1Electrospray Ionization Mass SpectrometryJessica GilmanCourtney Mashburn17 September 2002Chemistry 5181“Many users tend to view ESI as a ‘Black Box,’ because sources of instability, background, interference, competition, and suppression are not always understood.”2Outlinel Introductionl Ionization Processl Introduction of Ions into MSl Operational Conditions and Parametersl Solvent and Analyte Characteristicsl Sensitivity and Detection Limitsl Tandem Techniquesl SummaryIntroductionl ESI allows for large, non-volatile molecules to be analyzed directly from the liquid phasel Used for:l Mass determination of biomoleculesl Analysis and sequencing of proteins and oligonucleotidesl Analyzing drugs, pesticides, and carbohydratesl Long chain fatty acids3Electrospray Ionization ChamberAPI-Electrospray occurs at 760 torrProducts = neutrals, ions, and clusters of ionsIonization Mechanismsl Coulomb Fission : l Assumes that the increased charge density, due to solvent evaporation, causes large droplets to divide into smaller droplets eventually leading to single ions.l Ion Evaporation: l Assumes the increased charge density that results from solvent evaporation causes Coulombicrepulsion to overcome the liquid’s surface tension, resulting in a release of ions from droplet surfaces4Charging the Analytel Charge Separationl Gas-phase ions are formed when the droplets from the Taylor Cone evaporate and the ions carrying excess charge are released into the gas-phase.l Adduct Formationl Polar molecules that do not have acidic or basic groups can be charged through formation of adducts with various ions.l Negative ion ESI: form adducts with Cl-ionsl Positive ion ESI: form adducts with Na+, Li+, NH4+, or other cationic speciesl Problem: High [salt] causes background interference.Charging the Analyte, Cont’dl Gas-Phase Reactions:l Ionized at atmospheric pressure.l Generally through gas-phase proton transfer.l Proton goes to species with higher gas-phase proton affinity l Analyte must have higher proton affinity than the solvent.l Electrochem Redox Rxns: l “The continuous flow of charge from the metallic contact to the sample solution must occur via an electrochemical reaction at that contact.”l Positive ion ESI: oxidationl Negative ion ESI: reduction5Pneumatically-Assisted ESIl Must separate ions from neutrals and establish complete desolvation.l Use a neutral sheath gasl Aids in droplet formation and desolvation.l Off-Axis Positioningl Max amount of desolvated analyte l Selects against un-evaporated dropletsIntroducing Ions into the MSl Use a dry N2“curtain” gas:l Charged species penetrate the curtain because they are electrostatically attracted toward the orifice by an electric field gradient.l Use a heated metal capillary interfacel Aids in desolvation and declustering of ions from neutrals.6ESI Operating Conditionsl ESI operation depends on the ability to balance many variables simultaneouslyl Self-adjusted flow rate of samplel Self-adjusted voltage between power supply and contact with solutionl Capillary tube parameters:l Non-conductive: fused silica tubingl Conductive: metalized glass capillariesl Inner diameter = flow ratel Outer diameter = Taylor coneInstrumental Parametersl Stable and effective ESI spray conditions:l Taylor cone has constant shapel Constant stream of droplets from Taylor conel Nebulizing gas and solution flow rates l Applied voltage, viscosity, and dielectric constantl Distance between spray capillary and counter electrodel Methanol or pneumatic assistance is requiredl High water content means high surface tensionl Higher voltages must be applied7Analyte Characteristicsl Surface-active analytes have a higher responsel They follow charge during fissioning processl ESI response is directly related to:l Nonpolar surface areal ∆G transfer from NP to P solutionsl Reverse-phase HPLC retention timel Difference between analyte pH and solvent pHl If low response, change the molecular structurel Derivatitation can make the analyte more easily charged or can increase the surface activitySurface Activityl Equimolar amounts and all other variables equall Surface–inactive = Cs+l Surface–active = DTMA+l For quantitative work, calibration curves, etc. must be employedl Intensity of peak is not solely related to conc.8Solution Characteristicsl Variables that must be properly balanced:l Flow rate, applied voltage, conductivity, liquid surface tensionl Ideal solvent depends on applicationl Improves response and will not form clustersl Positive ion mode: 50% MeOH or Acetonitrilel Negative ion mode: Halogenated SolventsTypical ESI SpectrumPositive-Ion ModeNegative-Ion Mode9Ion Transmission and Sensitivityl Methods to improve sensitivity:l Improving ion transfer through MSl Improve efficiency of charging ESIanalytesl The limiting factor in sensitivity with ESI is the transfer of ions through the MS.Higher Sensitivity is Better!!!!Sources of Non-linearityl Background Interference:l Chemical noise results from mass analysis of charged species other than the analytel Heating droplets helps to drive off solvent and reduces interferencesl Random Noise:l Ubiquitous throughout MS spectruml Possible Sources:l Ions reach detector without going through mass analysisl Passage of large, highly-charged clusters impacting the detector, creating a shower of fragments with various m/z ratios10Improving Detection Limitsl To Lower Concentrations:l Limited to concentrations above 10-8M l Remove chemical noise if possiblel Selected reaction monitoringl Increase in sensitivityl To Higher Concentrations:l Limited to concentrations lower than 10-5Ml More efficient charging of gas-phase analytel More effective evaporation techniquesl When analyte concentration exceeds the excess charge, the ESI response should level offl Competition for a limited resource – Space and Excess Charge ESI Coupled with LC or CEl ESI is not a separation technique per sel It can be used as a “detector” when coupled with separation techniquesl Separation protocols must be modifiedl Ideal LC/CE solvent may match ideal ESI solventl Volatile buffers composed of weak acids and bases are used instead of non-volatiles which can cause signal suppressionl Ion-pairing reagents must be eliminated11Summaryl Advantagesl High mass samples l Non-volatile moleculesl Large mass range (105Da)l Low fragmentation l Determination of m.w.l Cheaper mass analyzersl Direct analysis of liquidsl Compatible