Date of Award

Spring 2024

Project Type

Dissertation

Program or Major

Chemistry

Degree Name

Doctor of Philosophy

First Advisor

Anyin Li

Second Advisor

Christopher Bauer

Third Advisor

John Tsavalas

Abstract

The analysis of analytes using electrospray ionization (ESI) and mass spectrometry (MS) has become a widely used tool for the detection and characterization of a wide class of chemical species. Because of its utilization for liquid phase sample and its application to a wide range of flow rates, ESI-MS can be coupled with liquid chromatography (LC) to offer additional separation, desalting, and sample pre-concentration prior to detection. Recent improvements in ESI performance have come from investigating ways to improve the ionization behavior of wide classes of analytes by reducing the liquid flow rate, initially generated droplet size, and ionization current. These factors are closely related and are unified with the Pfeifer-Hendricks equation, where a decrease in one of these factors is proportional to a decrease in the others. With a focus on decreasing the ionization current there is an expected decrease in flow rate and droplet size, eventually leading to a sufficiently small droplet and sample concentration, where only one analyte will be bound to each droplet and ionization efficiency is improved due to less suppression from other species which compete for charging.This dissertation outlines efforts to control and characterize these behaviors using the femtoelectrospray (femtoESI) mode of ionization. This mode is defined by its ultra-low ionization current (10s-1000s femtoamperes) which operates at a low flow rate (0.1-100s pL/min) and because of this flow rate it is hypothesized to produce smaller charged droplets compared to other modes of ionization with higher currents and flow rates. The ionization current from a femtoESI source is so low that multiple Faraday cage designs had to be built, utilized, and characterized to properly measure the currents and investigate the properties of the novel ionization mode. The first current measurement scheme used a picoammeter capable of measuring the ultra-low current with a baseline of 40 fA and measuring continuous ESI signals on the order of 100-1000 fA. A second-generation scheme, using a preamplifier with a high frequency oscilloscope observed pulsed ESI within the femtoESI mode with pulse widths (100s µs) and heights (100s pA) not previously reported for ESI. The ionization efficiency of analytes in a mixture has been of concern to scientists because not all species obtain charge in the same mechanism, especially in analyte mixtures where some analytes can compete for charge and suppress the ionization of other analytes. Although a universal ionization efficiency method has not been developed, the work in this dissertation outlines that small molecules, peptides, glycans, and proteins can be analyzed using the femtoESI mode to enhance the signal of analytes with low ionization efficiency. Along with improving the ionization efficiency, the femtoESI mode is also shown to decrease the average charge state of species which obtain multiple charges in ESI which allows for more facile detection of macromolecules in their native conformation. This is especially important for the analysis of proteins where the native conformation can be retained throughout the ESI process while other modes will partially/completely denature proteins or produce a mixture of native and denatured charge state.

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