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Gas-Phase Ions Produced by Freezing Water or Methanol for Analysis using Mass Spectrometry Vincent S. Pagnotti, Shubhashis Chakrabarty, Beixi Wang, Sarah Trimpin, and Charles N McEwen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac500132j • Publication Date (Web): 26 Jun 2014 Downloaded from http://pubs.acs.org on July 4, 2014
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Analytical Chemistry
Gas-Phase Ions Produced by Freezing Water or Methanol for Analysis using Mass Spectrometry Vincent S. Pagnotti,1+ Shubhashis Chakrabarty,1+ Beixi Wang,2 Sarah Trimpin,2 Charles N. McEwen*1 1
University of the Science, Department of Chemistry and Biochemistry, Philadelphia, PA 2
Wayne State University, Department of Chemistry, Detroit, MI
Abstract Introducing water or methanol containing a low concentration of volatile or nonvolatile analyte into an inlet tube cooled with dry ice linking atmospheric pressure and the first vacuum stage of a mass spectrometer produces gas-phase ions even of small proteins that can be detected by mass spectrometry. Collision induced dissociation experiments conducted in the first vacuum region of the mass spectrometer are consistent with analyte ions being protected by a solvent cage. The charges may be produced by processes similar to those proposed for charge separation under freezing conditions in thunderclouds. By this process, the surface of an ice pellet is charged positive and the interior negative so that removal of surface results in charge separation. A reversal of surface charge is expected for a heated droplet surface, and this is observed by heating rather than cooling the inlet tube. These observations are consistent with charged supercooled droplets or ice particles as intermediates in the production of analyte ions under freezing conditions. Keywords: Mass spectrometry, freezing water, ionization, lightning initiation, cold ionization, inlet ionization, peptides
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Introduction Mass spectrometry (MS) has greatly benefited from charged solvent droplets which provide a vehicle for transferring nonvolatile compounds such as proteins into the gas phase as ions for mass analysis.1,2 Electrospray ionization (ESI), for example, produces charged droplets by subjecting solution exiting a capillary to a high electric field.3
Pneumatic methods of
producing charged droplets such as aerosol formation,4 or using a high velocity nebulizing gas in sonic spray ionization,5 and even applying an electric field to levitated droplets6-8 can also produce ions for analysis in MS. Obstructions placed in the path of high velocity droplets9,10 and particles11 have been shown to increase ion abundance. Thermal methods such as passing a solution through a heated tube at atmospheric pressure,12 through a tube linking atmospheric pressure and the first vacuum stage of a mass spectrometer,9 or through a hot tube directly into vacuum13 produces ions for MS analysis, potentially from charged droplets or particles. Similar mass spectra were obtained without a voltage by introducing solid small molecule matrices containing analyte into a heated mass spectrometer inlet tube,14 or by laser ablation of matrix:analyte in vacuum.15
These methods are also proposed to involve highly charged
particles or matrix droplets as intermediates. The size distributions of particles produced upon laser ablation or mechanical shock have been measured.16-18 It has been proposed that matrixassisted laser desorption/ionization (MALDI) involves charged matrix:analyte clusters.19-23 Mechanisms for bare ion formation from charged solvent droplets were proposed by Dole24 and by Iribarne and Thomson.4 The ion evaporation model described by Iribarne and Thompson is generally accepted to be the operational mechanism for observing bare ions from small analyte molecules in ESI-MS, while the residue model by Dole is believed responsible for observation of larger gas-phase ions.25 In either model, charged droplets undergoing solvent evaporation will concentrate surface charge, and when the Rayleigh limit is reached, coulomb fission releases prodigy droplets having ca. 2% of the parent droplet mass and ca. 15% of its 2 ACS Paragon Plus Environment
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charge.26-28 Recently, Konermann proposed a modification of the ion evaporation model for conversion of denatured proteins from solution to the gas phase.29 These mechanisms have also been suggested to be responsible for multiply charged ions in solvent assisted ionization inlet (SAII) MS.9
Abundant analyte ions, even of nonvolatile large molecules, have been produced in SAII by simply introducing solvent, including water, containing analyte into the intermediate pressure of a heated inlet tube linking atmospheric pressure and the first vacuum region of a mass analyzer. One hypothesis for ion production in a heated inlet tube is that a thermal gradient between the surface and the interior of solvent droplets induces a charge gradient within the droplets.20 The gradient has been suggested to be the result of autoprotolysis equilibrium (2H2O ↔ H3O+ + OH-) being shifted to the charged products at increased temperature and to the faster diffusion of the proton relative to the hydroxide ion.30-32 Charged prodigy droplets have been proposed to be produced from the parent droplet by removal of surface charge as occurs in bubble bursting in liquid water.33,34 Bubble formation has been implicated as a means of charge separation in warming supercooled water droplets.35,36 An indication that bubbles are important in ion formation in SAII was obtained by carbonation of the solution resulting in increased ion abundance.37 Recently, it was shown that matrix assisted ionization inlet (MAII),14 which requires a heated inlet tube, can be extended to matrix assisted ionization vacuum (MAIV),38-40 in which a solid matrix such as 3-nitrobenzonitrile (3-NBN) produces bare gas-phase ions from compounds as large as the 66 kDa bovine serum albumin protein for analysis by MS by simply placing the solid matrix:analyte sample into vacuum. With 3-NBN as matrix, abundant analyte ions with similar charge states to ESI and MAII are produced.38 Without heat, ions must be generated from the solid matrix rather than a liquid droplet as in ESI. Sublimation in combination with matrix fracturing is proposed to produce gas-phase charged matrix:analyte particles, possibly by 3 ACS Paragon Plus Environment
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the same process that causes 3-NBN to triboluminescence.41 In triboluminescence, sometimes called fractoluminescence, crystal cracking is believed to cause charge separation at the separated surfaces.41,42 The gas-phase charged particles splintered from the matrix surface then release bare analyte ions upon matrix sublimation. The experiments with 3-NBN along with the fact that ice is implicated in charge generation in thunderclouds43 and is known to triboluminescence42 convinced us to look for the similar process for SAII; freezing solvent as a means of ion formation for use in MS. Charged ice particles have been produced by collisions in the laboratory44 and observed visually,45 as well as photographed spontaneously leaving the surface of freezing water droplets;46 apparently a result of a splintering process.46,47 Ice has been used in MS as a matrix in laser ablation,48,49 matrix-assisted laser desorption electrospray ionization,50 MALDI51,52 and secondary ion mass spectrometry.53-55 Cold ESI (coldspray) ionization of solvents with high organic content56 and with water solutions,57 as well as evaporation of liquid nitrogen as a nebulizing gas to produce ions from solution without use of a voltage58 have shown advantages relative to high temperature ionization. Lowering the ESI nebulizing gas temperature is effective in the analysis of DNA56,59 and small proteins preserving their native structure57. Under certain conditions using ESI, it is possible to produce frozen water droplets by evaporative cooling.60 Here we show results from cooling rather than heating the atmospheric pressure to vacuum MS inlet to produce analyte ions from water, water:methanol mixtures, and methanol passed through the inlet tube for analysis by MS. A cold process from which bare ions are produced might be analytically useful for analysis of thermally labile molecules and non-covalent complexes by MS, and has the potential to provide fundamental insights into ionization processes for nonvolatile compounds. To the best of our knowledge, this is the first example of producing ions for MS analysis simply by cooling solvents, including water. Experimental 4 ACS Paragon Plus Environment
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Materials: HPLC grade methanol, myoglobin (equine heart), lysozyme from (chicken egg white), ubiquitin (from bovine erythrocytes), insulin (bovine), 4-methoxybenzyl chloride and pyridine were obtained from Sigma Aldrich (St. Louis, MO). Angiotensin I, angiotensin II, and bradykinin were obtained from Anaspec Inc. (Freemont, CA). Tetra-butylammonium iodide was purchased from Lancaster Synthesis (Windham, NH) HPLC-grade water was purchased from EMD chemicals Inc. (Gibbstown, NJ). All compounds, solvents, and gases were used without further purification.
Rain water was collected directly into 20 mL glass vials placed over clean
aluminum foil at two locations during active thunderstorms.
A rural sample was collected
outside of Scranton, PA, and an urban sample was collected in Philadelphia, PA. 1-(4methoxybenzyl) pyridinium chloride was prepared by heating a solution containing dry pyridine and 4-methoxybenzyl chloride to 60 ºC for 3 hours. Instrumentation: Initial experiments were conducted using a homebuilt apparatus made with standard glass labware and diagrammed in Scheme 1. A rotary pump was used to evacuate the apparatus which had a 12 cm stainless steel tube (1mm OD X 0.6mm ID) linking atmospheric pressure and the evacuated region. This tube could be heated to as hot as 350 °C or cooled to as low as -78 °C.
An electrometer capable of measuring picoamps (Keithley Instruments,
Cleveland, OH) was used to monitor current on the inlet tube relative to a downstream electrode when solvent was passed through the inlet tube. In these experiments, both solvent injection (3 μL) into the inlet using a syringe and continuous solvent flow using the procedure shown in Scheme 1 were used. Continuous flow required a high percentage of methanol to prevent clogging the tube with ice. SAII was performed in the previously described manner using a Thermo Fisher Orbitrap Exactive mass spectrometer with the ion source removed.9 For cold ionization, this procedure was modified by replacing the commercial inlet tube with one that extended 2.5 inches external to the instrument (1mm OD X 0.6mm ID) allowing a section of the external portion to be cooled by direct application of dry ice while the interior portion of the inlet could be controlled with 5 ACS Paragon Plus Environment
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instrument software from 25 – 450 °C. The dual inlet tube provided instrument vacuum nearly identical to that provided by the commercial inlet. With the exception of the HCD collision gas which is always on, all gases to the ion source were set to zero as was the voltage applied to the ESI capillary. The temperature of the interior inlet was controlled by the ‘capillary temperature’ setting as noted in the text. All other voltages were those obtained by tuning using ESI (capillary 60 V, tube lens 75 V, skimmer 18 V. For fragmentation studies the ‘in-source CID’ voltage was adjusted from 0 V to 100 V as noted. An LTQ Velos (Thermo Scientific) was also used to perform cold ionization MS. The inlet of the mass spectrometer was fabricated to also have an external inlet extension. The exterior inlet tube was cooled with dry ice while the interior section of the tube was heated as noted in the text. All mass spectra were obtained in the positive ion mode. Sample solution (5 pmol μL-1) was introduced into the inlet using a Chemyx syringe pump (Stafford, TX) through fused silica tubing (220 μm OD X 180 μm ID), or a pipette (1 μL injected).
In all cases the solution was introduced at or very near the inlet tube opening.
Solution solvents were chosen based on whether or not freezing was desired. In continuous flow experiments, methanol was typically chosen to avoid clogging the inlet with frozen solution which occurs with high water content. Alternatively, sample could be introduced by freezing a water solution (2 µL) on dry ice and immediately holding the sample within 1 mm of the LTQ Velos inlet entrance aperture. The analyte solution became an ice ball when applied to the dry ice and continued to shrink during data acquisition. With this method, the external capillary was wrapped by ice bags to keep it cold, while the interior inlet was set to 40 °C (peptide) or 150 °C (protein). Results and Discussion In SAII,9 charge states nearly identical to those produced by ESI are obtained when solution is introduced into the heated inlet tube of a mass spectrometer without application of 6 ACS Paragon Plus Environment
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voltage. A potential mechanism for producing ESI-like charged droplets involves a thermal gradient within each droplet and separation of charge by selective removal of the surface, by for example, bubble formation.20 If a thermal gradient is responsible for charge separation using a heated inlet, then a cold inlet tube might also generate charge by this mechanism. A thermal gradient in freezing water droplets has been proposed as the source of charge generation in thunderstorms.43 The efficiency of the charge separation process can be gauged by the buildup of charge in thunderclouds, which in a matter of minutes produces numerous lightning strikes.61,62 One means of producing charge separation in thunderclouds is believed to be the result of separation of ice particles from the surfaces of freezing graupel (small hail pellets) having excess positive charge at the surface.43 When a portion of the surface is ejected during collisions with ice pellets,62,63 or by splintering,46,47 smaller positively charged ice particles are formed which are separated from the heavier now negatively charged graupel by the natural updraft.43
Bubbling caused by expelled air during freezing has also been proposed as a
mechanism for carrying away surface charge,36 similar to bubbling carrying away charge from boiling.1,20,34,64 In the thermal gradient mechanism, protons and hydroxyl ions produced by dissociation of water are more abundant under warm conditions (pKw = 14.167 at 20 °C and 12.432 at 90 °C)65, but protons diffuse more readily introducing an imbalance of charge.31,32 For this mechanism, the surface charge is expected to reverse in going from a hot to a cold inlet tube. Charge reversal has also been reported for water droplets believed to freeze under vacuum conditions.35 To test the charge reversal hypothesis, the homebuilt glass enclosure shown in Scheme 1 was used. A metal inlet tube linking atmospheric pressure and the vacuum region evacuated using a rotary pump can be cooled to as low as -78 °C (dry ice) or heated to 350 °C. A fused silica capillary delivers solvent to just inside a metal inlet tube, or solvent can be reproducibly injected into the tube using a syringe. The rapid flow of air from atmospheric pressure to vacuum through the inlet tube is believed to help nebulize the solvent into droplets 7 ACS Paragon Plus Environment
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which are either cooled or heated in traversing from atmospheric pressure to the evacuated region. Under these conditions, a metal electrode downstream of the inlet tube and connected to an electrometer registers current flow when charged droplets, or ions, strike the electrode. When the inlet tube is cooled to between -20 °C and -78 °C a significant temperature dependent (10-8 - 10-10 amps) negative charge is registered on the downstream electrode when water or methanol is introduced into the inlet and a positive charge is registered on the inlet tube demonstrating charge separation. On the other hand, if the inlet tube is heated, the readout reverses so that the downstream electrode reads positive and the inlet tube reads negative. These results are consistent with surface charge reversal. Because of the importance attributed to freezing droplets in charge generation in thunderclouds, numerous studies involving charge separation in water have previously been conducted.34,66-68 Studies under subliming conditions show ice particles have a cold surface and excess positive surface charge, which is also true for freezing water droplets, but water droplets at room temperature or warmer have a negative surface charge.43,69 Based on these studies, if solvent droplets are a source of charge, the inlet tube must register the droplet/particle surface charge and the droplet/particle bulk charge is measured downstream. Measuring the surface charge at the inlet tube is expected if charge transfer occurs from the particle/droplet surface upon a close encounter with the inner wall of the inlet tube. However, if charge separation occurs by, for example splintering off surface ice particles,46 aerodynamic breakup,34 or bubble formation,34 then the higher diffusion rates of these smaller charged droplets or particles from the surface relative to the larger parent droplet/particle is an alternative way to efficiently carry the surface charge to the walls of the inlet tube by diffusion.
This is in agreement with
experiments by Zilch, et al. in which solvent droplets produced by sonic spray and passing through a capillary were predominately positively charged, which was attributed to aerodynamic breakup of the droplet separating the surface negative charge in small prodigy droplets from the positively charged parent droplet.33 8 ACS Paragon Plus Environment
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Experiments similar to those described above using the apparatus of Scheme 1 were transferred to an Orbitrap Exactive mass spectrometer that has an inlet tube linking atmospheric pressure and the first vacuum stage of the mass analyzer which can be heated to create a thermal gradient within droplets passing through the tube. As has been reported, introducing water and other solvents into a hot inlet tube produces ions of dissolved analyte that are detected in a mass spectrometer as gas-phase bare analyte ions.9 The ions produced using a heated inlet tube are nearly identical in charge states to those produced by ESI.9,70 Few ions are observed by MS from analyte in water or water/organic solvent mixtures when the inlet is at or near room temperature.9 This is consistent with experiments with the apparatus of Scheme 1 in which little ion current (
