Grantee Research Project Results
Final Report: Development of Field-Portable Chromatographic Immunoassays
EPA Grant Number: R826253Title: Development of Field-Portable Chromatographic Immunoassays
Investigators: Hage, David S.
Institution: University of Nebraska at Lincoln
EPA Project Officer: Aja, Hayley
Project Period: January 1, 1998 through December 31, 2000
Project Amount: $277,762
RFA: Exploratory Research - Environmental Chemistry (1997) RFA Text | Recipients Lists
Research Category: Water , Land and Waste Management , Air , Safer Chemicals
Objective:
Immunoassay methods receive a great deal of attention for environmental testing due to their selectivity, low cost, and ease of use. However, most current immunoassays for pesticides and pollutants are based on manual methods that have only moderate precision and difficulties in differentiating between the analysis of interest and related compounds. Recent studies suggest that these problems can be overcome by using high-performance immunoaffinity chromatography (HPIAC), coupled with reversed-phase liquid chromatography (RPLC). This research project explored the development of field-portable systems that use this approach. The overall objective of this research project was to design portable and multi-analyte HPIAC/RPLC systems that could be used in field testing for environmental analysis. The specific objectives were to: (1) design and evaluate a field-portable system for the HPIAC/RPLC analysis of atrazine and related triazine compounds; (2) modify the field-portable system for the simultaneous detection of atrazine and 2,4-D (or alachlor) by mixed-bed HPIAC columns; and (3) develop and evaluate multiple HPIAC column method for the simultaneous analysis of atrazine, alachlor, and 2,4-D by the field-portable system.
Summary/Accomplishments (Outputs/Outcomes):
The first part of this research project involved the construction of a prototype. This prototype was a field-portable HPIAC/RPLC system for atrazine and triazine herbicides. This system was developed by using anti-atrazine monoclonal line AM7B2 from the Berkeley Monoclonal Antibody facility as the ligand within the HPIAC column, a Nucleosil 300-3 C18 silica precolumn, and an Alltech Rocket RPLC analytical column. The samples were applied to the HPIAC column in pH 7.0, 0.10 moles per liter (M) phosphate buffer and eluted with a pH 2.5, 0.10 M phosphate buffer. This latter buffer also was used for capture and reconcentration of the analytes on the Nucleosil precolumn. The reconcentrated analytes were eluted from the precolumn and onto the analytical column with a pH 4.0, 0.10 M phosphate buffer containing 27 percent 2-proppanol in the mobile phase. The eluting peaks were detected online at 229 nanometers (nm) by using a flow-through UV/Vis absorbance detector.
This system was powered by a portable 120 volt generator and was operated by a laptop computer. The system was mounted on a cart for greater ease of transport. The entire apparatus could be operated by a single person and transported from one location to the next by a minivan or small truck. The method could selectively remove and determine atrazine, simazine, cyanazine, and other related compounds with a throughput of 10 minutes per injection, or 15 minutes total analysis time. The method was found to yield reproducible behavior with a precision under normal climatic conditions of 1-2 percent. The lower limit of detection for routine measurements was 0.3 ppb for atrazine and related compounds. The linear range extended up to approximately 20 parts per billion (pbb), and the dynamic range extended to 100 ppb. However, these values could be adjusted to higher or lower levels by altering the sample volume that was applied to the system. The results of this technique for atrazine yielded a correlation with a standard method based on gas chromatography and mass spectrometric detection. Studies using this method to examine the distribution of herbicides in an environmental setting are now in progress. Work also has been completed in which this approach has been used to examine the adsorption isotherms of triazine compounds and their major degradation products to various solid materials. These materials might be used to remove such agents for the treatment of drinking water.
A second component of this work investigated the extension of the same method to 2,4-D and other chlorophenoxyacetic acid herbicides. This utilized an anti-2,4-D HPIAC column, which was developed using E2/G2 monoclonal antibodies. The RPLC columns were similar to those used in the triazine studies. In optimizing an HPIAC/RPLC system for these compounds, a systematic study was conducted in which kinetic and retention data were obtained for both the HPIAC and RPLC columns. These results were evaluated with respect to peak shape, analysis recovery, and method selectivity. A first-order dissociation process described the release of captured analytes from the immunoaffinity columns, where the rate of dissociation was dependent on the elution conditions. The passage of analytes from the immunoaffinity support onto the RPLC columns resulted in exponentially-modified Gaussian peaks. The effects of changing the elution buffer composition, flow rate, and type of RPLC column on these peaks were studied. This allowed the recovery and selectivity of the eluted analytes to be optimized. Data from these studies were used to characterize and develop a field-portable immunoaffinity/RPLC system for 2,4-D and related compounds. This approach has been tested in the laboratory, with analysis times similar to those seen for the triazines, and will be used in field studies during the upcoming growing season.
It was concluded from this study that it was feasible to create a field-portable system for herbicide analysis based on HPIAC/RPLC. The anlaysis times were compatible with those required for onsite field studies and the limits of detection were in the range that allowed the direct detection of triazine herbicides in river water and drinking water samples. The studies performed with 2,4-D indicate that the same type of method can be applied to other analytes with the substitution of other types of antibodies within the HPIAC column. Furthermore, a general technique was developed for obtaining information on the key items to consider in the optimization of such columns (i.e., binding and dissociation rates). Further work in this area and in the development of new HPIAC columns should allow the extension of this approach to other analytes of environmental interest.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
| Other project views: | All 15 publications | 3 publications in selected types | All 2 journal articles |
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Nelson MA, Gates A, Dodlinger M, Hage DS. Development of a portable immunoextraction-reversed-phase liquid chromatography system for field studies of herbicide residues. Analytical Chemistry 2004;76(3):805-813. |
R826253 (Final) |
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Nelson MA, Papastavros E, Dodlinger M, Hage DS. Environmental analysis by on-line immunoextraction and reversed-phase liquid chromatography: optimization of the immunoextraction/RPLC interface. Journal of Agricultural and Food Chemistry 2007;55(10):3788-3797. |
R826253 (Final) |
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Supplemental Keywords:
liquid chromatography, immunoassay, triazines, 2,4-D, field-portable analytical methods., Toxics, Air, pesticides, Contaminant Candidate List, Engineering, Chemistry, & Physics, liquid chromatography, field portable systems, solutes, triazine, triazine compounds, Triazines, human exposure, Alachlor ESA, atrazine, multi-analyte systems, chromatographic immunoassays, alachlorProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.