Electron Ionization (EI) Analysis
Electron ionization (EI), is the oldest and most established of the ionization techniques. Electron, ionization is achieved through the interaction of an analyte with an energetic electron beam that results in the loss of an electron from the analyte and the production of a radical cation.
M + e- --> M+ + 2e-
EI is the most appropriate technique for relatively small (m.w.<700) neutral organic molecules which can easily be promoted to the gas phase by heating 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 both thermally and energetically stable. Traditionally a major percentage of the analytes ionized by EI are not completely energetically stable and a portion of the analytes break apart or fragment as a relaxation pathway to remove the excess internal energy obtained during ionization. Fragmentation is not necessarily a bad thing since fragmentation provides structural information as well as a "finger print" means of identification. The EI ionizing energy typically used is 70 eV to promote the greatest sensitivity and to produce molecular and fragment ions used for chemical characterization and identification. Generally, the electron energy is variable between 0 and 100 eV. The ionization energy can be lowered to 15-30 eV in order to reduce fragmentation and/or permit the observation of molecular ions of more energetically labile species. However, reducing the ionizing energy reduces the overall ionization efficiency/sensitivity significantly[1], and as a result, requires more sample than at 70 eV.
Electron Ionization (EI) Analysis
Several books on basic interpretation of electron ionization mass spectra are available.[6,7] Reference libraries of mass spectra, in the form of the NIH/EPA Mass Spectral database and others, are available.[8] Two printed copies of the NIH/EPA Mass Spectral Database (Standard mass spectra of approximately 44,000 compounds) are available in the Laboratory, as well as being available for computer search. A copy of the Wiley Registry containing over 125,000 compounds is also available for searching of EI mass spectra online. Additional references that might be helpful to data interpretation are provided.[9-11] The book, Mass Spectrometry of Organic Compounds,[11] is an encyclopedic collection of fragmentation rules on a wide variety of classes of organic compounds and might also be helpful.
Chemical ionization (CI) Analysis
Chemical ionization[12,13] evolved as a means to ionize compounds to energetically labile for traditional EI analysis. Conceptionally, CI can be thought of as EI performed in the presence of a large excess of a reagent gas. The excess reagent gas is more likely to interact with the EI electron beam than the analyte. Munsen and Field reported that an analyte present in a relatively small amount (1:1,000) in a gas plasma (reagent gas) generated by EI underwent gas-phase acid/base types of reactions, i.e. proton transfer.[12] The gas-phase chemical reactions result in the removal or donation of a charged species (generally H+) from the reagent gas to the analyte to yield a pseudo-molecular ion (positively or negatively charged). The probability of proton exchange is based on the gas phase basicities and proton affinities of the analyte and the reagent gas.
M + [Reagent gas + H]+ --> [M + H]+ + Reagent gas
M + [Reagent gas - H]- --> [M - H]- + Reagent gas
The reaction energies of the gas-phase acid/base reactions are approximately 5 to 10 eV, which is significantly lower than EI. The lower energy ionization associated with CI results in more abundant molecular ions. Although CI is capable of generating molecular weight information from labile species, it still requires that the sample be volatile which could hinder the detection of thermally unstable analytes. Typical reagent gases used in CI are methane, isobutane, ammonia, and hydrogen. An extended list is provided by Watson.[1] CI is most applicable to analytes that are moisture sensitive and/or reactive with hydroxyl groups present in many common FAB matrices.
Chemical Ionization Schematic
Gas Chromatography - Mass Spectrometry
Basic information regarding the GC/MS experiment has been reported.[14,15] This technique allows for the on-line separation of complex mixtures of volatile species (nonvolatile if derivatized) followed immediately by mass selective detection. GC/MS provides a higher degree of specificity than traditional GC detectors and has been extensively utilized in environmental, petroleum, synthetic organic, and biological applications. Most modern GC/MS experiments are performed using capillary chromatographic columns to minimize pumping requirements of the mass spectrometers and to exploit the higher separation efficiencies of these smaller diameter columns. Capillary GC columns are utilized for their high resolving power since these columns can have plate numbers in excess of 100,000 per meter. These columns are typically 30 meters in length, internal diameters of approximately 250 mm, and coatings of < 1 µm. Most GC/MS analyses are performed using EI, but GC/MS using CI ionization can be performed but is more difficult.
Fast Atom Bombardment
Fast atom bombardment (FAB) as a mechanism for ionization was first described in the scientific literature in 1982[16-18] and rapidly became a very successful technique in mass spectrometry. FAB reached this elevated level of popularity by providing an efficient means to analyze polar, ionic, thermally labile, energetically labile, and high molecular weight compounds that are not amenable to normal EI/CI analysis. Thus, FAB found an extreme utility in the analysis of polar biomolecules and natural products. Ionic compounds can be analyzed by FAB, whereas they are intractable by EI. Depending upon whether the cation or the anion is of interest, either positive-ion or negative-ion FAB/MS analyses can be performed.
In the FAB experiment, a sample that has been dissolved in a suitable matrix [19-21] is inserted into the mass spectrometer and bombarded with 4-10 keV Ar or Xe atoms. The use of a liquid matrix yields more intense and longer-lived analyte signals than those generated by solid state, secondary ion mass spectrometry (SIMS).
Following ionization, the selected positive or negative ions are extracted, accelerated, and then mass analyzed. The FAB mass spectrum is characterized by peaks corresponding to matrix cluster ions, analyte ions, ions representing impurities, and ions of other matrix modifiers (e.g., trifluoroacetic acid) that were added in an attempt to increase the analyte ion abundance. The observed positive pseudo-molecular ion peaks are typically adducts formed with cations (i.e., [M+H]+ and [M+Na]+), whereas typical negative pseudo-molecular ions observed are [M-H]-. These adduct ions are generated by desorption of pre-formed ions in solution and/or gas phase cation exchange similar to that described in the CI section.
Liquid secondary ion mass spectrometry (LSIMS) is a similar particle desorption technique to FAB and differs only in the nature of its primary particle source. LSIMS uses a higher enegy primary ION beam (generally 10 to 50 keV Cs+) whereas FAB uses neutral Ar or Xe as the primary particle. Higher energies are used to compenstate for deaccleration of the charged primary particle as it enters the electrostatic field of the ion source. Early beliefs were that use of charged primary particles would induce detrimental charging of the electrically isolated or "floated" probe resulting in undesireable electrical discharges, however time has shown that LSIMS has several advantages over FAB including lower source pressures due to origination of the primary ion by thermal desorption of a solid as apposed to a gas fed saddle field gun used in FAB. The LSIMS source also has the added advantage of focusing and steering of the charged primary particle beam using electrostatic potentials that do not affect the neutral FAB particles.
Successful ionization by FAB is deeply dependent on the matrix selected for the analysis. The successful matrix must meet several requirements. The primary requirement is that the sample MUST be soluble in the matrix. In addition, the matrix must be a low volatility solvent which will not rapidly evaporate in the high vacuum system of the mass spectrometer. Thus, the matrix/sample will maintain its liquid nature in the vacuum system. Cook and coworkers [20] discuss the physical and chemical properties of common matrices while Gower [21] presents a summary of matrices reported in the literature and the compound classes with which they have been successful. Several successful matrices which have been widely used are glycerol, thioglycerol, m-nitrobenzyl alcohol, 18 -crown-6 crown ether, 2-nitrophenyloctyl ether, sulfolane, diethanolamine, and triethanolamine. Fenselau and Cotter summarize the mechanisms and fundamentals of FAB in a recent review.[22]
Matrix Assisted Laser Desorption Ionization Time of Flight
A relatively new ionization technique is matrix-assisted laser desorption ionization, more commonly referred to as MALDI. This desorption technique first introduced in 1988 by Hillenkamp and co-workers[25] is very similar to FAB, but it utilizes photons instead of particles to desorb analyte molecular ions, [M+H]+, from a crystalline matrix. The primary role of the matrix is to absorb the incident radiation which results in rapid heating of the crystal lattice on a time scale (femtoseconds) that is faster than thermal equilibration of the matrix-analyte lattice. This process results in desorptionor transfer to the gas phase of matrix and intact analyte ions. The process responsible for ionization is still under current investigation and discussion but revolves around cation transfer similar to CI and FAB. MALDI, utilized in conjunction with time-of-flight (TOF) mass spectrometry, has the ability to generate molecular ions from pmol to fmol quantities of species with molecular weights in excess of 100 kDa. This is a very distinct advantage over FAB performed on magnetic sector instruments. The capability of MALDI-TOF mass spectrometry to analyze such large molecules has found a high degree of usefulness in the analysis of high mass biomolecules[27-29]. The resolution limitation of TOF mass spectrometry is the only limiting factor of this technique, but this limitation is quickly decreasing due to advances in instrumental technologies.[30] To date, we have successfully utilized MALDI-TOF to study molecules such as large peptides and porphoryn based molecules.
MALDI Desorption/Ionization