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INTRODUCTION OBJECTIVE The purpose of this experiment is to perform spectrophotometric determination for nitrite on a provided river water sample. BACKGROUND Nitrite is a prevalent form of nitrogen, which is highly unstable in water. This is due to the fact that a bacterium (nitrobacter) converts NO2 to nitrate (NO3) rapidly, so that it can be a feed for many forms of bacteria and aquatic life. Because nitrite does not react well with some human biological processes1, it must be controlled. By the Canadian Drinking Water Guidelines, it is recommended that nitrite levels should not exceed 3.2 mg/L2 and nitrate levels should not exceed 45 N. High levels of nitrite can be reduced with the improvement of biological filtration and dilution by water change. The toxicity of nitrite present in samples of freshwater can be reduced with the addition of certain salts. Spectroscopy is a procedure widely used in analytical chemistry and environmental/ freshwater chemistry. The principle of spectrophotometry employs the analysis of the amount of light absorbed by a sample substance. It is compared to the calibration curve created from several standard solutions of known specific ion concentrations, which allows for one to determine the concentration of the unknown sample substance.

See WHO/SDE/WSH Nitrate & Nitrite in Drinking Water: Background document for development of WHO Guidelines for Drinking-water Quality p. 9-10

Canadian Drinking Water Guidelines:


METHODS AND MATERIALS PROCEDURE Please refer to CHEM 2560 Water Quality Analysis for Engineers 2010 Laboratory manual Experiment 3 for the detail procedure this experiment followed APPARATUS Please refer to CHEM 2560 Water Quality Analysis for Engineers 2010 laboratory manual Experiment 3 for a list of materials used in this experiment EXPERIMENTAL PROCEDURE To proceed to analysis through the spectrophotometer, it is required to prepare a set of standard solutions of the following nitrite concentrations: C2 (Standard concentration 0 of Nitrite/ g/L) Volume of 100 g/L Nitrite 0 stock solution required (mL) 20 10 40 20 60 30 80 40 100 50

Solutions were prepared by pipetting (with the volumetric pipette) of the required volumes of nitrite stock solution (stated above). These amounts were transferred directly into 50 mL volumetric flasks. The samples were then filled up to the 50 mL mark with deionized water and 2 mL of nitrite sensitive colour reagent was added to each flask. The solutions were left to stand for about 20 minutes for colour development and taken over to the spectrophotometer for analysis. Using the spectrophotometer required one to fill the cuvette with standard deionized water, and calibrate it to zero absorbancy by turning the absorbance calibration dial. After this, each sample was filled at least way in the cuvette and placed in the spectrophotometer. The absorbancies were taken from the spectrophotometer at = 543 nm (previously set by lab instructor). Note: It was important to wipe the cuvette tubes thoroughly prior to each reading to prevent the straying of light or interference.

DATA AND RESULTS EXPERIMENT RESULTS TABLE 1 NITRITE STANDARD SOLUTIONS Nitrite Volume of 100g/L Color standards NO2/N (mL) reagent (g/L) (mL) 0 0 2 20 10 2 40 20 2 60 30 2 80 40 2 100 50 2 Volume of deionized water (mL) 50 40 30 20 10 0

TABLE 2 SPECTROPHOTOMETRIC RESULTS MEASURED AT = 543 nm 2A Standard Solutions Concentration of Standard Absorbance (A) % Transmittance (%) Solution (g/L) 0 0 100. 20 0.064 86.3 40 0.136 73.1 60 0.203 62.7 80 0.266 54.2 100 0.315 48.4 Trial 1 2 2B - Unknown Sample Concentration of Absorbance Unknown Solution Sample (g/L) 47.20 0.155 52.49 0.172 % Transmittance 70.0 67.3


Calibration Curve - Absorbance (A) vs. Nitrite Concentration (

g/L )

y = 0.0032x + 0.0034 R = 0.9971

TABLE 3 CALIBRATION CURVE (Absorbance vs. Concentration) R2 Slope (m m) Y-intercept (y y) 0.997 0.00321 0.00009 0.00343 0.00528 TRANSMITTANCE CURVE EXPONENTIAL RELATIONSHIP
Chart Title

TABLE 4 - FINAL RESULTS OF UNKNOWN SAMPLE Trial Absorbance (A) % Transmittance, (%) 1 0.155 70.0 2 0.172 67.3 Average 0.164 68.6 SAMPLE CALCULATIONS

Concentration c c (g/L) 47.2 2.5 52.5 2.8 49.8 2.6

Preparing standard nitrite solutions Given a stock solution of 100 g/L & a set of 50 mL volumetric flasks, the following nitrite standards must be prepared (g/L): 0 20 40 60 80 100 By the dilution equation: C1V1 = C2V2 Solve for V1, (i.e. in the case of preparing 60 g/L stock solution) V1 = (60 g/L) (50 mL)/ (100 g/L) 30 mL of 100 g NO2-/L stock solution is required Determining Concentration by Graphical Analysis Trial 1 of Unknown Sample From graph: A = 0.00321c + 0.00343 Concentration of Unknown: C = (Absorbance intercept) / slope C = (0.155 0.00343) / 0.00321 = 47.1975 mg NO2/L Propagation of Errors Trial 1 of Unknown Sample Given: Absorbance uncertainty = +/- 0.02 A (From Least Squares Method on Excel) Slope Uncertainty = +/- 0.00009 A*L/g y- intercept uncertainty = +/- 0.00528 A Concentration Uncertainty C = [absorbance + intercept + (slope/slope)]*C C= [0.02 +0.00528 +(0.00009/0.00321)]* 47.1975 g/L C= 2.52 g/L Therefore, C= (47.2 2.5) g/L Determining % Transmittance for Trial 1 of Unknown Sample From Beers Law: A = -log10 (%) So, (%) = 100 * 10-A = 100 * 10-0.155 69.998% 70 % Average Nitrite Concentration of Unknown Sample (C1 + C2) = (47.1975 + 52.49) g/L 49.8 g/L

DISCUSSION From this experiment, the concentration of NO2- was determined to be 49.8 g/L (Range of 47.2 52.5 g/L). The Canadian Guidelines for Drinking water recommends that the concentration of nitrite should not exceed 3.2 mg/L. Therefore, with regards entirely to nitrite concentration, the unknown sample is considered to be biologically safe to drink. This does not consider other factors however. The fact that NO2 is a highly unstable ion, it was possible that the results obtained from this lab report does not reflect the true concentration of the sample analyzed. Since NO2 is a highly unstable ion, it is important to analyze the samples immediately after they are prepared. Unfortunately, since a spectrophotometer was not accessible for every bench group, this was impossible to achieve. Having to share the instrument with many other analysts meant that it was important to work efficiently (i.e. prepare all standard solutions prior to using the instrument). This doesnt allow for one to analyze each sample quickly enough to ensure that the ions have not already dissociated. Also, each solution had different times to settle (i.e. solution prepared first most likely has higher ion dissociation than the last one prepared). As a result, the issue of not being able to analyze each standard solution immediately after they are prepared imposed a major source of error in this experiment. To improve this error, the laboratory could have divided its members into larger groups. Otherwise, more equipment was required to be readily available and accessible to all lab group members. This couldve greatly affected experiment data, which would have given a lower yield of NO2- than what was actually present (assuming ion dissociation). Because of this, it is very possible that the sample does not meet drinking water regulations. A few other sources of error in this experiment included a possibly scratched cuvette (affecting absorbance values obtained), poor pipetting and inaccurate equipment (i.e. the dropper used to add the colour reagent used in this experiment). Data couldve been affected by the inconsistencies imposed by these sources, which were not accounted for. Extra precautions in measurements taken were to be made when preparing the solutions to have minimized such errors. It is also important to be consistent (i.e. adding the same amount of colour reagent to each standard solution) in order to obtain accurate results. It is not possible to know for sure (unless further analysis is made on the sample) if the nitrite concentration is accurate. The reason why it is important for wastewater engineers to make further analysis on such samples is because high levels of nitrite can be biologically harmful to humans (see reference #2). Such examples of nitrites harmful interactions with the human body include cases of methaemoglobinaemia (a condition which causes the body cells to be deprived of sufficient oxygen), caused by low levels of nitrite in infants and nitrites ability to react with nitrosatable compounds in the human stomach to form N- nitroso compounds a carcinogenic causing compound. Also, as discussed in the introduction of this report, nitrite can easily be converted to nitrate (the most stable prevalent form of nitrogen) from nitrobacteria and cyanobacteria. Since nitrate has many more

harmful biological effects on the human body, it is important to keep levels of nitrite and nitrate controlled as the two prevalent forms of nitrogen are directly related. 3

CONCLUSION From this experiment, it was determined that the concentration of the unknown river water sample to be (49.8 2.6) g/L from methods of spectrophotometry. This is seen accepted as safe drinking water from the Canadian Drinking Water guidelines, which recommends that safe drinking water should not exceed 3.2 mg/L. Restated below are the final results of this experiment. TABLE 4 - FINAL RESULTS OF UNKNOWN SAMPLE Trial Absorbance (A) % Transmittance, (%) 1 0.155 70.0 2 0.172 67.3 Average 0.164 68.6 Concentration c c (g/L) 47.2 2.5 52.5 2.8 49.8 2.6

REFERENCES 1) Wei - CHEM 2560 - Water Quality Analysis for Engineers Laboratory Manual 2010 Experiment 3; p. 9-10 2) Sawyer N.S. & McCarty P.L. (2003) Chemistry for Environmental Engineering, McGraw-Hill Book Company, 5th Chapter 25 (p. 631-648), p.476 3) Canadian Drinking Water Guidelines: 4) Skoog, West, Holler & Crouch Fundamentals of Analytical Chemistry - Chapter 24, p. 718 5) WHO/SDE/WSH Nitrate & Nitrite in Drinking Water: Background document for development of WHO Guidelines for Drinking-water Quality p. 9-10

WHO/SDE/WSH Nitrate & Nitrite in Drinking Water: Background document for development of WHO Guidelines for Drinking-water Quality p. 9-10



APPENDIX B ASSIGNED LAB-RELATED TEXTBOOK QUESTIONS Many spectrophotometers have 2 adjacent scales, one registering % transmission and the other registering absorbance. What numerical value on the latter scale corresponds to 70 on the former? Solution: Absorbance = 2 log (Transmittance %) Absorbance = 2 log (70%) Absorbance = 0.1549