2.2. Ionization Methods in MS


Ionization Methods in mass spectrometry

Dr.  Lhoëst G.J.J.

ESI            CYPA     Ionspray        #Nanosray source      Chip-based ESI         FAB      CF-FAB      MALDI           Photoionisation     lamma         REMPI     CI       NCI        EI  


APCI and ESI Mode of ionization

In the JEOL Lcmate, components for the APCI method are standard equipment. Components for the ESI method and the FAB and FritFAB ionization are optional.

In the APCI method, the sample solution is vaporized under atmospheric pressure and ionized by corona discharge.

APCI is a chemical-ionization technique which uses gas-phase ion-molecule reactions at atmospheric pressure. Primary ions are produced by corona discharge on a solvent spray. The chromatographic eluate ( from 0.1 to 1 ml/min) is directly introduced into a pneumatic nebulizer, where it is converted into a thin fog by a high speed air or nitrogen beam. Droplets are then displaced by the gas flow through a carefully controlled heated quartz tube , known as a desolvation/vaporization chamber. The hot gas (120°C) and the compounds leaving this tube and arriving at the reaction area of the source are chemically ionized (under atmospheric pressure) via proton transfer. The evaporated mobile phase acts as the ionising gas and yields (M+H)+ ions. The electrons needed for primary ionization result from corona discharge or negatron emitters. Thermal decomposition is kept to a minimum and ionization of the substrate is very efficient. Differential pumping over several several stages separated by skimmers, maintains a good vacuum and allows entry of as many as ions as possible to the analyzer.

The APCI ionization method has the following features :

1. It is a soft ionization method generating few fragment ions.

2. The method is compatible with mobile-phase fow rates ranging from

    0.1 to 1 ml/min.

3. It is suited to measurement of compounds with molecular

   weights of approximately 1,500 Da or less.

4. Compounds having medium polarity can be measured with

    high sensitivity.

5. The ions created under atmospheric pressure are effectively

  transferred into high vacuum.


Electrospray mode of ionization             

An electrospray (ES) is produced by applying a strong electric field, under atmospheric pressure, to a liquid passing through a capillary tube with a weak flux (1-10 µl /min).                                                          

                                                                                     JEOL Lcmate    

The electric field is obtained by applying a potential difference of 3-6 kV between the capillary and the counter electrode, which are separated by 0.3-2.0 cm. This field induces a charge accumulation at the liquid surface located at the end of the capillary, which will then break up to form highly charged droplets.

As the solvent contained in these droplets evaporates, they shrink to a point where the repelling coulombic forces come close to their cohesion forces, thereby causing their " explosion". Eventually, after a cascade of further "explosion" the highly charged ions desorb. Electrospray can also be used in the case of molecules without any ionisable sites, through the formation of sodium, potassium, ammonium and other adducts. A variation of this method known as" micro-electrospray" uses flow rates of a few nl/min. This enables sensitivities in the range of a few hundred attomoles to be achieved.

The ESI mass spectra normally correspond to a statistical distribution of consecutive peaks characteristic of multiply charged molecular ions (M + nX)n+ where X = mass of adduct and n = number of charges avoiding the contributions from dissociations or from fragmentations.

Consider an ion with charge n1 whose mass to charge ration is m1 Th, issued from a molecular ion with mass M Da and an ion with charge n2 whose mass to charge ration is m2 Th with                                                                                                                                                                                                                        Cytochrome C + Glucagon                   

m2 > m1 and n1 > n2.                            

m= m/z of a given peak

M = molecular weight of the analyte

X = mass of adduct (eg H+, Na+ ...)

n = number of charges         TOP

Ionspray source            

Ionspray, which is another form of electrospray, creates charged droplets by using a potential difference and a pneumatic nebulizer in order to accept higher flow rates and to produce a spray that is less dependent on the nature of the liquid phase.  The

ionspray technique consists of pumping the sample solution through a pneumatic nebulizer, which is maintained at a high voltage so as to form a fog of charge droplets, even if the flow rate being used is high.  The ions are then ejected from the droplets which evaporate, via a low-energy process that does not produce fragments.  Flow rates are ca. 50-200 µl min-1 .  A grounded screen lens acts as a spray splitter and thus inhibits the condensation of droplets that make the spray unstable at higher flow rates.  In this way, rates of up to 2 ml min-1 are then possible.


Illustration of the application ranges of different mass spectrometric / chromatographic coupling methods.


 NANOSPRAY source (Jeol Lcmate )  

 Because the signal intensity in ESI is       concentration dependent,ie almost independent of the flow rate employed, absolute intensity ca, can be increased up to a 1000-fold when the residence time of the analytes in the source is extended by reducing the flow rate to 10 to 50 nL/min.  Various nano-ESI sources are available commercially. Several analytical characteristics of nano-ESI , e.g tolerance for relatively high salt concentration may be explained by a model which depend on the size of the initial droplets.  A 1 to 2 µL sample will flow for 30 to 120 min providing more than adequate time to optimize critical experimental conditions  e.g. collision energy in MS/MS analysis of selected individual peptide ions of a mixture.



Microfabricated, Silicone Chip-Based Electrospray Ion Source        

Aiming to further increase the sensitivity with concurrent reduction of        cross-contamination of analytes, multiple ESI sources were made as parallel etched channels on microchips. Electrospraying was performed directly from the chip,using flow rates of 100 to 200 nL/min through the channels, the sensitivity limit for the protein angiotensin was 60 fmol/µL.  The chip, made of glass, was designed with nine parallel channels.  Each channel was connected to two wells, which allowed for fluid filling and sample injection into the channel. The fabrication process included thorough cleaning of the glass with low sodium content , metal deposition (first 300 A° Cr and then 1000 A° Au), photolithography, chemical etching, and thermal bonding.  Smooth channels were obtained by isotropic etching with a solution of HF/HNO3/H2O (20:14:66 v/v). For a 6 min etching time, the channel width was 60 µm and the depth 25 µm.  The channel lengths varied from 3.5 to 5 cm depending on their location on the glass chip.  The microchip device was enclosed by thermal bonding to a flat glass disk containing the wells.  The thermal bonding was accomplished over 3h at 620°C, after a temperature ramp of 10°C/min from room temperature.  A comparison of scanning electron microscopic photographs before and after bonding showed no noticeable alteration in channel shape.

Interface of Microchip to ESI-MS        

A three - dimensional stage was constructed to hold the microchip and to align sequentially each channel outlet with the MS inlet for optimum electrospray performance and sensitivity. The distance from the microchip channel exit to the MS inlet was between 3 and 8 mm.  The surface of the outlet edge was coated with a hydrophobic reagent, either Imunogen (Calbiochem, La Jolia, CA) or n-octyltriacetoxysilane (Sigma, St Louis, MO), to prevent the analyzed solution from spreading from one exit port to another.  With this configuration, the channel outlet could be visually aligned with the MS inlet, the interface was operating at atmospheric pressure and room temperature.

Multiplexed Electrospray          

A novel LC interface has been devised for the parallel analysis of four or eight LC streams (single component or mixtures) with negligible crosstalk.   Another recently developed interface was described and consisting of a simple microchip-ESI device that can be used for rapid MS analysis. A replaceable nanospray tip is attached to the end of a micromachined channel and the voltage necessary to generate the electrospray is applied to a fluid reservoir on the chip. The system has some similarities to conventional nanospray sources in that it operates at very low flow rates and does not require any pressure assistance for fluid delivery. Unlike conventional nanospray sources, however, the microchip coupled to the nanospray tip can be used to inject discrete samples and perform separations prior to detection.  The microchip may be used as a sample delivery device enabling, for example, rapid, repetitive analysis of the same sample, to monitor changes as a function of time, or high-throughput analysis from a chip with multiple sample ports, injecting each sample individually in an automated fashion.  One difficulty associated with a microchip device, where the spray is established directly from the planar edge of the chip, is that the fluid has a tendency to spread over the surface prior to the onset of electrospray reducing sensitivity and the efficiency of separations that may be performed on the device.  The hybridized structure consisting of a nanospray tip coupled to a microchip eliminated these problems.  The tips are also easy to replace extending the lifetime of the microchip if desired.  Fluid is continuously delivered at low flow rates ( 0.3-0.5 nL sec-1) solely by applying the electrospray voltage at one of the fluid reservoirs on the chip.


In the fast atom bombardment (FAB) ion source, ionization is accomplished by bombarding the sample, which is dissolved in a special liquid matrix with a high-energy atom beam.   This results in molecular sputtering leading to the desorption of secondary ions of the sample, from the liquid- vacuum interface, into the gas phase.  The ion beam is extracted from the ion source and mass analyzed, usually by quadrupole or magnetic sector analyzers in single or multistage combinations. The related fast ion bombardment technique, also known as liquid secondary-ion mass spectrometry (LSIMS), employs a stream of energetic ( ~ 30 keV) heavy ions (usually cesium) to accomplish the ionization.  FAB sources may be static or dynamic (continuous flow FAB).  In the static mode the beam of atoms, obtained in a special gun, bombards a conventional direct inlet probe modified to have a tip (ribbon of metallic or nonmetallic material ) upon which µL size drops of the dissolved material have been placed.  The primary atom beam of neutral argon or xenon (more energetic) to be used for bombarding the target is obtained in special FAB guns.  An argon or xenon ion beam obtained by EI is focused into a collision chamber where the energetic argon ions (~5 to 8 keV translational energy) become rapidly neutral atoms upon exchanging their charges with neutral argon or xenon atoms.  The remaining argon or xenon ions are deflected from the beam by a potential placed on a set of electrodes, while the momentum of the neutral argon or xenon atoms propels them to the probe. 

 Ar+. (fast) +  Argon (thermal) ---------   Ar (fast) + Ar+. (thermal)

  Because FAB only ejects ions into the gas phase that are already present in ionic form in the solution or ionized by proton transfer, the selection of the proper liquid matrix is critical.  Obvious requirements are that the sample must be soluble in the matrix and have a high diffusion rate through the matrix toward the surface.   Other chemical and physical considerations include acid/base characteristics, purity, vapor pressure, viscosity and surface tension.   Most FAB work has been done with glycerol, thioglycerol and m-nitrobenzyl alcohol N-formyl- 2-aminoethanol for non polar biomolecules  and diethanolamine (negative ions) as the matrix because they dissolve many polar compounds and have low volatility.  When sample-matrix interaction occurs, alternative matrix materials have to be explored.   

Effluents from HPLC can be analyzed by FAB with the continuous - flow FAB technique where the eluate is continuously mixed with the matrix and flows to the FAB target by passing through a needle where the tip is bombarded in a pulsed or continuous manner.    

                        MS50 CF-FAB probe design

Matrix-assisted Laser Desorption ionization               

Matrix-assisted laser desorption ionization (MALDI) is currently the most important technique for rapidly depositing enough energy into involatile analytes in the condensed phase to facilitate their ionization and desorption in the gas phase in one continuous step.  MALDI differs from FAB in four respects :                                          

a) The analytes are in a matrix and co-crystallized into solid phase rather than being dissolved and analyzed in a liquid matrix.

b) The analytes are exposed to an intense laser light rather than to a beam of energetic atoms or ions.

c) The laser light is applied in pulses of short duration in contrast to exposure to a continuous beam of energetic atoms or ions.

d) The analyte is ionized by energy transfer from the matrix rather than being "sputtered or ripped" from a liquid matrix.

In a typical MALDI analysis, a 10 µM solution is made  of the analyte in water or other appropriate solvent, and a 1 µL aliquot ( ~ 10 pmol ) is added to an ~ 104-fold excess of a matrix solution that has already been placed onto the target.  The solution is dried and allowed to crystallize so that it is comprised of microcrystals of a matrix into which the analytes are embedded.  The target is placed, through a vacuum interlock, into the path of the laser beam in the ion source.  An important practical advantage is the possibility of placing many samples in individual wells on a single target allowing for rapid analysis of multiple samples.  In commercial Maldi sources, the pulsed laser light of < 10 nsec duration is often produced by a nitrogen laser at 337 nm or a Q-switched Nd: YAG laser ( with tripled or quadrupled fundamental wavelength at 354 or 266 nm).  The laser light is focused onto the solid sample that contains both the matrix and the analyte.  The exposure usually consists of 50 to 200 single shot pulses.  Each pulse leaves a small crater in the target region. As the fluence (irradiance) is increased, a sharp threshold is reached at which analyte ions suddenly appear, e.g 10 mJ/cm2 (10 ns pulse width) for proteins.             Initially the matrix for MALDI was nicotinic or cinnamic acid or a simple derivative of these compounds.  Experimentation revealed that the matrix material had major effects on the efficiency of ionization.  In addition of being a good absorber of energy at the wavelength of the laser , the matrix has several distinct roles:

1. absorption of most of the laser light, thus minimizing sample damage by the laser     irradiation.

2.  vaporization when irradiated, thus causing some of the co-crystallized analytes to      vaporize by transferring energy in a controlled manner (ablation step)

3. participation in the ionization of the vaporized analytes in the gas phase.

4. serving as a co-crystallization agent for the analyte.

5. reducing intermolecular forces and minimizing the aggregation of the analyte         molecules by being present in a vast excess over the sample.

Matrices such as nicotinic, cinnamic, 4-methoxy cinnamic acid or 3-hydroxypicolinic acid, are typically low molecular weight organic acids.  

In TOF mass spectrometers, the gas phase ions produced in MALDI ion sources are subjected to controlled extraction and acceleration to a fixed energy by a strong electrostatic field (25 to 30 KeV).  The ion packets thus generated are focused by plates at high voltages (Einzel lenses) into the field-free region of the flight tube of a TOF analyzer for subsequent determination of the m/z values of the separated ions. TOF anlyzers are well suited to handle the ion packets produced by the discontinuous MALDI process.  Ion traps and FTICR analyzers have also been employed.  

There at least six advantages of MALDI ionization with TOF analysis:

1. very high mass range, to 1 MDa or higher

2.  high sensitivity at low pmoles and often to amole levels

3. suitability for mixture analysis because the technique is not specially subject to suppression or discrimination effects or to formation of multicharge states.

4. ability to do analysis in minutes (not including sample preparation)

5. little or no analyte fragmentation

6. reduced need for involved sample purification because a reasonable tolerance for salts and other common biochemical additives, often to mM concentrations.

Background ions from the matrix may be a problem for analytes with MW < 1000 Da

The recent ability to conduct MALDI at atmospheric pressure ionization (AP-MALDI) involves the pneumatically assisted transfer of ions from the region at atmospheric pressure to high vacuum by a stream of nitrogen. Sample preparation for AP-MALDI (including matrix selection) is similar to that with conventional MALDI.  When used with an orthogonal acceleration-TOF analyzer, the advantages of AP-MALDI include the possibility of using the same instrument for both ESI and MALDI analyses.  A disadvantage is increased sample consumption with respect to vacuum MALDI. 





Direct Laser Desorption Ionization

In this technique, analyte molecules are desorbed as intact molecules with the aid of laser beams of 1 to 200 ns duration pulses, focused on 10 to 300 µm o.d. spots at a variety of incident angles (30° to 75°).  Ion sources have been designed with a variety of lasers, including UV lasers, such as nitrogen (337 nm), excimer (at various wavelengths in the 193 to 351 nm range) and IR emitting lasers.  Instruments providing direct laser desorption, such as the laser microprobe analyser ( e.g LAMMA),       have been used to study the composition of thin (0,1 mm) biological sections, prepared in the same way as for transmission electron microscopy.

Resonance - Enhanced Multiphoton Ionization (REMPI)

In this technique the energy for ionization is obtained by the simultaneous absorption of two or more UV laser photons.  The two-photon energy must exceed the ionization energy of the analyte.  The efficiency of the two-photon absorption , i.e, the yield of ionization, may be increased by several orders of magnitude if the photon energy (laser wavelength) is in resonance with the analyte's UV-spectroscopic transition.  In addition to providing high sensitivity, REMPI is also highly selective because only those compounds can be analyzed that exhibit the required specific UV transition at the laser wavelength applied.  REMPI is a soft ionization technique, hence there is virtually no fragmentation.  Since REMPI is a pulsed type method of ionization, TOF is an ideal mass analyzer.  REMPI is a two-dimensional technique, combining both optical and mass selectivity.  Advantages are high selectivity, sensitivity and speed.  Because it takes only ~ 20 to 40 µs to obtain a spectrum, the method is well suited for time-resolved analyses of target compounds or specific compound classes in complex samples.  An application is the real-time on-line monitoring of aromatic pollutants in waste incinerator flue gases.  



Chemical ionisation has become very popular for structural elucidation since it was first developed in the mid-1960s.  Here, the concept of ionisation relies on the interaction of ions with neutral molecules and the further production of new ions.  In general, the amount of fragments ions produced is much less than in electron-impact ionisation , since little internal energy is imparted on the ionised molecule.  Also for this reason, CI is considered as a soft ionisation technique. A schematic representation of a chemical-ionisation ion source is shown in the next figure. 

If ionization occurs with methane at a pressure between 0.1 and 1.0 torr by electron impact (EI), a molecular ion is initially created.

  CH4 + e ------- CH4+. + 2e

Since the methane is at a high pressure, there is a distinct possibility of the molecular ion colliding with another methane molecule.  When this happens the most likely reaction is as follows:

                          CH4+. + CH4  -----------    CH3. +  CH5+

Therefore, if a small amount of the sample to be analyzed is introduced into the ion source in the vapour phase, it is the species CH5+ that acts as the means of ionization of the analyte which can react as follows:

                        CH5 +  M  ------------       CH4  +   (MH)+    

The quasi-molecular ion (MH)+ , will have an m/z value which is 1 amu greater than the molecular ion and has a much lower internal energy than the molecular ion produced by electron-impact ion sources.  Fragmentation observed in CI are minimized compared to fragmentation observed in electron impact as illustrated for proline.  Modern mass spectrometers have combined CI and EI ion sources that can be readily changed by the turning of a handle.  In CI the concentration of methane is at least 1000 times higher than the one of the sample. There is a much higher probability of the sample molecule  colliding with a CH5+ ion than with an electron.  In the CI spectra other quasi-molecular than (M+H)+ may be produced resulting from ion-molecule reactions leading to other reactant ions which are able to react differently with the sample.

                              CH4 +.     -----------------    CH3+  +  H+

The fragment ion, CH3+ , may itself undergo an ion-molecule reaction to form the C2H5+ ion :

                          CH3+  +  CH4   ----------------    C2H5+ + H2

The C2H5+ ion can also produce a quasi-molecular ion as can ethylation of the sample molecules :

  C2H5+  + M  -----------    (M+H)+ +  C2H4   or    M + C2H5+ ---------    (M + C2H5)

When the proton affinity of M is lower than that of CH4, the following reactions may take place, resulting in a quasi-molecular ion of 1 mass unit less than the molecular ion.  This reaction is predominant in the higher alkanes. 

                      CH5+ + M   ---------------     (M - H)+  +  CH4  + H2   

and                C2H5+ +  M   ----------    (M - H)+  +  C2H6 

Other reactant gases that are used in CI are ammonia and isobutane .When ammonia is used as a reagent gas, analytes that are more basic than NH3 yield abundant (M + H)+ ions by proton exchange while less basic analytes yield (M + NH4)+ ions by attachment.  In the latter case, the most abundant ion in the spectrum is 18 Da higher than M.  Beneficial results may be obtained by exploring a variety of ion-molecule reactions through consideration of the functionalities of the analyte and judicious selection of the reagent gas.

Negative ion CI (NCI)     is, in most instances, a misnomer because it does not involve an ion-molecule reaction but occurs when an analyte with several electronegative atoms captures low-energy electrons present in the CI plasma.  True negative chemical ionization involving a chemical reaction is rarely used.



Historically, electron ionization (EI, electron impact, electron bombardment) has been the most commonly used method to ionize relatively small (< 1.2 kDa) molecules which can be vaporized without thermal decomposition ( ~ 1 mL volume, stainless steel, maintained at ~ 200 °C to prevent sample condensation)