Table VI. Effect of Time of Incubation on Determination of Inhibitors 1 X 10~sAf Ag(I) or Hg(II) Time of AE/At incubation, min. mv./sec. Blank 1.50 0.0 1.40 1.0 0 2.0 0 5.0 0
(A)
1.2
ug./ml. p-Chloromercuribenzoic Acid or o-Iodosobenzoic Acid Blank 1.50 0.0 0 2.0 0 5.0 0 10.0 0 20.0 0
standard curves must be run at the same temperature. A constant temperature bath was used to regulate the temperature to within ±1° C. Effect of Ions. The effect of various ions on the slope of the depolarization curves is shown in Table V. Cations that bind to sulfhydryl groups greatly inhibit the enzyme, and thus must be absent in enzyme Concentrations 1 X 10_5Jf assays. of silver(I) and mercury(II) completely inhibit the enzymatic oxidation, and 10~5M concentrations of copper(II),
iron(II), zinc(II), and magnesium(II)
approximately a 50% inhibition of the enzyme. A method of analysis for these cations by enzymatic inhibition is thus possible. After incubation times of 1 minute, silver(I) and mercury(II) may be quantitatively determined in concentrations as low as 2.5 X 10~7M (Table VI). Phosphate ion, in concentrations up to 10_3Jli, does not affect the results, but cyanide which is electrochemically active [and is a known inhibitor of xanthine oxidase (1) ] does, and should be cause
preincubated with enzyme for good sensitivity (Table VI). The proposed electrochemical method for xanthine oxidase offers a rapid, specific, sensitive method for the determination of this enzyme and for the determination of inhibitors of this enzyme, provided the identity of these materials is known. Since only a very limited number of compounds and ions inhibit xanthine oxidase, the method is specific and sensitive for the determination of these materials.
absent.
Effect of Inhibitors. Since xanthine oxidase has an active —SH group, any compounds that tie up this group will inhibit its enzymatic action on hypoxanthine, and hence may be sensitively determined. Two inhibitors such sulfhydryl-binding studied were o-iodosobenzoic acid and p-chloromercuribenzoic acid. Studies on the effect of substrate, pH, and time of incubation upon the determination of these compounds, revealed that a 1.0 X 10-5Af solution of hypoxanthine in Macllvaine buffer, pH 6.76, using a 0.1-unit-per-ml. solution of xanthine oxidase in 25 ml. of solution, yielded best results. Under these conditions, incubation times of 2, 5, 10, and 20 minutes yielded no advantage over nonincubation, and none of the compounds need be
LITERATURE CITED
(1) Ball, E. G., J. Biol. Chem. 128, 51 (1939).
(2) Elving, P., Olson, E. C., J. Am. Chem.
Soc. 79, 2697 (1957). (3) Guilbault, G. G., Kramer, D. N., Cannon, P. L., Anal. Biochem. 5, 208 (1963). (4) Guilbault, G. G., Kramer, D. N., Cannon, P. L., Anal. Chem. 34, 1437 (1962). (5) Guilbault, G. G., Tyson, B. C., Kramer, D. N., Cannon, P. L., Ibid., 35, 582 (1963). (6) Horecker, B. L., Heppel, L. A., J. Biol. Chem. 178, 683 (1949). M., Ibid., 167, 429 (7) Kalckar, . (1947). (8) Kramer, D. N., Cannon, P. L., Guilbault, G. G., Anal. Chem. 34, 843 (1962).
Received for review August Accepted December 18, 1963.
6,
1963.
Brucine Method for Determination of Nitrate in Ocean, Estuarine, and Fresh Waters DAVID JENKINS and LLOYD L. MEDSKER Sanitary Engineering Research Laboratory, University of California, Berkeley, Calif.
A
technique
which
uses
brucine
for the determination of nitrate
natural waters and employs controlled heating and chloride masking is described. It is not significantly influenced by variations in chloride concentration between 0 and 20 grams per liter of chloride. The method gives highly reproducible results, and in the range 0.05 to 0.8 mg. per liter nitrate nitrogen, the color produced bears an essentially linear relationship to the nitrate concentration. The standard curve can be reproduced from day to day using the same reagents and test conditions. Recoveries of added nitrate from natural waters are quantitative but the method is not recommended for untreated sewages from which recoveries are poor. although often present in
concentrations of less than 0.1 mg. Nitrate,
per liter, is the principal form of nitro-
610
·
ANALYTICAL CHEMISTRY
gen in most natural bodies of water.
In quality surveys of estuarine and
in
offshore waters it is necessary to use a nitrate determination which is unaffected either by high concentrations of chloride or by wide fluctuations in the
chloride concentration.
The 11th edi-
tion of Standard Methods (3) lists two procedures for nitrate determination in water—the phenol disulfonic acid method and tentatively the brucine method. In the former, chloride exerts a strong interference while the latter method is not recommended for nitrate concentrations of less than 1 mg. per liter. The ivork presented in this paper describes an attempt to adapt the brucine method to concentrations of nitrate below 1 mg. per liter and to control the conditions of the reaction
that reproducibility and linearity may be obtained in samples containing from 0 to 20 grams per liter of chloride. The reactions of brucine and strychnine compounds with nitrate had so
recorded by the turn of the century (5, 6), but it is only recently that the quantitative potential of the reaction has been recognized. However, past experience in this laboratory had shown that the brucine method as described in Standard Methods (3) gave erratic, unreproducible results with standards as well as samples. It was therefore necessary to run a large number of standards with each group of samples. In an attempt to increase the sensitivity of the method to nitrate concentrations below 1 mg. per liter, the sample volume was increased from 2 to 5 ml. while keeping the volumes of A typthe other solutions the same. ical standard curve obtained using this method is shovm in Figure 1 (curve A). There is still a strong curvature below 1 mg. per liter of nitrate and the erratic nature persists. By close control of the reaction conditions and by use of a high concentration of chloride to mask chloride variations been
NOí-N, MG./L.
Figure 1. Typical standard curves for standard method (A) and for modified technique (B) using monitored brucine
in the samples, a reproducible method has been developed for nitrate concentrations from 0.05 to 0.8 mg. per liter. EXPERIMENTAL
Spectrophotometer, Apparatus. Bausch and Bomb Spectronic 20 or equivalent with adaptor to hold 1-inch (2.5-cm.) tubes. Stirred water bath at 100° C. Stock standard nitrate solution, 100 mg. per liter, 0.7218 gram potassium nitrate per liter of distilled water. Standard nitrate solution, 10 mg. per liter. Just prior to use dilute 10 ml. of stock standard nitrate solution to 100 ml. Sodium chloride solution, 30% aqueous solution. Sulfuric acid solution. Add 500 ml. of concentrated sulfuric acid to 125 ml. of distilled water. Cool before using and keep tightly stoppered to prevent absorption of moisture. Brucine-sulfanilic acid reagent. Dissolve 1 gram of brucine sulfate and 0.1 gram of sulfanilic acid in 70 ml. of hot distilled water. Add 3 ml. of concentrated hydrochloric acid, cool, and make up to 100 ml. This solution is stable for several months. The pink color that develops dees not affect its usefulness. Procedure. The development of the brucine nitrate yellow color is carried out in the 1-inch (2.5-cm.) colorimeter tubes so that transfer steps are avoided. If the samples are turbid or colored a sample blank consisting of sample, acid, and sodium chloride solution should be read before the addition of the brucine sulfate solution. The samples and standards are read against a reagent blank consisting of sulfuric acid solution, brucinesulfanilic acid solution, sodium chloride solution, and distilled water in place of the sample. Carefully pipet 10 ml. of sample containing between 0.5 to 8.0 ¿ig. nitrate into 1-inch colorimeter tubes held in a suitable rack in a cool water bath. Add 2 ml. of 30% sodium chloride solution and mix well by swirling. Allow the contents of the tubes to reach
Figure 2. Effect of Cl on brucine-nitrate color production using standard and modified methods
the temperature of the water bath. Pipet 10 ml. of sulfuric acid solution into each tube, mix by swirling, and allow the contents of the tubes to reach thermal equilibrium again. Add 0.5 ml. of brucine-sulfanilic acid solution to each tube, and mix thoroughly. Remove the rack of tubes from the cold water bath and place in a boiling water bath for 20 minutes. Remove the tubes from the boiling water bath and immerse them in the cold water bath to bring the temperature to between 15 to 25° C. This inhibits any further color change. Wipe off the tubes with tissue and read the absorbance at 410 µ in a spectrophotometer. Treat nitrate standards in the range 0.05 to 0.8 mg. per liter as described above; include standards of 0.05, 0.10, 0.5, and 0.8 mg. nitrate per liter. If a large number of samples which have absorbances falling on the straight line portion of the standard curve are mx + b analyzed, the equation y may be used where absorbance y nitrate nitrogen, mg. per liter x the intercept on the ordinate of b the extended linear portion m slope of straight line portion obtained by dividing the projection of the straight line portion of the standard curve on the ordinate by its projection on the =
=
=
=
=
abscissa
Thus
x
=
---
DISCUSSION
Acid Concentration. Evidence has been presented (1, 2) which indicates
that the yellow color developed by brucine and nitrate varies with the acid concentration and will not take place in
a solution of less than 25% sulfuric acid. Using the acid mixture of 500 ml. of concentrated sulfuric acid plus 75 ml. of distilled water recommended by Standard Methods (3), erratic results have been obtained with the brucine sulfate samples tested in this laboratory. By using a less concentrated sulfuric acid mixture consisting of 500 ml. of concentrated sulfuric acid plus 125 ml. of distilled water, satisfactory results have been obtained with all but one of the brucine sulfate samples
tested.
Heating. Much of the erratic nature of the standard method can be attributed to uncontrolled heating obtained during color development. The only recommendation made in Standard Methods (3) that would control the temperature of the reaction is to use beakers of similar heat capacity. The order of addition of reagents suggested in the standard method (sample, brucine, then acid) is not conducive to heat control. This means that a largely uncontrolled generation of heat takes place in the presence of the sample and brucine. To control the amount of VOL. 36, NO. 3, MARCH 1964
·
611
1. Effect of CD on BrucineNitrate Color Production Using Standard Method for Nitrate (Nitrate, 0.7 mg. N per liter)
Recovery of Added NO30 from Various Samples Using Modified Technique with 3 Lots of Brucine N03 as N, mg. per liter Type of sample Added Cl“ Brucine Present Total recovered, Source lot Added recovered gram/liter % initially
Table
C1-,
grams/liter 0
0.18 0.45 1.8 4.5 9.1 18 27 36 46 00
Per cent of color produced at O grams
Cl “/liter 100 104 97 89 85 95 88 78 81 69 63
Table II. Comparison of Accuracy of Standard and Modified Methods for NOg Determination N03 as N, mg. per liter Standard Modified method 0.32 0.40 0.25 0.36 0.29 0.22 0.31 0.25 0.30 0.30 Mean 0.30 Std. dev. 0.053 Rel. std. dev. 17.7
method 0.28 0.29 0.29 0.29 0.28 0.29 0.29 0.28 0.29 0.29 0.287 0.0048 1.7
heat supplied to the reaction, the modified technique alters the order of reagent addition to sample, acid, then brucine. The cooled reaction mixture is transferred to a stirred and boiling water bath, in containers spaced so that they are evenly heated, and a controlled amount of heat is supplied for color development. The water bath should be of sufficient capacity that the reaction mixture is completely covered with boiling water and is not drastically reduced in temperature wffien the cool samples are introduced. After a 20-minute reaction time, the reaction mixture is cooled rapidly to room temperature before reading the developed color. Chloride Effect. Although Standard Methods (3) states that chloride ion does not interfere with the brucine determination of nitrate, evidence has been produced which shows that increasing the chloride concentration produces a decrease in brucine-nitrate color development for the standard method (Table I, Figure 2). In the technique presented here the color produced for a given nitrate concentration remains constant with respect to
612
·
ANALYTICAL CHEMISTRY
Table III.
San Pablo Bay water
14.3
I II III
0.18 0.19 0.19
0.20 0.20 0.20
0.38 0.39 0.40
100 100 105
North San Fran-
15.8
I II III
0.13 0.14 0.14
0.20 0.20 0.20
0.33 0.33 0.35
100 95 105
I II III
0.07 0.07 0.08
0.20 0.20 0.20
0.26 0.26 0.28
95 95 100
cisco Bay water
Sacramento River water °
0.010
0.002 mg. added to each sample.
chloride only in the presence of chloride concentrations of between 27 to 50 grams per liter (Table I, and Figure
Using the modifications of heating control and chloride masking it should be possible to obtain a reproducible standard curve (Figure 1, curve B) which is essentially linear between 0.05 to 0.8 mg. per liter of nitrate. Within the linear range wffien a 1-inch light path is used, the curve should have a slope of between 0.4 and 0.8 absorbance units per milligram per liter of nitrate. A reagent blank of less than 0.015 absorbance unit should be obtained using a 1-inch light path. The brucinenitrate color produced should be stable in the cooled sample for 30 minutes or longer. (No brucine sulfate tested in this study has shown more than a 5% increase of absorbance during 30 min-
ment through a brucine-nitrite reaction, sulfanilic acid is added to ensure that no significant interference occurs with 0.5 mg. per liter nitrite. An attempt to decrease nitrite interferences by increasing the amount of sulfanilic acid in the brucine sulfate-sulfanilic acid solution was not successful because this tended to reduce the slope of the standard curve and lessen the sensitivity to nitrate. Calcium (100 mg. per liter), magnesium (100 mg. per liter), ferrous or ferric iron (1 mg. per liter) do not interfere. Accuracy and Precision. The modified technique was compared with the brucine method presented in Standard Methods (3) for the analysis of an estuarine water containing 17.7 grams per liter of chloride. The results of replicate analyses, presented in Table II shoiv that the Standard method (3) gave a mean of 0.30 mg. per liter with a relative standard deviation of 17.7%. The modified method had a mean of 0.287 mg. per liter and a relative standard deviation of 1.7%. The limit of detectability of the proposed technique is 0.01 mg. nitrate nitrogen per liter using a 10-ml. sample and the optical system described herein. Recovery. The recovery of added nitrate from estuarine water and river
utes.)
W'ater was
2).
It is therefore possible to mask out any effect chloride may have on the modified technique by adding a large amount of chloride to the reaction mixture, before color development. Screening Test. Different lots of brucine sulfate may vary considerably in their behavior in the nitrate test. Each bottle of brucine sulfate should be checked with standard nitrate solution for conformance with the
following criteria.
It will not be necessary to check brucine sulfate frequently for it is
possible to carry out about 200 determinations with 1 gram of the material. Interferences. Greenberg et at. (2) v. arned of the possible deleterious hydrolysis of organic nitrogen compounds at low' (
