Spontaneous Charge Separation and Sublimation Processes are Ubiquitous in Nature and in Ionization Processes in Mass Spectrometry
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Ionization processes have been discovered by which small and large as well as volatile and nonvolatile compounds are converted to gas-phase ions when associated with a matrix and exposed to sub-atmospheric pressure. Here, we discuss experiments further defining these simple and unexpected processes. Charge separation is found to be a common process for small molecule chemicals, solids and liquids, passed through an inlet tube from a higher to a lower pressure region, with and without heat applied. This charge separation process produces positively- and negatively-charged particles with widely different efficiencies depending on the compound and its physical state. Circumstantial evidence is presented suggesting that in the new ionization process, charged particles carry analyte into the gas phase, and desolvation of these particles produce the bare ions similar to electrospray ionization, except that solid particles appear likely to be involved. This mechanistic proposition is in agreement with previous theoretical work related to ion emission from ice.
KeywordsSublimation/evaporation Spontaneous charge separation Temperature/pressure relationship Charged particles Microscopy
In matrix assisted ionization (MAI) mass spectrometry (MS) , typically analyte is mixed in low concentration with a high concentration solution of a specific matrix compound such as 3-nitrobenzonitrile (3-NBN) , or one of the more than 40 other matrix compounds reported [3, 4]. The solution is allowed to dry and then exposed to the sub-atmospheric pressure of the mass spectrometer, although drying is not a requirement as it will occur when the sample is introduced to sub-atmospheric pressure. Without the use of additional energy input such as a laser, high voltages, or heat, and without the need for nebulizing or desolvation gases, as little as a few femtomoles of analyte are sufficient to obtain excellent quality mass spectra with detection limits of a few attomoles [5, 6, 7]. All MAI matrix compounds visually sublime under the conditions of a successful experiment [3, 4]. When introduced directly from atmospheric pressure, the ionization event is observed for several seconds, with increasing time being associated with colder conditions. At intermediate and low pressure (high vacuum) at room temperature, the duration increases to minutes, and under some condition to more than 30 min, with increasing time associated with lower pressure and minimal gas flow across the sample, or use of more matrix [3, 8, 9, 10]. Regardless of the method of sample introduction, when a solid or solution matrix is employed, the charge states of analyte ions closely match those obtained by electrospray ionization (ESI) [11, 12]. The terminologies inlet ionization and vacuum ionization, without implying differences in mechanism, are used to differentiate whether the sample is introduced into the inlet directly from atmospheric pressure or inserted through a vacuum lock directly into the mass spectrometer.
It has been shown that a matrix can be either a solvent or a small molecule compound that is a solid at room temperature [13, 14]. More matrix compounds produce analyte ions when introduced to an inlet aperture from atmospheric pressure compared with direct insertion into intermediate pressure, even when both methods are at or near room temperature, which is believed to be the result of higher gas flow in the former [3, 4, 15]. Interestingly, it was shown that introducing neat sample into a heated inlet tube produces protonated molecular ions of the sample with minimal fragmentation without addition of a matrix . In this way, singly-charged ions of the drug levaquin and multiply-charged ions of the protein myoglobin were observed. Freezing water or methanol, and mixtures thereof, containing analyte within a cold inlet tube, or just outside using dry ice, also produces analyte ions with ESI-like charge states [4, 15, 17]. Analogously, organic small molecules that are liquids at room temperature produced ions after being cooled to the solid state .
A critical step in any ionization method is separating positive from negative charge carriers. The exceptional sensitivity observed with some of the matrix compounds from which spontaneous ionization is achieved  strongly suggests an efficient non-statistical charge separation process. One such non-statistical process results in luminescence upon surface fracturing and is referred to as triboluminescence or fractoluminescence [18, 19]. These processes are also known to occur with fluids, so called sonoluminsecence and hydroluminescence, sub-categories of triboluminescence [20, 21], and of a variety of other means and conditions [22, 23, 24, 25, 26, 27, 28, 29, 30, 31]. The light observed is due to a discharge across oppositely charged surfaces. A significant fraction of compounds (over 50%) are predicted to show triboluminescence . Both frozen water and frozen methanol are known triboluminescence compounds [32, 33]. Triboluminescence has been well studied at and above atmospheric pressure [18, 34, 35], but fewer studies describe this charge separation process at sub-atmospheric pressure [36, 37, 38]. An overarching consensus in the literature is that although charge separation is natural and simply produced, understanding the processes involved is limited due to its complexity of influencing parameters.
In the absence of a neutralizing discharge, crystal fracturing is a potential mechanism for charge separation in MAI. The first, and still the best, MAI matrix to produce analyte ions without application of thermal energy is 3-NBN. This and other matrices [3, 4] are known to produce a triboluminescence discharge when fractured . With 3-NBN, a pure dinitrogen discharge is observed in air, which is nearly identical to the spectrum observed from lightening. This result is achieved because the absorption of 3-NBN crystals in the spectral region of a dinitrogen discharge (337 nm) is poor, making photon driven ionization of the matrix, as has been suggested for MALDI , highly unlikely. Further, thermal desorption of bare ions from a surface requires substantial energy to overcome the image charge attraction . A logical mechanistic proposition for MAI is that crystal fracturing produces both the necessary charge separation and the gas-phase particles from which bare ions are produced [1, 16]. An advantage of considering a fracturing process is that the energy necessary to break the crystal also potentially provides a physical means to propel the charged particles into the gas phase.
If particles are the carriers of analyte into the gas phase as ions, we need to ask what is the evidence for particles, how are gas-phase charged particles produced simply by exposing the matrix to sub-atmospheric pressure, are the particles composed of matrix molecules or residual solvent, and what is the mechanism for analyte ion release from the particles. Herein we discuss efforts to understand the processes whereby even proteins, at least as large as bovine serum albumin (BSA), 66 kDa [2, 42], are transported from the solid phase into the gas phase highly charged without application of external energy. Answering more detailed questions may provide a path to even higher sensitivity and utility of these new ionization processes developed into methods for MS, as recently shown [43, 44, 45], but also because these processes, previously unknown, have potential impact beyond MS.
All chemicals including the analytes BSA and angiotensin I, the matrices 3-nitrobenzonitrile (3-NBN) and 2-methyl-2-nitro-1,3-propanediol (MNP) (Scheme 1), and the solvents acetonitrile (ACN) and HPLC water were obtained from Sigma-Aldrich. The mass spectrometers used were Thermo Scientific Orbitrap Exactive and LTQ Velos, and Waters SYNAPT G2 and G2S. The atomic force microscopy (AFM) instrument was a Bruker Veeco diInnova. The Quartz Crystal Microbalance was from Gamry (eQCM 10M). The microscope used was a Nikon TiE (Eclipse) confocal microscope equipped with a CSU-X spinning disk confocal scan head (Yokogawa).
A mixture of leucine enkephalin, angiotensin I, angiotensin II, and bovine insulin at ca. 10 μM in a concentrated 3-NBN solution in 1:1 ACN:H2O was dried in a Chemglass (Chemglass Life Sciences, Vineland, NJ, USA) CG-3034-02A sublimation apparatus overnight. The bottom of the apparatus was immersed in an oil bath heated to 95 °C to the upper level of the matrix after it was connected to the condensation tube and pumped to a pressure of ca. 0.18 Torr with a rotary pump. Matrix crystals were noted to form on the walls above the matrix. The crystals observed using a Nikon microscope range 1–20 μm and were similar in appearance to crystals observed to fracture in microscopy experiments described below. Crystals were removed from different levels above the matrix and inserted into the skimmer inlet of a Waters SYNAPT G2 mass spectrometer to collect mass spectra. In a second experiment, a concentrated solution of 3-NBN (ca. 1 gram) in 7:3 ACN:water was added to the bottom of a test tube and allowed to dry overnight similar to the above experiment. A syringe needle was suspended ca. 1 cm above the matrix and the test tube with syringe needle was placed in a sand bath heated to 70 °C and the entire setup was placed in a vacuum desiccator pumped by a rotary pump. Matrix collected on the syringe was analyzed by MAI using an Orbitrap Exactive mass spectrometer.
In another experiment, a Corning glass Pasteur pipette (Sigma-Aldrich) was connected to a rotary pump, and 3 μL of a saturated solution of 3-NBN in 1:1 ACN:H2O was allowed to dry for 1 min on the tip of a 10 μL syringe before being inserted into the small opening of the pipette. A laboratory microscope was used to monitor at 10× what occurs when the matrix is placed inside the narrow tube.
Two different optical microscope experiments were performed. In one, matrix with bovine insulin (10 pm μL–1) was dried on a microscope slide and observed over 28 min. In the second, matrix solution in 7:3 ACN:H2O was dried on a microscope slide that was placed above a second microscope slide with a 2 mm spacing. The top slide was gently heated with a laboratory heat gun while the bottom slide was cooled with ice. When crystals formed on the bottom slide, they were placed under a microscope and pictures taken every 10 s for 3 min. In both cases, the crystals were observed to spontaneously fracture, and occasionally crystal particles landed in the frame of view.
In the AFM experiment, the 3-NBN matrix was either dissolved into a 220 μM solution of BSA in 7:3 ACN:H2O and 3 μL was placed on a silicon wafer to dry, or 3 μL of a 200 mg mL–1 ACN solution of 3-NBN was placed on a silicon wafer. The scanned image size of a selected crystal was 7 × 9 μm and the time to raster in each direction was 8.5 min for a total time of 120 min.
Charge Separation Inlet Tube Detection Devices
Another study using Instrument 2 collected material onto a clean graphite sheet that replaced the detector. 3-NBN matrix solution was combined with BSA using typical MAI conditions. The sample was placed on a glass plate using a pipette and, while still rather wet, was affixed to the flared entrance to the heated inlet tube by the pressure differential. After the material was desorbed from the glass plate, the experiment was stopped and the instrument disassembled to collect the graphite sheet assembly from the vacuum chamber to be observed by AFM. More detailed information regarding materials used, sample introduction, and experimental parameters can be found in the Supporting Information.
The Gaussion09  program package was used for all the calculations. The structures of charged and neutral matrix molecules were optimized with density functional theory (DFT) employing the long-range corrected hybrid functional LC-ωPBE. The standard Pople type 6-311++G(d, p) basis set was used for all the atoms. Harmonic vibrational frequencies were calculated to ensure that the optimized structures correspond to real local minima.
Results and Discussion
Charged Particles as Precursors to Bare Ions
The physical force provided by vapor pressure would need to be able to overcome van der Waals forces, and for aromatic matrices, also possibly Pi–Pi interactions, and, most importantly, the electrostatic attraction between the sum of the charges in the exiting particle and the image charge on the original surface. In the case of a charged particle, the electrostatic attraction is greatly decreased relative to a bare ion because of the charge separation caused by the matrix solvating the charges and increasing the distance between opposite charges. Additionally, the surface area-to-charge ratio is much larger in the particle than in a bare ion, substantially decreasing the force per unit area necessary to expel the particle from the surface. Release of bare gas-phase ions from the gas-phase particles would then occur through sublimation or evaporation of the matrix, depending if the matrix is a solid or liquid [1, 16], similar to the ESI charge residue model [11, 12].
So far, particle emission has not been visualized, possibly suggesting that they are nanometer in size and below the diffraction limit of visible light. A quartz crystal microbalance was therefore used to detect weight changes of matrix crystals under sub-atmospheric pressure conditions. If particles are formed slowly enough and are large enough, instead of a smooth weight loss expected from sublimation, step changes in weight loss might be observed as particles leave the surface. While we indeed detected weight loss, using typical MAI conditions and 3-NBN as matrix, step changes are not observed (Supplementary Figure S2). The particles may be too small to be observed above the noise, or they exit the surface sufficiently rapidly, similar to droplets leaving the emitter tip of sonic spray  or ESI, to give the appearance of a continuous process, as is also observed in the continuous and prolonged signal from the mass spectrometer detector when the matrix sample is inserted into an intermediate pressure vacuum enclosure [3, 9, 43]. Of course, another explanation would be that under the conditions of the experiment, particles are not expelled from the surface. The microbalance and mass spectral results suggest that in MAI, the large matrix particles that have been observed with optical microscopy to migrate over a distance are not observed in the microbalance experiment, or in pulses of ion current in the mass spectrometer. The best explanation for these observations is that similar to ESI, ionization is a continuous process, but unlike ESI, the MAI sample is limited and ionization ceases when the matrix is depleted even though most of the analyte may remain .
A number of experiments were carried out in which the 3-NBN matrix containing a mixture of bovine insulin, angiotensin I, angiotensin II, and leucine enkephalin was sublimed. In one set of experiments, a common sublimation apparatus was used (Supplementary Figure S4). Matrix was collected on the walls just above the oil bath, as well as further away from the heat source. All of the collected crystals, when exposed to the inlet of a mass spectrometer (LTQ Velos), gave multiply-charged signals for the analytes. However, these crystals were micron sized, similar to what is observed in the microscope study described above and likely not the particles from which gas-phase ions are directly produced. A similar experiment using a heated sand bath and a vacuum chamber with a syringe needle suspended about 1 cm from the matrix:analyte mixture was used. The syringe needle was observed through a microscope and no visual matrix particles were observed to be collected. The sublimate that collected on the syringe needle (Supplementary Figure S5) was scraped off and analyzed using MAI on an Orbitrap Exactive mass spectrometer. The peptide, angiotensin I, was in the initial 3-NBN solution at 100 μM concentration, an amount ca. 1000× that needed to obtain MAI mass spectra in which the peptide ions are the most abundant. The doubly- and triply-charged ions for the peptide were observable using MAI, but the representative signals were within the chemical noise in the mass spectra. This experiment suggests that the amount of analyte relative to matrix that transfers to the cold finger is reduced by >1000×, an indication that sublimation is dominant and the ion formation process leaves most of the analyte behind. However, this experiment is not definitive because particles containing the analyte would need to travel more than a centimeter against gravity, and it also cannot be ruled out that crystal cracking, as observed in the microscope studies, might propel crystals of matrix with analyte sufficient distance to stick to the needle, without visual detection, and produce the observed results. After the sublimation of matrix, the analyte was readily detected from the bottom of the flask. These results relate well with MAI probe experiments where completely subliming the 3-NBN matrix containing analyte and re-applying just matrix solution produced analyte ions through 20 repeats of this procedure , and is reminiscent of MALDI experiments showing significant analyte remains on the MALDI plate after complete laser ablation of the matrix . This is also consistent with MALDI experiments that show between 1:1000 and 1:1,000,000 analyte molecules are converted to detectable ions . These results suggest that in MAI and MALDI, there is considerable room for increasing sensitivity.
An interesting observation was made during sublimation of solid matrix freshly crystallized from ACN:water onto the tip of a syringe needle. Inserting the syringe needle with matrix into a Corning glass Pasteur pipette and pumping from the larger end of the pipette with a rotary pump resulted in initial observation, using a microscope, of a clear liquid collecting on the inside of the glass near the syringe needle tip (Supplementary Figure S6). The liquid quickly pumped away but suggests the possibility that solvent droplets are the source of analyte ions, which would readily explain the similarity to ESI. This observation is consistent with the hypothesis that the included solvent is involved in the MAI process. However, a number of experiments suggest charged liquid droplets are not the source of observed analyte ions. For example, the observation of collected clear liquid is from freshly prepared matrix and analyte, yet analyte ions are observed from well-dried matrix, including freeze drying, and 120 min under vacuum conditions (Supplementary Figure S7). Further, the observation of liquid is momentary, yet inserting matrix on the tip of a syringe needle into the heated (70 °C) inlet tube of the Orbitrap Exactive produces a burst of ions that diminish upon leaving the matrix in the inlet for over 1 min. The conditions of flowing gas and temperature of 70 °C are expected to remove solvent capable of producing droplets unless incorporated within the matrix crystals, yet upon removing the syringe barrel with matrix:analyte from the inlet, an even larger analyte signal is observed (Supplementary Figure S8). Pockets of water within the clear 3-NBN crystals are not observed by optical microscopy. Thus, it is not obvious how charged solvent droplets would be produced in the quantity to produce the prolonged ion current that has been observed. However, a protic solvent, such as water or methanol, is critical for successful ionization of nonvolatile analyte using MAI. One logical explanation is that the analyte retains its solution charge state in the crystal, implying that it also retains its solvent shell. This has also been suggested for MALDI matrices, and using electron microscopy, a high density of cavities were observed in 2,5- and 2,6-dihydroxybenzoic acid crystals, which was speculated to be the result of incorporated matrix with associated solvent [51, 52].
Even though direct observation of particles has not been obtained, there is reasonably strong circumstantial evidence that charged particles carry analyte into the gas phase and upon matrix sublimation release the ionized gas-phase analyte ions. The evidence is especially strong with less volatile matrices that require high inlet tube temperature to observe bare ions. Early work using commercial sources together with less volatile MAI and LSI matrices showed results indicating that final desolvation of the analyte ions occurs as late as in the mass analyzer [15, 16, 56]. It is important to note that using the more volatile 3-NBN matrix with the commercial atmospheric pressure (ESI) or intermediate pressure (MALDI) sources provided comparable MS/MS results using collision induced dissociation (CID) and electron transfer dissociation (ETD) to those obtained with ESI, presumably indicating the charged clusters of this matrix, which more readily sublimes, are desolvated in time to use the full capabilities of the commercial mass spectrometer .
This is similar to producing ions by freezing water:methanol mixtures in which a common feature is the inability to readily fragment ions in the ‘in-source’ fragmentation region, or to select ions for fragmentation without first providing thermal energy or collisions with surfaces, or gases, presumably to remove matrix molecules from the bare ions . This is not surprising because as early as in the 1960s, Latham and Stow calculated that ion evaporation from an ice surface agrees with experimental results when the ion is removed in a particle . With less volatile matrix compounds, the charged clusters can travel long distances before they fully desolvate, often with the aid of collisions and electric fields [15, 16, 56, 59, 60, 61].
Charge Separation by Traversing Particles Through an Inlet Tube
In order to further study the charge separation processes, an inlet tube, previously built for high efficiency transmission and collection of ions produced by ESI , was employed. One of the objectives was to quantify charge separation when particles of various compounds are passed through an inlet tube, and to determine which compound properties might contribute to improved charge separation.
To relate the generation and separation of charge in the inlet region to analyte ions detected in the mass spectrometer, a preparative mass spectrometry instrument was used (Instrument 1, Supplementary Scheme S1). While the preparative capability was not used, this instrument was particularly useful because it is capable of measuring absolute ion currents midway through the instrument at several positions (e.g., after leaving the rf-ion guides or before entering the TOF-mass spectrometer), and obtaining the corresponding mass spectra. For MAI experiments, the ESI ion source is operated without voltages applied to the capillary. In this mode, only the flowing gas can transport ions (and other particles) to the ion optics.
Using the inlet tube and 3-NBN as matrix, mass spectra similar to those previously reported were obtained for both positive and negative ions. Upon introduction of the matrix:analyte mixture, several short pulses (less than 1 s) of ion current are detected, which coincide with the detection of ion intensity in the mass spectrometer. The currents generated using a pipette tip or spatula under various solution condition are in the range of 10–100 picoamp (Supplementary Figures S9–S11), considerably lower than ESI currents of the same substances, which are detected at nanoamp intensity. Part of the reason for higher ion abundance in ESI is because of higher background signal.
To complement the MS measurements with charge separation data, the same ion source design was used in a setup where the ion currents flowing in the ion source chamber were detected (Instrument 2, Supplementary Scheme S2). Ions or charged particles impinging either the inlet tube or the detector plate are detected separately. The electrically isolated transfer capillary and a detector plate, about 3 cm downstream from the transfer capillary exit and in line with the inlet tube, were separately connected to electrometers.
The matrix 3-NBN created one of the strongest signals of all tested compounds. When crystals or powder from the original container are inserted using a pipette tip, a signal of several nanoampere is observed. The signal is positive on the detector but negative on the capillary, an indication that the charge separation takes place inside the capillary. Most compounds show signals on both capillary and detector with different intensity. While all signals are individual in shape because of the undefined nature of the injected powders/droplets, the magnitude of the signals is rather reproducible. Heating the inlet and/or dissolving the compound in ACN:water followed by drying, identical to sample preparation protocols in MAI, increases the detected current for most compounds studied (Supplementary Table S1). The detected signals varied greatly between compounds, with only a few compounds producing strong signals at room temperature, whereas most require a heated inlet to produce a significant signal.
Depending on the compound, and sometimes conditions employed, the current readings at the transfer tube and downstream detector plate were mostly of opposite polarity, e.g., positive/negative or negative/positive, but interestingly some compounds produced positive/positive or negative/negative readings on the transfer tube and detector, respectively. The readings of the same sign on both the inlet and the detector may suggest that particles of these compounds enter the inlet from the pipette tip with a net charge much larger than the current generated by charge separation, which leads to a reading similar to positive ion ESI. The majority of typical MALDI matrices showed positive/positive signs on the detectors (Supplementary Table S1).
The intensity detected for most compounds was in the order of 100 pA, with a few compounds such as 3-NBN reaching significantly higher currents in the nanoampere range. A particularly interesting finding was that the injection of 3-NBN powder from secondary vials, shipped from the USA to the lab in Germany, resulted in very low current readings on both detectors, whereas the 3-NBN available in the lab, purchased from the same vendor and used directly from the bottle, gave high current readings (Figure 5). Dissolving the matrices that produced low current in ACN:water and drying in room air for several min before passing through the inlet produced some of the strongest current reading observed in the study. We speculate on two possible reasons for this outcome: one was that the repackaged matrix shipped by airfreight lost water content during shipment, or that crystal morphology changed due to high temperature differences during the transport. Whatever the correct reason here, other results have shown that morphology, at least for 3-NBN, is important for the ionization process [3, 4, 15, 18, 19, 39]. What is clear from these results is that charge separation is a feature common in particles passing through an inlet tube from a higher to a lower pressure region and that heating the inlet tube is usually associated with increased charge separation. Other than the increased heat, dipole moments appear to correlate with successful charge separation, at least at low inlet tube temperature (Supplementary Table S1). This relates well with triboluminescence [18, 19].
Using Instrument 2, it was possible to observe the sublimation of a macroscopic 3-NBN deposit whereby particles are expelled from the crystal into the gas phase. Glass plates with various matrices including 3-NBN and typical MALDI matrices (Supplementary Figure S15), applied from solution and fixed to the conical shaped inlet tube entrance by the pressure differential, were used to visually observe small changes in the matrix while simultaneously recording the current collected on the inlet tube and detector. Small particles of matrix on the glass plate sublime over a duration of minutes and slowly disappeared without detection of current (Supplementary Figure S13). Only when a piece of matrix material is observed to dislodge from the glass surface is a signal detected. Warming the atmospheric pressure side of the glass plate with a heat gun increased the rate of sublimation and the rate at which pieces of matrix could be seen leaving the glass plate, both visually observed and recorded as a current signal. These processes are displayed in Supplementary Movies S3 and S4 for BSA with 3-NBN and 2,5-DHB matrix, respectively. While the sublimation of the matrix or evaporation of solvent at the glass/matrix interface might provide the pressure to crack the matrix material and propel particles into the inlet tube, the generation of the detected ions appears to predominantly take place inside the transfer capillary.
In addition to solid compounds, solvents traversing through the pressure differential within the inlet tube create charge separation as measured on the electrodes. Acetonitrile gave the lowest reading (Figure 5). These findings using liquids relate most closely to solvent-assisted ionization (SAI) [13, 62, 63], and work from the groups of Jarrold and Ewing reporting increased charge upon passing droplets produced by e.g., sonic spray through an inlet tube into vacuum . These authors measured an average charge of 12,000 e on droplets having a radius of 2–3 μm. Such droplets would need to be about 1/10th this diameter to reach the Rayleigh limit. In SAI, increasing inlet tube temperature increases the analyte ion abundance, which directly relates to the increase in current measured on the inlet tube and detector in the experiments conducted here when the inlet tube was heated to 160 °C. SAI is preferred for some compounds even at low inlet tube temperatures relative to MAI using 3-NBN, as demonstrated for 1,5-diaminonapthalene (Supplementary Figure S21). Horan and Johnston recently reported achieving exceptional sensitivity for aerosol droplets passed through an inlet tube heated as high as 950 °C .
Although previous results have shown that at high inlet tube temperatures [13, 14, 15, 16, 61, 62, 63] many of the compounds studied here assist in producing bare analyte ions as measured by MS, few of the compounds that show charge separation near room temperature produce analyte ions that are observed with the mass spectrometer. One likely factor is the ability to desolvate the charged particles to release the bare ions, but other factors are also important. In order to observe monomeric analyte ions, it is necessary that the final particle before release of the gas-phase ion be small enough to carry only a single analyte molecule/ion. Another factor might be the ability to transfer a proton to or from the analyte in the presence of the matrix compound. These and other studies show that a multitude of other factors are important, such as temperature, crystal morphology, solvent, additives, gas flow, collisions, and pressure on either side of the transfer tube. Understanding this simple to perform and sensitive ionization process, a challenge theoretically, may require thinking beyond what has been previously modeled for ionization processes [11, 12, 66, 67, 68, 69, 70], but there is room for great improvement in sensitivity with better knowledge of the processes involved in transferring solid-phase molecules to gas-phase ions without application of external energy input.
Overview 1: Key Findings Relative to MAI Mechanism
• Gas-phase ion production is inefficient relative to sublimation
• Gas-phase ion molecule reactions cannot produce highly-charged ions
• Energy is not available to remove multiply-charged ion from matrix surface
• Alternative is removal of charged droplets or particles
• Freshly crystallized matrix contains sufficient solvent to form liquid droplets
• Protic solvent is necessary for ionization of nonvolatile compounds
• Analyte ions are produced from dried matrix with exceptional sensitivity
• Ionization selectivity determined by the matrix and not the solvent
• Ionization is predominately by protonation even in salt solutions
• Particles have not been directly observed
• Means for producing matrix particles sufficiently small to contain a single analyte molecule
• Circumstantial evidence for charged particles as analytecarrier is presented
One concern from the beginning of the discovery of MAI was why, with all the sublimation experiments conducted over the years, was the phenomena in which large molecules are transported into the gas phase not previously reported as this would not be conducive to purification. The answer appears to lie in the inefficiency of particle emission, which leaves the great majority of nonvolatile compounds behind after complete sublimation, and yet the MAI method has sensitivity comparable to ESI and MALDI [2, 5, 6, 7]. There are great opportunities for improving this technology ahead.
This material is based in part upon work supported by the National Science Foundation under Grant NSF CHE-1411376 to S.T., and NSF Phase II 1556043 to MSTM, LLC. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.
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