ANALYTICAL EDITION
June 15, 1942
TABLE I. ANALYSIS OF ACETASILIDE Series I
(Calcd. X = 10.3717,) Series I1
%
7c
%b
10.27 10.2s 10.263
10.25 10.24
10.33 10.34 IO.32
...
Series I11
A\.. 10.29
mined in grams of mercury and is subtracted from the weight of displaced mercury (the loss in weight of A ) . The percentage of nitrogen is given by the formula:
yo s
=
100
x
523
the weight azotometer, with satisfactory results. The sample of acetanilide nas ILIallinckrodt’s U. S. P. powder recrystallized twice from xater. I t s melting point was 114.20”, and a cooling curve proved it to be homogeneous. A series of ten Kjeldahl determinations with the acetanilide averaged 10.35 per cent, and the same number of determinations b y the Dumas method using a n old and somewhat corroded Pregl azotometer averaged 10.45 per cent. All the results given in Table I are for analyses by the junior author, and represent the valid results of three series of four determinations each.
lclinowledgment The authors wish to express their thanks to the Atmater Reseaxh Fund for financial assistance and to the Coming Glass Korks for construction of the apparatus.
(wt. of Hg displaced - n t . of blank) X NZreduction factor density of Hg X wt. of sample
Results Acetanilide is the only substance whose analysis is reported in this paper, inasmuch as the method Only with the cO1lection of nitrogen produced b y a standard combustion procedure. If the combustion method is valid for anv substance when a volumetric azotometer is used, it should- be equally acceptable in the present scheme. Research samples of materials of established structure have been analyzed using
Literature Cited (1) Milner and Sherman, IXD. ESG.CHEY.,ANAL.ED.,8, 331 (1936). (2) Kiederl and Ni .derl, “Organic Quantitative Microanalysis”,
2nd ed., pp. 79-100, Kew York. John Wiley & Sons, 1943. (3) p. ”, (4) Pregl and Fyleman, “Quantitative Organic Microanalysis”, pp. 79-94, Philadelphia. P. Blakiston’s Son & Co., 1924. ( 5 ) Trautr, o., Mllikrochemie, 9,300 (1931).
Determination of Ethyl Alcohol bv Microdiffusion J
THEODORE WINNICK, Department of Surgery, Wayne University College of llledicine, Detroit, %rich.
L
EVISE and Bodansky (6) have pointed out that most
current methods for the determination of ethyl alcohol in tissue and body fluids employ a solution of potassium dichromate in sulfuric acid for oxidation of the alcohol to acetic acid, and that these methods differ only in the means of separating the alcohol from the tissue or fluid, and in measuring the partial reduction of the dichromate. I n addition to distillation and aeration methods, several procedures based upon desiccation of the sample have been developed. These are modifications of Widmark’s alcohol method (8), which employs a 50-ml. glass-stoppered flask with a small cup suspended from the bottom of the stopper. The alcohol passes by gaseous diffusion from the sample contained in the cup into a solution of dichromate in sulfuric acid in the bottom of the flask. The quantity of alcohol oxidized is determined by reducing the excess dichromate with potassium iodide, and titrating the liberated iodine with standard thiosulfate solution. I n Cavett’s modification of this method ( I ) , the excess dichromate is titrated with ferrous sulfate solution containing methyl orange. The present method substitutes the Conway microdiffusion unit ( 2 ) for the Widmark flask. The alcohol diffuses from a blood or urine sample placed in the outer chamber of the unit into a solution of potassium dichromate in sulfuric acid in the central chamber. The excess dichromate is determined iodometrically, as in the method of Widmark. The apparatus is inexpensiye and simple to operate, so t h a t a large number of determinations may be run simultaneously. I n addition, the Conwvay unit has the advantage of being applicable to a variety of biochemical determinations ( 2 , 9).
Experimental Analyses are performed on SIZESOF SAMPLES FOR AI~ALYSIS. blood or urine samples containing approximately 1 to 1.5 mg. of ethyl alcohol. Since the concentration of alcohol in blood
and urine is roughly proportional to the degree of intoxication (4),the size of the sample taken for analysis is varied according to the apparent condition of the subject as follows: 1 ml. for mild or doubtful, 0.5 ml. for moderate, and 0.25 ml. for severe intoxication. COLLECTIOX A S D hTEASUREMENT O F SAMPLES FOR ANALYSIS. Blood and freshly voided urine specimens are collected in tubes which are t,ightly fitted xith rubber stoppers. The tubes for blood samples contain oxalate or citrate. The author has confirmed Cavett’s observation ( 1 ) that the blood or urine can stand for 24 hours in the ice box without appreciable change in alcohol concentration. The 0.25 ml. pipet is made from capillary tubing of about 1-mm. bore, and is calibrated for content. In measuring samples with, this pipet, about 0.1 ml. of water is drawn up first, leaving a small air space at the tip, and then the pipet is filled to the calibration mark with blood or urine. The air space between the two columns prevents mixing of the fluids. When the pipet is emptied, the water washes out the film of blood or urine left along the inner wall of the pipet. MICRODIFFUSION PROCEDURE.The glass cover plate is suitably smeared with “cello-seal” (Fisher Scientific Co.), a lubricant which does not liquefy at the temperatures employcd (25’ to 50”). Approximately 0.5 ml. of 0.4 N potassium dichromate in 10 W sulfuric acid is delivered with a volumetric pipet into the central chamber of the unit. A constant time of drainage is employed. The exact volume or concentration of the dichromate solution need not be known, since blank analyses are run with each series. The blood or urine sample is pipetted quickly into the outer chamber, and the vessel is sealed immediately with the greased glass cover plate. The unit is rotated to spread the fluids over the floors of the chambers. After the unit has stood for 2 hours a t 50”, 6 hours a t 37”, or 10 hours at 25” (or for longer times), the cover plate is removed and the dichromate solution is diluted with approximately 1 ml. of water. Then about 0.5 ml. of 3 M potassium iodide solution is added with stirring to the centrak solution, and the liberated iodine is titrated a t once with standard 0.1 N thiosulfate solution. (The large excess of potassium iodide reduces the loss of iodine vapor from the solution before addition of thiosulfate. Tests showed that 0.4 to 0.5 per crnt of the iodine is lost per minute if the central solution is allowed to stand after addition of the potassium iodide. Accordingly,
Vol. 14, No. 6
INDUSTRIAL AND ENGINEERING CHEMISTRY
524
TABLE
.Alcohol in Blood
7
0.5 1.0 2 0 4.0 6.0 8.0 10.0
in the blood and urine of persons following anesthesia, were also tested. in Urine-Analyses were performed at 37' on 1-ml. portions of solutions (or dispersions) containing 50 to 100 mg. of the organic compound to be tested per 100 ml. of water. 37.5 ... 72 23 No appreciable quantity of dichromate 99 I 1 .. 81 in the central chamber was reduced when .. 103 solutions of the following substances ... ... were placed in the outer chamber: benzene, chloroform, carbon tetrachloride, naphtha, and trichloroethylene. Ten to 20 per cent of the dichromate was reduced -when methyl alcohol, isopropyl alcohol, amyl alcohol, octyl alcohol, isoamyl acetate, acetaldehyde, and propionaldehyde were tested, indicating that the vapors of these substances were readily oxidized by the dichromate solution. Accordingly, the drunkenness caused by these compounds cannot be differentiated readily from ethyl alcohol intoxication by the present method, as is possible in the case of the preceding group. Diethyl ether was oxidized slowly by the dichromate. This compound is not likely to interfere seriously \vith the ethyl alcohol determinations, since ethyl alcohol is present a t a relatively much higher level during intoxication and is more rapidly oxidized by the dichromate.
I. RECOVERY O F ETHYL ALCOHOL ADDEDTO BLOOD AND
26.5 57 80 92 97.5
...
27.5 48 79.5 86.5 93 102
30 60.5 84
96 99
.. ..
47 79.5 9s
..
7
41 70 103
... ... ... ...
--.llcohol
...
44.5
48 66 5 94 5 97 5 101
82 92.5 100,s
. .
...
if most of the thiosulfate is added less .than a minute after the liberation of the iodine, the amount of iodine. lost is, negligible,) The thiosulfate is added rapidly with efficient stirring, until nearly all of the iodine is reduced, and then dropwise until the color of the solution is light yellow-green. A drop of 1 per cent starch solution is added, and the titration is completed to the point a t which the deep blue starch-iodine color is replaced by the light blue-green color of the chromic ion Owing to the small volume of the solution being titrated, this color change is very abrupt. About 2 ml. of thiosulfate solution are required in the blank analyses, so that the final volume of fluid in the central chamber is never greater than 4 ml. The central chambers of the units used in this study have capacities of 4.5 to 5 ml BLANKANALYSIS. An analysis is performed nith water in place of blood or urine in the outer chamber. In the author's experience, blank analyses usually agreed to within 0.01 ml. (0.5 per cent). CALCULATION. The volume of thiosulfate solution which corresponds to the quantity of dichromate reduced, and to the quantity of alcohol oxidized to acetic acid, is given by the difference between the volumes of thiosulfate required in the blank analysis, VB, and in the alcohol determination, VA. Since 1 ml. of 0.1 N thiosulfate is equivalent to 1.152 mg. of alcohol, the milligrams of alcohol per 100 ml. of blood or urine equal ( V B - Va) X 1,152 X 100 Volume of sample analyzed
RECOVERY OF ETHYL ALCOHOL ADDEDTO BLOODAND URINE Standard solutions were prepared by adding weighed quantities of 97.5 per cent ethyl alcohol (percentage determined by specific gravity measurement) to urine and to a 1 per cent sodium chloride solution. The alcohol was weighed in sealed glass bulbs, which were broken beneath the surfaces of the solutions, so that no alcohol vapor could escape. Three standard solutions, containing 65.5, 155, and 341 mg. of alcohol per 100 ml. of blood, were prepared by diluting blood (found previously to contain no alcohol) nith suitable volumes of the sodium chloride solutions, which contained 1705 mg. of alcohol per 100 ml. Series of determinations with varying reaction times were made a t three different temperatures on aliquots of the blood and urine solutions. Table I s h o w that 97.5 to 103 per cent of the added alcohol was recovered from the blood or urine after reaction periods of 2 hours at 50°, 6 hours at 37", or 10 hours at 24-25'. The analyses were performed on 1-ml. portions of the solutions, except for the blood containing 341 mg. and the urine containing 374 mg. of alcohol per 100 ml.; 0.25-ml. samples were analyzed in these two series. Duplicate determinations usually agreed to within about 2 per cent. Approximately two-thirds of the dichromate solution was reduced by the alcohol in most cases. INTERFERING SUBSTANCES.Of the volatile compounds which may be encountered in body fluids, acetone need not be considered, since it is stable even in boiling dichromate solution. A number of organic compounds, important in industry, are known to produce conditions resembling ethyl alcohol intoxication when sufficient amounts of their vapors are inhaled. Tests were made with some of these substances to determine whether they could be distinguished from ethyl alcohol in the present method. Chloroform and diethyl ether, two common anesthetics which may linger in small amounts
URINE
TABLE11. CONCENTRATION OF ETHYLALCOHOL IN BLOOD AND URINEOF INTOXICATED PERSONS Apparent Degree of Intoxication Mild t o moderate Moderate t o severe
Very severe, "dead drunk"
Alcohol Found Blood Urine Mg./100 ml. M g . / l O O ml 132, 135 185, 188 260, 261 159, 163 219, 224 276, 289 162, 170 170, 176 269, 276 188, 190 236, 254 267, 278 249, 260 313, 316 304, 310 442, 452 380. 387 468, 480 520, 547 .....
Blood and Urine L e v e l s SORMAL. The blood and urine of six individuals who had taken no alcoholic beverages for several days contained no measurable amounts of alcohol. When 1-mi. samples were analyzed, the thiosulfate titrations ranged from 1.905 to 1.94 ml., as ccmpared with 1.925 ml. for the blank titration Cavett ( I ) and Gibson and Blotner (3)found traces of alcohol. 0 to 6 mg. per 100 ml. of blood, and 0 to 2 mg. per 100 ml. of urine, in persons who had taken no alcohol. These levels are probably close to the limits of sensitivity of these methods, and insignificant compared to the values encountered in intoxication. ALCOHOL LEVELSIN BLOODAND URINEDURING INTOXICATION. The results in Table I1 conform fairly well to the general classification of intoxication in terms of ethyl alcohol concentrations (4). The subjects were persons brought into the Emergency Service of the Detroit Receiving Hospital. I n most cases the duplicate values agree to within about 3 per cent. The urinary levels of alcohol are considerably higher than the corresponding blood concentrations in the cases studied. Jetter (5) has made this same observation, and states that this is particularly true for cases in which the bladder was previously emptied. RATE O F DISAPPEARANCE O F -4LCOHOL FROM BLOOD. Sewman, Lehman, and Cutting ( 7 ) showed that the alcohol concentration in blood falls in a linear fashion with increasing time, following the intravenous administration of ethyl alco-
June 15, 1942
ANALYTICAL EDITION
TABLE 111. RATEOF DISAPPEARANCE OF ETHYL ALCOHOLFROM BLOODO F INTOXICATED P E R S O N Time after .%lcohol in Taking First B:ood Sample Blood Hours . V ~ . / 1 0 0ml 0 304, 310
3 6.5 9 12
214. 256 164, 168 9.5, 97 32, 38
Condition of Subject Moderate t o severe intoxication. speaks with difficulty UncooDerative. fiehtine restraints Quiet i n d sleepyFairly rational Rational
hol to dogs. It was thought of interest to make a series of similar measurements with a human subject, t o illustrate further the use of the present method over a wide range of alcohol concentration. Table I11 gives the results obtained for a 12-hour period with a patient mho is classified in the “moderate to severe” range of intoxication in Table 11. If the alcohol values are plotted against the corresponding times, the points fall along a straight line.
Summary The Conway microdiffusion unit has been adapted to the determination of ethyl alcohol in blood and urine. The alcohol diffuses from the sample in the outer chamber of the unit into the central chamber, where it is oxidized b y a solution of potassium dichromate. The excess dichromate is determined iodometrically
525
Some illustrative alcohol concentrations in blood and urine during intcxication are re,ported. A number of industrially important organic compounds whose vapors can cause drunkenness resembling ethyl alcohol int’oxication were tested for possible interference with the method.
Acknowledgment The writer appreciates the advice and ass.oLance given bin1 by C. G. Johnston and I. B. Taylor, relative to the clinical phase of this study. He wishes also to thank C. P. McCord and G. C. Harrold, of the Industrial Hygiene Department, Chrysler Corporation, Detroit, for supplying some of the organic compounds tested, and for helpful information.
Literature Cited (1) Cavett, J. W., J . Lah. Clin. M e d . , 23, 543-6 (1938). (2) Conway, E. J., “Microdiffusion Analysis and Volumetric Error”, London, Crosby Lockwood and Son, 1939; Biochem. J . , 27, 419-34 (1933). (3) Gibson, J. G., and Blotner, H., J . Biol. Chem., 126, 551-9 (1938). (4) Goodman, L.. and Gilman, A., “Pharmacological Basis of Therapeutics”, New York, Macmillan Co., 1941. (5) Jetter, W.W., Quart. J . Alcohol, 2, 512-43 (1941). (6) Levine, H., and Bodansky, M., Am. J . Clin. Path., Tech. Section 3, 159-73 (1939). (7) Nemman, H. W., Lehman, A . J., and Cutting, W.C., J . PharmaC O ~ . .61, 58-61 (1937). (8) Widmark, E. M.P., Biochem. Z . , 131, 473-84 (1922). (9) Winnick, T., J . B i d . Chem., 141, 115-20 (1941): 142, 461-66 (1942). AIDED by a grant from the 3IcGregor Fund.
Semimicrodetermination of Carbon Using the Van Slyke-Folch Oxidation Mixture R . \I. MCCHEADY AND W. Z. HASSID, Division of Plant Nutrition, b-niversity of California, Berkeley, Calif.
V AS
SLYKE and Folch (4) pointed out that all the wet carbon combustion methods hitherto employed were unsatisfactory because the oxidizing mixtures used did not give quantitative results with the more difficultly combustible compounds. For this reason these methods did not find a place in the organic laboratory. X e t combustion methods were tried in this laboratory employing either a n iodic acid (3) or chromic acid (2) oxidizing mixture, but the results were unsatisfactory. While theoretical results could be obtained with many organic compounds, certain substances such as sugar acetates and some organic acids invariably gave low results. The Van SlykeFolch (4) manometric method, in which a n oxidizing mixture, consisting of fuming sulfuric, phosphoric, chromic, and iodic acids was used, gave excellent results with all the compounds tried. Equally satisfactory results were obtained using the Van Slyke-Folch oxidizing mixture, and a simple apparatus, whereby the carbon dioxide evolved was absorbed in alkali and weighed. Inasmuch as the Van Slyke manometric apparatus is not available in many laboratories, the following procedure, requiring simple manipulation and inexpensive equipment, is described. Reagents Van Slyke-Folch combustion mixture: 25 grams of chromium trioxide, 5 grams of potassium iodate, 167 ml. of sirupy phosphoric acid (specific gravity 1.7); and 333 ml. of fuming sulfuric acid (20 per cent free sulfur trioxide) are placed in a 1-liter Pyrex Erlerimever flask provided with a ground-glass stopper. The
open flask containing the mixture is heated over a wire gauze with a flame, until the temperature reaches 140” to 150” c‘. During heating, the flask is occasionally rotated to assist in the solution of the chromic anhydride. When a temperature of 150” c‘. has been reached, the flame is removed, the flask covered with an inverted beaker, and the mixture allowed to cool to room teniperature. The glass stopper is then inserted, and an inverted beaker is kept over the stopper to prevent the solution from being contaminated with dust. Potassium iodate, reagent grade, pulverized. Dehydrated phosphoric acid, prepared from 85 per cent acid by boiling.
Apparatus The apparatus is shown in Figure 1. Tubes A A are filled with soda lime and serve to obtain carbon dioxide-free air. The reaction flask, B , has a 15-1111. capacity and is connected to a reflux condenser, C, with a 14/35 standard taper glass joint. The construction of an Allihn condenser is modified (Figure 2) to contain a cold-water column inserted into its center. This modification greatly increases the efficiency of the condenser. A 5-ml. capacity cup, D, with a stopcock, b, forming part of the condenser, serves to introduce the oxidizing mixture into the reaction flask. Stopcock b is greased with viscous dehydrated phosphoric acid. The rest of the stopcocks are greased with ordinary stopcock grease. E is a bubble counter containing 0.5 ml. of concentrated sulfuric acid. A small glass-wool plug is inserted into each of its arms to trap any sulfuric acid which might splash over to tube F or condenser C. F is filled with granular zinc (30-mesh) to trap any volatile acids which form in the determination. The moisture-absorption tube, G, is filled with Anhydrone (rnagnesium perchlorate) leaving 1-em. space at each end for a glssswool plug. Previous to use in the apparatus, a slow stream of
