ANALYST, SEPTEMBER 1986, VOL. 111 1033 Study of Organic Interferences in the Spectrophotometric Determination of Nitrite Using Composite Diazotisation - Coupling Reagents George Norwitz and Peter N. Keliher" Chemistry Department, Villanova University, Villanova, PA 19085, USA A study was made of organic interferences in the spectrophotometric determination of nitrite by the diazotisation - coupling technique using three composite reagents [sulphanilamide and N41- naphthy1)ethylenediamine (NED); sulphanilic acid and NED; and 4-nitroaniline and NED]. Many organic substances interfere, usually causing low results. The interference is usually less with the 4-nitroaniline - NED and sulphanilamide - NED methods than with the sulphanilic acid - NED method. The interferents tested included aliphatic amines (primary, secondary and tertiary), aromatic amines (primary, secondary and tertiary), various phenolic compounds and miscellaneous organic compounds (sucrose, dextrose, lactic acid, succinic acid, acetamide, acetanilide, ethylenediamine tetraacetate, cholesterol, rennin, dodecyl sodium sulphate, acetophenone, urea, citric acid, caffeine, saccharin, morpholine, L-asparagine, gelatin, benzoic acid, formaldehyde, cinchonine, nicotinic acid, trypsin, creatine, starch, albumin, gum tragacanth, casein, formic acid, sorbic acid, ascorbic acid and acetaldehyde).The effect of detergents and soap was also examined. Large amounts of water-miscible solvents (methanol, ethanol, acetone and glycerin) can be tolerated. Water-immiscible solvents do not affect the colour.Keywords: Nitrite determination; organic interferences; composite diazotisation - coupling reagents; spectrophotometry The authors have previously formulated conditions for the spectrophotometric determination of nitrite using three com- posite diazotisation - coupling reagents [sulphanilamide and N-( 1-naphthy1)ethylenediamine (NED); sulphanilic acid and NED; and 4-nitroaniline and NEDl.1 Recently, they have studied inorganic interferences in the methods.2 The purpose of the work reported here was to study organic interferences. The problem of organic interferences is important because nitrite is frequently determined in the presence of organic materials, as in the characterisation of waters, wastes, food and chemical processes. No comprehensive study of organic interferences in the spectrophotometric determination of nitrite by the diazotisation - coupling technique has been made and even qualitative data are ~ c a n t .3 . ~ Experimental Reagents The organic compounds used in this investigation were purchased from Eastman Kodak or Aldrich Chemical. The detergents and soap were ordinary commercial products. Standard nitrite solution A , 1 ml = 100 pg of N02-N. Sodium nitrite (0.4926 g) was dissolved in water and diluted to 1 1 in a calibrated flask. Sodium nitrite solution B, 1 ml = 1.00 pg of N02-N. This was prepared fresh daily from standard nitrite solution A. Sulphanilamide - NED reagent. Sulphanilamide (2.5 g) was dissolved in 650 ml of 1 M hydrochloric acid, 30 ml of 0.20% NED solution were added and the solution was diluted to 1 1.Sulphanilic acid - NED reagent. Sulphanilic acid (5.00 g) was dissolved in a mixture of 750 ml of water and 35 ml of 1 M hydrochloric acid by heating, the solution was cooled to room temperature, 25 ml of 0.20% NED solution were added and the solution was diluted to 1 1. 4-Nitroaniline - NED reagent. 4-Nitroaniline (2.50 g) was dissolved in 300 ml of sulphuric acid (P + 1) by heating, the solution was cooled to room temperature, 50 ml of 0.20% NED solution were added and the solution was diluted to 1 1. * To whom correspondence should be addressed. The NED solution, sulphanilamide - NED and sulphanilic acid - NED reagents were stored in brown bottles in a refrigerator. The 4-nitroaniline - NED reagent was stored in a brown bottle at room temperature. Preparation of Calibration Graphs Appropriate aliquotsl of standard nitrite solution B were transferred into 50-ml calibrated flasks, 10 ml of composite reagent were added from a graduated cylinder, the volume was diluted to the mark and the solution was mixed.The absorbance was measured against the reagent blank at the following wavelengths after the following times: sulphanil- amide - NED, 542 nm and 15 min; sulphanilic acid - NED, 541 nm and 30 min; and 4-nitroaniline - NED, 542 nm and 10 min. The absorbance was plotted against micrograms of N02-N per 50 ml. Study of Organic Interferences Standard nitrite solution B (5 ml) and water were added to 50-ml calibrated flasks. Various amounts of interferent solution were then added and the solution was allowed to stand for about 10 min.Composite reagent (10 ml) was added, the solution diluted to the mark and the absorbance measured as described under Preparation of Calibration Graphs. After consideration of the many analyses for each interferent for each of the three composite reagents, the tolerance limit for the interferent (the limit beyond which the interferent produced an error greater than 0.015 absorbance unit) and the effect of the interferent beyond the tolerance limit were established. Results and Discussion Aliphatic Amines The results for the interference of aliphatic amines, aromatic amines, phenolic compounds, miscellaneous organic com- pounds and typical detergents and soap are shown in Table 1. Aliphatic amines can cause low results with all three reagents.The tolerance limit for the aliphatic amines with the1034 ANALYST, SEPTEMBER 1986, VOL. 111 Table 1. Tolerance limits for organic interferences. ide - NED = sulphanilamide - NED; ic - NED = sulphanilic acid - NED; 4N - NED = 4-nitroaniline - NED Tolerance limit/mg per 50 ml Effect beyond tolerance limit? Interferent Added as* Aliphatic amines: Methylamine . . . . . . 4% W soln. Ethylamine . . . . . . . . 5% W soh. Dimethylamine . . . . . . 5% W soln. Diethylamine . . . . . . 5% W soln. Trimethylamine . . . . . . 2.4% W soh. Triethylamine . . . . . . 1% W soln. Aromatic amines: An i 1 in e . . . . . . . . 4-Chloroaniline . . . . . . 2,4-Dichloroaniline . . . . Anthranilic acid (2-aminobenzoic acid) . . Anthranilamide (2-aminobenzamide) .. . . Naphthionic acid (Camino-l-naph t hal- enesulphonicacid) . . . . N,N-Dimethylaniline . . . . N-Methylaniline . . . . Phenolic compounds: Phenol . . . . . . . . Salicylicacid . . . . . . Resorcinol . . . . . . . . 3,4-Xylenol . . . . . . . . Pyrogallol . . . . . , . . Carvacrol . . . . . . . . Vanillin . . . . . . . . 1 -Naph tho1 . . . . . . . . 1 Yo M soln. 1% M soln. 1 YO M soh. 0.25% M soh. 0.25% M soln. 0.25% M soh. 5% M soh. 5% M soh. 2% W soln. 0.5% M soln. 0.2% W soh. 0.2% M soln. 0.2% W soln. 0.2% M soln. 0.2% M soln. 0.2% M soln. Miscellaneous organic compounds: Sucrose . . . . . . . . 10% W soh. Dextrose [(+)-glucose] . . . . 10% W soh. Lactic acid . . . . . . . . 10% W soh. Succinic acid . . . . . . 5% W s o h Acetamide . . . . .. . , 10% W soh. Acetanilide . . . . . . . . 0.5% W soln. Ethylenediaminetetra- acetate (Na,salt) . . . . 1% W soh. Cholesterol . . . . . . . . 2% W soln. Rennin . . . . . . . . 1% W soh. Dodecyl sodium sulphate . . 2% W soh. Acetophenone . . . . . . Acetophenone Urea . . . . . . . . . . 5% Wsoln. Citric acid . . . . . . . . 5% W soh. Caffeine . . . . . . , . 2% W soln. Saccharin . . , . . . . . 0.4% W soln. Morpholine . . . . . . . . 1 YO W soln. L-Asparagine . , . . , , 0.5% W soln. Gelatin . . . . . . . . 1% W soh. Benzoic acid . . . . . . 0.1 YO W soh. Formaldehyde . . . . . . 0.5% W soh. Cinchonine . . . . . . . . 0.05% W soln. Nicotinic acid . . . . . . 0.1 YO W soh. Trypsin . , . . . . . . 0.02% W soh. Creatine hydrate . . . . . . 0.1% W soln. Starch (potato) .. . . . . 0.5% W soh. Albumin (egg) . . . . . . 0.1% W soh. Gum tragacanth . . . . . . 0.1 YO W soh. Casein . . . . . . . . . . 0.1 YO in 0.01 M KOH Formic acid . . . . . . . . 0.05% W soln. Sorbic acid . . . . . . . . 0.05% W soh. Ascorbic acid . . . . . . 0.05% W soln. Acetaldehyde . . . . . . 0.05% W soln. ide - NED ic - NED 100 150 30 150 25 100 10 10 10 1 .o 7.5 0.2 50 100 400 50 1.5 2 0.05 3 10 0.2 3000 3000 1500 1500 1000 150 300 600 300 400 300 500 50 60 75 50 20 15 2.0 3 15 10 6 3 12.5 12.5 5 5 0.02 0.1 0.2 0.02 3 10 2 5 10 15 5 5 5 0.3 2 0.15 10 10 100 50 1.5 0.5 0.05 2 5 0.2 2000 2000 500 1500 1000 150 200 600 300 300 300 300 30 30 75 50 10 15 1 .o 3 5 10 6 3 10 10 5 5 0.02 0.1 0.1 0.02 4N - NED 800 900 900 1500 150 300 100 50 100 10 50 0.4 200 150 100 75 5 0.2 0.05 3 15 0.2 3000 3000 2000 1500 1500 150 300 600 300 400 300 50 400 200 75 75 50 75 2.5 40 2.5 5 6 5 25 12.5 5 5 0.05 0.05 0.2 0.1 ide - NED Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low --j: Low Low - - - - - - Low 9 Low Low Low Low Low Low Low Low Low Low Low High (colloid) High (colloid) High (colloid) High (colloid) 7 Low Low Low Low - - ic - NED Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low - - Low - - Low 5 Low Low Low Low Low Low Low Low Low High Low Low High (colloid) High (colloid) High (colloid) High (colloid)7 Low Low Low Low - 4N - NED Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low - - Low Low - - - - Low 0 Low Low Low Low Low Low Low Low Low High Low Low High (colloid) High (colloid) High (colloid) High (colloid) 7 Low Low Low Low -ANALYST, SEPTEMBER 1986, VOL.111 Table l-continued 1035 Interferent Detergents and soap: ‘‘All’’ detergent (Lever Brothers) . “Sparkleen” detergent “Ivory” soap (Fisher Scientific) . . . . (Proctor and Gamble) . . Tolerance limithg per 50 ml Effect beyond tolerance limitt Added as* ide - NED ic - NED 4N - NED ide - NED ic - NED 4N - NED 0.1% W soln. 10 10 10 High, High, High, low low low (colloid) (colloid) (colloid) 0.1% W soln. 10 111 111 Low Low Low 0.1% W soln. 0.03)) 0.0311 0.0311 High, High, High, low (ppt.) low (ppt.) low (ppt.) * W = Water solution and M = methanolic solution; all solutions mlV. t “Low” refers to a negative absorbance error greater than 0.015 in the presence of 5.00 pg of N02-N and “high” refers to a positive absorbance error greater than 0.015 in the presence of 5.00 pg of N02-N.$ -, The effect beyond the indicated tolerance limit could not be established because of the limited solubility of the interferent in the additive solution. 0 Larger amounts did not affect the colour but caused difficulty because of floating droplets. 7 Precipitate also formed. 11 See text. three reagents increases markedly in the order sulphanilic acid - NED, sulphanilamide - NED and 4-nitroaniline - NED and is surprisingly high for the 4-nitroaniline - NED reagent. In the sulphanilamide - NED and 4-nitroaniline - NED methods (but not the sulphanilic acid - NED method), more primary and secondary aliphatic amine can be tolerated than tertiary aliphatic amine.In all the methods, larger amounts of the ethylamines (primary, secondary and tertiary) can be toler- ated than the corresponding methylamines. The interference from aliphatic amines is probably mainly caused by the reaction of the aliphatic amine with the nitrite after the addition of the acidic composite reagent. In neutral solution, primary, secondary and tertiary aliphatic amines would merely form amine nitrite salts with the nitrite.5 However, in the presence of acid, primary aliphatic amines would react with nitrite to produce nitrogen, secondary aliphatic amines would react to produce nitroso compounds and tertiary aliphatic amines would still only form amine nitrite salts.5 It would seem from these reactions that tertiary aliphatic amines would interfere less than primary and secondary aliphatic amines but, as indicated above, this does not happen in most instances.It is understandable why the ethylamines (primary, secondary and tertiary) would interfere less than the corre- sponding methylamines, as the ethylamines contain a lower percentage of nitrogen. The 4-nitroaniline - NED method is clearly the method of choice for the determination of nitrite in the presence of aliphatic amines. Aromatic Amines Aromatic amines can cause low results in all three methods and the interference is moderate or strong depending on the particular aromatic amine and the method. The tolerance limit for aromatic amines with the three reagents increases in the order sulphanilic acid - NED, sulphanilamide - NED and 4-nitroaniline - NED, Primary aromatic amines interfere more than secondary and tertiary aromatic amines in all the methods.There is no easy explanation for this. In neutral solution, primary, secondary and tertiary aromatic amines would be expected to form amine nitrite salts with the nitrite (although for the secondary and tertiary aromatic amines this salt formation would be limited by the low solubility of these amines in water). In acidic solution, primary aromatic amines would react with nitrite to form diazonium salts, secondary aromatic amines would react to form N-nitroso compounds and tertiary aromatic amines would react to form p-nitroso compounds.5 In the methods, it might be expected that the interfering primary aromatic amines would be diazotised and then coupled with the NED, but this is by no means certain.Aromatic amines could possibly interfere by acting as coupling agents in place of the NED. However, in the 4-nitroaniline - NED and sulphanilamide - NED methods such coupling seems unlikely (it is known that aromatic amines can act as coupling agents only in neutral or very weakly acidic soh- tions6). The 4-nitroaniline - NED method is the method of choice for the determination of nitrite in the presence of primary, secondary and tertiary aromatic amines. The prob- lem of the determination of nitrite in the presence of primary aromatic amines is encountered in the analysis of wastes from industrial diazotisation processes. Phenolic Compounds Phenolic compounds can cause low results with all three methods.Phenol shows only moderate interference whereas substituted phenols usually produce strong interference. Generally, the interference from phenolic compounds is less with the 4-nitroaniline - NED and sulphanilamide - NED methods than with the sulphanilic acid - NED method. Probably the most important cause of the interference of phenolic compounds is the nitrosation or oxidation of the phenolic compounds by the nitrite after the addition of the composite reagent. It is known that such reactions readily take place in acidic solutions.5 As would be expected, the interference is especially strong with a compound such as 1-naphthol that is easily nitrosated or a compound like pyrogallol that is easily oxidised.It is not believed that phenolic compounds interfere by acting as coupling agents in place of the NED (phenolic compounds can act as coupling agents only in neutral or alkaline solution.6) Either the 4-nitroaniline - NED or sulphanilamide - NED method is the method of choice for the determination of nitrite in the presence of phenolic com- pounds. Miscellaneous Organic Compounds The extent of the interference from miscellaneous organic compounds is varied. Low results are usually produced by an excess of the interferent. Large amounts (greater than 150 mg per 50 ml) of sucrose, dextrose, lactic acid, succinic acid, acetamide, acetanilide, ethylenediaminetetraacetate (diso- dium salt), cholesterol, rennin, dodecyl sodium sulphate and acetophenone can be tolerated in all the methods.Large amounts of urea can be tolerated in the sulphanilamide - NED and sulphanilic acid - NED methods and large amounts of1036 ANALYST, SEPTEMBER 1986, VOL. 111 Table 2. Interference of water-miscible solvents. Abbreviations etc. as in Table 1. Tolerance lirnithl per 50 ml Effect beyond tolerance limit Solvent ide - NED ic - NED 4N - NED ide - NED ic - NED 4N - NED Methanol . . . . 5 5 20 High Low Low Ethanol . . . . 10 10 20 High Low Low Acetone . . . . 10 10 25 High Low Low Glycerin . . . . 10 10 20 Low Low Low citric acid and caffeine can be tolerated in the 4-nitroaniline - NED method. Moderate amounts (15-75 mg per 50 ml) of saccharin, morpholine, L-asparagine and gelatin can be tolerated in all the methods. Small amounts (usually several milligrams per 50 ml) of benzoic acid, formaldehyde, cincho- nine, nicotinic acid, trypsin, creatine hydrate, starch, albu- min, gum tragacanth and casein can be tolerated in all the methods. Trace amounts (in some instances less than 0.1 mg per 50 ml) of formic acid, sorbic acid, ascorbic acid and acetaldehyde can cause low results in all the methods. Formic acid, sorbic acid, ascorbic acid and acetaldehyde are all used as food additives,’ so the tolerance limits should be of practical interest.In general, it is believed that the cause of the low results from most of the miscellaneous organic compounds is nitro- sation or oxidation of the compounds by the nitrite, particu- larly after the addition of the composite reagent. However, a reaction between the organic compound and the diazotised aromatic amine of the composite reagent or even a reaction between the organic compound and the dye cannot be ruled out, The conditions and mechanism involved in the oxidation of organic compounds are different from those involved in the oxidation of inorganic compounds.The high results caused by starch, albumin and gum tragacanth are due to the colloidal nature of the solutions of these substances. Reasonably accurate results can be obtained in the presence of several times the recommended limits for these substances by making the absorbance measurements against a blank solution not containing the composite reagent. Casein interferes because of the colloidal nature of its solution and because a precipitate is formed on adding the composite reagent.Satisfactory results cannot be obtained by filtering solutions containing starch, albumin, gum tragacanth and casein. Generally, the 4-nitroaniline - NED and sulphanilamide - NED methods will tolerate greater amounts of the miscellaneous organic com- pounds than the sulphanilic acid - NED method. However, the interference from urea is much greater with the 4-nitroaniline - NED method than the other two methods (probably because of the greater acidity of the 4-nitroaniline - NED reagent). Detergents and Soap The interference from detergents and soap is diverse. As indicated in Table 1, the maximum limit for “All” detergent is 10 mg per 50 ml for all the methods and the maximum limit for “Sparkleen” detergent is 1 mg per 50 ml for all the methods.When 10 mg of “Sparkleen” per 50 ml were present, the recoveries in the sulphanilamide - NED, sulphanilic acid - NED and 4-nitroaniline - NED methods were 91,22 and 8l%, respectively. The maximum limit for “Ivory” soap is 0.03 mg per 50 ml for all the methods. When more than this amount is present, a curdy precipitate (stearic acid) is produced on adding the composite reagent. However, if the precipitate is filtered off after adding the composite reagent, up to 3 mg of the soap can be tolerated. More than 3 mg of the soap causes incomplete colour development in all the methods. Either the 4-nitroaniline - NED or sulphanilamide - NED method is recommended for the determination of nitrite in the presence of detergents and soap. Solvents The interference from water-miscible solvents is shown in Table 2.It can be seen that large amounts of water-miscible solvents can be tolerated and the tolerance limit for the solvents tested increased in the order glycerin, methanol, ethanol and acetone. The tolerance limits for water-miscible solvents is greater for the 4-nitroaniline - NED method than the other two methods. The effect of water-immiscible solvents (chloroform, hexane, toluene, ethyl acetate and diethyl ether) was also tested. These solvents were found not to affect the colour or extract it and do not interfere if they are separated from the sample solution by use of a separating funnel before or after the development of the colour. For all the organic substances tested in this work the recovery of nitrite obtained at the recommended limits for the organic interferents was approximately 95 YO and the reprodu- cibility at these limits was good.On adding an increasing amount of interferent beyond the recommended limit, the error for the recovery of nitrite tended to increase gradually but the results became somewhat erratic (this would be expected by the nature of the interference). The rate of increase of the error for the recovery of nitrite on adding the increasing amount of interferent varied for the 61 organic substances for the three methods. Organic Compounds in Distilled Water We conducted almost all of our work using reagent-grade (Fisher Scientific) distilled water. However, we also per- formed some work using water that is distilled for the Villanova University general chemistry laboratories by means of a high-capacity still.We found that the absorbance obtained for 5 pg of N02-N was consistently lower (by about 0.02 absorbance unit) when using the Villanova distilled water. This necessitated the preparation of a different calibration graph. We believe that the cause of the low results is organic matter, which is not removed in a large-scale distillation. The blank obtained using this water was insignificant and the pH was about the same as that of reagent water, ca. 6.5. The water distilled on a large scale did not contain detectable amounts of inorganic materials and boiling it (to remove gases) made no difference in the results for nitrite. The standard permanga- nate test for organic matter, which consists of acidification with sulphuric acid, addition of potassium permanganate and noting any decrease in colour after a few minutes standing, is not sufficiently sensitive to detect small amounts of organic matter. References 1. 2. Norwitz, G., and Keliher, P. N., Analyst, 1984, 109, 1281 Norwitz, G., and Keliher, P. N., Analyst, 1985, 110, 689.ANALYST, SEPTEMBER 1986, VOL. 111 1037 3. 4. Williams, W. J., “Handbook of Anion Determination,” 1972, pp. 10, 66, 330 and 783. 5. Boltz, D. F., and Howell, J . A., “Colorimetric Determination of Nonmetals,” Wiley, New York, 1978, pp. 216-220. Butterworths, London, 1979, pp. 147-151. Noller, C . R., “Chemistry of Organic Compounds,” Third Edition, W. B. Saunders, Philadelphia and London, 1965, pp. 261-262,529-530 and 554. Fierz-David, H. E., and Blangey, L., “Fundamental Processes of Dye Chemistry,” Interscience, New York, 1949, pp. 7. National Research Council, “Food Chemicals Index,” Second Edition, National Academy of Sciences, Washington, DC, 6. 249-257. Paper A 61 77 Received March 7th, I986 Accepted April 29th, 1986