Nitrate is a polyatomic ion and is considered to be the only thermodynamically stable oxidation state of nitrogen in the presence of oxygen in seawater.2 It is considered to be a micronutrient controlling primary production in the euphotic surface layer in many sea areas. Nitrite is the intermediate compound in the microbial reduction of nitrate, or in the oxidation of nitrogen compounds, such as ammonia.2 The concentration of nitrite in nature is very low (< 0.1 µmol/dm3); however in the transition zone, the thin layers of high nitrite concentrations may occur (> 2 µmol/dm3) along with low concentrations of dissolved oxygen (< 0.15 cm3/dm3). Since the nitrogen in nitrite has an oxidation state of +3, it makes it thermodynamically unstable relative to the other nitrogen containing molecules in seawater, such as nitrogen gas, ammonia, ammonium, and nitrate.1Due to this instability, nitrite is considered to be an intermediate in the nitrogen cycle in seawater. Therefore, nitrite is easily oxidized to a more stable compound, such as is nitrate in a process called nitrification which involves two reactions:2
2NH3+ → (reaction 1)
+ → (reaction 2)
In reaction 1, ammonia is oxidized into nitrite by reacting with dissolved oxygen in seawater.Once the nitrite is formed, it is further reacted with more dissolved oxygen to form a stable oxidation state of nitrogen, which is nitrate as shown in reaction 2.1In this experiment, the Griess Reaction method is used for the determination of nitrite in seawater. This procedure is modified from the method developed by Bendschneider and Robinson. This reaction involves converting nitrite into a highly colored azo-dye compound. First, the nitrite in solution is allowed to react with sulfanilamide ion in an acidic solution leading to the formation of a diazo compound.The product is then allowed to react with N-(1-naphthyl) ethylenediamine (NED) to form a highly colored azo dye.
In the Griess Reaction, it is very important to add the reagents sequentially because the sulfanilamide and NED compete for nitrite.2 The sulfanilamide should be added to the sample first, and allowed to stand for 2 to 8 minutes, followed by adding the NED solution to reach the azo dye solution.2 The highly colored azo dye increases the sensitivity and selectivity into which its concentration can be determined by using the Spectrophotometer 20+. Spectrophotometric measurements of the absorbance are performed at 543 nm. Determination of the concentration of nitrite in seawater can then be calculated by incorporating Beer’s Law, seen in equation 1, by generating an external calibration plot of absorbance vs. concentration, where A is the absorbance, ε is the molar absorptivity in L/mol-1cm-1, b is the path length in cm, and c is the concentration of the analyte in mol/L-1. An external calibration curve was then generated, as the solutions were not known to have a matrix effect.1
A = εbc(equation 1)