332
Anal. Chem. 1980, 52, 332-335
Determination of Nitrate and Nitrite at the Parts per Billion Level by Chemiluminescence Robert D. Cox Department of Chemistry, The University of Iowa, Iowa City, Iowa 52242
relatively large amounts of nitrate or nitrite, under the appropriate kinetic conditions the reactions are presumed to be applicable to much lower amounts of nitrate or nitrite. This presentation is directed toward the development of analytical methodology for nitrate and nitrite by chemical reduction to nitric oxide and subsequent determination via its chemiluminescent reaction with ozone. Equations for the respective half reactions are:
Nitrite and nitrate are reduced to nitric oxide which is determined via its chemiluminescence reaction with ozone. Nltrke Is selectively reduced under mild conditions and the total nitrate-nitrite content is determined by stronger reduction conditions. Suspended material and color do not interfere and hence the removal of these before analysis is not required. A detection limit of 0.05 pg/L nitrite was obtained using a 20mL sample and the detection limit obtained for nitrate was 5 pg/L using a 5-mL sample. The relative standard deviation was 2 % for nitrite at the 10 pg NO,-N/L level and 3 % for nitrate at the 100 pg NO,-NIL level. These results were compared to standard methodology for accuracy, precision and sensitivity.
+ 4H+ + 3e = NO + 2 H z 0 NO2- + 2H+ + e = NO + HzO
NO3-
E" = +0.96 V E" = + L O O V
Although the standard reduction potentials for nitrate and nitrite are similar, it has been demonstrated that nitrite can be reduced under much milder conditions than nitrate (25). By correctly choosing the reaction conditions, nitrite can be determined selectively. The total nitrate-nitrite content can be determined using stronger reducing conditions and nitrate can be calculated by difference. In addition, nitrate may be determined by removing nitrite and subsequently reducing nitrate to nitric oxide.
Nitrate and nitrite are ubiquitous anions which occur in a wide array of natural systems. In the environment both species are produced in the nitrification process, in which ammonia is oxidized by certain soil bacteria. Nitrate occurs in abundance to this level because of the use of nitrate salts as fertilizers. Also, nitrite results through the reduction of nitrate by denitrification bacteria. This process is though to be important in the occurance of nitrite in the human intestine (1). Humans are exposed to nitrates and nitrites through the ingestion of vegetables, water, and cured meats ( 2 ) . Nitrite, in high levels, can be fatal to infants causing a condition known as methemoglobinemia (3). At the present time, of importance is the relationship of nitrate and nitrite to the formation of N-nitrosamines ( 4 ) . Generally, colorimetric methods are used for the determination of nitrates and nitrites ( 5 , 6). The most common method for nitrite involves the reaction of nitrite with sulfanilamide in acidic solution to yield a diazonium salt which is coupled with an aromatic amine to produce a highly colored azo compound (7). Methods for the spectrophotometric determination of nitrate are generally based on nitration of a phenolic type compound (8-lo), oxidation of an organic compound by nitrate (6),or reduction of nitrate to nitrite and determination via the sulfanilamide method (6). These methods have been reported to be subject to various interferences, thus requiring elaborate clean-up procedures when working with biological media (11, 12). Ion chromatography has been applied to the determination of nitrate and nitrite in environmental samples (13). Although this method is more selective than those based on colorimetry, it is limited by a lack of sensitivity. Both nitrate and nitrite have been determined volumetrically or manometrically by reduction to various gaseous species (14-21). An ultraviolet absorption method for nitrate by reduction to ammonia has been published (22). Methods have also been described in which nitrate and nitrite were determined by thermal reduction to nitric oxide followed by chemiluminescence detection (23,24).In general the chemical reduction of nitrate required highly acidic conditions except when vanadium(I1) was used as a reducing agent. Although volumetric methods are applicable only to samples containing
EXPERIMENTAL Apparatus. A flow diagram of the system is presented in Figure 1. Helium was used as a carrier gas at a nominal flow rate of 170 mL/min. Teflon tubing ('/,-inch 0.d.) was used for
all gas lines. The reaction vessel and degassing apparatus was similar to that used by Drescher and Frank (26) except that the 2-cm cylindrical frit was replaced by a 10-mm disk-type frit to increase the nitric oxide degassing efficiency. The degassing apparatus was located inside an exhaust hood to isolate irritating acid vapors released when changing reaction vessels. Nitric oxide produced was measured using a Bendix 8101-B Oxides of Nitrogen Analyzer operated in the bypass mode to detect only NO. A scrubber was placed between the degassing apparatus and the NO, Analyzer t o trap acid fumes. The scrubber consisted of a 13-mm by 120-mm glass tube packed with approximately 5 g of anhydrous sodium carbonate (Matheson, Coleman and Bell Chemical Co.). Glass wool was wound in the interior of the tube to permit easier gas flow. The scrubber was heated at 130 "C for several hours before use. For nitrate determinations only, an additional cold trap consisting of a miniature impinger in an ice water cold bath was placed in line before the scrubber to remove excess water vapor. The signal from the NO, Analyzer was processed with a strip chart recorder and electronic integration. Two additional traps filled with activated charcoal were installed between the exhaust outlet of the NO, Analyzer and the vacuum pump t o prevent deterioration of pump seals. Choice of Reducing Agents. Table I presents the reducing agents which have been reported for the determination of nitrate and nitrite by reduction to various gaseous species. Although there was a wide choice of reactants, coupling these methods with chemiluminescencedetection involved several requirements. The criteria used for the choice of reducing agents were: (1) the species produced must be nitric oxide, (2) the reaction must be rapid and quantitative at low nitrate and nitrite levels, and (3) the reagent should be of low volatility. Because of its vapor pressure, mercury was eliminated as a viable reducing agent. In addition, vanadium(I1) was not considered since this reagent required timely preparation ( 1 7 ) . Of the other reducing agents, it was found that Fe(I1) and Mo(VI), and Ti(II1) provided the desired properties
a
0003-2700/80/0352-0332$01.00/0 1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 2, FEBRUARY 1980
x
F“=l
IAI
(KI
U
rs
Figure 1. Diagram of Instrumentation. A. Helium carrier gas, B. flow regulator, C. degassing apparatus and sample container, D. cold trap (nitrate only),E. vapw scrubber, F. Teflon filter, G. Bendix NO, Analyzer, H. oxygen, I. pump, J. strip chart recorder, K. electronic integrator
Table I. Reduction of Nitrate and Nitrite to Gaseous Species species pro-
reducing agent nitrate iodideiiodinc Fe(I1) Fe( II)/Mo(VI) Ti(II1) Hg V(I1) hydroquinone1 Mo( VI) sulfanilic acid sulfamic acid urea formic acid Ti( 111) nitrite Hg, F e W , Ti(II1) iodide hydroquinone formic acid sulfamic acid urea
acid used
duced
ref.
concd HC1 6MHCl 6MHCl concd HC1 concdH,SO, 3MHC1 6MHC1
NO NO NO NO NO NO NO
14 15 16 15 15, 16 17 14
concd HC1 concd HCl concd HCI
N,O
17
40% NaOH
N,O
19
N,O N,O NH,
20 21
22
NO NO NO 3 M HCl 3 M HCl
NO N,
N,
21
25 25
for the reduction of nitrate to nitric oxide at the parts-per-billion level. For the nitrite reaction, iodide ion in a weakly acidic medium was the most efficient reducing agent. Reagents and Chemicals. Anhydrous potassium nitrate (J. T. Baker Chemical Co.) and sodium nitrite (J.T. Baker Chemical Co.) were used t o prepare standard solutions of the respective anions. For nitrate determinations, solutions of reagent grade 4 % ferrous ammonium sulfate and 2 % ammonium molybdate (Matheson, Coleman and Bell Chemical Co.) in water were prepared daily. Titanium trichloride (technicalgrade) was purchased as a 20% solution from J. T. Baker Chemical Co. and diluted to 5% for use. Sulfuric acid “suitable for mercury determination” was obtained from J. T. Baker Chemical Co.
For nitrite, 0.2 M sodium iodide (Matheson, Coleman and Bell Chemical Co.) and glacial acetic acid (J. T. Baker Chemical Co.) were used to provide the proper reducing conditions. For both methods, high purity water was obtained by passing distilled water through two IWT Research Model 1ion-exchange columns connected in series (Illinois Water Treatment Co.). Procedures. Nitrite. A 20-mL aqueous sample, containing not more than 1.5 pg of nitrite, was placed in the reaction vessel. Added to this was 1 mL of 0.2 M NaI and 3 mL of acetic acid. The reaction vessel was placed on the degassing apparatus and the nitric oxide swept into the chemiluminescence analyzer. Quantitative evaluation of samples was obtained by comparison with standards on the basis of peak areas. Nitrate. For nitrate determinations, a 5-mL sample containing not more than 1.25 pg of nitrate was used. Added to this, in order, 1mL of 4% ferrous ammonium were 5 mL of concentrated H8O4, sulfate, and 1 mL of 2 % ammonium molybdate. The nitric oxide produced was measured as previously described. One milliliter of 5% titanium trichloride can be substituted for the ferrous ammonium sulfate and ammonium molybdate reagents.
RESULTS A N D D I S C U S S I O N Nitrite. When using a 20-mL sample, a linear calibration from 0.05 ppb NOz- to 75 ppb NO, (0.015 to 25 ,ug/L NO,-N) was obtained. The detection limit of 0.05 ppb NOz- corresponds to 1ng of nitrite in a 20-mL sample. The upper range was extended to 1.5 ppm NOz- by using a 1.-mL sample. Typical peak half-widths were approximately 20-25 s. The chemiluminescence method was compared to the N(1-napthy1)ethylenediaminecolorimetric method recommended by the USEPA and APHA-AWWA-WPCF for the determination of nitrite in water and wastes (5, 6). This method is reported to be highly selective for nitrite and a working range of 0.01 to 1.0 mg/L nitrite nitrogen (NO,-N) is quoted. Standard solutions with nitrite concentrations ranging between 0.10 and 0.0060 mg NO,-N/L were prepared and five replicates for each sample were analyzed using both methods. The results are presented in Table 11. The relative errors for the colorimetric method ranged from 6.7% to 83% while the precision ranged from 6% to 16%. I t should be noted that the concentration of sample D is a t the stated detection limit for the colorimetric method and that of sample E is below the detection limit for the N-(1-napthy1)ethylenediamine method. The chemiluminescence method for nitrite produced relative errors between 1% and 5% while precision data fell within 1.5% of the actual concentration values. This method proved to be very selective over nitrate. At a lo4 M excess of nitrate, no interference was observed. Several N-nitroso compounds were tested and only N nitrosodiphenylamine was denitrosated to produce NO under these conditions. Compounds such as alkyl nitrites which decompose under acidic conditions to produce nitrite also are considered to be an interference. Nitrate. For nitrate determinations, a linear calibration between 5 ppb and 250 ppb NO3- (1-55 pg/L NO,-N) was obtained using a 5-mL sample. In the case of nitrate, the detection was limited by impurities in the ammonium molybdate and ferrous ammonium sulfate reagents. The signal for the reagent blank usually corresponded to an equivalent
Table 11. Comparison of Colorimetric and Chemiluminescent Methods for Nitrite Determination sample
nitrite concn, mg NO,-NIL
A
0.10
B C D E
0.060 0.020 0.010 0.0060
colorimetric concn, mg NO,-N/L 0.12 i 0.02= 0.064 i 0.004 0.023 2 0.003 0.013 ? 0.002 0.011 i 0.001
333
relative error, % 20
6.7 15
30 83
Mean concentration values and standard deviations are for 5 determinations.
chemiluminescent
concn, mg NO,-NIL 0.101
f
0.001a
0.0624 i 0.0203 f 0.0103 f 0.0063 f
0.0006 0.0003 0.00006 0.00004
relative error, 70 1
4 1.5 3.4 5.7
334
ANALYTICAL CHEMISTRY, VOL. 52, NO. 2, FEBRUARY 1980
Table 111. Comparison of Colorimetrica and Chemiluminescent Methods for Nitrate Determination sample
nitrate concn, mg NO,-NIL 2.0
A B C D E a
colorimetric concn, mg NO,-N/L
relative error, %
chemiluminescent concn, mg NO,-NIL
15
2.1 i 0.16 1.06 i 0.03 0.684 i 0.008 0.106 i 0.003 0.0452 I 0.0006
1.7 I 0.2b 0.8 i 0.2 0.4 ?: 0.1 0.07 i 0.06
1.0
0.60 0.10 0.040
20
33 30 25
0.03 * 0.02
Brucine colorimetric method.
relative error, 9% 5 6
14 6
13
Mean concentration values and standard deviations are for 5 determinations.
Table IV. Determination of Nitrite and Nitrate nitrate concn, mg NO,-NIL 0.00300
nitrite concn, mg NO,-NIL
0.00600 0.0120
0.0240
0.00300 0.00600
0.0120 0.0240
nitrite determined, mg NO -NIL 0.00300 0.00608 0.0122 0.0242
Table V. Comparison of Analytical Methods for Nitrate and Nitrite sensitivity, range, mg NO,mg NO,method N02-NIL NO,-NIL
total NO,/NO, determined, mg NO ,-NO ,-NIL 0.00635
nitrate by difference, mg NO,-NIL 0.00335
0.0128
0.00672
0.0241 0.0488
0.0119 0.0246
-
interference
ref.
oxidizing and reducing agents, chlorine, Fe(I1) and Fe(III), chloride, nitrite oxidizing and reducing agents, chlorine, Fe(II1) nitrite quantitatively, particulate material organic material, chloride, nitrite organic material, chloride, nitrite bromide, phosphate, large quantities of organic material nitrite quantitatively
6
Kitrate brucine
0.1
0.1-2
chromotopic acid
0.05
0.05-5
cadmium reduction/N-(1naphthyl)-ethylenediamine 3,4-xylenol ion chromatographic
0.01
0.01-1.0 0.02-0.4
0.1
0.1-10
chemiluminescence
0.001
0.001-0.055
sulfanilic acid/N-(1-naphthyl)ethylenediamine chemiluminescence
0.01
0.01-1.0
0.000015
0.0003-0.025
0.02 0.2
0.2-0.8
6, 8
5 9 10
13
Nitrite
of 10 to 15 ppb NO3- in solution. Since this blank signal appeared in all standards, linear plots were obtained and a concentration of 5 ppb NO3- could be observed. This background signal could be decreased by mixing and degassing the reducing agents prior to use; however, this procedure became time consuming and was necessary only when detection of nitrate below 5 ppb was desired. Typical peak half-widths of 40-45 s were obtained using the described conditions. T h e nitrate chemiluminescence method was compared to the Brucine colorimetric method which is recommended for the determination of nitrate in water and wastes by the USEPA ( 5 ) and is considered a “tentative method” by the APHA-AWWA-WPCF (6). The results are presented in Table 111. The concentrations obtained using the Brucine method were all below actual concentrations with relative errors ranging from 15 to 33%. Precision for the first three samples was between 10 and 25% and significantly increased for the two lowest concentration solutions. The concentrations of samples D and E were prepared a t and below the stated detection limit, respectively ( 5 , 6). For the chemiluminescence method, relative errors were between 5 and 14% and precision was less than 5% for five replicate observations of each concentration. Mixtures. During the three-electron reduction of nitrate to nitric oxide, it is presumed that nitrite is an intermediate species, Le., nitrate is first reduced to nitrite followed by the one-electron reduction of nitrite to nitric oxide. If this is the case, then under the conditions presented for nitrate deter-
chlorine, Fe( 111)
5
mination, nitrate and nitrite should be reduced to nitric oxide in equimolar amounts. Solutions of various amounts of nitrate and nitrite were prepared and analyzed for nitrate and nitrite as presented in Table IV. Nitrite values were obtained using selective iodide reduction. Total nitratenitrite content was determined using stronger reducing conditions and nitrate was calculated by difference. The total amount of nitrate and nitrite found experimentally was within 3% of the calculated amount of nitratenitrite present, showing that both species are reduced to nitric oxide under these conditions. Table V presents a comparison of the features of common methods and the proposed chemiluminescence methods for the determination of nitrate and nitrite. Additional steps have been included in most of the methods to remove interfering species. Color and turbidity interfere in all spectrophotometric procedures and must be removed prior to determination of the desired species. Of the methods for nitrate, only the brucine and cadmium reduction methods are recommended by the USEPA for water analysis ( 5 ) . The brucine, chromotropic acid, and cadmium reduction methods are listed as “tentative” by the APHA-AWWA-WCPF (6). The sulfanilamide/N-(1-naphthy1)ethylenediamineprocedure for nitrite is the only one recommended by either agency.
CONCLUSIONS Methods have been developed for the determination of nitrate and nitrite a t the parts-per-billion level. A summary
Anal. Chem. 1980,
52,335-339
White, J. W. J . Agric. FoodChem. 1975, 23,891. Swann, P. F. J . Sci. Fd. Agric. 1975. 26,1761. Panalaks, T.; Iyengan, J. R.; Sen, N. P. J . Assoc. OM.Anal. Chem. 1973, 56,621. "Methods for the Chemical Analysis of Water and Wastes": USEPA: €PA-625-1 16-74-003, 1974; pp 197-205. "Standard Methods for the Examhatiin of Water and Wastewater", 14th ed.; APHA-AWWA-WPCF: Washington, D.C., pp 418-436. Sawicki, E.; Stanley, T. J.; Pfaff, J.; D'Amico, A. Talanta 1963. 10, 641-655. West, P. W.; Lyles, G. L. Anal. Chim. Acta 1960, 23, 227-232. Johnson, C. M.; Ulrich, A. Anal. Chem. 1950, 22, 1526. Holler, A. C.; Huch, R. V. Anal. Chem. 1949, 21, 1385. Wegner, T. N. J . Dairy Scl. 1972, 55,642. Follett, M. J.; Ratcliff. P. W. J . Sci. Fd. Agrlc. 1963, 14, 138-144. Molik, J.; Puckett, R.; Williams, D.; Sawickl, E. Anal. Lett. 1976, 9. 653-663. Hassan, S. S. M. Analyst (London) 1971, 96, 59-61. Awad, W. I.; Hassan, S.S. M. Talanta 1969, 76, 1383-1391. Van Slyke, D. D.; Lomonte, A. F. Microchem. J . 1969, 14, 609-626. Hassan, S. S. M.; Zaki, M. T. M. Fresenius' Z. Anal. Chem. 1976, 282, 138. Hassan, S. S. M. Anal. Chim. Acta 1972, 60, 442-446. Hassan, S. S. M. Anal. Chim. Acta 1971, 53, 449-451. Hassan, S. S. M. Microchim. Acta 1970, 6. 1109-1 115. Awad, W. 1.; Hassan, S. S. M.;Zaki, M. T. M. Talanta 1971, 78, 219-224. Cresser, M. C. Anavst (London) 1977, 702, 99-103. Spicer, C. W.; Schumaker, P. M. Kouyoumjian, J. A,; Joseph, D. W. Sampling Methodology for Atmospheric Particulate Nitrates", EPA/ 60012-781067, 1978. Moskowitz, A. H. "Particle Size Distribution of Nhate Aerosols in the Los Angeios Area Basin", €PA/600/3-77/053, 1977. Hassan, S. S. M. Anal. Chim. Acta 1972, 58,480-483. Drescher, G. S.; Frank, C. W. Anal. Chem. 1976, 50, 2118-2121.
Table VI. Conclusions nitrate
nitrite
sensitivity 5 ppb NO;, (5-mL sample) range 5-250 ppb NO,., (5-mL sample) 0.025-1.25 ppm NO;, (1-mL sample) precision reproducibility within 5% selectivity not selective over nitrite or alkyl nitrites and nitrates time 5-15 min per sample
0.05 ppb NO;, (20-mL sample) 0.1-75 ppb NO,., (20-mL sample) 0.02-1.5 ppm NO;, (1-mL sample) reproducibility within 2%
not selective over N-nitrosodiphenylamine or alkyl nitrites 5 min per sample
of the features of each method is presented in Table VI. Sensitivity of the chemiluminescence method for nitrite is 600 times lower than that of the colorimetric method. Chemiluminescent determination of nitrate is 100 times more sensitive than the brucine method and 10 times more sensitive than other recommended colorimetric procedures. In the studies presented, the chemiluminescence methods were shown to provide greater accuracy and precision than the colorimetric methods. The chemiluminescence methods also do not require removal of suspended material or color which interferes with colorimetric methods. Compounds such as alkyl nitrates or nitrites which would decompose under acidic conditions to produce nitrate or nitrite would also be detected with the chemiluminescence methods. Determination time is approximately 5 min per sample. Nitrate determinations on samples requiring dilution may be somewhat longer.
LITERATURE CITED (1) Tannenbaum, S. S.;Sinskey, Cancer Inst. 1974, 53,79.
S. J.; Weisman, M.; Bishop, W. J. J . Natl.
335
RECEIVED for review August 10, 1979. Accepted November 12, 1979. This research was partially supported through a grant in the form of a fellowship through Shell Foundation, Inc. This research was performed under auspices of the Environmental Chemistry Section, Institute of Agricultural Medicine and Environmental Health, Department of Preventive Medicine and Environmental Health, College of Medicine, The University of Iowa, Oakdale, Iowa.
Trace Chromium Determination by Furnace At om ic Absorption Spectrometry Following Enrichment by Extraction Sherman S. Chao' and E. E. Pickett" Department
ofBiochemistry,
University of Missouri, Columbia, Missouri 652 1 1
A procedure Is described for wet ashing small samples of bidogical materials and extracting chromium in a form suitable for determination by graphite furnace atomic absorption spectrometry. Wet ashing is done with mixed nitric, perchloric, and sulfuric acids. No loss of chromium occurred in wet-ashing yeast grown in medium containing %r. Chromium(V1) is complexed with methyltricaprylylammonium ion and extracted into a small volume of solvent. Recovery in the extraction step was measured with the use of "Cr. Satisfactory values were found for chromium in two NBS Standard Reference Materials. The method can be used to measure 10 ng of chromium in biological material with 6 % RSD. Chromium retained on any acid-insoluble residue may be a large fraction of the total and is best determined by arc emission spectrography.
Determination of chromium in the sub-part-per-million range in small amounts of biological samples has become 'Present address: Nalco Chemical Co., 6216 West 66th Place, Chicago, Ill. 60638. 0003-2700/80/0352-0335$01 .OO/O
important because of its involvement in the glucose tolerance factor (1-3) and in cardiovascular disease (3). There is thus a need for methods which can take advantage of the great sensitivity of graphite furnace atomic absorption (GFAA) but which avoid the matrix effects so often found by that technique. Good results for chromium in serum (3, 4 ) , in wheat ( 5 ) , and in pancreas and sugar products (2) have been obtained by GFAA without preliminary chemical separation. However, recent interlaboratory comparisons of chromium analyses showed quite poor agreement (6, 7). Moreover, several works have reported losses of chromium during ashing of biological materials (8-11), while others have found no losses or have shown how losses could be avoided in certain kinds of samples (12-16). Conflicting results were reported for several ashing methods and recoveries differed for inorganic and organically-bound chromium. This paper presents results of studies of the conditions which cause loss of chromium. Conditions were established for the wet ashing of plant and animal tissues without loss. An extractive enrichment procedure, based on published methods, is compared with other methods and is shown to be 0 1980 American Chemical Society
