1 Chapter 12 Mass Spectrometry ( 質譜 ) Organic Chemistry, 7 th Edition L. G. Wade, Jr. 藥學系 藥物分析科 許秀蘊 教授

2 學習目標 1. 光譜學簡介 2. 質譜儀之原理 3. 質譜儀之應用

3 Introduction Spectroscopy is a technique used to determine the structure of a compound. Most techniques are nondestructive (it destroys little or no sample). Absorption spectroscopy measures the amount of light absorbed by the sample as a function of wavelength.

4 Types of Spectroscopy Infrared (IR) spectroscopy measures the bond vibration frequencies in a molecule and is used to determine the functional group. Mass spectrometry (MS) fragments the molecule and measures the masses. Nuclear magnetic resonance (NMR) spectroscopy detects signals from hydrogen atoms and can be used to distinguish isomers. Ultraviolet (UV) spectroscopy uses electron transitions to determine bonding patterns.

5 Electromagnetic Spectrum Examples: X rays, microwaves, radio waves, visible light, IR, and UV. Frequency and wavelength are inversely proportional. c =, where c is the speed of light (3 x cm/sec). Energy per photon = h, where h is Planck ’ s constant, 6.62 x kJ sec.

6 The Spectrum and Molecular Effects

7 Mass Spectrum

8 Mass Spectrometry Molecular weight can be obtained from a very small sample. It does not involve the absorption or emission of light. A beam of high-energy electrons breaks the molecule apart. Destructive technique, the sample cannot be recovered. The masses of the fragments and their relative abundance reveal information about the structure of the molecule.

9 Mass Spectrometry 1. Ionizes molecules in a high vacuum 2. Sorts the ions according to their mass 3. Records the abundance of ions of each mass

10 Mass spectrometry -1 Molecules (or atoms) are bombarded by electrons, and become ionized Mass spectrometer separates individual particles (ionized atoms or molecules) M + e - [M] e -

11 Mass spectrometry -2 The ions are then accelerated in an electric field at a voltage V. Energy of particle = e V (e ： charge). Kinetic energy = ½ mv 2 (m: mass of particle) e V = ½ mv 2 Velocity of a particle depends on its mass.

12 Mass spectrometry -3 Charged particles enter at right angles to a homogeneous magnetic field B. Particles move in a circle The radius of the circular path depends on the accelerating voltage V, the magnetic field, ratio m/e.

13 Mass Spectrometer

14 1. Inlet system All samples first be converted to the gaseous state Inlet system heated up to 400 o C Unstable samples should not be exposed excessively high temp The rate which the sample enters the ionization chamber remains constant

15 2. Ionization Electron Impact Electron Spray Ionization (ESI) (large molecules) Fast Atom Bombardment (FAB) (molecular ion; sample dissolved in a high- boiling solvent/glycerol) Field Ionization (high electrostatic field) : M + Chemical Ionization (mix sample with a low MW gas / methane)

16 Radical Cation Formation When a molecule loses one electron, it then has a positive charge and one unpaired electron. This ion is therefore called a radical cation. M + e - [M]. + (molecular ion) + 2e -

17 Electron Impact Ionization A high-energy electron can dislodge an electron from a bond, creating a radical cation (a positive ion with an unpaired e - ). Other fragments can be formed when C — C or C — H bonds are broken during ionization. Only the positive fragments can be detected in MS.

18 3.Accelerating Chamber Ionized particles enter the accelerating cell, under the influence of the accelerating voltage V Under fixed voltage, only one mass would reach the detector In order to scan the entire mass range of fragments, the voltage is varied at a constant rate

19 4. Drift Chamber (Magnetic Field Path) Accelerated fragmented ions enter the drift chamber and come under the influence of the magnetic field The drift chamber is under a high vacuum to prevent fragment from colliding with each other

20 5. Detector Most common detector: electron multiplier Fragments from the drift chamber arrive at the electron – emissive surface of a dynode amplifier A collision releases several electrons Electrons are then accelerated to a second such surface Process is continued until a cascade of electrons arrives at the collector 中間極放大器

21 Analytical Sequence 1. gas sample is bled very slow at a constant rate into ionization chamber (ionized and fragmented) 2. A beam of electrons causes molecules to ionize and fragment. 3. Accelerating voltage (V) ： The mixture of ions is accelerated 4. Magnetic field: Ions passes through a magnetic field, where the paths of lighter ions are bent more than those of heavier atoms. By varying the magnetic field, the spectrometer plots the abundance of ions of each mass. 5. The exact radius of curvature of an ion's path depends on its mass-to-charge ratio, symbolized by m/z. In this expression, m is the mass of the ion (in amu) and z is its charge. 6. Collector

22 Separation of Ions A beam of electrons causes molecules to ionize and fragment. The mixture of ions is accelerated and passes through a magnetic field, where the paths of lighter ions are bent more than those of heavier atoms. Only the cations are deflected by the magnetic field. Amount of deflection depends on m/z. The detector signal is proportional to the number of ions hitting it. By varying the magnetic field, ions of all masses are collected and counted.

23 Analytical Uses of Mass Spectrometry 1. Elemental Analysis 2. Molecular Analysis 3. Protein Sequencing Analysis 4. Special Equipment for GC-MS Combinations 5. GC-FTIR-MS Systems (separation; functional group; MW) 6. Thermal Analysis-MS interface (physical measurements with temperature change)

24 Interpretation of Mass Spectra 1. Identification of the Parent Mass 2. Counting Oxygens 3. Compounds with Hetero Elements 4. Clusters of Halogens 5. Using Electron Impact 6. Rings Plus Double Bonds 7. Important Neutral Species

25 The Mass Spectrum In the spectrum, the tallest peak is called the base peak and it is assigned an abundance of 100%. The % abundance of all other peaks are given relative to the base peak. The molecular ion (M + ) corresponds to the mass of the original molecule.

26 Mass Spectrum

27 Isotopic Abundance

28 Recognizable elements in the Mass Spectrum Br M+2 as large as M + Cl M+2 a third as large as M + I I + at 127; large gap N odd M +, some even fragments S M+2 large than usual (4% of M + ) Isotopes: present in their usual abundance. Hydrocarbons contain 1.1% C-13, so there will be a small M+1 peak.

29 Mass Spectrum with Sulfur Sulfur has three isotopes: 32 S (95%), 33 S (0.8%), and 34 S (4.2%). The M + peak of ethyl methyl sulfide has an M+2 peak that is larger than usual (about 4% of M + ).

30 Mass Spectrum with Chlorine Chlorine is a mixture of 75.5% 35 Cl and 24.5% 37 Cl. The molecular ion peak M + is 3 times higher than the M+2 peak.

31 Mass Spectrum with Bromine Bromine is a mixture of 50.5% 79 Br and 49.5% 81 Br. The molecular ion peak M+ has 79 Br be as tall as the M+2 peak that has 81 Br.

32 Fragmentation Patterns in MS Ionization R ： R ’ + e - [R. R ’ ] e - radical cation (molecular ion) Fragmentation [R. R ’ ]. + R + +. R’ cation fragment radical fragment (observed) (not observed)

33 Mass Spectra of Alkanes Fragmentation of the Hexane Radical Cation

34 Mass Spectrum of n-Hexane Groups of ions correspond to loss of one-, two-, three-, and four-carbon fragments.

35 Fragmentation of Hexane [CH 3 CH 2 CH 2 --CH 2 CH 2 CH 3 ] +. CH 3 CH 2 CH CH 2 CH 2 CH 3 Hexane radical cation propyl cation propyl radical(43) M + 86 detected at m/z 43 not detected [CH 3 CH 2 CH 2 CH 2 CH 2 --CH 3 ] +. CH 3 CH 2 CH 2 CH 2 CH CH 3 Hexane radical cation pentyl cation methyl radical(15) M + 86 weak at m/z 71 not detected [CH 3 CH 2 CH 2 CH 2 CH 2 --CH 3 ] +. CH 3 CH 2 CH 2 CH 2 CH CH 3 Hexane radical cation pentyl radical methyl cation(15) M + 86 not detected (too unstable) X The stability of the cation is more important than the radical.

36 Mass Spectra of Alkanes More stable carbocations will be more abundant.

37 Fragmentation of Branched Alkanes The most stable carbocation fragments form in greater amounts.

38 Fragmentation Giving Resonance-Stabilized Cations [R － CH ＝ CH － CH 2 － R ’ ] +. [R － CH ＝ CH － + CH 2 R － + CH － CH ＝ CH 2 ] allylic cation +. R’

39 Chapter 12 Mass Spectra of Alkenes Resonance-stabilized cations favored.

40 Fragmentation Giving Resonance-Stabilized Cations Benzylic cations

41 Fragmentation Giving Resonance-Stabilized Cations Ketones & aldehydes ： loss of alkyl groups to give acylium ions

42

43 Mass spectra of Alcohols Fragmentation Splitting Out a Small Molecule

44 Mass Spectra of Alcohols Alcohols usually lose a water molecule. M+ may not be visible.

45 MW=144

46 The GC-MS A mixture of compounds is separated by gas chromatography, then identified by mass spectrometry.

47 High Resolution MS Masses measured to 1 part in 20,000. A molecule with mass of 44 could be C 3 H 8, C 2 H 4 O, CO 2, or CN 2 H 4. Using a mass with more significant figures would help identify the correct formula. For example, let ’ s say the compound we are looking for has mass of , pick the correct structure from the table:

48

49 Analytical Uses of Mass Spectrometry 1. Elemental Analysis 2. Molecular Analysis 3. Protein Sequencing Analysis 4. Special Equipment for GC-MS Combinations 5. GC-FTIR-MS Systems (separation; functional group; MW) 6. Thermal Analysis-MS interface (physical measurements with temperature change)

50 Common Fragmentation Patterns-1

51 Common Fragmentation Patterns-2

52 Common Fragmentation Patterns-3

53 Common Fragmentation Patterns-4

54 學習目標 1. 光譜學簡介 2. 質譜儀之原理 3. 質譜儀之應用 藥學系 藥物分析科 許秀蘊 教授