Mass Spectrometry. A truly interdisciplinary and versatile analytical method

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1 Mass Spectrometry A truly interdisciplinary and versatile analytical method MS is used for the characterization of molecules ranging from small inorganic and organic molecules to polymers and proteins. With MS we might be able to determine the molecular weight of a molecule ion, the elemental composition of a molecule ion, and the presence of certain functional groups. for mechanistic studies of gas-phase (ion) chemistry. This is also relevant for a better understanding of the chemistry in our atmosphere and in space. as a mass detector coupled to a GC, HPLC, capillary electrophoresis (CE), and thermo-gravimetric analysis. as an integrated mass detector in many high vacuum systems.

2 UofW seminar speaker January 2002 The Gas-phase ion chemistry of cluster ions Dr. Paul M. Mayer Assistant Professor Chemistry Department University of Ottawa

3 History of MS Applications 1899 Early Mass Spectrometry; 1956 Identifying Organic Compounds with MS; 1964 GC/MS 1966 Peptide Sequencing 1974 Extraterrestrial Mass Spectrometry 1990 Protein Structure 1991 Non-Covalent Interactions with ESI 1992 Low Level Peptide Analysis 1993 Oligonucleotide Sequencing 1993 Protein Mass Mapping/Fingerprinting 1996 MS of a Virus 1999 Desorption/Ionization on Silicon 1999 Isotope-Coded Affinity Tags

4 MS Celebrities 1 st MS Joseph John Thomson ( ) Cambridge University, Great Britain; Nobel Prize in Physics 1906 Ion Chemistry Francis William Aston ( ) Cambridge University, Great Britain; Nobel Prize in Chemistry 1922 ESI of Biomolecules John B. Fenn (1917) Ion Trap Technique Virginia Commonwealth Wolfgang Paul University, Richmond, ( ) Virginia University of Bonn, Germany; Nobel Prize in Physics 1989 Fragmentation Mechanisms Fred W. McLafferty (1923) Cornell University Ithaca, New York Peptide Sequencing using MS Klaus Biemann (1926) MIT, Cambridge, MALDI Massachusetts Franz Hillenkamp (1936) University of Münster, Germany Mechanisms and Applications R. Graham Cooks (1941) Department of Chemistry, Purdue University West Lafayette, Indiana Mechanism of MALDI & ESI Michael Karas (1952) University of Frankfurt, Germany

5 Basic Concepts A mass spectrometer is an instrument that produces ions and separates them in the gas phase according to their mass-to-charge ratio (m/z). Today a wide variety of mass spectrometers is available but all of these share the capability to assign mass-to-charge values to ions, although the principles of operation and the types of experiments that can be done on these instruments differ greatly. Basically, a mass spectrometric analysis can be envisioned to be made up of the following steps: Sample Introduction Evaporation and Ionization Mass Analysis Ion Detection/Data Analysis Samples may be introduced in gas, liquid or solid states. In the latter two cases volatilization must be accomplished either prior to, or accompanying ionization. Many ionization techniques are available to produce charged molecules in the gas phase, ranging from simple electron (impact) ionization (EI) and chemical ionization (CI) to a variety of desorption ionization techniques with acronyms such as FAB (fast atom bombardment), PDI (plasma desorption ionization), ESI (electrospray ionization) and MALD (matrix assisted laser desorption). The following pages are in part from

6 Sample Introduction Mass spectrometers are operated at reduced pressure in order to prevent collisions of ions with residual gas molecules in the analyzer during the flight from the ion source to the detector. The vacuum should be such that the mean free path length of an ion, i.e., the average distance an ion travels before colliding with another gas molecule, is longer than the distance from the source to the detector. For example, the mean free path length of an ion is approximately one meter at a pressure of 5x10-5 torr, i.e. about twice the length of a quadrupole instrument. Thus, the introduction of a sample into a mass spectrometer usually requires crossing of a rather large pressure drop, and several means have been devised to accomplish this. Gas samples may be directly connected to the instrument and metered into the instrument via a needle valve. Liquid and solid samples can be introduced through a septum inlet or a vacuumlock system. However, when connecting continuous introduction techniques like gas chromatography (GC), high performance liquid chromatography (HPLC) or capillary electrophoresis (CE), special interfacing becomes imperative to prevent excessive gas load.

7 Ionization Methods The ionization method is chosen criteria are often the following: 1. Physical state of the sample 2. Volatility and thermal stability of the sample 3. Type of information sought Comparison EI, CI, and DI are suitable for high resolution MS; EI works well only for thermally stable and volatile samples; CI, SI, and DI cause much less fragmentation (normally no radicals are formed); DI and SI must be combined with tandem mass detection (MS-MS) to extract more structural information from fragmentation; Organic Structural Spectroscopy by Lambert, Shurvell, Lightner

8 purely physical processes! Electron Impact Ionization (EI)

9 The ionization energies of many compounds are on the order of 7-14 ev but electron energies of 70 ev are often chosen for EI-MS to achieve higher signal intensities and to avoid changes in the mass spectrum with small changes in electron energy. The energy domain: 1 J (kg m 2 s -2 ) = x ev 1 J mol -1 = x 10-5 ev Energies of chemical bonds are typically between 100 and 600 kj mol -1 (C-H = 435 kj mol -1 ; C-O = 356 kj mol-1, C-N = 305 kj mol -1 ), which are equivalent to 1-6 ev The time domain: A 70 ev electron has a velocity of about 5 x 10 8 cm s -1 and transits a molecule of 1 nm length in 2 x s, while a typical bond vibration requires >10-12 s; thus, the molecular conformation remains unchanged as the electronic excitations occurs (Franck-Condon principle) The degree of fragmentation depends on the internal energy deposition in the molecular ion and on the resistance of the molecular structure to bond cleavage (fragmentation).

10 Schematic representations of an electron ionization ion source Schematic representation of an electron ionization ion source. M represents neutral molecules; e - = electrons; M + = the molecular ion; F + = fragment ions; V acc = accelerating voltage; and MS = the mass spectrometer analyzer.

11 about every 1/1000 molecule is ionized the sample is heated up until a sufficient vapour pressure is obtained only cations are measured sample pressure in the ion source is about 10-5 torr

12 Schematic Representation of a Sector Mass Spectrometer Spectroscopic Methods in OC by Hesse, Meier, Zeeh

13 Mass Analysis for Magnetic Sector MSs Magnetic sectors deflect accelerated beams of ions, with the degree of deflection depending on the mass, the charge, and the velocity of the ion beam. The speed of ions is described by: v = (2zU / m) 1/2 z = ionic charge, m = ion mass, U = acceleration potential (V) The mass separation is given by: r m = mv / zb (cm) B = magnetic field strength (T) Both equations can be combined to give the fundamental equation of MS: m/z = B 2 r 2 m / 2U U and B are kept constant: m/z = constant r 2 m U and r m are kept constant: m/z = constant B 2 For typical values of B max = 1.5 Tesla, r m = 30 cm, and U = 5 kv, the maximum value for m/z is 1953 Da/charge; Metastable ions are generated by fragmentation after acceleration but before entering the magnetic sector. The position of their diffuse peaks in the mass spectrum is given by: m * = m 22 /m 1 z 1 /z 2 2 Example: 1,2,3,4-tetrahydrocarbazole (M = 171) looses ethylene and a metastable peak at /171 = is observed;

14 Advantages & Disadvantages of EI-MS Organic Structural Spectroscopy by Lambert, Shurvell, Lightner

15 Contrasting degrees of fragmentation depending on the chemical structure Radical cations of aromatic and unsaturated conjugated hydrocarbons resist fragmentation more than radical cations of saturated hydrocarbons; Why?

16 CH 3 C=O + + OCH 2 CH2CH 2 CH 3 Change of ev Lowering the electron beam energy from 70 ev to 15 ev does not necessarily result in the observation of a molecular ion peak CH 2 =CHCH 2 CH CH 3 COOH It, however, does change the probability of different fragmentation pathways. Here, the H- rearrangement wins at the expense of singlebond cleavage.

17 The Molecular Ion Index of hydrogen deficiency (degree of unsaturation) The index of hydrogen deficiency is the number of pairs of hydrogen atoms that must be removed from the corresponding saturated formula to produce the molecular formula of the compound of interest. The index is the sum of the number of rings, double-bonds, and twice the number of triple bonds. Compounds can contain C, H, N, O, halogen, and S. Index = tetravalent (C, Si) - ½ monovalent (H, halogen) + ½ trivalent (N, P) + 1 Bivalent atoms such as O and S do not contribute. What s about H 3 C- SO 3 H? The nitrogen rule Molecular ions containing odd numbers of nitrogen must have an odd number m/z. Example The compound with the molecular formula C 7 H 7 NO has an index of? What are possible molecules? Note that a benzene ring counts for an index of 4, 3 double bonds and one ring!

18 The isotopic signature recognition of elements [M] +

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21 Calculation of an approximate intensity distribution based on the natural isotope abundance Organic Structural Spectroscopy by Lambert, Shurvell, Lightner

22 * * average weight of natural mixture of isotopes Organic Structural Spectroscopy by Lambert, Shurvell, Lightner

23 Molecular formula determination by exact mass measurements Examples The calculated isotope ratios for CO, N 2, and C 2 H 4, all of molar mass 28, are as follow: CO 100 (M) 1.14 (M+1) 0.2 (M+2) N 2 C 2 H (M) 0.76 (M+1) 100 (M) 2.26 (M+1) 0.01 (M+2) The calculated isotope ratios for C 3 H 6 and CH 2 N 2, both of molar mass 42, are as follow: C 3 H (M) 3.39 (M+1) 0.05 (M+2) CH 2 N (M) 1.87 (M+1) 0.01 (M+2)

24 Calculated exact masses for high resolution MS Cl isotope patterns

25 Calculated isotopic distribution for protonated bovine insulin. The chemical molecular weight based on elemental atomic weights is Da. The molecular weight based on the most abundant isotope of each element is Da, and the molecular weight based on the most abundant isotopic form of the molecule is Da. Different isotope distributions that match the molecule mass Da