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Simultaneous determination of nitrite and nitrate in various samples using flow-injection spectrophotometric detection
Microchemical Journal 69 Ž2001. 159᎐166
Simultaneous determination of nitrite and nitrate in various samples using flow-injection spectrophotometric detection A. Kazemzadeha,U , Ali A. Ensafi b a
Materials and Energy Research centre P.O. Box 14155-4777, Tehran, Iran b College of Chemistry, Isfahan Uni¨ ersity of Technology, Isfahan, Iran
Received 7 November 2000; received in revised form 10 January 2001; accepted 16 January 2001
Abstract The direct spectrophotometric method has been developed for the simultaneous determination of nitrite and nitrate by flow-injection analysis. The method is based on the catalytic effect of nitrite on the oxidation of pyrogallol red ŽPGR. by bromate in acidic media and the decrease in absorbance of the system at 465 nm. The injected sample is split into two streams. One of the streams is directly treated with the above reagents and then is passed to the sample flow cell of the spectrophotometer. The decrease in absorbance at 465 nm is due to the nitrite. The other stream is passed through a reductor micro column containing copperized-cadmium, where nitrate reduces to nitrite and then the sample was treated with the mixed reagents and was passed through the same cell of the spectrophotometer. The total nitrite concentration initially plus that produced was determined. The influence of reagents concentrations and manifold parameters were studied. The effect of potential interfering ions was examined. Nitrite and nitrate can be determined for the range of 0.003᎐2.00 and 0.030᎐2.00 g mly1, respectively. The sampling rate of analyses was 20 " 3 hy1 , with detection limits 0.001 and 0.010 g mly1 for nitrite and nitrate, respectively. Nitrite and nitrate were determined in food and water samples by the proposed method with satisfactory results. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Nitrite; Nitrate; Simultaneous; FIA; Spectrophotometry
U
Corresponding author. Tel.: q98-21-8771626-7; fax: q98-21-8773352. E-mail address: [email protected] ŽA. Kazemzadeh.. 0026-265Xr01r$ - see front matter 䊚 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 6 - 2 6 5 X Ž 0 1 . 0 0 0 7 2 - 8
160
A. Kazemzadeh, A.A. Ensafi r Microchemical Journal 69 (2001) 159᎐166
1. Introduction The determination of nitrate ion is an important factor in the analysis of food and natural waters. Nitrite and nitrate is intimately involved in the overall nitrogen cycle in soil and higher plants w1x. An upper limit of 45 g mly1 has been proposed on drinking water since the excessive amounts only lead to methemoglobinalmia in infant’s w2x. Nitrite formed during the biodegradation of nitrate and ammonical nitrogen or nitrogenous organic matter is an important indicator of faecal pollution of natural water. The determination of nitrite is of general importance because of its harmful impact on human health. The toxicity of nitrite is primarily due to its interaction with blood pigment to produce methemoglobinalmia. The reaction between nitrite and secondary or tertiary cumene leads to the formation of N-nitroso compounds, some of which are known to be carcinogenic, tetratogenic and mutagenic w3᎐5x. Most of the flow-injection methods for the simultaneous determination of nitrite and nitrate are based on the diazo-coupling reaction w6᎐9x ŽGriess method. or liquid᎐liquid extraction w10x. All these methods have a high limit of detection ŽLOD ) 20 ng mly1 . andror use toxic reagents. According to our knowledge, only one paper is based on the oxidationrreduction system for the simultaneous determination of nitrite and nitrate w11x. The proposed method is based on the catalytic effect of nitrite on the oxidation of pyrogallol red by bromate in acidic solution. Nitrate is reduced to nitrite by using two sequential column Žcopper and copper-coated cadmium. w12x and the total nitrite contents Žnitrite initial plus nitrite produced from nitrate . determined by the proposed method. The method is fast, simple and sensitive for the simultaneous determination of nitrite and nitrate.
grade with high purity. Double distilled water was used throughout. Nitrite standard solution Ž1000 g mly1 . was prepared by dissolving 0.1371 g of dried Žfor 4 h at 105᎐110⬚C. sodium nitrite ŽMerck. in distilled water and diluted to 100 ml in a standard flask. A pellet of sodium hydroxide was added to prevent the liberation of nitrous acid and 1 ml of chloroform to inhibit bacterial growth. Working standard solution was freshly prepared by diluting the stock solution with distilled water. Nitrate standard solution Ž1000 g mly1 . was prepared by dissolving 0.1500 g of dried Žfor 1 h at 105᎐110⬚C. sodium nitrate ŽMerck. in distilled water in a 100-ml standard flask. The solution was treated with a few drops of chloroform and kept in a refrigerator for preservation. Sodium bromate solution Ž0.060 M. was prepared by dissolving 2.2635 g of NaBrO 3 ŽMerck. in distilled water and diluting to 250 ml in a standard flask. Pyrogallol red solution Ž4.75= 10y5 M. was prepared by dissolving 0.0150 of the dye ŽAldrich. in 20 ml of ethanol solution and diluting to 100 ml with distilled water in a 100-ml standard flask. 2.2. Apparatus A diagram of the flow system employed is shown in Fig. 1. The decrease in absorbance was measured with a Shimadzu Model 6AV, equipped with a flow-through cell of Ž20-l inner volume. 1.0-cm optical path, its output was connected to a varian strip-chart recorder ŽModel 9176.. A 12chanel peristaltic pump ŽDesaga, Model PLG. with four sillicon rubber tubes Ž1.0-mm i.d.. was used. PTFE mixing joints and PTFE tubing Ž1.0mm i.d.. was used for the connections and for the mixing coil. The controlled water bath ŽGallenKamp, BLG, 220V. was used at a given temperature of 30 " 0.1⬚C. Sample solutions were injected with a Rheodyl sample injector system.
2. Experimental 2.3. Column reactor 2.1. Reagents All chemicals used were of analytical-reagent
The reduction column ŽR8. was made of a glass tube Ž10 cm = 3 mm i.d.. filled with copperised
A. Kazemzadeh, A.A. Ensafi r Microchemical Journal 69 (2001) 159᎐166
161
Fig. 1. Schematic representation of the FIA manifold for the simultaneous determination of nitrate and nitrite.
cadmium granuls ŽMerck. of particle diameter 0.3᎐1.50 mm held in position with glass woll plugs. The copper column ŽR5. was made of a glass tube Ž20 cm = 3 mm i.d.. filled with copper particles of diameter 0.5᎐1.50 mm. The columns were coated batchwise with copper by pumping a solution of 0.1 M EDTA at pH of 6.80 and 0.1% ŽWrV. copper sulfate through the column. The reduction column was regenerated by passing EDTA copper sulfate solution Žby replacing. the ammonium chloride solution for 10 min. 2.4. Real sample analysis 2.4.1. Preparation of food samples For the meat products Žbeef., 2.00 g of sample was mixed with sand and homogenised in a mortar. The thoroughly mixed sample was then taken in a 100-ml beaker and digested carefully following the method recommended by the AOAC w13x. For the flour samples, 2.00 g of the sample was taken in a 150-ml beaker and mixed with 80 ml of doubly-distilled water. The beaker was placed in a water bath at 40⬚C and the contents digested for 15 min following the method recommended by the AOAC w13x. For cheese samples, any wax coating was removed along with the outer portion of cheese rind and any surface mould. For the very hard cheeses ŽTabriz Cheese with less than 40% moisture., the samples were diced into - 6-mm cubes and mixed thoroughly. The sample was ground 3 times. Soft cheese ŽGorgan Cheese with more
than 40% moisture., sample was mixed well. All cheeses were stored in the glass jars. A cheese water slurry was prepared. The sample was blended in a high-speed blender at high-speed until smooth, 6 g of the slurry was weighted accurately and placed in a 100-ml beaker and digested using the method recommended by the AOAC w13x. 2.5. Procedure Water solution ŽR1., 0.14 M sulfuric acid solution ŽR2., 1.40= 10y2 M bromate solution ŽR3. and 4.75= 10y5 M pyrogallol red ŽR4., previously controlled at the appropriate temperature Ž30⬚C. at a rate of 32.45 ml hy1 for each channel via a peristaltic pump as shown in Fig. 1. The standard samples containing Ž0.003᎐1.50 g mly1 NOy 2, . were injected into a 0.030᎐2.00 g mly1 NOy 3 carrier stream Ždistilled water. at point S ŽFig. 1.. The sample was split into two streams. One was directly treated with the other reagents Žbromate acid and pyrogallol red and passed to the sample flow cell of the spectrophotometer, where the decrease in absorbance at 465 nm is a measure of the initial nitrite concentration Žfirst peak... The other stream was passed through the reduction micro columns of copper ŽR5. and the copperized cadmium ŽR6. where nitrate was reduced to nitrite. The sample was then treated with the mixed reagent via reaction coil ŽR8, 180 cm. and the overall mixture was passed to the same cell of the spectrophotometer. The decrease in absorbance due to nitrite and nitrate was measured Žsecond
162
A. Kazemzadeh, A.A. Ensafi r Microchemical Journal 69 (2001) 159᎐166
peak.. Thus, the nitrate contents were determined from the difference values. The concentration of nitrite and nitrate were evaluated from the peak height by using the calibration curve prepared from the results obtained on standards.
3. Results and discussion Pyrogallol red is a dye that can be oxidised by strong oxidation agents such as bromate w14,15x, hydrogen peroxide w15x and peroxidisulfate w16x. The oxidation of pyrogallol red by bromate is very slow, whereas in the presence of trace amount of nitrite it undergoes a fast reaction Žsee Scheme 1.. In order to optimise the flow-injection method, the influences of reagent concentrations, temperature and manifold variables on the magnitude of the peak height were studied. 3.1. Optimisation of the reagent concentrations and temperature Preliminary tests were carried out with the aid of different flow assemblies to select the optimal manifold configuration. The assembly in Fig. 1 was so selected to produce the best compromise between peak height and peak shape. The effect of reagent concentrations and temperature were studied with fixed manifold parameters, i.e. pump flow rate of 32.45 ml miny1 Žfor each channel ., 0.050 g mly1 NOy and 2 , sample volume of 200 l 0.050 g mly1 NOy 3 and the length of reaction coil ŽR8. of 180 cm at 30⬚C. Various acids such as hydrochloric, sulfuric and
Scheme 1.
Fig. 2. Effect of sulfuric acid concentration on the reaction rate. Conditions: PGR, 4.75= 10y5 M; Bromate, 0.014 M; Nitrite, 0.050 g mly1 ; Nitrate, 0.050 g mly1 ; temperature 30⬚C.
phosphoric acid at the same concentration were tested and sulfuric acid was found to be the best acid for the system. Different concentration of sulfuric acid was tested in the range of 0.020᎐0.200 M ŽFig. 2.. The results show that the best sensitivity was achieved with 0.140 M sulfuric acid. At higher concentrations of acid, the sensitivity is reduced. This is due to fast blank reaction which causes the catalytic effect of nitrite to diminish. Fig. 3 shows the effect of pyrogallol red concentrations on the peak height. The results showed that the pyrogallol red concentration higher than 5.00= 10y6 M has no effect in peak heights. Thus, 4.75= 10y5 M of the ŽPGR. was selected as the optimum concentration. The influence of bromate concentration was also studied in sensitivity ŽFig. 4., by increasing bromate concentration up to 1.40= 10y2 M, the peak height was increased. At the higher concentration of bromate, the blank reaction Žwithout nitrite and nitrate . is so fast, thus the rule of catalytic effect of nitrite is diminished. From the results, 1.40= 10y2 M bromate is the optimum value. The oxidation of pyrogallol red by bromate in
A. Kazemzadeh, A.A. Ensafi r Microchemical Journal 69 (2001) 159᎐166
Fig. 3. Effect of pyrogallol red concentration on the sensitivity. Conditions: Bromate, 0.014 M; Nitrite, 0.050 g mly1 ; Nitrate, 0.050 g mly1 ; temperature 30⬚C; sulfuric acid 0.140 M.
acidic media can occur at a suitable temperature. The results show that by increasing the temperature up to 30⬚C, the peak height increases, whereas before, it decreased at a higher temperature. Thus, the oxidation temperature of 30⬚C was selected for further study.
163
reduction column naturally depends on the amount of oxidising material passing through it. The life of column was over 36 h in continuous analysis. The copperized reduction column can be reactivated on-line by replacing the distilled water with the EDTA-copper sulfate solution. The peak height depends on the residence time of the sample zone in the system, e.g. on the total flow rate and length of the reduction coil. The effect of the pump flow rate was checked over the range of 17.22᎐70.50 ml hy1 for each channel. The signal was decreased as the flow rate increased. This is due to the fact that the residence time of reagents mixture is decreased at the higher flow rate and, hence, reduces the nitrate conversion to nitrite. In addition, the consumption of pyrogallol red is also decreased and causes a decrease in the peak height from the results, 32.45 ml hy1 for each channel, which was found to be the optimum value. The sample volume injected into the carrier line has a significant effect on the peak height. The signal increases with increasing sample volume up to 200 l and remains nearly constant
3.2. Influence of manifold ¨ ariable The optimisation of the proposed flow injection manifold was carried out in the presence of the optimum reagent concentrations and 0.050 g mly1 nitrite and nitrate at 30⬚C. The optimum length of the reduction column was set at 5.0, 10.0, 15.0 and 20.0 cm, with 0.050 g mly1 nitrate by using the reduction column ŽR5 and R6., respectively. For each situation, the percentage reduction was determined by comparison of the peaks plateau achieved with the corresponding 0.050 g mly1 nitrite solution, processed in the same way. A column length of 10᎐20 cm was chosen for R5 and R6. The reduction was complete at these lengths with the reproducible results and low back-pressure. The lifetime of the
Fig. 4. Effect of bromate concentration on the peak heights. Conditions: PGR, 4.75= 10y5 M; Nitrite, 0.050 g mly1 ; Nitrate, 0.050 g mly1 ; temperature 30⬚C; sulfuric acid 0.140 M.
164
A. Kazemzadeh, A.A. Ensafi r Microchemical Journal 69 (2001) 159᎐166 Table 1 Calibration range, detection limit and reproducibility for the determination of nitrite and nitrate Characteristics
Nitrite
Nitrate
Linear range Žg mly1 . Detection limit Žg mly1 . Precision ŽRSD.a 0.010 Žg mly1 . NO2 y and 0.020 Žg mly1 . NO3y 0.050 Žg mly1 . NO2 y and 0.050 Žg mly1 . NO3y 0.50 Žg mly1 . NO2 y and 0.50 Žg mly1 . NO3y
0.003᎐1.50 0.001
0.030᎐2.00 0.010
1.60%
1.64%
1.20%
1.26%
1.10%
1.14%
a
For 10 replicate measurement.
creasing length of the reduction coil from 100 to 300 cm increased the sensitivity due to the longer residence time for the reaction mixture. Thus, 180 cm ŽFig. 1, R8. was selected as the optimum length. 3.3. Determination of nitrite and nitrate
Fig. 5. Recording obtained in the flow-injection simultaneous determination of nitrite and nitrate. Concentration Žng mly1 .: Ža. 100, 100; Žb. 200, 200; Žc. 300, 300; Žd. 400, 400; Že. 500, 500 nitrite and nitrate, respectively.
for increasing volume up to 314 l. A sample volume of 200 l was chosen for further experiments. The influence of the reaction coil length on sensitivity was studied for constant flow rate. In-
The calibration curve was obtained from the results of several nitrite and nitrate standards assayed by the proposed flow-injection procedure and shown in Fig. 5. The performance of both analytes was compared in Table 1. In this table, the detection limit was calculated at 3 times the standard deviation of the peak height measured for 10 injection of 0.050 g mly1 nitrite solution. 3.4. Effect of interfering ions The potential effect of interfering ions on the
Table 2 Effect of interfering ions on the determination of 0.100 g mly1 nitrite Species Kq, Naq, KHP, Citrate, Tartrate, Al3q, Fy CNy, B4 O72y, Liq, Zn2q, Ni2q ClO3 y, NO3 y Co3q CH3 COOy, S2 O32y FeŽIII., BIŽIII. SCNy, Iy, Bry, CrŽIV., Agq, HgŽII., HgŽI., IO4y, AsŽIII., FeŽII., VO3y
Tolerance limit ŽWionrWno2y . 1000 500 50 10 0.50
- 0.20
A. Kazemzadeh, A.A. Ensafi r Microchemical Journal 69 (2001) 159᎐166
165
Table 3 Determination of nitrite and nitrate in food samples Samples
Sausage Salami Flour Tabriz cheese Gorgan cheese a
Concentration of nitrite founda Žg mly1 .
Concentration of nitrate founda Žg mly1 .
Proposed method
Standard method
Proposed method
Standard method
0.152" 0.004 0.092" 0.003 0.250" 0.004 0.030" 0.002 0.020" 0.003
0.154" 0.003 0.091" 0.004 0.252" 0.003 0.028" 0.003 0.022" 0.004
0.070" 0.003 0.048" 0.004 ᎐ 1.75" 0.004 2.160" 0.003
0.072" 0.002 0.050" 0.003 ᎐ 1.78" 0.004 2.145" 0.004
Mean for five determination.
Table 4 Determination of nitrite and nitrate in enviromental water samples Concentration of nitrite a Žg mly1 .
Concentration of Nitratea Žg.mly1 .
Proposed method
Standard method
Proposed method
Standard method
Karaj water Sample 1 Sample 2
0.04" 0.002 0.03" 0.003
0.03" 0.002 0.025" 0.003
1.05" 0.001 1.54" 0.002
1.06" 0.001 1.50" 0.003
Khazar sea water Sample 1 Sample 2
0.04" 0.001 ᎐
0.05" 0.001 ᎐
0.50" 0.001 0.20" 0.002
0.50" 0.001 0.20" 0.002
Sample
a
Mean for five determination.
oxidation reaction between pyrogallol red and bromate was also studied with 0.050 g mly1 nitrite concentration. The results are shown in Table 2. The results showed that many cations and anions cannot interfere with nitrite and nitrate with the proposed method. 3.5. Determination of nitrite and nitrate in real samples To check the applicability of the method, the determination of nitrite and nitrate in various samples such as meat product Žbeef., flour sample, cheese and environmental samples were carried out. The results are shown in Tables 3 and 4. The results show good reproducibility and accuracy in comparison to the standard method w13x.
Acknowledgements The authors are thankful to the Isfahan Uni-
versity of Technology Research Council and materials and energy research centre for the support of this work. References w1x H.O. Buckman, N.C. Brady, The Nature and Properties of Soils, 7th Edition, Macmilan, New York, 1969 ŽChapter 16.. w2x A.D. Eaton, L.S. Clesceri, A.E. Greenberg, Standard Methods for the Examination of Water and Wastewater, 19th Edition, American Public Health Association, Washington, 1995. w3x I.A. Wolff, A.E. Wasserman, Science 177 Ž1972. 15. w4x P.F. Swann, J. Sci. Food Agric. 26 Ž1975. 1761. w5x R.D. Cox, C.W. Frank, J. Anal. Toxicol. 6 Ž1982. 148. w6x J.L.F.C. Lima, A.O.S.S. Rangel, M.R.S. Souto, J. Agric. Food Chem. 43 Ž1995. 704. w7x A. Kojla, E. Gorodkiewicz, Anal. Chim. Acta 302 Ž1995. 283. w8x M.T. Oms, A. Cerda, V. Cerda, Anal. Chim. Acta 315 Ž1995. 321. w9x M.J. Ahmed, C.D. Stalikas, S.M. Karyannis, M.I. Karayannis, Talanta 43 Ž1996. 1009.
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w10x M. Gallego, M. Silva, M. Valcarcel, Fresenus J. Anal. Chem. 323 Ž1986. 50. w11x M. Trojanowicz, W. Matuszewski, B. Szostek, Anal. Chim. Acta 261 Ž1992. 391. w12x B. Bermudez, A. Rios, M.D. Luque de Castro, M. Valcarcel, Talanta 35 Ž1988. 810. w13x K. Helrich ŽEd.., Official Method of Analysis of the
Association of Official Analytical Chemists, 15th Edition, Association of Official Analytical Chemists, Arlington, VA, 1990. w14x A.A. Ensafi, E. Shams, Talanta 41 Ž1994. 1651. w15x A.A. Ensafi, E. Shams, Indian J. Chem. 34A Ž1995. 1005. w16x A.A. Ensafi, K. Zarei, Fresenius J. Anal. Chem. 358 Ž1997. 475.
Simultaneous determination of nitrite and nitrate in various samples using flow-injection spectrophotometric detection A. Kazemzadeha,U , Ali A. Ensafi b a
Materials and Energy Research centre P.O. Box 14155-4777, Tehran, Iran b College of Chemistry, Isfahan Uni¨ ersity of Technology, Isfahan, Iran
Received 7 November 2000; received in revised form 10 January 2001; accepted 16 January 2001
Abstract The direct spectrophotometric method has been developed for the simultaneous determination of nitrite and nitrate by flow-injection analysis. The method is based on the catalytic effect of nitrite on the oxidation of pyrogallol red ŽPGR. by bromate in acidic media and the decrease in absorbance of the system at 465 nm. The injected sample is split into two streams. One of the streams is directly treated with the above reagents and then is passed to the sample flow cell of the spectrophotometer. The decrease in absorbance at 465 nm is due to the nitrite. The other stream is passed through a reductor micro column containing copperized-cadmium, where nitrate reduces to nitrite and then the sample was treated with the mixed reagents and was passed through the same cell of the spectrophotometer. The total nitrite concentration initially plus that produced was determined. The influence of reagents concentrations and manifold parameters were studied. The effect of potential interfering ions was examined. Nitrite and nitrate can be determined for the range of 0.003᎐2.00 and 0.030᎐2.00 g mly1, respectively. The sampling rate of analyses was 20 " 3 hy1 , with detection limits 0.001 and 0.010 g mly1 for nitrite and nitrate, respectively. Nitrite and nitrate were determined in food and water samples by the proposed method with satisfactory results. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Nitrite; Nitrate; Simultaneous; FIA; Spectrophotometry
U
Corresponding author. Tel.: q98-21-8771626-7; fax: q98-21-8773352. E-mail address: [email protected] ŽA. Kazemzadeh.. 0026-265Xr01r$ - see front matter 䊚 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 6 - 2 6 5 X Ž 0 1 . 0 0 0 7 2 - 8
160
A. Kazemzadeh, A.A. Ensafi r Microchemical Journal 69 (2001) 159᎐166
1. Introduction The determination of nitrate ion is an important factor in the analysis of food and natural waters. Nitrite and nitrate is intimately involved in the overall nitrogen cycle in soil and higher plants w1x. An upper limit of 45 g mly1 has been proposed on drinking water since the excessive amounts only lead to methemoglobinalmia in infant’s w2x. Nitrite formed during the biodegradation of nitrate and ammonical nitrogen or nitrogenous organic matter is an important indicator of faecal pollution of natural water. The determination of nitrite is of general importance because of its harmful impact on human health. The toxicity of nitrite is primarily due to its interaction with blood pigment to produce methemoglobinalmia. The reaction between nitrite and secondary or tertiary cumene leads to the formation of N-nitroso compounds, some of which are known to be carcinogenic, tetratogenic and mutagenic w3᎐5x. Most of the flow-injection methods for the simultaneous determination of nitrite and nitrate are based on the diazo-coupling reaction w6᎐9x ŽGriess method. or liquid᎐liquid extraction w10x. All these methods have a high limit of detection ŽLOD ) 20 ng mly1 . andror use toxic reagents. According to our knowledge, only one paper is based on the oxidationrreduction system for the simultaneous determination of nitrite and nitrate w11x. The proposed method is based on the catalytic effect of nitrite on the oxidation of pyrogallol red by bromate in acidic solution. Nitrate is reduced to nitrite by using two sequential column Žcopper and copper-coated cadmium. w12x and the total nitrite contents Žnitrite initial plus nitrite produced from nitrate . determined by the proposed method. The method is fast, simple and sensitive for the simultaneous determination of nitrite and nitrate.
grade with high purity. Double distilled water was used throughout. Nitrite standard solution Ž1000 g mly1 . was prepared by dissolving 0.1371 g of dried Žfor 4 h at 105᎐110⬚C. sodium nitrite ŽMerck. in distilled water and diluted to 100 ml in a standard flask. A pellet of sodium hydroxide was added to prevent the liberation of nitrous acid and 1 ml of chloroform to inhibit bacterial growth. Working standard solution was freshly prepared by diluting the stock solution with distilled water. Nitrate standard solution Ž1000 g mly1 . was prepared by dissolving 0.1500 g of dried Žfor 1 h at 105᎐110⬚C. sodium nitrate ŽMerck. in distilled water in a 100-ml standard flask. The solution was treated with a few drops of chloroform and kept in a refrigerator for preservation. Sodium bromate solution Ž0.060 M. was prepared by dissolving 2.2635 g of NaBrO 3 ŽMerck. in distilled water and diluting to 250 ml in a standard flask. Pyrogallol red solution Ž4.75= 10y5 M. was prepared by dissolving 0.0150 of the dye ŽAldrich. in 20 ml of ethanol solution and diluting to 100 ml with distilled water in a 100-ml standard flask. 2.2. Apparatus A diagram of the flow system employed is shown in Fig. 1. The decrease in absorbance was measured with a Shimadzu Model 6AV, equipped with a flow-through cell of Ž20-l inner volume. 1.0-cm optical path, its output was connected to a varian strip-chart recorder ŽModel 9176.. A 12chanel peristaltic pump ŽDesaga, Model PLG. with four sillicon rubber tubes Ž1.0-mm i.d.. was used. PTFE mixing joints and PTFE tubing Ž1.0mm i.d.. was used for the connections and for the mixing coil. The controlled water bath ŽGallenKamp, BLG, 220V. was used at a given temperature of 30 " 0.1⬚C. Sample solutions were injected with a Rheodyl sample injector system.
2. Experimental 2.3. Column reactor 2.1. Reagents All chemicals used were of analytical-reagent
The reduction column ŽR8. was made of a glass tube Ž10 cm = 3 mm i.d.. filled with copperised
A. Kazemzadeh, A.A. Ensafi r Microchemical Journal 69 (2001) 159᎐166
161
Fig. 1. Schematic representation of the FIA manifold for the simultaneous determination of nitrate and nitrite.
cadmium granuls ŽMerck. of particle diameter 0.3᎐1.50 mm held in position with glass woll plugs. The copper column ŽR5. was made of a glass tube Ž20 cm = 3 mm i.d.. filled with copper particles of diameter 0.5᎐1.50 mm. The columns were coated batchwise with copper by pumping a solution of 0.1 M EDTA at pH of 6.80 and 0.1% ŽWrV. copper sulfate through the column. The reduction column was regenerated by passing EDTA copper sulfate solution Žby replacing. the ammonium chloride solution for 10 min. 2.4. Real sample analysis 2.4.1. Preparation of food samples For the meat products Žbeef., 2.00 g of sample was mixed with sand and homogenised in a mortar. The thoroughly mixed sample was then taken in a 100-ml beaker and digested carefully following the method recommended by the AOAC w13x. For the flour samples, 2.00 g of the sample was taken in a 150-ml beaker and mixed with 80 ml of doubly-distilled water. The beaker was placed in a water bath at 40⬚C and the contents digested for 15 min following the method recommended by the AOAC w13x. For cheese samples, any wax coating was removed along with the outer portion of cheese rind and any surface mould. For the very hard cheeses ŽTabriz Cheese with less than 40% moisture., the samples were diced into - 6-mm cubes and mixed thoroughly. The sample was ground 3 times. Soft cheese ŽGorgan Cheese with more
than 40% moisture., sample was mixed well. All cheeses were stored in the glass jars. A cheese water slurry was prepared. The sample was blended in a high-speed blender at high-speed until smooth, 6 g of the slurry was weighted accurately and placed in a 100-ml beaker and digested using the method recommended by the AOAC w13x. 2.5. Procedure Water solution ŽR1., 0.14 M sulfuric acid solution ŽR2., 1.40= 10y2 M bromate solution ŽR3. and 4.75= 10y5 M pyrogallol red ŽR4., previously controlled at the appropriate temperature Ž30⬚C. at a rate of 32.45 ml hy1 for each channel via a peristaltic pump as shown in Fig. 1. The standard samples containing Ž0.003᎐1.50 g mly1 NOy 2, . were injected into a 0.030᎐2.00 g mly1 NOy 3 carrier stream Ždistilled water. at point S ŽFig. 1.. The sample was split into two streams. One was directly treated with the other reagents Žbromate acid and pyrogallol red and passed to the sample flow cell of the spectrophotometer, where the decrease in absorbance at 465 nm is a measure of the initial nitrite concentration Žfirst peak... The other stream was passed through the reduction micro columns of copper ŽR5. and the copperized cadmium ŽR6. where nitrate was reduced to nitrite. The sample was then treated with the mixed reagent via reaction coil ŽR8, 180 cm. and the overall mixture was passed to the same cell of the spectrophotometer. The decrease in absorbance due to nitrite and nitrate was measured Žsecond
162
A. Kazemzadeh, A.A. Ensafi r Microchemical Journal 69 (2001) 159᎐166
peak.. Thus, the nitrate contents were determined from the difference values. The concentration of nitrite and nitrate were evaluated from the peak height by using the calibration curve prepared from the results obtained on standards.
3. Results and discussion Pyrogallol red is a dye that can be oxidised by strong oxidation agents such as bromate w14,15x, hydrogen peroxide w15x and peroxidisulfate w16x. The oxidation of pyrogallol red by bromate is very slow, whereas in the presence of trace amount of nitrite it undergoes a fast reaction Žsee Scheme 1.. In order to optimise the flow-injection method, the influences of reagent concentrations, temperature and manifold variables on the magnitude of the peak height were studied. 3.1. Optimisation of the reagent concentrations and temperature Preliminary tests were carried out with the aid of different flow assemblies to select the optimal manifold configuration. The assembly in Fig. 1 was so selected to produce the best compromise between peak height and peak shape. The effect of reagent concentrations and temperature were studied with fixed manifold parameters, i.e. pump flow rate of 32.45 ml miny1 Žfor each channel ., 0.050 g mly1 NOy and 2 , sample volume of 200 l 0.050 g mly1 NOy 3 and the length of reaction coil ŽR8. of 180 cm at 30⬚C. Various acids such as hydrochloric, sulfuric and
Scheme 1.
Fig. 2. Effect of sulfuric acid concentration on the reaction rate. Conditions: PGR, 4.75= 10y5 M; Bromate, 0.014 M; Nitrite, 0.050 g mly1 ; Nitrate, 0.050 g mly1 ; temperature 30⬚C.
phosphoric acid at the same concentration were tested and sulfuric acid was found to be the best acid for the system. Different concentration of sulfuric acid was tested in the range of 0.020᎐0.200 M ŽFig. 2.. The results show that the best sensitivity was achieved with 0.140 M sulfuric acid. At higher concentrations of acid, the sensitivity is reduced. This is due to fast blank reaction which causes the catalytic effect of nitrite to diminish. Fig. 3 shows the effect of pyrogallol red concentrations on the peak height. The results showed that the pyrogallol red concentration higher than 5.00= 10y6 M has no effect in peak heights. Thus, 4.75= 10y5 M of the ŽPGR. was selected as the optimum concentration. The influence of bromate concentration was also studied in sensitivity ŽFig. 4., by increasing bromate concentration up to 1.40= 10y2 M, the peak height was increased. At the higher concentration of bromate, the blank reaction Žwithout nitrite and nitrate . is so fast, thus the rule of catalytic effect of nitrite is diminished. From the results, 1.40= 10y2 M bromate is the optimum value. The oxidation of pyrogallol red by bromate in
A. Kazemzadeh, A.A. Ensafi r Microchemical Journal 69 (2001) 159᎐166
Fig. 3. Effect of pyrogallol red concentration on the sensitivity. Conditions: Bromate, 0.014 M; Nitrite, 0.050 g mly1 ; Nitrate, 0.050 g mly1 ; temperature 30⬚C; sulfuric acid 0.140 M.
acidic media can occur at a suitable temperature. The results show that by increasing the temperature up to 30⬚C, the peak height increases, whereas before, it decreased at a higher temperature. Thus, the oxidation temperature of 30⬚C was selected for further study.
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reduction column naturally depends on the amount of oxidising material passing through it. The life of column was over 36 h in continuous analysis. The copperized reduction column can be reactivated on-line by replacing the distilled water with the EDTA-copper sulfate solution. The peak height depends on the residence time of the sample zone in the system, e.g. on the total flow rate and length of the reduction coil. The effect of the pump flow rate was checked over the range of 17.22᎐70.50 ml hy1 for each channel. The signal was decreased as the flow rate increased. This is due to the fact that the residence time of reagents mixture is decreased at the higher flow rate and, hence, reduces the nitrate conversion to nitrite. In addition, the consumption of pyrogallol red is also decreased and causes a decrease in the peak height from the results, 32.45 ml hy1 for each channel, which was found to be the optimum value. The sample volume injected into the carrier line has a significant effect on the peak height. The signal increases with increasing sample volume up to 200 l and remains nearly constant
3.2. Influence of manifold ¨ ariable The optimisation of the proposed flow injection manifold was carried out in the presence of the optimum reagent concentrations and 0.050 g mly1 nitrite and nitrate at 30⬚C. The optimum length of the reduction column was set at 5.0, 10.0, 15.0 and 20.0 cm, with 0.050 g mly1 nitrate by using the reduction column ŽR5 and R6., respectively. For each situation, the percentage reduction was determined by comparison of the peaks plateau achieved with the corresponding 0.050 g mly1 nitrite solution, processed in the same way. A column length of 10᎐20 cm was chosen for R5 and R6. The reduction was complete at these lengths with the reproducible results and low back-pressure. The lifetime of the
Fig. 4. Effect of bromate concentration on the peak heights. Conditions: PGR, 4.75= 10y5 M; Nitrite, 0.050 g mly1 ; Nitrate, 0.050 g mly1 ; temperature 30⬚C; sulfuric acid 0.140 M.
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A. Kazemzadeh, A.A. Ensafi r Microchemical Journal 69 (2001) 159᎐166 Table 1 Calibration range, detection limit and reproducibility for the determination of nitrite and nitrate Characteristics
Nitrite
Nitrate
Linear range Žg mly1 . Detection limit Žg mly1 . Precision ŽRSD.a 0.010 Žg mly1 . NO2 y and 0.020 Žg mly1 . NO3y 0.050 Žg mly1 . NO2 y and 0.050 Žg mly1 . NO3y 0.50 Žg mly1 . NO2 y and 0.50 Žg mly1 . NO3y
0.003᎐1.50 0.001
0.030᎐2.00 0.010
1.60%
1.64%
1.20%
1.26%
1.10%
1.14%
a
For 10 replicate measurement.
creasing length of the reduction coil from 100 to 300 cm increased the sensitivity due to the longer residence time for the reaction mixture. Thus, 180 cm ŽFig. 1, R8. was selected as the optimum length. 3.3. Determination of nitrite and nitrate
Fig. 5. Recording obtained in the flow-injection simultaneous determination of nitrite and nitrate. Concentration Žng mly1 .: Ža. 100, 100; Žb. 200, 200; Žc. 300, 300; Žd. 400, 400; Že. 500, 500 nitrite and nitrate, respectively.
for increasing volume up to 314 l. A sample volume of 200 l was chosen for further experiments. The influence of the reaction coil length on sensitivity was studied for constant flow rate. In-
The calibration curve was obtained from the results of several nitrite and nitrate standards assayed by the proposed flow-injection procedure and shown in Fig. 5. The performance of both analytes was compared in Table 1. In this table, the detection limit was calculated at 3 times the standard deviation of the peak height measured for 10 injection of 0.050 g mly1 nitrite solution. 3.4. Effect of interfering ions The potential effect of interfering ions on the
Table 2 Effect of interfering ions on the determination of 0.100 g mly1 nitrite Species Kq, Naq, KHP, Citrate, Tartrate, Al3q, Fy CNy, B4 O72y, Liq, Zn2q, Ni2q ClO3 y, NO3 y Co3q CH3 COOy, S2 O32y FeŽIII., BIŽIII. SCNy, Iy, Bry, CrŽIV., Agq, HgŽII., HgŽI., IO4y, AsŽIII., FeŽII., VO3y
Tolerance limit ŽWionrWno2y . 1000 500 50 10 0.50
- 0.20
A. Kazemzadeh, A.A. Ensafi r Microchemical Journal 69 (2001) 159᎐166
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Table 3 Determination of nitrite and nitrate in food samples Samples
Sausage Salami Flour Tabriz cheese Gorgan cheese a
Concentration of nitrite founda Žg mly1 .
Concentration of nitrate founda Žg mly1 .
Proposed method
Standard method
Proposed method
Standard method
0.152" 0.004 0.092" 0.003 0.250" 0.004 0.030" 0.002 0.020" 0.003
0.154" 0.003 0.091" 0.004 0.252" 0.003 0.028" 0.003 0.022" 0.004
0.070" 0.003 0.048" 0.004 ᎐ 1.75" 0.004 2.160" 0.003
0.072" 0.002 0.050" 0.003 ᎐ 1.78" 0.004 2.145" 0.004
Mean for five determination.
Table 4 Determination of nitrite and nitrate in enviromental water samples Concentration of nitrite a Žg mly1 .
Concentration of Nitratea Žg.mly1 .
Proposed method
Standard method
Proposed method
Standard method
Karaj water Sample 1 Sample 2
0.04" 0.002 0.03" 0.003
0.03" 0.002 0.025" 0.003
1.05" 0.001 1.54" 0.002
1.06" 0.001 1.50" 0.003
Khazar sea water Sample 1 Sample 2
0.04" 0.001 ᎐
0.05" 0.001 ᎐
0.50" 0.001 0.20" 0.002
0.50" 0.001 0.20" 0.002
Sample
a
Mean for five determination.
oxidation reaction between pyrogallol red and bromate was also studied with 0.050 g mly1 nitrite concentration. The results are shown in Table 2. The results showed that many cations and anions cannot interfere with nitrite and nitrate with the proposed method. 3.5. Determination of nitrite and nitrate in real samples To check the applicability of the method, the determination of nitrite and nitrate in various samples such as meat product Žbeef., flour sample, cheese and environmental samples were carried out. The results are shown in Tables 3 and 4. The results show good reproducibility and accuracy in comparison to the standard method w13x.
Acknowledgements The authors are thankful to the Isfahan Uni-
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