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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

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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

10101010

Source AssemblySource Assembly

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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

303

182

272

82

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EI HARDWARE ADAPTATIONS EI HARDWARE ADAPTATIONS AND APPLICATIONSAND APPLICATIONS

15151515

Source AssemblySource Assembly

16161616

Auxiliary Transfer LineAuxiliary Transfer Line

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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

2020

21212121

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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ExamplesExamples

Clarke’s Analysis of Drugs and Poisons

24242424

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

25252525

EI GENERAL MAINTENANCEEI GENERAL MAINTENANCE

26262626

EI Maintenance ScheduleEI Maintenance Schedule

27272727

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

28282828

Foreline Pump Oil ChangeForeline Pump Oil Change

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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

30303030

EI Vent Valve LocationEI Vent Valve Location

31313131

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

32323232

EI Vent Valve LocationEI Vent Valve Location

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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

34343434

MSD ChamberMSD Chamber

35353535

EI Source RemovalEI Source Removal

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EI Source PartsEI Source Parts

37373737

EI Source PartsEI Source Parts

--------------------------

38383838

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

39393939

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

40404040

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.

41414141

EI Filament ReplacementEI Filament Replacement

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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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CI THEORYCI THEORY

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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

46464646

Reagent GasesReagent Gases

Most Common:Most Common: MethaneMethane AmmoniaAmmonia IsobutaneIsobutane

Less Common:Less Common: COCO22

HydrogenHydrogen FreonFreon TrimethylsilaneTrimethylsilane Nitric OxideNitric Oxide MethylamineMethylamine

47474747

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)

48484848

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.

49494949

Reagent Gas

Proton

Target Analyte

ProtonProton Transfer MechanismTransfer Mechanism

++ ++

++ ++e--++

2e--

50505050

Proton AffinitiesProton Affinities

51515151

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

52525252

Reagent Gas

Proton

Target Analyte

Hydride Abstraction MechanismHydride Abstraction Mechanism

++ ++

++ ++e--++

2e--

--+

53535353

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

54545454

Reagent Gas

Target Analyte

Addition MechanismAddition Mechanism

++

++ ++e--++

2e--

+

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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

56565656

Reagent Gas

Target Analyte

Charge Exchange MechanismCharge Exchange Mechanism

++ ++

++ ++e--++••

2e--

57575757

Examples of Positive CIExamples of Positive CI

                                         (Proton Transfer)

                                  (H − Abstraction)

                                  (Adduct Formation)

                               (Charge Exchange)

Primary Ion Formation

Secondary Ion Formation

58585858

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.

59595959

Example of Methyl StearateExample of Methyl Stearate

60606060

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

63636363

Electron Capture MechanismElectron Capture Mechanism

++

++ ++e--(230 eV)(230 eV)

++e--

(thermal)(thermal)

Reagent Gas

Halogen

Target Analyte

-•

64646464

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

65656565

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

67676767

Ion Pair Formation MechanismIon Pair Formation Mechanism

++

++ ++e--(230 eV)(230 eV)

++e--

(thermal)(thermal)

Reagent Gas

Halogen

Target Analyte

+ ++-

++e--

68686868

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

69696969

Ion Pair Formation MechanismIon Pair Formation Mechanism

++

Halogen

Target Analyte

-

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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

71717171

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

72727272

THC THC ExampleExample

73737373

Example of Negative CIExample of Negative CI

Endosulfan MW = 404

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CI HARDWARE ADAPTATIONSCI HARDWARE ADAPTATIONS

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Instrument Schematic Instrument Schematic

76767676

CI Source SchematicCI Source Schematic

-------------------------------------

77777777

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

78787878

79797979

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

80808080

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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Tune Limits for CITune Limits for CI

82828282

83838383

CI APPLICATIONCI APPLICATION

84848484

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

85858585

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

86868686

CI GENERAL MAINTANENCECI GENERAL MAINTANENCE

87878787

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

88888888

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

89898989

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

90909090

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

91919191

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

92929292

ESI THEORYESI THEORY

93939393

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

9494

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

9595

ESI Schematic 1ESI Schematic 1

9696

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

9797

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

9898

ESI Schematic 1ESI Schematic 1

http://www.waters.com/webassets/cms/category/media/other_images/primer_ms_Id_AP%20ESI%20figure.jpg

9999

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

100100100100

ESI Schematic 2ESI Schematic 2

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1853331/

101101

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

102102

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

103103

ESI ChamberESI Chamber

http://www.chromacademy.com/resolver/feb2011/fig05.jpg

104104104104

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!!!

105105

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

106106

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

107107

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

108108108108

ESI HARDWARE ADAPTATIONS ESI HARDWARE ADAPTATIONS AND APPLICATIONSAND APPLICATIONS

109109109109

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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PregabalinPregabalin

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FDAA derivatives

[M+H]+ = 412.4

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AldicarbAldicarb

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ESI GENERAL MAINTENANCEESI GENERAL MAINTENANCE

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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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AlprazolamAlprazolam

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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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APCI THEORYAPCI THEORY

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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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APCI MechanismAPCI Mechanism

http://www.chm.bris.ac.uk/ms/theory/apci-ionisation.html

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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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Polarity ChartPolarity Chart

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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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Remember!!!Remember!!!

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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 APCI HARDWARE ADAPTATIONS AND ADAPTATIONS AND

APPLICATIONSAPPLICATIONS

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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 GENERAL MAINTENANCEAPCI GENERAL MAINTENANCE

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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

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