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Mass Spectrometry and Related Techniques 2 Lecture Date: February 27th, 2008 Ion and Particle Spectrometry 2 - Outline  Atomic and Molecular Mass Spectrometry – Skoog et al. Ch 11 and 20.  Please read this additionalreference: – R.Aebersold and D. R. Goodlett, “Mass Spectrometry in Proteomics”,Chem. Rev., 2001, 101, 269-295.  Ion Mobility Spectrometry – If interested, see:  G. W.Eiceman, Critical Rev. Anal. Chem., 1991, 22, 471-489.  D. C. Collins and M. L. Lee, “Developments in ion mobility spectrometry – mass spectrometry”,Anal. Bioanal. Chem., 2002, 372, 66-73. 1 Homework Problems  If you read March’s paper on ion traps: – Whatis resonant excitation? Summarizehow resonant excitation is used in typical ion trap MS experiments.  If you read the Russell and Edmondson paper on MALDI-TOF and accurate mass: – Summarizethe advantages and disadvantages of MALDI-TOF (with DE and reflection) versus FTICR (including ESI-FTICR), especially in biochemical applications.  If you read the Aeberold and Goodlett proteomics paper: – Why is MS used so heavily in the study of post-translational modifications? Briefly describe an application to phosphopeptide sequence determinations.  If you read the Sleno and Volmer ion activation methods paper: – Pick any two of the ion activationprocesses described in the paper (e.g. in Table 1), describe how it works and the approximateenergies involved,and list one advantage Applications of Mass Spectrometry  Interpretationof mass spectra is the key to most applications of the technique  Information contained in a mass spectrum: – Molecular weight (viaexact or mono-isotopic mass). Usually obtained though a suitably accurate measurement of:  M+• (the molecularion, an odd-electronspecies)  [M+H]+ and [M-H]- (the protonated/de-protonatedmolecule, an even-electron species)  In sometechniques, can be confirmedby [M+Na]+, [M+K]+, [M+NH4]+,dimers, trimers,and other adducts, etc… – Molecular formula – Ionizationenergies – Isotopic incorporation (ex. 13C, 14C, 2H, 3H…) – Fragmentationand ion stability 2 Quasi-equilibrium Theory  Once we make an ion, what happens to it?  In EI, and similar techniques: the ionizingelectron has littlemass and high KE, so it barely movesthe molecule that it hits but leaves it in a higher rotational/vibrational state.  Ionizationenergies can sometimesbe determinedfromion intensities. Diagram from Strobel and Heineman, Chemical Instrumentation,A SystematicApproach,Wiley, 1989. Molecular Structural Analysis: Fragmentation  Fragmentationcan also be used to determine structure – commonfragmentationpathways and rearrangementscan be predicted in many cases Cl  General rules: - More stable carbocationsare more stablefragments (ex. tertiary carbocations are morestablethan primary) O p-chloro-benzophenone - Resonance can stabilize fragments,ex. allylic carbocationsand benzyl/tropyliumions - Loss of small,neutral, stablemolecules is favored Figure from R. M. Silverstein, Spectrometric Identification of Organic Compounds,6th Ed., Wiley, 1998. 3 Molecular Structural Analysis:  Isotope patterns can be used to determine molecular structure Isotope Patterns M (100%) – Example: the well-known methods of calculating(M+1) and (M+2) intensities – Especially useful for detecting chlorine, bromine,sulfur, silicon and many other elementswith characteristic profiles  Isotope patterns can also be used to extract out “isotope incorporation profiles”for labeled compounds – Examples:13C, 14C, 2H, 3H-labeled moleculesfor metabolism studies – Applicationsin isotope chemistry include the detection of stable and radioactive isotopes in synthetic products and in nuclear chemistry. M+1 (19.28%) M+2 (33.99%) M+3 (6.21%) 215 220 m/z O Cl p-chloro-benzophenone Molecular Structural Analysis: Accurate Mass  Nuclide masses are not integers. Example: Four things that weigh “28” amu: – CO, 27.9949 – 14N2,28.0062 – CH2N, 28.0187 – C2H4, 28.0312  m/z measurements to four decimal places or higher are needed  Accurate mass analysis is often used as a final confirmation of structure, or for unravellingcomplex fragmentation 4 Molecular Structural Analysis: Mass Defects Mass Defect (Da) 0.02 2H 0.01 1H 0 13C 14N 15N Atomic Mass Defects (All Different) -0.01 12C 16O Mass Defect = -0.02 Atom Mass – Nearest Integer -0.03 Every C H N O S mass is unique! 31P 32S -0.04 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Mass (Dalton) Picturecourtesy Prof. Alan Marshall, FSU/NHMFL Molecular Structural Analysis: MS-MS, and MSn  Step 1 – mass selection of an ion formed in the source  Step 2 – dissociation of the parent ion via collisions  Step 3 – mass analysis of the dissociated “daughter” ions  Step 4 – repeat… + + + + + Mass Analyzer 1 Collisions Mass Analyzer 2 + + + + + MassAnalyzer and Collision Chamber 5 ... - tailieumienphi.vn
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