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.

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   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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