UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Systematic study of dopants for use in atmospheric pressure photoionization mass spectrometry Smith, Derek Robert 2008

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2008_fall_smith_derek_robert.pdf [ 2.71MB ]
Metadata
JSON: 24-1.0061683.json
JSON-LD: 24-1.0061683-ld.json
RDF/XML (Pretty): 24-1.0061683-rdf.xml
RDF/JSON: 24-1.0061683-rdf.json
Turtle: 24-1.0061683-turtle.txt
N-Triples: 24-1.0061683-rdf-ntriples.txt
Original Record: 24-1.0061683-source.json
Full Text
24-1.0061683-fulltext.txt
Citation
24-1.0061683.ris

Full Text

SYSTEMATIC STUDY OF DOPANTS FOR USE IN ATMOSPHERIC PRESSURE PHOTOIONIZATION  MASS  SPECTROMETRY  by  DEREK ROBERT SMITH  B . S c , University of British Columbia, 2005  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF  M A S T E R OF S C I E N C E  in  T H E F A C U L T Y OF G R A D U A T E STUDIES (Chemistry)  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A (Vancouver)  July 2008  © Derek Robert Smith, 2008  Abstract Atmospheric pressure photoionization (APPI) is an effective ionization technique for the analysis of low polarity and nonpolar compounds using liquid chromatography/mass spectrometry. Ions are produced through a mechanism which begins with initial photoionization of a primary reagent, termed a "dopant", followed by either proton transfer or charge exchange with the analyte(s). This thesis regards improving the ionization efficiency of APPI by identifying new dopant candidates that can increase the breadth of compounds amenable to APPI, and/or improve the ionization efficiency for compounds that are already amenable to A P P I . The desired properties for a dopant candidate include high ionization energy (IE) and low reactivity of its photoions with solvent and dopant neutrals. Reactivity tests for 25 substituted-benzene compounds with substituents ranging from strongly electron withdrawing (EW) to strongly electron donating (ED) were performed. Results showed that E D groups decreased reactivity and IE while E W groups increased reactivity and IE; an exception was i f the E D group was itself acidic. O f the compounds tested, 2,4-difluoroanisole and 3-(trifluoromethyl)anisole showed the best potential as dopants for charge exchange. These dopants - along with two other novel dopants, bromo- and chlorobenzene - were compared with established dopants (toluene, anisole, and a toluene/anisole mixture) for charge exchange ionization of polycyclic aromatic hydrocarbons (PAHs). Bromo- and chlorobenzene both showed significant improvement in ionization efficiency compared with previously established dopants due to their relatively low reactivity with the solvent and high IE. It was also found that the improved performance for higher IE P A H s , when using anisole, diluted to 0.5% with toluene, was possibly due to the presence of an impurity in anisole. O f the dopants tested, bromobenzene/2,4-difluoroanisole (99.5:0.5 v/v) was determined to be the best overall for charge exchange ionization.  T A B L E OF CONTENTS  ABSTRACT  ii  TABLE OF CONTENTS  iii  List of Tables  v  List of Figures Abbreviations  vi vii  Acknowledgements  viii  Co-Authorship Statement  CHAPTER I  1.1 1.2 1.3 1.4 1.5  INTRODUCTION  Atmospheric Pressure Chemical Ionization Electrospray Ionization Atmospheric Pressure Photoionization Aims of the Study Bibliography  ix  1  2 3 4 10 12  CHAPTER n INVESTIGATION OF SUBSTITUTED-BENZENE DOPANTS FOR CHARGE EXCHANGE IONIZATION OF NONPOLAR COMPOUNDS BY ATMOSPHERIC PRESSURE PHOTOIONIZATION 16  2.1 Introduction 2.2 Experimental 2.2.1 Chemicals 2.2.2 Instrumentation 2.2.3 Method 2.3 Results and Discussion 2.3.1 Results for Substituted-Benzene Compounds 2.3.2 Hypothesis on the Effects of Substituents on Reactivity 2.3.3 Discussion of Results for Substituted-Benzene Compounds 2.3.4 Results for Fluoro-Substituted Anisoles 2.3.5 Discussion for Fluoro-substituted Anisoles 2.4 Conclusions 2.5 Bibliography  16 18 18 19 19 20 21 26 27 30 31 32 34  C H A P T E R III COMPARISON OF DOPANTS FOR CHARGE EXCHANGE IONIZATION OF NONPOLAR POLYCYCLIC AROMATIC HYDROCARBONS WITH REVERSED-PHASE L C APPI-MS 36  3.1 Introduction 3.2 Experimental 3.2.1 Chemicals 3.2.2 Instrumentation 3.2.3 Method 3.3 Results and Discussion 3.3.1 Group 1 Dopants and Supplementary Infusion Experiments 3.3.2 Discussion of Group 2 Dopants 3.3.3 Discussion of Group 3 Dopants 3.4 Conclusions 3.5 Bibliography  CHAPTERIV  4.1  36 41 41 42 44 45 47 53 55 56 58  CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE RESEARCH... 61  Bibliography  65  List of Tables  Table 2.1  Thermodynamic data and experimental results for dopant candidates  21  Table 3.1  List of P A H s examined  42  Table 3.2  Gradient timetable  43  Table 3.3  Average relative peak area for P A H s examined  46  List of Figures  Figure 1.1  Schematic of the PhotoSpray™ APPI source  5  Figure 2.1  Mass spectrum of methyl benzene carboxylate  22  Figure 2.2  Mass spectra of toluene  23  Figure 2.3  Mass spectra of hexafluorobenzene  24  Figure 2.4  Mass spectra of 1,3,5-trimethylbenzene, chloro- and bromobenzene  25  Figure 2.5  Mass spectra of 3-(trifluoromethyl)anisole and 2,4-difluoroanisole  31  Figure 3.1  Sample chromatogram of the P A H s  45  Figure 3.2  Extracted ion chromatograms for naphthalene  47  Figure 3.3  Results for supplementary infusion experiments  49  Abbreviations  APCI API APPI D/D^" Da DFA DFT ED ESI eV EW GC HPLC HV l/f IE LC/MS M/M^' MH^ MS MW m/z PA PAH(s) S SIM TFMA X XIC  atmospheric pressure chemical ionization atmospheric pressure ionization atmospheric pressure photoionization dopant / dopant photo ion dalton 2,4-difluoroanisole protonated dopant cation electron donating electrospray ionization electron volt electron withdrawing gas chromatography high performance liquid chromatography high voltage impurity / impurity cation ionization energy liquid chromatography/mass spectrometry analyte / analyte cation protonated analyte cation mass spectrometry / mass spectrometer molecular weight mass-to charge ratio halogen substitution product cation proton affinity polycyclic aromatic hydrocarbon(s) solvent selected ion monitoring 3-(trifluoromethyl)anisole halogen extracted ion chromatogram  Acknowledgements  A thank you goes out to Dr. Michael Blades and Dr. Damon Robb for their guidance and insight throughout my graduate studies. I am also gratefiil to Dr. Guillaume Bussiere for kindly allowing me to borrow his lab's H P L C equipment.  Co-Authorship Statement  The content in chapters two and three was a collaboration between Derek R. Smith, Damon B . Robb, and Michael W. Blades. Design, performance, and data analysis of the research was done by Derek R. Smith. Writing of the manuscript was done by Derek R. Smith and Damon B . Robb. Funding for the research was provided for by Michael W . Blades.  CHAPTER I INTRODUCTION  High performance liquid chromatography (HPLC) is a powerful analytical technique that can be used to separate a large variety of compounds, based on retention time, from low molecular weight drugs, to proteins. H P L C is often combined with mass spectrometry (MS), which allows for an additional dimension of separation, in this case, based on the mass-to-charge ratios of the analyte. The use of atmospheric pressure ionization (API) allows for the eluent to be infUsed directly from the H P L C into the ionization source of the mass spectrometer. Three A P I methods are typically used with L C / M S : atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI) and atmospheric pressure photoionization (APPI). The research presented in this thesis is focused on A P P I and its use with L C / M S . O f interest is expanding the range o f compounds amenable to APPI through the use of dopants, which are used to enhance ionization efficiency in APPI; the use of dopants in APPI is also referred to as dopant assisted-APPI.  The first chapter of this thesis presents a review of current A P I methods, focusing on A P C I , ESI and APPI, followed by a summary of the research objectives. The second chapter presents the research that was conducted on the identification of new dopants for APPI in an effort to broaden the ionization capabilities of this ionization technique [1]. Two  dopant  candidates  from  this  study,  2,4-difluoroanisole,  and  3-  (trifluoromethyl)anisole were included in an investigation on the ionization efficiency of these dopants in the analysis of polycyclic aromatic hydrocarbons (PAHs) by L C / M S [2], and is presented in chapter three. The thesis concludes with a summary of the results of these two studies and a brief discussion of the direction of fliture research.  1.1 Atmospheric Pressure Chemical Ionization  Atmospheric pressure chemical ionization is a common method o f ionization, which can be used with L C / M S and was originally developed in the early 70's by Homing et al. [3, 4]. A s the name suggests, ionization occurs through chemical ionization, in a reaction chamber which is maintained at atmospheric pressure, and is external to the low pressure region of the mass spectrometer. In the first A P C l sources, ionization was initiated by electrons, emitted from the beta decay of Ni-63. This has since been replaced by a corona discharge needle, which has the benefit o f improving sensitivity in addition to being less hazardous [5]. The source is heated in order to aid in vapourization of the solvent stream. Nitrogen is typically used as the nebulizing and auxiliary gas. The corona discharge initiates ionization o f the nitrogen gas to form nitrogen cations, which can in turn ionize water vapour in the source to form protonated water clusters that can then undergo proton transfer with an analyte.  Due to proton transfer being the primary ionization mechanism, compounds that can be ionized by A P C I are limited to those with a medium to high proton affinity. Compounds which have basic functional groups will tend to be more efficiently ionized  than compounds without these substituents; low polarity compounds tend not to be efficiently ionized. A P C I also has a mass limit for the molecules that it can ionize, considered to be below -2000 D a [6].  1.2 Electrospray Ionization  Electrospray ionization is another common A P I method that can be used with L C / M S . ESI was first developed by Dole et ah, and later combined with mass spectrometry by Yamashita and Fenn [7, 8]. In ESI, the solvent stream is first introduced into the source through a capillary, maintained at high voltage (-3000 - 5000 V ) . The solvent stream is then dispersed into a mist of multiply charged droplets which can eventually form the ions that are observed by the mass spectrometer. One theory of the ionization process involves evaporation of the solvent from the charged droplet. A s the droplet shrinks in size, the electric field at the surface is able to overcome the surface tension, a Coulombic explosion can occur, and smaller droplets are formed. This process can then repeat with the smaller droplets until a point where ions of analytes in the droplets are produced and can enter the mass spectrometer. A n alternative theory of the ionization process has been proposed where during evaporation of the solvent, the charge to surface area ratio will increase until the repulsive forces between the charges overcome the binding energy of the ion, which can then leave the drop [9, 10].  Regardless of which ionization description may be more accurate, in practice, ESI has become a powerful method for characterization of high molecular weight compounds  because of formation of multiply charged ions, which allows such compounds, for example proteins with weights exceeding 100,000 Da, to fall within the mass range accessible to commonly used mass spectrometers (0 - 5000 Da). Disadvantages of ESI include the formation of cluster and adduct ions with salts that may be in the sample, limiting the salt content of a solution to be ionized by ESI to less than 10'^ M . Compounds amenable to ESI are generally limited to those that already exist as charged species in solution and neutral/polar compounds that can be protonated (positive ion mode) or deprotonated (negative ion mode). Since the ions that are formed in ESI are typically protonated cations (often multiply charged), this makes ESI as an inefficient method for ionizing low polarity or nonpolar compounds [6, 11].  1.3 Atmospheric Pressure Photoionization  Atmospheric pressure photoionization (APPI) was developed concurrently by Robb et al. and Syage et al. as a new ionization method for use with L C / M S [12, 13]. A significant advantage of A P P I over ESI and A P C I is its ability to ionize both polar and nonpolar compounds [12, 14]. A s such, APPI has the potential to replace these A P I methods for many L C / M S applications. Despite the name, ionization of the analytes in APPI is primarily due to ion-molecule reactions between analyte molecules and primary reagent ions which are generated by direct photoionization of the primary reagent rather than through direct photoionization of the analyte. If there is not a suitable primary reagent present in the majority of the sample stream, one can be added intentionally postcolumn; the added compound is termed a dopant. The dopant can then undergo direct  photoionization, generating primary reagent ions that can result in the ionization of analytes through either proton transfer or charge exchange (electron transfer) pathways. This method of A P P I is known as dopant assisted-APPI.  A commercially available A P P I source known as the PhotoSpray^m, used for the work described in this thesis, was first developed by Robb et al. and consists of separate inlets for the dopant and sample stream which passes through a heated nebulizer probe [12]. A source designed by Syage et al. called the PhotoMate™ is similar but is orthogonal in design and is not part of the focus of this thesis [13]. The design of the PhotoSpray'''''^ is similar to that of an A P C I source, with the corona needle replaced with a krypton discharge lamp which is used as the photon source. Nitrogen is used for the nebulizer, auxiliary and lamp gas (also termed purge gas). A schematic of this source is shown in Figure 1.1.  curtain gas  -HV  lamp current poi«er suppi/  . " i-:^_^-*':™L;-irr  HV return  •  •'i-  curtain pfale  -—i:'?^}^ ,  •^,.»  •^.^j ; orificepiate  HV power supply  i: • i ';  :  i 1 :  lamp gas  nebulizer gas liquid sampie slresm auxiliary gas (with doparît)  :-: '  ^=EE=^ ' Quartz vaporiser tube  source block  teater  primary ionization region  reactioftlransport region  Figure 1.1: Schematic of the PhotoSprayTM APPI Source  starsd-oft rod  ;  The sample stream and dopant are introduced separately and combined in the source block. The mixture is transported to the primary ionization region via the auxiliary gas. The heater is maintained at a temperature typically around 400°C, and is used, in conjunction with the nebulizer gas to vaporize the sample and dopant mixture. Lamp gas is used to keep the window between the lamp and ionization region free of contamination. The dopant can be ionized by direct photoionization and can then ionize the analyte through various ion-molecule reactions. A n additional benefit is the similarity in physical design between the APPI source and the ESI and A P C I sources makes these sources  interchangeable and can be attached  to a mass  spectrometer without  modifications.  In dopant assisted-APPI, ionization can occur through either proton transfer or charge exchange mechanisms. In both cases, the ionization mechanism begins with the direct photoionization of the dopant (D) by 10 eV photons generated from the lamp to form dopant photoions (D"^').  D—!^D^'+e-  IE(D)<^v(10eV)  (1.1)  Two of the first dopants that were identified were toluene and acetone. It was found that when used under reversed-phase conditions ion-molecule reactions with high proton affinity (PA) solvent molecules (S) such as acetonitrile or methanol were favoured, forming protonated solvent clusters (see reaction 1.2). The solvent clusters could then react by proton transfer with analytes (M) with a high P A , shown in reaction 1.3 [12, 15].  >[D-H]*+S„H" S„H^+M  >MH^+/7S  PA (S)>PA(D^")  (1.2)  P A (M) > P A (S„)  (1.3)  In the cases of low P A solvents such as hexane, chloroform, or water, charge exchange ionization was favoured (see reaction 1.4) [15].  >M^* + D  ifIE(M)<IE(D^')  (1.4)  As such, the ionization of nonpolar compounds, or low P A compounds through charge exchange, would be limited to normal-phase conditions when using toluene or acetone as dopant.  It was not until the identification of anisole as a dopant for A P P I that charge exchange ionization of nonpolar compounds could be efficiently achieved under typical reversed-phase conditions [16]. Anisole photoions were found to be unreactive with reversed-phase solvents and could therefore be used for charge exchange ionization for compounds o f low ionization energy (IE) and low P A . The low IE of anisole (8.2 e V ) ' however, is a limiting factor that restricts the range of analytes that can be ionized to those o f low IE. While anisole is limited by its IE, a significant advantage was the increase in ionization efficiency of up to 100 times compared with other dopants.  ' All IE data are from the NIST Chemistry WebBook, unless otherwise indicated [17].  Bromobenzene (IE 9.00eV) and chlorobenzene (IE 9.07eV) were recently identified as an alternative for anisole, however the photoions of these dopants were found to be only partially stable under reversed-phase conditions [18]; to my knowledge, however, other than the conference presentation of vanDam and Bruins, there have been no reports on the use of either bromobenzene or chlorobenzene as an APPI dopant. A recent publication reported the potential use of a dopant mixture, combining anisole and toluene in an attempt to optimize the dopant for analysis of polycyclic aromatic hydrocarbons (PAHs) by L C / M S [19]. The optimized mixture was reported to be a 99.5:0.5 (v/v) ratio o f toluene to anisole. While this was able to improve sensitivity for PAHs with higher IE without significantly lowering sensitivity of lower IE PAHs, it was still limited to ionization of relatively low IE PAHs due to the reactivity of toluene photoions with the solvent, and the low IE of anisole.  A P P I has been used successfully for a large variety of applications which are either not accessible or inefficiently performed by either ESI or A P C I . A number of studies have looked at the use o f APPI in the place of other A P I techniques for use in drug discovery and have shown that A P P I can ionize a larger percentage of the small molecule compounds of interest, and in many cases, improve sensitivity. A n example of this is a recent study regarding the analysis of idoxifene, an estrogen blocker used in cancer treatment, which found APPI to be up to eight times more sensitive for idoxifene compared with A P C I [20]. Additional studies looking at a wide range of small molecule drugs including antidepressants, central nervous system active drugs, P-adrenal blockers and a number of small molecule drug candidates showed APPI to be more sensitive than  ESI or A P C I for a majority of compounds [14] as well as lower sensitivity to matrix ion suppression [21]. Other drug molecules that have been successfully analyzed using APPI include various steroids, such as sterols [22] and anabolic steroids [23], and peptide drugs such as cyclosporine A [24]. In each of these studies toluene was used as the dopant.  APPI has also found use in a variety of health and environmental related applications. Flavonoids are a naturally occurring compound found in various plants and food products such as citrus, tea, and dark chocolate and are of interest due to their antioxidative properties. APPI, using toluene as dopant, has been compared with ESI and A P C I for the analysis of a number of flavonoids which were chosen to represent three subgroups: flavonols, flavones, and catechins [25]. Both positive and negative ion modes were tested for each A P I method, with results showing comparable limits of detection with all techniques, which indicates that while APPI can be used in place of other A P I methods, there is room for improvement. Another group of compounds that are of interest due to health and environmental concerns are P A H s , due to their toxicity and carcinogenic effects, as well as being contributors to air pollution [26-29]. P A H s have been ionized with A P P I using both toluene and anisole as dopants, as well as using mixtures o f the two dopants [19, 30]. The analysis of P A H s contained in various matrices including sediment [31], and cigarette smoke [32], have also been reported; in both cases toluene was used as the dopant.  1.4 Aims of the Study  The underlying objective o f this research is to improve upon the ionization efficiency of the APPI source as well as increasing the range of compounds it can ionize when used under typical reversed-phase conditions. The research will focus on the use o f dopants and identification o f new dopants rather than changing the fundamental design of the ion source. Research will also be limited to improvement of A P P I when used in positive ion mode. One o f the limitations of A P P I mentioned previously was the range o f nonpolar analytes that can be ionized by charge exchange due to dopants with low IE such as anisole. While other dopants such as toluene, bromo- and chlorobenzene have a higher IE, their photoions have been shovra to react with reversed-phase solvents and are therefore no longer available for charge exchange ionization. B y identifying new, high IE dopants that have photoions which are unreactive with reversed-phase solvents, the breadth o f compounds amenable to A P P I could be significantly increased.  The first objective o f this research was to identify new dopant candidates for APPI that would be suitable for charge exchange ionization of nonpolar compounds when used under reversed-phase  conditions. This was accomplished through an initial  screening of a number of substituted-benzene compounds. The results of this screening led to the development of a hypothesis on the effect o f substituents on the reactivity of the substituted-benzene photoions. This hypothesis was used as a basis to select a number of fluoro-substituted anisole compounds as additional test candidates. O f the compounds tested, 2,4-difluoroanisole, and 3-(trifluoromethyl)anisole were identified as the top  dopant candidates and were used in the second stage of the research. The results of the first study are discussed in detail in Chapter 2 of the thesis.  With the identification of two new dopants, the second stage of this research was an evaluation of the effectiveness of these dopants compared with previously established dopants. The utility of the dopants was evaluated by examining the ionization efficiency of nonpolar compounds in a reversed-phase L C / M S application. P A H s were chosen as the analytes because they can be considered to be representative of nonpolar compounds, and P A H s are a class of compounds not easily ionized by other A P I methods [33-35]. In addition to this, a supplementary infusion experiment was performed to more closely examine the effects of different ratios of toluene to anisole in the dopant mixture. The results of this research are discussed in detail in Chapter 3 of the thesis.  Bibliography  1.  Robb, D.B., Smith, D.R., and Blades, M . W . , "Investigation of substituted-benzene dopants for charge exchange ionization o f nonpolar compounds by atmospheric pressure photoionization". Journal of the American Society of Mass Spectrometry, 2008. 19, (7), p955-963.  2.  Smith, D.R., Robb, D.B., and Blades, M . W . , "Comparison of dopants for charge exchange ionization of nonpolar polycyclic aromatic hydrocarbons with reversedphase L C - A P P I - M S " . submitted to Analytical Chemistry, 2008.  3.  Homing, E.C., Homing, M . G . , Carroll, D.I., Dzidic, I., and Stillwell, R . N . , "New picogram detection system based on a mass spectrometer with an external ionization source at atmospheric pressure". Analytical Chemistry, 1973. 12, (6), p936-943.  4.  Carroll, D.I., Dzidic, I., Stillwell, R . N . , Homing, M . G . , and Homing, B.C., "Subpicogram detection system for gas phase analysis based up on atmospheric pressure ionization (API) mass spectrometry". Analytical Chemistry, 1974. 46, (6), p706-710.  5.  Carroll, D.I., Dzidic, I., Stillwell, R . N . , Haegele, K . D . , and Homing, B . C . , "Atmospheric pressure ionization mass spectrometry: corona discharge ion source for use in liquid chromatograph-mass spectrometer-computer analytical system". Analytical Chemistry, 1975. 47, (14), p2369-2373.  6.  Ardrey, R . E . , "Liquid Chromatography-Mass Spectrometry: A n Introduction". 2003: John Wiley and Sons Ltd.  7.  Dole, M . , Mack, L . L . , Hines, R . L . , Mobley, R.C., Ferguson, L . D . , and Alice, M . B . , "Molecular beams of macroions". The Journal of Chemical Physics, 1968. 49, (5), p2240-2249.  8.  Yamashita, M . and Fenn, J.B., "Electrospray ion source. Another variation on the free-jet theme". Journal of Physical Chemistry, 1984. 88, (20), p4451-4459.  9.  Iribame, J.V. and Thomson, B . A . , "On the evaporation of small ions from charged droplets". Journal of Chemical Physics, 1976. 64, (6), p2287-2294.  10.  Thomson, B . A . and Iribame, J.V., "Field-induced ion evaporation from liquid surfaces at atmospheric pressure". Journal of Chemical Physics, 1979. 71, (11), p4451-4463.  11.  Hayen, H . and Karst, U . , "Strategies for the liquid chromatographic-mass spectrometric analysis o f non-polar compounds". Journal of Chromatography A, 2003. 1000, (1-2), p549-565.  12.  Robb, D . B . , Covey, T.R., and Bruins, A . P . , "Atmospheric pressure photoionization: A n ionization method for liquid chromatography-mass spectrometry". Analytical Chemistry, 2000. 72, (15), p3653-3659.  13.  Syage, J.A., Evans, M . D . , and Hanold, K . A . , "Photoionization mass spectrometry". American Laboratory, 2000. 32, (24), p24-29.  14.  Cai, Y . , Kingery, D . , McConnell, O., and Bach, A . C . , II, "Advantages o f atmospheric pressure photoionization mass spectrometry in support o f drug discovery". Rapid Communications in Mass Spectrometry, 2005. 19, (12), pi7171724.  15.  Kauppila, T.J., Kuuranne, T., Meurer, E . C . , Eberlin, M . N . , Kotiaho, T., and Kostiainen, R., "Atmospheric pressure photoionization mass spectrometry. Ionization mechanism and the effect o f solvent on the ionization of naphthalenes". Analytical Chemistry, 2002. 74, (21), p5470-5479.  16.  Kauppila, T.J., Kostiainen, R., and Bruins, A . P . , "Anisole, a new dopant for atmospheric pressure photoionization mass spectrometry o f low proton affinity, low ionization energy compounds". Rapid Communications in Mass Spectrometry, 2004. 18, (7), p808-815.  17.  Linstrom, P.J. and Mallard, W.G., NIST Chemistry WebBook, NIST Standard Reference Database Number 69. 2003, National Institute o f Standards and Technology.  18.  van Dam, A . and Bruins, A.P., New dopants for atmospheric pressure photoionization under reversed phase liquid chromatography conditions, in 2Ist Montreux Symposium. 2004: Montreux, Switzerland.  19.  Itoh, N . , Aoyagi, Y . , and Yarita, T., "Optimization o f the dopant for the trace determination of polycyclic aromatic hydrocarbons by liquid chromatography/dopant-assisted atmospheric-pressure photoionization/mass spectrometry". Journal of Chromatography A, 2006. 1131, (1-2), p285-288.  20.  Yang, C . and Henion, J., "Atmospheric pressure photoionization liquid chromatographic-mass spectrometric determination o f idoxifene and its metabolies in human plasma". Journal of Chromatography A, 2002. 970, (1-2), pl55-165.  21.  Hsiech, Y . , Merkle, K . , Wang, G., Brisson, J.M., and Korfmacher, W . A . , "Highperformance liquid chromatography-atmospheric pressure photoionization/tandem mass spectrometric analysis for small molecules in plasma". Analytical Chemistry, 2003. 75, (13), p3122-3127.  22.  Varga, M . , Bartok, T., and Mesterhâzy, À., "Determination o f ergosterol in Fusarium-'mfccted wheat by liquid chromatography-atmospheric pressure photoionization mass spectrometry". Journal of Chromatography A, 2006. 1003, (2), p278-283.  23.  Leinonen, A . , Kuuranne, T., and Kostiainen, R., "Liquid chromatography/mass spectrometry in anabolic steroid analysis - optimization and comparison o f three ionization techniques: electrospray ionization, atmospheic pressure chemical ionization and atmospheric pressure photoionization". Journal of Mass Spectrometry, 2002. 37, (7), p693-698.  24.  Wang, G . , Hsieh, Y . , and Korfmacher, W . A . , "Comparison o f atmospheric pressure chemical ionization, electrospray ionization and atmospheric pressure photoionization for the determination o f cyclosporin A in rat plasma". Analytical Chemistry, 2005. 77, (2), p541-548.  25.  Rauha, J.P., Vuorela, H . , and Kostiainen, R., "Effect o f eluent on the ionization efficiency o f flavonoids by ion spray, atmospheric pressure chemical ionization, and atmospheric pressure photoionization mass spectrometry". Journal of Mass Spectrometry, 200L 36, (12), pi269-1280.  26.  Rubin, H . , "Historical review: Synergistic mechanisms in carcinogenesis by polycyclic aromatic hydrocarbons and by tobacco smoke: a bio-historical perspective with updades". Carcinogenesis, 2001. 22, (12), pl903-1930.  27.  Toriba, A . and Hayakawa, K . , "Biomarkers o f exposure to polycyclic aromatic hydrocarbons and related compounds". Journal of Health Science, 2007. 53, (6), p631-638.  28.  Lev^as, J., " A i r pollution combustion emissions: Characterization o f causative agents and mechanisms associated with cancer, reproductive, and cardiovascular effects". Mutation Research, 2007. 636, (1-3), p95-133.  29.  Brody, J.G., Moysich, K . B . , Humblet, O., Attfield, K . R . , Beehler, G.P., and Rudel, R . A . , "Environmental pollutants and breast cancer: Epidemiologic studies". Cancer, 2007. 109, (12, Suppl.), p2667-2711.  30.  Impey, G . , Kieser, B . , and Alary, J.F. The analysis of polycyclic aromatic hydrocarbons (PAHs) by LC/MS/MS using a new atmospheic pressure photoionization source, in 49th ASMS Conference on Mass Spectrometry and Allied Topics. 2001. Chicago, IL.  31.  Moriwaki, H . , Ishitake, M . , Yoshikawa, S., Miyakoda, H . , and Alary, J.F., "Determination o f polycyclic aromatic hydrocarbons in sediment by liquid chromatography-atmospheric pressure photoionization-mass spectrometry". Analytical Sciences, 2004. 20, (2), p375-377.  32.  Ding, Y.S., Ashley, D.L., and Watson, C H . , "Determination o f 10 carcinogenic polycyclic aromatic hydrocarbons in mainstream cigarette smoke". Journal of Agricultural and Food Chemistry, 2007. 55, (15), p5966-5973.  33.  Airiau, C . Y . , Brereton, R . G . , and Crosby, J., "High-performance liquid chromatography/electrospray tandem mass spectrometry o f polycyclic aromatic hydrocarbons". Rapid Communications in Mass Spectrometry, 2001. 15, (2), pl35-140.  34.  van Leeuwen, S . M . , Hayen, H . , and Karst, U . , "Liquid chromatographyelectrochemistry-mass spectrometry o f polycyclic aromatic hydrocarbons". Analytical and Bioanalytical Chemistry, 2004. 378, (4), p917-925.  35.  Moriwaki, H . , "Liquid chromatographic-mass spectrometric methods for the analysis o f persistent pollutants: Polycyclic aromatic hydrocarbons, organochlorine compounds, and perfluorinated compounds". Current Organic Chemistry, 2005. 9, (9), p849-857.  CHAPTER II INVESTIGATION  OF SUBSTITUTED-BENZENE  DOPANTS FOR  CHARGE EXCHANGE IONIZATION OF NONPOLAR COMPOUNDS BY ATMOSPHERIC PRESSURE PHOTOIONIZATION*  2.1 Introduction  Atmospheric pressure photoionization (APPI) allows for the ionization of low polarity and nonpolar compounds under reversed-phase L C conditions through the use of charge exchange ionization between a dopant photoion and the analyte neutral [1-4]. A s discussed in the previous chapter, the mechanism for charge exchange ionization begins with direct photoionization of the dopant, forming the primary reagent ions. The primary reagent ions, or dopant photoions, can then undergo charge exchange with analyte neutrals i f certain conditions are met. One condition is the ionization energy (IE) of the dopant photoion must be greater than that of the analyte. In addition to this, for efficient ionization, the dopant photoions must not be consumed by other species that may be present, for example, solvents, impurities, or dopant neutrals. Consumption of the dopant photoion can lead to lower sensitivity for the analyte(s) being observed. The primary objective at this stage of the research was to identify new dopants that have potential for charge exchange ionization of nonpolar analytes under typical reversed-phase L C conditions. A secondary objective was to determine what factors affect reactivity of ' A version of this chapter has been published. Robb, D. B.; Smith, D. R.; Blades, M . W. "Investigation of substituted-benzene dopants for charge exchange ionization of nonpolar compounds by atmospheric pressure photoionization." J. Am. Soc. Mass Spectrom, 2008, 19, p955-963.  dopant photoions, and to use this information in the process of identifying potential dopant candidates.  One of the first dopants identified for APPI was toluene (IE 8.828 eV)^ and has been shown to be able to ionize compounds by charge exchange in the presence of low proton affinity solvents such as hexane or chloroform, typical normal phase L C solvents [3, 6]. Under these conditions, toluene photoions were unreactive and available for charge exchange ionization. However, in the presence of high proton affinity solvents such as methanol or acetonitrile, commonly used in reversed-phase L C , the toluene photoions were consumed making this dopant unsuitable for charge exchange. A n alternative to normal phase solvents is to reduce the flow rate of the reversed-phase solvent so that reactions with the toluene photoions are not driven to completion [1]. A second alternative is to use anisole (IE 8.2 eV) whose photoions are unreactive with reversed-phase solvents and therefore available for charge exchange ionization [2]. However, the low IE of anisole limits the compounds it can ionize by charge exchange to those o f low IE. A final option is to use either bromobenzene (IE 9.00 eV) or chlorobenzene (IE 9.07 eV), however, their photoions are only partially stable under reversed-phase conditions [7].  In this work, a broad range of substituted-benzene compounds with substituents ranging from strongly electron donating (ED) to strongly electron withdrawing (EW) were examined for their potential as APPI dopants. Two tests were performed on each compound using an APPI source and a single quadrupole mass spectrometer. Test 1 All IE data are from the NIST Chemistry WebBook, unless otherwise indicated [5]  involved introducing only the potential dopant into the source in order to isolate dopant photoions for Test 2 and to evaluate its reactivity with dopant neutrals. Test 2 introduced a 60:40 methanol/water (v/v) solvent at typical L C flow rates in addition to the dopant candidate in order to evaluate the reactivity of the dopant photoions with the solvent. Compounds whose photoions are at least partially unreactive with its neutrals and the solvent have potential to be a dopant for charge exchange ionization. A high IE is also required in order to have any possibility of improvement over anisole (currently the most effective dopant for charge exchange ionization) with respect to increasing the range of compounds amenable to APPI. The results of this experiment are presented and discussed below, along with a hypothesis on the effects of the substituents on the proton transfer reactions of the dopant photoions.  2.2 Experimental 2.2.1 Chemicals 1,3,5-trimethylbenzene, 2,4-difluoroanisole, 3-fluoroanisole, pentafluoroanisole (97+%), phenol, and (trifluoromethoxy)benzene were from Acros Organics (Morris Plains, NJ). Benzene was from E M D Chemicals (Norwood, OH). Methanol and toluene were from Fisher Scientific (Fair Lawn, NJ). Anisole, bromobenzene, fluorobenzene, nitrobenzene, and N,N-dimethylaniline were from Fluka (Switzerland). Acetophenone and biphenyl (both purities unstated) were from Matheson Coleman and Bell (Norwood, OH). 3-(trifluoromethyl)anisole, aniline, benzaldehyde, benzonitrile, hexafluorobenzene, methyl  benzene  carboxylate,  chlorobenzene,  m-xylene,  phenyl  acetate,  and  (trifluoromethyl)benzene were from Sigma-Aldrich (St. Louis, M O ) . Deionized water  was obtained from an in-house generator. A l l chemicals were of the highest purity available, > 99%, and were used as received unless otherwise indicated.  2.2.2 Instrumentation A first-generation PhotoSpray''"'^ A P P I source from M D S Sciex (Concord, Ontario, Canada) was used. The source was powered with a custom ITV supply (Electrical Services Shop, Chemistry Department, U B C ) . A lamp current of 0.760 m A was used for all experiments. The nebulizer temperature was maintained at a temperature of400°C. The flow rates o f the auxiliary and lamp gas (also known as purge gas) were set at 1.0 L min'', and the flow rate of the nebulizer gas was set at 1.9 L min''. Liquid nitrogen boil-off from Praxair (Mississauga, Ontario, Canada) was used for all the gases.  The mass spectrometer was a prototype, single-quadrupole insfrument from M D S Sciex (circa 1995) and is closely related to the A P I 100 series instruments. The PhotoSprayi""^ source was compatible with the atmosphere-vacuum interface of the mass spectrometer and was mounted without modification. The orifice plate and focusing ring voltages were set to 10 and 50 "V respectively, in order to minimize collision-induced dissociation. Each test utilized a scan range of 50-250 Da, with a step size of 0.1 Da, a dwell time o f 2 ms, and 10 scans were averaged.  The dopants and solvent were delivered separately using syringe pumps from Harvard Apparatus (Holliston, M A ) . 2.2.3 Method  Two reactivity tests were used to determine the suitability of each dopant candidate. Test 1 involved the introduction of neat dopant into the source at a flow rate of 0.2 iiL min"' in order to isolate dopant photoions and assess their reactivity with dopant neutrals. Two of the dopant candidates, phenol and biphenyl, are solids at room temperature, and were dissolved in methanol to concentrations of 1 M and 0.2 M respectively. The phenol and biphenyl solutions were delivered at flow rates of 2 and 10 laL m i n ' respectively resulting in molar flow rates approximately equivalent to that of 0.2 iiL min"' of neat benzene. The low flow rate was necessary in order to minimize impurities added to the source, since for a number of compounds, higher flow rates led to reactions with impurities which consumed the dopant photoions. Test 2 involved the introduction of 60:40 methanol:water (v/v) at 200 ^iL min"' in addition to the dopant (same flow rate as Test 1) in order to assess reactivity of dopant photoions with the solvent in typical reversed-phase conditions. Only the dopant candidates that passed Test 1 were subjected to Test 2. A dopant candidate was deemed to have passed a test i f the relative intensity o f its photoions was significant under test conditions.  2.3 Results and Discussion  A summary of the experimental results for the 25 compounds tested is presented in Table 2.1. Compounds are split into two groups: the main group consists of substitutedbenzene compounds with a wide range of E W (higher IE) and E D (lower IE) substituents, while the second group consists of fluoro-substituted anisole compounds. The two groups are listed in order o f decreasing IE. The results presented for Test 1 and Test 2 indicate  the relative peak intensities for the dopant photoion (D^*) as well as the product ions of three reactions which were observed and will be discussed below.  Table 2.1 Thermodynamic data and experimental results Test 1 Compound nitrobenzene  Substituent(s)  S2H*  123  100  93  <1  100  0  186 103  9.90 9.73  100 <1  < 1 100  < 1  39  100  -  -  -  CF, CHO COOCH3  146  9.69  100  < 1  100  0  acetophenone  -  -  benzene fluorobenzene chlorobenzene bromobenzene  (trifluorometliyl)benzene benzaldehyde methyl benzene carboxylate  F(x 6) CN  Test 2 '  MU' (Da) IE (eV)'-'' 9.94  hexafluorobenzene benzonitrile  NOj  d  106  9.50  1  3 67  136  9.32  2  100  COCH3  120 78 96 113 157  <1 100 100 100 100  100  H(x6) F Cl Br  9.28 9.24 9.20 9.07 9.00  -  2 6 3 10  2 < 1 46 31  100 20 100 100  0 100 69 22 0  CH3  92  8.83  100  5  <1  100  phenyl acetate  OCOCH3  136  8,60  < 1  100  -  -  -  m-xylene  CH3 (x 2)  106  8.55  100  10  < 1  100  0  9  toluene  phenol  OH  94  8.49 8.40  43  <1 100  0  120  100 100  100  CH3 (X 3)  43  0  anisole  OCH3  108  8.20  100  25  100  7  0  biphenyl  C5H3  154  8.16  100  48  100  3  0  aniline  NH2  93  7.72  100  21  100  < 1  0  1,3,5-trimethylbenzene  N,N-dimethylaniline pentafluoroanisole (trif]uoromethoxy)benzene 3-fluoroanisole 3-(trifluoromethyl)anisole 2,4-difluoroanisole  N(CH3)2  121  7.12  100  28  100  < 1  0  F (x 5), O C H 3  198  9.10  <1  < 1  -  -  -  OCF3  162  9.1 =  100  9  39  39  0  F, O C H 3  126  8.40  90  2  75  1  0  CF3, O C H 3  176  n.a.  100  10  100  20  0  F (x2), O C H 3  144  n.a.  100  12  100  6  0  ^All ionization energy (IE) data are from reference [5] unless otherwise stated. "n.a" means data not available. °1E from reference [18] Results presented are relative peak intensities corrected for '"'C ' "-" means Test 2 was not performed because the D*' could not be isolated in Test 1  2.3.1 Results for Substituted-Benzene Compounds Twenty substituted-benzene compounds were selected and included in the main group of compounds. In Test 1, the dopant photoion was isolated for all but five o f these compounds. Acetophenone, benzonitrile, benzaldehyde, methyl benzene carboxylate, and phenyl acetate all showed minimal signal intensity for their respective photoions. The  loss of dopant photoions appeared to be due primarily to self-protonation of the dopant (D), shown in reaction 2.1;  D^'+D  >(D-H)'+DH^  (2.1)  A n example of this is shown in Figure 2.1, which shows a mass spectrum for methyl benzene carboxylate with a base peak of 137 Da, consistent with the product ion of reaction 2.1. In addition to these five compounds, nitrobenzene also showed significant signal intensity for the protonated dopant ion, but also shows a base peak of the desired radical cation. A s such, nitrobenzene was included in the 15 compounds considered to pass Test 1.  100  Co-  80  ^  60  '35 a «  e  40  20  . 1 0 50  75  100  1—uM.  125  150  .  tl^  175  200  ,  225  1  .  250  tn/z Figure 2.1: Test 1 mass spectrum of methyl benzene carboxylate. The spectrum shows the product ion of reaction 2.1, the protonated dopant ion DH^.  O f the main group of compounds, 15 were included in Test 2 to assess the reactivity o f their photoions under typical reversed-phase conditions. A significant source  of photoion consumption was through protonation of the solvent (S), a reaction that has been observed previously with toluene [8]:  D^* +nS  >(D-H)* + S „ H ^  (2.2)  Benzonitrile, benzene, m-xylene, nitrobenzene, phenol, and toluene each had their photoions completely consumed by this reaction. Example spectra of toluene are shown in Figure 2.2.  50  75  100  125  150  m/z  175  200  225  250  50  75  100  125  150  175  200  225  250  m/z  Figure 2.2: Toluene spectra for (a) Test 1 and (b) Test 2. The Test 2 spectrum suggests reaction of the dopant photoion,  ", into the protonated methanol dimer S 2 H * , a product of reaction 2.2.  A third reaction which resulted in depletion of dopant photoions involved what could be attributed to the substitution of a halogen(s) (X) on the benzene ring for one or more methoxy groups forming a product ion, P"^':  D^'+nS  (2.3)  >nHX + P"  A n example of this is seen with hexafluorobenzene as shown in Figure 2.3. The base peak at 210 Da is consistent with the substitution of two fluorine atoms with two methoxy groups. It can also be seen that solvent protonation plays a role in consumption of the hexafluorobenzene photoions. Fluorobenzene was another compound that had its photoions consumed primarily by reaction 2.3.  100 -  a  80  F  60  / A  40  / \  20 0• • 50  f  1 .•..| 75  ^_4  L 4 ^ ' - - t - ^ -,100  125  150  175  200  m/z  ^ 225  250  50  75  100  125  150  175  200  225  250  m/z  Figure 2.3: Hexafluorobenzene spectra for (a) Test 1 and (b) Test 2. The Test 2 spectrum suggests reaction of the dopant photoion into the product ions of both reaction 2 and 3, the protonated methanol dimer, S 2 H * , and substitution product, P"', respectively.  Of  the  main group  dopants  included in Test  2,  only  bromobenzene,  chlorobenzene, and 1,3,5-trimethylbenzene had a significant signal corresponding to D"^*, and an IE greater than that of anisole. It should also be noted that significant loss of dopant photoions was seen in all three of these compounds due to solvent protonation, as well as substitution of a halogen in the cases of bromo- and chlorobenzene; this can be seen in Figure 2.4.  50  75  100  125  150  175  200  225  250  m/z  50  75  100  125  150  175  200  225  250  75  m/z  100  125  150  175  200  225  250  m/z  Figure 2.4: Test 2 spectra of (a) 1,3,5-trimethylbenzene, (b) chlorobenzene, and (c), bromobenzene. A l l spectra show the dopant photoion, D**, as well as the product ion of reaction 2.2 chlorobenzene both show the substitution product of reaction 2.3, (P**).  (S2H*).  Bromo- and  Despite this, the dominance of their respective photoions and relatively high IE compared with anisole, indicate these compounds show the properties desired for new dopant candidates for charge exchange ionization. Anisole, aniline, biphenyl, and N , N dimethylaniline all showed minimum loss of dopant photoions due to reaction 2.2, however with an IE less than or equal to that of anisole, these were only included to verify a hypothesis, developed over the course of this research.  2.5.2 Hypothesis on the Effects of Substituents on Reactivity During the course of this research, a hypothesis was developed to explain why the photoions of certain substituted-benzene compounds were being consumed while others remained stable. The hypothesis relates to the effects of substituents on the reactivity of the dopant photoion: E W substituents increase the tendency o f a substituted-benzene photoion to donate a proton to dopant and/or solvent neutrals, while E D substituents have the opposite effect, decreasing the tendency for proton donation, unless the substituent itself is acidic. This hypothesis allows for dopant candidates to be selected in a rational manner. It is also consistent with a previous study where E W substituents including F, CF3, C N , and NO2 were found to increase the acidity of the benzene ring [9] as well as a number of studies examining the tendency of E W substituents to increase reactivity of the phenyl radical [10-13].  The basis o f this hypothesis stems from the deprotonation of unsubstituted benzene radicals, which is described in a detailed study on hydration o f ionized aromatics [14]. O f importance is how adjacent C - H a-bonds on the benzene ring are either  weakened or strengthened by the retraction or addition, respectively, of electron density in the benzene ring. A weak C - H o-bond can increase the tendency of proton transfer from the benzene ring to another species due to the bond being more easily broken. Conversely, a strong C - H a-bond will make proton transfer less favourable. This explains the tendency of why E W substituents enhance reactivity through donation of a proton as well as the opposite effect of E D substituents. The exception of acidic E D substituents stems from electron donating weakening the R - H bonds in the substituent itself, causing an increase in acidity and a tendency for proton donation from the substituent rather than from the benzene ring.  In addition to affecting reactivity of mono-substituted-benzene compounds, substituents will also affect the IE o f these compounds. It is known that E W substituents will increase IE, while E D substituents will decrease IE relative to that of benzene [15, 16]. In cases o f multiple substituents, the effect appears to be additive. It is important to note that while E W substituents will increase IE, which is desired, the hypothesis predicts that there will be the undesired effect of increased reactivity. Therefore, one is forced to optimize between low reactivity and high IE by using E D substituents to offset the undesired effects of E W substituents.  2.3.3 Discussion of Results for Substituted-Benzene Compounds Starting with the results of Test 1, the hypothesis predicts that dopants with E W substituents will likely undergo proton transfer with dopant neutrals. It was observed that of the dopants that underwent significant self-protonation all but one have E W  substituents, which is consistent with the hypothesis; the one exception to this was phenyl acetate. It was also seen that not all the dopants with E W substituents underwent proton transfer. A reason why some dopants with an E W substituent self-protonate while others do not may be attributed to the dopant being substituent-protonating rather than ringprotonating due to a presence of a basic substituent. The basic substituent leads to a higher gas phase basicity that does not reflect the basicity of the benzene ring, but rather the substituent itself The E W property o f the substituent in turn weakens the C - H abonds on the benzene ring. The mechanism for self-protonation was presumed to be proton transfer from an acidic benzene ring of a dopant photoion to the basic substituent group of a dopant neutral [17]. A l l the compounds that were observed to self protonate in Test 1 were substituent-protonating with the exception of phenyl acetate. However, a reason for why phenyl acetate self-protonates could not be had due to uncertainty of whether it is a ring or substituent-protonating dopant.  Turning to the results of Test 2, nitrobenzene and (trifluoromethyl)benzene, both with E W groups had their dopant photoions consumed and a base peak of S2H^ was observed; this is consistent with the proposed hypothesis. This was also observed with benzene; the mechanism for proton transfer from benzene to solvent molecules such as water has been previously documented [14]. Before considering compounds with weakly E D substituents it should be noted that bromine and chlorine have been considered as weakly E D despite halogens being usually considered as EW.^ A s can be seen in Table  ^ The reason for classifying bromine and chlorine as ED was based on their effect on IE, resulting in chlorobenzene and bromobenzene having an IE less than that of benzene. This was attributed partly to the simultaneous donation and withdraw of electron density through the jt and a systems [18]. The removal of an electron from the n system would lower electron repulsion which could facilitate electron donation. This  2.1 as well as in Figure 2.4, while both bromo- and chlorobenzene were observed to react with the solvent forming S2H"*", the reaction was to a lesser degree than that observed with the benzene photoions. This suggests that the bromine and chlorine  substituents  strengthen the C - H a-bonds, which is what the hypothesis predicts E D substituents should do and also supports their classification as E D substituents. In the cases o f 1;,3,5trimethylbenzene, m-xylene, phenol and toluene, while having E D substituents they were observed to react significantly with the solvent. This can be explained by the substituents themselves being acidic. In the case of 1,3,5-trimethylbenzene, the reaction was observed to occur to a lesser extent than the other three dopants. This can be explained by the presence o f three E D groups which increases electron density in the benzene ring to the extent that a significant increase in electron repulsion is felt making the C - H o-bonds in the methyl groups stronger and less prone to proton transfer to a solvent neutral compared with toluene and m-xylene where there are only one and two methyl groups present, respectively.  The third reaction leading to consumption of dopant photoions, which is seen in Test 2, is the apparent substitution of a halogen(s) on the benzene ring for one or more methoxy groups. This was the primary reaction causing loss of photoions in the cases of hexafluorobenzene  and fluorobenzene  and to a lesser extent  with bromo- and  chlorobenzene. It was speculated that this was due to nucleophilic substitution, with methanol acting as the nucleophile and the halogen as the leaving group [18]. WTiile this may conflict with bromine and chlorine being considered as E D , they can also be  classification has also been done previously in a study on ionization potentials for disubstituted benzenes by DiLabio et al. where chlorine was considered as ED [16]  considered as E W due to their electronegativity which may play a role in the extent that the halogen substitution occurs; this reaction was most prominent with fluorobenzene, and least prominent with bromobenzene indicating a possible correlation between electronegativity and the extent of reactivity.  The compounds which were unreactive in both tests include aniline, anisole, biphenyl,  and N,N-dimethylaniline.  These  compounds all have non-acidic, E D  substituents, and are therefore consistent with the hypothesis. In the case of the biphenyl cation, one of the CeHs groups could be considered as an E D substituent with the sharing of delocalized electrons. The low IE o f biphenyl is also consistent with characterizing the CeHs substituent as E D . While these compounds do not make suitable dopants for charge exchange ionization due to their low IE, they support the hypothesis and can serve as a starting point for a more rational selection of dopant candidates, utilizing their stability and adding E W substituents in order to increase IE to a level more suitable for applications with APPI involving high IE analytes.  2.3.4 Results for Fluoro-substituted Anisoles Based on the hypothesis developed, five compounds predicted to be suitable candidates for charge exchange ionization were selected. This second group of compounds is listed in Table 2.1 in order of decreasing IE. The use of fluoro-substituted anisole compounds with strongly E D and E W groups were tested in order to attempt to increase IE while maintaining low reactivity with dopant and solvent neutrals. With the exception of pentafluoroanisole, each of the compounds passed Test 1. When subjected to  Test 2, (trifluoromethoxy)benzene  and 3-f!uoroanisole both showed strong signal  intensity for their respective photoions while the former showed some reactivity with the solvent; 3-fluoroanisole had minimal reactivity with the solvent. In the cases of 3(trifluoromethoxy)anisole and 2,4-difluoroanisole, the base peak was the  dopant  photoion, with minimal reaction with the solvent neutrals; their spectra are shown in Figure 2.5a and 2.5b respectively.  50  75  100  125  150  175  200  225  250  50  75  100  125  m/z  150  175  200  225  250  m/z  Figure 2.5: Test 2 spectra of (a) 3-(trifluoromethyl)anisole and (b) 2,4-difluoroanisole. Both spectra show base peaks for the dopant photoion D* and low signal for the protonated methanol dimer,  S2H*.  23.5 Discussion for Fluoro-substituted Anisoles As  shown  in  Table  2.1,  2,4-difluoroanisole,  3-fluoroanisole,  3-  (trifluoromethyl)anisole, and (trifluoromethoxy)benzene each produced base peaks of their respective photoions, showing minimal reaction with dopant neutrals. Each of these compounds have E W substituents, used to increase IE, and a strong E D substituent, predicted by the hypothesis to lower the tendency for reactivity. The results show good support for the hypothesis. However, the photoions for pentafluoroanisole could not be isolated in Test 1. It is believed that the presence of five fluorine substituents on anisole  produces a significantly larger E W effect on the benzene ring than seen with the other four compounds. This would have the effect o f significantly lowering the electron density on the benzene ring, which may attract nucleophiles that could consume its photoions.  In the case o f Test 2, when compounds were subjected to typical reversed-phase L C conditions, 2,4-difluoroanisole, 3-fluoroanisole, and 3-(trifluoromethyl)anisole were dominated by their dopant photoion peaks, lending further support for the hypothesis. (Trifluoromethoxy)benzene was found to react partially with the solvent via reaction 2.2. This would seem to indicate that the replacement of the hydrogens on the methoxy group with fluorines (considered electronegative) hinders the E D ability of the trifluoromethoxy group, making the compound more prone to reactions with solvent neutrals.  2.4 Conclusions  In this chapter, a total o f 25 substituted-benzene dopants for charge exchange ionization under typical reversed-phase L C conditions were examined. Results confirmed the potential of bromo- and chlorobenzene as suitable dopants, as well as identifying 1,3,5-trimethylbenzene, 3-(trifluoromethyl)anisole, and 2,4-difluoroanisole as possible dopant candidates. Over the course of this research, a hypothesis was developed that would allow for more rational selection of compounds to be tested as dopants. This hypothesis was well supported by the data collected, and allowed for selection of a number o f fluoro-substituted anisole compounds that were identified as potential dopants. However,  due  to  the  consumption  of  the  1,3,5-trimethylbenzene,  3-  (trifluoromethyl)anisole, and 2,4-difluoroanisole photoions at higher dopant flow rates, possibly due to the presence of impurities, the top candidates were determined to be bromo- and chlorobenzene. Due to the high IE and stability of the photoions of bromoand chlorobenzene under higher dopant flow rates and reversed-phase conditions, as well as the availability of these compounds at a high purity, they both meet the criteria for charge exchange dopants and there appears to be little benefit in further pursuing a search for new dopants. The next stage in this research was to evaluate their effectiveness in the ionization of nonpolar compounds, compared with other established dopants in a L C / M S application  Bibliography  1.  Robb, D . B . and Blades, M . W . , "Atmospheric pressure photoionization for ionization o f both polar and nonpolar compounds in reversed-phase L C / M S " . Analytical Chemistry, 2006. 78, (23), p8162-8164.  2.  Kauppila, T.J., Kostiainen, R., and Bruins, A . P . , "Anisole, a new dopant for atmospheric pressure photoionization mass spectrometry of low proton affinity, low ionization energy compounds". Rapid Communications in Mass Spectrometry, 2004. 18, (7), p808-815.  3.  Kauppila, T.J., Kuuranne, T., Meurer, B . C . , Eberlin, M . N . , Kotiaho, T., and Kostiainen, R., "Atmospheric pressure photoionization mass spectrometry. Ionization mechanism and the effect o f solvent on the ionization of naphthalenes". Analytical Chemistry, 2002. 74, (21), p5470-5479.  4.  Impey, G . , Kieser, B . , and Alary, J.F. The analysis of polycyclic aromatic hydrocarbons (PAHs) by LC/MS/MS using a new atmospheic pressure photoionization source, in 49th ASMS Conference on Mass Spectrometry and Allied Topics. 2001. Chicago, IL.  5.  Linstrom, P.J. and Mallard, W.G., NIST Chemistry WebBook, NIST Standard Reference Database Number 69. 2003, National Institute o f Standards and Technology.  6.  MuUer, A . , Mickel, M . , Geyer, R., Ringseis, R., Eder, K . , and Steinhart, H . , "Identification of conjugated linoleic acid elongation and p-oxidation products by coupled silver-ion H P L C APPI-MS". Journal of Chromatography B, 2006. 837, (l-2),pl47-152.  7.  van Dam, A . and Bruins, A.P., New dopants for atmospheric pressure photoionization under reversed phase liquid chromatography conditions, in 21st Montreux Symposium. 2004: Montreux, Switzerland.  8.  Robb, D . B . and Blades, M . W . , "Effects of solvent flow, dopant flow, and lamp current on dopant-assisted atmospheric pressure photoionization (DA-APPI) for L C - M S . Ionization via proton transfer". Journal of the American Society of Mass Spectrometry, 2005. 16, (8), pl275-1290.  9.  Meot-Ner, M . and Kafafi, S.A., "Carbon Acidities o f Aromatic Compounds". Journal of the American Chemical Society, 1988. 110, (19), p6297-6303.  10.  Ramirez-Arizmendi, L . E . , Guler, L . , Joseph, F.J., Thoen, K . K . , and Kenttâmma, H.I., "Hydrogen atom abstraction and addition reactions o f charged phenyl radicals with aromatic substrates in the gas phase". International Journal of Mass Spectrometry, 2001. 210/211, (1-3), p511-520.  11.  Tichy, S.E., Thoen, K . K . , Price, J . M . , Ferra, J.J., Jr, Petucci, C.J., and Kenttamma, H.I., "Polarity of the transition state controls the reactivity of related charged phenyl radicals toward atom and group donors". Journal of Organic Chemistry, 2001. 66, (8), p2726-2733.  12.  Heidbrink, J.L., Thoen, K . K . , and Kenttamma, H.I., "Polar effects on iodine atom abstraction by charged phenyl radicals". Journal of Organic Chemistry, 2000. 65, (3), p645-651.  13.  Heidbrink, J.L., Ramirez-Arizmendi, L . E . , Thoen, K . K . , Guler, L . , and Kenttamma, H.I., "Polar effects control hydrogen-abstaction reactions of charged, substituted phenyl radicals". Journal of Physical Chemistry A, 2001. 105, (33), p7875-7884.  14.  Ibrahim, Y . M . , Meot-Ner, M . , Alshraeh, E . H . , Samy El-Shall, M . , and Scheiner, S., "Stepwise hydration o f ionized aromatics. Energies, structures of the hydrated benzene cation, and the mechanism of deprotonation reactions". Journal of the American Chemical Society, 2005. 127, (19), p7053-7064.  15.  Crable, G.F. and Keams, G . L . , "Effect o f substituent groups on the ionization potentials of benzenes". Journal of Physical Chemistry, 1962. 66, p436-439.  16.  DiLabio, G . A . , Pratt, D . A . , and Wright, J.S., "Theoretical calculation o f ionization potentials for disubstituted benzenes: Additivity vs non-additivity of substituent effects". Journal of Organic Chemistry, 2000. 65, (7), p2195-2203.  17.  Lau, Y . K . and Kebarle, P., "Substituent effects on the intrinsic basicity of benzene: proton affinities o f substituted benzenes". Journal of the American Chemical Society, 1976. 98, (23), p7452-7453.  18.  Robb, D.B., Smith, D.R., and Blades, M . W . , "Investigation of substituted-benzene dopants for charge exchange ionization of nonpolar compounds by atmospheric pressure photoionization". Journal of the American Society of Mass Spectrometry, 2008. 19,(7), p955-963.  CHAPTER III COMPARISON IONIZATION  OF OF  DOPANTS NONPOLAR  FOR  CHARGE  EXCHANGE  POLYCYCLIC  AROMATIC  HYDROCARBONS WITH REVERSED-PHASE L C - A P P I - M S '  3.1 Introduction  A significant benefit of atmospheric pressure photoionization (APPI) compared with atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) is its ability to effectively ionize both low polarity and nonpolar compounds [1, 2]. The use of a dopant allows for the ionization of these compounds in both normal phase [3] and reversed-phase chromatographic conditions [2, 4, 5]. In the initial stage of this research (see Chapter 2), a number o f substituted-benzene compounds were tested to assess their potential as dopants for APPI with the goal of identifying a dopant that will increase the range o f compounds that amenable to A P P I . Two compounds, 2,4difluoroanisole ( D F A ) and 3-(trifluoromethyl)anisole ( T F M A ) , were selected from the previous study as top dopant candidates for A P P I and used in the next stage in this research, evaluating their efficiency for ionizing low polarity and nonpolar compounds under reversed-phase conditions. The dopant candidates, (trifluoromethoxy)benzene and 3-fluoroanisole were excluded due to partial reactivity with the solvent; 1,3,5trimethylbenzene was also excluded due to poor ionization efficiency [6]. T F M A and ' A version of this chapter has been submitted for publication. Smith, D. R.;Robb, D. B.; Blades, M . W. "Comparison of dopants for charge exchange ionization of nonpolar polycyclic aromatic hydrocarbons with reversed-phase LC-APPI-MS." submitted to J. Am. Soc. Mass Spectrom.,  D F A were compared against currently established dopants for their efficiency in ionization of a representative sample of polycyclic aromatic hydrocarbons (PAHs).  PAHs  were chosen as analytes  for this investigation because they  are  characteristic of nonpolar compounds in general and are not easily ionized by other A P I methods. In addition, the analysis of P A H s is of importance as they have been considered by the international scientific community to be a potential threat to human health; these compounds have known toxicity and carcinogenic effects, as well as being contributors to air pollution [7-10]. O f the P A H s selected for testing, all 16 are included on the U S A Environmental Protection Agency (EPA) list of priority-pollutant chemicals [11], and 12 are included on the Canadian Priority Substances List 1 [12, 13]. Currently there are a number o f methods employed for the analysis of P A H s . O f these, the most prevalent are gas chromatography (GC) combined with electron impact ionization mass spectrometry [14, 15], and liquid chromatography (LC) combined with either fluorescence or mass spectrometry with various A P I methods [16-18]. A significant advantage of L C over G C is its ability to fully resolve all 16 P A H s included in the E P A list of priority pollutants. However, the use of ESI or A P C I - L C / M S , as mentioned previously, is not well suited for the ionization of P A H s due to their inefficient ionization of low and nonpolar compounds. Through the use of a dopant, APPI can offer a more effective method for ionizing these classes of compounds.  Although the majority of analytes should in principle, be able to be ionized by direct photoionization, it has been well established in a number of publications that the  ionization efficiency of direct photoionization is extremely poor [2, 5, 19-21]. A more effective method of ionization, utilizes charge exchange ionization involving ionmolecule reactions between the dopant photoion and analyte. This raises an important requirement for the dopants to be efficient: the dopant photoions must not react with solvent, dopant neutrals, or trace impurities which may be present. This can result in the loss of photoions, which will, in turn, lower the overall ionization efficiency. Toluene was one of the original dopants identified by Robb et al. and its photoions have been found to react with reversed-phase  solvents such as methanol or acetonitrile at  conventional L C solvent flow rates (>200 [iL min'') making it unsuitable for charge exchange ionization. In fact, toluene has been found to promote proton transfer ionization under typical reversed-phase conditions through the formation of protonated solvent ions [22]. Recently, anisole has been identified as an alternative to toluene because its photoions are stable in the presence of methanol and acetonitrile and therefore available for charge exchange ionization [5]. In addition, anisole was found to give improved analyte signal intensity compared with toluene for most compounds tested; however this was limited to compounds o f low ionization energy (IE). This raises an additional requirement for charge exchange: the primary reagent ion must have an IE significantly above that of the analyte. The relatively low IE o f anisole (IE 8.20 eV)'^ limits the range of compounds amenable to charge exchange using anisole as the dopant. A s an example, naphthalene (IE 8.14 eV) is a compound which anisole cannot efficiently ionize.  In an effort to improve ionization efficiency of nonpolar compounds, Itoh et al. investigated various toluene/anisole solutions and tested their efficiency in ionizing a ^ All IE data are from the NIST Chemistry WebBook, unless otherwise indicated [22].  sample o f the 16 P A H s using the E P A standard reference solution [20]. The optimal ratio of toluene/anisole was identified as 99.5:0.5 v/v (0.5% anisole). B y using a dopant mixture, direct photoionization will occur primarily with the bulk dopant. The dopant which is diluted can be ionized by charge exchange ionization with the photoions of the bulk dopant. The analyte can then be ionized by the ions of both dopants. This can be used in cases where properties o f two dopants are desired, or to dilute impurities which may be present in one dopant. For the majority of the P A H s tested, using 0.5% anisole as the dopant resulted in slightly lower peak areas for the M"^ ' ion of the P A H s compared with neat anisole, although still significantly higher than the signal attained using neat toluene. Exceptions to this were naphthalene, acenaphthylene, fluorene, phenanthrene, and fluoranthene. In the case of naphthalene, a compound not effectively ionized by anisole, the analyte signal intensity when using 0.5% anisole was found to be comparable to the intensity observed when using toluene as dopant, while for the other compounds, the difference was more significant. Although the 0.5% anisole solution is an overall improvement compared with the neat dopants, it is nevertheless limited by the reactivity of toluene photoions and the low IE of anisole, and as a result, sacrifices the signal strength o f a number of P A H s in order to ionize all 16 P A H s .  Bromo- and chlorobenzene have been examined as A P P I dopants for reversedphase L C conditions, using testosterone as an analyte, and found to be more effective in charge exchange ionization than anisole; this was attributed to the higher IE of bromoand chlorobenzene [24]. These two compounds showed significant potential as dopants  for charge exchange ionization due to their high IE of 9.07 and 9.00 eV respectively but are limited by the partial stability of their photoions under reversed-phase conditions.  From the initial research presented in chapter two, the fluoro-substituted anisole compounds, D F A and T F M A , were identified as potential candidates for promoting charge exchange ionization under reversed-phase L C conditions. A t solvent flow rates of 200 jdL min'' and low dopant flow rates o f 0.2 [iL min'', the spectra of D F A and T F M A were dominated by their respective photoions with only minimal peaks due to ionmolecule reactions with the solvent (see Figure 2.5); these two compounds have yet to be tested in any L C / M S application. Advantages o f these fluoro-substituted anisole compounds include stability in reversed-phase conditions at low flow rates and a suspected high IE.  The second stage o f this research examined the ionization efficiency of D F A and T F M A for charge exchange ionization under reversed-phase conditions, using the 16 P A H s included in the E P A standard reference solution as representative analytes for low polarity compounds. The ionization efficiency o f the D F A and T F M A were compared with previously established dopants. Experimental tests were performed for each dopant, using a reversed-phase, gradient elufion, and a commercial A P P I source with selected ion monitoring (SIM) using a triple-quadrupole mass spectrometer. O f importance is the ability of the dopant to efficiently ionize analytes with high IE, which would broaden the range of compounds that can be analyzed by this method. The results of this experiment are presented and discussed below. In addition to these tests, supplementary infusion  experiments were performed to fLirther examine the effect o f the toluene/anisole ratio on ionization efficiency of the P A H s .  3.2 Experimental 3.2.1 Chemicals For the main L C / M S experiments, a standard reference solution o f 16 PAHs (EPA 610  Polynuclear Aromatic Hydrocarbons M i x , Cat. N o . 4S8743) from Supelco  (Bellefonte, P A ) was used. The solution was diluted lOOOx in 60:40 methanol/water (v/v); the P A H s are listed in alphabetical order in Table 3.1 along with their molecular weights (MW), IE, and final concentration. For the supplementary infusion experiments, individual 500 p,g mL"' stock solutions of pyrene, benzo[a]pyrene,  naphthalene,  acenaphthylene, fluorene, phenanthrene, and fluoranthene were prepared in hexanes; sample solutions at 5 fig m L ' ' were prepared from each o f the stock solutions with 90:10 methanol/water (v/v), with exception of naphthalene, which was diluted to 50 ng mL"' as a higher concentration was required due to poor ionization. The individual P A H s were from Sigma-Aldrich (St. Louis, M O ) , except for pyrene which was from Fluka (Buchs, Switzerland). The dopants investigated were D F A from Acros Organics (Morris Plains, NJ), bromobenzene and anisole from Fluka, chlorobenzene and T F M A from SigmaAldrich (St. Louis, MO), and toluene from Fisher Scientific (Fair Lawn, NJ). D F A and T F M A were not used neat because when used neat at normal flow rates, their photoions were consumed, due to what is believed to be reactions with impurities present in the chemical. Therefore D F A and T F M A were delivered in the form of dilute bromo- and chlorobenzene solutions (this will be discussed further in the Results and Discussion).  Solvents used were H P L C grade acetonitrile, methanol, and hexanes from Fisher Scientific and deionized water from an in-house generator. Apart from the dilutions, all chemicals were used as received.  Table 3.1 Molecular weight, concentration, and ionization energy of each P A H examined Compound acenaphthene acenaphthylene anthracene benzo[a Janthracene benzo[a]pyrene benzo[6 ]fluoranthene benzolghi ]perylene benzo[Â: Jfluoranthene chrysene dibenzo[a, h Janthracene fluoranthene fluorene indeno[ 1,2,3-câf Jpyrene naphthalene phenanthrene pyrene  MW(Da) Conc.(ng/mL) lECeV)'" 154 1.0 7.75 152 2.0 -8.1 0.1 7.44 178 228 0.1 7.45 252 0.1 7.12 252 0.2 n.a. 276 0.2 7.17 252 0.1 n.a. 228 0.1 7.60 278 0.2 7.39 202 0.2 7.90 0.2 166 7.91 276 0.1 n.a. 128 1.0 8.14 178 0.1 7.89 202 0.1 7.43  "n.a." means data not available All ionization energy (IE) data are from reference [22]  3.2.2 Instrumentation The L C experiments were performed using an 1100 series Hewlett Packard (Santa Clara, C A ) H P L C with an Inertsil ODS-P, 250 mm x 2.1 mm L D . , 5 |im column, from G L Sciences Inc. (Tokyo, Japan). The L C experiments were performed at room temperature using 90:10 methanol/water (v/v) (solvent A ) , and acetonitrile (solvent B) with a two step gradient elution, shown in Table 3.2.  Table 3.2 Gradient timetable Time (min) 0:00 1:00 7:00 11:00 42:00  %A 100 100 60 0 0  %B 0 0 40 100 100  The flow rate was held constant at 200 | i L min" for the duration of the run. A n 18 minute flush at 100% A was performed before each run to re-equilibrate the column and flush out the L C system. A 10 [iL volume of sample was injected using the integrated injection valve on the mass spectrometer. For the supplementary infusion experiments, 90:10 methanol/water (v/v) (190 ^ L min'') and the analyte solution (10 \iL min"') were delivered separately with syringe pumps from Harvard Apparatus (Holliston, M A ) . The analyte solution and solvent were combined in a tee prior to mixing with the dopant. For all experiments, the dopant was delivered at 20 \iL min"' using an integrated syringe pump on the mass spectrometer. The dopant was combined with the solvent before entering the ion source using a tee (post-column for the L C experiments) in order to avoid a suspected carry-over problem with the dopant port of PhotoSpray''"'^; the dopant port was plugged during the experiments.  The A P P I source used was a prototype PhotoSpray'^'^ source (identical to the commercially available source) used with an A P I 3200 Series triple-quadrupole mass spectrometer from Applied Biosystems/MDS Sciex (Concord, Ontario, Canada). For all experiments the probe's heated nebulizer temperature was maintained at 400 °C, and the ion transfer voltage was set to 700 V . The nebulizer and lamp gas were set at 60 and 20 psi respectively. The orifice potential (declustering potential) was set at 10 V , a potential  lower than is normally used, in order to minimize in-source fragmentation of any protonated P A H s . The lower orifice potential was necessary to ensure that any analyte cations detected were the result of charge exchange ionization, and not the result of proton transfer ionization of the analyte followed by deprotonation due to a high orifice potential, which would promote fragmentation. For the L C / M S experiments, a Q l Multiple Ion scan was used. Masses scanned were 128, 152, 154, 166, 178, 202, 228, 252, 276, and 278 Da, which correspond to the M"^* ions for the P A H s examined. The dwell time for each mass was set to 100 ns. For the supplementary infusion experiments, all scan parameters were the same, except Q l Single Ion scans were performed with a dwell time of 200 ns for a period o f 1 minute.  3.2.3 Method Each dopant was evaluated for its ability to ionize each of the 16 P A H compounds in the analyte mixture. Because the aim of the experiment was to compare sensitivities attained with each dopant, it was necessary to compare absolute peak intensity, requiring the monitoring o f intra- and interday drift in the response of the instrument. A s such, the dopants were divided into multiple groups: (1) toluene, anisole, and toluene/anisole; (2) chlorobenzene, chlorobenzene/TFMA, and chlorobenzene/DFA; (3) bromobenzene, bromobenzene/TFMA,  and  bromobenzene/DFA; and  an  additional  group  (4)  toluene/anisole, chlorobenzene and bromobenzene for monitoring of interday drift. Each of the dopant mixtures were at 99.5:0.5 (v/v). One group of dopants was tested each day. Intraday drift was monitored by obtaining a chromatogram for each dopant in the group, and then the series was repeated for a total of three chromatograms for each dopant.  obtained at different times throughout the day. Toluene was an exception as its chromatograms were obtained three times in a row at the start of the day due to the long time required to eliminate anisole residue in the system, and the large effect anisole has on response. In addition to the L C / M S experiments, supplementary infusion experiments were performed to examine the effect o f the toluene/anisole ratio on the response of select P A H s .  3.3 Results and Discussion A l l 16 P A H s were fully resolved with retention times ranging from approximately 5 to 40 minutes. A n example chromatogram of the P A H mixture is shown in Figure 3.1.  0.5 0.4  4-  0.3  o  3-  h  i  0.2  m  0.1  S 2  H Il  0.0  25  P  0  " A  f\ A 35  30  1f I h 0 0  10  i Ji  A  1  A  m ll  n  20  n 40  30  Retention Time (min.) Figure 3.1 Chromatogram of the 16 PAHs (M"') with bromobenzene/DFA (99.5:0.5 v/v) as dopant, a) naphthalene; b) acenaphthylene; c) acenaphthene; d) fluorene; e) phenanthrene; f) anthracene; g) fluoranthene;  h)  pyrene;  i)  benzo[a]anthracene;  j)  chrysene;  k)  benzo[b]fluoranthene;  1)  benzo[k]fluoranthene; m) benzo[a]pyrene; n) dibenzo[a,/i]anthracene; o) indeno[l,2,3-cJ]pyrene; and p) benzo[g, A, /] pery lene.  The results for the three dopant groups tested, in addition to direct photoionization (no dopant) are presented in Table 3.3. It can be seen that significant improvement in the signal strength o f naphthalene and acenaphthylene was achieved using either the group 2 or group 3 dopants compared with the previously established dopants in group 1.  Group 1 Dopants  Naphthalene Acenaphthylene Fiuorene Fluoranthene Phenanthrene Acenaphthene Chrysene Benzo[a]anthracene Anthracene Pyrene Benzo[b]fluoranthene Benzo[k]fluoranthene Benzola]pyrene Dibenzo[a,h]anthracene Indeno[l,2,3-cd]pyrene BenzofK,h,ilpervlene  0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0%  3% 4% 3% 2% 2% 1% 1% 1% 1% 2% 3% 3% 3% 3% 3% 3%  0% 1% 66% 6% 8% 100% 18% 100% 100% 100% 90% 100% 100% 100% 100% 100%  4% 42% 60% 47% 46% 42% 43% 37% 45% 57% 75% 68% 69% 68% 69% 70%  85% 82% 74% 65% 60% 59% 58% 51% 48% 50% 56% 51% 54% 49% 50% 50%  Group 3 Dopants  Group 2 Oopants  100% 94% 89% 85% 83% 82% 85% 78% 73% 77% 83% 73% 80% 73% 74% 72%  89% 100% 100% 100% 100% 98% 100% 90% 80% 81% 85% 75% 78% 71% 72% 73%  81% 81% 73% 71% 69% 73% 76% 71% 75% 81% 86% 77% 81% 74% 75% 72%  95% 93% 88% 88% 88% 97% 95% 89% 89% 92% 100% 88% 92% 84% 84% 84%  79% 94% 93% 92% 95% 92% 97% 87% 82% 82% 85% 75% 78% 70% 70% 72%  Table 3.3 Average relative peak areas for the PAHs examined. PAHs are order in increasing IE.  As an example, extracted ion chromatograms (XIC) for naphthalene (128 m/z) are presented in Figure 3.2, presented in order o f relative peak intensity for each of the dopants tested.  ;^  / /  /  #  / /  Jf  if-  I  //  1  [1  as  11  20  0  11 1  V  1  v_  1  ^  1  Figure 3.2 XIC for naphthalene (M*'), listed in order of relative peak intensity  The remaining P A H s displayed less dramatic improvements in sensitivity using any of the dopant candidates compared with established dopants. Direct A P P I was ineffective for all analytes tested. Regarding intraday drift, results showed the standard deviation of the peak area to be quite low ranging from 4% to <1%; the interday drift, determined from Group 4 showed up to a 10% difference in peak area from results obtained when the dopant was tested in its original group. Therefore comparisons within the groups will be more certain than those between groups. The results for the remaining dopant groups are presented and discussed in fiirther detail below along with details of the supplementary infiision experiments performed.  3.3.1 Discussion of Group 1 Dopants and Supplementary Infusion Experiments The first set of dopants consisted of toluene, anisole, and 0.5% anisole. The relative peak areas for each analyte, ordered by decreasing IE, are listed in Table 3.3. It was observed that the analytes could be grouped into three categories based on their  response to these dopants; these categories appear to correlate well with the IE of the analytes. The first category is comprised o f only naphthalene (IE 8.14 eV), which has the highest IE of all the analytes, and gave no response with anisole as the dopant, and comparable responses with either toluene or 0.5% anisole. The second category is comprised of acenaphthylene (IE -8.1 eV), fluorene (IE 7.91 eV), fluoranthene (IE 7.90 eV), and phenanthrene (IE 7.89 eV), all of which showed a significant increase in response when using the 0.5% anisole dopant compared with either toluene or anisole. The remaining 11 P A H s make up the final category and all have an IE less than or equal to 7.75 e V ^ These P A H s have the lowest IE and showed the strongest response when using anisole as dopant and the weakest response with toluene.  To more fully evaluate the effect of the ratio o f toluene to anisole, infusion experiments were performed using a series of toluene/anisole solutions with ratios ranging from 100:0 to 0:100 (v/v). A l l the P A H s in the first and second category were included; pyrene and benzo[a]pyrene were chosen to represent the 11 P A H s comprising the lower IE P A H s . The results of this experiment are shown in Figure 3.3.  The IE of benzo[b]anthracene, benzo[k]fluoranthene, and indeno[l,2,3-cd]pyrene were not available, but show results that are similar to PAHs of known, low IE.  P e r c e n t A n i s o l e (%)  Percent A n i s o l e (%)  Figure 3.3 Relative signal intensity for supplementary infusion experiments. Toluene:anisole solutions tested consisted of 100:0, 99.99:0.01, 99.98:0.02, 99.95:0.05, 99.9:0.1, 99.8:0.2, 99.5:0.5, 99:1, 98:2, 95:5, 90:10, 80:20, 50:50, and 0:100 (v/v). (a) Relative signal intensity for pyrene and benzo[a]pyrene, representative of low IE PAHs. (b) Relative signal intensity for the high and mid IE PAHs and (c) data from (b), plotted on a logarithmic scale for clarity.  As seen in Figure 3.3, there is an optimal ratio of toluene to anisole depending on which analyte is being ionized. A s seen in Figure 3.3a, pyrene and benzo[a]pyrene rapidly reach a maximum signal intensity and begin to plateau as the proportion o f anisole in the dopant increases. This is a behaviour that is undoubtedly different for the remaining PAHs, as shown in Figure 3.3b. In the cases of the higher IE PAHs, the optimal signal strength is quickly achieved, and is followed by a continuous drop in signal (a more significant drop for naphthalene and acenaphthylene) as the proportion of anisole in the  dopant increases. This is seen more clearly in Figure 3.3c, which shows the data from Figure 3.3b plotted using a logarithmic scale on the x-axis.  The results for pyrene and benzo [a]pyrene, which represent the lower IE PAHs, are well supported by previous study by Kauppila et al. (2004) which demonstrated the higher efficiency of anisole for low IE compounds compared with toluene. The results for naphthalene, acenaphthylene, fluorene, fluoranthene, and phenanthrene, the five P A H s with the highest IE, on the other hand, show an optimal ratio of toluene:anisole, after which, the addition of anisole results in lower signal intensity. A t the two extremes, 0% anisole (ie neat toluene), and 100% anisole, the results are still consistent with the study by Kauppila et al. (2004). Naphthalene and acenaphthylene, both with a high IE, show poor ionization efficiency when using neat anisole as a dopant compared with neat toluene.  A t slightly  lower IE, fluorene  (7.91 eV),  fluoranthene  (7.90eV), and  phenanthrene (7.89eV) all show significantly improved ionization efficiency with the use of anisole. When looking at the use of a toluene/anisole solution, an apparent correlation can be seen between the amount of anisole required for optimal ionization efficiency and IE of the analyte. Naphthalene, with the highest IE, achieves optimal ionization when using 0.1% anisole, whereas phenanthrene, with the lowest IE of the five P A H s achieves optimal ionization with 5% anisole. Acenaphthylene, fluorene, and fluoranthene fit this trend well, as can be seen in Figure 3.3c.  A possible explanation for the behaviour observed in the supplementary infusion experiments involves the presence of an impurity'* in anisole and the ion-molecule reactions that can occur as a result. The increase in analyte intensity observed as the proportion o f anisole in the dopant is increased follows from anisole being a more effective dopant than toluene. A drop in signal intensity of the analyte would indicate a loss of the primary reagent ions, in this case either toluene and/or anisole photoions. The amount of toluene photoions present is being lowered due to reactivity with the solvent, as well as less toluene present in the dopant mixture. The loss of anisole photoions could be the result of charge exchange with an impurity (I) with an IE < 8.2 eV:  [anisole]^* +1 ^ [anisole] + T '  (3.1)  The concentration of impurities would increase as the amount of anisole is increased, thereby driving reaction 3.1 and reducing the number of anisole photoions available for charge exchange ionization with an analyte. Although this reaction reduces the number of primary photoions, the impurity ions formed as a result could ionize an analyte i f the IE of the impurity is greater than that of the analyte; the impurity would effectively be acting as a dopant:  r*+M^M^*+I  (3.2)  It seems likely that if an impurity was present and is responsible, it is more likely to be an artifact of the synthesis/manufacturing of anisole rather than contamination, since this behaviour was also observed in the study by Itoh et al. and the anisole was supplied by different chemical companies (this assumes that the same method for producing anisole is used).  The results of the higher IE PAHs shown in Figure 3.2b are consistent with this hypothesis. The high IE of these PAHs could make the theimodynamics for reaction 3.2 unfavourable and therefore the reagent ions (anisole) that can be used for charge exchange ionization would decrease as the percentage o f impurities in the dopant mixture is increased, consuming the anisole photoions. At the very low percentages of anisole, there are simply insufficient amounts of primary reagent ions for efficient ionization; the ionization efficiency would approach that o f toluene as the percentage of anisole is lowered. The shift in the optimal levels of anisole can be explained by the observation that in general, the greater the difference in IE between the anisole and the analyte, the greater the ionization efficiency. A s such, the higher IE compounds such as naphthalene or acenaphthylene could experience a greater effect from consumption of anisole photoions. In addition, specifically for naphthalene and acenaphthylene, the ionization would be primarily driven by toluene since toluene was shown to be a more efficient dopant for these specific analytes; increasing the percentage o f anisole results in a decrease of toluene, thereby significantly reducing reagent ions formed by toluene. In the cases of pyrene and benzo[a]pyrene, compounds representing the low IE PAHs, the plateau can be explained by the occurrence of reaction 3.2. While anisole photoions would be consumed by charge transfer with the impurity, the impurity, in turn, can undergo charge transfer with the analyte, negating the initial effect.  This would suggest that the IE of the impurity would be in the range of 7.89 eV (the lowest IE o f the five P A H s showing an optimal ratio o f toluene to anisole) and 7.75 eV (the highest IE o f the compounds showing highest efficiency with neat anisole). A  possible impurity was observed in the mass spectrum at 122 Da. A s the percentage of anisole increased, the signal intensity at 122 D a also increased, nearing the levels where the detector was saturated with ions. A possible impurity would be a methyl anisole compound, which has a molecular weight o f 122 g mol''. In particular, 2-methylanisole has an IE o f 7.9 eV, which can meet the proposed IE requirements and has the appropriate molecular weight. However, more evidence would have to be collected in order to support this hypothesis.  Returning to the overall efficiency of the Group 1 dopants, the results have been discussed thoroughly by Itoh et al. and will not be discussed further [20]. In general, of the two neat dopants, only toluene was able to ionize all 16 PAHs, albeit relatively inefficiently; anisole did offer significant improvement in sensitivity for all the P A H s tested except for naphthalene and acenaphthylene. This was resolved by Itoh et al. by combining toluene and anisole into a mixture of 0.5% anisole, which was empirically determined to be the optimal dopant mixture. These results were confirmed by the data obtained this study.  3.3.2 Discussion of Group 2 Dopants The second group of dopants was comprised of bromobenzene, and D F A and T F M A , both diluted to 0.5% with bromobenzene. The reason for not using either D F A or T F M A in their neat form was due to loss of their respective dopant photoions vv-hen the dopants were used at normal dopant flow rates (20 |aL min"'); this was believed to be due to reactions with impurities in the dopant. The dopant was therefore diluted using another  dopant, similar to what was done with toluene and anisole. The dopants were diluted using bromobenzene as it had a high IE and would not only generate its own photoions, but could also ionize the dopant by charge exchange. It was believed that this could enhance the ionization efficiency when compared with neat bromobenzene. This was also done with chlorobenzene since it has a similar IE to bromobenzene and make up the group 3 dopants. The dilution of 0.5% was not optimized and was based on the optimal ratio of toluene to anisole.  The results for the Group 2 dopants are shown in Table 3.3. In each case, all compounds were effectively ionized and there is significant improvement in the ionization efficiency of the higher IE PAHs compared with the Group 1 dopants. The higher IE of these dopants allowed for more efficient charge exchange ionization of analytes with higher IE. There was minor loss in sensitivity observed for the P A H s with a low IE when comparing the Group 3 dopants with anisole. Analyte peak areas dropped to values as low as 48% o f the peak area obtained with anisole. This indicates that there is a point beyond which increased difference in IE between the dopant and the analyte no longer leads to improved charge exchange between the dopant photoions and analyte neutrals.  Both the D F A and T F M A mixtures showed improvement in ionization efficiency compared with neat bromobenzene. However, the minor improvement in sensitivity was attained at the expense of method complexity involving preparation of the dopant mixture. This brings into question whether or not the higher sensitivity attainable with  either dopant mixture warrants the extra time required for dopant preparation, when neat bromobenzene can be used without any further preparation. In addition, the use o f either D F A or T F M A introduces additional ions that can cause interference with isobaric compounds, which, depending on the application, may or may not be an issue. If extra sensitivity is required, either dopant mixture could be considered depending on the application. Overall, bromobenzene/DFA was considered to be the best dopant for charge exchange, giving strong ionization efficiency across the entire range of P A H s .  3.3.3 Discussion of Group 3 Dopants The third group of dopants tested included neat chlorobenzene, and D F A and T F M A each diluted to 0.5% with chlorobenzene. Results of for this group are shown on Table 3.3 and were similar with those of the Group 2 dopants. Each dopant was able to efficiently ionize all 16 P A H s including those of high IE. Chlorobenzene showed minor improvement over bromobenzene, and of the two would appear to be the optimal dopant for charge exchange ionization. However, the significant difference in m/z between bromo- and chlorobenzene, allows bromobenzene to be used in place of chlorobenzene in the analysis of compounds that are isobaric with chlorobenzene, without significant loss in analyte sensitivity.  In the cases of the fluoro-substituted anisole mixtures, slight improvement over the neat chlorobenzene dopant was observed. Either mixture could therefore be used for improved sensitivity if the additional preparation time and the presence of either the D F A or  TFMA  cations  are  not  an  issue.  O f the  three  dopants  in this  group.  chlorobenzene/TFMA shows the best overall ionization efficiency for all the P A H s . It is important to note that since the fluoro-substituted anisole mixtures were diluted to 0.5% based on the toluene/anisole mixture rather than through optimization, it may be possible that the sensitivity could be improved fiirther by altering the dopant ratios. If significant improvement in sensitivity can be attained, it could justify the additional preparation time required for the dopant mixtures.  3.4 Conclusions  The potential of bromo-and chlorobenzene, and four dopant mixtures as dopant candidates for the promotion of charge exchange ionization for low polarity and nonpolar analytes was explored and compared against currently established dopants. The relative efficiency in ionizing a representative sample of 16 P A H s in typical reversed-phase L C conditions was used to evaluate the different dopants. O f the dopants tested, bromo- and chlorobenzene showed the best overall performance and ease o f use (compounds can be used with out further preparation) compared with other dopants and dopant mixtures. Their high IE and availability at high purity make them promising dopants for other applications  involving  charge  exchange  ionization.  Addifionally,  bromo-  and  chlorobenzene could be substituted for one another in order to eliminate interference with isobaric compounds. The attempt to improve sensitivity by mixing either bromo- or chlorobenzene  with  one  of the  fluoro-substituted  anisole  compounds  showed  bromobenzene/DFA to be the best overall dopant, but minor improvement was obtained at the expense of increased preparation time as well as the introduction of an additional  compound that increases complexity of the mass spectra. Use o f either bromo- or chlorobenzene is likely to provide sufficient ionization efficiency, and therefore, pursuing further research into the optimization of these mixtures would appear to be of limited benefit.  3.S Bibliography  1.  Hayen, H . and Karst, U . , "Strategies for the liquid chromatographic-mass spectrometric analysis o f non-polar compounds". Journal of Chromatography A, 2003. 1000, (1-2), p549-565.  2.  Cai, Y . , Kingery, D . , McConnell, O., and Bach, A . C . , II, "Advantages o f atmospheric pressure photoionization mass spectrometry in support of drug discovery". Rapid Communications in Mass Spectrometry, 2005. 19, (12), pl7171724.  3.  Kauppila, T.J., Kuuranne, T., Meurer, E.G., Eberlin, M . N . , Kotiaho, T., and Kostiainen, R., "Atmospheric pressure photoionization mass spectrometry. Ionization mechanism and the effect of solvent on the ionization of naphthalenes". Analytical Chemistry, 2002. 74, (21), p5470-5479.  4.  Hsiech, Y . , Merkle, K . , Wang, G., Brisson, J.M., and Korfmacher, W.A., "Highperformance liquid chromatography-atmospheric pressure photoionization/tandem mass spectrometric analysis for small molecules in plasma". Analytical Chemistry, 2003. 75, (13), p3122-3127.  5.  Kauppila, T.J., Kostiainen, R., and Bruins, A . P . , "Anisole, a new dopant for atmospheric pressure photoionization mass spectrometry o f low proton affinity, low ionization energy compounds". Rapid Communications in Mass Spectrometry, 2004. 18, (7), p808-815.  6.  Smith, D.R., The investigation of compounds as potential dopants in dopant assisted - atmospheric pressure photoionization mass spectrometry, in Department of Chemistry. 2005, University of British Columbia: Vancouver.  7.  Rubin, H . , "Historical review: Synergistic mechanisms in carcinogenesis by polycyclic aromatic hydrocarbons and by tobacco smoke: a bio-historical perspective with updades". Carcinogenesis, 2001. 22, (12), pl903-1930.  8.  Brody, J.G., Moysich, K . B . , Humblet, O., Attfield, K . R . , Beehler, G.P., and Rudel, R . A . , "Environmental pollutants and breast cancer: Epidemiologic studies". Cancer, 2007. 109, (12, Suppl.), p2667-2711.  9.  Lewtas, J., " A i r pollution combustion emissions: Characterization o f causative agents and mechanisms associated with cancer, reproductive, and cardiovascular effects". Mutation Research, 2007. 636, (1-3), p95-133.  10.  Toriba, A . and Hayakawa, K . , "Biomarkers o f exposure to polycyclic aromatic hydrocarbons and related compounds". Journal of Health Science, 2007. 53, (6), p631-638.  11.  United States Environmental Protection Agency. Priority chemicals and chemical fact sheets. 2007 [cited 2008 January 18]; Available from: http://www.epa.gov/wastemin/chemlist.htm.  12.  Environment Canada. Canadian Environmental Protection Act Environmental Registry - Priority Substances List L 1994 [cited 2008 January 21]; Available from: http://ec.gc.ca/CEPARegistry/subs_list/Priority.cfm.  13.  Environment Canada and Health Canada, Canadian Environmental Protection Act: Priority Substances List Assessment Report - Polycyclic Aromatic Hydrocarbons. 1994, Minister of Supply and Services Canada.  14.  Veyrand, B., Brosseaud, A . , Ludovic, S., Vincent, V . , Monteau, F., Marchand, P., Andre, F., and le Bizec, B . , "Innovative method for determination o f 19 polycyclic aromatic hydrocarbons in food and oil samples using gas chromatography coupled to tandem mass spectrometry based on an isotope dilution approach". Journal of Chromatography A, 2007. 1149, (2), p333-344.  15.  Wang, W., Meng, B . , L u , X . , L i u , Y . , and Tao, S., "Extraction of polycyclic aromatic hydrocarbons and organochlorine pesticides from soils: A comparison between Soxhlet extraction, microwave-assisted extraction and accelerated solvent extraction techniques". Analytica ChimicaActa, 2007. 602, (2), p211-222.  16.  Moriwaki, H . , Ishitake, M . , Yoshikawa, S., Miyakoda, H . , and Alary, J.F., "Determination of polycyclic aromatic hydrocarbons in sediment by liquid chromatography-atmospheric pressure photo ionization-mass spectrometry". Analytical Sciences, 2004. 20, (2), p375-377.  17.  Moriwaki, H . , "Liquid chromatographic-mass spectrometric methods for the analysis of persistent pollutants: Polycyclic aromatic hydrocarbons, organochlorine compounds, and perfluorinated compounds". Current Organic Chemistry, 2005. 9, (9), p849-857.  18.  Lien, G.W., Chen, C . Y . , and Wu, C F . , "Analysis of polycyclic aromatic hydrocarbons by liquid chromatography/tandem mass spectrometry using atmospheric pressure chemical ionization or electrospray ionization with tropylium post-column derivatization". Rapid Communications in Mass Spectrometry, 2007. 21, (22), p3694-3700.  19.  Robb, D . B . , Covey, T.R., and Bruins, A . P . , "Atmospheric pressure photoionization: A n ionization method for liquid chromatography-mass spectrometry". Analytical Chemistry, 2000. 72, (15), p3653-3659.  20.  Itoh, N . , Aoyagi, Y . , and Yarita, T., "Optimization of the dopant for the trace determination of polycyclic aromatic hydrocarbons by liquid chromatography/dopant-assisted atmospheric-pressure photoionization/mass spectrometry". Journal of Chromatography A, 2006. 1131, (1-2), p285-288.  21.  Varga, M . , Bartok, T., and Mesterhâzy, Â., "Determination o f ergosterol in Fusarium-'mfected wheat by liquid chromatography-atmospheric pressure photoionization mass spectrometry". Journal of Chromatography A, 2006. 1003, (2), p278-283.  22.  Robb, D . B . and Blades, M . W . , "Effects o f solvent flow, dopant flow, and lamp current on dopant-assisted atmospheric pressure photoionization (DA-APPI) for L C - M S . Ionization via proton transfer". Journal of the American Society of Mass Spectrometry, 2005. 16, (8), pi275-1290.  23.  Linstrom, P.J. and Mallard, W.G., NIST Chemistry WebBook, NIST Standard Reference Database Number 69. 2003, National Institute o f Standards and Technology.  24.  van Dam, A . and Bruins, A.P., New dopants for atmospheric pressure photoionization under reversed phase liquid chromatography conditions, in 21st Montreux Symposium. 2004: Montreux, Switzerland.  CHAPTER IV CONCLUSIONS  AND  RECOMMENDATIONS  FOR  FUTURE  RESEARCH  Atmospheric pressure photoionization (APPI) using a dopant allows for the analysis of both polar and nonpolar compounds by L C / M S [1, 2]. O f interest was the analysis of nonpolar compounds, a class of compounds that is not efficiently ionized by either atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) [3]. Nonpolar compounds can be ionized using APPI through charge exchange ionization with the dopant photoions. Under normal phase conditions toluene can be employed as a dopant for charge exchange [4, 5]. However, when using reversed-phase solvents, the use of toluene for charge exchange ionization is limited to low solvent flow rates such as those employed in capillary L C methods [6]. A n alternative to toluene is anisole, which is unreactive with reversed-phase solvents. However, the low ionization energy (IE) of anisole limits the ionization process to low IE analytes. The underlying objective of this thesis was to increase the breadth of analytes amenable to APPI, and to improve ionization efficiency of compounds that can already be analyzed by A P P I . This was achieved by identifying new dopant candidates, requiring a high IE and low reactivity of the dopant photoions with reversed-phase solvents. Successful candidates were compared with current dopants in the analysis of a standard mixture of polycyclic aromatic hydrocarbons (PAHs) using L C / M S .  The search for new dopant candidates began with reactivity tests on a wide range of substituted-benzene compounds, with substituents ranging from strongly electron donating (ED) to strongly electron withdrawing (EW). The results of this test showed that both the tendency o f a dopant's photoions to be consumed through proton transfer reactions and its IE were related to the E D and E W properties of its substituents. It was found that E D groups would decrease reactivity and IE, while E W groups increased reactivity and IE; an exception to this was i f the E D group was acidic. This observation allowed for a more rational selection o f dopant candidates. Bromo- and chlorobenzene were confirmed as having strong potential for use as charge exchange ionization dopants, both having a good compromise between low reactivity while maintaining a high IE. A number of fluoro-substituted anisoles, where E W fluoro groups were used to counteract the E D methoxy group, also showed potential as dopants for A P P I . O f the fluorosubstituted anisoles tested, 2,4-difluoroanisole ( D F A ) and 3-(trifluoromethyl)anisole ( T F M A ) showed the greatest potential as dopant candidates.  With the identification of D F A and T F M A , as well as confirmation o f bromobenzene and chlorobenzene as dopant candidates, the next stage o f this research was a comparison o f these compounds with currently established dopants. P A H s were chosen as analytes since they are both characteristic of nonpolar compounds in general and are not easily ionized by either A P C I or ESI. A comparison of the ionization efficiency of the dopants and dopant mixtures tested indicated the best overall for ionizing the P A H s was bromobenzene/DFA (99.5:0.5 v/v), which was able to efficiency ionize all 16 P A H s and showing a greater ionization efficiency than toluene/anisole  (99.5:0.5 v/v). A n alternate dopant that could also ionize all PAHs, but at slightly lower efficiency, would be either bromo- or chlorobenzene. Bromo- and chlorobenzene also have the benefit of being available commercially in high purity form, and not requiring any additional preparation prior to its use as a dopant; these dopants are also easily substituted for one another when dealing with isobaric compounds. While the solutions involving D F A and T F M A were greater in overall efficiency then bromo- and chlorobenzene, they have a disadvantage of requiring preparation of the dopant, as well the introduction of additional ions that can cause interference with isobaric compounds. Results of the supplementary infusion experiments performed indicate that for higher IE PAHs there is an optimal ratio of toluene to anisole which depends on the IE of the P A H . This was hypothesized to be caused by the presence of an impurity in anisole, which consumes the anisole photoions, however does not affect lower IE P A H s due to charge transfer between the impurity ions and the P A H . A potential impurity was identified at 122 Da, and could be a methyl anisole, specifically 2-methylanisole, which has an appropriate IE at 7.9 eV. However, this was not proven and further research would have to be done to confirm this hypothesis.  It has been shown that both bromo-and chlorobenzene can be used for charge exchange ionization of nonpolar compounds and could be used as general-purpose dopants for this type o f application. Attempts to improve ionization efficiency by mixing fluoroanisoles with bromo- or chlorobenzene resulted in limited improvement, and research into the optimization of the mixtures may be of minor benefit. In addition, there may be limited benefit in pursuing further discovery of new dopants considering the  efficiency of bromo- and chlorobenzene. Future research into APPI would involve demonstrating the potential o f these novel dopants as well as further improving the ionization efficiency of A P P I in general. With regards to demonstrating the capabilities of APPI, research should entail an examination of the ionization efficiency of bromo- and chlorobenzene with different compound classes as well as with compounds of higher IE in order to assess their use as general-purpose dopants. This can be done in conjunction with a comparison o f A P P I against A P C I and ESI and would be beneficial in determining the relative efficiency o f these three ionization methods when using these new dopants with APPI.  4.1 Bibliography  1.  Robb, D . B . , Covey, T.R., and Bruins, A . P . , "Atmospheric pressure photoionization: A n ionization method for liquid chromatography-mass spectrometry". Analytical Chemistry, 2000. 72, (15), p3653-3659.  2.  Cai, Y . , Kingery, D . , McConnell, O., and Bach, A . C . , II, "Advantages o f atmospheric pressure photoionization mass spectrometry in support o f drug discovery". Rapid Communications in Mass Spectrometry, 2005. 19, (12), p i 7171724.  3.  Hayen, H . and Karst, U . , "Strategies for the liquid chromatographic-mass spectrometric analysis o f non-polar compounds". Journal of Chromatography A, 2003. 1000,(1-2), p549-565.  4.  Kauppila, T.J., Kuuranne, T., Meurer, E . C . , Eberlin, M . N . , Kotiaho, T., and Kostiainen, R., "Atmospheric pressure photoionization mass spectrometry. Ionization mechanism and the effect o f solvent on the ionization of naphthalenes". Analytical Chemistry, 2002. 74, (21), p5470-5479.  5.  Muller, A . , Mickel, M . , Geyer, R., Ringseis, R., Eder, K . , and Steinhart, H . , "Identification o f conjugated linoleic acid elongation and P-oxidation products by coupled silver-ion H P L C APPI-MS". Journal of Chromatography B, 2006. 837, (l-2),pl47-152.  6.  Robb, D . B . and Blades, M . W . , "Atmospheric pressure photoionization for ionization of both polar and nonpolar compounds in reversed-phase L C / M S " . Analytical Chemistry, 2006. 78, (23), p8162-8164.  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.24.1-0061683/manifest

Comment

Related Items