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Enantiomeric separations of natural compounds and metal complexes and the detection of anions using positive mode electrospray ionization mass spectrometry

Dissertation
Author: Molly Melissa Warnke
Abstract:
This dissertation focuses on two different areas of analysis: liquid chromatography (LC) enantiomeric separations and the detection of anions using electrospray ionization mass spectrometry (ESI-MS) and LC-ESI-MS. Enantiomeric separations of two distinct classes of chiral compounds were investigated. The first class is pterocarpans, which are isoflavanoids with cis -fused benzopyran benzofuranyl structures. In the reverse phase mode of operation, all pterocarpan enantiomers could be separated using cyclodextrin based chiral stationary phases (CSPs) with hydroxypropyl-β-cyclodextrin, acetyl-β-cyclodextrin, and γ-cyclodextrin showing the broadest enantioselectivity. Two macrocyclic glycopeptide CSPs, ristocetin A and vancomycin also proved useful in the reverse phase mode. Not as many separations were achieved in the normal phase mode for either set of CSPs. Chiral extended metal atom chains (EMACs) were first synthesized by F.A. Cotton and co-workers as the smallest possible molecular wires. It was not possible to resolve these enantiomers by crystallization and derivatization was impossible. The vancomycin macrocyclic glycopeptide stationary phase proved to be the best approach for separating these unusual chiral entities. Partial or baseline separation of 5 helical EMAC racemates was achieved in the polar organic mode or normal phase mode chromatography. In chapter 4, vibrational circular dichroism (VCD) is used to assign the absolute configuration of Ni 3 (dipyridylamine) 4 Cl 2 . Negative mode ESI-MS of anions can be problematic due to spray stability and background noise issues. The use of a positively charged reagent which pairs with the anion allows for the detection of anions in positive mode ESI-MS. Singly charged anions can be paired with dicationic reagents, while doubly charged anions can be paired with tricationic reagents to result in an overall +1 charged complex. In this dissertation, a variety of linear tricationic reagents were examined to determine which structural features are important for anion detection. The application of linear tricationic reagents and previously reported trigonal tricationic reagents were then applied to the detection of a larger variety of divalent anions (e.g. disulfonates, dicarboxylates) and bisphosphonates, which are a class of drug used to treat bone diseases. The use of MS-MS and LC-ESI-MS are also discussed.

TABLE OF CONTENTS

ABSTRACT ………………………………………………………………………………..

iv

CHAPTER 1. GENERAL INTRODUCTION …………………………………………...

1

1.1

Thesis o rganization……………………………………………………………. 1 1.2

Enantiomeric separations ………………………………………………………. 1 1.3

Macrocyclic chiral s tationary p hases …………………………………………. 2 1.4

Vibrational c ircular d ichroism ………………………………………………… 5 1.5

Analysis of anions using mass spectrometry …………………………………… 6 1.6

References ……………………………………………………………………... 11

CHAPTER 2. USE OF NATIVE AND DERIVATIZED CYCLODEXTRIN BASED AND MACROCYCLIC GLYCOPEPTIDE BASED CHIRAL STATIONARY PHASES FOR THE ENANTIOSEPARATION OF PTEROCARPANS BY HPLC …………… 19

Abstract ……………………………………………………………………………. 19

2.1 Introduction…………………………………………………………………… 20

2.2 Experimental …………………………………………………………………... 22

2.2.1 Materials …………………………………………………………….. 22

2.2.2 Equipment …………………………………………………………… 22

2.2.3 Calculations …………………………………………………………. 23

2.3 Results and Discussion…………………………………………………………23

2.4 Conclusions ……………………………………………………………………. 26

2.5 Acknowledgements ……………………………………………………………. 27

2.6 References ……………………………………………………………………... 27

CHAPTER 3. ENANTIOMERIC SEPARATION OF EXTENDED METAL ATOM CHAIN COMPLEXES: UNIQUE COMPOUNDS OF EXTRAORDIN ARILY HIGH SPECIFIC ROTATION …………………………………………………………………. 37

Abstract ……………………………………………………………………………. 37

3.1 Introduction……………………………………………………………………. 37

3.2 Materials and Methods ………………………………………………………… 39

3.3 Results and Discussion…………………………………………………………41

3.4 Conclusion……………………………………………………………………...43

3.5 Acknowledgements ……………………………………………………………. 44

3.6 References ……………………………………………………………………... 44

CHAPTER 4. RESOLUTION OF ENANTIOMERS IN SOLUTION AND DETERMINATION OF THE CHIRALITY OF EXTENDED METAL ATOM CHAIN S …………………………………………………………………………………... 53

Abstract ……………………………………………………………………………. 53

4.1 Communication………………………………………………………………... 53

4.2 Acknowledgement …………………………………………………………….. 57

iii

CHAPTER 5. EVALUATION OF FLEXIBLE LINEAR TRICATIONI C SALTS AS GAS - PHASE ION - PAIRING REAGENTS FOR

THE DETECTION OF DIVALENT ANIONS IN POSITIVE M ODE ESI - MS ………………………………………………. 66

Abstract ……………………………………………………………………………. 66

5.1 Introduction……………………………………………………………………. 67

5.2 Experimental …………………………………………………………………... 69

5.3 Discussion……………………………………………………………………... 70

5.4 Conclusions ……………………………………………………………………. 76

5.5 References ……………………………………………………………………... 76

CHAPTER 6. THE EVALUATION AND COMPARISON OF TRIGONAL AND LINEAR TRICATIONIC I ON - PAIRING REAGENTS FOR

THE DETECTION

OF ANIONS I N POSITIVE MODE ESI - MS …………………………………………... 84

Abstract ……………………………………………………………………………. 84

6.1 Introduction……………………………………………………………………. 85

6.2 Experimental …………………………………………………………………... 87

6.3 Results and Discussion…………………………………………………………88

6.4 Conclusions ……………………………………………………………………. 96

6.5 Acknowledgements ……………………………………………………………. 97

6.6 References ……………………………………………………………………... 97

CHAPTER 7. POSITIVE MODE ELECTROSPRAY IONIZATION MA SS SPECTROMETRY OF BISP HOSPHONATES USING DI CATIONIC AND TRICATIONIC ION - PAIRING AGENTS …………………………………………… 112

Abstract …………………………………………………………………………... 112

7.1 Introduction…………………………………………………………………... 113

7.2 Experimental …………………………………………………………………. 114

7.3 Results and Discussion………………………………………………………..116

7.4 Conclusions …………………………………………………………………... 122

7.5 References ……………………………………………………………………. 123

CHAPTER 8. GENERAL CONCLUSIONS ………………………………………….. 132

ACKNOWLEDGEMENTS ……………………………………………………………..136

APPENDIX A …………………………………………………………………………… 137

APPENDIX B …………………………………………………………………………… 149

iv

ABSTRACT

This dissertation focuses on two different areas of analysis: liquid chromatography (LC) enantiomeric separations and the detection of anions using electrospray ionization mass spectrometry (ESI - MS) and LC - ESI - MS.

Enantiomeric separations of two distinct classes of chiral compounds were investigated. The first class is pterocarpans, which are isoflavanoids with cis - fused benzopyran benzofuranyl structures. In the reverse phase mode of operation, all pterocarpan enantiomers could be se parated

using cyclodextrin based chiral stationary phases (CSPs) with hydroxypropyl - β - cyclodextrin, acetyl - β - cyclodextrin, and γ - cyclodextrin showing the broadest enantioselectivity. Two macrocyclic glycopeptide CSPs, ristocetin A and vancomycin also prove d useful in the reverse phase mode. Not as many separations were achieved in

the normal phase mode for either set of CSP s .

Chiral

extended metal atom chains (EMACs) were first synthesized by F.A. Cotton and co- workers as the smallest possible molecular wir es. It was not possible to resolve these enantiomers by crystallization and derivatization was impossible. T he vancomycin macrocyclic glycopeptide stationary phase proved to be the best approach for separating these unusual chiral entities . Partial or base line

separation of 5 helical EMAC racemates

was achieved in the polar organic mode or normal phase mode chromatography. In chapter 4, vibrational circular dichroism (VCD) is used to assign the absolute configuration of Ni 3 (dipyridylamine) 4 Cl 2 .

Negative mod e ESI - MS of anions can be problematic due to spray stability and background noise issues. The use of a positively charged reagent which pairs with the anion allows for the detection of anions in positive mode ESI - MS. Singly charged anions can be

v

paired wit h dicationic reagents, while doubly charged anions can be paired with tricationic reagents to result in an overall +1 charged complex. In this dissertation, a variety of linear tricationic reagents were examined to determine which structural features are i mportant for anion detection. The application of linear tricationic reagents and previously reported trigonal tricationic reagents were then applied to the detection of a larger variety of divalent anions (e.g. disulfonates, dicarboxylates) and bisphosphon ates, which are a class of drug used to treat bone diseases. The use of MS - MS and LC - ESI - MS are also discussed.

1

CHAPTER 1

INTRODUCTION

1.1

THESIS ORGANIZATION

High performance liquid chromatography (HPLC) and electrospray ionization mass spectrometr y (ESI - MS) are two v ery important methods used in analytical laboratories . This dissertation presents research in two areas: enantioselective

HPLC separations/applications and the ESI - MS analysis of anions in the positive mode. This introduction presents a

brief overview of both areas. It is followed by six chapters, each on a manuscript either published or submitted for publication. The final chapter presents the general conclusions from both research areas.

1.2 ENANTIOMERIC

SEPARATIONS

The resolution of e nantiomers is very important, especially in the pharmaceutical industry. Although enantiomers have identical chemical and physical properties in an achiral environment, they can have different pharmacological, toxicological, metabolic, and pharmacokinetic properties within the chiral environment of biological systems. In 1992, due to advances in enantiomeric LC separations, the Food and Drug Administration (FDA) issued guidelines for the development of sterioisomeric drugs . If a drug is to be developed as a

racemate, the effects of each single enantiomer and the racemate must be determined [2]. Enantiomeric

separations are also important for evaluating the products of asymmetric synthes e s and the evaluation of the enantiomeric composition of naturally occurr ing molecules, which are of increasing interest as new drugs or new drug leads [3].

Analytical techniques such as gas chromatography (GC), HPLC, supercritical fluid chromatography (SFC), and capillary electrophoresis (CE) are routinely used for

2

enantiomer ic separations [4]. HPLC is used most often in industry because it is robust and offers good reproducibility. HPLC and SFC are the favored techniques for preparative scale separations. There are over 100 chiral stationary phases (CSP) available commercial ly. The most important classes based on structure are macrocyclic, pi - pi association, and polymeric CSPs [5]. Macrocyclic CSPs will be discussed in the following section.

1.3 MACROCYCLIC CHIRAL S TATIONARY P HASES

Macrocyclic CSPs include three groups of chi ral selectors: chiral crown ethers, cyclodextrins, and macrocyclic glycopeptides. The cyclodextrin- based chiral selectors account for a vast majority of GC and CE enantioseparations. Cyclodextrins are also important HPLC CSPs, especially in reverse phase a nd polar organic mode s . Cyclodextrins and macrocyclic glycopeptides stationary phases will be discussed in the sections below.

1.3.1 Cyclodextrin based CSPs

Native α, β, and γ cyclodextrins are macrocyclic compounds formed from 6, 7, or 8 α - 1,4- linked D - glucose units respectively. The shape of a cyclodextrin is like a hollow, truncated cone (Fig. 1)

[4]. This cavity size increases as the n umber of glucose units increases . The interior cavity of the cyclodextrin is hydrophobic while the exterior rim s

are

hydrophilic. Inclusion complexes form in aqueous or hydro- organic solutions when nonpolar molecules or nonpolar moieties are attracted more strongly to the hydrophobic interior of the cyclodextrin than to the mobile phase [6,7].

The first successful cyclode xtrin based CSP involved binding cyclodextrin to silica gel via an ether linkage and was introduced by Armstrong [7]. This CSP was the first that could be used in reverse phase mode and separated many compounds. Later studies led to a more thorough underst anding of separation mechanisms [6,8]. A minimum of three points of

3

interaction are required for chiral recognition, with cyclodextrins and any other CSP. In the reverse phase mode, an inclusion complex must be formed between the analyte and the cavity. Al so, the chiral center of the molecule should be positioned near the exterior rim of the cyclodextrin so that interactions between the analyte and the mouth of the cyclodextrin are possible. These interactions include hydrogen bonding, dipolar, and steric i nteractions. For chiral discrimination, at least one of these interactions needs to be different for each enantiomer. Derivatized cyclodextrin based CSPs offer additional sites for interactions leading to chiral recognition. The hydroxypropyl - β - cyclodextrin (Cyclobond I 2000 RSP) has been shown to separate compounds not separated on the native β - cyclodextrin CSP [9]. Cyclodextrins derivatized with aromatic groups are effective for separations in the normal phase mode [10,11]. Nonpolar solvent molecules occupy the cyclodextrin cavity in the normal phase mode, therefore π - π interactions, dipole stacking, and hydrogen bonding interactions are important for chiral recognition [ 10] . Polar organic mode chromatography, where the mobile phase consists of mainly acetonitrile can also be used with cyclodextrin CSPs. In this mode, the solvent occupies the cyclodextrin cavity and the analyte resides on top of the chiral selector, so that hydrogen bonding is maximized [12].

1.3.2 Macrocyclic Glycopeptides

Macrocyclic glycopeptide based CSPs have also been shown to separate a wide variety of chiral compounds [13]. The commercially available CSPs are those based on the macrocyclic glycopeptide antibiotics vancomycin, ristocetin A, teicoplanin, and teicoplanin a glycone. All of these chiral selectors have similar peptide backbone s , multiple stereogenic centers, and functionalities such as carboxylic acids, amines, sugar moieties, and aromatic

4

rings [14]. The teicoplanin aglycone is the only chiral selector without saccharide groups attached. Along with a variety of functional groups, these chiral selectors have a secondary structure in the form of a twisted “C” shaped basket that is relatively non- polar [15,16]. Interactions between an analyte molecule and the func tional groups or hydrophobic cavity of the macrocyclic glycopeptide based CSP can lead to enantioselectivity.

The macrocyclic glycopeptide CSPs can be used in all mobile phase modes: reverse phase, normal phase, and polar organic [17]. In the

reverse phase mode, electrostatic interactions and hydrophobic interactions are thought to be the most important for chiral recognition [13]. In the normal phase mode, the polar functional groups and aromatic rings of the CSP provide the interactions needed for both r etention and chiral recognition. The dominant analyte - CSP interactions include hydrogen bonding, pi - pi interactions, dipole stacking, steric repulsion and sometimes electrostatic interactions. In the polar organic mode, the dominant interactions between th e analyte and CSP are hydrogen bonding, electrostatic, dipolar, and steric interactions [18]. Due to the dominance of different types of interactions in these three chromatographic modes, chiral recognition can vary dramatically. Hence, very different type s of chiral molecules can be separated in one mode vs. another. Method development with macrocyclic glycopeptides is often simplified by the fact that the stationary phases are complementary. If a partial separation is obtained on one stationary phase, it is likely the analyte

can be baseline separated on a related glycopeptide CSP [17,19]. Chapters 2 and 3 focus on the use of macrocyclic glycopeptide and cyclodextrin based CSPs for the separation of natural product analogs and chir al metal complexes.

5

1.4 VIBRATIONAL

CIRCULAR DICHROISM

Just as the separation of chiral molecules is necessary in many fields, structural characterization of enantiomers is also important. Absolute configuration and conformations of a chiral molecule are important factors in determining pharmaceutical activity [ 3 ]. Circular dichroism and optical rotation are two well known and highly used methods for absolute configuration determination [20,21], but they rely on the comparison of the measured rotation sign to related compounds of known absolute configuration. Incorrect assignments can be made, even with the number of useful correlations, rules, or procedures available [22 ]. X - ray crystallography can be used for the determination of absolute configuration. This method requires a

single crystal of sufficient size and quality, which can be difficult to obtain. Vibrational circular dichroism (VCD) circumvents these problems by correlating an optical activity measurement to accurate quantum mechanical calculations, which leads to the

direct determination of the absolute configuration of a molecule. For traditional CD, electronic transitions are probed in the visible spectral region. The same principles are applied in VCD, but in the mid- IR region with fundamental vibrational frequenci es being probed [23 ].

VCD was discovered in the 1970s [24,25 ] and instrumentation has been commercially available since 1997 [22]. For assignation of absolute configuration by VCD, experimental VCD and IR spectr a

are measured using an instrumental config uration as shown in Figure 2. Next, the vibrational absorption and CD spectra must be predicted using quantum mechanical programs. A widely used theoretical level is the density functional method with the B3LYP functional and 6- 31G* basis set [25]. For a m olecule with a single conformation, the starting geometry can be obtained in a straight - forward

manner through a molecule - building

6

program. Either of the two possible absolute configurations is arbitrarily chosen as a starting point and the geometry is fur ther optimized to obtain the minimum energy geometry. Vibrational absorption and CD intensities for all vibrational bands are then calculated for the chosen absolute configuration. A spectrum can be predicted based on the calculated band positions and inte nsities. If the predicted VCD band signs match the experimentally observed VCD band signs, then the absolute configuration of the experimentally investigated molecule is assigned the configuration of the enantiomer used in the calculations [2 5 ]. If the pre dicted signs and experimental signs are opposite, then the opposite absolute configuration is assigned. Figure 3 shows the comparison of the experimental VCD spectrum and the predicted spectrum for both enantiomers of (+) - 3- chloro - 1- butyne. It is clear tha t the VCD signs of the experimental spectrum match that of the (R) - enantiomer, hence (+) - 3- chloro- butyne is (R) - 3- chloro - butyne. The VCD

spectrum

of molecules that can exist as multiple - conformers is

more complicated as the relative populations of conforme rs must be taken into account for the calculations. A population- weighted VCD spectrum is generated from individual conformer calculations and compared to the experimentally observed VCD spectrum to assign absolute configuration, as for single - conformer mo lecules [2 5]. Chapter 4 is a report of the use of VCD to assign the absolute configuration to the enantiomers of one of the metal complexes

(Ni 3 (dipyridylamine) 4 Cl 2 ) separated in Chapter 3.

1.5 ANALYSIS OF ANIONS USING MASS SPECTROMETRY

Anion analysis is important in many areas of study, especially involving environmental samples, biological tissues and fluids, and foods and beverages. Separation techniques are often employed with such complex matrices to separate ions of interest from potential interferen ces in the matrix. Ion chromatography and capillary electrophoresis are

7

the most common separation methods used for ion analysis. Reverse phase mode chromatography can be used if the anion is sufficiently hydrophobic. Ion selective electrodes have also bee n used for anion detection. Most anions have little UV absorbance and unless derivatized, direct detection with a UV detector can be difficult.

Conductivity detection is used frequently for ion chromatography detection because it is a universal detector [2 6 ]. Analytes are detected when a difference in conductance between the ion and the background electrolyte is measured. Direct detection occurs when the conductance of the analyte is larger than that of the background, while

when

analyte conductance le ss th an that of the background indirect detection

must be used . Lower concentrations and conductivity of the background electrolyte lead to lower baseline noise, which improves detection sensitivity. Because of this, mo st conductivity detection is

performed usi ng background suppression. In this method the background electrolytes such as sodium hydroxide or sodium carbonate are converted to species of low conductance, like water or H 2 CO 3 , by cation exchange of the counterion for hydrogen ion . Suppression technology has been thoroughly reviewed [27,28 ]. Conductivity detection is a universal and useful method for anion detection, but it does not offer any structural information and lower limits of detection are required for some analyses.

Ion- selective electrodes have also been used for ion chromatography detection [ 29- 31 ]. These electrodes detect selected analytes, even in complex matrices, however the use of ion- selective electrodes with ion chromatography is not very common. Because of the high selectivity of th e ion- selective electrode, the information obtained in conjunction with a separation technique might be the same as that obtained by flow injection analysis [26]. In recent years, improvements in sensitivity and limits of detection have been made with ion-

8

selective electrodes [3 2], however they are most often used without a separation technique and in non- sample limited applications.

The use of mass spectrometry as a detection method for anion analysis is growing in popularity. The mass spectrometer can di scriminate between anionic species and/or provide structural information about the analytes, providing a second dimension of analysis. Inductively coupled plasma (ICP) and atmospheric pressure ionization methods (API), most often ESI, are m ethods of major importance for detection in ion chromatography. ICP interfaces are compatible with traditional ion chromatography flow rates (1 mL/min) and can detect several elements with great sensitivity [3 3 ,3 4 ]. ICP - MS is a very popular method for detecting metallic a nd halogenated species [35- 4 5 ]. While ICP is useful for el emental analysis, the high temperature used leads to the complete destruction of the analyte, ruling out any other structural information. Identification of unknown analytes is difficult using ICP, however API techniques, ESI and atmospheric pressure chemical ionization (APCI) can be used to elucidate structural and identity information of analytes from the resulting mass spectrum.

ESI is a useful ionization technique for many classes of analytes. Briefly, in electros pray ionization, liquid flow (effl uent) is pumped through a capillary which has an applied voltage ( + 2- 6 kV). This current flow

creates charge separation at the surface of the liquid, thereby producing a “Taylor cone” protruding from th e capillary tip. Droplets that contain an excess of charge (positive or negative depending on the polarity of the capillary voltage) will then detach from the end of the Taylor cone

(Fig. 4) . These droplets eventually yield “naked” ions for analysis by mass spectrometry via one of two generally accepted mechanisms [4 6, 47]: the charge residue model (CRM) and the ion evaporation model (IEM).

9

More detailed descriptions of the electrospray ionization process can be found elsewhere [4 6- 48 ].

The inherent negat ive charge of anions would seem to make the use of negative mode ESI - MS detection

an obvious choice . While negative mode ESI is the most straightforward method for detecting an ions, there are some drawbacks. The n egative ion mode is more prone to corona di scharge than the positive ion mode. Corona discharge is an electrical discharge resulting from the ionization of a fluid surrounding a conductor. In ESI, corona discharge occurs when the high concentration of electrons on the capillary lead to ionization o f molecules around the capillary. The large quantity of ionized molecules leads to significant background interferences and poor spray stability [48 ]. If corona discharge persists, it can lead to arcing, which not only leads to a reduction in spray current , but can also damage the electrical components of the instrument [4 8 ]. Yamashita and Fenn noticed that the onset of arcing occurred at lower applied potentials in negative ion mode than in positive ion mode [49].

Corona discharge can be reduced by the us e of electron - scavenging gases [5 0,51 ] and/or halogenated solvents [52- 54 ]. Halogenated solvents have a high relative electron affinity and can thus “capture” electrons, which reduces or eliminates corona discharge and increases spray stability. Butanol [5 1] and 2- propanol [48 ] have also been recommended for use as LC - MS solvents when negative mode is used. These solvents have higher proton affinities than traditional LC so lvents like water and methanol. Electron scavenging gases, such as oxygen or SF 6 , wor k in a similar manner. Commercial instruments today often use nitrogen from liquid N 2

dewars or generators which makes adding high electron affinity gases more difficult. Along with the difficulties of using electron scavenging gases, the

10

halogenated solve nts and alcohol modifiers referred to above are not commonly used in reverse phase chromatography or ion chromatography. A sensitive, positive mode ESI - MS method to determine anions using common HPLC solvents would be very beneficial. Recently, a method w as developed to detect singly charged anions using positive mode ESI - MS by pairing the anion with a dicationic reagent to create a positively charged complex [ 55 - 58]. The use of tricationic reagents which pair with divalent anions for the detection of a +1 complex also has been reported recently [ 59]. Using positive mode ESI - MS avoids the spray stability problems of negative mode ESI - MS. Beyond this, pairing the anion with the dicationic or tricationic reagent has other benefits. For example, monitoring of the anion/cation pair moves the detected species to a higher mass region where there is lower background noise. Also, anions of low mass may be moved well above the low mass cutoff of quadrupole instruments (e.g., ion traps). In addition, the pairing reage nts may be used to differentiate between the analyte of interest and an interference of the same m/z [5 7 ].

The final three chapters of this dissertation focus on the expansion of positively charged reagents for the detection of anions by positive mode

ESI - MS . In chapter 5, a variety of linear tricationic reagents are evaluated with a small number of divalent anions to determine what types of charge groups and alkyl chain lengths make ideal linear pairing agents. Chapters 6 and 7 apply the superior linear t rications from chapter 5 and the best rigid, trigonal trications from a previous study [59] to the detection of a wider range of divalent anions and bisphosphonate drugs. Also in these chapters, MS/MS was used to lower the background and therefore the limi ts of detection for many of the anions presented.

11

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Full document contains 156 pages
Abstract: This dissertation focuses on two different areas of analysis: liquid chromatography (LC) enantiomeric separations and the detection of anions using electrospray ionization mass spectrometry (ESI-MS) and LC-ESI-MS. Enantiomeric separations of two distinct classes of chiral compounds were investigated. The first class is pterocarpans, which are isoflavanoids with cis -fused benzopyran benzofuranyl structures. In the reverse phase mode of operation, all pterocarpan enantiomers could be separated using cyclodextrin based chiral stationary phases (CSPs) with hydroxypropyl-β-cyclodextrin, acetyl-β-cyclodextrin, and γ-cyclodextrin showing the broadest enantioselectivity. Two macrocyclic glycopeptide CSPs, ristocetin A and vancomycin also proved useful in the reverse phase mode. Not as many separations were achieved in the normal phase mode for either set of CSPs. Chiral extended metal atom chains (EMACs) were first synthesized by F.A. Cotton and co-workers as the smallest possible molecular wires. It was not possible to resolve these enantiomers by crystallization and derivatization was impossible. The vancomycin macrocyclic glycopeptide stationary phase proved to be the best approach for separating these unusual chiral entities. Partial or baseline separation of 5 helical EMAC racemates was achieved in the polar organic mode or normal phase mode chromatography. In chapter 4, vibrational circular dichroism (VCD) is used to assign the absolute configuration of Ni 3 (dipyridylamine) 4 Cl 2 . Negative mode ESI-MS of anions can be problematic due to spray stability and background noise issues. The use of a positively charged reagent which pairs with the anion allows for the detection of anions in positive mode ESI-MS. Singly charged anions can be paired with dicationic reagents, while doubly charged anions can be paired with tricationic reagents to result in an overall +1 charged complex. In this dissertation, a variety of linear tricationic reagents were examined to determine which structural features are important for anion detection. The application of linear tricationic reagents and previously reported trigonal tricationic reagents were then applied to the detection of a larger variety of divalent anions (e.g. disulfonates, dicarboxylates) and bisphosphonates, which are a class of drug used to treat bone diseases. The use of MS-MS and LC-ESI-MS are also discussed.