Mass Spectrometry

Mass SpectrometryMass Spectrometry A.) IntroductionA.) Introduction::

Mass Spectrometry (MS)Mass Spectrometry (MS) measures the atomic or molecular weight of a ion from the measures the atomic or molecular weight of a ion from the separation based on its mass to charge ratio (separation based on its mass to charge ratio (m/zm/z))

- - elemental composition of matterelemental composition of matter- - structures of inorganic, organic and biological moleculesstructures of inorganic, organic and biological molecules

- qualitative and quantitative composition of complex mixtures- qualitative and quantitative composition of complex mixtures- isotopic ratios of atoms in the sample- isotopic ratios of atoms in the sample

One of the One of the MOSTMOST Routinely used Analytical Techniques Routinely used Analytical Techniques

Mass SpectrometryMass Spectrometry Nobel Laureates:Nobel Laureates:

Joseph John ThomsonJoseph John ThomsonPhysics 1906Physics 1906first mass spectrometerfirst mass spectrometer

Francis William AstonFrancis William AstonChemistry 1922Chemistry 1922mass spectrometry of isotopesmass spectrometry of isotopes

Wolfgang PaulWolfgang PaulPhysics 1989Physics 1989quadrupole and quadrupole and quadrupole ion trap MSquadrupole ion trap MS

John B. FennJohn B. FennChemistry 2002Chemistry 2002electrospray ionization of electrospray ionization of biomoleculesbiomolecules

Koichi TanakaKoichi TanakaChemistry 2002Chemistry 2002Matrix-assisted laserMatrix-assisted laserDesoprtion/ionization (MALDI)Desoprtion/ionization (MALDI)

A Long and Continuing History of AchievementsA Long and Continuing History of Achievements

Mass SpectrometryMass Spectrometry A

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Qualitative Information

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Mass Spectrometry Data • Qualitative Analysis- Molecular Weight Determination- Structure Determination

• Quantitative Analysis- Biotechnology

analysis of proteins & peptides analysis of oligonucleotides

- Pharmaceutical drug discovery, combinatorial chemistry pharmokinetics, drug metabolism

- Clinicalneonatal screening, hemoglobin analysisdrug testing

- Environmental water, food, air quality (PCDs etcs)

- Geological oil composition

-Toxicology- Forensics

Mass SpectrometryMass Spectrometry Advantages Over Atomic Optical SpectrometricAdvantages Over Atomic Optical Spectrometric

• Detection limits three orders of magnitude Detection limits three orders of magnitude betterbetter

• Remarkably simple spectra that are unique Remarkably simple spectra that are unique and easily interpretedand easily interpreted

• Ability to measure isotopic ratiosAbility to measure isotopic ratios

DisadvantagesDisadvantages

• Instrument costs are two to three times Instrument costs are two to three times higherhigher

• Instrument drift that can be as high as 5-Instrument drift that can be as high as 5-10% per hour10% per hour

• Interference effectsInterference effects

Atomic Weights in MSAtomic Weights in MS

• Discriminates among the masses of isotopesDiscriminates among the masses of isotopes

- differs from other analytical techniquesdiffers from other analytical techniques

• Atomic mass units (amu) or daltons (Da)Atomic mass units (amu) or daltons (Da)

- Relative scale: Relative scale: exactly 12 amu exactly 12 amu

- amu or Da equals 1/12 mass of amu or Da equals 1/12 mass of

1.66054x101.66054x10-24-24 g/atom g/atom

• All All measured masses relative to measured masses relative to

- has a mass 2.91407 timeshas a mass 2.91407 times

- 12.0000 Da x 2.91407 = 34.9688 Da 12.0000 Da x 2.91407 = 34.9688 Da atomic weight = 34.9688 g/mol atomic weight = 34.9688 g/mol

• Exact Mass (Exact Mass (mm))

- Sum of specific set of isotopes within compoundsSum of specific set of isotopes within compounds

1212CC11HH44: : mm = 12.00000 x 1 + 1.007825 x 4 = 16.031 Da = 12.00000 x 1 + 1.007825 x 4 = 16.031 Da

1313CC11HH44: : mm = 13.00335 x 1 + 1.007825 x 4 = 17.035 Da = 13.00335 x 1 + 1.007825 x 4 = 17.035 Da

1212CC11HH3322HH11: : m m = 12.00000 x 1 + 1.007825 x 3 + 2.0140 x 1 = 17.037 Da = 12.00000 x 1 + 1.007825 x 3 + 2.0140 x 1 = 17.037 Da

3-4 significant figures to right of decimal3-4 significant figures to right of decimal

• Nominal Mass (Nominal Mass (mm))

- Whole number precision in mass measurementWhole number precision in mass measurement

1212CC11HH44: : mm = 12 x 1 + 1 x 4 = 16 Da = 12 x 1 + 1 x 4 = 16 Da

1313CC11HH44: : mm = 13 x 1 + 1 x 4 = 17 Da = 13 x 1 + 1 x 4 = 17 Da

1212CC11HH3322HH11: : m m = 12 x 1 + 1 x 3 + 2 x 1 = 17 Da= 12 x 1 + 1 x 3 + 2 x 1 = 17 Da

C126C126

C126C3517 C126C3517

Atomic Weights in MSAtomic Weights in MS• Chemical atomic weight or average atomic weightChemical atomic weight or average atomic weight

AA11, A, A22, A, Ann :atomic masses in Da :atomic masses in Da

pp11, p, p22, p, pnn :fractional abundance of each isotope :fractional abundance of each isotope

n n :number of isotopes :number of isotopes

• Weight of interest for most purposesWeight of interest for most purposes

• Sum of chemical atomic weights for the atoms in the compound formulaSum of chemical atomic weights for the atoms in the compound formula

- CHCH44 ( (mm) =12.01115 + 4 x 1.00797 = 16.0434 Da) =12.01115 + 4 x 1.00797 = 16.0434 Da

- typical atomic masses in periodic tabletypical atomic masses in periodic table

n

1innnn2211 pApApApAA

Boron: 10B 23% 11B 100%B (m) = (23x10 + 100x11)/123 = 10.81

Zirconium: 90Zr 51.5% 91Zr 11.2% 92Zr 17.1 % 94Zr 17.4% 96Zr 2.8%Zr (m) = (51.5x90 + 11.2x91 + 17.1x92 + 2.8x96)/100 = 91.22

Molecular Formulas from Exact Molecular WeightsMolecular Formulas from Exact Molecular Weights

• Requires identification of molecular ion peakRequires identification of molecular ion peak

• Exact mass needs to be determinedExact mass needs to be determined High resolution instruments High resolution instruments

detect mass differences of a few thousands of amudetect mass differences of a few thousands of amu

- Purine, CPurine, C55HH44NN44 ( (mm = 120.044) = 120.044)

- Benzamidine, CBenzamidine, C77HH88NN22 ( (mm = 120.069) = 120.069)

- Ethyltolune, CEthyltolune, C99HH1212 ( (mm = 120.096) = 120.096)

- Acetophenone, CAcetophenone, C88HH88O (O (mm = 120.058) = 120.058)

Molecular ion peak is 120.070 ± 0.005 Molecular ion peak is 120.070 ± 0.005 only C only C77HH88NN22 is possible formula is possible formula

Precision of a few parts per Precision of a few parts per millionmillion is routinely possible is routinely possible

Molecular Formulas from Isotopic RatiosMolecular Formulas from Isotopic Ratios

• Low-resolution instrument Low-resolution instrument only differentiate whole number masses only differentiate whole number masses

• Requires sufficiently intense molecular ion peakRequires sufficiently intense molecular ion peak

• Requires accurate heights for (M+1)Requires accurate heights for (M+1)++ and (M+2) and (M+2)++

CC66HH44NN22OO44

1313CC 6 x 1.08 = 6.486 x 1.08 = 6.48

22HH 4 x 0.0015 = 0.060%4 x 0.0015 = 0.060%

1515NN 2 x 0.37 = 0.74%2 x 0.37 = 0.74%

1717OO 4 x 0.04 = 0.16%4 x 0.04 = 0.16%

(M+1)(M+1)++/M/M+ + = 7.44%= 7.44%

CC1212HH2424

1313CC 12 x 1.08 = 12.96%12 x 1.08 = 12.96%

22HH 24 x 0.0015 = 0.36%24 x 0.0015 = 0.36%

(M+1)(M+1)++/M/M+ + = 13.32%= 13.32%

Resolution in MS (R)Resolution in MS (R)

• differentiate between massesdifferentiate between masses

R = m/R = m/mm

oror

• Higher the number the better the resolutionHigher the number the better the resolution 500,000 is better than 500500,000 is better than 500

Resolution of 4000 would resolve peaks occurring at m/z values of:Resolution of 4000 would resolve peaks occurring at m/z values of: 400.0 and 400.1400.0 and 400.1

40.00 and 40.0140.00 and 40.01

m2)mm(

R21

Example 22: Calculate the resolution to differentiate (a) C2H4+ (m = 28.0313)

and CH2N+ (m = 28.0187) and (b) N2+ (m = 28.0061) and CO+ (m =

27.9949).

Identification of Identification of odd electronodd electron ions – The Nitrogen Rule ions – The Nitrogen Rule

• Based on an anomaly in the relationship between the atomic weights and Based on an anomaly in the relationship between the atomic weights and valences of the common elementsvalences of the common elements

• An organic molecule containing the elements C, H, O, S, P or halogen An organic molecule containing the elements C, H, O, S, P or halogen

- odd nominal mass if it contains an odd number of nitrogen atomsodd nominal mass if it contains an odd number of nitrogen atoms

- even nominal mass if it contains an even number of nitrogen atoms (including 0)even nominal mass if it contains an even number of nitrogen atoms (including 0)

Element At Wt Valence

H 1 1

C 12 4

N 14 3

O 16 2

F 19 1

S 32 2

Cl 35/37 1

Compound MW

CO2 44

CO 28

CH4 16

HCO2H 46

HCl 36/38

HCN 27

N2 28

NH3 17Even nominal mass even valenceOdd nominal mass odd valence

Nitrogen is the exception

Hexane: C6H14

Mass of molecular ion: 86D = 1+1/2((6x(4-2) + 14x(1-2)) = 0

Hexene: C6H12

Mass of molecular ion: 84 D = 1+1/2((6x(4-2) + 12x(1-2)) = 1

Basic MS Instrument DesignBasic MS Instrument Design• Atomic Atomic Mass Spectrometry involves the following stepsMass Spectrometry involves the following steps ::

- atomizationatomization- conversion of atoms to ionsconversion of atoms to ions

most ions are single chargemost ions are single charge- separation of ions based on mass-to-charge ratio (separation of ions based on mass-to-charge ratio (m/zm/z))- Counting the number of ions of each type or measuring the ion currentCounting the number of ions of each type or measuring the ion current

• Principal ComponentsPrincipal Components- Vacuum system Vacuum system maintain low pressure(10 maintain low pressure(10-5-5 to 10 to 10-8-8 torr) torr)- Inlet: introduce Inlet: introduce -amount of sample into ion source-amount of sample into ion source- Ion source: sample converted into gaseous ion by bombardment with:Ion source: sample converted into gaseous ion by bombardment with:

ElectronsElectrons PhotonsPhotons IonsIons MoleculesMolecules Thermal/electric energyThermal/electric energy

- Positive/negative ions acceleratedPositive/negative ions accelerated

into mass analyzerinto mass analyzer- Mass analyzer: disperse ions (Mass analyzer: disperse ions (m/zm/z))- Transducer: convert beam of ions to Transducer: convert beam of ions to

electrical signalelectrical signal

Types of Atomic and Molecular MSTypes of Atomic and Molecular MS• Thermal ionization & Spark source Thermal ionization & Spark source first MS first MS

• Inductively coupled plasma (ICP) Inductively coupled plasma (ICP) current common approach current common approach- Differ by types ion sources and mass analyzerDiffer by types ion sources and mass analyzer

MS Theory:MS Theory:

1.) 1.) Mass analyzers use electric and magnetic fields to apply a force on charged particlesMass analyzers use electric and magnetic fields to apply a force on charged particles

FF = m = maa (Newton's second law) (Newton's second law)

F F = e(= e(EE+ + v x Bv x B) (Lorentz force law)) (Lorentz force law)

where:where:

FF - force applied to the ion - force applied to the ion m - mass of the ion m - mass of the ion

aa - acceleration - acceleration ee - ionic charge- ionic charge

v x B - v x B - vector cross productvector cross product EE - electric field - electric field

of the ion velocity and the applied of the ion velocity and the applied

magnetic field magnetic field

2.)2.) Force is therefore dependent on both Force is therefore dependent on both massmass and and chargecharge- spectrometers separate ions according to their - spectrometers separate ions according to their mass-to-charge ratiomass-to-charge ratio ( (m/zm/z))

- not by mass alone- not by mass alone

Inlet Systems:Inlet Systems:

• Introduce sample into ion source with minimum loss of vacuumIntroduce sample into ion source with minimum loss of vacuum• Spectrometer equipped with multiple inlets for different sample types Spectrometer equipped with multiple inlets for different sample types

Batch InletBatch Inlet Direct Probe InletDirect Probe Inlet Gas ChromatographyGas Chromatography Liquid ChromatographyLiquid Chromatography

External (Batch) Inlet Systems:External (Batch) Inlet Systems:• SimplestSimplest• Gas & liquid samples (bp < 500Gas & liquid samples (bp < 500ooC)C)• Sample heated (<400 °C) in small external ovenSample heated (<400 °C) in small external oven• Sample pressure 10Sample pressure 10-4-4 to 10 to 10-5-5 torr torr• Vapor admitted to ionizer through valveVapor admitted to ionizer through valve• Gas stream added to analyteGas stream added to analyte


Direct probe inlet system for solidsDirect probe inlet system for solids

Direct ProbeDirect Probe• Solids and non-volatile samplesSolids and non-volatile samples

- Less sample is required & wasted (few ng)Less sample is required & wasted (few ng)- sample held on the surface of glass or aluminum capillary tube, fine wire or small cupsample held on the surface of glass or aluminum capillary tube, fine wire or small cup

• Sample vial inserted through air-lock into ionizer chamberSample vial inserted through air-lock into ionizer chamber- Lock system minimizes amount of air that must be pumped from systemLock system minimizes amount of air that must be pumped from system

• Vial heated to vaporize sampleVial heated to vaporize sample

• Vial can be reduced to capillary or surface plate for small quantitiesVial can be reduced to capillary or surface plate for small quantities

The direct probe is only ¼" in diameter.The direct probe is only ¼" in diameter. Probe moves through various lock system stages permits Probe moves through various lock system stages permits for a step-wise increase in the vacuumfor a step-wise increase in the vacuum


Inlet Systems:Inlet Systems:• LC & GC coupled to mass spectrometerLC & GC coupled to mass spectrometer

• Permits separation and determination of components for complex mixtures Permits separation and determination of components for complex mixtures Requires specialized inlet systemsRequires specialized inlet systems Major interface problem – carrier gas dilutionMajor interface problem – carrier gas dilution Jet separator (separates analyte from carrier gas)Jet separator (separates analyte from carrier gas)

- Lighter carrier gas deflected by volumeLighter carrier gas deflected by volume- Heavier sample travels in straight lineHeavier sample travels in straight line

Ion Sources:Ion Sources:• Formation of gaseous analyte ions Formation of gaseous analyte ions • Mass spectrometric methods are dictated by ionization techniquesMass spectrometric methods are dictated by ionization techniques• Appearance of spectrum highly dependant on ionization techniqueAppearance of spectrum highly dependant on ionization technique• Gas-phase Gas-phase

Sample first vaporized then ionizedSample first vaporized then ionized Thermally stable compounds boiling points < 500Thermally stable compounds boiling points < 500ooCC MW < 100 amuMW < 100 amu

• DesorptionDesorption Solid or liquid directly converted to gaseous ionSolid or liquid directly converted to gaseous ion MW as large as 10MW as large as 1055 daltons daltons

Type Name and Acronym Ionizing Process

Gas Phase Electron Impact (EI) Exposure to electron stream

Chemical Ionization (CI) Reagent gaseous ions

Field Ionization (FI) High potential electrode

Desorption Field Desorption (FD) High potential electrode

Electrospray Ionization (ESI) High electric field

Matrix-assisted desorption ionization (MALDI) Laser beam

Plasma Desorption (PD) Fission fragments from 252Cf

Fast Atom Bombardment (FAB) Energetic atomic beam

Secondary Ion Mass Spectrometry (SIMS) Energetic beam of ions

Thermospray Ionization (TS)

Ion Sources:Ion Sources:

• Hard sources Hard sources Sufficient energy so analyte are in highly excited energy stateSufficient energy so analyte are in highly excited energy state Relaxation involves rupture of bondsRelaxation involves rupture of bonds

- Produces fragment ions with Produces fragment ions with mm//zz < molecular ion < molecular ion

- Kinds of functional groups Kinds of functional groups structural information structural information

Hard Ionization

Ion Sources:Ion Sources:• Soft sourcesSoft sources

Cause little fragmentationCause little fragmentation Mass spectrum consists of molecular ion and only few, if any, other peaksMass spectrum consists of molecular ion and only few, if any, other peaks Accurate massAccurate mass

Soft Ionization

Ion Sources:Ion Sources:• Electron-Impact Source (EI) Electron-Impact Source (EI)

Sample heated to produce molecular vaporSample heated to produce molecular vapor Bombard with a beam of electronsBombard with a beam of electrons

- Electrons emitted from heated tungsten or rhenium filamentElectrons emitted from heated tungsten or rhenium filament- Electrons accelerated by a potential of 70VElectrons accelerated by a potential of 70V- Path of electrons and molecular ion at right anglesPath of electrons and molecular ion at right angles

Form positive ions Form positive ions electron beam expels electron due to electrostatic electron beam expels electron due to electrostatic repulsionrepulsion

M + M + ee-- M M●●++ + 2 + 2ee--

Not very efficient Not very efficient one molecule in a million ionized one molecule in a million ionized Positive ions attracted to first slit by small potential 5VPositive ions attracted to first slit by small potential 5V High potential applied at accelerator plates 10High potential applied at accelerator plates 1033 to 10 to 1044 V V

- Generates molecular ion velocityGenerates molecular ion velocity

Ion Sources:Ion Sources:• Electron-Impact Source (EI) Electron-Impact Source (EI)

Hard source 50V higher energy than chemical bondHard source 50V higher energy than chemical bond Highly excited vibrational and rotational stateHighly excited vibrational and rotational state

- Electron beam does not increase translational energyElectron beam does not increase translational energy Relaxation results in extensive fragmentationRelaxation results in extensive fragmentation

- Large number of positive ions of various massesLarge number of positive ions of various masses- Typically less mass than molecular ionTypically less mass than molecular ion- Lower mass ions called Lower mass ions called daughter ionsdaughter ions- Sometimes molecular ion not presentSometimes molecular ion not present

Ion Sources:Ion Sources:• Electron-Impact Source (EI)Electron-Impact Source (EI)

Base peak Base peak most intense peak most intense peak- Usually a daughter ion or fragment ionUsually a daughter ion or fragment ion

Peaks at MW Peaks at MW greatergreater than molecular ion than molecular ion- Same chemical formula but different isotope compositionSame chemical formula but different isotope composition- Size of peak depends on relative natural abundance of isotopesSize of peak depends on relative natural abundance of isotopes

Collision Product peak (M+1)Collision Product peak (M+1)++

- Collision transfers a hydrogen atom to the ion to generate a protonated Collision transfers a hydrogen atom to the ion to generate a protonated moleculemolecule

- Second order reaction Second order reaction depends on concentration depends on concentration- Increases with increase in pressureIncreases with increase in pressure

Ion Sources:Ion Sources:• Electron-Impact Source (EI)Electron-Impact Source (EI)

AdvantagesAdvantages- Good sensitivityGood sensitivity- Fragmentation Fragmentation unambiguous identification of analytes unambiguous identification of analytes

DisadvantagesDisadvantages- Need to volatize sample Need to volatize sample thermal decomposition before ionization thermal decomposition before ionization- Fragmentation Fragmentation disappearance of molecular ion peak disappearance of molecular ion peak

- MW not determinedMW not determined

Ion Sources:Ion Sources:• Chemical Ionization Source (CI) Chemical Ionization Source (CI)

Electron Impact and Chemical Ionization are Interchangeable in a Electron Impact and Chemical Ionization are Interchangeable in a SpectrometerSpectrometer

Chemical Ionization is the second most common procedure for generating Chemical Ionization is the second most common procedure for generating ionsions

Gaseous atoms from the sample are:Gaseous atoms from the sample are:- Heated from a probeHeated from a probe- Collide with ions produced reagent gas bombarded by electronsCollide with ions produced reagent gas bombarded by electrons

- Usually positive ions are usedUsually positive ions are used Need to modify electron beamNeed to modify electron beam

- Add vacuum pump capacityAdd vacuum pump capacity- Reduce width of slit for mass analyzerReduce width of slit for mass analyzer

- Allow a reagent pressure of 1 torr in ionization areaAllow a reagent pressure of 1 torr in ionization area- Keep pressure below 10Keep pressure below 10-5-5 torr in analyzer torr in analyzer

Concentration ratio of reagent to sample is 10Concentration ratio of reagent to sample is 1033 to 10 to 1044

- Electron beam preferentially interacts with reagent instead of sampleElectron beam preferentially interacts with reagent instead of sample

Ion Sources:Ion Sources:• Chemical Ionization Source (CI)Chemical Ionization Source (CI)

Soft SourceSoft Source Methane is common reagentMethane is common reagent

- Also use propane, isobutane and ammoniaAlso use propane, isobutane and ammonia- Reacts with high-energy electron beam to generate several ionsReacts with high-energy electron beam to generate several ions- CHCH44

++, CH, CH33+ + (~90% of product) and CH(~90% of product) and CH22

++

- React with other methane moleculesReact with other methane molecules

CHCH44++ + CH + CH44 CH CH55

++ + CH + CH33

CHCH33++ + CH + CH44 C C22HH55

++ + H + H22

Collisions Between sample molecule and CHCollisions Between sample molecule and CH55++ & C & C22HH55

++ are highly reactive are highly reactive- Involve proton or hydride transferInvolve proton or hydride transfer

CHCH55++ + MH + MH MH MH22

++ + CH + CH44 proton transferproton transfer

CC22HH55++ + MH + MH MH MH22

++ + C + C22HH44 proton transferproton transfer

CC22HH55++ + MH + MH M M++ +C +C22HH66 hydride transferhydride transfer

- Proton transfer Proton transfer (M + 1) (M + 1)++

- Hydride transfer Hydride transfer (M – 1) (M – 1)++

- CC22HH55++ transfer (M + 29) transfer (M + 29)++

EIHard IonizationCI Soft

Ionization

Ion Sources:Ion Sources:• Matrix-Assisted Laser Desorption/Ionization (MALDI)Matrix-Assisted Laser Desorption/Ionization (MALDI)

Accurate MW for polar biopolymersAccurate MW for polar biopolymers- DNA, RNA, ProteinsDNA, RNA, Proteins- Few thousands to several hundred thousand DaFew thousands to several hundred thousand Da

Sample is mixed with large excess of radiation-absorbing matrix materialSample is mixed with large excess of radiation-absorbing matrix material

Solution is evaporated onto solid surfaceSolution is evaporated onto solid surface Sample exposed to pulsed laser beamSample exposed to pulsed laser beam

- Sublimation of analyte ionsSublimation of analyte ions- MS spectra recorded between laser beam pulsesMS spectra recorded between laser beam pulses


Low background noiseLow background noise- At high MW, matrix causes significant background at low MWAt high MW, matrix causes significant background at low MW

Absence of fragmentationAbsence of fragmentation Multiple charged ions (+2, +3)Multiple charged ions (+2, +3) Observe dimers trimersObserve dimers trimers Mechanism is not completely understoodMechanism is not completely understood

- Matrix compound must absorb the laser Matrix compound must absorb the laser radiationradiation- Soluble enough in sample solvent to be Soluble enough in sample solvent to be present in large excesspresent in large excess- Analyte should not absorb laser radiationAnalyte should not absorb laser radiation

Fragmentation will occurFragmentation will occur

Simulation of MALDI


MALDI spectra are greatly influenced by type of matrix, solvent and additiveMALDI spectra are greatly influenced by type of matrix, solvent and additive- At high MW, matrix causes significant background at low MWAt high MW, matrix causes significant background at low MW

Dariusz Janecki et al. (2002) ASMS

Ion Sources:Ion Sources:• Electrospray Ionization (ESI)Electrospray Ionization (ESI)

One of the most important techniques for analyzing biomoleculesOne of the most important techniques for analyzing biomolecules- Polypeptides, proteins and oligonucleotidesPolypeptides, proteins and oligonucleotides- Inorganic species synthetic polymersInorganic species synthetic polymers- MW >100,000 DaMW >100,000 Da

Uses atmospheric pressure and temperatureUses atmospheric pressure and temperature Sample pumped through a stainless steel capillarySample pumped through a stainless steel capillary

- Rate of a few Rate of a few ls per minutels per minute

- Needle at several KVs potentialNeedle at several KVs potential Creates charged spray of fine dropletsCreates charged spray of fine droplets


Passes through desolvating capillaryPasses through desolvating capillary- Evaporation of solventEvaporation of solvent- Attachment of charge to analyte moleculeAttachment of charge to analyte molecule- Molecules become smaller Molecules become smaller charge density becomes greater charge density becomes greater desorption of desorption of

ions into ambient gasions into ambient gas


Little fragmentation of thermally fragile biomoleculesLittle fragmentation of thermally fragile biomolecules Ions are multiply chargedIons are multiply charged

- mm//zz values are small values are small

- Detectable with quadrupole with mass range of 1500 or lessDetectable with quadrupole with mass range of 1500 or less Average charge state increases ~linearly with MWAverage charge state increases ~linearly with MW

- MW determined from peak distributionMW determined from peak distribution

Ion Sources:Ion Sources:• Fast Atom Bombardment Sources (FAB)Fast Atom Bombardment Sources (FAB)

Major role for MS studies of polar high molecular-weight speciesMajor role for MS studies of polar high molecular-weight species Soft Ionization techniqueSoft Ionization technique

- MW > 10,000MW > 10,000- Structural information for MW ~3,000Structural information for MW ~3,000

Samples are in a condensed stateSamples are in a condensed state- Glycerol solution matrixGlycerol solution matrix- Liquid matrix helps reduce lattice energyLiquid matrix helps reduce lattice energy

Ionized by bombardment with energetic (several keV) xenon or argon Ionized by bombardment with energetic (several keV) xenon or argon atomsatoms- Very rapid sample heatingVery rapid sample heating- Reduces sample fragmentationReduces sample fragmentation

Positive & negative analyte ions are sputtered from the surfacePositive & negative analyte ions are sputtered from the surface- Desorption processDesorption process- Must overcome lattice energy to desorb an ion and condense a phaseMust overcome lattice energy to desorb an ion and condense a phase- ““healing” the damage induced by bombardmenthealing” the damage induced by bombardment

Ion Sources:Ion Sources:• Fast Atom Bombardment Sources (FAB)Fast Atom Bombardment Sources (FAB)

Beam of fast energetic atoms are generated by:Beam of fast energetic atoms are generated by:- Passing accelerated argon or xenon ions from an ion source through a Passing accelerated argon or xenon ions from an ion source through a

chamberchamber- Chamber contains argon or xenon atoms at 10Chamber contains argon or xenon atoms at 10-5-5 torr torr- High-velocity ions undergo a resonant electron-exchange reaction without High-velocity ions undergo a resonant electron-exchange reaction without

substantial loss of translational energysubstantial loss of translational energy

Ar+* + Ar0 ----------------> Ar+ + Ar0*

Production of “fast atoms”Charge transfer

Accelerated argon ion from “ion gun”

Ground state argon atom

“slow ion”

“fast atom”

Focusing

Extraction plate

Analyte ion beam(secondary ions)

Probe tipAnalyte metrix

Atom beam

Example 23: Identify the ions responsible for the peaks in the following mass spectrum for 1,1,1,2-tetrachloroethane (C2H2Cl4):

Cl

Cl

Cl

Cl

MW = 167.85

Mass analyzers:Mass analyzers:• Double-Focusing analyzerDouble-Focusing analyzer

Two devices for focusing an ion beamTwo devices for focusing an ion beam- Electrostatic analyzerElectrostatic analyzer- Magnetic sector analyzerMagnetic sector analyzer

Ions accelerated through slit into Ions accelerated through slit into curved curved electrostatic fieldelectrostatic field- Focus beam of ions with narrow band of kinetic energies into slitFocus beam of ions with narrow band of kinetic energies into slit

Ions enter curved magnetic fieldIons enter curved magnetic field- Lighter ions deflected more than heavier ionsLighter ions deflected more than heavier ions


Curved magnetic field of 180, 90 or 60 degreesCurved magnetic field of 180, 90 or 60 degrees- Vary magnetic field strength or accelerating potential to select for ions of different massVary magnetic field strength or accelerating potential to select for ions of different mass

Kinetic energy of accelerated ion (before magnetic field)Kinetic energy of accelerated ion (before magnetic field)

where:where:V = voltageV = voltage vv = ion velocity = ion velocity ee = electronic charge = electronic charge

(1.60x10(1.60x10-19-19C)C)zz = ion charge = ion charge

Path through magnet depends on the balance of two forces:Path through magnet depends on the balance of two forces:- Magnetic force (FMagnetic force (FMM) and centripetal force (F) and centripetal force (Fcc))

where:where:B = magnetic field strengthB = magnetic field strength r = radius of curvature of magnetic sectorr = radius of curvature of magnetic sector

Substitute velocity into kinetic energy equationSubstitute velocity into kinetic energy equation

Select masses by varying Select masses by varying BB by changing current in magnet by changing current in magnet

2mv2

1zeVKE

r

mvBzev

2

r

mvF

2

c

cM FF

BzevFM

m

Bzerv

V2

erB

z

m 22

Mass analyzers:Mass analyzers:• Quadrupole mass analyzerQuadrupole mass analyzer

More compact, less expensive, ruggedMore compact, less expensive, rugged High scan rate High scan rate spectrum in < 100ms spectrum in < 100ms

• Four parallel cylindrical rods serve as electrodes Four parallel cylindrical rods serve as electrodes Opposite rods are connected electricallyOpposite rods are connected electrically

- One pair attached to positive side of variable dc sourceOne pair attached to positive side of variable dc source- One pair attached to negative side of variable dc sourceOne pair attached to negative side of variable dc source

Variable radio-frequency ac potential (180Variable radio-frequency ac potential (180oo out of phase) applied to each out of phase) applied to each pair of rodspair of rods

• Ions accelerated through space between rodsIons accelerated through space between rods Potential of 5 to 10 VPotential of 5 to 10 V ac and dc voltages increased simultaneously with ratio being constantac and dc voltages increased simultaneously with ratio being constant All ions without specific All ions without specific m/zm/z strike rods and become neutral strike rods and become neutral

- only ions having a limited range of only ions having a limited range of mm//zz reach transducer (detector) reach transducer (detector)

Mass analyzers:Mass analyzers:• Quadrupole mass analyzerQuadrupole mass analyzer• Positive rodsPositive rods

Alternating ac causes ions to converge during positive arc and diverge Alternating ac causes ions to converge during positive arc and diverge during negative arcduring negative arc- If ions strike rod during negative arc If ions strike rod during negative arc neutralized and removed by neutralized and removed by

vacuumvacuum Striking a rod depends on:Striking a rod depends on:

- rate of movement through rodrate of movement through rod- Mass to charge ratioMass to charge ratio- Frequency and magnitude of the ac signalFrequency and magnitude of the ac signal

dc current effects momentum of ionsdc current effects momentum of ions- Momentum directly related to square-root of massMomentum directly related to square-root of mass- More difficult to deflect heavy ions than lighter ionsMore difficult to deflect heavy ions than lighter ions- Prevents heavier atoms from striking rodsPrevents heavier atoms from striking rods

High-pass mass filterHigh-pass mass filter

Mass analyzers:Mass analyzers:• Quadrupole mass analyzerQuadrupole mass analyzer• Negative rodsNegative rods

In the absence of ac, all positive ions drawn to rods In the absence of ac, all positive ions drawn to rods annihilated annihilated Offset for lighter ions by acOffset for lighter ions by ac Low-pass mass filterLow-pass mass filter

• For ions to pass through the rods (band of ions): For ions to pass through the rods (band of ions): Significantly heavy to pass positive rodsSignificantly heavy to pass positive rods Significantly light to pass negative rodsSignificantly light to pass negative rods

- One pair attached to positive side of variable dc sourceOne pair attached to positive side of variable dc source- One pair attached to negative side of variable dc sourceOne pair attached to negative side of variable dc source

• Adjusting ac & dc moves the center of band of ions which pass the rodsAdjusting ac & dc moves the center of band of ions which pass the rods

Mass analyzers:Mass analyzers:• Time of Flight (TOF) Mass AnalyzersTime of Flight (TOF) Mass Analyzers• Ions generated by bombardment of the sample with a brief pulse of:Ions generated by bombardment of the sample with a brief pulse of:

Electrons, secondary ions, laser-generated photonsElectrons, secondary ions, laser-generated photons Frequency of pulse 10 to 50 kHz, duration of pulse 0.25 Frequency of pulse 10 to 50 kHz, duration of pulse 0.25 ss

• Ions accelerated by electric field pulse 10Ions accelerated by electric field pulse 1033 to 10 to 1044 V V Same frequency of ionization pulse, but lags behindSame frequency of ionization pulse, but lags behind

• Accelerated particle enter Accelerated particle enter field-freefield-free drift tube drift tube Ions enter tube with Ions enter tube with samesame kinetic energy kinetic energy Ion velocity vary inversely with massIon velocity vary inversely with mass

- Lighter particles arrive at detector before heavier particlesLighter particles arrive at detector before heavier particles- Flight times are 1 to 30 Flight times are 1 to 30 ss- Requires fast electronicsRequires fast electronics

Peak broadening due to variability in ion energies and initial positionPeak broadening due to variability in ion energies and initial position- Limits resolution compared to magnets and quadrupoleLimits resolution compared to magnets and quadrupole- Less widely used than quadrupoleLess widely used than quadrupole

Advantages: unlimited mass range, rapid data acquisition, simplicity, ruggedness, Advantages: unlimited mass range, rapid data acquisition, simplicity, ruggedness, ease of access to ion sourceease of access to ion source

Transducers (detectors) for Mass Spectrometry:Transducers (detectors) for Mass Spectrometry:• Electron MultipliersElectron Multipliers

Detects positive ionsDetects positive ions Similar to photomultiplier used in UV/visSimilar to photomultiplier used in UV/vis

- Each dynode held at successfully higher voltageEach dynode held at successfully higher voltage Dynodes have Cu/Be surfacesDynodes have Cu/Be surfaces

- Burst of electrons emitted when struck with energetic ions or electronsBurst of electrons emitted when struck with energetic ions or electrons Electron multiplies contain upwards of 20 dynodesElectron multiplies contain upwards of 20 dynodes

- Typical current gain of 10Typical current gain of 1077

Continuous-Dynode electric multiplierContinuous-Dynode electric multiplier- Glass that is heavily doped with leadGlass that is heavily doped with lead- Potential of 1.8 to 2 V across length of transducerPotential of 1.8 to 2 V across length of transducer- Trumpet shapedTrumpet shaped- Ions strike surface near entrance that ejects electronsIons strike surface near entrance that ejects electrons- Electrons then skip along surface ejecting more electrons with each impactElectrons then skip along surface ejecting more electrons with each impact- Typical current gain of 10Typical current gain of 1055 to 10 to 1088

Rugged, reliable with high current gainsRugged, reliable with high current gains Positioned directly at the exit slit of magnetPositioned directly at the exit slit of magnet

- Ions have enough energy to eject electronsIons have enough energy to eject electrons- Requires accelerator with quadrupole MSRequires accelerator with quadrupole MS

Transducers (detectors) for Mass Spectrometry:Transducers (detectors) for Mass Spectrometry:• Faraday CupFaraday Cup

hollow collector, open at one end and closed at the other, used to collect hollow collector, open at one end and closed at the other, used to collect beams of ionsbeams of ions- incident ion strikes the dynode surface which emits electronsincident ion strikes the dynode surface which emits electrons- induces a current which is amplified and recordedinduces a current which is amplified and recorded

Surrounded by cageSurrounded by cage- Prevents the escape of reflected ions and ejected secondary electronsPrevents the escape of reflected ions and ejected secondary electrons

Inclined with respect to ion beamInclined with respect to ion beam- Particles striking or leaving the electrode are reflected away from entranceParticles striking or leaving the electrode are reflected away from entrance

Connected to ground potential through a large resistorConnected to ground potential through a large resistor- Ions striking plate are neutralized by flow of electrons from resistorIons striking plate are neutralized by flow of electrons from resistor- Causes a potential drop across resistorCauses a potential drop across resistor

Independent of the energy, mass or chemical nature of ionIndependent of the energy, mass or chemical nature of ion Inexpensive and simple mechanical and electronic deviceInexpensive and simple mechanical and electronic device Disadvantages:Disadvantages:

- Need for a high-impedance amplifierNeed for a high-impedance amplifier Limits speed at which spectrum can be scannedLimits speed at which spectrum can be scanned

- Less sensitive than electron multipliersLess sensitive than electron multipliers

Atomic Mass Spectra and Interferences:Atomic Mass Spectra and Interferences:• Spectroscopic interferenceSpectroscopic interference

An ionic species in the plasma has the same An ionic species in the plasma has the same mm//zz values as an analyte values as an analyte Isobaric interferenceIsobaric interference

- Two elements that have isotopes with nearly the same massTwo elements that have isotopes with nearly the same mass- Differ by less than 1 amuDiffer by less than 1 amu

113113InIn++ overlaps with overlaps with 113113CdCd++ and and 115115InIn++ overlaps with overlaps with 115115SnSn++

- Isobaric interference occurs with the most abundant and most sensitive Isobaric interference occurs with the most abundant and most sensitive isotopeisotope 4040ArAr++ overlaps with overlaps with 4040CaCa++ (97%) need to use (97%) need to use 4444CaCa++ (2.1%) (2.1%) 5858NiNi++ overlaps with overlaps with 5656FeFe++ need to use need to use 5656FeFe++

- Exactly predictable from abundance tablesExactly predictable from abundance tables

Polyatomic ion interferencePolyatomic ion interference- More serious than isobaricMore serious than isobaric- Polyatomic species form from interactions with species in plasma, matrix or Polyatomic species form from interactions with species in plasma, matrix or

atmosphereatmosphere- Several molecular ions can form and interfereSeveral molecular ions can form and interfere- Typically observed for Typically observed for mm//zz < 82 amu < 82 amu- Potential interference: Potential interference: 4040ArAr2+2+, , 4040ArHArH++, , 1616OO22

++,,

HH221616O,O,1616OHOH++, , 1414NN++

- Serious interference: Serious interference: 1414NN22++ with with 2828SiSi++, ,

NOHNOH++ with with 3131PP++, , 1616OO22+ with + with 3232SS++,,

4040ArOArO++ with with 5656FeFe++, , 4040ArAr22

++ with with 8080SeSe++

- Correct with blankCorrect with blank

Atomic Mass Spectra and Interferences:Atomic Mass Spectra and Interferences:• Spectroscopic interferenceSpectroscopic interference

Oxide and Hydroxide species interferenceOxide and Hydroxide species interference- Most seriousMost serious- Oxides an hydroxides of analyte, matrix solventOxides an hydroxides of analyte, matrix solvent plasma gasesplasma gases- MOMO++ and MOH and MOH++ ions ions- Formation depends on injector flow rate, RF power,Formation depends on injector flow rate, RF power,

sampler skimmer spacing, sample orifice size,sampler skimmer spacing, sample orifice size,plasma gas composition, oxygen eliminationplasma gas composition, oxygen eliminationsolvent removal efficienciessolvent removal efficiencies

Matrix EffectsMatrix Effects- Occur at concentrations > 500 to 1000 Occur at concentrations > 500 to 1000 g/mlg/ml- Reduction in analyte signal, sometimes signal enhancementReduction in analyte signal, sometimes signal enhancement- General effectGeneral effect- Minimized by using more dilute solutions, altering sample introduction Minimized by using more dilute solutions, altering sample introduction

procedure or by separating out offending speciesprocedure or by separating out offending species- Use internal standard to correct effectUse internal standard to correct effect

Affect of pesticide response to matrix effects

Add protectant to remove matrix effect

Analytical Sciences 2005, 21, 1291Analytical Sciences 2005, 21, 1291

Fourier Transform (FT) MS:Fourier Transform (FT) MS:• Improves signal-to-noise ratiosImproves signal-to-noise ratios• Greater SpeedGreater Speed• Higher sensitivity and resolutionHigher sensitivity and resolution• Requires an Requires an Ion TrapIon Trap

Ions are circulated in well-defined orbits Ions are circulated in well-defined orbits for extended periodsfor extended periods

Ion cyclotron resonance (ICR)Ion cyclotron resonance (ICR)- Ions in a magnetic field circulate in a Ions in a magnetic field circulate in a

plane perpendicular to the direction of the plane perpendicular to the direction of the fieldfield

- Angular frequency of this motion Angular frequency of this motion cyclotron frequency (cyclotron frequency (cc))

Ion trapped in a magnetic field can Ion trapped in a magnetic field can absorb energy from an ac electricabsorb energy from an ac electric fieldfield- Frequency of field must match cyclotron Frequency of field must match cyclotron

frequencyfrequency- Absorbed energy increases velocity of Absorbed energy increases velocity of

ion and radius without affecting ion and radius without affecting cc

m

zeB

r

vc

Fourier Transform (FT) MS:Fourier Transform (FT) MS:• Detection of ICRDetection of ICR

Ions circulating between plates Ions circulating between plates induces current between platesinduces current between plates- Image currentImage current- Non-destructiveNon-destructive- Decays over a few seconds Decays over a few seconds

through collisionthrough collision

Decay over time of the image Decay over time of the image current after applying an RF pulse current after applying an RF pulse is transformed from the time is transformed from the time domain into a frequency domaindomain into a frequency domain

Ions of two different m/q ratios excited on resonance for the same amount of time with the same excitation voltage. Ion [A] has the lower m/q ratio and thus has a higher cyclotron frequency. Ion [B] has the higher m/q ratio and thus a lower cyclotron frequency.

Mass Spectrometry

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