The objective of this experiment was to prepare Beer-Lambert calibration curves for different forms of nitrogen and to determine the concentration of nitrate, nitrite, and ammonia ions in different water samples from the Biscayne Bay.
Introduction
Nitrogen is the most abundant element in the Earth atmosphere. In is not surprising that it is one of the most crucial elements not only to human life but all life on our planet. The chemistry of nitrogen is rather diverse because it can exist in different oxidation levels – from +3 to -5. Due to its large impact on the biosphere, the circulation of nitrogen is given significant attention. The totality of processes happening to nitrogen in biosphere and atmosphere is called the nitrogen Cycle. The brief scheme of this process is depicted in Fig 1. There are four principal processes in this cycle, which are namely fixation, decay, nitrification, and denitrification (Lab Manual 68).
The process of nitrogen fixation consists in turning atmospheric nitrogen into ammonium is the most challenging to human technology. Nitrogen is present in the atmosphere in the form of diatomic gas N2 with three bonds between the nitrogen atoms. The bonds in this compound should be broken in order to convert nitrogen into the soluble form are indeed very strong. Under conditions close to normal nitrogen gas only reacts with Lithium to form Li3N, with some of the transition metal complexes and with oxygen in hydrogen with the help of enzymes in nitrogen-fixing bacteria (Flowers et al. 1029). In general, nitrogen fixation also occurs in lightning where at high temperatures nitrogen gas oxidizes to form nitrogen oxide (II) which is then converted to nitrogen oxide (IV) which in its turn dissolves in water and are carried to the soil with precipitation but its contribution is insignificant compared to bacteria’s. With the rise of chemical technology, humans also contribute to nitrogen fixation (with the production of ammonia from atmospheric nitrogen).
Figure 1. nitrogen Cycle (Sprent 6)
The decay process takes place when the reduced organic matter is processed by bacteria and as a result, ammonium is produced by microorganisms. Via exchange and digestion processes, nitrogen is then transferred to other organisms.
Nitrification also happens with the help of bacteria and consists in oxidizing ammonium to nitrite ion. The nitrite ion can be further oxidized to nitrate ion.
Denitrification happens when by various means nitrogen is reduced from nitrates to nitrogen gas. The processes in nitrogen cycle are strongly interconnected, and while some of them may, in theory, be carried separately, all processes always occur in nature together (Sprent 6).
The identification, measurement, and control of nitrate and nitrite levels in water and foods is an important task due to the effect they exert on human health. Nitrates and nitrates are without a doubt necessary for healthy functioning of a human organism. They participate in a number of processes and produce some beneficial effects when used for food and water treatment. For instance, they retard the microbial spoilage of meats, inhibit the growth of Clostridium botulinum – the cause of lethal foodborne infection botulism, and contribute to the control of other pathogens (The Health Effects Of Nitrate, Nitrite, And N- Nitroso Compounds 1-2). However, the effect they exert on human health strongly depends on the concentration of these compounds as well as on the other compounds in combination with which they are consumed. The direct effect of consumption of excess quantities of nitrates and nitrates include the development of methemoglobinemia which consists in the decrease of hemoglobin’s capability to transfer oxygen. The carcinogenic effect of these ions is also considered and researched nowadays (Nitrate And Nitrite In Drinking Water 12). However, the danger of increased concentrations of nitrates and nitrates in the environment also consists in their ability to interact with amides and amines in plants and nitrite-preserved foods to form N-nitroso compounds which are known for their strong carcinogenic effect (The Health Effects Of Nitrate, Nitrite, And N- Nitroso Compounds 1-1).
The experimental method employed in this experiment is called spectroscopy. It is grounded on the fact that some chemical compounds can absorb the photons of visible light and, thus, have distinct and constant color, intensity of which can be measured using special instruments - spectrophotometers. This process is described with the combined Beer-Lambert’s law which can be stated as follows. “The absorbance A of a solution is linearly related to the concentration c, of the absorbing species, and the path-length [l], of the radiation in the absorbing medium” (Krupadanam 128). Or when expressed with mathematical means it looks as follows.
A=εlc=logI0I1
where ε is molar absorptivity of a given substance, I0, I1 – intensity of light prior and after the interaction respectively. It is very convenient for analytical purposes because molar absorptivity is constant for a given substance at given conditions and if the cuvette length is constant, the relationship between absorbance and solute concentration is linear. The analysis is typically carried in two steps. Firstly, the calibration graph is produced for a given substance at given conditions. For this, the absorbance of several samples with initially known concentration is measured. Based on the data the graph of absorbance vs. concentration is constructed. Then the second step (the analysis itself) is carried out. The absorbance of a solution with an unknown concentration is measured and then using the calibration curve, the concentration of the substance in the unknown sample is found.
Procedure
First of all, the initial preparations of the laboratory ware and materials for the experiment were performed. The sample solutions for producing calibration graphs were prepared, and the concentration of nitrogen was found in all the samples. The solutions were stored in labeled plastic bottles for further usage. The colorimeter was operated with the use of the MicroLab program. The timer was prepared for the experiment.
Ammonia (Salicylate Method)
Into a clean screw-cap test tube 10 mL of deionized water were added and the content of one Ammonium Salicylate Pillow. The test tube was covered and shaken to dissolve all the powder. Then the timer was set for three minutes and started. When the timer ended, the content of one Ammonia Cyanurate Reagent Powder Pillow was added to the test tube; it was shaken again, and the timer was set started for 15 minutes. When the time elapsed, the content of the test tube was transferred into a cuvette and put inside the colorimeter. READ BLANK was pressed in the program interface, and the blank value was recorded.
Nitrite (Diazotization Method)
Into the clean screw-cap test tube, 10 mL of deionized water were transferred. The content of the NitriVer Nitrite Reagent Powder Pillow was added to the test tube, it was covered and thoroughly shaken to dissolve the powder. The timer was set to twenty minutes and started. When the time elapsed, the solution was transferred into a cuvette; the cuvette was wiped with Kimwipes and put inside the colorimeter. The READ BLANK was pressed, and the blank value was recorded.
Nitrate (Cadmium Reduction Method)
Into the clean screw-cap test tube, 10 mL of deionized water were put, and the content of one NitraVer Nitrate Reagent Powder Pillow and the timer was started. The timer was set for a minute and during this time the sample was vigorously shaken. After one minute had passed, the sample was set to rest, and the timer was started again, bit this time for five minutes. As the time passed, the sample was transferred into a cuvette; the cuvette was wiped clean from the outside with Kimwipes. It was then put inside the colorimeter, and the blank sample was recorded by pressing the READ BLANK. The calibration curve was produced by using the previously prepared samples. Starting with the most dilute sample with regard to nitrogen, 10 mL of the sample solution were transferred into the cuvette. The content of one NitraVer Nitrate Reagent Powder Pillow was added into the cuvette and the timer set for one minute was started. During this minute, the covered cuvette was vigorously shaken. After one minute had passed, the cuvette was set to rest for five minutes which were also measured using the timer. As five minutes elapsed, the cuvette was put inside colorimeter and sample number, and nitrogen concentration were entered into the program. The same procedure was repeated for all the sample solutions in the order of increasing nitrogen concentration. After all the samples had been analyzed, the calibration curve was produced.
The water samples, in which the concentration of nitrogen was measured were treated the same way as the standard solutions of the corresponding ion. For each water sample, three measurements were performed.
References
Flowers, Paul et al. Chemistry. Print.
Krupadanam, G. L. David. Analytical Chemistry. Hyderabad: Universities Press, 2001. Print.
Nitrate And Nitrite In Drinking Water. Washington D.C.: National Academy Press, 1995. Print.
Sprent, Janet I. The Ecology Of The nitrogen Cycle. Cambridge [Cambridgeshire]: Cambridge University Press, 1987. Print.
The Health Effects Of Nitrate, Nitrite, And N- Nitroso Compounds. Washington, D.C.: National Academy Press, 1981. Print.
