IUPAC Terms and Definitions in Mass Spectrometry The third Draft Document released in August 2006 by the IUPAC task group on MS Terms has fixed the basic definitions to be adopted and those to be abandoned in the MS field: Unified atomic mass unit, u - A non-si unit of mass defined as one twelfth of the mass of one atom of 12 C in its ground state and equal to 1.6605402(10) 10-27 kg. The term atomic mass unit (amu) is deprecated. The Dalton (Da) is usually accepted. Nominal mass - Mass of an ion or molecule calculated using the mass of the most abundant isotope of each element rounded to the nearest integer value and equivalent to the sum of the mass numbers of all constituent atoms (mass number: sum of the number of protons and neutrons in an atom, molecule or ion)
Exact mass - Calculated mass of an ion or molecule containing a single isotope of each atom, most frequently the lightest isotope of each element, calculated from the masses of these isotopes using an appropriate degree of accuracy. Monoisotopic mass - Exact mass of an ion or molecule calculated using the mass of the most abundant isotope of each element. Average mass - Mass of an ion or molecule calculated using the average mass of each element weighed for its natural isotopic abundance.
mass formula nominal exact average C 3 H 8 O 60 60.05754 C 2 H 4 O 2 60 60.02112 Accurate mass - experimentally determined mass of an ion that is used to determine an elemental formula. Note: accurate mass and exact mass are NOT synonymous. The former refers to a measured mass, the latter to a calculated mass.
Isotopic distribution: carbon Natural abundance (%): 12 C: 98.90 13 C: 1.10 14 C: 0.0000000001 100 90 80 70 60 50 40 30 20 10 0 12 C 1 13 100 90 80 70 60 50 40 30 20 10 0 120 10 12 C, no 13 C C 10 9 12 C, 1 13 C 121 122 100 90 80 70 60 50 40 30 20 10 0 1,200 1,202 C 100 1,204 1,206 100 90 80 70 60 50 40 30 20 10 0 12,000 12,010 C 1000 8 12 C, 2 13 C 12,020 12,030 Adapted from http://ms.mc.vanderbilt.edu/tutorial.htm
Isotopic distribution: effects on large molecules When molecules with high molecular weight are considered, the difference between exact mass and average mass becomes progressively higher: Alanine oligo/poly-peptides
The available mass resolution can influence significantly the appearance of the isotopic distribution, as shown in the following example for glucagon, a 29-amino acid polypeptide hormone: High resolution Low resolution
Ionization Techniques Several techniques are available for the generation of charged molecules either in the gas phase, from volatile and non-volatile analytes, or from solid/liquid samples, under vacuum or in atmospheric conditions: Electron ionization (EI) Chemical Ionization (CI) Field ionization (FI) Nebulization Ionization Thermospray Ionization (TSI) Electro-Spray Ionization (ESI) Field Desorption (FD) Plasma Desorption (PD) Fast Atom Bombardment (FAB) Matrix Assisted Laser Desorption (MALDI) Desorption/Ionization on Silicon (DIOS) Desorption Electro-Spray Ionization (DESI)
The ionization source is one of the key components of a mass spectrometer:
Gas phase vs. Desorption ionization (for molecular mass spectrometry) Gas phase: the sample is first vaporized and then ionized, so that the method is applicable only to volatile compounds, typically with molecular meight lower then 1 KDa. Desorption: applicable to nonvolatile and thermally unstable samples (although volatiles can also be used). Hard vs. soft ionization methods Hard methods: energy transferred to analyte molecules is very high and produces ions in highly excited energy states. Relaxation produce estensive fragmention. Soft methods: low energy transferred; little (if any) fragmentation observed.
Soft vs hard ionization: effect on mass spectra The appearance of mass spectra obtained for a given compound depends strongly on the method adopted for ion formation:
Effect of ionization technique When possible, different ionization techniques should be used for the same analyte, since the information provided by each one is often complementary to that obtained from the others.
Pressure requirements for ionization sources Most ionization methods in mass spectrometry require that a relatively low pressure is achieved inside the ionization chamber. cm/s 1 s 2 volume (covered in 1 s) = 2 Actually, the relative motion between colliding molecules must be considered
Magnitude of relative velocity: Average velocities must be considered, since molecules do not travel at the same speed:
Vacuum Technology When electrons, atoms/molecules or ions are involved in the ionization of analyte molecules (like in EI, CI and FAB), the estimated low mean free paths imply that collisions occur between ionizing particles and neutral species, thus lowering the energy of the formers and, consequently, ionization efficiency. Rotary vane pumps usually provide vacuum conditions suitable for low pressure ionization (10-3 /10-4 torr)
Lower pressures (down to 10-7 torr), usually required for mass analysers and ion detectors, can be achieved using different devices, like turbomolecular or oil diffusion pumps. Turbomolecular pump Oil diffusion pump
Desirable properties for ion sources/ion beams generation of ion beams compatible with analyzer geometry compliance with detector sensitivity (10-7 - 10-14 A) generation of ion beam with small energy spread high ionization efficiency selectivity against unwanted ions bipolarity (ability to generate either positive or negative ions) ability to ionize also high molecular weight compounds control of fragmentation absence of memory effects stability of ion emission with time minimization of chemical procedures required before MS analysis (sample pretreatment)
Electron (impact) ionization (EI) IUPAC definition of Electron Ionization:
Scheme and representation of a EI source
Exploded view of VG Trio 2000 electron (chemical) ionization source 7 - ion exit plate Focus plate Source block 4 ion extraction plate
Quadrupole entrance Vacuum lock GC transfer line LC interface Solid probe Diffusion pump
Focus plate Ion beam exit
Solid probe; LC interface (Particle Beam) trap (anode) repeller filament (cathode) GC column inlet
In a EI source electrons are emitted by a hot W or Re filament, due to thermoionic effect, and accelerated towards a anode target by a potential difference. Along their path electron interact with gaseous molecules coming from the sample introduction system (including the outlet of a GC column). Actually, due to the dimension difference, their interaction with a molecule is not an impact in the common meaning of this word. Ionization efficiency (IE) is the ratio between the number of ions formed and that of neutral molecules entering the source. Electron current and energy have a remarkable influence on the ionization efficiency.
Filament emission characteristics A roughly ohmic behaviour is observed until the filament blows, due to excessive heating
Aspects related to electron energy Electron energies typically adopted in EI range between 20 and 70 ev. At least 10 to 20 ev are usually transferred to a neutral molecule during interaction with these electrons. Since the ionization potential of an organic molecule is typically lower than 10 ev, the interaction leads to the generation of a molecular ion, M. +, having excess energy on it, responsible for its subsequent fragmentation. It is worth noting that the wavelengths related to ionizing electrons ( = h/mv) are in the range 2.7-1.4 Å (20-70 ev). At very high energies the wavelength becomes very small and the molecule becomes transparent to the electron. In other words, there is not enough time for the electron-neutral interaction and energy transfer to occur.
A key parameter in determining the ionization efficiency is the ionization cross-section, that is increased with electron energy: The number of positive ions generated by EI is given by: x + where + = cross section for positive ionization, N 0 = gas density, N e = number of electrons, x = electron path.
Actually ionization is just one of several phenomena occurring during electron-neutral interactions. Their cross-sections for CF 4 are shown in the following graph: Note that ionization is relevant only between 20 and 600-700 ev electron energies.
A detailed graph of CH 4 ionization cross-sections as a function of electron energy, calculated by different authors, is shown in the following:
The plot of Ion current vs. electron energy clearly resembles the trend observed in the rising part of the cross-section vs energy plot. Three regions can be distinguished: A) threshold region, principally molecular ions produced B) production of fragment ion becomes important C) routine operation for EI: mostly fragment ions observed
Ionization efficiencies for different compounds have qualitatively similar trends with respect to electron energy, yet the absolute values can be orders of magnitude different. The number of ions generated by EI (mostly fragments) is generally maximum for electron energies comprised between 50 and 100 ev.
Time scales of electron ionization The time available for the electron-neutral interaction is strongly dependent on the electron energy. 70 ev electrons have velocities higher that 5 10 6 m/s; if a 10 Å (10-9 m) diameter is assumed for a molecule, a transit time of 2 10-16 s can be easily estimated. This time scale is consistent with that pertaining to electronic transitions, whereas vibrational transitions require at least 10-12 s. The Franck-Condon principle can be applied: the molecule nuclei remain frozen in their positions during the interaction. Only later internal energy excess will lead to fragmentation, eventually occurring outside the source (Post Source Decay, PSD):
Among activation methods used in mass spectrometry, EI is the one involving the shortest times at all: In the scheme green and blue (dotted) boxes represent timescales for vibrational and electronic excitation, respectively:
Energy transfer and distribution accompanying ionization Ionization of a molecule occurs very rapidly, thus the bond lengths in the molecule are assumed not to change during the ionization process. The ionization process obeys the Franck-Condon principle, which states that electronic transitions occur with no change in nuclear configuration. This corresponds to vertical transitions in the following diagram, relevant to a diatomic molecule: Transition a shows ionization from the ground state of the molecule to a higher vibrational state of the corresponding ion.
If the transition occurs to a state whose energy is higher than the dissociation energy (D) then the ion will be fragmented. In the case of transition b the ground state of the ion is dissociative, thus no intact molecular ion will be formed. When polyatomic molecules are considered, several different fragmentation pathways can occur. ABCD.+ A + + BCD. A. + BCD + fragm. BC + + D. rearrang. CD. + AB + A. + B + fragm. ADBC.+ A + + DBC. A + + B.
Unimolecular reactions Reactions in an EI source are assumed to be unimolecular, since the pressures are too low to enable significant interactions between molecules and ions. Types of ions detected 1) Molecular ions (precursor/parent) reach the detector without fragmentation 2) Product ions (fragment) - fragment by decomposition reactions in the source 3) Metastable ions ions that fragment by decomposition reactions after leaving the source
Factors dictating the fragmentation extent (and hence the appearance of the spectrum): - the internal energy available in the molecular ion; - the time taken for the ion to be transmitted from the source to the detector. Time scales The time factor needs to be considered since different processes occur with different time scales or rate constants; the rate constants for various types of reaction have to be considered in relation to the time spent by ions in the source and analyzer regions of the spectrometer. Residence time of ions in the source: 10-6 s 5 10-6 s, depending on source parameters and accelerating voltage. Time taken for an ion to traverse the mass analyzer region: ca. 10-5 s. Decomposition times are expressed in terms of a rate constant, which can be expressed as the reciprocal of the ion lifetime, e.g. the rate constant for a decomposition of an ion with a lifetime of 10 µs is 10 5 s -1.
1) Fragment ions will only be detected if the parent ions possess sufficient energy for decomposition; the rate constant for decomposition is > ~10 4 s -1. 2) Parent ions will be detected only if the rate constant for decomposition is < 10 4 s -1. 3) For rates close to 10 5 s -1, metastable decompositions occur between the source and detector. 4) For rate constants of 10 6 s -1 or greater, decompositions occur in the source, giving rise to fragment ions.
Ionization events can be classified as: soft, if little excess energy is transferred to the ionised molecule: a molecular ion is formed; hard, if a significant fragmentation occurs.
The distinction can be made also by looking at potential energy curves:
Fragmentation is less pronounced when lower electron energies are adopted, thus favouring the molecular ion presence. On the other hand, the absolute intensity is somewhat reduced at lower electron energies, suggesting a lower ionization efficiency. 70 ev 15 ev
Electron energy affects the internal energy distribution of the molecule interacting with an electron. The model internal energy distribution P( ) resulting from interaction of an organic molecule with 70 ev electrons can be depicted as follows: The maximum internal energy that an ion can acquire in this case is 70 ev minus the ionization energy (IE). Actually most ions acquire small internal energies.
Lower electron energies imply a modification of the internal energy distribution for the molecule involved in ionization: As a consequence, the fragmentation routes available to the parent ion are different according to the electron energy. Such information can be easily described by two complementary approaches: the so-called breakdown curves, i.e. plots of fragment ion abundances as a function of the internal energy of the originally generated ion; the Wahrhaftig diagrams.
Breakdown curves Breakdown curves can be determined by effecting interactions of the compound of interest with electrons having a single energy, scanned over an interval, and recording the relative intensity of generated fragments. 70 ev The correlation between breakdown curves and EI spectrum is shown for n-propanol.
Wahrhaftig diagram for a generic ABCD. + ion In the upper side of a Wahrhaftig diagram (developed by the American chemist Austin L. Wahrhaftig) the distribution of internal energies for the parent ion is reported: In the lower side the probability of a specific fragmentation pathway is shown in the form of a rate constant: IE(M) = ionization energy
A rate constant of ~10 6 s -1 or greater is necessary for ion-source decomposition (lifetimes ~ 10-6 s or less): log K = 6 on the curve for the.. reaction M + AD + defines the. minimum M + internal energy. required for AD + formation,. indicated as E s (AD + ). E s is usually somewhat higher than the reaction critical energy, E 0 ; the latter is defined as the difference between the zero-point energy of M +. and that of the activated complex for the AD +. generation. AD +. M +. In the diagram m* represents a metastable ion, the corresponding required miminum internal energy is indicated as E m.
Vertical lines traced for each E s value divide the total area underlining the P(E) curve into different regions: each region area is related to the abundance of different generated ions Note that the area relevant to ions. like AD + and AB + refers to their initial amount, as they could eventually generate further fragments. A similar scenario is observed for metastable ions, like F*: If the ionizing electron energy is lowered, the relative proportion of M +. ions that do not dissociate will increase, although the total signal in the MS spectrum will be lower.
Thermodynamic vs kinetic effects The observed Wahrhaftig diagram shows a peculiar effect: the reaction of lowest critical energy does not necessarily produce the mostabundant ion. In particular, [AB + ] > [AD +. ] although the M +. AD +. process has a lower critical energy than M +. AB+. This finding can be explained if the transition states for the two processes are compared: In the rearrangement forming. AD + two new bonds are formed, offsetting the energy required to cleave A B and C D. The critical energy for AB + formation is higher, since this requires B C cleavage:
On the other hand, the steric requirements for AD +. formation are much more stringent than those for AB + formation. It can be said that the first process has the more favorable enthalpy, whereas the second has the more favorable entropy. For higher-energy ABCD +. ions, the B C bond dissociation can take place whenever sufficient energy accumulates in this bond. For AD +. formation, the energy requirements must be met at the same time that A and D are within bonding distance, which is true only for a small proportion of all possible conformations of ABCD +.. These offsetting enthalpy and entropy effects generally lead to a substantial number of competing primary reactions as well as consecutive secondary and further reactions, thus the mass spectrum of a large molecule can shown even hundreds of peaks. Small changes in molecular structure result in large differences in peak abundances. For this reason, when interpreting an unknown spectrum it is helpful to study the spectra of closely related compounds.
Effect of molecular structure When the same electron energy is adopted, the fragmentation pattern is significantly affected by the molecular structure, even when isobaric species (i.e. having the same molecular weight) are considered: As a general rule, molecules characterized by the presence of aromatic rings, especially condensed ones, do not undergo a significant fragmentation.
Due to: the purely physical nature of the phenomenon the involvement of gas-phase unimolecular reactions electron ionization is characterized by high reproducibility, as shown in the following comparison between n-propanol EI spectra (70 ev):
Quasi-equilibrium theory (QET) The quasi-equilibrium theory (QET) provides a generally accepted physical description of mass-spectral behavior, usually able to explain breakdown curves or Wahrhaftig diagrams. According to this theory, the basic steps of the ionization-fragmentation process are: 1) ionization of the molecule, taking place in approximately 10-16 s, yielding the excited molecular ion without change in bond length (a Franck-Condon process); 2) except for the smallest molecules, rapid transitions between all the possible energy states of the molecular ion, leading to a "quasi-equilibrium" before ion decomposition; 3) decomposition of the molecular ion, depending only on its structure and internal energy, and not on the method used for the initial ionization.
Exceptions to QET are usually found for: 1) dissociations involving small molecules, having fewer, more widely separated states 2) excited electronic states that are similarly "isolated" 3) very fast decompositions (<10-11 s). The basic parameters involved in QET are: the thermochemical appearance energy, AE: the minimum energy necessary to produce a fragment from the ground-state neutral molecule.; the critical energy, E 0 : the minimum internal energy of M +. required for the decomposition to yield a specific fragment. Note that AE = IE(M) + E 0 where IE = ionization energy; the rate constant k for molecular ion decomposition, involved in the kinetic relation: ln[m +. ]0 /[M +. ] = kt.
Within QET a E S value reported into Wahrhaftig diagrams corresponds to the internal energy of precursor ions which lead to an equal probability of leaving the ion source (i.e. equal rate constants) for adjacent ions along the P(E) curves. The E s -E 0 difference is defined as: kinetic shift when referred to the fragment having the. lowest E 0 value (AD + in this case) competitive shift, when referred to higher E 0 fragments (like AB + ) Kinetic shift Competitive shift The kinetic shift varies between 0.01 and 2 ev; the competitive shift can be higher.
Metastable ions The decompositions of metastable ions (MI) in a field-free drift region have rate constant values just below the minimum required for ion-source decomposition, i.e. in the 10 5-10 6 s -1 range. In the Wahrhaftig diagram shown before metastable ions arise from molecular ones having internal energies comprised between E m (AD +. ) and E s (AD +. ):
In a mass spectrum metastable ions appear as weaker, broader peaks occurring at non-integer m/z values (m*): The following equation can be written for an ion M 1+ that decomposes to M 2 + in the field free region of the mass spectrometer: M 1+ = M 2+ + (M 1 - M 2 ) The M 1 -M 2 difference corresponds to the mass of the neutral fragment released during decomposition.
If v 1 and v 2 are the velocities for M 1+ and M + 2, respectively, and the assumption is made that also the neutral species has a v 2 velocity, conservation of momentum leads to the following equation: m 1 v 1 = m 2 v 2 + (m 1 - m 2 ) v 2 which implies that: m 1 v 1 = m 1 v 2 v 1 can be calculated if the M 1 ion charge, z 1, and the accelerating potential, V, are known: z 1 V = ½ m 1 v 1 2 On the other hand, the ion actually entering the mass analyser is M 2. If a magnetic sector is used, the following equation applies: m 2 v 2 2 /r = B z 2 v 2
v 1 and v 2 can be calculated from the last two equations : v 12 = (2z 1 V)/m 1 v 22 = B 2 z 22 r 2 /m 2 2 Since v 1 = v 2 and z 1 = z 2 = z, we can write: (2zV)/m 1 = (B 2 z 2 r 2 )/ m 2 2 m 2 2 /(m 1 z) = B 2 r 2 /2V The last equation shows that the M 2+ ion arising from the decomposition of M 1+ behaves like an ion with mass m 22 /m 1, that is lower than m 2. For example, for m 1 = 60 and m 2 = 59 (loss of hydrogen), m 22 /m 1 = 58.02. Due to the large energy dispersion characterising the generated ion, a peculiar large peak is observed in the mass spectrum: Metastable M 2 + M 2 + M 1 + m/z
In several cases metastable ions can provide useful information about fragmentation patterns. The electron ionization spectrum of benzene provides an interesting example: 76 In this case the m/z = 78 is related to the molecular ion (M 1 ), whereas m/z 77 is referred to the phenyl carbocation (C 6 H 5+ ), M 2, arising from H loss. If such loss occurred also in the field-free region of the mass spectrometer a metastable ion with m/z = 77 2 /78 = 76.0 would be observed. Such a peak is actually found in the spectrum. In many cases both M 2+ ions and the corresponding metastable ions are observed in EI spectra.
The procedure can be reversed: if the m/z ratios of metastable ions are known, masses for M 2+ ions can be retrieved starting from those of M 1 + ions: m* = m 22 /m 1 m 2 = (m* m 1 ) 1/2 Further metastable ions, observed in the benzene spectrum at m/z 74.1 and 34.7, lead to m 2 values 76.02 and 52.02, respectively, that are also found directly in the EI spectrum. Such values suggest further fragmentations for the molecular ion: C 6 H 4 C 6 H +. 76 6 78 +. + H 2 C 4 H 4 +. + C 2 H 2 52
The importance of heteroatoms in electron ionization Heteroatoms may have non bonding electrons, which are more easily removed to form ions, as shown for formaldehyde: The probability of removal of non bonding electrons is related to the heteroatom: S > N > O
Example of heteroatom influence: electron ionization fragmentation pathways for 2-hexanone:
A summary of Pros and Cons of Electron Ionization