Ion mobility spectrometry is a powerful technique for the study related to molecule. The work of tow major applications are introduced in this paper. The first application is the optimization of parameters in CCS. The accurate calculation of the collision cross section for multiple molecules is a long-time interested topic in the research for substances detection in micro scale. No reliable analytical approach to calculate the collision cross section has been established to date. Different approaches rely on different mechanism will provide different results in significant extent. This work introduce a method for the determination of parameters in the Lennard Jones potential. Experimental data combined with numerical computation was the fundamental strategy during the optimization of the parameters. In the experiment, electrospray is used as the ion source of IMS while a nebulizer was utilized to electrify the aromatic compounds. New parameters show no less accuracy and equal efficiency while can explain the physical meaning of the collision more clearly. The second application is the trace detection of explosives with very low concentration. The detection of explosives is an important topic in security, while the detection will be difficult due to the low vapor pressure of explosives. In this work, two types of devices are designed for the trace detection of explosives at an extremely low concentration. TNT is selected as the explosives in the experiment. The experiment succeed to reach a sensitivity of 1 part per quintillion, and even find out a linear relationship between the logarithm of TNT concentration and TNT vapor pressure.

One of the main goals in structural biology is to understand the folding mechanisms and three-dimensional structure of biomolecules. Many biomolecular systems adopt multiple structures as a function of their microenvironment, which makes them difficult to be characterized by traditional structural biology tools (e.g., NMR, X-ray crystallography). As an alternative, complementary tools that can capture and sample multiple conformations needed to be developed. In the present work, we pioneered the application of a new variant of ion mobility spectrometry, trapped ion mobility spectrometry (TIMS), which provides high mobility resolving power and the possibility to study kinetically trapped intermediates as a function of the starting solution (e.g., pH and organic content) and gas-phase conditions (e.g., collisional activation, molecular dopants, hydrogen/deuterium back-exchange). When coupled to mass spectrometry (TIMS-MS), action spectroscopy (IRMPD), molecular dynamics and biochemical approaches (e.g., fluorescence lifetime spectroscopy), a comprehensive description of the biomolecules dynamics and tridimensional structural can be obtained. These new set of tools were applied for the first time to the study of Flavin Adenine Dinucleotide (FAD), Nicotineamide Adenine Dinucleotide (NAD), globular protein cytochrome c (cyt c), the 31 knot YibK protein, 52 knot ubiquitin C terminal hydrolase (UCH) protein, and the 61 knot halo acid dehydrogenase (DehI) protein.

The complexity of biological matrices makes the detection and quantification of compounds of interest challenging. For successful targeted or untargeted identification of compounds within a biological environment, the use of complementary separation techniques is routinely required; in many situations, a single analytical technique is not sufficient. In the present dissertation, a multidimensional analytical technique was developed and evaluated, a combination of new sample preparation/extraction protocols, liquid chromatography, trapped ion mobility and mass spectrometry (e.g., LC-TIMS-MS and LC-TIMS-MS/MS). The performance of these techniques was evaluated for the detection of polybrominated diphenyl ethers metabolites, polychlorinated biphenyls metabolites in human plasma, opioid metabolites in human urine, and lipids in Dictyostelium discoideum cells. The new workflows and methods described in the body of this dissertation allows for rapid, selective, sensitive and high-resolution detection of biomarkers in biological matrices with increased confidence, sensitivity and shorter sample preparation and analysis time.

The use of laser desorption as a sample introduction method for solid phase microextraction (SPME) has been investigated in this research project. Three different types of analytical instruments, mass spectrometry (MS), ion mobility spectrometry (IMS) and gas chromatography (GC) were employed as detectors. The coupling of laser desorption SPME to these three instruments was constructed and described in here. <br /><br /> Solid phase microextraction/surface enhanced laser desorption ionization fibers (SPME/SELDI) were developed and have been coupled to two IMS devices. SPME/SELDI combines sampling, sample preparation and sample introduction with the ionization and desorption of the analytes. Other than being the extraction phase for the SPME fiber, the electro-conductive polymer coatings can facilitate the ionization process without the involvement of a matrix assisted laser desorption/ionization (MALDI) matrix. The performance of the SPME coatings and the experimental parameters for laser desorption SPME were investigated with the SPME/SELDI IMS devices. The new SPME/SELDI-IMS 400B device has a faster data acquisition system and a more powerful data analysis program. The optimum laser operation parameters were 250 <em>&mu;J</em> laser energy and 20 <em>Hz</em> repetition rate. Three new SPME coatings, polypyrrole (PPY), polythiophene (PTH) and polyaniline (PAN) were developed and evaluated by an IMS and a GC. The PPY coating was found to have the best performance and was used in most of the experiments. The characteristics of the PPY and the PTH SPME/SELDI fiber were then assessed with both IMS and MS. Good linearity could be observed between the fiber surface area and the signal intensity, and between the concentration and the signal intensities. <br /><br /> The ionization mechanism of poly(ethylene glycol) 400 (PEG) was studied with the SPME/SELDI-IMS 400B device. It was found that the potassiated ions and sodiated ions were both present in the ion mobility spectra. The results obtained with quadrupole time-of-flight (QTOF) MS confirmed the presence of both potassiated and sodiated ions. This result suggested that cationization is the main ionization process when polymers are directly ionized from the PPY coated silica surface. Four PEGs with different average molecular weights and poly(propylene glycol) 400 were also tested with this SPME/SELDI device. The differences between the ion mobility spectra of these polymers could be used for the fast identification of synthetic polymers. <br /><br /> The SPME/SELDI fibers were then coupled to QTOF MS and hybrid quadrupole linear ion trap (QqLIT) MS, respectively. Improved sensitivity could be achieved with QqLIT MS, as the modified AP MALDI source facilitated the ion transmission. The application of method for analysis of urine sample and the bovine serum albumin (BSA) digest were demonstrated with both PPY and PTH fibers. The LOD for leucine enkephalin in urine was determined to be 40 <em>fmol &mu;L^-1</em> with PTH coated fiber; and the LOD for the BSA digest was 2 <em>fmol &mu;L^-1</em> obtained with both PTH and PPY fibers. <br /><br /> A new multiplexed SPME/AP MALDI plate was designed and evaluated on the same QqLIT MS to improve the throughput, and the performance of this technique. The experimental parameters were optimized to obtain a significant improvement in performance. The incorporation of diluted matrix to the extraction solution improved the absolute signal and S/N ratio by 104X and 32X, respectively. The incorporation of reflection geometry for the laser illumination improved the S/N ratio by more than two orders of magnitude. The fully optimized high throughput SPME/AP MALDI configuration generated detection limit improvements on the order of 1000-7500X those achieved prior to these modifications. This system presents a possible alternative for qualitative proteomics and drug screening. <br /><br /> Laser desorption SPME as a sample introduction method for the fast analysis of non-volatile synthetic polymers was also demonstrated here. The coupling of laser desorption SPME to GC/FID and GC/MS was performed, and the advantage of laser desorption over traditional thermal desorption was demonstrated in this research. Laser desorption PEG 400 was observed more effcient than thermal desorption. Good separation was obtained even with a 1-m or 2-m column. These results demonstrate the potential of laser desorption SPME as a sample introduction method for the fast GC analysis of non-volatile compounds such as synthetic polymers.

Drift tube ion mobility spectrometry (IMS) is a powerful, post-ionization separation that yields structural information of ions through an ion-neutral collision cross section. The ion-neutral collision cross section is governed by the collision frequency of the ion with the neutral drift gas. Consequently, ions of different size will have different collision frequencies with the gas and be separated in the drift cell. A significant challenge for IMS, however, is to separate ions with very similar collision cross sections, requiring higher resolution ion mobility spectrometers. Resolution in IMS is of utmost importance for the separation of complex mixtures, e.g. crude oil samples, proteolytic digests, positional isomers, and ion conformers. However, most methods employed to increase mobility resolution significantly decrease ion transmission through the mobility device. Herein, a periodic-focusing DC ion guide drift cell (PDC IG) is presented to display its potential capabilities for higher mobility resolution with increased ion transmission. The PDC IG utilizes unique electrode geometry compared to the conventional uniform field electrode design. Electrode geometry can be defined by the electrode inner diameter (d), thickness (t), and spacing (s). Specifically, the ratio of d : t : s is equal to, or very near, 1:1:1. The PDC IG electrode design creates a non-uniform (fringing) electric field-especially near the electrode walls. The design also causes variations in the radial electric field which provides an effective RF as ions move through the device and a radially confining effective potential that improves ion transmission through the device. In this dissertation the analytical utility of the PDC IG drift cell for ion mobility separations will be explored. The radial focusing properties of the device will be presented along with studies of electrode geometry and its effect on ion mobility resolution and ion transmission through the drift cell. PDC IG drift cell length is also examined to determine its effect on mobility resolution and ion transmission. Finally, the PDC IG drift cell device is coupled to an orthogonal-acceleration time-of-flight mass spectrometer as well as a modular, PDC IG drift cell being adapted to a commercial qTOF mass spectrometer for IM-MS experiments.

Drift time ion mobility spectrometry (DTIMS) is a rapid post ionization gas-phase separation technique that distinguishes between compounds based on their differences in reduced mass, charge and collisional cross-section while under a weak, time-invariant electric field. Standalone DTIMS is currently employed throughout the world for the detection of explosives, drugs and chemical-warfare agents. The coupling of IMS to MS (IM-MS) has enabled the performance of time-nested multidimensional separations with high sample throughput and enhanced peak capacity, allowing for the separation of ions not only based on their mass/charge (m/z) ratios, but also their shape. This allows for the elucidation of valuable structural information that can be utilized for determining gas phase ion conformation and differentiation between closely related ionic species. Over the past decade, these advances have transformed IM-MS applications and instrumental designs into one of the most rapidly growing areas of mass spectrometry. The work presented in this thesis is aimed at the development and subsequent characterization of a novel high-resolution resistive-glass atmospheric pressure DTIMS, and the application of this prototype DTIMS to the detection of environmentally relevant compounds. A review of the different types of ion mobility spectrometers, their principles of operation, and the advantages and disadvantages of each type are presented in Chapter 1. Chapter 2 describes the design and development of our prototype resistive glass DTIMS. A detailed description of the IMS hardware, including the ion sources, custom-built control computer, pulsing electronics, data acquisition system, and the timing schemes developed to operate the instrument in standalone DTIMS, multiplexed DTIMS, and IM-MS mode, are presented. Chapter 3 presents an initial characterization of the performance of a prototype resistive glass DTIMS under a wide range of instrumental parameters and also characterizes the radial ion distribution of the ions in the drift region of the spectrometer. Chapter 4 addresses the lack of sensitivity in DTIMS and explores ion trapping and multiplexing methods, introduces the principles of multiplexing and describes an extended multiplexing approach that encompasses arbitrary binary ion injection waveforms with variable duty cycles. Chapter 5 presents a detailed theoretical and experimental study of the separation power of our DTIMS and presents an evaluation of the field homogeneity and the performance of the ion gate.

Field asymmetric waveform ion mobility spectrometry (FAIMS) is a gas phase atmospheric pressure separation technique that exploits the difference in the mobility of ions in alternating low and high electric fields as they are carried between two electrodes. In this thesis, a miniaturised FAIMS separation step has been applied to increase selectivity, enhance sensitivity and improve the quality of mass spectral data for rapid, high-throughput protein and peptide analysis. In Chapter 2, charge state separations were used to generate pseudo-peptide mass fingerprint data by FAIMS-MS, permitting confident protein identification using ESI sample introduction as an alternative to MALDI-TOF-MS methods. In addition, pre-cursor ions were targeted prior to MS/MS analysis. Chapter 3 describes the analysis of intact proteins by miniaturised FAIMS-MS. Multiple charge states of intact proteins were separated on the basis of differences in differential mobility. Higher charge states were found to be transmitted at similar CVs suggesting that the miniaturised FAIMS device was separating ions on the basis of 3D structure. In addition, multiple species could be observed at the same m/z suggesting the presence of different protein conformers. In Chapter 4, miniaturised FAIMS was used to select ions on the basis of differential mobility prior to in-source collision-induced dissociation CID, LC and ToF-MS analysis for qualitative and quantitative analysis of peptides mixtures. This was applied to the analysis of co-eluting model peptides and tryptic peptides derived from human plasma proteins, allowing precursor ion selection and CID to yield product ion data suitable for peptide identification via database searching.

This dissertation mainly focuses on analytical method development for characterization of proteins, glycoproteins and glycans using the recently developed ion mobility spectrometry (IMS) techniques and various electron activated dissociation (ExD) tandem mass spectrometry methods. IMS and ExD have become important techniques in structure analysis of biomolecules. IMS is a gas-phase separation method orthogonal to liquid chromatography (LC) fractionation. ExD is capable of producing a large number of structurally informative fragment ions for elucidation of structural details, complementary to collision-induced dissociation (CID). We first applied the selected accumulation-trapped IMS (SA-TIMS)-electronic excitation dissociation (EED) method to analyze various mixtures of glycan isomers. Glycan linkage isomers with linear or branched structure were successfully separated and subsequently identified. Theoretical modeling was also performed to gain a better understanding of isomer separation. The calculated collisional cross section (CCS) values match well with the experimentally measured ones, and suggested that the choice of metal charge carrier and charge state is critical for successful IMS separation of isomeric glycans. In addition, a SA-TIMS-electron capture dissociation (ECD) approach was employed to study gas-phase protein conformation, as the ECD fragmentation pattern is influenced by both the charge distribution and the presence of various non-covalent interactions. We demonstrated that different conformations of protein ions in a single charge state could produce distinct fragmentation pattern, presumably because of their differences in tertiary structures and/or proton locations. The second part describes characterization of glycoproteins using LC-hot ECD. To improve the cleavage coverage of glycopeptides, hot ECD, a fragmentation method utilizing the irradiation of high-energy electrons, was optimized for both middle-down and bottom-up analyses of glycopeptides, including peptides with multiple glycosylation sites. Hot ECD was shown to be an effective fragmentation technique for sequencing of glycopeptides, even for ions in lower charge states. In addition, the online LC-hot ECD approach was applied to characterize extensively modified glycoproteins from biological sources in which all glycosylation sites could be unambiguously determined. This study expands the applications of IMS, CID and ExD to structural analysis of various biomolecules, and explores the analytical potential of combining them for investigation of complex biological systems, in particular, enzyme mechanisms.

If the analyses of trace components in complex organic samples are to be optimised, then these compounds must be isolated either physically or chemically from surrounding matrices. Ion mobility spectrometry (IMS) is an analytical technique used worldwide for the detection of on-site trace compounds. The technique can be optimised to isolate the target species from complex matrices through both physical separation, based on the mobility of the analyte ions at ambient pressure, and chemical discrimination through preferential ionisation of the target. Optimisation of the latter is commonly achieved through doping the spectrometer with a selective reagent gas, termed a dopant. The chemical processes required to optimise the responses of target analytes are dependent on the identity and concentration of the dopant. As such, a variety of dopants have been successfully implemented in ion mobility spectrometers. The technology for the deliverance of dopants in IMS is commonly through permeation sources, which provide a stable chemical environment in the ion mobility cell. Althoughrelatively inexpensive and durable, these devices are difficult to change and generally deliver a single dopant concentration. As a result, only one type of chemistry is possible and the responses cannot be optimised for a range of analytical applications. Such limitationsbecome increasingly significant when IMS is hyphenated to a chromatograph where a range of different dopant conditions may be sought over the course of a chromatographic run. This thesis sought to overcome these limitations through the development and implementation of piezoelectric dispensers, interfaced directly to the transport gas regions of IMS cells. The study demonstrates for the first time the ability to use piezoelectric dispensing as a dopant introduction methodology in IMS for controlling and calibrating a range of dopant chemistries. 2-butanol, acetone, dichloromethane, 1-chlorohexane, 4-heptanone and 1-bromohexane were the candidate dopants chosen for the studies, covering a wide range of physical and chemical properties. The novel technology was used to dispense the target dopants into IMS cells at concentration ranges over three orders of magnitude. Dopant chemistries were achieved within three seconds from the point of dispensing, administered in drop-ondemand formats, and could be delivered either transiently or at steady-state concentrations. The concept was validated through integrated spectral dopant responses. In transient control, dynamic linear relationships of R2 = 0.991 - 0.998 were achieved between dispensed dopant mass and peak area. Under continuous operation, the RSD of the dopant level was < 18% for all dopants. Clear out times for dopant responses were in the order of 3-5 seconds, indicating negligible hysteresis effects. The study also proved the concept of controlling monomer and dimer ion chemistries from 2-butanol actuations when interfaced to a differential mobility spectrometer at mass fluxes between 21 - 1230 ng m-3 , and the simultaneous control of dopants in negative and positive ionisation modes to RSDs <10%. This thesis describes the techniques used to optimise the piezoelectric dispensing of the full dopant range, as well as the full design protocols required to interface to mobility spectrometers. The outcomes from these studies provide a realisation for piezoelectric dispensers as a future mainstream dopant introduction technique for the analysis of complex samples.

Sampling and preconcentration techniques play a critical role in headspace analysis in analytical chemistry. My dissertation presents a novel sampling design, capillary microextraction of volatiles (CMV), that improves the preconcentration of volatiles and semivolatiles in a headspace with high throughput, near quantitative analysis, high recovery and unambiguous identification of compounds when coupled to mass spectrometry. The CMV devices use sol-gel polydimethylsiloxane (PDMS) coated microglass fibers as the sampling/preconcentration sorbent when these fibers are stacked into open-ended capillary tubes. The design allows for dynamic headspace sampling by connecting the device to a hand-held vacuum pump. The inexpensive device can be fitted into a thermal desorption probe for thermal desorption of the extracted volatile compounds into a gas chromatography-mass spectrometer (GC-MS). The performance of the CMV devices was compared with two other existing preconcentration techniques, solid phase microextraction (SPME) and planar solid phase microextraction (PSPME). Compared to SPME fibers, the CMV devices have an improved surface area and phase volume of 5000 times and 80 times, respectively. One (1) minute dynamic CMV air sampling resulted in similar performance as a 30 min static extraction using a SPME fiber. The PSPME devices have been fashioned to easily interface with ion mobility spectrometers (IMS) for explosives or drugs detection. The CMV devices are shown to offer dynamic sampling and can now be coupled to COTS GC-MS instruments. Several compound classes representing explosives have been analyzed with minimum breakthrough even after a 60 min. sampling time. The extracted volatile compounds were retained in the CMV devices when preserved in aluminum foils after sampling. Finally, the CMV sampling device were used for several different headspace profiling applications which involved sampling a shipping facility, six illicit drugs, seven military explosives and eighteen different bacteria strains. Successful detection of the target analytes at ng levels of the target signature volatile compounds in these applications suggests that the CMV devices can provide high throughput qualitative and quantitative analysis with high recovery and unambiguous identification of analytes.