THALLIUM POISONING I. T H E DÉTECTION OF THALLIUM IN BIOLOGIC MATERIAL ALEXANDER O. GETTLER AND LOUIS WEISS From the Chemical Laboratories of Washington Square Collège, New York University, and of the Chief Médical Examiner's Office of New York City * Before proceeding to the actual détection of thallium, ail organic matter in the tissue must be destroyed by oxidative processes. The following procédures hâve been described for this purpose. 1. Dry ashing in the présence of an excess of NaOH. On heating, the gummy alkaline mass has a tendency to spatter, causing loss of material. It therefore becomes a tedious and time consuming opération to dry the alkaline mixture. Thallium salts are easily volatile, even in the présence of alkali, and therefore the ashing must be conducted at a dull red heat. The ashing is never complète at this température even after many hours of heating, and therefore the acid extract of the ash is often colored. The components causing the color, as well as the large quantity of salts that was introduced, interfère with many of the analytical procédures for thallium. 2. Digestion with HCl and KClOs. This procédure, known as the Fresenius and von Babo method, is so well known that a description of same may be omitted. The method is very tedious, time consuming and requires continued attention. The destruction of the organic matter is never complète, and the final solution contains excessive amounts of salts which interfère in the analysis for thallium. 3. Digestion with HN03 + HySOi. This method has replaced the HC1-KC103 digestion to a large extent. The procédure is well known and a description of same is not necessary. One advantage is that no salts are added. Although this method is less time consuming than the previous ones, it still takes at least two to three hours of continued attention. 4. Digestion with HNOs + H2SOt + HClOi. By using a HN03 + HC104 mixture after the HNOs + H2SO4 treatment, the time for the complète destruction of the organic material is greatly reduced. No salts of any kind are introduced. The final solution is free of ail organic material, and is perfectly colorless. Our experiments hâve proven that this is the best method for the complète destruction of the organic material in tissue. A detailed description of the procédure will follow. Qualitative tests for the détection of thallium. There are two kinds of thallium ions, the monovalent thallous ion (Tl+), and the trivalent thallic ion (T1++"1"). The thallic ion is very unstable, and spontaneously changes into the thallous ion. On dissolving thallium in minerai acids (even nitric acid) the thallous ion is always produced. Oxidizing agents such as bromine, chlorine or aqua regia convert the thallous ion into the unstable thallic ion. The properties of the thallous ion simulate those of the monovalent éléments, and that of the thallic ion the trivalent éléments. The following tests for the détection of thallium in aqueous solution may be found in the literature. Thallous ion. 1. HCl produces a white precipitate of T1C1. The test is not a sensitive one, since T1C1 is fairly soluble. The test is not spécifie for thallium, since AgCl, HgCl and CuCl also corne down as white précipitâtes. 2. HBr produces a pale yellow, almost white, precipitate of TIBr. The test is much more sensitive than the previous one, but it is not spécifie for thallium since pale yellow AgBr and HgBr also precipitate. 3. HI produces a pale yellow precipitate of TH. This is the most sensitive test with halide ions. The test is not spécifie for thallium since yellow Agi, Pbl2, Hgl, red Hgl2 322 THALLIUM POISONING. I 323 (easily soluble in excess of reagent) and white Cul (discolored by iodine if Cu++ was présent) also precipitate. 4. Na2Cr04 in acetic acid solution produces a yellow precipitate of Tl2Cr04. The test is not very sensitive, nor is it spécifie for thallium since other ions such as Ag+, Pb++ and Ba++ are also precipitated as the red and yellow chromâtes respectively. Many reducing agents and large quantities of ammonium salts also interfère with the test. 5. H2S in alkaline solution produces a black precipitate of T12S. Many other metallic ions also yield black insoluble sulfides. 6. H2PtCl8 produces a yellow precipitate of Tl2PtCl6. The test is not sensitive nor is it spécifie for thallium, since K+ and NHi^ ions, which are always présent in tissue digests also, give a yellow precipitate. 7. Na3Co(N02)6 produces a red precipitate of Tl2NaCo(N02)6 in neutral or slightly acid solution. The test is not sensitive enough for the détection of very small quantities of thallium, and K+ and NHÎ ions, which are always présent in tissue digests, interfère with the test since they yield yellow précipitâtes. 8. KMn04 in sulfuric acid solution is decolorized by Tl+ ions. Ali reducing agents will do this. 9. (Kl + SbCU) reagent produces orange red crystals of 3 T1I-2 SbLj. The test is not sensitive. Quite often the test results negatively although small quantities of thallium are known to be présent. 10. Organie reagents (yielding colors or précipitâtes) such a thionilid, quinalizarin, dithizone and mercaptobenzothiazole) hâve been used for the détection of thallous ions. The tests with thèse organie reagents are not spécifie since many other metallic ions also yield colored compounds or précipitâtes. When working with digests of biologieal material the iron and phosphates interfère with some of thèse color reactions. Thallic ion. The thallous ion must first be oxidized to the thallie state. This is done by adding bromine water, dropwise, until the solution attains a yellow color. The excess bromine is then removed by adding a few drops of phénol solution. 1. NaOH or NEUOH produces a brown precipitate of Tl(OH)3. Many other cations also give précipitâtes with thèse reagents, and those of Ag, Fe and Mn are also brown. In the case of tissue digests, the phosphates of Ca, Mg and Fe will also precipitate on the addition of the alkali. 2. When Kl is added to a solution containing thallic ions, free iodine is liberated. Cu++, Fe+++, AsOé2 and oxidizing agents in gênerai will do the same. 3. Organie reagents (yielding colored compounds) such as benzidine acétate, dimethylp-phenylene diamine-HCl, a-naphthol and a-naphthylamine hâve been used for the détection of the Tl+++ ion. The tests are not spécifie, because other ions, notably Fe+++, Cu"1""1", Co++ and oxidizing agents in gênerai give similar colors or interfère in some other way. Thallium, dry reaction. If a speck of thallium halide, on a platinum wire, is inserted into the outer cône of a non-luminous gas flame, a characteristic emerald green flame is emitted, which lasts but a fraction of a second. When viewed through a spectroscope, one observes a green line at 535.0 im». Barium also gives a greenflame,and one of its visible lines is at 534.7 nnt. Large quantities of salts, especially sodium salts, greatly interfère with the test when small quantities of thallium are présent. By means of the spectrograph, thallium can be definitely detected by means of two of the several lines in its spectrum (2918.3A and 3519.2A). The instrument however is very expensive, and the spectrograms must be read by an experienced spectroscopist. On studying the various tests outlined above it is évident that no one test (except the spectrographic) is definite proof for the présence of thallium. Then again the tests must never be applied to the tissue digest directly, because of the many interfering components that may be présent, such as : (1) A high sulfuric aeid content. (2) Large quantities of Na, K, NH4, Ca, Mg, Fe and PO4 ions, which are always présent in tissues. 324 ALEXANDER O. GETTLER AND LOUIS WEISS (3) Ocoasionally ions which interfère with, or simulate the tests for thallium, such as Ag, Hg, Pb, Cu, Bi, As, and Sb may be présent in the tissues as a resuit of poisoning or therapeutic treatment. The first step therefore is the séparation of the thallium from thèse interfering components. Our experiments hâve proven that the removal of the latter from small quantities of thallium (expected range 0.1 mg. to 5.0 mg.) by précipitation of the interfering ions is not advisable for two reasons. The small amount of thallium présent may be entirely lost by virtue of co-precipitation, and even with the best methods for the removal of the iron, traces of the latter will always remain, and interfère with some of the reactions for thallium. A much better procédure is to precipitate the thallium from the other ions that are présent. The method finally adopted for the isolation and détection of thallium présent in a tissue digest follows. The digestion of biologie material. 50 Gm. of finely ground tissue or blood are placed into a 500 ml. Erlenmeyer flask, and 100 ml. of concentrated nitric acid are added. After allowing the mixture to stand in a warm place, with occasional stirring, for one hour the flask and contents are placed on a steam bath and allowed to remain until ail of the tissue is dissolved and évolution of N 0 2 has virtually ceased, about two hours. The contents are then permitted to cool slowly to room température. In order to avoid explosive reactions during the perchloric acid digestion, any solidified fat is filtered off, through glass wool, and discarded. The nitrate is collected in a 300 ml. Kjeldahl flask. When analyzing bone, 50 Gm. are placed into a large beaker, 100 ml. of nitric acid are added, the beaker covered with a watch glass, and placed upon the steam bath. When the bone is entirely dissolved, the slowly cooled yellow solution is filtered through glass wool into a beaker. 10 ml. of concentrated sulfuric acid are slowly added, with stirring. A copious precipitate of calcium sulfate forms. After allowing to stand about one hour it is filtered into a 300 ml. Kjeldahl flask. When analyzing urine samples, large volumes (one liter if possible) should be used. The sample is evaporated to a syrupy consistency on the steam bath. 100 ml. of concentrated nitric acid are then gradually and very carefully added, since a violent reaction ensues. The contents are permitted to remain on the steam bath until a clear yellow solution results, which is then transferred to a 300 ml. Kjeldahl flask. The nitric acid digest of tissue, blood, bone or urine in the Kjeldahl flask, after adding 6 ml. of concentrated sulfuric acid and several small glass beads, is boiled over a free flame. When ail of the nitric acid has been boiled off, charring begins, and is generally accompanied by frothing. It is well to lower the flame at this point, and then to increase it again when the charred liquid has ceased frothing. If during the charring the contents solidify, allow to cool, and add 2 ml. of concentrated sulfuric acid and continue heating. When the charred liquid has ceased to froth, the flame is raised, and a digestion mixture, composed of two volumes of 66 per cent perchloric acid and one volume of concentrated nitric acid, is added dropwise. When 0.5 ml. of the digestion mixture has been added (dropwise), further addition is deferred until frothing ceases again. Additional one half ml. portions are thus added dropwise until the color of the hot liquid has changed from black to red to yellow and often to colorless or white. THALLIUM POISONING. I 325 About ) seven ml. of the nitric-perchloric mixture are consuméd for 50 Gm. of tissue, and if added during an interval of approximately ten minutes will give the desired resuit. When there is no further change in color, the nitric-perchloric treatment is stopped. The heating is continued until sulfur trioxide fumes evolve. The flask and contents are allowed to cool slowly to room température. Ten ml. of water are added, and again boiled until sulfur trioxide fumes are given off. This treatment is repeated once more, in order that ail traces of nitric acid and oxides of nitrogen are destroyed and expelled. The contents are then allowed to cool slowly to room température, and 25 ml. of distilled water are added. The final solution should be colorless when permitted to cool slowly to room température. The diluted digest is nltered into a large test tube, and used for the précipitation of the thallium. The détection of thallium in the diluted tissue digest. Sulfur dioxide is bubbled through the solution until it is saturated with the gas. One-half ml. of hydriodic acid is added dropwise, the bubbling of sulfur dioxide being continued during the addition and five minutes thereafter. If no turbidity is observed, thallium is absent, and the analysis is discontinued. Should a turbidity or precipitate form, allow to stand not more than one hour, then collect the precipitate by centrifuging, and discard the supernatent liquid. The formation of a precipitate, even if pale yellow, at this stage does not mean that thallium is présent, for Hg and Cu will also precipitate. At times yellow colloidal sulfur may form due to the réduction of the S0 2 by HI. Some analysts hâve made the error of considering the formation of a yellow cloud or precipitate as a final positive test for thallium. The precipitate in the centrifuge tube is washed twice with 2 ml. portions of HI solution saturated with S0 2 and then once with dilute ammonia solution and once with a little acétone, in order to remove contaminating précipitâtes. The washed precipitate remaining in the centrifuge tube is dissolved in 2 ml. of dilute nitric acid (1 to 3), transferred to a small watch glass, and evaporated to dryness on the steam bath. A few drops of water are added, and again evaporated to dryness, in order to remove ail traces of free nitric acid. The residue should now consist of white thallous nitrate. It is dissolved in 1 ml. of distilled water. If not perfectly clear the solution is centrifuged. The supernatant solution containing the thallium is divided into five approximately equal portions (about 0.2 ml. each) which are placed into micro centrifuge cônes and used for the following tests. Thallium is a unique élément, in that its thallous ion behaves in many ways as the éléments of Group I and of Group V, and its thallic ion behaves as the metals of Group III. In order to definitely prove the présence of thallium we hâve therefore chosen tests characteristic of each of the three groups mentioned. 1. Précipitation as thallous bromide. By means of a capillary tube, sulfur dioxide is bubbled through the solution in the centrifuge cône and then one drop of HBr solution is added, the bubbling continued for a few seconds. The precipitate that forms is centrifuged, so that its color may be better seen. Thallous bromide is very pale yellow, almost white. 2. Précipitation as thallous iodide. The test is done in the same way as the 326 ALEXANDER O. GETTLEK AND LOUIS WEISS bromide test, except that HI solution is used instead of HBr. Thallous iodide has a pale yellow color. 3. Flame test. A portion of the precipitated and centrifuged halide from either of the above two tests is placed on the tip of a platinum wire, and introduced into the outer cône of a non-luminous gas flame. A characteristic green color of short duration may be observed. If observed through a hand spectroscope, a green line will be seen in the spectrum. 4. Précipitation as thallous chromate. One drop of dilute ammonia, and one drop of dilute potassium chromate are added to the solution to be tested. A yellow precipitate of thallous chromate should resuit. 5. Précipitation as thallic hydroxide. The thallium is oxidized to the thallic state by adding bromine water dropwise until a yellow color persists. After five minutes, ammonia solution is added dropwise until alkaline. A brown precipitate indicates thallic hydroxide. 6. Précipitation as thallous cobalti-nitrite. A small drop of sodium cobaltinitrite reagent is added, then warmed to 50 C and allowed to cool slowly. A scarlet red crystalline precipitate of thallous cobalti-nitrite should form if thallium is présent. In order to obtain the best results with the cobalti-nitrite test, the reagent should be prepared as foUows: Dissolve 20.7 Gm. of N a N 0 2 and 29.1 Gm. of Co(N0 3 ) 2 -6H 2 0 in 50 ml. of distilled water, and add one ml. of 6N acetic acid. Let the mixture stand for 24 hours, then filter and dilute to 100 ml. The reagent will keep for several weeks. Using the method as described we fiave been able to isolate in pure form, and obtain ail six tests which are necessary for establishing the présence of thallium, in biological material which contained as little as 0.05 mg. of thallium in 50 Gm. of tissue. This sensitivity is ample from a toxicologie standpoint. The smallest quantity we found in our séries of deaths due to thallium poisoning was 0.1 mg. in 50 Gm. of tissue. SUMMARY 1. Various methods for the digestion of biologie material are reviewed. 2. The method of digestion which the authors hâve found to be the least time consuming, and at the same time yielding a solution free of ail organic substances is described in détail. 3. The various qualitative tests for thallium are critically reviewed. 4. A detailed description for the détection of thallium in biologie material is given.
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