Unravelling complex systems: development and applications of NMR and MS methodology

Abstract

Complex mixtures are notoriously difficult to analyse as they contain hundreds or even thousands of compounds that cannot be easily separated using conventional chromatographic techniques. This makes the traditional approach of isolating single compounds and identifying them impossible. Instead, high-resolution techniques, namely nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) can be used to obtain information. The thesis focuses on improving existing methodology for the analysis of complex mixtures using NMR and MS as well as applying these techniques on complex mixtures with everyday life significance.

Improvements to 1H-detected 13C–13C double quantum experiments, known as ADEQUATE, are presented. These experiments allow for the carbon skeleton of a molecule to be traced out, which is beneficial in structure elucidation, however, they have poor sensitivity as they rely on molecules containing coupled pairs of 13C nuclei at a natural abundance of around 1-in-8,100. This requires high sample concentrations and long experiment times and hence these methods are not commonly used in the analysis of complex mixtures. A modification involving the refocussing of 1JCH coupling prior to 13C–13C double quantum coherence evolution is presented which doubles the sensitivity of these experiments. A 2-fold sensitivity enhancement is equivalent to a 4-times reduction in experiment time; hence the modified experiments allow 13C–13C double quantum spectra to be recorded overnight instead of over multiple days. Additionally, ADEQUATE spectra are known to contain artefacts at HSQC frequencies at comparable intensities to the desired correlations. The modifications described above reduce these artifacts substantially. Another modification of ADEQUATE, the clean ADEQUATE is presented, which comprehensively eliminates these artifacts, reducing the chance for data misinterpretation.

In search for sensitivity gains in NMR, the SHARPER (Sensitive, Homogeneous And Resolved PEaks in Real time) acquisition technique is applied. In this, the application of short spin-echo intervals in combination with non-selective pulses is used to remove the chemical shift modulation of signals spanning thousands of Hertz. The impressive sensitivity gains from collapsing multiplets, parts of, or even complete spectra open new possibilities for characterisation of molecules and their mixtures. SHARPER has numerous applications in solidand solution-state NMR spectroscopy, some of which are presented here. Firstly, the SHARPERINADEQUATE experiment achieves a 3-fold increase in sensitivity by collapsing the multiplets acquired in this experiment. Further acquisition and processing methods, such as oversampling, the use of NUS and removal of the imaginary FID, result in an up to 10-fold increase in sensitivity compared to the original INADEQUATE experiment acquired for the same time. The SHARPER method was also extended to solid-state NMR experiments. Besides the expected sensitivity gains, the CP-SHARPER experiment allows the measurement of sharp, intense signals even without the use of MAS. An example is the CP-SHARPER-INADEQUATE which applies the sensitivity gains to solid-state structure elucidation problems.

A specific complex mixture analysed in this work was the disinfection by-products (DBPs) produced during the addition of chloramine to drinking water. Chloramine kills pathogens; however, it also reacts with dissolved organic matter and anthropogenic contaminants producing DBPs, which are known to be toxic and carcinogenic. Although the levels of some DBPs are regulated, the majority of these compounds are unknown and it has been shown that the total toxicity of DBPs cannot be accounted by regulated compounds alone.

Methodology for MS formula assignment for compounds containing 15N and halogens was developed. Statistical analysis of the assigned formulae showed that the number of compounds, the diversity of the mixture, and the chlorine count increase during the chloramination reaction. The complex reaction mixture was investigated as a network of reactions using PageRank and Reverse PageRank algorithms. Independent of the MS signal intensities, the PageRank algorithm calculates the formulae with highest probability at convergence of the reaction; these were chlorinated and nitrated derivatives of the starting materials. The Reverse PageRank revealed that the most probable chemical transformations in the complex mixture were chlorination and decarboxylation. These agree with the data obtained from INADEQUATE NMR spectra and literature data, indicating that this approach could be applied to gain insight into reaction pathways taking place in complex mixtures without any prior knowledge.