V O L U M E 2 8 , NO. 1 0 , O C T O B E R 1 9 5 6
It is somewhat surprising that eutectic melting takes place under the circumstances described, in view of the lack of an intimate mixture of the two components. However, the eutectic is very sharp in some cases, as shown by the photomicrographs of Figure 1. This series of pictures shoms unmistakable melting of Xylon type 6 with p-nitrophenol a t approximately 40’ C. below the melting point of p-nitrophenol and about 140’ C. below the melting point of Kylon type 6 . Figure 1, a, shows t x o filaments surrounded by fragments of p-nitrophenol crystals just before melting has begun. I n Figure 1, b, melting has begun as evidenced by the curved, slightly sir-ollen, and irregular outline of the filaments, and the rounding off and coalescing of pnitrophenol fragments. As the temperature continues to rise the melting proceeds faster and more obviously as shonm in Figure 1, c and d. If a t this point the slide is shifted to show a field containing an excess of p-nitlophenol, the fibers can be seen to dissolve completely a t temperatures well belorn7 the melting point of p-nitrophenol I t IS believed that the relatively high vapor pressure of the p-nitrophenol is responsible for the successful eutectic melting, the fibers being bathed with vapor of p-nitrophenol Tithin the confines of slide and cover glass. Of a
1589
number of compounds surveyed as possible reference reagents, only those which exhibited a strong tendency to sublime also exhibited eutectic melting. The results obtained also indicate the potential utility of fusion methods for other studies with synthetic fibers. For example, the change in the sign of elongation that occurs when Orlon is heated indicates structural changes in the nature of a second order transition. LITERATURE CITED (1) Am. Assoc. Textile Chemists Colorists, Tentative Test RIethod 20-53, “Identification and Quantitative Separation of Fibers.” (2) Am. SOC.Testing Materials, D 276-49, “Standard Method of
Identification of Fibers in Textiles.” Kofler, L., Kofler, A., “hlikro-Methoden,” Universitatsverlag Wagner Ges. m.b.H., Innsbruck, 1948. (4) Luniak, B., “Identification of Textile Fibres,” Pitman & Sons, London, 1953. (5) hlcCrone, W. C., Mikrochemie tser. Mikrochim. Acta 38, 476
(3)
(1951).
RECEIVED for review February 10, 1956. Accepted June 15, 1956. Presented a t Meeting-in-Miniature. Cleveland Section, ACS, February 15, 1956.
Microbiological Determination of Nitrate G. B. GARNER, J. S. BAUMSTARK, M. E. MUHRER, and W. H. PFANDER Departments of Agricultural Chemistry and Animal Husbandry, University of Missouri, Columbia, M o . Nitrate determinations have lacked specificity in many methods described in the literature. The specificity of the test reported is based on the reduction of nitrate to nitrite by a microbiologically produced nitrate reducLase. The method is simple and equipment is easily assembled. The nutritional requirements of the microorganisms are met by trypticase. Kitrate may be determined in the presence of many compounds known to interfere in other methods. The range of the test is from 2 to 20 y of potassium nitrate, with a reproducibility of 1 0 . 1 2 y.
The applicability of the enzymic reduction of nitrate for analytical use has been suggested in studies (9,11) on the purified enzyme “nitrate reductase.” Many organisms, such as E. coli, are capable of nitrate reduction and have been used for cell-free nitrate reductase preparations. This laboratory found i t more convenient to use a microorganism isolated from the rumen of a sheep to produce nitrate reductase in each tube than to attempt purification, preservation, and use of the enzyme. This method has been successfully employed in determining nitrate in silage, forage, hay, rumen fluid, and urine. APPARATUS
A
POSITIVE correlation between a qualitative test for nitrate and the toxicity of forages produced in drought areas (8) led t o a need for a rapid, quantitative method for determining nitrate. hlany methods described in the literature are based on determining nitrate nitrogen by difference or by extraction and nitration of organic compounds. The Devarda method ( 1 ) has been extensively used. It requires either a separation of the nitrate nitrogen from the total water-soluble nitrogen or a total nitrogen determination and a standard Kjeldahl determination. The actual distillation step in the Devarda method is rather difficult. The Robertson method ( d ) , like the Devarda method, is not applicable in the presence of cyanamide or urea. The limitation and precautions for these methods have been studied ( 5 ) . Kitration of organic compounds 2,4-xylenol ( 7 ) , and 3,4-xylenol such as phenoldisulfonic acid (4), ( 3 ) in the presence of sulfuric acid has been used. Without extreme precautions, results may be high in the presence of carbonaceous material, if the temperature becomes elevated, but this may be partially overcome by repeated extraction. A polarographic method for the determination of nitrate has been described (6) for concentrations in the range of 1.0 X 10-5 to 2.5 X 10-8 mole per liter. A method (IO) for reducing nitrate to nitrite uses zinc dust in an acid medium. 1-Saphthylaminesulfanilic acid is not so satisfactory as S(1-naphthyl)-ethylenediaminesulfanilic acid as a stable color-producing reagent (12).
The photometer was constructed with a photocell and highgain amplifier for use with narrow band filters. A set of matched test tubes, 13 X 100 mm., having a light path of 11 mm. was selected. The desired wave length of 5500 A. was approached by the use of Corning filters 3384, 5120, and 9780. REAGENTS
All reagents were prepared from analytical grade chemicals in double-distilled water. The color-developing reagent is similar to the reagent described by Saltzman ( I d ) . The reagents are stable for a t least 2 months if refrigerated. Stock Reagent Solution. To prepare 0.1 % 11’( 1-naphthyl)ethylenediamine dihydrochloride, dissolve 0.1 gram of the reagent in 100 ml. of water. Working Reagent Solution. Dissolve 5 grams of sulfanilic acid in 700 ml. of warm water, cool, and add 170 ml. of glacial acetic acid followed by 20 ml. of the above stock solution. Dilute to 1liter. Standard Potassium Nitrate Solution. Standard Stock Solution. Prepare the standard solution in such a manner as to give 0.2 mg. of potassium nitrate per ml. of solution. Working Standard Solution. Dilute 5 ml. of the stock solution to 100 ml. Triple-Strength Trypticase Medium. Dissolve 3 grams of trypticase and 1.5 grams of sodium chloride in 100 ml. of distilled water. Autoclave a t 15 pounds per square inch for 15 minutes. Trypticase may be obtained from the Baltimore Biological Laboratory, Inc. (BBL). 0.2M Phosphate Buffer. Dissolve 1.362 grams of potassium dihydrogen phosphate and 4.157 grams of disodium hydrogen phosphate in 500 ml. of water (pH 7 ) .
ANALYTICAL CHEMISTRY
1590 I.o
standard nitrate solution. Potassium nitrate concentration per tube ranges from 2 to 20 y . To each of these tubes add sufficient water to bring volume to 2 ml. Finall add 0.5 ml. of triplestrength trypticase, followed by 0.2 m? of the inoculum, and bring the final volume to 2.7 ml. Prepare tubes containing material to be assayed as follows: To duplicate tube add 0.2, 0.5, 1.0, 1.5, and 2 ml., respectively, of the sam le solution (diluted to approximately that of the standard sohion). To each tube add sufficient water to bring the volume to 2 ml. Finally add 0.5 ml. of triple-strength trypticase, followed by 0.2 ml. of the inoculum, bringing the final volume to 2.7 ml. Place the tubes in an incubator set a t 39' C. for exactly 5 hours. The timing is critical, as shown in Figure 1. At the end of 5 hours, add 5 ml. of the N ( 1-naphthyl)-ethylenediaminesulfanilic acid solution per tube. Maximum color develops in 15 to 20 minutes. The color is stable and the tubes can be stoppered and read a t a convenient time. Set the photometer a t 100% transmittance with water a t 5500 A. Run a blank with each standard to correct for the slight turbidity and endogenous nitrite of the organisms.
.?
4
E
Hu I
E 5
5
ai 0
CALCULATIONS
U
The photometric densities of the samples and standards minus the photometric densities of the blank are plotted on log log paper.
.a
Figure 1. Photometric densities of nitrite derivative as influenced by incubation time Microgram levels of potassium nitrate per tube. D , 20
A , 2; B , 5; C, 10;
CULTURE OF MICROORGANISM
The organism was isolated from the rumen of a sheep. It has been identified only as far as the following: gram-positive, sporeforming bacillus which utilizes dextrose, fructose, and starch and reduces nitrate to nitrite. I t s nutritional requirements are met by either trypticase (BBL), a pancreatic digest of casein, or yeast dextrose agar. Organism ATCC No. 12480 may be obtained from the American Type Culture Collection, 2029 M St., N. W., Washington, D. C. Inoculate a sterile medium consisting of 1 gram of trypticase (BBL), 0.5 gram of sodium chloride, and 0.1 gram of sodium nitrate per 100 ml. of water from a yeast dextrose agar slant containing organism ATCC No. 12480. Subculturing the organism without the presence of nitrate leads to a decreased nitrate reductase activity. This is overcome by the addition of nitrate to the slant or by subculturing in the above medium two or three times. After the nitrate reductase activity has been brought to a maximum by subculturing, return the organism to a slant, because repeated subculturing will allow the adaptive enzyme "nitrite reductase" to be formed. If this occurs, the sensitivity of the test is markedly lowered and reproducibility becomes difficult. Allow inoculum to grow overnight a t 33" to 40" C. Spin the resulting bacterial suspension down and wash re eatedly with either dilute 0.2144 phosphate buffer or sterile 0.9gsodium chloride solution until free of nitrites. Usually three or four washings are required. Then suspend the organisms in 0.2M phosphate buffer and adjust to a turbidity of a proximately 60% transmittance compared with water a t 6300 This inoculum should be prepared each day.
1.
SAMPLE PREPARATION
Extract nitrate from 5 to 10 grams of ground forage, silage, and hay by steaming for 10 to 15 minutes in a 500-ml. Erlenmeyer flask with approximately 250 ml. of distilled water. Before steaming bring the pH of the solutions to 7 by addition of sodium hydroxide. Then quantitative filter the suspension through a Buchner funnel and wash with approximately 150 ml. of warm water in several portions. Dilute the resulting solution to 500 ml. Urine samples must be diluted tenfold and rumen fluid fivefold to prevent inhibition of the organisms. PROCEDURE
Prepare standard nitrate tubes as follows. To duplicate tubes add 0.0, 0.2, 0.5, 1.0, 1.5, and 2.0 ml., respectively, of the working
L
I
2
51
MICROGRAMS OF KNO3 PER TUBE (STANDARD) i
I
I
I
0.2
I 0.5
I
I
I
I
I
IO
I
I 2.0
M L OF SOLUTION PER TUBE Figure 2.
Parallelism between standard curve, A, and curves for materials tested
The concentrations of the nitrate in the samples are then determined from the standard curve. As shown in Figure 2, the curvee plotted from reading of the samples should closely parallel the standard curve. If nonparallelism exists, it is due to interference, which must be corrected for either by dilution or removal of the inhibiting substances. Reading from graph corrected to 1-ml. aliquot X sample diluloo
factor matter.
% dry matter
=
KNOI equivalent per gram of dry
REPRODUCIBILITY AND ACCURACY
As shown in Table I, the reproducibility and accuracy are within a practical range for microbiological test based on eight replications. Further checks were obtained when equimolar quantities of nitrite and nitrate were run in parallel tests. After 5 hours' incubation the two series of tubes gave equal photometric densities indicating a complete conversion of the nitrate to nitrite a t a level of 10 per tube. Error due to the utilization of
1591
V O L U M E 28, NO. 10, O C T O B E R 1 9 5 6 Table I.
Range of Photometric Readings,
Standard Deviation coefficient Mean As As of Photometric photometric KiYOs, Variation, % T Density density Y % 59-61 0.2212 0.00625 0.105 2.83 5 41-43.6 0.379 0.0069 0.115 1.82 10 24.5-26 0.598 0.0071 0.119 1.19 20 11.5 0.939 0.0000~ 0.0 ... Same approximate error is not apparent, a s i t is read to nearest 0.5% T.
Amount of KNOI, Y 2
a
Reproducibility and Accuracy of Method
inhibit the organism or the extracellular enzyme. In order to obtain valid results, a given amount of nitrate is added to decreasing concentrations of test substance. The dilution necessary to o allow desired activity is found in the tube giving a 1 0 0 ~recovery of the added nitrate. The test can be performed in the presence of nitrite, provided the combined amounts of nitrite present a t the end of the incubation period are not greater than the highest level in the standard series. ACKNOWLEDGMENT
the nitrite by the organism is extremely small. It is therefore possible to determine nitrate in the presence of nitrite.
The authors are grateful to E. E . Pickett for the instrumcnt,ction and to Laura &I.Flynn for technical advice. LITERATURE CITED
Assoc. Offic. Agr. Chemists, “Official Methods of Analysis,” 7th
LIMITATION
ed., p.
Timing is important A standard curve must be obtained with each series of determinations to correct for variation in temperature, time, and enzyme activity. As in most microbiological tests, the greatest accuracy is attained in the central portion of the test range. The test has been run in the presence of 2 mg. of glucose per tube without affecting the quantitative relationship of nitrate conversion to nitrite, although the turbidity is increased. When the turbidity is increased, it may be either corrected for by a blank or eliminated by centrifugation. The organism is large and is easily spun out after treatment with the color reagent. Urea in concentrations as high as 8 mg. per ml. exhibited no detrimental effect. With rumen fluid a fivefold dilution is necessary to prevent inhibition. Nitrate content of urine has been successfully determined in a tenfold dilution. K i t h some samples of natural materials there may be substances present which
14, 1950.
Ibid., p. 15.
Balks, R., Reekers, I., Landwirtsch. Forsch. 6, 121-6 (1954). Barnett, A . J. G., “Silage Fermentation,” pp. 157-9, Academic Press, New York, 1954. Dickinson, W. E., AXAL.CHEM.26, 777-9 (1954). Hamm, R. E., Withrow, C. D., Ibid., 27, 1913-15 (1955). Jones, B. G., Underdown, R. E., Ibid.,25, 806-8 (1953). Muhrer, M. E., Case, A. A., Garner, G. B., Pfander, W. H., J . A n i m a l Sci. 14, 1251 (1955). Kason, A,, Evans, I€. J., J . BioZ. Chem. 202, 655-73 (1953). Kelson, J. L., Kurta, L. T., Bray, R. H., ANAL.CHEM.26, 1081-2 (1954).
Nicholas, D. J. D., Kason, Alvin, hIcElroy, W. D., J. Biol. Chem. 207, 341-51 (1954). Saltaman, B. E., ANAL.CHEM.26, 1949-54 (1954). RECEIVED for review April 19, 1956. Accepted June 15, 1956.
Contribution from the Missouri Agricultural Experiment Station, Journal Series No 1617. Publication approved by the director.
Detection and Identification of Clinically Important Barbiturates LEO LEVI Food and Drug Laboratories, Department o f National Health a n d w e l f d r e , Ottawa, O n t , Canada CHARLES
E. HUBLEY
Defence Research Chemical Laboratories, Defence Research Board, Ottawa, Ont., Canada Clinically important barbiturates in commercial preparations and biological materials can be made to react with aqueous copper sulfate-pyridine solutions to give characteristic dark purple-colored derivatives of the composition (NCHCHCHCHCH), L
Cu(OCNHCOCR’R“CON),. Chemical I
A
evidence
pre-
I
sented suggests that the metathetical reaction proceeds via the interaction of a negatively charged, enolized barbituric acid ion with a positively charged copperpyridine complex ion. In accordance with this mechanism the dissociation constants of the barbituric acids are a major factor governing product yields. The reaction becomes more sensitive as the copper sulfatepyridine ratio in the reagent is increased or the pyridine-water ratio of the system is decreased. Infrared absorption data suggest that in the complex the barbiturate is bonded to the central metal atom through the carbonyl oxygen in the 2 position. The compounds show unique features throughout the region studied, 4000 to 650 cm.-’, and hence, the method affords a high degree of specificity for detecting and characterizing these drugs.
T
HE toxicologist and forensic chemist are often concerned with
the detection and estimation of barbituric acid derivatives, which “are the organic poisons most frequently encountered in the toxicological examination of organs and body fluids from cases autopsied in the Medical Examiner’s Office’’ ( 2 6 ) . The barbiturates can be identified by inspection of the infrared absorption spectra of their copper-pyridine complexes, as well as the infrared absorption spectra of the free barbituric acids recovered from the complexes. Because of the ease with which the derivatives can be prepared and the barbiturates recovered, the method should prove a valuable tool for the microchemical identification and characterization of clinically important barbiturates. MICROCHEMICAL REACTION WITH AQUEOUS COPPER SULFATE-PYRIDINE SOLUTIONS
I n 1931 the Dutch chemist Zwikker ( 2 7 ) prepared a crystalline derivative of the commonly used sedative Verona1 (5,5-diethyl barbituric acid) by reaction of the barbiturate with an aqueous solution of copper sulfate in pyridine. He assigned to the compound the formula (barbital)&u(pyridine)z. The metal was determined by the Bruhns method after wet ashing the complex in a sulfuric-nitric acid system. The barbiturate was recovered by extraction with a chloroform-ethyl acetate mixture following
