As you may recall from my earlier posts, I analyze three nutrients (nitrate, nitrite, and ammonium). I’ve already talked in detail about nitrate, so today’s post is dedicated to our nitrite experiment. Get ready for a lot of chemistry 🙂
Nitrite, like nitrate, is a polyatomic ion made up of nitrogen and oxygen, but it has only 2 oxygen atoms instead of the three of nitrate. Therefore, nitrite’s formula is NO2–. We’re analyzing nitrite concentrations in the seawater because nitrite is an intermediate (it’s both a product and a reactant) in many biological processes in the nitrogen cycle.
What I find really cool is that we use a completely different method to test for each of the nutrients. For NO2– we measure absorbance of light to tell us a solution’s concentration. Absorbance is, quite simply, how much light is absorbed by a solution. Think of it like blocking the sun with curtains. Dark curtains will absorb a lot of the light, and therefore stop it from entering your room ,while light curtains will allow much more light through Solutions work in the exact same way – deeply colored solutions will absorb light while colorless solutions allow light to pass through. We can measure how much light passes through a solution with a spectrophotometer.
Seawater (which contains nitrite) is naturally colorless, so in order to produce color, we need to react it with compounds that will cause a color change. To do this, we add sulfanilamide and napthylethylenediamine (NED) to 10mL of our nitrite standards and all of our samples. These compounds, when reacted with nitrite, form a magenta-colored product. Standards and samples with greater nitrite concentrations produce the most color, and are therefore the deepest, while the most colorless standards and samples have the least amount of nitrite. The reaction occurs according to the following mechanism:
I know this looks a lot more complicated that what you’re used to, but that’s because sulfanilamide and NED are organic compounds, so they’re drawn using their structure, not just their formulas. The nitrite (circled in red) first reacts with the sulfanilamide (underlined in green). Then, the product of that reaction (underlined in gray) reacts with NED (underlined in blue) to make an azo compound (boxed in magenta), which is a double-bonded nitrogen compound. It’s this azo compound that provides the color for the absorbance analysis – and the color is magenta.
Once we’ve added the sulfanilamide and NED to the samples, we can start analyzing them. We fill a cuvette with the standard or sample we want to test, then put it in the machine. The spectrophotometer will shine light at a particular wavelength through the sample and measures how much passes through, then displays how much is absorbed. We use 543nm for the nitrite analysis because this is the where the azo compound absorbs the most light, as seen from the graph below (adapted from Promega). 543nm is green light, which is best absorbed by our magenta solution (blue and red light go right through the solution, so they are not helpful).
The reason a spectrophotometer works is due to the Beer-Lambert Law, which states that absorbance (A) is directly correlated to concentration (C) when multiplied by a constant (k) and the path length (L, the distance the light must travel) – A = k × L × C. Since the constant and path length are unchanged, the only factor that affects the absorbance is the concentration. If you look at the picture of the cuvettes, the darkest magenta on the right will have the highest absorbance, while the lightest one on the left will have the smallest absorbance.
Just like with the nitrate analysis, we use the absorbance values from the standards solutions to make a standard curve; then, when we plot the samples’ absorbance values to that curve, we can determine the nitrite concentration of each of our samples. This gives the researchers on the expedition an idea of the biological processes happening at each depth sampled.
Science is awesome. 🙂
Until next time,