12.5. CONCLUSIONS Journal of Agricultural and Food Chemistry 52 (23),

7180e7186.
Archak, S., Lakshminarayanareddy, V., Nagaraju, J., 2007.
With the variety of separation modes available High-throughput multiplex microsatellite marker assay
and the ease of automation, CE shows increasing for detection and quantification of adulteration in Basmati
promise in the field of food analysis, and rice (Oryza sativa). Electrophoresis 28 (14), 2396e2405.
continued advancement will likely result in Baena, B., Cifuentes, A., Barbas, C., 2005. Analysis of
carboxylic acids in biological fluids by capillary elec-
increased throughput and selectivity. In terms of
trophoresis. Electrophoresis 26 (13), 2622e2636.
future directions, the recent reviews of CEeMS Bean, S.R., Lookhart, G.L., 2001. High-performance capillary
applications suggest the combination of CE selec- electrophoresis of meat, dairy, and cereal proteins.
tivity with MS sensitivity offers the greatest capa- Electrophoresis 22 (19), 4207e4215.
bilities as an analytical technique. This is likely Benito, I., et al., 1999. Detection of bovine whey proteins by
on-column derivatization capillary electrophoresis with
spurred by the widespread application of
laser-induced fluorescence monitoring. Journal of
HPLCeMS techniques for protein and peptide Chromatography A 841 (1), 105e114.
analysis, with electrophoresis replacing chroma- Bilitewski, U., et al., 2003. Biochemical analysis with
tography as the separation mode. CE has already microfluidic systems. Analytical and Bioanalytical
demonstrated its capabilities as the workhorse for Chemistry 377 (3), 556e569.
Bonoli, M., et al., 2003. Fast determination of catechins and
the Human Genome Project, and as more nucleic
xanthines in tea beverages by micellar electrokinetic
acid separations are required for food analysis, chromatography. Journal of Agricultural and Food
the well-established CGE methods should prove Chemistry 51 (5), 1141e1147.
to be very valuable to the field, despite a relative Boyce, M.C., 2007. Determination of additives and organic
lack of current applications. Reports of microchip contaminants in food by CE and CEC. Electrophoresis 28
(22), 4046e4062.
electrophoresis applications are increasing, with
Braddock, R.J., Bryan, C.R., Burns, J.K., 2001. Capillary
a mere 1-year interval between recent reviews electrophoresis analysis of orange juice pectinesterases.
(Escarpa et al., 2007; Escarpa et al., 2008). This is Journal of Agricultural and Food Chemistry 49 (2),
not unexpected, since the microchip represents 846e850.
the latest evolution of the electrophoresis plat- Bruin, G.J.M., et al., 1991. Optimization and evaluation of
the performance of arrangements for UV detection in
form, but it is somewhat surprising that the
high-resolution separations using fused-silica capil-
most common form of detection has been EC laries. Journal of Chromatography A 559 (1e2), 163e181.
when LIF is utilized in the majority of microchip Campa, C., et al., 2006. Overview on advances in capillary
applications (Karlinsey, 2008). It is expected in electrophoresis-mass spectrometry of carbohydrates:
the near future that more integrated devices or a tabulated review. Electrophoresis 27 (11), 2027e2050.
Cao, L., Wang, H., Zhang, H., 2005. Analytical potential of
mTAS (micro-total analysis systems) will be
6-oxy-(N-succinimidyl acetate)-9-(2’-methoxycarbonyl)
designed and employed for food samples, fluorescein for the determination of amino compounds
utilizing the ability to combine sample pretreat- by capillary electrophoresis with laser-induced fluores-
ment steps to introduce fluorescent labels. In cence detection. Electrophoresis 26 (10), 1954e1962.
addition, the promise of portability and miniatur- Carvalho, A.Z., da Silva, J.A.F., do Lago, C.L., 2003. Deter-
mination of mono- and disaccharides by capillary elec-
ized instrumentation for field-testing suggests
trophoresis with contactless conductivity detection.
that microchip analysis could find use in food Electrophoresis 24 (12e13), 2138e2143.
diagnostics for safety, quality, and authenticity. Caslavska, J., Gebauer, P., Thormann, W., 1994. Fractionation
of human serum proteins by capillary and recycling iso-
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C H A P T E R

12.1. Introduction 375 12.2.6. Capillary Isoelectric Focusing 388

12.1. INTRODUCTION strength (7.25 V cm1), chosen to reduce

Chemical Analysis of Food: Techniques and Applications

further developments by Hjertén (1967) using During electrophoresis, a steady state is

12.1.1. Theory mp ¼ q$ð6phrÞ1 (12.5)

(a) (b) or resistive heating), a phenomenon where

(a) polymer coating (b)

buffer inlet buffer outlet

FIGURE 12.3 Electroosmotic flow. (a)

(a) migration (b) (c) 175

Electric field (V/cm, dotted line)

+ – 101 151 100

(iii) Non GM-maize 25

zone electrophoresis in polytetrafluoroethy- is static due to the strong interaction with

12.2.2. Micellar Electrokinetic

Relative Fluorescence Units

E 450 455 460 465 470 475 480

(c) pH gradient (low to high) 0

sample, and the absorbance A is directly related a photodiode or a photomultiplier tube. In

Year Subject References

2005 CEeMS in food analysis Simó et al. (2005b)

CEeMS of amino acids in foods Simó et al. (2005a)

CE in routine food analysis Castaneda et al. (2005)

Analysis of biologically active amines by CE Chiu et al. (2006)

CE in bioanalysis Kostal et al. (2008)

Analysis of sulfonamides by CE Hoff and Kist (2009)

Analytical applications of CEeCL Lara et al. (2010)

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

12.5. CONCLUSIONS Journal of Agricultural and Food Chemistry 52 (23),

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