anion is a potential interference for the others. If more than one of these anions is present in a sample, some type of separation would have to be devised. Oxidizing substances present would lead to high results by oxidation of the iodide added while reducing agents would cause low results by reacting with some of the iodine formed. However, cadmium iodide, which is used in the linear starch reagent, is resistant to oxidation (13). However, for those samples where there are no potential interferences, the mercuric iodate method should be advantageous. General Aspects of the Methods. The extent of reaction between all of these anions and mercuric iodate is quite high as can be seen by comparing the molar absorptivity values for iodate ion and a specific anion, corrected for the dilution factor. The reactions are in the range of 75-80z complete in 10-15 minutes. It is necessary to ensure good mixing to have the reaction be as complete as possible in minimum time. Separation of the excess solid from the solution prior to developing the color is of extreme importance. Filtration and centrifugation were both tried. Mercuric iodate seems to be somewhat easier to remove than mercuric chloranilate. Centrifugation is somewhat easier and faster and usually is sufficient. Results were possibly somewhat better for the lower levels when both separation methods were used. Different concentration levels for these anions could be determined by simply using different dilution factors. Effective molar absorptivities and concentration ranges are reported for 5-100 and 24-100 dilutions. In one trial, a 2-100
dilution was used and Beer’s law followed for chloride ion from 2-36 ppm, the effective molar absorptivity being 1800. Another approach to the problems of a high blank, other than a dilution procedure, is to add a constant amount of a sodium thiosulfate solution to each sample solution to remove an amount of iodine equivalent to that in the blank. This procedure was used in the method employing silver iodate (6). This was tried in one series of measurement for sulfite ion in which water was used as solvent and a 5-100 dilution used. The effective molar absorptivity for sulfite was 4800, Beer’s law being followed over the range of about 2-30 ppm sulfite. If interfering anions are not present or can be removed, this method for the determination of anions should be useful for accurate measurements at very low levels. The procedure is simple, reasonably fast, and requires a minimum of chemicals and manipulation. Reagents used are stable and impurities are not troublesome, as is sometimes the case with mercuric chloranilate (21). The color formed is also stable for a matter of hours. The possibility of adjusting the sensitivity by using different dilution factors should also be advantageous.
RECEIVED for review June 23, 1972. Accepted September 11, 1972. The authors express their appreciation to the Robert A. Welch Foundation of Houston, Texas for support of this Research. This work was part of the M.A. thesis of Willie L. Hinze, Sam Houston State University, May 1972.
Ethchlorvynol Reagent for Functional Groups Detection Frank F. Fiorese, Joerg N. Pirl, Paul R. Manio, and Anthony Carella’ Department of Public Health, Bureau of Toxicology, 1800 West Fillmore Street, Chicago, Ill. 60612
ETHCHLORVYNOL (l-chloro-3-ethyl-l-penten-4-yn-3-ol) is a hypnotic drug marketed under several trade names the most common being Placidyl. Several procedures for its detection have already been reported (1-4); Wallace and Washburn (2,5) pointed out that in the presence of strong acids such as HCI, the carbonyl derivative of ethchlorvynol, 1-pentyne-3one, is formed. This property could be appropriately utilized for analytical purposes as we found in this laboratory that in the presence of concentrated orthophosphoric acid, ethchlorvynol adsorbed to Florisil, will react with carbamates, primary aromatic amines, hydrazine, indole derivatives, and some sulfur-containing compounds to form different colored products. In this communication, we report data gathered with respect to the interactions of ethchlorvynol with representative classes of various chemical compounds.
Table I. Results of Interaction between Different Functional Groups and Ethchlorvynol Reactive Color Color in Color Chemical functional in CHC1, on in family group CHC13 standing EtOH Green Dark Discolors Carbamates //O R-CH,-C--SH: green Primary Red Yellow Dark aromatic yellow amines
1 Present address, Office of the Chief Medical Examiner, 520 First Avenue, New York, N.Y.
EXPERIMENTAL
(1) C. J. Umberger, Office of the Chief Medical Examiner, 520 First Ave., New York, N.Y., personal communication, 1963. (2) J. F. Wallace, W. T. Wilson, and E. Dahal, J. Forensic Sci., 9, 342 (1964). (3) . , E. J. Alaeri, L. G. Kostas. and M. A. Luonogo, - . Amer. J . C1k. Parhof., 38, 125 (1962). (4) A. W. Davidson, J. Ass. Ofic. Aizal. Chem., 53, 834 (1970). (5) W. H. Washburn and F. A. Scheske, ANAL.CHEM.,29, 346 (1957). 388
Apparatus. All reactions were carried out on a 7 X 0.4 cm cylindrical glass tubing plugged at one end with glass wool and filled two-thirds with activated Florisil (60-100 mesh). Reagents. All reagents used were of analytical grade: 2 g per cent ethchlorvynol in ACS chloroform (ethchlorvynol reagent); concentrated orthophosphoric acid (OPA) and 95 ethanol.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
Table 11. Detection of Ethchlorvynol by Representatives of Chemical Families Detection Detection limit in limit in Chemical family Representative CHC13,Mg Color in CHC13 ethanol, fig Carbamate Meprobamate 3 Green ... Primary aromatic amine Benzocaine 50 Yellow 3 Hydrazine 100 Yellow 30 Nardil Indole Ergotrate 3 Blue ...
Table 111. Results of Interaction between Some Nonphenothiazine Sulfur Derivatives and Ethchlorvynol Chemical Malathion Tapazole Glutathione Thiobarbituric acid Methapyrilene
Color in CHC1, Red Pink violet Red-green Green-red Green
Procedure. Florisil was activated by heating at a temperature of 200 "Cfor 2 hours and cooled or preserved in a vacuum desiccator prior to use. Two-drop aliquots of a chloroform solution of the sample to be analyzed were added to the top of two identical Florisil columns. Three drops of the ethchlorvynol reagent were then added t o each column followed by the addition of 3 drops of concentrated OPA. After 1 minute, 5 drops of 95% ethanol/water or other polar solvent such as dimethyl sulfoxide were added to one of the columns, the other served as a control. The colors developed were recorded immediately and 5 minutes later. To determine their stability, the intensities of the color produced were scored with a system of +'s at both times. RESULTS AND DISCUSSION
The best results were obtained with concentrated OPA since other acids such as sulfuric, nitric, hydrochloric, perchloric, and acetic did not perform as well either in their concentrated or diluted forms. I n addition, Florisil proved to be the best adsorbent when compared to cellulose, Silica Gel G, infusorial earth, aluminum oxide, and many synthetic resins; even though Florisil had to be activated first, suggesting that excess moisture may interfere initially with these reactions. The action of Florisil is as yet not completely understood. I t is unlikely that it serves only a n adsorbent function since other known adsorbents were ineffective; probably its basic property may be a n additional asset to the efficiency of these reactions. Furthermore, none of these reactions occurred when conducted in solutions in test tubes or o n Whatman No. 1 filter paper. The ethchlorvynol reagent was prepared in chloroform for convenience but low petroleum distillates and other nonpolar
Color in EtOH Colorless Red Violet Discolors
solvents were found also to be adequate. In these solvents, the control test is colorless whereas with aromatic solvents the blank is violet. The chloroformic solution of ethchlorvynol is stable for several weeks a t room temperatuie; however, polymerization sets in eventually, yielding a dark-brown solution which should be discarded. The functional groups reacting with the reagent and the colors of the respective products are shown in Table I. Ethchlorvynol itself can be detected by many members of the aforementioned chemical families as shown in Table 11. Carbamates. Four types of carbamic acid derivatives were investigated; derivatives of monocarbamic acid (hydroxyphenamate); dicarbamic acid (meprobamate); N-substituted carbamic acid (carisoprodol); and cyclic carbamic acid (metaxalone). The reactivity of the carbamates is related to the alkyl group, 1" alkyl > 2" alkyl > 3" alkyl. Carbamates having tertiary alkyl groups are strongly affected by mineral acids and hydrolyze largely under the stated reaction conditions resulting in poor color formation. The detection limit for meprobamate, which was the most reactive carbamate studied, was 3 pg. Primary Aromatic Amines. The primary aromatic amines studied belonged to two main families: anesthetics and sulfa drugs. Sensitivity was directly proportional to solvent solubility and to the number of amino groups present in the molecule. The colors produced after addition of 9 5 x ethanol/water or dimethyl sulfoxide varied from red to redviolet (Table I). The detection limit for benzocaine, which was the most reactive amine studied, was 3 pg. Sulfur-Containing Compounds. Table 111 gives the results of the colors produced between a number of sulfur-containing. nonphenothiazine chemicals. The results with phenothiazine derivatives were anomalous since a pink to dark violet color was obtained without the ethchlorvynol reagent, but this color disappeared on addition of 95 ethanol. Hydrazine Drugs. All the hydrazine derivatives studied gave a yellow color with the reagent (Table IV) except the monosubstituted aromatic hydrazines which yielded red or violet colors upon the addition of alcohol, water, or dimethyl sulfoxide. The detection limit for phenelzine, which was the most reactive hydrazine studied, was 3 pg.
Table IV. Results of Interaction between Representative Hydrazine Derivatives and Ethchlorvynol Hydrazine derivative Formula Color in CHC1, Color in EtOH Iproniazid Yellow Yellow p-PyCONHNHCH(CHa)z Nialamide Yellow Yellow p-PyCONHNHCHzCHKONHCHzPh Isocarbazid Phenelzine Isoniazid Phenylhydrazine
PhCHZNHNHCOC=NOCH( CH3)CHz PhCHzCHzNHNHz p-PyCONHNHz PhNHNH2
Yellow Yellow Yellow Yellow
Yellow Red Yellow Violet
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Tab!e V. Results of Interaction between Representative Indole Derivatives and Ethchlorvynol Name LSD maleate Ergotrate maleate Reserpine Methysergide Rescinnamine Yohimbine Meprobamate : Reference
Color in chloroform Initially Later Violet Light blue Pink Violet Violet Light brown Green
Green-blue Green-blue Violet Green-blue Blue Violet Dark green
Indole Derivatives. Table V shows the colors obtained for a number of indole-containing drugs. LSD, as well as other n-substituted lysergic acid amides produced a violet color initially during the reaction but changed to a more stable bluish green. Once a color was established o n addition of 95 ethanol, it remained stable during the 5-minute experimental period. ACKNOWLEDGMENT
We are grateful for the excellent technical assistance of Gale Rosenquist and for helpful discussions with Edet E. Inwang. RECEIVED for review March 30, 1972. Accepted August 21, 1972.
New Kinetic Method for Determination of Ultramicro Quantities of Organic Substances Determination of Amino Acids (Glycine, DL-Serine, DL-Phenylalanine, DL-Glutamic Acid, and L-Arginine) T. J. JanjiC and G . A. MilovanoviC Chemical Institute, Faculty of Sciences, University of Belgrade, Belgrade, Yugoslavia A NEW KINETIC method for the determination of ultramicro quantities of amino acids is described in this paper. The catalytic activity of copper in the reaction of oxidation of pyrocatechol violet by hydrogen peroxide has been found to decrease in the presence of ultramicro quantities of amino acids, because of the formation of 1 :1 complexes. Since the decrease in catalytic activity turned out to be proportional to the quantity of amino acid present, a method whereby amino to acids can be determined in concentrations from 2.0 x 8.0 X 10-6Mhas been developed. There are many detailed kinetic studies concerning the determination of ultramicro quantities of inorganic ions ( I ) . I n contrast, there is a considerably smaller number of reports dealing with kinetic methods for the determination of ultramicro quantities of organic compounds. The best investigated are the enzyme-catalyzed reactions that have been used for the determination of enzymes themselves (2-6), substrates (7-10), activators ( I I ) , and inhibitors (12, 13).
In order to extend the kinetic methods of analysis to the field of organic compounds, we have decided to investigate changes (decrease or increase) in the rate of some simple metal-catalyzed reactions caused by changes in the catalytic activity of metal ion, due to the formation of metal complexes with organic substance added. We have anticipated that the change in catalytic activity would be dependent o n the quantity of the complex formed, which could provide a basis for evaluating the quantity of the organic substance present. It should be mentioned that such an idea was first put forward by Yatsimirskii (14). However, this author merely pointed out that the complex should be stable and catalytically inactive, but did not cite any experimental data in support of it. Generally speaking the reversible reaction of the formation of 1:l complex between a metal catalyst and a compound capable of coordinating it can be presented by the following general reaction scheme:
(1) K. B. Yatsimirskii, “Kineticheskie Metodi Analiza,” I1 Izd.,
M + L ~ M L
Izdatelstvo “Khimia,” Moscow, 1967. (2) W. J. Blaedel and G. P. Hicks, Anal. Biochem., 4,476 (1962). (3) W. H. Marsh, B. Fingerhut, and E. Kirsh, Clin. Chem., 5, 119 (1959). (4) M. K. Schwartz, G. Kessler, and 0. Bodansky, Ann. N . Y . Acad. Sci., 87,616(1960). ( 5 ) G. D. Winter, ibid., p 875. (6) B. Fingerhut, R. Feryola, W. H. Marsh, and J. B. Levine. ibid., 102,137(1962). (7) . , W. J. Blaedel and G. P. Hicks. in “Advances in Analytical Chemistry and Instrumentation,” Vol. 3, C. N. Reilley, Ed., Interscience, New York, N.Y., 1964, p 105. (8) H. V. Malmstadt and T. P. Hadjiioannou, ANAL.CHEM.,34, 455 (1962). (9) Zbid.,35: 14(1963). (10) 0. N. Kramer, P. L. Cannon, and G. G. Guilbault, ibid., 34, 842 (1962). (11) G. P. Hicks, Ph.D. thesis, University of Wisconsin, Madison,
where M is the catalyst, L is the ligand added in a subequivalent quantity in relation to catalyst M to prevent the formation of complexes with higher ligand-metal ratios; M L is the 1:1 complex possessing lower or higher catalytic activity than catalyst M. M indicates the catalyst in its active form, n o matter whether it is a hydrated metal ion o r any other species that can be formed in solution, or a mixture of them. Therefore, it follows that M L can have a more complex composition, too. L represents a ligand in all its forms present in a n acidbase equilibrium system. Considering the complexity of such systems, charges of ions are omitted. Applying the law of mass action to Reaction 1 and denoting the total concentration of each component as [MI, [ML], and [L] we obtain:
Wis.. 1963. (12) H. W. Linde. ANAL.CHEW.31. 2092 (1959). (13) 0. N. Kramer, P. L. Cannon, and GI G. Guilbault, ibid., 34, 1437 (1962).
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(14) K. B. Yatsimirskii, “Kineticheskie Metodi Analiza,” I1 Izd., Izdatelstvo “Khimia,” Moscow, 1967, p 92.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 2, FEBRUARY 1973
