Colorimetric Determination of Nitrate and Nitrite in Foods

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Laboratory Procedure
LPFC-126
December 1983

Health Protection Branch Laboratories
Bureau of Chemical Safety
Ottawa

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Definition: This method is applicable to the determination of nitrate and nitrite in foods in accordance with Table XI of Division 16 of the Food and Drug Regulations.

Scope: This method has been tested in one laboratory on samples of meat products, cheeses and 6 different vegetables (1). The average recovery of sodium nitrite added at levels ranging from 10 to 30 ppm was 95% and recovery of sodium nitrate added at levels from 30 to 400 ppm was 94%. In a subsequent set of experiments involving the recovery of nitrite from cured meats, the overall recoveries of added sodium nitrite ranged between 80 and 114% with an average of 95.4% (2).

THE ANALYST SHOULD BECOME FAMILIAR WITH THIS METHOD USING STANDARDS AND SPIKED SAMPLES BEFORE UNDERTAKING ANALYSIS OF UNKNOWNS.

Principle: The sample is extracted with distilled water and the aqueous extract clarified with zinc hydroxide. Nitrate is reduced to nitrite on a spongy cadmium column (the nitrite originally present is unaltered); sulfanilic acid is diazotised by the nitrite and coupled with N-(1-naphthyl)-ethylenediamine dihydrochloride to form a pink azo dye the absorbance of which is measured at 550 nm. For those samples containing both nitrite and nitrate, the nitrite content is first determined from the unreduced sample filtrate, and then the total nitrite content (existing nitrite and nitrite formed from nitrate) from the column eluate. The nitrate content is then calculated by difference.

Caution: All chemicals should be handled with care. For details respecting chemical hazards, refer to the precautionary notes described in Chapter 51, Laboratory Safety, of the current edition of Official Methods of Analysis, Association of Official Analytical Chemists, Washington, D.C., or other recognized texts respecting laboratory safety.

Apparatus :

Precautions:

Reagents:

Colorimetric Determination of Nitrate and Nitrite in Foods

4. Cadmium sulphate solution, 0.14 M.

5. Zinc metal, ca 10 cm sticks.
6. Sulfanilic acid.
7. Sulfanilic acid solution, 1% in 30% acetic acid.
8. N-(1-naphthyl)-ethylenediamine dihydrochloride (Marshall's Reagent),
0.1% in 60% acetic acid.
9. Color reagent.
10. Sodium nitrate standard solutions:
I. Stock solution, 1 mg/mL.
II. Working solution, 10 mg/mL.
11. Sodium nitrite standard solutions:
I. Stock solution, 500 mg/mL.
II. Working solution, 5 mg/mL.

Procedure: A. Preparation of Standard Curve for Sodium Nitrite

1. Add 0.0, 1.0, 2.0, 4.0, 6.0 and 10.0 mL of NaNO₂ working solution to separate 50 mL volumetric flasks.

2. Add 10 mL color reagent, dilute to volume with H₂O, mix, and let stand for 25 min in the dark.
3. Set the spectrophotometer at 550 nm.
4. Read standards in the cuvettes provided against blank (0 standard addition, see Note 5).
5. Prepare a calibration curve by plotting absorbance against mg NaNO₂/50 mL.
6. The absorbance range should extend from 0 to 0.6 approx. (Note 1).

B. Preparation of Modified Jones Reductor
7. Place 3-5 Zn sticks each in two 800 mL beakers containing 500 mL CdSO₄ solution.
8. Remove Zn sticks every 2-3 hr (or as soon as a thick layer of Cd forms) and scrape off the spongy metallic Cd by rubbing sticks against each other (Caution: Cd must be kept covered with aqueous solution at all times).
9. Decant after 6-8 hr.
10. Wash deposits with two 500 mL portions of distilled H₂O.
11. Transfer Cd with H₂O to a high-speed blender.
12. Blend 2-3 sec.
13. Retain 8-40 mesh particles.
14. Repeat blending to increase yield of particles.
15. Wash particles with 0.1 N HCl.
16. Stir occasionally with a glass rod.
17. Leave overnight in acid.
18. Stir once to degas.
19. Decant.
20. Wash with two 500 mL portions of H₂O.
21. Fill the modified Jones Reductor with H₂O.
22. Insert a glass wool plug.
23. Add Cd to a depth of 8-10 cm.
24. Drain occasionally during filling but do not allow liquid level to fall below top of Cd bed (Note 7).
25. Eliminate bubbles in Cd bed by tapping sides of column.
26. Wash Cd column with 25 mL NH₄Cl buffer and drain to top of Cd bed.

Colorimetric Determination of Nitrate and Nitrite in Foods

C. Testing Efficiency of Cadmium Column
27. Mix 6 mL NaNO₃ working solution (10 mg/mL) and 5 mL NH₄Cl buffer and pour on the Cd column (Note 8).
28. Adjust flow rate to 3-5 mL/min.
29. After column empties, wash with 15 mL H₂O.
30. Collect the eluate and wash in a 50 mL volumetric flask.
31. Add 5.0 mL 60% acetic acid (Note 6).
32. Add 10 mL color reagent.
33. Dilute to volume with H2O and mix.
34. Let stand in dark for 25 min.
35. Prepare a blank in the same manner by taking 6 mL of H₂O through steps 27 and 34 (Note 9).
36. Read extracts in spectrophotometer at 550 nm.
37. The NaNO₂ concentration, as determined from the standard curve, should be ca 48.7 mg/mL if 100% conversion occurs (Note 2).
38. If there is less than 90% conversion, recondition the column by passing 25 mL 0.1 N HCl followed by two 25 mL portions H₂O and 25 mL
NH₄Cl solution and repeat Steps 27-37 (Note 3).
39. Check efficiency of each column once a week.

D. Sample Preparation
40. Remove any outer coating or outer portion of sample and any surface mould.

E. Extraction Procedure
41. Weigh ca 100g sample.
42. Cut into very small pieces.
43. Mix thoroughly by hand or homogenize using a blender.
44. Weigh out a 10 g sample from above homogenate.
45. Blend with 70 mL water and 12 mL 2% NaOH solution in blender until smooth (~5 min).
46. Transfer slurry into a 200 mL volumetric flask.
47. Rinse blender with 30-50 mL H₂O and add to the vol flask.
48. Mix well by swirling.
49. Take out 1-2 drops of suspension from the flask and check pH with pH paper.
50. If pH is on the slightly alkaline side heat the contents in a water bath (50°- 60°C) until the temperature of the suspension reaches close to 50°C.
51. If the pH is less than 8 add additional amounts of 2% NaOH solution until pH rises to 8-10, and then heat as above.
52. Occasionally swirl the contents while heating.
53. Maintain temperature at ca 50°C for an additional 10 min, mixing occasionally.
54. Add 10 mL ZnSO₄ solution.
55. Mix by swirling.
56. If no white precipitate of Zn(OH)₂ becomes visible after the addition of ZnSO₄ solution add 2 to 5 mL 2% NaOH solution and mix (avoid excess addition of 2% NaOH solution).
57. Cool to room temperature in H₂O bath.
58. Dilute to volume with H₂O and mix thoroughly.
59. Filter through fluted paper (Whatman no 1 or preferably no 41) (Note 4) discarding first 20 mL filtrate, into 250 mL glass-stoppered flask.
60. Refilter if extract is not clear.

F. Determination of Nitrite
61. Transfer a 10 mL aliquot of filtrate to a 50 mL volumetric flask.

62. Add 10 mL color reagent (Note 6).
63. Dilute to volume with water.
64. Mix.
65. Let stand in dark for 25 min.

G. Determination of Nitrate plus Nitrite
66. Mix a second 10 mL aliquot of filtrate with 5 mL NH₄Cl buffer and pass through the modified Jones Reductor (Note 8).
67. Wash the column with ca 15 mL H₂O.
68. Collect the eluate and wash in a 50 mL volumetric flask.

70. Dilute to volume with H₂O.
71. Mix.
72. Leave in dark for 25 min.

H. Preparation of Reagent Blank 73. Carry 70 mL of H2O through Steps 45-72 (Note 9).
I. Calculations (Note 5)
74. After color development read the unreduced blank and sample filtrates against the blank described in Step 4.
75. Read the reduced blank and sample eluates against the same blank.
76. Subtract the blank values from the corresponding sample figures.
77. Determine the concentration of nitrite in each case from the standard curve.
78. Calculate the concentration of nitrite in the unreduced and reduced sample filtrates.
79. The difference between the two values is a measure of the nitrate concentration.
80. Finally, calculate the concentrations of nitrite and nitrate in the original sample.

Colorimetric Determination of Nitrate and Nitrite in Foods

81 The following is a typical example:

Example:

I. NaNO₂ determination:

From Standard Curve A, 0.005 = 0.5 mg/50 mL. Since this was from a 10 mL aliquot, The total in 200 mL = (200/10) ´ 0.5 = 10 mg.

Since the original sample wt was 10g,
The NaNO₂ concentration is 10 mg/10 g = 1 mg/g = 1 ppm.

II. NaNO₃ determination:

From Std Curve A, 0.105 = 14.75 mg/50 mL. From "I" we know the sample NaNO₂ concentration to be 0.5 mg/50 mL, then the total NaNO₂ from NaNO₃ is 14.75-0.5 = 14.25 mg/50mL.

Since this is from 10 mL aliquot, in 200mL solution NaNO₂ from NaNO₃ = (200/10) ´ 14.25 = 285 mg.

Since the original sample wt was 10g, conc NaNO₂ from NaNO₃ is 285 mg/10 g = 28.5 mg/g or 28.5 ppm.

The conversion factor from NaNO₂ (MW 69) to NaNO₃ (MW 85) is 1.23.

Therefore, NaNO₃ concentration is 28.5 ´ 1.23 = 35 ppm.

Note: If the results are beyond the range of the standard curve, then dilutions must be made. The aqueous extract may be diluted with H₂O before Steps 61 and 66 and a 10 mL aliquot of the diluted solution should be taken for the analysis. Do not dilute the final extracts after color development has occurred. Appropriate dilutions must also be made to the sample blanks.

Note 1: Check full calibration curve once a month. Each day, while analyzing a sample, take a 6 mL NaNO₂ working standard solution, add 9.0 mL NH₄Cl buffer and
5.0 mL 60% acetic acid and immediately add 10 mL color reagent and determine the absorbance as described. If the absorbance is much different (more than 10%) from what it should be, appropriate corrections should be made to the standard curve before calculating the results. Alternatively, a new standard curve from freshly prepared working standards should be established.

Note 2: 85 mg (1 m mole) NaNO₃ • ⁶⁹ mg (1 m mole) NaNO₂.

Note 3: Recondition the column in the same manner after each analysis and store the Cd under NH₄Cl buffer between analyses.

Note 4: Take one piece of filter paper from top, center and bottom of the box. Fold the three sheets and place in filter funnel. Wash paper with ca 25 mL of distilled H₂O. Analyse filtrate for nitrite and nitrate as described in method. If high readings are obtained, use a different box or brand of filter paper.

Note 5: Each day before reading extracts, standardize the spectrophotometer at 0 absorbance using a solution of 9 mL NH4Cl buffer and 10 mL of color reagent (no nitrite) diluted to 50 mL with H2O as described in Step 4.

Note 6: Since nitrite is unstable in acidic solutions the color reagent should be added immediately after adding the 60% acetic acid solution.

Note 7: When the column is drained the liquid level should be just at (± 2 mm) top of the Cd bed. To achieve this, the delivery end of siphon should be about the same level as the top of Cd bed. Determine the volume of liquid held in Cd bed plus that in the siphon. The total should be 4 ± 0.2 mL.

Note 8: The Food Research Division found that the Cd bed holds about 4.0 mL buffer. Five mL added + 4.0 mL from column makes a total of 9.0 mL buffer in this case. For this reason, the column should be drained to top of Cd bed just before addition of nitrate solutions. Since the intensity of the final color depends on the pH, the amount of total buffer in each case must be strictly controlled.

Note 9: If high blank is obtained for NO3 on Cd column follow the following washing schedule to remove absorbed residual NO3 from Cd column:
Pass 50 mL 1 N NaOH through Cd column.
Wash column with distilled water until the effluent is close to neutral (pH 7 - 8).
Then pass 25 mL 0.1 N HCl and again wash column with H2O.
Finally, wash column with 25 mL NH4Cl buffer.
Then proceed from Step 66 or Step 27.
This should be done each day (early morning) and the columns be kept ready for use later on. Omit the above steps if columns are brand new, i.e. being tested for the first time.

References:

1. Sen, N.P., Donaldson, B., J. Assoc. Official Anal. Chem., 61, 1389 (1978).
2. Sen, N.P., Lee, Y.C., McPherson, M., J. Assoc. Official Anal. Chem., 62, 1186 (1979).
3. Sen, N.P., and McPherson, M., J. Food Safety, 1, 247 (1978).
4. Hamilton, J.E., J. Assoc. Official Anal. Chem. , 59, 284 (1976).

Colorimetric Determination of Nitrate and Nitrite in Foods

Method Evaluation

Table 1 shows the recoveries of nitrate and nitrite added to a variety of foods. Table 2 shows the results of a subsequent experiment performed in the same laboratory involving the recovery of added nitrite to cured meats.

TABLE 1

^a Uncorrected for efficiency of Cd column.

^b Corrected for 90% efficiency of Cd column.

Colorimetric Determination of Nitrate and Nitrite in Foods

TABLE 2

^a pH of an aqueous suspension of 10 g sample + 100 mL water after homogenization in a blender.