ANALYTICAL CHEMISTRY
1748 TabIe IV. Interference of Other Solvents in Sudan 111 Method Ap arent ’% gound cello% BuOH solve 10.2 0.0 0.5 2.0 0.0 0.0
Other Solvent _____ Methanol Ethanol
10.0 2.0 1.0
~
2-Propanol
~
0.0 0.0
I+ 0.2 0.0
Acetone
10.0 2.0 1.0
0.0 0.0 0.0
1+ 0.2 0.0
1+ 0 2 00
0 2 0 2 00
Dioxane
100 2 0 1 0
1+
0 2 00
0 0 00 00
0.0
1-Propanol 10 0 2 0 1 0
Apparent % Found Celloyo BuOH solve 10.0 If 0.0 2.0 0.2 0.0 1.0 0.0 0.0
Other Solvent
Advantages. The Sudan 111 method is sensitive and reproducible to i 0 . 2 % . Moreover, a complete analysis may be run in 10 to 15 minutes. Little interference is encountered from other solvents when they are present in percentages comparable to butanol and Cellosolve. Finally, no added difficulties were encountered in analyzing over 10,000 waste stream samples by this method, when dissolved salts, amines, and oils were present. Reproducibility of this test in the hands of laboratory personnel has been confirmed by hundreds of reruns, which almost invariably yielded identical results. LITERATURE CITED
(1) Christman, J. F., Cunningham, G. L., Jr., Stain Technol. 28, 275-8 (1953).
(2) Lange, N.A . , “Handbook of Chemistry,” Handbook Publishers,
Sandusky, Ohio, 1952.
bled, and analyzed according to the procedure above. The only solvents that gave a positive test a t concentrations of 1% or less were butanol and Cellosolve.
(3) Trubey, R. H., Christman, J. F., Stain Technol. 27, 87-92 (1952). (4) Werner, H. J., Christman, J. F., Zbid., 27,93-6 (1952). RECEIVED for review December 22,1Q56. Accepted July 23,1956. Meeting in-Xiniature, New York Section, ACS, New York, N. Y., March 1956.
Semiquantitative Specific Test Paper for Glucose in Urine J. P. COMER Analytical Research Department, Eli Lilly and Co., Indianapolis 6, lnd.
Data are presented to show that a test paper consisting of the enzymes glucose oxidase, catalase, and horseradish peroxidase, F.D.C. yellow No. 5 dye, and otolidine, when made froin standardized enzymes, is 96% accurate in the range from 0 to 2% glucose. The paper is dipped into the specimen, removed, and compared after 1 minute to a color chart. Results are not influenced by the normal variation of pH and temperature of the urine, or by the presence of common drugs or metabolites in the urine. This test is more sensitive and more selective for the lower concentrations of glucose than is the Benedict’s test. The paper is stable if protected from light.
Table I. Patient 1
2 3 4 5 6
7 8 9 10 11 12 13 14
15 16
T
H E use of paper as a medium for analytical reagenh has been accelerated with the routine use of filter paper chromatography and electrophoresis and with the availabilit,y of general literature references on spot tests in the text by Feigl (3). A paper test for glucose in urine, which would have a simplicity and accuracy similar t o the paper for pH measurements (Hydrion paper, Micro Essential Laboratories, Brooklyn 10, N. Y.), has been desired for many years. However, the measurement of the nonspecific reducing properties of glucose by the usual chemical reagents coated on paper has not been practical because of instability of such reagents on paper under normal storage conditions. Keston (6) has described a novel idea for the simultaneous use of two enzymes for testing for glucose. The reactions involved are: Glucose
H,Oz
+
glucose
0 2
oxidase
+ o-tolidine
gluconic acid
horse-radish peroxidase
+ Hz02
blue color
17 19 l8 20
RIedications Used for Diabetic and Nondiabetic Patients under Test Medication Liver extract Vitamin Bn Insulin. dieitoxin Dieihylstilobestrol Aspirin, phenobarbital Aspirin, phenacetin, caffeine Digitoxin, ethinamate Reserpine, lactose, protoveratrine Aspirin, ethinamate, 3-o-toloxy-1,2-propanediol Tricyclamol sulfate, multiple vitamins, aspirin, phenacetin, caffeine. ethinamate. dioxvline Ethinamate, folic acid Insulin, chloral hydrate, quinidine Lente insulin, digitoxin Aspirin (arthritis dosage) Secobarbital quinine sulfate Tricsclamol.’ magnesium trisilicate, aluminum hydroxide, reserpine Theamin Carbacrylamine resin, thenylpyramine Digitoxin, multiple vitamins, erythromycin Cortisone
Glucose oxidase (2) is a specific enzyme that catalyzes the oxidation of glucose with oxygen into gluconic acid and hydrogen peroxide. The hydrogen peroxide formed in the presence of o-tolidine and the enzyme horse-radish peroxidase forms a blue color that has not been identified. Glucose oxidase has been used previously for the manometric determination of glucose in biological materials by Keilin and Hartree (4) and for the titrimetric determination of glucose in corn sirup by Whistler and coworkers (8). The mechanism of the action of glucose oxidase is amply discussed by Bentley and Neuberger ( I ) , and the specificity by Keilin and Hartree (6). The isolation and properties of horse-radish peroxidase are reported by Theorell ( 7 ) . Work was started in this laboratory to develop a test paper for glucose based on the reactions described by Keston (6),that would
1749
V O L U M E 2 8 , N O . 11, N O V E M B E R 1 9 5 6 Table 11. Statistical Study of the Accuracy of Glucose Test Paper
0
0.10
100
0.25
97
1
3
93 -
0.50
2
91
2 7 a Gnderscored figures represent per cent correct from 300 samples a t each concentration, or a total of 1500 samples.
6
2
98
be simple, specific, give a greater differentiation in the lower concentrations than Benedict's test, and be suitable for commercial praduction. There was developed from this work a test paper impregnated with the enzymes glucose oxidase, horseradish peroxidase, o-tolidine, and F.D.C. yellow No. 5 dye. This is now commercially available from Eli Lilly and Co. as TesTape. TEST PAPER
The test paper can be prepared in the laboratory by dipping analytical grade filter paper into 450 ml. of a 4470 alcohol-water solution containing 1.9 grams of o-tolidine, 54,000 units of glucose oxidase, 34,000 P.Z. (purpurogallin zahl) units of horse-radish peroxidase, and 0.42 gram of F.D.C. yellow Yo. 5 dye, adjusted to p H 5.0 with formic acid. The data presented here are from experiments performed with the commercially available paper prepared from carefully standardized enzymes by machine impregnation and drying. Comparisons were made with a color chart which comes with the paper and is graduated in yellow for negative glucose, light green for O.lO%, dark green for 0.257,, blue-green for 0.50%, and dark bluc for 2 7 , glucose. Minor changes are necessary in the color chart for each laboratory preparation of paper because of variations in the hand dipping process. The paper is dipped into the urine specimen, removed, and, after 1 minute, the darkest area is compared to the color chart. DISCUSSION
One of the first considerations of the possible sources of error for the semiquantitative use of the enzyme test paper was the influence of the p H variation of the urine. The buffering action of the paper was found to be sufficient, however, by conducting tests a t p H values from 2 to 9 (in increments of 1 p H unit). 4 t glucose concentrations of 0.10, 0.25, 0.50, and 2%, no differences were observed between the amount of glucose present and that found. Temperature is known to alter the rate of enzymatic reaction, but there was no noticeable change in the results using the test paper under conditions of normal variation in room temperature. The temperature study was extended to specimen and room temperatures of 6", 25O, 37", and 50" C. for the 0.10 and 2 % glucose concentrations, and there was still no variation from the color chart. This remarkable result may possibly be explained by the presence of another enzyme, catalase, which appears as a controlled impurity in the glucose oxidase. 2H202
2H20
+
0 2
Because the catalase is competitive with the peroxidase for the hydrogen peroxide, it may act as a governing agent on the test paper. Most of the variables such as pH, temperature, enzyme inhibitors, and accelerators, which change the rate of the reaction of oxidase and peroxidase, similarly change the rate of reaction of the catalase, resulting in only a small net change in the amount of peroxide available for the color reaction.
Several drugs were dissolved or suspended in urine containing 0.5% glucose. The urine was tested with the paper; no variation was observed from the 0.5% reading for glucose. The following drugs were used: Sulfathiazole Theophylline Thenylpyramine Tricyclamol sulfate Reserpine Protoveratrine maleate Erythromycin Dihydrostreptomycin Penicillin potassium Vitamin A acetate Vitamin BIZ a-Tocopherol Folic acid Menadione Xicotinamide Kicotinic acid Pyridoxine hydrochloride Riboflavin
Acetophenetidin Acetylsalicylic acid Amobarbital sodium Atropine sulfate Caffeine Cholesterol Diethylstilbestrol Ephedrine sulfate Hyoscine hydrobromide Methyltestosterone Morphine sulfate p-Aminosalicylic acid Procaine hydrochloride Secobarbital sodium Sulfadiazine Sulfamethazine Sulfamerazine Sulfapyridine
The concentration of the drugs was 10 mg. per ml. of urine, with the exception of reserpine, vitamin B12, folic acid, and riboflavin, for which the concentration was 0.1 mg. per ml. of urine. The screening of several thousand urine specimens from nondiabetic people has revealed no false positive reactions To test further for false positive reactions, urine specimens were tested from both diabetic and nondiabetic patents under the various medications listed in Table I and no false positive reactions were observed with the test paper. Benedict's test was negative for all the patients with the exception of No. 14, which gave a trace reaction for glucose. Urine solutions containing sucrose, galactose, lactose, xylose, and fructose gave no positive reaction. Ascorbic acid above 0.05% retards the color formation a t the 0.10% glucose level. A false positive reaction is obtained with the Benedict's test with urine containing 0.05% ascorbic acid. To ascertain the accuracy of the test paper, six panels of 10 people assayed a total of 1500 urine samples with a random distribution of 300 samples for each glucose concentration. The results of this study are shown in Table 11; the over-all accuracy was 96%. The data shown in Table I11 illustrate one of the basic differences in the test paper and the Benedict's test. Five people were allowed to familiarize themselves with the technique but not the color chart of both methods. They then tested 35 samples with random distribution of five samples for each of seven concentrations. The greater differentiation of the test paper for the 0.10, 0.25, and 0.50% glucose concentrations is evident, because most of the samples were called 1+ (approximately 0.25%) by the Benedict's method. The Benedict's test gave an accuracy of 72, 68, and 80% for the 1, 2, and 3% levels, respectively, indicated by the approximate 2 + , 3 + , and 4+ system of the Benedict's chart.
Table 111. Comparison of Glucose Test Paper and Benedict's Test on Urine Solutions of Known Concentration % Glucose Present 0 0.10 0.25
0.50 1 2 3
% Glucose Reportedo Tes-Tape 0, 1 0.10, 1 0.25, 1 0.50, 4 14 0.50, 11 22 = 2, 3 25 = 2 24 23 22 21
= = = = =
= = = = = =
0.50 0,
-
Benedict = neg 1 = 0 . 2 5 24 = l + : '
24
0.10, 2 = 0 . 5 0 23 = 1 + , 0.25 21 = 1 + , 2 18 = 2 + , 0.50 17 = 3 + , 20 = 4 + ,
1 1
2 4 6 8 5
=
I+
0
= 2+ = 2+ = 1+,
1 = 3+
=2+ 3+
=
a Number of observations for five persons testing 35 samples with random distribution of five samples for each concentration.
1750
ANALYTICAL CHEMISTRY
Table IV. Comparison of Glucose Test Paper and Benedict's Test on Urine Specimens in Diabetic Clinic Test Paper Glucose, 07 No.
Keg.
Trace
+
++ +++ ++++
Benedict's Test
every 15 seconds diiring thP s c ~ o n Jminiitr. In the timing int,erval the moist paper can Lie foldrd and himy from the edge of the specimen container 01' plared in order on adhesive cellophane tape. The stability of the paper, as indicated by t,esting at intervals over a period of 6 months, was found t,o be satisfactory at room temperature, 37" C, and 50" C. when protect,ed from light. Direct sunlight causes a gradual deterioration of the paper, but the opaque plastic dispenser, in which the commercially available paper is packaged, serve8 to prot'ect it. LITERATURE CITED
Urine specimens received at a diabetic clinic were tested with the Benedict's test and the test paper. Table IT' lists the number of observations for the test pnper and the corresponding observations for the Benedict's test. Again, better differentiation is evident by the test paper at the lon-er concentrations and by the Benedict's test for the range from 1 to 2% not covered by the color chart for the test paper. For better differentiation R ith the test paper at higher glucose concentrations, dilutions of the urine are necessary. The test paper can be dipped conveniently into the specimens as they are received in the normal variety of containers. Four determinations can be made every 2 minutes by dipping one strip cvery 15 seconds during the first minute and reading one strip
(1) Bentley, R., S e u b e r g e r , .4.,Riochem. J . 45, 584 (1949). (2) C o u l t h a r d , C. E., Michaelis, R., S h o r t , W. F., Sykes, G., Skrimshire, G. E. H., S t a n d f a s t , -1. F. B , Birkinshaw, J. H., Raist r i c k , H., I b i d . , 39, 24 (1948). (3) Feigl, Fritz, "Qualitative Analysis b y Spot T e s t , Inorganic a n d Organic Applications," 3 r d English ed., Elsevier, K e w York, 1947. (4) Keii;, D., H a r t r e e , E. F , Biochem. J . 42, 230 (1948). (5) Ibid., 50, 331 (1952). ( 6 ) Keston, .1.S., A b s t r a c t s of P a p e r s , 129th N e e t i n g , ACS, Dallas, Tex., p . 31c, -4pril 1956. (7) Theorell, H u g o , -4rkio Kenai, Mineral. Geol. 2, 1 (1942). Hougli, I,., I I y l i n , J. IT., ASAL. CHEM.25, 1215 (8) Whistler, R. L.,
(1953).
RECEIVED for review March 14, 1956. Accepted August 3, 1956. Dir-ision of A~ialyticalChemistry, 1 2 9 t h Neeting, ACS, Dallas, Tex., April 1956.
Boron Hydride Monitoring Devices Employing a Triphenyltetrazolium Chloride Reagent L. J. KUHNS, R. H. FORSYTH, and J. F. MAS1 Callery Chemical Co., Callery, Pa.
Two instruments may be used to monitor atmospheres contaminated with boron hydrides: a portable, field model with a hand-operated pump and a continuous analyzer which automatically records boron hydride concentration. Both were designed to make use of the nonspecific reduction of triphenyltetrazolium chloride by boron hydrides to form the red-colored formazan. Metered air samples are passed through filter paper or cloth tape impregnated with the reagent solution. The red color produced is measured by visual means with the field model and by a differential reflectance photometer with the automatic instrument. The instruments w-ere calibrated with diborane, pentaborane, and decaborane; although they are relatively insensitive to diborane, very low concentrations of pentaborane and decaborane can be detected. The field model can detect 0.1 p.p.m. of decaborane and 0.5 p.p.m. of pentaborane, while the automatic instrument is capable of detecting 0.1 p.p.m. of either compound.
T
HE high toxicity of boron hydrides makes it desirable to have monitoring devices for the detection of very low concentrations of these compounds in areas where they may contaminate the atmosphere. The instruments described here are capable of detecting pentaborane and decaborane at least at the maximum allowable concentrations (MAC). For decaborane this concentration is less than 1 p.p.m. (8) and for pentaborane may be set at less than 0.2 p.p.m. ( 6 ) . It was first established by Hill that triphenyltetrazolium chloride (TTC) in alkaline solntion is reduced by boron hydrides to
form the red-colored formazan and it was employed by him in the quantitative estimation of these compounds ( 3 ) . The color reaction is not specific for boranes; methods are reported in the literature for its use in the quantitative determination of sugars and of enzyme (dehydrogenase) activity (5, 7 ) . Although it is not specific, the high sensitivity of the reagent for these compounds justifies its use. A reagent developed in this laboratory is employed as the detecting element for the instruments described in this paper. The reagent, which contains triphenyltetrazolium chloride, quinoline, pyridine, and water, is particularly suited for monitoring devices of this kind. It has a low volatility of the necessary alkalinity without being as unstable as an aqueous alkaline solution of the salt. The salt and its reduced form are both soluble in the solution, whereas the reduced form precipitates out of an aqueous solution. A silver nitrate reagent is reported by Etherington and McCarty ( 2 ) for boron hydride detection with a monitoring device. Instruments similar to the automatic recording analyzer described here are available commercially for the detection of compounds other than boron hydrides (hydrogen sulfide analyzer, Rubicon Co., Philadelphia 32, Pa.; Microsensor, Vitro Corp., 233 Broadway, New York 7 , N. Y.) Some parts of the devices reported on in this paper had to be fabricated from stainless steel and Teflon, because of the action of the reagent on other metals, rubber, etc. FIELD MODEL
The portable model consists of the sensing unit and the handoperated vacuum pump (Figure 1). The stainless steel sensing
