UC Riverside

The goal of this dissertation is to develop improved analytical methods for the separation and characterization of ionic analytes while gaining insights into their underlying mechanism. The work focused reversed-phase ion-pair high-performance liquid chromatography (RPIP-HPLC) and capillary isotachophoresis (cITP) separations of the highly charged molecules, heparin and heparan sulfate. Furthermore, this work will explore methods to improve the sensitivity of NMR, with a focus on both microcoil and microslot NMR probes.

RPIP-HPLC is an important method for the separation of ionic solutes using lipophilic ions, referred to as ion-pairing reagents (IPR), as mobile phase modifiers to aid in their retention on a hydrophobic stationary phase. However, the fine details of the RPIP separation mechanism are still being debated. The described work investigates the role that competition between ion-pairing reagents plays in the separation of structural isomers of heparin and heparan sulfate (HS) disaccharides, and how factors such as IPR concentration and buffer pH affect the resolution of their anomeric forms.

NMR spectroscopy can yield a vast amount of structural information about a compound without destroying the sample. However, NMR is often limited by poor sensitivity and for samples containing more than one compound of interest, the spectra

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obtained can quickly become convoluted, complicating interpretation. To solve these problems we use the pre-concentration/separation method cITP coupled with microcoil NMR detection. The first part of this work uses doxepin as a model compound to study both the type and strength of interactions that occur during cITP. This work focuses on the binding interactions of doxepin with the buffer modifier Beta-cyclodextrin and the role that the counterion acetate plays in the cITP process.

The insights gained in the doxepin cITP study were then used to develop an improved cITP-NMR method for the analysis of heparin and HS derived oligosaccharides, focusing on designing a buffer system that reduces the effects of current induced resonance broadening that occurs when cITP is coupled to microcoil NMR. Finally, this work demonstrates the coupling of cITP with a new microslot NMR probe design which eliminates the deleterious affects of the magnetic field produced by the separation.