In the Laboratory
ELISA and GC–MS as Teaching Tools in the Undergraduate Environmental Analytical Chemistry Laboratory
W
Ruth I. Wilson, Dan T. Mathers, and Scott A. Mabury* Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6, Canada; *
[email protected] Greg M. Jorgensen Department of Environmental Toxicology, University of California, Davis, CA 95615
Objectives Our objective in this lab experiment is to expose students to enzyme-linked immunosorbent assay (ELISA) and gas chromatography–mass spectrometry as complementary techniques for the analysis of pollutants in the environment. This experiment provides an excellent way to introduce the specific techniques of ELISA/GC–MS as well as the generic analysis principles of sample extraction and cleanup, detection, and quantification. ELISA has been utilized as a quantitative and a screening technique to determine levels of environmental contaminants in a variety of matrices. The strengths of this method include rapid screening of large numbers of samples at a low cost, minimal sample preparation, low limits of detection, and wide availability of assays for a range of chemicals of environmental interest. The main weaknesses of this method are cross reactivity with related compounds, lack of a separation technique, and interfering matrix components. GC–MS solves some of the weaknesses of the ELISA technique by providing a separation method and confirms the presence of an analyte by providing structural information. Furthermore, GC–MS can detect the presence of other chemicals of interest. Sample preparations required for this technique, time requirements, and cost hinder its applicability as a screening tool, whereas low limits of detection, obtaining structural information, and the lack of matrix interferences make it desirable for accurate results. The students are provided with a river water “sample” that was obtained from an area of high corn production and subsequently spiked with a known amount of atrazine, the atrazine degradation product desethylatrazine, and simazine, another commonly used herbicide.
N
N N
N
H
CH3 CH3
N H
Atrazine Cl
Cl N
N HN H
N
CH3 N
CH3
H
N
N H3C
N
N
H
Desethylatrazine
N H
Simazine
Materials and Methods
River Water Spike Pesticides were obtained from ChemService (West Chester, PA). Three liters of river water was spiked to a concentration of 106 ng/mL atrazine, 152 ng/mL desethylatrazine, and 198 ng/mL simazine. The respective cross-reactivities for the RapidAssay (Strategic Diagnostics) antibodies are reportedly 22% for desethylatrazine and 15% for simazine. SPE Extraction and GC–MS Analysis Two C18 SPE cartridges (500 mg; Supelco) were prepared by passing two-column volumes of ethyl acetate, methanol, and water before loading of the aqueous river water sample (100 mL). Elution was with 2 mL of ethyl acetate (50:1 sample concentration), which was subsequently passed through a Pasteur pipet loaded with sodium sulfate to remove residual water. The spiked sample was analyzed by GC–MS on a PE Autosystem XL with a PE TurboMass; the column was a DB 17, 30 m × 0.25 mm and 0.25 µm film thickness. Ions monitored were atrazine 58, 215; desethylatrazine 172, 187; simazine 158, 186. Quantitation was by external standardization.
ELISA Analysis The RaPID Assay for Atrazine was obtained from Strategic Diagnostics (Newark, DE) and the RPA-III Field System was used for measuring the absorbance of the final colored product (450 nm). These antibodies are incorporated with a magnetic particle that allows easy manipulation for stepwise reaction and wash steps to be performed. The river water was diluted 1 mL to 100 mL with Milli-Q water and the analysis was done in triplicate. QC samples and standards were prepared according to methodology supplied with the antibodies, with calibration by external standardization.
Cl
H3CH2C
The ratio of degradation products to atrazine is adjusted to result in a significant cross-reactivity toward the antibody, yielding an artificially high value for the ELISA determination. The actual concentration of atrazine and each of the degradation products or related triazines was determined by GC– MS after SPE extraction and concentration.
CH3
Hazards There are no significant hazards involved in carrying out this experiment.
JChemEd.chem.wisc.edu • Vol. 77 No. 12 December 2000 • Journal of Chemical Education
1619
In the Laboratory
Results and Discussion The utility of this experiment as a teaching tool lies in the lessons learned from comparing two methods in their ability to identify and quantify atrazine in water. Students had the opportunity to experience firsthand the strengths and weaknesses of each method as well as to learn the fundamentals of operation for ELISA and GC–MS, basic MS fragmentation theory, isotopic contributions to mass spectra, the nitrogen rule, use of antibodies as analytical tools, environmental degradation processes and pathways, and the occurrence of toxic chemicals in natural waters. ELISA results for the concentration of atrazine in the water sample are given in Table 1. The QC value obtained was 3.3 ± 0.38 ng/mL (N = 11). The atrazine value obtained by ELISA is within the range of the theoretical value for the concentration of atrazine and the cross-reacting desethyl and simazine triazines; we do not rule out that there were other cross-reactive substances in the water, although no atrazine was found in the water prior to spiking. These results illustrate both the strengths of ELISA assays as rapid, easy to do with simple equipment, and sensitive, and the weakness of crossreactivity with other compounds of similar structure. The cross-reactivity exhibited by ELISA can potentially be viewed as a strength if the objective is to have a quick evaluation of triazine contamination. Results obtained for atrazine, desethylatrazine and simazine by GC–MS SIM are also shown in Table 1. Students were able to directly observe the enhanced quality of information obtained from the GC–MS in terms of analyte identification, but at the cost of a significant amount of effort for extracting and concentrating the sample. The differential sensitivities of the two methods were exemplified by the 1:100 dilution required for ELISA and the 50:1 concentration required for the GC–MS. This experiment clearly showed the utility of ELISA for rapid analysis, or for surveying a large number of samples in order to maximize the utility of the added effort and value of performing GC–MS on a particular sample.
!
Table 1. Concentrations of Triazines Found in Water Sample Triazine
True Value
Method ELISAa
GC–MS SIMb
Concentration/(ng mL᎑1) Atrazine
106.0
126 ± 43.6
Desethylatrazine
152.0
126 —
122.9 ± 18.7
97.9 ± 17.1
Simazine
198.0
126 —
175.8 ± 51.9
± RSD; N = 11. b Value ± RSD; N = 10. a Value
Useful References Thurman, E. M.; Goolsby, D. A.; Meyer, M. T.; Mills, M. S.; Pomes, M. L.; Kolpin, D. W. Environ. Sci. Technol. 1992, 26, 2440. Gruessner, B.; Shambaugh, N.C.; M. C. Watzin. Environ. Sci. Technol. 1995, 29, 251. Meulenberg, E. P.; Mulder, W. H.; Stoks, P. G. Environ. Sci. Technol. 1995, 29, 553. Kelter, P. B.; Grundman, J.; Hage, D. S.; Carr, J. D.; Mauricio, C. J. Chem. Educ. 1997, 74, 1413. Anderson, G. L.; NcNellis, L. A. J. Chem. Educ. 1998, 75, 1275.
Acknowledgments This experiment was developed and carried out in the ANALEST facility kindly supported by Perkin Elmer Canada, Analytical Instruments Division. The diligent efforts of Bruce Hammock and Adam Harris at UC-Davis and a number of teaching assistants at U of T and UC-Davis are gratefully acknowledged. W
Supplemental Material
Student and teacher instructions are available in this issue of JCE Online.
CAUTION
Experiments, laboratory exercises, lecture demonstrations, and other descriptions of the use of chemicals, apparatus, instruments, computers, and computer interfaces are presented in the Journal of Chemical Education as illustrative of new or improved ideas of concepts in chemistry instruction and are directed at qualified teachers. Although every effort is made to assure and encourage safe practices and safe use of chemicals, the Journal of Chemical Education cannot assume responsibility for uses made of its published materials. Many chemicals are hazardous. Precautions for the safe use of hazardous chemicals and directions for their proper disposal are described in the Material Safety Data Sheets and on the labels. We strongly urge all those planning to use materials from our pages to make choices and to develop procedures for laboratory and classroom safety in accordance with local needs and situations.
1620
Journal of Chemical Education • Vol. 77 No. 12 December 2000 • JChemEd.chem.wisc.edu
