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Organic Chemistry, 5th Edition L. G. Wade, Jr. Chapter 12 Mass Spectrometry Jo Blackburn Richland College, Dallas, TX Dallas County Community College District 2003, Prentice Hall 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. • The masses of the fragments and their relative abundance reveal information about the structure of the molecule. => Chapter 12 2 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-). H H H C C H H H H H H H e- + H C C+ H C C H H H H H H H C+ Chapter 12 H H H C H H => 3 Separation of Ions • 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. => Chapter 12 4 Mass Spectrometer Chapter 12 5 => The Mass Spectrum Masses are graphed or tabulated according to their relative abundance. Chapter 12 6 => The GC-MS A mixture of compounds is separated by gas chromatography, then identified by mass spectrometry. Chapter 12 7 => High Resolution MS • Masses measured to 1 part in 20,000. • A molecule with mass of 44 could be C3H8, C2H4O, CO2, or CN2H4. • If a more exact mass is 44.029, pick the correct structure from the table: C3H8 C2H4O CO2 CN2H4 44.06260 44.02620 43.98983 44.03740 => Chapter 12 8 Molecules with Heteroatoms • Isotopes: present in their usual abundance. • Hydrocarbons contain 1.1% C-13, so there will be a small M+1 peak. • If Br is present, M+2 is equal to M+. • If Cl is present, M+2 is one-third of M+. • If iodine is present, peak at 127, large gap. • If N is present, M+ will be an odd number. • If S is present, M+2 will be 4% of M+. => Chapter 12 9 Isotopic Abundance 81Br => Chapter 12 10 Mass Spectrum with Sulfur => Chapter 12 11 Mass Spectrum with Chlorine Chapter 12 12 => Mass Spectrum with Bromine Chapter 12 13 => Mass Spectra of Alkanes More stable carbocations will be more abundant. Chapter 12 14 => Mass Spectra of Alkenes Resonance-stabilized cations favored. Chapter 12 15 => Mass Spectra of Alcohols • Alcohols usually lose a water molecule. • M+ may not be visible. Chapter 12 16 => Identifying Molecular Ions •Potential question; Is the largest m/z the molecular ion or is it a prominent fragment from an even heavier molecule? •Increase sample loading •In EI, can lower the beam voltage (make the M•+ less energetic, perhaps more long-lived.) •Logical interval between significant peaks and suspected M•+ . i.e. the loss of 3-14 mass units is unusual, as is loss of 19-25 (except F). Loss of 33, 35, 38 also unusual. However a loss of 15, 18, 31 is good evidence for a molecular ion. •Switch to CI, vary reagent gas. Positive, negative probes. Check for CI adduct ions. e.g. C2H5+ , CH5+, C3H5+ •Find MW by other method Other compounds present may give ions that deceive us. May be more detectable. •Prepare derivative MS intensities are problematic The “Nitrogen Rule” •Molecules containing atoms limited to C,H,O,N,S,X,P of even-numbered molecular weight contain either NO nitrogen or an even number of N •This is true as well for radicals as well. • Not true for pre-charged, e.g. quats, (rule inverts) or radical cations. •In the case of Chemical Ionization, where [M+H]+ is observed, need to subtract 1, then apply nitrogen rule. •Example, if we know a compound is free of nitrogen and gives an ion at m/z=201, then that peak cannot be the molecular ion. ElectronImpact and ChemicalIonization EI CI Sometimes too energetic for molecular ion to survive Stronger, more reliable molecular ions Rich harvest of fragment ions Fewer fragments “fingerprint” nature of fragment patterns lends itself to database library searches Can choose different reagent gasses and exploit chemistry, giving different fragmentation. e.g. NH3/ND3 Adduct ions give support to identities EI CI Nitrogen rule works but inverted Can do negative ion Mass Spec When would you use CI, EI? EI CI •When “fingerprint” is needed for Identification by comparison screening in databases •When rapid, reliable identification of molecular ion is needed. •Trace analysis •Forensics •Environmental •Total unknowns, e.g natural products •LC-MS •Following a synthetic chemistry route, tentative impurity ID •Biological samples, other fragile or sensitive to decomposition; Drug or other metabolite ID •When reagent gas chemistry is key, e.g. exchange D in for H •Fragment homology within a series, e.g. of natural products •Minimize fragmentation, get most intensity in molecular ion. How can I tell which (EI or CI) was run? Chemical Ionization Adduct ions higher m/z than MH+, ,[M+C2H5]+ ,[M+C3H5]+ [M+NH4]+ Large molecular ion Relatively few fragment ions Electron Impact No ions higher m/z than M•+ Smaller M•+ intensity Rich family of fragment ions The “Rule of 13” as an aid to guessing a molecular Formula Take the Weight of ion, divide by 13 This answer is N, for (CH)N and any numerical remainder is added as H e.g.; 92 92/13 = 7 with remainder = 1; C7H8 weighs 92. This is our candidate formula Can evaluate other alternative candidate formulas possessing heteroatoms. For each member of the list below, replace the indicated number of CHs in the above answer Hetero substitution CH replacement Hetero substitution CH replacement O CH4 P C2H7 N CH2 S C2H8 O+N C2H6 O+S C4 F CH7 I C10H7 Si C2H4 Cl,Br (use isotopes) Analyzing Ion Clusters: a way to rule candidate structures in or out Mass spectrometry “sees” all the isotopomers as distinct ions An ion with all 12C is one mass unit different from an ion with one 13C and the rest 12C Since the isotope distribution in nature is known* for all the elements (13C is 1.1%), the anticipated range and ratios of ions for a given formula can be predicted and calculated Follows a binomial expansion: e.g.; for N carbon atoms (%12C + %13C)N Clusters of Ions Spaced by unit mass The Nominal mass is m/z of the lowest member of the cluster. This is the isotopomer that has all the C’s as 12C, all protons as 1H, all N’s as 14N, etc. Each peak is for the same molecular formula Different peaks because there are some molecules with 13C, 2H etc. Especially significant for Cl, Br m/z Isotope Patterns in Ion Clusters Here are two molecular ions of nearly the same m/z. One of them is “carbon-rich”, and has a larger number of 13C’s The other, presumably has proportionately, more heteroatoms C24H50 C12H22O11 Why is this Important? A rule of thumb, made possible by knowing the isotopic abundance is that the number of C in a formula is given by: N= Intensity M 1 x90.1 M C10 All 12C C100 1 13C 2 13C 1 13C From this, it is clear that for large or macromolecules, there will be practically no population having all 12C or even only 113C Fragmentation EI [M·]+ A+ + B· (neutral) or B+ + A· Better carbocation wins and predominates (“Stevenson’s Rule”) CI [M+H]+ PH+ + N (neutral) The “Even Electron Rule” dictates that even (non-radical) ions will not fragment to give two radicals (pos• + neutral•) (CI) Reading a Mass Spec from the M+• Down (EI) Fragment Due to loss of… Interpretation M+• -1 -H• Aldehydes, tert. Alcohols, cyclic amines M+• -2 Multiple -H• Secondary alcohols M+• -3 Multiple -H• Primary alcohols M+• -4 to -13 (doubtful) Consider contaminants M+• -14 (doubtful) CH2• , N• not good losses M+• -15 CH3• Available methyl groups, methylesters M+• -16 O• Peroxides M+• -17 OH• Alcohols, phenols, RCO2H M+• -18 H2O alcohols M+• -19 -F• M+• -20 -HF M+• -21 to -25 No peaks expected M+• -26 HCCH M+• -27 •HC=CH2 or HCN HCN from pyridine, anilines M+• -28 CO or CH2=CH2 Check for McLafferty R&R How Do I go about using Mass Spec Data for Unknowns? First, get the molecular weight Identify prime, smaller mass losses like water, etc. Now stop. Don’t worry about the fragments till you have some candidate structures Based on NMR, IR get some notions of structure candidates or partial structures, functional groups Now go back to MS, predict some fragments your structure will give, calculate the molecular weights and check MS Back and forth with other data, to corroborate or refute a possible structure. Nominal Mass Here for example is a list of the compounds in the Merck Index (9th ed) that weigh nominally, 200 Exact mass measurements can easily distinguish These instruments (and other) can generate exhaustive lists of possible structure formulas near the exact mass value. Example, m/z’s for 157 molar mass: 157 Formula M+1 M+2 MM e/o dbr HN2O8 1.05 1.60 156.9732 e 1.5 HN10O 3.75 0.20 157.0337 e 5.5 H3N3O7 1.41 1.40 156.9971 o 1 H3N11 4.12 0.00 157.0576 o 5 H5N4O6 1.78 1.20 157.021 e 0.5 H7N5O5 2.14 1.00 157.0448 o 0 CHO9 1.45 1.80 156.9619 e 1.5 CHN8O2 4.15 0.43 157.0224 e 5.5 CH3NO8 1.81 1.60 156.9858 o 1 CH3N9O 4.51 0.24 157.0463 o 5 CH5N2O7 2.17 1.41 157.0096 e 0.5 CH5N10 4.88 0.04 157.0702 e 4.5 CH7N3O6 2.54 1.21 157.0335 o 0 C2HN6O3 4.55 0.66 157.0111 e 5.5 C2H3N7O2 4.91 0.47 157.0350 o 5 C2H5O8 2.57 1.61 156.9983 e 0.5 C2H5N8O 5.27 0.28 157.0589 e 4.5 C2H7NO7 2.93 1.42 157.0222 o 0 C2H7N9 5.64 0.09 157.0827 o 4 C3HN4O4 4.94 0.89 156.9998 e 5.5 C3H3N5O3 5.31 0.70 157.0237 o 5 C3H5N6O2 5.67 0.51 157.0476 e 4.5 C3H7N7O 6.03 0.33 157.0714 o 4 C3H9N8 6.4 0.14 157.0953 e 3.5 C4HN2O5 5.34 1.11 156.9885 e 5.5 C4H3N3O4 5.70 0.93 157.0124 o 5 C4H5N4O3 6.07 0.74 157.0362 e 4.5 C4H7N5O2 6.43 0.56 157.0601 o 4 C4H9N6O 6.79 0.38 157.0840 e 3.5 C4H11N7 7.16 0.20 157.1078 o 3 C5HO6 5.74 1.32 156.9772 e 5.5 C5H3NO5 6.10 1.15 157.0011 o 5 C5H5N2O4 6.46 0.97 157.025 e 4.5 C5H7N3O3 6.83 0.79 157.0488 o 4 C5H9N4O2 7.19 0.61 157.0727 e 3.5 C5H11N5O 7.55 0.44 157.0965 o 3 C5H13N6 7.92 0.26 157.1204 e 2.5 C6HN6 8.84 0.33 157.0264 e 9.5 C6H5O5 6.86 1.19 157.0136 e 4.5 C6H7NO4 7.22 1.02 157.0375 o 4 C6H9N2O3 7.59 0.84 157.0614 e 3.5 C6H11N3O2 7.95 0.67 157.0852 o 3 C6H13N4O 8.31 0.50 157.1091 e 2.5 C6H15N5 8.68 0.32 157.1329 o 2 C7HN4O 9.23 0.58 157.0151 e 9.5 C7H3N5 9.60 0.41 157.0390 o 9 C7H9O4 7.98 1.07 157.0501 e 3.5 C7H11NO3 8.35 0.90 157.0739 o 3 C7H13N2O2 8.71 0.73 157.0978 e 2.5 C7H15N3O 9.07 0.56 157.1217 o 2 C7H17N4 9.44 0.40 157.1455 e 1.5 C8HN2O2 9.63 0.81 157.0038 e 9.5 C8H3N3O 9.99 0.65 157.0277 o 9 C8H5N4 10.36 0.49 157.0516 e 8.5 C8H13O3 9.11 0.96 157.0865 e 2.5 C8H15NO2 9.47 0.80 157.1104 o 2 C8H17N2O 9.83 0.64 157.1342 e 1.5 C8H19N3 10.20 0.47 157.1581 o 1 C9HO3 10.03 1.05 156.9925 e 9.5 C9H3NO2 10.39 0.89 157.0164 o 9 C9H5N2O 10.75 0.73 157.0403 e 8.5 C9H7N3 11.12 0.57 157.0641 o 8 C9H17O2 10.23 0.87 157.1229 e 1.5 C9H19NO 10.59 0.71 157.1468 o 1 C9H21N2 10.96 0.55 157.1706 e 0.5 C10H5O2 11.15 0.97 157.029 e 8.5 C10H7NO 11.51 0.81 157.0528 o 8 C10H9N2 11.88 0.66 157.0767 e 7.5 C10H21O 11.35 0.79 157.1593 e 0.5 C10H23N 11.72 0.64 157.1832 o 0 C11H9O 12.27 0.90 157.0654 e 7.5 C11H11N 12.64 0.75 157.0892 o 7 C12H13 13.40 0.84 157.1018 e 6.5 C13H 14.32 0.97 157.0078 e 13.5 Clearly, some are not realistic! Calculated mass distributions IMASS for Mac OSX Version 1.0 (v2A15) © 2000 - 2002, Urs Roethlisberger, Isotopic Element Massesand Atomic Weights:Lide, D.R., Ed., CRC Handbook of Chemistry and Physics,74th Ed., CRC Press, Boca Raton FL,(1993) Isotope Distribution:Rockwood, A. L., Van Orden, S. L.,Smith, R. D.,Anal. Chem. , 67, 2699, (1995) •iMass is freeware. •Contact: [email protected] Fragment Ions •The Game is, to rationalize these in terms of the structure •Identify as many as possible, in terms of the parent structure •Generally, simply derived from the molecular ion •Or, in a simple fashion from a significant higher mw fragment. •Simply, here means, ions don’t fly apart, split out neutrals and then recombine. •Fragments will make chemical sense •A good approach is the “rule of 13” to write down a molecular formula for an ion of interest. •Especially in EI, we only identify major fragments Chemical Ionization Fragmentation Loss of neutral molecules, small stable, from MH+ Loss of neutrals from protonated fragments Subsequent reprotonation after a loss Typically there is no ring cleavage (needs radical) or two bond scissions. Depends highly on ion chemistry specifically acidbase (proton affinities) Some popular cleavages Cleave at a branch point. Loss of radical or other neutral to provide a more stable cation + H3C CH3 CH3 H3C Cleave to a heteroatom (capable of supporting positive charge) Note the use of “half arrow” for one-electron movements. e.g homolytic cleavage + CH3 H3C C + Obs. in Mass Spec RO: CH3. neutral RO + RO : Obs. in Mass Spec Resonance stabilized + neutral Some examples Primary alcohols, m/z=31 CH2=OH+ Primary amines, m/z=30 CH2=NH2+ Commonly encountered ElectronImpact fragments O + 29 H CH2 + 43 H 77 H + CH2 + N 92 H H + H 91 + McLafferty Rearrangements Radical cations localized on keto-type oxygen give cleavage The mechanism limits this to EI fragmentation Needs a H atom on a sp3 carbon Ketones, esters, carboxylic acids all give McLafferty products H + O• R1 R2 H + O R1 • Note the use here, of the “half arrow” to represent “1-electron flow” The new radical cation is stabilized by resonance •• • OH + R2 Loss of neutral alkene Important example of McLafferty R&R OH + • OH m/z = 60 Seen for primary carboxylic acids Non-Sequential Losses O CH3 M+-CH3CO M+-CH3 MW=152 CH3 Hydrocarbons Weak [M•]+ Intense CnH2n+1 Good 43 m/z = C3H7 protonated cyclopropane 57 m/z = C4H9+ 71 m/z = C5H11 Hydrocarbon chains characterized by successive losses of m/z=14 (clusters) Cleavage to C=O groups + :O . :O + + neutral Prominent for ketones :O: + Obs. in mass spec. Acylium ions are resonance-stabilized CH3C=O+ m/z=43 Example O O O C O O O + + + M+• -45, loss of ethoxy radical Example + O O M+• -43; also tropylium ion Cleave to Heteroatoms like O, N R + R :O. • neutral : O. : + + Heterolytic cleavage Observed in Mass Spec provided that a good stabilized carbocation can form Rearrangements and fragmentations to give good Carbocations CH 2+ Benzylic cation (stabilized C+ H including “tropylium” H+C ion m/z=91 CH 2 CH 2 CH+ Good cleavage to aromatic rings Example Br Tropylium ion Bromine pattern Carboxylic acids H present?; can give McLafferty R&R to alkene plus CH2=C(OH)(OH)•+ at m/z=60 Loss of water, especially in CI Loss of 44 is loss of CO2 m/z=45 suggests OC–OH+ Amines N•+ -R• N + R Cyclic amines will lose adjacent H•, form iminium ion In CI, NH+ can eliminate adjacent alkene, reprotonate Silyl Ethers O+ • Si Loss of CH3• from Si Loss of R• in cleavage Loss of •CR3 then CH3• to (CH3)2Si=OH+ m/z=75 Total loss of carbinol to (CH3)3Si+ m/z=73 H transfer in heterosubstituted Anisoles OR Loss of CH2O + OR H OCH3 +• Extra H transfer mediated by adjacent heteroatom H H Nitroaromatics m/z= 93 Loss of •N=O + N O + O O• Loss of CO Aromatic! m/z=65 Good test for aryloxy CH+ (this can form from lots of different origins) Sulfur Compounds Fortunately there is an [M+2]+ of 4% for the natural abundance of 34S. This is diagnostic for S vs 2x16O Aliphatic thiols can split out H2S, [M-34] Alpha cleavage at carbon bearing the sulfur in thiols, thioethers, similar to ethers, etc. R -R• S • + S + The “retro Diels-Alder” Cleavage Cyclohexenes, with favorable 6-membered transition state. Can include heteroatoms (N,O, driven by keto-enol like stability. Observed! + • + • + Typically you see both. More stable cation will predominate Also works for hetero-substituted (e.g. make enol) Both EI (shown) and in Chemical Ionization. (protonated molecular ion, cleave, then reprotonation + • + Observed! An Example from Terpenoid Chemistry + • + + • 12-Oleanene m/z 204 A good example for Retro Diels Alder fragmentation HO EI Mass Spectrum HO 4-terpineol + mz 68 MW 154 + + mz 86 O Double bonds can isomerize OH MW=396 -cleavage following double bond migration m/z118 136-water m/z136 C9H12O+ Mass Spectral “shifts” Note highly conserved regions; series of related compounds Losses down to ions common in series. Variation can not influence the fragmentation or introduce new fragmentation, e.g. internal fission not possible for homologs Using the Information in Ion Clusters--Halogens The paired appearance flags the ions as to the number of halogens 35Cl 37Cl CH3Cl CHCl3 One chlorine Three chlorines Fragment ions with the same halogen count preserve the pattern 79Br 81Br 81Br 81Br CH3Br CHBr3 One bromine Three bromines 2 1