intensity Lecture 8: Mass Spectrometry Relative abundance m/z 1
Ethylbenzene CH 2 CH 3 + m/z = 106 CH 2 + m/z = 91 C 8 H 10 MW = 106 CH + m/z = 77 + 2 2
What information can be obtained from a MS spectrum? Molecular weight, isotopes Molecular structure Bond cleavage Bond energy C 8 H 8 O 4 MW = 180.16 3
Brief history of mass spectrometry J. J. Thomson built MS prototype to measure m/z of electron (Nobel Prize in 1906) MS concept first put into practice by Francis Aston, a physicist working in Cambridge England in 1919. Designed to measure mass of elements, Aston awarded Nobel Prize in 1922 1948-52 - Time of Flight (TOF) mass analyzers introduced 1955 - Quadrupole ion filters introduced by W. Paul, also invents the ion trap in 1983 (wins 1989 Nobel Prize) 1968 - Tandem mass spectrometer appeared Mass spectrometers are now one of the most powerful analytical tools in chemistry 4
5 Mass analyzer principles Magnetic force: Centripetal force: V e z mv E 2 2 1 Kinetic energy of ion: r mv F c 2 zevb B v q F V e r B z m 2 2 2 v B + F
Mass spectrometry Create gas-phase molecules, fragment and ionize molecules, determine mass of the fragments. Spectrum is characteristic of molecule. One can identify molecules by comparison with library. Relatively sensitive - need nanograms of material Very important in forensics Very important in pharmaceutical industry 6
Mass spectrometry instruments Ionization source convert analyte to gaseous ions. Mass separator physically separates or filters specific masses Detector detects the ions after being filtered. Ionization source Mass separator Mass Detector 7
Typical ion sources for mass spectrometry 8
Ion sources: electron impact ionization (small molecules, 1-1000 Daltons, structure) Sample introduced into instrument by heating it until it evaporates Gas phase sample is bombarded with electrons coming from rhenium or tungsten filament (energy = 70 ev) Molecule is shattered into fragments (70 ev >> 5 ev bonds) Fragments sent to mass analyzer 9
Electron Impact Ionization Molecules collide with electrons, knocking an electron from molecule M + e - (~70 ev) Μ + + 2e - (about 10-4 % ionized) The molecule has lost an electron, but otherwise is intact. This ion is called the parent ion. It is a hard ionization method (70 ev» chemical bond). Molecules are electronically, vibrationally, and rotationally excited. High-energy electrons fragment the molecule M + e - D + + N + 2e - Electron impact ionization usually makes a highly fragmented product and little parent ion is produced. 10
Ion sources: chemical ionization Relative of electron impact ionizer. Produces very little fragmentation in an ion. It is called a soft ionization method. Useful to determine empirical formula. Chemical ionization gas is introduced into the ionization chamber at ~ 1 torr. Methane is a common example. Methane is ionized by the electron beam, creating a lot of methane cations CH 4 + e - CH 4 + + 2e - These methane cations collide with a sample molecule, and they undergo ion-molecule reactions CH 4 + + M CH 3 + MH + The instrument is identical to the electron impact ionization chamber, only with a bit of gas thrown in. 11
ESI MALDI NMR 12
Ion sources: matrix-assisted laser desorption ionization (MALDI) This technique is very popular for the study of proteins. The 2002 Nobel Prize in Chemistry was awarded to Koichi Tanaka for its development. Soft ionization technique, good for structure determination. Sample is mixed with a solid organic acid. The mixture is coated on the end of a metal rod, which is inserted into a vacuum system. Blast sample with laser pulse Produce gas-phase molecular ions Not quantitative Repeller plate Sample Laser Ions Metal plate Acceleration grids 13
Ion sources: electrospray ionization Another soft ionization method that is often used to couple liquid chromatography to mass spectrometry for analysis of proteins. The 2002 Nobel Prize in Chemistry was awarded for to John Fenn for its development. Sample is dissolved in acetone or methanol and pumped through glass tube. A high voltage is applied across the tube to form a very fine spray forms at the tip of the tube. Droplets are charged. Charge to volume ratio increases as droplets evaporate. Eventually, droplets undergo Coulombic explosion, creating smaller droplets. Process repeats, created gas-phase ions. www.magnet.fsu.edu 14
Electrospray ionization Taylor cone of liquid forming at the end of the metal capillary. I. G. Loscertales et al. Science, 295, 1695-1698 15
Electrospray ionization The high voltage is applied from the capillary tip to a plate at the entrance to the mass spectrometer. The spray forms a cone that is incident on the entrance. Vacuum pumps reduce the pressure in the mass spectrometer. A skimmer isolates the ions from the spray and sends them to the mass analyzer. capillary mass analyzer High voltage Entrance to the mass spectrometer 16
Mass Resolution Resolution, R, in mass spectrometry is defined as R m m Example What resolution is needed to discriminate glutamine + (C5O3N2H10) and lysine + (C6O2N2H15)? Calculate based on major isotopic component. 17
Mass Analyzer: magnetic sector 18
Mass Analyzer: magnetic sector The earliest mass analyzers were magnetic sectors. Kinetic energy of ion: E 1 2 mv 2 z ev Magnetic force: F qv B zevb Centripetal force: 2 mv F c r m z 2 B r 2V 2 e 19
Mass Analyzer: Time of Flight It is usually used with MALDI, where the laser creates a pulse of ions. Ions are accelerated by electric field applied to plates. The ions enter a field-free region, where they travel at some velocity, which is related to the mass of the ion. 20
A time of flight instrument accelerates ions through a potential of 1000 V, down a 1-meter long flight tube. How long does it take a m/z = 1,000 Da ion to reach the detector? What about 1050 Da? 21
Mass Analyzer: Quadrupole Mass Analyzer Relatively compact and inexpensive. Ions are accelerated through a region surrounded by four rods. Radio-frequency (~3 khz to 300 GHz) electric field is applied to poles. Ions wiggle in strange paths as they move along the quadrupole. Only ions with a particular M/z ratio can pass through the quadrupole mass analyzer, and the M/z depends on the radio frequency applied to the poles. 22
Mass Analyzer: Triple Quadrupole Mass Analyzer Collision gas Ion source 1 st analyzer 2 nd analyzer Detector Ion selection Collision cell Fragment ion selection Sample inlet All ions Sample ion of selected m/z tandem mass spectrometry or MS/MS. Fragment ions of selected ion The first quadrupole acts as a mass analyzer to select one ion from a mixture. That ion passes to second quadrupole, which contains some low-pressure gas, such as Ar or N 2. Ions collide with this neutral gas, becoming fragmented in the process, generating daughter ions. The third quadrupole obtains the mass spectrum of the daughter ions generated in the second quadrupole. It is very useful to determine molecular structure. 23
Mass Analyzer: Ion Trap Mass Analyzer Similar to a quadrupole A RF field is applied to focus ions. Made from three pieces of metal. The left and right plates are hyperbolic end-caps. The end caps have a hole drilled in them to let ions in and out. The end caps are surrounded by a circular ring electrode, which also has a hyperbolic cross-section. Ions of specific M/z are trapped by applying an appropriate radio frequency electric field to the caps and circular electrode. Ring Electrode Spacer rings Inlet focusing Ions in Trapped ions Exit lens Ions out Entrance endcap electrode Exit endcap electrode 24
Isotopic Patterns The isotopic pattern is characteristic of the element. For example, bromine has two stable isotopes, 81 Br with 49% natural abundance and atomic weight 80.916289 and 79 Br with 51% natural abundance and atomic weight 78.918336. Example. Bromodiphenyl acetic acid has a nominal molecular weight of 291.14. The molecule can be made from many different isotopes. The most likely formulae for the parent ions are 12 C 14 16 O 2 79 Br 1 H 11 and 12 C 14 16 O 2 81 Br 1 H 11, with molecular weights of 289.99844 and 291.996394 AMU, respectively, with nearly equal amounts of each. 25