mass spectrometry with electrospray ionization from an adjustable

Mass Spectrometry with Electrospray

Ionization from an Adjustable Gap

Patrik Ek

Licentiate Thesis

School of Chemical Science and Engineering

Department of Chemistry

Division of Analytical Chemistry

KTH, Royal Institute of Technology

Stockholm, Sweden, 2008

AKADEMISK AVHANDLING som med tillstånd av Kungliga Tekniska Högskolan i Stockholm

framlägges till offentlig granskning för avläggande av teknologie licentiatexamen torsdag 29 maj 2008, kl. 10.00 i sal E2, Lindstedtsvägen 3, Stockholm.

Avhandlingen presenteras på svenska.

ii

Mass Spectrometry with Electrospray Ionization from an Adjustable Gap

Patrik Ek

Thesis for the degree of Licentiate of Technology in Chemistry KTH, Royal Institute of Technology School of Chemical Science and Engineering Department of Chemistry Division of Analytical Chemistry Stockholm, Sweden, 2008 ISBN 978-91-7178-926-6 TRITA-CHE Report 2008:24 ISSN 1654-1081 Copyright © Patrik Ek, 2008 All rights reserved for the summary part of this thesis, including all pictures and figures. No part of this publication may be reproduced or transmitted in any form or by any means, without prior permission in writing from the copyright holder. The copyright for the appended journal papers belongs to the publishing houses of the journals concerned.

Printed by US-AB, Stockholm, 2008

iii

ABSTRACT In this thesis the fabrication and analytical evaluation of two new electrospray emitters utilized for mass spectrometry analysis is presented. The emitters are based on a new concept, where the spray orifice can be varied in size. The thesis is based on two papers. All present-day nanoelectrospray emitters have fixed dimensions. The range of the applicable flow rate for such an emitter is therefore rather limited and exchange of emitters may be necessary from one experiment to another. Optimization of the signal of the analyte ions is also limited to adjustments of the applied voltage or the distance between the emitter and the mass spectrometer inlet. Furthermore, clogging can occur in emitters with fixed dimensions of narrow orifice sizes. In this thesis, electrospray emitters with a variable size of the spray orifice are proposed. An open gap between two thin substrates is filled with sample solution via a liquid bridge from a capillary. Electrospray is generated at the end point of the gap, which can be varied in width. In Paper I, electrospray emitters fabricated in polyethylene terephthalate have been evaluated. Triangular tips are manually cut from the polymer film. The tips are mounted to form a gap between the edges of the tips. The gap wall surfaces are subjected to a hydrophilic surface treatment to increase the wetting of the gap walls. In Paper II, silicon electrospray chips with high precision are fabricated and evaluated. A thin beam, elevated from the bulk silicon chip is fabricated by means of deep reactive ion etching. The top surfaces of the beams of two chips act as a sample conduit when mounted in the electrospray setup. An anisotropic etching step with KOH of the intersecting <100> crystal planes results in a very sharp spray point. The emitters were given a hydrophobic surface treatment except for the hydrophilic gap walls. For both emitter designs, the gap width has been adjusted during the experiments without any interruption of the electrospray. For a continuously applied peptide mixture, a shift towards higher charge states and increased signal to noise ratios could be observed when decreasing the gap width. The limit of detection has been investigated and the silicon chips have been interfaced with capillary electrophoresis.

Patrik Ek Stockholm, 2008

Keywords: Electrospray ionization, mass spectrometry, nanoelectrospray, ESI, MS, chip, emitter, gap, liquid bridge, nozzle, peptides

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SAMMANFATTNING I denna avhandling presenteras tillverkningen och den analytiska utvärderingen av två nya elektrosprayspetsar som används för masspektrometrisk analys. Spetsarnas funktion baseras på ett nytt koncept där arean, från vilken elektrosprayen genereras, är varierbar i storlek. Avhandlingen grundar sig på två vetenskapliga artiklar. Alla dagens nanoelektrosprayspetsar har låsta dimensioner. För sådana spetsar är det tillämpbara flödeshastighetsområdet tämligen begränsat och byte av spetsar kan vara nödvändigt från experiment till experiment. Optimering av signalen från analytjonerna är också begränsad till justeringar av applicerad spänning eller avståndet mellan spetsen och masspektrometerns insläpp. Dessutom kan spetsar med små inre dimensioner täppas igen. I denna avhandling presenteras elektrosprayspetsar med varierbar storlek på spraymynningen. En öppen spalt mellan två tunna substrat fylls med provlösning via en vätskebrygga från en kapillär. Elektrospray genereras vid mynningen av spalten, vilken kan varieras i bredd. I Artikel I (Paper I) utvärderas elektrosprayspetsar tillverkade i polyetylen-tereftalat. Triangulära spetsar skärs manuellt från en polymerfilm och monteras så att en spalt bildas mellan spetsarnas kanter. Spaltväggarnas ytor utsätts för en ytbehandling för att göra dem mer hydrofila och öka vätningen av spaltväggarna. I Artikel II (Paper II) utvärderas elektrosprayspetsar som tillverkats med hög precision i kisel. En tunn balk, upphöjd från ett kiselchip, tillverkas med hjälp av djupetsning med DRIE. Ovansidorna av de två chipens balkar fungerar som en kanal för provlösningen när de monterats i experimentuppställningen. Ett anisotropiskt etssteg med KOH av de korsande <100> kristallplanen resulterar i en mycket skarp spraypunkt. Spetsarna gavs en hydrofob yta genom ytbehandling, bortsett från de hydrofila spaltväggarna. För båda designerna av spetsar har spaltbredden justerats under pågående experiment utan avbrott i elektrospraygenereringen. För en kontinuerligt tillförd peptidblandning observerades ett skifte mot högre laddningstillstånd samt ökat signal-till-brusförhållande när spaltbredden minskades. Detektionsgränsen har utvärderats och kiselchipen har sammankopplats med kapillärelektrofores.

Patrik Ek Stockholm, 2008

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CONTENTS

1 LIST OF PUBLICATIONS......................................................... 1 2 INTRODUCTION ................................................................... 2 3 MASS SPECTROMETRY........................................................... 3

3.1 Electrospray ionization ......................................................... 3 3.2 Ion trap mass analyzer ........................................................ 6

4 CAPILLARY ELECTROPHORESIS................................................ 7 4.1 Principles of CE ................................................................... 7 4.2 CE-ESI-MS interfaces........................................................... 9

5 A BRIEF SURVEY OF ELECTROSPRAY EMITTERS ......................... 10 6 NEW CONCEPT OF ELECTROSPRAY FROM AN ADJUSTABLE GAP ...... 13

6.1 Adjustable Gap Electrospray from PET-Tips ............................14 6.1.1 Fabrication .......................................................................... 14 6.1.2 Setup ................................................................................. 16 6.1.3 Initial experiments ............................................................... 16 6.1.4 ESI-MS experiments ............................................................. 17

6.2 Adjustable Gap Electrospray from Silicon Chips.......................22 6.2.1 Fabrication .......................................................................... 22 6.2.2 Setup ................................................................................. 24 6.2.3 ESI-MS experiments ............................................................. 25 6.2.4 CE-MS experiments .............................................................. 26

7 CONCLUSIONS AND FUTURE PERSPECTIVES.............................. 28 8 ACKNOWLEDGEMENTS ......................................................... 30 9 ABBREVIATIONS................................................................ 31 10 REFERENCES..................................................................... 32 APPENDICES: PAPER I-II

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1 LIST OF PUBLICATIONS This thesis is based on the following publications, which will be referred to in the text by their Roman numerals: I Electrospray Ionization from a Gap with Adjustable Width

P. Ek, J. Sjödahl and J. Roeraade Rapid Communications in Mass Spectrometry, 20, 3176-3182, 2006

II Electrospray Ionization from an Adjustable Gap between two Silicon Chips P. Ek, T. Schönberg, J. Sjödahl, J. Jacksén, C. Vieider, Å. Emmer and J. Roeraade Manuscript, to be submitted, 2008

Reprints are published with kind permission of the journal. The contributions of the author of this thesis to these papers are: I All experiments and major part of the writing II All experiments (except chip fabrication) and major part of the writing

Parts of the work in this thesis have also been presented at conferences:

o Electrospray Ionization from a Gap with Adjustable Width P. Ek, J. Sjödahl and J. Roeraade Poster presented at 20th International Symposium on Microscale Bioseparations, Amsterdam, the Netherlands, January 22-26, 2006 Second prize, Best Poster Award

o Electrospray Ionization from a Silicon Emitter with an Adjustable Gap T. Schönberg, P. Ek, J. Sjödahl, J. Roeraade and C. Vieider Poster presented at 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, µTAS, Paris, France, October 7-11, 2007

o Electrospray Ionization from an Adjustable Gap between two Silicon Chips P. Ek, T. Schönberg, J. Sjödahl, J. Jacksén, C. Vieider, Å. Emmer and J. Roeraade Poster presented at 22nd International Symposium on Microscale Bioseparations and Methods for Systems Biology, Berlin, Germany, March 9-13, 2008


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2 INTRODUCTION In the past decades mass spectrometry (MS) has experienced a tremendous development. The technique is now utilized for a broad range of applications in the average laboratory.1 Still, each day researchers around the world encounter the limitations of present-day mass spectrometers when trying to solve intriguing analytical problems. Therefore, the need for new technologies and methodologies is always present to solve these problems with improved detection, identification and quantification of molecules of interest.2 Two main developments of mass spectrometry has been the soft ionization techniques, electrospray ionization (ESI)3, 4 and matrix-assisted laser desorption/ionization (MALDI)5, 6. These ionization techniques revolutionized the field of analytical chemistry, especially bioanalytical chemistry, since it was now possible to ionize large non-volatile molecules without fragmentation.7 Today, ESI is widely used in combination with separations such as liquid chromatography and capillary electrophoresis, while MALDI is more commonly used for discrete analysis of single compounds or mixtures.8 ESI-MS is today a technique of central importance for biological research and is also used in drug discovery, screening of combinatorial libraries, analysis of proteins, oligonucleotides, carbohydrates and lipids.9 In ESI, the ionization is so gentle, that it is even possible to ionize proteins without disrupting non-covalent interactions. When this was discovered, it opened an enormous field of research, e.g. on conformational changes, protein folding and receptor-ligand complexes.10 Since the introduction of protein analysis utilizing ESI-MS in the late 1980’s, bioanalytical applications and questions have to a large extent fuelled the development of new MS components.11 The main issues for ESI-MS developers have been to increase the sensitivity, throughput and versatility to handle complex samples.2 These issues are easily exemplified when considering the field of proteomics, i.e. the systematic analysis of the proteins in a tissue or cell. Proteomics present a field of particular complexity. Large variation of components are encountered at concentration ranges that by far exceeds the dynamic range of today’s mass spectrometers.8, 12 Approximately 50-70% of all expressed proteins appear at a minute-abundance.13 To detect and identify these molecules, new techniques with improved sensitivity are of particular importance. The need for high-throughput analyses is obvious if detailed information from a whole proteome of thousands of compounds is desired.8

3

3 MASS SPECTROMETRY Mass spectrometry as an analytical technique dates back to 1912 when Sir J.J. Thomson constructed the first mass spectrometer, which at that time was called a parabola spectrograph.14 Thomson was able to obtain early mass spectra corresponding to molecules of oxygen and nitrogen and atoms of carbon among other species. Since that day a large number of mass spectrometry instruments have been developed and present-day mass spectrometers differ widely in instrumental configuration.15 However, all instruments comprise the following fundamental elements: An ion source; a mass analyzer and a detector. In the ion source, as the name implicates, ions are generated. In the mass analyzer, the ions are separated by their mass-to-charge ratio (m/z) and subsequently, in the detector the ions are detected by their m/z and abundance. Computer programs are nowadays used to control the instruments and process the data. The readout from a mass spectrometer can be used to accurately identify a molecule by its molecular mass, determine its structure and measure its abundance. The ion source and mass analyzer, electrospray ionization and ion trap respectively, utilized in the work comprised in this thesis are further described in the next two sections.

3.1 Electrospray ionization In electrospray ionization, ions are transferred from solution to the gas phase. The basic principles of electrospray are used in many different fields of application such as spray painting of cars, drug delivery by inhalation and electrostatic spray deposition of pesticides on crops.16, 17 During the last decades, electrospray has also gained a major interest in bioanalytical chemistry when utilized as an ion source for mass spectrometers.12 Electrospray ionization was first presented by Dole in 196818, partly based on previously shown principles by e.g. Zeleny19 and Taylor20. Dole’s group succeeded in generating an electrospray of a solution containing polystyrene molecules with molecular weights of 51 and 411 kDa. However, the use of ESI took a gigantic leap during the 1980s when ESI was successfully included as an ion source to MS by Fenn and co-workers3, 4, 21 and approximately at the same time by Aleksandrov and co-workers22. Fenn et al. showed mass determinations of proteins with higher molecular weight than the detector mass range. This was possible due to the fact that non-fragmented multiply charged ions could be generated with electrospray ionization, allowing for separation and detection of heavy biomolecules within the range of a few thousand m/z. The soft ionization without fragmentation of the analyte molecules is a key feature for the extensive use of electrospray in the analysis of non-volatile chargeable molecules such as proteins12, nucleic acids23 or whole viruses24. Fenn shared a Nobel Prize for his work on ESI in 2002.


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The fundamentals of the electrospray ionization process have been described in several books and reviews. One of the most comprehensive, is the book Electrospray Ionization Mass Spectrometry – fundamentals, instrumentation & applications, consisting of 15 review chapters with numerous references to published works.9 The following condensed explanation of the ESI process is based on this book (especially Chapter 1-4) and references therein. The ionization process in ESI takes place at atmospheric pressure and therefore ESI is called an atmospheric pressure ionization (API) technique. To understand electrospray ionization one can consider a conductive hollow needle containing a solution of an electrolyte at low concentration as well as charged analyte molecules. The open end of the needle is positioned facing a counter electrode of a mass spectrometer. An electrical field is established between the end of the needle and the planar counter electrode. The value of the electric field (E) experienced at the tip of the electrospray needle, can be calculated from the approximate relationship:

( )rdrVE⋅⋅

=4ln

2 [Eq. 1]

where V is the voltage difference between the emitter and the counter electrode, r is the radius of the emitter, and d is the distance between the emitter and the counter electrode. The electrical field forms a charge distribution of anions and cations. For positive ESI mode, i.e. when the potential at the needle tip is higher than the potential at the counter electrode, the positive ions are attracted towards the counter electrode. The cations accumulate at the liquid surface and due to repulsions between the cations the meniscus of the liquid extends to a curvature. A further increase in the electric field destabilizes the liquid curvature and a cone-jet develops (Figure 1). The equation for the required electrical field that forms the jet onE can be written as:

2/1

0

cos2⎟⎟⎠

⎞⎜⎜⎝

⎛≈

rEon ε

θγ [Eq. 2]

where γ is the surface tension of the solvent, ε0 is the permittivity of vacuum, r is the radius of the emitter, and θ is the half-angle of the so-called Taylor cone. Equation 2 together with Equation 1 leads to the equation for onset voltage ( onV ):

( )rdrVon /4ln2cos

2/1

0⎟⎟⎠

⎞⎜⎜⎝

⎛≈

εθγ [Eq. 3]

For positive ionization, the jet emerging from the Taylor cone carries a large excess of positive ions. When positive ions are extracted from the needle tip, electrons must be provided to maintain charge balance in the electrical circuit. This will occur in an electrochemical oxidation process at the liquid-metal surface of the conductive needle. The electrospray process can therefore be considered to behave like a kind of electrolytic cell. Possible reactions are oxidation of negatively charged species in the solution or conversion of atoms from the metal to positive metal ions according to: 4OH – (aq) → O2 (g) + 2H2O (l) + 4e – 2H2O (l) → O2 (g) + 4H+ (aq) + 4e – M (s) → Mn+ (aq) + ne– (in metal)

5

The release of metal ions into the solution in terms of adducts to the analyte molecules can often be neglected. During electrospray runs, an electrical current is monitored as a quantitative measurement of the total number of excess positive ions that leave the capillary.

Figure 1: Schematic of the electrospray ionization process.

The cone-jet breaks up into small droplets and due to coulombic repulsions these droplets are driven away from each other (Figure 1). When exposed to the open air, the solvent evaporates, which leads to a decreased droplet radius and an increased charge density (due to constant charge during the evaporation). At a certain point called the Rayleigh limit, the electrostatic repulsion exceeds the preserving force of the surface tension and the droplet disintegrates into smaller droplets. The disintegration is referred to as coulombic fission or droplet-jet fission. In the fission process, a number of offspring droplets about one order of magnitude smaller than the parent droplet are ejected. The generated daughter droplets will undergo a similar shrinkage and fission process. Repeated evaporation and fission of parent and daughter droplets leads to a droplet population with the shape of a plume. Finally, gas-phase ions are formed from the very small droplets. Two different models have been proposed and discussed for this process: The charged residue model (CRM) of Dole18 and the ion evaporation model (IEM) by Iribarne and Thomson25. In the charge residue model, the gas-phase ions are suggested to be formed as a result of continuous droplet fission until each droplet only contains one excess ion. The ion evaporation model predicts that gas phase ion emission occurs directly from small droplets. Disregarding the exact process, the gas phase ions then traverse through the inlet hole of the MS.


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3.2 Ion trap mass analyzer In a general API-MS instrument the pressure difference between the ion source and the mass analyzer is several orders of magnitude.26 The risk of losing certain ions of interest before they reach the mass analyzer is obvious. Nevertheless, instrument developers have developed brilliant pressure-reducing regions and ion-focusing components that confine the ions of interest and provide the mass analyzer region of the MS with a stream of these ions.15 Ion optics and pressure reducing components will however not be highlighted further here. The mass analyzer comprised in the MS instrument utilized for the present work is a quadrupole ion trap (QIT) mass analyzer. Quadrupole ion traps were first invented in 1953 by Paul and Steinwedel at the University of Bonn.27 Three years later a German patent included the three-electrode arrangement of an ion trap. Paul shared a Nobel Prize in Physics for his work on radio frequency (RF) ion traps in 1989. Earlier during the 1980s ion traps gained importance when they were incorporated in mass spectrometers.28 An ion trap consists of two hyperbolic end cap electrodes and a donut shaped ring electrode (Figure 2).15

Figure 2: Schematic, showing the general construction of a quadrupole ion trap mass analyzer. A cross-section can be seen to the left and a tilted semi-3D view to the right.

The electrodes are configured to store ions entering from the ion source in a restricted volume, usually denoted a “trap”.29 In positive MS mode, a small negative voltage offset is applied to the electrodes to inject the electrosprayed positive ions through a small ion injection hole in the first end cap electrode. By applying a RF voltage to the electrodes, a three-dimensional quadrupole field is created. The field is referred to as a quadrupole field since the cross-section of the ion trap resembles the cross-section of a linear quadrupole (Figure 2). For a set of ions inside the ion trap, the quadrupole field forms stable trajectories for ions of a pre-determined m/z or the entire m/z range of interest, while unwanted ions are ejected or collided with the walls.30 The quadrupole field traps ions by continuously forcing them toward the center of the trap volume. In the last step, the mass-analysis step, the trapped ions are sequentially ejected out of the chamber through an ejection hole of the second end cap electrode to the detector. The signal from the detector is then transferred to the data system for conversion into a mass spectrum where the signal intensity versus m/z is presented.

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4 CAPILLARY ELECTROPHORESIS Separation of charged species under the influence of an electric field has been known for over a century, but it was in the beginning of the 1980s when Jorgenson and Lukacs presented results from separations in narrow bore capillaries that the potential of capillary electrophoretic separations became evident.31 Narrow bore capillaries resulted in a high electrical resistance, which allowed for the use of high voltages and therefore fast and highly efficient separations were obtained.

4.1 Principles of CE The following section is based on the publication by Kok and references therein.32 The general principle of electrophoretic separations is that charged particles in a solution are influenced by an electric field and migrate with different velocities depending on their charge and/or size. By applying an electrical field gradient, an electrostatic force is created. This force is proportional to the electrical field strength and the net charge of a particle. The force results in a migration of the particle towards the electrode with a charge opposite to that of the particle. However, a counteracting viscous force proportional to the velocity is experienced by the particle. When these forces are balanced the particle moves with a constant velocity ( iv ) through the solution (assuming a spherical particle):

i

ii r

Eqv

⋅⋅

=πη6

[Eq. 4]

where iq is the net charge of the particle, E is the electric field strength, η is the viscosity of the solution and ir is the radius of the particle. For a given ion and medium an ionic mobility ( iμ ) can be defined as:

Evi

i =μ [Eq. 5]

By combining Equation 4 and Equation 5 the mobility can be explained as:

i

ii r

q⋅

=πη

μ6

[Eq. 6]

This shows that small and highly charged species have higher mobilities than large and low-charged species and therefore all these species can be separated in the electrophoretic process. For capillary electrophoresis in a fused silica capillary, the capillary wall surface usually possesses an excess of negative charges (SiO– ) under aqueous conditions (at pH>2-3). This negative surface attracts positive counter ions to the wall in a so-called double layer. When a voltage gradient is applied along the length of the capillary, the cations start to migrate towards the cathode. This movement drags the bulk solution towards the cathode and forms a flat-profiled flow called electroosmotic flow (EOF).


8

An electroosmotic mobility ( EOFμ ) can be defined similarly to the ionic mobility:

EvEOF

EOF =μ [Eq. 7]

Most often the electroosmotic mobility is larger than the ionic mobilities of the analytes in a solution. In such cases, all species migrate towards the cathode with an apparent mobility ( appμ ) corresponding to the sum of the electroosmotic mobility and the ionic mobility:

EOFiapp μμμ += [Eq. 8]

All analytes can then be separated and determined in one run. Analyte ions attracted towards the inlet electrode migrate behind the EOF, neutral analytes migrate with the EOF and analyte ions attracted towards the outlet electrode migrate in front of the EOF. In this thesis the most common mode of CE operation was employed; capillary zone electrophoresis (CZE).32 In CZE the capillary is filled with an electrolyte buffer and the sample solution is introduced into the capillary by means of a small plug injection. When the CE separation voltage is applied, the analyte ions in the sample plug move with different mobilities, thereby forming zones of ions of a certain charge and size. In this work, the sample was injected hydrodynamically by an overpressure at the sample vial. Although this contributes to a somewhat broadened sample plug due to the parabolic flow profile of a pressure injection, a representative plug of the sample solution is injected into the capillary without discrepancies.32 The injected sample volume is in the nanoliter-range, which makes CE suitable for the analysis of minute volumes of material.33

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4.2 CE-ESI-MS interfaces CE shows extraordinary resolving power and complements well with the selectivity and identification possibilities obtained with MS.33 For on-line hyphenation of CE and MS, ESI is the primary method of choice for ionization, due to a facilitated transfer of the analytes from the CE separation capillary to the MS via the ESI interface.34 CE-ESI-MS is an interesting technique with important applications in genomics, proteomics, glycomics, biomarker discovery and for drug analysis among other fields.35, 36 A drawback when interfacing CE with MS, is that standard background electrolyte buffers normally used in CE are not always MS compatible, due to a high salt content or too low volatility.33 The buffers used, must therefore be carefully chosen to optimize the CE without disturbing the electrospray. One of the key issues to consider when interfacing CE with ESI-MS is the requirement for electrical connections for the two electrically driven liquid flow systems. For previously presented CE-MS interfaces mainly two methods for the electrical contact have been proposed; 1) Voltage connection via a conductive support liquid (sheathflow) and 2) Voltage connection directly applied to the CE capillary in a sheathless mode.33 In sheathflow interfaces, the CE capillary outlet is surrounded by a sheath liquid that is mixed with the CE eluent at the end of the capillary where the electrical circuit is completed. Usually the sheath liquid contains volatile acids and/or organic solvents to facilitate the ESI process. The sheath liquid can be used for optimization of the signal of the analyte in the electrospray process.37 Sheath flow interfaces are robust and easy to use and have reached a dominant position in commercial interfaces. Other liquid support systems have included a liquid junction between the CE capillary and the ESI needle. The support liquid is added via a Tee junction, in which the electrical contact is completed.38 However, sheath liquids such as acetonitrile have been reported to increase the risk of clogging at the electrospray point due to swelling of the protective polyimide layer on the CE capillary.39 Another disadvantage is the addition of a sheath liquid in terms of ionization efficiency. The sheath liquid inevitably dilutes the eluting analytes from the CE capillary. This is one of the main reasons for the use of sheathless CE-ESI-MS interfaces. Lack of sheath liquid can increase the sensitivity 10-fold.40 Several strategies have been developed for closing the electrical circuit, such as coating the capillary outlet with a conductive layer of metal or polymer or insertion of a conductive wire into the outlet of the CE capillary.33, 41, 42 Often the fabrication of these interfaces has been time-consuming. The durability of the interfaces is a crucial issue and intense research has been carried out in this area.43 Recently an etched porous junction near the spray tip, based on the principles of Moini44, has been utilized for the electrical contact in a commercialized sheathless interface manufactured by Beckman Coulter Inc.


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5 A BRIEF SURVEY OF ELECTROSPRAY EMITTERS Early ESI devices utilized a (typically) 100 µm inner diameter metal capillary needle.3 An electrolyte-containing analyte solution was pumped through such needles at low µL/min flow rates. When the needle was coupled to a high potential relative to the counter electrode of the MS inlet, droplets with an initial diameter of more than 1 µm were generated.45 To manage these high liquid flow rates and desolvate the droplets effectively, instrumental configurations such as heated inlet regions or countercurrent flows of dry gas have been used.11 Due to the mismatch between the size of the electrospray plume and the narrow MS inlet hole, only a small part of the droplets from the electrospray plume reaches the first stage of reduced pressure in the mass spectrometer.46 The typical size ESI needles mentioned above are still in use today, but apart from these, other electrospray emitters have emerged. It was known at an early stage, that the initial droplet size from an emitter depends on the flow rate and the conductivity of the solution (as well as other conditions).47 Low flow rates and high conductivities lead to small droplets. This led a number of groups to develop electrospray emitters with reduced flow rates. Early efforts were made by Gale and Smith who demonstrated high signal stability and sensitivity for flow rates in the range of low nL/min, utilizing fused silica capillaries (5-20µm ID) etched to an electrospray tip.48 Emmet and Caprioli presented a micro-electrospray ionization source based on electrospray with flow rates in the mid-range of nL/min from a fused silica capillary end.49 They reported on increased sensitivity compared to the performance of conventional electrospray sources. Both these inventions employed liquid feed systems, where the sample solution was delivered to the spray capillary by means of e.g. pumping or the flow from a separation column. Another miniaturized emitter, referred to as a nanoelectrospray (nESI) emitter, was presented by Wilm and Mann.50, 51 Also this emitter operated at low nL/min flow rates, but with the important distinction from the previously mentioned emitters that the electrospray process itself determined the flow rate without assisting liquid feed systems. Borosilicate glass capillaries were used as spray capillaries with one end drawn to result in a spray orifice of approximately 1-10 µm. Electrical contact was established via a gold-coating on the spray end of the capillary. It was possible to generate electrospray using lower voltages than with conventional ESI. The reduced flow rate and the narrow needle dimensions resulted in initial droplets of approximately one magnitude smaller size compared to droplets generated utilizing conventional ESI, i.e. a droplet size in the nm range. The generation of smaller initial droplets leads to less coulombic fissions before the droplets are desolvated and the spray needle can be positioned closer to the inlet orifice of the MS. Despite the lower applied voltages and the lower mass flow per time unit, more intense signals are observed.51, 52 Furthermore, nanoelectrospray shows a higher tolerance towards salt contaminations and facilitates stable spraying of solvents with high surface tensions, such as water. The invention of nanoelectrospray has extended the applicability of ESI in analytical MS in a significant way.46

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The next milestone in the history of electrospray emitters was when microfabrication techniques were utilized for the fabrication. With the miniaturized ESI emitters, the importance of precision in the fabrication increased, since possible dead volumes become more significant and spray tip variations can influence the appearance of the mass spectrum.53 With microfabrication technologies it is possible to fabricate highly reproducible structures with sub-micrometer precision. Furthermore, the flow rates typically obtained in micromachined channels are on the order of low nL/min, which is compatible with nESI.53 Micromachining technologies can be utilized for many different materials, such as glass or silicon. The material for an electrospray emitter must be carefully chosen to minimize possible sample adsorption on the microdevice channel surfaces, which in general have large surface-to-volume ratios. Furthermore, the material must provide sufficient chemical stability to avoid the formation of analyte adducts from the microdevice itself. Early ESI microdevices were glass chips with open end channels, where electrospray was generated from blunt ends.54, 55 However, hydrophilic chips with blunt ends resulted in uncontrolled spray cones both in terms of size and position along the microchip end. Attempts were made to solve this problem by means of hydrophobic treatments, which were successful to some extent.54-56 Another approach was to insert fused silica capillaries or electrospray needles into the holes of microchannels. This approach was presented almost simultaneously by a number of groups57-60, and is still utilized. However, the attachment of capillaries into microchips is a rather time-consuming and difficult to automate. Furthermore, the interfacing should not result in any dead volumes or contamination.53 Therefore, several groups have fabricated microchips with integrated pointed electrospray emitters in order to simplify the fabrication, reduce potential sources of error and facilitate mass production. Microfabrication technologies employed in the fabrication of electrospray emitters from silicon substrates have the potential of high precision fabrication in automated batch processes. Silicon nozzles in arrays and matrices for high-throughput analysis were fabricated by Schultz et al.61 This concept is now widely used in a commercialized emitter manufactured by Advion BioSystems. Furthermore, thin-walled nozzle arrays fabricated in silicon and silicon dioxide have been described by Sjödahl et al.62 A drawback of silicon emitters is the need for an advanced and expensive facility for the fabrication. This is one of the main reasons for the extensive use of other materials, such as polymers, in emitter fabrication. Polymers have the advantage of low cost. Moreover, the wide range of polymer materials combined with many techniques for modification of materials has made polymers become the obvious choice for many groups. A hollow parylene nozzle extending from a silicon substrate was fabricated by Licklider et al. in an attempt to present reliable electrospray nozzles by a simple batch procedure.63 In another approach, microchannels were fabricated by plasma etching of polyimide and sealed with polyethylene terephthalate (PET) and cut to a pointed electrospray tip.64 Polyimide is nowadays also used in a commercial chip-based emitter manufactured by Agilent Technologies. Both polycarbonate and


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polymethylmethacrylate (PMMA) have been used in electrospray emitters where the nozzles have been mechanically grinded and polished to its pointed structure.65-68 Nevertheless, mechanical treatment of the material can result in small particles that possibly can disturb the electrospray or clog the emitter. Polydimethylsiloxane (PDMS) is suitable for replication by casting, and has been used in two emitter designs. In both emitters a microchannel was fabricated and subsequently manually cut to its final pointed shape.69, 70 Ageing of the PDMS and lack of long-term stability can however not be ruled out.70, 71 Sharp pointed emitters have also been manufactured from the negative photo resist SU-8 with on-chip CE separations interfaced with ESI-MS.72, 73 An interesting concept was presented by Le Gac et al., who based their emitter design on the principle action of a fountain pen. They fabricated a reservoir for the sample solution, which was coupled to a slot in which the sample was transported by capillary and electrostatic forces to the exit of a pointed nib, where the electrospray was generated. Based on this design, nibs have been fabricated in both the negative photo resist SU-8 and in silicon.74, 75

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6 NEW CONCEPT OF ELECTROSPRAY FROM AN ADJUSTABLE GAP

Drawn fused silica needles, fabricated with similar techniques as presented by Wilm and Mann51 are today still extensively used. They are simple and inexpensive to fabricate, but the fabrication often results in needles that lack in precision and reproducibility of the dimensions. Such needles are sometimes blocked and need to be opened by means of breaking the needle tip. Tapered fused silica needles with carefully controlled dimensions are also commercially available. Such needles are more reproducible but are also more expensive. Furthermore, obtaining durable conductive coatings of the electrospray needles has not been straightforward and intensive research on this subject has been performed.76 As outlined in the previous section, a large number of electrospray emitters fabricated with micromachining technologies have also been proposed. However, many of the proposed designs are basically only novel in terms of using a new material or fabrication procedure compared to previous designs. One fundamental characteristic that is common for all of the contemporary emitters is that the dimensions of the spray nozzles are fixed. Emitters with fixed dimensions of the orifice have a number of drawbacks. First, the risk of clogging of the needle is significant for nozzles with narrow dimensions. Nozzles with fixed dimensions present almost no possibilities to circumvent such clogging. Clogged nozzles may have to be discarded with loss of an emitter and valuable sample as a consequence. Second, when needles with fixed dimensions are utilized, optimization of the strength and stability of the analyte signal is limited to adjustments in voltage and distance from the MS counter electrode. Finally, emitters with fixed dimensions inevitably have a limited applicable flow range. The work comprised in this thesis presents the fabrication and analytical evaluation of electrospray emitters that utilize a new concept, where the electrospray orifice has a variable size. The basic idea is to form a narrow gap, in the micrometer range, between the edges of two thin substrates and fill the gap with sample solution by capillary forces, while the electrospray is generated from the end point of the gap. The area from which the electrospray is generated is defined by the height and the width of the gap and by adjusting the gap width, the area of the spray orifice is varied. For optimal filling of the sample solution into the gap, a high aspect ratio-gap (height/width) with smooth and hydrophilic gap walls should be employed. Spontaneous imbibition is obtained when

whw θcos⋅≤ [Eq. 9] where w is the gap width, h is the height of the gap wall and θw is the contact angle between the gap wall and the sample solution.77 The relationship is valid assuming that gravity effects can be neglected and for smooth gap walls. To enhance the confinement of the sample solution to the gap, the surrounding surfaces should be hydrophobic. The spray point needs to be sharp and pointed to confine the spray cone to the area between the gap walls. A pointed shape of the emitter provides a higher electrical field strength for a given applied electrospray voltage than in the case of a blunt end.


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6.1 Adjustable Gap Electrospray from PET-Tips In Paper I an ESI emitter fabricated in polyethylene terephthalate (PET) is presented.

6.1.1 Fabrication

For the fabrication of the emitter, a 36 µm thick PET-film was used. A number of polymers were tested, and PET was chosen since initial tests showed that the PET-film was rigid enough to allow for fabrication of tips that could protrude a few millimeters from a support material without uncontrolled deflecting. Additionally, no significant background signals were observed. Briefly the fabrication procedure was as follows (Figure 3):

1. A rectangular piece of PET-film was cut out from a larger film, using a micro-scalpel. In this cutting it was important that one of the sides had a sharp 90° edge. (In initial experiments the next and final step was step 5.)

2. Then, the top and bottom planar surfaces of the PET-

film piece were covered in a clamp (the dark grey PMMA-plates), with the edges of the PET-film uncovered (light grey).

3. This device was then exposed to a hydrophilization

treatment by means of oxygen plasma etching78 or deposition of silicon dioxide79. The incorporated hydrophilic compounds are represented by the black dots.

4. Only the outer edges of the PET-film were hydrophilized,

due to the covering of the top and bottom surfaces by the clamps, which were then removed.

5. The treated PET-film was cut along X1 and the sides Y

and Z, which resulted in a tip with triangular shape.

6. Side X was thus hydrophilic while all other surfaces were hydrophobic, due to the hydrophobic properties of the polymer.

Figure 3: Process flow of the PET-tip fabrication.

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In the initial experiments with the PET-tips, the gap wall edges were untreated. However, the hydrophobic nature of the gap walls resulted in poor wetting. Therefore, a surface treatment with oxygen plasma etching was carried out (Figure 4). In the hydrophilization process, reactive oxygen species are incorporated onto the surface of the PET-film in a plasma oven.78 The contact angle between sample solutions and the PET-surface decreased to roughly 40% compared to the contact angles for native PET. This improved the imbibition of the gap significantly. However, the long-term storage stability was rather poor and therefore another hydrophilization treatment was employed. New tips were fabricated with a thin layer of silicon dioxide deposited on the gap wall surface using a similar hydrophilization process as for the oxygen plasma, but with the distinction that a mixture of oxygen and hexamethyldisiloxane (HMDSO) was added in the plasma oven.79 The resulting SiO2-treated tips could be stored for several weeks without any noticeable loss of performance.

Figure 4: Schematic of the plasma oven, in which the surface treatment process was performed.

The essential feature of the fabrication is that an optimized imbibition of the gap is obtained due to the hydrophilic treatment, while maintaining hydrophobic surfaces of the surrounding parts of the gap. This maximizes the confinement of the liquid to the gap. This straightforward approach results in a selective surface treatment.


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6.1.2 Setup

The PET-tips were glued on supporting microscope glass slides, with the tips protruding the edges of the glass slides, which were mounted on translation stages (Figure 5). The tips were aligned with the thin 90° sharp hydrophilic edges opposing each other, to form the gap. The sample solution was applied via a liquid bridge from a capillary connected to a syringe pump. The sample solution was electrically grounded via a stainless steel union.

Figure 5: Schematic, showing two PET-tips mounted in the setup for adjustable gap electrospray generation. The enlarged insert shows the gap and the spray point between the two PET-tips. (Paper I)

6.1.3 Initial experiments

In initial experiments with the surface treated PET-tips, a bench-top setup was constructed where two oxygen-plasma-treated tips were mounted as shown in Figure 5. The tips were facing a copper wire, serving as a counter electrode, instead of facing the MS inlet counter electrode (as in Figure 5). Electrospray was generated at the end point of the gap when negative high voltage was applied to the copper wire. A sample solution of 50:50 (v/v) water:methanol was employed. Using the translation stages the width of the gap could be varied, thus varying the spray orifice size. In Figure 6 photographs of the electrospray from three different gap widths are shown. From this experiment it was evident that electrospray generation from an adjustable gap between the PET-tips was feasible.

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Figure 6: Electrospray generated from the PET-tips utilizing three different gap widths; (a) 20 µm, (b) 10 µm and (c) 5 µm. (Paper I)

6.1.4 ESI-MS experiments

Further evaluations of the PET-tips were carried out with SiO2-treated tips mounted in front of the MS. In MS experiments, using a sample solution of 10 µM angiotensin I, the gap width was gradually increased while monitoring the peptide signal. The total signal-to-noise ratio (S/N) was calculated as the sum of the S/N ratios for singly, doubly and triply charged angiotensin I ions. When the gap width was gradually increased from 1 to 36 µm, a decrease of the total S/N ratio was observed (Figure 7). This was expected since the generation of an electrospray from a larger spray orifice should result in larger initial droplet size, leading to a lower sensitivity and ionization efficiency.51 The observed trend is somewhat diverted, when observing the results obtained at a gap width of 16 µm. This could be due to a slight change in behavior of the spray, which is dependent on orifice size, voltage and flow rate among other conditions.80, 81 With more accurate current measuring instruments and high-speed cameras, it should be possible to investigate this further.

Figure 7: Total signal-to-noise ratio of angiotensin I ions obtained with different gap widths. Sample solution: angiotensin I (10 µM) in 50:50 (v/v) methanol:1mM formic acid in water. (Paper I)


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Furthermore, from the spectra generated for each gap width, it could be observed that the charge state distribution of the multicharged peptide ions varied. A weighted average charge state (ACS) was calculated from each spectra according to:

∑∑= iii IINACS [Eq. 10]

where iN is the number of charges of the peptide corresponding to peak i, and iI is the absolute intensity of peak i. For increased gap widths, a shift towards lower charge states was observed (Figure 8). However, it should be noted that the flow rate was increased when increasing the gap width, which also might have affected the ACS and the total S/N ratio.51, 82 It has also previously been shown for electrospray generated with nESI needles, that the orifice size influenced the appearance of the mass spectra.82

Figure 8: Weighted average charge state calculated from spectra generated with different gap widths. Sample solution: angiotensin I (10 µM) in 50:50 (v/v) methanol:1mM formic acid in water. (Paper I)

To compare the analytical performance of the adjustable gap emitter with commercially available high-quality nESI needles, two separate experiments were performed where the values of the operating parameters were kept constant. The spray orifice area of a 2 µm gap width was roughly equal to the orifice area of a 10 µm ID nESI needle. Equal flow rates were applied to the two systems and the distance from the spray point to the counter electrode was also equal. The ACS of the two spectra were roughly equal but a higher S/N ratio was observed for the data obtained from the adjustable gap emitter compared to the data obtained with the nESI needle (Figure 9).

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Figure 9: ESI mass spectra of 10 µM angiotensin I obtained with (a) PET-tips with a 2 µm gap width and (b) a 10 µm ID nESI needle. (Paper I)

This led us to suggest that the increased S/N ratio was due to a concentrating effect of the sample solution resulting from an evaporation of the solvent in the open configuration of the liquid bridge and the adjustable gap (Figure 10).

Figure 10: Liquid bridge interface between the sample capillary and the gap between the PET-tips. The possible evaporation is shown with arrows.

In an off-line experiment, the interface of the liquid bridge was mimicked. Two syringes were aligned in juxtaposition to form a gap (Figure 11). A sample solution flow was applied from one syringe to the gap, while draining the liquid from the gap by suction with the other syringe. When balance between the two flows is obtained, and the shape of a liquid bridge is stable, the difference between the applied flow rate and suction flow rate is accounted for by the evaporation rate.


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Figure 11: Photograph of the evaporation experiment in which two syringe needles are positioned in juxtaposition with a liquid bridge of sample solution in between. (Paper I)

For a sample solution of 50:50 (v/v) water:methanol and experimental conditions equivalent to those employed in the MS experiments of Figure 9, an evaporation of approximately 40% of the solvent was observed. This means that the electrosprayed flow rate is lower than the originally applied flow rate from the syringe pump. This should therefore result in an increased S/N ratio. However, due to the different volatilities of water and methanol, the evaporation should also change the composition of the electrosprayed sample solution. By injecting the content of the suction syringe into a gas chromatograph it could be measured that the content of the sample solution was 60:40 (v/v) water:methanol, compared to 50:50 (v/v) original content (Figure 12). Electrospraying such a sample solution, which has a higher water content and consequently higher surface tension of the generated droplets, could possibly counteract the effect of increased S/N ratios. If the evaporation could be controlled in a reproducible way, the liquid bridge interface could offer an interesting on-line concentrating that possibly might increase the sensitivity further. Controlled evaporation could perhaps be accomplished in a closed environment with accurately regulated conditions of the entrapped gas volume.

Figure 12: Gas chromatogram showing to the left; the original 50:50 water:methanol content and to the right; the 60:40 water:methanol content after evaporation of the solvent at the liquid bridge interface.

The conclusion of the experiments carried out with electrospray generated from an adjustable gap between two PET-tips is that it was possible to generate electrospray from one and the same emitter with different spray orifice sizes for different flow rates. The tips were inexpensive and their fabrication was based on a straightforward procedure. However, the manual cutting of the PET-tips resulted in a limited precision of the tips. Multiple cuttings were occasionally needed before satisfactory tips were obtained.

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Although an ultra fine micro knife of carbon steel was used, cutting the polymer sometimes resulted in a disrupted tip shape. Figure 13 shows micrographs of two PET-tips, both in need for a repeated cutting. A logical development was to improve the emitter fabrication. It was obvious that an automated microfabrication process would have the best potential for manufacturing more precise and reproducible emitters.

Figure 13: Scanning electron microscopy images showing PET-tips cut with a scalpel. Both tips are in need for further cutting before satisfactory tips are obtained.


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6.2 Adjustable Gap Electrospray from Silicon Chips It is well known that micromachining technologies offer excellent precision and batch fabrication possibilities of microdevices. Therefore silicon micromachining technologies were utilized to fabricate reproducible high precision electrospray chips. In Paper II, the fabrication and analytical evaluation of chips employed for generation of electrospray from an adjustable gap is shown. The main focus in the fabrication process was to improve the sharpness of the spray point as well as the smoothness of the gap walls compared to the PET-tips.

6.2.1 Fabrication

For the design of the new electrospray chips, the general requirements outlined in the beginning of Section 6 were taken in consideration. The chips should be sufficiently large to be handled and mounted in the electrospray setup. The chips should include a thin protruding substrate that could be mounted to form a narrow gap. Furthermore, the thin substrate should have a very sharp pointed end and a smooth gap wall surface. The starting material used in the fabrication was a silicon wafer with the crystal planes in the orientation of <100>. When such a material is anisotropically etched, using KOH, it forms perfectly smooth tilted etched walls of 54.7 degrees angles. From the bottom side of the silicon wafer such tilted grooves were etched. Each groove defined the front side of one chip and the back side of another chip. The top side of the wafer was patterned in two steps. The first photo resist patterning and plasma etching defined a thin beam consisting of a layer of silicon oxide and silicon nitride. The second patterning and deep reactive ion etching (DRIE) outlined the outer dimensions of the chip structure from the wafer bulk. Then the second mask was removed, which revealed a silicon oxide and silicon nitride beam in the shape as defined by the first patterning. A second DRIE process etched down the entire chip apart from this thin beam. This resulted in a through-etched chip with a high and thin elevated beam centred at each chip. Each wafer contained 630 chips. Further details of the fabrication process are provided in Paper II. In the initial fabrication procedure, the chips were individually separated from the wafer in a dicing-step. However, particles generated during the dicing can remain on the chip surface, even after extensive washing. Clogging with particulate matter during electrospray is then an obvious risk. Other groups have experienced problems with clogging and have proposed solutions to these. For example, etching of the fused silica capillaries without tapering the internal dimensions have been proposed, or to incorporate frits near the capillary outlet.83, 84 The adjustable gap emitter provides a unique opportunity to rectify clogging simply by widening the gap and release the particulate matter. But to reduce the potential risk of disturbance of the electrospray due to adhered silica particles, a second fabrication process generation was developed, where the process did not involve any dicing. The chips were attached to the wafer via a hinge at one of the sides of the chip bulk material. The hinge could easily be snapped off,

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giving an electrospray chip ready for mounting. Figure 14 shows a section of the wafer comprising five chips, each attached to the wafer by the hinge.

Figure 14: A photograph of five electrospray chips attached to the wafer by a hinges.

In Figure 15 scanning electron microscopy (SEM) images of an electrospray chip are shown. The thin and high aspect ratio-beam has a pointed front end and a blunt back end. The pointed end of the tilted etched wall constitutes the spray point while the blunt back end facilitates the sample application from a capillary to the gap. An important feature is that the spray point of the beam is formed between the intersecting crystal planes of the <100> silicon, which results in an extremely sharp edge. The sharpness is highly reproducible in the fabrication process, since it is based on the inherent crystalline properties of the silicon. In the event of over-etching in the processing of the silicon, the spray point still remains sharp. Although the sides of the beams are a somewhat rough due to the scallops formed during the DRIE etching (Figure 16), the gap wall surface is the perfectly smooth, originally polished top surface of the silicon wafer. The precision of the gap wall surfaces allows for an alignment of the chips to form a gap of perfectly uniform dimensions and facilitates an optimized imbibition.

Figure 15: SEM images showing a silicon electrospray chip. A tilted top view to the left, a side view to the upper right and a close-up of the sharp spray edge to the lower right. (Paper II, modified)


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Figure 16: SEM image showing the back end of a beam. The DRIE-process results in scallops (wave-like structures) due to the sequential blocking and etching.

6.2.2 Setup

The bottom surface of a chip was glued onto the long edge of a pointed microscope glass slide. The SiO2 surfaces of the chip are hydrophilic. Therefore, all other surfaces of the chip, apart from the gap wall surface were subjected to a hydrophobic surface treatment to create conditions which confine the liquid to the gap. The surfaces were treated with the hydrophobizing agent GlassClad 18. After the surface treatment, two chips were mounted in juxtaposition, aligned to form a narrow gap between the hydrophilic top edges of the beams (Figure 17), in a similar setup as for the PET-tips (Figure 5). Further details are given in Paper II. This device was then aligned in front of the MS inlet. The sample solution was fed to the rear end of the gap from a capillary with a conically shaped end (Figure 18). The use of a conical outlet end of the capillary resulted in a more narrow liquid bridge than that obtained with a capillary with a blunt end (as in the setup with the PET-tips). This might affect the evaporation since a different surface-to-volume ratio can be obtained. However, this has not yet been investigated further.

Figure 17: Schematic, showing two chips aligned to form a gap between the beam edges.

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Figure 18: Sample solution applied to the gap via a liquid bridge from a sample capillary. The sample solution is electrically grounded via the stainless steel union.

6.2.3 ESI-MS experiments

When sample solution was fed to the rear end of the gap, a liquid bridge was immediately formed between the outlet end of the capillary and the gap. The liquid spontaneously filled the gap and electrospray was generated as soon as the high voltage was applied to the counter electrode. The sample solution was electrically grounded via a stainless steel union connected to the capillaries as shown in Figure 18. For a given flow rate, the signal strength and stability of the analyte ions could be optimized by varying the gap width, the voltage and the distance from the spray point to the MS inlet. Gap widths could be gradually increased from 1 µm to 25 µm, without any interruption of the electrospray. In an experiment where a sample solution containing six peptides was continuously fed, using two different gap widths, the S/N ratio and ACS followed the same trends as for the experiments with the PET-tips. As has been described, the need for high sensitivity is an essential issue. To verify the performance of the new electrospray emitter, the silicon chips were continuously fed with a 10 nM sample solution of the peptide Insulin Chain B (oxidized). During the electrospray experiment, a clear and continuous signal from the triply charged peptide ion was observed (Figure 19). The relative detection limit calculated for a S/N ratio of 3 was 4 nM. This limit of detection is approximately one order of magnitude better than that obtained with the PET-tips, and compares well with the state-of-the-art.64, 66, 67, 69

Figure 19: Mass spectrum of a sample solution of 10 nM Insulin Chain B (oxidized) in 49.5:50:0.5 (v/v) water:methanol:formic acid. (Paper II)


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6.2.4 CE-MS experiments

Since the liquid bridge interface proved to be a straightforward way of applying the sample solution from a capillary to the gap, it seemed reasonable to anticipate that the adjustable gap chips could be interfaced with a separation capillary in the same way. The advantages of CE separations combined with ESI-MS detection have already been mentioned in Section 4.2. The liquid bridge and the adjustable gap chips operate in an open configuration. Therefore, to accomplish a CE-ESI-MS interface, only an electrical connection between the two electrically driven flow systems was required. We constructed the electrical connection interface by sputtering a layer of Au/Pd onto the conical outlet end of a CE capillary (Figure 20). A conductive silver adhesive was applied onto the Au/Pd layer to ensure a durable electrical connection. This is a slightly modified procedure compared to the methods which has been previously presented.42 Electrical ground was connected to this interface, both for the ESI and the CE processes. Grounding the ESI emitter is the most robust way of circumventing any interferences between the CE current (which is in the µA range) and the MS current (which is in the nA range).34 In this open interface, the use of a conical outlet end of the capillary is crucial to reduce the risk of introducing dead volumes and extra column bandbroadening of the separation. Another common source of bandbroadening is adsorption of the analyte ions to the separation capillary wall. For the positively charged peptides in the acidic solutions, which were employed in this study, adsorption will be excessive on the negatively charged capillary wall. However, this can be rectified by imparting a positive charge to the capillary wall. This was accomplished by a pre-treatment of the capillary with the cationic polymer Polybrene. The cationic polymer binds to the negatively charged wall surface and the sample solution in the capillary therefore experiences a positive net charge of the capillary wall. The Polybrene treatment results in a reversed EOF compared to the EOF obtained using a bare capillary wall surface. Both the inlet of the CE and the counter electrode of the MS therefore operated at negative voltages. The outlet end of the CE capillary was interfaced to the rear end of a pair of electrospray chips. Once the liquid bridge had been established after applying high voltage to the CE inlet, the flow of CE eluent through the gap was balanced by applying the electrospray voltage and adjusting the gap width. The balance between the two electrically driven liquid systems is monitored by the shape and size of the liquid bridge. A sample solution of six peptides was analyzed using this configuration. In Figure 21, extracted ion electropherograms for the different peptides are shown together with corresponding mass spectra.

Figure 20: CE eluent applied to the gap via a liquid bridge from a conductive capillary outlet.

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Figure 21: Extracted ion electropherograms and corresponding mass spectra for six peptides analyzed with CE-MS. (Paper II)

In conclusion it can be stated that it was feasible to generate an electrospray from a gap between two silicon chips. The gap width is adjusted to optimize the analyte signal for an applied flow rate. Even more, the adjustable gap chips have been interfaced with capillary electrophoresis via a liquid bridge from the CE capillary.


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7 CONCLUSIONS AND FUTURE PERSPECTIVES In this thesis a conceptually new idea of generating an electrospray from a variable spray orifice size has been presented. It has been shown that electrospray emitters could be fabricated either by a manual procedure, or by means of micromachining using a batch process. A stable nanoelectrospray has been generated for different gap widths and different applied flow rates. The electrospray performance has been optimized during operation by varying the width of the gap. For the two emitter designs an improved sample solution imbibition and sample confinement to the gap has been obtained by surface treatments. The analytical performance of the adjustable gap emitters is comparable with the results obtained with commercial nESI emitters. The open interface between the gap and a sample capillary via a liquid bridge is straightforward and allows for interfacing of one and the same emitter with different capillaries. The adjustable gap has been interfaced with a CE capillary and it is reasonable to assume that other separation columns, such as liquid chromatography columns, could be interfaced. This would be very interesting to investigate since the demand for flexible ESI emitters is vast and LC-MS is an important analytical technique.85 A strong urge in ESI research is to further enhance the sensitivity. Therefore, it would be appealing to further decrease the size of the electrospray. With the precise fabrication process utilized for the silicon chips it should be possible to reduce the width of the beam and by employing more accurate translation stages it should be possible to align chips with a controllable sub-micrometer gap width. It is likely that this would reduce the size of the electrospray and generate smaller droplets, conceivably in the low nanometer range. Electrospray generated from pulled fused silica needles of sub-micrometer diameters with flow rates operating in the pL/min range have previously been accomplished.86 Furthermore, a slot in the nanometer range has previously been fabricated for electrospray generation.87, 88 However, no MS data have been shown using this emitter, which might be due to particular problems, when using such small dimensions. For emitters with sub-micrometer orifice dimensions, clogging is an obvious risk.86 Electrospray generated from an adjustable gap operating at these dimensions would of course also run the risk of clogging problems, but this should easily be remedied by widening the gap for a short while without loss of sample or the whole emitter. It would probably be advantageous to align such emitters closer to the MS inlet hole to increase the efficiency.46 Although the onset voltage for the electrospray generation likely would be lower for an emitter operating at pL/min flow rates, the risk of possible electrical discharges when approaching the counter electrode of the MS must be considered. Therefore, it might be beneficial to fabricate emitters with minimum surrounding bulk materials, to reduce possible shielding of the electric field. To implement a picospray device, imaging possibilities and setup must be further enhanced.

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To reduce the use of unions and minimize the time for sputtering of the CE capillaries, a simplified and improved electrical connection at the CE-MS interface would be advantageous. This could perhaps be possible by using a doped part of the chips as an inherent electrode. It would also be desirable to increase the throughput of analyses performed with the adjustable gap emitters. This could be enhanced by the use of an automated setup where the chips are mounted in a static holder, keeping the chips in exact position at all times, only allowing a variation of the gap width. Furthermore, the optimization of the signal could perhaps be simplified by creating a feedback loop for example between the electrospray current and the gap width.


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8 ACKNOWLEDGEMENTS I would like to express my gratitude to my supervisors; Professor Johan Roeraade and Associate professor Åsa Emmer for assistance and guidance in my work. A special thanks to Johan Sjödahl for inspiring discussions and important help during my initial time as a Ph D student. Also, I send my appreciation to my colleagues; Johan Jacksén, Johan Pettersson Redeby, Jonas Bonn, Mårten Stjernström, Catharina Silfwerbrand-Lindh, Henning Wiberg, Musie Araia and of course to former colleagues at Analytical Chemistry; Martin, Alf, Fredrik, Lisa among others. Moreover, I would like to thank Tommy Schönberg at Acreo AB. Huge thanks to my family for endless support and to my friends for making my spare time so valuable! For financial support I would like to thank the Swedish Science Council as well as the Nanochemistry program, funded by the Swedish Foundation for Strategic Research.

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9 ABBREVIATIONS

ACS Average charge state API Atmospheric pressure ionization CE Capillary electrophoresis CRM Charged residue model CZE Capillary zone electrophoresis DRIE Deep reactive ion etching EOF Electroosmotic flow ESI Electrospray ionization HMDSO Hexamethyldisiloxane ID Internal diameter IEM Ion evaporation model m/z Mass-to-charge ratio MALDI Matrix-assisted laser desorption/ionization MS Mass spectrometry nESI Nanoelectrospray PDMS Polydimethylsiloxane PET Polyethylene terephthalate PMMA Polymethylmethacrylate QIT Quadrupole ion trap RF Radio frequency S/N Signal-to-noise ratio SEM Scanning electron microscopy v/v Volume-to-volume ratio


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