Anal. Cham. 1989, 61, 2715-2718
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Nanogram Nitrite and Nitrate Determination in Environmental and Biological Materials by Vanadium(III) Reduction with Chemiluminescence Detection Robert S. Braman* and Steven A. Hendrix
Anal. Chem. 1989.61:2715-2718. Downloaded from pubs.acs.org by EASTERN KENTUCKY UNIV on 01/24/19. For personal use only.
Department of Chemistry, University of South Florida, Tampa, Florida 33620 Our work agrees with that of Ellis and Vogel (5) who report reduction of nitrates to ammonium ion by carefully prepared vanadium(II) in hot acidic solutions protected from air oxygen. The stronger reducing agents such as chromium(II) (6) and alkaline titanium(III) (7) apparently also reduce nitrates to ammonium ion. In our work, solutions containing vanadium(II) mixed with vanadium(III) did not produce NO from nitrates or nitrites. Use of vanadium(III) as the nitrate reducing agent is examined here and applied to the analysis of a variety of environmental samples and human fluids. Comparison is made to the acidic iron(II)-molybdate reduction method (1-3).
Nitrite In environmental water samples Is reduced at room temperature to nitric oxide In acidic medium containing vanadium(III). Nitrate Is also rapidly reduced after heating to 80-90 °C. Nitric oxide Is removed from the reaction solution by scrubbing with helium carrier gas and Is detected by means of a chemiluminescence NO, analyzer. Nanogram detection limits are obtained. The method has the advantage of not requiring highly acidic solutions for nitrate reduction and has been applied to the analysis of a variety of environmental waters, sediment, plant materials, and human urine and blood serum.
EXPERIMENTAL SECTION Apparatus. Solutions being analyzed for nitrites or nitrates
INTRODUCTION
by reduction reactions producing NO were degassed by using helium which was then passed into a Bendix Model 8101 chemiluminescence analyzer. The apparatus arrangement used is shown in Figure 1. The analyzer inboard flow rate was controlled by a micrometering valve set to approximately 200 mL/min. The helium degassing flow rate was set at approximately 120 mL/min, while oxygen make-up gas to the “T” was set at 100 mL/min. A single bubbler containing 1-2 M NaOH at room temperature was used to remove any acidic gases from the inboard flow into the detector. The “T” system avoids the problem of matching the flow of the analysis stream from the reaction chamber to the inboard flow demanded by the detector. Cold trapping is not necessary in this apparatus arrangement because the mix of dry make-up gas with the saturated carrier gas produces a relative humidity near 60%. Chemiluminescent analyzers were originally designed for use in continuous analyses of ambient air and so can accommodate high-humidity air. The detector was used in the NO only mode. The output signal was recorded by means of a Linear Instruments, Inc., Model 252A integrating recorder. Several sizes of reaction chambers were used in the development of the method ranging in size from a 50-mL microimpinger to a 300-mL round-bottomed flask. Flasks in the 50-100-mL range gave the most rapid responses. Volumes of reducing reagent needed varied from 20 to 50 mL depending upon the amount necessary to cover the bubbler frit. Reducing Reagents. Solutions of vanadium(III) approximately 0.10 M, also 1-2 M in HC1, were produced by reduction of acidic 0.10 M solutions of vanadyl sulfate using a Jones reductor. Generally, the solution developed a pink-purple color during this step indicating the presence of vanadium(II). Redox titration of the reduced solution using standard cerium (IV) confirmed that approximately 10-90% of the vanadium present was vanadium(II) depending upon the contact time of the vanadium solution with the reductor. Figure 2A is a typical titration of a reduced vanadium sample. The vanadium(II) was converted to vanadium(III) by bubbling air or oxygen through the solution. Figure 2B gives the titration curve for the vanadium(III) solution thus prepared. The absence of vanadium(II) in the solution can be determined by measuring the solution potential with the stronger reducing vanadium(II/III) couple having a potential of E° -0.256 V versus standard hydrogen electrode and the weaker reducing vanadium(III/IV) couple having a solution potential of E° +0.359 V versus standard hydrogen electrode or, more simply, by determining the response of the system to nitrate. Solutions containing vanadium(II) did not produce NO from nitrite or nitrate standards added using the analysis procedure.
The chemiluminescence detector-based method for trace nitrites and nitrates in aqueous samples was first reported by Cox (1) and was later applied to analyses of seawater by Garside (2,3). Because of the sensitive nature of the detector, it was possible to analyze samples containing nanomolar concentrations or nanogram amounts of nitrite and nitrate ions. The chemiluminescence analysis method, then, is of importance in environmental analyses, chemical oceanography, and other applications where trace nitrite and nitrate data are needed. Parts-per-billion concentrations can be analyzed with milliliter sample volumes while parts-per-million and higher concentrations can also be determined by using microliter range sample sizes or by dilution. The earlier methods (1,2) used an acetic acid-potassium iodide mixture at room temperature for nitrite reduction to nitric oxide. Ferrous ammonium sulfate with ammonium molybdate in hot, approximately 50% concentrated sulfuric acid, was used for reduction of nitrate plus nitrite. Nitrate was determined as the difference between analyses of the same sample by the two methods. Extensive trapping of the analyte carrier gas was needed to prevent introduction of acidic gases into the NO, detector.
REDUCTION METHODS We have studied the use of vanadium (III) as a reductant for nitrate and find that it has substantial advantages. It is more reactive as a reducing agent than iron(II)-molybdate and can be used at far lower acidities. As was found, sequential, multiple large volume water samples (10-100 mL) can be analyzed by using the same reduction solution, a feature not achieved by the iron(II)-molybdate reduction method which requires fresh, blanked reduction solution for each sample of substantial volume. Addition of large water samples reduces the acidity of the iron (Il)-molybdate reagent to the point that it no longer reduces nitrates. Vanadium(II) was mentioned as a possible reducing agent in the initial work by Cox (1), making reference to work by Hassan and Zaki (4) who reported that it reduces nitrate to nitric oxide. As will be shown, our work indicates that this is incorrect. Vanadium(III), not vanadium(II), reduces nitrate to nitric oxide. The earlier work (4) did not verify the presence of vanadium(II) in the reducing agent nor the presence of NO as the gas evolved. 0003-2700/89/0361-2715$01.50/0
=
=
©
1989 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 24, DECEMBER 15, 1989
·
He
In
nitrite samples
l
are added to the degassed reducing solution at temperature and are then degassed until all is removed. A series of samples can be analyzed by using the same vanadium(III) solution despite dilution of the vanadium(III) as the samples are added. Procedure—Nitrite + Nitrate. Nitrates are rapidly reduced by vanadium(III) at 80-95 °C. Consequently, the vanadium(III) reagent is added to a reaction flask and heated to this temperature range until the residual nitrate is removed. Samples are then added to the blank solution and the NO signal is recorded. Since nitrites are also reduced, this analysis gives nitrites + nitrates. Procedure—Nitrite and Nitrate. Nitrites can be separately determined at room temperature and the results subtracted from the analysis of nitrite + nitrate. Alternatively, a hot degassed vanadium(III) solution can be cooled to room temperature and the sample added. This analysis gives nitrite only. The solution is then placed in the hot water bath with no helium carrier gas flowing for 2 min and is then degassed while heating to obtain the nitrate signal. The vanadium(III) solution can then be cooled in an ice bath to be ready for the next sample. This temperature cycling procedure provides for direct analysis of both nitrite and nitrate in the same sample. Procedure—Sediment Analysis. Samples of essentially 98% calcium carbonate ocean sediments were analyzed directly for nitrite + nitrate. In this case, the vanadium(III) reagent was prepared in 4 M HC1 to allow for depletion of the acid by reaction with carbonates during analysis. Sediment samples of 100-300 mg were weighed and added to the blanked, hot reducing solution directly through the top of the reaction flask. The apparatus was immediately reassembled and the analysis performed. The carbonate dissolved quickly with no significant change in response time. The carbon dioxide produced did not give a signal in the NOj detector with most probably being removed by the NaOH bubbler trap. Some 34 sediment samples were analyzed in sequence utilizing the same vanadium(III) reductor solution. Nitrate calibrations performed following these analyses showed no degradation in response. Procedure—Human Fluids, Leaves, and Tobacco. Urine and serum responded in a manner similar to that of other fluids. Since nitrite and nitrate samples are in the parts-per-million concentration range, samples of 10-100 mL can be injected directly into a blanked, hot vanadium(III) reduction solution or into a blanked room temperature vanadium(III) solution. Plant leaves were simply dipped into the reduction solution and then removed. The apparatus was then reassembled and degassed for analysis. A single dipping was sufficient to remove 90% of the surface nitrate present on a leaf. Tobacco samples in the 1-5 mg range were added directly to the reducing solution for analysis. Alternatively, samples were weighed and soaked in water in a 100-mL volumetric flask and small volumes of the solution injected or pipetted into the reaction flask for analysis. room
B NOx
detector
Filter i
Vanadium
_l
Gil)
solution
Recorder
2 M NaOH
solution
Figure 1. Apparatus arrangement.
+800 +700 +600 +500
+400 CO CO
>
+300
1
+200
B
+100
I
1 0
-100
-200 -300
0
12
16
20
24
28
32
Volume (mL) Figure 2. Potentiometric titration of (A) V0S04 solution after passage through a Jones reductor and (B) vanadium(II) solution after exposure
to oxygen.
Vanadium(II) is reported to reduce nitrates to ammonia (7) in earlier work using this as a reducing agent for titrimetric analysis of nitrates. Acidic solutions of vanadium(IV) did not reduce nitrate to NO, consequently, vanadium(III) is the active reducing agent. Prepared solutions of vanadium(III) appeared to maintain their reducing character over at least several months. The initial potentiometric assay was 0.0704 M versus 0.0654 M 7 months later. The acidic reducing agents used by Cox (1) and Garside (2) were also prepared in order to compare the two methods. These were concentrated sulfuric acid, 4% ferrous ammonium sulfate, and 2% ammonium molybdate. To 5 mL of sample was added 5 mL of concentrated sulfuric acid, 2 mL of 4% ferrous ammonium sulfate, and 2 mL of 2% ammonium molybdate. This solution was degassed in a hot water bath at 80-90 °C. Successive samples of microliter range amounts can be analyzed by using the hot acidic iron(II)-molybdate reductant. If larger water volumes are to be analyzed, i.e. 5-10 mL, then new blank reagent solutions must be used for each sample to avoid loss of response by decrease in
acidity through dilution. Procedure—Nitrites. Nitrites are rapidly reduced to NO at room temperature by the vanadium(III) reagent. Nitrates are only very slowly reduced at room temperature. Consequently,
RESULTS AND DISCUSSION Vanadium(III) Concentration Study. The effect of vanadium(III) concentration on the reduction of nitrate was determined by analyzing standard nitrate samples over a range of vanadium(III) concentrations produced simply by diluting the starting 0.066 M reduction solution with deionized water. Results indicate that reduction occurs at a reasonable rate (4-5 min/analysis) down to approximately 0.001 M vanadium(III). A similar response effect was obtained for nitrite at room temperature. Table I gives the results for these sets of analyses.
Temperature Effect Study. The effect of temperature
the rate of nitrate reduction was determined by analysis of a series of standards at varying water bath temperatures, the results of which are given in Table II. The indication is that water bath temperature should be approximately 85-95 °C for rapid reaction. Under these conditions analyses require 2-5 min per sample. Typical responses for nitrite + nitrate at varying water bath temperatures are shown in Figure 3. Nitrite reacts at room temperature and equally well at the on
elevated temperatures.
ANALYTICAL CHEMISTRY, VOL. 61, NO. 24, DECEMBER 15, 1989
Table I. Effect of Vanadium(III) Reductor Solution Concentration on Nitrite and Nitrate Response” [V3+], M
NOf-N, ng
NOs--N, ng
0.063 0.043 0.0315 0.0210 0.0145 0.00741 0.00394 0.00091
77.13 74.71 73.56 89.14 79.49 73.49 75.16 78.05
72.22 76.54 77.10 77.10 84.69 75.42 64.71 83.42
II
00
77.59 ± 5.14
II
Table II. Effect of Water Bath Temperature Reduction
60 66
7.7 7.8 6.5 4.2
71
79
time”
[V3+]6
NOf-N, ppm
0.6 1.0 2.0 3.0
0.033 0.031 0.030 0.029
8.41 ± 0.30 5.27 ± 1.23
NOf-N, ppm 8.01 8.49 7.79 6.39
± ± ± ±
0.05 0.17 0.11 0.15
Average of duplicate 8.00 ppm standard N03 injections, hot, and 8.00 ppm standard N02~ injections, cold. 6 After dilution with ”
2
M NaOH.
Table IV.
76.41 ± 6.26
10-mL injections of 8.00 ppm standard solution. measured base line to base line; NOf, NOf.
response
1.5 1.6 2.0 1.6 2.7 3.2 3.9 6.2
pH
00
“
water bath temp, °C
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Table III. Effect of Vanadium(III) Reductor Solution Acidity on N03~ and N02" Response”
time6 1.5, 1.6, 1.6, 2.1, 2.1, 1.6, 2.0, 2.2,
·
on
6
In minutes,
a.
Determination of N03~ + N02" as Nitrogen in Hillsborough River Water
total solution vol, mL
NO f + NOf-N, ppm
time”
70 90 110 130 150 170 190 210 230 250
0.13 0.13 0.13 0.13 0.13 0.13 0.12 0.12 0.10 0.11
4.5 4.1 4.3 3.9 4.0 4.0 4.0 3.4 3.2 3.0
Nitrate
water bath temp, °C
response
85 90 98
3.0 2.4 2.1
time”
In minutes, measured base line to base line, 10-juL injections of 8.00 ppm standard nitrate solution. 0
b.
Vanadium(III) Acidity Effect Study. The acidity of the vanadium(III) reductor solution was adjusted to several different values by addition of 2 M NaOH. The solution was then used to analyze standard nitrite and nitrate solutions in order to determine the effect on response. Nitrite standards were analyzed by use of room temperature vanadium(III) solution while the nitrate standards were analyzed by use of hot vanadium(III) solution. The results, given in Table III, indicate that nitrate response diminishes beginning at approximately pH 1 and disappears altogether at pH 2 and higher. Nitrite response begins to diminish at approximately pH 2. Adjusting the vanadium(III) reductor solution to pH 0.6 required the addition of approximately 20 mL of 2 M NaOH doubling the initial vanadium(III) solution volume. This and further additions of 2 M NaOH did not reduce the vanadium(III) concentration to the point where its activity was significantly decreased. Nitrate and nitrite response can be restored by the addition of HC1 to the reductor solution. Interferences. The possibility of nitro-organic compounds producing a positive nitrite or nitrate response was investigated by analyzing several of these compounds using the nitrite + nitrate procedure. iV-Nitrosodimethylamine, 1-nitrosopyrrolidine, 4-nitrosophenol, 2-nitrosotoluene, and nitro-
as
Nitrogen in Tampa Bay Seawater
total solution vol, mL
N02--N, ppb
time”
75 100 125 150 175 200 225 250 275 300
1.56 1.65 1.69 1.68 1.57 1.56 1.77 1.73 1.69 1.66
6.0 5.6 5.2 5.0 4.4 4.2 4.0 3.6 3.4 3.4
“
Figure 3. Effect of water bath temperature on N03~ + N02“ response time.
Determination of NOf
In minutes, measured base line to base line.
benzene produced no detectable response. Also, no degradation of sample materials (i.e. vegetation or human fluids) to produce volatile organic compounds or other gases detectable by the NO* analyzer was observed. Response Curves and Detection Limits. The response curves for nitrite and nitrate are the same within statistical variation. Two curves obtained by using the same reduction
solution
are
N03" Sb
=
N02Sh
=
N (ng) = 7.997(x) 0.075 ±0.048, S, = ±0.514 (N = 4)
as
as
-
N (ng)
=
±0.181, Sr
=
7.598W + 0.549 ±2.130 (.N = 4)
where (x) is the integrator count per response, Sb is the standard deviation of the slope, and Sr is the standard deviation of the residuals. A lower detection limit of 2-3 ng of nitrate or nitrite-nitrogen was found. Sequential Analyses of Large Water Samples. Two sets of sequential analyses were performed to further determine the effect of dilution on analysis response. The possibility of analyzing, in sequence, a series of large volume water samples was demonstrated by using 20-mL samples of Hillsborough River water (Tampa, FL). The nitrite + nitrate procedure was used. A 300-mL reaction flask was used starting with 50 mL of 0.0645 M vanadium(III). Ten samples were
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 24, DECEMBER 15, 1989
·
Table V. Comparison of the Iron(II) Molybdate Method and the Vanadium(III) Method in Analysis of Various Environmental Samples
sample0
A B
iron(II) molybdate6 N03- + N02N
