Preparative Mass Spectrometry Applications in Nanomaterials and Organic Synthesis Michael Wleklinski Purdue University

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12-2016 Preparative mass spectrometry applications in nanomaterials and organic synthesis Michael Wleklinski Purdue University

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PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance

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(QWLWOHG PREPARATIVE MASS SPECTROMETRY APPLICATIONS IN NANOMATERIALS AND ORGANIC SYNTHESIS

Doctor of Philosophy )RUWKHGHJUHHRI

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Graham Cooks

Zoltan K. Nagy

Garth J. Simpson

Eric Barker

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PREPARATIVE MASS SPECTROMETRY APPLICATIONS IN NANOMATERIALS

AND ORGANIC SYNTHESIS

A Dissertation

Submitted to the Faculty

Purdue University

Michael Wleklinski

In Partial Fulfillment of the

Requirements for the Degree

Doctor of Philosophy

December 2016

Purdue University

West Lafayette, Indiana

For Hemingway

iii

ACKNOWLEDGEMENTS

I am thankful for all those before me who laid the foundation of my path. First, I thank my family, for their patience and love have guided me through this process. My mother is by far the kindest and most reliable person I know. She has done everything she can to help me, my friends, or anyone. My father provided a lot of practical guidance throughout my life, which has kept me grounded. Finally, I thank my sister Amy, whose wisdom about the small things in life smoothed out many rough patches for me.

I am extremely grateful to my advisor, Dr. R. Graham Cooks for being active in my research while maintaining such a hectic schedule. His guidance on research projects is just as valuable to me as is his opinion on literature. His knowledge of history, especially the history of Indiana is admirable. A special thanks goes to Professor Garth

Simpson, who serves as a committee member and also introduced me to long distance bike rides through our various adventures to Turkey Run. I thank Dr. George Devendorf,

Dr. Thomas Adams, and Dr. Thomas Arnold who ignited my passion for chemistry and history.

I am especially thankful for my fellow members of Aston Labs. I will do my best here to thank each individual in a unique way. First I thank Dr. Santosh Soparawalla, Dr.

Jonell Samberson, Dr. Dahlia Campbell, Dr. Sheran Oradu, Dr. Paul Hendricks, Dr.

Fatkhulla Tadjimukhamedov, Dr. Sandilya Garimella, and Dr. Josh Wiley who first welcomed me into the lab and provided guidance on all the problems of a first year. Next,

I wish to thank Dr. Abraham Badu-Tawiah, Dr. Ryan Espy, and Dr. Jon Dagleish for helping to train and establish my place in the lab. I’d like to thank various members of the lab for their contribution to my growth as a scientist including Dr. Jacob Shelley, Dr.

Kevin Kerian, Caitlin Falcone, Dr. Fred Jjunju, Dr. Pu Wei, Dalton Snyder, Kiran Iyer,

Karen Yanell, Dr. Hsu-Chen Hsu, Dr. Soumabha Bag and Dr. Zane Baird. I owe a special thanks to Dr. Christina Ferreira who has helped keep me sane during all of the DARPA reports.

I would like to thank the people most critical to my PhD success. A special thanks goes to Jobin Cyriac who taught me all I know about vacuum systems and helped introduce me to a culture I love and admire. Ryan Bain and Steve Ayrton deserve a thanks for putting up with my stubbornness and willingness to always make a joke to try and cheer me up. I thank Chris Pulliam for providing inspiration to finish my last Turkey

Run bike ride. Next, I wish to thank Dr. Anyin Li, who at first felt like a total cold stranger, but after multiple trips homes and deep philosophical discussion feels like a brother. I sincerely thank Adam Hollerbach, as there isn’t a nicer person I’ve ever meet and still am thankful for the three months you watched and cared for Hemingway.

Finally, from Aston labs I thank my two classmates Dr. Alan Jarmusch and Dr. Xin Yan, who are amazing scientists with whom I am proud to be part of the same graduating year.

Now to thank my extended research family I’ve met during my many travels. First many thanks go to Professor T. Pradeep as your guidance on research during my time in

India was invaluable. I’d also like to thank you for introducing me to East of Eden, a

v book I have come treasure. I’d like to thank the rest of Professor Pradeep’s lab including

Anirban Som, K. R. Krishnadas, Atanu Ghosh, Soujit Sengupta, Shridevi Bhat, Avijit

Baidya, Sandeep Bose, and P. Gautham who helped me with various experiments during my time in India. A special thanks to Rahul Narayanan and Depanjan Sarkar for contributing directly to and providing guidance in my own work. To Vidhya

Subramanian and Dr. Ananya Baksi who are my big sisters and made sure I stayed safe during my visit. I am also grateful that you took care of my sister during her visit. I’d like to thank Dr. Stephan Rauschenbach and Sabine Abb who treated me like family and taught me so much in only a week. Most importantly I thank Pamela Pruski who I meet early on at Purdue and has stayed a close friend throughout my PhD.

A special thanks goes to the newest people I have had the opportunity to work with a result of the DARPA project. I thank Professor Thompson, Professor Nagy,

Professor Barker, and Professor Grama who contributed to making the project a possibility and help me understand how to work with such a diverse team. Thanks goes to Zinia Jaman, Sam Ewan, Dr. Seok-Hee Hyun who all contributed great effort to this project. I miss the late night “pharming” that Brad Loren and Dr. Andy Koswara contributed to and hope one day we can do it again.

My thanks go to all the support staff who made my time at Purdue as painless as possible. This includes Betty Dexter, Ned Gangwer, Steven Barker, Aaron Harkleroad,

Craig MacDonald, Suzy Gustafson, Darci DeCamp, Randy Repogle, Sarah Johnson, and

Leann Houmes who helped me countless tasks. Special thanks goes to Brandy

McMasters who answered every single question I ever had and taught me every detail

vi about budgeting. I also like to thank Steve Rudolph and Susan McCreery for making every bit of Bindley Biosciences enjoyable.

I’d like to thank Dr. Jon Amy for being an inspiration in both science and in society. The stories you have shared with me provide new inspiration for life. Lastly, I thank Erica Baker for your patience and love during the end of my PhD.

vii

TABLE OF CONTENTS

Page

LIST OF TABLE ...... x

LIST OF FIGURES ...... xi

ABSTRACT ...... xvii

CHAPTER 1. INTRODUCTION ...... 1

1.1 Overview ...... 1 1.2 Electrospray Ionization ...... 4 1.2.1 Droplet Based Ion Formation...... 5 1.3 Preparative Mass Spectrometry ...... 8 1.3.1 Ion Soft Landing ...... 8 1.3.2 Preparative Electrospray ...... 9 1.3.3 Ion/Surface Interactions ...... 9

CHAPTER 2. ZERO VOLT IONIZATION AND ITS MECHANISM ...... 11

2.1 Introduction ...... 11 2.2 Experimental Details ...... 14 2.2.1 Zero Volt Paper Spray and Instrumentation ...... 14 2.2.1.1 Chemicals and Materials ...... 14 2.2.1.2 Zero Volt Paper Spray ...... 14 2.2.2 Computational Resources ...... 17 2.3 Results and Discussion ...... 18 2.3.1 Characteristics of Zero Volt Paper Spray Mass Spectra ...... 18 2.3.2 Source of Protons, pH Effect, and Low Voltage Effect ...... 22 2.3.3 Analyzing Organic Salt/Organic Analyte Mixtures by Zero Volt Paper Spray, kV Paper Spray, and Nanoelectrospray ...... 27 2.3.4 Overview of Ionization Mechanism for Zero Volt Paper Spray ...... 31 2.3.4.1 Aerodynamic Breakup ...... 31

viii

Page

2.3.4.2 Initial Droplet Conditions for Evaporation and Columbic Fission Cycles ...... 32 2.3.4.3 Droplet Evaporation to Rayleigh Limit ...... 33 2.3.4.4 Droplet Fission and Progeny Droplets ...... 34 2.3.4.5 Analyte Ion Formation ...... 36 2.3.5 Single Analyte Simulation ...... 38 2.3.6 Experimental and Simulated Results and Mechanistic Considerations for Multianalyte Mixtures ...... 40 2.4 Conclusion ...... 44

CHAPTER 3. AMBIENT PREPARATION AND REACTIONS OF GAS PHASE SILVER CLUSTER CATIONS AND ANIONS ...... 46

3.1 Introduction ...... 46 3.2 Experimental Details ...... 49 3.3 Results and Discussion ...... 53 3.3.1 Unheated silver salts ...... 53 3.3.2 Heated Silver Salts ...... 57 3.3.3 Mechanism ...... 60 3.3.4 Reactivity of Silver Cluster Cations ...... 64 3.3.4.1 Alcohol Reactivity ...... 64 3.3.4.2 Methyl Subsituted Pyridine Reactivity ...... 67 3.3.4.3 Ligand Exchange ...... 70 3.3.4.4 Hydrocarbon Reactivity ...... 74 3.3.4.5 Oxidation ...... 75 + + 3.3.4.6 Reactions of Agn and Oxidized Agn with Ethylene and Propylene...... 77 3.3.5 Reactivity of Silver Cluster Anions ...... 80 3.3.5.1 Formation of [Agn(OH)]- ...... 80 3.3.6 Ion soft Landing ...... 81 3.4 Conclusions ...... 83

CHAPTER 4. CAN ACCELERATED REACTIONS IN DROPLETS GUIDE CHEMISTRY AT SCALE? ...... 85

4.1 Introduction ...... 85 4.2 Experimental Details ...... 88 4.2.1 Droplet Reactor Experiments ...... 88 4.2.2 Offline Droplet Reactor ...... 89 4.2.3 Leidenfrost Droplet Experiments ...... 90 4.2.4 Mass Spectrometry ...... 91 4.2.5 Synthesis of Diazepam Precursor ...... 91 4.2.6 Diphenhydramine ...... 91

Page

4.2.7 Atropine ...... 92 4.2.8 Microfluidics ...... 92 4.2.9 Chemicals and Reagents ...... 92 4.3 Results and Discussion ...... 93 4.3.1 Synthesis of Diazepam Intermediate ...... 93 4.3.2 Synthesis of Diphenhydramine ...... 99 4.3.3 Synthesis of Atropine ...... 102 4.4 Conclusions ...... 105

CHAPTER 5. OUTLOOK AND FUTURE DIRECTIONS ...... 107

5.1 Laboratory Automation ...... 107 5.1.1 Continuous Electrospray ...... 108 5.1.2 Neutral Droplet Emitter ...... 108 5.1.3 Grand Challenge ...... 110

LIST OF REFERENCES ...... 112

VITA ...... 124

LIST OF PUBLICATIONS ...... 126

LIST OF TABLE

Table ...... Page

Table 3.1 Reaction products of Ag+ with various reagentsa ...... 67

+ a Table 3.2 Reaction products of Ag3 with various reactants ...... 67

Table 3.3 Reaction products of Ag+ with various methyl-substituted pyridinesa ...... 70

+ a Table 3.4 Reaction products of Ag3 with various methyl-substituted pyridines ...... 70

+ Table 3.5 Reaction products of Ag3 with acetone, acetontrile, acetone followed by acetonitrile, and acetonitrile followed by acetonea ...... 73

LIST OF FIGURES

Figure ...... Page Figure 2.1 a) Overview of the zero volt paper spray process. The distance between the front edge of the paper and the MS inlet is 0.3 - 0.5 mm. No voltage is applied to either the paper or the MS inlet capillary. The suction force of the MS inlet causes the release of analyte-containing droplets, which are sampled by the mass spectrometer. b) Sampling and detections procedures. Photographs of the inlet region c) without and d) with solvent. A solvent spray or stream is generated when solvent is applied to the paper...... 16

Figure 2.2 Panels A-D are consecutive images of the spray process occurring at 0 volts. The spray is illuminated with a red laser pointer and captured on a Watec Wat-704R camera. Panels A-D show a droplet event over the course of 4 consecutive scans. The time elapsed is around 100 milliseconds. Panels E and F are the mass spectrum of 50 ppm tributylamine and its corresponding ion chronogram. Tributylamine was added in a continuous manner at 15 µL/min through a fused silica capillary...... 17

Figure 2.3 Mass spectra recorded using zero volt PS of three samples examined in the positive ion mode: a) 1 ppm tributylamine; b) 8 ppm cocaine c) 1 ppm terabutylammonium iodide and three samples examined in the negative ion mode d) 10 ppm 3,5-dinitrobenzoic acid, e) 10 ppm fludioxonil; f) 10 ppm sodium tetraphenylborate. Methanol and water (v/v 1:1) were used as spray solvent for all samples. Both positive and negative signals were recorded with much lower intensities compared with kV PS and nESI. * Indicates the presence of background species...... 20

Figure 2.4 Positive ion mode MS/MS data for tributylamine, cocaine, and tetrabutylammonium iodide taken by kV paper spray, nano-electrospray ionization, and zero volt paper spray ...... 21

Figure 2.5 Negative ion mode MS/MS data for 3,5-dinitrobenzoic acid, fludixonil, and sodium tetraphenylborate taken by kV paper spray, nano-electrospray ionization, and zero volt paper spray...... 22

xii

Figure ...... Page

Figure 2.6 Zero volt PS mass spectra of 1 ppm tributylamine using a) methanol/water (v/v 1:1) and b) deuterated methanol/water (v/v 1:1) as solvent. When deuterated solvent is used, [M+D]+ becomes the major peak...... 23

Figure 2.7 Analysis of aromatic heterocycles, guanine (top, m/z 152), adenine (middle, m/z 136), thymine (middle, m/z 127), and Pyridine (bottom, m/z 80) with 0 V paper spray under both neutral (left) and acidic conditions (right)...... 25

Figure 2.8 Analysis of three different amines. Tetramethyl-1,4-butanediamine (top, m/z 145), diisopropylamine (middle, m/z 102), and methyl amine (bottom, m/z 32). All samples are dissolved in methanol at neutral with pH 7. Proton affinities are obtained from NIST Webbook...... 26

Figure 2.9 Mass spectra of 50 ppm diphenylamine (DPA) on a paper substrate at 0 V and 1 V, respectively. Note the difference in scales and the fact that the m/z 170 signal intensity is about 1.5 times higher than that at 0 V...... 26

Figure 2.10 Mass spectra of a mixture of 9 ppm cocaine and 0.1 ppm tetrabutylammonium iodide using a) nESI, b) kV PS, and c) zero volt PS...... 28

Figure 2.11 Mass spectra of 5 µL of a mixture of 9 ppm morphine and 0.1 ppm tetrabutylammoniumiodide using a) nESI, b) kV PS, and c) zero volt PS. Mass spectra of 5 µL of a mixture of 36 ppm 3,5-dinitrobenzoic acid and 5 ppm sodium tetraphenylborate using d) nESI, e) kV PS, and f) zero volt PS. The relative intensity of tetrabutylammonium signal to morphine in zero volt PS is much higher than in nESI and kV PS in both cases. The same is true of the ratio of tetraphenylborate to 3,5-dinitrobenzoic acid for zero volt PS as compared to nESI and kV PS...... 29

Figure 2.12 Simulation results of Weber number of methanol droplets. Using this information, it is assumed that droplets may have diameters between 1-4 µm after aerodynamic breakup...... 37

Figure 2.13 The number of ionized molecules vs. concentration for 2 micron (bottom) and 4 micron (top) droplets. The simulation was run at three different surface activities...... 39

Figure 2.14 Ionization efficiency vs. concentration of 2 micron (bottom) and 4 micron (top) droplets. The simulation was run at three different surface activities...... 40

xiii

Figure ...... Page

Figure 2.15 a) Cocaine to tetrabutylammonium cation ratio dependence in positive ion mode for zero volt PS and nESI. Cocaine concentration is held constant at 1 ppm, while tetrabutylammonium iodide concentration changes. b) The relative surface activity of cocaine calculated according to the experimental data in a). c) Ratio dependence of 3,5-dinitrobenzoate to tetraphenylborate in negative ion mode for zero volt PS and nESI. 3,5-Dinitrobenzoic acid concentration is held constant at 20 ppm, while the sodium tetraphenylborate concentration changes. d) Relative surface activity of 3,5-dinitrobenzoic acid calculated according to the experimental data in c). Surface activity of the salt is assumed to be 1 for all simulations...... 43

Figure 2.16 Cocaine to tetrabutylammonium iodide ratio dependence in positive ion mode for zero volt PS and nESI. a) Tetrabutylammonium iodide concentration is held constant at 0.1 ppm, while cocaine concentration changes. b) Relative surface activity of cocaine calculated to match the experimental ratio in part a). c) Sodium tetraphenylborate is held constant at 5 ppm, while 3,5-dinitrobenzoic acid concentration changes. d) Relative surface activity of 3,5-dinitrobenzoic acid calculated to fit experimental data in part c). Surface activity of the salt is assumed to be 1 for simulations...... 44

Figure 3.1 Apparatus for the production of silver cluster cations and anions ...... 50

Figure 3.2 Apparatus for studying atmospheric pressure ion/molecule reactions of silver clusters (cations/anions) with various reagents ...... 52

Figure 3.3 Apparatus for performing atmospheric pressure ion/molecule ligand exchange reactions of silver clusters with various reagents ...... 52

Figure 3.4 Apparatus for performing ion/molecule reactions using either a gas, low temperature plasma, or both. The low temperature plasma is used to generate reactive species to oxidize silver cluster cations ...... 53

Figure 3.5 Positive ion mode mass spectra of A) unheated and B) heated electrosprayed silver acetate. Negative ion mode for C) unheated and D) heated electrosprayed silver fluoride solution. The numbers above each peak in A) indicate the number of silver atoms, ligands, and water molecules present. The numbers above each peak in B) and D) indicate the number of silver atoms...... 55

Figure 3.6 Negative ion mode mass spectra of A) unheated and B) heated silver acetate. Positive ion mode of C) unheated and D) heated silver benzoate. Negative ion mode mass spectra of E) unheated and F) heated silver benzoate. Positive ion mode mass spectra of G) unheated and H) heated silver fluoride. The numbers above each peak indicate the number of silver atoms, ligands, and water present, and the absence of the final number indicates zero water molecules are present ...... 56

xiv

Figure ...... Page

+ + + 2+ + 2+ Figure 3.7 Tandem MS of A) Ag5 , B) Ag7 , C) Ag8 /Ag16 , D) Ag10 /Ag20 , E) + 2+ + 2+ 2+ Ag11 /Ag22 , F) Ag13 , G) Ag19 , and H) Ag21 ...... 58

------Figure 3.8 Tandem MS of A) Ag7 , B) Ag9 , C) Ag11 , D) Ag12 , E) Ag13 , F) Ag14 , - - - - G) Ag15 , H) Ag16 , I) Ag17 , and J) Ag18 ...... 59

Figure 3.9 Selected ion chronograms for ions of interest in the formation of silver cluster cations from a silver acetate precursor. At time zero the heating is turned on and temperature slowly rises to 250 Celsisus over the course of a few minutes ...... 61

Figure 3.10 Tandem MS tree for ions observed when silver acetate is subjected to 100-150 C and harsh in source conditions. Species in black text are observed in the full MS, while species appearing in red are only observed by tandem MS. Red asterisks indicate ions that reversibly bind H2O in the ion trap ...... 62

Figure 3.11 Reaction of silver cluster cations with A) ethanol, B) 1-propanol, C) isopropyl alcohol, D) tert-butyl alcohol, E) acetone, and F) acetonitrile. Tandem MS for selected ions are shown as insets. M stands for the reactant of interest. Peaks at m/z 105, 337, and 365 are common background ions with the first originating from the spray and the latter arising from the cotton swab ...... 66

Figure 3.12 Reaction of silver cluster cations with A) pyridine, B) 2-ethylpyridine, C) 3-ethylpyridine, D) 4-ethylpyridine, E) 3,4-lutidine, F) 2,6-lutidine, G) 2,5- lutidine, H) 3,5-lutidine, and I) 2,4,6-trimethylpyridine. M stands for the reactant of interest ...... 68

+ Figure 3.13 Tandem mass spectrometry data for [Ag3(C8H11N)3] (left) and + [Ag3(C7H9N)3] (right) where 2,4,6-trimethylpyridine and 3,5-lutidine are the 2 3 4 respective neutral reactants. A) MS , B) MS , C) MS of [Ag3+(2,4,6- + 2 3 4 + trimethylpyridine)3] . D) MS , E) MS , F) MS of [Ag3+(3,5-lutidine)3] . M stands for the reactant of interest ...... 69

2 + 2 + Figure 3.14 A) MS of [Ag3+C2H3N+NH3] , B) MS of [Ag3+C2H3N+(NH3)2] , 2 + and C) MS of [Ag3+(C2H3N)2+NH3] for the reaction of silver clusters with first ammonia (from ammonium hydroxide) then acetonitrile ...... 72

Figure 3.15 Full MS for the reaction of silver clusters with first 2,6-lutidine then acetonitrile. Inset: Blowup of m/z 350-700 ...... 72

2 + 2 Figure 3.16 A) Full MS, B) MS of [Ag3 + (C3H6O) + (C2H3N)] , and C) MS of + + [Ag3 + (C3H6O)2] for the reaction of silver clusters with Ag3 with first acetone, than acetonitrile ...... 73

Figure ...... Page

Figure 3.17 Analysis of A) Hexadecane, B) Isocetane, C) Squalane, and D) Ultragrade 19 oil using silver cluster spray. The inset of C) is the MS2 of [Ag+Squalane]+ ...... 75

Figure 3.18 Mass spectra of silver cluster cations generated from silver fluoride A) + without ozone and B) with ozone. C) Shows the selected ion chronogram of Ag3 + (top) and Ag3O (bottom) in the presence and absence of ozone. D) High mass + accuracy measurements confirm the formation of Ag3O ...... 76

Figure 3.19 Reaction of silver cluster cations with A) ethylene and B) ethylene and ozone ...... 78

+ Figure 3.20 MS fragmentation tree for [Ag5(C2H4)O2] . Arrows indicate a single stage of CID ...... 79

Figure 3.21 Reaction of silver cluster cations with A) propylene and B) propylene and ozone ...... 80

Figure 3.22 Mass spectra of silver cluster anions with the ESSI sprayer inserted A) 1cm and B) 2-3 mm inside the heating tube. Red and blue numbers respectively indicate silver atoms and OH molecules...... 81

Figure 3.23 Positive ion mode mass spectra of A) unheated and B) heated silver acetate. Positive ion mode of C) unheated and D) heated silver benzoate. Positive ion mode mass spectra of E) unheated and F) heated silver fluoride. All these mass spectra were collected on a custom surface science instrument. The numbers above each peak indicate the number of silver atoms and ligands present ...... 82

Figure 4.1 Methods used to perform microdroplet reactions, based on ESI either a) with on-line product analysis by MS or b) with sprayed droplet deposition and subsequent off-line MS product analysis ...... 89

Figure 4.2 Leidenfrost droplet reaction methodology. The reaction mixture is added using a Pasteur pitette and the hot surface causes its levitation. The droplet temperature is usually 10 – 20 C below the solvent boiling point...... 90

Figure 4.3 Solvent screening for the amide synthesis of 3 from chloroacetyl chloride 2 in batch and in charged droplets. Acceleration rate is calculated as previously discussed ...... 94

Figure 4.4 Synthesis of 3 in ACN using a) chloroacetyl chloride and b) bromoacetyl chloride in droplet reactor and microfluidics (µ-Fl) ...... 95

Figure 4.5 Synthesis of 3 in toluene using a) chloroacetyl chloride and b) bromoacetyl chloride in charged droplets and microfluidics ...... 95

xvi

Figure ...... Page

Figure 4.6 Synthesis of 3 in toluene using chloroacetyl chloride a) and bromo acetylchloride b) in Leidenfrost, charged microdroplets and microfluidics ...... 97

Figure 4.7 Synthesis of 3 in ACN using a) chloroacetyl chloride and b) bromoacetyl chloride in Leidenfrost, charged droplets and microfluidics ...... 98

Figure 4.8 Charged microdroplet (a,c) and microfluidic (b,d) reaction telescoping for diphenhydramine synthesis in two solvents (ACN, toluene) ...... 100

Figure 4.9 Droplet reactor and microfluidic reaction telescoping for diphenhydramine synthesis in ACN ...... 101

Figure 4.10 Droplet reactor and microfluidic reaction telescoping for diphenhydramine synthesis in toluene ...... 101

Figure 4.11 Charged microdroplet (a,c) and microfluidic (b,d) reaction telescoping for atropine synthesis using potassium methoxide or 1,5-diazabicyclo[4.3.0]dec-5- ene ...... 104

Figure 4.12 Comparison of relative conversion to atropine using three different bases in droplet reactor and microfluidics ...... 105

Figure 5.1 Methods for scale up of preparative electrospray reactions. a) Continuous Venturi solvent spray with recycling electrospray apparatus b) commercial (HTX Imaging, Inc.) matrix sprayer set up for reaction with analytes on a surface. c) Data showing accelerated reaction of p-methoxyaniline with pyrylium salt using the matrix sprayer (acceleration factor is 300) ...... 110

xvii

ABSTRACT

Wleklinski, Michael Ph.D., Purdue University, December 2016. Preparative Mass Spectrometry Applications in Nanomaterials and Organic Synthesis Major Professor: R. Graham Cooks.

Preparative mass spectrometry was traditionally a vacuum based experiment where mass selected ion beams were deposited onto surfaces; however, recently ions have been

manipulated in the open air to perform accelerated synthesis. The work of this

dissertation focuses on the generation of charged ions from microdroplets and their use in

organic synthesis and the preparation of atomically precise metal nanoclusters. First the

mechanism for charging in the zero volt paper spray will be presented. This works builds

a computational model based on the statistical fluctuation of positive and negative

charges based on the theory of Dodd. It is found through experiment and simulation that

in zero volt paper spray and presumably other zero volt methods the most surface active

species are preferentially ionized.

Charged microdroplets are exploited in this dissertation for two very different

applications but in seemingly similar ways. First metal salts are electrosprayed, heated,

and subjected to harsh in-source conditions to cause the reduction of the metal salts into

atomically precise metal clusters. This method is capable of producing atomically precise

silver cluster cations and anions as well as bimetallic silver-palladium complexes.

Microdroplets are also exploited to screen chemical pathways to common pharmaceutical

xviii including atropine and diphenhydramine. The two step synthesis of diphenhydramine from benzhydrol was performed in charged microdroplets and confirmed in microfluidics. While the fundamental mechanism of reaction in microdroplets and microfluidics might differ useful connections were found in the cases of atropine, diphenhydramine, and diazepam. It is envisioned that this methodology could impact drug discovery as a rapid platform for the screening of chemical pathways to both old and new molecules. The droplet reaction was extended to a “super reagent” form where a reagent of choice is sprayed onto a variety of substrate contained in a well plate or on paper. This method allows for rapid screening with hopes of screening 1000’s of reaction conditions per hour.

CHAPTER 1. INTRODUCTION

1.1 Overview

Mass spectrometry (MS) traces back to the early 20th century when the first

experiments were performed by J.J. Thomson using a “parabola spectrograph”[1] and

Francis William Aston measuring isotopes.[2, 3] The general workflow for a mass

spectrometry experiment to this date remains relatively unchanged. Ions are generated

using an ionization source, analyzed based most commonly on their m/z, and then

detected. This entire experiment is typically done under vacuum, where the physics and

motions of ions have been mathematically solved.

Organic mass spectrometry originally concentrated on volatile compounds, the vapors of which were subjected to electron impact, a process that lead to electronic excitation, ionization or both. The great merit of electron ionization (EI) lay in two facts: the data were highly reproducible as they depended on the physical processes just described occurring within a set time interval. This also meant that useful libraries of EI mass spectra could be generated and used in an approximately instrument independent fashion. The other advantage was that the combination of hard and soft ionization events produced by the statistical nature of the electron/molecule collision, meant that most spectra contained molecular ions (radical cations) from which molecular weight

2 information could be obtained as well as fragment ions which were characteristic of molecular structure. Since most organic compounds are not volatile, the practice of organic mass spectrometry frequently resorted to chemical derivatization – as in the derivatization of amino acids to create methyl esters or alternatively their N-acyl derivatives and also quite often, isotopic labeling, to help elucidate fragmentation mechanisms.[4] Derivatization was also used to make compounds more amenable to gas

chromatography which at that time used normal rather than reverse stationary phases.

Two capabilities came together to allow direct, rapid analysis of complex mixtures

of organic compounds (volatile and non-volatile) by mass spectrometry. They were (i)

mass-analyzed ion kinetic energy spectrometry, MIKES[5, 6], a method of characterizing

ions of given m/z ratio by isolating them from a mixture of ions and then performing an operation, typically collision-induced dissociation (CID)[7-9], that generated a

characteristic set of fragment ions from each precursor ion and (ii) chemical ionization

(CI) a method of ionization that allowed control of ion internal energy including the

ability to cause soft ionization so as to minimize fragmentation during ion formation.

Together these two capabilities allowed a complex mixture of neutral molecules to be

separated into a mixture of ions, the structures of which reflected those of the original

compounds in the mixture.[10] When ions of particular m/z ratios were selected from the

ionized mixture by mass analysis and then subjected to CID, a secondary mass spectrum

would be recorded from which the nature (connectivity) of the corresponding neutral molecule could be inferred. This process depended on creating an ionic surrogate of each neutral compounds of interest; to do this successfully, ionization had to be gentle.

The analysis of complex mixtures by collisional activation has continued to grow and is arguably the most common mass spectrometry experiment. Today instead of chemical ionization, electrospray and matrix-assisted laser desorption ionization

(MALDI) are the most common soft ionization method and also allow for ionization outside the vacuum system of the mass spectrometer.[11-15] The type of analyzer changed

from sectors to quadrupoles and ion traps.

Besides the obvious analytical uses of MS, the use of ions for synthetic purposes

forms a field called preparative MS. Preparative MS originates from the Manhattan

Project that sought to separate 235U from 238U. Racetracks of calutrons were

manufactured in order to produce the amount of uranium necessary for the atomic

bombs.[16] The explicit modification of surfaces with low kinetic energy ions is termed

ion soft landing (SL).[17, 18] Soft landing has been demonstrated for a variety of

application including the preparation of protein microarrays[19] and nanoparticles[20].

Recently preparative mass spectrometry has included reactions of ions in microdroplets,

where ordinary organic reactions are accelerated by orders of magnitude.[21-25] The major

objective of this dissertation is to show how preparative mass spectrometry is applicable

in the synthesis of organics and nanomaterials. Microdroplets under appropriate

conditions are shown to act as vessels to accelerate and screen chemical pathways to

common pharmaceuticals, such as diphenhydramine and atropine.[25] Alternatively,

electrospray microdroplets are demonstrated as a cluster ion source that is capable of

transforming metal salts into their corresponding naked metal cluster ions.[26]

1.2 Electrospray Ionization

The fundamental idea of electrospray ionization comes from the theory of Lord

Rayleigh who calculated the amount of charge an isolated liquid droplet could contain.[27]

Various researchers went on to discover and visualize the different modes of electrospray, as well as, understand the role of electric filed in creating the deformed

droplet shape of a Taylor cone.[28-30] It took about 100 years from Lord Rayleigh’s

discovery for electrospray to be realized a powerful tool for modern mass spectrometry

when John Fenn demonstrated its ability to transfer high molecular weight non-volatile

compounds from solution into the gas phase gently.[12, 13] This work along with

MALDI[14] was recognized with a Nobel Prize in chemistry for their ability to analyze

biological macromolecules. The title of Fenn’s Nobel lecture was, “Electrospray Wings

for Molecular Elephants”.[12]

Electrospray is an atmospheric ionization technique, where a solution of interest is

infused into a capillary with a high voltage applied. Generally, the capillary is around 100

µm, infusion rates of 1-1000 µl/min, high voltage of 1-5 kV. A sheath gas is also

introduced to help in the breakup and desolvation of droplets.[31-33] This is especially

important for higher flow rate applications where insufficient desolvation can lead to

poor ionization efficiencies. A highly efficient variant of ESI, called NanoESI was

developed to improve sensitivity and reduce sample consumption.[34] The size of

capillaries is typically less than 10 µm with corresponding flow rates in the nL/min range.

Due to the small size of the capillary emitter, clogging of the emitter is a major concern

along with obtaining a stable electrospray.

1.2.1 Droplet Based Ion Formation

Electrospray revolutionized the “omics” field with its ability to gently analyze biological macromolecules and its ability to easily interface with liquid chromatography.

Scientists have strived to develop a complete mechanistic understanding of electrospray; however, many questions still remain on the intricacies of the droplet and ion formation in electrospray.[31] The generally accepted mechanism for electrospray is the production

of microdroplets (nanodroplets in the case of nESI) from a Taylor cone, followed by

droplet evaporation and fission until droplets reach a size where naked gas phase ions are generated via ion evaporation or charge residue model.[32] of A combination simulation

methods including molecular dynamics and Monte Carlo have been used to understand

both the initial steps (droplet evaporation and breakup) and the final steps (generation of gas phase ions).[32, 35]

The electrochemical nature of electrospray is partially responsible for the initial

charging of droplets emitting from the Taylor cone.[36, 37] In positive ion mode typically

protons, sodium ions, or potassium ions are the typical charge carriers. Additionally,

during droplet formation aerodynamic forces between the emerging droplets and sheath

gas can cause droplets to break apart and charge. These aerodynamic forces break apart

droplets until they reach a size on the order of 1 to 4 µm where the aerodynamic forces

are no longer strong enough to cause further droplet breakup.[38, 39] Aerodynamics forces are described by the weber number, which is defined as

Eq. 1.1

where ρg is the gas density, Vg is the gas velocity, Vd is the droplet velocity, Dd is the

diameter of the droplet, and σ is the surface tension of the solvent.[40] During the

aerodynamic breakup process, there is a very large chance that the positive charges and

negative charges will be unevenly separated, that is to say, many of these progeny

droplets will be (slightly) charged. The extent of charging is unknown, but has been reported to be large in some cases.[38]

After the droplet becomes stable to aerodynamic forces, it will evaporate and cool

until it reaches it Rayleigh Limit.

∗ Eq. 1.2 ∗∗∗

Here Dq is the charge on the droplet, e is elementary charge, ε0 is the permittivity of a

vacuum, and γ is the solvent surface tension. At the Rayleigh limit a droplet undergoes

fission and produces progeny droplets. Accordingly, the size of precursor and progeny

droplets was calculated according to these equations:

1∆ ∗ Eq. 1.3

∆ Eq. 1.4

where Npd is the number of progeny droplets taken to be 10, Dpd is the diameter of the

progeny droplets, and Δm = 0.02. The values of these numbers are taken from literature

but their exact values depend on the specific chemical system.[35] The number of analytes

7 in each progeny droplet was defined by a Poisson distribution based on the concentration of analytes in the outer region of the droplet. The position of an analyte within the droplet is determined by its surface activity, S. Surface activity is a number between 0 and 1 describing the probability of a molecule being at the surface or the interior of the droplet.

Larger values of surface activity mean the analyte competes more strongly for surface sites. The evaporation/coulombic fission process continues until all droplets reach a size a sufficiently small size suitable for ionization. (<10 nm). Ions are produced from droplets smaller than 10 nm by the ion emission mechanism or by the charge residue model, which will be further discussed below.[32]

Ions in electrospray are typically generated by one of two models: ion evaporation

and charge residue. Ions of low molecular weight are transferred as ions into the gas phase from solution via the ion evaporation model. When a nanodroplet is sufficiently small, low molecular weight ions can be directly ejected from the droplet surface, forming gas phase ions. Molecular dynamics simulations show that the ion is often ejected as a single ion with a solvation shell.[32] Therefore, solvated ion could be

interpreted as a progeny droplet. Ions of higher molecular weight, such as globular

proteins are brought into the gas phase by the charge residue model. As the globular

protein is too large to eject from the nanodroplet, other small ions will be released until

the droplet evaporates completely. At this point the remaining charge is on the globular

species.[32]

1.3 Preparative Mass Spectrometry

In this section, three distinct methods of preparative mass spectrometry will be discussed. The first will be the traditional approach using mass selected ion beams in a technique called soft landing.[18] Next, preparative electrospray will be discussed, where

the microdroplet environment of electrospray is used to accelerate organic reactions.[23, 24]

Finally ion/surface reactions will be introduced, where the generation of unique

nanomaterials has been observed.[20]

1.3.1 Ion Soft Landing

Ion soft landing (SL) is a promising approach to preparative mass

spectrometry.[18] In this experiment, mass-selected hyperthermal energy ion beams are

non-destructively deposited onto surfaces. The technique was first demonstrated in 1977

by the collision of sulfur containing ions with surfaces.[41] Mass selection, control of ion

translational energy and characterization of the prepared surface are critical to the success

of SL. Chemical characterization of the surface after SL including information on the

charge state, molecular structure and nature of the binding to the substrate is important to

the utilization of this procedure for surface tailoring and this needs to be achieved by in

situ methods of analysis.

SL allows surface modification with control over spatial resolution and the nature

of the landed ion. Recent reports on SL include preparation of prototype protein micro-

arrays,[19] surfaces with potential catalytic activity,[42] surfaces covered with size-selected sub-nanometre metal clusters,[43-45] and surfaces with covalently immobilized

proteins/peptides/complex species,[46-48] or nano motifs.[49-51] In addition, utilization of

9 well-defined experimental conditions to achieve SL for fundamental studies has been of interest.[43, 52, 53]The ability to produce complex chemical forms in the gas phase for

transfer to the surface represents an advantage over alternative surface modification

procedures, like chemical vapor deposition (CVD), atomic layer deposition (ALD) and

thin film coating, which are less chemically specific although they may have other

advantages.[54, 55] Recently soft landing has been extended to the ambient environment.[56]

1.3.2 Preparative Electrospray

Charged microdroplets produced by ESI are known to accelerate reaction rates as

the solvent evaporates because of changes in concentration, pH, surface/volume ratios,

and interfacial effects.[21, 57-60] The acceleration factors can be remarkably large.[61] A diverse number of reactions have accelerated including, Claisen-schmidt condensation,

Michael addition, mannich condensation, and Hantzsch synthesis of 1,4- dihydropyridines.[58, 62-64] This effect has been exploited in desorption electrospray

ionization to perform rapid derivatization of ketosteroids.[24] Charged micrdroplets under

appropriate conditions (heat, harsh in-source conditions) can be used as a reductive

environment.[26] The topic of accelerated reactions in droplets, including evidence that partial solvation of reagents at interfaces contributes to the orders of magnitude reaction rate acceleration that can be seen, has recently been reviewed.[23]

1.3.3 Ion/Surface Interactions

Ion/surface reactions occurring under ambient conditions can be used for the direct modification of surface properties.[65] They are of intrinsic interest for the similarities and

10 differences in the ion/surface chemistry that occurs under vacuum vs. in the ambient environment. NanoESI was used to synthesize both metallic nanoparticles and metallic nanobrushes.[20, 65, 66] In order to generate these materials, either metal ions are formed in

the electrospray process, which at the grounded surface undergo reduction and form

aggregates. The source of metal ions is either a metal salt doped into the solution, or

electrolytic generation of metal ions from a corresponding metal wire. The shape and size

of the metal nanomaterials can be controlled by changing the deposition surface or the

exposure time.[20] Metallic nanobrushes made from electrospray deposition are capable of capturing bacteria,[66] while metallic nanoparticles made from electrolytic deposition can

be used as highly active SERS substrates.[20]

CHAPTER 2. ZERO VOLT IONIZATION AND ITS MECHANISM

2.1 Introduction

Mass spectrometry (MS) is a powerful analytical technique because of its high

sensitivity, selectivity, and speed. Ionization, the first step in MS analysis, plays a pivotal

role in the experiment. The applicability of MS to complex samples examined without

sample pretreatment is the key feature of desorption electrospray ionization (DESI)[67]

and other ambient ionization methods.[68, 69] These ionization methods have been used in

various fields including drug discovery, metabolomics, forensic science and biofluid

analysis. Ambient ionization methods which avoid the use of a high voltage have obvious

advantages, especially for in vivo analysis.[70]

The first voltage-free spray ionization method, thermospray, was developed by

Vestal et al. in the 1980s as an interface for LC/MS.[71, 72] In this experiment thermal energy was used to help release solvent and create ionized analytes. A few years later, another important voltage-free spray ionization method (SSI), was introduced by

Hirabayashi.[73, 74] In this experiment a high speed gas flow is used to break up the bulk

solution into droplets, which go on to evaporate and generate gaseous ions. A variety of

compounds[75-77] can be ionized by SSI and it has been used for in vivo analysis when

combined with ultrasonic aerosolization.[70] Desorption sonic spray ionization[78] (DeSSI,

also referred to as

12 easy ambient sonic-spray ionization, EASI[79, 80]) is another example of a zero voltage sprayionization method. It represents a particular mode of operation of DESI which removes the high voltage. In SSI and EASI ionization, pneumatic forces play an important role. Another voltage-free ionization method, solvent assisted inlet ionization

(SAII) is applicable in LC/MS.[81-83] Here, as in thermospray, both thermal energy and

pneumatic forces appear to contribute to ionization. In addition to these voltage-free

spray ionization methods, ultrasound produced by piezoelectric devices has also been

used for spray ionization.[84, 85] A low frequency ultrasonicator has been used to analyze

biomolecules and monitor organic reactions, reducing background noise by decreasing

interference from background electrochemical reactions.[86, 87] In 2013, Chung-Hsuan

Chen et al. reported yet another new spray ionization method, Kelvin spray ionization,[88]

which required no external electric energy, the system itself producing a voltage.

Paper spray (PS), first reported in 2009,[89] has proven practically useful for

qualitative and quantitative analysis. The use of paper as the substrate in PS allows

ionization of compounds of interest while leaving some of the other components of

complex matrices adsorbed to the paper; this feature makes it suitable for the

quantification of therapeutic drugs in blood.[90, 91] Paper cut to a sharp point ensures that a

kilovolt applied potential will generate an electric field high enough to cause field

evaporation of charged analyte-containing solvent droplets; these droplets evaporate and

undergo coulomb explosion and perhaps solvated ion emission to give ESI-like mass

spectra.[32] A recent variant on the PS method uses paper impregnated with carbon nanotubes as a way to achieve the necessary field strengths while applying only a low voltage.[92]

We show in this study that by removing the applied voltage entirely, a zero volt form of PS can be performed. This experiment, which is phenomenologically similar to the SAII method, retains the advantages of the paper substrate by allowing complex mixtures to be examined directly without chromatography while removing the electric field and dispensing with the strong pneumatic forces needed in the pneumatically assisted ionization methods of SSI and EASI. In zero volt PS, as in SAII, the vacuum provides a sufficient pneumatic force. Our results demonstrate that zero volt PS gives both positive and negative ions just as does conventional kV PS and nESI, albeit with much lower signal intensities. The reduction in signal intensities roughly parallels that between EASI and DESI. Qualitative mechanisms, which are not fully understood, have been proposed for other zero volt methods, but this paper seeks to develop a quantitative model for zero volt paper spray. Simulations, based on the theory of Dodd,[93] have been

performed to gain insights into the ionization mechanism. The proposed mechanism

includes charge separation during droplet formation due to statistical fluctuations in

positive and negative ion distributions[77] after aerodynamic droplet breakup as described

by Jarrold and coworkers.[38] Subsequent solvent evaporation and coulombic fission

processes follow the accepted ESI mechanisms.

2.2 Experimental Details

2.2.1 Zero Volt Paper Spray and Instrumentation

2.2.1.1 Chemicals and Materials

Deionized water was provided by a Milli-Q Integral water purification system

(Barnstead Easy Pure II). Morphine and cocaine were purchased from Cerilliant (Round

Rock, Texas). Methanol was from Mallinckrodt Baker Inc. (Phillipsburg, NJ). Deuterated methanol and water were provided by Cambridge Isotope Laboratories (Tewksbury,

MA). The paper used as the spray substrate was Whatman 1 chromatography paper

(Whatman International Ltd., Maidstone, England). All samples were examined in methanol solution except where noted.

2.2.1.2 Zero Volt Paper Spray

As is shown in Figure 2.1 the experimental details of zero volt PS were a little different from those previously reported for kV PS.[89] The choice of paper shape for kV

PS is important to generate the required electric fields for ionization; however, the choice of paper shape for zero volt PS is less important than the orientation of the paper relative to the MS inlet. Therefore, a rectangular piece of paper cut to 8 mm by 4 mm and held in place by a toothless alligator clip (McMaster-Carr, USA Part 7236K51) was used, with the center of a straight edge being placed closest to the inlet and on the ion optical axis.

This arrangement made it easier to control the distance between the paper and the inlet than in the case of a paper triangle (Figure 2.1a), increasing the reproducibility of the

15 experiment. A xyz-micrometer moving stage (Parker Automation, USA) was used to set the distance between the front edge of the paper and the MS inlet in the range 0.3 mm to

0.5 mm. A camera (Watec Wat-704R) was used to help in positioning the paper and to observe the spray which was illuminated by a red laser pointer. No voltage was applied to the paper or the capillary of the MS, instead the spray was generated by the pneumatic forces at play near the MS inlet.

Figure 2.1b depicts the typical workflow. Typically, 5 μL of sample dissolved in methanol was loaded onto the paper and allowed to dry. During drying, the paper was positioned appropriately with respect to the MS. A methanol and water solvent (1:1 v/v, applied to the paper in three 7 μL aliquots) was used to generate the spray and detect signal. For each 7 μL aliquot of solvent, the signal lasted for about 10 s. Micropipette tips were used to load solvent onto the paper. Sample solutions could also be directly applied to the paper to generate spray. Figures 2.1c and 2.1d are photographs taken without and with solvent on the paper, respectively. Clearly, droplets are observed only in the presence of solvent. The spray process was monitored using a 30 Hz camera and details are shown in Figure 2.2. Note that there are similarities to the experiments described by

Pagnotti and coworkers.[81, 82]

Figure 2.1 a) Overview of the zero volt paper spray process. The distance between the front edge of the paper and the MS inlet is 0.3 - 0.5 mm. No voltage is applied to either the paper or the MS inlet capillary. The suction force of the MS inlet causes the release of analyte- containing droplets, which are sampled by the mass spectrometer. b) Sampling and detections procedures. Photographs of the inlet region c) without and d) with solvent. A solvent spray or stream is generated when solvent is applied to the paper.

2.2.2 Computational Resources

All programs used in the simulation of zero volt PS were coded in Python 3.4.2 and computed using computational resources provided by Information Technology at Purdue

Research Computing (RCAC) on the Carter supercomputer. Smaller codes were tested on a small desktop computer (core i3).

2.3 Results and Discussion

2.3.1 Characteristics of Zero Volt Paper Spray Mass Spectra

A variety of samples was used to test the ionization capabilities of zero volt PS.

As shown in Figure 2.3, both positive and negative spectra were obtained, although the

signal intensities were about two orders of magnitude lower than those of nESI and kV

PS spectra. The MS/MS results for zero volt PS were almost identical to those for the

same ions generated by nESI and kV PS (Figure 2.4 and 2.5). These results show that the

range of analytes to which zero volt PS is applicable is very similar to kV PS and nESI,

but the ionization efficiency is much lower.

As noted in the Introduction, ionization without application of a voltage has been

observed using several methods. This makes it important to seek to understand the

fundamental processes that lead to the formation of ions at zero volts. It is expected that

such an enquiry for zero volt PS might be of some later use in guiding mechanistic

studies of the other methods. A simple experiment was performed to determine the

maximum distance between the paper and MS inlet that still allows observation of signal.

It was found that without external forces and with the instrument and paper used, the

paper must be within 1 mm of the inlet for the observation of a spray and the

corresponding ion signal. At larger distances, an external force such as an applied

voltage or additional pneumatic force is needed. A distance of 0.3 - 0.5 mm was chosen

19 for subsequent experiments to optimize signal intensity and lower its fluctuations. The spray process was monitored using a 30 Hz camera in an experiment that used 50 ppm of tributylamine fed continuously onto the paper at a flow rate of 15 µl/min. The emission of individual droplets occurs rapidly enough that the chronogram appears to be continuous when observed using an ion injection time of 100 ms (Figure 2.2). By illuminating the spray with a handheld red laser pointer the spray process could be videographed. Figure 2.2 a-d shows the suction of one droplet over the course of 4 consecutive images. This indicates that a single suction event occurs in a time on the order of ~100 ms. This experiment was repeated using manual additions of solvent (7 µl) and similar droplet events are observed. The important finding is that signal is observed only when a droplet event is recorded by the camera, indicating that droplets are necessary to produce gas phase ions.

Figure 2.4 Positive ion mode MS/MS data for tributylamine, cocaine, and tetrabutylammonium iodide taken by kV paper spray, nano-electrospray ionization, and zero volt paper spray

Figure 2.5 Negative ion mode MS/MS data for 3,5-dinitrobenzoic acid, fludixonil, and sodium tetraphenylborate taken by kV paper spray, nano-electrospray ionization, and zero volt paper spray.

2.3.2 Source of Protons, pH Effect, and Low Voltage Effect

Figure 2.6 shows the zero volt PS spectrum of 1 ppm tributylamine using methanol:water 1:1 and deuterated methanol:water 1:1 as solvents, respectively (Figure

2.6a and 2.6b). When methanol/water was used, m/z 186 ([M+H]+) was the dominant peak; accompanied by an isotopic signal at m/z 187. However, when deuterated methanol/water was used, m/z 187, [M+D]+, was dominant and m/z 188 is its isotopic peak. These results indicate that the protons mainly come from the solvent and that the normal acid/base equilibria occurring in bulk solution are ultimately responsible for the

23 ions seen in the mass spectra. In Figure 2.6b, there is still a small peak of m/z 186 while in Figure 2.6a ions of m/z 185 are virtually absent, indicating that a small proportion of tributylamine is still ionized as [M+H]+ when deuterated solvents are used. Possible sources of the proton include autoionization (2M ⇌ [M+H]+ + [M-H]-), gas phase water molecules and residual protic compounds in the instrument.

The effects of solvent pH on the spectra have been tested using a representative compound, tributylamine. The intensity of the signal for the protonated molecule is high at pH 7, but when the pH is raised to 10, the intensity drops to zero. Further tests were done by using a series of aromatic heterocyclic compounds (pyridine, guanine, thymine,

24 and adenine) at neutral and acidic pH (Figure 2.7). The results show that all four compounds ionize well from acidic solution; pyridine and adenine also ionize at neutral pH, while guanine and thymine do not. These observations are consistent with expectations based on acid/base solution equilibria. The pKa values of the conjugate

acids of thymine and guanine are 0 and 3.2 respectively, while the pyridine and adenine

values are significantly higher, 5.25 and 4.1, respectively.[94] A series of basic amines

including tetramethyl-1,4-butanediamine, diisopropylamine, and methyl amine, was also analyzed to investigate the effect of proton affinity (Figure 2.8). Tetramethyl-1,4- butanediamine, has the highest proton affinity (1046.3 kJ/mol) among these analytes, and also the highest absolute MS signal intensity. Diisopropyl amine (971.9 kJ/mol) is second in PA and methyl amine (899 kJ/mol) has the lowest PA and MS signal. Basicity in the gas phase and in solution appear to play important roles in determining the types of ions and conditions (pH) under which they will be observed, similar to the effects observed in electrospray.[95]

To investigate the role low - as opposed to zero - voltages can have on ionization,

paper spray experiments were performed using diphenylamine and low and zero volts.

The results demonstrate that the small signal at zero volts rises measurably (by a factor of

1.5) upon providing 1 volt on the paper (Figure 2.9), and increases by a factor of 2.5 on

raising the potential to 10 volt (data not shown). The addition of even a low voltage

supplies additional charges, which increases ionization efficiency.

2.3.3 Analyzing Organic Salt/Organic Analyte Mixtures by Zero Volt Paper Spray, kV Paper Spray, and Nanoelectrospray

A mixture containing 9 ppm cocaine and 0.1 ppm tetrabutylammonium iodide was examined by nESI, kV PS and zero volt PS. The results are shown in Figure 2.10a-c.

For nESI and kV PS, cocaine (protonated molecule, m/z 304) is the dominant peak, while the signal intensity of tetrabutylammonium (m/z 242) is only about 2% of that of cocaine.

For zero volt PS, m/z 304 is still dominant, but the relative intensity of tetrabutylammonium (m/z 242) is much higher than in nESI and kV PS (about 50% relative abundance). The trend is even more obvious in the results of 9 ppm morphine/0.1 ppm tetrabutylammonium iodide (Figure 2.11a,b,c). The data for nESI (Figure 2.11a) and kV PS (Figure 2.11b) show the signal for morphine (m/z 286) to be the base peak, while the relative abundance of tetrabutylammonium (m/z 242) is only about 2% in both cases.

By contrast, in the zero volt PS result, it is the ion m/z 242 that constitutes the base peak, while the relative abundance of the protonated morphine ion is only about 10% (Figure

2.11c). An analogous effect was observed in the negative ion mode, by analyzing a mixture of 36 ppm sodium tetraphenylborate and 3,5-dinitrobenzoic acid (Figure 2.11 d,e,f).

Figure 2.10 Mass spectra of a mixture of 9 ppm cocaine and 0.1 ppm tetrabutylammonium iodide using a) nESI, b) kV PS, and c) zero volt PS.

To account for these results we note the well-known fact both in nESI and in conventional kV PS that the signal intensity is closely related to the concentration of the analyte, at least in the lower concentration range where the available number of charges is sufficient to convert all analyte into the ionic form. The observation that zero volt PS is ca. two orders of magnitude less efficient than kV PS and nESI is interpreted simply as the result of the limited number of charges provided by the statistical droplet breakup process versus direct solvent charging. However, this does not explain the large differences between the cocaine/tetrabutylamonium and morphine/etrabutylamonium ion signals in zero volt PS vs. the conventional PS and nESI methods. The aqueous pKb of

cocaine is 5.39 (15), and morphine is slightly higher, 5.79 (25 ). This means that

morphine should produce somewhat fewer ions than cocaine even when their absolute

concentrations are the same. However, the main reason for the low relative intensity of

morphine in zero volt PS is likely the lower surface activity of morphine compared to

cocaine.[96] Evidence for this comes from the fact that when mixed with the very surface

active compound tetrabutylammoniumiodide, suppression of ionization is much more

obvious for morphine than for cocaine in zero volt PS. In kV PS and nESI, the influence

of surface activity is not as severe as in zero volt PS since their ionization efficiencies are

so high that most of the analytes in the droplets can be ionized and pushed to the droplet

surface. A parallel result is observed in negative ion mode where the surface active

tetraphenylborate signal is relatively enhanced in zero volt PS as compared to the kV PS

and nESI experiments. Given these qualitative explanations we can now test then using a

quantitative model of the ionization mechanism.

2.3.4 Overview of Ionization Mechanism for Zero Volt Paper Spray

It is well known that most analytes that can be ionized by ESI (or nESI) or by PS are Bronsted acids or bases. For a basic compound M dissolved in a solvent (S), a certain amount of M exists in the ion pair form (normally as solvent-separated ion pairs) because of the equilibrium:

M+ S ⇌ [M+H]+ + [S-H]-

For negative ion generation from of an acidic compound N, the equilibrium is:

N + S ⇌ [N-H]- + [S+H]+

For an ionic compound, say CA, there exists a dissociation equilibrium:

CA ⇌ C+ + A-

It is these solution-phase ion pairs which can go on to be evaporated and detected in zero

volt PS as positively or negatively charged ions.

2.3.4.1 Aerodynamic Breakup

When sufficient solvent is applied, droplets are pulled from the filter paper by the

suction of the instrument. Typically a few µL of sample is added before each suction

event suggesting that the initial droplets will be at least of similar volume. The droplets,

initially at zero velocity enter a high speed gas flow (170 m/s) due to the suction of the

inlet and experience an aerodynamic force.[38] This force causes the droplet to

simultaneously accelerate and breakup. The droplet will continue to breakup while its

Weber number is larger than 10.[40, 97] The weber number is defined by

Eq. 2.1

where ρg is the gas density, Vg is the gas velocity, Vd is the droplet velocity, Dd is the

diameter of the droplet, and σ is the surface tension of the solvent.[40] This suggests that

droplets will primarily breakup due to aerodynamic forces until they either accelerate to

the velocity of the surrounding gas or reach a certain size. There is evidence from charge

detection mass spectrometry that water droplets produced by either sonic spray ionization

or vibrating orifice aerosol generator reach a common size of about 2.5 µm after traveling

through the inlet.[97] This is also approximately the average size measured for kV PS

mass spectrometry.[98] This suggests that methanol droplets should undergo a similar

phenomenon, but in fact could be smaller due to the reduced surface tension of methanol as compared to water. Using this information, it is assumed that droplets may have diameters between 1-4 µm after aerodynamic breakup (Figure 2.12).

2.3.4.2 Initial Droplet Conditions for Evaporation and Columbic Fission Cycles

Aerodynamic breakup determines that droplets will have diameters between 1 and

4 µm and this serves as the initial diameter of droplets modeled in this section. The

number of analytes in a droplet was calculated based on initial analyte concentration and

its dissociation constant to determine the number of ions it will produce. Only ions can be

separated into detectable quantities by mass spectrometry, thus solution phase neutrals

are ignored in this model. The initial droplet charge was modeled by the statistical

fluctuations of positive and negative ions present in the total population of ions. For a

33 droplet containing n ions, of which the ions are either positively or negatively charged, the overall charge is modeled by a binomial distribution (Eq. 2.2).

; , 1 Eq. 2.2

For this distribution, p is the probability of an ion being charged (either positive or negative), n is the number of ions, and z is number of positive charges. The initial number of positive and negative ions is on average, equal; however, statistical fluctuations in the positive and negative ions will produce some net charge. This is simulated by using a binomial random number generator with parameter p = 0.5 and n is the previously calculated number of ions. The initial charge is found by subtracting the number of negative ions from the positive ions.

2.3.4.3 Droplet Evaporation to Rayleigh Limit

With the droplet’s initial parameter set (size, charge, number of analytes), evaporation is allowed to occur. The temperature of the droplet was kept constant at 298

K to ease the computation time required. This is justified by the fact that droplets will

cool evaporatively,[99] but will also be warmed by collisional activation, so the

temperature will drop initially but may rise later on, thus an accurate model for

temperature will be difficult to obtain over the droplet size range of the simulation (4 µm

– 10 nm).[100-102] The droplet is allowed to evaporate until it reaches the Rayleigh limit

diameter.[27, 33]

∗ Eq. 2.3 ∗∗∗

Here Dq is the charge on the droplet, e is elementary charge, ε0 is the permittivity of a

vacuum, and γ is the solvent surface tension. Surface tension was estimated using a

regression method developed by Jasper et al.[103, 104]

2.3.4.4 Droplet Fission and Progeny Droplets

Upon reaching the Rayleigh limit, droplets undergo fission and lose mass and

charge in the form of progeny droplets. At this point columbic fission occurs with most

reports indicating a small mass loss, Δm, (2%) from the precursor droplet and large charge loss, Δq, (15%).[105-107] From this the diameter of the precursor and progeny

droplets can be calculated, assuming that on average 10 progeny droplets are generated in

a fission event. The exact number of progeny droplets generated is unknown, but 10 is

within the range of typical values reported.[108-110] Accordingly the size of precursor and

progeny droplets was calculated according to these equations:

1∆ ∗ Eq. 2.4

∆ Eq. 2.5

where Npd is the number of progeny droplets taken to be 10, Dpd is the diameter of the

progeny droplets, and Δm = 0.02. At the time of fission only ions that are close to the

35 surface are allowed the possibility of being transferred to a progeny droplet. A volume fraction, Vf, is specified as the volume which can be considered for transfer to progeny droplets. In this simulation it is taken to be 15% of the total volume, but the exact value is unknown. The position of a solvated ion within a droplet is determined by its surface

activity, S. Surface activity is a number between 0 and 1 describing the probability of a

molecule being at the surface or the interior of the droplet. This is modeled by a binomial

distribution, similar to equation 2.2, except that p = S, n is the number of ions, and z is the

number of ions found in the outer region of the droplet. Thus when S = 1 all ions are

located in the outer region, and when S = 0, none are located in the outer region. Any ions

free of their respective counter charge are assumed to be in the outer region of the

droplet. The average number of ions, NIP, and charges, Nq, per progeny droplet are calculated from (Eq. 2.6) and (Eq. 2.7)

∗ ∗ Eq. 2.6

∗∆ Eq. 2.7

where CIP and Cq are the number concentration of ions and charges in the outer region of

the droplet. The number of ions transferred to progeny droplets can be modeled by a

[35] Poisson distribution. The number of ions, Nanal-IP, and charges, Nanal-q is chosen

randomly from a Poisson distribution.

∗ , Eq. 2.8 !

The same equation is used for Nanal-q with the appropriate substitutions. At this

point, more random charging can occur due to the statistical fluctuations of positive and

negative ions present in the total population of positive and negative ions. This is

modeled in the same manner as described in the initial droplet conditions section (Eq

2.2). With this information, the charge of the progeny droplet is calculated by subtracting

the total population of positive ions from negative ions. This same methodology is

completed for all the other progeny droplets, and then the conditions of the precursor

droplet are updated based on the total number of ions consumed by the progeny droplets.

All droplets (precursor and progeny) larger than 10 nm then undergo more

evaporation/fission cycles until all droplets reach 10 nm in size.

2.3.4.5 Analyte Ion Formation

Once all droplets have reached 10 nm in size the simulation ends. At this time

each droplet is analyzed for charge to determine the number of ionized analytes. For

example, a droplet containing a +2 charge is assumed to have two ionized molecules.

Note that in the simulation the actual ionization event is not modeled explicitly. Gas

phase ions could be produced by either the charge residue model or the ion evaporation

model. This counting process is repeated for all the droplets of size <10 nm and then

ionization efficiency can be calculated. Typically, 5,000 – 50,000 precursor droplets are

modeled to obtain an estimate of ionization efficiency and total number of ionized molecules. Alternatively, this model can be applied to droplets containing multiple

37 analytes, in which case multiple analyte ratios can be calculated. Note that multiple charges on the small analytes of interest are very unlikely and this possibility is ignored.

Figure 2.12 Simulation results of Weber number of methanol droplets. Using this information, it is assumed that droplets may have diameters between 1-4 µm after aerodynamic breakup.

Scheme 2.1 Overview of the ionization mechanism of zero volt PS ionization including representations of the aerodynamic breakup process and the droplet evaporation/coulombic fission. In both steps statistical distribution of charge to the fragments is assumed.

2.3.5 Single Analyte Simulation

Simulations were run with 2 µm droplets to investigate the possible limits of detection of zero volt PS. Both sizes lead to indicated limits of detection between 10-7 and

10-8 M (Figure 2.13), based on the assumption of being able to detect a single ion. The

detection limit determined from simulation is calculated from the ionization efficiency,

which is defined as the ratio of the number of ions generated vs. the total number of

molecules used.[111] Qualitatively, 18 ppb (4.87*10-8 M) of tetrabutylammonium iodide

could be detected experimentally, which is in good agreement with the estimate of

detectability by simulation. The simulation was also repeated at three different surface

activities and it was found that the number of ionized molecules decreases as the surface

activity decreases (Figure 2.14). Surface activity has been reported to have a similar

effect on the ionization efficiency. [31, 112]

Figure 2.13 The number of ionized molecules vs. concentration for 2 micron (bottom) and 4 micron (top) droplets. The simulation was run at three different surface activities.

Figure 2.14 Ionization efficiency vs. concentration of 2 micron (bottom) and 4 micron (top) droplets. The simulation was run at three different surface activities.

2.3.6 Experimental and Simulated Results and Mechanistic Considerations for Multianalyte Mixtures

A series of mixtures of cocaine and tetrabutylammonium iodide were analyzed with zero volt PS. In Figure 2.15a the amount of tetrabutylammonium iodide was varied while the amount of cocaine was held constant at 1 ppm. A similar experiment was performed using 3,5-dinitrobenzoic acid and sodium tetraphenylborate but analyzed in negative ion mode. In Figure 2.15c the amount of sodium tetraphenylborate was changed while the concentration of 3,5-dinitrobenzoic acid was held constant at 20 ppm. At each point, the ratio of (de)protonated molecular ion to salt was calculated. Simulations were

41 run in which the more surface active compound (salts in this case) was assumed to have a surface activity of 1 and surface activity of (de)protonated molecular ion relative to the salt was varied until the simulated ratio matched within 1 % error of the experimental ratio.

In Figure 2.15a, as the amount of tetrabutylammoiun iodide decreases the ratio of cocaine to tetrabutylammoiun iodide increases for both zero volt PS and nESI. Note that nESI has larger ratios than zero volt PS. This is because the kV applied voltage provides protons[33, 113] which can serve to ionize the cocaine, but will not cause additional

ionization of the tetrabutylammonium iodide salt. Thus the measured ratio becomes

closer to the concentration ratio, with differences being due to intrinsic ionization and detection efficiency. We applied the experimental intensity ratios of zero volt PS to our simulation and calculated the relative surface activity trend. The results are shown in

Figure 2.15b. As the amount of tetrabutylammonium iodide decreases the relative surface

activity of cocaine is calculated to increase. This makes sense since as the amount of

tetrabutylammoium idodie decreases more cocaine will move towards the droplet surface

and can compete against the tetrabutylammonium cation for surface sites.[114] Tang et al.

developed a model, which suggests that at low concentrations, 10-8 to 5*10-6 M, the ratio

of analyte ion signals is dependent upon the relative surface activities of the two analytes.[96] This agrees with our simulation results very well. Figure 2.15c,d display the

same general trend observed in Figure 2.15a,b As the concentration of sodium tetraphenylborate decreases the ratio of 3,5-dinitrobenzoic acid to sodium tetrphenylborate decreases (Figure 2.15c), while the relative surface activity of 3,5- dinitrobenzoic acid increases (Figure 2.15d). A similar set of experiments was performed

42 except that the concentration of the analyte was varied and the salt concentration was held constant (Figure 2.16).

In Figure 2.16a, the amount of tetrabutylammonium iodide was held constant at 0.1 ppm, while that of cocaine was changed. In Figure 2.16a the ratio of cocaine to tetrabutylammonium iodide increases as the concentration of cocaine increases. Figure 2.16b shows a similar trend to

Figure 5b, but since the amount of cocaine is increased the calculated relative surface activity of cocaine increases. Again this is because as the cocaine concentration increases, more cocaine can occupy the surface increasing its relative surface activity. In Figure 2.16c, sodium tetraphenylborate was held constant at 5 ppm and 3,5-dinitrobenzoic acid was varied. The data in

Figure 11c are consistent with those of Figure 2.16a in that as the concentration of the analyte increases the ratio of analyte to salt signal increases. Additionally, the relative surface activity of

3,5-dinitrobenzoic acid increases (Figure 2.16d) as the analyte concentration increases. The only noticeable difference is in the nESI results of Figure 2.16c, which show a drop in the ratio of 3,5- dinitrobenzoic acid to sodium tetraphenylborate in spite of its higher surface activity.

Figure 2.15 a) Cocaine to tetrabutylammonium cation ratio dependence in positive ion mode for zero volt PS and nESI. Cocaine concentration is held constant at 1 ppm, while tetrabutylammonium iodide concentration changes. b) The relative surface activity of cocaine calculated according to the experimental data in a). c) Ratio dependence of 3,5- dinitrobenzoate to tetraphenylborate in negative ion mode for zero volt PS and nESI. 3,5- Dinitrobenzoic acid concentration is held constant at 20 ppm, while the sodium tetraphenylborate concentration changes. d) Relative surface activity of 3,5-dinitrobenzoic acid calculated according to the experimental data in c). Surface activity of the salt is assumed to be 1 for all simulations.

2.4 Conclusion

Chemical analysis at zero volts from paper substrates has been demonstrated. Zero

volt PS gives both positive and negative ion signals, and allows detection of similar

45 compounds to those seen by kV PS and nESI, but with lower ionization efficiency. In spite of the low efficiency, the process is intrinsically very gentle and is more sensitive to surface active compounds. A mechanism for zero volt PS has been proposed based on the statistical fluctuation of positive and negative ions in droplet solutions during the course of droplet breakup. This model has been used to predict a detection limit similar to that observed experimentally. In the case of multiple analytes, the simulation is also able to calculate the relative surface activity of both positive and negative ions, such as cocaine and 3,5-dinitrobenzoic acid. The relationship of this model to other zero volt spray ionization mechanism is not known but is of great future interest, especially for the inlet ionization experiments[81, 82] and the nanowire spray[115] methods. Besides the

understanding of its mechanism, zero volt PS also has potential application advantages: it

could be used to study systems where there is a need to avoid the influence of external

electric fields and at the same time to use paper to eliminate the complex matrix, such as

in living organisms analysis.

CHAPTER 3. AMBIENT PREPARATION AND REACTIONS OF GAS PHASE SILVER CLUSTER CATIONS AND ANIONS

3.1 Introduction

Transition metals exhibit unique physical, optical, and chemical properties both as

bulk materials and in the nanoscale. Silver nanomaterials have applications in water

purification[116], catalysis[44], and surface enhanced Raman spectroscopy[65, 117], among

many others. The properties of atomically precise metal clusters are even more

fascinating as the addition or removal of a single atom affects the electronic[118] and

chemical[119] properties of the cluster. Atomically precise metal clusters can be generated

both in solution and in the gas phase. Mass spectrometry (MS) is a critical tool for

preparing and understanding the unique properties of metal clusters both in the gas

phase[120, 121] and when deposited onto surfaces.[44, 122, 123]

The production of gas-phase metal clusters has been demonstrated using a variety

of ionization sources, including magnetron sputtering,[124] laser ablation,[125-128] gas aggregation,[129, 130] cold reflux discharge[131], pulsed arc cluster ion sources,[132, 133] and

electrospray ionization (ESI).[134-139] These sources produce positive, negative, and

neutral clusters over a wide size range with a seemingly unlimited choice of elemental

compositions.[140] Flow tube reactors[124, 141, 142] and ion traps[143, 144] are common ways of

+/- studying metal clusters in the gas phase. Small silver clusters (Agn n < 20), the topic of

interest

47 in this paper, have been studied extensively in the gas phase due to their value in partial oxidation and catalytic reactions.[119, 136, 137, 141, 145-151]

+ The silver cluster, Ag4H has been used to mediate the carbon-carbon coupling of

allyl bromide.[136] In the overall reaction, three molecules of allyl bromide are needed to

+ + generate [Ag(C3H5)2] , [Ag3Br3], and CH2=CHCH3. The product, [Ag(C3H5)2] loses

C6H10, most likely 1,5-hexadiene, upon collisional activation, which represents an example of gas phase carbon-carbon bond coupling.[58, 152] Silver has long served as a

+ catalyst for the partial oxidation of ethylene to ethylene oxide and Ag2O has been

investigated as a model for silver-mediated olefin epoxidation.[150] Roithová and Schröder

note clean O-atom transfer reactions with ethylene. Propylene undergoes allylic H-atom

abstraction and other oxidation pathways but C-H abstraction from propylene leads to the

formation of unwanted byproducts, preventing high selectivity in the formation of

propylene oxide. [153]

The physical and chemical properties of anionic silver clusters in the gas phase

have also garnered attention. Measurements on the binding energies of O2 and

[145, observation of cooperative binding of O2 to silver cluster anions have been reported.

146] - Recently, Luo et al. noted the enhanced stability of Ag13 towards etching with O2 due

[141] to a large spin excitation energy. The reaction of O2 with CO in the presence of

- anionic silver clusters was found to be strongly cluster-size dependent with only Ag7 ,

- - [119] - Ag9 , and Ag11 serving as potential catalytic centers. Luo et al. found that Ag8 reacts with chlorine through a harpoon mechanism[148] and reported that silver cluster anions

- - [149] activate C-S bonds in ethanethiol to produce such products as AgnSH and AgnSH2 .

While extensive literature exists on the chemistry of gaseous silver clusters in vacuum, there is also a growing community interested in studying their properties on surfaces. For example, size-selected silver cluster cations with different energies were deposited onto Pt(111) and the surface topography was measured with scanning tunneling microscopy. When the energy was less than 1eV/atom, it was possible to non- destructively deposit the clusters.[154] Palmer et al. deposited size-selected silver clusters

[155] + on graphite as a method for preparing nanostructured surfaces. Recently, Ag3 deposited on alumina was found to be a highly selective catalyst for the epoxidation of

[44] propylene by O2. Size selected-silver clusters were deposited on passivated carbon in order to understand the discharge process in lithium-oxygen cells. The size of the deposited silver clusters greatly affected the morphology of lithium peroxide, indicating that precise control of sub-nanometer features on a surface might improve battery technology.[156]

In this study, we describe a novel and facile method for the production of naked metal cluster ions by electrospray ionization (ESI) and cluster heating in air. When the appropriate silver salt is subjected to these conditions, it is possible to generate silver

cluster cations and anions without use of lasers or collision-induced dissociation (CID).

Physiochemical properties are elucidated by subjecting the clusters in the open air to ion/molecule reactions; for example, they can be oxidized in the presence of ozone. The silver clusters can also be used for the analysis of hydrocarbons or to study ligand exchange processes. Finally, it is demonstrated that this ionization source can be coupled to an ion soft landing instrument to perform catalytic studies.

3.2 Experimental Details

Silver fluoride, silver acetate, silver benzoate, 2-ethylpyridine, 3-ethylpyridine, 4-

ethylpyridine, 3,4-lutidine, 2,6-lutidine, 2,5-lutidine, 3,5-lutidine, hexadecane, isocetane,

squalane, and ethanol were purchased from Sigma Aldrich, USA. HPLC grade methanol

was purchased from Mallinckrodt Baker Inc., Phillipsburg, NJ. Deionized water was

provided by a Milli-Q Integral water purification system (Barnstead Easy Pure II).

Deuterated solvents, such as D2O and CD3OD were purchased from Cambridge Isotope

Laboratories (Tewksbury, MA). Tert-butyl alcohol, 1-propanol, ammonium hydroxide,

and pyridine were purchased from Mallinckrodt Chemicals (St. Louis, MI). Isopropyl

alcohol, acetic anhydride, and acetone were purchased from Macron (Center Valley, PA).

2,4,6-trimethylpyridine was purchased from Eastman chemical company (Kingsport,

TN). All chemicals were used as received without further purification.

A home-built electrosonic spray ionization (ESSI) source was coupled to a heated

drying tube as shown in Figure 3.1.[157, 158] The heated drying tube is made from 316L stainless steel (Amazonsupply.com, part # S0125028T316SAL 6', O.D. 1/8”, I.D. 0.069”) and coiled twice to a diameter of 5.5cm and to a total length of 43 cm. The stainless steel

loop was wrapped with heating tape (Omega, part # FGR-030). The ESSI source was

typically positioned between 3 mm outside to 3 mm inside the heating loop, the position

being chosen such that the maximum ion signal was obtained. Independent positioning of

the ESSI sprayer and heating loop was achieved by using two xyz micrometer moving

stages (Parker Automation, Rohnert Park, CA). The distance between the heating loop

and linear ion trap mass spectrometer (LTQ, Thermo Scientific, San Jose, CA) inlet was

50 typically between 1 to 10 mm. At shorter distances, the ion signal was higher as more of the electrospray plume entered the MS inlet.

Figure 3.1 Apparatus for the production of silver cluster cations and anions

In a typical experiment, a metal salt was dissolved in 1:1 methanol:water at a concentration of 1 mM. Spray was initiated using a voltage of +/- 5 kV and N2 nebulizing gas (105 psi pressure). The electrospray plume entered the heating loop, which was set to

250 . The temperature was controlled by the voltage supplied using an autotransformer and monitored by a thermocouple. The ions exiting the loop were analyzed by a LTQ mass spectrometer using the following parameters: capillary temperature 200 °C, capillary voltage +/- 140 V, tube lens voltage +/- 240 V, maximum injection time 100 ms, and an average of 3 microscans. Automatic instrument tuning was used to optimize the potential applied to all other lenses. For collision-induced dissociation (CID), normalized collision energies of 25-35% (manufacturer’s unit), Mathieu parameter qz values of 0.25-0.35, and isolation windows were typically 2 m/z units larger than the

+ isotopic distribution (e.g. for Ag6 an isolation window of 14 mass units was used). In

certain experiments, high mass accuracy (<5 ppm) was achieved using a hybrid LTQ-

Orbitrap mass spectrometer (LTQ-Orbi, Thermo Scientific, San Jose, CA). The mass

resolution was set to 30,000 with a typical injection time between 250 ms and 5 s

depending on the initial ion intensity. Tandem MS Orbitrap experiments utilized the same

conditions as the LTQ.

To perform ion/molecule reactions, the distance between the heating loop and

inlet was typically set at 1cm. Vapors of interest were introduced via a cotton swab held

between the heating loop and MS inlet (Figure 3.2). In order to perform sequential

ion/molecule reactions, a short piece of metal tubing (2.5 cm) was placed between the

inlet and heating loop (Figure 3.3). One neutral reagent was introduced into the first gap

(between the heating loop and the additional metal tubing) and the second reagent was

introduced into the second gap (between the added metal tube and the inlet). In addition,

some experiments were performed using ozone which was generated by low temperature

plasma (LTP) (Figure 3.4). The low temperature plasma source used has been described

previously.[159, 160] Gases were introduced using Swagelok couplings at a flow rate

between 0.5-1 L/min.

Figure 3.2 Apparatus for studying atmospheric pressure ion/molecule reactions of silver clusters (cations/anions) with various reagents

Figure 3.3 Apparatus for performing atmospheric pressure ion/molecule ligand exchange reactions of silver clusters with various reagents

3.3 Results and Discussion

3.3.1 Unheated silver salts

The electrospray of solutions of metal salts has long been known to produce

+ - species of the type [CnAn-1] and [CnAn+1] where C is the cation and A is the anion of the salt.[161] Consistent with this, the mass spectrum of silver acetate recorded with the heating loop turned off shows the formation of simple salt clusters (Figure 3.5A). This

+ spectrum is characterized by a weak geometric magic number for [Ag5(CH3COO)4] and

+ [Ag8(CH3COO)7] , the enhanced stability likely being associated with 3x3 and 3x5 units.

For smaller clusters (Ag3 and smaller), some hydration is present. For larger clusters

+ ([Ag4(CH3COO)3] and larger), dissociation (by CID) of the mass-selected ion is

dominated by the loss of Ag2(CH3COO)2 units, which likely occurs in two separate

steps.[137] Smaller hydrated clusters undergo water loss followed by ejection of

+ AgCH3COO while the cluster [Ag2(CH3COO)] undergoes decarboxylation in agreement

with observations by O’Hair et al.[162] Negative ion mode electrospray produces a series

- of ions [Agn-1(CH3COOn)] , which do not exhibit any geometric magic numbers (Figure

3.6A). Dissociation of silver acetate cluster anions is by loss of Ag(CH3COO). Silver

benzoate exhibits similar clustering to that of silver acetate except that the geometric

+ magic numbers are replaced by an abundant ion [Ag7(C7H6O2)6] (Figure 3.6 C,E). Silver

fluoride exhibits a unique unheated mass spectrum. In the positive ion mode, naked silver

+ + + + cluster cations (e.g. Ag3 ,Ag5 , Ag7 , Ag9 ) along with various oxidized and hydrated

cations are observed (Figure 3.6G). In the negative ion mode, extensive clustering of

silver with multiple counter anions is observed (Figure 3.5C).

3.3.2 Heated Silver Salts

The mass spectra of the various silver salts changed significantly when the heating loop was set to 250oC. Regardless of the silver salt chosen, the positive ion mode

mass spectra were all essentially identical (Figure 3.5B, Figure 3.6D,H). These spectra

+ 2+ are dominated by the naked metal clusters Agn and Agn . All species undergo expected

fragmentations (Figure 3.7), including the loss of Ag2 from odd-numbered clusters,

2+ 2+ [163-166] coulombic explosion for Ag16 , and monomer loss from other Agn ions. The

+ + expected odd/even alternation and magic numbers at Ag3 and Ag9 was also

[167] + 2+ observed. Thus, the fragmentation behavior of the Agn and Agn ions is the same,

regardless of the starting material, viz. silver benzoate or silver acetate. Moreover, during heating there is no evidence for the formation of silver hydride clusters. Interestingly,

- only silver fluoride has the ability to generate negatively charged clusters, Agn (Figure

3.5D). Silver acetate and benzoate do not produce silver cluster anions, but instead

produce mainly organic fragments, presumably because of the stability of the organic

counter anion (Figure 3.6B,F). The fragmentation of small silver cluster anions (n < 12)

matches that reported in the literature (Figure 3.8).[168] Larger silver cluster anions

typically lose neutral Ag, which has not been reported in literature. For the silver cluster

- [141] anions, the expected odd/even alternation and magic number at Ag7 is observed.

+ + + 2+ + 2+ Figure 3.7 Tandem MS of A) Ag5 , B) Ag7 , C) Ag8 /Ag16 , D) Ag10 /Ag20 , E) + 2+ + 2+ 2+ Ag11 /Ag22 , F) Ag13 , G) Ag19 , and H) Ag21

------Figure 3.8 Tandem MS of A) Ag7 , B) Ag9 , C) Ag11 , D) Ag12 , E) Ag13 , F) Ag14 , G) Ag15 , - - - H) Ag16 , I) Ag17 , and J) Ag18

3.3.3 Mechanism

The above experiments used heat as well as in-source collisions to transform silver acetate and other silver salts into silver cluster cations and anions. Ready access to these clusters ions facilitated the study of their formation and dissociation as a source of information on their structure and reactivity. Possible intermediates responsible for the formation of silver cluster cations from silver acetate were investigated by monitoring the total ion chronogram while the temperature of the loop was ramped from room temperature to 250oC over the course of a few minutes (Figure 3.9). At low temperatures,

+ the salt clusters [Agn(CH3COO)n-1] were observed. At intermediate temperatures (100 -

o + 150 C), new species were observed in the MS, including [Ag9(CH3COO)6O] ,

+ + [Ag8(CH3COO)5O] , and [Ag7(CH3COO)4O] , indicating losses of acetic anhydride

(C4H6O3), eq.(1). For smaller clusters, an additional water molecule was present on the

+ + cluster, as in [Ag6(CH3COO)3OH2O] and [Ag5(CH3COO)2OH2O] . This may simply be

the result of addition of residual water to coordinatively unsaturated metal complexes by

ion/molecule reactions in the ion trap, a known process. An alternative to this pathway, the loss of ethenone (C2H2O) followed by water, is considered less likely.

+ + Agn(CH3COO)n-1  [Agn(CH3COO)n-1-2mOm] + mC4H6O3 Eq. 3.1

Thermochemical considerations are useful in deciding on likely decomposition

pathways. For example if n =3, then the process shown in (1) forms one molecule acetic

+ [169] anhydride and Ag3O . The formation of acetic anhydride is exothermic by -572.5 kJ/mol, which almost 250 kJ/mol more exothermic than the alternative pathway,

61 suggesting that acetic anhydride formation is the favored pathway leading to oxidized silver cluster cations.

Figure 3.10 provides a chart of all ions observed in the full scan mass spectra along with their relationships as established by CID experiments for the decomposition of the silver acetate ions in the positive ion mode. All ions observed in the mass spectra can be explained as possible products of equation (1) when water and oxygen are allowed to arrive or leave from the cluster. When the heating experiment is performed at the highest temperatures, the formation of simple silver cluster cations and oxidized silver cluster cations was observed. Once the final temperature was reached and some time had passed, the formation of primarily naked metal clusters was observed.

The thermochemical mechanism for cationic cluster formation extends to other silver salts such as the benzoate, which is assumed to undergo thermal dissociation by

+ similar routes to give silver cation clusters. A method for the production of Agn and Agn-

+ 1H from cluster ions generated from precursor solutions containing silver nitrate and

either glycine or N,N-dimethyl glycine has been reported by O’Hair, using ion trap

CID.[137] The choice of precursor compounds is important in these experiments as it can

+ - allow the production of [Cn+1An] and [Cn-1An] where A is an anion with favorable redox

properties. For example, glycine contains both an amine and deprotonated carboxylic group, which appear to meet these redox criteria.[137] It should be noted that under the

conditions of the O’Hair study, silver acetate did not produce silver cluster cations upon

CID.[137] The heating loop utilized in our work dissociates clusters to this extent at

atmospheric pressure, but the similarities and differences between the ion trap CID and

ambient heating experiments are not well understood.[56]

Anionic cluster formation can be considered in a similar vein. The loss of fluorine could be by hydrolysis to give the enthalpically favored HF or in a redox process to give the less favored F2. The favored formation of the odd-numbered silver anion clusters

(Figure 3.5D) is simply due to their greater stability than their even-numbered counterparts.[141]

3.3.4 Reactivity of Silver Cluster Cations

3.3.4.1 Alcohol Reactivity

Atmospheric pressure ion/molecule reactions were carried out between organic

solvents (ethanol, 1-pronanol, isopropyl alcohol, tert-butyl alcohol, acetone, and

acetonitrile) and silver cluster cations generated from silver acetate. Typically, an organic

solvent of interest was added to a cotton swab and introduced into the region after the

heating tube and before the MS inlet (Figure 3.2). A summary of the reactions can be

found in Table 3.1, Table 3.2, and Figure 3.11. Monomeric Ag+ reacts to form mainly

+ + + [AgL] and [AgL2] , except in the case of acetonitrile where the only product is [AgL2] .

+ + + The cluster Ag3 reacts to form [Ag3L] and [Ag3L2] , except that acetonitrile behaves

+ + differently in forming [Ag3L2] and [Ag3L3] . This reactivity differs from reported gas

+ + phase reactions, where [AgL2] and [Ag3L3] are the terminal products for all reagents

studied except ethanol, which was reported to be unreactive.[170] The differences in reactivity are not unexpected given that our experiments were performed in air, and the literature data refers to vacuum, and there are also differences in reaction time. The

maximum interaction time in our experiments was on the order of a single scan, ca. 0.2-

0.5 s, compared to possibly 60 s reported in the literature. The ion/molecule reactions

under vacuum do not provide efficient third body stabilization for smaller ions,

explaining the difference in reactivity with ethanol.[170] Tandem mass spectrometry data

+ on the reaction products (e.g. [Ag3L2] , Figure 3.11 A,C,E,F) indicate that the reagents

+ serve as simple ligands. The reaction product, for example [Ag3L2] , loses one ligand for

+ each stage of tandem MS to form bare Ag3 . Simple ligation is the main result observed

in these reactions. There appears to be relatively good agreement between silver clusters

with alcohols obtained under vacuum and in the open air. Due to the inability to mass

select the clusters before reaction, it is possible that some of the reaction products form from the larger clusters which react with accompanying losses of Agn moieties. The loss of Ag upon ligand addition for even-numbered silver cluster cations is an example of such a reaction that has been previously reported.[170, 171]

Table 3.1 Reaction products of Ag+ with various reagentsa + + + Reactant Ag [AgL] [AgL2] Ethanol 9.7% 14% 100% 1-propanol 4.4% 6.7% 100% Isopropyl Alcohol 24.5% 13.2% 100% Tert-Butyl Alcohol 18.2% 3.6% 100% Acetone 24.8% 28.5% 100% Acetonitrile 9.4% N.R. 100% a L refers to the respective reagent

+ a Table 3.2 Reaction products of Ag3 with various reactants + + + + Reactant Ag3 [Ag3L] [Ag3L2] [Ag3L3] Ethanol 100% 75.4% 57.3% N.R. 1-propanol 33.0% 48.4% 100% N.R. Isopropyl Alcohol 100% 44.2% 73.% N.R. Tert-Butyl Alcohol 38.6% 11.0% 100% N.R. Acetone 100% 48.5% 51.8% N.R. Acetonitrile 44.8% N.R. 57.5% 100% a L refers to the respective reagent

3.3.4.2 Methyl Subsituted Pyridine Reactivity

Atmospheric pressure ion/molecule reactions were carried out between methyl-

substituted pyridines and silver cluster cations generated from silver benzoate (Figure

3.12, Table 3.3,3.4). Ligation of the neutral reagents to the silver cluster was observed

+ + with the dominant products being [AgL2] and [Ag3L3] except in the case of pyridine

itself and 2,4,6-trimethylpyridine which also produced species with a smaller number of

+ ligands. The nature of the interaction between Ag3 and the neutral reagent was

confirmed by CID (Figure 3.13) which showed the sequential loss of ligands from the

+ + species [Ag3(C8H11N)3] and [Ag3(C7H9N)3] generated from 2,4,6-trimethylpyridine and

+ 3,5-lutidine. This confirms that the neutrals are acting as simple ligands on Ag3 .

Figure 3.12 Reaction of silver cluster cations with A) pyridine, B) 2-ethylpyridine, C) 3- ethylpyridine, D) 4-ethylpyridine, E) 3,4-lutidine, F) 2,6-lutidine, G) 2,5-lutidine, H) 3,5- lutidine, and I) 2,4,6-trimethylpyridine. M stands for the reactant of interest

+ Figure 3.13 Tandem mass spectrometry data for [Ag3(C8H11N)3] (left) and + [Ag3(C7H9N)3] (right) where 2,4,6-trimethylpyridine and 3,5-lutidine are the respective 2 3 4 + 2 neutral reactants. A) MS , B) MS , C) MS of [Ag3+(2,4,6-trimethylpyridine)3] . D) MS , 3 4 + E) MS , F) MS of [Ag3+(3,5-lutidine)3] . M stands for the reactant of interest

Table 3.3 Reaction products of Ag+ with various methyl-substituted pyridinesa + + + Reactant Ag [AgL] [AgL2] Pyridine 1.0% 6.3% 100% 2-ethylpyridine 0.1% 1.4% 100% 3-ethylpyridine 0.9% 1.8% 100% 4-ethylpyridine 0.09% 0.9% 100% 3,4-Lutidine 1.8% 2.5% 100% 2,6-Lutidine 3.6% 1.2% 100% 2,5-Lutidine 2.2% 2.0% 100% 3,5-Lutidine 3.1% 1.4% 100% 2,4,6- 1.2% 10.6% 100% trimethylpyridine a L refers to the respective reagent

+ a Table 3.4 Reaction products of Ag3 with various methyl-substituted pyridines + + + + Reactant Ag3 [Ag3L] [Ag3L2] [Ag3L3] Pyridine 28.8% 44.1% 73.8% 100% 2-ethylpyridine N.R. N.R. N.R. 100% 3-ethylpyridine N.R. N.R. N.R. 100% 4-ethylpyridine N.R. N.R. N.R. 100% 3,4-Lutidine N.R. N.R. N.R. 100% 2,6-Lutidine N.R. N.R. N.R. 100% 2,5-Lutidine N.R. N.R. N.R. 100% 3,5-Lutidine N.R. N.R. N.R. 100% 2,4,6- 23.2% 52.5% 51.0% 100% trimethylpyridine a L refers to the respective reagent

3.3.4.3 Ligand Exchange

+ It is obvious from the previous examples that ligation of Ag3 is easily achieved.

+ A further test was made to determine if the generation of mixed-ligand Ag3 species was

possible. A slightly modified version of the apparatus of Figure3.2 was used so that

multiple neutral reagents could be introduced separately (Figure 3.3). The system was

71 tested with three mixtures of ligands: ammonia (from ammonium hydroxide) and acetonitrile, 2,6-lutidine and acetonitrile, and acetone and acetonitrile. For the first set of reagents, ammonia and acetonitrile, a variety of mixed ligand species was observed,

+ + + including [Ag3+C2H3N+NH3] , [Ag3+(C2H3N)2+NH3] , and [Ag3+C2H3N+(NH3)2] as

confirmed by MS/MS (Figure 3.14). 2,6-Lutidine and acetonitrile also generated a mixed

+ ligand species (Figure 3.15), [Ag3+C7H9N+C2H3N] ; however, this was the only mixed

+ + ligand species observed in this case. In addition, [Ag3+(C7H9N)2] and [Ag3+(C7H9N)3]

+ were formed in higher intensity than [Ag3+C7H9N+C2H3N] , indicating a preference

towards binding 2,6-lutidine. To qualitatively test the effect of the sequence used to

introduce neutral reagents, acetone and acetonitrile were used. Acetone was allowed to

interact first with the cluster spray followed by the acetonitrile and vice versa (Table 3.5

and Figure 3.16). When acetone was allowed to react first, it was possible to generate a

mixed species as well as species containing either acetone or acetonitrile. This was in

stark contrast to the case where acetonitrile was the first reagent as it produced only species with acetonitrile ligands; i.e. ligand displacement did not occur due to stronger binding to this ligand.

2 + 2 + Figure 3.14 A) MS of [Ag3+C2H3N+NH3] , B) MS of [Ag3+C2H3N+(NH3)2] , and C) 2 + MS of [Ag3+(C2H3N)2+NH3] for the reaction of silver clusters with first ammonia (from ammonium hydroxide) then acetonitrile

Figure 3.15 Full MS for the reaction of silver clusters with first 2,6-lutidine then acetonitrile. Inset: Blowup of m/z 350-700

2 + 2 Figure 3.16 A) Full MS, B) MS of [Ag3 + (C3H6O) + (C2H3N)] , and C) MS of [Ag3 + + + (C3H6O)2] for the reaction of silver clusters with Ag3 with first acetone, than acetonitrile

+ Table 3.5 Reaction products of Ag3 with acetone, acetontrile, acetone followed by acetonitrile, and acetonitrile followed by acetonea Compound Acetone Acetonitrile Acetone, then Acetonitrile, [Ag3 + (C3H6O)x + Only Only Acetonitrile then Acetone + (C2H3N)y] [1,0]   N.R. [2,0]   N.R. [3,0] N.R. N.R. [0,1]    [0,2]    [0,3]    [1,1]  N.R. [2,1] N.R. N.R. [1,2] N.R. N.R. a Checkmarks indicate the presence of a species as determined by the appropriate MS/MS experiment.

3.3.4.4 Hydrocarbon Reactivity

Silver cationization is known to selectively ionize unsaturated compounds,

including polycyclic aromatic hydrocarbons and alkenes.[172-175] A series of hydrocarbons

was analyzed using ambient ion/molecule reactions in the same manner as other neutral

+ + reagents (Figure 3.17). Both cationization by Ag and Ag3 occurred for hexadecane, isocetance, and squalane. Cationization of a pump oil (Ultragrade 19) sample was also demonstrated, but the identity of the silver adducts is unknown. The fact that heat was required to generate the silver cluster spray is advantageous for the analysis of heavy

hydrocarbons, which are difficult to vaporize and often have low vapor pressures. The

convenient ionization of saturated hydrocarbons in the ambient environment is a potential

analytical application of this methodology.

Figure 3.17 Analysis of A) Hexadecane, B) Isocetane, C) Squalane, and D) Ultragrade 19 oil using silver cluster spray. The inset of C) is the MS2 of [Ag+Squalane]+

3.3.4.5 Oxidation

Oxidized silver clusters can serve as model catalysts for partial oxidation

[150] + reactions, including epoxidation . Roithová and Schröder produced Ag2O in vacuum

+ + from the dissociation of Ag2NO3 into NO2 and Ag2O . Another possible approach to

+ simple oxides is the direct oxidation of Agn clusters, a possibility indicated by the reaction between neutral silver clusters and ozone.[176] Ionic metal cluster oxidation was achieved by utilizing a low power plasma (LTP) source (Figure 3.18), which under

76 appropriate conditions serves as a source of ozone.[159, 177] Silver cluster cations were generated from silver fluoride and allowed to interact with the generated ozone (Figure

3.18B). A series of oxidized metal clusters was observed which contained multiple oxygen atoms as well as water molecules. The simplest and most notable reaction was the

+ + transformation of Ag3 into Ag3O . It is observed that silver clusters containing at least one oxygen atom can react with water too. It seems that water is only reactive towards already oxidized clusters, since naked silver cluster cations have been reported to be unreactive towards water.[170] (The water is likely adventitious water in the ambient environment or in the ion trap used for mass analysis.)

Figure 3.18 Mass spectra of silver cluster cations generated from silver fluoride A) without + + ozone and B) with ozone. C) Shows the selected ion chronogram of Ag3 (top) and Ag3O (bottom) in the presence and absence of ozone. D) High mass accuracy measurements + confirm the formation of Ag3O .

+ + 3.3.4.6 Reactions of Agn and Oxidized Agn with Ethylene and Propylene

Supported silver trimers have been demonstrated to be catalysts for the direct

epoxidation of propylene using gaseous oxygen.[44] In our study, odd-numbered silver cluster cations interacted with ethylene to produce mono-, bi-, and tri-ligated species

(Figure 3.19A). No ligation was observed for even-numbered clusters, either due to their inertness or to the loss of Ag upon reaction to produce the favored odd-numbered cluster.

+ A small amount of [Ag3(C2H4)3O] was observed, which presumably arises from reaction

+ with the small amount of Ag3O present within the spray. With the addition of ozone

from the LTP source, the mass spectra change significantly. The intensity of

+ + [Ag3(C2H4)3O] increases dramatically and a species [Ag5(C2H4)4O2] is observed (Figure

3.19B). It is interesting to note that there appears to be a cooperative effect in the binding

+ of ethylene to oxidized clusters as Ag3 binds one or two ethylenes, but it appears that

+ Ag3O binds only three ethylenes. The same phenomenon occurs for the binding of one,

+ + two, or three ethylenes to Ag5 but Ag5O2 only binds four ethylenes. Cooperative

binding has been reported for O2 and CO with anionic silver clusters, but no such reports

[119, 145] + + exist for cationic silver clusters. [Ag3(C2H4)3O] and [Ag5(C2H4)4O2] were

subjected to CID to identify any potential epoxide products; however, only the products

+ of ligation were observed. The tandem MS of [Ag5(C2H4)4O2] (Figure 3.20) is

interesting as the loss of two ethylene molecules is accompanied by the addition of a

single water molecule, and the loss of three ethylenes is accompanied by the addition of

two water molecules.

Figure 3.19 Reaction of silver cluster cations with A) ethylene and B) ethylene and ozone

+ Figure 3.20 MS fragmentation tree for [Ag5(C2H4)O2] . Arrows indicate a single stage of CID

Similar experiments were performed with propylene as the reagent gas. In the absence of ozone, propylene exhibits nearly identical reactivity to ethylene (Figure 3.21).

+ The reactivity of ozone and propylene is similar to ozone and ethylene for the Ag3 cluster but differs for the larger clusters (Figure 3.21B). A variety of species containing

+ + + propylene, oxygen, and water are observed for both Ag5 and Ag7 . [Ag7(C3H6)O] is particularly interesting because it fragments to lose neutral C3H6O, propylene oxide or a structural isomer.

Figure 3.21 Reaction of silver cluster cations with A) propylene and B) propylene and ozone

3.3.5 Reactivity of Silver Cluster Anions

3.3.5.1 Formation of [Agn(OH)]-

The reactivity of silver cluster anions generated from silver fluoride was examined. The position of the ESI source relative to the heating tube was altered to form an ambient discharge. Without the discharge, naked metal silver clusters are observed

(Figure 3.22). With the discharge, the even-numbered clusters (especially n = 6, 8, and

- 10) gained an OH group to form [AgnOH] , possibly due to reaction with hydroxyl

[178] - radicals created by the discharge. The amount of [AgnOH] varies with cluster size but generally decreases as n increases. Deuterated 1:1 methanol:water was used as the spray

+ solution in an attempt to identify the source of the OH. The intensity of [AgnOD] was at

- most 30% of the [AgnOH] intensity, indicating that the source of OH must be from

elsewhere, such as atmospheric water.

3.3.6 Ion soft Landing

As a proof of principle, the metal cluster ion source was coupled to a homebuilt ion

soft landing instrument.[42, 117] Each of the three silver salts was electrosprayed and similar mass spectra were obtained as previously described (Figure 3.23). For unheated silver acetate and silver benzoate, the same type of salt clustering was observed as previously seen (Figure 3.5A). A similar effect was observed for silver fluoride, but the exact number of silver and amount of oxygen or water bound could not be determined

82 with the given instrument resolution. Upon heating, all three salts produced a range of

+ + clusters from Ag3 to Ag15 without any even-numbered clusters. This set of ions was

deposited onto a gold surface and a current of 10 pA could be achieved. Efforts are

underway to improve the current of the source so it will be possible to study mass-

selected ions on surfaces.

3.4 Conclusions

The major significance of this study lies in the development of a simple

methodology to produce anionic and cationic metal clusters in the ambient environment.

In addition (1) Insights into the mechanisms of formation of the cluster ions was obtained

by studying effects of ambient heating and from thermochemical considerations. The

formation of a series of odd-numbered silver anions is most remarkable. (2)

Ion/molecule reactions at atmospheric pressure revealed a rich chemistry of the silver

cluster cations and anions. Examples of this include, ligation, ligand exchange,

cationization of alkanes and olefins, and oxidation reactions. Ligand exchange chemistry

shows the relative strength of different ligands towards silver cluster cations, which may

be of use in choosing ligands for capping atomically precise clusters. (3) The potential

analytical utility of the cluster ions was demonstrated by silver cationization of saturated

+ alkanes. (4) The use of ozone to produce oxidized clusters especially Ag3O was

demonstrated; these silver clusters are especially important as partial oxidation catalysts.

In future work, attempts will be made to operate these ambient sources so as to

produce a narrower range of cluster ions, to select clusters of a particular size by ion

- - mobility and to explore further the chemistry of such anionic species Agn and AgnOH .

Extensions of this approach to the generation of cluster ions from other metals will also

+ + - be attempted. For example, it is already known that Pdn , PdnOy , and PdnOy (where

from a mixture of metal salts is also expected to be straightforward. Given the existence of a rationale for the loss of the counterion as a small stable molecule (acetic anhydride in the case of acetates), other counterions will be selected to optimize cluster ion formation.

The most interesting aspect of the new capabilities provided will be the investigation of processes of industrial significance using metal clusters and metal oxide clusters, which have some of the structural features believed to be important in full scale heterogeneous catalysis (certain metal cluster sizes and compositions).[121, 179, 180] The fact that these

species can be generated in air compensates to a degree for the fact that they are not

atomically precise.

CHAPTER 4. CAN ACCELERATED REACTIONS IN DROPLETS GUIDE CHEMISTRY AT SCALE?

4.1 Introduction

This study is part of a larger project, the overall goal of which is to develop an

automated scalable and continuous synthesis system. A key objective is to test possible

synthetic pathways quickly on a small scale seeking a go/no-go result. We “spot test”

particular routes using a chemical pruning step which employs reaction acceleration in

droplets with independent mass spectrometric analysis. A simple yes/no answer to product or (in multistep reactions) intermediate formation is sought using the charged droplet reactor. We use electrospray (ESI) for both synthesis and analysis with careful

control of parameters to avoid unwanted reaction during analysis.[21, 23, 58]

Charged microdroplets are produced by ESI. It is known that reaction rates increase as the solvent evaporates because of changes in concentration, pH, surface/volume ratios, and interfacial effects.[21, 57-60] The acceleration factors can be

remarkably large.[61] A recent review covers the topic of accelerated reactions in droplets,

including evidence that partial solvation of reagents at interfaces contributes to the orders of magnitude reaction rate acceleration that can be seen.[23] The hypothesis

Investigated here is that the accelerated reactions that occur in droplets might assist in rapidly evaluating reactivity in microfluidic systems.

A second method of producing droplets is based on the Leidenfrost effect.[181] It has recently been shown that accelerated organic reactions occur in Leidenfrost droplets.[22] These droplets differ from ESI based droplets in that they are i) larger ii) net

neutral and iii) involve elevated temperatures. The difference in droplet size means that

larger amounts of reagent can be studied, but the surface/volume ratio is greatly

decreased. The measured reaction acceleration factors for three previously studied

Leidenfrost reactions, hydrazone formation, Katritsky pyrylium to pyridinium conversion

and Claisen-Schmidt condensation, are about an order of magnitude.[22]

Note that we do not expect to be able to transfer optimized conditions exactly

from the droplet scale to the microfluidics scale, in part because of uncertainty about the

origins of acceleration effects in the two systems. We do expect that these optimized

conditions will represent a starting point for efficient optimization of the conditions in the

microfluidics reactor. We also expect that information on reaction intermediates and

mechanisms might be acquired from the study of droplet reactions. This information is

already being obtained in experiments in which the degree of desolvation of the initial

droplets is varied by changing the distance that the droplets travel before analysis.[58]

Information obtained from the droplet reactor on experimental parameters including

solvent, catalyst, pH, etc. is also readily acquired and we examine how transferable this

information is in optimizing the microfluidics flow reactor.

The identification of suitable pathways to target molecules is just one step

towards an online, automated flow-through synthesizer, for which the groundwork has

87 been laid by several groups. Notable are the mole-scale, end-to-end, continuous manufacturing pilot plant developed by MIT/Novartis,[182] the refrigerator sized,

reconfigurable, on demand synthesizer of pharmaceuticals of MIT,[183] the nanomole-

scale robotic high-throughput synthesizer of Merck[184] and the automated synthesis

laboratory of Eli Lilly.[185] The mole-scale MIT synthesizer was used to produce aliskiren

hemifumarate in tablet form, from a complex intermediate in a continuous fashion.[182]

The Merck nanomole system was used to screen 1,500 reactions per day to identify

potential candidate reactions for large scale synthesis. The Lilly system combined

automated synthesis with analysis performed remotely controlled in real-time, to produce

gram-scale products.

The main question underlying this study is whether droplet reactions may be used to predict chemical reactivity in flow chemistry systems, in particular in microfluidics.

The mechanism for acceleration observed in microdroplets is certainly different from that in microfluidics in that evaporation is not significant in microfluidics; however, interfacial effects may still play an important role especially in droplet microfluidics.[186,

187] The speed of data acquisition in droplets makes this approach attractive. Note that

false negatives (predict no reaction, but reaction can be observed) is not expected to be a

serious problem because there are usually many available routes to test. On the other

hand, a false positive result will lead to wasted effort in seeking an analogous flow

reaction. Note, too, that use of droplets for a simple yes/no regarding occurrence of

reaction represents only one level of enquiry, even though it is the most important one.

As will be seen in the results now to be discussed, information on reaction conditions is

88 also obtained, although the quality of this information remains to be evaluated further by studying more cases.

4.2 Experimental Details

4.2.1 Droplet Reactor Experiments

These experiments used electrospray ionization (ESI) by spraying the reaction

mixture either i) directly into the MS or ii) onto a collection surface before taking up the

residue in solvent and performing ESI-MS product analysis. Figure 4.1 illustrates these

two options, which are distinguished by the fact that i) is virtually instantaneous (10 – 15

s) while ii) can take a few minutes, but ii) is more versatile in that the reaction and

analysis occur in separate steps, more product is formed, and the procedure allows

temperature and other conditions to be varied and optimized. In both cases, the primary

question being asked is whether the desired reaction occurs or not. Products, byproducts

and residual reagents were identified from mass spectra recorded at unit resolution while

tandem mass spectrometry (MS/MS) was used to confirm identifications.

Figure 4.1 Methods used to perform microdroplet reactions, based on ESI either a) with online product analysis by MS or b) with sprayed droplet deposition and subsequent off-line MS product analysis

4.2.2 Offline Droplet Reactor

Offline droplet experiments were performed using a homebuilt electrospray

ionization source. In the cases of atropine and diazepam, the reagents were premixed and

subjected to offline electrospray at 10 µl/min with +5kV voltage and 100 PSI N2. For diphenhydramine, reagents were mixed inline using a mixing tee and offline electrospray was performed under the same conditions. After the electrospray deposition was complete, reaction product was rinsed from the collection surface and then analyzed by nanoESI. Samples were diluted at least 100-fold before analysis in order to quench the reaction and ensure no further reaction could occur during the analysis step. For two- step reactions, the washed material was drawn back into a syringe and mixed with the second step reagent and then electrosprayed, collected, and washed as before.

4.2.3 Leidenfrost Droplet Experiments

Reactions in Leidenfrost droplets[22] differ in that these are i) larger ii) net neutral and iii) involve elevated temperatures. Reaction mixtures were added in aliquots over a 2 min period to maintain a constant droplet volume (Figure 4.2). The droplet (ca. 2 mm diameter) was levitated in a petri dish atop a heater with a surface temperature of 400 –

500 C [CARE!]. Reactions occurred at temperatures close to, but below, the boiling

point of the solvent.[188]

4.2.4 Mass Spectrometry

Mass spectral analysis of reaction products was performed using an LTQ ion trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) with nanoESI ionization. All product samples (spray, Leidenfrost, or flow reactions) were diluted 1:100 into acetonitrile before analysis unless otherwise noted. The distance between the tip of the spray emitter and ion transfer capillary to the MS was held constant at ca. 1 mm.

Experiments were performed using borosilicate glass pulled to a ca. 1-3 um aperture. A spray voltage of either positive or negative 2.0 kV was used for all analysis. Positive ion mode was used for all chemical analysis unless otherwise noted. Product ion (MS/MS) spectra were recorded using collision induced dissociation (CID) with a normalized collision energy of 25 (manufacturer’s unit).

4.2.5 Synthesis of Diazepam Precursor

Reactions were performed with 100 mM 5-Chloro-2-(methylamino)benzophenone and 100 mM 2-haloacetyl chloride (halo = Cl, Br) dissolved in either toluene or acetonitrile. Solutions were either mixed prior to use (droplet reactor, Leidenfrost) or mixed online (microfluidics) to give a final reaction concentration of 50 mM. Other conditions were explored as indicated in the results section.

4.2.6 Diphenhydramine

Reactions were performed in a two-step manner. First, 500 mM benzhydrol was mixed with 500 mM mesyl chloride, then in the second step 20 equivalents of

92 dimethylaminoethanol was used in the droplet reactor, while 1 equiv was used for microfluidics. Other conditions were explored as indicated in the results section.

4.2.7 Atropine

The atropine intermediate was first synthesized by reacting 1 M phenylacetyl chloride and 1 M tropine dissolved in DMA. For the second step, 7 equiv of base in

DMA and 7 equiv of formaldehyde in H2O was used. Other conditions were explored as

indicated in the Results section.

4.2.8 Microfluidics

Microfludic reactions were performed using a Labtrix S1 system from Chemtrix,

Ltd. The Chemtrix system is comprised of syringe pumps to deliver regents, microfluidic

chips, a Peltier element, back pressure regulator, and a collection carousel. For the

synthesis of diphenhydramine a homebuilt Peltier controlled system coupled with the

Chemtrix microfluidic platform was used to allow for multi-step reactions to be performed with control over the temperature of each step.

4.2.9 Chemicals and Reagents

All chemicals were purchased from Sigma Aldrich and used without further

purification.

4.3 Results and Discussion

4.3.1 Synthesis of Diazepam Intermediate

The charged droplet and microfluidic based synthesis of amide 3, generated by N-

acylation of 1 with the 2-haloacetyl chloride 2, was examined due to its importance as a

synthetic step in the pathway to diazepam (Scheme 4.1). An electrospray droplet reactor was used to evaluate potential solvents for the N-acylation reaction using chloroacetyl

chloride. Offline charged droplet reactions were performed using a mixture of 1 and 2 (X

= Cl) in various solvents, and conversion to product as analyzed by ESI MS was

compared to a 30-minute batch reaction (Figure 4.3). Before analysis, samples are

quenched in order to ensure no further reaction by diluting the collected product into the

solvent used in the prior step. The results indicate that there is significant acceleration of

the reaction when the solvent is DMF, ACN, or toluene. Acceleration in microdroplets is

associated with evaporation and is proposed to be due, in part, to intrinsic rate

acceleration at the interface.[60] These initial results encouraged a more extensive reaction

screening, where the effect of the chosen 2-haloacetyl chloride (Cl or Br) and the solvent

(ACN, toluene) was investigated using a droplet reactor. Interestingly, the droplet reactor data indicated nearly complete conversion of starting materials to product for both the chloro and bromo starting materials in acetonitrile (Figure 4.4) and in toluene (Figure

4.5). Starting material 1, is observed at m/z 246 and product, 3 appears in either its protonated (X = Cl, m/z 322-326 or X = Br, m/z 366-370) or sodiated (X = Cl, m/z 344-

348 or X = Br, m/z 388-392). A small amount of SN2 product (m/z 322) was observed for

bromoacetyl chloride in ACN but not in toluene. In the case of the chloro starting

material (2), the SN2 and acylation products have identical molecular formulae and are not differentiable; however, the presence of only small amounts of the SN2 product using bromo starting material, provides evidence that the chloro reacts mainly to form the desired acylation product.

Figure 4.3 Solvent screening for the amide synthesis of 3 from chloroacetyl chloride 2 in batch and in charged droplets. Acceleration rate is calculated as previously discussed

Figure 4.4 Synthesis of 3 in ACN using a) chloroacetyl chloride and b) bromoacetyl chloride in droplet reactor and microfluidics (µ-Fl)

Figure 4.5 Synthesis of 3 in toluene using a) chloroacetyl chloride and b) bromoacetyl chloride in charged droplets and microfluidics

Flow experiments were performed using the same concentrations as in the droplet reactor, while screening the effect of temperature for a fixed residence time of 30 seconds. High conversion to 3 was observed with chloroacetyl chloride in both solvents at 50°C. More interestingly, a major difference was observed with bromoacetyl chloride in ACN, wherein a major amount of SN2 reaction product was observed, especially at

higher temperatures (100 and 150°C). The presence of a by-product (ion m/z 288) arising

from the initial SN2 reaction product was confirmed by NMR and MS/MS (data not shown). The droplet reactor predicted formation of the desired intermediate, which was observed in flow. However, under higher temperature conditions in flow, the proportion of SN2 product increased. This difference can be explained by the fact that evaporative

cooling occurs during flight in the droplet reaction at room temperature, thus reducing the

reaction temperature.[189] Nonetheless, the droplet reaction demonstrated reaction

feasibility and showed that specific solvents (ACN, toluene) are better than others a fact

reiterated under microfluidic conditions for this transformation.

The occurrence of accelerated organic reactions in Leidenfrost droplets[22] is at least in part a surface property (partial solvation of reagent molecules at the surface reduces activation energies). Consistent with this, and the smaller surface/volume ratios of Leidenfrost droplets, acceleration factors are smaller than in electrosprayed microdroplets. However, the larger droplets (0.5 mL volume) mean that conditions in the

Leidenfrost droplets are closer to those in microfluidic solutions and in the bulk, so the predictive power of Leidenfrost droplet reactions might be even greater than that of electrospray generated droplets. The lack of a formal charge also strengthens the expected analogy with scaled-up chemistry.

This expectation is met when one considers data for the first step of the diazepam synthesis using bromoacetyl chloride and chloroacetyl chloride. First, consider the mass spectra recorded for charged droplets in ACN and in toluene versus those for Leidenfrost droplets in the same two solvents (Figure 4.6, Figure 4.7). The assignment of m/z is the same as figure 4.4 and scheme 4.1. The conversion in the Leidenfrost experiment is not as great (more starting material seen) as in the charged droplet reactions, but both methods give almost exclusively the desired acylation intermediate as opposed to the SN2 product. There is not a large difference in the results for ACN versus toluene as solvent, except that the conversion is slightly higher in ACN. If we now consider the difference

between microfluidic flow and Leidenfrost droplet data, we find remarkable similarities.

Microfluidic synthesis at 50°C results primarily in desired acylation product as is the case

in the Leidenfrost droplets. One difference is in the formation of a minor species seen at

m/z 260 in the Leidenfrost case.

Figure 4.6 Synthesis of 3 in toluene using chloroacetyl chloride a) and bromo acetylchloride b) in Leidenfrost, charged microdroplets and microfluidics

Figure 4.7 Synthesis of 3 in ACN using a) chloroacetyl chloride and b) bromoacetyl chloride in Leidenfrost, charged droplets and microfluidics

Scheme 4.1 Reaction of 1 and 2 (X = Cl, Br) to form amide 3.

The ion m/z 260 is believed to be due to a ring closure product resulting from acylation. The uncharged Leidenfrost droplets closely mirror the chemistry in microfluidics, except when the temperature in the microfluidics reaction is greatly elevated. Under these circumstances different byproducts are generated as SN2 becomes

more competitive.

The complete synthesis of diazepam in Leidenfrost droplets was demonstrated by

adding 7 or 70 equivalents of NH3 to intermediate 3 (not isolated) in a telescoped

reaction. The chloro intermediate was unable to produce diazepam in quantity. However,

the bromo intermediate produced diazepam (confirmed by MS/MS) in agreement with a

reported flow synthesis.[183]

4.3.2 Synthesis of Diphenhydramine

The ability of droplet reactivity to guide chemistry at scale was investigated for another important drug, diphenhydramine. The flow based synthesis of diphenhydramine

was demonstrated by the reaction of dimethylaminoethanol (DMAE) with chlorodiphenylmethane.[12] This synthesis featured 100% atom economy; however, chlorodiphenylmethane is an expensive starting material, which motivated an effort to develop a more cost effective process by replacing chlorodiphenylmethane using a

commodity starting material, benzhydrol, 4. To synthesize diphenhydramine 5, benzhydrol (4) was converted to the corresponding mesyl ester, which was subsequently

treated with DMAE to produce 5 (Scheme 4.2). The droplet reactor synthesis of 5 was

demonstrated by performing two sequential charged droplet reactions in either a toluene

or acetonitrile solvent system. First benzhydrol and mesyl chloride were sprayed to

100 produce the mesyl ester. This material was recovered and re-dissolved before introduction of the second reagent, dimethylaminoethanol (20 equivalents) and repetition of the spray process (Figure 4.8). The MS analysis of the two-step spray product indicated that ACN is overall a better solvent for the synthesis of diphenhydramine, 5

(m/z 256), than toluene. Unreacted DMAE (m/z 90), mesylated DMAE (m/z 168), and a dimer of DMAE with methanesulfonic acid (m/z 275) were observed with the charged microdroplets. A similar trend was observed in flow (Figure 4.9, 4.10), where optimized conditions gave diphenhydramine in 35% and <1% yield in ACN and toluene, respectively, using one equivalent of dimethylaminoethanol. Good agreement is observed between the charged droplet reactor and flow in this synthesis.

Figure 4.8 Charged microdroplet (a,c) and microfluidic (b,d) reaction telescoping for diphenhydramine synthesis in two solvents (ACN, toluene)

101

Figure 4.9 Droplet reactor and microfluidic reaction telescoping for diphenhydramine synthesis in ACN

Figure 4.10 Droplet reactor and microfluidic reaction telescoping for diphenhydramine synthesis in toluene

102

Scheme 4.2 Mesylation of 4 followed by reaction with dimethylaminoethanol (DMAE) to form diphenhydramine,5.

4.3.3 Synthesis of Atropine

The charged droplet and microfluidic synthesis of atropine also was achieved by telescoping two reaction steps. The intermediate ester 8 (Scheme 4.3) was prepared from the commercially available starting material tropine 6 and phenylacetyl chloride 7. The intermediate 7 was used without further purification for the aldol condensation reaction to produce the final product, atropine. The first step was optimized for solvent and reactant stoichiometry. The droplet reactor indicated dimethylacetamide as the best solvent and this was confirmed in microfluidics (data not shown). Using the unpurified intermediate ester 8 (m/z 260), a base screen with the droplet reactor determined the effectiveness of three bases in synthesizing atropine, 9 (m/z 290). Each base was successful in the droplet reactor, producing significant amounts of atropine (and byproducts), with 1,5- diazabicyclo[4.3.0]dec-5-ene being the most effective (Figure 4.11, Figure 4.12). In flow, each base produced atropine (and byproducts) with 1,5-diazabicyclo[4.3.0]dec-5-ene again being the most efficient. There is also some agreement between flow and charged

103 droplets on the type and extent of byproduct formation. For example, using 1,5- diazabicyclo[4.3.0]dec-5-ene, atropine and its dehydration product (m/z 272, 10) are observed. However, with MeOK the same type of byproducts could be observed, but their proportions were quite different. (Scheme 4.3). The major byproduct in flow, characterized by m/z 304, 11, is barely formed in charged droplets. One possible reason for this difference is that formaldehyde with its high vapor pressure escapes the droplets rapidly obviating formation of this byproduct. Finally, good agreement between the two methods was observed with NaOH, where the ,-unsaturated product (m/z 272, 10) is the major byproduct for both droplet reactor and microfluidics. Thus, for the synthesis of atropine, the charged droplet reactor was useful in guiding the choice of solvent and base.

104

Figure 4.11 Charged microdroplet (a,c) and microfluidic (b,d) reaction telescoping for atropine synthesis using potassium methoxide or 1,5-diazabicyclo[4.3.0]dec-5-ene

105

Figure 4.12 Comparison of relative conversion to atropine using three different bases in droplet reactor and microfluidics

Scheme 4.3 Esterification reaction of 6 with 7 to synthesize 8 followed by base catalyzed aldol condensation with formaldehyde to synthesize atropine, 9.

4.4 Conclusions

This study provides evidence for the importance of MS, not only in the traditional sense as an analytical method, but as a fast, predictive means to perform small scale continuous synthesis. The data encourage the use of spray ionization as a method of

106 screening for successful reaction pathways in flow reactions. At a secondary level, we find some parallels in the favored catalysts, solvents, mole ratios of reagents, and other operating conditions. However, little is known of droplet reaction mechanisms (an important topic in its own right), so extrapolation from conditions that favor reactions in nanodroplets to microfluidic chemistry may not be simple or universal. Nevertheless, as we show in this study, a useful guide to the global aspects of flow chemistry is obtained in these cases.

Limitations in further extending this approach to reaction screening (and to on-line reaction monitoring) are to be found in the size, cost and complexity of commercial mass spectrometers, many of the features of which are unnecessary for this type of study.

What is needed for the purposes described here is a small, portable, unit resolution, low mass/charge range (to m/z 1000) instrument which has ambient ionization and tandem mass spectrometry capabilities. A recent review of miniature MS instruments[190] describes a few systems of this type.

107

CHAPTER 5. OUTLOOK AND FUTURE DIRECTIONS

5.1 Laboratory Automation

In pursuit of the most robust and reproducible results, there is a natural desire to

remove human interaction when possible from the screening of a chemical reaction

space. High throughput assays have been used by both biological and chemical

community for many years.[184, 191] Early attempts at automation including the pioneering work of Professor Phil Fuchs and Gary Kramer. There system included a sample delivery system that was based on a model railroad system.[192, 193] More sophisticated systems

have been developed over the years including the automated gram scale synthesis lab of

Lilly[185] and the nanomole high-throughput device of Merck.[184] The main components

of each of these devices are sample preparation (fluid handling), reaction, sample

analysis, and sample interpretation. Herein strategies for automated methods of running

and analyzing reaction mixture will be discussed.

The goal of this automation approach is to reduce the workload of the organic

chemist in the laboratory (or completely remove it) and instead allow them to focus on

more high-value decisions, such as the choice of molecular structure for a subsequent

biological study. The development of such automation could socialize organic synthesis.

108

5.1.1 Continuous Electrospray

Electrospray has been shown to be an excellent preparation method for many organic reactions; however, it is not expected that all reactions will complete within the short millisecond reaction time.[23] One way to circumvent this would be to increase the

number of times reactions occur. Additionally, evaporation has been shown to play an

important role in the acceleration of reactions in microdroplets; however, a byproduct of

the evaporation process is the generation of large quantities of solvent waste. A way for

the collection of the droplets and evaporated solvent is desirable, as it is economical and

avoids undesirable solvent waste. In this manner, if a reaction is incomplete, the sprayed

material can be collected and resprayed in order to be processed one or more times. That

provides unreacted molecules a chance to react and allows incomplete reactions to be

driven to completion, thereby increasing a yield of a chemical reaction. One version of

the device is pictured in (figure 5.1a), and the primary components are an electrospray

device, droplet collector, liquid degasser, condenser, and solvent pump. These

components allow for a way to continuously process a reaction mixture with electrospray

maximizing product yield and saving solvent.

5.1.2 Neutral Droplet Emitter

Another format that will also be useful, both for mechanistic studies and for

synthesis is the use of a commercial (HTX Imaging, Inc.) matrix sprayer (figure 5.1b). In

preliminary experiments (figure 5.1c) we have found this to be an effective way of

performing reaction acceleration in droplets, showing similar acceleration factors to those

previously reported.[22, 194] The device conveniently allows variation in flow rate,

109 position of the spray tip, and temperature of the reagent spray. Droplets generated are on the same size scale as ESI and can be easily varied. We are especially interested in using it as a source of “super-reagents” to react with analytes on surfaces, in solution or in other sprayed droplets. Note that the experiment differs from reactive DESI both in the charge on the droplet and also in their low momentum. Nonetheless, the system gives high flow rates (100 µL/min) and concentrations of reagents can be relatively high (0.1 M) The system will be particularly useful for testing whether reactions can be effective at an interface in cases where droplets containing both reagents are not splashed from the surface (as is the case in reactive DESI). Alternatively, the “super-reagent” spray can be used to rapidly investigate the reactivity of multiple substrates in a well based format allowing rapid discovery of new chemistry. Preliminary results using an array of different reagents on a paper substrate are highly encouraging.

110

5.1.3 Grand Challenge

The following question has stimulated much of the discussion in this section,

“Can small molecule discovery be fully automated?” This idea stems back to the beginning of my graduate career when an insightful undergraduate remarked that organic chemists seemingly have all the tools to make any molecule possibly, it is only a matter of expending the effort to make said molecule. Organic chemistry is certainly a discipline where the idea of chemical intuition and chemical hands is highly valued. To date some

108 drug-like organic molecules have been synthesized, but the total size of the space is

111

1060.[195] For even the simplest reactions there are easily more than 108 reaction

conditions possible (i.e. temperature, solvent, catalysis, pH, etc.).[196] It has also been

estimated that the number of transformations for a single chemical is estimated to be 80

meaning the size of chemical space is frustratingly vast.[197]

The development of an autonomous high-throughput reaction screening device

could revolutionize organic chemistry, not only to screen reaction conditions but to learn

the subtle rules and art of synthesis. Will it be possible to use machine learning and other

advanced techniques to allow a computer to both hypothesize chemical pathways, test

chemical pathways, and ultimately refine and discovery new chemical pathways? I

believe the answer is yes as evidenced by other machine learning projects.[198, 199]

Achieving even a portion of this goal would allow the removal of the organic chemist from the laboratory altogether and allow them to spend time on the more important problem, which is deciding which molecules of the 1060 we should be synthesizing.

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179. Yoon B, Häkkinen H, Landman U, Wörz AS, Antonietti J-M, et al. 2005. Science 307: 403-7

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181. Josserand C, Thoroddsen ST. 2016. Annual Review of Fluid Mechanics 48: 365- 91

182. Mascia S, Heider PL, Zhang H, Lakerveld R, Benyahia B, et al. 2013. Angewandte Chemie International Edition 52: 12359-63

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189. Gibson SC, Feigerle CS, Cook KD. 2014. Analytical Chemistry 86: 464-72

190. Snyder DT, Pulliam CJ, Ouyang Z, Cooks RG. 2016. Analytical Chemistry 88: 2- 29

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194. Yan X, Augusti R, Li X, Cooks RG. 2013. ChemPlusChem 78: 1142-8

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VITA 124

VITA

Michael Wleklinski was born in Munster, Indiana and raised in South Bend,

Indiana. He developed a passion for history and chemistry during high school while attending the Indiana Academy for Science, Mathematics, and Humanities from which he graduated in 2007. During high school Michael participated in his first research experience under the guidance of Dr. George Devendorf. Michael was awarded a modest

$300 research grant to study the role of dipole moments and polarizabilities in non- additive solution densities and presented this work at a regional chemical society meeting in Covington, KY.

Michael began his undergraduate education at Purdue University, pursuing a triple major in Chemistry, Mathematics, and Statistics. Throughout his undergraduate education, Michael was awarded multiple scholarships including Legacy Foundation Inc.

Scholarship, Purdue Alumni Association of St. Joseph Valley Scholarship, and Frederick

C. Mennen Memorial Scholarship. Michael was also employed as a quality control chemistry intern for Cargill corn milling North America and a steam plant chemistry intern for Betchel Marine Propulsions Corporation. Michael was exposed to programming through an undergraduate scientific computing class, and was able to utilize these skills during his time as undergraduate researcher for Professor Susanne

Hambrusch in computer science and then Professor Adam Wasserman in chemistry.

125

Michael was an active member of the Purdue student cluster competition, which used high performance computing to solve specific science problems culminating in a contest in Portland, Oregon where 48 hours were allowed to churn through simulated data. In the spring of 2011, Michael began doing research with Aston Labs under the direction of R.

Graham Cooks. Michael graduated from Purdue in the spring of 2011 and after developed an immense interest in mass spectrometry decided to pursue doctoral studies at Purdue

University under the direction of Professor R. Graham Cooks.

Throughout Michael’s graduate career he had the opportunity to study, present and perform research in South Korea, Germany, and India. Michael’s research focused on the use of preparative mass spectrometry for applications including the production of nanomaterials and organic synthesis. This includes the development of an atmospheric pressure ion source for the production atomically precise metal clusters and progress towards an automated droplet reactor. Michael co-authored ten peer reviewed publication, including three first author papers, and has filled two U.S. patents. Michael research was publicly recognized multiple times with travel awards (Jere Koskinen, and

The 6th Journal of Mass Spectrometry Award) and a fellowship (Purdue Research

Foundation Research Assistantship) Michael was an active part of the community having

partipated in a 24-hour bicycle race raising money for to support the court appointed

special advocates (CASA) for kids fund. Michael raised over $1k qualifying him as a

grand champion.

LIST OF PUBLICATIONS 126

LIST OF PUBLICATIONS

1. Wleklinski, M.; Falcone, C.; Loren, B.; Jaman, Z.; Iyer, K.; Ewan, H. S.; Hyun, S.-H.; Thompson, D.; Cooks, R. G. Can Accelerated Reactions in Droplets Guide Chemistry at Scale? European Journal of Organic Chemistry, 2016, Ahead of Print

2. Li, Y.; Wang, J.; Zhan, L.; Wleklinski, M.; Wang, J.; Ziong, C.; Liu, H.; Zhou, Y.; Nie, Z. The bridge between thin layer chromatography-mass spectrometry and high- performance liquid chromatography-mass spectrometry: The realization of liquid thin layer chromatography-mass spectrometry (LTLC–MS). Journal of Chromatography A 2016, 1460, 181-189.

3. Sarkar, D.; Mahitha, M. K.; Som, A.; Li, A.; Wleklinski, M.; Cooks, R. G.; Pradeep, T. Metallic nanobrushes made using ambient droplet sprays. Advanced Materials 2016, 28 (11), 2223-2228.

4. Narayanan, R.; Sarkar, D.; Som, A.; Wleklinski, M.; Cooks, R. G.; Pradeep, T. Anisotropic Molecular Ionization at 1 V from Tellurium Nanowires (Te NWs). Analytical Chemistry 2015, 87, 10792-10798.

5. Wleklinski, M.; Sarkar, D.; Hollerbach, Adam; Pradeep, T; Cooks, R.G. Ambient preparation and reactions of gas phase silver cluster cations and anions. Physical Chemistry Chemical Physics 2015, 17, 18364-18373.

6. Wleklinski, M.; Li, Y.; Bag, S.; Sarkar, D.; Narayanan, R.; Pradeep, T; Cooks, R.G. Zero volt paper spray ionization and its mechanism. Analytical Chemistry 2015, 87, 6786-6793.

7. Cooks, R. G.; Jarmusch, A. K.; Wleklinski, M. Direct analysis of complex mixtures by mass spectrometry. International Journal of Mass Spectrometry 2015, 377, 709- 718.

8. Espy, R. D.; Wleklinski, M.; Yan, X.; Graham Cooks, R. Beyond the flask: Reactions on the fly in ambient mass spectrometry. TrAC Trends in Analytical Chemistry 2014, 57, 135-146.

127

9. Dalgleish, J. K.; Wleklinski, M.; Shelley, J. T.; Mulligan, C. C.; Ouyang, Z.; Graham Cooks, R. Arrays of low-temperature plasma probes for ambient ionization mass spectrometry. Rapid Communications in Mass Spectrometry 2013, 27, 135-142.

10. Cyriac, J.; Wleklinski, M.; Li, G.; Gao, L.; Cooks, R. G. In situ Raman spectroscopy of surfaces modified by ion soft landing. Analyst 2012, 137, 1363-1369.

128

Article

pubs.acs.org/ac

Zero Volt Paper Spray Ionization and Its Mechanism † # † # † ‡ ‡ ‡ Michael Wleklinski, , Yafeng Li, , Soumabha Bag, Depanjan Sarkar, Rahul Narayanan, T. Pradeep, † and R. Graham Cooks*, † Department of Chemistry and Center for Analytical Instrumentation Development, Purdue University, West Lafayette, Indiana 47907, United States ‡ DST Unit of Nanoscience (DST UNS) and Thematic Unit of Excellence (TUE), Department of Chemistry, Indian Institute of Technology Madras, Chennai, Tamil Nadu 600 036, India

*S Supporting Information

ABSTRACT: The analytical performance and a suggested mechanism for zero volt paper spray using chromatography paper are presented. A spray is generated by the action of the pneumatic force of the mass spectrometer (MS) vacuum at the inlet. Positive and negative ion signals are observed, and comparisons are made with standard kV paper spray (PS) ionization and nanoelectrospray ionization (nESI). While the range of analytes to which zero volt PS is applicable is very similar to kV PS and nESI, differences in the mass spectra of mixtures are interpreted in terms of the more significant effects of analyte surface activity in the gentler zero volt experiment than in the other methods due to the significantly lower charge. The signal intensity of zero volt PS is also lower than in the other methods. A Monte Carlo simulation based on statistical fluctuation of positive and negative ions in solution has been implemented to explain the production of ions from initially uncharged droplets. Uncharged droplets first break up due to aerodynamics forces until they are in the 2−4 μm size range and then undergo Coulombic fission. A model involving statistical charge fluctuations in both phases predicts detection limits similar to those observed experimentally and explains the effects of binary mixture components on relative ionization efficiencies. The proposed mechanism may also play a role in ionization by other voltage-free methods.

ass spectrometry (MS) is a powerful analytical technique method. It represents a particular mode of operation of DESI M because of its high sensitivity, selectivity, and speed. which removes the high voltage. In SSI and EASI ionization, Ionization, the first step in MS analysis, plays a pivotal role in pneumatic forces play an important role. Another voltage-free the experiment. The applicability of MS to complex samples ionization method, solvent assisted inlet ionization (SAII) is − examined without sample pretreatment is the key feature of applicable in LC/MS.15 17 Here, as in thermospray, both 1 desorption electrospray ionization (DESI) and other ambient thermal energy and pneumatic forces appear to contribute to 2,3 ionization methods. These ionization methods have been ionization. In addition to these voltage-free spray ionization fi used in various elds including drug discovery, metabolomics, methods, ultrasound produced by piezoelectric devices has also fl forensic science, and bio uid analysis. Ambient ionization been used for spray ionization.18,19 A low frequency ultra- methods which avoid the use of a high voltage have obvious sonicator has been used to analyze biomolecules and monitor advantages, especially for in vivo analysis.4 fi organic reactions, reducing background noise by decreasing The rst voltage-free spray ionization method, thermospray, interference from background electrochemical reactions.20,21 In was developed by Vestal et al. in the 1980s as an interface for 2013, Chung-Hsuan Chen et al. reported yet another new spray LC/MS.5,6 In this experiment thermal energy was used to help ionization method, Kelvin spray ionization,22 which required no release solvent and create ionized analytes. A few years later, external electric energy, the system itself producing a voltage. another important voltage-free spray ionization method (SSI) fi 23 was introduced by Hirabayashi.7,8 In this experiment a high Paper spray (PS), rst reported in 2009, has proven speed gas flow is used to break up the bulk solution into practically useful for qualitative and quantitative analysis. The droplets, which go on to evaporate and generate gaseous ions. use of paper as the substrate in PS allows ionization of − A variety of compounds9 11 can be ionized by SSI, and it has compounds of interest while leaving some of the other been used for in vivo analysis when combined with ultrasonic aerosolization.4 Desorption sonic spray ionization12 (DeSSI, Received: March 11, 2015 also referred to as easy ambient sonic-spray ionization, Accepted: May 29, 2015 EASI13,14) is another example of a zero voltage spray ionization Published: May 29, 2015

Analytical Chemistry Article components of complex matrices adsorbed to the paper; this feature makes it suitable for the quantification of therapeutic drugs in blood.24,25 Paper cut to a sharp point ensures that a kilovolt applied potential will generate an electric field high enough to cause emission of charged analyte-containing solvent droplets; these droplets evaporate and undergo Coulomb explosion and perhaps solvated ion emission to give ESI-like mass spectra.26 A recent variant on the PS method uses paper impregnated with carbon nanotubes as a way to achieve the necessary field strengths while applying only a low voltage.27 We show in this study that by removing the applied voltage entirely, a zero volt form of PS can be performed. This experiment, which is phenomenologically similar to the SAII method, retains the advantages of the paper substrate by Figure 1. a) Overview of the zero volt paper spray process. The allowing complex mixtures to be examined directly without − chromatography while removing the electric field and distance between the front edge of the paper and the MS inlet is 0.3 0.5 mm. No voltage is applied to either the paper or the MS inlet dispensing with the strong pneumatic forces needed in the capillary. The suction force of the MS inlet causes the release of pneumatically assisted ionization methods of SSI and EASI. In ffi analyte-containing droplets, which are sampled by the mass zero volt PS, as in SAII, the vacuum provides a su cient spectrometer. b) Sampling and detections procedures. Photographs pneumatic force. Our results demonstrate that zero volt PS of the inlet region c) without and d) with solvent. A solvent spray or gives both positive and negative ions just as does conventional stream is generated when solvent is applied to the paper, as shown in kV PS and nESI, albeit with much lower signal intensities. The detail in a video in the Supporting Information. reduction in signal intensities roughly parallels that between EASI and DESI. Qualitative mechanisms, which are not fully positioning the paper and to observe the spray which was understood, have been proposed for other zero volt methods, illuminated by a red laser pointer. No voltage was applied to the but this paper seeks to develop a quantitative model for zero 28 paper or the capillary of the MS, instead the spray was volt paper spray. Simulations, based on the theory of Dodd, generated by the pneumatic forces at play near the MS inlet. have been performed to gain insights into the ionization Figure 1b depicts the typical workflow. Typically, 5 μLof mechanism. The proposed mechanism includes charge sample dissolved in methanol was loaded onto the paper and separation during droplet formation due to statistical allowed to dry. During drying, the paper was positioned fl 11 uctuations in positive and negative ion distributions after appropriately with respect to the MS. A methanol and water aerodynamic droplet breakup as described by Jarrold and co- μ 29 solvent (1:1 v/v, applied to the paper in three 7 L aliquots) workers. Subsequent solvent evaporation and Coulombic was used to generate the spray and detect signal. For each 7 μL fi ssion processes follow the accepted ESI mechanisms. aliquot of solvent, the signal lasted for about 10 s. Micropipette tips were used to load solvent onto the paper. Sample solutions ■ EXPERIMENTAL DETAILS could also be directly applied to the paper to generate spray. Chemicals and Materials. Deionized water was provided Figures 1c and 1d are photographs taken without and with by a Milli-Q Integral water purification system (Barnstead Easy solvent on the paper, respectively. Clearly, droplets are Pure II). Morphine and cocaine were purchased from Cerilliant observed only in the presence of solvent. The spray process (Round Rock, Texas). Methanol was from Mallinckrodt Baker was monitored using a 30 Hz camera and details are shown in Inc. (Phillipsburg, NJ). Deuterated methanol and water were Figure S1 and the Supporting Information videos. Note that provided by Cambridge Isotope Laboratories (Tewksbury, there are similarities to the experiments described by Pagnotti MA). The paper used as the spray substrate was Whatman 1 and co-workers.15,16 chromatography paper (Whatman International Ltd., Maid- Computational Resources. All programs used in the stone, England). All samples were examined in methanol simulation of zero volt PS were coded in Python 3.4.2 and solution except where noted. computed using computational resources provided by In- Zero Volt Paper Spray. As is shown in Figure 1, the formation Technology at Purdue Research Computing experimental details of zero volt PS were a little different from (RCAC) on the Carter supercomputer. Smaller codes were those previously reported for kV PS.23 The choice of paper tested on a small desktop computer (core i3). shape for kV PS is important to generate the required electric Instrumentation. Mass spectra were acquired using a fields for ionization; however, the choice of paper shape for Thermo Fisher LTQ mass spectrometer (Thermo Scientific zero volt PS is less important than the orientation of the paper Inc., San Jose, CA). The MS inlet capillary temperature was relative to the MS inlet. Therefore, a rectangular piece of paper kept between 150 and 200 °C except where noted, and the tube cut to 8 mm × 4 mm and held in place by a toothless alligator lens voltage and the capillary voltage were held at zero volt for clip (McMaster-Carr, USA Part 7236K51) was used, with the both positive and negative ion detection. Collision-induced center of a straight edge being placed closest to the inlet and on dissociation (CID) was used to carry out tandem mass the ion optical axis. This arrangement made it easier to control spectrometry analysis on precursor ions mass-selected using the distance between the paper and the inlet than in the case of windows of 1.5 mass units. To record the corresponding kV PS a paper triangle (Figure 1a), increasing the reproducibility of spectra, 3.5 kV and 2.0 kV were used in the positive and the experiment. A xyz-micrometer moving stage (Parker negative ion modes, respectively, while for nESI 1.5 kV was Automation, USA) was used to set the distance between the used in both polarities. The same CID conditions were used for front edge of the paper and the MS inlet in the range 0.3 mm to the analysis of the same sample regardless of the ionization 0.5 mm. A camera (Watec Wat-704R) was used to help in method.

6787 DOI: 10.1021/acs.analchem.5b01225 Anal. Chem. 2015, 87, 6786−6793 130

Analytical Chemistry Article ■ RESULTS AND DISCUSSION camera in an experiment that used 50 ppm of tributylamine fed fl μ Characteristics of Zero Volt PS Mass Spectra. A variety continuously onto the paper at a ow rate of 15 L/min. The of samples was used to test the ionization capabilities of zero emission of individual droplets occurs rapidly enough that the volt PS. As shown in Figure 2, both positive and negative chronogram appears to be continuous when observed using an ion injection time of 100 ms (Figure S 1f). By illuminating the spray with a hand-held red laser pointer the spray process could be videographed. Figure S1 a-d shows the suction of one droplet over the course of 4 consecutive images. This indicates that a single suction event occurs in a time on the order of ∼100 ms. This experiment was repeated using manual additions of solvent (7 μL), and similar droplet events are observed. Movies of the continuous and discrete methods of analysis are included in the Supporting Information. The important finding is that signal is observed only when a droplet event is recorded by the camera, indicating that droplets are necessary to produce gas phase ions. Source of Protons, pH Effect, and Low Voltage Effect. Figure 3 shows the zero volt PS spectrum of 1 ppm

Figure 2. Mass spectra recorded using zero volt PS of three samples examined in the positive ion mode: a) 1 ppm tributylamine; b) 8 ppm cocaine, and c) 1 ppm terabutylammonium iodide and three samples examined in the negative ion mode: d) 10 ppm 3,5-dinitrobenzoic acid, e) 10 ppm fludioxonil, and f) 10 ppm sodium tetraphenylborate. Methanol and water (v/v 1:1) were used as spray solvent for all samples. Both positive and negative signals were recorded with much lower intensities compared with kV PS and nESI. * Indicates the presence of background species. Figure 3. Zero volt PS mass spectra of 1 ppm tributylamine using a) methanol/water (v/v 1:1) and b) deuterated methanol/water (v/v 1:1) as solvent. When deuterated solvent is used, [M + D]+ becomes spectra were obtained, although the signal intensities were the major peak. about 2 orders of magnitude lower than those of nESI and kV PS spectra. The MS/MS results for zero volt PS were almost tributylamine using methanol:water 1:1 and deuterated identical to those for the same ions generated by nESI and kV methanol:water 1:1 as solvents, respectively (Figure 3a and PS (Figures S2 and S3). These results show that the range of 3b). When methanol/water was used, m/z 186 ([M + H]+) was analytes to which zero volt PS is applicable is very similar to kV the dominant peak, accompanied by an isotopic signal at m/z PS and nESI, but the ionization efficiency is much lower. 187. However, when deuterated methanol/water was used, m/z As noted at the beginning of the paper, ionization without 187, [M + D]+, was dominant, and m/z 188 is its isotopic peak. application of a voltage has been observed using several These results indicate that the protons mainly come from the methods. This makes it important to seek to understand the solvent and that the normal acid/base equilibria occurring in fundamental processes that lead to the formation of ions at zero bulk solution are ultimately responsible for the ions seen in the volts. It is expected that such an enquiry for zero volt PS might mass spectra. In Figure 3b, there is still a small peak of m/z 186, be of some later use in guiding mechanistic studies of the other while in Figure 3a ions of m/z 185 are virtually absent, methods. A simple experiment was performed to determine the indicating that a small proportion of tributylamine is still maximum distance between the paper and MS inlet that still ionized as [M + H]+ when deuterated solvents are used. allows observation of signal. It was found that without external Possible sources of the proton include autoionization (2 M ⇌ forces and with the instrument and paper used, the paper must [M + H]+ +[M− H]−), gas phase water molecules, and be within 1 mm of the inlet for the observation of a spray and residual protic compounds in the instrument. the corresponding ion signal. At larger distances, an external The effects of solvent pH on the spectra have been tested force such as an applied voltage or additional pneumatic force is using a representative compound, tributylamine. The intensity needed. A distance of 0.3−0.5 mm was chosen for subsequent of the signal for the protonated molecule is high at pH 7, but experiments to optimize signal intensity and lower its when the pH is raised to 10, the intensity drops to zero. Further fluctuations. The spray process was monitored using a 30 Hz tests were done by using a series of aromatic heterocyclic

6788 DOI: 10.1021/acs.analchem.5b01225 Anal. Chem. 2015, 87, 6786−6793 131

Analytical Chemistry Article compounds (pyridine, guanine, thymine, and adenine) at neutral and acidic pH (Figure S4). The results show that all four compounds ionize well from acidic solution; pyridine and adenine also ionize at neutral pH, while guanine and thymine do not. These observations are consistent with expectations based on acid/base solution equilibria. The pKa values of the conjugate acids of thymine and guanine are 0 and 3.2, respectively, while the pyridine and adenine values are significantly higher, 5.25 and 4.1, respectively.30 A series of basic amines including tetramethyl-1,4-butanediamine, diisopropylamine, and methyl amine was also analyzed to investigate the effect of proton affinity (Figure S5). Tetramethyl-1,4- butanediamine has the highest proton affinity (1046.3 kJ/mol) among these analytes and also the highest absolute MS signal intensity. Diisopropyl amine (971.9 kJ/mol) is second in PA, and methyl amine (899 kJ/mol) has the lowest PA and MS signal. Basicity in the gas phase and in solution appear to play important roles in determining the types of ions and conditions (pH) under which they will be observed, similar to the effects observed in electrospray.31 To investigate the role low - as opposed to zero - voltages can have on ionization, paper spray experiments were performed using diphenylamine and low and zero volts. The results demonstrate that the small signal at zero volts rises measurably (by a factor of 1.5) upon providing 1 V on the paper (Figure S6) and increases by a factor of 2.5 on raising the potential to 10 V (data not shown). The addition of even a low voltage supplies additional charges, which increases ionization efficiency. Analyzing Organic Salt/Organic Analyte Mixtures by Zero Volt PS, kV PS, and nESI. A mixture containing 9 ppm cocaine and 0.1 ppm tetrabutylammonium iodide was examined Figure 4. Mass spectra of a mixture of 9 ppm cocaine and 0.1 ppm by nESI, kV PS, and zero volt PS. The results are shown in tetrabutylammonium iodide using a) nESI, b) kV PS, and c) zero volt Figure 4a-c. For nESI and kV PS, cocaine (protonated PS. molecule, m/z 304) is the dominant peak, while the signal intensity of tetrabutylammonium (m/z 242) is only about 2% of that of cocaine. For zero volt PS, m/z 304 is still dominant, conventional PS and nESI methods. The aqueous pKb of but the relative intensity of tetrabutylammonium (m/z 242) is cocaine is 5.39 (15 °C), and morphine is slightly higher, 5.79 much higher than in nESI and kV PS (about 50% relative (25 °C). This means that morphine should produce somewhat abundance). The trend is even more obvious in the results of 9 fewer ions than cocaine even when their absolute concen- ppm morphine/0.1 ppm tetrabutylammonium iodide (Figure trations are the same. However, the main reason for the low S7a,b,c). The data for nESI (Figure S7a) and kV PS (Figure relative intensity of morphine in zero volt PS is likely the lower S7b) show the signal for morphine (m/z 286) to be the base surface activity of morphine compared to cocaine.32 Evidence peak, while the relative abundance of tetrabutylammonium (m/ for this comes from the fact that when mixed with the very z 242) is only about 2% in both cases. By contrast, in the zero surface active compound tetrabutylammonium iodide, suppres- volt PS result, it is the ion m/z 242 that constitutes the base sion of ionization is much more obvious for morphine than for peak, while the relative abundance of the protonated morphine cocaine in zero volt PS. In kV PS and nESI, the influence of ff ion is only about 10% (Figure S7c). An analogous e ect was surface activity is not as severe as in zero volt PS since their observed in the negative ion mode, by analyzing a mixture of 36 ionization efficiencies are so high that most of the analytes in ppm sodium tetraphenylborate and 3,5-dinitrobenzoic acid the droplets can be ionized and pushed to the droplet surface. A (Figure S7 d,e,f). parallel result is observed in negative ion mode where the To account for these results we note the well-known fact surface active tetraphenylborate signal is relatively enhanced in both in nESI and in conventional kV PS that the signal intensity zero volt PS as compared to the kV PS and nESI experiments. is closely related to the concentration of the analyte, at least in Given these qualitative explanations we can now test them the lower concentration range where the available number of using a quantitative model of the ionization mechanism. ffi charges is su cient to convert all analyte into the ionic form. Overview of Ionization Mechanism for Zero Volt PS. It The observation that zero volt PS is ca. 2 orders of magnitude is well-known that most analytes that can be ionized by ESI (or ffi less e cient than kV PS and nESI is interpreted simply as the nESI) or by PS are Bronsted acids or bases. For a basic result of the limited number of charges provided by the compound M dissolved in a solvent (S), a certain amount of M statistical droplet breakup process versus direct solvent exists in the ion pair form (normally as solvent-separated ion ff charging. However, this does not explain the large di erences pairs) because of the equilibrium: between the cocaine/tetrabutylammonium and morphine/ +⇌ ++ + − − tetrabutylammonium ion signals in zero volt PS vs the M S[MH][SH]

6789 DOI: 10.1021/acs.analchem.5b01225 Anal. Chem. 2015, 87, 6786−6793 132

Analytical Chemistry Article

Scheme 1. Overview of the Ionization Mechanism of Zero Volt PS Ionization Including Representations of the Aerodynamic a Breakup Process and the Droplet Evaporation/Coulombic Fission

aIn both steps statistical distribution of charge to the fragments is assumed.

For negative ion generation from of an acidic compound N, the and diameter for each droplet were specified, but the charge of equilibrium is each droplet was randomly assigned based on a theory 28 +⇌ −− + + + described by Dodd. To determine the initial charge, the N S [N H] [S H] number of ions an analyte forms in solution was calculated For an ionic compound, say CA, there exists a dissociation based on the initial concentration and dissociation constant of equilibrium: the analyte. Statistical fluctuations in the number of positive ⇌++ − and negative ions in each droplet were modeled by a binomial CA C A distribution It is these solution-phase ion pairs which can go on to be n f znp=−ppzn−z evaporated and detected in zero volt PS as positively or (; , )z (1 ) negatively charged ions. () (2) In zero volt PS, a droplet experiences aerodynamic forces as where p is the probability of an ion being charged (either it is pulled into the mass spectrometer by the suction of the positive or negative), n is the number of ions, and z is number vacuum system. These aerodynamic forces break apart droplets of positive charges. The difference in ion polarity count μ until they reach a size on the order of 1 to 4 m where the determines the initial charge. It should be noted that charge is aerodynamic forces are no longer strong enough to cause 29,33 assumed to be carried only by analytes added to the solution. further droplet breakup. Aerodynamics forces are described The droplet then evaporates until its diameter reaches the fi by the Weber number, which is de ned as Rayleigh limit (3).38,41 2 ρ ()VVD− 1/3 g gdd ⎛ De22* ⎞ We = ⎜ q ⎟ σ (1) D = ⎜ ⎟ R ⎝ (8πεσ2** * )⎠ ρ 0 (3) where g is the gas density, Vg is the gas velocity, Vd is the droplet velocity, D is the diameter of the droplet, and σ is the d Here DR is the diameter of the droplet at the Rayleigh limit, Dq surface tension of the solvent.34 The droplet will continue to ε is the charge on the droplet, e is elementary charge, 0 is the break up while its Weber number is larger than 10 (Figure σ 34,35 permittivity of a vacuum, and is the solvent surface tension. S8). During the aerodynamic breakup process, there is a At the Rayleigh limit a droplet undergoes fission and very large chance that the positive charges and negative charges produces progeny droplets. Accordingly the size of precursor will be unevenly separated, that is to say, many of these progeny and progeny droplets was calculated according to these droplets will be (slightly) charged. The extent of charging is equations unknown. However, because the initial Weber number in zero DmD=−Δ*1/3 volt paper spray is larger than 1000 a catastrophic breakup of d (1 ) R (4) the initial droplet occurs to produce progeny droplets (Figure S8).34,36 After aerodynamic breakup it is assumed that droplets 1/3 ⎛ Δm ⎞ will undergo multiple rounds of evaporation and Coulombic D = ⎜ ⎟ D fi pD ⎜ N ⎟ R ssion until they are ionized by either of the main ESI models, ⎝ pd ⎠ (5) the charge residue model or the ion evaporation model37,38 or 26 its close analog, solvated ion emission. A schematic of the where Npd is the number of progeny droplets taken to be 10, Δ overall mechanism is shown in Scheme 1. The model used here Dpd is the diameter of the progeny droplets, and m = 0.02. to describe evaporation and fission is similar to other The number of analytes in each progeny droplet was approaches used to model nESI based on Monte Carlo determined from two Poisson distributions: the concentration 39 methods, except that droplet charging is determined by of ions, Nanal‑IP (both positive and negative), and the nonsymmetrical fragmentation as described by Dodd.28 While concentration of free ions in the outer region of the droplet, 28,40 the charging calculated by Dodd is small, there is enough Nanal‑q. The position of a solvated ion within the droplet is charge to allow for the Coulombic fission of most 1 to 4 μm determined by its surface activity, S. Surface activity is a number droplets to occur after an appropriate evaporation time. Note, between 0 and 1 describing the probability of a molecule being too, that we assume statistical breakup and resulting charge at the surface or the interior of the droplet. Larger values of distributions in the fragments during the early aerodynamic surface activity means the analyte competes more strongly for phase of bulk breakup but are aware of the fact that this is surface sites. Surface activity is modeled by a binomial simply a first approximation. Simulations have been done based distribution, similar to eq 2, except that p = S, n is the number on the above postulated mechanism. The initial concentration of ions, and z is the number of ions found in the outer region of

6790 DOI: 10.1021/acs.analchem.5b01225 Anal. Chem. 2015, 87, 6786−6793 133

Analytical Chemistry Article

Figure 5. a) Cocaine to tetrabutylammonium cation ratio dependence in positive ion mode for zero volt PS and nESI. Cocaine concentration is held constant at 1 ppm, while tetrabutylammonium iodide concentration changes. b) The relative surface activity of cocaine calculated according to the experimental data in a). c) Ratio dependence of 3,5-dinitrobenzoate to tetraphenylborate in negative ion mode for zero volt PS and nESI. 3,5- Dinitrobenzoic acid concentration is held constant at 20 ppm, while the sodium tetraphenylborate concentration changes. d) Relative surface activity of 3,5-dinitrobenzoic acid calculated according to the experimental data in c). Surface activity of the salt is assumed to be 1 for all simulations.

39 ff the droplet. The number of ions, Nanal‑IP, and charges, Nanal‑q, There are di erences, but there are also strong similarities in is chosen randomly from a Poisson distribution. the ionization mechanism of zero volt PS and conventional kV PS. Both processes involve the key steps of (i) droplet −NN eNIP* anal‐ IP formation and (ii) droplet breakup. Droplet formation is mostly f(;)NN= IP anal‐ IP IP N ! due to pneumatic forces in zero volt paper spray, whereas it is anal‐ IP (6) mostly due to electrical forces at kV paper spray. There exists a continuum in behavior between zero volt PS and kV PS as Here NIP is the average number of ions close to the surface. evidenced by the low voltage PS data cited above. Droplet The same equation is used for Nanal‑q with the appropriate substitutions. For the progeny droplets, additional charging can breakup starts with a mechanical breakup process which may or arise from the statistical fluctuations in the number of positive may not occur in kV PS and is then followed by processes and negative ions, and this is modeled in the same manner as analogous to those of kV PS. above (2). The evaporation/Coulombic fission process Single Analyte Simulation. Simulations were run with 2 continues until all droplets reach a size of 10 nm. At 10 nm, μm droplets to investigate the possible limits of detection of zero volt PS. Both sizes lead to indicated limits of detection ions free of their counter charge are considered ionized (i.e., − − considered to undergo subsequent rapid desolvation), which is between 10 7 and 10 8 M (Figure S8), based on the a simplification of the actual processes that leads to ion assumption of being able to detect a single ion. The detection formation. Ions are produced from droplet smaller than 10 nm limit determined from simulation is calculated from the by the ion emission mechanism or by the charge residue ionization efficiency, which is defined as the ratio of the model.26 The formation of gas-phase ions from 10 nm droplets number of ions generated vs the total number of molecules − is not explicitly modeled here. A more detailed explanation is used.42 Qualitatively, 18 ppb (4.87 × 10 8 M) of provided in the Supporting Information. tetrabutylammonium iodide could be detected experimentally,

6791 DOI: 10.1021/acs.analchem.5b01225 Anal. Chem. 2015, 87, 6786−6793 134

Analytical Chemistry Article which is in good agreement with the estimate of detectability by fluctuation of positive and negative ions in droplet solutions simulation. The simulation was also repeated at three different during the course of droplet breakup. This model (developed in surface activities, and it was found that the number of ionized detail in the Supporting Information) has been used to predict molecules decreases as the surface activity decreases (Figure a detection limit similar to that observed experimentally. In the S10). Surface activity has been reported to have a similar effect case of multiple analytes, the simulation is also able to calculate on the ionization efficiency.43,44 the relative surface activity of both positive and negative ions, Experimental and Simulated Results and Mechanistic such as cocaine and 3,5-dinitrobenzoic acid. The relationship of Considerations for Multianalyte Mixtures. A series of this model to other zero volt spray ionization mechanisms is mixtures of cocaine and tetrabutylammonium iodide were not known but is of great future interest, especially for the inlet analyzed with zero volt PS. In Figure 5a the amount of ionization experiments15,16 and the nanowire spray47 methods. tetrabutylammonium iodide was varied, while the amount of Besides the understanding of its mechanism, zero volt PS also cocaine was held constant at 1 ppm. A similar experiment was has potential application advantages: it could be used to study performed using 3,5-dinitrobenzoic acid and sodium tetraphe- systems where there is a need to avoid the influence of external nylborate but analyzed in negative ion mode. In Figure 5c the electric fields and at the same time to use paper to eliminate the amount of sodium tetraphenylborate was changed, while the complex matrix, such as in living organisms analysis. In the concentration of 3,5-dinitrobenzoic acid was held constant at course of this work we have learned of related unpublished 20 ppm. At each point, the ratio of (de)protonated molecular paper spray ionization experiments done by Dr. Akira ion to salt was calculated. Simulations were run in which the Motoyama of Shiseido Company. more surface active compound (salts in this case) was assumed to have a surface activity of 1 and surface activity of ■ ASSOCIATED CONTENT (de)protonated molecular ion relative to the salt was varied *S Supporting Information until the simulated ratio matched within 1% error of the Additional information as noted in text. The Supporting experimental ratio. Information is available free of charge on the ACS Publications In Figure 5a, as the amount of tetrabutylammoiun iodide website at DOI: 10.1021/acs.analchem.5b01225. decreases the ratio of cocaine to tetrabutylammoiun iodide increases for both zero volt PS and nESI. Note that nESI has ■ AUTHOR INFORMATION larger ratios than zero volt PS. This is because the kV applied 38,45 Corresponding Author voltage provides protons which can serve to ionize the *E-mail: [email protected]. cocaine but will not cause additional ionization of the Author Contributions tetrabutylammonium iodide salt. Thus, the measured ratio # becomes closer to the concentration ratio, with differences These authors contributed equally. being due to intrinsic ionization and detection efficiency. We Notes applied the experimental intensity ratios of zero volt PS to our The authors declare no competing financial interest. simulation and calculated the relative surface activity trend. The results are shown in Figure 5b. As the amount of ■ ACKNOWLEDGMENTS tetrabutylammonium iodide decreases the relative surface The support of the National Science Foundation CHE 1307264 activity of cocaine is calculated to increase. This makes sense is acknowledged. T.P. acknowledges DST for funding. D.S. and since as the amount of tetrabutylammoium idodie decreases R.N. acknowledge UGC for their research fellowship. Yafeng Li more cocaine will move toward the droplet surface and can is a visiting student from the Institute of Chemistry, Chinese compete against the tetrabutylammonium cation for surface Academy of Sciences, and she acknowledges the Chinese sites.46 Tang et al. developed a model, which suggests that at Scholarship Council for financial support. low concentrations, 10−8 to 5 × 10−6 M, the ratio of analyte ion signals is dependent upon the relative surface activities of the ■ REFERENCES 32 two analytes. This agrees with our simulation results very (1) Takats,́ Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science well. Figure 5c,d display the same general trend observed in 2004, 306, 471−473. Figure 5a,b. As the concentration of sodium tetraphenylborate (2) Monge, M. E.; Harris, G. A.; Dwivedi, P.; Fernandez,́ F. M. Chem. decreases the ratio of 3,5-dinitrobenzoic acid to sodium Rev. 2013, 113, 2269−2308. tetrphenylborate decreases (Figure 5c), while the relative (3) Ambient Ionization Mass Spectrometry; The Royal Society of − surface activity of 3,5-dinitrobenzoic acid increases (Figure Chemistry; 2015; pp P001 508. (4) Schafer,̈ K.-C.; Balog, J.; Szaniszlo,́ T.; Szalay, D.; Mezey, G.; 5d). A similar set of experiments was performed except that the ́ ́ ́ concentration of the analyte was varied, and the salt Denes, J.; Bognar, L.; Oertel, M.; Takats, Z. Anal. Chem. 2011, 83, 7729−7735. concentration was held constant (Figure S11). This produced (5) Blakley, C. R.; Carmody, J. J.; Vestal, M. L. Anal. Chem. 1980, 52, similar conclusions, and a detailed explanation is provided in 1636−1641. the Supporting Information. (6) Blakley, C. R.; Vestal, M. L. Anal. Chem. 1983, 55, 750−754. (7) Hirabayashi, A.; Sakairi, M.; Koizumi, H. Anal. Chem. 1994, 66, ■ CONCLUSION 4557−4559. (8) Hirabayashi, A.; Sakairi, M.; Koizumi, H. Anal. Chem. 1995, 67, Chemical analysis at zero volts from paper substrates has been − demonstrated. Zero volt PS gives both positive and negative ion 2878 2882. (9) Hirabayashi, A.; Hirabayashi, Y.; Sakairi, M.; Koizumi, H. Rapid signals and allows detection of similar compounds to those seen − ffi Commun. Mass Spectrom. 1996, 10, 1703 1705. by kV PS and nESI but with lower ionization e ciency. In spite (10) Hirabayashi, Y.; Hirabayashi, A.; Takada, Y.; Sakairi, M.; of the low efficiency, the process is intrinsically very gentle and Koizumi, H. Anal. Chem. 1998, 70, 1882−1884. is more sensitive to surface active compounds. A mechanism for (11) Ozdemir, A.; Lin, J.-L.; Wang, Y. S.; Chen, C.-H. RSC Adv. zero volt PS has been proposed based on the statistical 2014, 4, 61290−61297.

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(12) Haddad, R.; Sparrapan, R.; Eberlin, M. N. Rapid Commun. Mass Spectrom. 2006, 20, 2901−2905. (13) Haddad, R.; Sparrapan, R.; Kotiaho, T.; Eberlin, M. N. Anal. Chem. 2008, 80, 898−903. (14) Haddad, R.; Milagre, H. M. S.; Catharino, R. R.; Eberlin, M. N. Anal. Chem. 2008, 80, 2744−2750. (15) Pagnotti, V. S.; Inutan, E. D.; Marshall, D. D.; McEwen, C. N.; Trimpin, S. Anal. Chem. 2011, 83, 7591−7594. (16) Pagnotti, V. S.; Chubatyi, N. D.; McEwen, C. N. Anal. Chem. 2011, 83, 3981−3985. (17) Wang, B.; Trimpin, S. Anal. Chem. 2014, 86, 1000−1006. (18) Wu, C.-I.; Wang, Y.-S.; Chen, N. G.; Wu, C.-Y.; Chen, C.-H. Rapid Commun. Mass Spectrom. 2010, 24, 2569−2574. (19) Zhu, H.; Li, G.; Huang, G. J. Am. Soc. Mass Spectrom. 2014, 25, 935−942. (20) Chen, T.-Y.; Chao, C.-S.; Mong, K.-K. T.; Chen, Y.-C. Chem. Commun. 2010, 46, 8347−8349. (21) Chen, T.-Y.; Lin, J.-Y.; Chen, J.-Y.; Chen, Y.-C. J. Am. Soc. Mass Spectrom. 2010, 21, 1547−1553. (22) Ozdemir, A.; Lin, J.-L.; Gillig, K. J.; Chen, C.-H. Analyst 2013, 138, 6913−6923. (23) Wang, H.; Liu, J.; Cooks, R. G.; Ouyang, Z. Angew. Chem., Int. Ed. 2010, 49, 877−880. (24) Manicke, N. E.; Yang, Q.; Wang, H.; Oradu, S.; Ouyang, Z.; Cooks, R. G. Int. J. Mass Spectrom. 2011, 300, 123−129. (25) Espy, R. D.; Teunissen, S. F.; Manicke, N. E.; Ren, Y.; Ouyang, Z.; van Asten, A.; Cooks, R. G. Anal. Chem. 2014, 86, 7712−7718. (26) Konermann, L.; Ahadi, E.; Rodriguez, A. D.; Vahidi, S. Anal. Chem. 2013, 85,2−9. (27) Narayanan, R.; Sarkar, D.; Cooks, R. G.; Pradeep, T. Angew. Chem., Int. Ed. 2014, 53, 5936−5940. (28) Dodd, E. E. J. Appl. Phys. 1953, 24,73−80. (29) Zilch, L. W.; Maze, J. T.; Smith, J. W.; Ewing, G. E.; Jarrold, M. F. J. Phys. Chem. A 2008, 112, 13352−13363. (30) Yen, T.-Y.; Judith Charles, M.; Voyksner, R. D. J. Am. Soc. Mass Spectrom. 1996, 7, 1106−1108. (31) Ehrmann, B. M.; Henriksen, T.; Cech, N. B. J. Am. Soc. Mass Spectrom. 2008, 19, 719−728. (32) Tang, L.; Kebarle, P. Anal. Chem. 1993, 65, 3654−3668. (33) Wang, R.; Allmendinger, P.; Zhu, L.; Gröhn, A.; Wegner, K.; Frankevich, V.; Zenobi, R. J. Am. Soc. Mass Spectrom. 2011, 22, 1234− 1241. (34) Krzeczkowski, S. A. Int. J. Multiphase Flow 1980, 6, 227−239. (35) Wierzba, A. Exp. Fluids 1990, 9,59−64. (36) Pilch, M.; Erdman, C. A. Int. J. Multiphase Flow 1987, 13, 741− 757. (37) Iribarne, J. V.; Thomson, B. A. J. Chem. Phys. 1976, 64, 2287− 2294. (38) Kebarle, P.; Verkerk, U. H. Mass Spectrom. Rev. 2009, 28, 898− 917. (39) Hogan, C. J., Jr.; Biswas, P. J. Am. Soc. Mass Spectrom. 2008, 19, 1098−1107. (40) Knochenmuss, R. Mass Spectrometry 2013, 2, S0006−S0006. (41) Rayleigh, L. Philos. Mag. Ser. 5 1882, 14, 184−186. (42) Murray, K. K.; Boyd, R. K.; Eberlin, M. N.; Langley, G. J.; Li, L.; Naito, Y. Pure Appl. Chem. 2013, 85, 1515−1609. (43) Cech, N. B.; Enke, C. G. Anal. Chem. 2000, 72, 2717−2723. (44) Cech, N. B.; Enke, C. G. Mass Spectrom. Rev. 2001, 20, 362− 387. (45) Kertesz, V.; Van Berkel, G. J. Anal. Chem. 2007, 79, 5510−5520. (46) Enke, C. G. Anal. Chem. 1997, 69, 4885−4893. (47) Narayanan, R.; Sarkar, D.; Som, A.; Cooks, R. G.; Pradeep, T. 2015, Unpublished.

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Ambient preparation and reactions of gas phase silver cluster cations and anions† Cite this: Phys. Chem. Chem. Phys., 2015, 17,18364 Michael Wleklinski,a Depanjan Sarkar,b Adam Hollerbach,a Thalappil Pradeepb and R. Graham Cooks*a

Electrospray ionization of metal salt solutions followed by ambient heating transforms the resulting salt clusters into new species, primarily naked ionic metal clusters. The experiment is done by passing the clusters through a heated coiled loop outside the mass spectrometer which releases the counter-anion while generating the anionic or cationic naked metal cluster. The nature of the anion in the starting salt determines the type of metal cluster observed. For example, silver acetate upon heating generates + only positive silver clusters, Agn , but silver fluoride generates both positive and negative silver clusters, +/ Agn (3 o n o 20). Both unheated and heated metal salt sprays yield ions with characteristic geometric and electronic magic numbers. There is also a strong odd/even eﬀect in the cationic and anionic silver clusters. Thermochemical control is suggested as the basis for favored formation of the observed clusters, with anhydride elimination occurring from the acetates and fluorine elimination from the fluorides to give cationic and anionic clusters, respectively. Data on the intermediates observed as the temperature is ramped support this. The naked metal clusters react with gaseous reagents in the open air, including methyl substituted pyridines, hydrocarbons, common organic solvents, ozone, ethylene, and propylene. Argentation of hydrocarbons, including saturated hydrocarbons, is shown to occur and serves Received 16th March 2015, as a useful analytical ionization method. The new cluster formation methodology allows investigation of Accepted 11th June 2015 ligand–metal binding including in reactions of industrial importance, such as olefin epoxidation. These DOI: 10.1039/c5cp01538c reactions provide insight into the physicochemical properties of silver cluster anions and cations. The potential use of the ion source in ion soft landing is demonstrated by reproducing the mass spectra of www.rsc.org/pccp salts heated in air using a custom surface science instrument.

1. Introduction is a critical tool for preparing and understanding the unique properties of metal clusters both in the gas phase7,8 and when Transition metals exhibit unique physical, optical, and chemical deposited onto surfaces.2,9,10 properties both as bulk materials and in the nanoscale. Silver The production of gas-phase metal clusters has been demon- nanomaterials have applications in water purification,1 catalysis,2 strated using a variety of ionization sources, including magnetron and surface enhanced Raman spectroscopy,3,4 among many sputtering,11 laser ablation,12–15 gas aggregation,16,17 cold reflux others. The properties of atomically precise metal clusters are discharge,18 pulsed arc cluster ion sources,19,20 and electrospray even more fascinating as the addition or removal of a single ionization (ESI).21–26 These sources produce positive, negative, atom aﬀects the electronic5 and chemical6 properties of the and neutral clusters over a wide size range with a seemingly cluster. Atomically precise metal clusters can be generated unlimited choice of elemental compositions.27 Flow tube both in solution and in the gas phase. Mass spectrometry (MS) reactors11,28,29 and ion traps30,31 are common ways of studying metal +/ clusters in the gas phase. Small silver clusters (Agn n o 20), the topic of interest in this paper, have been studied extensively a Department of Chemistry and Center for Analytical Instrumentation Development, Purdue University, West Lafayette, Indiana 47907, USA. in the gas phase due to their value in partial oxidation and 6,23,24,28,32–38 E-mail: [email protected] catalytic reactions. b + DST Unit of Nanoscience (DST UNS) and Thematic Unit of Excellence (TUE), The silver cluster, Ag4H has been used to mediate the carbon– Department of Chemistry, Indian Institute of Technology Madras, carbon coupling of allyl bromide.23 In the overall reaction, three Chennai 600 036, India molecules of allyl bromide are needed to generate [Ag(C H ) ]+, † 3 5 2 Electronic supplementary information (ESI) available: Additional experimental Q + setups, mass spectra of unheated and heated silver salts, tandem MS of silver [Ag3Br3], and CH2 CHCH3.Theproduct,[Ag(C3H5)2] loses C6H10, cluster cations and anions, mass spectra of silver cluster cation/anion reactivity, most likely 1,5-hexadiene, upon collisional activation, which repre- 39,40 and tabular results of silver cluster reactivity. See DOI: 10.1039/c5cp01538c sents an example of gas phase carbon–carbon bond coupling.

Silver has long served as a catalyst for the partial oxidation of ionization (ESI) and cluster heating in air. When the appropriate + ethylene to ethylene oxide and Ag2O has been investigated as a silver salt is subjected to these conditions, it is possible to generate model for silver-mediated olefin epoxidation.37 Roithova´ and silver cluster cations and anions without the use of lasers or Schro¨der note clean O-atom transfer reactions with ethylene. collision-induced dissociation (CID). Physiochemical properties are Propylene undergoes allylic H-atom abstraction and other oxida- elucidated by subjecting the clusters in the open air to ion/molecule tion pathways but C–H abstraction from propylene leads to the reactions; for example, they can be oxidized in the presence of formation of unwanted byproducts, preventing high selectivity in ozone. The silver clusters can also be used for the analysis of the formation of propylene oxide.41 hydrocarbons or to study ligand exchange processes. Finally, it is The physical and chemical properties of anionic silver clusters demonstrated that this ionization source can be coupled to an in the gas phase have also garnered attention. Measurements on ion soft landing instrument to perform catalytic studies. the binding energies of O2 and observation of cooperative binding 32,33 of O2 to silver cluster anions have been reported. Recently, Luo et al. noted the enhanced stability of Ag13 towards etching with 2. Experimental 28 O2 due to a large spin excitation energy. The reaction of O2 with 2.1 Chemicals and materials CO in the presence of anionic silver clusters was found to be Silver fluoride, silver acetate, silver benzoate, 2-ethylpyridine, strongly cluster-size dependent with only Ag7 ,Ag9 ,andAg11 6 3-ethylpyridine, 4-ethylpyridine, 3,4-lutidine, 2,6-lutidine, serving as potential catalytic centers. Luo et al. found that Ag8 reacts with chlorine through a harpoon mechanism35 and reported 2,5-lutidine, 3,5-lutidine, hexadecane, isocetane, squalane, that silver cluster anions activate C–S bonds in ethanethiol to and ethanol were purchased from Sigma Aldrich, USA. HPLC 36 grade methanol was purchased from Mallinckrodt Baker Inc., produce such products as AgnSH and AgnSH2 . While extensive literature exists on the chemistry of gaseous Phillipsburg, NJ. Deionized water was provided by a Milli-Q silver clusters in vacuum, there is also a growing community Integral water purification system (Barnstead Easy Pure II). interested in studying their properties on surfaces. For example, Deuterated solvents, such as D2O and CD3OD were purchased size-selected silver cluster cations with diﬀerent energies were from Cambridge Isotope Laboratories (Tewksbury, MA). tert-Butyl deposited onto Pt(111) and the surface topography was measured alcohol, 1-propanol, ammonium hydroxide, and pyridine were with scanning tunneling microscopy. When the energy was less purchased from Mallinckrodt Chemicals (St. Louis, MI). Isopropyl than 1 eV per atom, it was possible to non-destructively deposit alcohol, acetic anhydride, and acetone were purchased from the clusters.42 Palmer et al. deposited size-selected silver clusters Macron (Center Valley, PA). 2,4,6-Trimethylpyridine was purchased on graphite as a method for preparing nanostructured surfaces.43 from Eastman chemical company (Kingsport, TN). All chemicals + were used as received without further purification. Recently, Ag3 deposited on alumina was found to be a highly 2 selective catalyst for the epoxidation of propylene by O2. Size selected-silver clusters were deposited on passivated carbon in 2.2 Experimental methods order to understand the discharge process in lithium–oxygen A home-built electrosonic spray ionization (ESSI) source was cells. The size of the deposited silver clusters greatly affected coupled to a heated drying tube as shown in Fig. 1.45,46 The heated the morphology of lithium peroxide, indicating that precise drying tube is made from 316L stainless steel (Amazonsupply.com, control of sub-nanometer features on a surface might improve part # S0125028T316SAL 60,O.D.1/800,I.D.0.06900) and coiled twice battery technology.44 to a diameter of 5.5 cm and to a total length of 43 cm. The stainless In this study, we describe a novel and facile method for steel loop was wrapped with heating tape (Omega, part # FGR-030). the production of naked metal cluster ions by electrospray The ESSI source was typically positioned between 3 mm outside

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to 3 mm inside the heating loop, the position being chosen This spectrum is characterized by a weak geometric magic + + such that the maximum ion signal was obtained. Independent number for [Ag5(CH3COO)4] and [Ag8(CH3COO)7] , the enhanced positioning of the ESSI sprayer and heating loop was achieved stability likely being associated with 3 3 and 3 5 units. For

by using two xyz micrometer moving stages (Parker Automation, smaller clusters (Ag3 and smaller), some hydration is present. + Rohnert Park, CA). The distance between the heating loop and For larger clusters ([Ag4(CH3COO)3] and larger), dissociation linear ion trap mass spectrometer (LTQ, Thermo Scientific, San (by CID) of the mass-selected ion is dominated by the loss of 24 Jose, CA) inlet was typically between 1 to 10 mm. At shorter Ag2(CH3COO)2 units, which likely occurs in two separate steps. distances, the ion signal was higher as more of the electrospray Smaller hydrated clusters undergo water loss followed by ejec- + plume entered the MS inlet. tion of AgCH3COO while the cluster [Ag2(CH3COO)] undergoes In a typical experiment, a metal salt was dissolved in 1 : 1 decarboxylation in agreement with observations by O’Hair methanol : water at a concentration of 1 mM. Spray was initiated et al.50 Negative ion mode electrospray produces a series of ions using a voltage of 5kVandN2 nebulizing gas (105 psi pressure). [Agn(CH3COO)n+1] , which do not exhibit any geometric magic The electrospray plume entered the heating loop, which was set numbers (Fig. S4A, ESI†). Dissociation of silver acetate cluster

to 250 1C. The temperature was controlled by the voltage supplied anions is by loss of Ag(CH3COO). Silver benzoate exhibits similar using an autotransformer and monitored by a thermocouple. The clustering to that of silver acetate except that the geometric + ions exiting the loop were analyzed by a LTQ mass spectrometer magic numbers are replaced by an abundant ion [Ag7(C7H6O2)6] using the following parameters: capillary temperature 200 1C, (Fig. S4C and E, ESI†). Silver fluoride exhibits a unique unheated capillary voltage 140 V, tube lens voltage 240 V, maximum mass spectrum. In the positive ion mode, naked silver cluster + + + + injection time 100 ms, and an average of 3 microscans. Automatic cations (e.g. Ag3 ,Ag5 ,Ag7 ,Ag9 ) along with various oxidized and instrument tuning was used to optimize the potential applied hydrated cations are observed (Fig. S4G, ESI†).Inthenegativeion to all other lenses. For collision-induced dissociation (CID), mode, extensive clustering of silver with multiple counter anions normalized collision energies of 25–35% (manufacturer’s unit), is observed (Fig. 2C).

Mathieu parameter qz values of 0.25–0.35, and isolation windows were typically 2 m/z units larger than the isotopic distribution (e.g. 3.2 Heated silver salts + for Ag6 an isolation window of 14 mass units was used). In certain The mass spectra of the various silver salts changed significantly experiments, high mass accuracy (o5 ppm) was achieved using when the heating loop was set to 250 1C. Regardless of the silver a hybrid LTQ-Orbitrap mass spectrometer (LTQ-Orbi, Thermo salt chosen, the positive ion mode mass spectra were all essen- Scientific, San Jose, CA). The mass resolution was set to 30 000 tially identical (Fig. 2B, Fig. S4D and H, ESI†). These spectra are + 2+ with a typical injection time between 250 ms and 5 s depending dominated by the naked metal clusters Agn and Agn .Allspecies on the initial ion intensity. Tandem MS Orbitrap experiments undergo expected fragmentations (Fig. S5, ESI†), including the

utilized the same conditions as the LTQ. loss of Ag2 from odd-numbered clusters, coulombic explosion 2+ 2+ 51–54 To perform ion/molecule reactions, the distance between for Ag16 , and monomer loss from other Agn ions. The + the heating loop and inlet was typically set at 1 cm. Vapors of expected odd/even alternation and magic numbers at Ag3 and + 55 interest were introduced via a cotton swab held between the Ag9 was also observed. Thus, the fragmentation behavior of the + 2+ Published on 15 June 2015. Downloaded by Purdue University 27/10/2016 14:23:43. † heating loop and MS inlet (Fig. S1, ESI ). In order to perform Agn and Agn ions is the same, regardless of the starting material, sequential ion/molecule reactions, a short piece of metal tubing viz. silver benzoate or silver acetate. Moreover, during heating there (2.5 cm) was placed between the inlet and heating loop (Fig. S2, is no evidence for the formation of silver hydride clusters. Interest- ESI†). One neutral reagent was introduced into the first gap ingly, only silver fluoride has the ability to generate negatively (between the heating loop and the additional metal tubing) and charged clusters, Agn (Fig. 2D). Silver acetate and benzoate do the second reagent was introduced into the second gap (between not produce silver cluster anions, but instead produce mainly the added metal tube and the inlet). In addition, some experi- organic fragments, presumably because of the stability of the organic ments were performed using ozone which was generated by low counter anion (Fig. S4B and F, ESI†). The fragmentation of small temperature plasma (LTP) (Fig. S3, ESI†). The low temperature silver cluster anions (n o 12) matches that reported in the literature plasma source used has been described previously.47,48 Gases (Fig. S6, ESI†).56 Larger silver cluster anions typically lose neutral Ag were introduced using Swagelok couplings at a flow rate upon collisional activation, which has not been reported in litera- between 0.5–1 L min 1. ture. For the silver cluster anions, the expected odd/even alternation 28 and magic number at Ag7 is observed.

3. Results/discussion 3.3 Mechanism The above experiments used heat as well as in-source collisions 3.1 Unheated silver salts to transform silver acetate and other silver salts into silver The electrospray of solutions of metal salts has long been known cluster cations and anions. Ready access to these clusters ions + to produce species of the type [CnAn1] and [CnAn+1] where C is facilitated the study of their formation and dissociation as a the cation and A is the anion of the salt.49 Consistent with this, source of information on their structure and reactivity. Possible the mass spectrum of silver acetate recorded with the heating loop intermediates responsible for the formation of silver cluster turned oﬀ shows the formation of simple salt clusters (Fig. 2A). cations from silver acetate were investigated by monitoring the

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Fig. 2 Positive ion mode mass spectra of (A) unheated and (B) heated electrosprayed silver acetate. Negative ion mode for (C) unheated and (D) heated electrosprayed silver fluoride solution. The numbers above each peak in (A) indicate the number of silver atoms, ligands, and water molecules present.

Published on 15 June 2015. Downloaded by Purdue University 27/10/2016 14:23:43. The numbers above each peak in (B) and (D) indicate the number of silver atoms.

total ion chronogram while the temperature of the loop was process shown in eqn (1) forms one molecule acetic anhydride + 57 ramped from room temperature to 250 1Coverthecourseofafew and Ag3O . The formation of acetic anhydride is exothermic minutes (Fig. S7, ESI†). At low temperatures, the salt clusters by 572.5 kJ mol 1, which almost 250 kJ mol 1 more exother- + [Agn(CH3COO)n1] were observed. At intermediate temperatures mic than the alternative pathway (ESI†), suggesting that acetic (100–150 1C), new species were observed in the MS, including anhydride formation is the favored pathway leading to oxidized + + + [Ag9(CH3COO)6O] ,[Ag8(CH3COO)5O] ,and[Ag7(CH3COO)4O] , silver cluster cations. † indicating losses of acetic anhydride (C4H6O3), eqn (1). For smaller Fig. S8 (ESI ) provides a chart of all ions observed in the full clusters, an additional water molecule was present on the cluster, scan mass spectra along with their relationships as established + + as in [Ag6(CH3COO)3OH2O] and [Ag5(CH3COO)2OH2O] .Thismay by CID experiments for the decomposition of the silver acetate ions simply be the result of addition of residual water to coordinatively in the positive ion mode. All ions observed in the mass spectra can unsaturated metal complexes by ion/molecule reactions in the ion be explained as possible products of eqn (1) when water and trap, a known process. An alternative to this pathway, the loss of oxygen are allowed to arrive or leave from the cluster. When the

ethenone (C2H2O)followedbywater,isconsideredlesslikelyas heating experiment is performed at the highest temperatures, discussed in the ESI.† the formation of simple silver cluster cations and oxidized silver

+ + cluster cations was observed. Once the final temperature was Ag (CH COO) - [Ag (CH COO) O ] + mC H O n 3 n 1 n 3 n 1 2m m 4 6 3 reached and some time had passed, the formation of primarily (1) naked metal clusters was observed. Thermochemical considerations are useful in deciding on The thermochemical mechanism for cationic cluster formation likely decomposition pathways. For example if n = 3, then the extends to other silver salts such as the benzoate, which is

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assumed to undergo thermal dissociation by similar routes to from the larger clusters which react with accompanying losses + give silver cation clusters. A method for the production of Agn and of Agn moieties. The loss of Ag upon ligand addition for even- + Agn1H from cluster ions generated from precursor solutions numbered silver cluster cations is an example of such a reac- containing silver nitrate and either glycine or N,N-dimethyl glycine tion that has been previously reported.59,60 has been reported by O’Hair, using ion trap CID.24 The choice 3.4.2 Methyl substituted pyridine reactivity. Atmospheric of precursor compounds is important in these experiments as it pressure ion/molecule reactions were carried out between methyl- + can allow the production of [CnAn1] and [CnAn1] where A is substituted pyridines and silver cluster cations generated from an anion with favorable redox properties. For example, glycine silver benzoate (Fig. S10, Tables S3 and S4, ESI†). Ligation of the contains both an amine and deprotonated carboxylic group, neutral reagents to the silver cluster was observed with the 24 + + which appear to meet these redox criteria. It should be noted dominant products being [AgL2] and [Ag3L3] except in the case that under the conditions of the O’Hair study, silver acetate of pyridine itself and 2,4,6-trimethylpyridine which also produced did not produce silver cluster cations upon CID.24 The heating species with a smaller number of ligands. The nature of the inter- + loop utilized in our work dissociates clusters to this extent at action between Ag3 and the neutral reagent was confirmed by atmospheric pressure, but the similarities and differences CID (Fig. S11, ESI†) which showed the sequential loss of ligands + + between the ion trap CID and ambient heating experiments from the species [Ag3(C8H11N)3] and [Ag3(C7H9N)3] generated are not well understood.58 from 2,4,6-trimethylpyridine and 3,5-lutidine. This confirms that + Anionic cluster formation can be considered in a similar vein. the neutrals are acting as simple ligands on Ag3 . The loss of fluorine could be by hydrolysis to give the enthalpically 3.4.3 Ligand exchange. It is obvious from the previous + favored HF or in a redox process to give the less favored F2. The examples that ligation of Ag3 is easily achieved. A further test + favored formation of the odd-numbered silver anion clusters was made to determine if the generation of mixed-ligand Ag3 (Fig. 2D) is simply due to their greater stability than their even- species was possible. A slightly modified version of the apparatus numbered counterparts.28 of Fig. S1 (ESI†) was used so that multiple neutral reagents could be introduced separately (Fig. S2, ESI†). The system was tested 3.4 Reactivity of silver cluster cations with three mixtures of ligands: ammonia (from ammonium 3.4.1 Alcohol reactivity. Atmospheric pressure ion/molecule hydroxide) and acetonitrile, 2,6-lutidine and acetonitrile, and reactions were carried out between organic solvents (ethanol, acetone and acetonitrile. For the first set of reagents, ammonia 1-pronanol, isopropyl alcohol, tert-butyl alcohol, acetone, and and acetonitrile, a variety of mixed ligand species was observed, + + acetonitrile) and silver cluster cations generated from silver including [Ag3 +C2H3N+NH3] ,[Ag3 +(C2H3N)2 +NH3] ,and + † acetate. Typically, an organic solvent of interest was added to a [Ag3 +C2H3N + (NH3)2] as confirmed by MS/MS (Fig. S12, ESI ). cotton swab and introduced into the region after the heating 2,6-Lutidine and acetonitrile also generated a mixed ligand + tube and before the MS inlet (Fig. S1, ESI†). A summary of the species (Fig. S13, ESI†), [Ag3 +C7H9N+C2H3N] ; however, this reactions can be found in Tables S1, S2 and Fig. S9 (ESI†). was the only mixed ligand species observed in this case. In + + + + + Monomeric Ag reacts to form mainly [AgL] and [AgL2] , except addition, [Ag3 +(C7H9N)2] and [Ag3 +(C7H9N)3] were formed + + in the case of acetonitrile where the only product is [AgL2] . The in higher intensity than [Ag3 +C7H9N+C2H3N] , indicating a + + + Published on 15 June 2015. Downloaded by Purdue University 27/10/2016 14:23:43. cluster Ag3 reacts to form [Ag3L] and [Ag3L2] , except that preference towards binding 2,6-lutidine. To qualitatively test + + acetonitrile behaves differently in forming [Ag3L2] and [Ag3L3] . the effect of the sequence used to introduce neutral reagents, This reactivity differs from reported gas phase reactions, where acetone and acetonitrile were used. Acetone was allowed to + + [AgL2] and [Ag3L3] are the terminal products for all reagents interact first with the cluster spray followed by the acetonitrile studied except ethanol, which was reported to be unreactive.59 and vice versa (Table S5 and Fig. S14, ESI†). When acetone was The differences in reactivity are not unexpected given that our allowed to react first, it was possible to generate a mixed species experiments were performed in air, and the literature data refers as well as species containing either acetone or acetonitrile. This to vacuum, and there are also differences in reaction time. The was in stark contrast to the case where acetonitrile was the first maximum interaction time in our experiments was on the order reagent as it produced only species with acetonitrile ligands; of a single scan, ca. 0.2–0.5 s, compared to possibly 60 s reported i.e. ligand displacement did not occur due to stronger binding in the literature. The ion/molecule reactions under vacuum do to this ligand. not provide efficient third body stabilization for smaller ions, 3.4.4 Hydrocarbon reactivity. Silver cationization is known to explaining the difference in reactivity with ethanol.59 Tandem selectively ionize unsaturated compounds, including polycyclic + 61–64 mass spectrometry data on the reaction products (e.g. [Ag3L2] , aromatic hydrocarbons and alkenes. A series of hydro- Fig. S9A, C, E and F, ESI†) indicate that the reagents serve as carbons was analyzed using ambient ion/molecule reactions + simple ligands. The reaction product, for example [Ag3L2] ,loses in the same manner as other neutral reagents (Fig. S15, ESI†). Both + + + one ligand for each stage of tandem MS to form bare Ag3 . cationization by Ag and Ag3 occurred for hexadecane, isocetance, Simple ligation is the main result observed in these reactions. and squalane. Cationization of a pump oil (Ultragrade 19) sample There appears to be relatively good agreement between silver was also demonstrated, but the identity of the silver adducts is clusters with alcohols obtained under vacuum and in the open unknown.Thefactthatheatwasrequiredtogeneratethesilver air. Due to the inability to mass select the clusters before cluster spray is advantageous for the analysis of heavy hydro- reaction, it is possible that some of the reaction products form carbons, which are difficult to vaporize and often have low

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vapor pressures. The convenient ionization of saturated hydro- be catalysts for the direct epoxidation of propylene using gaseous carbons in the ambient environment is a potential analytical oxygen.2 In our study, odd-numbered silver cluster cations inter- application of this methodology. acted with ethylene to produce mono-, bi-, and tri-ligated species 3.4.5 Oxidation. Oxidized silver clusters can serve as model (Fig. S16A, ESI†). No ligation was observed for even-numbered catalysts for partial oxidation reactions, including epoxidation.37 clusters, either due to their inertness or to the loss of Ag upon + Roithova´ and Schro¨der produced Ag2O in vacuum from the reaction to produce the favored odd-numbered cluster. A small + + + dissociation of Ag2NO3 into NO2 and Ag2O . Another possible amount of [Ag3(C2H4)3O] wasobserved,whichpresumablyarises + + approach to simple oxides is the direct oxidation of Agn clusters, from reaction with the small amount of Ag3O present within the a possibility indicated by the reaction between neutral silver spray. With the addition of ozone from the LTP source, the mass 65 + clusters and ozone. Ionic metal cluster oxidation was achieved spectra change significantly. The intensity of [Ag3(C2H4)3O] + by utilizing a low power plasma (LTP) source (Fig. 3), which increases dramatically and a species [Ag5(C2H4)4O2] is observed under appropriate conditions serves as a source of ozone.47,66 (Fig. S16B, ESI†). It is interesting to note that there appears to be a Silver cluster cations were generated from silver fluoride and cooperative eﬀect in the binding of ethylene to oxidized clusters as + + allowed to interact with the generated ozone (Fig. 3B). A series of Ag3 binds one or two ethylenes, but it appears that Ag3O binds oxidized metal clusters was observed which contained multiple only three ethylenes. The same phenomenon occurs for the + + oxygen atoms as well as water molecules. The simplest and most binding of one, two, or three ethylenes to Ag5 but Ag5O2 only + + notable reaction was the transformation of Ag3 into Ag3O .Itis binds four ethylenes. Cooperative binding has been reported for

observed that silver clusters containing at least one oxygen atom O2 and CO with anionic silver clusters, but no such reports exist 6,32 + + can react with water too. It seems that water is only reactive for cationic silver clusters. [Ag3(C2H4)3O] and [Ag5(C2H4)4O2] towards already oxidized clusters, since naked silver cluster were subjected to CID to identify any potential epoxide products; cations have been reported to be unreactive towards water.59 however,onlytheproductsofligationwereobserved.Thetandem + (The water is likely adventitious water in the ambient environ- MS of [Ag5(C2H4)4O2] (Fig. S17, ESI†) is interesting as the loss of ment or in the ion trap used for mass analysis.) two ethylene molecules is accompanied by the addition of a single + + 3.4.6 Reactions of Agn and oxidized Agn with ethylene and water molecule, and the loss of three ethylenes is accompanied propylene. Supported silver trimers have been demonstrated to by the addition of two water molecules. Published on 15 June 2015. Downloaded by Purdue University 27/10/2016 14:23:43.

Fig. 3 Mass spectra of silver cluster cations generated from silver fluoride (A) without ozone and (B) with ozone. (C) Shows the selected ion chronogram + + + of Ag3 (top) and Ag3O (bottom) in the presence and absence of ozone. (D) High mass accuracy measurements confirm the formation of Ag3O .

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Fig. 4 Reaction of silver cluster cations with (A) propylene and (B) propylene and ozone.

Similar experiments were performed with propylene as the 3.5 Reactivity of silver cluster anions reagent gas. In the absence of ozone, propylene exhibits nearly 3.5.1 Formation of [Agn(OH)] . The reactivity of silver cluster identical reactivity to ethylene (Fig. 4). The reactivity of ozone anions generated from silver fluoride was examined. The position + and propylene is similar to ozone and ethylene for the Ag3 of the ESI source relative to the heating tube was altered to form cluster but diﬀers for the larger clusters (Fig. 4B). A variety of an ambient discharge. Without the discharge, naked metal species containing propylene, oxygen, and water are observed silver clusters are observed (Fig. 5). With the discharge, the + + + for both Ag5 and Ag7 . [Ag7(C3H6)O] is particularly interesting even-numbered clusters (especially n = 6, 8, and 10) gained an because it fragments to lose neutral C3H6O, propylene oxide or OH group to form [AgnOH] , possibly due to reaction with a structural isomer. hydroxyl radicals created by the discharge.67 The amount of Published on 15 June 2015. Downloaded by Purdue University 27/10/2016 14:23:43.

Fig. 5 Mass spectra of silver cluster anions with the ESSI sprayer inserted (A) 1 cm and (B) 2–3 mm inside the heating tube. Red and blue numbers respectively indicate silver atoms and OH molecules.

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[AgnOH] varies with cluster size but generally decreases as counterions will be selected to optimize cluster ion formation. n increases. Deuterated 1 : 1 methanol : water was used as the The most interesting aspect of the new capabilities provided spraysolutioninanattempttoidentifythesourceoftheOH.The will be the investigation of processes of industrial significance + intensity of [AgnOD] was at most 30% of the [AgnOH] intensity, using metal clusters and metal oxide clusters, which have indicating that the source of OH must be from elsewhere, such as some of the structural features believed to be important in atmospheric water. full scale heterogeneous catalysis (certain metal cluster sizes and compositions).8,69,70 The fact that these species can be 3.6 Ion soft landing generated in air compensates to a degree for the fact that they As a proof of principle, the metal cluster ion source was coupled are not atomically precise. to a homebuilt ion soft landing instrument.4,68 Each of the three silver salts was electrosprayed and similar mass spectra were obtained as previously described (Fig. S18, ESI†). For unheated Acknowledgements silver acetate and silver benzoate, the same type of salt clustering was observed as previously seen (Fig. 2A). A similar effect was Financial support from the Separations and Analysis Program, observed for silver fluoride, but the exact number of silver and Office of Basic Energy Sciences, US Department of Energy, amount of oxygen or water bound could not be determined with DE-FG02-06ER15807 is acknowledged. TP acknowledges DST the given instrument resolution. Upon heating, all three salts for funding. DS acknowledges UGC for their research fellowship. + + produced a range of clusters from Ag3 to Ag15 without any Zane Baird is thanked for technical assistance. even-numbered clusters. This set of ions was deposited onto a gold surface and a current of 10 pA could be achieved. Efforts are underway to improve the current of the source so it will be possible to study mass-selected ions on surfaces. References

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