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Detection and Determination of 1,4,5,6,7,8,8=Heptachloro3a,4,7,7a=tetrahydro=4,7=methanoindene PERCY B. POLEN AND PAUL SILVERMAN’ Research Department, Velsicol Corp., Chicago 11, I l l .
The need for a sensitive and selective means for evaluating trace residues of the insecticide 1,4,5,6,i,8,8 heptachlor0 -3a,4,i,7amtetrahydro-4,7-methanoindene (heptachlor) on food crops prompted this investigation. Heptachlor in benzene or hexane solutions reacts with a reagent composed of 0.5 M ethanolamine and 0.5 i M potassium hydroxide in butyl Cellosolve to produce a pink to violet color. By photometric measurement of this color at or near 564 mp, heptachlor can be determined within a mean
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HI< need foi adequate methods of detecting traces of com-
niercial insecticides is well recognized. -4 colorimetric method has been developed to meet this need for the insectiride 1,1,.5,R,i,8,8-heptachloro-3a,4,i,Ta-tetrah\ tll0-1,7heptachlor, niethanoindenv (I). CL
I I:ucellent voluiiirtric (3) and gravimetric ( 1 ) niethotls air known for estimating heptachlor through its reaction with silver ealts t o form silver chloride. These methods depend upon the high reactivity of the allylic chlorine atom attached t o the fivemembered ring. Whereas the procedures give highly accurate results, they lack the specificitv required for wide applications in the field. The method, which is the subject of this paper, is highly sprcific and selective for heptachlor.
relatibe error of +1.5 to 2.Oqo. Reproducibility is dependent upon stabilizing the reagent which exhibits an aging effect. .4s little as 3 micrograms of heptachlor can be detected visually, and by special techniques 3 micrograms can be estimated satisfactorily. Other common insecticides, with the exception of technical chlordan, do not interfere. This method can be successfully applied to the estimation of heptachlor iesidues on food crops to which this compound has been applied as an insecticide.
the mouth of the tube during t,he first 3 minutes of heating contribute t,o the snioot,h evaporation of t.he volatile solvent and consequently to the reproducibility of the determination.) At this point the color is fully developed. To detect heptachlor it is merely necessary now to observe whether the pink to violet color has formed. If necessary, the color of this mixture may be compared x i t h a blank similarly treated. By using sniall volumes in the color-forming reaction, as little as 5 micrograms of heptachlor can be visually detected. To make quantitative estimation, the colored solution is prepaxed for colorimetric or spectrophotometric examination hy dilution t o an accurately known volume with 95570 ethyl alcohol. The dilution may be made to 5, 10, or 25 ml., depending upon the intensity of the color developed and the instrument with which the examination is to be made. The main consideration here is to ohtain solutions whose transniittancies are between ca. 25 and i 5 % , The transmitt,ancy or absorbancy may be determined against 95% ethyl alcohol as a blank. I n order to minimize the effect of fading, which had been observed at higher concentrations, photometric r’eadings should he made a t a standardized time int,erval follo~~-ing the removal of the mixture from the heat-
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JJ-hen solutions of heptachlor are treated with a special reagent, a color visually similar t o t h a t of aqueous potassium permanganate is developed. I n concentrated solutions this color is deep violet, while in dilute solutions it is pink. Alcoholic solutions of the colored material exhibit a strong absorption peak a t 564 m p (Figure 3). Preferably, the heptachlor is treated in the form of its solution in benzene or hexane.
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- DAYS Figure 1. Effect of Reagent .4ge upon Absorbancy TtME
The reagent is a solution of ethanolamine (Carbide and Cai hon Chemicals Co., Kew York, S . Y.) and potassium hydroxidr, each 0.5 molar, in conimercial butyl Cellosolve (Carbide and Carbon Chemicals Co.). For convenience in preparing the reagent, the potassium hydroxide may be introduced as a 50% aqueous solution. Briefly, the test is carried out in the following manner: Equal volumes, usually 1 ml., of the heptachlor test solution and the reagent are mixed in a test tube. The tube is heated for 15 minutes in a vigorously boiling water bath, and is then cooled rapidly to room temperature by immersion in cold water. (The addition of a silicon carbide chip and the directing of a stream of dry air over 1
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ing bath; a 20-minute interval has been found convenient and satisfactory. The concentration is then estimated from a standard calibration curve. (Analytical grade heptachlor for standards is available from the Velsicol Corp., 330 East Grand Ave., Chicago 11, Ill.) Concentration may also be estimated by applying the method of internal compensation, or the “increment procedure” ( 2 ) , wherein calculations are based upon results obtained by addition of known amounts of heptachlor t o the solution being assayed. To attain the best precision and accuracy, the test should be
Present address, Helene Curtis Industries, Inc., Chicago, Ill.
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ANALYTICAL CHEMISTRY
734 carried out under controlled conditions in accordance with the considerations set forth in the following paragraphs. EFFECT OF HISTORY O F REAGENT
It was observed that as the age of the reagent increases, thc intensity of the color developed from it also increases. This effect is sh0n.n in Figure 1.
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Figure 2.
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obvious that significant heptachlor losses occur. This does not mean that hexane may not, be used as a n extracting solvent, but it emphasizcs the necessity of correcting for the losses resulting from the concentration of fairly large volume^ of solution. Evaporation of benzene solutions of heptachlor also results in similar losses. I t may be seen from the upper curve, however, t h a t recover!' is virtually quantitative when pentane (Skellvsolve a) solutions are treated in a similar nianiicr. It has been found practical, where the method has been applied to natural products, t o extract with pentane, concentrate thcextract, perform chromatography, and finally evaporate all the pent,ane without significant loss of heptachlor. T h e residue niay then be taken u p in the required amount of bcneene for performing t,he color reaction. This procedure minimizes the uncertainty of incurring evaporation losses and brings the heptachlor t o t'he color reaction in that solvent which appears to yield colors of greatest absorbancy. T h e sanie solvent used for the color reaction must be used for preparing the standard reference solutions from which the calibration curves are to bv made.
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Recovery of Heptachlor upon Evaporation of Solutions
T h e solid line represents the absorbancies (or optical densities) obtained from a single Concentration of heptachlor with a continually aging reagent. T h e intensity of the color produced increases until the reagent is about one month old: after this, the intensity remains constant. For maximum sensitivity and rrproducibility, t,herefore, the reagent should be aged in a closed glass vessel for a t least one month. Calibration curves made before the reagent has attained its maximum scnsitivity will not be consistent from day to day, and daily calibrations will therefore he required. It is not always convenient, however, to make a reagent one month in advance of its contemplated use. This difficulty niay be partially overcome by heating freshly prepared reagent for 5 hours a t 50" C. in a closed g!ms system. -411accelerated aging effect results, as shown by the dashed linr in Figure 1. The illustrat,ion shows the effect of treaatment applied at about 5 (lays, but a similar effrct result,s from treatment of the fresh reagent. The increased senpitivity which results remains constant for about. 7 t o 10 days, hut aftw that period of s t a l d i t y the spontaneous aging trend seems to resume. T h e expedient of heat treatment makes possible the use of a reagent for reproducible results shortly after its preparation. LYhere a reagent of greatest effectiveness is required, t'he spontaneously aged one is preferred. Calibrations may var!from batch t o batch of reagent similarly prepared and treated. The information reported below is based upon the use of a Einyle stock of spontaneously aged reagent. HEPTACHLOR TEST SOLUTIONS
Solutions in a volatile solvent such as benzene or hexane (Skellysolve B) are most suitable for analysis. LVhen solvents whose boiling points are over 100" C. are used, they are not volatilized during the test and as a result the color develops extremely slowly or not, at all. Benzene solutions give the greatest intensity of color aniong a number of common volatile solvents t,ried; hexane solutions are second in this respect. I n the application of the test to problems involving extraction, chromatography, and concentration, precautions are necessary in the use of benzene or hexane as solvents. Figure 2 shows what si likel$ t o occur when comparatively large volumes of hexane extracts, for esample, are concentrated. I n this figure, the recovery of heptachlor is plotted against the initial volumes of solutions-tnat were evaporated to ca. 10 ml. on a steam bath. It is
The, concentration of heptachlor in the test solution must also be considered. I n Figure 3 are plots of the absorption spectra obtained from various concentrations of heptachlor. T h e number associated with each curve represents the concentration in milligrams per milliliter of heptachlor from which the color was developed. A tenfold dilution of the original heptachlor concentration \vas made with 95% ethyl alcohol for the final absorbancy ieadings The strong absorption peak at 564 mp niay br noted.
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Figure 3.
Absorption Spectra of Solutions from Color Test
I n Figure 3 absorbancies have been plotted on a logarithmic scale. Had a single absorbing material been formed throughout the range of concentrat,ions, thew curves should all be of the same shape (4). This condition is not completey fulfilled. The minor absorption peak a t 485 to 490 nip otiet,rved a t higher concentrations disappears in the lower curves and only a small hump remains in its place. The difference in the elevation between the peaks and the troughs in each curve of this famil!- ma:- also be noted. From these considerations, it is concluded that more than one light-absorbing species is formed. This emphasizes the need for carrying out t,he procedures in a standardized manner with respect to concentrations, heating time, etc., in order t o assure consistency of results. K i t h the concentration chosen lor this illustration, t,he curves should be evenly spaced if a straight-line relationship between concentration and absorbancies exists. This is not the case for the whole range. The peak a t 564 mp was chosen'as a good point a t which t o
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make colorimetric observations, as it appears t o be generally least susceptible t o the concentration effects. CALIBRATION CURVES
Figure 4 is a plot of absorbancies zs. heptachlor concentrations in benzene test solutions. This is a calibration curve for a specific. set of operating conditions. The solid curved line represent. actual data; the dashed line is, loosely speaking, a Beer’s law plot. For the broad range illustiated, there is agreement between thi. two with a mean error of i5c7,, whereas in the region betwerii 0.15 and 0.45 mg per ml. a mean error of about 1.5% is observed T h e requirements of the particular application t o which the t w t is placed and the characteristics of t h e particular instrument in use would largely dictate whether the assumption of a straightline relationship iq juqtified.
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and are especially suited to the needs of the heptachlor color reaction. T h e Bausch and Lomb instrument when used with the nominal 560 m r filter for heptachlor evaluation exhibited about i 5 % of the sensitivity that was observed with t h e Beckman instrument. Heptachlor analyses have also been satisfatorily carried out using t h e standard broad band filters but a t the espense of sensitivity. INTERFERENCES
The color reaction has been applied t o mixtures of heptachlor containing other commercial insecticides such a s DDT, toxaphene parathion, aldrin, and benzene hexachloride. None of these was found to interfere when readings were made a t or near 564 mp. The t,est cannot be used to differentiate between heptachlor and technical chlordan, however, because the latter contains materials which give similar color reactions. Parathion forms a lemon-yellow color with the reagent, but this does not int,erfere when the readings are taken a t the suggested wave length. Visual masking may occur, but only when parathion is present in great excess over heptachlor in a mixture of the two. A PPLIC.4TION S
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T y p i c a l Calibration Curve
An important differentiation regarding two aspects of coneentration-color linearity must he made. Over the twentyfold range depicted on t,he graphj absorhancs!. does not bear a stricti!. straight-line relationship to the original heptachlor concentration. but the colored material once fornied will, upon successive dilutions, exhibit absorbancies in conformance x i t h Beer’s law. For example, a t a given dilution, the absorbancy developed for 1.0 mg. per ml. of heptachlor will not be exactly twice as great as that from 0.5 mg. per ml. On the other hand, a twofold dilution of thti concentrated colored solution nil1 result in an absorbancy csactl!one half its original value. Should t h e test’, therefore, be carried out on a n unknown which gives color5 too deep to permit malting colorimetric readings, the colored solution may be diluted until suitable absorbancies are obtained. The data thus collected will indicate roughly the degree to which the original test solution must be diluted to bring i t into the optinium concentrat,ion range (0.15 t o 0.45 nig. per nil.). It is desirable wherever possible t,o tlivide a sample into two portions and t o make preliminary tests to determine whrther further dilution or concentration of the teat I more accurate analysis may then be solution may be required. : obtained. Such analyses carried out upon synthetic mixtures have given hept,schlor values with a mean error of 1 1 . 5 t o 2 . 0 7 the amount present. INSTKU~IEUTS
T h e quantitat,iveestimation of heptachlor has beeil satislactorily carried out, not only on the Becknian Model DU rpectrophotometer, but also on the Bausch and Lonib lIonochromatic colorimeter and the Photovolt Lumetron colorimet,er. The Bausch and Lonib instrument is especially well suited t o routine application, affording high sensitivity and specificity because of its design for use with interferencetype filters. These filters are preferred because of their relatively narrow transmittance bands and because they can be obtained with these bands a t any selected wave length of light. Special filters transmitting a t 560 mp are now available
Prc~liiuinarywork has been done on the application of thii. color reaction to the est,imation of hepbachlor in milk and in various ratv and canned fruits and vegetables ( 3 ) . Further work is in progress to extend the scope of these applications. Certain substance? which are carried in t,he estract,s cause complications if not reniovcd before t,he color react,ion, but, they are successfully separated from the heptachlor by simple chromat,ography in short columns with alumina (adsorption grade, 80- to 200-mesh, Fischrr Scientific Co., St. Louis, 1 2 0 . ) or Florex (XXS grade, Floridin c‘o., JYarren, P a . ) as adsorbents. T h e results thus far itre encouraging. The method is being modified to measure quantitatively smaller quantities of heptachlor. Uncompleted work has indicated that through the use of small volumes of solutions i n cievrloping the color, satisfactor)- measurements can he made in the rangr of 3 micrograms of heptachlor ( S ) . SUMMARY
I h t r(wdt8 are obtained when the color reaction is carritd out with rt.agent that has h e n aged for a t least one month or is given an accelerated aginp’through heat treatment. Thp heptachlor t w t solution should be in a concentration range of about 0.05 to 1 .O mg. per nil., anti prrLfei,ably,0.15 t o 0.45 mg. per nil. in a volatile solvent,. In carrying out, the, color-forming procedure a heating time of 15 minutes in boiling water is suggested in order to obtain maximum color formation. Colorimetric readings should be made a t some standardized time interval after the heat,ing procedure is completed, so that fading errors are minimized. Photometric observations should be made on an alcoholic solution of thv colored body a t or m a r 361 m p . ACKNOWLEDGMENT
T h e authors express appreciation to Iforton Kleiman for his interest and helpful discussions during the course of this work. LITERATURE CITED
(1) Kleiman, hT., Coldman, A , , and Paluch, C., unpublished work. ( 2 ) Moss, AI. L., “Analytical -4bsorption Spectroscopy,” edited hy hI. G. Mellon, pp. 17-19, Kew York, John Wiley & Sons. 1950.
(3) Ordas, El. P., and Smith, Victor C., unpublished work. (4) Stearns, E. I., “.lnalytical Absorption Spectroscopy,” edited by RI. G. Mellon, pp. 307-10, New York, John Wiley & Sons, 1950. RECEIVED for review April 22, 1951. Accepted J a n u a r y 7, 1952. Presented before t h e Division of Agricultiiral and Food Chemistry, Pesticide Subdivision, Symposium on Methods of Analysis for Micro Quantities of Pesticiden. at t h e 119th Meeting of the AMERICAS CHEVICAL SOCIETY, Boston, Mass.
