11 ionization sources drug chemistry training may 31-june 2, 2011 by: rachel beck, dftcb
TRANSCRIPT
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Ionization SourcesIonization Sources
Drug Chemistry TrainingDrug Chemistry Training
May 31-June 2, 2011May 31-June 2, 2011By: Rachel Beck, DFTCBBy: Rachel Beck, DFTCB
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OverviewOverview
IonizationIonization DefinitionDefinition
TheoryTheory
ApplicationApplication
General MaintenanceGeneral Maintenance
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What is Ionization?What is Ionization?
Energy necessary to remove an electron from a Energy necessary to remove an electron from a neutral atom.neutral atom.11
The complete removal of an electron from an atom The complete removal of an electron from an atom following the transfer of energy from a passing following the transfer of energy from a passing charged particle.charged particle.22
Any process by which electrically neutral Any process by which electrically neutral atoms/molecules are converted to electrically atoms/molecules are converted to electrically charged atoms /molecules (ions).charged atoms /molecules (ions).33
1. http://hyperphysics.phy-astr.gsu.edu/hbase/chemical/ionize.html
2. http://www.ndt-ed.org/EducationResources/CommunityCollege/Radiography/Physics/Ionization.htm
3. http://www.britannica.com/EBchecked/topic/293007/ionization
The formation of charged species (analyte The formation of charged species (analyte ions) for the purpose of identification and ions) for the purpose of identification and quantification by:quantification by:
Addition or loss of a protonAddition or loss of a proton Adduct additionAdduct addition Loss of an electron (single electron)Loss of an electron (single electron) Charge ExchangeCharge Exchange Bond dissociationBond dissociation
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Classification of IonizationClassification of Ionization
Two classes of ionizationTwo classes of ionization Hard ionization Hard ionization
Impart sufficient energy to analyte molecules so they Impart sufficient energy to analyte molecules so they are in an excited energy state. are in an excited energy state.
Relaxation involves repeated rupture of bonds Relaxation involves repeated rupture of bonds producing fragment ionsproducing fragment ions
Soft ionizationSoft ionization Causes little fragmentationCauses little fragmentation Very simple spectra as a resultVery simple spectra as a result
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Ionization SourcesIonization Sources
4 major types of ionization sources4 major types of ionization sources EI: EI: EElectron lectron IImpactmpact CI: CI: CChemical hemical IIonizationonization ESI: ESI: EElectrolectroSSpray pray IIonizationonization APCI: APCI: AAtmospheric tmospheric PPressure ressure CChemical hemical IIonizationonization
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EI TheoryEI Theory Gaseous Analytes and carrier gas are
bombarded with electrons from the filament. Filament = 70eV
The collisions cause the analyte to lose an electron yielding a positively charged molecular ion. Molecular ion – charged form of the
molecule/analyte The molecular ion will fragment further to
reduce the excess energy. Extensive fragmentation
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Ionization Mechanism
++ ++e-- 2e--++••
Analyte
++++ ••
+++•+•
Clarke’s Analysis of Drugs and Poisons
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EI MechanismEI Mechanism
http://www.chromacademy.com/resolver-november2010_Understanding_GCMS_part_1.asp
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Ion MovementIon Movement Ions are accelerated out of the ionization chamber
towards the mass analyzer by the repeller. Repeller 0 to + 42.8 V dc (dc = direct current)
The ions will pass through the draw out lens and cylinder Ground potential 0 V
The ions will then pass through the ion focus lens and entrance lens to the mass analyzer Ion focus 0 to -127 V dc
Typically -70 to -90 V dc
Entrance Lens = 4.4V dc + offset + (gain x mass) Entrance offset 0 to -64 V dc (-20 V is typical) Entrance Gain 0 to -128mV/amu (0 to -40 mV/amu typical)
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Electron IonizationElectron Ionization
Only positive ions are formed Singly charged
Sample must be volatile Limited to low molecular weight compounds
approximately 600 Da or less Hard Ionization technique
Extensive fragmentation Low abundance of molecular ion
Typically used in conjunction with a mass analyzer
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Cocaine Example
http://www.chromacademy.com/resolver-november2010_Understanding_GCMS_part_1.asp
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272
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EI Temperature SettingsEI Temperature SettingsRemember !!
Ions are in the vapor phase so high temperatures are needed.
Auxiliary transfer line Limits 0 – 350 ºC Typically set to 280 ºC
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TuningTuning Tune with PFTBA (Perfluorotributylamine) Auto Tune
Adjusts all values to reach maximum abundances Has minimum values
Standard Spectra Tune (Target Tune) Set to reach specified ratio targets for specific ion Has both minimum and maximum values
Quick Tune Adjusts the mass assignments, peak widths, and abundances. Does not change ion ratios
Can also tune for specific spectral results DFTPP or BFB Target Tunes Tune to meet preset ratios
http://www.chem.agilent.com/Library/Support/Documents/f05036.pdf
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Auto Tune CriteriaAuto Tune Criteria
Mass assignments should be within 0.2 atomic mass units (amu) 69, 219, 502
Relative Abundance 100, > 40, and > 2 %
respectively Peak widths should be
+/- 0.1 of the target (0.5 or 0.6)
Isotope Assignments should be within 0.1 amu 70, 220, 503
Isotope Intensities approximately 1.08, 4.32,
10.09 respectively Should be less than 10 %
water, oxygen and nitrogen (18, 32, 28 respectively)
Agilent ChemStation Rev. B. 01.00; Help/Contents/Introduction to Tuning/Tune Report
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TuningTuning
DFTPP Target Tune Tunes using PFTBA but
looks for set relative abundances for EPA regulation
BFB Target Tunes Again tunes PFTBA to
set relative abundances for EPA regulation
http://www.chem.agilent.com/Library/Support/Documents/f05011.pdf
http://www.chem.agilent.com/Library/Support/Documents/f05010.pdf
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EI Application AdvantagesEI Application Advantages
Analyte must be in gas phase Limit of 600 Da
Can be used to ionize any molecule that can lose an electron
Most widely used ionization method and is typically coupled with a mass analyzer
Extensive reproducible fragmentation Abundance of reference spectra available
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EI Application DisadvantagesEI Application Disadvantages
Limitations on analyte introduction Molecular weight 600 Da
Extensive fragmentation Breakdown of molecular ion Common small m/z between several compounds
Phentermine
Methamphetamine
Clarke’s Analysis of Drugs and Poisons
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Foreline Pump OilForeline Pump Oil Weekly checks of the pump oil should be performed
Oil should be clear Oil level should be between the two markings
An oil change should occur at a minimum every six months Vent the mass spectrometer detector Release the drain plug Drain used oil out Refill with fresh oil
Oil level should be located between the two markings
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PFTBA RefillPFTBA Refill The PFTBA levels should be checked every
six months As needed refill the tuning compound
Vent the MSD The PFTBA level should be just below the internal
tube Holds approximately 70 uL
After refill, purge the air out of the vial and valve Software setting turns off analyzer voltages and
opens the CI calibration valve for several minutes
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EI Source CleaningEI Source Cleaning Vent instrument according to hardware manual
Allow time for source and quadrupole to cool Oven off Auxillary Tranfer line off Turbo pump shut down
Shut the mass spectrometer detector (MSD) off when prompted by the computer Turn off carrier gas if using hydrogen
Unplug the foreline/rough pump Release the vacuum from the vent valve
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EI Source CleaningEI Source Cleaning Disconnect the side board control cable and the
source power cable Remove leads and unscrew the source Remove the source There are 7 key parts to clean on the source
Source body Repeller Draw out lens Draw out cylinder Ion focus Entrance lens Interface socket
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EI Source CleaningEI Source Cleaning Cleaning options for source parts
Scrub with microgrit slurry Use a series of solvent for sonication
Water, methanol, acetone, and hexane or Dichloromethane, acetone, and methanol
Typically performed every 6 months with oil change unless needed sooner Tune profile peak splitting Increasing electron multiplier voltage
Can not exceed 3000 Loss of sensitivity
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During EI Source CleaningDuring EI Source Cleaning Examine ion source parts for replacement
Ceramics broken – insulate source components for proper voltage to be maintained
Leads broken – supply energy to heater assembly Inspect filaments for replacement
One burned out Extensive stretching of the coils
May need to lubricate the side plate o-ring Allows for air tight seal
May need to replace vent valve o-ring
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Source CleaningSource Cleaning Reassemble the source and install it back in the
instrument. Plug in the rough pump
Allow 5 to 10 minutes for rough pump to come up to speed Ensure vent valve is closed Initiate pump down in software Turn on the MSD and transfer line Allow the pump down procedure 2 hours to equilibriate
After 10-15 mins. turbopump should be up to 80% or better Delay heating of oven, source, and quadrupoles until absence
of leak is verified.
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EI SummaryEI Summary Only positive ions are formed Sample must be volatile
Limited to low molecular weight compounds approximately 600 Da or less
Hard Ionization technique Due to extensive fragmentation low abundance of molecular ion
Typically used in conjunction with a mass analyzer
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Chemical IonizationChemical Ionization The reagent gas (present in large excess) is The reagent gas (present in large excess) is
ionized by electron impact and the reagent gas ionized by electron impact and the reagent gas ions collide with the gaseous analyte creating ions collide with the gaseous analyte creating ions.ions.44
More than one collision can occur creating More than one collision can occur creating multiply charged analytes.multiply charged analytes.
Both positive and negative analytes are formed Both positive and negative analytes are formed in the same way.in the same way.
Considered to be a soft ionization technique.Considered to be a soft ionization technique.
4. Harrison, Alex G. Chemical Ionization Mass Spectrometry. Ch 1
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CI VisualizationCI Visualization
http://www.chromacademy.com/resolver-november2010_Understanding_GCMS_part_1.asp
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Reagent GasesReagent Gases
Most Common:Most Common: MethaneMethane AmmoniaAmmonia IsobutaneIsobutane
Less Common:Less Common: COCO22
HydrogenHydrogen FreonFreon TrimethylsilaneTrimethylsilane Nitric OxideNitric Oxide MethylamineMethylamine
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Positive CIPositive CI 4 processes explain the ionization products4 processes explain the ionization products
Proton TransferProton Transfer Hydride Abstraction Hydride Abstraction Addition/ Adduct FormationAddition/ Adduct Formation Charge Exchange Charge Exchange
Not as sensitive (higher detection limits)Not as sensitive (higher detection limits)
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Proton TransferProton Transfer
BHBH++ + M → MH + M → MH++ + B + B B = reagent gas B = reagent gas M = analyteM = analyte
Dependent upon the proton affinity of analyteDependent upon the proton affinity of analyte If proton affinity of reagent gas is larger than If proton affinity of reagent gas is larger than
analyte no proton transfer will occur.analyte no proton transfer will occur.
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Reagent Gas
Proton
Target Analyte
ProtonProton Transfer MechanismTransfer Mechanism
++ ++
++ ++e--++
2e--
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Hydride AbstractionHydride Abstraction
RR++ + M → [M–H] + M → [M–H]++ + RH + RH
A hydride ion is the anion of hydrogen (negatively A hydride ion is the anion of hydrogen (negatively charged)charged)
Contains two electrons and one protonContains two electrons and one proton Molecular ion will have m/z of M-HMolecular ion will have m/z of M-H Exothermic reactionsExothermic reactions Increased fragmentationIncreased fragmentation
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Reagent Gas
Proton
Target Analyte
Hydride Abstraction MechanismHydride Abstraction Mechanism
++ ++
++ ++e--++
2e--
--+
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AdditionAddition Thermodynamically favoredThermodynamically favored Known as adduct reactions (association)Known as adduct reactions (association) Reagent gas ions are reactive enough to Reagent gas ions are reactive enough to
combine with the analyte molecules.combine with the analyte molecules. Ex. [M+CEx. [M+C22HH55]]+ + corresponds to M+29 m/zcorresponds to M+29 m/z
More prevalent for ammonia due to the high More prevalent for ammonia due to the high proton affinityproton affinity
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Charge ExchangeCharge ExchangeRR++·· + M → M + M → M++·· + R + R
A radical is created when a single electron is lost/stripped A radical is created when a single electron is lost/stripped leaving an unpaired single electronleaving an unpaired single electron Radicals are unstable and very reactiveRadicals are unstable and very reactive
Reagent gases:Reagent gases: Noble gases: helium, neon, argon, krypton, xenon, and radon Noble gases: helium, neon, argon, krypton, xenon, and radon NitrogenNitrogen Carbon Dioxide/Carbon Monoxide, Carbon Dioxide/Carbon Monoxide, HydrogenHydrogen Other gases that do not react “chemically” with the analyteOther gases that do not react “chemically” with the analyte
Uncommon mechanismUncommon mechanism
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Reagent Gas
Target Analyte
Charge Exchange MechanismCharge Exchange Mechanism
++ ++
++ ++e--++••
2e--
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Examples of Positive CIExamples of Positive CI
(Proton Transfer)
(H − Abstraction)
(Adduct Formation)
(Charge Exchange)
Primary Ion Formation
Secondary Ion Formation
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Importance of Positive CIImportance of Positive CI
Used for the ionization of any drug or compound that contains functional groups that will readily accept protons Bronsted Lowery Bases R-NHR-NH22 + H + H++ = R-NH = R-NH33
++
Examples: Methadone, Cocaine, Methamphetamine, etc.
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Negative CINegative CI Analyzer voltage polarities are reversedAnalyzer voltage polarities are reversed
Repeller voltage is negativeRepeller voltage is negative Ion focus and entrance lens voltages are positiveIon focus and entrance lens voltages are positive
4 Common Mechanisms4 Common Mechanisms Electron captureElectron capture Dissociative electron captureDissociative electron capture Ion pair FormationIon pair Formation Ion-molecule reactionsIon-molecule reactions
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Negative CINegative CIR (g) + eR (g) + e–– (230eV)(230eV) → R → R++ + e + e––
(thermal)(thermal)
Reagent gas ions are not negativeReagent gas ions are not negative The thermal eThe thermal e-- interacts with the analyte interacts with the analyte
Lower energyLower energy Lower detection limits due to no reagent gas Lower detection limits due to no reagent gas
ions being detectedions being detected COCO2 2 is the most common buffer gasis the most common buffer gas Common applications for the analysis of Common applications for the analysis of
benzodiazepinesbenzodiazepines
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Electron CaptureElectron Capture
MX + eMX + e–– (thermal)(thermal) → MX → MX––··
Thermal electrons create radical molecular ionsThermal electrons create radical molecular ions Works well with heteroatom molecules Works well with heteroatom molecules Increases sensitivity 10 to 1000 times higher than Increases sensitivity 10 to 1000 times higher than
positive CIpositive CI Very sensitive to water and oxygen Very sensitive to water and oxygen
Causes slower ion-molecule reactions which decreases Causes slower ion-molecule reactions which decreases sensitivitysensitivity
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Electron Capture MechanismElectron Capture Mechanism
++
++ ++e--(230 eV)(230 eV)
++e--
(thermal)(thermal)
Reagent Gas
Halogen
Target Analyte
-•
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Dissociative Electron CaptureDissociative Electron Capture
MX + eMX + e––(thermal)(thermal) → M → M·· + X + X––
Lower abundance of molecular ionLower abundance of molecular ion
The thermal electrons cause the dissociation of The thermal electrons cause the dissociation of molecules creating a radical and an ion.molecules creating a radical and an ion.
Decreased sensitivityDecreased sensitivity
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Dissociative Electron Capture Dissociative Electron Capture MechanismMechanism
++
++ ++e--(230 eV)(230 eV)
++e--
(thermal)(thermal)
Reagent Gas
Halogen
Target Analyte
• ++ -
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Ion Pair FormationIon Pair Formation
MX + eMX + e––(thermal)(thermal) → M → M++ + X + X¯̄ + e + e––
Electron is not capturedElectron is not captured Reaction of a molecule forming both positive and Reaction of a molecule forming both positive and
negative ions.negative ions.55
http://mass-spec.lsu.edu/msterms/index.php/Ion-pair_formation
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Ion Pair Formation MechanismIon Pair Formation Mechanism
++
++ ++e--(230 eV)(230 eV)
++e--
(thermal)(thermal)
Reagent Gas
Halogen
Target Analyte
+ ++-
++e--
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Ion Molecule ReactionsIon Molecule Reactions
M + XM + X–– → MX → MX––
XX-- is halogen or OH is halogen or OH
Occur in the presence of water, oxygen, or Occur in the presence of water, oxygen, or other contaminantsother contaminants
2 to 4 times slower than electron capture2 to 4 times slower than electron capture Compete with electron capture reactionsCompete with electron capture reactions
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Importance of Negative CIImportance of Negative CI
Used with any analyte that contains functional groups that readily donate protons Bronsted Lowery Acids
R-COR-CO22H = R-COH = R-CO22--
R-OH = R-OR-OH = R-O-- Examples: Benzodiazepines, THC
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Flunitrazepam ExampleFlunitrazepam Example
http://www.lhl.uab.edu:15022/science/article/pii/S0379073896020622http://webbook.nist.gov/cgi/cbook.cgi?Spec=C1622624&Index=0&Type=Mass
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CI SourceCI Source The CI source is very similar to the EI source but The CI source is very similar to the EI source but
only has the entrance lens in common.only has the entrance lens in common. The electron entrance and ion-exit holes are very The electron entrance and ion-exit holes are very
small 0.5 mmsmall 0.5 mm Makes pressurization of the ionization chamber possibleMakes pressurization of the ionization chamber possible
Source Body, Draw out Plate, and Repeller are at the Source Body, Draw out Plate, and Repeller are at the same potential same potential ramped during tune to find optimal settingramped during tune to find optimal setting
Reduction of the entrance lens slit width Reduction of the entrance lens slit width These differences allow ionization pressures of 1 torr These differences allow ionization pressures of 1 torr
and analyzer pressures below 10and analyzer pressures below 10-5-5 torr. torr. 760 Torr = 1 atm = 760 mmHg760 Torr = 1 atm = 760 mmHg
The addition of vacuum pump capacityThe addition of vacuum pump capacity
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Operating TemperatureOperating Temperature Always bring instrument up in Positive CI mode (no Always bring instrument up in Positive CI mode (no
troubleshooting available in Negative CI mode) with troubleshooting available in Negative CI mode) with methanemethane
Typical EI: Source at 230 Typical EI: Source at 230 ºC,ºC, Quadrupole 150 Quadrupole 150 ºC,ºC, Auxillary at 280Auxillary at 280ºCºC
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TuningTuning Load PCICH4.ULoad PCICH4.U Tuning compound Tuning compound
perfluoro-5,8-dimethyl-3,6,9-trioxidodecane perfluoro-5,8-dimethyl-3,6,9-trioxidodecane (PFDTD)(PFDTD)
AutotuneAutotune Abundance of m/z 19 < 50% of m/z 17 Abundance of m/z 19 < 50% of m/z 17 No peak at m/z 32 (ONo peak at m/z 32 (O22)) Electron Multiplier Voltage Electron Multiplier Voltage >/=>/= 2600 indicates 2600 indicates
problemproblem No tune performance criteriaNo tune performance criteria
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Application Application CI sources are coupled to detectors (typically CI sources are coupled to detectors (typically
mass analyzers) and used for the identification mass analyzers) and used for the identification and quantification of target analytes.and quantification of target analytes.
CI produces little fragmentation causing an CI produces little fragmentation causing an increased intensity in the molecular ion (Mincreased intensity in the molecular ion (M++)) Allows for accurate mass determinationsAllows for accurate mass determinations More accurate identification of molecules that More accurate identification of molecules that
fragment extensivelyfragment extensively Example sympathiomemetic aminesExample sympathiomemetic amines
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Positive CI vs. Negative CI Positive CI vs. Negative CI ReviewReview
Positive CI is used for molecules with Positive CI is used for molecules with functional groups that readily accept protons.functional groups that readily accept protons.
Bronsted Lowery BasesBronsted Lowery Bases R-NHR-NH22 + H + H++ = R-NH = R-NH33
++
Negative CI is used for molecules with Negative CI is used for molecules with functional groups that readily donate protons.functional groups that readily donate protons.
Bronsted Lowery AcidsBronsted Lowery Acids R-COR-CO22H = R-COH = R-CO22
-- R-OH = R-OR-OH = R-O--
http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm
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CI Source MaintenanceCI Source Maintenance Source cleaning is more frequentSource cleaning is more frequent Cleaning the CI sourceCleaning the CI source
Use a wooden toothpick to gently clean out the Use a wooden toothpick to gently clean out the electron entrance hole on the source body and the electron entrance hole on the source body and the ion exit hole on the draw out plateion exit hole on the draw out plate
Do not use halogenated solvents (Group 17)Do not use halogenated solvents (Group 17) Allow 2 hours for CI to bake out when using Allow 2 hours for CI to bake out when using
PCI PCI Overnight bake out if using Negative CIOvernight bake out if using Negative CI
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CI Filament MaintenanceCI Filament Maintenance One active filament (# 1) and one dummy One active filament (# 1) and one dummy
filamentfilament Filament lifetime factorsFilament lifetime factors
Maintain proper vacuumMaintain proper vacuum Set solvent delay such that the filament remains off Set solvent delay such that the filament remains off
during solvent elutionduring solvent elution Higher electron energy reduces lifetimeHigher electron energy reduces lifetime Higher emission current reduces lifetimeHigher emission current reduces lifetime
Filament stretching IndicationsFilament stretching Indications High Electron Multiplier Voltage (EMV)High Electron Multiplier Voltage (EMV) Linear electron energy rampLinear electron energy ramp
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CI Rough Pump MaintenanceCI Rough Pump Maintenance
Ammonia use causes more frequent pump Ammonia use causes more frequent pump maintenancemaintenance Change pump oil recommended every 2 to 3 Change pump oil recommended every 2 to 3
monthsmonths Air ballasting for an hour ever day (operating Air ballasting for an hour ever day (operating
temperature) will remove the ammonia and extend temperature) will remove the ammonia and extend oil lifeoil life
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Reagent Gas related MaintenanceReagent Gas related Maintenance
Use ultra high purity reagent gasesUse ultra high purity reagent gases The gas purifier should be replaced ever 4 The gas purifier should be replaced ever 4
tankstanks Large leaks will reduce the metal on the oxygen Large leaks will reduce the metal on the oxygen
and moisture traps.and moisture traps. Water contamination will dramatically decrease CI Water contamination will dramatically decrease CI
sensitivitysensitivity
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CI SummaryCI Summary Low energyLow energy Easily identifiable molecular ion (higher Easily identifiable molecular ion (higher
sensitivity)sensitivity) Useful for determining molecular weights of Useful for determining molecular weights of
compoundscompounds Reagent gas selection depends on analyteReagent gas selection depends on analyte Positive and Negative CI possiblePositive and Negative CI possible Molecular Weight limited to 10Molecular Weight limited to 1033 Da Da
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ESI OverviewESI Overview
Electrospray ionization is the mechanism for creating gas phase ion from solution for mass spectrometry analysis
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1853331/
http://projects.nfstc.org/tech_transition/lcms/Presentation/Day%201/Day%201%20MSMS%20theory.ppt#275,10,Electrospray: Overview
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ESI TheoryESI Theory
Three steps for gas-phase ion production Production of charged droplets at needle tip Desolvation and Coulomb Fission
Desolvation - shrinkage of charged droplets due to solvent evaporation
Coulomb fission - repeated charge-induced droplet disintegrations
Gas-phase ion production Occurs at atmospheric pressure
Electrospray Ionization Mass Spectrometry: Fundamentals Instrumentation & Application. Richard B. Cole
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Production of Charged DropletsProduction of Charged Droplets A voltage of 2-5 kV is applied to the needle
creating the charges. A potential field difference is created in the air
between the needle tip and the counter electrode The electric field creates a charge distribution which
leads to essentially a field-free conditions inside the solution.
This field-free condition allows the ions to separate In positive mode; positive ions move towards
meniscus and negative away from meniscusElectrospray Ionization Mass Spectrometry: Fundamentals Instrumentation & Application. Richard B. Cole
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Production of Charged DropletsProduction of Charged Droplets The mutual repulsion overcomes the surface
tension of the liquid and the surface begins to expand forming a cone. Most common is the Taylor cone In positive mode the counter electrode (curtain plate)
draws positive ions towards itself. Excess charge at the cone overcomes surface
tension and a jet of small charged droplets is formed.
The use of a nebulizing gas promotes droplet formation
Electrospray Ionization Mass Spectrometry: Fundamentals Instrumentation & Application. Richard B. Colehttp://www.ncjrs.gov/pdffiles1/nij/grants/230032.pdf
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ESI Schematic 1ESI Schematic 1
http://www.waters.com/webassets/cms/category/media/other_images/primer_ms_Id_AP%20ESI%20figure.jpg
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DesolvationDesolvation Solvent evaporation at constant charge leads to
droplet shrinkage and electric field increase. With decreasing droplet radius there is an
increasing repulsion within the droplet that overcomes the surface tension
Droplet-jet fission occurs at the Rayleigh limit Rayleigh limit is the condition at which the
electrostatic repulsion within the droplet is equal to the force of the surface tension
The droplet will expel ions to regain stability Droplets undergo fission at or near the Rayleigh
limit.Electrospray Ionization Mass Spectrometry: Fundamentals Instrumentation & Application. Richard B. Cole
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CoulombCoulomb FissionFission Droplet-jet fission is generally referred to as
coulombic fission Droplet dividing into particles Uneven fission Leads to a loss of 2% in droplet mass and 15 % in droplet
charge
http://projects.nfstc.org/tech_transition/lcms/Presentation/Day%201/Day%201%20MSMS%20theory.ppt#275,10,Electrospray: Overview
Electrospray Ionization Mass Spectrometry: Fundamentals Instrumentation & Application. Richard B. Cole
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Gas Phase Ion ProductionGas Phase Ion Production Three theoretical models
Charged Residue Model Ion Evaporation Model Ion Emission Model
Appropriate model depends on analyte size Importance
The solvent is completely evaporated and the remaining charges and analytes combine to create gas phase ions
Can be multiply charged species
Paul Kebarle and Udo H. Verkerk. (2010) Reactive Intermediates: MS Investigations in Solution
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ESIESI Softest Ionization Technique
Produces little fragmentation Concentration dependent Volatile buffers and mobile phases increase
ionization efficiency Performed at atmospheric pressure Both positive or negative mode Lower flow rates ION SUPPRESSION!!!
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Ion SuppressionIon Suppression Charge Thief Results from inefficient droplet formation Causes
Nonvolatile buffers or salts (phosphates) Nonvolatile materials in mobile phase ion pairing Higher molecular weight ions suppressing smaller
analytes More prominent early in the LC run, but can
occur at any time Emphasizes the need for good chromatography
Co-elution can cause suppressionhttp://projects.nfstc.org/tech_transition/lcms/Presentation/Day%201/Day%201%20MSMS%20theory.ppt#276,11,ESI: Production of Charged Droplet
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Positive ESIPositive ESI Best suited for basic compounds that form HCl
salts Protonation – addition of a proton
[M+H]+
[M+nH]n+
Addition of ammonium ion [M+NH4]+
+ + + Molecule
Proton
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Negative ESINegative ESI Best suited for acidic compounds that readily
form Na+ salts Loss of a proton
[M-H]-
[M-nH]-n
Addition of acetate ion [M+CH3COO-]-
- + -Molecule
Proton
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Ionization ChamberIonization Chamber Example of turbospray
source Different hardware
arrangement due to large flow rates.
Probe is located centrally Turbo heaters are at 45º
on either side of probe Counter electrode is at a
90º to ion path
http://www.freepatentsonline.com/6759650-0-large.jpg
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ESI ProbeESI Probe
Parts Retaining ring to fasten
the probe to the probe tower
Electrode adjustment nut to set length of electrode tip extension
Electrode tip through which samples are sprayed into the chamber.
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ESI Hardware OptimizationESI Hardware Optimization Installing probeInstalling probe
Insert the probe into the orifice of the probe housingInsert the probe into the orifice of the probe housing Align the hole on the probe with the alignment pin at Align the hole on the probe with the alignment pin at
the top of the housingthe top of the housing Gently push down on the source probe to ensure Gently push down on the source probe to ensure
aligning the contacts with those in the toweraligning the contacts with those in the tower Turn the bronze retaining ring over the probe, push it Turn the bronze retaining ring over the probe, push it
down to engage its thread with the threaddown to engage its thread with the thread Source exhaust system will not function without Source exhaust system will not function without
proper probe installationproper probe installation
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ESI Hardware OptimizationESI Hardware Optimization
Two Essential parametersTwo Essential parameters Probe positionProbe position Electrode tip adjustmentElectrode tip adjustment
Position of sprayPosition of spray Affects sensitivity Affects sensitivity Signal stabilitySignal stability
Compound and flow rate dependentCompound and flow rate dependent
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ESI Probe PositioningESI Probe Positioning Set horizontal and vertical axis positions to 5Set horizontal and vertical axis positions to 5 Infuse sampleInfuse sample Adjust probe position in small increments while Adjust probe position in small increments while
monitoring the signal to find optimum positionmonitoring the signal to find optimum positionLC Flow Rate Operational
Range
Parameter 5 to 50 uL/min 200 uL/min 1000 uL/min 5 to 3000 uL/min
Probe Vertical Axis Position
5 to 10 mm 0 to 5 mm 0 to 5 mm 0 to 13 mm
Probe Horizontal axis position
3 to 8 mm 5 to 8 mm 5 to 8 mm 0 to 10 mm
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Ion Source ParametersIon Source Parameters Curtain gas (CUR) [35psi]
– pure N2 that flows between orifice and curtain plate. Should be optimized at
highest possible pressure Range 10 to 50 psi
IonSpray Voltage –applied between the needle and orifice that ionizes and nebulizes the liquid flow Positive mode 4 – 5.5 kV Negative mode -3 – -4 kV
http://projects.nfstc.org/tech_transition/lcms/Presentation/Day%201/Day%201%20MSMS%20theory.ppt#304,42,Source Parameters
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Ion Source ParametersIon Source Parameters Ion Source Gas 1 (GS1)
[50psi]– the nebulizer gas pressure facilitates droplet formations Higher flow rate means
higher GS1 Range 20 to 60 psi
Ion Source Gas 2 (GS2) [55psi] – heater gas pressure aids in solvent evaporation Intersects the nebulized
liquid stream at 90º Range 30 to 70 psi
http://projects.nfstc.org/tech_transition/lcms/Presentation/Day%201/Day%201%20MSMS%20theory.ppt#304,42,Source Parameters
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ApplicationApplication
Typically coupled with a mass analyzer for identification and quantification of analytes.
Positive mode used for Sympathiomemetic amines, benzodiazepines,
aldicarb, pregabalin Negative mode used for
Aspirin, valproic acid, steroids Hydroxyl containing analytes
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Benzodiazepine Positive ESIBenzodiazepine Positive ESI
http://www.youngin.com/application/an-0607-0100en.pdf
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Electrospray ProbeElectrospray Probe Wipe down the end of the probe with methanol:water
(50:50) Can also be soaked or sonicated
Wipe down the end of the electrode with methanol Replace needs as needed
Loss in sensitivity Clogged needle – no spray Salt deposits on end of electrode – droplet formation on side of
electrode Drifting Retention Times Increased back pressure Ghosting peaks
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Electrospray HeatersElectrospray Heaters
Turbo heaters should be replaced as needed Separation of cover from heater unit Exposure of heating beads Will cause inefficient ionization and may skew
MRM ratios Spray appears wet – source can’t reach setpoint Spray is to one side
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Electrospray Needle TubingElectrospray Needle Tubing
Red peak tubing connecting the injection loop to the spray needle
Should be replaced as needed
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Electrospray Counter ElectrodeElectrospray Counter Electrode Should be cleaned frequently. Residual effluent from mobile phase and
unionized particles are deposited on the front plate
Reduces sensitivity Clean:
methanol, water, soap solution, water, methanol Sonicate as needed:
water, methanol, isopropanol
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APCIAPCI Analytes and mobile phase (eluent) are sprayed by a Analytes and mobile phase (eluent) are sprayed by a
pneumatic nebulizer and desolvated at >300 pneumatic nebulizer and desolvated at >300 ºC.ºC. Desolvation is the conversion of liquid eluent into gaseous Desolvation is the conversion of liquid eluent into gaseous
dropletsdroplets A Corona discharge creates NA Corona discharge creates N22
++ and N and N44+ + ions through ions through
electron ionization.electron ionization. NN22
++ and N and N44+ + ions collide with solvent creating ions collide with solvent creating
secondary ions.secondary ions. The secondary ions are responsible for the ionization The secondary ions are responsible for the ionization
of the analytes.of the analytes.
corona discharge: an electrical discharge brought on by the ionization of a fluid surrounding a conductor, which occurs when the potential gradient (the change in the strength of the electrical field) exceeds a certain value.
The term "pneumatic" is defined as 'of or relating to or using air or a similar gas'.
The word "nebulizer" is derived from the Latin "nebula" meaning mist and is defined as 'an instrument for converting a liquid into a fine spray'.
Therefore, a pneumatic nebulizer is literally an instrument for converting a liquid into a fine spray that uses a gas as the driving force.
http://www.inorganicventures.com/tech/icp-operations/nebulizers-spray-chambers-torches
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APCIAPCI
Chemical ionization that take place at Chemical ionization that take place at atmospheric pressure.atmospheric pressure.
Ionization occurs in the gas phase.Ionization occurs in the gas phase.
Less soft ionization technique than ESILess soft ionization technique than ESI
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APCIAPCI
Products consist of molecular ion species and Products consist of molecular ion species and adducts (little fragmentation).adducts (little fragmentation).
Thermal and volatility requirements limit Thermal and volatility requirements limit molecular mass to 1500 daltonsmolecular mass to 1500 daltons
Ideal for nonpolar or medium polarity solventsIdeal for nonpolar or medium polarity solvents
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Primary and Secondary IonsPrimary and Secondary Ions
http://www.chm.bris.ac.uk/ms/theory/apci-ionisation.html
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Mobile Phase SolventsMobile Phase Solvents
Reversed Phase Reversed Phase (non-polar stationary phase in column)(non-polar stationary phase in column)
AcetonitrileAcetonitrile MethanolMethanol WaterWater
Normal phase Normal phase (polar stationary phase in column)(polar stationary phase in column)
TolueneToluene HexaneHexane DichloromethaneDichloromethane
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Positive APCIPositive APCI Positive Ion APCI can occur as follows:
Proton Transfer: H3O+ + M → (M+H)+ + H2O Also CH3OH2
+, CH3CNH+, NH4+
Adduct Attachment: NH4+ + M → (M+ NH4)+
Also CH3OH2+, CH3CNH+, H3O+
Remember:Remember:
Dependent upon proton affinity of analyteDependent upon proton affinity of analyte
Remember:Remember:
Reagent gas ions are reactive enough to combine Reagent gas ions are reactive enough to combine with the analyte molecules.with the analyte molecules. Ex. [M+CEx. [M+C22HH55]]+ + corresponds to M+29 m/zcorresponds to M+29 m/z
Charge exchangeCharge exchange
Remember:Remember:
RR++·· + M → M + M → M++·· + R + R Reagent gases:Reagent gases:
gases should not react “chemically” with the analyte gases should not react “chemically” with the analyte (ex. Noble)(ex. Noble)
Uncommon mechanismUncommon mechanism
++ ++
++ ++e--++
2e--++
++ ++e--++
2e--
+
++ ++
++ ++e--++••
2e--
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Negative APCINegative APCI
Negative Ion APCI can occur as follows: Proton Abstraction: OH- + M → (M - H)- + H2O
Also CH3O-, CH2CN-
Adduct Addition: CH3COO- + M → (M+CH3COO)-
Also Cl-, HCOO-
Electron CaptureElectron Capture
Remember:Remember:
MX + eMX + e–– (thermal)(thermal) → MX → MX––··
Increases sensitivity 10 to 1000 times higher than PCIIncreases sensitivity 10 to 1000 times higher than PCI Very sensitive to water and oxygen Very sensitive to water and oxygen Works well with heteroatom moleculesWorks well with heteroatom molecules++
++ ++e--(230 eV)(230 eV)
++e--
(thermal)(thermal)
-•
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APCI Hardware OptimizationAPCI Hardware Optimization
Conversion from ESI Conversion from ESI to APCIto APCI Switch out the probeSwitch out the probe Change the software Change the software
settings in accordance settings in accordance with the Operator’s with the Operator’s ManualManual
Turn the corona Turn the corona discharge needle discharge needle aroundaround
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APCI Hardware OptimizationAPCI Hardware Optimization Installing probeInstalling probe
Insert the probe into the orifice of the probe housingInsert the probe into the orifice of the probe housing Align the hole on the probe with the alignment pin at Align the hole on the probe with the alignment pin at
the top of the housingthe top of the housing Gently push down on the source probe to ensure Gently push down on the source probe to ensure
aligning the contacts with those in the toweraligning the contacts with those in the tower Turn the bronze retaining ring over the probe, push it Turn the bronze retaining ring over the probe, push it
down to engage its thread with the threaddown to engage its thread with the thread Source exhaust system will not function without Source exhaust system will not function without
proper probe installationproper probe installation
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APCI Hardware OptimizationAPCI Hardware Optimization
Allow to source to heat for 30 minutes prior to Allow to source to heat for 30 minutes prior to starting liquid sample flow.starting liquid sample flow. Eliminates possible solvent vapor condensationEliminates possible solvent vapor condensation
APCI probe must be heated slowly to avoid APCI probe must be heated slowly to avoid thermal shock to heating elementthermal shock to heating element
Curtain plate orifice should remain clear of Curtain plate orifice should remain clear of solvent or solvent dropssolvent or solvent drops
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ApplicationApplication
Typically, coupled with modified ESI source Typically, coupled with modified ESI source on an LCMSMS system.on an LCMSMS system.
Used for ionization of low molecular weight Used for ionization of low molecular weight pharmaceuticalspharmaceuticals Limit: less than 1500 Da Limit: less than 1500 Da
Not suitable for thermally labile compoundsNot suitable for thermally labile compounds Thermally Labile – unstable in the presence of heatThermally Labile – unstable in the presence of heat Clorazepate conversion to NordiazepamClorazepate conversion to Nordiazepam Carbamates - AldicarbCarbamates - Aldicarb
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APCI CleaningAPCI Cleaning
Electrode CleaningElectrode Cleaning The electrode is housed in the probeThe electrode is housed in the probe
Should be cleaned with decreased performanceShould be cleaned with decreased performance Clean with 50:50 methanol/water solutionClean with 50:50 methanol/water solution
Wipe or sonicateWipe or sonicate
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APCI Cleaning ContinuedAPCI Cleaning Continued
Ionization Spray ChamberIonization Spray Chamber Clean daily or as symptom present.Clean daily or as symptom present.
SymptomsSymptoms Loss of sensitivityLoss of sensitivity Excessive peak tailingExcessive peak tailing
Clean with mobile phase or mixture of IPA/HClean with mobile phase or mixture of IPA/H22OO
Ionization Spray ShieldIonization Spray Shield Wipe down with IPA when cleaning chamber and Wipe down with IPA when cleaning chamber and
capillarycapillary
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APCI Cleaning ContinuedAPCI Cleaning Continued
Vacuum CapillaryVacuum Capillary Outer end of capillary can be wiped without Outer end of capillary can be wiped without
venting systemventing system Corona NeedleCorona Needle
Clean with observedClean with observed Decrease in sensitivityDecrease in sensitivity Decreased signal stability Decreased signal stability Increased corona voltageIncreased corona voltage
Wipe with IPAWipe with IPA
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CI vs. APCICI vs. APCI
CICI Soft ionization (more Soft ionization (more
gentle than EI)gentle than EI) Liquid samples are Liquid samples are
vaporized and then vaporized and then ionizedionized
Low mass range <10Low mass range <103 3 DaDa Instrumental set-up more Instrumental set-up more
complex (requires complex (requires venting) and preventative venting) and preventative maintenance required maintenance required frequently.frequently.
APCIAPCI Soft ionization (Harsher Soft ionization (Harsher
than ESI)than ESI) Liquid samples are Liquid samples are
nebulized and then nebulized and then ionized.ionized.
Larger mass range up to Larger mass range up to 1500 Da1500 Da
Instrumental set-up easy Instrumental set-up easy switching mode and less switching mode and less time consuming time consuming maintenancemaintenance
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SummarySummary Ionization ScaleIonization Scale
ESI – softest ionization technique (3000 Da or less)ESI – softest ionization technique (3000 Da or less) Good for polar and ionic compoundsGood for polar and ionic compounds Large molecular weightLarge molecular weight
APCI – soft ionization technique (1500 Da or less)APCI – soft ionization technique (1500 Da or less) Good for non polar compoundsGood for non polar compounds Lower molecular weight compoundsLower molecular weight compounds
CI - soft ionization technique (10CI - soft ionization technique (1033Da or less)Da or less) Good for molecular weight determinationsGood for molecular weight determinations
EI – hard ionization technique (600 Da or less)EI – hard ionization technique (600 Da or less) Can ionize anything in gas phase that can loose an electronCan ionize anything in gas phase that can loose an electron
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Har
dnes
s
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ReferencesReferences Agilent Technologies Hardware Manual http://www.home.agilent.com/agilent/home.jspx?cc=US
&lc=eng Electrosray Ionization Mass Spectrometry:
Fundamentals Instrumentation and Application. Richard B. Cole
www.appliedbiosystems.com Reactive Intermediates: MS Investigations in Solution.
Paul Kebarle and Udo H. Verkerk