United States
Environmental Protection
Agency
Environmental Sciences Division
P.O. Box 93478
Las Vegas, NV 89193-3478
July 1999
OFFICE OF RESEARCH AND DEVELOPMENT
TECHNOLOGY SUPPORT PROJECT
r/EPA Capillary
Electrophoresis
for
Environmental
Monitoring
Capillary, Capillary
Inlet Cr \ LJetector j^^^l Inlet
I Electrolyte I
^ML ^/> Buffer x^ J^K
Reservoir I ^Reservoir
The Need
The Use
The Analytical Chemistry
Research Program of the
National Exposure Research
Laboratory's Environmental
Sciences Division (ESD) is
developing new methods for
determining toxic and hazar-
dous chemicals in samples
from hazardous waste sites.
This research is guided by
several goals for analytical
methods:
• Shorter analysis time to
reduce costs and improve
quality control procedures.
• Improved separations per-
formance and applicability to
a wide spectrum of analytes,
including nonvolatiles, as
compared with current tech-
niques based on capillary
gas chromatography (GC).
• Field-screening capability to
achieve faster results and
better coordination between
sampling and analytical
workers.
• "Green" chemistry tech-
niques that reduce the gen-
eration of laboratory waste
(e.g., low solvent consump-
tion) while simultaneously
reducing personnel exposure
to toxic chemicals.
• Simple technology, export-
able to foreign countries and
applicable to a broad range
of analytes in a continuous
monitoring format.
These goals are summed up
by the phrase "cheaper, better,
and faster," and are being met
by an innovative separations
technology called capillary
electrophoresis (CE) that is
new to environmental analysis.
Traditional methods for intro-
ducing a sample into an
analytical device have various
drawbacks. Liquid introduction
of samples and liquid chroma-
tographies and electrophoretic
separations are the more
universally applicable
techniques since they do not
depend on volatility of analytes
or have molecular weight
limitations. Thermally labile
and polar compounds often
deposit in the injector systems
of gas chromatographs (even
cold on-column retention gap
systems) to degrade chroma-
tography, precision, and
quantitative accuracy. High
performance liquid chromato-
graphy (HPLC) has attempted
to fill the need for liquid state
separations, but its application
to ionic organics, neutral
hydrophobic compounds, and
inorganic ions has not been
universal. CE is a separations
technique that can meet the
goals stated above while filling
a central, cross-cutting role in
analytical chemistry for polar
volatiles, most semivolatiles,
nonvolatiles (e.g., herbicides),
inorganic cations, inorganic
anions, and biomarkers (i.e.,
indicators of exposure). Intro-
duced in 1981, CE is now
firmly established as the tech-
nique of choice for pharma-
ceutical and biomedical
analysis.
CE is easily interfaced with
optical detection methods
based on Uv-visible absorp-
tion, indirect detection (UV or
fluorescence), and laser-
induced fluorescence (LIF)
detection.
CE methods that are applica-
ble to routine problems are
emerging, and EPA-approved
CE methods are anticipated
shortly. CE technology is
widely developed commercial-
ly, and EPA staff at ESD are
confident that current CE
methods are sufficiently robust
to provide valuable contribu-
tions to environmental assess-
ment at the present time.
CE instrumentation is simple
(see illustration in the brochure
header). A fused silica
capillary (typically 0.050 or
0.075 mm x 27 to 57 cm)
connects two buffer (elec-
trolyte) reservoirs. A high-
voltage power supply (ca. 30
kV) connects the reservoirs via
the buffer-filled capillary. The
technique has been micro-
miniaturized as "CE on a chip",
and it is capable of adaptation
to continuous monitoring
applications based on fast
separations (see page two of
this fact sheet).
197CMB98.FS-39
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The Use CE analyte bands travel with
continued fiat profiles that produce
extremely high resolutions
(see Figure 1). Reported
Cross-sectional flow profile
due to electroosmotic flow
Cross-sectional flow profile
due to hydrodynamic flow
Figure 1. Flow Profiles
values usually range from
250,000 to 1,000,000 theoret-
ical plates, with exceptional
values up to 2.7 million.
These values exceed those
obtained with other liquid
phase techniques such as
HPLC, and equal or surpass
the best capillary GC tech-
niques. This extremely high
resolution permits separation
of many more analytes on a
given column, eliminating
chromatographic interferences.
Figure 2 illustrates some of the
principles involved in CE and
micellar electrokinetic chroma-
tography (MEKC) which
involves the addition of sur-
factant molecules to the buffer
solutions. MEKC (also called
MECC) was introduced by
Terabe et al. in 1984. Terabe
et al. also introduced applica-
tions of cyclodextrins and urea
in MEKC for improving separ-
ations involving hydrophobic
molecules. Electrophoresis
(i.e., the migration or mobility
of ions in an electric field)
accounts for the movement of
ions of the appropriate charge
toward the cathode or anode
in narrow bands. The electro-
phoretic flow is shown in
Figure 2 by a smaller, dark
arrow. In addition, an
electroosmotic (EO) flow exists
that transports bulk liquid with
buffer from one reservoir to
the other depending on
conditions. Usually, for bare
silica, an excess of mobile
positive charge exists in
solution because of the
ionization of silanol groups on
the silica surface. The EO
flow is illustrated by the large,
white arrow. This flow is
characterized by a flat, piston-
like profile rather than the
parabolic flow characteristic of
pressure-driven systems. This
flat-flow profile results in
extremely narrow peaks and
high efficiency in CE. The
separation of neutral analytes
under MEKC is based on their
affinity for micelles
(aggregates of surfactant
molecules) that migrate under
these conditions. These
micelles are considered to
form a pseudo-stationary
phase. Another capillary
format using EO flow,
electrokinetic chromatography
(EKC), involves the use of
packed capillary columns with
C18 derivatized silica particles
forming the stationary phase.
The fact that CE is based on
electromigration of ions means
that the technique can be of
great value in determining in-
organic ion concentrations.
The U.S. EPA Region VII has
approved a method for deter-
mining hexavalent chromium
(Cr(VI)). Ionic organic applica-
tions developed in the phar-
maceutical and biomedical
areas include separation of
proteins and amino acids.
Applications to environmental
organic ions include determin-
ation of acids, phenols, and
amines. Dr. William Brumley
and co-workers at the EPA
ESD are actively pursuing
research in CE and MEKC
separations of such organic
analytes as sulfonic acids,
carboxylic acids, benzidines
(substituted p,p'-diaminobi-
phenyls), phenols, anilines,
and PNAs.
The combination of ion-based
CE and MEKC CE provides a
nearly universal analyte separ-
ation methodology. Two valu-
able characteristics of CE in
developing routine methods
are separation speed and sen-
sitivity. Brumley and Brown-
rigg (1994) report high-speed
CE separations of four substi-
tuted benzidines in a little
over two minutes. For
samples that do not require
extensive preparation,
= Surfactant
(negative charge)
= Analyte
= Electroosmotic Flow
= Electrophoresis
Figure 2. Illustration of the components of MEKC
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The Use replicate sample analyses can
continued be performed to provide better
confidence in analytical
results. Because the detector
is on-column, there is no
band-broadening and signal
loss due to "dead volume"
mixing in the detector. This
gives CE extremely low
analyte mass detection limits.
Absolute mass sensitivity of
detection by optical spectro-
scopy (no preconcentration or
field amplification) is about
10-6 M to 10-7 M in a sample
by UV or indirect UV or indir-
ect fluorescence detection.
For a relative molecular mass
of 100, this calculates to 1 pg
to 100 fg on-column for a 10
nl_ injection. In the case of
LIF, the detection limit may
approach 10-11 M to 10-14 M in
a sample resulting in 10 ag to
10 zg injected on-column.
Reaching these mass sensi-
tivities presents one of the
greatest challenges in the de-
velopment of CE/MS.
Lack of detection sensitivity
under UV is one of the major
factors that has limited the
development of environmental
applications of CE. Although
detection in CE is very mass
sensitive (i.e., the absolute
amount of substance on-
column), concentration limits
of detection are substantially
higher because of the small
injection volume typically used
(ca. 1 to 10 nl_). This can be a
serious limitation for environ-
mental analysis, whereas for
microenvironment studies
(e.g., a single cell) it is an
advantage. Various approach-
es for overcoming the limita-
tions of nl_ injection volumes
are discussed below. These
include both sample handling
and improving detector sensi-
tivity (e.g., using LIF). One
approach is to use preconcen-
tration techniques such as
solid-phase extraction before
injecting samples. Additional
approaches have coupled C18
columns with CE columns for
preconcentration. An alter-
native approach is to use CE
techniques to concentrate
analytes. These can involve
coupled columns and a tech-
nique called isotachophoresis.
Another approach is to use
field amplification during sam-
ple injection to concentrate
analyte ions. These tech-
niques can lead to factors of
100 to 1000 improvement in
detection limits.
CE is a highly leveraged ana-
lytical tool in terms of invest-
ment because of biomedical
and pharmaceutical research.
It is thus assured of continu-
ous and rapid development.
Current improvements in
absorption detection, such as
degenerate four-wave mixing,
promise lowering detection
limits to 10-8 M.
Derivatization strategies to
take advantage of LIF sensi-
tivity (as low as 6 molecules
on-column) are currently
underway at ESD.
Additional leveraging in envi-
ronmental applications is
being sought through inter-
agency agreements. One of
the tasks currently being con-
sidered is parallel processing
of sample streams via bundled
capillaries. Sample through-
put could be increased 10-fold,
for example, with 10 capillaries
operating simultaneously with
detection. Partners with EPA
are also being sought for
development of CE/MS.
The Status Current developments in
CE/MS at ESD and elsewhere
focus on electrospray ioniza-
tion with quadrupoles, double
focusing instruments, ion
traps, and time-of-flight mass
spectrometers. Currently, a
cooperative agreement be-
tween ESD and an external
institution is awaiting imple-
mentation (Ms. Tammy Jones,
Project Officer).
Laboratory evaluations and
research efforts have resulted
in at least one EPA CE meth-
od for hexavalent chromium
that was approved in Region
VII in March 1994. Dr. W.C.
Brumley, Dr. Wayne Garrison,
ERL-Athens, as well as other
EPA and independent
researchers, have performed
considerable research into the
application of CE to environ-
mentally important analytes.
The results have been suffi-
ciently successful that the next
step is to apply the technology
to real-world samples. Once
the methods have been de-
monstrated on these types of
samples, EPA staff are inter-
ested in soliciting requests to
perform CE analyses in a field
setting. To submit environ-
mental samples for CE anal-
ysis or to be considered for a
CE field demonstration, con-
tact Dr. Brumley or Mr. Ken
Brown, listed at the end of this
sheet.
References Anon., Introduction to Capillary Electrophoresis, Vol. I, Beckman Instruments, Fullerton, CA, 1991.
Anon., Standard Operating Procedure No. 3124.3B: Determination of Hexavalent Chromium in Soil
Using Capillary Electrophoresis, U.S. EPA, Region VII, Jan. 1994.
Brumley, W.C., "Qualitative analysis of environmental samples for aromatic sulfonic acids by high-
performance capillary electrophoresis," J. Chromatogr., 603, 267, 1992.
Brumley, W.C. and C.M. Brownrigg, "The electrophoretic behavior of aromatic-containing organic
acids and the determination of selected compounds in water and soil by capillary electrophoresis,"
J. Chromatogr, 646, 377, 1993.
-------
References
Continued
Brumley, W.C. and C.M. Brownrigg, "Applications of MEKC in the determination of benzidines
following extraction from water, soil, sediment, and chromatographic adsorbents," J. Chromatogr.
ScL, 32,69, 1994.
Gaitonde, C.D. and P.V. Pathak, "Capillary zone electrophoretic separation of chlorophenols in
industrial waste water with on-column electrochemical detection," J. Chromatogr., 514, 389, 1990.
Terabe, S., Micellar Electrokinetic Chromatography, Vol.
1992.
I, Beckman Instruments, Fullerton, CA,
Yik, Y.F., C.P. Ong, S.B. Khoo, H.K. Lee, and S.F.Y. Li, "Separation of selected PAHs by using high
performance capillary electrophoresis with modifiers," Envir. Mon. Assess., 19, 73, 1991.
For Further For more information about applying CE to
Information environmental problems, contact:
Dr. William C. Brumley
U.S. Environmental Protection Agency
National Exposure Research Laboratory
Environmental Sciences Division
P.O. Box 93478
Las Vegas, NV 89193-3478
Tel.: (702) 798-2684
For information about evaluating CE at a
hazardous waste site (Superfund or RCRA) or
for analysis of samples at a field site, contact:
Mr. J. Gareth Pearson, Director
Technology Support Center
U.S. Environmental Protection Agency
National Exposure Research Laboratory
Environmental Sciences Division
P.O. Box 93478
Las Vegas, NV 89193-3478
Tel.: (702) 798-2270
XO^OA,.^
'QtOGY^
The Technology Support Center fact sheet series is developed by Clare L. Gerlach, with technical
contributions in the CE sheet by Nelson R. Herron, Ph.D., Lockheed Martin, Las Vegas.
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