Mass SpectrometryA 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.

Dr. Paul M. MayerAssistant ProfessorChemistry DepartmentUniversity of Ottawa

UofW seminar speaker January 2002

The Gas-phase ion chemistry of cluster ions

1899 Early Mass Spectrometry;1956 Identifying Organic Compounds with MS;1964 GC/MS1966 Peptide Sequencing1974 Extraterrestrial Mass Spectrometry1990 Protein Structure1991 Non-Covalent Interactions with ESI1992 Low Level Peptide Analysis1993 Oligonucleotide Sequencing1993 Protein Mass Mapping/Fingerprinting1996 MS of a Virus1999 Desorption/Ionization on Silicon1999 Isotope-Coded Affinity Tags

History of MS Applications

MS Celebrities

1st MSJoseph John Thomson (1856 - 1940) Cambridge University, Great Britain; Nobel Prize in Physics 1906

Ion ChemistryFrancis William Aston(1877 - 1945)Cambridge University, Great Britain; Nobel Prize in Chemistry 1922

Ion Trap TechniqueWolfgang Paul(1913 - 1993) University of Bonn, Germany; Nobel Prize in Physics 1989

ESI of BiomoleculesJohn B. Fenn (1917)Virginia Commonwealth University, Richmond, Virginia

Fragmentation MechanismsFred W. McLafferty(1923)Cornell UniversityIthaca, New York

Peptide Sequencing using MSKlaus Biemann (1926)MIT, Cambridge, Massachusetts

MALDIFranz Hillenkamp (1936)University of Münster, Germany

Mechanisms and ApplicationsR. Graham Cooks (1941)Department of Chemistry, Purdue UniversityWest Lafayette, Indiana

Mechanism of MALDI & ESIMichael Karas (1952)University of Frankfurt, Germany

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 AnalysisSamples 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 http://ms.mc.vanderbilt.edu/tutorials/ms/

Basic Concepts

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 vacuum-lock system. However, when connecting continuous introduction techniques like gas chromatography (GC), high performance liquidchromatography (HPLC) or capillary electrophoresis (CE), specialinterfacing becomes imperative to prevent excessive gas load.

Sample Introduction

Ionization Methods

The ionization method is chosen criteria are often the following:1. Physical state of the sample2. Volatility and thermal stability of the sample3. 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

Electron Impact Ionization (EI)

purely physicalprocesses!

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 m2 s-2) = 6.24145 x 1018 eV1 J mol-1 = 1.03641 x 10-5 eVEnergies 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 108 cm s-1 and transits a molecule of 1 nm length in 2 x 10-16 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).

Schematic representation of an electron ionization ion source. Mrepresents neutral molecules; e- = electrons; M+· = the molecular ion;F+ = fragment ions; Vacc = accelerating voltage; and MS = the mass spectrometer analyzer.

Schematic representations of an electron ionization ion source

sample pressure in the ion source is about 10-5 torr

about every 1/1000 molecule

is ionized

only cationsare measured

the sample is heated up until a sufficient vapour

pressure is obtained

Schematic Representation of a Sector Mass Spectrometer

Spectroscopic Methods in OC by Hesse, Meier, Zeeh

Mass Analysis for Magnetic Sector MSsMagnetic 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: rm = mv / zB (cm)B = magnetic field strength (T)Both equations can be combined to give the fundamental equation of MS: m/z = B2 rm2 / 2UU and B are kept constant: m/z = constant rm

2

U and rm are kept constant: m/z = constant B2

For typical values of Bmax = 1.5 Tesla, rm = 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* = m2

2/m1 z1/z22

Example: 1,2,3,4-tetrahydrocarbazole (M = 171) looses ethylene and a metastable peak at 1432/171 = 119.6 is observed;

Organic Structural Spectroscopy by Lambert, Shurvell, Lightner

Advantages & Disadvantages of EI-MS

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?

Lowering the electron beam energy from 70 eVto 15 eV does not necessarily result in the observation of a molecular ion peak

It, however, does change the probability of different fragmentation pathways.Here, the H-rearrangement wins at the expense of single-bond cleavage.

CH2=CHCH2CH3+

+ CH3COOH

CH3C=O+ + •OCH2CH2CH2CH3

Change of eV

The Molecular IonIndex 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) + 1Bivalent atoms such as O and S do not contribute. What’s about H3C-SO3H?

The nitrogen ruleMolecular ions containing odd numbers of nitrogen must have an odd number m/z.

ExampleThe compound with the molecular formula C7H7NO 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!

The isotopic signature – recognition of elements

[M]+•

Organic Structural Spectroscopy by Lambert, Shurvell, Lightner

Calculation of an approximate intensity distribution based on the natural isotope abundance

Organic Structural Spectroscopy by Lambert, Shurvell, Lightner

* average weight of natural mixture of isotopes

*

Molecular formula determination by exact mass measurements

Examples

The calculated isotope ratios for CO, N2, and C2H4, all of molar mass 28, are as follow:CO 100 (M) 1.14 (M+1) 0.2 (M+2)N2 100 (M) 0.76 (M+1)C2H4 100 (M) 2.26 (M+1) 0.01 (M+2)

The calculated isotope ratios for C3H6 and CH2N2, both of molar mass 42, are as follow:C3H6 100 (M) 3.39 (M+1) 0.05 (M+2)CH2N2 100 (M) 1.87 (M+1) 0.01 (M+2)

Cl isotope patterns

Calculated exact masses for high resolution MS

Calculated isotopic distribution for protonated bovine insulin. The chemical molecular weight based on elemental atomic weights is 5734.6 Da. The molecular weight based on the most abundant isotope of each element is 5728.6 Da, and the molecular weight based on the most abundant isotopic form of the molecule is 5733.6 Da.

Different isotope distributions that match the molecule mass 5733.6 Da