Final Report: Capillary Immunophoresis for Environmental Monitoring

EPA Grant Number: R823292
Title: Capillary Immunophoresis for Environmental Monitoring
Investigators: Lee, Cheng S.
Institution: University of Maryland , Iowa State University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1995 through September 1, 1998
Project Amount: $269,984
RFA: Exploratory Research - Environmental Biology (1995) RFA Text |  Recipients Lists
Research Category: Biology/Life Sciences , Health , Ecosystems


The proposed capillary immunophoresis as a novel environmental monitoring tool combines the strengths of capillary electrophoresis (ease and speed of analyses) and the biospecificity and sensitivity of the antigen-antibody reaction. In analogy to a two-dimensional separation system, micellar electrokinetic capillary chromatography (MEKC) as the first separation dimension to separate the structurally related pollutants based on their differential partitioning with the micellar phase. Environmental pollutants separated by MEKC can be detected in the presence of other sample components by immunocomplex formation in a post-capillary immunoreactor. Immunoreaction as the second dimension utilizes the antibody's cross-reactivity for the recognition of triazines with a common structural element (epitope). The direct sensing of the (triazines) antigen-antibody reaction is achieved by monitoring the fluorescence polarization of atrazine hapten-fluorochrome conjugate in a competitive immunoassay format.

This research was a fundamental effort in the development of capillary immunophoresis as a novel analytical technique for the identification of unknown contaminants in complex sample matrix. More specifically, we studied: (1) the effect of electroosmotic control on the separation of contaminants in MEKC, (2) the quantitative dependence of solution ionic strengths and capillary dimensions on the effectiveness of mass transfer and mixing of pollutants with antibodies in the post-column immunoreactor, and (3) the direct sensing of the antigen-antibody reaction by measuring the fluorescence polarization of the hapten-fluorochrome conjugate in an on-column laser-induced fluorescence detector. The integration of MEKC with online immunodetection for multiresidue analysis of environmental pollutants was demonstrated using triazine herbicides as the model system.

Summary/Accomplishments (Outputs/Outcomes):

Mechanistic Studies of Partial-Filling Micellar Electrokinetic Chromatography. The need for coupling MEKC with post-capillary antigen-antibody reaction initiates the development of partial-filling MEKC. In comparison with conventional MEKC, only a small portion of the capillary is filled with a micellar solution for performing the separation in partial-filling MEKC. Analytes first migrate into the micellar plug, where the separation occurs, and then into the leading electrophoresis buffer, which is free of surfactants. A theoretical model is proposed for predicting the separation behavior of triazine herbicides in partial-filling MEKC. The comparisons between conventional and partial-filling MEKC in terms of separation efficiency and resolution of triazine herbicides are presented and discussed. The optimization techniques, possible applications, and advantages of partial-filling MEKC are similarly addressed (Nelson WN, Lee CS. Analytical Chemistry 1996;68:3265).

Online Micellar Electrokinetic Chromatography-Electrospray Ionization Mass Spectrometry Using Anodically Migrating Micelles. Online MEKC-electrospray ionization mass spectrometry (ESI-MS) is demonstrated for the analysis of chlorotriazine herbicides. In this study, the micellar velocity is directly manipulated by the adjustment of electroosmosis rather than the electrophoretic velocity of the micelle. The electroosmotic flow is adjusted against the electrophoretic velocity of the micelle by changing the solution pH in MEKC. The elimination of MEKC surfactant introduction into ESI-MS is achieved with an anodically migrating micelle, moving away from the electrospray interface. The effects of moving surfactant boundary in the MEKC capillary on separation efficiency and resolution of triazine herbicides were investigated. The mass detection of herbicides sequentially eluted from the MEKC capillary was acquired using the positive electrospray mode (Yang L, Harrata KA, Lee CS. Analytical Chemistry 1997;69:1820).

Micellar Electrokinetic Chromatography-Mass Spectrometry. The combination of MEKC with MS is very attractive for the direct identification of analyte molecules, for the possibility of selectivity enhancement, and for the structure confirmation and analysis in a MS-MS mode. The direct coupling of MEKC with MS can present problems due to the effect of nonvolatile MEKC surfactants on MS performance, including the loss of analyte sensitivity and ion source contamination. The possibility of offline coupling between MEKC and matrix-assisted laser desorption/ionization (MALDI)-MS remains to be investigated. Various approaches for online coupling MEKC with ESI-MS, including the use of high-molecular-mass surfactant, an electrospray-chemical ionization (ES-CI) interface, a voltage switching and buffer renewal system, partial-filling micellar plug, and anodically migrating micelles, were reviewed and evaluated. The use of an ES-CI interface is most promising for routine operation of online MEKC-MS under the influence of nonvolatile salts and surfactants. The use of a high-molecular-mass surfactant allows the formation of a micellar phase at very low surfactant concentrations and avoids the generation of a high level of background ions in the low m/z range. Alternatively the application of a partial-filling micellar plug and anodically migrating micelles eliminate the introduction of MEKC micelles into the ESI-MS system. It is possible to directly transfer the conventional MEKC separations to partial-filling MEKC-ESI-MS and MEKC-ESI-MS using anodically migrating micelles without any instrument modifications (Yang L, Lee CS. Journal of Chromatography?A 1997;780:207).

Postcapillary Fluorescence Polarization Immunodetection of Triazine Herbicides in Capillary Electrophoresis. Postcapillary fluorescence polarization immunodetection has been implemented for online detection of capillary electrophoresis. In this study, electrophoretic separation was used to speciate within a chemical class (such as triazine herbicides) that is selectively detected by the postcapillary immunoreaction without any prior sample treatment or cleanup. A new postcapillary reactor is developed for performing immunoreaction in a competitive format. A polarization-modulated laser-induced fluorescence detector is constructed for continuous measurement of fluorescence polarization of reaction mixtures in the detection capillary. It is further shown that many postcapillary reaction schemes require the use of hydrodynamic (instead of electric field) mobilization in the detection capillary (Cooper BT, Cao Y, Lee CS. Journal of Chromatography?A 2000 [in press]).


This work demonstrated online coupling of MEKC with both ESI-MS and postcapillary fluorescence polarization immunodetection for selective and sensitive analysis of triazine herbicides. We have achieved the use of electrophoretic separations to speciate within a chemical class that is selectively detected by fluorescence polarization immunodetection without any prior sample treatment or cleanup. Several observations can be made about postcapillary reaction in capillary electrophoresis. First, despite the simplicity and potentially higher efficiency of electroosmotic mobilization, many postcapillary reaction schemes require hydrodynamic mobilization. The exceptions are when the reaction does not appreciably change the electrophoretic mobility of the detected species, or when the reaction occurs so quickly that detection can occur almost immediately after elution from the separation capillary.

In this study, we employed hydrodynamic mobilization because the immunoreaction (for practical reactant concentrations) was relatively slow and because the reactors and products had significantly different electrophoretic mobilities. Second, quantitative precision of analyte determination requires careful control of reagent stoichiometry in the postcapillary reactor. Finally, some loss of separation efficiency necessarily accompanies any postcapillary reaction schemes. However, at least for hydrodynamic mobilization, the dominant source of peak broadening is parabolic flow in the detection capillary.

Because fluorescence polarization immunoassay has been developed for a broad variety of small-molecule analyte classes, the combination of capillary electrophoresis with fluorescence polarization immunodetection can potentially find wide applicability. Perhaps the largest obstacle to applying widespread use of capillary electrophoresis-fluorescence polarization immunodetection technique is the lack of commercially available postcapillary detection. This situation can change quickly as miniaturization technologies advance.

Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other project views: All 9 publications 3 publications in selected types All 3 journal articles
Type Citation Project Document Sources
Journal Article Nelson WM, Lee CS. Mechanistic studies of partial-filling micellar electrokinetic chromatography. Analytical Chemistry 1996;68(18):3265-3269. R823292 (Final)
  • Abstract from PubMed
  • Journal Article Yang LY, Lee CS. Micellar electrokinetic chromatography–mass spectrometry. Journal of Chromatography A 1997;780(1-2):207-218. R823292 (Final)
  • Abstract from PubMed
  • Other: Science Direct PDF
  • Journal Article Yang LY, Harrata AK, Lee CS. On-line micellar electrokinetic chromatography-electrospray ionization mass spectrometry using anodically migrating micelles. Analytical Chemistry 1997;69(10):1820-1826. R823292 (Final)
  • Abstract from PubMed
  • Supplemental Keywords:

    water, groundwater, health effects, organics, chemicals, toxics, environmental chemistry, analytical, measurement methods, agriculture., Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Environmental Chemistry, Chemistry, Monitoring/Modeling, Biology, environmental monitoring, traizine, environmental measurement, antigen-antibody reaction, gas chromatography, capillary immunophoresis, immune system response, herbicides

    Progress and Final Reports:

    Original Abstract
  • 1996
  • 1997