Electrospray principle and applications

Electrospray principle and applications

The electrospray ion source was created in 1984 by John Fenn[1] to apply to biological and medical compounds such as pesticides, nucleic acids and penicillin. The interest of this ion source is the lack of fragment ion, it is simple, fast and sensitive that requires fewer samples. The ions are formed continually from the aqueous phase allowing coupling on line with liquid chromatography (LC-MS), the fragmentation is controlled in the area between the outlet of the capillary, skimmer I and skimmer II (or between the areas of ​​the electrostatic lenses). This ion source is soft ionization allowing the observation of pseudo-molecular ions, which is not the case of electron ionization[2]. In addition, while the molecules are being ionized, weak bonds such as non-covalent bond present in the molecule can be preserved. One of the most advantages of the electrospray source is that it forms multicharged ions, consequently the mass range is reduced allowing better resolution.

       Since the invention of this ion source which has opened a new dimension for mass spectrometry, researchers have continuously improved[3] this ionization/desorption method. Advances have also been made with the adaptation of the ion source to various analyzers (Time Of Flight (TOF)), quadrupole, ion trap, Fourier transform spectrometer (FT/ICR, Orbitrap), ion mobility IMS). The development of this ion source has been rewarded in 2002, when John Fenn receives the Nobel Prize in Chemistry.

I – Sample flow from the ion source to the analyzer

            The analyst diluted in a suitable solvent is injected into the introducing metal capillary of the ion source ESI (Fig. 1) by means of a syringe or liquid chromatography. The typical solvents for electrospray ionization are prepared by mixing water with volatile organic compounds (e.g. methanol, acetonitrile). The flow rate used is in the range of few nL/min (nano-spray) to a few hundred μL/min and the temperature imposed on the nebulization and evaporation gas is generally between 30 and 300° C. Note that this gas is not needed if the flow rate is very low as in the case of nano-spray.

electrospray process

Fig. 1: Diagram of an electrospray ion source, the desolvation zone, and the charged ion transfer zone.

      Under the pressure of a nebulizing gas (N2) and possibly gas arriving at the counter-current, the sample solution is vaporized in an area at atmospheric pressure in which a strong electric field is present. This electric field is produced from a potential difference (0.8kV to 6kV) applied between the outlet of the introduction capillary and the counter-electrode (Fig. 2). In this area, either solvated ions after desorption or charged aggregates are produced, their charge state are positive or negative, depending on the sign of the potentials applied to the capillary.

     Then, these charged species reaches a transfer area through a capillary metallic. The capillary acts as an interface between the atmospheric pressure zone of the ion source and the low pressure zone. As a result, the ions undergo a pressure gradient between the outlet of the transfer capillary and the desolvated ion transfer zone.

     Between the transfer capillary and the first cone (skimmer 1), a potential difference (ddp) accelerates these solvated ions and charged aggregates, which will undergo collisions to complete the desolvatation. Indeed, this ddp increases the speed of the solvated ions that undergo a large number of collisions (but with a low mean free path). This means that the ions have little space to accelerate therefore the collisions are not very energetic. Generally, in this area, the ions are not totally free of solvents because the collisions are not energetic enough. These solvents can be removed in a lower pressure zone where the ions are accelerated with a greater mean free path, so the collisions are more energetic, thus eliminating all solvent molecules. However, an excessive increase of the dp in this region, transfer enough internal energy to the charged species which can cause their fragmentation, therefore, fragment ions can be formed in this area.

            Finally, the solvent free ions are refocused by means of a set of multi-polar bars to be ejected from the desolvation region of the ion source to the analyzer. This geometry is commonly used to optimize ion transmission.

II Principles of electrospray ionization/desorption

II.1 – Ionization/desorption process

             Electrospray at atmospheric pressure is based on a succession of physicochemical phenomena. Their description remains simple.

             First, the positive (or negative) electric field causes an accumulation of negative (or positive) charges on the surface of the liquid (Fig. 2), in electrospray process, the ions are already formed in the phase liquid.

electrospray process, formation of charged species

Fig. 2: Principle of electrospray ionization/desorption in the case of formation positive ion experiment.

       Under the effect of the movements of these charges, and the surface tension, the droplets deform to take the form of a cone (called Taylor cone). As soon as Coulomb pulses between positive charges exceed the surface tension of the liquid, a jet of charged droplets is emitted from the tip of the cone. The diameter of these charged droplets is around few micrometers. It depends on the value of the electric potential, the flow rate of the solution, the nature of the solvent and the temperature of the gas N2.

The solvent evaporates from a charged droplet until it becomes unstable upon reaching its Rayleigh limit which is defined by the following equation (equation 1). At this point, the droplet deforms as the electrostatic repulsion of like charges, in an ever-decreasing droplet size, becomes more powerful than the surface tension holding the droplet together. At this point the droplet undergoes Coulomb fission, whereby the original droplet explodes creating many smaller, and more stable droplets. The new droplets undergo desolvation and subsequently further Coulomb fission. During the fission, the droplet loses a small percentage of its mass along with a relatively large percentage of its charge.

equation 1(equation 1)

qRayleigh : Charges moyenne de la goutte.       γ: Tension de surface du solvant.

rmax : Rayon de la goulette.                  εo: Permittivité du vide.

At this stage, the best-known ion production in gas phase model according to the following steps is the following.

            In 1968, Dole et al.[4] present a model known as the theory of charge residue (Fig. 3). According to their ideas, the production of ions process is based on the fission continues of the droplets alternating with the evaporation of the solvent. The total evaporation of solvent, from droplets with a certain critical radius, leads to the birth of an intact molecule solvent-free and with several charges. The second ion production model is, the gas-phase ions form after the remaining solvent molecules evaporates, leaving the analyte with the charges that the droplet carried.

 ( [M + nH]n+ in case of a positive ionization, where M represents the analyst).

Model production ion

 Fig. 3: Diagram relating to the residual charge theory of Dole et al (S represents a solvent molecule)

      There are other models to explain the production of ions, according to these models, the ions are ejected from the droplet[5], or ejected directly from the Taylor cone[6].

            To date, the models have not been defined, and remain complementary. Nevertheless, the charge residue theory and the ion evaporation model are the most cited, as shown in Kebarle and Peschke’s review[7]. According to this review, it could be the combination of two major models represents the reality of the phenomenon. In this case, the model of ion evaporation would be the last stages of the theory of charge residue.

II.2 Large distribution charge state

    Electrospray is a technique for observing ions of the same molecule at various charge state. This distribution results from the solvent used (preformed ions in solution), from the conformation(s) adopted by the molecule, and from the ionization/desorption process of the molecules which are analyzed (mild desolvation conditions).

    Depending on the pH of the solution, the molecules will be more or less charged. This phenomenon is more evident when the molecules are folded, they are maintained by non-covalent interactions, or even associated with other molecules. When the pH value decreases, the non-covalent interactions become weaker, the molecules are unfolded and able to carry a greater number of charges[8],[9], more acidic and basic regions are accessible. Moreover, low pH conditions increase the number of protons on the surface of the droplets during the ionization/desorption process.

    The nature of solvent influences also the conformation of the molecules and therefore the accessibility of the region which can carry a charge. Several researches on proteins demonstrated that the greater proportion of organic solvent in solution, the more biological molecules will be denatured[10],[11],[12] so that they can carry more charge.

     Generally at the first skimmer of the electrospray ion source, the molecule of interest will acquire its final charge state. In an ionized aggregate molecule/solvent, the molecules will have more or less facility for capturing charges. When the aggregate reaches the skimmer, the hydrogen bonds between the ion and the solvent are broken. During this desolvation, the charges will be distributed between the solvent and the ion according to their proton affinity (Diagram 1). In conditions of mild desolvation (low potential difference), high charge states will be favored. On the contrary, under hard desolvation conditions (higher potential difference), low charge states will be observed. Thus, the internal energy of ion can be controlled by changing the potential difference. With reference to the condition of desolvation, a minimum of energy is required, but the application of a high voltage to the skimmer can induce cleavage of covalent bonds. A compromise is therefore necessary between desolvation of ions and preservation of their intact form. It is sometimes possible to observe ion/solvent aggregates[13] under low voltage conditions. This highlights the ability of the electrospray ionization/desorption technique to preserve fragile complexes.

transition state of ion in electrospray

Diagram 1: Schematic representation of the two types of desolvation (soft and hard) of a charged aggregate molecule/solvent. M represents the species of interest and S represents the solvent. The case presented proposes arbitrarily: AP (S)> AP ([M + H] + (AP proton affinity).

     The temperature of ion source plays also a role in the number of charge carried by the analyst. An increase in temperature allows better desolvation of charged aggregates and ions by thermal evaporation, this allows working at low values ​​of voltage of the skimmer. On the other hand, under such conditions, the non-covalent bonds inter and intra molecule(s) break apart, which releases basic site and can carry charge[14],[15].

      So far we have described only the protonation of the molecules. However, Electrospray also produces cationic and anionic adducts, allowing molecules that do not have an ionizable site to be ionized. The production of multicharged ions is one of the assets of electrospray because it makes possible the observation of a very high molecular weight. For a mass spectrum bas resolution, the charge state can be calculated by the equations (2).

i : Charge state.

M: masse de la molécule chargé i.

Mi+1 : masse de la molécule chargée (i+1).

However, this equation becomes obsolete if the mass spectrum is recorded with high resolution allowing the separation of the isotopic peaks. Indeed, for a monocharged molecule, the isotopic peaks are spaced by 1 Th. For a charge state n, the peaks are spaced by 1/n Th. Software based on deconvolution algorithms allow to do this calculation very quickly, automatically, and displays the result obtained on the mass spectrum.

Limit of electrospray

The physicochemical propriety of solvent plays an important role in the quality of the spray (Table 1). It is well known that organic solvents, methanol for example, produce better spray than water, because methanol is more volatile than water. This property is related to low surface tension of methanol solvent, and also by its viscosity. The surface tension characterizes the cohesive force of the solvent. As for the viscosity, when its value is low, it is possible to ensure a smooth and homogeneous flow of the liquid. In addition, the ability of a solvent to separate positive and negative electrical charges is directly related to the quality of aerosol formation in an electric field. As a result, a solvent with high dielectric constant will be a good candidate.

Solvent Surface tension γ(en mN.m-1; à 25°C) Viscosity η(en Cp; à 25°C) Dielectric constant ε(en C2.J.m-1; à 25°C)
Water 72 0.9 78
Acetonitrile 28 0.4 36
Methanol 22 0.5 33

Table 1 : Solvent parameters

    For samples in aqueous medium, the use of a nebulizing gas (nitrogen) arriving co-axially with the injection needle facilitates nebulization of the sample (Fig. 4). Such geometry allows increasing the injection rate of the sample, facilitating for example, the combination of the ion source with liquid chromatography. This pneumatic assistance was introduced for the first time in the electrospray ion source[16] in 1987 by P. Bruins and his co-workers. Currently, the majority of commercialized ion source electrospray allow the use of pure electrospray and electrospray assisted pneumatically.

Fig. 4: scheme of introduction needle of electrospray ion source. 

On the other hand, the quality of the ESI mass spectra also depends on the parameters specific to the analyzers coupled to the ion source. They intervene on the sensitivity, the resolution, the mass range, the mass accuracy. It should be noted that the quality of an electrospray spectrum also depends on the stability of the flow, the external conditions, and the parameters of mass spectrometer.

[1] Yamashita, M. and J.B. Fenn. 1984. Electrospray ion-source—another variation on the free-jet theme. J. Phys. Chem. 88:4451-4459.

[2] Edmond de Hoffman; Vincent Stroobant (2001). Mass Spectrometry: Principles and Applications (2nd ed. ed.). John Wiley and Sons

[3] Irina Manisali, David D.Y. Chen, Bradley B. Schneider Electrospray ionization source geometry for mass spectrometry:past, present, and future Trends in Analytical Chemistry, Vol. 25, No. 3, 2006.

[4] Malcolm Dole, L. L. Mack, R. L. Hines, R. C. Mobley, L. D. Ferguson, and M. B. Alice Molecular Beams of Macroions J. Chem. Phys. 49, 2240 (1968)

[5] Iribarne, J. V.; Thomson, B. A. On the evaporation of small ions from charged droplets Journal of Chemical Physics, Volume 64, Issue 6, pp. 2287-2294 (1976).

[6] Siu k. w. m. ; Guevremont r. ; le Blanc j. c. y. ; O’brien r. t. ;Berman s. s. ; Is droplet evaporation crucial in the mechanism of electrospray mass spectrometry? Organic mass spectrometry 993, vol. 28, no5, pp. 579-584

[7] Paul Kebarle and Michael Peschke, On the mechanisms by which the charged droplets produced by electrospray lead to gas phase ions Analytica Chimica Acta Volume 406, Issue 1, 1 February 2000, Pages 11-35

[8]Swapan K. Chowdhury, Viswanatham Katta, and Brian T. Chait Probing Conformational Changes in Proteins by Mass Spectrometry Am. Chem. Soc. 1990, 112, 9012

[9] 1. Lars Konermann, D. J. Douglas Equilibrium unfolding of proteins monitored by electrospray ionization mass spectrometry: distinguishing two-state from multi-state transitions, Rapid Communications in Mass Spectrometry, Volume 12, Issue 8, pages 435–442, 30 April 1998

[10] Carol V. Robinson, Evonne W. Chung, Birthe B. Kragelund, Jens Knudsen, Robin T. Aplin, Flemming M. Poulsen, and Christopher M. Dobson Probing the Nature of Noncovalent Interactions by Mass Spectrometry. A Study of Protein−CoA Ligand Binding and Assembly J. Am. Chem. Soc., 1996, 118 (36), pp 8646–8653.

[11] Kodali Ravindra Babua, Annie Moradiana and D. J. Douglas The methanol-induced conformational transitions of β-lactoglobulin, cytochrome c, and ubiquitin at low pH: a study by electrospray ionization mass spectrometry Journal of the American Society for Mass Spectrometry Volume 12, Issue 3, March 2001, Pages 317-328

[12] Samalikova M, Grandori R. Testing the role of solvent surface tension in protein ionization by electrospray. J Mass Spectrom. 2005 Apr;40(4):503-10.

[13] Sébastien Guillaumonta, Jeanine Tortajadaa, Jean-Yves Salpina, and Al Mokhtar Lamsabhib Experimental and computational study of the gas-phase interactions between lead(II) ions and two pyrimidic nucleobases: Uracil and thymine International Journal of Mass Spectrometry Volume 243, Issue 3, 1 June 2005, Pages 279-293

[14] le Blanc j. C. Y.; Beuchemin d; siu k. W. M. ; Guevremont r. ; Berman s. S. ; hermal denaturation of some proteins and its effect on their electrospray mass spectra Organic mass spectrometry 991, vol. 26, no10, pp. 831-839

[15] Matthias Mann, Chin Kai Meng, John B. Fenn, Interpreting mass spectra of multiply charged ions Anal. Chem., 1989, 61 (15), pp 1702–1708

[16] Andries P. Bruins, Thomas R. Covey, Jack D. Henion Ion spray interface for combined liquid  chromatography/atmospheric pressure ionization mass spectrometry Anal. Chem., 1987, 59 (22), pp 2642–2646

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