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Yat Li Department of Chemistry & Biochemistry University of California, Santa Cruz

CHEM 146C_Experiment #5 Measuring Gas-phase Basicities of Amino Acids using an Ion Trap Mass Spectrometer. Yat Li Department of Chemistry & Biochemistry University of California, Santa Cruz. Objective. In this laboratory experiment, we will learn:. The principle of mass spectrometry (MS)

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Yat Li Department of Chemistry & Biochemistry University of California, Santa Cruz

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  1. CHEM 146C_Experiment #5 Measuring Gas-phase Basicities of Amino Acids using an Ion Trap Mass Spectrometer Yat Li Department of Chemistry & Biochemistry University of California, Santa Cruz

  2. Objective In this laboratory experiment, we will learn: The principle of mass spectrometry (MS) How to perform MS and MS/MS analysis How to measure the gas-phase basicities of amino acids using MS/MS technique

  3. Mass spectrometer Ions with different mass-to-charge ratio (m/z) can be separated and analyzed by mass spectrometer ½ mv2 = zV Hzv = mv2/r m/z = H2r2/2V m: mass of the ion z: charge of the ion H: applied magnetic field r: radius of arc of deflection V: applied accelerating voltage

  4. Ionization methods 1. Electron impact method (electron) 2. Chemical ionization method (ions) M + e M+ + 2e NH4+ + RH  RH2+ + NH3 3. Fast Atom Bombardment (atom) 4. Electrospray (for marcomolecules such as proteins)

  5. Instrumentation of mass spectrometer Single focusing MS Double focusing MS Quadrupole MS Time-of-Flight MS

  6. Determination of molecular mass of ions An acidic solution containing two amino acids, Amino acid A: Phe Amino acid B: Leu Phe-H+ MS Spectrum: Phe-H-Leu+ Leu-H+

  7. Ion separation and analysis using MS/MS technique AHB+ ions are separated and further analyzed by MS/MS. MS/MS Spectrum: Phe-H+ Leu-H+

  8. Kinetics in the fragmentation of ions kf At equilibrium, AH+ + B BH+ + A kb DG: free energy of the process R: ideal gas constant Teff: effective temperature at ion dissociation keq: equilibrium constant DG = -RTefflnkeq k1 k3 AH+ + B AHB+ BH+ + A k4 k2 (proton-bound dimer) k3[AHB+] = k4 [BH+][A] k1[AH+][B] = k2[AHB+] and At equilibrium, keq = k1k3/k2k4 = [BH+][A] / [AH+][B]

  9. Kinetics in the fragmentation of ions keq = k1k3/k2k4 = [AH+][B] / [BH+][A] k1 ≈ k4 k1 and k4 refer to collision rates between ions and neutral molecules keq= k3 / k2 D(DG) = -RTefflnkeq = -RTeffln(k3/k2) = RTeffln(k2/k3) k3 k2 AH+ + B AHB+ BH+ + A The ratio is equal to the product ratio, which is to a good approximation equal to the ratio of the intensities for these two ions in the MS/MS spectrum: D(DG) = RTeffln([[AH+] / [BH+])

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