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Direct Analysis in Real Time Ionization for High-Resolution Mass Spectrometry

Permanent Link: http://ufdc.ufl.edu/UFE0041066/00001

Material Information

Title: Direct Analysis in Real Time Ionization for High-Resolution Mass Spectrometry
Physical Description: 1 online resource (219 p.)
Language: english
Creator: Rummel, Julia
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: ambient, dart, fticrms, ionization, irmpd, tofms
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Direct Analysis in Real Time (DART) is an ionization method for mass spectrometry (MS) that was introduced by Cody and coworkers in 2005. This source operates by passing helium or nitrogen between a high voltage needle and a counter electrode to produce an electrical discharge. Additional electrodes are present to remove all but the excited-state gas atoms/molecules. The excited species produced by the DART source may either ionize analytes directly or may interact with ambient components to create reagent ions that may ionize analyte molecules. Analytes are inserted directly into the stream of excited gas atoms or molecules that exit the DART source for desorption and ionization. Because little or no sample preparation is required and analyses are performed in ambient conditions, DART-MS offers rapid analyses of a variety of compound classes with minimal effort. For the current work, a DART source was fabricated and interfaced to several different mass spectrometers. The source was optimized and its analytical figures of merit were determined with analytes representative of illicit drugs, explosives, pesticides, and chemical warfare agents. Sensitivity and signal stability of the mass spectrometers were improved with the addition of a passively sampling flared inlet modification. A commercial actively sampling inlet modification was added and compared to the passive design. The utility of the custom-built DART source was demonstrated with a number of different applications with little or no sample preparation. Analytes were sampled from a wide variety of surface types including glass, metal, soil, sand, money, food products, and more. Matrixes such as ocean water, wine, and dimethyl sulfoxide caused suppression of analyte signal, while salt content and the presence of other solvents showed little or no effect. The addition of dopants to the sampling region was explored as a method of modifying ionization characteristics and expanding DART capabilities. The mechanistic factors governing DART sensitivity were systematically studied. With volatile compounds, proton affinity was found to be the most important determinant of sensitivity. In addition to the mechanisms previously proposed in the literature, evidence of a self-protonation mechanism was observed. A spatial variation in the mechanism of ion formation was demonstrated with polycyclic aromatic hydrocarbons. Generally, the pathways of DART ionization appeared to fall into the class of mechanisms operative in atmospheric pressure chemical ionization (charge exchange, proton transfer, etc.). DART was coupled to several FT-ICR MS instruments. The highest resolving powers ever reported with DART-MS analyses were demonstrated. The capability of the FT-ICR MS to do extended ion trapping was exploited to conduct infrared multiple photon dissociation experiments for spectroscopic structural determination of DART-ionized molecules.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Julia Rummel.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Eyler, John R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041066:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041066/00001

Material Information

Title: Direct Analysis in Real Time Ionization for High-Resolution Mass Spectrometry
Physical Description: 1 online resource (219 p.)
Language: english
Creator: Rummel, Julia
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: ambient, dart, fticrms, ionization, irmpd, tofms
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Direct Analysis in Real Time (DART) is an ionization method for mass spectrometry (MS) that was introduced by Cody and coworkers in 2005. This source operates by passing helium or nitrogen between a high voltage needle and a counter electrode to produce an electrical discharge. Additional electrodes are present to remove all but the excited-state gas atoms/molecules. The excited species produced by the DART source may either ionize analytes directly or may interact with ambient components to create reagent ions that may ionize analyte molecules. Analytes are inserted directly into the stream of excited gas atoms or molecules that exit the DART source for desorption and ionization. Because little or no sample preparation is required and analyses are performed in ambient conditions, DART-MS offers rapid analyses of a variety of compound classes with minimal effort. For the current work, a DART source was fabricated and interfaced to several different mass spectrometers. The source was optimized and its analytical figures of merit were determined with analytes representative of illicit drugs, explosives, pesticides, and chemical warfare agents. Sensitivity and signal stability of the mass spectrometers were improved with the addition of a passively sampling flared inlet modification. A commercial actively sampling inlet modification was added and compared to the passive design. The utility of the custom-built DART source was demonstrated with a number of different applications with little or no sample preparation. Analytes were sampled from a wide variety of surface types including glass, metal, soil, sand, money, food products, and more. Matrixes such as ocean water, wine, and dimethyl sulfoxide caused suppression of analyte signal, while salt content and the presence of other solvents showed little or no effect. The addition of dopants to the sampling region was explored as a method of modifying ionization characteristics and expanding DART capabilities. The mechanistic factors governing DART sensitivity were systematically studied. With volatile compounds, proton affinity was found to be the most important determinant of sensitivity. In addition to the mechanisms previously proposed in the literature, evidence of a self-protonation mechanism was observed. A spatial variation in the mechanism of ion formation was demonstrated with polycyclic aromatic hydrocarbons. Generally, the pathways of DART ionization appeared to fall into the class of mechanisms operative in atmospheric pressure chemical ionization (charge exchange, proton transfer, etc.). DART was coupled to several FT-ICR MS instruments. The highest resolving powers ever reported with DART-MS analyses were demonstrated. The capability of the FT-ICR MS to do extended ion trapping was exploited to conduct infrared multiple photon dissociation experiments for spectroscopic structural determination of DART-ionized molecules.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Julia Rummel.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Eyler, John R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041066:00001


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PAGE 20

Overview

PAGE 21

Mass Spectrometry

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Ionization

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Direct Analysis in Real Time Literature Review Introduction

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DART Function

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DART Mechanism Ionization

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Other considerations Applications

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Biological applications et al Forensic science and homeland security applications

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Other application areas

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Related Ionization Techniques Corona discharge Dielectric barrier discharges and RF plasmas

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. in situ Glow discharges

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DART Literature Review Concluding Remarks High Resolution Mass Spectrometry

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oa oa

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Timeof Flight Mass Spectrometry Overview m z d V e t d m zeV

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oa oa

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Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Overview

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f, f = ezB m m z = eB f T

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High-Resolution Mass Spectrometry Discussion Conclusions Scope of Thesis

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Mass Spectrom. Rev. 2001, 20,

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Introduction Experimental Chemicals and R eagents

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Instrumentation DART source

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Optical studies design

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Mass spectrometer

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Sample introduction

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Results and Discussion DART Optimization Flow rate tests High voltage tests

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Disk and grid electrode tests

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Other Considerations Gas temperature Sample placement

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Inlet Modifications Flared capillary extender description and introduct ion

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Vapur description and introduction

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Comparison of Inlet Modifications

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Inlet Modification Optimizations Flared capillary extender optimization Flare angle. Helium flow rate and DARTto flare distance.

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Vapur optimization Ceramic tube length and DART to Vapur distance.

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Ceramic tubeto capillary distance and helium flow rate.

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Background Ions

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Signal Abundance and Variation Signal abundance. Signal variation.

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Figures of Merit

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Blank m Blank Inlet Modification Comparison Conclusions

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General Conclusion

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71 Figure 2 -1. Cutaway diagram of the custom-built DART source. Figure 2 -2. Possible configurations of DART source with fiber optic probe. The fiber optic is aimed (a.) down the bore of the DART source or (b.) orthogonal to bore of DART source. Figure 2 -3. Helium DART emission spectrum. This spectrum was collected using the configuration shown in Figure 2-2 a.

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72 Figure 2-4. Pictures of the motorized sample introduction stage. The stage is shown (a.) with metal sample rods inserted into the sample tray and (b.) in position with the instrument. Figure 2-5. Gas flow rate optimization performed (a.) optically and (b.) with the mass spectrometer. Each plot in (b.) represents the responses of a single dodecylamine sample.

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73 Figure 2-6. Discharge needle voltage optimization performed (a.) optically and (b.) with the mass spectrometer. Each plot in b. represents the responses of a single dodecylamine sample. Figure 2-7. Disk and grid electrode optimizations performed (a. and c.) optically and (b. and d.) with the mass spectrometer. Each plot in b. and d. represents the responses of a single dodecylamine sample.

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74 Figure 2-8. Effect of gas temperature on analyte detection. Fentanyl (a.), is optimally analyzed at a higher temperature than methamidophos (c.) as indicated by their temperature profiles (b. and d., respectively). Figure 2-9. Diagram and results of sample positioning study. A diagram of how the sample placement was adjusted is presented (a.) as well as the response of dodecylamine as a function ion sample distance (b.).

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75 Figure 2-10. Diagram of flared capillary extender with end of DART source. The red arrows depict gas flow lines. A section of the extender is cut away to show the canted coil spring. Figure 2-11. Extracted ion chromatograms of dodecylamine (a.) without and (b.) with the flared capillary extender. Figure 2-12. Diagram of the Vapur interfaced to the DART source with red arrows depicting DART gas flow lines. The drying gas flow lines will be shown in Figure 2-13. Add text 1.5e6 No Flared Capillary Extender a. b. 8e5 Intensity, cts 0.45 0.35 0.25 0.15 0.05 0.55 0.65 0.75 0.85 1.5e6 With Flared Capillary Extender 1.1e6 Intensity, cts 0.45 0.35 0.25 0.15 0.05 0.55 0.65 0.75 0.85 Time, min Time, min 7e5 6e5 5e5 4e5 3e5 2e5 1e5 0.0 1.4e6 1.3e6 1.2e6 1.1e6 1.0e6 9e5 7e5 6e5 5e5 4e5 3e5 2e5 1e5 0.0 1.4e6 1.3e6 1.2e6 1.0e6 9e5 8e5

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76 Figure 213. Diagram of Vapur without and with the drying gas diverter. Red arrows represent the flow of drying gas from the TOF. Figure 214. Photographs of the quiet cabinet (a.) closed and (b.) open, displaying the diaphragm pump. Figure 215. Flared capillary extender optimization studies. The studies were used to optimize (a.) flare angle and (b.) DARTto flare distance.

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77 Figure 216. Less complicated Vapur optimization studies. The signal abundances of 10 ng caffeine samples were plotted as a function of (a.) the ceramic tube length and (b.) the DARTto Vapur distance. Figure 217. Additional Vapur optimization studies. The signal abundance of 10 ng caffeine samples was monitored as a function of (a. c.) tube to inlet distances and (d. f.) the helium flow rate.

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78 Figure 218. (a.) Primary contaminant in the DARTMS background and comparison of background spectra of the (b.) flared capillary extender and Vapur (c.) without and (d.) with the drying gas diverter.

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79 Figure 219. Comparisons of signal variation and signal abundance using the Vapur and flared capillary extender. Extracted ion chromatograms of separate 1 ng caffeine samples are plotted in a. and b. Mass spectra of 1 ng caffeine samples are plotted in c. and d. Background ions are marked with (*).

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80 Figure 220. Calibration curves generated to compare figures of merit of the flared capillary extender and the Vapur. Trend lines are not drawn through data points that were past the linear region of the curve.

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81 CHAPTER 3 SELECTED APPLICATIONS Introduction One of the greatest benefits of DART as an ionization technique is its versatility. Not only can it ionize compounds from a wide variety of surfaces, but it can be used to examine many different classes of compounds with a wide range of polarities. Figure 3 1 presents compound classes and groups that have been detected with the custom built DART sourced described in Chapter 2. While many of these compound classes were purchased from chemical companies, several were synthesized by students in the UF Chemistry Department. The structures included in the chalcones group were synthesized by students in the UF undergraduate organic chemistry teaching labs. This experiment aptly demonstrated both the directness (absolutely no sample preparation was done) and rapidity of analyses by DARTMS. With the aid of Dr. Soledad Cerutti processing the data, approximately 250 chalcone samples were analyzed in a little over 4 hours. In addition to analyzing many different compound types, a number of applications were performed with either real samples that originated from somewhere other than a chemical company or were performed with reaction mixtures to demonstrate the potential utility of DARTMS to the synthetic chemists in the UF Chemistry Department. Much of the work presented here was done as proof of concept, demonstrating the general applicability of DART MS. These selected applications also demonstrate that the custom built DART source is capable of the same types of analyses as a commercial version. Additional attention will be paid to studies involving the analysis of

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82 thin layer chromatography plates and detection of nicotine from fingerprints with DARTMS. Experimental Instrumentation The Agilent 6210 TOF (Agilent Technologies, Wilmington, DE) was used for all TLC plate studies. Its setup and usage are described in Chapter 2. The custom built DART source and flared capillary extender described in Chapter 2 were also employed in this work. Chemicals and Materials HPLC grade methanol was purchased from Burdick and Jackson (Muskegon, MI). HPLC grade methylene chloride and ACS grade hexane and ethyl acetate were purchased from Fisher Scientific (Fair Lawn, NJ). All solvents were used without further purification. Theophylline and caffeine were purchased from Sigma Aldrich (St. Louis, MO). Goodies Powder was purchased from a local grocery store. Cyclam and a reaction mixture containing diethyl 2(dodecylamino)terephthalate were synthesized by Ian Rummel of the UF Chemistry Department. All TLC experiments were performed with Whatman 20 x 20 cm Aluminum backed Flexible Silica TLC Plates with UV254 indicator (catalog no. 4420222) cut to sizes ranging from 0.5 x 1 cm to 3 x 6 cm. Thin Layer Chromatography Plates Thin layer chromatography (TLC) separates multi component solutions into spots that vary according to the compounds retention times. When solution components are known or suspected, as is often the case in fields like forensic science or food science, TLC spots ca n be identified by comparing the spot positions, related to the retention times, to the retention times of known materials. Synthetic chemists,

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83 however, often face the challenge of separating materials from solutions that contain multiple unknown components for which no standards are available, making it difficult to ascertain the nature of the resulting TLC spots. In this situation, additional analytical techniques are required to ascertain the nature of the TLC spots. As demonstrated in the following paragraphs, DARTMS can be used to rapidly identify unknowns from TLC plates with a high degree of certainty. Very shortly after this work was completed but prior to publishing, Morlock et al. reported DART analyses of high performance TLC plates.84 Explanation and Method At least two different methods have been presented for analyzing TLC plates with DART. Morlock et al. were able to analyze compounds from TLC plates by mounting the plates in a holder and tilting them 160 so that an exposed edge (cut into an eluted sample spot) could be introduced into the sampling region of a commercial DART source.84 Kusai et al. employed a glycerol matrix to enhance detection of compounds from TLC plates.106 Presented here is a somewhat simpler method that employs a higher gas temperature than typically used in DARTMS analyses of small molecules. A number of different sampling geometries were tested, but the best results were achieved by horizontally positioning the plates against the end of the DART source (diagram m ed in Figure 32). In this configuration, both the top and the bottom of the plate were exposed to the hot gas, which was thought to enhance thermal desorption. The glycerol method was also attempted here, but was dismissed because of the very high background ion count it produced.

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84 deposited in total. Some TLC plates were incubated in chambers with varying amounts of ethyl acetate and hexane and others were spotted and analyzed without incubation. Eluted samples were visualized with a UV lamp and marked with a pencil. Sample spots were cut from the TLC plates in roughly 0.5 x 1.5 cm strips with DART sampling occurring across the short side of the strip. Heating effects As mentioned above, one of the most important factors in detecting analytes in this study was the gas temperature. Deposited on nonporous substrates such as glass, organic molecules with masses below 200 Da are typically desorbed with DART gas temperatures below 250 C. Dodecylamine, as discussed in Chapter 2, does not require heating for DARTMS analyses. However, as demonstrated in Figure 33, additional heating is required for desorption from TLC plates. Appreciable protonated dodecylamine signal was not detected until the gas temperature was raised to 300 C and drastic improvement was observed by raising the temperature above 400 C. This disparity in required gas temperatures in TLC plates versus smooth samples is not surprising. With smooth sample holders such as glass, very little interaction occurs between the analyte and its substrate. The porous TLC substrate offers an increased surface area allowing additional van der Waals interactions as well as other intermolecular attractions depending on the stationary phase to occur. Storage effects Though testing a TLC plate immediately after a separation is achieved is ideal, there are no doubt times when this is not convenient. To test the effects of storage time and, consequently, exposure to air, TLC plates were analyzed within minutes of

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85 preparation and one month later. The monthold plate was stored in a drawer uncovered. As Figure 34 demonstrates, little change was observed between the spectrum of a freshly prepared caffeineloaded TLC plate and that of a monthold plate. A fragment of caffeine at m/z 138 was observed with both of the plates, but it was likely formed by thermal degradation rather than exposure to air. Mixtures Goodys Powder. As an example of a commercial pharmaceutical product separated with TLC and analyzed with DARTMS, Goodys Powder was tested. The active ingredients of this over thecounter pain medication are acetaminophen, aspirin, and caffeine. After incubation, three spots were visualized with a UV light. Of those, one was identified as caffeine and another was identified as acetaminophen. The mass spectrum of the second spot is given in Figure 35. With internal mass calibration using the phthalate fragment at m/z 149, subppm mass accuracy was achieved with the protonated acetaminophen. The exact mass of this and all other compounds identified with accurate mass are given in Table 31. No ions other than the expected background ions were detected in the third spot. If this spot contained aspirin (a.k.a. acetylsalicylic acid) detection in positive mode would be unlikely because the compound would probably deprotonate to form a negative ion. Unfortunately, the instrument was not operable in negative mode when this test was done. Synthetic reaction mixture. To prove the technique could be used in a capacity beneficial to synthetic chemists, a TLC plate used to separate components of a reaction mixture was analyzed. With exposure to UV light, eight different components fluoresced. After direct analysis of each of the spots, the sixth was found to contain the desired product, diethyl 2(dodecylamino) terephthalate. A photograph of the TLC plate

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86 and the mass spectrum co rresponding to the sixth spot are given in Figure 36. This study provided information on the purity of the sample and allowed the researcher to better estimate when the desired product would elute during a largescale preparative column separation of the reacti on mixture. Whole TLC plate analysis. Ideally, DART MS could be used to analyze TLC plates with no prior visualization step (typically with a UV lamp). As a proof of concept, a TLC plate lane was spotted with caffeine, theophylline, cyclam, and adenine at various positions. For analysis, the TLC plate was slowly slid between the DART source and the capillary. Because the motorized stage was not yet built, this was done by hand. A total ion chromatogram (TIC) and the extracted ion chromatograms for each of the compounds are shown in Figure 37. Though varying degrees of sensitivity and fragmentation were observed, each analyte was detected. Cyclam was detected with the lowest signal to noise ratios, which could have resulted from improper sample positioning. A moving sample introduction stage equipped with a device for mounting TLC plates could reduce this effect and generally reduce the hands on time by the operator. By knowing the rate at which the samples were introduced and the starting positi on, correlation between chromatogram peaks and location on the plate would be trivial. TLC Plates Study Conclusion A method has been presented for the analysis of thin layer chromatography plates using the custom built DART source. A high gas temperature was demonstrated to effectively desorb the analytes used in this study. Though probably compounddependent, analysis of fresh and old TLC plates produced similar results. With internal mass calibration, compounds from TLC separated mixtures were succ essfully

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87 indentified with accurate mass. These studies proved that, like the commercial system, the custom built DART source is capable of desorbing and ionizing analytes from TLC plates. Nicotine Detection from a Smokers Fingerprints The goal of another proof of concept study was to use DARTMS to determine if a person had been smoking without testing his or her blood or urine. Though one method would be to test the breath of a person, it was decided that a better demonstration of DART as a surface ionization technique would be to test skin excretions. Fingerprints were thought to be an ideal source of skin excretions because they could be obtained in a completely non invasive manner, preserved for later use if required, and could be analyzed without exposing the test subject to the 250 C helium exiting the DART source. The smoker who participated in this study was an interesting candidate because he typically handles machinery and industrial lubricants, which could contribute significantly to the matrix of the fingerprints. It should be noted that the smokers participation in this study was completely voluntary and that the smoker was not required to smoke any additional cigarettes than he would have smoked in a normal day. No incisions or bodily fluids were taken for this study. Fingerprint collection was achieved by rolling a glass pipette between a smokers index finger and thumb. No additional sample preparation was performed. Samples were directly analyzed by inserting the print loaded area of the pipette into the gas stream of the DART source. Analyses were performed after smoking with and without hand washing.

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88 Fingerprint Analysis Fingerprints immediately after smoking No hand washing. The first sample was taken moments after the smoker had smoked a cigarette prior to washing his hands. As shown in Figure 38, protonated nicotine was detected in low abundance. In much greater abundance were several peaks attributed to known industrial solvents or phthalates (industrial plasticizers) As will be demonstrated in Chapter 5, DART ionization is a competitive process. The presence of the many contaminants on the smokers fingers may have caused some signal suppression when this mass spectrum was acquired. Hand washing prior to smoking a cigarette. To solve the problem of the contamination observed above, the smoker washed his hands immediately prior to his next cigarette. Shown in Figure 39, a slightly larger nicotine signal was detected and the previously observed contaminants were greatly reduced with this combination of hand washing and smoking. The protonated nicotine peak observed here had better signal to noise than in the last spectrum and better mass accuracy (0.6 ppm error versus 6.1 ppm error in the previous spectrum). Han d washing after cigarette. To determine if the nicotine observed above was excreted from the smokers fingers or transferred directly from the cigarettes, the smoker washed his hands immediately after smoking but prior to sample collection. Upon analysis of the finger prints, no nicotine was detected (Figure 310). Only the contaminants shown in the previous spectrum were detected here. The series of peaks from 200 to 300 Da were suspected to come from soap used for the hand washing.

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89 Fingerprints twenty minutes after smoking Approximately 20 minutes after the previous spectrum was acquired, additional fingerprints were collected. Some differences were observed between this spectrum (Figure 311) and the one acquired 20 minutes prior (Figure 310). Fir st, the relative distributions of a number of the background ions changed. An explanation for this could be that the sample introduction was slightly different. If the finger print coated pipette was held closer to the DART source, it would have been exposed to higher temperatures, which could promote desorption of the larger mass contaminants. The more important difference between the two spectra was that a low abundance nicotine peak was observed. As the smoker had not smoked a cigarette in 20 minutes and had limited his interaction with the items he typically handles, the detected nicotine was probably excreted from his fingers. Fingerprint Study Conclusions A method for detecting nicotine from the fingerprints of a smoker was presented. With various combinations of smoking and handwashing, varying nicotine signals were detected. As expected, the signal to noise ratio of protonated nicotine was affected by the presence of contaminants like machine lubricants. Handwashing completely depleted the nicotine signal, but weak signals attributed to nicotine were regained after 20 minutes and were suspected to be the result of skin excretions. This proof of concept study demonstrated the utility of DARTMS in the analysis of components from fingerprint s. This study should be repeated on fingerprints distributed on surfaces, first directly analyzed from glass slides and later analyzed from collected prints. One might take the direction described by Benton et al. of doping fingerprint dust with sorbent

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90 m aterials, followed by DART MS analysis.107 It would also be very interesting to perform the same analyses using other pharmaceuticals or illicit drugs. Miscellaneous Appli cations Food Products An area where DART MS may see increased application in coming years is the food industry. Two examples are DART analyses of food products are given in Figure 312. The first spectrum was obtained by directly analyzing melted Dove dark chocolate with DART MS. The primary alkaloid produced by cocoa beans, theobromine, and the listed ingredient, vanillin, were detected. Though caffeine detection was also expected, the overly abundant peak seen in this spectrum was suspected to originate from sample carry over instead of the chocolate. The other spectrum in Figure 312 demonstrates that both the protonated molecule and the protonbound dimer of limonene can be detected when directly analyzing orange peels. The dimerization demonstrates how highly concentrated the limonene is on the peels surface. Interestingly, limonene was not detected when the flesh of the same orange was analyzed by DARTMS (not shown). Pharmaceuticals The subject area where DARTMS has seen the most utilit y thus far is pharmaceuticals analysis. Drugs are often very good candidates for DARTMS analysis because they tend to be small molecules (less than 1000 Da) that possess functionalities amenable to DART ionization (amines, carbonyls, phenyl groups, etc. ). Two examples of pharmaceuticals analyzed with the custom built DART source are presented in Figure 313. In the first spectrum, an Excedrin Tension Headache tablet with its outer coating removed was positioned between the DART source and the mass

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91 spec trometer. By doing so, the two active ingredients (caffeine and acetaminophen) were detected as protonated molecules as well as a protonbound adduct of the two compounds. The twenty dollar bill spectrum is a reproduction of the most famous DART MS analy sis. With no special sample preparation, the edge of a twenty dollar bill was inserted into the DART gas stream. Confirmed with accurate mass, cocaine and nicotine were detected in high abundance. The presence of cocaine was not surprising as it has been detected on currency in a number of studies employing various analytical methodologies.108-113 Flavors and Fragrances With DART MS, one can rapidly see that pure vanilla extract and imitation vanilla purchased from a grocery store are different. Figure 314 contains spectra obtained by directly analyzing pure vanilla extract, imitation vanilla extract, Mexican vanilla extract, and, for comparison purposes, a vanillascented Little Tree air freshener. Peak identifications were based on exact masses. Of the four spectra, only the pure vanilla extract and the Mexican vanilla displayed a peak identified as protonated vanillin (sometimes called methylvanillin). It is not surprising that the synthetic vanilla fla voring, ethylvanillin, appeared in the spectra of the imitation vanilla and the air freshener. However, as ethylvanillin does not occur naturally, it was suspicious that the Mexican vanilla also contained the compound. The ethylvanillin detected in the M exican vanilla could not be attributed to carry over because it was analyzed in an entirely different week than the other samples, after the mass spectrometer inlet was cleaned several times. This indicated that the Mexican vanilla extract was artificiall y flavored. Ammoniated and deprotonated species of a simple sugar (either glucose or

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92 fructose) were detected in the vanilla extract and imitation vanilla but not in the unsweetened Mexican vanilla or the air freshener. General Conclusion Several different applications of the custom built DART MS have been presented in this chapter. Almost no sample preparation was done in any of the above studies, demonstrating the utility of DART as a rapid screening technique. However, making DART MS more than just a screening technique, highly specific information was obtained and identifications were made with a high degree of certainty because the DART source wa s combined with high resolutionmass spectrometry (i.e. the TOF used in this work). These studies demonstrated that the custom built DART source can be used in the same ways as the commercial DART source and that, like the commercial version, it can be applied to analyses in a wide variety of areas.

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93 Table 3-1 Compounds of interest exact m assesa Ion Specie Compound [M+H]+ [M+NH4]+ [M H]+ [2M+H]+ Vanillin 153.0546 Ethyl vanillin 167.0703 Glucose or Fructose 198.0972 179.055 Limonene 137.1325 273.2577 Caffeine 195.0877 Theobromine 181.072 Nicotine 163.123 Co caine 304.1543 Acetaminophen 152.0706 a. All masses are numbers represent mass -to -charge ratios, expressed in terms of Da.

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94 Pharmaceuticals Theophylline Acetaminophen Loratidine Spiperone Polycyclic Aromatic Hydrocarbons Anthracene Chrysene Pyrene Perylene Rubrene Organometallics and Coordination Complexes (4 dodecanamidophenyl) Platinum Oligomer mercury(II) chloride Pesticides Methamidophos Acephate Dimethoate DEET Chemical Warfare Agent Simulants Dimethyl Diisopropyl 2 Chloroethylethylsulfide m ethylphosphonate methylphosphonate Explosives 1,3 Dinitrobenzene 2,4 Dinitrotoluene Tetryl RDX Flavors and Fragrances Vanillin Methyl Salicylate Limonene Allicin Toxic Industrial Compounds Acrolein Formaldehyde Allylamine Compounds of Biological Relevance: Nucleotide Bases, Amino Acids, Carbohydrates, Metabolites Adenine Lysine Sucrose Crotonic Acid Lauric Acid Chalcones and Related Compounds Chalcone 1 Chalcone 8 Hydrogenated Chalcone 5 Figure 3-1. Compound classes/groups analyzed by DART and example structures. Theophylline Acetaminophen Loratidine Spiperone Theophylline Acetaminophen Loratidine Spiperone Methamidophos Acephate Dimethoate DEET Acrolein Formaldehyde Allylamine Acrolein Formaldehyde Allylamine Acrolein Formaldehyde Allylamine Adenine Lysine Sucrose (4 Platinum Oligomer mercury(II) chloride

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95 Figure 3-2. Diagram of the TLC plate sampling configuration. Figure 3-3. Effects of gas heater temperature on TLC plate analyses. Responses of protonated dodecylamine are given. Figure 3-4. Effects of extended TLC plate storage. Spectra of a (a.) fresh TLC plate and a (b.) month-old TLC plate containing caffeine are presented.

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96 Figure 3-5. DART-MS analysis of the second spot from a Goodies Powder-loaded TLC plate. Figure 3-6. Photograph of TLC separation of synthetic organic reaction mixture and corresponding mass spectrum.

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97 Figure 3-7. Whole TLC plate analysis by DART-MS. The total ion chromatogram (a.) and corresponding extracted ion chromatograms of (b.) adenine, (c.) cyclam, (d.) theophylline, and (e.) caffeine are presented. Figure 3-8. Spectrum of smokers fingerprint after cigarette but before washing hands. Protonated nicotine is marked with (*).

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98 Figure 3-9. Spectrum of smokers fingerprint after cigarette, hands washed before. Figure 3-10. Spectrum of smokers fingerprint after cigarette, hands washed afterward.

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99 Figure 3-11. Spectrum of smokers fingerprint 20 minutes after cigarette. Protonated nicotine is marked with (*). Figure 3-12. Examples of DART-MS analyses of food products. Mass spectra acquired by analyzing melted chocolate bar and an orange peel are presented. Figure 3-13. Examples of DART-MS analyses of pharmaceuticals. Mass spectra from the analysis of a pain reliever and a twenty-dollar bill are presented.

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100 Figure 3-14. Examples of DART-MS analyses of different vanilla flavorings and a vanilla air freshener. Note: V stands for vanillin, E stands for elthylvanillin, and G stands for glucose or fructose.

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101 CHAPTER 4 SUBSTRATE EFFECTS Introduction An important consideration in developing analytical methods is the analyte matrix. Many analytical methods incorporate several preparative steps for extracting, purifying, and preconcentrating samples prior to the actual analysis. Often described as their greatest advantage, ambient ionization techniques like DART can be performed with a minimal number of preparative actions (sometimes none). However, when little or no sample preparation is done, the technique must overcome numerous obstacles that may hinder the analysis including the means by which the sample is introduced and the presence of compounds that can compete for ionization. In the follow ing sections, the effects of analyte substrate surface, material, and matrix in DART analysis will be discussed. Included in this discussion are examples of how the ambient environment might affect and effect ionization of analyte molecules. Experimental Instrumental The Agilent 6210 TOF mass spectrometer was used for all TLC plate studies. Its setup and usage are described in Chapter 2. The custom built DART source described in Chapter 2 was applied to all of the studies presented here. The flared c apillary extender was used for all cases except the signal suppression studies. The Vapur modified the TOF inlet throughout the suppression studies. The gas temperature was varied according to application and will be specified in the discussion of each study. The motorized stage described in Chapter 2 was used for sample introduction for all data that are plotted on an Excel graph.

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102 Chemicals and Materials HPLC grade methanol, acetonitrile, water, and isopropanol were purchased from Burdick and Jackson (Muskegon, MI). Methylene chloride (HPLC grade), hexane, ethyl acetate, dimethyl sulfoxide, ammonium hydroxide, nitric acid, sodium chloride, sodium acetate, and ammonium acetate were purchased from Fisher Scientific (Fair Lawn, NJ). All solvents were used without further purification. Glycerol was purchased from Mallinckrodt (St. Louis, MO). Caffeine, spiperone, 2,6dinitrotoluene, 1,3dinitrobenzene were purchased from Sigma Aldrich (St. Louis, MO) and were dissolved in methanol (caffeine and spiperone) or acetonitrile (2,4dinitrotoluene, 1,3dinitrobenzene). Dimethyl methylphosphonate and diisopropyl methylphosphonate were purchased from Alfa Aesar (Ward Hill, MA) and dissolved in methanol. Dimethoate and methamidophos were donated by Northrop Grumman Corporation (Baltimore, MD) and prepared in methanol. The Nitroaromatics and Nitramine Explosives in Drinking Water mixture (Catalog no. 33900) was purchased from Restek (Bellefonte, PA) and diluted in acetonitrile. Deuterated water was purchased from Cambridge Isotope Laboratories (Andover, MA). Ocean water was collected from the Canaveral National Seashore (Titusville, FL). Cab Cube cabernet sauvignon was purchased from a local Target store. Surface Effects Explanation and Method It was demonstrated with thin layer chromatography plates in Chapter 3 that surface morphology can play a role in the desorption of an analyte during DARTMS analysis. The influences of substrate and surfaces were studied with the following compounds: fentanyl, dimethyl methylphosphonate (DMMP), 2,4 dinitrotoluene

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103 (DNT), and dimethoate. These analytes were chosen because they are of Department of Homeland Security interest, representing an illicit drug, a nerve agent simulant, an explosive, and a pesticide. In addition to determining the effects of surface on analyte detection, a goal of these studies was to determine the analytical figures of merit of this DART source. Calibration curves were generated to evaluate the sensitivity (calibration curve slope), limit of detection, and linearity for each of the analytes. Internal standards (IS) that were similar to the analytes in volatility and ionization properties were used to reduce signal variability and responses were reported in terms of analyteto IS extrac ted ion chromatogram peak area ratios. Six replicates were analyzed for each datapoint. All four analytes were tested on smooth glass and smooth metal surfaces. The liquid analyte, DMMP, was also tested on rough glass and rough metal surfaces. Prelimin ary tests with solid analytes indicated that sampling from the rough surfaces resulted in a 10x reduction in signal abundance and a decision was made to forego generation of calibration curves with dimethoate, fentanyl, and DNT on rough surfaces for the sake of time. Information on the target analytes along with their selected internal standards and DART gas temperatures are presented in Table 41. Substrate preparation and sample introduction. Glass and stainless steel rods with diameters of approximately 2 mm were obtained from the UF Chemistry Glass and Machine Shops. The rods were cut to lengths of 6.5 cm. From their respective cutting processes, the ends of the glass rods possessed rough surfaces and the metal rods possessed smooth surfaces. Measures were taken to obtain smooth and rough surfaces of both substrate types. To create smooth glass surfaces, half of the glass

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104 rods were held in a Bunsen Burner flame for a short period of time until their ends were smooth but not rounded. To create rough metal surfaces, the metal rods were sanded by the Machine Shop. The ends of all of the rough rods appeared to have been sandblasted with a homogenous granular surface (no long striations were visible). All smooth rods ends were smooth (no obvious markings) and flat (no rounding from the flaming process). The rods were inserted into the tray of the sample introduction stage as described in Chapter 2 and samples were deposited onto the flat ends facing upward prior to analysis. Samples were driven in front of the DART source with the stage motor operated at its maximum rate and then stopped. A camera aimed from above the DART source sampling region and a video monitor were used to monitor the position of the sample posts so that all samples could be brought to the same location for analysis. Analyte responses were monitored with the real time chromatograms plotted by the Agilent TOF operating software while each sample rested in front of the DART source. Samples were driven out of the sampling region after their analyte responses returned to the baseline. Sample rods were soaked with methanol or acetonitrile and heated with the DART gas between analyses for cleaning. All analytes except DMMP were allowed to dry prior to analysis. DMMP was immediately analyzed to prevent sample losses due to evaporation. Results and Discussion The calibration curves generated with analyte and each substrate are presented in Figure 41. The figures of merit for each analyte/substrate combination are listed in Table 42. The limit of detection was calculated according to the following equation:105 Blank m. (4 1)

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105 In equation 41, Blank is the standard deviation of the blank and m is the slope of the calibration curve. Linear ranges were calculated by subtracting the limit of detection from the highest sample quantity where the calibration curve was still considered linear (R2 > 0.99) assuming linearity through the LOD Based on the preliminary tests, smooth surfaces were determined to be better for analysis of the solid analytes (DNT, dimethoate, and fentanyl) than rough surfaces. The rough surfaces were not disregarded for DMMP because it is a liquid and more volatile than the other compounds. The added surface area of the rough surfaces was hypothesized to provide better control of the thermal desorption of DMMP. This would perhaps allow more DMMP to be desorbed while the sample was positioned correctly between the DART source and the inlet of the mass spectrometer and not earlier in the process of moving the sample into the DART stream. When a comparison was done, however, very little difference in any of the figures of merit was seen between the two surface morphologies or the two material types. This at least indic ated that the rough surface did not hinder the analysis of the semi volatile liquid, DMMP. Though differences in the figures of merit were detected for dimethoate, fentanyl, and DNT on glass and metal, neither glass nor metal rods consistently produced better results. Slightly calibration better slopes with both substrates were achieved with dimethoate than with fentanyl, but the limits of detection were similar for both compounds because the background levels at m/z 230 (dimethoate [M+H]+) were hi gher and more variable than those at m/z 337 (fentanyl [M+H]+). The dramatically worse results obtained with DNT may have resulted from less efficient negative ion formation by DART. Another contributor is the poorer sensitivity of the TOF when run in negative

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106 mode. Additionally, the 13C isotope of the largest background contaminant, suspected to be deprotonated glucose, had the same nominal mass as the [M H]ion of DNT. This compound increased both the abundance and variation in blank measurements. The reduction in sensitivity due to instrument problem s and background contributions seems especially possible when considering that Song et al. reported an LOD of about 4.5 pg for nitroaromatic explosives with negative mode DARTMS using a JEOL TOF.65 Conclusion from Surface Effects Studies The surface studies showed little difference between glass and metal substrates for the three compounds that are solids at room temperature. For DMMP, a semi volatile liquid, detection was independent of the surface material and morphology (this statement is limited to smooth versus rough surfaces and does not include porous surfaces). These similarities are actually a good testament to the versatility of DART and its utility in the analysis of analytes from different substrate surfaces. The other goal of this study, to establish the analytical figures of merit for this technique, was accomplished for four different model compounds. The results of the analytes forming positive ions (fentanyl, dimethoate, and DMMP) reported in Table 42 are similar to those reported for the commercial DART source.50,54,72 Alternative Surfaces Explanation and Method As was demonstrated in Chapters 1 and 2, the greatest advantage of DARTMS is the ability to introduce and analyze samples directly. Substrates such as pharmaceutical tablets and plant material lend themselves to fairly simple introduction because they either can fit directly into the sampling region or small segments can easily be excised and placed into the DART gas stream. In applications where

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107 pesticide analysis is required or in the event of a chemical warfare attack, the analytes of interest may be distributed over a large area and sample substrates may include sand, soil, concrete, or other materials in the immediate vicinity. In an effort to explore detection from alternative media, target analytes were spiked onto sand, soil, concrete, cotton swabs, aluminum foil, and more. Target analytes in these studies included pesticides, illicit drugs, nerve agent simulants, volatile organic compounds, metabolites, and explosives. In the following paragraphs, the analysis of dimethoate, a pesticide, and dimethyl methylphosphonate, a nerve agent simulant, from a number of alternative media will be described. Because of the anticipated real world variability of the surfaces in question, the purpose of these studies was to establish the feasibility of direct detection from the various surfaces rather than to establish a detailed set of figures of merit. Sample Preparation and Analysis Sand and soil samples. Sample solutions, 1 mg/mL in concentration, were autopi petted into samples of sand or soil for a final concentration of 50 ppm (w/w). To better distribute the analyte, the pipette tip used for sample deposition was used to stir the sand/soil. Samples were allowed to dry after thorough distribution. To retrieve the analytes from the soil/sand matrix, a cotton swab wet with methanol was stirred throughout the mixture until it appeared to be coated. After they had dried, the swabs were shaken and blown briefly to assure that no sand or soil particles could fall off. Immediately prior to analysis, the sampleloaded swabs we methanol and introduced by hand into the DART gas stream. The DART heater was set to 350 C for sand samples and 450 C for soil samples.

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108 Concrete samples. Sample solutions in 1 mg/mL concentrations were deposited onto chunks loaded area. The DART source was tilted 10 downward to sample from the glycerol covered concrete (diagramed in Figure 42) for analysis. Activated carbon. For vapor collection, pure solid or liquid samples were placed in open 1.5 mL Eppendorf tubes that were allowed to stand upright inside 30 mL scintillation vials. Resting in the bottom of the scintillation vials was a thin layer of activated carbon (20 mg). For sample collection, the scintillation vials were closed and incubated at room temperature for 30 minutes (DMMP) to 8 hours (dimethoate). Approximately a quarter of the activated carbon was loaded between the two mesh pieces of the sample holder for direct analysis as pictured in Figure 43. The hollow sample holder consists of an outer brass section into which two pieces of stainless steel mesh are placed. Another brass section screws into the outer brass section to hold the pieces of mesh in place. The sampleloaded brass holder was inserted between the DART source and the mass spectrometer inlet for analysis, allowing the DART gas stream to pass through it (Figure 44). Results and Discussion Sand and soil. Spectra acquired with dimethoateloaded sand, soil, and concrete samples are shown in Figure 4-5 As mentioned above, elevated gas temperatures (450 C vs. 350 C for sand) were required to detect samples from soil. This was attributed to the increased porosity of the soil compared to sand that led to an increase in sample adsorption. Results with DMMP (and many of the other compounds tested) were comparable but are not displayed. The signal to noise ratios presented

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109 here are not ideal, but these data demonstrate that DARTMS can be used to analyze sand and soil. With further optimization of the sample retrieval and introduction, these spectra could probably be improved and levels lower than 50 ppm (w/w) could be detected. Similar results were observed when the sand and soil samples were analyzed in the holder designed for sorbent analyses. Concrete. A mass spectrum of dimethoate analyzed from concrete is presented in Figure 46 a. To detect analyte molecules from concrete it was necessary to apply a liquid matrix to the area suspected to contain the analyte molecules. The glycerol matrix was suspected to dissolve the analytes from the concrete surface prior to exposure to the hot DART gas stream, thus less heating was required for analyte desorption than with dry concrete. It is also possible that analyte molecules were desorbed from the surface from the sheer force of the rapidmoving helium atoms passing over it. Detection of dimethoate and many of the other compounds tested was not possible without both the glycerol matrix and a gas temperature of 450 C. The disadvantage of using the liquid matrix addition technique in DART analysis is that signal suppression will occur if the analyte has a lower proton affinity than the matrix. For example, if any of the analytes used in this study had proton affinities lower than 874.8 kJ/mol (the proton affinity of glycerol), suppression would be very likely.66 Background subtraction using a glycerol blank improved the quality of the spectra obtained by this method. A comparison of dimethoate mass spectra with and without background subtraction is presented in Figure 46. Because of the shape of the concrete samples and the normally tight geometry of the sampling region, it was necessary to tilt the DART source downward approximately

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110 10 toward the samples (refer to Figure 42). Now all new commercial DART sources are equipped with movable stages for adjusting the sampling geometry.114 Again, the signal to noise values detected were less than desirable, but these experiments did demonstrate that DARTMS could be used to detect analytes from concrete. Activated carbon. A mass spectrum displaying protonated dimethoate analyzed from activated carbon is presented in Figure 47 a. It should be noted that the activated carbon never came into contact with the dimethoate sample bed, but instead was exposed to dimethoate vapors in a closed chamber for 8 hours. When the morevolatile DMMP was incubated with activated carbon for just 30 minutes, an intense signal of the protonated DMMP molecule and its protonbound dimer were detected, Figure 46 b. Additional ammoniatedDMMP species were also observed. These results demonstrate that analytes may be detected from sorbent materials. Instead of loading exposed sorbent into a sample holder, an action that may promote sample carry over, it is easy to envision premade sorbent packs that could be hung in a room to monitor air purity or worn on clothing to monitor the presence of nerve agents. After exposure for a given time, the packs could be analyzed by DART MS. Conclusions Regarding Alternative Substrates Although different operating conditions and some instrumental modifications were required for each, the abil ity to perform DART MS analyses from sand, soil, concrete, and sorbent materials was demonstrated. As indicated by the varying degrees of success, further development and optimization studies would be required to establish these methods for routine analys es.

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111 Suppression Studies As discussed in Chapter 1, DART is among several ionization techniques considered to be governed by a mechanism akin to atmospheric pressure chemical ionization (APCI). Being an APCI technique, various competing chemical reactions can occur among analyte molecules, contaminants, and reagent ions during DART ionization. Depending on the ionization characteristics of the substances involved, varying proportions of the desired target analyte ions may be observed. Signal suppressio n is said to have occurred if, for example, fewer analyte molecules are protonated because a higher proton affinity contaminant is present and competing for available protons. This phenomenon will be discussed further in Chapter 5. Other effects may also cause apparent ion suppression, such as a decrease in analyte desorption due matrix components. In the following studies, the target analytes caffeine and methamidophos were combined with various matrices to observe suppression. Their structures are pr esented in Figure 48. Caffeine and methamidophos were chosen for this study because they are small molecules known to both desorb and ionize well by DART and because they are representative of the types of analytes typically analyzed with DARTMS (drugs, pesticides, etc.). Caffeine and methamidophos solutions were combined with matrix solutions for final target analyte concentrations of 5 ppm each. Three categories of matrices were tested here: salt, solvent, and real matrices. Sample solutions wer e analyzed for each datapoint. Sums of the different ion species responses (protonated molecule abundance plus ammoniated molecule abundances) of each analyte are plot ted. All analyses were performed with the gas heater set to 250 C.

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112 Salt Matrices Explanation and method. The suppressive effect of inorganic salts has been discussed in studies involving matrix assisted laser desorption ionization (MALDI), surface -assi sted laser desorption ionization (SALDI), and electrospray ionization (ESI). 115-118 With DART, the presence of salts co crystallized with the analytes was suspected to reduce analyte volatility either by physical submersion in the salt matrix (with a resultant difficulty in desorption) or by ion pair formation. To determine if the presence of salts has an effect on DART detection, solutions of 5ppm caffeine and methamidophos were combined with varying quantities of sodium chloride (NaCl), sodium acetate (NaAc), and ammoni um acetate (AmAc). Results and discussion. The responses of caffeine and methamidophos relative to their average unspiked (no additives) responses are plotted as a function of salt concentration in Figure 49. Variation was observed in the responses of both analytes. Somewhat unexpectedly, only NaAc appeared to impact the response of caffeine, but not methamidophos. As no suppression was observed with NaCl or AmAc with methamidophos, this effect was disregarded. Because no discernable trend was detected with the nonvolatile NaCl, it was concluded that the presence of salts at these levels (up to 25 mg/mL) does not reduce analyte volatility. The only salt that appeared to affect the response of both analytes was ammonium acetate. With both metham idophos and caffeine, significant improvements in signal were achieved with an AmAc concentration of 12.5 mg/mL. The response of methamidophos further improved with 25 mg/mL, but did not with caffeine. Upon plotting the ammoniated and protonated species of methamidophos separately, a more pronounced effect was seen with the ammoniated ion than with the protonated specie

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113 (Figure 410). These data indicate that the observed signal enhancement was due to a dopant effect, which will be discussed later in this chapter. This was not the case for caffeine because ammonium adducted ions were not observed. Solvent Matrices Explanation and method. Non conductive solvents are known to suppress signal in electrospray ionization (ESI).119 Because it does not rely on an electric field gradient or a conductive nebulizer for ionization, DART ionization should be unaffected by solvent conductivity. However, as the solvent can effectively be considered a contaminant competing with the analyte for ionization, its composition is of concern. In the following studies, the effects of various solvents were tested. The analytes dissolved only in methanol were considered unspiked. Other solvents commonly used in ESI were tested, including isopropanol and acetonitrile. To determine if conductivity was important, hexane was combined with the analytes. A solvent with a moderate proton affinity (PA), ethyl acetate (PA = 835.7 kJ/mol), and a solvent with a higher proton affinity, dimethyl sulfoxide (DMSO, PA = 884.4 kJ/mol), were tested to evaluate their competitive effects.66 Table 43 presents the proton affinities and boiling points of the solvent matrices applied here. To assure solvent presence during the desorption/ionization event, samples were not allowed to dry prior to analysis and instead were deposited onto the glass rods immediately before introduction into the DART gas stream. Results and discussion. The responses of caffeine and methamidophos relative to their average unspiked (no additives) responses are plotted as a function of added solvent percentage in Figure 411. With concentrations up to 10 percent, none of the solvents except for DMSO produced a significant reduction in analyte signal. The

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114 responses of both caffeine and methamidophos were reduced significantly with 50 percent hexane. This effect was not attributed to solvent conductivity but rather poor sample deposition. It was considerably more difficult to consistently aspirate and dispense equal volumes using the autopipette with this concentration of hexane and visibly les s volume was deposited onto the sample rods at this point in the experiment. The most dramatic effects were observed with a DMSO spiked matrix. A significant decrease in signal was observed with a DMSO concentration of 1 percent with both caffeine and methamidophos. The analyte signals were nearly depleted with 10 percent DMSO. At 50 percent (not plotted), the signal abundances of caffeine and methamidophos were too low to be recorded by the data analysis software. Two characteristics of DMSO may have affected the responses of caffeine and methamidophos. First, because of its high proton affinity, DMSO could better compete for protons than the other solvents. Second, DMSO has a much higher boiling point than the other compounds. Though the DART gas was heated to 250 C, a temperature that should have been adequate for volatilization of any of the solvents, DMSO would have taken longer to desorb than the other solvents. This could have also reduced the thermal desorption rate of caffeine and methamidophos. The high proton affinity and low volatility of DMSO combined to create very poor sampling conditions for caffeine and methamidophos. Real Matrices Explanation and method. The final set of matrices tested was intended to mimic real samples, w hich are often complex and require some kind of preparation. Ocean water, urine, and wine are examples of matrices that may cause problems due to their high salt content and/or presence of other compounds. Additionally, these

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115 matrices hold relevance in commonly studied areas including environmental science, biological/metabolomics, and food science and will all likely be interrogated with DARTMS in the near future. In the same manner as the salt and solvent suppression studies, the real matrices were combined in varying amounts with the caffeine and methamidophos solutions. The samples were deposited onto glass rods, allowed to dry, and introduced into the sampling region for analysis. Results and discussion. The responses of caffeine and methamidophos relative to their average unspiked responses are plotted as a function of added real matrix percentage in Figure 412. All three matrices eventually caused reduction in the signals of caffeine and methamidophos. Above all, the urine was expected to cause the most dramatic reductions because of both its high salt content (the results in the salt suppression study disprove this idea) and because of its high content of urea, which has a proton affinity of 873.5 kJ/mol.120 The spectrum of urine in Figure 413 demonstrates that urea was so concentrated in the sample that it readily formed abundant protonbound dimers. Interestingly, combined with 1 percent wine, an increase in methamidophos abundance was observed. This was suspected to be due to either instrumental variation of the TOF being used for this study or resultant of some kind of controlled release of the analyte molecules from their wine substrate that may have been absent with the less complicated matrices. This effect was not retained when the wine concentration was increased to 10 percent. The experiments with these three matrices demonstrated that DARTMS is subject to signal suppression. At only 10 percent of added matrix, significant reductions in caffeine and methamidophos signal abundances were detected. One can infer the

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116 sort of results that would be attained if caffeine or methamidophos were analyzed by DART MS in matrices made up of 100 percent ocean water, urine, or wine. It is obvious from these data that a preliminary extraction may be necessary in various real world situations. Thus, a limitation of DART MS has been presented. Conclusions from Suppression Studies These studies provided information on DARTs resistance and susceptibility to matrix suppression. Unlike other ionization techniques, DART showed no susceptibility to salt suppression and no dependence on solution conductivity. In cases where ocean water, urine, and wine are analyzed, the directness of DART may be limited by the need for a prepurification to reduce signal suppression. Atmospheric Effects Another area that can heavily influence the analytical responses of DARTMS is the ambient environment. Unmodified, this environment consists of air, water vapor (humidity), and the hot gas exiting the DART source. Though generally adequate for analyzing many types of molecules, occasionally it may be useful to modify the air with an additional compound, or dopant, to improve detection. Presented below are a few examples of how the detection of certain analytes may be altered with the addition of a dopant. In the first two examples, this was accomplished by opening containers of the dopant producing liquids in the vicinity of the DART source and sampling region. In the last example, a cotton swab soaked in the dopant of choice was inserted into the sampling region along with the analyte. The gas temperatures ranged from 250 to 350 C for the three studies.

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117 Ammonium Hydroxide In positive ion mode, the most common ions other than protonated species are ammoniated species. These adducts are suggested to be formed from interaction with ambient ammonium that has escaped from open containers of ammonium hydroxide.1 Figure 414 gives a comparison of mass spectra collected with 250 pg of methamidophos before and after a container of ammonium hydroxide was opened near the source region. Because of low instrumental sensitivity at the time of analysis, methamidophos signals in both spectra were low. However, with the addition of ammonium vapor, an ammoniated methamidophos ion was detected, the abundance of the protonated methamidophos molecule was increased, and the noise was generally reduced. The ammonium adduct was expected and in many cases can be used to enhance sensitivity of an analyte. Ammonium adducts of oxygencontaining compounds are commonly observed with chemical ionization when ammonia was used as a reagent gas.121 As is probably the case with methamidophos, if an analyte has a proton affinity greater than ammonia (PA = 853.6 kJ/mol), the molecule can also receive protons from the ammonium ions.66 For this reason, the abundance of the protonated molecule also increased. The reduction in noise was less expected but can be explained by suppression. If the compounds responsible for making up the observed background have lower proton affinities than ammonia, their ionization (protonation) could be suppressed. Methylene Chloride and Nitric Acid The negative ion work in this research has primarily focused on the detection of nitroaromatic explosives. For example, calibration curves generated with 2,4-

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118 dinitrotoluene (DNT) and 1,3dinitrobenzene (DNB) were presented earli er in this chapter. Little effort was required to detect the deprotonated DNT or the electronattached DNB ions. However, when the explosives hexahydro1,3,5trinitro 1,3,5triazine (a.k.a. RDX) and tetryl were analyzed, as demonstrated with the Restek e xplosives mixture in Figure 415 a., they were not detected in an unmodified ambient environment. The spectrum in Figure 415 b. shows the Restek explosives mixture analyzed in negative ion mode with both methylene chloride and nitric acid containers open near the DART source. When only a container of methylene chloride was opened near the sampling region, chlorideadducted RDX ions were detected. Upon opening a container of nitric acid near the DART source, nitrate adducts were detected with both RDX an d tetryl. As discussed in the DART Mechanisms section of Chapter 1, the addition of halogenated solvents and volatile acids may enhance detection or they may reduce sensitivity. The electronegative halogenated solvent vapors may preferentially capture thermal electrons, which could reduce the detection of compounds such as DNB. The acids may preferentially donate protons and reduce the formation of [M H]ions, such as those formed by DNT. Deuterium Oxide Another way one may modify the ambient environment about the DART source is with the addition of deuterium oxide (D2O). This is compound is not a dopant in same sense as the compounds discussed above, but it is certainly capable of modifying the ions formed during a DARTMS analysis. Figure 416 presents spectra of cyclam analyzed without and with a D2Osoaked cotton swab held in the DART gas stream. In the presence of an H/D exchange reagent, such as D2O, exposed protons bound to nitrogen or oxygen may exchange with deuterium atoms. With four exch angeable

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119 protons, the ion distribution of cyclam drastically changed in the presence of D2O (Figure 416 b.). Vail et al. have used the H D exchange process to characterize the structures of unknowns.83 Atmospheric Effects Study Conclusions As demonstrated above, the addition of dopants can dramatically modify the ionization processes that occur with DART. All of the dopants presented above have benefits and drawbacks. In positive ion mode, ammonium can enhance detection of analytes or it can compete for available protons and suppress ionization. In negative ion mode, methylene chloride and nitric acid allow detection of RDX and tetryl, but they can also suppress ionization of analytes that form Mand [M H]species. Finally, the addition of D2O can provide information useful in structural elucidation but it can also greatly complicate spectra. General Summary The effects of analyte substrate, matrix, and surrounding environment were explored in this chapter. Little difference was seen between molecules desorbed from glass or metal, but substrate roughness was important in the analysis of solids. The ability to analyze alternative substrates such as sand, soil, and concrete was established. Some of the governing factors in matrix suppression included matrix volatil ity and proton affinity. Finally, the importance of ambient gas composition in ionization properties was demonstrated.

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120 Table 41. Analyte names and experimental information for figures of merit studies with various substrates. Calibration Compound Molecular Weight (Da) Internal Standard (IS) Amount of IS on Substrate (ng) Gas Temperature (C) Fentanyl 336.4705 Spiperone 2.5 350 Dimethoate 225.2574 Methamidophos 2.5 250 2,4 DNT a 182.1335 1,3 DNB b 7.5 330 DMMP c 124.0755 DIMP d 2.5 100 a. Abbrevi ation for dinitrotoluene c. Abbreviation for dimethyl methylphosphonate b. Abbreviation for dinitrobenzene d. Abbreviation for diisopropyl methylphosphonate Table 42. Results from figures of merit studies with various substrates. Compound and Substra te Slope (ng 1 ) Linear Range a LOD b (pg) Fentanyl Smooth Glass 0.877 1.3 19 Fentanyl Smooth Metal 1.17 2.7 7.7 Dimethoate Smooth Glass 1.98 3.9 8.8 Dimethoate Smooth Metal 1.83 2.8 28.9 2,4 DNT c Smooth Glass 0.227 2.3 305 2,4 DNT c Smooth Metal 0.316 2.0 565 DMMP d Smooth Glass 0.642 2.9 34.1 DMMP d Smooth Metal 0.524 2.9 38 DMMP d Rough Glass 0.659 2.6 30.2 DMMP d Rough Metal 0.568 2.9 35.1 e. Linear range is expressed in orders of magnitude f. Abbreviation for limit of detection g. Abbreviation for dinitrotoluene h. Abbreviation for dimethyl methylphosphonate Table 43. Solvents used in solvent suppression studies.66 Solvent Proton Affinity, kJ/mol Boiling Point, C Acetonitrile 779.2 81.7 Isopropanol 793.0 82.4 Hexane N/A 68. 8 Ethyl Acetate 835.7 77.1 Dimethyl Sulfoxide 884.4 190.9

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121 Figure 41. Calibration curves generated for substrate effects studies. Note: smooth glass is abbreviated as SG, smooth metal is abbreviated as SM, rough glass is abbreviated as RG, rough metal is abbreviated as RM, and internal standard is abbreviated as IS. See Table 42 for compound abbreviations.

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123 Figure 45. Dimethoate analyzed from sand and soil. Figure 46. Dimethoate analyzed from concrete with a glycerol matrix. Mass spectra are presented (a.) with and (b.) without background subtraction.

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124 Figure 47. Spectra of compounds analyzed from activated carbon. Dimethoate (a.) was incubated with the activated carbon for 8 hours. DMMP (b) was incubated for only 30 minutes. Figure 48. Structures of caffeine and methamidophos, compounds used for suppression studies.

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125 Figure 49. Results from salt suppression tests. Spiked analyte responses are plotted relative to their unspiked responses as a function of salt concentration. AmAc refers to ammonium acetate, NaAc refers to sodium acetate and NaCl refers to sodium chloride. Figure 410. Variation in methamidophos signal with ammonium acetate concentration. The protonated specie had an m/z of 142 (blue) and the ammoniated specie had an m/z of 159 (red).

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126 Figure 411. Results from solvent suppression tests. Spiked analyte responses are plotted relative to their unspiked responses as a function of added solvent percentage. Figure 412. Results from real matrix suppression tests. Spiked analyte responses are plotted relative to their unspiked responses as a function of added matrix percentage. Real matrices include urine, red wine, and ocean water. Figure 413. Mass spectrum of urine.

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127 Figure 414. Comparison of methamidophos mass spectra acquired (a.) without and (b.) with a container of ammonium hydroxide open near the sampling region. Fig ure 415. Negative ion mode DARTMS spectra of the Nitroaromatics and Nitramine Explosives in Drinking Water mixture analyzed (a.) without dopants and (b.) with containers of methylene chloride and nitric acid open near the sampling region. Other peaksare identified: A is dinitrotoluene (isomer is unknown), B is amino4,6dinitrotoluene, C is trinitrobenzene, and D is trinitrotoluene.

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128 Figure 416. Comparison of cyclam mass spectra acquired (a.) without and (b.) with deuterium oxide present in thesource sampling region. In the deuterium exchanged spectrum (b.), each D before the M in a peak label represents an exchanged deuterium.

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129 CHAPTER 5 STUDIES OF THE POSITIVE ION FORMATION MECHANISMS OF HELIUM DART Introduction In recent years, there have been many reports of the use of Direct Analysis in Real Time (DART) ionization in a wide array of applications. The ionization mechanisms of DART, however, have not been fully delineated, though suggestions have been made by Cody et al. and others.1,48,65 This chapter presents experiments carried out to further elucidate the mechanism of DART ionization. Because detailed discussion is given in Chapter 1, only a brief overview of the DART ionization mechanism will be given here. DART ionization originates from the interaction of an analyte with species created in a gaseous electric discharge where electrons, radical ions, and metastable neutrals are generated.56 Ionized species are removed from the gas stream so that only metastable gas atoms or molecules exit the DART source.1 From this point, the ionization processes include competing reactions that are governed by analyte gas phase basicities and acidities, ionization energies, electron affinities, discharge gas type, and dopants present in the sampling region (added intentionally or not). Most reported applications of DART have employed helium as the discharge gas. The electrical discharge in the helium produces electronically excited He(23S) atoms with an electronic energy of 19.8 eV. He(23S) is sufficiently energetic to directly ionize most organic compounds via Penning ionization to form radical cations (reaction 5.1).66 He(23S) + M He(11S) + M+ + e(5.1) The energetic He(23S) atoms have also been shown to react with background gases (e.g. H2O, O2) generating a variety of reagent ions that may further react with and

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130 ionize the sample.48 In the original DART publication, Cody et al. observed a background of positively charged water clusters up to 14mers that may be produced by sequential reactions as summarized below .1 He(23S) + n H2O [(H2O)n-1H]++ OH+ He(11S) (5.2) Analyte molecules are most commonly protonated during DART ionization and this is thought to occur via gas phase proton transfer from the protonated water clusters formed as indicated in reaction 52.1 The proton affinity (PA) of water is 691 kJ/mol and the PA of its dimers is considerably higher, 808 kJ/mol. 60,122 The PAs of larger ionized water clusters are even greater.123 For the proton transfer mechanism to be operative the PA o f the analyte must be greater than that of the ionizing reagent. As [M+H]+ ions are often observed in positive mode DART MS, the PA of an analyte should play an important role in governing whether it will be ionized by protonation. In this work, mechanistic studies were directed toward understanding the factors that govern the formation of positively charged compounds in DART analyses. First, lowmass background ions were monitored to examine the similarity between those seen with a commercial DART source and with the custom built source and to examine their variation as a function of gas temperature, the position of the DART source, and grid electrode voltage. Next, a series of experiments were performed to systematically study the effects of analyte and competing analyte PAs and analyte volatility. These experiments included analyte sensitivity monitoring as a function of the analytes PA and as a function of the PAs of possible ionization suppressors. Competition experiments between pairs of analytes with different PAs were also carried out to observe suppression. To determine if a competitive desorption mechanism might be

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131 operative during DART analyses, competition experiments between pairs of analytes with different boiling points were performed. Another set of experiments was conducted to demonstrate and determine the spatial variation in DART ionization mechanisms. Lastly, efforts that involved enclosing the sampling region were made to observe the Penning ionization mechanism. Experimental Chem icals and Reagents HPLC grade methanol was purchased from Burdick and Jackson (Muskegon, MI). Methylene chloride was purchased from Fisher Scientific (Fair Lawn, NJ). All solvents were used without further purification. Aniline, cyclohexanone, benzaldehyde, 2,5 dimethylpyrrole, 1methyl-2pyrrolidionone, piperazine, 2,6lutidine, and triethylamine were purchased from Sigma Aldrich (Milwaukee, WI) and diluted in methanol. Polycyclic aromatic hydrocarbons (PAHs) including anthracene, 1,2benzanthracene, biphenylene, chrysene, naphthacene, perylene, and triphenylene were purchased from Sigma Aldrich (Milwaukee, WI) and were dissolved in methylene chloride. Deuterated water was purchased from Cambridge Isotope Laboratories (Andover, MA). Deuterated anthracene was purchased from Isotec (Miamisburg, OH) and was dissolved in methylene chloride. Helium, nitrogen (research grade and UHP), air, and liquid nitrogen were purchased from Airgas (Gainesville, FL) Instrumentation Mass spectrometer and DART s ource The Agilent 6210 timeof flight (Agilent Technologies, Wilmington, DE) mass spectrometer (TOF) discussed in Chapter 2 was used for all experiments. Though normally operated at 250 Vp-p, the RFonly transfer octapole ion guide was operated at

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132 65 Vp-p to enable detection of low mass background ions. The custom built DART source described in Chapter 2 was used in these studies and operated as described. The DART source was heated with a heater removed from an Agilent atmospheric pressure photoionization source. The Vapur inlet modification was used to interface the DART source to the TOF inlet as described in Chapter 2. Sample Introduction and Experimental Design Background ion monitoring No samples were introduced when the low mass background ions were monitored. To establish similarity between it and the commercial DART source, the custom built DART source was operated with normal voltages and a normal helium flow rate (see Chapter 2) with its heater was set to 200 C. Spectra were acquired for two minutes at a time for this experiment. Additionally, the background ions were monitored as the following operating parameters were varied: DARTto ceramic tube (of the Vapur interface) distance, temperature, and grid electrode voltage. In the distance experiment, the gas temperature was held constant at 200 C. In the temperature experiment, the DARTto ceramic tube distance was maintained at 7.5 mm. Both the temperature and distance were held constant as the grid electrode voltage was varied. For t hese experiments, signal was averaged for 30 seconds to generate a mass spectrum and 3 spectra were averaged for each data point. Studies related to sample proton affinity Several different experiments were conducted to study the effects of analyte PA and the PAs of other compounds competing for ionization. Unless otherwise stated, the following conditions were used in all of the PA studies. The DART heater was set to

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133 225 C. The motorized stage and glass posts described in Chapter 2 were used to introduce samples into the DART source the sampling region at a rate of approximately 1 mm/sec. Immediately before analysis, samples from Table 51 were deposited in 0.5 stream. In all cases, the analytes from Table 51 were introduced without drying to prevent sample losses from evaporation. Four replicates were run for each data point in all of the PA studies. Calibration curves. To determine sensitivity as a function of analyte proton affinity, analytes from Table 51 were introduced in concentrations of 6.25, 12.5 and 25 slopes were averaged for sensitivity determinations. To determine if the presence of a competing reagent with a different proton affinity could cause a general reduction in the sensitivity of a given analyte, possible suppression agents were added to the calibrant solutions of aniline, 1methyl-2pyrrolidinone, and 2,5dimethylp yrrole. The concentration of the suppression agent in dimethylpyrrole, and 2,6lut idine. Calibration curves of 2 methyl-1pyrrolidinone (at agents: 2,5dimethylpyrrole, piperazine, and triethylamine. Calibration curves of 2,5dimethylpyrrole (at con following suppression agents: aniline, 1methyl-2pyrrolidinone, and 2,6lutidine. The

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134 slope of each compounds threepoint competitive calibration curve was tracked as the type of spiked compound (and proton affinity) was changed. Competition experiments. Analytes were introduced alone and paired with other competitor compounds from Table 51 to determine if any PA based competition would occur. First, equimolar competition experiments w ere conducted with each Next, unequal molar competition experiments were carried out with the target analytes aniline, 2,5dimethylpyrrole, and 2,6roduced with competitors from Table 1 with both higher and lower proton affinities. The competitor to target analyte molar ratios used included 0:1, 1:1, 10:1, 50:1, and 100:1. Studies related to sample boiling point Analytes from Table 52 were run alone and in pairs to determine if a competitive desorption mechanism could occur. Solutions of compounds from Table 52 were region. The following sample types were analyzed: aniline alone, anthracene alone, 4fluorobenzamide alone, aniline with 4fluorobenzamide, and aniline with anthracene. Aniline was run alone twice because the tests occurred over two days. All solutions had s were taken for each compound and the mixtures at gas temperatures between 100 and 400 C, a range extending from below the boiling point of aniline to above the boiling points of anthracene and 4fluorobenzamide. The proton affinities and boiling points of these compounds are given in Table 52.

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135 Mechanism mapping experiments Mechanism mapping experiments were conducted to demonstrate and determine the spatial distribution of ionization mechanisms that occur as the distance between the DART source exit and the sample is varied (explained further in the Results and Discussion Section). A deuterated water ( D2O)humidified controlled environment glass chamber was fabricated to perform these studies; it is shown in Figure 51 connected to the DART source and the Vapur inlet modification. The chamber was equipped with a row of holes spaced approximately 4 mm apart for sample introduction at different distances from the DART source. A vaporizer designed to mimic a gas chromatograph inlet was used to introduce D2O vapor through the hole at the bottom of the chamber. It was equipped with a septum through which an electrospray transfer line was inserted and a gas connection to provide unidirectional flow of D2O vapor toward its exit. D2O was introduced into the 0.5 L/min) of nitrogen was used to guide the vapor into the chamber. All openings of the glass chamber but the sample hole in use were sealed with Teflon tape during the experiments. Glas s melting point tubes were dipped into 100 ppm to 1 mg/mL solutions of the PAHs in Table 5 3, allowed to dry, and introduced into the sampling holes of the glass chamber by hand. Samples were held in place for approximately 3 seconds. Alternating sampling holes starting at the one closest to the DART source were used for mapping. Enclosed sampling region experiments It was understood that in order to observe the Penning ionization mechanism (and formation of M+ ions) for many highPA analytes, the sampling region needed to be enclosed to prevent exposure to advantitious water. This was first done by inserting a 1

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136 in. length of Teflon tubing (1/8 in. i.d.) with a hole for sample introduction cut into it between the DART and the Vapur ceramic tube. The hot helium passing through the tube was believed to eliminate moisture and prevent protontransfer from protonated water clusters. A diagram of this design is presented in Figure 52. A second enclosure was created with a cut 1mL autopipette tip placed between the DART source and the Vapur, diagrammed in Figure 53. A 1.5 mm diameter hole was cut into the pipette tip for introduction of external gases, such as air, ultrahigh purity nitrogen, and additional helium. An electrospray transfer line was inserted into a much smaller hole (less than 0.5 mm diameter) for sample introduction. Injections of 1 introduced into the pipette enclosure via this line. Samples were vaporized and ionized inside the enclosure. Results and Discussion Background Ion Monitoring Establishment of source similarity In a recent publication, Cody discussed background ions observed with the commercial DART source.48 These include a series of protonated water clusters, NO+, and O2 +. As shown in Figure 54, these same background ions were also observed with the custom built DART source employed in this work. It should be noted that the relative distribution of these ions can be tailored as the operator desires by adjusting the transfer optics of the mass spectrometer. The similarity of background ions and ions observed in the analyses of various compounds in this work to those observed with a commercial DART source led to the belief that the custom built source in this w ork is

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137 adequately similar to the commercial version to perform mechanism studies that may be related to either source design. An important series of ions that has received almost no discussion in the literature are the ammoniated water clusters observed in this spectrum. We have also observed these clusters using both helium and nitrogen with a commercial DART source. Recent ab initio quantum chemistry calculations performed by scientists at ENSO, Inc. using GAMESS and ACES II/ACES III software packages indicated that these species have relatively high PAs.124-126 The PA of [H2O + NH4]+ was computed to be 905.4 kJ/mol and the PAs of larger clusters increased as a function of the number of water molecules included. PAs of this magnitude could play a major role suppressing the proton transfer reactions that should ionize lower PA analytes and adversely affect sensitivity. Background ion monitoring with DART parameter variation Temperature variation. Figure 5-5 contains plots of identified water clusters as a function of gas temperature. Both the absolute and relative abundances are plotted to give perspective of the overall amounts and relative amounts of the different species. A couple of observations can be made from these plots. First, a slight decrease in abundance of the ammoniated water clusters was observed as a function of gas temperature, implying that their presence is not (or only mildly) temperature dependent. Second, the protonated water clusters continuously increased in abundance through 400 C. This result implied that their formation was temperature dependent, which is somewhat counter intuitive if one considers that solvent (water molecules) evaporation should increase with increasing temperatures.

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138 DART to Vapur distance variation. Plots of the changes in water cluster abundances as a function of DARTto Vapur distance are given in Figure 56. A distance of 15 mm produced the overall greatest response both in terms of absolute abundance and relative abundance. This result, however, is not consistent with the D ART to Vapur distance studies reported in Chapter 2, which indicated that the greatest analyte sensitivities were achieved with a distance of 5 mm. Though the absolute abundances of all species decreased when distances were greater than 20 mm, the relative abundances of all ammoniated species generally increased or leveled off (at 20 mm) as the distance was increased. A possible explanation is that detection of the ammoniated species was less affected by the general decrease in temperature in the sampling region (especially nearest the Vapur) as the distance was increased. This explanation is consistent with results from the study presented in the previous section on temperature variation. Grid electrode voltage variation. Plots of the changes in water cluster abundances as a function grid electrode voltage are given in Figure 57. Though there were some differences in the relative amounts of the different species, the absolute abundances of the all water clusters varied in approximately the same manner as the grid voltage was changed. All of the water clusters decreased in abundance as the voltage was raised through 500 V followed by a plateau in abundance variation bey ond that point. This indicates that the changes seen were a function of ion transmission and not ionization mechanism. Harris et al. reported that higher grid voltages could result in greater ion velocities that caused a distortion in ion trajectory (away from the inlet) and hindered detection.49 A similar effect was observed with the water clusters in this study.

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139 Proton Affinity Studies A number of studies were performed to demonstrate the relationship between an analytes PA and its sensitivity and detectability. These studies addressed the impact of PA on sensitivity and the competition for available protons that may occur between analytes with differing PAs. The compounds listed in Table 51 were used in these studies and were chosen for their diversity in PA and their similarity in volatility. The analyte boiling points were considered acceptable if they were below 250 C and within the temperature range easily attained by the DART source heater. Calibration curves for sensitivity determinations Unspiked calibrant solutions. Short calibration curves were generated to track DART sensitivity as a function of analyte PA. Example calibration curves generated for 1methyl-2pyrrolidinone are shown in Figure 58 a. Curves were also generated for aniline, benzaldehyde, 2,5dimethyl pyrrole, 2,6lutidine, and triethylamine. As seen in Figure 58 b., DART sensitivity steadily increases as the PA of the analyte is increased. These data demonstrate that proton transfer is a dominant mechanism in DART ionization. Spiked calibrant solutions. Short calibration curves generated from calibrant solutions with added suppression agents were generated to determine if sensitivity could be altered by the presence of another compound. The variation in sensitivities of aniline, 2,5dimethyl pyrrole, and 1methyl-2pyrrolidinone are shown in Figure 59 a. through c. Below each of the sensitivity plots are the intensities of the spiking agents as the concentrations of the calibrants were varied in Figure 59 d. through f. The sensitivities of aniline and 2,5dimethylpyrrole decreased significantly as a function of the suppression agents PA. The sensitivities when 1methyl-2pyrrolidinone

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140 was paired with higher PA suppression agents were lower than when it was with a lower PA agent. However, its sensitivity did not change significantly between the two higher PA agents, even though their proton affinities are separated by nearly 40 kJ/mol. Likewise, some of the responses from the spiking agents show a slight decrease in intensity, but the changes were not significant with the target analyte concentrations used. These data indicate that the sensitivity (change in signal versus changes in concentration) of these analytes was affected by the presence of higher PA compounds. Competition between analytes of varying proton affinities will be further explored in the next section. Equimolar proton affinity competition experiments Gas phase proton transfer from Penningionized protonated water clusters is the only pathway for protonation that has been proposed in the literature. If this is indeed the only means of protonation, the assertion that there is a finite number of protons available for donation should be true.1,65 To test this hypothesis and the competitive nature of compounds with differing PAs, equimolar amounts of the target analytes were allowed to compete for available protons and, thus, ionization. The responses of the target analytes were monitored as a function of the PA of the competing compounds. Re sults from selected competition experiments are shown in Figure 510. No discernable trend was detected. Competing compounds with both higher and lower PAs affected the ionization of the target analyte in the same manner. An observation from these results is that similar amounts of compounds with varying PAs have little effect on each others signal intensities at the concentrations measured. This can be considered an advantage of this ionization technique. One explanation for this may be that there was no interaction between the analytes and their competitors. This would be

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141 dependent on the number densities and collisional cross sections of the molecules i nvolved. If the number density of the ionized water clusters or other ionizing media was sufficiently high, then analytes in these relative amounts could be ionized free of interference. Similar results were seen when samples were volatilized prior to io nization. Unequal -m olar proton affinity competition experiments Due to the absence of suppression seen in the equimolar PA competition experiments greater concentrations of suppression agents, or competitors, were used to effectively reduce the ionization of the target analytes. By increasing the concentration of the competitors, the likelihood of the target analyte interacting with the competitor or being in closer proximity to effectively compete for ionizing protons was increased. Target analytes aniline, 2,5dimethylpyrrole, and 2,6concentrations were introduced with varying amounts of competitors from Table 51 with both higher and lower proton affinities. The spiked target analyte response relative to that of the unspiked target analyte response was tracked as a function of the competitor to target analyte ratio. The results of these studies are given in Figure 511. In all cases, the lower PA competitor compound caused no significant suppression of the target analyte. Also, in all cases a 10:1 or greater ratio of higher PA competitor produced significant suppression of the target analyte. These data indicated that the presence of a higher quantity of a competing compound does not suppress ionization of the target analyte unless the competitor has a higher PA than the target analyte.

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142 Boiling Point Effects In the conjunction with the work presented in this dissertation and in several other cases, thermal desorption has been demonstrated to be the dominant mechanism for the tra nsport of analytes into the vapor phase during DARTMS analyses.38,50,127 For this reason, analyte volatility can play a major role in the sensitivity of a given compound. All DART sources are equipped with adjustable gas heaters that make desorption of high boiling point compounds, such as explosives, possible. Pairs of compounds with nearly the same proton affinity but very different boiling point s were analyzed separately (to establish a baseline) and simultaneously to determine if there might be competition between the molecules while in (or just entering) the gas phase. Data were taken for each compound and the mixtures at temperatures between 100 and 400 C in order to sample at temperatures less than and greater than the boiling points of each of the compounds in a mixture. As shown in Figure 512, similar responses were observed for all of the analytes with or without a competitor. These data highlighted the importance of using an appropriate temperature setting for all components in a sample, which may be a difficult task depending on the complexity of the sample. At temperatures significantly below their boiling point, compounds such as anthracene and 4fluorobenzamide appeared to be suppressed. It can be concluded from these data that analyte boiling point is less important to DART sensitivity than proton affinity as long as there is sufficient analyte vapor. Mapping Experiments Figure 513 demonstrates that both molecular ions (M+) and protonated molecules ([M+H]+) are formed during DARTMS analyses of polycyclic aromatic hydrocarbons. Additionally, it has been shown that the relative abundance ratio of the

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143 species varies as a function of the distance from the exit of the DART source to the sample (DART to sample distance ).64 At first glance, this appears to be only a competition between Penning ionization or chargeexchange involving ionized ambient components, which result in the formation of the molecular ion, and proton transfer by the mechanisms discussed above (protons are donated from protonated water clusters). An alternative route for protonation that has seen almost no discussion in the DART community is self -p rotonation, as represented in reaction 53. M+ + M MH+ + [M H] (5 3) This reaction is said to be ubiquitous for many classes of compounds and in many cases the rate constant for self protonation is comparable to the calculated capture collision rate constant.63 Evidence for this mechanism occurring in DART ionization is shown in Figure 514 where deuterium adducts were observed during the analysis of deuterated anthracene. No other source of deuterium was present during this study. By creating a D2Ohumidified environment around the sampling region, efforts were made to delineate the source of protons (protonated water clusters vs. analyte molecules) and the spatial distribution of the operative ionization mechanisms. With this configuration, it was postulated that observed protonation was the result of analyteto analyte proton transfer, whereas observed deuteration was resultant of deuterium donation from deuterated D2O clusters, i.e. [(D2O)n+D]+ species. The environment was considered to be adequately saturated with D2O when the background ion dioctylphthalate was deuterated. Dioctylphthalate (structure shown in Figure 515 (a.) was considered a good litmus for the degree of deuteration because it has no

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144 exc hangeable protons, meaning all adducted deuterium ions were known to result from a transfer from the [(D2O)n+D]+ species Its spectra without and with a D2O enviroment are shown in Figures 515 (b.) and (c.), respectively. The PAHs used in this study, a long with their gas phase ion energetics, are listed in Table 53. Figure 516 demonstrates the change in the ion distribution that was detected at each DARTto sample distance with 1,2benzanthracene. Both the protonated and deuterated species increased relative to the molecular ion up to a 20 mm distance, where the abundances of the two ions were nearly equal. Similar responses were obtained with all of the PAHs tested and the molecular ionto deuterated molecule ratio for each PAH is plotted against the DART to sample distance in Figure 517. All of the PAHs tested showed an increase in deuteration as the DARTto sample distance was increased. Sampling at the 20 mm position was performed in addition to the alternating sample introduction holes in the chamber for all of the PAHs because there appeared to be an inflection point between regions of low deuteration and higher deuteration at this distance. As this behavior was exhibited with all of the PAHs, a few observations may be made. First, these data demonstrate that three mechanisms occur during ionization of these molecules: Penning ionization/chargeexchange with ionized ambient components, self protonation, and gas phase proton (or in this case deuterium) transfer from ionized water clusters. Under normal circumstances where analyses are performed in ambient conditions, the relative contribution of the latter two mechanisms is unknown. It should also be noted that it was possible to observe molecular ions here because PAHs have relatively low ionization energies and losses of charge by charge-

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145 exchange processes are unlikely. Second, from the point closest to the DART source to a distance of less than 20 mm, ionization via interaction with ambient water clusters is not the dominant mechanism it was not until after 20 mm that this became true. Enclosed Sampling Region Experiments As part of a series of experiments originally designed to demonstrate Penning ionization with DART, the sampling region was enclosed to prevent interference from p rotonated water clusters. Using the Teflon enclosure, background levels were tracked as the voltage of the DART grid electrode was varied with the source in the following configurations: open, closed but with the hole open, and completely closed (hole blocked with glass rod). Average total currents of 20second scans are plotted in Figure 518 The levels seen when voltage was applied in the closed configurations demonstrate that the species responsible for protonation were not originating from the heli um. Several of the compounds were analyzed with the source in its open and closed configuration and in all cases sample signals were completely depleted upon closing the source (results not shown). These results led to the hypothesis that either Penning ionization was not occurring in the closed configuration or that ion charge was being lost prior to the analytes reaching the mass analyzer. Unfortunately, the method of closing the source (with a small bore Teflon tube that did not fit well between the D ART source and the Vapur ceramic tube) left doubt in the validity of these possible conclusions. Next, the pipette tip enclosure was used to both create a better seal and to allow more open space inside the enclosure. Experiments were conducted with the source in a closed configuration as shown in Figure 53 and in an open configuration, where the DART source was placed 5 mm from the large end of the pipette tip.

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146 2Octanone test The analyte 2octanone has an ionization energy of 9.75 eV, which is below the internal energy of the metastable helium atoms (19.8 eV) that are responsible for Penning ionization. It also has a lower ionization energy than molecular oxygen (12.07 eV) and should be able to undergo charge exchange with ionized oxygen. Despite t hese facts, 2octonone signals were only detected when the source was in an open configuration. To determine if the loss of ion signal was caused by decreased ion transfer or via another mechanism, water and air were introduced into the enclosure to induc e ionization. Ion signals were only detected when air was added. A total ion chromatogram and corresponding mass spectra from an analysis of 2octanone with periodic air introduction are shown in Figure 519. Ion signal was reproducibly detected when air was introduced into the enclosure but not when only water was int roduced. Ions exhibiting protonadduction were detected despite there being little or no source of water for protonated water cluster formation. Additional compounds Additional compounds were tested in a manner similar to that described above for 2 octanone and additional gases were introduced as the external gas. The first set of experiments was performed to demonstrate that the effects seen were not unique to 2octanone. The TICs from enclosedsource experiments with benzaldehyde, 2,6lutidine, triethylamine, and hexafluorobenzene are shown in Figure 520. The three analytes with proton affinities above that of water and the water dimer were ionized when air was introduced. However, no expected ions were observed with hexafluorobenzene. Though this molecule has a PA that makes it unlikely to protonate,

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147 hexafluorobenzene should be able to Penning ionize or undergo charge transfer from ionized molecular oxygen or nitrogen. Additional gases In Figure 521, the TICs from enclosedsource analyses of 2octanone using purified air, ultrahigh purity nitrogen, and helium as external gases are given. Ionization was observed with 2octanone when purified air and nitrogen were added but not when helium was introduced. The same results were seen with benzaldehyde, 2,6lutidine, and triethylamine. When only helium was introduced as the external gas, Penning ionization of all of the molecules should have been observed. Instead no ion signal was detected. It should be noted the presence of the electrical plasma at the needle electrode was confirmed by measuring its current (it was the same as the current measured when ions were detected) and by visually observing it with the addition of a window to the gas lines. Because Penning ionization was not observed, one may conclude that another ambient constituent is required to allow ion detection. Since the nitrogen alone as the external gas allowed ion detection, water and oxygen were shown to be unnec essary. Neither the purified air nor the ultrahigh purity nitrogen reduced the amount of protonation that occurred (mass spectra not shown). Though with the purified air, one may argue that the cold trap did not effectively eliminate water vapor, if any water vapor was present in the ultrahigh purity (99.999% pure) nitrogen, it was so low in abundance that water clusters were not detected. A likely conclusion to be drawn from these results is that the molecules were undergoing self protonation upon exc itation or after chargeexchange with another ambient component. Again, although Penning ionization or charge exchange should have been possible, hexafluorobenzene was not detected

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148 with any of the gases used. Unlike the other analytes employed in these studies, hexafluorobenzene has no protons available for self-protonation. Ionization Mechanism Conclusions The positive-mode ionization mechanism studies performed in this work demonstrated that several different pathways contribute to the DART ionization process. These mechanisms and the means by which they were observed are discussed below. Dominant Mechanism 1: Gas-Phase Proton Transfer Though the proton affinity experiments did not provide information on the source of the proton-donating species, they did demonstrate that gas-phase proton transfer is a dominant mechanism in positive-mode DART ionization. Sensitivity with or without competition from another analyte was heavily dependent on analyte proton affinity. The boiling point study reaffirmed this conclusion, because a volatility-based competitive relationship was never observed. During the mapping studies three different types of ions were observed for most of the PAHs involved: molecular ions, protonated ions, and deuterated ions. The first of these were expected because PAHs have been known to form molecular ions in Powell laboratory for some time.11 The last was also expected because the samples were analyzed in a D2O-humidified environment. A more rapid increase in deuteration was observed for samples positioned farther than 20 mm from the DART source exit. At points closer than this distance, self-protonation was demonstrated. The formation of protonated molecules in the closed source configuration, which should have been devoid of water vapor, also supports this conclusion. Demonstrated Proton-Transfer Avenues: 1. Proton-transfer with water clusters: M + [(H2O)n + H]+ [M+H]+ + nH2O

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149 2. Self-protonation: M+" + M MH+ + [M H]" Dominant Mechanism 2: Molecular Ion Formation Seven different polycyclic aromatic hydrocarbons in the mapping experiments formed molecular ions. Knowledge of how Penning ionization produces ions, the ionization energies of the various analytes involved, and the energy of the metastable helium produced by a DART source dictate that Penning ionization should be possible with DART ionization. However, Penning-ionized molecules were not detected in the enclosed source studies when, again, this pathway should have been possible. It was not until nitrogen or air was added that any ions were seen. Results from these experiments indicate that something other than Penning ionization allowed molecular ions to be detected. The other options for molecular ion formation are charge-exchange or photoionization. The latter seems unlikely because samples are ionized several inches from any light source. It is entirely possible that the energetic metastable species produced by the DART source immediately react with ambient components upon exiting and produce the mentioned charge-exchange agents. The absence of any type of detectable ionization when helium was added as an external gas was most perplexing, but further supported the conclusion that ionized ambient components are responsible for molecular ion formation. Demonstrated Molecular Ion Formation Avenues: 1. Charge-exchange: M + CE+" M+" + CE *CE=charge exchange agent General Conclusion Through a series of experiments, the mechanism of ionization of DART was systematically studied. The impact of proton affinity (both of the analyte and other compounds in its vicinity) on analyte sensitivity and detectability was demonstrated.

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150 General Conclusion Through a series of experiments, the mechanism of ionization of DART was systematically studied. The impact of proton affinity (both of the analyte and other compounds in its vicinity) on analyte sensitivity and detectability was demonstrated. Progress was made in this endeavor with the observation of pathways that had seen little or no discussion in the literature for positive mode DART, such as chargeexchange and self protonation. Additionally, the sourceenclosure experiments exposed the fact that DART ionization may not be as simple as previously discussed in the literature and that something other than Penning ionization may be responsible for the production of ions with helium DART. In all cases, the pathways of DART ionization appeared to be firmly rooted in atmospheric pressure ch emical ionization.

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.

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153 Figure 53. Final closedsource configuration. A pipette tip was used to make a connection between the DART source and the Vapur. Figure 5-4 Background ion spectrum from the custom built DART source. The inset is zoomed in on the masses ranging from 29 to 33 Da.

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156 Figure 59. Competitive sensitivities plots. The changes in sensitivities of (a.) aniline, (b.) 2,5 dimethylpyrrole, and (c.) 2methyl-1pyrrolidine are shown.The change in signal of each of the spiked compounds as the concentration of the named analyte changes are shown in (d.) through (f.). Figure 510. Example results of equimolar proton affinity competition experiments performed with 2methyl-1pyrrol idinone and triethylamine. Abundances of the target analytes are plotted against the difference in proton affinities of the target vs. that of the competitors. The red dots represent the response of the unspiked analyte. Note: Proton Affinity Differenc e = PACompetitor PATarget.

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157 Figure 511. Results of unequal molar proton affinity competitions performed with aniline, 2,5dimethylpyrrole, and 2,6lutidine. The abundances of the target analytes spiked with a competing compound are plotted relative tothe average unspiked target analyte signal abundances. Competitions between the analyte and competitors with lower proton affinities are plotted in blue and the competitions with a higher proton affinity competitor are plotted in red. Figure 512. Ana lyte responses as a function of gas temperature during boiling point studies. The signal abundances of aniline, anthracene, and 4fluorobenzamide are plotted as a function of gas temperature. Blue data points represent the named analyte analyzed alone andred data points represent the named analytes in competition.

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159 Figure 515. Structure and mass spectra of dioctylphthalate. As seen above in (a.), dioctylphalate has no exchangeable protons. The isotope distribution of dioctylphthalate may be seen (b. ) when there is no deuterium oxide introduced (c.) and when the chamber is saturated with D2O humidity. Note: although this was the highest degree of deuteration that was ever seen for dioctylphalate; it was seen very consistently throughout experiments. Figure 516. Variation in 1,2benzanthracene iondistribution as a function of distance. Mass spectra of 1,2 benzanthracene at varying distances from the DART source are presented.

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160 Figure 517 Variation in PAH isotope distribution as a function of distance. The deuterated moleculeto molecular ionratios of seven PAHs are plotted as the sampleto DART distance was varied. Figure 518. Background ion currents as a function of grid voltage. Background currents for the three different source configurations with the Teflon tube enclosure are shown.

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161 Figure 519 Total ion chromatogram (TIC) and extracted mass spectra from an analysis of 2octanone inside the sampling region enclosure.Arrows are drawn from the regions of the TIC from which the mass spectra were extracted. The blue line indica tes the time when the source was moved into the closed configuration, green lines represent when air was allowed to flow into the enclosure, and red lines denote when the airflow was stopped.

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162 Figure 520 Total ion chromatograms of compounds analyzed in the source enclosure with periodic air introduction. Blue lines indicate when the source was closed. Green lines indicate when the air was turned on. Red lines indicate when the air is turned off. Figure 521 Total ion chromatograms of 2octonone analyses in the enclosure with purified air, ultrapure nitrogen, and helium introduced. Blue lines indicate when the source was closed. Green lines indicate when theexternal gas flow was began. Red lines indicate when theexternal gas flow was ended.

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163 CHAPTER 6 THE COUPLING OF DART TO FT ICR MASS SPECTROMETERS FOR ULTRAHIGHRESOLUTION MASS ANALYSIS Introduction To date, DART sources have been interfaced only to mass analyzers of low to moderate resolving power. Fourier transform ion cyclotron resonance m ass spectrometry (FTICR MS) is uniquely advantageous over other mass analyzers because it provides higher resolving power and higher mass accuracy, and can isolate and observe ions for extended periods.100 FTICR MS is thus well suited for analysis of complex mixtures containing multiple ions of the same nominal mass, as well as for tandem mass spectrometry. Combining ambient ionization methods such as DART with FTICR MS can provide an approach for analyses that are rapid, highly selective, and informationrich. This chapter presents the first coupling of a DART source, custom built at the University of Florida, with two FTICR mass spectrometers. Experimental Chemicals and Reagents HPLC grade methanol was purchased from Burdick and Jackson (Muskegon, MI). Methylene chloride was purchased from Fisher Scientific (Fair Lawn, NJ). Both solvents were used without further purification. Polycyclic aromatic hydrocarbons (PAHs) 1,2 benzanthracene and 9,10diphenylanthracene were purchased from Aldrich (Milwaukee, WI) and were dissolved in methylene chloride at 100 ppm (w/v) and dried on glass pipettes. Theophylline and bergamottin were purchased from Sigma Aldrich (St. Louis, MO) and dissolved in methanol at 10 ppm (w/v). Diisopropyl methylphosphonate (DIMP) was purchased from Alfa Aesar (Ward Hill, MA) and

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164 vapors from an open container of it were introduced into the sampling region. A crude oil sample, NIST Standard Reference Material 2722, a heavy, sweet (low sulfur) crude oil, obtained from the National Institute of Standards and Technology ( Gaithersburg, MD), was analyzed with no dilution. Fresh ruby red grapefruit ( citrus paradisi) and habanero peppers ( capsicum chinense ) were purchased from a local grocery store and washed with tap water. Mass Analyzers 4.7 T FTICR MS. The first FTICR MS instrument to which this ionization source was interfaced was a Bruker Daltonics (Billerica, MA) BioApex II 4.7 T FT ICR MS. Daily tuning of ion focusing optics and mass calibration were performed with electrospray ionization (ESI) with ESI Tuning Mix (Agilent Technologies, Wilmington, DE). A voltage of 3500 to 4000 V was applied to the entrance electrode of the glass capillary for ESI (the spray needle was held at ground potential). After tuning, in preparation for the DART analyses, the capillary voltage was dropped to 1100 V and the hexapole accumulation period was raised from 0.51 second to 24 seconds depending on the sample. All other parameters were left unc hanged. 9.4 T FTICR MS. A custom built FTICR mass spectrometer equipped with a 22 cm horizontal room temperature bore 9.4 Tesla magnet (Oxford Corp., Oxney Mead, UK) controlled by a modular ICR data station (MIDAS)128 provided the highest reported resolving power achieved with DART ionization. Ions passed through a heated metal capillary into an RFonly octapole, then through a quadrupole to a second octapole where they were accumulated for 12 seconds. Helium gas in the accumulation octapole served to collisionally cool the ions prior to transfer through a 200 cm rf only octapole ion guide into a Penning ion trap (10 cm i.d. x 30 cm long).129 Ions were

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165 excited to their cyclotron radius by broadband frequency sweep (chirp) excitation. TOFMS. An Agilent 6210 TOF ( Wilmington, DE) instrument was used for comparatively lower resolution studies at the University of Florida. The orthogonal geometry of the flight tube enables mass resolving power up to 18,000. Sampling with 2 GHz analogto digital (A/D) conversion at the detector allows mass resolving power up to approximately 6,000 at the lowest tune mass (118 Da), while sampling with 4 GHz A/D conversion allows mass resolving power above 10,000. Tuning and mass cal ibration with the instruments autotune function were performed approximately every two days by use of ESI and the Agilent ESI Tuning Mixture. Custom -B uilt DART Source The DART source described in Chapter 2 was used in this work. The copper heating block into which four Watlow Firerod cartridge heaters were inserted was used for heating the gas. The heater was monitored and controlled with a Micromega temperature controller. Unless otherwise stated, the source parameters were as follows: needle electrode: +3 kV, ring electrode: +100 V, grid electrode: +250 V, heating block temperature: 100450 C (depending on sample), and gas flow rate: 3 L/min. The source was aimed directly into the inlet of each of the three mass spectrometers from a distance of approximately ~2.5 cm. The DART source was interfaced to all three mass spectrometers with a 30 flared capillary extender (refer to Chapter 2 for more information). Results and Discussion Resolution Comparison of a TOF and an FTICR Mass Spectrometer Although a tenscan DART -FTICR mass spectrum takes about 30 seconds to acquire, the method is nevertheless attractive for reliable identification of multi -

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166 component samples with a higher degree of specificity than the use of instruments capable of lower resolving powers. Figure 61 shows mass spectra obtained simultaneously for solid theophylline ([M+H]+ = 181.0720 Da) and DIMP ([M+H]+ = 181.0988 Da) vapors with DART TOF (Figs. 61 a. and 61 b.) and DARTFTICR MS (Fig. 61 c.). With 2 GHz A/D conversion, the TOFMS was unable to achieve the mass resolving power of 6,700 required to separate these peaks and instead one broad peak with a mass that is approximately the mean of the two masses was observed (Figure 61 a.). For 4 GHz A/D conversion, the TOFMS displayed peaks for the protonated 50% 10,000 (Figure 61 b.). Although not baselineresolved, the mass errors of the observed peaks were l ess than 10 ppm. Figure 61 c. shows that the 4.7 T FTICR MS easily achieved baseline resolution for these species. The resolving power for the theophylline and DIMP protonated molecules was ~71,000 and ~62,000, respectively, with mass errors of 5.5 ppm and 2.8 ppm. FTICR MS demonstrated improved mass accuracy and far superior resolving power than TOF MS. Polycyclic Aromatic Hydrocarbon Studies Both molecular radical cations and protonated molecules may be observed with DART ionized PAHs. To resolve M+ containing one 13C from monoisotopic [M+H]+, a resolving power of 51,000 for 1,2benzanthracene and 74,000 for 9,10diphenylanthracene is required. The 9.4 T FTICR MS displays resolving power greater than 300,000 for both analytes (Figure 62 b. and 62 d.). The 4.7 T FTICR MS resolved M+ containing one 13C from monoisotopic [M+H]+ of 1,2 benzanthracene with m/ m50% 60,000 (Figure 62 c.), but failed to resolve the analogous peaks for 9,10diphenylanthracene (Figure 62 f.). Where the species were resolved, comparable

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167 mass accuracies (3.5 ppm or less) were observed with both instruments for the two ion types (Figure 62 b. and 62 c.), although improvement may be possible with instrumental calibration. Differences in the apparent distributions of the 1,2benzanthracene and 9,10diphenylanthracene isotopes (Figure 62 a. and 62 d.) can be attributed to the differences in the relative abundances of the two ion types. The 4.7 T FTICR MS can produce resolving power greater than 100,000 for most ions below 1000 Da.100 The sub60,000 resolving power seen in Figure 6-2 c. and 62 f. may result from the increased pressure in the analyzer cell caused by the DART gas flowing into the mass spectrometer. In this situation, collisions with background gas atoms could damp the transient response signal and reduce the resolving power. However, the effect could also be attributed to a number of other factors including space charging, imperfections in the excitation conditions, and ion cloud irregularities. As demonstrated by the 4.7 T FTICR mass spectra in Figure 63, varying the distance between the sample and the inlet allows one to observe different types of ions from PAH molecules. When a 1,2benzanthracene sample deposited on a glass pipette was placed less than 1 mm from the exit of the DART source, the primary ionization mechanism appeared to be either direct Penning ionization or charge transfer with molecular oxygen, and an oddelectron molecular ion was produced. When the sample was positioned 5 mm from the exit, a mixture of molecular ions and protonated molecules w as seen. Finally, when the sample was 40 mm from the source, only protonated species were observed. This experiment was the first demonstration that there are at least two mechanisms by which PAH molecules may ionize with DART and presents an interesting direction one may take in performing DART mechanism studies.

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168 Chapter 5 showed that similar behavior was also observed with se veral other PAH molecules Real Samples Due to its ultrahigh mass resolving power and accurate mass capability, FTICR MS presents an enhanced utility for the analysis of unknown species. As for TOFMS, a wide range of samples from a variety of matrices may be analyzed by DART FT ICR MS. To date, drug tablets, food items, cigarettes, gaseous samples, pesticides, and chemical warfare simulants have been analyzed; the only difference in the experimental design was that the sample was held in place in front of the FTICR inlet longer than for the TOFMS. Three examples of the utility of accuratemass/high resolution DART FTICR MS for the analysis of two simple food products and a complex crude oil sample are presented below. Grapefruit By direct analysis from a pipette inserted into the flesh of grapefruit, the spectrum in Figure 64 was obtained with the 4.7 T FTICR MS. A nearb y opened container of ammonium hydroxide resulted in ammonium adducts for some simple carbohydrates. Based on their accurate masses, some of those masses are likely due to fructose and glucosan (i.e., a polysaccharide that yields glucose on hydrolysis) which have been previously observed in grapefruit.130 Because the isomer types of these masses are unknown, the identifications of fructose and glucosan are tentative. Bergamottin (cal culated [M+H]+ = 339.1591 Da), which has been identified as a cytochrome P450 inhibitor and is the cause of grapefruit consumption relatedpharmacokinetic interactions was also observed in low abundance.131 The identification of bergamottin was further confirmed by analysis of a pure standard (not shown). The resolving powers achieved

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169 for the low abundance ions such as ammoniated fructose and protonated bergamottin were ~30,000 and ~20,000. Pepper Direct analysis of the vein of a habanero pepper revealed a protonated molecule and a protonbound dimer of capsaicin (Figure 65). The added trapping that occurs with the 4.7T FTICR MS both in the hexapole, where ions are first accumulated, and in the analyzer cell increases the likelihood of ionion and ionmolecule interactions and the formation of protonbound dimers. This increased propensity for dimer formation has also been observed with dilute solutions of mass standards such as caffeine and the pesticide methamidophos. Based on its accurate mass, m/z 322 likely resulted from oxidation of capsaicin (calculated [M+O+H]+ = 322.2013 Da). The resolving power for the protonated capsaicin was ~50,000. Crude oil By use of a syringe pump for introduction directly into the heated gas stream exiting the DART source, NIST Heavy Sweet crude oil could be analyzed continuously and for a very long time. With 100 scans summed, a distribution of hydrocarbons that is characteristic of crude oil samples was observed by 4.7 T FTICR MS (Figure 6 6). Because it is a standard reference material, the NIST Heavy Sweet crude oil was expected to contain species ranging from 200900 Da. With DART ionization, only the volatile, low molecular weight species in this sample were detected. Changes in the heating of the DART source, syringe positioning, or crude oil flow rate could extend the upper mass limit by improving analyte desorption. Even taking into account the limited mass range, the mass spectrum obtained was less complex than those reported in other studies of crude oil based on other ionization

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170 techniques.132 One reason is the limited mass resolution of the 4.7 T FTICR MS. As the inset in Figure 67 reveals, many poorly resolved clusters of peaks appear throughout the spectrum. Although the sample is clearly complex, unambiguous identification of most of the components was impossible. Dramatically better results have been reported with a 9.4 T FTICR MS.132 The present mass spectral simplicity may also result from the competitive nature of DART ionization; once in the gas phase, the most easily ionized species will dominate. For example, the ionization of species with low proton affinity may be suppressed entirely by the presence of species with higher proton affinity. Use of dopants, such as ammonium hydroxide, in the sampling region may facilitate ionization of a broader range of analytes. The combination of DART with the 4.7 T FTICR MS was less successful than desired for this crude oil sample. Improved results can probably be achieved with a higher resolving power instrument and samplespecific DART source optimization. Conclusions A custom built direct analysis in real time ionization source was successfully coupled to each of two different Fourier transform ion cyclotron resonance mass spectrometers for the first time. It should be noted that a compact DART source, such as the one discussed in this work, is advantageous because it can easily be adapted to any mass spectrometer with an atmospheric pressure inlet. This design makes this device easily transportable and provides a wide range of options for source and sample positioning. The superior resolving power of FTICR MS was demonstrated by comparing mass spectra of the isobaric species DIMP and theophylline. Although TOFMS of fers faster analysis, it may not be able to resolve isobaric masses in complex samples. By

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171 analyzing different PAH molecules, the highest mass resolving power yet reported for DART ionization was achieved. A spatial variation in the ionization mechanisms that occur between the DART source and the mass spectrometer was noted for the first time (this work occurred prior to the work reported in Chapter 5). Mass spectra of several analyte types analyzed by DART FTICR MS were also reported, demonstrating that this technique may be useful for a wide variety of applications.

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177 CHAPTER 7 STRUCTURAL ELUCIDATION OF DART IONIZED NERVE AGENT SIMULANTS WITH INFRARED MULTIPLE PHOTON DISSOCIATION SPECTROSCOPY Introduction Compound identification with DARTMS is usually accomplished by measurement of analytes exact masses.133-135 Though H/D exchange was applied in one report, typically any structural elucidation in DARTMS experiments has been gained by performing source collision induced dissociation (CID)17,83 Source CID is induced by increasing the electric field gradient between the capillary exit and the skimmer in the first vacuum stage of a mass spectrometer. As the pressure is relatively high (up to about 1 torr) at this point in the mass spectrometer, many collisions occur between the analyte ions and the background gas molecules, causing the internal energy of the ions to increase to their dissociation threshold.136 Though the dissociation can be tuned to some degree by varying the voltages of the capillary and skimmer, source CID remains nonspecific because the ion of interest is not mass selected prior to fragmentation.137-139 When multi component mixtures are analyzed without prior separation, as is the case with DARTMS, source CID can create very complicated mass spectra that are difficult to interpret. Mass sp ectrometers capable of tandem in space or tandem in time experiments may be used to selectively isolate and fragment parent ions of interest. Two fragmentation methods that may be employed in the cell region the 4.7 T FTICR MS at UF are sust ainedoff resonance irradiationcollision induced dissociation (SORI CID) and infrared multiple photon dissociation (IRMPD).140-144 With SORI CID, the pressure inside the cell is raised to allow more collisions and the ion of interest is excited to a frequency offset from its cyclotron frequency. In this process, the selected ions

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178 alternately increase and decrease in cyclotron radius (and kinetic energy) and undergo many collisions.145 A slow internal energy buildup in the molecule eventually leads to dissociation. The dissociation method focused on in the work reported in this chapter is IRMPD. This technique is performed by directing an infrared (IR) laser beam into the cell of a mass spectrometer to irradiate trapped ions. After absorbing each photon, the mass selected ions undergo a process known as intramolecular vibrational energy redistribution (IVR) and the absorbed energy is distributed throughout the other vibrational modes of the molecules.140-142,146 Because the initial absorbing mode retur ns to ground state after IVR, the process can be repeated with one wavelength until dissociation occurs.140 This process is depicted in Figure 71. IRMPD is advantageous as a method of dissociation because it can be performed without raising the pressure of the analyzer cell region (and not sacrificing mass resolving power) and because it is wavelength dependent and can be used to gain spectroscopic (and thus structural) information. In the work discussed here, IRMPD action spectroscopy was performed on DART ionized molecules with both the Free Electron Laser for Infrared Experiments (FELIX)147 at the FOM Institute for Plasma Physics Rijnhuizen in Nieuwegein, The Netherlands and an optical parametric oscillator (OPO) laser at UF. Photons are generated in FELIX by oscillating free electrons through a series of oppositely poled magnets known as an undulator (Figure 72).147 The spacing of the magnets in the undulator and the energy of the electrons dictate the wavelength of the light exiting the laser. The radiation exiting FELIX is composed of 30 to 50 mJ macropulses, each of

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179 which is composed of hundreds of micropulses spaced 1 ns apart.147 FELIX is operated with 5 or 10 macropulses being generated per second (i.e. 5 or 10 Hz). The great advantage of FELIX is that it can be tuned over a very wide range of wavelengths OPO lasers generate different wavelengths of light by pumping a beam of light generated by another laser into a nonlinear optical crystal.148 The pump beam is converted into two lower frequency waves by means of nonlinear optical interaction with the crystal. The outputted wavelengths may be tuned by directing the pump laser through different positions of the crystal, a.k.a. poling periods, and/or by varying the temperature of the crystal. Infrared multiple photon dissociation spectra were generated for dimethyl methylphosphonate (DMMP) and diisopropyl methylphosphonate (DIMP) with the goal of elucidating the position of protonation on the two compounds (structures are given in Figure 73). Based on their structures, it is most likely that protonation will occur either on the phosphonyl oxygen atom of these molecules or one of the singly bonded oxygen atoms. These compounds have been widely used to mimic the chemical and physical properties of the class of chemical warfare agents known as G agents such as sarin and tabin without the associated toxological properties.149-152 As demonstrated in the previous chapters of this dissertation, DMMP and DIMP are readily ionized by DART. Making them ideal for a study coupling DART and IRM PD spectroscopy, these compounds are semi volatile liquids that can be used in large quantities for long periods of time without contaminating the source region of a mass spectrometer because the hot DART gas continually cleans whatever surface it strikes.

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180 Density functional theory calculations were performed to compute theoretical IR spectra of the DIMP and DMMP with the protons in each possible position. Experimental Chemicals and Reagents Dimethyl methylphosphonate (DMMP) and diisopropyl methylphosphonate (DIMP) were purchased from Alfa Aesar (Ward Hill, MA) and dissolved in methanol in concentrations of 100 ppm to 1 mg/ml depending on instrument stability. Arginine, 18crown 6, methanol, and water were supplied by the Molecular Dynamics group at the FOM Institute for all experiments in their facility. Arginine and 18crown 6 were dissolved in 1:1 mixtures of water and methanol in 1 mM concentrations. HPLC grade methanol was purchased from Burdick and Jackson (Muskegon, MI) and used without purifica tion for all experiments at UF. Instrumentation Custom built DART source The custom built DART source discussed in Chapter 2 was used in this work. The power supplies discussed in Chapter 2 were used to power the electrodes of the source for the experiments at UF. The relatively small size of this DART source made it simple to transport (on flights to Europe) and to couple to any mass spectrometer with an atmospheric pressure inlet. Heating was done with an insulated coil heater removed from an Agilent atmospheric pressure photoionization source. The heater was powered with variable AC transformers typically operated to at 2030 percent of their maximum power. At the FOM Institute, a high voltage power supply normally used with the electrospray ionization source was used to power the needle electrode and the counter

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181 electrode was grounded. Another power supply was used to apply potential to the grid electrode. Because no significant changes in ionization have been seen at UF when varying the voltage of the ring electrode no voltage was applied during these studies. Unless otherwise stated, the DART source parameters used were as follows. The needle electrode was set to 3 kV. The grid electrode was set to +250 V. The gas heater temperatures ranged from 100 to 200oC. The helium flow rate was held at 2.5 L/min. Infrared l asers Preliminary dissociation of the protonbound dimers of DIMP and DMMP was performed at both locations with fixedwavelength CO2 lasers. At UF, powers ranged from 0.8 to 2 watts depending on the sample and the alignment of the laser. DIMP and DMMP were irradiated for 0.6 seconds so that little or no dimer was detected with either molecule. At FOM, DIMP required irradiation with the CO2 laser attenuated to 4 watts for 0.5 seconds and DMMP required irradiation with the CO2 laser attenuated to 8 watts for 0.5 seconds to eliminate protonbound dimers. A Linos OS 4000 OPO laser was used at UF to generate an infrared spectrum -1) for DIMP. This OPO laser is equipped with a continuous wave (cw) neodymium yttrium aluminum garnet pump laser and a periodically poled lithium niobate crystal for generating different wavelengths. Because of the low power of the OPO laser (maximum powers were about 40 mW because only one of the outputted light beams was properly aimed into the cell at the time these experiments were performed), preliminary heating of the molecules with the C O2 laser was required to bring the molecule to the dissociation threshold. To do so, the molecule (DIMP) was given enough CO2 laser irradiation to initiate fragmentation of the monomer

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182 (a fragment appeared with 1 to 2 percent of the abundance of protonated monomer). After this point, the protonated DIMP monomers were irradiated for an additional 4 seconds with the OPO laser. It should be noted that production of fragments with beyond 12 percent of the abundance of the monomer was OPO wavelength dependent and was not observed unless DIMP was undergoing a vibrational mode transition. The OPO laser was not powerful enough at that time to dissociate DMMP. FELIX was used to generate infrared spectra ranging in the wavelength range from (560 to ~1600cm-1) The laser was operated at 5 Hz, achieving maximum powers of 250 mW (50 mJ per macropulse). After preliminary dissociation of their protonbound dimers with either SORI CID or irradiation from a CO2 laser, protonated DIMP monomers were irradiated by FELIX for 3 seconds and protonated DMMP monomers were irradiated for 5 seconds. FTICR MS at UF Studies at UF were performed with a Bruker Daltonics (Billerica, MA) BioApex II 4.7 T FTICR MS. Daily tuning of ion focusing optics and mass calibration were performed with electrospray ionization (ESI) using ESI Tuning Mix (Agilent Technologies, Palo Alto, CA). Voltages of 3500 to 4000 V were applied to the entrance electrode of the glass capillary for ESI (the spray needle was held at ground potential). After tuning, in preparation for the DART analyses, the capillary voltage was dropped to 1100 V and the hexapole accumulation time was raised to 2 to 4 seconds depending on the sample. The spray shield used for the ESI was replaced with the flared capillary extender and the DART source was aimed directly at the inlet for analyses. All other parameters of the FTICR MS were left unchanged. After ionization and injection of protonated DIMP dimers into the ICR cell, several

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183 steps occurred to create an IRMPD action spectrum. First, ions were irradiated with the CO2 laser to dissociate the protonbound dimers and to preliminarily energize the molecule. Next, the protonated monomer was isolated in the cell with a swept frequency ejection pulse to eliminate all other ions. Last, the protonated monomer was irradiated with the OPO laser. This process was repeated for wavelengths between 2.7 (3430 to 3700 cm-1). The experiment was repeated with DMMP with various times for each lasing event, but an IR spectrum was not generated due to inadequate fragmentation. Five mass spectral scans were averaged at each wavelength. FTICR MS at the FOM Institute A custom built 4.7 T FTICR MS was u sed for experiments at the FOM I nstitute.153 Tuning was done daily with a Waters Zspray electrospray source using 18crown 6 and arginine solutions. After tuning, the ESI source housing and sprayer were removed and the DART source was aimed directly at the skimmer inlet for all DART experiments (pictured in Figure 74 a.). Ions were accumulated in the hexapole for 5 to 6 seconds prior to transport into t he ICR cell. Proton bound analyte dimers were dissociated prior to IR spectral acquisition using either a CO2 laser or SORI CID in the ICR cell to produce protonated monomers for both of the species. After ionization and injection of protonated DIMP or DMMP dimers into the ICR cell, seve ral steps occurred to create an IRMPD action spectrum First, ions were dissociated either by irradiation with the CO2 laser or with SORI CID. After CO2 irradiation, the ions were allowed to cool for one second. Next, t he protonated monomer was isolated in the cell with a stored waveform inverse Fourier transform (SWIFT) pulse. Last, the protonated monomer was irradiated with infrared photons produced by FELIX. This process was repeated for wavelengths between 6.25 to 17.8

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184 (560 to ~1600cm-1). Four mass spectral scans were averaged at each wavelength. Sample Introduction Two methods of sample introduction were used in these studies. For general tests to determine that the instrument was working properly, the tapered ends of glass p ipettes were dipped into sample solutions and held in place in the region between the DART source and the mass spectrometer inlets by hand. For IRMPD studies, samples were syringeinfused into the DART gas flow at flow rates of 5in depending on signal stability. This sample introduction configuration allowed samples to be introduced for hours at a time. The sampling configuration is pictured in Figure 74 b. Density Functional Theory Calculations Density functional theory (DFT) calculations were used to compute theoretical IR spectra of the two molecules to elucidate the location of protonation. Prior to DFT calculations, conformations of the molecules were generated in the Hyperchem software suite.154 Conformations were further optimized with the Gaussian 03 so ftware suite.155 Initial calculations were performed by Dr. Cesar Contreras using (B3LYP/6 31G(d )) method and basis set. Because the level of theory used in these calculations was determined to be too low, Dr. Jan Szczepanski performed additional calculations using the (MPW1PW91/6311++G(d,p) functional and basis set. Results and Discussion DFT Calculations The lowest energy structures of DIMP A, where the ionizing proton is attached to the phosphonyl oxygen, and DIMP B, where the proton is attached to the singly bonded oxygen, calculated with (B3LYP/6 31G(d)) and (MPW1PW91/6311++G(d,p), are given in Figure 75. Both levels of theory indicated that DIMP A was at least 22 kcal/mol

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185 lower in energy than DIMP -B An elongated (broken in appearance) bond between the protonbound singly bonded oxygen atom and the connecting carbon atom was generated with both levels of theory. If the ionizing proton was attaching to this location, the observed destabilization could be a driving force for the second fragmentation m echanism displayed in Figure 7-9 b., which will be discussed below. The appearance of double bonds between the phosphorous and the singly bonded oxygens when the proton is bound to the phosphonyl oxygen as in Figure 75 a. indicates increased stabilization of those bonds (not actually double bonds). A similar effect was seen with DMMP when the ionizing proton was attached to the phosphonyl oxygen (Figure 76). The results of the spectral calculations will be presented in conjunction with the experimental IRMPD spectral results. DIMP and DMMP Fragmentation Mass spectra of DIMP and DMMP dissociated with a CO2 laser are given in Figure 77. Further irradiation with the OPO laser tuned to 3643 cm-1 for 4 seconds was used to generate the DIMP spectrum (Figure 77 a.) and a short (1 second), highpower (12.5 W) irradiation event with the CO2 laser was used to generate the fragments in the DMMP spectrum (Figure 77 b.). As mentioned previously, no additional fragmentation was obtained by irradiating DMMP with the OPO laser. Regardless of the dissociation method, the same fragments were consistently seen with each molecule. The daughter ion assignments are based on exact mass. Protonated DMMP underwent a loss of methanol to yield one daughter ion with a mass of 93. A fragmentation mechanism of DMMP is given in Figure 78. Snyder et al. gave evidence of this mechanism by using chemical ionization with a deuterium oxide reagent gas to ionize the molecule.156 Upon CID in a triple quadrupole mass

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186 spectrometer, the [M+D]+ ions were fragmented to produce species with m/z 93. The authors stated that this result demonstrated that the added D+ cation was not retained during fragmentation and instead was part of the leaving methanol group (containing a singly bonded oxygen atom).156 It should be noted that twice as much power was required to dissociate the DMMP dimers as the DIMP dimers in these experiments. Li kewise, fragmentation with FELIX required 5 seconds of unattenuated irradiation compared to 3 seconds with DIMP. The fragmentation of DIMP was more complicated. Daughter ions with masses of 97 and 139 corresponding to neutral propylene losses were primarily detected. Very low abundances of m/z 79, corresponding to the loss of a water molecule from the m/z 97 ion, were also seen occasionally. These three fragments were also reported by Johnson et al. while using DIMP as a model compound in a study comparing the triple quadrupo le to the quadrupo le ion trap.157 Two fragmentation mechanisms that involve sequential dissociations are presented in Figure 7-9 The first is fairly simple, but requires the participation of another molecule (X) or part of the leaving group for donation of a proton in each step. The second mechanism was proposed by Snyder et al. and involves two McLafferty like rearrangement steps to produce each of the daughter ions.158,159 The loss of water from the m/z 97 ion follows. The first mech anism may favor protonation on the phosphonyl oxygen and the second mechanism supports protonation on one of the singly bonded oxygen atoms. Preliminary dissociation of DIMP and sample flow rate concerns Two methods were used to dissociate the protonbound DIMP dimer into protonated monomers. The first method was SORI CID. A comparison of a mass

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187 spectrum of DIMP without and with SORI CID is given in Figure 710. As shown in Figure 711 the quantity of dimer present after the SORI event appeared to be related to the sample introduction flow rate. As a result, both an abundant dimer and a large degree of monomer fragmentation could be observed simultaneously. This was believed to be due to an uneven distribution ion temperatures in the cell, resulting in fragmentation of some monomers while other dimer species were still dissociating into monomers. After an acceptable flow rate was found, stored waveform inverse Fourier transform (SWIFT) pulses were used to eliminate the fragments and remaining protonbound dimer following the SORI process. One FELIX IRMPD spectrum (one round of acquiring spectra while the molecule was irradiated over a range of wavelengths) of DIMP was acquired with SORI CID preliminary dissociation. All other IRMPD spectra (both with FELIX and the OPO laser) were acquired with preliminary dissociation occurring via irradiation with a CO2 laser. Little or no preliminary monomer fragmentation was seen when the CO2 laser was used prior to irradiation with FELIX. A one second cooling period was included between the two irradiation events to allow equilibration of the ions internal energies. IRMPD Spectra IRMPD spectra were generated by collecting mass spectra while protonated monomer ions were irradiated with an infrared laser whose output could be tuned over wavelengths in a given range. Fragmentation yield (F.Y.) was plotted as a function of wavenumber to generate each spectrum. Data points on the IRMPD graphs were determined by dividing the sum of the abundances of the fragments produced by the chosen protonated molecule by the sum of the fragments and parent abundances, as shown in equation 7.1.

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188 OH region of the DIMP IRMPD spectrum (3400 3700 cm-1) The OPO laser was used to interrogate the OH region of the IRMPD spectrum of DIMP. Upon inspection of the IRMPD spectral results, one narrow absorption band with its maximum at 3644 cm-1 was observed. Though observation of a single peak was in agreement with the calculated spectra using either level of theory, determining which calculated spectrum was most similar the experimental spectrum was not possible. DFT calculated vibrational frequencies are known to vary from the frequencies observed experimentally. Because the shift in frequencies is generally uniform throughout a region of the spectrum, a scaling factor is applied to the calculated values.160 Generally, with (MPW1PW91/6311++G(d,p), the experimental spectrum was closest to the spectrum calculated for DIMP B, whether the scaling factor applied to the calculated frequencies was 0.97, 0.98, or 1 (Figure 712 a.). Still, even greater scaling factors (i.e. multiplication of the calculated frequencies by a factor 0.96 or lower) have been applied to this region of the spectrum.161 For spectra generated with (B3LYP/6 31G(d)), the calculated spectra could be shifted to align the experimental peak to match either structure, depending on the scaling factor (Figure 712 b.). To further complicate the matter, Correia et al. stated that reliable scaling factors are unavailable for molecules containing a phosphorous group.162 This uncertainty and a general absence of other features in the spectrum led to the conclusion that this region was not diagnostic for DIMP and that the fingerprint region (below 1600 cm-1) should be examined at the FELIX facility. Further efforts to examine DMMP with the OPO laser were suspended for this reason.

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189 Fingerprint region of the DIMP IRMPD spectrum (500 1600 cm-1) Comparisons of the calculated IRMPD sp ectra ( (MPW1PW91/6311++G(d,p) ) and the IRMPD action spectrum generated with FELIX are given in Figure 713. All comparisons and assignments of vibrations will be made between the experimental spectrum and the spectra calculated with (MPW1PW91/6 311++G(d, p) theory. Table 71 (next page) lists dominant peaks from the experimental spectra and spectra generated with FELIX irradiation as well as some vibrational assignments. The experimental spectrum was very noisy and broad between 850 and 1200 cm-1, a re gion that likely corresponds to P OR and P=O stretches. However, the highest maximum of this conglomeration occurred experimentally at 1055 cm-1. The highest peaks in the lower energy, DIMP A, and the higher energy, DIMP B, calculated spectra were positioned at 1053 and 1006 cm-1 respectively. With all features considered, a marginally better correlation between the experimental spectrum and the DIMP A spectrum was present from about 860 to 930 cm-1 and the DIMP A feature at 1120 cm-1 may have also contributed to the broadened region right of the maximum at 1055 cm-1. The distinctive features between 1300 and 1500 cm-1 may be attributed to PCH3 bends but have overlapping peaks with both structures calculated spectra. The experimental low frequency peak at 697 cm-1 was broad enough that it might be attributed to contributions from either structure. With a visual comparison and a frequency to frequency comparison, the agreement between the experimental IRMPD spectrum generated with DIMP using FELIX as the light source and the DIMP A calculated spectrum was fairly convincing. This corresponded to attachment of the ionizing proton to the phosphonyl oxygen. However, several spectral features and the occasional appearance of the m/z 79

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190 fragment ion indicated that a population of DIMP ions with the ionizing proton bound to a singly bonded oxygen could not be ruled out. With these pieces of information, it seemed possible that both species were present in the ICR cell, though there was probably a greater population of phosphonyl oxygenprotonated DIMP species. Fingerprint region of the DMMP IRMPD spectrum (500 1600 cm-1) Comparisons of the calculated IRMPD spectra ( (MPW1PW91/6311++G(d,p)) and the IRMPD action spectrum generated with FELIX are given in Figure 713. Only three distinct features were detected in the experimental IRMPD spectrum of DMMP. The observed absence of features was probably due to the drastically smaller extent of IRMPD fragmentation for DMMP when irradiated; thus, a smaller difference in the fragment to total ion abundance ratio was observed at any given point than with the other analyte, DIMP. The highest peak in this spectrum (1095 cm-1) is similar in position to that observed with DIMP and, again, it more closely corresponds to lower energy structure, with the ionizing proton bound to the phosphonyl oxygen (DMMP A). The region from 750 to 970 cm-1 has no real definition and no conclusions can be drawn from it except that P OR and P=O stretches were observed (which would be expec ted with DMMPA or DMMPB). The experimental peak with its maximum at 1315 cm-1 is bracketed by a calculated DMMP B peak, whose frequency is 1305 cm-1, and a DMMP A peak, whose frequency is 1327 cm-1. The peaks relative height is more similar to the DMM PB peak at 1305 cm-1, though generally intensity is considered less important in IRMPD spectra than frequency.142 With respect to the most dominant absorption band, it appears that the theoretical DMMP A spectrum may agree somewhat better with the experimental spectrum than the theoretical DMMP B spectrum. However, due to the broadness

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191 throughout it, it is difficult to draw conclusions from the experimental IRMPD spectrum of DMMP other than, again, it appears there may have been a mixture of DMMP A and DMMP B in the ICR cell. Conclusion The coupling of a DART source to an FTICR MS to perform IRMPD studies was demonstrated for the first time. A novel sample introduction method (for DARTMS) involving syringe injection was employed that allowed analyte ions to be produced and detected for extended periods of time. Though used to generate IRMPD spectra in this application, the syringe introduction technique could also be very useful in situations where it would be desirable to average many spectra for signal to noise improvements. Based on IRMPD spectral comparisons and the observed daughter ions, it was concluded that a population of DIMP had an ionizing proton bound to the singly bonded oxygen, as previously implied in the literature.158 However, because the IRMPD action spec trum most closely corresponded with the calculated spectra of the lower energy structure, it was concluded that a greater population of DIMP ions had an ionizing proton bound to the phosphonyl oxygen atom. Because it is a more energetically stable structu re that requires more imputed energy for fragmentation, little information was gained in performing IRMPD with DMMP. It was concluded that, again, a mixture of the structures was present, though an inference was not made about the relative proportions of the two species.

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192 Table 71. Frequencies corresponding to dominant absorption bands in the experimental and calculated IRMPD spectra of DIMP. Dominant Bands Experimental (cm -1 ) DIMP A a (cm -1 ) DIMP B a (cm -1 ) Best Match b Vibrational assignment c 557 654 658 671 A 697 708 695 B 789 776 764 A POO 866 864 A 881 886 882 Both 916 965 923 B C C, POH, POH 1006 Swamped POH, POH, POC POH, POH, POC POH, POH, POC 1040 A/Swamped 1053 1055 1068 A 1096 Swamped 1110 1121 A 1130 Swamped 1175 Swamped 1264 1263 B POC, CH 3 P 1282 1311 1318 1325 1327 Both CH 3 P 1368 1393 1390 1397 Both COH, C H 1448 1465 1466 Both a. The (MPW1PW91/6-311++G(d,p) theory was used to calculate these frequencies. DIMP A is the lower energy structure, with the ionizing proton bound to the phosponyl oxygen. DIMP-B is the higher energy structure, with the ionizing proton bound to the singly -bonded oxygen atom. b. Refers to the structure (DIMP A or DIMPB) whose theoretical spectrum best matches the experimental spectrum at the corresponding frequency range. c. Vibrational absorption band assignments are based on reports by Correia et. al. and Brunol et al. Stretches are denoted with and deformations are denoted with 162 163

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194 Fig ure 73. Structures and possible protonation sites of (a.) DIMP and (b.) DMMP. Figure 74. Photographs of the DART source interfaced to the FT ICR MS at the FOM Institute. A zoomedout photo (a.) shows that a syringe pump was used for sample introduction and the syringe needle can be seen in the close up photograph (b.). b. a.

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196 Figure 77. Mass spectra showing fragmentation of (a.) DIMP and (b.) DMMP. Figure 78. Simplified fragmentation mechanism of DMMP. The dashed arrow denotes the proposed location of fragmentation upon irradiation.

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197 Figure 79. Proposed fragmentation mechanisms of DIMP. The proposed mechanism s involve (a.) retrieval of a proton from another molecule or part of DIMP after fragmentation and (b.) a McLafferty like rearrangement. The dashed arrow denotes the proposed location of fragmentation upon irradiation. a. b.

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199 Figure 712 IRMPD spectrum of DIMP acquired in the OH region overlapped with spectra calculated by (a.) (MPW1PW91/6 311++G(d,p) theory and (b.) (B3LYP/6 31G(d)) theory.

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202 CHAPTER 8 CONCLUSIONS AND FUTURE DIRECTIONS Summary of Presented Work Many different aspects of Direct Analysis in Real Time ionization for mas s spectrometry were studied. A custom built DART source was used for a number of different applications. Fundamental studies increased understanding of the mechanisms important in DART ionization. A novel coupling of the custom built DART source to mult iple FTMS instruments took advantage of the ultrahigh resolving power capabilities and the extended trapping capabilities of FTICR MS. An inexpensive custom built DART source was fabricated and studied. The source was optimized and low picogram detection limits were achieved with several analytes. The DART source was applied to a number of different applications with little or no sample preparation. These experiments demonstrated the wide utility of DART as a surface ionization technique and that the custom built DART source could be used in the same manner as the commercial DART source. These studies showed that DARTMS is particularly suitable for compound identification and rapid screening experiments. The effects of analyte substrate, matrix, and surrounding environment were explored. Little difference was seen between molecules desorbed from glass or metal, but substrate roughness was important in the analysis of solids. The ability to analyze alternative substrates such as sand, soil, and concrete was established and showed that preliminary extraction or purification may be warranted at times. Suppression effects were observed when analytes of interest were spiked into complex matrices. The possibility of modifying ionization characteristics and even expanding DART capabilities was demonstrated with the addition of dopants to the sampling region.

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203 The mechanistic factors governing DART sensitivity were systematically studied. The importance of proton affinity (both of the analyte and other compounds in its vicinity) in analyte sensitivity and detectability was demonstrated. Self protonation was definitively shown to be operative in DART ionization even though it had not been discussed previously in the DART literature. The source enclosure experiments introduced doubt to the widely held belief that Penning ionization is a dominant analyte ionization mechanism in helium DART. In all cases, the pathways of DART ionization appeared to fall into the class of mechanisms operative in atmospheric pressure chemical ionization (charge exchange, proton transfer, etc.). Its compact design allowed the custom built DART source to be interfaced to several different FTICR instruments. The utility of ultrahigh resolving power afforded by FTICR MS was dem onstrated by analyzing isobaric compounds and by distinguishing 13C molecular ion isotopes and [M+H]+ species of different polycyclic aromatic hydrocarbons. Infrared multiple photon dissociation was applied for spectroscopic structural determination of DA RT ionized molecules. Future Studies As illustrated in the above summary, the work presented in this dissertation covered a wide range of topics involving DART ionization. Further expansion of any one of these areas is possible. Though not discussed in this dissertation, nitrogen was also employed as a desorption/ionization gas with a commercial DART source in additional studies. In the future, argon should also be explored as a desorption gas because it is cheaper than helium and produces relatively highenergy metastable species (11.55 eV).164 A few more ideas that were either actually implemented to some degree during with this work or that were just dreamed of are presented below.

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204 Applications Crude oil analysis An analysis of crude oil with DARTFTICR MS was presented in Chapter 6. In this proof of concept study, the ions intensities and distribution were less than desirable. Future work in this area should include optimization of the sampling conditions and ambient environment about the DART source. To start, future researchers should find the optimal sample flow using a syringe pump, syringe needle position, and gas temperature. It may be beneficial to explore spray creation as venue for transporting the oil to the gas phase. Next, dopants should be explored to enhance ionization. Addition of ammonium hydroxide vapors may promote adduct formation. Addition of a volatile acid vapor, such as acetic acid, may enhance ionization of low proton affinity compounds. One may also consider adding dopants directly to the crude oil sample to ensure that interaction occurs between the analyte molecules and the dopants. Taking these steps should allow better desorption and ionization of the crude oil sample, thus generating mass spectra with more abundant ion signals spanning a wider range of masses. Reaction monitoring A couple of studies claiming to perform reaction monitoring with DART have been reported.38,165 Neither of these studies presented a real time analysis of the reaction mixtures because each involved sample retrieval prior to analysis. Real time reaction monitoring is possible and has been accomplished in the mass spectrometry laboratory where the research reported in this dissertation was done. With a couple of gas lines (one inserted in a reaction mixture and one leading from the containers headspace to the DART source sampling region) and a flow of nitrogen, gas phase reaction products

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205 were monitored in real time as they were produced. Generation of a spray of the liquid in a reaction mixture for analyzing less volatile components could be accomplished with a peristaltic pump and a nebulizer. Though these studies could be somewhat cumbersome to implement, the information they could provide would be invaluable and novel. This could be a very rewarding project for a new graduate student. Polycyclic aromatic hydrocarbons Studies from Chapter s 5 and 6 demonstrated that the distribution of PAH ions could be modified by adjusting the position of the sample relative to the DART source. There is interest in this class of molecules because they are believed to be responsible for spectral observations in interstellar clouds.166,167 As generation of protonated PAH species has been an experimental challenge,168 DART may be a suitable ionization source for IRMPD studies of these molecules. Conversely, IRMPD studies of DART ionized PAH molecules may provide information on their formation. Though several mechanisms are possible, the work reported here did not experimentally show how all molecular ions and protonated species are formed during analyses with DART (particularly those that do not appear to have interacted with ambient water clusters). For example, Vala et al. and others have presented evidence that these molecules undergo proton ejection.169-171 The reported studi es with deuterated anthracene ( Chapter 5) also support this hypothesis. Spectroscopic investigations could shed light on this subject. TSA s ecurity swabs An interesting project suitable for an undergraduate researcher is analysis of the swabs used by the Transportation Security Administration (TSA) at airports to collect samples for their ion mobility spectrometers (IMS). Though widely used across the

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206 nation for airport security, a common complaint about IMS instruments is their false positive rate.172,173 DART MS could either provide a highly specific alternative to IMS without sacrificing rapidity or it could complement IMS detection systems. In either case, it would be interesting to determine how DARTMS compares to traditional IMS systems for the analysis of drugs, explosives, and chemical warfare agents from swabs used to wipe surfaces like the inside of luggage or shoe soles. Continued Mechanism Studies Source enclosure experiments. At the conclusion of the mechanism studies presented in Chapter 5, the source enclosure studies indicated the possibility of additional mechanisms occurring in lieu of (or maybe in combination with) Penning ionization. A direction future researchers should take with this is to repeat the enclosure studies with varying amounts of other external gases. In particular, an experiment should be done where helium and nitrogen mixtures are introduced to determine if there is a correlation (possibly an inverse relationship) between the amount of helium and the signal abundance. This will determine if the helium itself is hindering ionization or if another mechanism is operative. Other inert atomic gases such as argon should also be introduced externally to determine if the loss of signal is the result of collisional deexcitation between helium atoms prior to interaction with the analytes (helium and argon do not have various vibrational modes to contain and distribute internal energy like bimolecular gases). If ion signals are detected, collisional deexcitation is not the answer. Combination Ionization Sources Though the minute size of the DART source used in this work simplified trans fer to various other mass spectrometers, it can also allow the device to be easily combined

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207 with other ionization sources for multi mode operation. All multi mode ionization sources are designed with a goal of broadening the range of capabilities of one or both ionization sources (possibly synergistically). This goal may be achieved by providing more efficient sample introduction (e.g. enhanced desorption) with one source followed by ionization with another source or by creating multiple venues for analyte ionization. Laser desorptionDART The DART source was originally built to serve as an atmospheric pressure chemical ionization (APCI) source that would be inserted into an atmospheric pressure matrix assisted laser desorption ionization source (MALDI) to create a combined laser desorption/ APCI source. This coupling was intended to broaden the range of analytes ionizable by desorption ionization on porous silicon (DIOS). The benefits of this combination source would be two fold. First, adding reagent ions to the source region atmosphere would provide more opportunities for desorbed materials to ionize, thus improving the sensitivity of DIOS (or MALDI). Second and derived from the first benefit, identification of the neutral species desorbed during the DIOS process might be possible, perhaps providing greater insight into the ionization mechanism(s) of DIOS. There was too little time to implement laser desorptionDART (LD DART) in the current work. However, because all of the necessary components (a DART source and a MALDI source) are already housed in the Powell laboratory at UF, a few simple modifications to the respective sources could make the implementation and application of LD DART a fruitful project for a future graduate student. DART combined with other ionization sources Other ionization sources could also be coupled with the DART source to enhance analysis capabilities. For example, it would be fairly simple to direct the DART gas

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208 stream through an electrospray ionization (ESI) nebulizer in place of nitrogen. By doing so, analytes exiting a high performance liquid chromatograph could be ionized both by ESI and APCI without modifying the geometry of the ESI spray head (as is the case with the current ESI/APCI multimode source). Another possibility that was explored for a while in conjunction with the current work was DART atmospheric pressure photoionization (DARTAPPI). This source was implemented by replacing the nebulizer in an APPI source with the DART source. This combination allowed low proton affinity analytes such as naphthalene to be desorbed without solvents and ionized by photon absorption. A similar idea that essentially combined desorption electrospray ionization (DESI) with APPI was reported by Haapala in 2007, but required solvents. Further method development would be necessary to take DART APPI from an idea to a versatile ambient ionization technique. General Conclusion Research with and about Direct Analysis in Real Time ionization is still in the early stages. Though many different topic areas utilizin g the custom built UF DART source were covered in this thesis, many more areas remain unexplored. Because of its simplicity and wide applicability, the use of DART MS as a powerful analytical tool will continue to grow.

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209 LIST OF REFERENCES (1) Cody, R. B.; Laramee, J. A.; Durst, H. D. 2297. (2) Watson, J. T. ; 3rd Ed.; Lipincott-Raven Publishers: Philadelphia, PA, 1997. (3) KiviEtelatalo, E.; Kostiainen, O.; Kokko, M. 205. (4) Helmig, D.; Greenberg, J. P. 123. (5) Nanita, S. C.; Pentz, A. M.; Grant, J.; Vogl, E.; Devine, T. J.; Henze, R. M. 797. (6) Augusti, D. V.; Carazza, F.; Augusti, R.; Tao, W. A.; Cooks, R. G. 3458. (7) Nicol, G.; Sunner, J.; Kebarle, P. 135. (8) Wang, H. Y.; Zhang, X.; Guo, Y. L.; Lu, L. 1561. (9) Tolmachev, A. V.; Udseth, H. R.; Smith, R. D. 970. (10) de Hoffmann, E.; Stroobant, V. ; John Wiley & Sons, Ltd.: West Sussex, England, 2007. (11) Yamashita, M.; Fenn, J. B. 4451. (12) Wilm, M. S.; Mann, M. 167. (13) Rockwood, A. L.; Busman, M.; Smith, R. D. 103. (14) Cech, N. B.; Krone, J. R.; Enke, C. G. 208. (15) Gaskell, S. J. 677. (16) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. 471. (17) Steiner, R. R.; Larson, R. L. 617. (18) Jackson, A. U.; Werner, S. R.; Talaty, N.; Song, Y.; Campbell, K.; Cooks, R. G.; Morgan, J. A. 272. (19) Talaty, N.; Takats, Z.; Cooks, R. G. 1624. (20) Haefliger, O. P.; Jeckelmann, N. 1361.

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210 (21) Shiea, J.; Huang, M. Z.; Hsu, H. J.; Lee, C. Y.; Yuan, C. H.; Beech, I.; Sunner, J. 3701. (22) Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C. 1712. (23) Nemes, P.; Vertes, A. 8098. (24) Haapala, M.; Pol, J.; Saarela, V.; Arvola, V.; Kotiaho, T.; Ketola, R. A.; Franssila, S.; Kauppila, T. J.; Kostiainen, R. 7867. (25) McEwen, C. N.; McKay, R. G.; Larsen, B. S. 7826. (26) Takats, Z.; Cotte-Rodriguez, I.; Talaty, N.; Chen, H. W.; Cooks, R. G. 1950. (27) Venter, A.; Nefliu, M.; Cooks, R. G. 284. (28) Van Berkel, G. J.; Pasilis, S. P.; Ovchinnikova, O. 1161. (29) Harris, G. A.; Nyadong, L.; Fernandez, F. M. 1297. (30) Nyadong, L.; Galhena, A. S.; Fernandez, F. M. 7788. (31) Kertesz, V.; Ford, M. J.; Van Berkel, G. J. 7183. (32) Cotte-Rodriguez, I.; Takats, Z.; Talaty, N.; Chen, H. W.; Cooks, R. G. 6755. (33) Haddad, R.; Sparrapan, R.; Eberlin, M. N. 2901. (34) Na, N.; Zhao, M. X.; Zhang, S. C.; Yang, C. D.; Zhang, X. R. 1859. (35) Chen, H.; Yang, S.; Wortmann, A.; Zenobi, R. 7591. (36) Andrade, F. J.; Shelley, J. T.; Wetzel, W. C.; Webb, M. R.; Gamez, G.; Ray, S. J.; Hieftje, G. M. 2646. (37) Harper, J. D.; Charipar, N. A.; Mulligan, C. C.; Zhang, X. R.; Cooks, R. G.; Ouyang, Z. 9097. (38) Smith, N. J.; Domin, M. A.; Scott, L. T. 3493. (39) Van Berkel, G. J.; Kertesz, V.; Koeplinger, K. A.; Vavrek, M.; Kong, A. N. T. 500.

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211 (40) Shelley, J. T.; Ray, S. J.; Hieftje, G. M. 8308. (41) Rezenom, Y. H.; Dong, J.; Murray, K. K. 226. (42) Martinez-Lozano, P.; Rus, J.; de la Mora, G. F.; Hernandez, M.; de la Mora, J. F. 287. (43) Faubert, D.; Paul, G. J. C.; Giroux, J.; Bertrand, M. J. 69. (44) Hiraoka, K.; Fujimaki, S.; Kambara, S.; Furuya, H.; Okazaki, S. 2323. (45) McLuckey, S. A.; Glish, G. L.; Asano, K. G.; van Berkel, G. J. 2312. (46) database, (accessed November, 2009) (47) "The DART MS Discussion Board." http://groups.google.com/group/dart-mass-spectrometer-users. (accessed September 2009) (48) Cody, R. B. 1101. (49) Harris, G. A.; Fernandez, F. M. 322. (50) Nilles, J. M.; Connell, T. R.; Durst, H. D. 6744. (51) Grange, A. H. 127. (52) Grange, A. H. 137. (53) Grange, A. H. 183. (54) Yu, S. X.; Crawford, E.; Tice, J.; Musselman, B.; Wu, J. T. 193. (55) Shelley, J. T.; Wiley, J. S.; Chan, G. C. Y.; Schilling, G. D.; Ray, S. J.; Hieftje, G. M. 837. (56) Fridman, A. A.; Kennedy, L. A. ; Taylor & Frances: Abingdon, Oxford, UK, 2004. (57) Penning, F. M. 818. (58) Searcy, J. Q.; Fenn, J. B. 1861. (59) Harrison, R. G.; Aplin, K. L. 199.

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219 BIOGRAPHICAL SKETCH Julia Diane Laney Rummel was born on October 2, 1981 and grew up in Livermore, Kentucky. In 2000, she matriculated at Eastern Kentucky University (EKU) to study forensic chemistry. While at EKU, Julia was encouraged to participate in a Research Experience for Undergraduates (REU) program and to consider graduate school. After doing an REU at the University of Florida (UF) and later doing an internship at the North Carolina State Bureau of Investigation Crime Laboratory in drug chemistry, Julia was convinced she should attend graduate school. In the fall of 2004, after receiving her Bachelors of Science in forensic chemistry, Julia started graduate work at UF. She worked under Dr. David Powell and Dr. John Eyler doing research on the ambient ionization technique, Direct Analysis in Real Time. In 2006, she married Ian Rummel. In December of 2009, Julia will graduate with a doctorate in analytical chemistry.