The Gas-Phase Chemistry of Novel Carbon-Rich Molecules

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The Gas-Phase Chemistry of Novel Carbon-Rich Molecules An Investigation by Means of Modern Mass Spectrometry ✵ Die Gasphasenchemie von Neuartigen Kohlenstoffreichen Molekülen Eine Untersuchung Mittels Moderner Massenspektrometrie

Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) zur Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von

ROLF W. KIRSCHBAUM

aus Nürnberg

Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 29. April 2016 Vorsitzender des Promotionsorgans: Prof. Dr. Jörn Wilms Gutachter / in: Prof. Dr. Thomas Drewello Prof. Dr. Carola Kryschi

Für Gréta.

  

Die vorliegende Arbeit entstand in der Zeit von Oktober 2011 bis September 2015.

Mein Dank gilt Prof. Dr. Thomas Drewello, Dr. Marc S. von Gernler, Dr. Jing Li, Dr. Leanne C. Nye, Ina D. Kellner, Thomas S. Neugebauer, Jakob F. Hitzenberger, Prof. Dr. Walter Bauer, Prof. Dr. Andreas Hirsch, Prof. Dr. Rik R. Tykwinski, Dominik Prenzel, Dr. Stephanie Frankenberger, PD Dr. Christoph Böttcher, Dr. Jörg Schönamsgruber, Dr. Boris Schade, Dr. Lennard K. Wasserthal, Chau Vinh, Prof. Dr. Ivana Ivanović-Burmazović, Markus Hausmann, Prof. Dr. Olga V. Boltalina, Prof. Dr. Steven H. Strauss, Dr. Theodor Milek, Dr. Christian Lübbert, Dr. Doris Segets, Prof. Dr. Wolfgang Peukert, Prof. Dr. Dirk Zahn, Dr. Ana-Maria Krestel, Dr. Günter Schmid, Prof. Dr. Dirk M. Guldi, Dr. Florian Kessler, Vera Warmbrunn, Dr. Christian Neiß, Prof. Dr. Andreas Görling, Xenia Kostrov, Dirk Harnisch, Gerd Gätzschmann, Irmgard Ismayer, Margit Hartmann, Beate Maaß, Gertrud Weiß, Martin Kolacyak, Andrea Buchner, Dr. Andreas Bayer, Anette Göbel, Dr. Frank Hauke, Friedhold Wölfel, Thomas Hofmann, Hermann Hauke, Viola Ziegler, Bernd Hofmann, Angelika Leistner, Corinne Wiegner, Dorjkhand Rudolph, Markus Paesler, Florian Warko, Bernd Kreß, Uwe Sauer, Hans-Peter Bäumler, Dr. Georg Brehm, Dr. Guido Sauer, Dr. Karin Mansyreff, Dr. Christain Ehli, Dr. Axel Kahnt, Rauda Träger, Margarete Dziallach, Wolfgang Donaubauer, Dr. Frank Hampel, der Deutschen Forschungsgemeinschaft (DFG) — SFB 953 ‘‘Synthetic Carbon Allotropes’’, der Graduate School Molecular Science (GSMS), Evelyn Abdoulaye, Christian Sprogar, Reinhard Pinzer, Dr. Willy Weiß, Beatrice Baars, Marion Simon, Margit Gotzler, Daniela Schramm, Thomas Loos und meiner Familie.

Contents ______

Contents

Contents ...... i Figures ...... iii Abbreviations & Symbols ...... v Introduction ...... 1 1 Aims ...... 1 2 Gas-Phase Ion Chemistry ...... 1 2.1 Ionisation ...... 1 2.1.1 Ionisation Energy (IE) ...... 1 2.1.2 Electron Affinity (EA) ...... 3 2.2 Ion Formation by Cation / Anion-Attachment ...... 3 2.2.1 Gas-Phase Basicity (GB) and Proton Affinity (PA) ...... 3 2.2.2 Gas-Phase Acidity (GA) ...... 4 2.2.3 Metal Cation Affinities ...... 4 3 Experimental Section ...... 6 3.1 Ion Sources ...... 6 3.1.1 ESI source...... 6 3.1.2 Matrix-Assisted Laser Desorption / Ionisation (MALDI) ...... 12 3.2 Ion Transfer (In ESI-Instruments) ...... 22 3.2.1 Glass Capillary ...... 22 3.2.2 Skimmer ...... 23 3.2.3 Ion Funnel ...... 23 3.2.4 RF-only Multipoles ...... 24 3.3 Mass Analysers ...... 25 3.3.1 Linear Quadrupole (Q) ...... 25 3.3.2 Quadrupole Ion Trap (QIT)...... 27 3.3.3 Time-of-Flight (TOF) Analyser ...... 33

i Contents ______3.4 Detector ...... 37 3.4.1 Channel Electron Multiplier (CEM, Channeltron) ...... 38 3.4.2 Micro Channel Plate (MCP) ...... 38 3.5 Isotope Pattern ...... 38 3.6 Resolution & Resolving Power[144] ...... 39 3.6.1 Resolution: 10 %-Valley Definition ...... 39 3.6.2 Resolution: Peak Width Definition ...... 39 3.6.3 Full Width at Half Maximum (FWHM) ...... 39 3.6.4 Resolving Power in Mass Spectrometry ...... 40 3.7 Instruments ...... 40

3.7.1 BRUKER esquire6000 ...... 40

3.7.2 BRUKER micrOTOF-Q II ...... 40

3.7.3 BRUKER Reflex IV ...... 41

3.7.4 SHIMADZU Axima Confidence ...... 41 4 Compounds ...... 42 4.1 Polyynes ...... 42 4.2 Fullerenes and Fullerene Derivatives ...... 42 4.3 Perylenetetracarboxylic Acid Diimides (PDIs) ...... 44 4.4 Polycationic [60]Fullerene Hexakis-Adduct ...... 45 4.5 Neutral Transition Metal Complexes (nTMCs) ...... 46 References ...... 47 Oral Contributions & Poster Presentations ...... 57 Papers & Patents ...... 59 Appendix ...... 63

ii Figures ______

Figures

Figure 1. Vertical and adiabatic transition in a diatomic molecule ...... 2 Figure 2. Scheme of an electrospray ionisation (ESI) ion source ...... 6 Figure 3. Different electrospray set-ups ...... 7 Figure 4. Scheme of an ESI ion source working as an electrolysis cell ...... 7 Figure 5. Different nebulisers ...... 8 Figure 6. Droplet-evolution in an ESI ion source ...... 10 Figure 7. Scheme of a solvated ion evaporating from a droplet ...... 12

Figure 8. Microtiter plate 384, ground steel (BRUKER)...... 13 Figure 9. Scheme of a matrix-asisted laser desorption / ionisation (MALDI) ion source...... 14 Figure 10. Structure of the electron transfer matrix DCTB ...... 20 Figure 11. Structures and names of prevalent MALDI matrices ...... 21 Figure 12. Ion transfer set-ups ...... 22 Figure 13. Ion transfer glass capillary with platinum coated ends ...... 22

Figure 14. Skimmer (BRUKER esquire6000) ...... 23 Figure 15. Scheme of an ion funnel ...... 23 Figure 16. Scheme of an RF-only octopole ...... 24 Figure 17. Scheme of a quadrupole ...... 25 Figure 18. Scheme of a quadrupole ion trap (QIT) ...... 28

Figure 19. aZ-qZ-stability diagram...... 29 Figure 20. Enlargement of the overlapping area A from Figure 19...... 30 Figure 21. Part of the stability area shown in Figure 20 ...... 31 Figure 22. Point of resonance...... 32 Figure 23. Scheme of an MS2 collision-induced dissociation (CID) experiment ...... 33 Figure 24. Scheme of a time-of-flight (TOF) analyser ...... 33 Figure 25. MALDI ion source with reflectron-time-of-flight (ReTOF) analyser ...... 34 Figure 26. Delayed extraction ...... 35

iii Figures ______

Figure 27. MALDI ReTOF mass spectrum of Tr*−(C≡C)10−Tr* ...... 36 Figure 28. Linear and curved channel electron multiplier (CEM, Channeltron) ...... 38 Figure 29. Micro channel plate (MCP) ...... 38 Figure 30. 10 %-valley definition for resolution in mass spectrometry...... 39 Figure 31. Full width at half maximum (FWHM)...... 40

Figure 32. Set-up of the BRUKER esquire6000 (ESI-QIT-MS)...... 40

Figure 33. Set-up of the BRUKER micrOTOF-Q II (ESI-Qq-TOF-MS)...... 41 Figure 34. Structures of polyynes of different chain-lengths ...... 42

Figure 35. Structure of C60-fullerene ...... 42

Figure 36. SCHLEGEL diagrams of C60-fullerene ...... 43

Figure 37. Structure of a C60F44-isomer with D2-symmetry ...... 43 Figure 38. Chemical Structures of the perylenetetracarboxylic acid diimides (PDIs) ...... 44 Figure 39. Structure of [60]Fullerene hexakis adduct ...... 45 Figure 40. Neutral transition metal complexes (nTMCs) ...... 46

iv Abbreviations & Symbols ______

Abbreviations & Symbols

2-AA Anthranilic Acid (= AA, MALDI Matrix) 3-AQ 3-Aminoquinoline (MALDI Matrix) AA Anthranilic Acid (= 2-AA, MALDI Matrix) acac Acetylacetonate ACCA α-Cyano-4-hydroxycinnamic Acid (= CHCA = α-CHCA = α-CCA = 4-HCCA, MALDI Matrix) Ann− Anion (n-fold negatively charged) BP Benzopyrene (MALDI matrix) Catn+ Cation (n-fold positively charged) α-CCA α-Cyano-4-hydroxycinnamic Acid (= CHCA = α-CHCA = 4-HCCA = ACCA, MALDI Matrix) CEM Channel Electron Multiplier CFR Curved Field Reflectron CHCA α-Cyano-4-hydroxycinnamic Acid (= α-CHCA = α-CCA = 4-HCCA = ACCA, MALDI Matrix) α-CHCA α-Cyano-4-hydroxycinnamic Acid (= CHCA = α-CCA = 4-HCCA = ACCA, MALDI Matrix) CI Chemical Ionisation CID Collision-Induced Dissociation CRM Charged Residue Model Da Dalton (1 Da = 1 u = 1.66053878 × 10−27 kg) 1,5-DAN 1,5-Diaminonaphthalene (MALDI Matrix) DC Direct Current DCTB trans-2-[3-(4-tert-Butylphenyl)-2-methyl-2-propenylidene]malononitrile DFG Deutsche Forschungsgemeinschaft (German Research Foundation) DGMS Deutsche Gesellschaft für Massenspektrometrie (German Mass Spectrometry Society)

v Abbreviations & Symbols ______DHB 2,5-Dihydroxybenzoic Acid (= DHBA = 2,5-DHBA, MALDI Matrix) 2,5-DHBA 2,5-Dihydroxybenzoic Acid (= DHB = DHBA, MALDI Matrix) DHBA 2,5-Dihydroxybenzoic Acid (= DHB = 2,5-DHBA, MALDI Matrix) DIT Dithranol (MALDI Matrix) DMAN 1,8-Bis(dimethylamino)naphthalene or N,N,N‘,N’-Tetramethyl-1,8-naphthalenediamine or Proton Sponge® DOF Degrees of Freedom e Elementary Charge (e = 1.6022 × 10−19 C) E Electric Field Strength or Energy EA Electron Affinity EI Electron-Impact Ionisation Er:YAG Erbium-Doped Yttrium Aluminium Garnet (Active Laser Medium) ESI Electrospray Ionisation ESPT Excited State Proton Transfer FWHM Full Width at Half Maximum GA Gas-Phase Acidity GB Gas-Phase Basicity H Laser Fluence HABA 2-(4-Hydroxyphenylazo)benzoic Acid (= HBABA, MALDI Matrix) HBABA 2-(4-Hydroxyphenylazo)benzoic Acid (= HABA, MALDI Matrix) 4-HCCA α-Cyano-4-hydroxycinnamic Acid (= CHCA = α-CHCA = α-CCA = ACCA, MALDI Matrix) HOMO Highest Occupied Molecular Orbital 3-HPA 3-Hydroxypicolinic Acid (= HPA, MALDI Matrix) HPA 3-Hydroxypicolinic acid (= 3-HPA, MALDI matrix) HV High Voltage I Intensity ICA Indole-2-carboxylic Acid IE Ion Evaporation IE Ionisation Energy IEM Ion Evaporation Model

IH Laser Irradiance IMMS Informal Meeting on Mass Spectrometry IMSC International Mass Spectrometry Conference vi Abbreviations & Symbols ______IR Infra-Red ISD In-Source Decay iTMC Ionic Transition Metal Complex kB BOLTZMANN constant Laser Light Amplification by Stimulated Emission of Radiation LDI Laser Desorption / Ionisation LMCO Low-Mass Cut-Off LSIMS Liquid Secondary Ionisation Mass Spectrometry m/z mass-to-charge ratio MALDI Matrix-Assisted Laser Desorption / Ionisation MBT 2-Mercaptobenzothiazole (MALDI Matrix) MCA Multiply Charged Anion MDQ 2-Methyldibenzo[f,h]quinoxaline MPI Multiphoton Ionisation MS Mass Spectrometry MS / MS Tandem Mass Spectrometry (= MS2) MS2 Tandem Mass Spectrometry (= MS / MS) MTP Microtiter Plate 9-NA 9-Nitroanthracene (MALDI Matrix) NA Nicotinic Acid (MALDI Matrix) Nd:YAG Neodymium-Doped Yttrium Aluminium Garnet (Active Laser Medium)

NDOF Number of Degrees of Freedom nTMC Neutral Transition Metal Complex OLED Organic Light-Emitting Diode OLEEC Organic Light-Emitting Electrochemical Cell P Time-of-Flight Instrument Parameter 9-PA 9-Phenylanthracene (MALDI matrix) PA Proton Affinity PDI Perylenetetracarboxylic Acid Diimide PEG Poly(ethylene Glycol) ppy 2-Phenylpyridine PS Polystyrene PSD Post-Source Decay

vii Abbreviations & Symbols ______PTSM Polskie Towarzystwo Spektrometrii Mas (Polish Mass Spectrometry Society) q Quadrupole Collision Cell

q MATHIEU Parameter Q Analytical Quadrupole Q Electric Charge QIT Quadrupole Ion Trap r Radius val R10 % Resolution (10 %-Valley Definition) wid R50 % Resolution (Peak Width Definition at 50 % Peak Height) ReTOF Reflectron-Time-of-Flight RF Radio Frequency

RI RAYLEIGH Instability SA Sinapic Acid (MALDI Matrix) SEM Secondary Electron Multiplier THAP 2,4,6-Trihydroxyacetophenone (MALDI Matrix) TOF Time-of-Flight Tr* Supertrityl Group u Unified Atomic Mass Unit (1 u = 1 Da = 1.660538921(73) × 10−27 kg)[1] U Direct Current Voltage UV Ultra-Violet V Radio Frequency Voltage z Charge Number

Greek Characters

α Solid-State Absorption Coefficient δ Laser Penetration Depth (= α−1, with α being the absorption coefficient)

ϱi Mass Concentration σ Rotational Symmetry Number

τ Laser Pulse Duration

Ω Auxiliary Frequency ω Main Frequency

viii Introduction ______

Introduction

1 Aims

The objectives of this work include i) the improvement of the present understanding of mass spectrometry-based processes and ion formation reactions accompanying the transition of molecular entity from the condensed phase into the gas phase, ii) the establishment of distribution / localisation of the charge(s) in the resulting ion and iii) elucidation of the structural features of the gaseous ion. The scientific tools applied to achieve these objectives comprise the use of soft ionisation methods such as matrix-assisted laser desorption / ionisation (MALDI) and electrospray ionisation (ESI) for objective i) in conjunction with tandem mass spectrometry in the form of collision-induced dissociation (CID) experiments for ii) and iii). The compounds under investigation are of particular importance in the context of new synthetic carbon allotropes and carbon-rich molecules and comprise compound classes such as polyynes, fullerene derivatives, dendrimeric perylenetetracarboxylic acid diimides (PDIs), acetate clusters and neutral transition metal complexes (nTMCs).

2 Gas-Phase Ion Chemistry

2.1 Ionisation

2.1.1 Ionisation Energy (IE)

The first ionisation energy (IE) of a molecule is the energy required to remove an electron in the gas phase from the highest occupied molecular orbital (HOMO) to infinity. It is usually in the range of 6 – 10 eV and is thus significantly higher than most bond energies (~3.6 eV for C−C). Depending on the type of experiment to measure the IE for a given molecule either the vertical or the adiabatic transition (Figure 1) is observed. Usually a bond is weakened by the removal of a binding electron from the HOMO. This results in an increased bond length, which manifests itself in a shift of the local potential energy minimum to a greater inter- nuclei distance (bond length). In this case vertical ionisation means an additional excitation of vibrational modes of the ion. Thus, the vertical IE is always greater than the adiabatic IE. According to the FRANCK-CONDON principle the vertical ionisation is much more likely than

1 Introduction ______the adiabatic ionisation. The excess energy in a vertical ionisation often causes the fragmentation of the molecular ion. There is only one way of excitation, if the geometry of the neutral molecule does not differ from the geometry of the molecular cation—vertical and adiabatic are identical (not shown in Figure 1).[2-3]

Figure 1. Vertical and adiabatic transition in a diatomic molecule. The lower potential energy curve represents the neutral state. The upper curve refers to the radical cation.

Ionisation of a molecule can be obtained by several different processes: i) bombardment of the target molecule with highly energetic electrons, ii) irradiation of the target with light of appropriate wavelength, iii) collisions of the target molecule with accelerated ions and iv) collisions with atoms of sufficiently high kinetic energy.[2]

i) M + e− → M+• + 2e− (1)

ii) M + ℎ휈 → M+• + e− (2)

iii) M + X+ → M+• + X• (3)

iv) M + X → M+• + e− + X (4)

2 Introduction ______2.1.2 Electron Affinity (EA)

The attachment of an electron to a neutral molecule or atom is mostly accompanied by energy release. The first electron affinity (EA1) is the amount of energy being released during this process (Reaction 5) and is therefore defined as the negative of the enthalpy of the electron attachment. The second electron affinity (EA2) relates to the electron attachment to an already singly negatively charged molecule or atom (Reaction 6). Referring to an electron loss, the anion is thermodynamically stable, if the EA is positive.

− −• M + e → M − Δ퐻 = 퐸퐴1(M) (5)

−• − 2− M + e → M − Δ퐻 = 퐸퐴2(M) (6)

Multiply charged anions (MCAs) are fairly rare gas-phase ions. The attachment of a second electron to a singly negatively charged ion is kinetically hindered by a COULOMB barrier.[4]

Even for the formation of thermodynamically stable dianions (EA2 > 0) the approaching electron experiences electrostatic repulsion until it has reached a distance of approximately 10−11 m. Thus, the formation of dianions by the mere attachment of an approaching electron in the gas phase is unlikely. However, if the MCAs are formed directly by desorption / ablation from the condensed phase (e. g. ESI and MALDI), the COULOMB barrier can preserve the existence even of thermodynamically unstable (EA2 < 0) MCAs. It prevents the electron from escaping. This behaviour is discussed for the first time in the context of fullerene dianions by Compton et al.[5]

2.2 Ion Formation by Cation /Anion-Attachment

An alternative way to furnish a molecule with a charge is to attach a small ion (e. g. H+, Na+, Ag+, Cl−, HCOO−, etc.) to the target molecule. The resulting adduct ion is called quasi-molecular ion. The attachment of an ion to the neutral molecule can occur in a gas-phase reaction or already in solution before the resulting neutral / ion-complex is transferred into the gas phase. One big advantage is the softness of this type of ion formation process. The mere attachment usually does not provide enough excess energy to induce fragmentation of the resulting quasi-molecular ion.

2.2.1 Gas-Phase Basicity (GB) and Proton Affinity (PA)

GB and the PA are thermodynamic quantities. GB refers to the negative of the GIBBS free energy change (GB = −ΔG0) for the protonation of a certain molecule M. GB applies to neutral molecules M (Reaction 7) as well as to anions M− (Reaction 8).

3 Introduction ______M + H+ → MH+ (7)

M− + H+ → MH (8)

PA refers to the negative of the enthalpy of the same reaction (PA = −ΔH0). Thus, the GB differs from the PA by the TΔS0 term according to Equations 9 and 10.

Δ퐺0 = Δ퐻0 − 푇Δ푆0 (9)

푃퐴 = 퐺퐵 − 푇Δ푆0 (10)

The TΔS0 term includes the entropy of basicity and is primarily governed by i) the entropy of the gas-phase proton (108.8 J mol−1 K−1)[6] and ii) the ratio of the rotational symmetry numbers

0 −1 + [7] σ (entropy of protonation = ΔSP = R × ln[σ(M) × σ (MH )] . The first term (i) is the largest and amounts to 32 kJ mol−1 at 298 K.

2.2.2 Gas-Phase Acidity (GA)

GA refers to the deprotonation reaction of a certain neutral BRØNSTED acid AH (Reaction 11).

AH → A− + H+ (11)

In contrast to GB, the GA is not unambiguously defined. It indicates either the change of the GIBBS free energy ΔG0 or of the enthalpy ΔH0 of reaction 11. The enthalpies of these reactions are numerically larger than the GIBBS free energies due to the TΔS0 term (typically 20 – 40 kJ mol−1, if no chelation effect is involved). The effect of temperature on the relative GAs in the temperature range typically applied in MS is only minor.[2]

2.2.3 Metal Cation Affinities

Protonation and deprotonation are commonly utilised in formation reactions in ESI and MALDI for analytes that contain basic or acidic sites, respectively (e. g. proteins / peptides, oligonucleotides). However, if the target molecule does not possess groups that facilitate acid-base chemistry, ion formation through molecule-metal-cation adduct can make the target molecule accessible to MS analysis. Even if the molecule under investigation is nonpolar, cationisation may occur due to the formation of coordination complexes. Prevalent metal cations belonging to the group of alkali metals are Li+, Na+, K+, Rb+ and Cs+, showing a high affinity to oxygen-rich compound classes like crown ethers or esters. Alkaline earth metals like Mg2+, Ca2+ and Ba2+ are also known to form molecule-metal-cation complexes. Transition

4 Introduction ______metal ions such as Fe2+ / 3+, Ni2+, Zn2+, Cu+ / 2+ and Ag+ are preferred metalation agents for compounds with good electron donor properties such as the nitrogen lone pair or a preferably conjugated or aromatic π-electron system. Applying metal-cation attachment in ESI in the positive-ion mode usually leads to the observation of singly and doubly charged ion species. Typically the metal salt is added to the sample solution before introducing it into the ESI source. The detected ions can most often be denoted as the following species: [M + CatI]+, [M + 2CatI]2+, [M + CatII]2+, [M − H + CatII]+ or more generally [M − nH + mCatI](m − n)+. Depending on the solvent, the pH of the solution, the polarity and the PA of the target molecule, protonated species are formed as well. Even mixed ion species of the type [M + H + CatI]2+ can be observed. If one metal ion binds to two or more molecules, aggregate ions of the type [nM + CatI]+ are formed. The aggregation of several deprotonated molecules and metal cations often results in the observation of cluster ions of the type [n(M − H) + mCatI](m − n)+ with m = n + 1 for singly, m = n + 2 for doubly charged species, etc. In the negative-ion mode multiple cationisation of easily deprotonated molecules may occur, resulting in the formation of the ion species [M − nH + mCatI](n − m)− with m = n − 1 for singly m = n − 2 for doubly negatively charged ions. Under MALDI conditions usually only singly charged ions are formed. The addition of monovalent metal salts to the sample solution typically results in the formation of [M + CatI]+. An increasing metal salt concentration gives rise to poly-cationisation leading to the observation of the species [M − H + 2CatI]+, [M − 2H + 3CatI]+, etc. Depending on the relative affinity of the analytes to protons or metal cations, the metalation competes with protonation. Diva- lent metal ions (Cat2+) typically form singly charged species either by reduction of the metal ion [M + CatI]+ or by deprotonation of the analyte molecule [M − H + CatII]+. This reduction of the metal cation in MALDI is typically occurring by attachment of the free electrons in the gas phase, which are released from the support during laser activation. Both types of ions are formed with proteins and polar polymers (e. g. PEG), whereas the reduction of the metal ion dominates for non-polar polymers (e. g. PS). Although multiple additions of divalent metal cations are also observed, these species are singly charged. An example for this behaviour is the formation of [M − 3H + 2CdII]+ with M = α-cyclodextrin.[8]

5 Introduction ______3 Experimental Section

3.1 Ion Sources

3.1.1 ESI source

The principle set-up of an ESI ion source is displayed in Figure 2. The source consists of a grounded stainless steel needle, through which the sample solution is introduced into the spray chamber under atmospheric pressure conditions applying a syringe pump. The spray, which originates from the needle tip, is converted into solvent-free ions (Figure 2). These ions are attracted by the electric counter potential which is applied to the orifice of the instrument. This small aperture is often the metal-coated end of a transfer glass capillary (see 3.2.1) and allows the ions to enter the first vacuum stage of the instrument (see 3.7.1 and 3.7.2) from where the ions are transferred to the analyser. In order to prevent neutral species from entering the mass spectrometer, a heated counter flow sheath gas (also: dry gas; often nitrogen) is directed coaxially with regard to the orifice of the instrument towards the spray plume (Figure 2). Collisions between the sheath gas molecules and solvated ions cause the solvent shell to be stripped off, yielding the bare ions. Ion cluster species of the types

[CatmAnn]+ and [AnmCatn]− (m = n + 1) are also destroyed by collisions with the sheath gas molecules.

a) b) Figure 2. Scheme of an electrospray ionisation (ESI) ion source. HV = High voltage a) Scheme of an ESI ion source (lateral cut-away view). b) Photo of the opened ESI ion source attached to a BRUKER es- quire6000 mass spectrometer.

6 Introduction ______Generating an ion beam requires three principal steps: i) the formation of charged droplets out of the sample solution, ii) the shrinking of the droplets by evaporation of solvent molecules and iii) the formation of gas-phase ions from small highly charged droplets.[2]

3.1.1.1 Spray Needle Arrangement In order to prevent the mass spectrometer from being contaminated by unwanted neutral components in the spray, the needle can be placed off axis (Figure 3b), diagonally (c) or or- thogonally (d). Further arrangements are described elsewhere.[2-3,9] Preferably the smaller droplets at the outer regions of the spray plume are converted into bare ions and introduced into the mass spectrometer. Thus, diagonal and orthogonal spray arrangements show great benefit concerning easier desolvation of these small and highly charged droplets. Addition- ally the spray is more stable, if it is pneumatically assisted.[9]

Figure 3. Different electrospray set-ups. a) on axis, b) off axis, c) diagonal, d) orthogonal.

3.1.1.2 Droplet-Formation Due to the small radius of the spray needle tip a strong electric filed is generated at the needle outlet, if a voltage is applied. The electric filed causes polarisation of the sample solution. Depending on the polarity, cations or anions are driven to the surface of the sample solution. The repulsion of equally charged ions results in the formation of the TAYLOR-cone (Figure 4).

Figure 4. Scheme of an electrospray ionisation ion source working as an electrolysis cell in the positive-ion mode.[2-3,9]

7 Introduction ______As soon as the electrostatic attraction between the ions and the counter electrode exceeds the surface tension of the sample solution, a small droplet separates from the TAYLOR-cone. For unassisted electrospray (Figure 5a) the droplet size depends primarily on the sample flow rate. For higher flow rates it additionally depends on the applied voltage. Small droplets are obtained for small flow rates.[10] Pneumatically assisted ESI (Figure 5b) comprises a coaxially directed gas flow nebulising the sample solution while leaving the spray needle and facilitating the use of less volatile solvents, higher flow rates and a lower electric field strength. The charge of the droplets is still generated by the electric field.[2] Nebulisation of liquids with high surface tension can be obtained by applying an ultrasonic nebuliser (Figure 5c). The large droplets obtained by this technique result in little ion yields. However, high flow rates (50 – 1000 μL min−1) can be employed, which is often applied to LC-MS experiments.[3]

Figure 5. Different nebulisers. a) pure electrospray, b) pneumatically assisted electrospray, c) ultrason- ically assisted electrospray.

3.1.1.3 Electrochemical Processes The ESI ion source can be seen as an electrolysis cell (Figure 4). In contrast to typical electrolysis the ions do not pass through a coherent medium but through vacuum or a gaseous atmospheric pressure medium. The spray needle is a thin metal capillary that acts as the working electrode. Due to the applied voltage, the metal-coated entrance of the glass transfer capillary acts as the counter electrode. The voltage is usually set to a value between 3 and 5 kV[9] resulting in a particular high electric filed strength (E ≈ 106 V m−1)[9] which causes charge separation within the sample solution at the needle tip. In the positive-ion mode, the voltage applied to the spray needle is higher than the voltage applied to the transfer capillary entrance. The resulting electric field causes the cations to move towards the surface of the sample solution which is exiting the needle outlet. The anions are directed towards the inner needle wall. Repulsive COULOMB forces between the cations near the surface cause an exten- sion of the solution surface in such a way that the meniscus turns into a cone—the TAYLOR- cone. If the electric field is strong enough, the TAYLOR-cone deforms to a jet which disinte- grates to small droplets with a positive net charge. The removal of positive charge from the sample solution requires a neutralisation of the remaining oppositely charged ions. Other- wise the remaining charge would generate a counter field which would inhibit the droplet

8 Introduction ______formation. The negatively charged ions are neutralised by a heterogeneous electron transfer from the anion to the needle.[2,9] In the negative-ion mode the processes described above take place with reversed polarities. Reactions 12 and 13 show possible oxidation reactions occurring in aqueous solutions of the ionic compound Cat+An− while the solution is leaving the stainless-steel spray needle. Due to the oxidative disappearance of anions, the resulting net charge of the droplets is always positive. The electric field between spray needle and the counter electrode (platinum coated glass capillary) accelerates the droplets towards the capillary entrance. The electrons formed by oxidation are absorbed by the stainless-steel spray needle.

An− ⇄ An• + e− (12)

+ − 2 H2O ⇄ 4 H + O2 + 4 e (13)

In cases where there are no ionic compounds dissolved in the sample solution the protons generated by oxidation of water (Reaction 13) can attach to the target molecule (e. g. peptide) and provide the required charge. If the oxidation potential of the target molecule is not too high, it might be oxidised directly (Reaction 14) by electron transfer to the spray needle or to solvent molecules.

M ⇄ M+• + e− (14)

In the negative-ion mode an excess of negative charge is required. The removal of cations (Reaction 15) can occur in analogy with Reaction 12. Direct reduction of the analyte molecule is possible as well. The electron supply for the reduction of the analyte can also occur through anodic corrosion of the steel spray needle (Reaction 16).

Cat+ + e− ⇄ Cat• (15)

Fe ⇄ Fe2+ + 2 e− (16)

3.1.1.4 Droplet Evolution The droplets that emanate directly from the TAYLOR-cone decrease in size by evaporation of neutral solvent molecules from the original droplet, while the electric charge of the droplet does not change. The shrinking droplet size causes an increasing COULOMBIC repulsion between the ions near the droplet surface. The droplet diminution continues until the stability boundary (RAYLEIGH-limit) is almost reached. The RAYLEIGH-limit defined by Equation 17, where Q is the charge, ε0 is the absolute permittivity of the vacuum and γ is the surface ten-

9 Introduction ______

sion of the droplet. The RAYLEIGH-limit describes the state where the COULOMBIC repulsion equals the attractive forces, which are caused by the surface tension γ.

2 2 3 푄 = 64휋 휀0훾푟 (17)

The droplet becomes instable and undergoes fission, if Equation 17 is valid for a certain electric charge Q and droplet radius r. Droplets reaching a radius of about 20 μm already dissociate at 80 % of the RAYLEIGH-limit (Figure 6).[10-12] Stimulated by spheroidal vibrations such a droplet forms a sort of filament which affords nearly 20 smaller droplets (jet fission). The original droplet loses ca. 2 % of its mass and 15 % of its charge. The remaining bigger droplet is stable until it again approaches the RAYLEIGH-limit through solvent evaporation.[11-12] This process is repeated until a certain droplet size is reached. However, up to now it was only observed for droplets with r > 1 μm and for non-polar solvents.[10]

Figure 6. Droplet-evolution in an electrospray ionisation ion source.[11]

3.1.1.5 Formation of Gas-Phase Ions from Small Droplets The Charged Residue Model (CRM)[13-17] and the Ion Evaporation Model (IEM)[18-19] describe the formation of gas-phase ions evolving from small and highly charged droplets whose genesis is depicted in Figure 6. Both mechanisms have been discussed extensively in the past.[20-28] According to the CRM the analyte ion emanates from COULOMB explosions[13] of very small droplets (r < 3 nm),[23] from which no more solvent can evaporate.[17] The IEM ex- plains the ion formation by evaporation of solvated ions (Figure 6) from bigger droplets (r = 10 nm) which carry some hundred unit charges (z = 102).[23] The CRM is based on the assumption that the gas-phase ions will be generated, when the droplet evolution (Figure 6) comes to the point where no further evaporation of the solvent

can occur. Dry ion species of the type [Catm(CatAn)n]m+ remain. The charge number m and the cluster size n will cover a wide range. Since the COULOMB fissions generate droplets of different size and charge, the large ion species (high m and n) are mainly generated from the parent droplets, while the offspring droplets produce small ion species (low m and n) including single ions Cat+. Due to the large number of small droplets created in every COULOMB fission, the high intensity of the small ion species with low m and n values (mainly Cat+, i. e. m = 1 and n = 0) will predominate the mass spectrum.

10 Introduction ______According to the IEM solvated ions evaporate directly from the droplet surface into the gas phase, if the droplet size is less than 10 nm in diameter. Compared to COULOMB explosions this process already occurs at lower charge states of the droplet (Equation 17). Iribarne & Thomson[18] have discussed the relationship between the droplet radius r and the energy bar- ‡ rier ΔG applying the EYRING-theory (Equation 18), where kIE is the specific reaction rate of the ion evaporation from the droplet surface, kB is the BOLTZMANN-constant and h the PLANCK-constant.

Δ퐺‡ 푘퐵푇 − 푘 = e 푘퐵푇 (18) 퐼퐸 ℎ

It was found that ion evaporation (IE) occurs, if the droplet reaches a size in the order of 10−8 m, which results in a total evaporation time in the order of μs. This time regime brings ‡ about a reaction rate of −dlnc / dt ≈ 106 s−1 leading to ΔG ≈ 40 kJ mol−1.[18] However, the IE competes with the RAYLEIGH instability (RI), hence for a given number of unit charges z the critical radius rIE must be reached before the critical radius rRI at which droplet fission occurs

(rIE > rRI). Considering Equation 17 the conditions determined by Equation 19 must apply.[18]

1 2 3 푄 (19) 푟퐼퐸 > ( 2 ) 64휋 휀0훾

It is assumed that the reaction rate is determined by the solvated ion leaving the droplet surface. During this reaction step, attractive and repulsive forces compete. The attraction is at- tributed to the ion charge and the polarisability of the droplet. Its range decreases strongly with increasing distance between the ion and the droplet surface whereas the repulsive COULOMB interactions show a greater range.

‡ ‡ ⃗ 0 Δ퐺 = Δ퐺 (퐸) − Δ퐺solv (20)

‡ The first term ΔG (E⃗⃗ ) is a function of the electric field perpendicular to the droplet surface, 0 which is determined by the above mentioned interactions. The second term ΔGsolv describes the GIBBS free solvation energy, which is usually relatively high. However, the fact that the ion leaves the droplet carrying a shell of solvent molecules with it reduces its contribution to the overall energy barrier.[17]

11 Introduction ______

Figure 7. Scheme of a solvated ion evaporating from a droplet according to the ion evaporation model.[18] a) Initial state: The excess charges are expected to be close to the droplet surface to achieve a minimum of repulsion. The charge carriers are small solvated ions. The radius of the ion including the solvent shell is the distance d from the charge centre to droplet surface. b) Transition state: A solvated ion has been released from the droplet surface. Typical values for watery droplets are x = 0.6 nm, r = 8 nm and z = 70.[18]

However, Equation 17 is valid only for a continuous charge distribution. If the droplet radii are in the range of several nanometres and there are only few charges close to the droplet surface, this approximation is not valid. Numerous studies into the mechanistic aspects of ESI revealed that both models CRM and IEM are border cases.[11,13,17-18,23,28] However, these results indicate a trend, in that the genesis of smaller gas-phase ions like Na+ is better explained by the IEM, whereas very small droplets containing bigger ions, such as the charged molecules of proteins or polymers tend to undergo COULOMB fission.[23]

3.1.2 Matrix-Assisted Laser Desorption / Ionisation (MALDI)

3.1.2.1 Sample Preparation The Sample is usually dissolved using a volatile solvent and an excess of matrix substance is added (Page 20). This solution is then placed directly on the microtiter plate (MTP, Figure 8). Evaporation of the solvent leaves a solid mixture of the analyte and the matrix substance behind.[29-30] The evaporation process can be accelerated by reducing the atmospheric pressure above the sample droplet. Faster drying reduces the size of the analyte / matrix crystals and increases crystal homogeneity, which entails higher reproducibility (in-spot and spot-to- spot). Another way of preparing the sample is to mix analyte and matrix directly on the MTP. In this case the matrix solution is added to the droplet of analyte solution, which was previously deposited on the MTP, before the solvent is evaporated. Different techniques (dried-droplet, thin-layer, thick-layer and sandwich-method) are described in the literature.[31]

12 Introduction ______

Figure 8. Microtiter plate 384, ground steel (BRUKER) for MALDI application.

3.1.2.2 Desorption and Ion Formation ● The Desorption Process Following the sample preparation, the MTP is transferred into the vacuum of the mass spectrometer, where sample ablation and ionisation are initiated by the use of a laser (Figure 9). Usually pulsed ultra-violet- (UV) or infra-red- (IR) lasers are used. The active laser medium of UV lasers is mostly nitrogen (337 nm) or Nd:YAG (355 nm, frequency-tripled or 266 nm, frequency-quadrupled), whereas Er:YAG is used for IR-lasers.[32-34] Due to an attenuator the laser intensity is adjustable. The attenuator is a rotary disc which absorbs laser light to a varying degree, depending on the angle it is set to. The laser beam is usually focused to result in a laser spot diameter which is in the μm-regime.[34] Typical parameters for a laser spot diameter of approximately 100 μm are: laser pulse duration τ = 0.5 – 20 ns, laser penetration depth into the sample δ = 50 – 300 nm, laser fluence H = 30 – 10,000 J m−2, laser irradiance

IH = 3 × 105 – 1 × 109 W cm−2.[35] The intensity of the applied laser light is either described by the laser fluence, which is the energy of one laser pulse divided by the laser spot area, or by the laser irradiance, which is the laser fluence divided by the laser pulse duration. Most of the MALDI-TOF mass spectrometers allow the adjustment of the so-called laser power, which is a percentage value of the maximum laser fluence of the applied laser. Direct photoexcitation of the analyte molecules is avoided if the matrix shows sufficiently high absorption at the laser wavelength so that the matrix can be photo excited in competition to the analyte. Pho- toexcitation of the matrix allows the study of analytes that do not absorb at the laser wavelength. Since the analyte is not directly laser-activated, it will experience less internal excitation and thus less fragmentation of the analyte molecules will occur. The absorption spectrum of the matrix molecule differs when going from solution to the solid state. Absorption bands are red-shifted and broadened.[36-39] Consequently, the solid matrix can still absorb the laser light, even when its absorption maximum in solution may slightly differ from the laser wavelength.

13 Introduction ______

Figure 9. Scheme of a MALDI ion source.

The quality of the MALDI mass spectrum usually increases with increasing absorption.[36] The threshold laser fluence at which ion generation can be detected decreases with an increasing laser-spot diameter,[34,40] which is also true for the desorption of neutral matrix molecules.[34] However, the onset of the ablation of neutral species already occurs at laser fluences 2 – 3 times lower than the threshold laser fluence for the detection of ionic species.[34,41-42] The reasons for the strong dependence on the laser-spot size have not yet been clarified. The correlation might be explained by the change in the ratio of molecular desorption and cluster desorption with an increasing cluster formation for smaller laser-spot sizes.[35,43] The signals generally gain intensity with an increasing laser fluence[34,44-49] until saturation is reached. After this point the ion abundance starts to drop, if the laser fluence is further increased. This is explained by fragmentation and / or detector saturation.[35] The laser energy per volume which is deposited in the sample is calculated according to the BOUGUER-BEER-LAMBERT law (Equation 21) with α being the solid-state absorption coefficient, x being the depth in the

sample material and H0 being the laser fluence at the sample surface (x = 0 m).[35]

푑퐸(푥) = 훼퐻 푒−훼푥 (21) 푑푉 0

For volume increments at the surface (x ≈ 0 m) Equation 21 reduces to Equation 22.

퐸 = 훼퐻 (22) 푉 0

For common laser spot sizes (50 – 200 μm in diameter) energy densities in surface-near volume increments (≈ 10 nm) are on the order of several tens to about hundred kJ mol−1. The combined heats of fusion and vaporisation of typical matrix materials are on the same order

14 Introduction ______of magnitude. Therefore, a predominant thermal component of the transition to the gas phase is mandatory.[34-35] The amount of thermal energy that is deposited in the sample at one single laser shot event strongly depends on the laser pulse duration. If the laser pulse is too long, the accumulated thermal energy is high enough to result in fragmentation of the sample and / or matrix molecules.[50] If the excitation of the matrix material happens fast, i. e. short laser pulses, still sufficient energy for the desorption process will be provided, but the accumulated thermal energy will be so little that the fragmentation rates are lower than those for the ablation. Fast fragmentation channels whose time constants are on the order of the phase transition result in the so-called “in-source-decay”[51] (ISD), while dissociation processes with time constants on the order of the flight times in a time-of-flight (TOF) analyser are called “post-source-decay”[52-53] (PSD).[35] In addition to short laser pulses the molar matrix / analyte ratio needs to be high enough to prevent too much energy transfer from the matrix lattice vibrations to the internal vibrational modes of the enclosed analyte molecules.[50,54] The phase transition itself, which depends on the laser and material properties, can be described by several models: Thermal desorption of individual molecules,[35] surface layer-by- layer evaporation / sublimation,[35,50,54-55] removal of chunky sample-material bits (volume ablation) by phase explosion[35,43,56] and volume ablation by laser-induced pressure pulses.[55] Surface layer-by-layer evaporation / sublimation affords mainly individual molecules and small metastable clusters. Phase explosion, on the other hand, would result in the ejection of large clusters or even chunks of sample material. The actual way of the phase transition depends strongly on the amount of energy which is deposited in the sample material. There- fore, the laser fluence plays a crucial role in the MALDI process. It is expected that a high laser fluence causes preferably volume ablation to take place. Since the ablation process depends on several parameters, there is no general model that describes the MALDI process accurately.[35] The lowest amount of analyte necessary per laser shot, which is required to generate a viable MALDI spectrum has been estimated to be in the low attomol range.[57-59] Absorption of IR-laser light results in vibrational excitation of the matrix molecules. The pe- culiarities of IR-MALDI are minutely discussed in the literature.[60-65]

● Ion Formation Mechanisms[66-68] Generally, the overall ion-to-neutral ratio in UV-MALDI is only about 10−4.[69-70] The mechanisms can be divided into two categories: primary and secondary ionisation processes. Pri- mary ionisation refers to the first ion generation which evolves directly from the neutral molecules of the target material (often matrix molecules).[71] Secondary ionisation describes the process of ion formation from neutrals (often analyte molecules) interacting with primarily formed ions (matrix).[68]

15 Introduction ______Primary Ionisation Reactions Usually the following types of ions are observed in the positive-ion mode: radical cations M+•, protonated pseudo-molecular ions MH+ and cationised pseudo-molecular ions MCat+. Radical anions M−•, deprotonated pseudo-molecular ions [M − H]− and anionised pseudo- molecular ions MAn− are observed in the negative-ion mode. Zenobi & Knochenmuss[66] have discussed seven ion formation mechanisms with regard to primary ionisation. However, these processes do not necessarily provide enough energy to ionise and transfer the sample molecules into the gas phase. It is believed that the physical expansion of the plume provides (collisions with neutrals) the energy to separate previously formed ion pairs to such distances that they are too far apart to recombine. So, the mechanisms discussed in the following describe only the initial processes of the formation of gas- phase ions.

i) Multiphoton ionisation (MPI). Ionisation could straightforwardly be explained by single molecule multiphoton ionisation (MPI) as indicated by Reaction 23.

n ℎ휈 M → M+• + e− (23)

Consistently, the observation of matrix radical cations[72] and electrons[46] emitted from the sample material is reported in literature. The wavelength dependence[73-74] shows clearly that direct laser excitation of a matrix molecule is required for efficient UV MALDI. The lifetime of the excited state is in the range of some nanoseconds so that further photons could be absorbed to yield the radical cation M+• (Reaction 24).

ℎ휈 m ℎ휈 M → M∗ → M+• + e− (24)

Due to typical MALDI irradiances of 106 – 107 W cm−2 being too low for a two-photon absorption in the second step (m ≥ 2, Reaction 24) it is unlikely to happen. Thus the absorption of a total of two photons has to provide sufficient energy to ionise the matrix molecule. Semi-em- pirical calculations afford IEs for the isolated DHB molecule of 8.54 eV at the minimum,[75] which is too high for to be provided by two nitrogen laser photons (7.36 eV). The following ionisation mechanisms have been developed to overcome the two-photon dilemma.[76]

ii) Energy Pooling. If two adjacent matrix molecules are excited separately, they can pool their energy gain to afford one single matrix radical cation (M+•, Reaction 25) or analyte radical cation (A+•, Reac-

16 Introduction ______tion 26). Such processes are known to be likely for closely packed chromophores as it is the case in the solid state or in clusters. [55,77-81]

2 ℎ휈 2 M → 2 M∗ → M + M+• + e− (25)

2 ℎ휈 A 2 M → 2 M∗ → 2 M + A+• + e− (26) iii) Excited-State Proton Transfer (ESPT). In contrast to energy pooling, ESPT requires only one photon. An excited matrix molecule M* exhibits a higher acidity than the non-excited molecule. Therefore it can act as a BRØNSTED acid and facilitate proton transfer to an adjacent matrix or analyte molecule (Reac- tions 27 and 28).

ℎ휈 A M → M∗ → (M − H)− + AH+ (27)

ℎ휈 M M → M∗ → (M − H)− + MH+ (28)

Significant changes in pK values are known for compounds endowed with aromatic amine or hydroxyl groups.[66,82-87] iv) Disproportionation Reactions. Many compounds yield ion signals in the positive-ion mode as well as in the negatvie-ion mode. A possible common origin of such ion pairs is indicated by the disproportionation reactions of strongly coupled excited-state molecule pairs as it is shown in the Reactions 29 and 30.[66,88]

n ℎ휈 2 M → (MM)∗ → (M − H)− + MH+ (29)

n ℎ휈 2 M → (MM)∗ → M−• + M+• (30)

The importance of such a concerted absorption / excitation process lies in its low activation barrier compared to a two-step process (e. g. ESPT), which will be limited by the activation energy of the first step.[66] v) Desorption of Preformed Ions. Charge-tagging by protonation or metalation in solution results in preformed ions. If these preformed ionic species survive the solvent evaporation process during sample preparation,

17 Introduction ______they will be thermally desorbed during / after laser irradiation.[89] Zhu et al.[90] found that the positive-ion signal of small proteins and peptides was enhanced if their chains had a basic amino acid incorporated. This effect is explained by pre-protonation of the peptides / proteins lowering the energy barrier for the ion formation.

vi) Thermal Ionisation. Thermal ionisation in the matrix bulk represents effectively a disproportionation reaction (Reaction 31). The extent of ionisation upon scattering at a surface can be described by the use of the SAHA-LANGMUIR Equation (Equation 32), where N+ is the number of positive ions leaving the surface per unit area per second and N0 is the number of neutral species emitted from the same surface in the same time. C is a constant factor near unity. EA and IE refer to the matrix.[91-92]

훥퐻 2 M → M− + M+ (31)

푁+ 퐸퐴−퐼퐸 = 퐶 ∙ 푒 푘퐵푇 (32) 푁0

The equation describes the temperature dependence of the degree of ionisation. It is based upon the assumption that thermal equilibrium with regard to all degrees of freedom (DOF) is established between adsorbed species on the surface and the surface itself.[66,91] However, the internal energy of desorbed neutral molecules in UV MALDI corresponds to effective temperatures of around 500 K.[70] Thermal desorption / ionisation at these temperatures is clearly not significant.[66]

vii) Pressure Pulses and Spallation Rapid mechanical stress caused by the MALDI laser pulse results in crystal breakage desorption, which in turn can cause electronic excitation leading to the emission of neutral molecules and ions. If thermal stress builds up faster than can be purged by emission of acoustic waves, ablation of matter accompanied by the emission of ions and electrons is caused.[66,93]

Secondary Ionisation Reactions in the MALDI Plume When the primary ions are formed, they are in a moderately hot and dense environment full of neutral matrix molecules and clusters. Consequently these ions will have experienced many collisions before being extracted into the drift tube of the TOF analyser. Under these circumstances secondary reactions lead to the formation of ions differing from the primarily formed ions. The plume reactions can be described by three major types of reaction: i) Proton transfer, ii) metal-cationisation and iii) electron transfer.

18 Introduction ______i) Gas-Phase Proton Transfer Matrix-Matrix reactions result in the formation of protonated (Reaction 33) and deprotonated matrix ions (Reactions 34 and 35).

M+• + M → MH+ + (M − H)• (33)

(M − H)• + e− → (M − H)− (34)

M + e− → (M − H)− + H• (35)

Protonated matrix ions (Reaction 33) can subsequently protonate analyte molecules if the PA of the analyte molecule is larger than the PA of the matrix (Reaction 36) neglecting entropic effects.[66,72] The attachment of an electron by a dehydrogenated matrix molecule (M − H)• results in the deprotonated species (M − H)−.[94] The deprotonated species can also be formed by a dissociative electron capture reaction as it was proposed originally as an ion-formation mechanism in liquid secondary ionisation mass spectrometry (LSIMS).[94] Matrix-analyte reactions lead to protonated or deprotonated analyte molecules as indicated by the Reactions (36 and 37).

MH+ + A → M + AH+ (36)

If the PA of the matrix M is considerably lower compared to the analyte’s PA, the exother- micity of the proton transfer will lead to increased fragmentation of the analyte quasimolecu- lar ion.[95-96] Deprotonated matrix molecules could induce a proton transfer from the neutral analyte A to (M − H)− as shown in Reaction 37.

(M − H)− + A → M + (A − H)− (37) ii) Gas-Phase Metal Cationisation Laser irradiation of peptide / matrix mixtures in the presence of free gas-phase sodium ions lead to significantly enhanced MNa+ signals compared to normal MALDI conditions.[97-98] Together with the finding of an intensity maximum of the pseudo-molecular ion when varying the delay time,[97] this is interpreted as evidence for a gas-phase metal attachment in the MALDI plume (Reaction 38).

M + Cat+ → MCat+ (38)

19 Introduction ______iii) Electron Transfer Electron transfer reactions can afford both positively and negatively charged ions, depending on the IEs and EAs of the matrix and the analyte. For the formation of positively charged ions of the analyte it is necessary that the matrix has a higher IE than the analyte. Single electron transfer from the analyte molecule to the primarily formed matrix radical cation will occur (Reaction 39).[99]

M+• + A → M + A+• with IE(M) > IE(A) (39)

The IE of the matrix is regarded as being equivalent to the recombination energy of the matrix radical cation, with which the reaction commences. Upon electron uptake the recombination energy is gained and used to ionise the analyte. This behaviour was found for some me- tallocenes using terthiophene or anthracene as an electron transfer matrix.[100] For the formation of radical anions, the EA of the matrix must be lower than the EA of the analyte according to Reaction 40.

M−• + A → M + A−• with EA(M) < EA(A) (40)

In combination with fullerene derivatives[101-105] and other carbon-rich compounds, trans-2-[3- (4-tert-Butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB, Figure 10) shows a broad applicability as electron transfer matrix, very often much better performing than other matrix materials. This can be assigned to its relatively high IE (8.54 eV)[99] on the one hand and to its relatively low EA (2.31 eV)[99] on the other hand. Additionally, the desorption / ionisation process is expected to be softer compared to other matrices, because of the lower laser fluences that are required for ion formation using DCTB.[101]

Figure 10. Structure, chemical and abbreviated name of the electron transfer MALDI matrix that was

used for all MALDI experiments in this work. IE = 8.54 eV[99] and EA = 2.31 eV,[99] λmax = 346 nm.[106]

3.1.2.3 Matrix Substances The matrix materials commonly employed in MALDI are depicted in Figure 11. Some of the matrix substances have been investigated regarding their propensity to cause fragmentation of the analyte molecule. It was found that the ion fragmentations of protonated glycoproteins

20 Introduction ______will be enhanced in the order of HPA < DHB < SA,[107] which is explained by the sublimation point of the matrix materials increasing in the same order.[77]

Figure 11. Structures, chemical / trivial / abbreviated names of prevalent MALDI matrices.

21 Introduction ______3.2 Ion Transfer (In ESI-Instruments)

Ions generated from an ESI ion source that works under ambient pressure conditions are always accompanied by neutral sheath-gas and solvent molecules leading to a relatively high pressure near the entrance orifice of the instrument. In order to allow for an accessible high vacuum stage, the instrument is usually partitioned into compartments connected to turbomolecular pumps. For the ion transfer into the high vacuum stage where the mass analyser— the actual mass spectrometer—is located, the ions require to be focused and guided by appropriate electric fields (Figure 12). The components described in the following comply with these requirements.

Figure 12. Ion transfer set-up of the a) BRUKER esquire6000 electrospray ionisation-quadrupole ion trap-mass spectrometer (ESI-QIT-MS). b) BRUKER micrOTOF-Q II electrospray ionisation-quadrupole- time-of-flight mass spectrometer (ESI-Qq-TOF-MS).

3.2.1 Glass Capillary

The glass capillary (Figure 13) functions as the entrance to the vacuum chamber. Its small inner diameter (0.5 mm) prevents most of the neutral gas from entering the instrument while voltages applied to the platinum coated endcaps generate an electric field that guides the ions into the instrument. The glass material has a very high electric resistance R = 1 GΩ.

Figure 13. Ion transfer glass capillary with platinum coated ends (BRUKER micrOTOF-Q II)

22 Introduction ______3.2.2 Skimmer

The Skimmer (Figure 14) removes part of the residual neutral molecules (sheath gas and solvent) from the ion pathway. Applying different electric potentials to the skimmer and the capillary exit (Figure 12a) leads to acceleration of the ions resulting in collisions with the neural molecules still present. This effect can either be exploited to desolvate the ions or, if the skimmer voltage is increased, to fragment the ions by collision-induced dissociation (skimmer CID). Isolation of a fragment ion species and further CID enable so-called pseudo- MS3-experiments. Real MS3 would require also the isolation of the initial precursor ion of the first dissociation step, which is in skimmer CID not the case.

Figure 14. a) Photo of a skimmer (BRUKER esquire6000, front view), b) scheme of a skimmer (lateral cut-away view).

3.2.3 Ion Funnel

a) b) Figure 15. a) Scheme of an ion funnel (lateral cut-away view). The vertical lines represent the ring electrodes which decrease in diameter from left to right. A radio frequency (RF) voltage is applied to the electrode array in order to produce a funnel shaped electric field which repels the ions so that they are focused on the central symmetry axis of the funnel. b) photo of an ion funnel (BRUKER micrOTOTF-Q II, front view).[108-110]

The ion funnel (Figure 15) focuses the ion beam due to its funnel shape and the repulsive effective potential resulting from the radio frequency (RF) voltages which are applied to the ring electrodes. Neutral molecules (sheath gas and sample) drift away from the ion beam and are removed by the turbomolecular pump. This set-up is able to transfer the ions more gently towards the high vacuum stage than the skimmer arrangement mentioned before. It

23 Introduction ______makes the whole ion-transfer process softer. However, the ion transfer can be made harsher, if there is a second ion funnel placed directly after the first ion funnel (dual-funnel system, Figure 12b). An increase of the voltage between the exit of ion funnel 1 and the entrance of ion funnel 2 accelerates the ions facilitating dissociative collisions with neutral species similar to skimmer CID (see 3.2.2).

3.2.4 RF-only Multipoles

The multipole guides a broad range of ions without substantial losses to the high-vacuum stage, where the analyser is located. The linear multipoles are usually built up of 4 (quadrupole), 6 (hexapole), 8 (octopole) or more cylindrical rod electrodes arranged in a circular pattern (Figure 16). Each of the two RF voltages with an angular phase shift of π radians is applied to every second electrode (Figure 16). Except for the quadrupole it is not possible to separate the ion motion into two orthogonal independent components. There is no exact solution for the equations of motion.[111] Different approximations are described in the literature.[111-116] Based on the measurement of transmission properties in crossed ion beam- molecular beam experiments, the electric potential within an octopole arrangement is de-

scribed by Equation 41 with R, Θ and Y being cylindrical coordinates. R0 is the free radius of

the octopole, Y is the position along the symmetry axis, ω is the frequency and V0 the peak amplitude of the RF voltage. Further details are discussed below (3.3.1 Linear Quadrupole (Q)).

푅 4 푉(푅, 훩) = 푉0cos(4훩)cos(휔푡) ( ) (41) 푅0

Figure 16. a) Scheme of an RF-only octopole (front view) showing the R-Θ-plane (cylindrical coordinates), b) scheme of an RF-only octopole (side view) showing Y-direction.

24 Introduction ______3.3 Mass Analysers

3.3.1 Linear Quadrupole (Q)

The potential Φ of an electric quadrupolar field can be described by the following expression, where R0 is the distance from the centre to one of the rod electrodes and α, β and γ are weighting constants for the X, Y and Z directions.

훷0 2 2 2 훷 = 2 (훼푋 + 훽푌 + 훾푍 ) (42) 2푅0

A potential which increases quadratically with the distance R causes a linear increase of the field strength in the same direction. These are the conditions necessary to bind a charged particle elastically to an axis or a point in space. The compliance of these conditions is achieved by satisfying the LAPLACE equation ΔΦ = 0.

휕2훷 휕2훷 휕2훷 Δ훷 = ∇2훷 = + + = 0 (43) 휕푋2 휕푌2 휕푍2

Consequently, the sum of the weighting constants has to be zero: α + β + γ = 0. For a two-dimensional field (linear quadrupole Q) the weighting constants are α = −γ and β = 0. The resulting expression for the potential is given by Equation 44.

훷0 2 2 훷 = 2 (푋 − 푍 ) (44) 2푅0

The linear quadrupole is built up of 4 parallel cylindrical rod electrodes assembled in a square configuration. Each of the two voltages which are applied to opposite electrodes is composed of the DC voltage U and the RF voltage V alternating with the frequency ω (Figure 17).

Figure 17. a) Scheme of a quadrupole (front view) showing the R-Θ-plane (cylindrical coordinates), b) scheme of a quadrupole (side view) showing Y-direction.

25 Introduction ______Ions which are exposed to such an undulating field experience attraction and repulsion depending on the momentary polarity of each electrode. A periodic change of the polarity

causes the ions to oscillate in the X-Z-plane while moving along the Y-axis. The force FX which affects an ion of the charge state z and the mass m in X-direction is described by Equa- tion 45.

휕2푋 휕훷 퐹 = 푚푎 = 푚 = −푧푒퐸 = −푧푒 (45) 푋 휕푡2 휕푋

with a being the acceleration, e being the elementary charge and E being the electric field

strength. If the electric potential Φ0 = U + V cos(ωt) which is applied to the electrodes is sub- stituted in Equation 44 and differentiated with respect to X, the potential gradient in X-direction is obtained (Equation 46).

휕훷 푋 = 2 (푈 + 푉cos(휔푡)) (46) 휕푋 푅0

Substitution of Equation 46 in Equation 45 and rearrangement results in Equation 47.

휕2푋 푧푒 2 + 2 (푈 + 푉cos(휔푡))푋 = 0 (47) 휕푡 푚푅0

The ion motion in Z-direction is derived analogously.

휕2푍 푧푒 2 − 2 (푈 + 푉cos(휔푡))푍 = 0 (48) 휕푡 푚푅0

The motion of the ions around the Y-axis is stable, if the amplitudes in X- and Z-direction are limited, so that they do not hit the electrodes. The conditions for this movement can be described in the form of the commonly accepted MATHIEU-equations (Equation 49)

휕2푋 = (푎 − 2푞 cos(2휏))푋 = 0 (49) 휕휏2 푋 푋

where a and q are dimensionless stability parameters. The parameter τ can be expressed as

τ = 1/2 × ωt. Substitution and rearrangement yields Equation 50.

휕2푋 휔2 = (푎 − 2푞 cos(휔푡))푋 = 0 (50) 휕푡2 4 푋 푋

26 Introduction ______Comparison of Equation 47 with Equation 50 yields an expression for the stability parameters aX and qX. The parameters aZ and qZ for the Z-direction are obtained analogously (Equa- tions 51 and 52).

4푧푒푈 푎푋 = 2 2 = −푎푍 (51) 푚푅0휔

2푧푒푈 푞푋 = − 2 2 = −푞푍 (52) 푚푅0휔

Depending on U, V and ω only ions within a certain range of mass-to-charge ratios are able to pass the linear quadrupole. Thus, it can be used as a mass filter for the isolation of the precursor ions in CID experiments.

3.3.2 Quadrupole Ion Trap (QIT)

A three dimensional quadrupolar electric field allows the storage of ions within certain boundaries. Applying this principle WOLFGANG PAUL has developed the QIT and was awarded the nobel prize in physics (together with HANS G. DEHMELT for the development of the penning trap).[117]

3.3.2.1 Fundamentals The QIT consists of two hyperboloid end-cap electrodes, with a hyperbolic ring electrode in between (Figure 18). Applying the voltage Φ0 = U + Vcos(ωt) to the ring electrode an undulating quadrupolar field is generated that keeps the ions on a trajectory within the QIT pre- venting them from hitting the electrodes or escaping from the QIT. The inner radius of the ring electrode is represented by R0 and half the distance between the end cap electrodes is represented by Z0. Both end-cap electrodes provide small holes for the ions to enter the trap and to be ejected, respectively. In order to satisfy the LAPLACE equation for a three-dimensional field (quadrupole ion trap QIT) the weighting constants are α = β = 1 and γ = −2 yielding Equation 53,

훷0 2 2 2 훷 = 2 2 (푋 + 푌 − 2푍 ) (53) 푅0 + 2푍0 which transforms to Equation 54 if expressed in cylindrical coordinates.

27 Introduction ______

a) b) Figure 18. a) Scheme of a quadrupole ion trap with the main RF voltage applied to the ring electrode

and the auxiliary RF voltage applied to the end-cap electrodes. Ω = 1/3 × ω (Ω = auxiliary RF, ω = main RF). b) Potential energy surface of a quadrupole ion trap (QIT). The cylindrical coordinates R, Z define the location of the ion in the trap.

훷0 2 2 훷 = 2 2 (푅 − 2푍 ) (54) 푅0 + 2푍0

Analogous to the linear quadrupole, the radial and axial motion of the ions in a three-dimensional quadrupolar field can be described as follows.

푑2푅 2푧푒 푚 2 + 2 2 (푈 + 푉 cos(휔푡))푅 = 0 (55) 푑푡 푚(푅0 + 2푍0 )

푑2푍 4푧푒 푚 2 − 2 2 (푈 + 푉 cos(휔푡))푍 = 0 (56) 푑푡 푚(푅0 + 2푍0 )

Comparison of the Equations 55 and 56 with Equation 50 yields the stability parameters. (Equations 57).

16푧푒푈 8푧푒푉 푎Z = −2푎R = − 2 2 2 푎푛푑 푞Z = −2푞R = 2 2 2 (57) 푚(푅0 + 2푍0 )휔 푚(푅0 + 2푍0 )휔

Solutions to the MATHIEU equation result in realms of stability and instability. The description of the motion of an ion species within the QIT applying the MATHIEU equation implies a term which reflects the force that acts on an ion residing in a quadrupolar electric field. This perception affords the determination of the two dimensionless MATHIEU parameters a and q, representing quantities containing the mass-to-charge ratio of the trapped ions, the dimensions of the QIT and the quantities and frequencies of the applied potentials.[117-118] However,

the DC voltage is usually not applied (U = 0 V) leading to 푎R = 푎Z = 0. The particular meaning of this is explained below (3.3.2.2 Stability Diagrams).

28 Introduction ______3.3.2.2 Stability Diagrams The equation of motion has two types of solutions resulting in i) instability-trajectories meaning that the ion of interest is not stable and can therefore not be stored in the QIT and ii) stability-trajectories leading to storage times being theoretically infinite. Whether the ion is stable or not depends solely on the two parameters a and q. The a-q-diagram (Figure 19) de- picts the stability realm for ions in a QIT.

Figure 19. a) aZ-qZ-diagram. Stability realms are displayed in light grey. b) aR-qR-diagram. Stability realms are displayed in dark grey. According to Equation 57, the aR- and qR-scale is twice as large as the aZ- and qZ-scale. c) aU-qU-diagram (aU = aR,Z and qU = qR,Z). Areas where the Z- and R-stable regions overlap (A, B, C, ...) represent solutions to the MATHIEU-equation, which allow ions to be stored in the quadrupole ion trap. An enlarged version of area A is shown in Figure 20.

Ions can be stored only, if the MATHIEU parameters a and q are within the stability boundaries with respect to the R- and Z-direction, i. e. where the R- and Z-stable regions overlap in the aU-qU-diagram (Figure 19c, red).[13,117-118] The range for stable mass-to-charge ratios can be adjusted by variation of the slope of the load line a / q = 2U / V (Figure 20).[117] The stability boundary βZ = 1 intersects the qZ-axis at qZ = 0.908. The mass-to-charge ratio which refers to this point is the lowest one that can be stored in the QIT under the corresponding conditions [118] (low mass cut off (LMCO)). The upper m/z limit is not sharply defined. The axial depth 퐷̅Z of the pseudo-potential well can be approximated according to Equation 58. It is inversely proportional to m/z.[118] The pseudo-potential well flattens out for increasing m/z. This leads to an increasing probability for the ions to escape before they are ejected and detected.

푞Z푉 퐷̅ ≈ (58) Z 8

29 Introduction ______

Figure 20. Enlargement of the overlapping area A from Figure 19c. In order to keep things simple only the Z-parameters are shown.

3.3.2.3 Secular Frequencies The ion movement is a superposition of slow secular vibrations with the fundamental fre-

quencies ωR,Z and very fast micro-movements. The frequency ω of the main RF voltage applied to the ring electrode corresponds to the fundamental frequency of the ion movement. The factor β (0 ≤ β ≤ 1), which determines the frequency, is a function of the MATHIEU parameters a and q.[117] The resulting ion movement has the general appearance of a LISSAJOUS curve. This is explained by the undulation of the quadrupolar field or the potential surface, respectively. This undulation can also be seen as a rotation of the saddle shaped potential surface. The ions perform approximate harmonic vibrations in R- and Z-direction. They behave as if they were moving on a potential surface that increases quadratically in all coordinates. The depth of the pseudo-potential well is determined by the amplitude of the applied voltage V and by the parameters a and q. The well in R-direction is as half as deep as in Z- direction.

3.3.2.4 Mass-Selective Instability Mode One possibility of using the QIT as mass analyser is to destabilise the ions successively in the order of their mass-to-charge ratio. First, all ions will be stored in the QIT. Since there is no

DC voltage applied (U = 0 V, aZ = 0), every m/z can be explicitly assigned to a qZ-value within

the range from βZ = 0 to βZ = 1 on the qZ-axis (Figure 20). A large mass-to-charge ratio refers to

a small qZ-value and vice versa (Equation 57, Figure 21a). An increase in the RF voltage V

leads to an increase in the qZ-value for all ions trapped, which means a right-shift of all ion species in the stability diagram (Figure 21b) and thus a consecutive ejection of the different

30 Introduction ______ions in Z-direction, beginning with the lowest m/z-value. Ion ejection happens at the stability border where qZ = 0.908 meaning βZ = 1.

Figure 21. Part of the stability area shown in Figure 20 at a) the RF-potential V1, which is smaller than b) the RF-potential V2. The increase in the main RF-potential V affords a shift of the trapped ions to higher qZ-values.

3.3.2.5 Resonant Excitation In order to cool the ions down and to concentrate them in the centre of the QIT, they are exposed to collisions with helium atoms (buffer gas). The kinetic energy is reduced to approximately 0.1 eV which translates to a temperature of roughly 800 K (Ekin = 1.5 × kBT). The ions diverge to less than 1 mm from the centre of the QIT.[118]

The axial and the radial ion movement, which are determined by the two components ωZ and ωR of the secular frequency ωU, can be stimulated independently from each other. Ap- plying an additional RF voltage (with the frequency Ω) to the end-cap electrodes results in a time-dependent increasing ion amplitude in Z-direction in the case of resonance (Ω = ωZ).[117] Resonant excitation can be applied to i) quickly eject certain ion species (e. g. m/z-scan by resonant ejection) and ii) to accelerate ions of interest within the helium buffer / collision gas for CID.

3.3.2.6 Resonant Ejection

Ejection occurs at qZ < 0.908, if the auxiliary RF, which is applied to the end-cap electrodes, accords with the axial component of the secular frequency ωZ of a certain ion.

훽Z 휔 = (n + ) 휔 n ∈ ℕ+ (59) Z 2 0

For the BRUKER esquire6000 the auxiliary RF is set to one third of the main RF (Ω = 1/3 × ω).

Equation 59 yields βZ = 2/3 for the fundamental (n = 0) secular ion motion in Z-direction under resonance conditions (Figure 22a). The qZ-value where βZ = 2/3 intersects the qZ-axis characterises the point of resonant ejection. The resulting gain in kinetic energy enables the resonant ion to escape from the quadrupolar trapping field in Z-direction (Figure 22b).

31 Introduction ______

Figure 22. a) Curve βZ = 2/3 intersects the qZ-axis at the point where resonance causes instability of the corresponding ion. b) Axial component (Z-direction) of the ion motion in the quadrupole ion trap for an ion achieving resonance.

An increase of the amplitude of the main RF voltage V at the ring electrode in the course of time lets ions with different mass-to-charge ratios consecutively come into resonance at

qZ < 0.908 (βZ = 2/3). Ejection occurs in the order of m/z and time-dependent detection of the abundance of the ejected ions yields a mass spectrum.

3.3.2.7 Collision-Induced Dissociation (CID) For CID experiments fast internal energy gain of previously isolated ion species is required without ejecting the ions from the trap. Resonant excitation of the ion movement in Z-direction leads to increasing kinetic energy resulting in internal energy gain due to momentum- exchange collisions with the buffer gas atoms. Changes in kinetic energy occur much faster (10−6 s) than changes in internal energy (10−3 s). Therefore, a short ion cooling period after each excitation period leads to rapid kinetic energy loss while the internal energy is practic- ally unaffected (kinetic cooling). Repeating this cycle allows for an incremental increase in internal energy resulting in enhanced fragmentation.[118]

3.3.2.8 Tandem Mass Spectrometry (MSn) The QIT facilitates tandem-in-time experiments, i. e. the single steps of the tandem-MS experiment occur subsequently in the course of time, however, in the same place. First, all ions over the whole m/z-range are stored and cooled down by collisions with the buffer gas atoms (Figure 23a). Second, all ions which are not of interest are ejected applying the mass-selective instability mode (page 30) to ions of smaller m/z-value than the ion of interest and applying multiple frequencies to the end-cap electrodes (resonant ejection) for the ejection of ions of higher m/z-value. What remains are the ions of interest within a narrow m/z-range (b). In a third step fragmentation is induced by resonant excitation of the isolated ions (c). Due to the differing m/z-ratios of the fragment ions they experience no further excitation. Instead, kinetic cooling occurs which allows the ions to focus at the centre of the QIT (d). Ejection of the ions in the order of their m/z-values (mass-selective instability mode with resonant ejection) and subsequential detection (e) affords the MS2-CID spectrum (f). n-fold repetition of the steps b – d leads to MSn-tandem mass spectrometry.

32 Introduction ______

Figure 23. Individual steps of an MS2 collision-induced dissociation (CID) experiment in the quadrupole ion trap (QIT).

3.3.3 Time-of-Flight (TOF) Analyser

3.3.3.1 TOF Principle All ions being accelerated in the MALDI ion source and carrying the same charge gain the same amount of kinetic energy. (Equation 60, Figure 24).

1 퐸 = 푒푧푉 = 푚푣2 (60) kin 2

Substitution of the velocity v for d / t and rearrangement yields an expression for the time an ion with a certain mass-to-charge ratio needs to pass through the drift zone (Equation 61). The time is directly proportional to (m/z)1 / 2.

푑 푚 푡 = √ (61) √2푒푉 푧

Thus, measuring the time an ion needs from leaving the ion source to being detected allows for the determination of its mass-to-charge ratio.

Figure 24. Scheme of a time-of-flight (TOF) analyser.

33 Introduction ______3.3.3.2 Reflectron (Ion Mirror) The MALDI process affords gas-phase ions being generated at different positions close to the sample surface. The difference in the starting position results in slightly different kinetic energies for ions having the same mass-to-charge ratio. In order to reduce the resulting peak broadening in the mass spectrum (linear mode), a reflectron acting as an ion mirror, can be used. It consists of an array of ring electrodes (Figure 25). The applied linear field gradient influences the flight path, i. e. the depth an ion plunges into the reflectron, according to its kinetic energy. Therefore, fast ions having high kinetic energy tread longer pathways than slower (low kinetic energy) ions of the same mass-to-charge ratio. The excess time of tarrying in the reflectron compensates for the higher ion velocity resulting in narrower peak shapes.

Figure 25. Matrix-assisted laser desorption / ionisation (MALDI) ion source with reflectron-time-of- flight (ReTOF) analyser. The reflectron, acting as an ion mirror, enhances the resolution by compen- sating kinetic energy spread.

Another design is the curved field reflectron (CFR). While the linear reflectron field requires in MS / MS experiments (PSD, page 35) the stepping down of the reflectron voltages and re- stiching of voltage regions for a complete PSD spectrum, this is not necessary with a CFR. The CFR focusses all daughter ions directly onto the detector and requires in principle only one “scan” to record the full PSD mass spectrum.[119-120] The CFR can also be coupled directly with a preceding high-energy collision cell, which required for the linear reflectron the development of tandem TOF instrumentation and the method of LIFT, which is described elsewhere.[121-123]

34 Introduction ______3.3.3.3 Delayed Extraction Depending on their genesis, the ions which are formed during the desorption / ionisation process (page 12), gain different amounts of kinetic energy, leading to different initial velocities for the same ionic species. In order to overcome the resulting peak broadening, a time delay is introduced before the acceleration voltage is applied to the source electrodes (delayed extraction, Figure 26a). Due to the drift during the delay, the ions have a different starting position when the electric field builds up (b). The different gain in kinetic energy during the ion extraction process compensates for the difference in initial velocity. This nar- rows down the time range in which ions of the same mass-to-charge ratio arrive at the detector (c) resulting in narrower peaks in the mass spectrum. Delayed Extraction is the com- mercial name for the dual stage acceleration method, developed by Wiley & McLaren[124] to enhance resolution in a linear TOF MS.

Figure 26. Delayed extraction. a) Drift of ions of the same species with different initial velocities v0 under field free conditions for several hundred nanoseconds. b) Different starting positions result in different energy gain. c) The higher initial velocity was compensated by a lower kinetic-energy gain (and vice versa) resulting in a narrower time distribution of the ions arriving at the detector.

3.3.3.4 Post-Source Decay (PSD)

If metastable ions fragment in the field-free region, both the neutral fragment species F•PSD and the charge carrying fragment species F+PSD still have the same velocity as the precursor ion species M+• (Reaction 62).

+• • + Mp → F푃푆퐷 + FPSD (62)

35 Introduction ______

Consequently, F•PSD and F+PSD will both hit the linear detector at the same time as M+• would do, if the instrument was operated in the linear mode (reflectron voltage off). The dissoci-

ation that had occurred remains hidden. However, F+PSD and M+• are repelled by the reflec-

tron field. On the one hand the F+PSD species spends less time in the reflectron than M+•, due

to its lower mass and thus lower kinetic energy. If M+• and thus its dissociation product F+PSD

was selected by an ion gate, the use of a reflectron can separate M+• and F+PSD in time, with

F+PSD arriving first at the detector. The result is a PSD spectrum with M+• as parent and F+PSD

as the daughter ion signal (MS2). On the other hand, the overall time-of-flight of F+PSD, which

has the same velocity as the heavier precursor ion species M+•, is larger than of F+ISD. F+ISD represents the in-source-decay (ISD) fragment ion, that has received the full acceleration

voltage as it was produced inside the ion source. The F+ISD ion is thus faster than F+PSD. If the PSD process is abundantly enough occurring, all three signals may be observable in the

“normal” mass spectrum (MS1) resulting in a spectrum, where the F+PSD-signal is located in

between the signals of F+ISD and M+• (Figure 27).

Figure 27. Matrix-assisted laser desorption / ionisation (MALDI) Reflectron time-of-flight (ReTOF)

mass spectrum of Tr*−(C≡C)10−Tr* / DCTB recorded in the positive-ion mode. tBu-loss (Δ(m/z) = 57.07 u) yields two signals caused by in-source decay (ISD) and post-source decay (PSD). The instrument parameter was determined to be P = 0.786(3).[125]

The change of the trajectories and velocities of the fragments due to the energy release during the fragmentation causes peak broadening and was not taken into account in the above

discussion.[126-127] Considering the above mentioned relation of M+•, F+PSD and F+ISD, a parameter P depending only on the extraction voltage and the instrument geometry can be calculated using a known dissociation reaction. In turn, the knowledge of this parameter P allows the validation of the correlation between any PSD-, ISD- and precursor ion signal (Equation 63).[125]

푚 +• − 푚 +• 푚 + M푃 √ M푃 F푃푆퐷 푃 = (63) 푚 +• 푚 + − 푚 + √ M푃 F푃푆퐷 F퐼푆퐷

36 Introduction ______3.3.3.5 Collision-Induced Dissociation (CID) The CID mechanism is divided into two steps. i) excitation of the precursor ion by collision with the collision gas and ii) the unimolecular decay of the excited precursor ion which can be described by the quasi-equilibrium or Rice-Ramsperger-Kassel-Marcus (RRKM) theory.[128-131] Principally the collisions are classified by the energy regime in which they occur: i) low energy collisions up to several hundred eV and high energy collisions in the range of keV. In order to estimate the internal energy that is transferred during single high-energy collisions, the laboratory frame energies have to be converted into the centre-of-mass frame applying Equation 64.[132-136]

푚푐표푙푙𝑖푠𝑖표푛 𝑔푎푠 퐸COM = 퐸LAB × (64) 푚푐표푙푙𝑖푠𝑖표푛 𝑔푎푠 + 푚푝푟푒푐푢푟푠표푟 𝑖표푛

High-energy collisions are often conducted under single collision conditions, while low-energy CID is mostly performed under multiple collision conditions. Fragmentation rates decrease drastically with an increasing ion size (i. e. number of oscillators / vibrational DOF). The internal energy necessary to dissociate large ions at observable fragmentation rates is significantly higher than the critical energy (the minimum internal energy of the precursor ion needed to overcome the energy barrier of the dissociation). The difference is called the “kinetic shift”. It characterises the internal energy above the critical energy necessary to drive the reaction fast enough to observe dissociation. In order to induce dissociation, the internal energy per oscillator (DOF) should be roughly the same for different ions of similar constitution. This is reflected in the fact that the internal energy necessary for dissociation increases almost linearly with the number of degrees of freedom (NDOF).[137] The internal energy a precursor ion stores is quickly redistributed to all its oscillators (vibrational DOF). In order to compare the internal excitation during collisions of different precursor ions one has to correct by dividing the collision energy by NDOF of the precursor ion.[137-144]

3.4 Detector

Ion detectors are usually secondary electron multipliers (SEM). The impact of an accelerated ion onto the detector surface, to which a voltage is applied, causes electrons to be emitted from the surface material (conversion dynode). The amount of the initial ions is increased by 6 – 9 orders of magnitude in order to produce a measureable current. This increase is achieved by accelerating the emitted electrons towards a second electrode (dynode) with a lower potential applied to it. Every electron hitting a dynode surface causes several electrons to be emitted from the dynode. An array of several discrete dynodes with a potential gradient affords the required signal enhancement.

37 Introduction ______3.4.1 Channel Electron Multiplier (CEM, Channeltron)

CEMs have the same working principle as SEMs with discrete dynodes. However, the CEM is a single tube with a voltage of approximately 2 kV applied to the ends of the detector (Figure 28). Linear CEMs work properly up to a multiplication of 104 (Figure 28a). Curved CEMs perform enhancements of the factor 108, due to shorter electron drift pathways and the resulting higher signal-to-noise ratio (Figure 28b).[3]

Figure 28. a) Linear channel electron multiplier (CEM, Channeltron). b) Curved CEM.

3.4.2 Micro Channel Plate (MCP)

MCPs are round plates (diameter 2 – 5 cm) having millions of small diagonal channels (diameter in the low μm-range). The current multiplication factor is about 104 (Figure 29a and b). Arrangements of two (chevron plate) or more plates above each other afford enhanced signals (106 – 107, Figure 29b and 108, Figure 29c).[3]

Figure 29. Micro channel plate (MCP). a) top view, b) lateral cut-away view, c) double layer MCP (chevron plate), lateral cut-away view, d) triple layer MCP, Z-stacked, lateral cut-away view.

3.5 Isotope Pattern

The natural occurrence of constant abundance ratios of the different isotopes of each element leads to a particular isotope pattern for each ion species. Consequently, the isotope pattern carries the information of the elemental composition of the detected ion species. By calculat- ing the pattern for a putative sum formula, it can be compared to the experimental result. This is a powerful tool which complements the mass determination (accurate mass measurement) of a certain ion species in the ion-identification process.

38 Introduction ______3.6 Resolution & Resolving Power[144]

3.6.1 Resolution: 10 %-Valley Definition

In order to determine the resolution according to the 10 %-valley definition, two signals of equal height which are separated by a valley at 10 % of the height of either peak are required (Figure 30).

Figure 30. 10 %-valley definition for resolution in mass spectrometry.

The resolution is then calculated according to Equation 65, with Δ(m/z) being the difference between the two peaks on the m/z axis and m/z referring to the ion of lower mass.

푚/푧 푅푣푎푙 = (65) 10 % Δ(푚/푧)

3.6.2 Resolution: Peak Width Definition

According to Equation 66 the resolution can also be calculated from a single peak (Figure 31) generated by singly charged ions at mass m, where Δm is the full width at a specified fraction of the peak height (0.5, 5, or 50 %).

푚 푚 푚 푅푤𝑖푑 = 표푟 푅푤𝑖푑 = 표푟 푅푤𝑖푑 = (66) 0.5 % Δ푚 5 % Δ푚 50 % Δ푚

3.6.3 Full Width at Half Maximum (FWHM)

The full width at half the intensity of a peak is also commonly used as a measure for the resolution (Figure 31). Here FWHM itself is the measure for the resolution (FWHM = Δ(m/z) wid at 50 % of the peak height) and is not to be confused with the resolution R50 %.

39 Introduction ______

Figure 31. Full width at half maximum (FWHM).

3.6.4 Resolving Power in Mass Spectrometry

“Resolving power in mass spectrometry is defined as the ability of an instrument or measurement procedure to distinguish between two peaks at m/z values differing by a small amount and expressed as the peak width in mass units”.[144]

3.7 Instruments

3.7.1 BRUKER esquire6000

Figure 32. Set-up of the BRUKER esquire6000 electrospray ionisation-quadrupole ion trap-mass spectrometer (ESI-QIT-MS).

3.7.2 BRUKER micrOTOF-Q II

The BRUKER micrOTOF-Q II operates a dual-funnel stage which can be used for collisional activation of the whole m/z-range similar to skimmer CID. This type of dissociation is re- ferred to as “in-source CID”. Mass selected ions can be studied in MS2-CID experiments. These are conducted in a quadrupole collision cell (q, Figure 33) which is located between the preceding analytical quadrupole (Q) and the subsequent orthogonal ReTOF mass analyser. The collision gas is nitrogen. This set-up allows to perform energy dependent dissociation experiments for the comparison of bond strengths. It is notable that, in contrast to the QIT, all fragment ions (and neutrals) may undergo further excitation and dissociation. This is

40 Introduction ______because the fragments generated in the collision cell keep hold of the velocity of their precursor ion resulting in further collisions with the collision gas.

Figure 33. Set-up of the BRUKER micrOTOF-Q II electrospray ionisation-quadrupole-time-of-flight mass spectrometer (ESI-Qq-TOF-MS).

3.7.3 BRUKER Reflex IV

The set-up of the BRUKER Reflex IV is depicted in Figure 25 (page 34). Further information on the instrument geometry can be found in the literature.[145]

3.7.4 SHIMADZU Axima Confidence

The set-up of the SHIMADZU Axima Confidence is principally the same as for the BRUKER Reflex IV. However, the Axima operates a curved field reflectron, which allows for the re- cording of PSD mass spectra by carrying out only one single MS2-experiment rendering the stepping of the reflectron voltage unnecessary. Furthermore the drift tube is oriented verti- cally.

41 Introduction ______4 Compounds

4.1 Polyynes

Figure 34. Polyynes of different chain-lengths with the protecting end groups Tr* = supertrityl and tBu = tertiary butyl group. Given m/z values refer to the most intense peak of the isotope envelope.

4.2 Fullerenes and Fullerene Derivatives

Buckminster fullerenes, which were discovered by SIR HAROLD KROTO[146] and RICHARD E.

SMALLEY,[147] are football-shaped molecules with Ih-symmetry consisting of 60 carbon atoms.

They are named after the architect RICHARD BUCKMINSTER FULLER. The structure of the C60-

fullerene is shown in Figure 35. The chemistry and properties of C60 are discussed elsewhere.[148-154]

Figure 35. C60-fullerene: a) LEWIS-structure, pentagon-centred. b) Ball-and-stick-model, pentagon- centred. c) LEWIS-structure, hexagon-centred. d) Ball-and-stick-model, hexagon centred.

42 Introduction ______Enumeration and the relative positional relationships of [6,6]-bonds are depicted in Figure 36.

Figure 36. SCHLEGEL diagrams of C60-fullerene: a) Positions of different [6,6]-bonds relative to the black [6,6]-bond. e = equatorial, trans-1 means the position axial to the black [6,6]-bond.[149-150] b) Enu- meration of the carbon atoms.[150,152,155]

Fluorofullerenes are fullerenes carrying a certain number (theoretical maximum = 60) of fluor atoms. The structure of C60F44 may serve as an example for the appearance of fluorofullerenes (Figure 37). The properties of fluorofullerenes are described in the literature.[156-160]

Figure 37. C60F44-isomer with D2-symmetry: a) SCHLEGEL diagram with pentagonal centre. b) Ball-and- stick model.[161]

43 Introduction ______4.3 Perylenetetracarboxylic Acid Diimides (PDIs)

Figure 38. Chemical Structures of the perylenetetracarboxylic acid diimides (PDIs) under investigation.

44 Introduction ______4.4 Polycationic [60]Fullerene Hexakis-Adduct

The polycationic C60-fullerene hexakis adduct is a fullerene derivative carrying twelve permanent positive charges, which are compensated by twelve bromide counter ions.

Figure 39. [60]Fullerene hexakis adduct. The net charge state depends on the bromide content. If the 12 positive pyridinium charges are compensated by n bromide ions (n < 12) the net charge state is +(12 − n). Given m/z values refer to the most intense peak of the isotope envelope.

45 Introduction ______4.5 Neutral Transition Metal Complexes (nTMCs)

Efficient organic light emitting diodes (OLEDs) employ an active multi-layer medium consisting of low-molecular-weight compounds like the neutral transition metal complexes (nTMCs) shown in Figure 40.

Figure 40. Neutral transition metal complexes (nTMCs) are used as active medium of organic light emitting diodes (OLEDs). a) Bis(2-methyldibenzo[f,h]quinoxaline)(acetylacetonate)iridium(III) = Ir(MDQ)2(acac). b) Tris(2-phenylpyridine)iridium(III) = Ir(ppy)3.

46 References ______

References

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56 Oral Contributions & Poster Presentations ______

Oral Contributions & Poster Presentations

Formation and Fragmentation of Sodiated Amino Acid Cluster Ions in the Gas Phase  Oral presentation at the Joint Conference of Polish Mass Spectrometry Society (PTSM) and German Mass Spectrometry Society (DGMS) 2012 in Poznań, POLAND.  Oral presentation at the 30th Informal Meeting on Mass Spectrometry (IMMS) 2012 in Olomouc, CZECH REPUBLIC.

How Electronic Properties Govern the Behaviour of Isolated Fluorinated Fullerene Dianions in the Gas Phase ✎ Poster presentation at the 2nd Erlangen Symposium on Synthetic Carbon Allotropes 2013 in Erlangen, GERMANY.

✎ Poster presentation at the BRUKER User Meeting 2014 in Berlin, GERMANY.

Ligand-Sphere Chemistry and Sphere-Sphere Interactions of Negatively Charged Alkoxylated Fullerenes  ✎ Oral presentation and poster presentation at the 32nd Informal Meeting on Mass Spectrometry (IMMS) 2014 in Balatonszárszó, HUNGARY. ✎ Poster presentation at the 20th International Mass Spectrometry Conference (IMSC) 2014 in Genève, SWITZERLAND.

✎ Poster presentation at the BRUKER User Meeting 2015 in Kassel, GERMANY. ✎ Poster presentation at the 3rd Erlangen Symposium on Synthetic Carbon Allotropes 2015 in Erlangen, GERMANY.

Gas-Phase Experiments with Polyynes: Laser Activation and Ag+-Induced Reactivity  Oral presentation at the 33rd Informal Meeting on Mass Spectrometry (IMMS) 2015 in Szczyrk, POLAND.

57 Oral Contributions & Poster Presentations ______Investigation of the Electronic Properties of Polyynes and Polyyne Rotaxanes by Silver Induced Gas-Phase Reactivity ✎ Poster presentation at the 3rd Erlangen Symposium on Synthetic Carbon Allotropes 2015 in Erlangen, GERMANY.

58 Papers & Patents ______

Papers & Patents

Publication 1 Synthesis and Aggregation Properties of Polycationic Perylenetetracarboxylic Acid Diimides Jörg Schönamsgruber, Boris Schade, Rolf Kirschbaum, Jing Li, Walter Bauer, Christoph Böttcher, Thomas Drewello, and Andreas Hirsch Eur. J. Org. Chem. 2012, 2012, 6179 – 6186, DOI: 10.1002/ejoc.201201062

The paper covers synthesis, structural characterisation and aggregation behaviour of highly water-soluble dendronised polycationic perylenetetracarboxylic acid diimides (PDIs) carrying 6, 9, and 18 preformed permanent positive charges. Due to the pyridinium termination, the water-solubility does not depend on the pH value. The compounds with the smaller first- generation dendrons show enhanced aggregation (π-π stacking of the perylene cores) in water, which is detained in the case of the bulkier second-generation dendrons. Although the charges are preformed and permanent, it is not trivial to obtain mass spectra from this compound class. The spray conditions had to be adjusted to allow ion formation by separation of the ion pairs. The publication comprises ESI-QIT-MS data of all target compounds covering charge states from 2+ to 7+.

Publication 2 Formation of Highly Charged Quasi-Molecular Ions of a Polycationic [60]Fullerene Hexakis-Adduct and Their Fragmentation Behavior in the Gas Phase Jing Li, Leanne C. Nye, Lennard K. Wasserthal, Chau Vinh, Rolf W. Kirschbaum, Ivana Ivanović-Burmazović, Andreas Hirsch, and Thomas Drewello Eur. J. Org. Chem. 2015, 2015, 2282 – 2290, DOI: 10.1002/ejoc.201500102

This investigation includes synthesis, structural characterisation and investigation of the gas- phase behaviour of a novel polycationic [60]fullerene hexakis-adduct comprising twelve preformed positive charges and covering a charge-state envelope from 3+ to 12+ in ESI-MS. CID experiments revealed three fragmentation pathways. Neutral losses result in daughter ions preserving the charge state of the highly charged precursor ion. Furthermore two different kinds of COULOMB explosions result in singly charged fargments and the corresponding charge reduced [60]fullerene hexakis-adduct fragment species.

59 Papers & Patents ______

Publication 3 Laser Desorption Mass Spectrometry of End Group-Protected Linear Polyynes: Evidence of Laser-Induced Cross-Linking Rolf W. Kirschbaum, Dominik Prenzel, Stephanie Frankenberger, Rik R. Tykwinski, and Thomas Drewello J. Phys. Chem. C 2015, 119, 2861 – 2870, DOI: 10.1021/jp5112444

The cross-linking of end group-protected linear polyynes of composition Tr*−(C≡C)n−Tr* and

tBu−(C≡C)6−tBu is studied by LDI-TOF MS. The abundant formation of oligomeric species upon laser activation and tandem MS reveal strong bonding within the oligomers. Com-

pared to the LDI-induced coalescence reaction of C60, the cross-linking of the polyynes is less energy demanding and more efficient. Smaller end groups and longer sp-carbon chains result in larger oligomers. Cross-linking is reduced if the solid sample is diluted by addition of the matrix substance DCTB. Comparison of CID experiments of Ag+-tagged polyyne dimers generated by MALDI and ESI provide evidence for the existence of different binding motifs.

Publication 4 Energy-Dependent Gas-Phase Fragmentation of Fluorofullerene Multiply Charged Anions (MCAs) Rolf W. Kirschbaum, Markus Hausmann, Olga V. Boltalina, Steven H. Strauss, and Thomas Drewello Phys. Chem. Chem. Phys. 2015, 17, 23052 – 23058, DOI: 10.1039/C5CP03112E

Long-lived di- and trianions have been formed from fluorofullerenes in the gas phase by electrospray ionisation. It was possible to study dianions with odd and even fluorine attain- ment, possessing even and odd electron configurations, respectively. Two complementary CID experiments have been carried out. MSn experiments in a QIT explain the connectivity of precursor ion to product ion and nth-generation product ions on the one hand and energy- dependent CID experiments with a Qq-TOF instrument on the other hand disclose the energetic demands of the different dissociation channels being inherent to the electron configuration of the respective precursor ion. Dianions with the less stable odd-electron configuration dissociate into species with the more stable even-electron configuration. Dianions with an odd electron count release a neutral F• atom to become an even electron system. Dianions with an even electron count undergo a more energy demanding COULOMB explosion into an even electron monoanion and an F− anion. The studied trianions behave accordingly.

60 Papers & Patents ______

Publication 5

On The Mechanism of Zn4O-Acetate Precursors Ripening to ZnO: How Dimerization is Promoted by Hydroxide Incorporation Theodor Milek, Rolf W. Kirschbaum, Marc S. v. Gernler, Christian Lübbert, Doris Segets, Thomas Drewello, Wolfgang Peukert, Dirk Zahn. J. Chem. Phys. 2015, 143 (064501), 1 – 4, DOI: 10.1063/1.4928190

This study describes Zn4O-ion clusters stabilised by acetate anions (AcO−). Ab initio calculations of acetate substitution by hydroxide ions are compared with mass spectrometry data. Though quantum calculations in the gas phase indicate strong energetic preference, no experimental evidence of stable Zn4O(OAc)6−x(OH)x clusters in ethanolic solutions could be observed. This apparent contradiction is rationalised by identifying the supportive role of hydroxide ions for the association of Zn4O(OAc)6 and Zn4O(OAc)5+ clusters. Mass spectrometry and quantum calculations hint at the stability of (Zn4O)2(OAc)12−x(OH)x dimers with x = 1, 2. Therein, the hydroxide ions establish salt-bridges that allow for the formation of additional Zn3 motifs with the OH above the triangle centre — a structural motif close to that of the ZnO-crystal. The association of Zn4O(OAc)6 clusters is thus suggested to involve AcO− → OH− substitution as an activation step, quickly followed by dimerisation and the subsequent agglomeration of oligomers.

Publication 6 Metal Ion Adducts of Neutral Phosphorescent Emitters for Use in Light-Emitting Organic Optoelectronic Components Thomas Drewello, Rolf Kirschbaum, Ana-Maria Krestel, Günter Schmid, Dirk Michael Guldi, Florian Kessler Applicant: Siemens Aktiengesellschaft, WO/2015/086400

Sufficient efficiency of OLEDs is so far obtained only by the use of an active multi-layer medium consisting of low-molecular-weight compounds like the neutral transition metal complexes (nTMCs). While the manufacturing of polymer-based OLEDs would principally be easier and more cost-effective, their efficiency and lifetime is falling short of expectations. The latest types of organic light emitting electrochemical cells (OLEECs) show that they can potentially combine cost-effective manufacturing with the advantages of low-molecular- weight compounds. Mass spectrometry gives proof that nTMCs show enhanced attractive interactions with alkali metal ions such as Li+, Na+, K+, Rb+ and Cs+. This discovery gives rise to the development of a new kind of positively charged ionic transition metal complexes (iTMC) which can be used as active materials in OLEECs. The patent claims the invention of a compound of the general formula M+(nTMC)x (M = Li, Na, K, Rb, Cs; x = 1, 2).

Appendix ______

Appendix

Due to copyright restrictions the publications are available on the internet:

Synthesis and Aggregation Properties of Polycationic Perylenetetracarboxylic P1 Acid Diimides Jörg Schönamsgruber, Boris Schade, Rolf Kirschbaum, Jing Li, Walter Bauer, Christoph Böttcher, Thomas Drewello, and Andreas Hirsch Eur. J. Org. Chem. 2012, 2012, 6179 – 6186 http://dx.doi.org/10.1002/ejoc.201201062

Formation of Highly Charged Quasi-Molecular Ions of a Polycationic P2 [60]Fullerene Hexakis-Adduct and Their Fragmentation Behavior in the Gas Phase Jing Li, Leanne C. Nye, Lennard K. Wasserthal, Chau Vinh, Rolf W. Kirschbaum, Ivana Ivanović-Burmazović, Andreas Hirsch, and Thomas Drewello Eur. J. Org. Chem. 2015, 2015, 2282 – 2290 http://dx.doi.org/10.1002/ejoc.201500102

Laser Desorption Mass Spectrometry of End Group-Protected Linear Polyynes: P3 Evidence of Laser-Induced Cross-Linking Rolf W. Kirschbaum, Dominik Prenzel, Stephanie Frankenberger, Rik R. Tykwinski, and Thomas Drewello J. Phys. Chem. C 2015, 119, 2861 – 2870 http://dx.doi.org/10.1021/jp5112444

63 Appendix ______

Energy-Dependent Gas-Phase Fragmentation of Fluorofullerene Multiply P4 Charged Anions (MCAs) Rolf W. Kirschbaum, Markus Hausmann, Olga V. Boltalina, Steven H. Strauss, and Thomas Drewello Phys. Chem. Chem. Phys. 2015, 17, 23052 – 23058 http://dx.doi.org/10.1039/C5CP03112E

On The Mechanism of Zn4O-Acetate Precursors Ripening to ZnO: P5 How Dimerization is Promoted by Hydroxide Incorporation Theodor Milek, Rolf W. Kirschbaum, Marc S. v. Gernler, Christian Lübbert, Doris Segets, Thomas Drewello, Wolfgang Peukert, Dirk Zahn. J. Chem. Phys. 2015, 143 (064501), 1 – 4 http://dx.doi.org/10.1063/1.4928190

Metal Ion Adducts of Neutral Phosphorescent Emitters for Use in Light- P6 Emitting Organic Optoelectronic Components Thomas Drewello, Rolf Kirschbaum, Ana-Maria Krestel, Günter Schmid, Dirk Michael Guldi, Florian Kessler Applicant: Siemens Aktiengesellschaft, WO/2015/086400 https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2015086400

64 Appendix ______Abstract This work contributes to the understanding of ion formation reactions accompanying the transition of molecules from the condensed phase into the gas phase. Furthermore, structural features of the gaseous carbon-based ions are elucidated. The applied scientific tools comprise the use of soft ionisation methods such as matrix-assisted laser desorption / ionisation (MALDI) and electrospray ionisation (ESI) in conjunction with tandem mass spectrometry in the form of collision-induced dissociation (CID) experiments. The compounds under investigation are of particular importance in the context of new synthetic carbon allotropes and carbon-rich molecules and comprise compound classes such as polyynes, fullerene derivatives, dendrimeric perylenetetracarboxylic acid diimides (PDIs), acetate clusters and neutral transition metal complexes (nTMCs). The studies cover i) the synthesis, structural characterisation and aggregation behaviour of highly water-soluble dendronised PDIs carrying 6, 9, and 18 preformed permanent positive charges, ii) the synthesis, structural characterisation and fragmentation behaviour of a novel polycationic [60]fullerene hexakis-adduct featuring twelve preformed positive charges and covering a charge-state envelope from 3+ to 12+ in ESI-MS, iii) the cross-linking of end group-protected linear polyynes of composition Tr*−(C≡C)n−Tr* and tBu−(C≡C)6−tBu by LDI-TOF-MS revealing strong bonding within the oligomers, iv) the investigation of long- lived fluorofullerene di- and trianions with even and odd electron configurations by two complementary CID experiments revealing the connectivity of precursor ion to the nth- generation product ions and disclosing the energetic demands of precursor ions with different electron configurations, v) the investigation of the supportive role of hydroxide ions for the transformation of zinc acetate clusters to Zn4O employing ESI-MS and ab initio calculations of acetate substitution by hydroxide ions, and vi) the enhanced attractive interactions of nTMCs with alkali metal ions such as Li+, Na+, K+, Rb+ and Cs+ giving rise to the development of a new kind of positively charged ionic transition metal complex (iTMC) for the potential use as active materials in organic light emitting electrochemical cells (OLEECs).

65 Appendix ______Zusammenfassung Die vorliegende Arbeit befasst sich mit den Mechanismen der Ionenbildung beim Übergang von Molekülen aus der kondensierten in die Gasphase, sowie der Strukturaufklärung gasförmiger, kohlenstoffbasierter Ionen. Die angewandten wissenschaftlichen Methoden umfassen sanfte Ionisationsverfahren wie Matrixunterstützte Laser-Desorption / Ionisation (MALDI) und Elektrosprayionisation (ESI) in Verbindung mit Tandemmassenspektrometrie in Form von stoßinduzierten Dissoziationsexperimenten (CID). Die untersuchten Verbin- dungen sind von besonderer Bedeutung im Zusammenhang mit neuen synthetischen Koh- lenstoffallotropen und kohlenstoffreichen Molekülen und umfassen Verbindungsklassen wie Polyine, Fullerenderivate, dendrimere Perylentetracarbonsäurediimiden (PDIs), Acetatclus- ter und neutrale Übergangsmetallkomplexe (nTMCs) . Die vorliegenden Studien umfassen i) die Synthese, strukturelle Charakterisierung und das Aggregationsverhalten gut wasserlöslicher dendrimerisierter PDIs, welche 6, 9 und 18 per- manente positive Ladungen tragen, ii) die Synthese, strukturelle Charakterisierung und das Fragmentierungsverhalten eines neuartigen polykationischen [60]Fulleren-Hexakis-Addukts mit 12 positiven Ladungen, wobei sich die Ladungszustände im ESI-MS-Experiment von 3+ bis 12+ erstrecken, iii) die Erforschung der chemischen Vernetzung von endgruppenge-

schützten, linearen Polyinen des Typs Tr*−(C≡C)n−Tr* and tBu−(C≡C)6−tBu, welche starke, kovalente Bindungen innerhalb der Oligomere offenbart, iv) die Untersuchung von langle- bigen Fluorofullerendi- und trianionen mit gerad- und ungeradzahliger Elektronenkonfigu- ration durch zwei komplementäre Stoßexperimente, welche die Abstammung verschiedener Generationen von Produktionen aufzeigt und den Energiebedarf für die Dissoziation von Vorläuferionen mit unterschiedlicher Elektronenkonfigurationen aufzeigen, v) die Untersu- chung der unterstützenden Rolle von Hydroxidionen für die Transformation von Zinkacetat-

clustern zu Zn4O unter Anwendung von ESI-MS und ab initio Berechnungen der Substitu- tion von Acetat- durch Hydroxidionen und vi) die starke Affinität von nTMCs zu Alkalime- tallionen, die zur Entwicklung einer neuen Art von positiv geladenen, ionischen Übergangs- metallkomplexen (iTMCs) für die Verwendung als aktive Materialien in organischen licht- emittierenden, elektrochemischen Zellen (OLEECs) ermöglichen könnten.