Electrospray ionization

The first electrospray ionization experiments were carried out by Malcolm Dole and his co-workers in the late 1960s.^92,101 The authors discovered the phenomenon of the generation of multiply charged molecule ions during electrospray ionization and successfully introduced a polystyrene polymer (average MW = 51,000 Da) into the gas phase as a charged species.¹⁰² Dole used a very dilute solution of the analyte and then nebulized it into extremely small droplets, to obtain many droplets that contained only one analyte molecule. Evaporation of the droplets led to a transfer of the analyte molecules into the gas phase. Subsequent work by Fenn and co-workers^94,103 confirmed and continued Dole’s approach by demonstrating that ESI-MS could be used very effectively for analysis of peptides and proteins with molecular mass m which could be much higher than m/z 1500 daltons.¹⁰⁴ Fenn was latter on recognized for his contributions by the award of the 2002 Nobel Prize for Chemistry.¹⁰⁵ This soft ionization technique in combination with mass spectrometry is today used as one of the commonly employed soft ionization techniques for the investigation of large biomolecules and many other analytes.⁹² The remarkable growth in popularity of this technique is representative of its powerful abilities to investigate species in solution even in very low concentrations and in complex mixtures.¹⁰⁶

The electrospray is a process which involves the creation of a fine aerosol of highly charged micro droplets in a strong electric field. The sample is passed through a highly charged capillary with a voltage, Vc, typically in the range of 2-5 kV in which the solution is distorted into a

I.3. Mass spectrometry and gas-phase ion chemistry

21 Taylor cone^*, producing a fine spray of highly charged droplets that evaporate due to the presence of the electric field with the aid of a stream of nitrogen, a process called nebulization. Because the spray capillary tip is very thin, the electric field Ec at the capillary tip is very high (Ec ≈ 100 V/m).

The value of the field at the capillary tip can be estimated with the approximate relationship:

𝐸_𝑐 = ^2𝑉𝑐

𝑟_𝑐ln(^4𝑑

𝑟𝑐) (1.1)

where Vc is the applied potential, rc the capillary outer radius, and d the distance from the capillary tip to the planar counter electrode.

The imposed field, Ec, will also partially penetrate the liquid at the capillary tip. When the capillary is the positive electrode (positive ion mode) some positive ions will drift toward the liquid surface and some negative ions will drift away from it until the imposed field inside the liquid is removed by charge redistribution. The accumulated positive charge at the surface leads to its destabilization since the positive ions are drawn down-field but cannot escape from the liquid.

The surface is then drawn down-field in such a way that a liquid cone is formed.

Under these conditions, the droplets break down and, while moving inside the source, their size is continuously being reduced. The charge density on the shrinking droplets builds up until surface tension is balanced by the Coulombic repulsion. This process is called the Rayleigh limit,^108,109 and the number, zR, of elementary charges e is given by

𝑧_𝑅 = ^8𝜋

𝑒 √𝜀₀𝛾𝑅³ (1.2)

where R is the droplet radius, ε0 is the vacuum permittivity, and γ is the surface tension. Droplets at the Rayleigh limit produce even smaller and highly charged offspring droplets via jet fission.

Repeated evaporation/fission events ultimately yield the final generation of ESI droplets with radii of a few nanometers. Gaseous analyte ions that are detected by MS are produced from these highly charged nanodroplets.105,110-112

*The Taylor cone was named after Sir Geofrey Ingram Taylor in 1964 before the appearance of the electrospray technique. He was one of the first to investigate the conditions under which a stable liquid cone can be observed in spray processes from which a jet of charged particles emanates above a threshold voltage.¹⁰⁷

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There are two possible mechanisms for the production of gas-phase ions in electrospray: the charge residue model (CRM) proposed by Dole et al.¹⁰¹ and the ion evaporation model (IEM) proposed by Iribarne and Thomson who worked with small ionic analytes such as Na⁺ and Cl -(Figure I.8).^113,114 The IEM typically prevails for relatively small ions (m/z < 3300)^115,116 whereas the CRM is more applicable for larger large globular species such as natively folded proteins.¹¹⁷

Figure I.8 A schematic representation of the possible pathways for ion formation from a charged liquid droplet.

The upper and the lower parts of the diagram illustrate the ion formation mechanisms depicted in the CRM of Dole et al. and the IEM of Iribarne and Thomson, respectively. (Adapted from Fenn B.¹¹²)

Dole's idea^† of how solute ions are formed was that the offspring droplets resulting from a first Rayleigh Instability would continue to evaporate solvent so that they too would undergo a Rayleigh Instability and disrupt. If the original solution was sufficiently dilute, a sequence of such evaporation–disruption episodes would ultimately produce droplets so small that each would contain only one solute molecule. As the last of the solvent evaporated from the ultimate droplet the remaining solute molecules would retain some of the droplet charge and thus become a free gas-phase ion. This process is known as the charged residue model (CRM) for ES ion formation.

CRM comes from the observation that ESI of globular proteins produces ions with a composition close to [M + zR H]^zR+,where zR is the Rayleigh charge of protein-sized water droplets. The IEM later proposed by Iribarne and Thomson is based on the fact that the electric field emanating from a Rayleigh-charged nanodroplet (with R < 10 nm) is sufficiently high to cause the ejection of small solvated ions from the droplet surface.105,109,118

The experimental results involved measurements of the relative abundance of the ions produced by ESI of solutions containing NaCl as the only solute. The authors found that there was a large

† “I got this idea from learning about the electrospraying of paint on to automobile bodies while working as a consultant for a paint company in Chicago” – Dole M.

I.3. Mass spectrometry and gas-phase ion chemistry

23 number of ion aggregates of the type [(NaCl)n(Na)m]^m+ the abundance of which decreased rapidly as n decreases. However, the lowest mass ion in that series, Na⁺ (n = 0 and m = 1), and the hydrated species Na(H2O)^k+ (k = 1-3) had thehighest abundances.^113,114 This observation led the authors to conclude that while large aggregate ions are probably due to a charged residue mechanism (CRM) type process, the abundant Na⁺ and Na⁺ hydrates must be formed through a different mechanism where sodium ions escape directly from the droplet’s surface.¹¹⁰ The applicability of one of the two theories to account for the generation of gas phase ions is yet not well established and at which stage it takes place, considering the size and the charge of the droplets.

The electrospray ion source is at very high pressure (atmospheric) with respect to the very low pressure that is required for ion separation by a mass analyzer, so the interface between the two involve a series of skimmer cones (acting as small orifices) between the various differentially pumped regions (Figure I.9). The ions are drawn into the spectrometer proper through the skimmer cones. A voltage can be applied (the cone voltage), which will accelerate the ions relative to the neutral gas molecules. This leads to energetic ion/neutral collisions and fragmentation due to what is termed collision induced dissociation (CID). The remaining bath gas is pumped away in stages (in order to attain the high vacuum necessary for separation of the ions) and the ions are focused through a lensing system into the mass analyzer.¹⁰⁶

Because ESI-MS is a very sensitive method, very low concentrations, 10^-7 to 10^-3 mol/L (M), of analyte can be used. Although methanol, acetonitrile or mixtures of acetonitrile-water and methanol-water are often used as solvents, other solvents such as toluene (that have a very low solubility for electrolytes) can also be used.

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Figure I.9 A schematic representation of the ESI-ion source.¹⁰⁶

I.3. Mass spectrometry and gas-phase ion chemistry

25 I.3.1.2. Laser desorption ionization/Laser ablation

Lasers in general are used in mass spectrometry for sample volatilization by laser desorption and sample ionization, atomization, and excitation. Any samples with known light absorptivity can be analyzed by laser ablation (LA) and ionization to provide elemental information. They are suitable for the production of intact gas-phase species from non-volatile, polar, high molecular weight, and thermally labile substances. The technique of laser sampling has many advantages, e.g. little sample size requirement and little or no sample preparation, low risk of reagent or solution waste, the avoidable introduction of contamination, and high spatial (lateral and in-depth) resolution. The power density deposited on the spot area can easily be controlled by adjustment of laser parameters (e.g., pulse duration, wavelength and energy) in comparison with other ionization sources. In combination with the proper detection system, it is an attractive technique for the elemental composition analysis of various samples through various spectroscopic methods. Some disadvantages of direct laser desorption ionization are a strong matrix effect, and the inability for the operator to control the sample volatilization and ionization processes independently. In 1988, Karas and Hillenkamp developed the Matrix-Assisted Laser Desorption Ionization method for the production of parent ions from large and very large non-volatile molecules.⁹⁵ This method was originally developed from LDI with the idea to add to the analyte a chemical matrix that strongly absorbs the wavelength of the desorption laser and promote efficient desorption of the fragile analyte molecules. MALDI has become a very successful method for soft ionization and is widely used for obtaining large ions in the gas phase such as biomolecules (DNA, proteins, peptides, sugars) and large organic molecules (polymers, dendrimers) which tend to be fragile and fragment when ionized by more conventional ionization methods.

Laser desorption (LD) and laser ablation are two laser–matter interaction regimes to be considered. LD results in emission of ions, atoms, and molecules without any substantial disturbance in the surrounding surface. LA implies a large scale disruption of surface and near-surface geometrical and electronic structure. LD and LA have to be viewed as the extremes of a continuum, which ranges from the emission of isolated neutrals or ions in the case of LD to the massive removal of material resulting from the collective effects of multiple photons irradiating the same spatial locale in the case of LA.

LA relies on the formation of a plume of ejected species, which follows the laws of plasma and gas dynamics, and produces a large number of ionized species. Collisions of ions with neutrals occurring in the gas-phase plume after LA lead to ion/molecule condensation reactions. On the

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other hand, ions and neutrals possess a significant amount of energy, which can result in dissociation reactions. Laser desorption ionization (LDI) and LA experiments are generally conducted on the same instruments since in both cases, ions are generated by laser irradiation of the sample surface. In LA experiments, the amounts of matter expelled from the surface are definitely greater than are observed under LDI conditions. Usually, LA refers to removing material with a pulsed laser, but it is possible to ablate material with a continuous wave laser beam if the laser intensity is high enough. LA is used in a widespread range of applications, including as a sampling procedure for the elementary chemical analysis of materials. Many analytical techniques makes use of LA such as LA Ion Mobility Spectrometry, Resonant LA, LA-Atomic Fluorescence Spectrometry and LA-Microwave Induced Plasma-Atomic Emission Spectrometry; among them the most popular ones are probably the LA Inductively Coupled Plasma (LA-ICP) techniques and the Laser Induced Breakdown Spectroscopy (LIBS).

LDI is more commonly used for the analysis of inorganic non-volatile solid materials.^119-121 The technique uses one single laser source (pulsed or CW) to perform desorption and ionization in a single step. A laser beam is focused directly on the solid sample usually deposited on a direct inlet probe. LDIMS was first used as an analogous technique to other methods for direct ion production such as Secondary Ion Mass Spectrometry (SIMS), Fast Atom Bombardment (FAB), or Plasma Desorption Mass Spectrometry (PDMS). In 1978, the group of Kistemaker published impressive LDIMS data of complex organic and bio-organic thermally labile substances such as sucrose (m/z= 342) and digitonin (m/z = 1228), setting a high mass record at this time with the latter substance.¹²² LDIMS has been widely used with a variety of lasers (pulsed and CW CO2 lasers, pulsed ruby, Nd glass, Nd:YAG, and N2 lasers), and with quadrupole, time-of-flight (TOF), FT-ICR and sector mass analyzers. Mass range and mass resolution of LDIMS instruments are essentially determined by the type of mass analyzer used.¹²³ The sensitivity is given by the product of the ionization efficiency and the instrument throughput. At very high laser power densities, the ionization efficiency can approach 100%, but this condition also causes extensive analyte fragmentation, broad ion energy distributions, and the presence of multiply charged species. For inorganic analysis using LDI/LA-FT-ICR, the ionization step is usually performed using a Nd-YAG laser. The diameter of the laser beam on the sample can be adjusted by means of the internal lens and an external adjustable telescope from 5 to several hundred micrometers, which corresponds to a power density ranging from 10⁶ to 4x10¹⁰ W.cm^-2. An example of the experimental sequence used for these analyses is as follows:¹²⁴

 Ions are formed by laser ablation in the source cell. During the ionization event, the conductance limit plate between the two cells and the source trap plate is fixed at a

I.3. Mass spectrometry and gas-phase ion chemistry

27 trapping potential (of typically 2 V or at a lower potential in some particular cases, down to 0.25 V).

 A variable delay period follows, typically close to 1 min, during which ion/molecule reactions can occur.

 Ions are then excited by a frequency excitation chirp and the resulting image current is detected, amplified, digitized, apodized and Fourier-transformed to produce a mass spectrum.

For practical applications, sensitivities in the pmol/cm² range have been reported for organic LDIMS.¹²⁵ Also, laser microprobe FT-ICR-MS, developed by Carre et al¹²⁴ has led to several further experiments in gas-phase ion chemistry. As an example, LA-FT-ICR-MS of UO3 or (NH4)2U2O7

allowed the formation of gas-phase molecular uranium oxide anions with compositions ranging from [UOn]^-, with n =2-4, to [U14On]^-, with n=32-35 by using a Nd:YAG laser (1064 nm) with an average focused power density of ca. 200 MW cm^-2. Single or multiple pulses on the same or on fresh spots yielded similar results.¹²⁶ Also, several negatively charged metal chalcogenide clusters, namely, sulfides^127,128 and oxides,^129-133 have been also generated by LDI/LA and studied by FT-ICR-MS. Furthermore, uranyl analogues, IrUO⁺, have too been prepared by direct LDI of a U/Ir alloy, and by oxidation of UIr⁺ with N2O and C2H4O. Properties of the formed bimetallic ions have been studied and they demonstrate direct actinide–transition metal bonding, and support the concept of “autogenic isolobality”.¹³⁴