For some time now I have been conducting a molecular census of fermented leaves. The goal here is to get a clearer picture of the microbial content in this niche and define core organisms and their potential for metabolizing nitrogenous compounds. We are specifically interested in looking at the conversion of nitrate to nitrite. Now, a homogenous sample preparation is fairly straight forward, however upon extraction ( --> Be it an hour of agitation in high purity water) of the plant material associated microorganisms, we obtain a colored solution that seems to interfere with the fluorometric readings in nitrite detection assays. Is there a way this substances (whatever they may be) can be separated from the matrix in order to avoid interference?
Hi, Angel -
It's difficult to answer your question without more information. In general, the answer could be relatively straightforward, since the interfering colors are probably from organic compounds, while nitrate and nitrite are inorganic, so it might be as simple as extracting the aqueous layer with immiscible organic solvents. But - see next paragraph - if you have micro-organisms - an organic solvent might be fatal.
It sounds as if you're interested in nitrate reductases in the plant-associated microorganisms;, which - see above - adds an additional level of complexity. So it would help if you could, for example, upload a flow-chart of your protocol, along with some details of your method for detecting nitrate/nitrite (and for isolating and culturing the micro-organisms).
You're project may be more complex than simply getting rid of whatever is interfering with the nitrate/nitrite analysis.
:)
The color interference in the matrix might be of consequence only if you are attempting a photometric quantification of the two molecules (nitrates/nitrites). Color interfence will be of no significance if you separate nitrates and nitrites by liquid chromatography (HPLC). It is fast and easy. Extract your samples in water by heating at 80C with gentle agitation for 20 min. Once cooled down, bring to final volume depending on appropriate dilution factor, which will depend on the range of concentrations anticipated. Prepare a mobile phase of 0.01M octylamine in 20%MeOH, pH adjusted to 6.5 with H3PO4. Use a typical C18 column (4.6 x 250mm; 5-micron) and a flow rate of 1 ml/min. Measure on a diode array detector (DAD) at 220 nm. Retention times should be roughly 7.6 min and 9.4 min for NO2 and NO3, respectively. Quantify on a calibration curve prepared with aqueous solutions of the two standards.
HPLC using anion exchange column seems to be the better solution to de
Angel:
You may use electrochemical biosensors for independent quantification of nitrite and nitrate. In principle, the complexity of your samples will not interfere with the process. If you find this strategy interesting, please take a look to some of my papers.
Good luck.
Angel,
Sorry to hear about what you're going through. I went through something very similar in trying to quantify nitrite and nitrate using the colurimetric Griess assay following fermentarion with beet root juice; of course, the colour of the beet root juice completely interfered with the assay.
As Maria said above, electrochemical detection would be perfect for you. If there's any way you can gain access to this type of equipment or similar (http://www.geinstruments.com/products-and-services/nitric-oxide-analyzer), that could solve a lot of problems.
Good luck.
Hi Angel. and Hi all
To determine of nitrite and nitrate HPLC is good method and its method was asserted by Kyriacou perfectly. Also, Dr Gabriela Almeida was proposed novel and simple determination.
I definitely agree with John S Wishnok, you have hard work as you know that nitrite is unstable intermediate ion.
Angel,
you could determine in a liquid sample the sum of nitrites + nitrates and nitrite using an Autoanayzer. Then, you can calculate the nitrate content by difference.
On the other hand, you could determine the total nitrogen content in the sample (solid or liquid).
I hope having been helpful.
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