ProcessesNeeded toGenerate a

MassSpectrum

1) Generate desolvated ions(Sources)

2) Transfer ions into massspectrometer vacuum(Interface Region)

3) Mass analyze ions(Quadrupoles)

4) Detect ions(Detector)

5) Process the data(Software)

Liquid ChromatographyCoupled to Tandem Mass

Spectrometry

The Source

The Source

Ionization Sources

Turbo IonSpray - IonSpray (pneumaticallyassisted Electrospray) option which utilizesa heated auxiliary gas flow.Heated Nebulizer - Atmospheric PressureChemical Ionization (APCI) source whichionizes compounds in the gas phase.

Ion Evaporation andFormation

++ +

++

++ ++ +

++ --

---

+++++

+ +- ---

+ +

+

++

- +++++-

IonSpray (ESI)

IonSpray: - Electrospray ionization (ESI)- Gentlest ionization technique- Applicable to polar and ionic substances

Multiply Charged Ions

Molecules which can be multiplyprotonated or deprotonated can exist inmore than one charge statePeaks at [M ± nH]n± (n = number ofcharges)

1.56e6 cps+Q1: from myoglobina

1060.5

1414.01542.5

1696.0

8 0 9 .0

8 4 8 .5

8 9 3 .0

9 4 2 .5

9 9 8 .01 1 3 1 .0

1 2 1 2 .0

1 3 0 5 .0

800 1000 1200 1400 1600m/z, amu

5.0e5

1.0e6

Inte

nsity

, cps

10+11+12+

13+14+

16+15+

etc.

Nebulizer

Primary Flash Zone

Secondary Flash Zone

Cool Zone

Heated Nebulizer(APCI)

Heated Nebulizer: - Atmospheric Pressure Chemical Ionization (APCI) - corona discharge - polar to non-polar thermally stable compounds

Corona Discharge Chemistry – Positive Ions1) EI on atmosphere causes e- removal from

N2 & O2 forming N2+• & O2

+• (primaryions)

2) In a complex series of reactions N2+• &

O2+• react with H2O & CH3OH forming

H3O+ & CH3OH2+ as reagent ions for CI.

3) H3O+ & CH3OH2+ donate protons to

analyte forming [M+H]+

Atmospheric Pressure ChemicalIonization (APCI)

GC-MS vs. LC-MS

IonSpray

APCI

GC-MS

IonicA

naly

te P

olar

ity

Molecular Weight101 102 103 10104 105

Non-Ionic

Ion ProductionAPI Chemistry

andExample Spectra

Ion ProductionAPI Chemistry

andExample Spectra

GAS PHASE Acid-Base Scalefor Positive Ions & Neutral Molecules

Reagent IonsWeak Base

HCOOH2+

CH3COOH2+

H3O+

CH3OH2+

C6H6OH2+

HCHOH+

N2OH+

CH5+

C2H5+

C6H6H+

CH3CNH+

C2H5OCOC2H6+

NH4+

C6H15NH+

Strong Base

• Addition of acetic acid or formic acid willincrease intensity in positive ion mode byhigher concentration of H+.

• Trifluoroacetic acid (TFA) works well inpositive ion mode at a concentration up to0.05%, but lingers for a long time in negativeion mode. (TFA ions are at m/z 113 and m/z227—dimer)

• Changing from acetonitrile to methanol mayincrease signal intensity.

Neutral MoleculesStrong acid

formic acidacetic acid

watermethanol

phenolformaldehyde

N2Omethane

ethanebenzene

acetonitrileethyl acetate

ammoniatriethylamine

Weak acid

Incr

easin

g A

cidi

ty

Reagent IonsStrong Base

NH2-

OH-

C6H5CH2-

CH3O-

C2H5O-

CH2CN-

CH3COCH2-

CH3S-

CH2NO2-

CN-

C6H5O-

CH3COO-

C6H5COO-

Cl-

Weak Base

• For R-COOH compounds use NH4ac buffer(pH = 6.4).

• Try addition of NH3 in case of neutral analytesin negative ion mode.

• Stay away from triethylamine, it collects in thesource!

GAS PHASE Acid-Base Scalefor Negative Ions & Neutral Molecules

Neutral moleculesWeak Acid

NH3

WaterToluene

MethanolEthanol

AcetonitrileAcetoneCH3SH

CH3NO2

HCNPhenol

Acetic acidBenzoic acid

HClStrong Acid

Incr

easin

g B

acic

ity

Ionization SuppressionCompounds That Cause Sensitivity Suppression– Salts can interfere with ionization and can cluster to

complicate spectrum (but also aid in identification)– Strong bases or quaternary amines can interfere with

positive mode analytes, e.g. Triethylamine (TEA)– Acids - Sulfuric/Sulfonic acids and TFA interfere in

negative mode experiments– Phosphate buffer and non-volatile ion pairing agents (e.g.

SDS) can cause severe suppression and complex spectraDimerization ([2M+H]+) can occur at highconcentration, leading to non-linearity duringquantitation– Dimer Signal at m/z = (MW*2)+1

– Can cause non-linearity at high concentrations

Progesterone in Positive Ion ESI-MS

[M+ Na]+

Effect of Phosphate Buffer8.78e7 cps–Q1 MCA (10 scans): from cck q1 -ve nano

254.43424.53

566.44

733.93

835.93

898.22 1000.42

1164.611 1 4 2 . 4 1

CCK dissolved in Phosphate Buffer

300 400 500 600 700 800 900 1000 1100m/z, amu

1e7

2e7

3e7

4e7

5e7

6e7

7e7

8e7

Inte

nsity

, cps

4.44e7 cps–Q1 MCA (10 scans): from CCK PURIFIED Q1 SACN 16.7

292.82 377.02

439.03497.03

570.44

700.63 798.63879.23

1142.03Phosphate Buffer removed from CCK sample

300 400 500 600 700 800 900 1000 1100m/z, amu

5.0e6

1.0e7

1.5e7

2.0e7

2.5e7

3.0e7

3.5e7

4.0e7

Inte

nsity

, cps

Adducts & Clusters formed in LC/MSAdduct Ion Formed Cause

Adduct*/ Ionic APCI ESI +/- m/z of Ion

Lithium [M+Li]+ Lithium Salts Ionic + [M + H + 6]+ Ammonia [M + NH4]+

Ammonia/ NH4OH Adduct + [M + H + 17]+

Water [M + H3O]+

Water/ Acids Adduct + [M + H + 18]+

Sodium [M + Na]+ Sodium salts Ionic + [M + H + 22]+ Methanol [M + H + CH3OH]+

Methanol in Solvent Adduct +/-

[M + H + 32]+ [M - H + 32]-

Chloride [M + Cl]- Chlorinated solvent Ionic - [M + 35 (& 37)]- Potassium [M + K]+ Potassium salts Ionic + [M + H + 38]+ Acetonitrile [M + H + CH3CN)+

Acetonitrile in solvent Adduct +/-

[M + H + 41]+ [M - H + 41]-

Formic Acid [M - H + HCO2H]- Formic Acid Adduct - [M - H + 46]- Acetic Acid [M - H + CH3CO2H]- Acetic Acid Adduct - [M - H + 60]- TFA [M - H + CF3CO2H]- TFA Adduct - [M- H + 114]-

* Adducts can often be broken up by using a higher DP voltage.

Ion TransferIon Transfer

Ion Path“Electronic Ion Ski SlopeElectronic Ion Ski Slope”

Lens (IQn) and rod (ROn) voltages are linkedKeep ions moving at correct speed through system For bestsensitivity, proper mass filtering

OR =45V

Q0= -10V RO1 =-11V

Moves with C.E.0V, SK at ground

RO3, moves withRO2 (-2-3V)

Q0

IQ1

ST RO1 ST2 RO3

DF

CEM

OR SK

EXITRO2 (LINAC)ST3

MS &MS/MS (tandem)

MS &MS/MS (tandem)

Quadrupole TheoryQuadrupole acts as a mass filter– Separates ions based on m/z ratio

Quad. made of 4 rods– “A” pole - vertical rods; “B” pole -

horizontal rods (by convention)DC & RF voltages are imposed:– U = (DC)A - (DC)B (FDC)– V = RF voltage (RFp-p)– V = Const.•M•r0

2•ν2

Note: M α V• ν = RF frequency– r0 = ½ distance between rods

Stability DiagramDC/RF

Operating Line(through the apices) →

V (RF)

U (D

C)

High

Unit

Low

MS OperationScan Modes

MS OperationScan Modes

Q1 MS Scans

Start/Stop Mode

Q3 Single-Stage MS Scans

Start/Stop Mode

MS/MS Scan ModesAvailable Scan Modes– Product Ion

Provides structural information– Precursor Ion

Provides structural information complementary toProduct Ion

– Neutral LossProvides compound class specificity

– MRMUsed for Quantitation

In all MS/MS modes, Q3 is a mass filter– Q3 may scan or be fixed at a given m/z– Q2 is “source” of product ions entering Q3

MS/MS – Product Ion Scanm1

+ fixed

m3+ scanned

MS/MS – Precursor Ion Scan

m3+ fixed

m1+ scanned

MS/MS – Neutral Loss Scan

m1+ scanned

m3+ scanned

δm

MS/MS – MRM(Multiple Reaction Monitoring)

Precursor ion fixed

Product ion fixed

Fragmentation(CAD)

FragmentationWhen an ion fragments the following formulas apply:– Positive mode: [Precursor]+ → Product+ + Neutral– Negative mode: [Precursor]- → Product- + Neutral

Two modes of fragmentation on Sciex Instruments:– CAD = Collisionally Activated Dissociation

Energy controlled by CE; occurs in Q2 (collision cell)Ions collide with N2 molecules (CAD gas), collision energy isconverted into vibrational energy, and bonds break

– CID = Collision-Induced DissociationEnergy controlled by DP; occurs between orifice and skimmerIons collide with N2 (from curtain gas) and solvent molecules,collision energy is converted…

Ion DetectionIon Detection

Detector & Signal Handling

CEM BiasVoltage (+)

DeflectorIon Path

Signal Output

1000 pF10 kV

1 Mž

CEM HornVoltage (-)

CEM

Signal HandlingBoard

System Controller(in System

Electronics Box)