Jack L. Pinkus and Lyle S. Goldman Clark universitv Worcester, Massachusetts 01610
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A Benzidine Rearrangement and the Detection of Trace Quantities of Blood A laboratory experiment in criminalistics
Formerly one of t h e classic experiments in t h e organic chemistry laboratory was t h e benzidine rearrangement. Benzidine (I) a n d i t s salts a r e considered dangerous carcinoe e n s in m a n causine an increased incidence of bladder cancer.1,2T h e ~ c c u p a ~ o nSafety al a n d H e a l t h Administration (OSHA) has classified benzidine as a hazardous substance a n d h a s promulgated rules a n d regulations (Federal O S H A S t a n d a r d s CFR 1910.93j) regarding i t s use and handling.3 Criminalists have utilized (I) or o-tolidine(3,3'-dimethylbenzidine) (II), as a presumptive blood-test reagent. Unfortunately, (11) is also considered to be a c a r c i n ~ g e n . ~ , ' Recently, the synthesis of 3,5,3',5'-tetramethyIbenzidine(V1) was described by Holland and coworkers (see the fig.).5 The hindered amine 2,6-dimethylaniline(II1)was oxidatively coupled with potassium ferricyanide to furnish the am compound (IV), which after reduction with zinc dust and ammonium chloride to the hydrazine (V), was converted to (VI) in the presence of sulfuric acid. The overall yield was about 15%. Animal studies, though limited, suggested that (VI) was non-carcinogenic. We have modified the work-up procedure by removing basic impurities in the oxidation step prior t o column chromatography thus allowing a less costly and facile purification. The rapidly eluted red azo compound (IV) is reduced and converted to (VI) with a variety of acid catalysts. We found for the fimt time that methanesulfonic acid is an efficient catalyst for tlre henzidine rearrangement. Compound (VI) is then utilized for the detection of blood. For comparison plant peroxidases are also tested. These peroxidases are found in the narticulate contentsof the cells of the ~ l a ntissue. t Thus plant juices aluwgivra nrgnrworonly a faint positive reaction. The plant pwoxida~wimnd. for example, in m i m , cahuagc, and porato areshwvn ro br heat l a h h (lInl0C,5 min, and can tw differentiated from a true hlood stain. Animal hemoglobin peroxidases are very stable substances. Blood stains many months old still give strong positive reactions. ~
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Experimental 2,6,2',6'-Tetramethylazobenzene ( I V ) In a 250-mI beaker dissolve 18.0 g (66.9 mmole) of potassium ferricyanide and 2.55 g (63.9 mmole) of sodium hydroxide pellets in 90 ml of distilled water. In a 50-ml beaker, place 1.20 g (9.9 mmole) of 2,6-dimethylaniline (111) and add 25 ml of 1N hydrochloric acid. Heat both solutions to 9&96'C using a Bunsen burner; then remove from the heat. Immediately add the solution of the amine salt to that of the potassium ferricyanide solution while stirring with a glass rod. A vigorous reaction occurs and a black tar forms on the surface. Stir the reaction mixture for 5 min; then cool in an ice bath. Pour the cold reddish-brown reaction mixture, including the black tar into a 250-ml separatory funnel. Extract with 2 X 50 ml of diethyl ether. (Caution: Make sure that all flames are extinguished before undertaking the extractions with ether.) Wash the ether extracts with 2 X 100 ml of 4 N hydrochloric acid. Discard the aqueous layer. Filter the ether layer through a Buchner funnel t o remove the precipitated amine salts. Triturate the precipitate with 20 ml of ether to extract residual product. Wash the combined ether solutions with 2 X 50 ml of water. Dry the ether solution over anhydrous sodium sulfate. Gravity filter (or decant) the ether into a 250-ml round-bottomed flask. Evaporate the ether under reduced pressure t o obtain about 0.6 g of a reddishbrown gum. Prepare 100 ml of a chloroform-hexane (1:4, vlv) solution. Alternativelv. mav be substituted for the ,. oetroleum ether (b.o.60-80°C) . hrxaw I'our h ml of the a c h m mixture into a 25. o r a 50-ml buret equipped prefcrnhly with a Teflon st ml methanol and 0.5 rnl saturated d i u m acetate aolutim. A few drops of acetic acid is then added.
Detection of Blood Stain Prepare blood and vegetable stains on cloth or filter paper. The vegetable stains should be fresh. Portions of the stained materials are distributed as unknowns to thestudents. Testingmay be done in two ways. Rub a tip of folded filter paper several times over a dried stain. Add twodropsof the test reagent to the folded filter paper tip. After s brief interval (30 s) to ensure that no color develops, one drop of 3% hydrogen peroxide is added. Alternatively, a section of stained cloth or filter paper is added to a small culture tube and covered with the test reagent. After agitating the tube for 1 min a drop of 3% hydrogen peroxide is added. An immediate deep blue coloration indicates a oositive reaction. If the stained sections in the tube are heated on the steam hath for5-IOminucrs prmr rtlrestiny, the lal,ile plant p e n w idaresnredewoyed while rhr true hlcxd stnln u,ill stillgiwa prwitiw test. ~~
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Volume 54, Number 6. June 1977 1 381
