V O L U M E 25, NO. 4, A P R I L 1 9 5 3
653
Table 11, and with the results of Malouf and White; hence, values in Table I1 are given to the nearest 0.05 microgram. DISCUSSION
An examination of Tables I and I1 s h o w that the two methods give approximately the same rhenium content. The precision of the modified method is slightly better. When no chloride ion was present in the solution to be analyzed by the modified Tribalat method, there was a greater loss of rhenium by adsorption on the calcium chloride used for drying the chloroform extract; accordingly, a 0.3 M initial chloride ion solution was used. The modified Tribalat method requires approximately 2 hours for analysis of a sample; hence, is shorter than the Malouf and White method. I n addition, it is much more economical from the standpoint of chemicals.
ACKNOW LEDG llENT
The authors wish to acknowledge financial assistance from the University of Utah Research Committee and from the University of Utah Kennecott research grant. They also wish to thank E. E. Malouf of the Kennecott Copper Co. for his valuable suggestions. LITERATURE CITED
(1) Geilrnan, IT.,JT‘rigge, F. IT.,and Weibke, F., Z. anory. allgem. Chem., 208, 220 (1932). (23 hlalouf, E. E . , and White, hf. G., A b i ~ 4CHEM., ~ . 23, 497-9 (1961). (3)
Snyder, Harold H., Ph.D. thesis, University of Wisconsin,
(4)
Tribalat, Suzanne, A n a l . Chi?%.Acta., 3, 113-26 (1949).
1946.
RECEIT-ED for rex-iew September 8, 1952.
.iccept?d December 6, 1952
A Modification of Winnick’s Method for the Raoid Determination of Ethyl Alcohol in Biological Flu/ds IRVING SCNSHINE
AND
ROBERT NEYAD
Cuyahoga County Coroner’s Ofice Laboratory, Znstitute of Pathology of Western Reserce University, and C‘nirersity Hospital, Cleveland, Ohio 4 ? * potentially ~
preventable deaths from trauma or disease have resulted from the erroneous assumption that the odor of alcohol combined with staggering gait and slurred speech. or with coma, is valid evidence that the individual is under the influence of alcohol. A simple qu:tutitative determination for ethyl alcohol in biological fluids is obviously desirable to aid in rapidly establishing a dingnosic of alcoholic intosication in the hospital emergency room. 80
-
70
-
OTHER ACCIDENTS
0
~ 6 0 -
HOME ACCIDENTS
P 10-
-
10 20-
ACCIDEWTS
HOMICIDES
nI SUICIDES
I I
Figure 1. Incidence of Alcohol in Violent Deaths in Cugahoga County, Ohio, 1943-51
Ethyl alcohol determinations are the most common laboratory analyses in forensic medicine (Figure 1). 1Iore than 45% of all individuals involved in vehicular fatalities are found to have recently ingested an alcoholic beverage. I t is reasonable to assum& that in many instances the resulting impairment of judgment and reflex response has contributed to the occurrence of these accidents. In Cuyahoga County (Cleveland) it has been found that 6170 of the victims of homicide and 30% of all suicides are under the influence of alcohol at the time of their death. Prompt and accurate determination of ethvl alcohol is essential to the administration of justice. The large metropolitan police laboratorv concerned with a surge of alcohol determinations after a week end of activity would benefit from a rapid test that could be done “en masse.” Sunierous methods have been described in the literature for the quantitative determination of ethyl alcohol. Most of these are based on its volatility and reducing power. I n the main these procedures separate the ethyl alcohol from other biological ma-
terial by distillation ( 4 , 6, 8). Some aeration procedures have been described ( 2 , 6 ) , but these are seldom used because they are not adapted to multiple analyses. Diffusion desiccation methods (1, 9 ) , particularly Widmark’s, have been favored on the European continent but are not used extensively in this country. The distillate (diffusate) is then oxidized, usually by potassium dichromate. Two procedures have been described using Conway ( 3 ) cells for the determination of alcohol. Winnick (10)placed the test material in the outer compartment of the Conway cell, and the alcohol which diffused reduced potassium dichromate which was placed in the inner compartment. The excess dichromate ion then was determined iodometrically. MacLeod ( 7 ) used a similar apparatus but absorbed the alcohol in alkaline potassium permanganate. T o determine the excess permanganate, the contents of the center well were reduced by thiourea in the presence of barium ions. Potassium carbonate was added to the outer section to hasten the diffusion of the ethyl alcohol. The present method also uses potassium dichromate in the central well of the Conway cell. The complete diffusion (evaporation) takes 20 minutes. Results are thus obtained quickly so that they have clinical value. The amount of alcohol present is determined colorimetrically. The contents of the center well are compared with prepared standards, or the optical density is measured with a photoelectric photometer. RE4GEYTS
Potassium Dichromate. Dissolve 4.262 grams of analytical reagent grade potassium dichromate in 100 ml. of distilled water. Carefully add, while cooling, 500 ml. of reagent grade sulfuric acid, and then dilute this mixture, while cooling, to 1000 nil. with distilled water. One nil. of this reagent is equivalent to one mg. of ethyl alcohol. Sodium Carbonate, 20% Solution. Dissolve 20 grams of sodium carbonate in water and dilute to 100 ml. Standard Alcohol Solutions. Dilute absolute ethyl alcohol with distilled water to known concentrations varying from 50 mg. to 600 mg. per 100 ml. solution. Dow Corning Silicon‘Stopcock Grease. PROCEDURE
Pipet 3.00 ml. of potassium dichromate into the center well of reservoir buret will facilitate the performthe Conway cell. (-4 ance of a large number of analyses.) .idd 1 ml. of 20y0 sodium carbonate to the outer compartment. Apply silicone stopcock
ANALYTICAL CHEMISTRY
654 Table I.
Recovery of Alcohol from Biological Fluids .-4l~oholFound (COnCn. % W t .VOl.)
Alcohol Added (Concn, % Wt. I Vol.)
Blood
0.06
0.06 0.06 0.16 0.16
0.15
0.?7
.
0.28 0.28
0.36
0.36 0.36
0.48
0.48 0.48
0 54
0.52 0.52
Urine 0.07 0.08 0.16 0.14 0.27 0.28 0.37 0.34 0.48 0.48 0.64 0.53
grease to the surface of the ground glass plate, and place it over the Conway cell so that only a small portion of the outer compartment is uncovered. Place 0.50 ml. of the fluid to be analyzed in the outer compartment through the uncovered area and completely cover the cell. Carefully tilt the entire cell several times t o allow the liquids to cover the surface. Then incubate the Conway cell a t 90' C. for 20 minutes in an oven or water bath. (-4&liter beaker containing a rack that holds 5 cells has been effectively used instead of the oven. Place water in the bottom of the beaker and heat. Control the temperature by placing a watch glass cover over the beaker top.) .4fter incubation, remove the cell from the oven (steam bath), and remove the lid. Compare the resultant color in the center well with prepared standards. A green color is immediate presumptive evidence of intoxication. For forensic purposes carefully remove the contents of the center well with a pipet, rinse the well twice with distilled water, and then dilute the mixture to 25 ml. Compare this solution with suitable standards, or read in a photoelectric photometer using a blue filter (450 mp). Determine a calibration curve by diluting a prepared blood sample containing 0.60% alcohol, and processing each sample in duplicate according t o the procedure given above. This curve may be used in evaluating unknown samples. .4t 450 mp this calibration curve is linear over the range 0 to 0.55% of ethyl alcohol (0-550 mg. per ml. of fluid). In cleaning the cells remove the excess silicone first. Rinse the cell thoroughly in cold water. Wipe the lids free of excess silicone. Then soak both lid and cell in detergent, rinse thoroughly with tap water and distilled water, and dry. (Occasionally toluene may be used to clean the lids before the soaking step.) EXPERKMENTAL
Known amounts of alcohol were added to blood and urine samples, and these samples were assayed for ethyl alcohol. Table I gives the data. These data indicate that the recovery of alcohol is within &0.02% of the amount added. Other data indicate that the precision of each measurement is within 3%. To ascertain the optimum conditions of time and temperature. a sample of blood containing 0.40% alcohol was processed in the Conway cells as described above. Twenty-four Conway cells were run simultaneously at a given temperature. At fixed time intervals a pair of cells were taken out of the constant temperature bath and analyzed. Figure 2 illustrates the data for 2 5 O , 36", 50°, and 90" C. All the reactions were completed in a measurable period of time. The temperature of choice is 90' C. because the reaction is complete in the shortest period of time. If a higher temperature is used, splattering occurs, and the analysis becomes unreliable. DISCUSSION
For optimum usefulness the quantitative determination of ethyl alcohol should give a reliable result within 30 minutes; be simple in technique and require inexpensive equipment; permit parallel analyses to be completed in a short period of time; require one standard solution; and be capable of accurate rapid performance by the average clinical technician.
The microdiffusion method satisfies the above criteria. This method employing the Conway cell has several advantages over those previously described. Winnick's method is limited to samples containing 1 to 1.5 mg. of alcohol and frequently necessitates repeat analyses to select the proper aliquot. His procedure requires 2 hours a t 50' C. or 10 hours at room temperature. A standard sodium thiosulfate solution must be prepared frequently and checked. The color change a t the end point is abrupt and overtitration is not uncommon. Inasmuch as less than 2 ml. of sodium thiosulfate Is required, a microburet is essential for precise and accurate work. MacLeod also used a Conway cell for the determination of alcohol. A 0.1-ml. sample is required. Consequently capillary blood is frequently used, whereas venous blood is the accepted material used in ethyl alcohol determination. Accurate measurement of 0.1 ml. is difficult. The procedure is carried out a t room temperature and requires 3 hours for completion. The range in this method, F-hich is 3 mg. of ethyl alcohol, is greater than that of Winnick's. This is still below many commonly occurring alcohol levels. The permanganate reagent is sensitive to dust and light, and thus is unsatisfactory for an occasional determination, The alkali used in preparing the permanganate reagent reduces it; therefore a correction must be made each time this reagent is prepared. The method is feasible for daily analyses where time is not a significant factor. I t is undesirable for the occasional analysis inasmuch as the reagents deteriorate in 2 to 3 weeks.
100
-
IO IO
5c 70 2 60 zc 5 0 -
5 40-
::30 I
::: n-
1-
0
I
I
I
'h
I
2 1IM (HOURS)
I
a
I
4
Figure 2. Time Required for Complete Alcohol Diffusion The present adaptation of the Conway cell for alcohol determination can be performed in an area of 2 square feet by personnel with no special training. A single reliable result is obtained in 25 minutes. Twenty-four multiple determinations can be made in 2 hours. In the latter instance a thermostatically controlled oven is essential to house all the cells simultaneously a t the given temperature. Where time is not a factor (Figure l), other temperatures may be used. Using a photoelectric photometer, the procedure is sufficiently reliable for forensic purposes. Duplicate determinations agree to within 3%. Recoveries from blood and urine are within 0102% of the given value. For clinical purposes it is sufficient to visually compare the contents of the inner compartment with prepared standards. The specificity of this reaction is well documented (6, 8, IO). Methanol, paraldehyde, formaldehyde, and acetone interfere. When these substances are present they may give a false positive reaction. Acetone can readily be determined in the plasma or urine. The presence of ketone bodies yields a dichromate reduction equivalent to not more than 0.05% ethyl alcohol. For forensic purposes all positive tests should be followed by an acetone determination. If the presence of methanol, paraldehyde, or formaldehyde is suspected, a distillation must be performed and the distillate tested for these substances. Ethyl alcohol may
655
V O L U M E 25, NO. 4, A P R I L 1 9 5 3 cause coma a t levels above 0.30%. Therefore the special tests are not essential in the hospital emergency room. LITERATURE
(1) Cavett, J. W., J . Lab. Clzn. Med., 23,543 (1938). (2) Chaikelis,a.S., and Floereheim, R. D., Ani. J. Clin. Pathol., 16,
180 (1946).
(3) Conway,E. G., “Microdiffusion Analysis and Volumetric Error,” London, Crosley Lockwood and Son, 1947. (4) Gettler, A. O., and Freireich, -4.W.,J . Bid. Chem., 92, 199
(1931).
Am. J . Clin. Pathol., 4, 182, (1934). ( 5 ) Heise, A. 4., (6) Kaye, S., and Stolman, .L,in “Clinical Laboratory Methods and Diagnosis,” by R.B. H. Grada-ohl, Vol. 11, 4th ed., p. 2010, St. Louis, C. U. Jlosby 8: Co., 1948. 171 MacLeod, L. D., J . Biol. Chem., 181,323 (1949). i8j Shupe, L. M., and Dubowski, K., .4m. J . Clin. Pathol., 111 press. Widmark, E. hl. P., Biochem. Z . , 131,473 (1922). (10) T i n n i c k , Theodore, IXD.ENG. CHEM.,ANAL. ED., 14, 523
(1942).
RECEIVED for review September 8, 19.52. hccepted November 12, 1952. Presented before the Division of Biological Chemistry a t the 122nd JIeeting of the .4MERrChs CHEMICAL SOCIETY, Atlantic City, N. J.
Two Specific Methods of Determining Copper in Soil and in Plant Material KUANG LU CHENG1 AND ROGER H. BRAY University of Illinois, Urbana, Ill. NUMBER of procedures for the colorimetric determination of Acopper in soil and in plant material have been published. Lagerwerff (8) surveyed the best known of these and recommended the diethanolamine method. Beeson and Gregory ( 1 ) preferred the carbamate method for determining copper in plants. Lundblad et al. (9) determined copper in soil by a carbamate method in which interferences from iron, cobalt, or nickel were prevented by a series of separations. Steenbjerg and Boken (16) precipitated copper on a cathode by electrolysis, dissolved it in acid, and determined it colorimetrically by adding an excess of concentrated ammonia. Dithizone is also commonly used for determining copper in soil and in plant material ( I S ) . However, the methods mentioned are subject to interference and should be carried out under carefully controlled conditions. The object of this paper is to propose two simple and specific methods for determining copper in soil and in plant material. These are an improved carbamate method and the biquinoline method.
IMPROVED CARBAMATE METHOD
Sodium diethyldithiocarbamate reacts with copper to give copper diethyldithiocarbamate. This salt has a golden brown color. This reaction has been considered one of the most sensitive for copper that has been developed. The compound formed in this reaction has been assigned the following formula (15):
S (C1HB)? = S-C
//L
CUQ
8’ In very dilute copper solutions a colloidal suspension suitable for colorimetric comparison is obtained ( S ) , especially if it is stabilized by gum arabic or similar substances. Since the copper carbamate is soluble in many organic solvents such as isoamyl alcohol, isoamyl acetate, broniobenzene, and carbon tetrachloride, an extraction procedure is often used to decrease the effect of interfering ions and to increase the sensitivity of the reaction. Most metals, other than calcium a.nd magnesium, may interfere if present in sufficient amounts. The chief interfering metals are iron, manganese, and nickel, and the method has been criticized ( 2 ) because of the many interferences. Many complexing reagents such as pyrophosphate, citrate, and ammonium hydroxide have been recommended for eliminating iron interference, provided the amount of iron is not too large (12). Interference by nickel and cobalt can be prevented by adding dimethylglyoxime to the sample solution before adding the ammonium hydroxide. The precipitate is separated by filtration or centrifugation (9). Cobalt remains in the aqueous layer, to which it 1
Present address, Commercial Sol\ents Corp , Terre Haute, Ind.
imparts an orange color not extracted by carbon tetrachloride. Manganese interferes to a considerable extent in the extraction procedure by imparting a pinkish color to the organic layer. This color is more or less unstable. The solution becomes virtually colorless if only small amounts of manganese are present, or if the solution is allowed to settle for a t least 1 to 2 hours. When appreciable amounts of iron, nickel, or manganese are present, copper cannot be determined by the carbamate method without some chemical separation ( 1 7 ) . The principle of employing sequestering reagents to complex metals, as used by Schwarzenbach and coworkers (141, has long been known and has been applied many times in the field of analytical chemistry (7,10-16,15). If a mixture of Versenate and citrate solution is used in the carbamate procedure for copper, all interference can be eliminated except that from bismuth, which is ordinarily present in negligible amounts in both soils and plants. This principle is utilized in the following procedure. Reagents. Carbamate solution. Dissolve 1 gram of sodium diethyldithiocarbamate in 100 ml. of redistilled water, and filter. Versenate and citrate mixture. Dissolve 20 grams of ammonium citrate and 5 grams of the disodium salt of (ethylene dinitrilo) tetraacetic acid (Versenate) in 100 ml. of redistilled water. Ammonium hydroxide, concentrated. Carbon tetrachloride. Procedure. Place 25 to 50 ml. of the solution to be analyzed, containing not more than 50 micrograms of copper, in a suitable separatory funnel. Add 10 ml. of the Versenate and citrate mixture and 1 to 2 ml. of concentrated ammonium hydroxide to obtain a pH of 7 to 10. Mix, and add 1 ml. of 1% carbamate solution. Then add exactly 10 nil. of carbon tetrachloride, stopper, and shake vigorously for more than 2 minutes. hllow the carbon tetrachloride layer to settle, and, when it is free from xater droplets, run it directly into a dry absorption cell. Then pour another 5 ml. of carbon tetrachloride into the separatory funnel, and shake for 1 minute to extract the remainder of the copper carbamate from the solution. rldd this second extract to the cell nhich contains the first extract. Usually the second extract is almost colorless. Measure the color in the combined extracts with a spectrophotometer a t 500 mp. with a 1-cm. light path length. Make a calibration curve in exactly the same manner with standards containing from 0 to 50 micrograms of copper. If the carbon tetrachloride extract is cloudy, clear it by centrifugation, or by adding 0.5 ml. of methanol.
ELIMINATION OF INTERFEREXCE B Y 1-ERSENATE. Versenate forms a soluble chelate complex with many hi- and trivalent metals in the following way: Na2H2Y4
+ &leT‘ +SaJ\IeT4 + 2H+
where Me represents bi- or trivalent metals. The stability of the metal Versenate complex varies with different metals. Since, in general, Versenate requires an alkaline reaction in order to
