Gas Chromatographic Determination of Environmentally Significant Pesticides Walter E. Rudzinski and S t e v e Beu Southwest Texas State University, San Marcos, TX 78666
In the instrumental analysis laboratory students are taught to use a gas chromatograph analyzing "samples" with little regard for the extensive sample preparation usually required prior to the injection of a sample. The student can obtain a false impression as far as the ease of analysis is concerned. In order to exemplify a common separation, identification, and quantitation scheme employed in the analysis of trace organic contaminants such as organochlorine pesticides, PCB's, nitrosamines and phthalate esters ( I ), we have chosen a procedure which can he adapted for the analysis of organophosphorus pesticides in orange juice (2). T h e following procedure may detect trace levels of pesticides in the environment if all the necessary precautions are taken to ensure the purity of all solvents and glassware (1,2, 3, 4); however, such scrupulous attention to detail is timeconsumine and difficult to achieve within the constraints of a n undergraduate laboratory. The following procedure has been modified so that the level of pesticide present in the sample is high and therefore will preclude having to worry about interferences and samule detectahilitv. The scheme has pedagogical value and shouli provide the uhdergraduate with some initial exverience in trace analvsis. T h e majorit" of the methods prescribed by the Environmental Protection Agency require that the indi\,idual verforming the analysisbe experienced or under the clos; supervision of such a qualified person. This procedure provides the undergraduate with some of the required experience. Summary of the Method A thoroughly mixed sample of orange juice is extracted with acetonitrile. An aliquot of the acetonitrile-water sample is extracted into pet ether. The pesticide residues are purified by chromatography on a Florisil column, eluting with a mixture of petroleum and ethyl ethers. The residues in the concentrated eluates are then measured and identified by gas chromatography. instrumentation The instrument used was a Tracor 5 6 P gas chromatograph equipped with flame ionization and electron capture detectors. T h e column used was glass, 1.8 m X 4 mm i. d. packed with 1.5% SP-225011.95% SP-2401 on 1001120 Supelcoportl. Other columns can he used for the analysis (3).The column should he conditioned overnight a t 245'C with a nitrogen flow of 30 mL/min. The recommended operating conditions when using the electron capture detector are injection port temperature 225"C, column temperature 215'C, detector temperature 350°C, carrier gas flow rate: 30 mL of Nalmin, and purge gas flow rate: 50 mL of Nzlmin. The recommended operating conditions when using the flame ionization detector are injection port temperature 2 2 5 T , column temperature 215OC, carrier gas flow rate: 30 mL of Nalmin (60 mL of Helmin), hydrogen gas flow rate: 30 mL/min, and air flow rate: 350 mllmin. A Houston 0mniscribe"recorder was used to obtain all the gas chromatographs.
614
Journal of Chemical Education
Experimental Add 50.0 g of orange juice spiked with pesticide2to 100mL ofaceFilter the tonitrile and mix thoroughly. Record the total volume (T). solution, using a Buchner funnel, through a layer of Celite (diatomaceousearth) coated on Whatman No. l filter paper. Collect the filtrateand record the volume ( F ) .Transfer the measured filtrate to a 500-mL semratorv funnel. Add 50.0 mL of oet ether and shake f& a few minutes. Add 5 m~ ofa sat;ratpd NaCl solution vieorouslv .. ~~~~. and lot1 mL dH,O and mu vigcrousls for nbour a minute. Thr Nxc'i solution is added in order to prevent the furmntim 01 an rmu.Gm. Let the 2 layers separate and discard the aqueous layer. Repeat 2 ~
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'1 ranskr the pet ethcr layer to a 100-ml.graduared cylinder and rend t h p volume I/',. Trnnsfertht pet ethcr too 11.0-mI.srpar3lorv funnel and add about 10 e ofanhvrlmu\ Sa&O1. . . Shake w ~ ~ ~ n ~ u s i v After letting the per ethcrdry for'l5 min,grau~tyfilter t h c ' ~ o l u r ~ o ~ , and collm the pet ether i n n 25 X 2(10 turn rest ru1,e. ('wcenrrarr the solution w i p l u m L 'This can be dme I,). hexing thp pet ether extrnct at 30DCover a water bath. Prepare a 22 mm id. Florisil column (chromatographictuhe with Teflonm stopcocks and coarse fritted plate or glass wool plug; 22 mm i.d. X 300 mm), containing 3 in. of packing (aftersettling)of activated FlorisiP topped with about % in. of anhydrous NazS04.Use about 40-50 mL of pet ether in order to initially wet the column. Do not let the column get dry. Prepare a set of eluting solvents by mixing ethyl e t h d with redistilled pet ether in the followingvolume/volume ratios: 6%eluting solvent (12 mL of ethyl ether: 188 mL of pet ether), 15%eluting solvent (30mL of ethyl ether: 170 mLofpet ether),5090elutingsolvent (100 mL of ethyl ether: 100 mL of pet ether). Transfer the pet ether concentrate to the column and rinse the sample tuhe with two 5-mL portions of pet ether. Elute the sample with the 6%elutingsolventand adjust the flowrate toapproximately 5 mLImin. Collect the eluent in a 250-mL Erlenmeyer flask. Label as 6%sample eluent. Next, rinse the column with the 15%eluting solvent and collect the eluent. Finally rinse the column with the 50%eluting -
' The packed column can be purchased from Supelco, inc. Supelco
Park, Beliefonte, PA 16823. The pesticides employed In this study were obtained from the manufacturers. Two grams of pure malathion were obtained from American Cyanarnid Co.. Agricultwai Division, P.O. Box 400, Princeton. NJ 08540: 1 a of ethion was obtained from FMC Corooration. Aariculof turai Chemical Division. 100 Niaaara St.. l e o.o r t .14105:i ' ~ ~~~. o-. - . ~ i d d~,~ oiazmon was oota ned born C ba-Geigy. Agricu t~raiDivision. PO. Box 11422, Greensooro. NC 27409 The OJality Asswance Section. Analytical Chernlslry Branch. ETOIdER- (MD-691. J.S. Environmental Protection Agency, Research Triangle Park,'NC 27711 wiil also supply 100 mg of the standard pesticides. 1 WLof pure pesticide added to the orangejuice wiil provide more than adequate response when using an electron-capturedetector, or a flame ionization detector. Florisil can be activated by heating overnight in a muffle furnace at 650°C. then heating at 13O0C for approximately 16 hr prior to
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Theelhyl ether must be Wee of peroxidesas'ndicated oy EM Oant rest strips (Test strips are available from EM Laooratorfesinc 500 Execut've Blvd.. Elmsford.NY 105231. Procedures recommended for removal of peroxides are provided with the test strips. After cleanup. 20 mL ethyl alcohol preservative must be added to each liter of ether.
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ta '1.60 mi". lr Diazimn
1~-14.93 min.
A
31"
L
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t ~ 748min. = la = 316 min.
2
4
15
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RETENTION TlME (MINI
Figlne 1. Gas Chromatogam (ECD)of a sample containing a mixlure of Dizimn. Malathion, and Ethion. Column: 1.8 m X 4 mm i. d. glass packed wilh 1.5% SP-225011.95% SP-2401 an 100H20 Supelcaport. Column temperature: 215'C: detector temperature: 350%. Carrier gas flow rate: 30 mL of N21min.; purge gas flaw rate: 50 mL of Ndmin. 1.0 fiL of pesticide added to 125 mL of orange juice; sample volume (v) - 0.005L; 1 fiL of sample injected into the gas chromatograph. Anenuation of Tracor 56W gas chromatograph = 200.
solvent. Cover the sample flasks with aluminum fail and refrigerate until readv to concentrate. .\diust the wiumedthr ~ulutimin thr 3 Erlenmeyer flaiksiurhar each llask rontntns approximately 3 m L i,lsdwnt.: Record the vulume (u). Inject a suitable aliquot (14fiL) of the concentrated 6%eluate into the gas chromatograph. In order todetermine "a suitable aliquot"vary the injection volume until the recorder responds, giving an approximate 50%of full scale deflection. Record the svrinee Reoeat , . volume. ~ the prwe~turefur all uf the lample; and for the pesticide standard mmturp.' For nll quantitative rnpasurpmmts thr detector mu.1 ire operated within its linrsr rang?, and thr detrcrur noise level i h d d be less than 2% of full scale
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Results and Discussion T h e results of the gas chromatographic analyses are depicted in the two figures. The pesticides in the eluate samples have been identified positively by comparing the chromatographic retention volume of the pesticide in the eluate with that obtained from the chromatogram of a known pesticide standard solution. Calculations 1) Calculate the calibration factor (CF) for each pesticide
where C represents the concentration of the corresponding uesticide in rcdL and A reuresents the area under the chromatographic peak. 2) Calculate the concentration of the pesticide in the sample
If the instructor wishes to reduce the size of the solvent peak when usina a flameionization detectw. it is suaaested that the eluent collected afte;chromatoaraohv on a ~lorisilcolumn be reduced to drvness , ,~ . and the sample rea~ssolvedin 5 mL of pestcide grade acetone. The sens t i v q of the detector in responding lo acetone is low, and therefore diazinon wi I not be as difficult 10 quant late. Tne pesticde standard mixture should also be prepared in acetone. The pesticide standard mixture can be prepared by weighing approximately 20 mg of pesticide and dissolving it in 100 mL of lsooctane.
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Lyt 4 6 RETENTION TlME (MINI
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Figure 2. Gas chromatogram (FID) of a sample containing a mixture of Diazinon (approximately 170 ppm). Malathion (approximately 170 ppm), and Elhion (aporoximatelv. 680 .oomb . . Column: 1.8 m X 4 mm i. d. alass oacked with 1.5% SP-22501195% SP-2401 on 1001120 Supelcopon Ca m n temwralae 2 15'C. Caner gas I ow rate. 60 mL of Helrnm: hyoroqen gas llov rate. 30 m-lm n, a r flow rate: 350 mL m8n The sample mxt.re contams 0 9 #L of D aman, 0.9 fiL of Malahion, and 3.6 pL of sample injected intothe gas chromatograph. input Range of the FID detector = 10; output altenualor = 1 (nominal input current = 5 X lo-" Amperes for full scale recorder output). syringe volume standard syringe volume sample 3) Calculate the micrograms of pesticide (MG) in the concentrated sample. Csmp~e= A,I.
X
CF X
where V is the volume of the concentrated eluent expressed in units of liters. 4) Calculate the micrograms of pesticide (Wpesticide)in the original sample taking into account losses while purifying and concentrating the sample.
where P, T, and F are experimentally determined volumes as described in the experimental section. 5) Calculate the concentration of pesticide in the original sample. C+.id.
=
Wpesticide total volume of orange juice
Acknowledgment I would like to thank the Welch Foundation for partial support of this work, Welch Grant No. A1 809. In addition, I would like to thank Drs. Fitch and Perry for their assistance in reviewing the manuscript. Literature Cited
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(11 "Guidelines Estsblishing Test Proredures for the Analyais of Pollutants; Proposal Roguiations."Fed. Reg., 44, No. 233, Oec. 8, 1979, p. 69501. (21 "Oflicial Methods of Analysis."11th Ed., Association of Official Analltics1 Chemists, Washingtun. D.C.. 1970,p.475. (3) "Analysis of Pesticide Residues in Human and Environmental Samples: U S Envimnmentai Pmtxtion Agency. HERLmTO R e m h Triangle Park, N.C. 27711, June 1977, Sections 3.4.8, and LO. (4) ''Method for Organophosphorus Penticides in Industrial Effluents: National Pollutant Dirrhaxe Elimination Swtem,Appendix A Fed. Reg, 38. No. 75, Nov. 28,1978.
Volume 59
Number 7
July 1982
615
