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Week 5 - New Roadblocks and Exciting Sucess
On Monday, I prepared a variety of solutions to test in the HPLC on Tuesday. Because the ascorbic acid had so obscured the 4-chloroaniline spike during last week’s HPLC run, I prepared less concentrated solutions, and also tested solutions of acetic acid and sodium hydrosulfite. Unfortunately, acetic acid did not dissolve the MnO2 much at all, and sodium hydrosulfite was insoluble in methanol, an important requirement for our HPLC setup. Therefore, I determined that low concentrations of ascorbic acid would be our best bet for creating usable HPLC data.
On Tuesday, I retested some old vials on the HPLC, and noticed the 4-chloroaniline peak coming out significantly later than we had previously experienced. This is concerning, because it suggested that either our 4-chloroaniline samples were degrading much faster than should be expected, or that something about our process was flawed for 4-chloroaniline. I tried ramping up the methanol content of our mobile phase, but this just widened the 4-chloroaniline peaks and made them difficult to read accurately. I consulted the HPLC troubleshooting guide, but was not able to find a satisfying solution.
On Wednesday, I did not have access to the HPLC, and so I spent the day refining our MnO2 synthesis procedure. Ali and I recently read a paper detailing methods to produce various morphologies of MnO2, and the method for our specific morphology was a bit more detailed than any previous procedure we had read. I selected the steps of this procedure that made sense for our purposes, and wrote up a new standard operating procedure. Additionally, Ali found a guide to HPLC use from another company that suggests buffering the mobile phase may be critical to prevent peak shifts. We tested the pH of our aliquots and found it to be about pH 4. The pKa of 4-chloroanilne is about 4, so it makes sense that small variations in pH could shift the balance of 4-chloroaniline between the protonated and deprotonated forms.
On Thursday and Friday, I created a phosphate buffer of pH 7, far from the pKa of 4 for 4-chloroaniline, and tested various old vials. Fortunately, the 4-chloroaniline peaks all read at around six minutes! This is the retention time we were expecting, and the homogeneity of the results told us that many of our previous perceived problems were likely the result of the mobile phase being unbuffered. While this has still not enabled us to produce any really beautiful kinetics data, it does clear up much of our previous confusion. Sometimes, moderate success is what you need to get excited about in the realm of science.
