Mass spectrometry is the study of systems causing the formation of gaseous ions, with or without fragmentation, which are then characteried by their mass to charge ratios (m/) and relative abundances. Mass Spectroscopy In MS, compounds are ionied, ionied molecule decomposes into smaller ions/radicals/radical-ions/ neutrals. One way to ionie molecules is to extract electrons from a molecule. Base peak Molecular Ion peak M+. The positively charged fragments produced are separated, based on their mass/charge (m/) ratio. M ioniation M+. fragmentation M+1 + M+.2+..+N1+ N2. +.. Parent ion/ Molecular ion daughter ions, radicals, neutral, Most of the ions has =+1 m/ = mass of the fragment. A plot of relative abundance vs. m/ of the charged particles is presented as the Mass Spectrum. Nominal mass Spectrum output is presented as a histogram. Fragmentation: M1+ + N1. M+. M1+ M3+ + N4 M2+. + N2 M+. N. N Radical ion (odd e) Neutral radical (odd e) Neutral (even e) M+ (even e) would not break up into a radical ion. Actual signal has peaks with a line width.
Isotope peaks: In mass spectroscopy the actual mass of fragments generated are determined. Therefore fragments with different isotopes are distinguished, e.g. Lead metal. For molecular fragments, the isotope peak abundance is dependent on the molecular constitution and the natural isotope abundance of the constituent elements. In mass spectroscopy the masses of individual ions are measured. Isotope peaks Mass and abundance of ions of each isotopic composition is measured!! not the average molecular mass. CH 4 13 2 peak M + 2 4 + 3 CH, HCH H13 CH+3, 2 H2CH+2 Unit mass spacing isotope peak M+1 Cluster isotope peak - negligible M+ 2.. Cl-CH2-CH2-S-CH2-CH2-Cl The existence of isotopes generates a cluster of peaks (isotope peaks). The Nominal mass is m/ of the lowest mass isotopomer, i.e. the member of the isotopes cluster that has all the C s as 12C, all protons as 1H, all N s as 14N,. Different peaks, because there are some molecules with etc. Especially significant for Cl, Br 13C, 2H Peaks are spaced by a unit mass m/ All peaks (cluster) are of the same molecular formula Isotopomers (isotopic isomers) are isomers having the same number of each isotopic atom but differing in their positions. Mass Spectrometer: Mass spectrometer has devices for each of the following; Sample Introduction Create gas-phase ions of sample Separate ions in space or time; based on m/ ratio accomplished by mass analyers. Detect of the quantity of ions/ current from each m/ ratio ion
Ioniation: If a quantity of energy is supplied to a molecule greater than the ioniation energy of the molecule, a molecular ion is formed M+. Electron Ioniation (Electron Impact, EI) Chemical Ioniation (CI) Electrospray Ioniation (ESI) Matrix Assisted Laser Desorption Ioniation (MALDI) Atmospheric Pressure Chemical Ioniation (APCI) Atmospheric Pressure Photo-ioniation (APPI) Atmospheric Pressure Laser Ioniation (APLI) Fast Atom Bombardment (FAB) Inductively Coupled Plasma (ICP) Electron impact ioniation Magnetic sector separation single focusing Mass Analyers: Magnetic Sector Mass Analyer (Single/Double Focusing ) Quadrupole Mass Filters Time-of-flight (TOF) Mass Analyer Ion Trap Mass Analyer Fourier-Transform Mass Spectrometry (FTMS) Ion Detection: Faraday Cup Electron Multiplier Photomultiplier Conversion Dynode The interior of the mass spectrometer must be evacuated. The ion source, mass filter/analyer, and detector are under vacuum so that the ions would move from the ion source to the detector without colliding with other ions and molecules. The mean-free path of a charged particle should be greater than the distance between ioniation and detection regions. Mass Spectrometer B -i A high vacuum is created with two pumps where a low-vacuum pump is connected to the output of a high-vacuum pump. F Evacuated system - 10-6 torr Diffusion pump(s) + rotary-vane rough pump Turbo-molecular pump(s) + rotary-vane rough pump Pressure (Torr) 760 1 Mean Free Path (m) 6.0x10-8 4.5x10-5 10-3 4.5x10-2 10-5 4.5 10-7 4.5x102 10-9 4.5x104 Electron impact ioniation (EI) 70V - + V Ion optics Ekin = ev = mv2/2 70eV high energy electrons, molecular ion - very energetic, low/no abundance.
Volatilied compound is ionied by electron impact. An electron beam is generated by a accelerating the electrons from a heated filament through an applied voltage. The electron energy is defined by the potential difference between the filament and the source housing and is usually set to 70 ev. A field keeps the electron beam focused across the ion source. Upon impact with a 70 ev electron, the gaseous molecule may lose one of its electrons to become a positively charged radical ion, daughter ions, etc. Magnetic sector mass analyer: V Depending on the lifetime of the excited state, fragmentation will either take place in the ion source giving rise to stable fragment ions, or on the way to the detector, producing metastable ions. m er 2 B 2 2V note: slits Ion source accelerates ions to a KE KE = ½ mv2 = ev In the magnet A 'repeller' serves to define the field within the ion source. Each m/ beam follows it s own path (r) for a given B and V in the magnetic sector (60 o/900). B r All ions are subsequently accelerated out of the ion source by an electric field produced by the potential difference applied to the ion source and a grounded Electrode, V. F = mv2 /r = Bev, Upon rearrangement r = mv/eb = (2Vm/e)1/2/B m/ = (eb2r2)/(2v) The m/ ratio of the ions that reach the detector can be varied by scanning either the magnetic field (B) or the applied voltage of the ion optics (V). i.e. by varying the voltage or magnetic field of the magnetic-sector analyer, the individual ion beams are separable spatially, radius of curvature is held constant. For specific V and B ions of unique m/ pass thro the magnetic sector and reaches the stationary detector. Variations of V and/or B causes fragments of different m/ value to reach the detector. Usually B is scanned to allow different m/ s to reach the detector sequentially generating the complete mass spectrum keeping V constant. e(v- V) e(v+ V) The distribution of a given mass by way of energy distribution of kinetic energy.
Magnetic Sector Mass Analyer: Double Focusing (EB) E B + - V mv 2 mv 2 and ev= r 2 2V m r ; r independent of in E; focussing! E ee Resolving Power 1. Defined in terms of the overlap (or valley ) between two peaks. For two peaks of equal height, masses m1 and m2, when there is overlap between the two peaks to a stated percentage of either peak height (10% is recommended), then the resolving power is defined as [m1/(m1 m2)]. The percentage overlap (or valley ) concerned must always be stated. R Actual signal has peaks with a line width. Imposes a limitation on the resolvability of consecutive peaks Example M M Minimum resolution necessary to separate N2+ and CO+ peaks? Exact masses: N2+ = 28.006158 amu CO+ = 27.994915 amu 10% R M 28 2490 M 0.011241 2. Example State the method of calculation when expressing resolving power, and the position of the lower peak.
Ioniation EI: Electrons in molecules occupy molecular orbitals and hence acquire the energy associated with such orbitals. To remove electrons from such orbitals and ionie the molecule energy is required. The energy required depends on the orbital of electron occupation namely the HOMO. Thus the ease of ioniation will depend on the types of electrons in the molecule. The molecular ion (dominant) is formed by the removal of the least tightly bound electron. Chemical ioniation (CI): Interaction of the molecule M with a reactive ionied reagent species (gaseous Bronsted acids). e.g.., EI of methane, generates CH4+ which then reacts to give the Bronsted acid CH5+; CH4+ + CH4 CH5+ + CH3 If M in the source has a higher proton affinity than CH 4, the protonated species MH+ will be formed by the exothermic reaction. M + CH5+ MH+ + CH4 CI is a softer ioniation process. M+. nearly nonexistent. Abundant M+. CI is a lower energy process than EI, results in less fragmentation and therefore a simpler spectrum with the parent/molecular ion intact.
Fast Atom Bombardment Ioniation: The sample droplet is bombarded with energetic atoms (Ar, Xe) of 8-10 kev kinetic energy. Ions (e.g., Cs+) can be used as the bombarding particle in a similar technique termed liquid secondary ion mass spectrometry (LSIMS) Beam collides with the sample and matrix molecules, producing positive and negative sample-related ions that can be accelerated into the mass spectrometer. Fast Atom Bombardment Used for polar organic compounds, acidic and basic functional groups. Basic groups run well in positive ioniation mode and acidic groups run well in negative ioniation mode. FAB analytes: peptides, proteins, fatty acids, organometallics, surfactants, carbohydrates, antibiotics, and gangliosides. Quadrupole Mass Analyer (spectrometer): 4 parallel, polished metal rods -[U+Vcos(ωt)] + [U+Vcosωt] y y x x Diagonal electrodes have potentials of the same sign U= DC voltage, V=AC voltage, ω= angular velocity of alternating voltage Behavior of electrical charges in electric fields. The path of charged particles depends on sie/mass differences. Larger masses have a higher inertia than a small mass. Apply a DC; make it (+) on two diagonal rods. -[U+Vcos(ωt)] + + [U+Vcosωt] 2r0 x + ions Parameters affecting motion: m/, U, V, r 0 and. + y
Superimpose an AC; V sin t with an amplitude V and a frequency. + U + Vsin t + ions + U + V sin t + ions + U + V sin t Lighter ions spirals out of the quadrupole (filters out). Apply a DC; make it (+) on the two rods. Superimpose an AC; V sin t with an amplitude V and a frequency. + U + V sin t Heavier ions travel straight to the detector - U - V sin t High Pass Filter Lower m/ crashes High mass pass through + ions m/ - U - V sin t Heavier ion spirals out of the quadrupole (filters out). - U - V sin t Low mass pass through + ions - U - V sin t Lighter ions travel straight to the detector. m/ Low Pass Filter higher m/ crashes
Narrow window pass High Pass Filter Narrow window pass High Pass Filter Low Pass Filter m/ m/ m/ m/ Low Pass Filter Quadrupole Mass Analyer (Spectrometer): Ions oscillate under the influence of the variable fields. y + Viewed down y x Combined DC and RF potentials on the quadrupole rods create a stable linear path and passes only a selected m/ ratio (resonant ion) at a time. All other m/ ions acquire unstable paths and spirals out. y y x The mass spectrum is obtained by varying the voltages on the rods and monitoring which ions pass through the quadrupole rods. Quadrupole mass (QM) analyer is a "mass filter".
The solution of equations of motion of ions traveling through a QM analyer shows that for an ion with a particular m/ to pass through, certain combinations of U and V must be obtained. Varying rod voltages (scanning the spectrum): a. scan ω while holding U and V constant b. scan U and V but keep the ratio U/V fixed Two functions a and q define a stable trajectory for which ions do not collide with the rods across a range of values of U and V. a 4 eu m 2 r02 2 2 ev m 2 r02 2 In principle QM analyer can be operated for a range of U and V values. If U and V are scanned such that U/V = constant, V>U then successive detection of ions of different m/ is achieved. Stability curve for an ion - m/ q= a 2U q V Graphically, e.g. the three stability curves represent values of U and V for which the masses m 1, m2 and m3 have stable trajectories through the quadrupole. Only those mass values on the operating line transmits. (U) (V) A given m/ ion travels thro the quadrupole if the values of U and V are in a segment of the operating line and bounded by the stability curve. (U) (V) The resolution is determined by the magnitude of U/V ratio, i.e the slope. Resolution of the mass analyer can be increased by increasing the slope of the line, U/V (= held const.), and that if U = 0 then ions of all m/ are transmitted. (U) (V) Because quadrupoles operate at lower voltages, they can be scanned at faster rates (~1000 a.m.u./s) than magnet based spectrometers. QMs are better detectors for LC-MS and GC-MS implementation.
MS/MS MS1 MS2 Dissociation region QqQ Scan with MS1 (only) turned on entire MS spectrum. Set (e.g. U and V) MS1 to filter fragment of interest, dissociate further in q (Q2) collision with Ar or N 2 the fragment selected in MS1 and mass analye by scanning with MS2. Time-of-Flight Mass Analyers (spectrometer): TOF measures the mass-dependent time required for ions of different masses to move from the ion source to the detector. L This requires that at the starting time t=0, (time ions leaves the ion source) to be well-defined. Ions are created by a pulsed method (MALDI), or by rapid electric field switching that serves as a 'gate' to release the ions from the ion source in a very short time. KE ev Time-of-Flight Mass Spectrometry, Principle mv 2 2 v L t m 2Vt 2 2 e L Ion Source - MALDI Laser +U Probe (Start) Ion Detector m/ MALDI Ion Source Field Free Drift Tube dsource dtof ttotal = tsource + ttof Metal plate +/- U ions Acceleration region t L 1 2eV m
Time-of-Flight Mass Spectrometers, General Organiation TC1 Linear TOF: L a Laser Prism Detectors BA2 Ioniing Probe (start) M3 Inlet BA1 Sample Plate M2 M1 Ion detector (MCP) Grids Ion Signals Flight Tube +/- U Camera Ion Selector Reflector Valves Pumps (Turbo) TC2 time Fore Pump Reflex MALDI TOF Mass Spectrometer Laser output ion mirror Reflection time-of-flight mass spectrometer Parent/Molecular Peak M: High Resolution MS: An molecular ion that has not lost/gained atoms. The nominal mass of which is calculated with the mass numbers of the predominant isotopes of atoms. Using mass number for isotopes of atoms is approximate. Actual mass of a given isotope deviates From this integer by a small but unique amount ( E = mc2). Relative to 12C at 1000000, the isotopic mass of 16O is 15.9949146 a.m.u., etc. Base peak: Base peak is the peak from the most abundant ion, which is often the most stable ion. High resolution mass spectrometers that can determine m/ values accurately to four/more decimal places, making it possible to distinguish different molecular formulas having the same nominal mass.
m/=74 Very short list. Isotope Accurate Mass MF Unsaturation CH2N2O2 C6H2 6.0 C3H3FO C2H3FN2 1-H 2-H 1.007825 14102 12-C 13-C 100000000 13.0033548 14-N 15-N 14.0030740 15.0001090 C3H6O2 1.0 16-O 17-O 18-O 15.9949146 16.9991306 17.9991594 C2H6N2O 1.0 C4H7F 1.0 C4H10O 0.0 C3H10N2 0.0 http://www.chem.queensu.ca/facilities/nmr/nmr/mass-spec/mstable3.htm m/=74 MF C2H2O3 Unsaturation Exact Mass C2H2O3 74.00040 CH2N2O2 74.01163 C6H2 6.0 74.01565 C3H3FO 74.01679 C2H3FN2 74.02803 C3H6O2 1.0 74.03678 C2H6N2O 1.0 74.04801 C4H7F 1.0 74.05318 C4H10O 0.0 74.07316 C3H10N2 0.0 74.08440 MF finder CHEMCALC