1Electrospray Ionization Mass Spectrometry

Jessica GilmanCourtney Mashburn

17 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.”2

OutlinelIntroductionlIonization ProcesslIntroduction of Ions into MSlOperational Conditions and ParameterslSolvent and Analyte CharacteristicslSensitivity and Detection LimitslTandem TechniqueslSummary

IntroductionlESI allows for large, non-volatile molecules to be analyzed directly from the liquid phase

lUsed for:lMass determination of biomoleculeslAnalysis and sequencing of proteins and oligonucleotides

lAnalyzing drugs, pesticides, and carbohydrateslLong chain fatty acids3

ElectrosprayIonization ChamberAPI-Electrosprayoccurs at 760 torrProducts = neutrals, ions, and clusters of ions

Ionization MechanismslCoulomb Fission : lAssumes that the increased charge density, due to solvent evaporation, causes large droplets to divide into smaller droplets eventually leading to single ions.

lIon Evaporation: lAssumes 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 surfaces4

Charging the AnalytelCharge SeparationlGas-phase ions are formed when the droplets from the Taylor Cone evaporate and the ions carrying excess charge are released into the gas-phase.lAdduct FormationlPolar molecules that do not have acidic or basic groups can be charged through formation of adducts with various ions.

lNegative ion ESI: form adducts with Cl-ions

lPositive ion ESI: form adducts with Na+, Li+, NH4+, or other cationic species

lProblem: High [salt] causes background interference.

Charging the Analyte, Cont’dlGas-Phase Reactions:lIonized at atmospheric pressure.lGenerally through gas-phase proton transfer.lProton goes to species with higher gas-phase proton affinity lAnalyte must have higher proton affinity than the solvent.lElectrochemRedoxRxns: l“The continuous flow of charge from the metallic contact to the sample solution must occur via an electrochemical reaction at that contact.”

lPositive ion ESI: oxidation

lNegative ion ESI: reduction5

Pneumatically-Assisted ESIlMust separate ions from neutrals and establish complete desolvation.

lUse a neutral sheath gaslAids in droplet formation and desolvation.lOff-Axis Positioning

lMax amount of desolvatedanalyte

lSelects against un-evaporated droplets

Introducing Ions into the MSlUse a dry N2“curtain” gas:

lCharged species penetrate the curtain because they are electrostaticallyattracted toward the orifice by an electric field gradient.

lUse a heated metal capillary interfacelAids in desolvationand declusteringof ions from neutrals.6

ESI Operating ConditionslESI operation depends on the ability to balance many variables simultaneously

lSelf-adjusted flow rate of samplelSelf-adjusted voltage between power supply and contact with solution

lCapillary tube parameters:

lNon-conductive: fused silica tubinglConductive: metalizedglass capillarieslInner diameter = flow ratelOuter diameter = Taylor cone

Instrumental ParameterslStable and effective ESI spray conditions:lTaylor cone has constant shapelConstant stream of droplets from Taylor cone

lNebulizinggas and solution flow rates

lApplied voltage, viscosity, and dielectric constantlDistance between spray capillary and counter electrode

lMethanol or pneumatic assistance is required

lHigh water content means high surface tensionlHigher voltages must be applied7

Analyte CharacteristicslSurface-active analyteshave a higher responselThey follow charge during fissioningprocesslESI response is directly related to:

lNonpolarsurface areal∆G transfer from NP to P solutionslReverse-phase HPLC retention time

lDifference between analyte pH and solvent pH

lIf low response, change the molecular structure

lDerivatitationcan make the analyte more easily charged or can increase the surface activity

Surface ActivitylEquimolaramounts and all other variables equal

lSurface–inactive = Cs+lSurface–active = DTMA+

lFor quantitative work, calibration curves, etc. must be employed

lIntensity of peak is not solely related to conc.