Mass Spectrometry. General Principles

Transcription

1 General Principles Mass Spectrometer: Converts molecules to ions Separates ions (usually positively charged) on the basis of their mass/charge (m/z) ratio Quantifies how many units of each ion are formed over a given period Provides: Molecular Weight Fragmentation information propane H H H H C C C H H H H m/z = 15 H H H C C C H H H m/z = 43 H ionization H H H H H H H C C C H H C C C H H H H H H H m/z = 44 m/z = 29 (molecular ion) H H H C C C H H H m/z = 28 relative intensity Mass Spectrum m/z

2 Basic Mass Spectrometer acceleration Many different methods for ionization and detection are known - useful for different types of molecules - vary in amount of energy delivered à impacts ionization / fragmentation

3 Spectrum Terminology base peak parent ion! parent ion: molecular ion ( [M] + ) or quasimolecular ion ( [M+H] +, [M+Na] +, etc.) base peak: the tallest peak in the spectrum

4 Ionization Methods Ionization Method Typical Analytes Sample Introduction Electron Impact (EI) Chemical Ionization (CI) relatively small, volatile relatively small, volatile GC or liquid/solid probe GC or liquid/solid probe Mass Range up to 0 Daltons up to 0 Daltons Ionization [mass] hard, [M] + if observed hard to soft; varies with carrier [M+H] + Fast Atom Bombardment (FAB) carbohydrates, organometallics, peptides, nonvolatile sample mixed in viscous matrix up to 6000 Daltons soft, but harder than MALDI, ESI [M+Na] +, [M+H] + Matrix Assisted Laser Desporption (MALDI) peptides, proteins, nucleotides sample mixed in solid matrix up to 500,000 Daltons soft [M+H] + Electrospray (ESI) peptides. proteins, nonvolatile HPLC or syringe up to 6000 Daltons soft [M+Na] +, [M+H] +

5 Ionization Methods Hard Ionization e M e M * FRAGMENTS e Soft Ionization H + (etc) + M MH

6 Ionization Methods Electron Impact (EI) Hard ionization method sample in Used for low MW, nonpolar compounds (MW <750) à sample must be volatile (& thermally stable) Method that is most commonly used for GC-MS Sample bombarded with high energy electrons à typically 70 ev Ejects one electron from sample à radical cation (molecular ion) à ionization potenial (IP) of an organic molecule is 8-15 ev Molecular ion is often small or not observed à fragmentation Fragmentation pattern is highly reproducible; unique to each compound à provides structural information

7 Ionization Methods Effect of electron energy on Impact of EI Spectrum

8 Ionization Methods Chemical Ionization (CI) Soft ionization method (more controlled) à some fragmentation observed Indirect ionization of sample Collisions between sample & gas ions cause proton transfers à produces [M+H]+ ions, not M+ ions (so parent is M+1) More controlled than EI à reduced fragmentation à greater sensititivty à typically ~5 ev energy transfer Good for molecular weight determination Provides less information about structure

9 Ionization Methods Chemical Ionization (CI) Ionization methods include proton transfer and adduct formation Results in formation of "quasimolecular" ion M + H 3 + [M+H] + + H 2 M + CH 5 + [M+H] + + CH 4 M + C 2 H 5 + [M+C 2 H 5 ] + M + (CH 3 ) 3 C + [M+H] + + (CH 3 ) 2 =CH 2 M + (CH 3 ) 3 C + [M+C(CH 3 ) 3 ] + M + NH 4 + [M+H] + + NH 3 +

10 Ionization Methods EI vs. CI O CH 3 O CH 3 O MW = 180 CH 3 EI CI (methane) CI (methane)

11 Ionization Methods EI vs. CI EI MW = 142 CI (methane) [M+H] +

12 Ionization Methods Chemical Ionization (CI) reagent gas reagent ion sample ion hard H 2 H 3 + [M+H] + CH 4 CH 5 +, C 2 H 5 +, C 3 H 5 + [M+H] +, [M+C 2 H 5 ] + i-c 4 H 10 C 4 H 9 + [M+H] +, [M+C 4 H 9 ] + soft NH 3 NH 4 + [M+H] + reagent gas proton affinity notes H kcal/mol produces significant fragmentation CH kcal/mol less fragmentation than H 2 ; can form adducts (CH 3 ) 3 CH 196 kcal/mol mild, selective protonation; little fragmentation; some adduct formation NH kcal/mol selective ionization; little fragmentation; some adduct formation

13 Ionization Methods Chemical Ionization (CI) carrier gas CH 4 O O MW = 196 CH 3 H H 3 C CH 3 NH 3

14 Ionization Methods Fast Atom Bombardment (FAB) Soft ionization method Sample mixed with a condensed phase matrix (glycerol) protects sample from excess energy Mixture ionized by bombarding with beam of high energy atoms à Xe or Ar (6-10 kev) Ionization from protonation ([M+H] + ) or cation attachment ([M+Na] + ) High resolution, exact mass determination is possible fast (6-10 KeV) beam of Xe, Ar or Cs ions M M M M M M M H or M Na secondary ionization and desorption from matrix

15 Ionization Methods Matrix Assisted Laser Desorption Ionization (MALDI) Soft ionization method Sample is mixed with a condensed phase matrix that contains a chromophore Mixture is ionized with a laser à proton tranfer from matrix to sample Charged molecules are ejected from matrix Little excess energy little fragmentation Good for large molecules (proteins, polymers, carbohydrates)

16 Ionization Methods Electrospray Ionization (ESI) Soft Ionization method Does not require vacuum pressures Sample solution pumped through a narrow, stainless steel capilliary à aerosol of charged droplets Solvent evaporates until electrostatic repulsion within droplets too great à Coulombic explosion Sample ions released into vapor phase [M+H] +, [M+Na] + Good for large molecules (proteins, polymers, carbohydrates)

17 Ionization Methods Electrospray Ionization (ESI) OH O CO 2 Et

18 Mass Analysis Magnetic Sector Mass Analyzer Magnetic field is used to deflect ions around a curved path Radius of curvature of an ion depends on m/z ratio and strength of magnetic field Ions with correct m/z pass through the detector Ions too heavy or too light do not make it through Can vary strength of magnetic field so that all ions can be detected Can increase resolution further by subjecting ions to an additional electric field double focusing mass analyzer double focusing mass analyzer

19 Mass Analysis Quadripole Mass Analyzer Ions fly through a tunnel of four charged rods Voltage of the rods is changed in order to focus ions Ions with correct m/z are able to fly through to detector less sensitive à low resolution mass spec

20 Determination of Molecular Weight Identification of the Molecular Ion e + M M + 2 e Don t forget about isotopes polyisotopic mass CH 4 12 CH 4 13 CH 4 MW = 16 MW = 17 monoisotopic mass m/z 16 17

21 Determination of Molecular Weight Effects of Isoptope Differences Relative Isotopic Abundances of Common Elements Element Isotope Relative Abundance Isotope Relative Abundance Isotope Relative Abundance Carbon 12 C 13 C 1.11 Hydrogen 1 H 2 H Nitrogen 14 N 15 N 0.38 Oxygen 16 O 17 O O 0.2 Fluorine 19 F Silicon 28 Si 29 Si Si 3.35 Phosphorous 31 P Sulfur 32 S 33 S S 4.4 Chlorine 35 Cl 37 Cl 32.5 Bromine 79 Br 81 Br 98 Iodine 127 I see also: Pavia Table 3.5

22 Determination of Molecular Weight Effects of Isoptope Differences Exact Masses of Isotopes Atomic Element Weight Nuclide Mass Hydrogen Carbon Nitrogen Oxygen Fluorine Silicon Phosphorous Sulfur Chlorine Bromine Iodine H D ( 2 H) 12 C 13 C 14 N 15 N 16 O 17 O 18 O 19 F 28 Si 29 Si 30 Si 31 P 32 S 33 S 34 S 35 Cl 37 Cl 79 Br 81 Br 127 I (std) see also: Pavia Table 3.4

23 atom exact mass unit mass Mass Spectrometry 12 C:! 1 H: Determination of Molecular Weight 14 N: Effects of Isoptope Differences!16 O: O N C 7 H 7 NO MW =

24 Determination of Molecular Weight isotope 13 C:! 2 H: 15 N:!17 O: relative abundance O N C 7 H 7 NO MW = [M+1] % [M+1] = [M+1] M (1.1 x # of C atoms) + (0.016 x # of H atoms)!+ (0.38 x # of N atoms) etc. x = x M

25 O isotope relative abundance Mass Spectrometry Determination of Molecular Weight Relative abundance of [M+1] N C 7 H 7 NO MW = C:! 2 H: 15 N:!17 O: % [M+1] = [M+1] M (1.1 x # of C atoms) + (0.016 x # of H atoms)!+ (0.38 x # of N atoms) etc. x = x M % [M+1] = [M+1] M (1.1 x 7 C atoms) + (0.016 x 7 H atoms)!+ (0.38 x 1 N atoms) + (0.4 x 1 O atom) x = x % [M+1] = [M+1] M x = x 121 relative abundance % % [M+1] = [M+1] M x = 8 x = 6.6% 121 [M+1] 6.6% (calc) BUT: 13 C most abundant of isotopes for every C, 1.1% are 13 C can estimate: 7C x 1.1% = 7.7% 7.7% (est)

26 Determination of Molecular Weight Effects of Isoptope Differences M+3 M + M+1 M+2 M+3 M + A. peptide with 96 carbon atoms B. insulin (257 carbon atoms)

27 Determination of Molecular Weight # carbons? 1. calculate relative % [M+1] 2. divide by abundance 13 C m/z relative intensity M + [M+1] 1. calculate relative % [M+1] 2. divide by natural abundance of 13 C % [M+1] = [M+1] M x = 4.0 x = 8% = 7

28 Determination of Molecular Weight Effects of Isoptope Differences Relative Isotopic Abundances of Common Elements Element Isotope Relative Abundance Isotope Relative Abundance Isotope Relative Abundance Carbon 12 C 13 C 1.11 Hydrogen 1 H 2 H Nitrogen 14 N 15 N 0.38 Oxygen 16 O 17 O O 0.2 Fluorine 19 F Silicon 28 Si 29 Si Si 3.35 Phosphorous 31 P Sulfur 32 S 33 S S 4.4 Chlorine 35 Cl 37 Cl 32.5 Bromine 79 Br 81 Br 98 Iodine 127 I see also: Pavia Table 3.5

29 Determination of Molecular Weight Calculation of Relative Intensities Isotopic peak distribution using ChemDraw N O Chemical Formula: C 7 H 7 NO Exact Mass: Molecular Weight: m/z: (.0%), (7.7%) O SiMe 3 Chemical Formula: C 8 H 18 OSi Exact Mass: Molecular Weight: m/z: (.0%), (8.9%), (5.1%), (3.3%), (1.0%) [M+1] [M+2] [M+3] [M+4]

30 Determination of Molecular Weight Effects of Isoptope Differences Relative Isotopic Abundances of Common Elements Element Isotope Relative Abundance Isotope Relative Abundance Isotope Relative Abundance Carbon 12 C 13 C 1.11 Hydrogen 1 H 2 H Nitrogen 14 N 15 N 0.38 Oxygen 16 O 17 O O 0.2 Fluorine 19 F Silicon 28 Si 29 Si Si 3.35 Phosphorous 31 P Sulfur 32 S 33 S S 4.4 Chlorine 35 Cl 37 Cl 32.5 Bromine 79 Br 81 Br 98 Iodine 127 I see also: Pavia Table 3.5

31 Determination of Molecular Weight natural abundance 35 Cl!%!! 37 Cl 32.5% ca. 3:1 ratio Effects of Isoptope Differences Cl MW = 78 [M] + 9.0% [M+2] 3.1%

32 Determination of Molecular Weight natural abundance 78 Br!%!! 80 Br 98% ca. 1:1 ratio Effects of Isoptope Differences Br MW = 122 [M] + 8.6% [M+2] 8.3%

33 Determination of Molecular Weight Effect of Isoptope Differences

34 Identification of the Molecular Ion Ease of Fragmentation less fragmentation more fragmentation aromatics alkenes unbranched hydrocarbons ketones amines esters ethers carboxylic acids branched hydrocarbons alcohols higher relative abundance of M + lower relative abundance of M +

35 Identification of the Molecular Ion Ease of Fragmentation Stability plays a factor in whether or not a molecular ion is observed Decreasing ability to give prominent M+: aromatics > conjugated alkenes > cyclic compounds > organic sulfides > alkanes > mercaptans Decreasing ability to give recognizable M+: ketones > amines > esters > ethers > carboxylic acids ~ aldehydes ~ amides ~ halides M+ is frequently not detectable from: aliphatic alcohols, nitrites, nitrates, nitro compounds, nitriles, highly branched compounds

36 Identification of the Molecular Ion Molecular Ion Requirements 1. Peak must correspond to ion of highest mass in the spectrum - excluding those from isotopic compounds (usually lower intensity) 2. Decreasing energy in the ionizing electron stream (EI) should result in an increase in relative intensity of the molecular ion 3. The ion must have an odd number of electrons. 4. The Nitrogen Rule must be obeyed - a molecule with an odd number of N atoms has an odd atomic mass - a molecule with an even number of N will have an even atomic mass 5. Observed fragmentation must be reasonable - ion must be capable of forming the fragment ions seen by loss of neutral ions. Unlikely losses [M-4] [M-14] [M-21] [M-25] [M-33], [M-37], [M-38] Losses that identify M + [M-15] CH 3 [M-18] H 2 O [M-31] CH 3 O [M-16], [M-18] likely only if O present

37 Determination of the Molecular Formula Molecular Ion Requirements

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