Table IIL Apparent Molar Absorptivities Anionn CN-
S$-
sopCeC1*04*-
e,
525 nmb 115 250c 150d
1 ,m 950
6,
330 nmb 11,500
32,000
16,800 22,800
Solvent was 1 :1 ethanol-water. Values are based on the anion concentration. c Solutions seemed to have a brown tint, possibly due to HgS which was not separated. d Sodium nitrate present at 8 x concentration. 4
b
ride ion with mercuric chloranilate (2, 3 ) . The organic solvent presumably promotes the reaction by lowering the dissociation of mercuric chloride and also lowers the blank by decreasing the solubility of mercuric chloranilate. These effects were not significant in the determination of sullite ion. The magnitude of the blank is an important factor in the choice of solvent or buffer. The blank can be decreased significantly by cleaning up the mercuric chloranilate as has been reported (5). It is likely that the extent of reaction could be improved in some buffers or solvent systems but the blank would probably increase in a similar manner. Sensitivity of the Reactions. Apparent molar absorptivities for cyanide, sulfide, and sulfite in both the UV and visible regions are presented in Table 111, Comparing the molar absorptivity values at 330 nm for these substances with that for chloranilic acid under the same conditions indicates that reaction between mercuric chloranilate and these anions is extensive at the low concentrations. The sulfite reaction is possibly slightly less than complete while the cyanide reaction would appear to be essentially complete, since two cyanide ions are required to release one chloranilate ion. The molar absorptivity for sulfide at the UV peak is considerably higher than would be expected from the stoichiometry of the reaction and the molar absorptivity of the chlor-
anilate ion. The reason for this is not readily apparent. Beer’s law is obeyed by this anion, however. At the higher concentrations of these anions, measuring the absorbance at 525 nm, the extent of reaction for cyanide and sulfide is rather low judging from the molar absorptivity values and comparing these to the value for chloranilic acid under the same conditions. The presence of finely-divided mercuric sulfide is evident in the sulfide reaction. In some instances sodium nitrate was added in an attempt to coagulate the sulfide. The sensitivity for cyanide and sulfide ions is somewhat less than for chloride ion, when these ions are determined using 1 :1 ethanol-water and the chloride determined in methyl cellosolve-water. On the other hand, the sulfite reaction seems to proceed to about the same extent at the higher concentrations as at the lower levels. The apparent molar absorptivity for this anion at 525 nm is very close to the value for chloranilic acid. The wavelength maxima and molar absorptivity values for a solution containing chloranilic acid are strongly pH dependent as it is possible to have the chloranilate ion, the acid chloranilate ion, or the free acid (3). In this work, reproducible results were obtained using an ethanol-water solvent without a buffer. Some work using various buffers showed a high blank while in other instances the extent of reaction with the mercuric chloranilate was less than without the buffer. Possibly some improvement might result if a buffer mixture were added after centrifuging. However, the estimate of extent of reaction of the anions with the mercuric chloranilate based on comparing molar absorptivity values of the solution with those for a chloranilic acid solution should be of some validity even without buffer since solvent and pH conditions are essentially the same.
RECEIVED for review January 25, 1971. Accepted March 19, 1971. The authors would like to express their appreciation to the Robert A. Welch Foundation of Houston, Texas, for support of this research.
Example of Flame Photometric Analysis for Methyl Parathion in Rat Whole Blood and Brain Tissue Joe Gabica, Joe Wyllie, Michael Watson, and W. W. Benson Idaho Community Studies on Pesticides, Idaho Department of Health, Statehouse, Boise, Idaho 83707 MONITORING OF ENVIRONMENTAL pesticide residues has become one of the more important practical applications of gas chromatographic analysis. Typically, gas chromatography is used to detect the presence of an increasingly great variety of chemically diverse pesticides by means of a relatively nonspecific electron capture detector, flame ionization detector, microcoulometer, or similar device. Unfortunately, the current wave of multiplicity in pesticide application presents the analyst with a need for greater specificity in his methodologies. It is possible for several chemically unrelated pesticide residues to be present in a given sample. If some of these compounds happen to behave similarly when subjected to a relatively diffuse analytical procedure such as electron capture detection, their presence could be 1102
ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
easily overlooked or misinterpreted. (For example : the simultaneous presence of p,p’-DDE and ethyl parathion would elicit only a single peak on an OV-1, 3x, gas chromatographic column in conjunction with an electron capture detector.) The recently increased use of organophosphate pesticides requires that a more specific means of detection be employed. As noted by other workers ( I , 2), the extreme toxicity of some of the organophosphates requires that detection methods be highly sensitive as well as highly ( I ) D. L. Pettijean and C . D. Lantz, J . Gas Chroniarogr., 1, 23 (1963).
(2) R. A. Vukovich, A. J . Triolo, and J. M. Coon, J . Agr. Food Chem., 17, 1190 (1969).
Methyl
Parathion
Methyl Parnhion
f
c
Figure 1. Representative chromatograph of electron capture detection of methyl parathion in rat whole blood
Figure 2. Representative chromatograph of flame photometric detection of methyl parathion in rat whole blood Column, SE-303 x
Column, OV-1 3%
specific. This report deals with the suitability of using gas chromatograph equipped with a flame photometric detector and phosphorus filter (thus making the apparatus sensitive only to compounds containing organically bound phosphorus) to specifically confirm the presence of the organophosphate insecticide, methyl parathion, in the blood and brain tissue of previously poisoned rats. EXPERIMENTAL
Procedure. Eighteen Sprague-Dawley adult male albino rats weighing from 250 to 325 grams were injected intraperitoneally with methyl parathion at a dose level of 8 mg/Kg. The injection vehicle was 25 per cent ethyl alcohol, in which the methyl parathion (100 per cent analytical reagent grade, U. S. Environmental Protection Agency Pesticide Research Laboratory, Perrine, Fla.) was mixed at a concentration of 10 mg/ml. A group of six identical rats served as controls, receiving comparable doses of injection vehicle alone. Each animal was sacrificed by decapitation five minutes after being injected. Blood was collected in heparinized tubes and kept frozen at -4 "C until subsequent analysis was performed. The brains were removed, frozen with dry ice, and stored in the same manner, Extraction and Analysis. The whole bloods were extracted for pesticides by the hexane extraction method reported previously by Dale et al. (3). Brains were extracted by a modified Mills method essentially that of Enos et al. (4). Pesticide analysis was by means of a Micro-Tek 220 gas chromatograph equipped with a Melpar flame photometric detector and phosphorus filter (5). The column type was (3) W. E. Dale, A. Curley, and C. Cueto, Life Sci., 5,47 (1966). (4) H. S. Enos, F. J. Baros, D. T. Gardero, and J. P. Wood, U.S.E.P.A. Pesticide Research Lab., P.O. Box 490, Perrine, Fla. 33157, in preparation, 1971, ( 5 ) S. Brody and J. E. Chaney, J . Gas Chromatogr., 4 , 4 2 (1966).
m
Figure 3. Representative chromatograph of electron capture detection of methyl parathion in rat brain tissue Column OV-1 3%
SE-30 3 %, o n Chromosorb XXX, 80-100 mesh, maintained at a temperature of 195 "C. Confirmatory analysis was performed on a n identical instrument, but using a twocolumn system and tritium parallel plate electron capture detectors. Column No. 1 was OV-1, 3%, o n Gaschrom Q, 80-100, while column No. 2 was OV-1, QF-1, on
2z,
ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
3z,
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Table I. Methyl Parathion Levels in Rat Whole Blood and Brain Tissue as Determined by Both Flame Photometric and Electron Capture Detection Whole blood Brain Exposed Methyl parathion, ppb Methyl parathion, ppb Rat number Wt, g Flame photometry Electron capture Flame photometry Electron’ capture 305 3 23 325 309 303 322 276 288 305 294 320 289 253 263 27 1 312 316 286 263-325 298
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Range Mean
23.0 3.0 15.0 69.0 138.0 117.0 92.0 169.0 87.0 86.0 145.0 40.0 87.5 55.0 25.0 17.5 17.5 107.5 3.0-169.0 71.9
46.0 3.7 19.0 32.0 145.0 187.0 106.0 130.0 73.0 67.0 118.0 34.0 57.5 86.5 47.5 20.0 20.0 92.5 3.7-187.0 71.4
405.0 68.0 97.0 1324.0 564.0 1297.0 125.0 388.0 189.0 94.0 686.0 410.0 520.0 448.0 85.0 25.0 82.5 650.0 25.0-1 297.0 414.3
... ... ... ... ... ...
... ... ...
...
... ...
... ... ...
... ... ...
Control 296 319 293 289 279 276
,..
... ...
...
... ... . I .
I . .
... ...
... ... ...
...
...
...
Since both methods of detection produced compatible results in whole blood, brain methyl parathion levels were not confirmed by electron capture. a
Methyl Pararhlon
Gaschrom Q , 80-100 mesh. Both columns were maintained at 195 “ C . ,and nitrogen was the carrier gas used in all cases. RESULTS AND DISCUSSION
A representative chromatograph showing electron capture detection of the extracted whole blood is seen in Figure 1. Although a methyl parathion peak is present, this relatively general means of detection also shows numerous other 1104
ANALYTICAL CHEMISTRY, VOL. 43, NO. 8 , JULY 1971
and flame photometry, respectively. Again, the latter method is more clear cut and free from interfering “trash” peaks. When comparing the electron capture chromatograph of blood analysis with that of brain, it is obvious that the !atter tissue presents a somewhat more ambiguous picture. This plus the difficulties traditionally encountered in the pesticide analysis of brain tissue makes flame photometry especially well suited for this purpose. Table I shows the quantitative results obtained by the two respective methods of detection. Reasonable confirmation
levels among individual animals is most likely due to the creation of considerable variability in dose-absorption rate by the use of intraperitoneal injection. In this study, both methods were found to be capable of detecting methyl parathion levels as low as 3.0 ppb (Table I). Although both flame photometry and electron capture are potentially sensitive to much lower quantities, flame photometry best satisfies the degree of specificity needed for authoritative determination of organophosphates. It
RECEIVED January 12, 1971. Accepted April I , 1971. This research was supported under contract by the Pesticide Cominiinity Studies Divisim, Pesticides ORce, EnviroDmental Pro!cc:icr: Agency, tZl;ough the Idaho St2te daeaarrmerit of Health.
is hoped that this example of one of the many possible applications of flame photometric detection will prove useful to investigators involved with toxicological and forensic problems, as well as t o those concerned with the monito-ing of environmental pesticide residues.
metric Pita
Determination of Perbaomates by J. R. Brand' a n d M. L. Smith Department of Chemistry, Kansas State Teachers Co?lege, Emparia, iYan. 6680;
THERECENT SUCCESSFUL preparation of perbromates ( I , 2) and the resulting interest in perbromate chemistry has created a need for a rapid, convenient, and precise method for perbromate determination. Previous analytical methods have been precise, but slow and tedious. One method ( 2 ) involved the reduction of B r 0 4 - to Br3- in a solution that was 11 M in HBr, followed by addition of KI and titration with standard thiosulfate. In another ( I ) , Br04- was reduced to Br- in 6M HCI solution by SnC12 [catalyzed by Mo(IV)]. Addition of C12 oxidized the Br- to BrCn in neutral cyanide solution. KI was then added followed by titration with standard thiosulfate. Still a third method (3) was suited to the determination of micro amounts of perbromate. I n this procedure, perbromate and crystal violet were extracted into chlorobenzene and a spectrophotometric measurement followed. Perbromate can be determined in the presence of macro amounts of other bromine anions with a precision af 1.5 to 2.0%with the latter method. The analysis developed in this research is based o n a previously reported ( 4 ) procedure in which perchlorate is determined by titration with tetraphenylarsonium chloride. The removal of perchlorate from solution was monitored with a perchlorate ion activity electrode. In this study, the perchlorate ion activity electrode has been shown to respond t o perbromate activity, and is used in the potentiometric titration of perbromate with standard tetraphenylarsonium chloride. EXPERIMENTAL
Reagents and Apparatus. Potassium perbromate was prepared and analyzed as described in a previous communication (5). Tetraphenylarsonium chloride hydrate was purchased from Aldrich Chemical Co., and dissolved in conductance water to make 0.05 molar solutions. Baker and Adamson reagent grade potassium perchlorate and potassium metaperiodate and Fisher ACS certified potassium bromate were all used without further purification. Each of these salts was dried to constant weight at 110 "C and stored over phosphorus pentoxide before use. All solutions were made with conductance water. A Beckman research model p H meter was used for all potentiometric measurements with the Orion Model 92-81 perchlorate ion activity electrode. Class A volumetric glassware was used throughout this study. 1
Author to whom correspondence should be addresed.
(1) E. H. Appelman, J. Amer. Chem. Soc., 90, 1900 (1958). (2) E. H. Appelman, I m r g . Chem., 8, 223 (1969). (3) L. C. Brown and G. E. Boyd, ANAL.CHEW, 42,291 (1970). (4) R. J. Baczuk and R. J. Dubois, ibid., 40, 685 (1968). (5) J. R. Brand and S. A. Bunck, J. Amer. Chem. Soi., 91, O j o c l ( 19 69).
i w
-log
hir
Figure 1. Responsz of the perchlorate electrode to other peshalates; potential in millivolts plotted cs. - log molarity for -- Wr04-, IO4-, and . . . . . c104 Procedure. Perhalate samples of about 0.2 millimole were used with a Class A 10-ml buret. All titrations were carried out in 50-mi beakrrs which contnined a magnetic stirring bar and the perhalate sa.mpie. 'i'he jotentisl developed at the electrodes was iound to decrease slow;y if the solution was not stirred. Rapid stirring caused air bubbles to collect on the membrane of the perchlorate e!ectrode and consequent erratic potential readings. With slow stirring, potentials stable to within 0.2 millivolt were developed in less than two seconds through most of the titration. Stabilization times increased to about one minute in the vicinity of the end point. A standard KC104 solution was used in determining the perbromate selectivity cocfic:ii:?t cif the perchlorate electrode. Three KBrO.$samples W L X 1 oiuwrrically diluted with this solution. Multiple E h l F readings with the electrodes in the KClO, solution and the mixtusrs allowed the selectivity coefficient io be calculated. RESULTS AND DISC LSSION
Electrode Response. The respoii:r of the perchlorate ion activity electrode to activity changrs of other perhalates was determined by measuring potentia's developed in solutions of 7,arious ,:om:eriti-&ti.c,lis af k2CW4> K B P ~ and ~ ~ , KlC4. As the p e r h o b k con;r;utr~tians dzcrtwed from 10-1 to 10-5 mcdar. th:: time rrquii-i:d fnx 'P.: ,:lectrode to attain a stable EMF zricreased f m n R f . m s c c m d s to nearly one minute. Plots of t h r e x p e r i n w t d data are givm in Figure 1. These pioh all lineat in the IO--: i c t IC--; m o h r concentration region, but :.D :xh:+%t some G I !uri. greater diluANALYTIGA? CHEMISTRY, VOL. 43, NO. 8,JULY 1971
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