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 ------- 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 ------- 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. ------- |