Protocol For Nitrite, Nitrate and Iron Determination

Protocol for Nitrite Determination

Principle: Nitrite reacts with Sulfanilamide to form a diazonium salt, which then couples with
N-(1-naphthyl)ethylenediamine to produce a pink-colored azo dye. The intensity of the color,
measured at 540 nm, is proportional to the nitrite concentration.
Chemicals Needed:
• Sulfanilamide
• N-(1-naphthyl)ethylenediamine dihydrochloride (NED)
• Hydrochloric acid (HCl, 1 M solution)
• Sodium nitrite (for preparing standards)
• Distilled water
Preparation of Reagents:
1. Sulfanilamide Solution (0.8% w/v):
o Dissolve 0.8 g of Sulfanilamide in 100 mL of 1 M HCl solution.
o Mix thoroughly until the sulfanilic acid is fully dissolved.
2. NED Solution (0.1% w/v):
o Dissolve 0.1 g of N-(1-naphthyl)ethylenediamine dihydrochloride in 100 mL of
distilled water.
o Store in a dark bottle to prevent degradation.
Preparation of Nitrite Standards:
1. Nitrite Stock Solution (1000 mg/L):
o Dissolve 1.5 g of sodium nitrite (NaNO2) in 1 L of distilled water.
2. Dilute Standards:
o Prepare standards with concentrations such as 0, 0.1, 0.2, 0.5, 1.0, and 2.0 mg/L
by diluting the stock solution:
Target
Final Volume with Distilled Water
Concentration Volume of Stock Solution (µL)
(mL)
(mg/L)

0 0 100

0.1 10 100
0.2 20 100
0.5 50 100
1.0 100 100
2.0 200 100

Procedure for Nitrite Determination:
1. Sample Preparation:
o Take 10 mL of your sample in a clean test tube or cuvette.
2. Reaction Setup:
o Add 1 mL of the Sulfanilamide solution (prepared with 1 M HCl) to the sample
and mix well.
o Add 1 mL of the NED solution and mix thoroughly.
o Allow the reaction to proceed for 10 minutes for full color development.
3. Measurement:
o Measure the absorbance of the sample at 540 nm using a spectrophotometer.
o Create a standard curve by plotting absorbance values of the prepared nitrite
standards against their concentrations.
o Use this curve to calculate the nitrite concentration in your sample by comparing
its absorbance to the standard curve.
Protocol for Nitrate Determination

Principle: Nitrate is reduced to nitrite, which then reacts with Sulfanilamide to form a
diazonium salt. This salt couples with N-(1-naphthyl)ethylenediamine to produce a pink-
colored azo dye. The absorbance at 540 nm is proportional to the nitrate concentration after
reduction.
Chemicals Needed:
• Zinc powder or granules (or another reducing agent like Devarda's alloy)
• Sulfanilamide
• N-(1-naphthyl)ethylenediamine dihydrochloride (NED)
• Hydrochloric acid (HCl, 1 M solution)
• Potassium nitrate (for preparing standards)
• Distilled water
1. Sulfanilamide Solution (0.8% w/v):
o Dissolve 0.8 g of Sulfanilamide in 100 mL of 1 M HCl solution.
o Mix thoroughly until the Sulfanilamide is fully dissolved.
2. NED Solution (0.1% w/v):
o Dissolve 0.1 g of N-(1-naphthyl)ethylenediamine dihydrochloride in 100 mL of
distilled water.
o Store in a dark bottle to prevent degradation.
Preparation of Nitrate Standards:

1. Nitrate Stock Solution (1000 mg/L):
o Dissolve 1.63 g of potassium nitrate (KNO3) in 1 L of distilled water.
o Prepare standards with concentrations such as 0, 0.1, 0.2, 0.5, 1.0, and 2.0 mg/L
by diluting the stock solution:
Target
Volume of Stock Final Volume with Distilled Water
Concentration
Solution (µL) (mL)
(mg/L)
0 0 100
0.1 10 100
0.2 20 100
0.5 50 100
1.0 100 100
2.0 200 100
Procedure for Nitrate Determination:

1. Reduction of Nitrate to Nitrite:
o Take 10 mL of your sample in a clean test tube.
o Add a pinch of zinc powder (or a few granules) as a reducing agent.
o Acidify the sample by adding 1 mL of 1 M HCl solution.
o Let the mixture stand for 15–30 minutes to allow the reduction of nitrate to
nitrite.
o After reduction, filter the sample to remove the zinc particles.
2. Reaction Setup:
o Take 10 mL of the filtered sample.
o Add 1 mL of the Sulfanilamide solution (prepared with 1 M HCl) to the sample
and mix well.
o Add 1 mL of the NED solution and mix thoroughly.
o Allow the reaction to proceed for 10 minutes for full color development.
3. Measurement:
o Prepare a standard curve with the nitrate standards, following the same
procedure, including the reduction step.
o Use this curve to calculate the nitrate concentration in your sample by
comparing its absorbance to the standard curve.
Protocol for Iron Determination

Principle: In this method, thioglycolic acid reduces ferric iron (Fe3+) to ferrous iron (Fe2+)
and forms a color complex with it. The complex forms a characteristic color under alkaline
conditions, which can be measured spectrophotometrically. Citric acid is used to buffer the
solution, and ammonium hydroxide is used to adjust the pH.
Chemicals Needed:
• Thioglycolic acid (TGA)
• Citric acid
• Ammonium hydroxide (NH_4OH, 25% solution)
• Iron standard solution (e.g., 1000 mg/L Fe)
• Distilled water
1. Thioglycolic Acid Solution:
o Use thioglycolic acid directly from the bottle. Typically, a few drops
(approximately 0.1 mL) per sample are sufficient for the reaction.
2. Citric Acid Solution (10% w/v):
o Dissolve 10 g of citric acid in 100 mL of distilled water.
3. Ammonium Hydroxide Solution (1 M):
o Dilute the concentrated ammonium hydroxide (25%) to 1 M by mixing
approximately 4 mL of the 25% NH_4OH with 96 mL of distilled water.
Preparation of Iron Standards:
1. Iron Stock Solution (1000 mg/L):
o dissolve 0.702 g of ferrous ammonium sulfate in 1 L of distilled water.
o Prepare standard solutions with concentrations of 0, 0.5, 1.0, 2.0, 3.0, and 5.0
mg/L by serially diluting the iron stock solution.
Target
Volume of Stock Final Volume with Distilled Water
Concentration
Solution (µL) (mL)
(mg/L)
0 0 100
0.5 50 100
1.0 100 100
2.0 200 100
3.0 300 100
5.0 500 100
Procedure for Iron Determination:

1. Sample Preparation:
o Take 10 mL of the sample in a test tube or a suitable container.
2. Reduction and Complex Formation:
o Add 2–3 drops (approximately 0.1 mL) of thioglycolic acid to the sample. This
will reduce Fe3+ to Fe2+ and form a complex.
o Add 2 mL of the 10% citric acid solution to prevent any precipitation and
maintain the solution's pH.
3. pH Adjustment:
o Slowly add a sufficient amount of the 1 M ammonium hydroxide solution to
adjust the pH to approximately 8-9. At this pH, the iron-thioglycolate complex
will form a visible color.
o Mix the solution well and allow it to stand for 10–15 minutes for the color to
fully develop.
4. Measurement:
o Prepare a standard curve by plotting the absorbance of the iron standards against
their concentrations.
o Use this standard curve to determine the iron concentration in your sample by
comparing its absorbance to the standard curve.

Protocol For Nitrite, Nitrate and Iron Determination

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Protocol for Nitrite Determination

1.0 100 100

2.0 200 100

Protocol for Nitrate Determination

Preparation of Nitrate Standards:

1.0 100 100

2.0 200 100

Procedure for Nitrate Determination:

Protocol for Iron Determination

1.0 100 100

2.0 200 100

3.0 300 100

5.0 500 100

Procedure for Iron Determination:

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