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Week 5: Reduction of trinitrotoluene in iron porphyrin
On Monday of week 5, Dr. Tratnyek, Ali and I had a meeting to discuss the results I obtained from my TNT trials. I ran six different trials two times each with six different concentrations of iron porphyrin, and I made a graph of the observed rate constant (k-observed) versus the concentration of iron porphyrin ([FeP] (M)).
From that graph, I was able to obtain a KFeP value=152.95, which falls very close to the extension of the line of best fit created by previous literature.
TNT was not one of the nitroaromatic compounds that Schwartzenbach measured in a reduction reaction with iron porphyrin and cysteine, so the ΔG0 is an estimated value, and the line of fit it falls on is a projection of the fit calculated in the 1990 Schwartzenbach paper.
We were excited to see that the KFeP of both TNT and NB fall close to the predicted fit, however, my TNT data does not look as nice as the resulting KFeP value looks. Firstly, I ran two trials of each FeP concentration, and in some cases, the k-observed values are vastly different from each other, which does not make sense. Another trend we are seeing is that the last few data points drop off to form a little bit of a ‘j’ curve at the end, which doesn’t exactly follow second order reaction kinetics. These two graphs illustrate the trends I have described:
This one is a good example of the k-observed values aren't very similar, see how the two fit lines aren't very close?
This next graph shows a really clear tendency for the last few time points to drop off in a 'j' curve shape.
For now, we have decided to move on as planned with the other nitroaromatic compounds, but I am running a couple of controls with the TNT to see if maybe the TNT is having a reaction with the cysteine as well as the iron porphyrin, which could cause those irregularities in the data. Dr. Tratnyek is also looking into whether or not he has seen these kinds of irregularities with the TNT before in different experiments. If the reaction kinetics are not second order, or if the TNT is reacting with the cysteine, then we would have to approach TNT differently. My next order of business for the week was to run trials with 4-chloronitrobenzene.
4-chloronitrobenzene or 4-CNB is one of the NAC’s that Schwartzenbach has data on, and Ali suggested that I analyze this compound next because it is less complicated than DNAN, and it shouldn’t have any of the problems that DNAN was having. Initially, I ran into some problems with the HPLC, and in order to describe them, I am going to have to tell you how the HPLC works.
High Performance Liquid chromatography separates a mixture of chemicals by passing a liquid sample through a column packed with adsorbent material, the different chemicals in the mixture interact differently with the adsorbent column, the ones with the weaker interactions elute out of the column faster, and the stronger interactions have a longer retention time. Because organic molecules adsorb UV light a differing wavelengths, the detector shines a UV light through the sample, and measures the amount of light absorbed. We just have to know at what wavelength to set the detector at. 254nm is where our nitroaromatic compounds absorb best.
Anyway, when I ran a calibration with 4-CNB, the HPLC chromatogram was nothing but noise, Which indicates either a problem with the wavelength, or that the 4-CNB has too long of a retention time. In this case, it was the retention time. 4-CNB doesn’t elute until around 12 minutes, and I was only running the HPLC for 10. It was a pretty quick fix, and I have been running 4-CNB trials for the rest of the week. Next week I will have the data from all of them.
