University of Pittsburgh

Mass Spectrometry Introduction

Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles. It is used for determining masses of particles, for determining the elemental composition of a sample or molecule. The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measurement of their mass-to-charge ratios by using the one of a variety of techniques (e.g EI/CI/ESI/APCI/MALDI). 

 A) Brief introduction to Electron Ionization (EI) technique
EI is the most appropriate technique for relatively small (m.w.<600) neutral organic molecules which can easily be promoted to the gas phase without decomposition, i.e. volatile. Since EI samples are thermally desorbed to the gas phase and subjected to the high energy of EI, analytes must be thermally stable. The gas phase molecules enter into the ion source where they are bombarded with free electrons emitted from a filament (Figure 1). The electrons bombard the molecules causing a hard ionization that fragments the molecule, and turn into positively charged particles called ions. This is important because the particles must be charged to pass through the analyzer. As the ions continue from the source, they travel through an analyzer (electromagnetic/quadrupole/the ion trap) that filters the ions based on mass to charge ratio. The filter continuously scans through the range of masses as the stream of ions come from the ion source. A detector counts the number of ions with a specific mass. This information is sent to a computer and a mass spectrum is created. The mass spectrum is a graph of the number of ions with different masses that traveled through the analyzer.

Schematic diagram of EIMS

Figure 1. Schematic diagram of EI Source

B) Chemical Ionization (CI) technique
CI technique is especially useful when no molecular ion is observed in EI mass spectrum of a compound, and also in the case of confirming the molecular weight of a compound. CI technique uses nearly the same ion source device as in EI, except, CI uses tight ion source, and a reagent gas (Figure 2).

Schematic diagram of CI interface

Figure 2. Schematic diagram of CI source

Reagent gas is first subjected to electron impact to yield reagent gas ions.  These initial reagent gas ions further undergo ion-molecule reactions with neutral reagent molecules (R) to yield reagent selective ions (reagent plasma, e.g., RH+). When sample is introduced, the sample molecules (A) undergo ion-molecule reactions with reagent plasma to produce sample ions.  In general, reagent gas molecules are present in the ratio of about 100:1 with respect to sample molecules. Pseudo-molecular ions, [A+H]+ (positive ion mode) or [A-H]- (negative ion mode) are often observed. Unlike in EI method, the CI process is soft ionization and yields abundant quasi-molecular ions, with less fragment ions.   

                   Positive ion mode: RH+ + A ------> AH+ + G

                   Negative ion mode: [R-H]- + A ------> [A-H]- + G

The fragmentation pattern of protonated molecules obtained under CI conditions may be different from that of the molecular ions observed under EI conditions. In CI mass spectrometry the molecules of a vaporized sample are ionized by a set of reagent ions (reagent plasma) in a series of ion-molecule reactions. The energy transferred by these reactions is lower than the energy imparted by electrons in EI source, and therefore fragmentation of the sample molecules is greatly decreased. For this reason CI mass spectrometry has been finding increasing use as a tool for the molecular weight confirmation and for elucidation of structure of variety of organic compounds including differentiation of isomeric compounds.  Generally hydrogen (H2), methane (CH4), isobutane (iso-C4H10) and ammonia (NH3) are used as reagent gases in CI mass spectrometry; with all these CI gases the compounds form protonated molecule ion in their CI spectra. 

C) Electrospray Ionization (ESI) technique

ESI technique involves spraying of a solution of the sample through a highly charged needle called ESI capillary which is at atmospheric pressure (Figure 3). The spraying process can be streamlined by using a nebulizing gas. The charged droplets are produced in which the positive or negative ions are solvated with solvent molecules. Heat gas or a dry gas, usually called as desolvation gas is applied to the charged droplets to cause solvent evaporation. The desolvation process decreases the droplet size, leads to the columbic repulsion between the charges present in the droplet and further the droplet fission leads to the formation of individual gas phase analyte ions (that critical point known as the Rayleigh limit), that are guided through ion optics into the mass analyzer. 

ESI can produce singly or multiply charged ions. The number of charges retained by a particular analyte depends on several factors such as the size, chemical composition, and higher order structure of the analyte molecule, the solvent composition, the presence of co-solutes, and the instrument parameters. For small molecules (< 2000 Da) ESI typically generates singly, doubly, or triply charged ions, while for large molecules (> 2000 Da) ESI can produce a series of multiply charged ions. 

Schematic diagram of a typical ESIMS source phenomenon operating in positive mode

Figure 3. Schematic diagram of Electro spray ionization process

ESI is very suitable for a wide range of biochemical compounds including peptides and proteins, lipids, oligosaccharides, oligonucleotide, bio-organic compounds, synthetic polymers, and intact non-covalent complexes. 

D) Atmospheric pressure chemical ionization (APCI) technique
PCI has also become an important ionization source because it generates ions directly from solution and it is capable of analyzing relatively non-polar compounds. Similar to electrospray, the liquid effluent of APCI (Figure 4) is introduced directly into the ionization source through APCI probe. Sample solution undergoes nebulization to form an aerosol spray of fine droplets and it is rapidly heated in a stream of nitrogen desolvation gas before emerging from the probe as a stream of desolvated/vaporized sample plume. Solvent/Reagent ions are formed in the region of the corona discharge needle. These ions react with analyte molecules to form singly charged protonated or deprotonated analyte ions.

Schematic diagram of APCI interface

Figure 4. Schematic diagram of APCI process

In general, proton transfer occurs in the positive mode to yield [A+H]+ ions. In the negative ion mode, either electron transfer or proton loss occurs to yield M- . or [A-H]- ions, respectively. The moderating influence of the solvent clusters on the reagent ions, and of the high gas pressure, reduces fragmentation during ionization and results in primarily intact quasi-molecular ions. Multiple charging is typically not observed presumably because the ionization process is more energetic than ESI.

E) Atmospheric solid Analysis probe ionization (ASAP)

ASAP ionization is used for the direct analysis of volatile, semi volatile, solid and liquid samples at atmospheric pressure, it is also known as one of the atmospheric pressure ionization (API) technique. This technique utilizes the heated nitrogen desolvation gas to vaporize the sample and a corona discharge for sample ionization and it is a useful replacement for the conventional EI/CI vacuum insertion solids probe. The sample is placed on surface of glass capillary tube and the glass tube inserted into source just adjacent to discharge corona needle which is provided 2 to 4kV. Reagent ions are generated at the region of corona needle due to voltage discharged process between molecules. Reagent ions react with analyte molecules to form ions.


Ion Formation


Schematic diagram of MALDI source

Figure 5. Schematic diagram of ASAP Process

F) Atmospheric pressure photo ionization (APPI)

APPI also one of the atmospheric pressure ionization (API) technique, it is good for low to moderately polar compounds like polycyclic aromatics, steroids, some mycotic toxins etc. APPI is relatively less popular ionization technique for LCMS instruments when it compared with ESI and APCI. In APPI technique samples are ionized by using UV light, molecules interacted with photon beam of UV light with vapors of nebulizer liquid solution. Analyte molecules (A) absorb a photon (hv) and become an electronically excited molecule. If the ionization energy (IE) of analyte molecules is lower than the energy of photon, then the analyte molecule releases energetic electron and become the radical cation.

A+hv → A* 

A* → A+∙ + e- (IE < hv) 

If IE of analyte is more than photon (hv), A* may undergo photodissociation, photon emission. In such cases, use the substance called Dopant (D) which is promoting the ionization of analyte.

D+hv →  D+

D+. + A → D + A+∙ if EAA > EAD   (Electron affinity)

D+. + S → [D-H]∙ + [S+H]+   if PAS > PAD  (Proton affinity) 

[S+H]+ → S + [A+H]+      if PAA > PAS

Schematic of a triple quadrupole mass spectrometer

Figure 6. Schematic diagram of APPI source

G) MALDI technique

Matrix-assisted laser desorption/ionization (MALDI) is a technique to allows the high molecular weight compounds such as organic macro molecules and labile bimolecular into the gas phase as intact ions. MALDI is one of the recent developments of soft ionization techniques in the field of mass spectrometry. It can desorb intact analyte molecular ions with relative masses up to 300KDa. In MALDI-MS analysis, the analyte is first co-crystallized with a larger excess of a matrix compound (CHCA, DBA, Sinapic acid etc), after which, on laser radiation of this matrix-analyte preparation results in desorption of the matrix as a plume, which carries the analyte along with it into gas phase (Figure 7). Thus the matrix plays a key role by strongly absorbing the laser light energy and causing, indirectly, the analyte to vaporize. The matrix also serves as a proton donor and acceptor, acting to ionize analyte in both positive and negative ionization modes, respectively. The TOF analyzers are typically used with the MALDI ionization source.

Figure 7. Schematic diagram of MALDI source

Figure 7. Schematic diagram of MALDI source