Uploaded March 2020 | Updated September 2026, 2 weeks ago
Testing a drinking water sample for Nitrite using a spectrophotometer.
Nitrite Using Spectrophotometer Method, rough draft, needs editing.
Significance:
Nitrite is significant is that ingestion of oxidizing agents such as nitrates or nitrites cause methemoglobinemia. Hemoglobin in human Red Blood cells is oxidized and converted to methemoglobin, and methemoglobin is unable to carry Oxygen through the blood stream. Nitrite is monitored and kept to less than 1mg/L.
Principle:
Nitrite combines with diazotized sulfanilamide with NED to make a reddish-purple azo dye at a pH of 2.0 to 2.5. This color is read spectrophotometrically. Absorbance is proportional to the amount of nitrite in the Test sample. This method is linear for samples up to 180ug/L N/L with a 1cm light path at 543nm.
Specimen:
Nitrite (NO2-) is unstable and wants to oxidize to nitrate. Because of instability, samples for nitrite should be tested immediately. If unable to test immediately, store samples up to 48 hours at 4 degrees Celsius to prevent bacterial conversion of Nitrite to Nitrate or Ammonia. Do not add acid to the Test sample or the nitrite will be converted to nitrate.
Materials:
1. Nitrite free water (Assume our DI water is nitrite free, although DI water should be run through a demineralizer column to remove nitrites present.)
2. Color Reagent, prepared before class, containing phosphoric acid, sulfanilamide, and NED [N-(1-naphthyl)-ethylenediamine dihydrochloride]
3. Stock Nitrite solution, 100ppm. Make sure you use the bottle of standard labeled NITRITE and not NITRATE. It is different!
4. Spectrophotometer set up for 1 cm light path at 543 nm absorbance. The 5cm light path (rectangle cuvettes) are appropriate for Nitrate levels between 5 and 50ug N/L
5. pH paper.
6. Pipettors with tips
Limitations:
1. Concentrations need to be very small, so care must be used when preparing standards. Prepared standards only last one day.
2. Interferences of other ions may occur because they can cause precipitation: Sb3+, Au3+, Bi3+, Fe3+, Pb2+, Hg2+, Ag+, chloroplatinate (PtCl62+), and metavanadate (VO32-).
3. Cupric ions may cause false low results.
4. Colored ions may interfere with color reaction.
5. NCl3 may cause a false red color change when color reagent is added.
Instrument verification:
Spectrophotometer is verified by using spectrophotometer standards and method provided.
Calibration:
Spectrophotometer is calibrated at the factory.
Quality Control:
1. A blank is used in the procedure.
2. If available a separate manufacturer control sample should be used to verify proper procedure is followed.
3. Nitrite stock solutions should be standardized. Our stock solution was standardized at the manufacturer.
Procedure:
Prepare calibrators:
1. Make a working standard by:
A. Dilute the 100 mg/L Nitrite standard, 25 mL of 100mg/L standard in 100mL of DI water to make a 250ug/L solution.
B. With that solution, dilute 50 mL in 250mL of DI water to make an intermediate solution of 5ug/L NO2-.
C. With the intermediate solution, dilute 10mL in 1000mL of DI water to make a 0.5ug of NO2- working standard solution.
2. Prepare standards using 0.5ug working standard solution: (next page)
Nitrite Standards - 50 mL volumetric flask
concentration (ug/L as N)
mL of 0.5ug/L Nitrite working sta
3. Add 2 mL of color reagent to 50 mL of the standards made.
4. Note: readings on the spec should be performed between 10 minutes and 2 hours following addition of color reagent. During this lag time, verify the spec is working properly (Verifying Photometric Accuracy, AKA Instrument Verification)
5. Produce calibration curve using the standards, absorbance versus ug/L as N
Procedure, test sample:
1. Use filter flasks and 0.45um diameter pore membrane filter to filter your sample if sample appears turbid.
2. Determine the pH of your sample using pH paper. Record pH. The pH needs to be between 5 and 9 S.U. If you need to adjust the pH, use 1.0 N HCl or 1.0 N ammonium hydroxide.
3. Add 2.0 mL of the color solution to 50 mL of your test sample.
4. Reading on the spec should be performed between 10 minutes and 2 hours following addition of color reagent.
5. Determine nitrite concentration of test sample using calibration curve. If your Test sample has a nitrite level above 180ug/L, then dilute your sample so result is within that range.
Calculations:
Interpretation of Subjective Results:
1. If results did not go as planned, think about the dilutions of the 100mg/L standard. There are three dilutions of the standard being made, and with each dilution, a little more error is introduced.
2. Test sample must be in range of the standards, and free of particles.
3. Light path may need to be changed from 1 cm to 5 cm to get a good standard “curve” which really should be a straight line.
Questions to test knowledge:
1. Why are parents
Testing a drinking water sample for Nitrite using a spectrophotometer.
Nitrite Using Spectrophotometer Method, rough draft, needs editing.
Significance:
Nitrite is significant is that ingestion of oxidizing agents such as nitrates or nitrites cause methemoglobinemia. Hemoglobin in human Red Blood cells is oxidized and converted to methemoglobin, and methemoglobin is unable to carry Oxygen through the blood stream. Nitrite is monitored and kept to less than 1mg/L.
Principle:
Nitrite combines with diazotized sulfanilamide with NED to make a reddish-purple azo dye at a pH of 2.0 to 2.5. This color is read spectrophotometrically. Absorbance is proportional to the amount of nitrite in the Test sample. This method is linear for samples up to 180ug/L N/L with a 1cm light path at 543nm.
Specimen:
Nitrite (NO2-) is unstable and wants to oxidize to nitrate. Because of instability, samples for nitrite should be tested immediately. If unable to test immediately, store samples up to 48 hours at 4 degrees Celsius to prevent bacterial conversion of Nitrite to Nitrate or Ammonia. Do not add acid to the Test sample or the nitrite will be converted to nitrate.
Materials:
1. Nitrite free water (Assume our DI water is nitrite free, although DI water should be run through a demineralizer column to remove nitrites present.)
2. Color Reagent, prepared before class, containing phosphoric acid, sulfanilamide, and NED [N-(1-naphthyl)-ethylenediamine dihydrochloride]
3. Stock Nitrite solution, 100ppm. Make sure you use the bottle of standard labeled NITRITE and not NITRATE. It is different!
4. Spectrophotometer set up for 1 cm light path at 543 nm absorbance. The 5cm light path (rectangle cuvettes) are appropriate for Nitrate levels between 5 and 50ug N/L
5. pH paper.
6. Pipettors with tips
Limitations:
1. Concentrations need to be very small, so care must be used when preparing standards. Prepared standards only last one day.
2. Interferences of other ions may occur because they can cause precipitation: Sb3+, Au3+, Bi3+, Fe3+, Pb2+, Hg2+, Ag+, chloroplatinate (PtCl62+), and metavanadate (VO32-).
3. Cupric ions may cause false low results.
4. Colored ions may interfere with color reaction.
5. NCl3 may cause a false red color change when color reagent is added.
Instrument verification:
Spectrophotometer is verified by using spectrophotometer standards and method provided.
Calibration:
Spectrophotometer is calibrated at the factory.
Quality Control:
1. A blank is used in the procedure.
2. If available a separate manufacturer control sample should be used to verify proper procedure is followed.
3. Nitrite stock solutions should be standardized. Our stock solution was standardized at the manufacturer.
Procedure:
Prepare calibrators:
1. Make a working standard by:
A. Dilute the 100 mg/L Nitrite standard, 25 mL of 100mg/L standard in 100mL of DI water to make a 250ug/L solution.
B. With that solution, dilute 50 mL in 250mL of DI water to make an intermediate solution of 5ug/L NO2-.
C. With the intermediate solution, dilute 10mL in 1000mL of DI water to make a 0.5ug of NO2- working standard solution.
2. Prepare standards using 0.5ug working standard solution: (next page)
Nitrite Standards - 50 mL volumetric flask
concentration (ug/L as N)
mL of 0.5ug/L Nitrite working sta
3. Add 2 mL of color reagent to 50 mL of the standards made.
4. Note: readings on the spec should be performed between 10 minutes and 2 hours following addition of color reagent. During this lag time, verify the spec is working properly (Verifying Photometric Accuracy, AKA Instrument Verification)
5. Produce calibration curve using the standards, absorbance versus ug/L as N
Procedure, test sample:
1. Use filter flasks and 0.45um diameter pore membrane filter to filter your sample if sample appears turbid.
2. Determine the pH of your sample using pH paper. Record pH. The pH needs to be between 5 and 9 S.U. If you need to adjust the pH, use 1.0 N HCl or 1.0 N ammonium hydroxide.
3. Add 2.0 mL of the color solution to 50 mL of your test sample.
4. Reading on the spec should be performed between 10 minutes and 2 hours following addition of color reagent.
5. Determine nitrite concentration of test sample using calibration curve. If your Test sample has a nitrite level above 180ug/L, then dilute your sample so result is within that range.
Calculations:
Interpretation of Subjective Results:
1. If results did not go as planned, think about the dilutions of the 100mg/L standard. There are three dilutions of the standard being made, and with each dilution, a little more error is introduced.
2. Test sample must be in range of the standards, and free of particles.
3. Light path may need to be changed from 1 cm to 5 cm to get a good standard “curve” which really should be a straight line.
Questions to test knowledge:
1. Why are parents
