6 H 2 0 in water) were added. After 10 minutes, 6 ml. of concentrated NH4OH were added followed by 15 ml. of dimethylglyoxime (1.5% in alcohol reagent, J. T. Baker Co.). At the end of 3 minutes the nickel dimethylglyoxime precipitate was filtered and washed several times with the 1M T\TaN03 solution. The filtrate was collected in a 250-ml. volumetric flask and brought up to volume with the same salt solution. X 50-ml. aliquot of the filtrate is pipetted into a 100-ml. volumetric flask and acidified with 2.5 ml. of concentrated HCl t o pH 1 to 2. After 5 minutes, 10 ml. of potassium dithiooxalate (o.25yO in water, Eastman) were added and the solution was brought up to volume with alcohol reagent. Colorimetric measurements were made after 5 minutes. The blank was made up of 100 ml. of the NaN03 solution and carried through the same procedure as the samples. 4ll measurements n-ere made on the Bausch and Lomb Spectronic 20 colorimeter a t wave length 509 mfi using the special 1-inch test tubes made for this particular colorimeter. DISCUSSION
The curve follows Beer's law in the range of 0 to 250 mg. per liter, and the reproducibility within a run is good. Maximum shift of the calibration curve
from day to day is 10%; therefore it is recommended that a calibration curve be carried out concurrently with the sample solutions to obtain maximum accuracy. Metals which form weaker complexes with DBDTTA than Ni+2, such as Ca+2, Mg+2, Sr+2, Ba+2, Mn+*, and Fe+2,will not interfere with the analysis if present in small quantities; whereas metals such as Co+', Fe+3, and Cu+* will react adversely. Peptization of precipitates being a property of nonionic surface-active agents (5) and possibly of this particular amphoteric agent, there exists a possibility that some of the nickel dimethylglyoxime will pass through the filter paper. In order to resolve this problem, a nonionic surfactant, Igepal Co-990 (nonylphenoxy polyoxyethylene ethanol, Antara Chemicals, New York, N Y . ) was added in different proportions to DBDTTA. There was no apparent interference or change in the final DBDTTA determination. On the basis of these experimental results, indications are that complete retention of the nickel dimethylglyoxine by the filter paper was achieved. A further check on the peptization of nickel dimethylglyoxime precipitate was carried out in the same manner in the
presence of a charged nonsequestering surfactant, Dowfax 2A1 (alkylsulfonated diphenyl oxide, The Dow Chemical Co., Midland, Mich.). As in the former, analysis showed no peptization of the nickel precipitate. At the present time, this procedure is being used exclusively for DBDTTA. Further work has been planned to extend the possibilities of this procedure to other amphoteric surfactants. LITERATURE CITED
(1) Darbey, *4., ANAL. CHEM.24, 373
(1952).
( 2 ) Chaberek, S., Martell, A. E., "Organic Sequestering Agents," pp. 572, 580,
Wiley, New York, 1959.
(3) .Schnepf, R. W., Ph.D. thesis, Columbia University, Dept. of Chemical
Engineering,New York 27, N. Y. (4) Schnepf, R. W., Gaden, E. L., Jr., Illirocanik, E. Y., Schonfeld, E., Chem. Eng. PTOQT. 5 5 , 42-6 (1959). (5) Schwarte, A. M., Perry, J. W., Berch, J., "Surface Active Agents and Detergents," Vol. 11, pp. 487-8, Interscience, New York, 1958. SARAH MOOK ERNEST SCHONFELD DIPENGHOSH Radiation Applications, Inc. Long Island City, N. Y.
WORKsupported by the U. S. Atomic Energy Commission under Contract AT(30-1)-2093.
Enzymatic Determination of Glucose Stabilization of Color Developed by Oxidation of o-Dianisidine SIR: The colorimetric method for the enzymatic determination of glucose using o-dianisidine as chromogen (3, 9) is specific and sensitive, but the color formed is unstable (2, C), a disadvantage which seriously limits its usefulness. The present work investigates the causes of the color instability and suggests some means to prevent it.
Using this system, many variables and procedures were investigated : temperature of incubation (20' to 50" C.);
PROCEDURE
One hundred and twenty-five milligrams of glucose oxidase (Miles-Ames Research Division) and 5 mg. of peroxidase (horseradish, Sigma Chemicals Co.) or an equivalent amount of the two enzymes in the form of Glucostnt (Worthington Biochemical Corp.) were dissolved in 99 ml. of 0.06M phosphate buffer a t pH 7.0; 10 mg. of o-dianisidine (Xutritionnl Biochemicals Corp.), disethyl alcohol, solved in 1 ml. of w c w added to make a 4 X l O - 5 X concentration of chromogen, and the solution was filtered. To 2.9 nil. of this reagent, 0.1 ml. of standard glucose solution (5.6 X 10-41V to 5.6 X 10-3Jl), or suitable volumes of Somogyi filtrate (8),were added and the mixture was inubntrd at 37' C. for 60 minutes.
I . * + . 0 2040 60
.
,
,
90 120 150 180
300
MINUTES
Figure 1. Effect of temperature of incubation on color development and decay Arrows indicate end of incubation and beginning of storage a t 20'C. Glucose concentration 6 X 10-6M
temperature a t which the samples were kept after incubation (storage temperature, 2" to 37" C.); concentration of enzymes over a 10-fold range; concentration of o-dianisidine (4 X 10-6M to 2 X 10-SM); molarity of the buffer (0,005N to 0 . 5 X ) ; pH of the system after incubation, modified by adding 20 pl, of HC1, H2S04,or KOH a t different concentration a t the end of the incubation period; substitution of 1 part of gum ghatti (1.85 grams of soluble gum ghatti in 1 liter of hot distilled water, autoclaved a t I 5-pound pressure for 20 minutes) for an equal volume of phosphate buffer, in 3 or 4 parts of reaction mixture; and extraction with isobutyl alcohol (2-methyl-1-propanol) a t the end of the incubation period, using 1 part of isobutyl alcohol to 1 to 5 parts of reaction mixture. Absorbance was measured in a Beckman DU spectrophotometer. In some experiments, enzyme and odianisidine nitrogen were determined by nesslerization. RESULTS AND DISCUSSION
When the described system is incubated for 1 hour a t 37' C. and a t pH 7.0, a reddish brown color with a maxiVOL. 33, NO. 1 , JANUARY 1961
* 151
1
I
a LL
0
10
.
w v)
a W
a v
w
15-
D I-
z
W V
20.
a w a
0 30
1
\\
30
60
90
120
150
180
MINUTES ~
Figure 3. Effect of addition of gum ghatti and extraction with isobutyl alcohol on color decay 0
120
60
IS0
Glucose concentration 4 X M. Experiments in buffer continued for 240 minutes, those with isobutyl alcohol for several weeks. Curves derived b y method of least squares. Number of experiments in parentheses. Mean values zk S.E.
MINUTES
Figure 2. Effect of changes in pH on decay of color developed at pH 7.0 Glucose concentration 4 X 10-6 M. Experiments at pH 7.0 continued for 240 minutes. Curves derived b y method of least squares. Points omitted to simplify drawing
mum absorption a t 450 mk develops and
a straight-line relationship is obtained between absorbanceand 2 to 8 X 10-6LV glucose concentrations. If the developed color is allowed to stand a t 20" C. and at p H 7.0, the absorbance decreases a t the rate of 4 to 10% per hour during the first 3 to 4 hours and a brown precipitate can be seen a t the bottom of the tube after 1- or 2-day standing. Since o-dianisidine is practically insoluble in water a t p H 7.0 and a n increase of dianisidine concentration in the mixture accelerates color decay, it seems very likely that this decay is due to a slow aggregation and precipitation of the colored substance itself. This phenomenon is not influenced by the temperature of storage after incubation, by the concentration of phosphate buffer, or by the concentration of glucose oxidase and peroxidase. On the other hand, color decay increases when the oxidation of o-dianisidine is accelerated by increasing the temperature of incubation (Figure l),suggesting that the rate of o-dianisidine oxidation may play a role in the aggregation phenomenon. The addition of acid or alkali after development of color a t pH 7.0 has a profound effect on color stability. Figure 2 shows that the color is stable for at least 3 hours at pH 10.0. The stability of color in acid or alkaline media may be due to the prevention of aggregation or to the formation of a true solution. The first phenomenon may occur a t pH 11.0, when the color decay is delayed but not fully prevented; the second a t pH 2.0, 152
ANALYTICAL CHEMISTRY
at which o-dianisidine and its oxidation product are soluble and no precipitation forms even after several weeks. The stability of color in a n acid medium confirms previous observations made with hydrochloric ( 1 ) and sulfuric acids (5, 7 ) . At p H 2.0 the
Table 1. Comparison of Guinea Pig Blood Glucose Measured with the Enzymatic Method (Gum Ghatti Added) and the Method of Nelson (6)
9%
Enzv- Differ.4nimala Nelsone maticc ence No. Sample' ( N ) (E) (N-E) 85
87
1 2 3
87 88 69
52 85 69
$6 $4 0
1 3
118 125 106
111 119 104
-I-6 +6 $2
1 2
100 125
3
1OG
98 125 107
$2 0 -1
1 2 3
81 150 144
78 150 144
$4 0 0
2
88
89
Animals selected a t random from series subjected to glucose tolerance tests. * Samples 1, 2, and 3 collected before and 1 and 2 hours after glucose load, respectively. Measurements performed on same Somogyi filtrate and expreseed as mg per 100 ml. of blood. 5
0
color becomes yellow and the absorption peak shifts to 400 mk. The addition of gum ghatti decreases the rate of oxidation and, accordingly, increases from 60 to 80 minutes the incubation time required for complete development of color, but prevents color decay almost completely for several hours (Figure 3) and extends the linearity of the calibration curve toward greater concentrations of glucose. Gum ghatti may act as a colloid stabilizer and, in addition, may reduce color decay by slowing down the rate at which the color had been previously formed. Extraction with isobutyl alcohol after incubation results in the formation of a stable color solution (Figure 3), having a satisfactory calibration curve. Nitrogen determination shows that the alcoholic phase contains all the o-dianisidine and its oxidation products and that the enz-mes remain in the aqueous phase. Since the amount of isobutyl alcohol required for the extraction is small in comparison with the volume of the reaction mixture, the color can be concentrated and the sensitivity of the method, accordingly, increased. Reproducibility of the procedures (modification of pH, addition of gum ghatti, or extraction with isobutyl alcohol) is limited only by the usual errors of manipulation and measurement of absorbance. I n a series of triplicate analyses of Somogyi filtrates using the same pipetting set, the standard deviation was less than 2%. Table I shows good agreement between blood glucose values obtained using one of the proposed modifications (gum ghatti) and the standard method of Nelson (6).
'I'he procedures based on the modification of pH or on the addition of gum ghatti are simple and can be used for routine work. The procedure based on isobutyl alcohol extraction is slightly more complicated, but may be used with advantage when a greater sensitivity is desired. LITERATURE CITED
(1) Cawley, L. P., Spear, F. E., Kendall,
R., Tech. Bull. Registry M e d . Technol. 29, 111 (1959).
(2) Guidotti, G., Colombo, J. P., FOB, P. P., Abstracts of Papers, 137th
(9) Teller, J. D., Abstracts of Papers,
130th Meeting, BCS, Atlantic City, J., September 1956, p. 69C.
Meeting, BCS, Cleveland, Ohio, Bpril 1960, p. 4c. (3) Huggett, A. St. G., Sixon, D. A., Lancet 273,368 (1957). (4) Jacobsen, L. N.,Scand. J . Clin. Lab. Invest. 12, 76 (1960). ( 5 ) McComb, R. B., Yushok, W. D., J . Franklin Inst. 265,417 (1958). (6) Selson, N., J . Bid. Chem. 153, 375 (1944). ( 7 ) Saifer, -4., Gerstenfeld, S., J. Lab. Clin. Med. 51, 448 (1558). (8) Somogyi, M., J . Bid. Chem. 160, 69 (1945).
?;.
GUIDOGUIDOTTI JEAN-PIERRE COLOMBO PIEROP. Foi, Department of Physiology and Pharmacology The Chicago Medical School Chicano 12. Ill. 0
,
AIDEDby grant A-552 from the National Institute of Arthritie and Metabolic Diseases, U. S. Public Health Service.
Spectrophotometric Determination of Hydroxyl Groups in Poly(propy1ene Glycols) SIR: Methods for the determination of hydroxyl content in poly(propy1ene glycols) by infrared spectroscopy have been published ( 2 , 3 ) . The method described needs no construction of standard working curves because it is, in effect, a direct spectrophotometric titration of the hydroxy compound with a standard solution of acetyl chloride. PROCEDURE AND EQUIPMENT
Equal samples of poly(propy1ene glycol), about 10 grams each (differing by not more than 10 mg.), are transferred into I5O-ml. iodine flasks, each of
x
~-
-.
I
I
Figure Near-infrared spectra of poIy(propyiene glycol) after reaction with acetyl chloride Absorbance measurements made at polnts on dotted line Background at point of arrow
which contains 25 to 35 grams of zinc metal (c.P. grade, 20-mesh granules). Sufficient toluene is added in each flask SO that the samples are quantitatively diluted to 40 ml. upon subsequent addition of different and definite amounts of 0.6M acetyl chloride in toluene solution. The toluene and the acetyl chloride solution are most accurately added with burets connected to the respective vessels for easy refilling and delivery. Six or seven samples are sufficient for a determination. Amounts of acetylating reagent are chosen to obtain absorbances between 0.2 and 0.8. The flasks are firmly stoppered, gently shaken, and placed in a 37" ==I 1' C. oil bath so that only their bases contact the oil. After 40 to 45 minutes they are removed from the bath and cooled to about the temperature of the cell compartment of the spectrophotometer. Spectra of each sample are recorded with a Beckman DK-2 spectrophotometer. A silica cell is employed with 0.52-mm. path. The settings are: reference, air; speed, 5/2X; scale, 0-1; sensitivity, 0.5; period, 0.2. As the temperature affects absorption by hydroxyl, care must be taken to keep constant temperature as far as feasible during scanning of samples for each determination (3). Allowing the sample in the cell to achieve thermal equilibrium with the compartment (about 2 minutes) yields reproducible and usable spectra. If a constanttemperature cell holder is available, it should be preferred. Absorbances are measured a t 2.87 to 2.88 microns, as shown in Figure 1. To determine the background absorbance, an excess of ca. 5 and 8 ml. of acetyl chloride solution is added t o each of two samples, respectively. Total absorbance is plotted us. milliliters of acetyl chloride solution. From the plot the number of milliliters of acetyl chloride solution required to react with the sample is obtained, as shown in Figure 2.
Then
where M W
= =
molarity of acetyl chloride and weight of sample
As any water in the sample is assumed to react completely with the acetyl chloride, correction for water is made as follows: (Mi. X M
-
%Ha0 X W/1.8)56.1 W = hydroxyl No.
The mater content is determined by the Karl Fischer method. It is assumed that the acetic acid formed by the hydrolysis of acetyl chloride does not react with the hydroxy compound, under the experimental conditions. For samples containing up to 0.27'0 water such correction yielded results in good agree-
0.6 -
ig a s 2 8 -
1
0.4
i 0.3
Figure 2. Typical spectrophotometric plot of poly(propy1ene glycol) titrated with acetyl chloride VOL. 33, NO. 1, JANUARY 1961
153
