Figure 5. Formaldehyde levels in urban air
(2) Atkinson, W. B., Stain Technol. 27, 153 (1952). (3) Barnes, E. C., Speicher, H. W., J . Ind. Hyg. Tozicol. 24, 10 (1942). (4) Blaedel, W. J., Blacet, F. E., IND. ENG.CHEM.,ANAL.ED. 13, 449 (1941). (5) Denigbs, G., Compt. rend. 150, 529 (1910). (6) Goldman, F. H., Yagoda, H., IND. ENG.CHEM.,ANAL.ED. 15, 377 (1943). (7) Hatch, T., Warren, H., Drinker, P., J . Ind. Hyg. 14, 301 (1932).
(8) Hoffpauir, C. L., O’Connor, R. T., IND. ENG. CHEM., AXAL. ED. 21, 420 (1949). (9) Jacobs, M. B., Eastman, E. L.,
Shepard, D. L., J. Am. Pharm. Assoc., Sci. Ed. 40, 365 (1951). (10) Kersey, R. W., Maddocks, J. R., Johnson, T. E., Analyst 65, 203 (1940). (11) Magill, P. L., Holden, F. R., Ackley, C., eds., “Air Pollution Handbook,” Sect. 3, p. 4, McGraw-Hill, New York,
OTHERSUBSTANCES.No significant interference occurred with 20-mg. amounts of formic acid (go%), glacial acetic acid, methanol, or ethanol. Formaldehyde Content of Urban Air. Samples were taken a t hourly intervals throughout the day in a
commercial area in Toronto, Canada. The sampling site was located a t street level, near a busy intersection. Two series of results are presented in
Figure 5. Diurnal variations occurred on all days on which the tests were run. The concentrations were greatest during the morning and late afternoon, and least during the early hours of the morning. LITERATURE CITED
(1) American Standards Association, New
York, N. Y. “Allowable Concentration of Formaldehyde,” (pamphlet), 1944.
1956. (12) Melekhina, V. P., Gigiena i Sanit. 8 , 10 (1958). (13) Ripper, M., Monatsh. Chem. 21, 1079 (1900). (14) Romijn. G.. 2. anal. Chem. 36. 19 f189fI.’ ’ (15) Schiff,’H., Ann. 140, 92 (1866). (16) West, P. W., Sen, B., 2.anal. Chem. 153, 177 (1956). (17) Wilson, W. L., “An ,,Automatic ~
Impinger for Air Sampling, Air Pollution Control Assoc., Meeting, Chattanooga, May 1954. RECEIVED for review July 1, 1960. Accepted November 25, 1960.
Spot Test Microdetermination of DDT and Its Related Compounds in Biological Materials AROKIASAMY IRUDAYASAMY and A.
R. NATARAJAN
Sfate Forensic Sciences Laborafory, Madras 3, India
b The nitrated product of DDT displays four distinct colors successively when treated with 20% alcoholic potassium hydroxide, and five distinct colors successively when acetone is added also. This reaction occurs for amounts as small as 1 pg. These tests are applied in the field of forensic toxicology for the identification of DDT and its related compounds, extracted from biological materials of human origin.
S
colorimetric methods have been described for the determination of D D T (3, 13) and its nitrated product. Schechter et al. ( I d ) used methanolic sodium methylate with a benzene solution of the nitrated product for the production of intense colors with a sensitivity of 10 fig. of D D T . Several variations have been suggested for the Schechter method (4, 7, 8). Alessandrini (1) substituted 1N alcoholic potassium hydroxide, while Iling and Stephenson (6) used alcoholic potassium hydroxide and ammoniacal hydroxylamine for sodium methylate. EVERAL
630
ANALYTICAL CHEMISTRY
Amsden and Walbridge (2) substituted isopropylamine for sodium methylate. Luis (6) obtained a residue of insecticidal properties from Stas-Otto extracts of the viscera of a poisoned person, which was confirmed as D D T after purification by chromatography on alumina (1). The production of color in solution is slow, requires warming, and the method is not sensitive when microquantities are present. Prickett et al. (9) described a modified Schechter method for the determination of D D T or methoxychlor with a lower limit of 5 fig. of either compound in 5 to 6 grams of fat. In cases of poisoning by DDT, often only trace amounts are left in biological materials, as the major part is either destroyed or removed from common toxicological specimens by the following means : vomit; degradation in the metabolism to related compounds; total degradation to simpler compounds and excretion through the kidneys and intestines; storage in fatty tissues, etc. Therefore, a definite chemical method
was needed by which microquantities of D D T and its decomposition products could be isolated from biological materials and identified. The purpose of this paper is to present a simple method for the isolation and identification of very small quantities of D D T and its metabolized products present in biological specimens, while examining several D D T poisoning cases of legal significance. Yo special apparatus or technique is involved. Specimens that are encountered are vomited matter, stomach wash, urine, excreta, stomach with its contents, intestines with their contents, liver, kidney, spleen, and other organs. They are expected to be preserved in rectified spirit for toxicological analysis. However, they are sometimes putrified, because of improper preservation by the personnel who collect the specimens or natural decomposition prior t o preservation, and such specimens are analyzed by the modified method with a slight decrease in sensitivity. The principal steps in the method are: extraction of D D T and related com-
pounds from biological materials like viscera, excreta, and other fluids; nitration; and spot test colorimetric determination of the tetranitro derivative, which forms the major part of the nitrated product. The tube test of Quintana Y Mari et al. (10) is modified with an increase in sensitivity. A method for the quantitative estimation of the nitrated product by modifying the method of Schechter et al. ( l a ) is being studied. REAGENTS
20% Alcoholic Potassium Hydroxide (w./v.). Dissolve 5 grams of analytical reagent grade potassium hydroxide in 2.5 ml. of distilled water, cool, add 22.6 ml. of absolute ethyl alcohol, and shake well. The reagent is stable for 24 hours. Kitrating Rlisture. Mix equal parts of concentrated sulfuric acid and fuming nitric acid. Absolute Ethyl Alcohol. Contains not less than 99.5% v./v. of ethyl alcohol. Rectified Spirit. Contains not less than 90% v./v. of ethyl alcohol. Aqueous Potassium Hydroxide. 1% w./v. All the other chemicals used were of analytical reagent grade.
I
Table 1.
PROCEDURE
For liquids such as vomit, stomach wash, urine, cerebrospinal fluid, etc. : Add enough rectified spirit so that the alcohol concentration is 75 to 8570 v./v. and allow to stand for not less than 12 hours with occasional shaking. For viscera such as stomach with its contents, intestines with their contents, liver, kidney, spleen, etc.: Cut the viscera into small pieces with a pair of scissors, add a suitable quantity of rectified spirit, and homogenize by means of a high speed macerator; add more rectified spirit if necessary so that the alcohol concentration is 7 5 to 85% v./v. and allow to stand for not less than 24 hours with occasional shaking. Filter the alcoholic extract and concentrate it to half its volume by heating on a water bath. Cool, and add an excess of acetone till the precipitation of protein and other colored bodies is complete; stir well and filter. Evaporate the filtrate on a water bath to dryness. Cool, treat the residue with 10 ml. of chilled nitrating mixture, transfer quantitatively to a test tube 8 inches long and inch in internal diameter, and heat in a boiling water bath for 1 hour. I n the absence of a large amount of organic matter reduce the duration of nitration considerably, up to hour. -4dd more nitrating mixture if carbonaceous matter is
present in the resulting product and heat further for ' / zhour. Cool the product, dilute it with 50 to 100 ml. of ice cold R-ater, and extract successively with 20 and 10 ml. of chloroform. Wash the combined chloroform extracts with 50 ml. of aqueous potassium hydroxide and thrice with 50 ml. of distilled water. Dry the chloroform extract over anhydrous sodium sulfate and evaporate on a water bath till the volume is reduced to about 1 ml., and drive off the remaining chloroform by a slow current of air and cool. Dissolve the residue containing the nitro derivative in a few drops of chloroform, transfer the solution to two depressions of a white spotting tile, and allow the solvent to evaporate. To one portion add 1 drop of 20% alcoholic potassium hydroxide and note a play of colors. -4rose color is formed which turns immediately to bright blue, gradually to green, and ultimately yellow. To the second portion add 1 drop of 20% alcoholic potassium hydroxide followed by 1 or 2 drops of acetone. The bright blue color formed changes to bright purple, gradually turns grey, and finally yellow. Modification of Extraction Procedure. I n the analyses of viscerae with a large amount of fat, putrified, or preserved in saturated sodium chloride solution, we recommend one more
Detection Limit of DDT in Biological Specimens
(Specimens were those received in connection with medico-legal cases, in which no poison was detected and which gave negative tests for DDT) Weight, Grams ~ _ _ - Sequence of Color Changes from Reaction0 of Nitrated Product of DDT to Which Known Amounts of DDT Were Added PreDDT. Microarams Tis- serva0.5 tive Total 0.1 sue Specimen \Tell-preserved stomach viash
...
...
loo*
Faint purple, yellowd
Purple, yellod
Stomach vith its contents, Expt. I Stomach with its contents, Expt. I1 Intestines and contents
50
5oc
100
Purple, brownd
Faint blue, purple, brownd
50
5oc
100
Purple, brownd
50
5oc
100
Faint purple, yellowd
Liver
80
20
100
No color change
Kidney
50
50
100
KO color change
Rose, blue, purple, grey, brown Rose, faint blue, purple, yellowd Rose, faint blue, purple, brownd No color change
Urine
...
.. .
lOOb
Faint purple, grey, yellowd
Rose, faint blue, purple, grey, yellow
50
5oc
100
No color change
Faint blue, purple, brown
50
50C
100
Faint purple, brownd
50
50"
100
Faint purple, brownd
Liver
60
40
100
Faint purple, brownd
Faint blue, purple, brownd Faint blue, purple, brownd Faint purple, brownd
Kidney
40
60
. 100
X o color change
PJ? color change
Putrified Stomach and contents Expt. I Stomach with its contents Expt. I1 Intestines and contents
a
Rose, blue, purple, grey, yellow Rose, blue, purple, grey, brown Rose, blue, purple, grey, brown Rose, blue, purple, grey, yellow Rose, blue, purple, grey, yellow Faint blue, purple, brownd Rose, blue, purple, grey, yellow Orange, blue, purple, grey, yellowe Orange, blue, purple, brown. Orange, blue, purple, brown. Orange, blue, purple, brown0 Faint blue, purple, brownd
Reacted with 1 drop of 20% alcoholic KOH followed by 1 or 2 drops of acetone.
* Weight of liquid specimen in spirit.
Weight includes liquid and semisolid contents of specimen. Other colors in sequence not distinguished; in most cases ultimate color was brown instead of yellow because of interference. Final extract was yellow or light brown and initial rose color therefore appeared orange; grey color after purple not distinguished in three cases. c
VOL. 33, NO. 4, APRIL 1961
631
step in the extraction proccdure. Evaporate the 7 5 to 85% alcoholic extract to dryness, cool, tease t h e residue thoroughly with 75 ml. of absolute ethyl alcohol, and filter. Concentrate the filtrate on a water bath to about 20 ml., cool, add an excess of acetone, and proceed as described before. DISCUSSION A N D RESULTS
Of the two tests described, the second one is more sensitive and was therefore applied to all the determinations. The course of the first reaction is explained by Schechter (11, 12), only sodium methylate has been replaced by potassium hydroxide. The second reaction, the chemistry of v, hich has not been studied, is a n application of the mell-known Janovsky reaction. although sodium methylate can also be used in place of potassium hydroxide for these spot tests, n i t h equal sensitivity, the latter was preferred because of the folloning advantages. The successive color changes are distinct and relatively slow, for close observation of the transition of colors, while in the case of sodium methylate the successive color changes are indistinct and too quick to follow; also, the reagent can be prepared easily. Applications. The tests are not specific for a n y single component, and are applicable to the nitrated products of P,P’-DDT and its degradation products, such as P,P’-DDE and P,P’-DDA; they are also applicable to D D D , D F D T , and methoxychlor. Detection Limit and Recovery. The loner limit of detection of D D T is
1 pg. (Table I). If the nitrated product is free from interfering radicals, quantities less than 1 fig. can be detected and the results reproduced. Recovery by this method is almost complete as compared to the color intensity of spots developed with standards. Quantities up to 3 or 4 pg. can be determined semiquantitatively by comparing the color intensity of the test spot with those simultaneously developed with standards. Interferences. Although 1 gg. of the nitrated product of D D T could be detected by t h e tests described, a t times t h e sensitivity is decreased slightly in t h e case of putrified viscera. The nitrated product of its extracts are contaminated with intensely colored degradation products of tissue despite t h e removal of proteins and colored bodies which are likely to mask the color tests. They are removed by precipitating them from the alcoholwater extract by the addition of excess acetone, and further by oxidation with nitric acid during nitration. Also, in the cases of liver and kidney tissue with fatty deposits and viscera of exhumed bodies, the final nitrated product is colored yellow or light brown and renders the test less sensitive. It is difficult sometimes to distinguish the sequence of colors, as some of the colors are nearly masked. I n such cases the modified procedure is recommended whereby the interference can be greatly minimized. Normal constituents of tissue and urinary excretions do not interfere with the color tests. In our laboratory, cases of D D T poison-
ing have been analyzed by the above method with satisfactory results. This method with suitable niodifications can be used to advantage in other fields such as residue analysis, where interfering substances are less when compared to biological materials. ACKNOWLEDGMENT
The authors thank Satesa T’edachalani of this laboratory for his assistance. LITERATURE CITED
(1) Alessandrini,
M. E., Arm. Chim. applicata 38,53-4 (1948). (2) Amsden, R. C., Ralbridge, D. J., J. Agr. Food Chem. 2, 1323 (1954). (3) ~. Claborn. H. V.. J . Assoc. Ofic. - 1 ~ . Chemists 29, 330-7 (1946). f41 Downing. G.. Norton. L. €3.. -&SAL. CHEM.23T1870-1 (195f). ( 5 ) Iling, E. T., Stephenson, W. H., Analvst 71, 310 (1946). (6) Luis, Policarpo, Rev. asoc hioqitim. arg. 17, 334-8 (1952). ( 7 ) Martin, J. T., Batt, R. F., -inn. Rept. Agr. Hort. Research Sta., Long Ashton, Brzstol 1953, 121 (1954). ‘ (8) Pontoriero, P. L., Ginsburg, J. M., J . Econ. Entomol. 46, 903-5 (1953). (9) Prickett, C. S., Kunze, F. M., Laug, E. P., J . -4ssoc. Ofic. Bgr. Chemasls 33, 880-6 (1950). (10) Quintana Y Mari. A,. Cid Caoella. ‘ AnaMaria, Cz~uderno’81,229-51 ((946): (11) Schechter, M. S., Haller. H. L., J . Am. Chem. SOC.66, 2129 (1944). (12) Schechter, M. S., Solowaq, S. B., Hayes, R. A., Haller, H. L., 1 s ~ ENG. . CHEM.,ANAL.ED. 17, 704-9 (1945). (13) Stiff, H. S., Jr., Castillo, J. C., Science 101, 440-3 (1945). \ - ,
~
for review May 24, 1960. AcRECEIVED cepted December 16, 1960.
An Absolute Method of Turbidimetric Ana lysis E. J. MEEHAN and W. H. BEATTIE’ School o f Chemistry, University o f Minnesota, Minneapolis 7 4, Minn.
b An absolute method of turbidimetric analysis is described which eliminates empirical comparison of known and unknown suspension, and is applicable to heterodisperse suspensions. It is based upon measurement of turbidity a t an experimentally determined wave length at which the turbidity is proportional to the reciprocal of the wave length. The observed turbidity at this wave length is related to weight concentration through the M i e scattering coefficient. An example is given of application to a silver bromide sol in which the precision of determination is a few per cent.
1 Present address, Shell Chemical Co., Box 211, Torrance, Calif.
632
ANALYTICAL CHEMISTRY
based upon turbidity traditionally depends on the comparison of intensity of light transmitted by a suspension of the unknown Kith that transmitted by a suspension of known concentration, both suspensions being prepared under identical conditions. I n nephelometric analysis a similar comparison is made of intensities scattered more or less at right angles to the incident beam. With modern instruments both kinds of measurements can be made precisely. The principle difficulty is that the intensity scattered by a given substance depends on the weight concentration and on the particle size and shape. The present discussion is restricted t o nonabsorbing spherical particles. For such particles with radii much smaller than the wave length, the scattering NALTSIG
per unit n eight concentration (specificscattering) increases with the cube of the radius. For particles much larger than the wave length the specific scattering is inversely proportional to the radius. For intermediate sizes t h r behavior is complex (10). Therefore, turbidimetric and nephelometric comparisons are meaninples. unless the particle size is the same in both suspensions. For heterodispersed suspensions the size distribution must be the same. As is well known, i t is difficult to form a suspension-e.g., barium a manner sulfate, silver chloride-in which yields reproducible particle sizes. illso, both the initial particle size and rate of change of size with time are subject to a variety of chemical and physical influences. It is evident why empirical turbidimetric and nephelo-
