Lecture 3:Ionization Techniques – Part IICU- Boulder CHEM 5181Mass Spectrometry & ChromatographyTaught by S. Kato / Slides from J. KimmelFall 2009Electrospray IonizationAtmospheric pressure ionizationEnables MS detection of large, non-volatile molecules (e.g., proteins) with no fragmentation (→Nobel Prize 2002)Search “ESI-MS” = 13,000 articlesFenn’s 1985 A Chem paper cited 845 timesLiquid elutes through a high voltage tip Coulombic explosions yield a continuous mist of bare, gas-phase ions (positive or negative)Conveniently coupled to liquid separations Characterized by multiply charged ionsNewobjective.comElectrospray Mechanism•An electrolytic analyte solution is pushed through the conductive end of capillary (id 10-100 um) at very low flow rate (0.1-10 uL/min) held a few mm from the entrance of the MS•High potential (2-4 kV) induces a strong electric field (106–107 Vm-1) •For positive field, cations will move towards the liquid surface and anions will move towards the conductive tip. •Repulsions between adjacent cationscombined with the pull of the cations towards the grounded MS inlet cause the surface to expand into a so-called ‘Taylor cone.’Gomez & Tang, Phys Fluids, 1994, 6:404–414ESI Mech (con’t)Balance induced E field and surface tension of liquidTip of the cone elongates into a filament, which breaks up and emits a stream of charged droplets towards the inlet of the mass spectrometer. Evaporation of solvent from the droplets increases the charge density.At the ‘Rayleigh limit,’ repulsion between cationsequal surface tension, causing ‘Coulombicexplosions’ that produce even finer droplets. This process of evaporation and explosion repeats until fully desolvated ions are released.The release of ions occurs either by repeated fission events until total evaporation of the solvent (Charge Residue Model) or by direct ion emission from a charged droplet (Ion Evaporation Model).Gomez & Tang, Phys Fluids, 1994, 6:404–414From Fig 13-18 LambertESI Mass SpectrumESI-MS of Cytochrome C, ~12,360 DaHigh charge states make m/zpracticle for most mass analyzer types.z can be determined by isotope distibution or sequence of peaks (see section 1.8.1 of De Hoffmann and HW #2)Isotope spacing= 0.067 = 1/15ESI Source DesignESI source must:1. Move ions from solution to the gas phase2. Transfer the gas-phase ions from atmospheric pressure to vacuum 3. Yield ion beam with maximum current and minimum kinetic energy distributionOn 1.• Stable spray requires user optimization• High flow rates may require nebulizing gas to form dropletsOn 2.• Heated drying gas + capillary encourage desolvation, and limits solvent analyte-adduct formation during expansion• Pumping speed places practical limit on size of entrance aperature• Transfer of ions between stages of decreasing pressure can result in a total ion loss on the order of four to five orders of magnitudeOn 3.• Harnessing expansion• Constant Velocity = high E distribution For discussion, see: “ESI Source Design and Dynamic Range Considerations,” A. P. Bruins, in “Electrospray Ionization Mass Spectrometry,” R. B. Cole, 1997.From de HoffmannControlled Current Electrolytic Flow CellFrom: Cech and Enke, Mass Spec Rev, 20, 362, 2001.•Electrical circuit to sustain ESI current : (+) Terminal to tip, to counter electrode, to (-) Terminal.•Electrolysis at electrodes maintains the charge balance to allow continuous production of charged droplets.•In order to supply demanded current, potential at electrode/solution interface has value permitting the oxidation process characterized by lowest oxidation potential in solution.•This process determines the total # of ions that can be produced per unit timeB-→ B + e-,etc.A++ e-→ Ae-e-oxidationreductionESI: Concentration Dependence• It is the excess charge in final droplets that imparts charge to gas-phase ions. (See Fig 1.24 in De Hoffmann)• ESI is sensitive to concentration, not flow. Because limiting current, IM, is dependent on oxidation process at tip.• ESI response can vary significantly among different analytes that have identical concentrations• For a system with one analyte, spray current for will depend on its concentration and a analyte-specific rate constant. IA= kA[A]• For system of two analytes, A and B, IT= IM= IA + IB And, currents proportional to relative desorption rates and signal responses are coupled. Complicates quantification. (See section 1.8.4 of de Hoffmann)• For any system, dynamic range limited at high end (~1 mM) by:– Limited amount of excess charge– Limited space on droplet surface– Ion suppression • Consider separations prior to ESI to maximize sensitivityNanosprayNew Objective SilicaTips. Tip i.d. range from 5 to 30 um.Flow rate: 20 to 1000 nL/min•10 – 100 nl/min flow rate with fine spray tip•Flow rate and droplets 100-1000 times smaller than conventional ESI•Large proportion of analyte available for desorption from surface. 2-3 time higher ion current than ESI at a given concentration•Smaller tip close to orifice: narrow dispersion of droplets yields better transfer in MS•Orders of magnitude (2+) improvement in efficiency (analyte detected / analyte sprayed)•At these flow rates, ESI becomes “mass flux sensitive” •Longer analysis times -- better SNR and/or more options in MS experimentSee: Wilm and Mann, A. Chem., 68, 1-8, 1996.EI and CI are methods for molecular analysis of gas phase sampleAPCI and ESI: molecular analysis of liquid phase Now, “Chemistry” of ESI.Summary(S. Kato, Fall 2009)Mass Spectrom. Rev. (2009)• • • • • • • • • • • •Sorry….Why Is Chemistry Important in ESI?MS method Ionizing reagent Ion polarity & typeMALDI-TOFESI-Quadrupole-TOF(or ESI-Ion trap)GC-Ion trapsolid matrix (CHCA, SA, DHB)protic solvent (CH3OH, H2O,…)additives (HCOOH, NaCl,…)electron+/-, closed shell+/-, closed shell+, open shell*(radical cation M+•)ESI chemistry (incl. fragmentation and CID):Con: Many parameters to optimizeFro: “Closed-shell chemistry”, relatively easy to understand and control*Fragmentation of radical cations will be discussed in a later lecture.(MH+, [M - H]-)(MH+, [M - H]-)What is Known about ES Ionization of Neutrals?Where and how neutral species get ionized?Similarity and dissimilarity between gas-phase and solution-phaseionization chemistries?• in solution, in the vanishing droplets, and in the gas