V O L U M E 25, NO. 7, J U L Y 1 9 5 3 TR.iTE. Transfer jo ml. of distillate into a 5o-nil. xessler tube (sample tube) and add 1 nil. of sodium alizarinsulfonat,e. Adjust acidity with 0.05 S hydrochloric acid until the equivalent of 2 ml. is present-that is, 2 ml. minus milliliters of sodium hydroxide required under Section 1. Add thorium nitrate solution (0.26 gram of tetrahydrate per liter) from a microburet until a faint pink color appears. When 50 ml. of distillate require from 2 t o 5 nil. of sodium hydroxide for neutralization, omit the hydrochloric acid addition to samplc distillate, hut add t o the standard tube the same quantity of acid as was found in the sample. 3. TITRATIOS OF FLUORIDE STANDARD.Pour 50 nil. of water into a 60-ml. Sessler tube (st,andard tube), add 1 ml. of sodium itlizarineulfonate, add 2 ml. of 0.05 .V hydrochloric acid to match the sample (see Section 2 ) , and add standard sodium fluoride solution equivalent t o about 80% of the fluoride present in the sample aliquot as indicated by the thorium nitrate required under Section 2. Mix thoroughly, add the same volume of thorium nitrate required for titration of the sample under Section 2, and again mix thoroughly. The standard t,ube will be more highly colored than the sample tube. From a microburet add sodium fluoride to the standard until the color matches that of the sample. Equalize the volumes in the two tubes and mix each solution thoroughly by inverting, allowing all air bubbles to escape before making color comparisons. Check thcx end point by adding 1 or 2 drops of standard sodium fluoride t o the standard tube. If the colors were matched, a d i d n c t failing of the color in the standard tube should occur.
1065
4. B L ~ \ ED I E T E R XITIOX. II~ Deduct :I blank obtained b v carrying the qame amounts of all reagents used through the entire process of afihing, fusion, distillation, and titration. With proper attention t o detail., total blanks of the order of 5 mic~ograms are obtainable. CALCULiTION. P.P.M. of F (dry baiis) = ml. of didillate net ml. of std. S a F X mp. of F / m I X ml,of aliouot X 1000 g. of total ash - % moisture) g. of ash fused g. of m n p l e X 100
(
LITERATURE CITED
of Official Agricultural Chemists, “ h I e t h o d s of .Inalysis,” p. 389-96. 1950. 12) Churchill. H. V..ISD.ENG.CHEJI...INAL. ED..17. 720 (1945). ( 3 ) Remmert, L. F.,’P;t~ks. T. D., Lawrence. 1.LI., and ;\lcBurney, E. H.. . ~ N . L I . . ( ’ H E M , . 25, 450 11953). (4) Willard, H. H., and \T-inter, 0. B., ISD. ESG. C‘HEX., SAL. En., 5, 7 (1933). (1) Association
RECEIVEDfor reviex Janiiary 1 7 . 1953. Accepted A p r i l 11, 1953. P r c sented before the Pittsburgh ronfrrence on Analytical C‘heiiiiatry and A P plied Spwtro.scopy. Pitt?hiirgli, Pa., March 2 to 6, 1933.
Determination of DDT and Related Substances in Human Fat 4RNOLD 31. MATTSON, J4SET T. SPILLiNE, CURTIS B 4 K E K , A Y D GEOKGE W. PEARCE Coiiiniccnicable Diseuse Center, Public Health Serrire, K‘.S. Department of Heulth, Education, and F’elfare, Box 769, Savannah, Ga. Preliminary e\idence of the occurrence of DDE as well as DOT i n human fat made it necessary to stud) the Schechter-Haller method critically as applied to fat containing DDT and the degradation product, DDE. Through empirical standardization it was possible to make differential determinations of DDT and DDE totaling 5 micrograms in unknown fat samples, with rough estimations down to 2 micrograms. 4 modified Davidow column was used in a rapid method €or isolating DDT and DDE. Among 50 samples all but 2 contained substantial qiiantities of DDE. The total DDT plus DDE ranged from 0 to 80 p.p.m., with DDE representing from 39 to 86% of the total. Chromatographic and spectrophotometric data provided e\ idence that the degradation prodnct i s TIDE.
I
S THIC c o u ~ s cof u study on the occurrence of DDT in human
fat it became clear that a detailed study of both the method of isolating DDT from fat and the final estimation by t,he Schechter-Haller procedure was necessary. This became more essential \\.hen evidence was obtained that a substantial quantity of DDK [ l.l-dichloro-2,2-bis(p-chlorophen~-l)ethylene] as well as D D T \\.as present in the samples. .4 preliminary report on this observation has been given hy Pearcr et a!.(8). The present paper reports the results of the study on the methods eniployed and .solile additional evidence as to the presence of DE)],; in human fat. SEP4RATION O F FAT
The isolation of DDT and related compounds from biological materials invariably has presented difficulties, espec’ially in dealing with fats or oils because of the pronounced solubility of chlorinated hydrocarbons in fat, solvents. The mo.st conimonly used methods for separating DDT from fat are bayed on a sulfuric acid treatment which partially sulfonates the fat and greatly reduces its affinity for DDT and thus permits rxtraction of the D D T in an inert solvent such as chloroform or c a h o n tetra-
chloride. I n the method ot &.herhter et ai. (f01.the opemtion i.. carried out in separator) funnels. Daviclow ( 3 ) has adapted the sulfuric acid treatment and solvent estrac.tion to a chroniatographic column. \Tith modifications, the Dsvidow rolunin has been found to be niorp rapid and consideralll>~ leps laborious in the writers’ experience. I11 the present work well as in that already report,ed, the modified Davidox column has been employed. The fat ~ a m p l e sv w e extracted in preparation for chromatographing as IoIIo\vR: Samples of known weight n-rye ground in a mortar with xnhydrous sodium sulfat,e and then transferred to 500-ml. gl. SQstoppered bottles uqing C.P. carbon tetrachloride :is solvent and transfer agent (50 to 70 nil. of carbon tetrachloride per 2 grams of fat). The bottles were shaken mechanically for a t least 2 hours. The contents xvere then filtered and the filtrate was chroiii:ttographed through previously prepared Davidow columns ( 2 ) . SCHECHTER-IIALLER A N L Y S I S
The Schcchter-Halier ( 1 1 ) method for determining D D T in micro quantities is now generally accepted as the best available. Various modifications have been introducecl from time to time and it is. doubtful whether any t v o laboratories follow exactly
ANALYTICAL CHEMISTRY
1066 the same procedure. Application to analysis of spray residues, where relatively large amounts of D D T are involved, has in general been successful; holvever, in the case of fats and tissues and other biological materials containing relatively small amounts of DDT, considerable trouble has been experienced in the application of the method. In this work the size of the samples collected was usually small (1 to 3 grams) and in most instances the amounts of D D T !yere of the order of 5 to 10 p.p.m. Thus, reasonably accurate measurement down to and below 5 micrograms was desired. The magnitude and variation in blank values are of great importance with these low amounts of D D T and very careful empirical standardization of the method becomes necessary to maintain variation in duplicate analyses suhstantially below 10%. Moreover, application of the tiyo-color equation of Knudson (6) is very unreliable unless light absorption due to blank is minimized and controlled. For these reasons preliminary factors affecting reagent blanks and blanks arising from use of the Davidow column have been cnrefullv evaluated and the method has been modified accordingly. FACTORS 4FFECTIYG B L 4 N K S
One cause of erratic and undesirably high blank values is the failure to remove all of the solvent before nitration. The state of apparent dryness of a particular sample is usuallj judged by odor and appearance. but the acuity of this judgment may varv from day to day for the same analyst and also betneen analysts. This variation can be prevented by adding a period of heating and aeration beyond the point of apparent dryness. It also has been found, as observed by Clifford (I), that heating the nitrated product just prior to solution in benzene and adding methylate to develop the color will help to maintain low and consistent blank values. The writers have adopted a prenitration heating time of 15 minutes a t about 75” C. with simultaneous gentle aeration and a postnitration heating period of 30 minutes a t --0 i n C. Using a 1-em. absorption cell in a Beckman llodel R Spectrophotometer, average reagent blanks at 520 and 597 mp were found to be 0.OOi and 0.004, expressed as optical density for a final volume of 6 ml. It also became evident that some blank effect was arising from the Davidow column. Blanks occurring from elution of carbon tetrachloride through the column have been evaluated and found to average 0.007 and 0.005 net at 520 and 597 mp. respectively. Thus, the combined column blanks and the SchechterHaller reagent blanks averaged 0.014 and 0.009. These values appear to be the average minimum blank values attainable by the procedure used by the writers. In terms of p,p’-DDE and p,p’-DDT these values represent about 1.5 and 1.0 micrograms. respectively. If one is attempting estimation of 5 micrograms of total D D T and DDE, the blank values are obviously of major significance and unless they are reasonably constant great errors will appear in the calculated amounts of D D T and D D E present. The average deviation from the mean blank values has amounted to 0.4 and 0.2 microgram of D D E and DDT, respectivelv. With this degree of deviation, there will be no excessive error, even though the blank represents a substantial quantity of D D E and DDT. One source of unusually high and variable reagent blanks encountered in this laboratory appears worth mentioning. The practice of cleaning glassware with aromatic solvents, such as xylene, is extremely hazardous. The last traces of these solvents are very difficult to remove from glass and apparently carry through the nitration, resulting in brownish yellow colors 15 hich absorb strongl:, throughout the spectrum. The exact evaluation of blanks arising from the fat itself has presented some difficulty. The amount of human or animal fat free of D D T or related compounds is extremely limited. With the experimental material available it was found that the fat blanks were low and reasonably constant from about 500 through 600 mp. This seems to agree w-ith the eaperience of Offner
and Calvary ( 7 ) ,who made an intensive study of this problem in connection with analysis of urine, liver. and other biological materials. Evidence that the fat blanks were low and substantially constant in the present study was obtained by analysis of composite fat samples, addition of known quantities of D D T and D D E to samples, and analysis after another method of fat elimination. In addition, several samples of human fat predatinq the introduction of D D T have been examined. DETERMINATION O F DDT AND RELATED C O M P O L I D S
Bbsorption spectra data (8)indicated that a substantial portion of the Schechter-Haller-positive compounds isolated from the human fat samples being examined was DDE, and that o,p’isomers were not present in abnormal amounts, as no pronounced absorption occurred a t 597 mp. I t seems probable that the o,p’ and p,p’ isomers would appear in fat in the proportion they occur in technical DDT-Le., 1 to 4 or 5, respectively (4). Although one can determine o,p’ and p , ~isomers ‘ by the SchechterHaller method by treating them as a two-component color system by the method of Knudson et al. ( 6 ) , resolving more than two components such as mixtures of o,p’-DDT, o,p’-DDE, p , p ‘ D D T , and p,p’-DDE is not feasible. For this reason it has been necessary to simplify the analysis by treating the isolated products as consisting predominantly of p,p’-DDT and p,p’-DDE. This simplification represents a practical solution to the complex problem of analyzing materials containing mixtures of o,p‘and p,p’-DDT and their degradation products.
Table I.
Extinction Coefficients for DDT and DDE
At W a r e Length 597 XIr, A K I A , p,p’-DDT Kidjp,p‘-DDE 0 067
At Ware Length 520 l f p , B p,p’-DDT K @ , p p’-DDE
KiR,
0 005
0 027
0 070
EVALUATION O F EXTIYCTIOY COEFFICIENTS
Table I presents the extinction coefficients for pure p , p ’ D D T and D D E after processing by the Schechter-Haller method as adapted in this laboratory ( 2 ) . D The values were calculated from the equation K = c where K = extinction coefficient D = optical density, 1-cm. absorption cells C = micrograms per ml. of colored solution Using the values in Table I in the Knudson equations: CI (micrograms of p,p’-DDT per nil. of colored solution) = 1.1 - (14Djgr - D ~ N ) and C2 (micrograms of p,p’-DDE per ml. of colored solution) = 5.9 - (2.5Dj2o-Dsg1) where Dare = optical density a t 520 nip Dig, = optical density a t 597 mp Since a total volume of 6 ml. of colored solution was used (2
Table 11. Analysis of Known Mixtures of p,p’-DDE and p,p’-DDT Sample
Sa 1 2 3 4 5 6 7
8 9 10 11
12 13
Added. 7 DDE DDT 0 10 1 9 2 8 4 6 6 4 8 2 0 20 20 0 4 16 8 12 8 8 12 8 16 4
DDE 10.3 8.8 7.7 7.1 4.4 1.9 20.0 0.0 17.0 12.0 7.3 8.5 4.0
Found, y D D T DDE f D D T 0.2 10.6 0.7 9.5 9.5 1.8 12.0 4.9 11.0 6.6 9.8 7.9 20.0 0.0 21.5 21.5 21.1 4.1 20.1 8.1 14.7 7.4 20.3 11.8 20.6 16.6
V O L U M E 2 5 , N O . I, J U L Y 1 9 5 3
1067
ml. of benzene plus 4 ml. of sodium methylate solution), the final equations as used were: C1(micrograms of p,p’-DDT per G ml. of colored solution) = 6.6 ( 1 4 0 s ~- & o ) (1) C2
(micrograms of p , p’-DDE per 6 ml. of colored solution) 35.4 (2.5D~20- 0 5 8 7 )
=
(2)
Known mixtures of the two components were run through the method in the absence of fat to test the reliability of these equations. Table I1 presents typical results of the analysis of various mivtures of the components. The data indicate satisfactory resolution of p,p’-DDT and p,p’-DDE and are believed to represent the normal variation that might be expected in using the Schechter-Haller method for analysis of a t-0-color system under more or less ideal conditions.
Table 111. Retention of p,p’-DDh, p,p’-DBP, p,p’-DDE, and p,p’-DDT by Davidow Column Material Added (50 Y) p,p’-DDh p,p’-DBP p,p’-DDE p,p’-DDT p,p‘-DDTa a Sot
Found, 1.1 4.0 50.5 48.7 50.6
y
Recovery. 2.2 8.0 101.0 97.0 101.2
cO
run through column.
BEHAVIOR OF D 4 V I W W COLUMN
As the Davidow chromatographic column yas being used to separate the fat, it was believed essential to determine whether it would retain D D E or other degradation products of DDT. Some representative tests are shown in Table 111. I t is apparent that p,p-DDA [bis(p-chloropheny1)acetic acid] and p,p’-DBP (4,4’-dichlorobenzophenone)are largely retained by the column, whereas D D E and, of course, D D T are passed. This behavior is further justification for basing calculations on only p,p’-DDE and p,p’-DDT hlthough there is no reason to suspect that the Davidoucolumn causes any significant degradation of DDT, nevertheless, experiments were conducted to demonstrate this point. A few samples of human and monkey fat were available which were apparently free of D D T and D D E as determined by prior analysis. Known quantities of p.p’-DDT were added to the samples and then they were processed through the complete procedure. Good recoveries of D D T were obtained in all cases. This evidence, plus the many knowns which have been run through the columns in the absence of fat from time to time, leaves little doubt that the Davidow column causes no significant degradation of DDT. T E S T S OF WETHOD 4 s APPLIED T O F4T
shows very good agreement. In other experiments one half of each of several fat samples was analyzed separately. The remaining halves were combined in a composite sample and also analyzed. Data on such experiments have been reported (8). These data as well as those in Table IV indicate the accuracy of the procedure adopted and also show that the blanks are essentially constant and being correctly evaluated. Laug et a/. ( 6 ) have published D D T analyses of human fat samples using the method of Prickett et al. (9),which involves an alkaline saponification to remove the interfering fat. This saponification dehydrohalogenates the D D T to DDE, which i j estimated as such by the Schechter-Haller method and then calculated as DDT. The method, of course, fails to differentiate between any D D E present in the fat before dehydrohalogenation and that arising from dehydrohalogenation of the D D T present. I t was of interest to compare this method of analysis with the one employed in the present study. Eluates of two different samples were analyzed by the writers’ procedure and also after treatment with alcoholic sodium hydroxide according to the method of Prickett et al. (9). One sample consisted of 15 grams of human fat, which was extracted with carbon tetrachloride and then passed through Davidow columns in five portions. The eluates TFere combined and the volume was reduced to 60 ml. Two 25-ml. aliquots ( 1 and 2, Table V), each representing 6.1 grams of fat, were used for analysis before and after dehydrohalogenation. The second sample was obtained by extracting and chromatographing a number of small fat samples containing from 2 to 20 p.p.m. of DDT and combining the eluates into a composite sample. Equal aliquots (3 and 4, Table V) of this composite solution representing 5.8 grams of fat were used for the analysis before and after dehydrohalogenation. All calculations were based on the twocolor equations given earlier.
Table IV.
Recovery of p,p’-DDE and p,p’-DDT Added t o Human Fat Sample Eluates Sample
1. 2.
3.
Found, DDT
13.3
9.7
+
Human fat A 10 micrograms D D E and 5 micrograms D D T Human fat A alone Recovery of added D D E and D D T D D E and D D T alone by analysis
Y
DDE f DDT 23.0
2.9 4.3 7.2 10.4
5.4
15.8
10.4
5.8
16.2
11.7
11.9
23.6
D D E and DDT. P.P.31.
e
+
Human fat B 5 micrograms D D E and 5 micrograms DDT 5. Human fat B alone Recovery of added D D E and D D T 6. D D E and D D T alone by analysis
4.
DDE
Human fat C 4-5 micrograms D D E and 5 micrograms DDT 8. Human fat C alone Recovery of added D D E and D D T 9. D D E and D D T alone by analysis
5.4 7.7 13.1 6.3
4.2
10.5
5.3
5 5
10.8
13.2
11.5
24.7
io
7.
7.9 6.3 14.2 -
‘3
In order to test further the accuracy and reliability of the pro5.3 5.2 10.5 cedure adopted, known quantities of p , ~ ’ - D D Eand p,p’-DDT 5.3 5.5 10.8 were added to aliquots of eluates of human fat samples. Typical results are presented Table V. Coniparative Results of DDT-DDE Analyses before and after Dehydroin Table IV. Samples 3, 6 , halogenation and 9 represent standards conMicrograms P.P.M.“ taining the same amount of Total Total % D D E of Total QS as D D E and D D T D D E and D D T as that added DDE DDT DDT DDE DDT DDT Originally Present to the respective fat eluates Single human fat sample 62 31.9 19.5 55.0 5.2 3.2 9.0 which were analyzed simul1. Before 2. After 51.8 0.0 57.6 ... ... 9.4 taneously with the eluates. Composite of human fat samples 59 22.4 15.9 40.8 3.9 2.7 7.0 3. Before Comparison of the figures 4. After 36.3 0.0 40.3 ... ... 6.9 showing micrograms recovered 6.1 gram of fat for each of samples 1 and 2. with the data on the stand5 . 8 gram of fat for each of samples 3 and 4. ards as determined by analysis
ANALYTICAL CHEMISTRY
1068
of two-color equations based on technical grade D D T and p,p’-DDE give an approximation of the magnitude of the effect. For this reason the following equations were derived and applied to some representative fat samples:
Table V shows that the total quantity of D D E and D D T calculated as DDT agrees extremely well both before and after dehydrohalogenation. This indicates that the method used does evaluate the amount of D D T present, as it is recovered quantitatively as DDE. Furthermore, it helps to substantiate that the component being measured as D D E is indeed D D E or other degradation products of D D T having similar absorption spectra.
hIicrograms of p, p’-DDE per 6 ml. of colored solution = 46.2 (1.93Djzo - D w ) (3) Micrograms of technical DDT per 6 ml. of colored solution = 7.7 (14Dsw - &a) (4) where D52o= optical density a t 520 mp
SUMMARY OF HUMAN FAT ANALYSES
Table VI presents a summary of the analysis of 50 human fat samples examined by the procedure adopted ( 2 ) . The samples were taken from different geographical areas and different occupational groups and are therefore not representative of any single group. Ninety-six per cent of the samples show detectable amounts of D D T and DDE. In no case was D D T found alone; D D E was always indicated as being present, to the extent of 39 to 86%. Some of these samales were obtained from individuals having known high exposure to DDT. It seems significant that even in these cases the materials isolated contained a high proportion of DDE. In addition to the 50 samples reported in Table VI, 53 samples have been analyzed using a less refined analytical procedure. In the latter group all but four samples contained SchechterHaller-positive materials and the average calculated D D E content was about 60% with a range of 40 to 90%. Among these samples one of particular interest was obtained from an individual who worked in a DDT formulation plant. The sample showed 122 p.p.m. of D D T and 127 p.p.m. of DDE. This suggests that DDT can be degraded to D D E in the human body, as it is not likely that this individual was exposed to any significant quantities of DDE. The percentages of D D E reported in Table VI are necessarily somewhat in error, as the values were calculated from equations based on p,p’-DDT and p,p’-DDE only. The SchechterHaller-positive materials occurring in human fat undoubtedly arise from technical grade D D T which may contain 15 to 20% o,p’-DDT. The latter isomer shows fairly strong absorption a t 520 mp, the wave length used to measure principally the DDE. The presence of o,p’-DDT in the fat samples will affect the values for both D D E and D D T when Equations 1 and 2 are used. While it is extremely difficult to evaluate this effect exactly, use
DSg7= optical density a t 597 mp Table VIII. Chromatographic Behavior of Various Nitrated Materials in Alumina Column Theoreticala,
Recovery,
Solvent 72 0 Acetone 95%acetone p , p ’ - ~ ~ ~ 68 0 Acetone 95% acetone Tech, DDT 68 0 Acetone 95% acetone o,p-DDT 17.5 Acetone 95% acetone a B y analysis before chromatography.
Y
%
0 0 58 8 67 0 0 0 66 7 0 0 17 0 0 0
0 81 99 0 9s 0 97 0
Material p 3p’- D D E
I -
4
Y
Recovery
il comparison of the data obtained using Equations 3 and 4 and 1 and 2 is presented in Table VII. The two sets of equationa were applied to the same optical density values observed for each sample, so that direct comparison is valid. It is noteworthy that there is only a relatively small change in the calculated parts per million of DDE, whereas the difference in parts per million of D D T is much more marked. The proportion of D D E percentagewise shows only a moderate difference. Thus, the use of p,p’-DDE as one of the primary standards appears to introduce no major error. In any case, it is likely that any o,p’D D E would be included with the p,p’-DDE, since their absorption characteristics are similar a t the measuring wave lengths employed. One may then consider that all DDE values reported in this work represent the total of both isomers. The most important aspect of the data in Table VI1 is the appreciable difference in the total D D T plus D D E calculated by the two methods. This difference is largely due to the fact that the extinction coefficients for p,p’-DDT and the technical product used as standards are markedly different a t 597 mp-i.e., 0.067 Table VI. Human Fat Analyses and 0.058, respectively. The lower value for the technical Range, NO. product is due to the presence of the o , p ’ isomer and probably D D T plus D D E , of DDE some non-Schechter-Haller-positive ,materials. A t 520 mp P.P.M. Samples Range, 70 S o t detectable 2 the presence of the o,p’ isomer produces a slightly higher extinc2-5 12 46162 tion coefficient in the case of the technical product, 0.030 as 6-9 19 39-86 10-19 7 43-63 compared to 0.027 for the p,p’-DDT. The net effect of the differ20-29 2 53-81 30-39 3 54-63 ences in the extinction coefficients of the two standards a t both 40-49 1 78 wave lengths is partially compensatory. However, the difference 50-59 1 56 60-69 2 65-74 a t 597 mp outweighs that a t 520 and thus tends to increase the 70-79 1 49 total D D T calculated. Total 50 I t would seem from this brief discussion that unless one knows the relative proportions of the major isomers present in a sample, thecorrect selection of primary standards is not possible. This Table VII. Comparison of Two Methods of Calculations Applied to Typical Human Fat Analyses means then that an arbitrary selection must be Ca1cd.b as Tech. D D T and p.p’-DDE Calcd.0 as p , p ’ - D D T and p, p’-DDE made when dealing with materials contaminated with technical DDT of unknown composition aa Sample DDE, DDT, DDE, DDE, DDT. DDDDETTTt D D E , KO. p.p.m. p.p.m. p.p.m. % p.p.m. p.p.m. p.p.m. % in the case of human fat. Perhaps the closest ap1 2.3 2.3 4.6 50 2.4 3.2 5.6 43 proximation of the total D D T and degradation 2 3.1 2.9 6.0 52 3.5 4.3 7.8 45 products present in foods or animal tissues could 3 6.3 6.7 13.0 48 6.7 9.7 16.4 41 be attained by use of p,p’-DDE and a mixture of 4 4.6 3.9 a. 5 54 5.2 5.6 10.8 48 9.9 23.1 57 6.8 18.2 63 13.2 5 11.4 o,p’- and p,p’-DDT in the ratio of 1 to 4 as pri3.2 6.4 50 6 2.9 2.3 5.2 56 3.2 mary standards, However, the writers believe it Equations 1 and 2. is most practical for routine purposes to use simply b Equations 3 and 4. p,p’-DDE and p,p’-DDT. There is some hope
-
DDDDT
1069
V O L U M E 25, NO. 7, J U L Y 1 9 5 3 Table IX. Analyses of Three-Component Mixture Containing 17.5,68, and 72 Micrograms of o,p'-DDT, p,p'-DDT, and p,p'-DDE
.
Recovery before, y opp'G plus p,p'-DDT p,p'-DDE 86.6 77.8
(Chromatography, alumina column) Solvent Recovery after, y 0,P'O
Acetone 95% acetone
plus p,p'-DDT
o,p'-DDTb
85.5
19.1
0.0
0.0
p,p'-DDTb
66.4 0.0
Recovery, % of Theoretical 101 108 100 109 98 a0.p' plus p,p'-DDT calculated as technical D D T in. combination with p p'-DDE. b Two-component equatioxv for mixtures of o,p'- plus p,p'-DDT applied 'to acetone eluate.
Table X .
p,p'-DDE 0.0
41.3 57
Resolution of Nitrated Schechter-Haller-Active Material from Animal and Human Fat Samples
(Chromatography, alumina column) Recovery before Resolution, y Solvent Recovery after Resolution Tech. p,p-DDE, Tech. p,p'-DDE, Tech. Type of Sample p,p'-DDE DDT Y DDT, Y % DDT, % 0.0 68.8 0 100 Acetone 68.8 Rat fat (DDT-fortified 24 4 ' diet) 95% acetone 15.7 0.0 64 0 Human fat (composite 54.4 42.2 Acetone 0.0 42.In 0 100 sample) 95% acetone 26.3 0.0 48 0 a D D T found to contain 16% o,p'-DDT and 84% p,p'-DDT calculated by tw-o-component equation for mixtures of 0 . P I - and p,p'-DDT.
of alleviating this general problem by use of chromatographic separations. Some progress has been made along this line and is described below. CHROMATOGRAPHIC EXPERIMENTS
The chromatographic separation of D D T and its degradation products has been investigated in this laboratory in connection with the present study as well as studies of the degradation of D D T by resistant houseflies. In general, satisfactory quantitative separations of p,p'-DDT and D D E as such have not been successful. A sharp separation of the nitrated products has been attained using 3 to 5 grams of 80- to 200-mesh absorption alumina (Fisher Scientific Co.) in columns 15 mm. in outside diameter and acetone as solvent. The products obtained after nitration by the Schechter-Haller method were taken up in 2 to 3 ml. of acetone and introduced to the column, using several more milliliters of acetone to transfer quantitatively onto the alumina. Enough acetone was then added to the top of the column so that 25 ml. of eluate could be collected. 4 n elution speed of 20 to 30 drops per ~. minute was maintained by use of a stopcock on the column. The acetone elution will remove o,p'-DDT, p,p'-DDT, and technical D D T quantitatively, is reTable XI. tained on the column. pjp'-DDE
eliminate possible confusion
to rat and human fat samples and some typical results are shown in Table X. The data have been calculated in terms of technical D D T and p,p'-DDE. The D D T appears quantitatively in the acetone eluates. A complete absorption spectrum of the Schechter-Haller color developed for the human fat acetone eluate was typical of that of technical DDT showing evidence of the presence of about 16%of o,p'-isomer. As in the case of knowns, the D D E was found in the 95% acetone eluates, although quantitative recovery was not realized. Complete absorption spectra of the recovered products coincided with that of pure p,p'DDE. Thesechromatographic data provide confirmatory evidence that D D E is present in the human fat samples and with the o,p' isomer.
FAT SAMPLES PREDATING DDT
The evidence presented here and elsewhere ( 8 )that D D T and a degradation product, probably DDE, occur in human fat is based entirely on spectrophotometric analysis of the SchechterHaller-positive materials isolated by means of the Davidow chromatographic column. There is no apparent reason to doubt the validity of this evidence, as the colors produced by the Schechter-Haller method are highly specific for D D T and closely related compounds. It is extremely unlikely that compounds native to human fat would give colors characteristic of D D T and its relatives. In support of this, samples of fat have been obtained from autopsy specimens collected prior to the advent of DDT. Analyses of three of these samdes are presented in Table XI. S o trace of Schechter-Haller colors could be detected. A very slight yellow color developed, which is typical of blanks. The absorption due to this yellow color has been calculated in terms of D D E and D D T in Table X I merely to show the order of magnitude of the fat blanks.
Analyses of Human Fat Samples Taken in 1938 and 1940 Wave Length,
Bfr This is illustrated by the data submitted in Equivalent of b Absorbance in Terms of Xet 520 597 Year Grams Corrected5 p,p'-DDE, p,p'-DDT, p,p'-DDE, p,p'-DDT, Table VIII. Taken of Fat absorbance Y p.p.m. p.p.m. At'tempts to elute the D D E from the column 1938 2,5 o,013 o,oo8 0.8 0.4 0.3 have not been totally successful. Only partial re1940 1.9 0.004 0,008 0.1 0.9 0.1 0.5 1940 1.6 0.010 0,009 0.7 1.0 0.4 0.6 covery has been realized sofar, using a great variety Corrected for reagent and Davidoivoolumn blanks. of solvents. Sinety-five per cent acetone-water b 40Schechter-Haller colors in evidence. has given the beet results, but from 20 to 50% of the _D D E is usually lost. This loss is not due to incomplete elution, as exhaustive elution with 95y0 aceDISCUSSION tone-water as well as with other solvents fails to give quantitative yield of the original nitrated DDE. Kevertheless, the separation In the preliminary report ( 8 ) of this study analysis of individual of the D D E from the D D T is quantitativeandvalid, since no D D E samples and composite samples made up of the =me samples appears in the first acetone eluate. Complete absorption spectra was presented. The composite analyses agree very well with the of the eluted materials after treatment with the methylate reagent sum of the individual analyses. These data have been suppleahow no evidence of contamination of the D D T fraction with D D E mented in the present report by two additional treatments. nor does the partially recovered D D E show evidence of anything First, known quantities of D D T and D D E have been added to other than DDE. In Table I X resolution of a mixture of o,p'fat samples and analyses made before and after such additions. DDT, p,p'-DDT, and p,p'-DDE by the technique is illustrated. The net D D E and D D T found by direct analysis and by subtraction of the known amQunts added after analysis in their presence The chromatographic technique developed has been applied Q
ANALYTICAL CHEMISTRY
1070 agree remarkably well (Table IV). Second, analysis by the writers' two-color treatment and by converting to D D E through dehydrohalogenation also shows excellent agreement (Table V). These three independent approaches would appear to be ample evidence that D D T plus a substantial proportion of the degradation product D D E is present in human fat. Supplementing these experiments are the data on the absorption spectra before and after dehydrohalogenation of the Schechter-Haller materials isolated (8). Many additional absorption spectra data have been collected by the writers, but have not been presented for the sake of brevity. These omitted data are in all cases confirmatory. It is also believed the chromatographic experiments are highly significant (Table X). Finally, the examination of fat samples predating the advent of D D T (Table XI) showed no evidence of the presence of Schechter-Haller-positive materials. The writers believe that DDT and D D E are contaminants of human f a t of the general population.
courtesy of A. J. Miller, Department of Pathology, School of Medicine, University of Louisville, Louisville, Ky. LITERATURE CITED
(1) Clifford, P. A,, J. Assoc. (2)
(3) (4) (5) (6)
Ofic.A g r . Chemists,30, 337-49 (1947). Communicable Disease Center, Technical Development Branch, Federal Security Agency, Savannah, Ga., Chemical Memorandum 1,1952. Davidow, B., J . Assoc. Ofic.Agr. Chemists,33, 130-2 (1950). Haller, H. L., et al., J . Am. Chem. Soc., 67, 1591-602 (1945). Knudson, H. W., Meloche, 1%'.V., and Juday, C., IND. ESG. CHEM., ANAL.ED.,12,715-18 (1940). Laug, E. P., Kunee, F. and Prickett, C. S., Arch. Ind. Hug.
and Occupational Med., 3,245-6 (1951). (7) Offner, R. R., and Calvary, H. O., J . Pharmacol. Erptl. Therap., 85.363-70 (1945). (8) Pearce, G. W:, Mattson, A. hl., and Hayes, W. J., Science, 116, 254-6 (1952). (9) Prickett, C. S., Kunze, F. M., and Laug, E. P., J . Sssoc. Ofic. A g r . Chemists,33,880-6 (1950).
ACKNOWLEDGMENT
(10) Schechter, M. S., Pogorelskin, M . A, and Haller, H. L., IND. ESG.CHEM.,ANAL.ED.,19,51-3 (1947). (11) Schechter, 11.S., Soloway, S.B., Hayes, R. A., and Haller, H. , L., Ibid., 17, 704-9 (1945).
The writers wish to express their appreciation to F. -4.Gunther and R. C. Roark for supplying the o,p'-DDT used in this work. The samples of fat predating D D T were supplied through the
RECEIVED for review February 18, 1953. Accepted April 2 5 , 1953. Presented before the Division of Agricultural and Food Chemistry. Pesticides Subdivision, at the 122nd Meeting of the AMERICAN CHEMICAL SOCIETY, Atlantic City, 37. J.
Nitrogen Compounds in Distillate Fuels LOYAL F. WARD, J R . ~R , . T. 3\IOORE, AND JOHN S. BALL Petroleum a n d Oil-Shale Experiment Station, Bureau of Mines, Laramie, Fyo. Some types of nitrogen compounds have been shown to cause instability in distillate fuels. An analytical scheme for types of nitrogen compounds in petroleum distillates has been based on a combination of methods for total nitrogen, basic nitrogen, and pyrrole nitrogen. This analysis has been made on 34 samples of distillate fuels chosen to be representative of geographical origin of the crude oil and of processing methods. With the exception of the California oils the total nitrogen content is below 0.05% for each oil. Differentiation between catalytically and thermally cracked stocks can be made on the basis of the types of nitrogen compounds present. A test for distinguishing among straight-run, catalytically cracked, and thermally cracked fuels is proposed.
T
HIRTY-four samples of distillate fuels representing production from different areas of the country and various methods of processing have been investigated for information as to types of nitrogen compounds present. -4 combination of methods for total nitrogen, basic nitrogen, and pyrrole nitrogen provides a systematic approach for obtaining this information. Results from this combination of methods show distinctive patterns for fuels obtained by different processing methods. The presence of nitrogen compounds, particularly pyrroles, has been shown (9) to affect adversely the storage characteristics of distillate fuels. This study of the types of nitrogen compounds in distillate fuels was made to provide a background for correlation with stability studies in progress on the same fuels. It also gives information on the types of nitrogen compounds present as a guide to further development of analytical methods. Comparatively little is known concerning the nitrogen compounds present in petroleum. Bailey and coworkers isolated ( I , 6) a series of pyridines and quinolines from straight-run kerosene. Sauer, Melpolder, and Brown (8) found carbazoles, indoles, pyrroles, pyridines, and quinolines in straight-run distillate fuel from Kuwait by a mass-spectrometer technique. Thompson, Symon, and Wankat (IO)have found pyrroles in virgin distillate 1 Present
addrem, Julius Hyman and Co., Denver, Colo.
fuel oils by a colorimetric method, Treibs (11)isolated porphyrins from various petroleums. Pyridines, quinolines. pyrroles, and nitriles have been identified also from shale oil (18 ) . -4nalytical methods for the determination of types of nitrogen compounds are far from satisfactory. With the exception of the California oils, the fuels studied had nitrogen contents of less than 0.05 weight % and extremely sensitive methods are needed. The most promising technique, that of Sauer, Melpolder, and Brown (8)) is time-consuming, being based on chromatography and mass spectrometry. A separation into basic and nonbasic classifications can be achieved by titration of the basic nitrogen compounds with perchloric acid (3, 6, 7), and a colorimetric method for the determination of pyrroles has been described (IO). The analysee described in this paper combine these methods to give results for basic nitrogen, nonbasic nitrogen, and pyrrole nitrogen. -4 test to distinguish between straight-run, thermally cracked, and catalytically cracked distillates is also suggested. SAMPLES
The 34 samples of distillate fuels were obtained by the Western Petroleum Refiners Association for a stability testing program being conducted a t the Petroleum Experiment Station of the Bureau of Mines, Bartlesville, Okla. These samples were selected 80 as to he representative of crude oil sources now in use in this
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