Electrospray Ionization

ESI is one of the most important ionization techniques for the coupling of a liquid chromatograph to a mass spectrometer. It is a method of choice for the analysis of labile biomolecules that can not be ionized by other techniques. In principle, ESI is the transfer of analyte, generally ionized in the condensed phase, into the gas phase as an isolated entity.

1) the column effluent from the LC containing analytes is pumped through a capillary which is held at high potential (2-6 kV) and nebulized. The applied voltage can be either positive or negative, depending on the analytes; nebulization is usually assisted pneumatically (except in nanospray);

2) the droplets that detach from a tip of the capillary contain an excess of positive or negative charge as a result of the applied high voltage;

3) electrical field gradient attracts charged droplets towards the entrance of the mass spectrometer;

4) charged analyte molecules are generated from the small charged droplets either by the charged residue model or by the ion evaporation model. Ion formation from the droplets is promoted by a flow of drying gas (usually heated nitrogen);

5) the ions, solvent vapor, and drying gas molecules are sampled through a capillary into a first pumping stage (0.08-0.75 Torr) where they are supersonically expanded;

6) the ions and some other neutral molecules are sampled via a skimmer into the second pumping stage (0.001-0.01 Torr) containing an ion focusing and transfer device (usually RF hexapole or octapole and a set of lenses);

7) ions enter the mass analyzer region (< 10-5 Torr).    

Ion Formation in ESI

Evaporation of solvent from charged droplets results in the reduction of diameter. "Coulomb explosion" (fission of a droplet) will occur at the point, called "Rayleigh limit" when the repulsion forces between charges in a droplet are sufficient to overcome the surface tension holding it together. Continuous depletion and fission of droplets lead to the concentration of charges and analyte molecules (or ions) in a drop and the eventual generation of ions by one of the possible mechanisms. An ion was formed by the charge residue model when the fission of charged small droplets stops at some point and the remaining solvent molecules were removed by evaporation. Large ions, like ions of proteins or synthetic polymers, are probably formed by the charge residue model. Smaller ions should be formed by the ion evaporation model. The ion evaporation model is made on the assumption that when charged droplets are reduced to a certain size, direct emission of ions to the gas phase occurs. In other words, the small ion is pushed out of a densely charged droplet. ESI is a softer ionization method than any other known ionization methods. In the ESI process, internal energy (vibrational and rotational) of a charged analyte is dissipated during the desolvation stage when loosely bound solvent molecules dissociate from the final charged analyte ion. Fragmentation of an ion (desired or not) however, can occur in a region between the capillary exit and skimmer where the gas pressure is sufficient to induce CID fragmentation at certain voltages.

Ions of analytes are generated in ESI either by charge separation or by adduct formation. Examples of ion formation in ESI of some common compounds:

Examples of ion formation in the positive ESI mode

R-NH2 + H+ → R-NH3+  (protonation)

C6H12O6 (carbohydrate moieties) + Na+ → C6H12O6Na+    (adduct formation)

Examples of ion formation in the negative ESI mode

R-COOH → R-COO─ + H+   (deprotonation)

C6H12O6 (carbohydrate moieties) + A─→ C6H12O6A─   (adduct formation, A= Cl─, CH3COO─, HCOO─ etc.)

The ESI process results in a simple mass spectrum with an intense molecular (pseudomolecular) ion and no structural information. This technique is particularly suited for polar organic compounds that cannot be analyzed by gas chromatography-mass spectrometry. High molecular weight compounds can be detected as multi-charged molecular ions. Ion fragmentation, an essential step for analyte characterization, can be carried out, for instance, in the analyzer region of the ion-trap mass spectrometer, or in the collision cell of the triple quadrupole or Q-Exactive mass spectrometer.