Transcript Slide 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 Objective In this laboratory experiment, we will learn: 1. The principle of mass spectrometry (MS) 2. How to perform MS and MS/MS analysis 3. How to measure the gas-phase basicities of amino acids using MS/MS technique 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 Ionization methods 1. Electron impact method (electron) M + e M+ + 2e 3. Fast Atom Bombardment (atom) 2. Chemical ionization method (ions) NH4+ + RH RH2+ + NH3 4. Electrospray (for marcomolecules such as proteins) Instrumentation of mass spectrometer Single focusing MS Double focusing MS Quadrupole MS Time-of-Flight MS 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+ 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+ Kinetics in the fragmentation of ions At equilibrium, AH+ + B kf kb BH+ + A DG = -RTefflnkeq AH+ + B k1 k2 AHB+ (protonbound dimer) At equilibrium, k1[AH+][B] = k2[AHB+] k3 k4 and DG: free energy of the process R: ideal gas constant Teff: effective temperature at ion dissociation keq: equilibrium constant BH+ + A k3[AHB+] = k4 [BH+][A] keq = k1k3/k2k4 = [BH+][A] / [AH+][B] 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) 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: AH+ + B k2 AHB+ k3 BH+ + A D(DG) = RTeffln([[AH+] / [BH+])