Mass Spectrometry Lecture Slides

Chem 425- Mass Spectrometry Tuesday and Thursday: 10am – 11:20am, C2-361 Richard W. Smith, Ph.D. [email protected] C2-264 (office) / C2-267 (lab) UW Mass Spectrometry Facility Course TA: Jonathan Martens [email protected] Office Hour: Friday, 12:30pm-1:30pm, C2-269B 1

Course Overview •

1. Introduction

•

2. History

•

3. Electron Ionization – The mass spectrometer and mass calibration – Electron Ionization (EI) • Fragmentation • QET • Elemental composition, isotopic species, mass resolution, accurate mass, z>1, rings plus double bonds, nitrogen rule • Interpretation of EI mass spectra

•

4. Chemical Ionization (CI and DCI) – Ion formation – Thermochemical considerations and reagent systems – Negative ion formation

2

Course Overview cont •

5. Field Ionization and Field Desorption (FI and FD)

•

6. Particle Bombardment – FAB, LSIMS and 252Cf

•

7. Laser Desorption Ionization and Matrix Assisted Laser Desorption Ionization (LDI and MALDI)

•

8. Inductively Coupled Plasma (ICP)

•

9. Atmospheric Pressure Ionization (API) – Electrospray (ESI) – Atmospheric Pressure Chemical Ionization (APCI) – Atmospheric Pressure Photo-Ionization (APPI) 3

Course Overview cont •

10. Mass Separation – Magnetic and electrostatic fields (B and E) – Quadrupoles (Q and QQQ) – 3D Quadrupole Ion Trap (QIT) – 2D linear Ion Trap – Time of Flight (Tof) – Fourier Transform Ion Cyclotron Resonance (FTICR)

•

11. Hyphenation in Mass Spectrometry – Gas Chromatography – Mass Spectrometry (GC-MS) – Liquid Chromatography – Mass Spectrometry (LC-MS) • Interfaces – MS-MS and MSn - Tandem Mass Spectrometry • Collision Induced Dissociation (CID) • BE, QQQ, 3D QIT, 2D QIT, QTof…… 4

Course Overview cont •

12. Quantitation – Primarily with QQQ in MRM/SRM mode

•

13. Ion detection and Focusing – Faraday cup, Electron multiplier, multichannel plate and photomultiplier

•

14. Summary

5

Course Overview cont •

70 minute mid-term exam during class (Thursday, February 28th) – 25%

•

150 minute final exam (to be scheduled) – 55%

•

10 Weekly Quizzes – total of 10%. Held at the end of class starting on Tuesday, Jan 15th (~10min) – 10 questions/quiz

•

– 7 to 10 correct answers

1 mark

– 3 to 6 correct answers

0.5 mark

– 0 to 2 correct answers

0 mark

12 minute seminar – 10%. Held weekly at the end of class starting on Thursday, Jan 17 – Teams of 2 prepare and present the material

6

Course Calendar January 2008 24 class sessions: Tuesday and Thursday – 10am to 11:20am in C2-361

Sun

6

Mon

7

Tues

Wed

Thurs

Fri

Sat

1

2

3

4

5

9

10

11

12

18

19

25

26

8 1. Introduction 2. History

13

14

15

3. EI

16

Quiz 1 3. EI

20

21

22

Sem 1 3. EI

23

Quiz 2 4. CI

27

28

29 Quiz 3 5. FI and FD

17 24 Sem 2 4. CI

30

31 Sem 3 6. Particle Bombardment

7

Course Calendar February 2008 Sun

3

Mon

4

Tues

5

Wed

6

Quiz 4 7. LDI & MALDI

10

11

12

18

19

24

25

26 Quiz 6 9. API

7

Fri

Sat

1

2

8

9

15

16

22

23

Sem 4 8. ICP

13

Quiz 5 9. API

17

Thurs

14 Sem 5 9. API

20 21 Reading Week 27

28

29

Mid-Term 8

Course Calendar March 2008 Sun

Mon

Tues

Wed

Thurs

Fri

Sat 1

2

3

4

5

Sem 6 10. Mass Sep

9

10

11

17

18

12

24

25 Sem 9 11. Hyphenation

30

8

13

14

15

21

22

28

29

Quiz 8 11. Hyphenation

19

Sem 8 11. Hyphenation

23

7

Quiz 7 10. Mass Sep

Sem 7 10. Mass Sep

16

6

20 Quiz 9 11. Hyphenation

26

27 Quiz 10 12. Quantitation

31 9

Course Calendar April 2008 Sun

Mon

Tues

Wed

1

2

Sem 10 13. Ion Focusing and Detection

Thurs 3

Fri

Sat

4

5

Sem 11 &12 14. Summary Q and A

6

7

8

9

11

12

16

10 Exams Start 17

13

14

15

18

19

20

21

22

23

24

25

26

27

28

29

30 10

Resources Text Books •

Mass Spectrometry – A Textbook (on 1 hour hold at Davis Library) Jurgen H. Gross - Springer 2004 – QD96M3G76

•

Mass Spectrometry: A Foundation Course K. Downard – Royal Society of Chemistry 2004 – QD96M3D69X2004

•

Chemical Ionization Mass Spectrometry – 2nd edition Alex G. Harrison – CRC Press 1992 – QD96M3H371992

•

Interpretation of Mass Spectra Fred W. McLafferty – University Science Books

Database website •

NIST Library of EI Mass Spectra: http://webbook.nist.gov/chemistry/name-ser.html

11

Resources Journals: •

Biomedical and Environmental Mass Spectrometry / Biological Mass Spectrometry

•

International Journal of Mass Spectrometry

•

Journal of the American Society For Mass Spectrometry

•

Mass Spectrometry Reviews

•

Journal of Mass Spectrometry

•

Rapid Communications in Mass Spectrometry

12

History • 1913: J. J. Thomson – parabola spectrograph – Detection of the neon isotopes 20 and 22 – Parallel electric and magnetic field – Detection on photoplate + Gas Discharge ion source

+ve and -ve ions recorded simultaneously! Focusing

Mass separation using magnetic and electric fields 1

Photoplate detector

J. J. Thomson: First Mass Spectrum of 20Ne and 22Ne Modern measurements of 20Ne:22Ne ~ 10:1

2

History •

1918 – 1920: Dempster and Aston (Nobel prize in 1922) – Focussing of ions – Higher resolution (≈ 600) – tandem electric and magnetic field – Allowed the detection of 21Ne (0.3% abundance)

3

History •

Late teens to early 30‘s, MS primarily used for isotopic analyis of the stable elements

•

1934: J. Mattauch and R. Herzog (Nobel prize) – Double focusing instruments > resolution 6500

•

1936: Secondary Ion Mass Spectrometry introduced

•

1940: Westinghouse Electric begins development of a portable MS for commercial sale

•

1942: E.O. Lawrence develops the “Calutron“ prep scale MS for separation of uranium isoptopes – 235U

•

1943: CEC installs 1st commercial MS at the Atlantic Refining Company

•

1945: CEC introduces 1st analogue computer for data analysis 4

History •

1946 -1947: 1st description of time-of-flight MS, Metropolitan Vickers and General Electric begins manufacturing MS

•

1948: ICR MS “Omegatron“ is developed

•

1952: 1st A/D converter for data aquisition (CEC)

•

1953: W. Paul → Quadrupole mass spectrometer and ion trap detectors – Nobel prize in 1989.

•

1954: 1st high resolution MS developed at Imperial Chemical Industries (ICI)

•

1956: PA determinations made by MS, McClafferty rearrangment described, steroids 1st analyzed by MS 5

History •

1957: GC/MS 1st demonstrated at Phillip Morris

•

1958: CEC introduces the 1st digitizer. Bendix TOFMS introduced

•

1959: peptides and oligonucleotides sequeunced at MIT

•

1960: Bendix introduces 1st direct insertion probe – CEC introduces 1st high res double focusing MS

•

1966: Tandem MS for ion-molecule studies is developed – Chemical Ionization is developed at ESSO (Munson and Field)

•

1968: Electrospray Ionization introduced at Northwestern U for studying macromolecules. Computer library searching used for identification of 6 unknown compounds

History •

1969: 1st GC/MS with integrated computer introduced

•

1971: relectron time-of-flight develpoed in Lenningrad

•

1974: M.B. Comisarow, A.C. Marshall – FT-MS. APCI interface is developed for LCMS

•

1975: mass spectrometers are placed on the Viking Mars missions

•

1979: Ion evaporation model developed (ESI)

•

1980: ICPMS is developed at Iowa State U. 1st commercial QQQs introduced.

•

1981: FAB introduced.

•

1983: A.L. Yergey, M.L. Vestal – Thermospray interface for LCMS announced. 7

History •

1984: Yamashita and Fenn – Electrospray (Nobel prize, 2002). 1st commercial ion trap is introduced. Particle Beam interface developed at GIT.

•

1986: LC interfaced to MS with pneumaticaly assisted ESI.

•

1988: Karas and Hillenkamp – MALDI

•

1994: Micro- and nano- ESI are introduced

•

In the past 10 years a wide variety of hybrid instruments have been introduced along with great improvements in instrument capabilities and sensitivity (attamole) 8

The Mass Spectrometric Process *Field free region with ion focusing

Sample Introduction

Ion source

*

Mass Analyzer(s)

Separates ions based on their m/z value Gas or condensed Some form of phase sample + ionization

*

Detector

Indirect detection

Ions

Integrated with some degree of computer control

1

The Mass Spectrometer •

Sample Introduction • Batch - direct insertion probe, desorption probe, heated inlet, infusion • Chromatographic – GC or LC (SFC)

•

All Ion sources produce either • •

•

Odd electron ions eg M+. (EI, charge exchange CI) Even electron ions eg [M+H]+, [M-H]- and/or adducts [M+Na]+ (CI, FAB/SIMS, ESI, APCI, APPI) Stability of these ions determines the observed mass spectrum 2

The Mass Spectrometer •

•

All Mass Analyzers •

Separate ions according to their mass/charge ratio (m/z)

•

This separation is accomplished in a variety of fashions eg by momentum (BE/EB), time of flight (Tof), selective transmission employing Rf and DC voltages (Q and IT), frequency of motion (FTICR)

•

MS/MS and MSn

•

Continuous beam (Q, BE/EB) and pulsed beam (IT, Tof, FTICR)

All Detectors • •

•

Conversion of +ve and –ve ions to 2o electrons or photons Indirect detection

Computer employed for instrument control, data acquisition and processing 3

JEOL HX110 Double Focusing Mass Spectrometer (EB)

ESA

Magnet

Tuning Console Source and Sample Introduction

Detector

4

Benchtop Quadrupole GC/MS system

MS Courtesy of Agilent

GC 5

Ion Types • Molecular ions: [M]+•, [M]-• are ions that arrive intact at the detector – Stable – k < 105s-1 • Fragment ions: [F]+ formed in the source by direct bond cleavage or rearrangement – Unstable k > 106s-1 • Metastable ions, m* are ions that fragment outside the ion source but before they arrive at the detector – 105s-1 < k < 106s-1

6

Ion Types Fragment ions M+.

Metastable ion M+. and fragment ions~0.3Da wide Metastable ion~3Da wide! 7

Ion Types • Odd electron (such as [M]+•) and even electron ions (such as [M+H]+) • Quasi-molecular ions, e.g. [M+H]+, [M-H]-, [M+NH4]+, [M+Na]+, [M+Cl]- …….. • Cluster ions, [2M+H]+, (proton bound dimer) • Isobaric ions (same nominal mass) • Isotopic species

8

Mass Calibration •

All mass spectrometers make ions, pass them through a region where they are separated according to their m/z ratio and finally detected

•

A mass calibration must be performed in order to convert arrival times of ions at the detector into a m/z value

•

This is achieved by introducing a reference compound(s) into the source who’s masses are known and then comparing this uncalibrated mass spectrum with a reference spectrum of the same compound(s). The patterns are matched and the data system then can, in real time, convert arrival times into m/z values.

•

Reference compounds are many and varied eg PFK, PEG, CsI and other clusters such as phosphoric acid. The compound(s) chosen will depend upon the ionization mode employed. 9

Mass Calibration •

Perfluorkerosene (PFK) with the JEOL HX110 62.3

69.0

380.94

344.6

542.9

493.1

Uncalibrated

Software algorithm

Uncalibrated ΔM at m/z 380.97 = 36.3Da

Calibrated

Calibrated ΔM at m/z 380.97 = 0.03Da 10

Electron Ionization – The Source Filament Source block

Sample in

S N Focus plates e-

Repeller Ion beam out M+. and fragment ions

Trap S N magnet

11

Electron Ionization • Source operates at high vacuum (10-6 mbar) and high temp (~200oC) • Electrons are produced by heating a filament with a few amps ie thermionic emission from a hot wire (rhenium or tungsten) • They are focused onto the trap using a magnetic field and voltage - usually 70V (can be changed) therefore e- are said to have 70eV translational energy • Trap: Collects the electrons on the far side of the source. Usually the trap current is set and the electronic circuitry adjusts the filament emission to maintain this value • Repeller: +ve relative to the source, pushes the +ve ions formed toward and out the exit slit where they are further accelerated into the mass analyzer region

12

Electron Ionization • Magnetic field causes e- to spiral increasing the chance of an interaction with the sample species – higher sensitivity • Sample is introduced and must be volatilized before ionization can occur – ie dilute gas phase unimolecular processes (same for EI and CI) 13

EI of Methyl Stearate at 70eV O O

Mwt=298.5095 Monoisotopic mass=298.2872

M+.

14

Electron Ionization • We need to understand the processes involved if we hope to use MS as a structural elucidation tool! • Ionization • Isotopic distribution • Accurate mass • Fragmentation 15

Ionization • one of the most common forms of ionization

AB + e1o

AB+. + e- + e1o

2o (slow)

A + + B.

etc

ΔHf (AB+.) = IP + ΔHf (AB) IP is the ionization potential of AB ie the energy required to remove one electron • note that radical cations (M+.) are initially formed

16

Ionization • the term electron “impact” should be avoided as the electron does not impact the molecule • 70 eV electrons have a deBroglie wavelength associated with them of about 1.4Angstrom which is on the order of a bond length. The interaction of the electron “wave” with the molecule causes a disturbance which causes excitation or ionization of the molecule • Removal of the e- from a molecule can be considered to occur at: σ-bond < π-bond < free electron pair

17

Probability of ionization as a function of electron energy

• Maximum ionization efficiency of organic ions at 20 – 50 eV • every species has it‘s own curve depending on it‘s ionization cross section • Excess energy ( ~ 1 – 8 eV) can lead to substantial fragmentation 18

Electron Energy •

•

•

some of the energy of the electron goes into ionizing the molecule (~7-12 eV). There is still more than enough energy available to cause extensive fragmentation of molecules - typical bond strengths are 1 – 4 eV. The electron energy chosen must be higher than the ionization potential (IP) of the compound the higher the electron energy, the more energy there is available for fragmentation and therefore a EI spectra of β-lactam using 70 and 15 higher degree of fragmentation is eV electrons. Note the intensity scale. usually observed. 19

Ionization •

•

σ/ The ionization potential (IP), is A2 Molecule the minimum energy required to remove an electron from a He 0.38 species. The ionization cross Ne 0.62 section (σ) is a measure of the Ar 3.52 relative ease of ionization of a Kr 5.29 molecule. In general, this is Xe 7.31 proportional to the polarizability CH4 4.30 of the molecule.

Polarizability is the relative tendency of the electron cloud of an atom to be distorted from its normal shape by the presence of a nearby external electric field

IP (eV)

Polarizability (10-24cm3)

24.59

0.21

21.56

0.40

15.76

1.64

14.00

2.48

12.13

4.04

12.61

2.59

C2H6

8.35

11.52

4.47

C3H8

11.1

10.94

6.30

C6H14

22.3

10.13

11.9

C6H6

16.9

9.24

10.3

C10H8

-

8.14

16.3 20

Appearance Energy (AE) • For sufficiently energetic electrons, ionization may be accompanied by bond cleavage:

AB + e- → A+ + B + 2e• Most of the energy exchanged creates electronic excitation (along with ionization). Almost no M+. ions will be in the vibrational ground state. “Some” of these vibrationally excited ions may be above the dissociation energy level • The appearance energy (AE) of A+ from AB is defined as the minimum electron energy at which A+ is formed from AB. AE measurements are very useful for determining standard heats of formation however requires specialized instrumentation.

AE = ΔHf (A+) + ΔHf (B) – ΔHf (AB)

21

Appearance Energy (AE) • For example: the appearance energy of CH2OH2+ by dissociative ionization of HOCH2CH2OH (ethylene glycol), also producing neutral formaldehyde (CH2O) was found to be 11.42 eV (JACS 1982, 104, 2931). What is the heat of formation of CH2OH2+ given that the heats of formation of ethylene glycol and formaldehyde are –387.6 and –108.7 kJ mol-1, respectively?

HOCH2CH2OH

AE = 11.42eV

CH2OH2+ + CH2O

1eV=96.485 kJ mol-1 Therefore 11.42 eV = 1102 kJ mol-1 22

Appearance Energy (AE) AE = ΔHf (CH2OH2+) + ΔHf (CH2O) – ΔHf (HOCH2CH2OH) or ΔHf (CH2OH2+) = AE - ΔHf (CH2O) + ΔHf (HOCH2CH2OH) ΔHf (CH2OH2+) = (1102 + 108.7 – 387.6) kJmol-1 = 823 kJmol-1 In fact, CH2OH2+ has been determined to be approximately 20 kJ mol-1 lower in energy than its conventional isomer CH3OH+ (ΔHf = 844 kJmol-1).

23

Fragmentation in EI • Both simple bond cleavages as well as complex rearrangements may take place after EI ie. simple bond cleavage, t-butyl chloride:

C(CH3)3Cl +e-

C(CH3)3+ +Cl. +2e-

24

Fragmentation in EI ie complex rearrangement (McLafferty Rearrangement) O + e-

C3H6O+ + C2H4 + 2e-

2-pentanone

H

distonic radical cation O .+

OH+

CH2 CH2

+

.CH

C 2

CH3

McLafferty Rearrangement: any fragmentation that can be described as a transfer of a γ-hydrogen to a double bonded atom through a six-membered transition state with β-cleavage. Occurs in saturated aldehydes, ketones and carboxylic acids Distonic Radical Cation: a radical cation in which the charge and radical sites are 25 separated

Fragmentation in EI Stevenson’s Rule: AB+ eA+ + B. + 2eor A. + B+ + 2eFor simple cleavage, the species of lower ionization energy will give the more abundant ion. Therefore: If IE(A) < IE(B) then IA+ > IB+ 31

EI of methanol

32 29 CH3+ IP=9.84eV

OH+ IP=13eV

26

EI of acetone, (CH3)2CO CH3CO+ IP=7eV

43

M+. m/z 58

CH3+ IP=9.84eV

27

Quasi-Equilibrium Theory (QET) • A theoretical approach to describe the unimolecular decomposition of ions • In most mass spectrometers, processes occur in the highly diluted gas phase therefore bimolecular reactions are rare • isolated ions are not in thermal equilibrium with their surroundings and can only internally redistribute energy by dissociation or isomerization (rearrangement) • The rate constant (k) of a unimolecular reaction is strongly influenced by the excess energy (Eex) of the reactants in the transition state • Removal of an electron can be considered to occur at a σ-bond, πbond or at a free electron pair • For charge localization: free electron pair > π-bond > σ-bond and 28 this is reflected in IE’s

Vertical Transitions Energy

M.+

A+ + B.

dissociation energy

M+. stable

IE

Ionization is very fast therefore termed to be a vertical ionization process.

M

r 0 r1

29

Dissociation coordinate

Take Away Messages from QET • EI occurs very fast ~ 10-16s and is a vertical process (FranckCondon principle) • The probability of a particular vertical transition from the neutral to a certain vibrational level is described by the Franck-Condon factors. • The larger r1 compared to r0, the more probable will be the generation of ions excited even above their dissociation level • Ionization tends to cause weakening of the bonding (bond lengthening) in the ion compared to it’s precursor neutral • The shape of the potential energy surface will also influence the 30 ions fate

Interpretation of EI spectra: Elemental Composition

The elemental composition: • The use of isotope peaks • High resolution for accurate mass determinations • Rings + Double Bonds • double bond equivalents (DBE)

31

Some Common Isotopic Species

Element Type is sometimes termed X, X+1, X+2 etc 32

Isotopic Classification of the Elements Some Definitions: •

Atomic number specifies the number of protons in the nucleus eg C is element 6 6C

•

Atoms with the same atomic number but with different number of 35Cl and 37Cl (3:1) neutrons are termed isotopes eg 17Cl

•

The mass number is the sum of protons and neutrons in an atom can be confusing for example both 18Ar and 20Ca have a mass number of 40

•

Elements are classified as:

A A+1 A+2 A-1 polyisotopic

19F, 31P, 127I 12C

and 13C 35Cl and 37Cl 6Li and 7Li Sn 10 isotopes 33

Isotopic Distributions Calculation of the abundance ratios of isotopic peaks of molecules containing two isotopes, where the isotope natural abundances are given as a and b and n is the number of this species in the molecule: (a + b)n = an +nan-1b + n(n-1)an-2b2/(2!) + n(n-1)(n-2) an-3b3/(3!) + …. (binominal equation) e.g. if n = 2: (a+b)² = a² + 2ab + b² For two Cl atoms where a = 100 (35Cl) and b = 32 (37Cl): 100² + 2•100•32 + 32² = 100 : 64 : 10 Likewise if n=3: and if n=4:

a3 + 3a2b +3ab2 + b3 a4 + 4a3b + 6a2b2 + 4ab3 +b4

34

Common Isotopic Distributions

12

m as

13

0 19

120

Si1

100

0

1199 1200 1201 1202 1203 1204 1205

Si2

100

%

0

m as

121

28

29

30

m ass 31

0

m ass

31

32

33

Si3

100

%

27

S1

100

S2

100

34

35

0

m ass

63

64

65

66

F1

100

%

S3

100

%

%

%

% 0

C100

100

%

C10

10

%

C1

10

m ass

0

95

96

98

m ass 99

F10

100

%

97

%

35 0

55

56

57

58

m ass

0 82

83

84

85

86

m ass 87

0

18

19

20

21

m ass

0 188

189

190

191

m ass

Common Isotopic Distributions Cl1

100

Cl2

100

%

%

0

34

35

36

37

38

m ass

Cl3

100

%

%

0 68

69

70

71

72

73

Br1

74

75

0 103

mass

104

105

106

107

108

Br2

109

110

111

112

mass

0

79

80

81

82

mass

100

100

100

%

%

%

0

157

158

159

160

161

140

141

142

162

163

mass

0 235

236

237

238

239

240

241

242

143

144

145

mass

146

Br4

%

78

139

Br3

100

0

Cl4

100

243

244

mass

0 314

315

316

317

318

319

320

321

322

323

324

mass

36

Isotopic Distributions at “High” m/z C200H320N50O50S10

100

@FWHM=1

100

FWHM=0.4 ~ 10,200 Resn %

%

0

0

4536

4538

4540

4542

4544

4546

4548

4550

4552

4554

4556

4536

4538

4540

4558

C192H290N40O40S5Cl5Br5

100

4542

4544

4546

4548

4550

4552

4554

4556

4558

mass

10vs16 amu

100

%

%

0 4524

0 4524

4526

4528

4530

4532

4534

4536

4538

4540

4542

4544

4546

4548

mass

37

4526

4528

4530

4532

4534

4536

4538

4540

4542

4544

4546

4548

mass

mass

Mass Resolution • Mass resolution: represents the ability to separate two adjacent masses. It measures the "sharpness" of the MS peak.

Resolution = M/ΔM Normally reported at 10% or 50% valley

•Mass accuracy: indicates the accuracy of the mass information 38 provided by the mass spectrometer

Mass Resolution 1188.5654

100

%

1189.5654

FWHM = 0.1

1190.5654 1191.5654 1192.5654

0 1188.5654

100

%

1189.5654

FWHM = 0.2

1190.5654 1191.5654 1192.5654

0 1188.5654

100

FWHM = 0.5

%

1189.5654

1190.5654 1191.5654 1192.5654

0 1188.6514

FWHM = 1

%

100

0

FWHM = 2

1189.1201

%

100

0 1183

1184

1185

1186

1187

1188

1189

1190

1191

1192

1193

1194

1195

1196

1197

mass 39 1198

Nominal vs Monoistopic* Mass Nominal mass

Acc mass

1H

1

1.00783

2H

2

2.01410

12C

12

12.00000

13C

13

13.00335

14N

14

14.00307

15N

15

15.00010

16O

16

15.99491

17O

17

16.99913

18O

18

17.99916

19F

19

18.99840

32S

32

31.97207

33S

33

32.97146

34S

34

33.96787

36S

36

35.96708

35Cl

35

34.96885

37Cl

37

36.96590

Av Mass 1.00798 12.01104 14.00676

% Rel Int 99.98 0.01 98.9 1.1 99.63 0.37 99.76

15.99933

0.04 0.2

18.99840

100* 95.02

32.06439

0.75 4.21 0.02

35.45274

75.77 24.23

40

Nominal vs Monoistopic vs Average Mass Take for example: C19H31N4O4Cl1,nominal mass = 414 Monoisotopic mass = 414.2034

100.0

80.0

60.0

Average mass = 414.9267 40.0

20.0

0.0 412.00

414.00

416.00

418.00

420.00

422.00

41

Nominal vs Monoistopic vs Average Mass Nominal Mass (A)

Average Mass (B)

Monoisotopic Mass (C)

C18H36O2

284

284.47724

284.27152

C17H32S1O1

284

284.50138

284.21737

A: different elemental composition but same nominal mass – isobaric ions B: calculated using C= 12.01104, H= 1.00798, O= 15.99933 and S= 32.06439 C: calculated using 12C=12.0000, 1H= 1.00783, 16O=15.99491 and 32S= 31.97207 42

Accurate Mass Determinations • High resolution enables isobaric species to be differentiated based on their non-integral accurate mass. • Consider the example below: all have a nominal mass of 28 and are therefore considered to be isobaric: CO = 27.9949 ΔM = 0.0112 M/ΔM = 2500 N2 = 28.0061 ΔM = 0.0252 M/ΔM = 1110 C2H4 = 28.0313

} }

These isobaric ions can be differentiated based on their accurate mass if sufficient mass resolution is employed

43

Accurate Mass Determinations • Most often employed to determine the elemental composition of an ion, usually [M]+. or [M+H]+ ion • Experimental error minimized by co-introducing a reference compound into the source along with the unknown ie Perfluorokerosene (CxFy) • PFK used as it is mass deficient ie 12C=12.00000 and 19F= 18.99840 • Error normally expressed as ppm or millimass units (1mmu=0.001amu)

( true mass - observed mass ) Mass Accuracy (ppm) = × 10 6 true mass

44

Mass Deficiency and Mass Sufficiency • The isotopic mass is very close but not equal to the nominal mass of that isotope: • Therefore the calculated exact mass of a molecule or of a monoisotopic ion equals it’s monoisotopic mass •

12C

is the only exception because the unified atomic mass (u) is defined as 1/12 of the mass of 12C, 1u=1.66055x10-27kg and 12C is arbitrarily assigned as 12.0000000

• As a consequence of these non-integer masses almost no combination of atoms will have the same calculated exact mass whereas they might have the same nominal mass, that is, they are isobaric 45

Mass Deficiency and Mass Sufficiency •

Mass Deficiency – the exact mass of an isotope or molecule is lower than it’s nominal mass – eg nominal mass = 352, exact mass = 351.9897

•

Mass Sufficiency – the exact mass of an isotope or molecule is higher than it’s nominal mass – eg nominal mass = 352, exact mass = 352.0347 – Only H, He, Li, Be, B and N are mass sufficient

•

Atomic masses up to O are slightly higher than the nearest whole number while O and above are slightly less than the nearest whole number 46

Proton, Neutron and Electron Mass • Mass of proton : 1.6726 x 10-27 kg • Mass of neutron: 1.6749 x 10-27 kg • Mass of electron: 0.00091x10-27 kg • Relative: proton : neutron : electron 1 : 1.00138 : 0.0005 Example 12C: 6 protons + 6 neutrons + 6 electrons (1s2.2s2.2p2) should = 12.01128 but in fact is 12.0107

WHY? 47

Mass Defect

•

The mass of an atom is less than the total mass of the constituent protons, neutrons and electrons • Mass defect, Δm = (Σmi) – matom and should be negative!

•

This missing mass can be explained by Einstein's theory of mass-energy equivalence, E = mc2 The deviations from whole numbers represents the energy required to bind the atomic nucleus together Also known as the Binding Energy, BE = Δmc2

• •

48

Mass Deficiency and Mass Sufficiency •

The larger the nucleus the more energy is required and the more mass deficient an isotope is: – 4He – 20Ne – 40Ar – 84Kr – 132Xe

•

4.0026 19.99244 39.96238 83.91152 131.90415

Relative to 12C = 12.00000

If the unified atomic mass (u) had been based on 1H = 1.00000 and not 12C/12 = 1.00000 (1H = 1.0078) then all isotopes would be mass deficient ie this is completely arbitrary and not based on some fundamental property of matter 49

Accurate Mass Determinations LR EI of mw=308 (Resn~950)

PFK

PFK

Resn~9200

308.1085 C16H20O4S1 1ppm error or 0.3mmu

PFK

50

Charge State (z>1): EI of Pyrene (70eV) doubly charged pyrene, M++ @ m/z 101 M+. 202

100

101.5

51

EI of Pyrene at low electron energy: ~20eV 1st

IE~7.4eV 2nd IE~16.6eV

M+. 202

Note: no M++ and no fragmentation

52

Multiply Charged Ions: z>>1 A

100

ions are 1amu apart %

A compound of mw A of formula CxHy

Z=1

(Mass resolution is constant ) mass 100

A/2

For example A=500.4

Z=2

• z=1, m/z=500.4/1 = 500.4

%

ions are 0.5 amu apart

• z=2, m/z=500.4/2 = 250.2 mass

• z=3, m/z=500.4/3 = 166.8

100

%

• z=10, m/z= ?

Z=3

A/3 ions are 0.333 amu apart

53 mass

Rings + Double Bonds Based on valence rules the following expression can be derived: imax

r+d = 1 +0.5 ΣNi(Vi-2) i

Where Ni is the # of atoms and Vi is the valence of an element Using 0.5x(Vi-2): For monovalent elements (H, F, Cl, Br, I) = -0.5 For divalent elements (O, S, Se) = 0 and so don’t contribute to DBE For trivalent elements (N,P) = +0.5 For tetravalent elements (C, Si, Ge) = +1

r+d = 1 - 0.5Nmono + 0.5Ntri + Ntetra + 1.5Npenta + 2Nhexa + …… Restriction to formulas of the general type, CxHyNzOn reduces the expression to the commonly cited form: r+d = x – 0.5y + 0.5z + 1

54

Rings + Double Bonds • a whole number for any OE ion • a non-integer for an EE ion • Significance – Informs about ion type (OE or EE) and basic structural info (number of rings + double bonds) – For EE ions, subtract 0.5 to get the right number of rings + double bonds – Sometimes called double bond equivalents (DBE)

55

Nitrogen rule • If a compound contains an even number of nitrogen atoms (0,2,4,6…), its molecular ion will be an even mass; and if it has an odd number (1,3,5…), it will have an odd mass • Why nitrogen? – With the exception of N, all elements having an odd # of valences also have an odd mass (H, P, F, Cl etc) – all elements having an even # of valences have an even mass (C, O, S, Si, etc) – N has an odd # of valences and an even mass • Value: places constraints on the numbers of nitrogens present • It will be the inverse for EE ions, (M+H)+

56

Interpretation of EI spectra: The molecular ion, M+. • Molecular ion, M+•, most valuable information (mass, isotopic distribution, elemental composition) • Odd electron ions, e.g. [M]+. ions, dissociate and form even electron fragment ions and neutral radicals (direct bond cleavages) or odd electron fragment ions and neutral molecules (rearrangement reactions). • Often not stable, not detectable M+• → CI, ESI • Requirements for an ion to be the molecular ion: •Highest mass in spectrum •Odd electron ion •High mass ions near M+• muss be explained by logical neutral losses mass losses of 4 –14 and 21-25 highly unlikely 57

Interpretation of EI spectra: The molecular ion, M+. • Application of the Nitrogen rule: – If a compound contains an even number of nitrogen atoms (0, 2, 4..) its molecular ion will have an even number. – If a compound contains an odd number of nitrogen atoms (1, 3, 5..) its molecular ion will have an odd number. • Relative abundance of molecular ion reflects structure and stability 58

Ion Stability 1,3-dimethyladamantane – C12H20 Dodecane – C12H26

M+.

M

+.

M+.

Perylene – C20H12

59

Guidelines for Understanding Ion Fragmentation • We specify the nature of the precursor ion as either .

– Odd electron ions (OE+ ) – Even electron ions (EE+)

• Favorability of ionization sites parallels bond stability (lowest ionization energy, highest proton affinity) – Sigma < pi, < nonbonding electrons (lone pairs)

• These ions can undergo the following processes, in various combinations – – – –

Radical-site initiation Charge-site initiation Rearrangements Charge retention/charge migration 60

Electron Ionization: basic mechanisms of ion fragmentation • Fragmentation in an EI source always unimolecular (low pressure, no collision) • Fragmentation stepwise (consecutive): [ABCD]+• → [ABC]+ + D•

[AB]+ + C• •

Fragmentation by direct bond cleavage (leads to even electron ions): [ABCD]+• → [AB]+ + CD• example: CH3COCH3+• → CH3CO+ + CH3•

•

or fragmentation by rearrangement (leads to odd electron ions): [ABCD]+• → [AD]+• + BC example: CH3COCH3+• → CH2=CO+. + CH4 61

Basic factors that influence the ion abundance •Stability of product ions •Stability of the neutral (neutral loss) Neutral can be a neutral molecule or a neutral radical, where the molecule is always more stable than the radical In general the heat of formation of the ion is much higher than that of the neutral (i.e. the ion is more important than the neutral) •Stevensons rule (1951): If their are two sets of ions and neutral radicals, such as [ABCD]+• → [ABC]+ + D• or [ABCD]+• → [ABC] • + D+ than the ion of lower ionization potential will be formed preferentially •Loss of the largest alkyl radical: If in an ion several different alkyl groups are bound to a carbon, the largest alkyl group is lost preferentially (stability of the neutral is of importance) 62

Basic Factors that Influence Ion Abundance: Loss of Largest Radical Example: 3-methyl-3-hexanol CH3 C2H5

C

+. C4H9

C2H5

OH

CH3

CH3

C4H9

C+ > C4H9

C+ > C2H5

C+

OH

OH

OH

m/z 73

m/z 87

m/z 101

73 43 87

101

no M+.

63

Radical site, charge site •

For the interpretation it is assumed in a simplistic approach that the ion has localized sites for the odd electron (radical site) and the charge (charge site - McLafferty). In reality the situation is more complex, where either the radical or the charge site may drive the reaction

Charge retention, charge +. migration O Assuming that upon ionization the C CH3 CH3 charge remains localized at a specific site, fragmentation may either lead to charge retention or charge migration

+

O CH3

C

retention CH3+

migration 64

Sigma (σ) bond cleavage • If upon ionization the electron is removed form a single (σ) bond, cleavage of this bond is favoured. This is e.g. a typical fragmentation with ionized alkanes. • Example – 2,2-dimethylpropane CH3-C(CH3)3 +e- → CH3•+C (CH3)3 → +C (CH3)3 + CH3• 57 H 3C

C(CH 3 )3 57

29

41 no M+.

65

Sigma (σ) bond cleavage n-decane – C10H22

fragments that can stabilize the charge better are preferred ie 30 > 20 > 10

3,3-dimethyloctane – C10H22 ** +

m/z113* +

*

m/z71** 66

Alpha (α) cleavage (radical site initiation) •

α- cleavage (radical site initiation) arises from the strong tendency for electron pairing: the odd electron is donated to form a new bond to the adjacent atom. This leads to the cleavage of the bond next to the α-atom CH3

.+ CH2 O

C2H5

. CH3 +

CH2

+ O

C2H5

•

The tendency to donate electrons to the adjacent bond increases in the following order: N > S, O, π, R• > Cl, Br > H.

•

α- cleavage is thus particularily pronounced with amines, but also observed with ethers, ketones, olefines, alkyl substituents, while is hardly observed with halides 67

General Procedure for the Interpretation of a Mass Spectrum 1.

Test for molecular ion identity: must be the highest peak in spectrum with even m/z value, if C, O, S, H, Cl, Br, and logical neutral losses

2.

Use isotopes (13C, 34S, 37Cl, 81Br) to help deduce the elemental composition

3.

If high resolution is available, deduce elemental composition

4.

Ring plus double bond

5.

Fragmentation pattern: important low mass series and primary neutral losses from M+.

6.

Library spectra (NIST, Wiley etc) 68

Q: Assuming these ions are molecular species and were acquired under accurate mass conditions, which is the correct molecular formula? C6F12O C22H46 309.1244 C15H20N3O2Cl C23H32N5O11S2 %

100

311.1244

310.1244

312.1244

0

315.9757

%

100

316.9757 0

310.3600

%

100

311.3600 0

310.0843

%

100

310.5843 0 308

309

310

311.0843

311

312

313

314

315

316

317

mass 318

69

?

70

Chemical Ionization (CI) • Positive Ion Chemical Ionization (Munson and Field, 1966) – Protonation by ion–molecule reaction or charge exchange (CE) – Reactant gas at ~ 0.1-1 Torr ionized by electron ionization → reactant ions formed by ion-molecule reactions → low energy electron transfer reactions (CE) – Analytes mixed in ion source with reactant gas Analyte : reactant gas = 1 : 102 – 104 – Ion source tight (higher pressure) to promote ion-molecule reactions 1

Chemical Ionization (CI) •

EI produces ions which are generally high in internal energy resulting in a large degree of fragmentation and in some cases prohibits the detection of the molecular ion.

•

If the pressure of a reagent gas (CH4 or NH3) in the ion source is sufficiently high (0.5 - 1 mbar) so that many collisions occur within the residence time of ions inside the source, then reactions may take place

•

This is called chemical ionization, a technique whereby ions may be produced with much less internal energy (sometimes with a known amount) and less fragmentation occurs.

•

The CI process most often yields even electron ions such as (M+H)+ or (M-H)- which fragment is different ways to odd electron ions. 2

The Source CI Source

EI Source Filament

S

Filament

N

Sample in

e-

Repeller

S N

Sample in

M+.

e-

Repeller

[M+H]+

Trap

Trap

S

S

N

N

Source pressure ~ 10-6 Torr

Source pressure ~ 0.1-1 Torr 3

Positive Ion Chemical Ionization Formation of reagent ions depends on pressure and residence time: Methane: CH4 + eCH4+. + CH4 CH3+ + CH4 CH5+ + M

Ammonia: NH3 + eNH3 + NH3+. NH4+ + M NH4+ + M

CH4+. + 2eCH5+ + CH3. C2H5+ + H2 MH+ + CH4

NH3+. + 2eNH4+ + NH2. MH+ + NH3 [M+NH4]+ 4

NH3CI – Formation of Reagent Ions NH3+. NH4+

NH3+.

Low pressure – EI conditions

mid pressure – ion/molecule reactions initiated

NH4+ high pressure – CI conditions The effect of ion source pressure is clearly seen! NH3NH4+ (NH3)2NH4+ 5

NH3CI – Formation of Reagent Ions NH4+

NH4+ Short residence time

Long residence time

NH3+. NH3NH4+

(NH3)2NH4+

•In these experiments, NH3 pressure is constant •Residence time is changed by increasing the voltage on the repeller 6

Reagent Ion Intensity vs Source Pressure +

CH

4

+ 5

CH5+

Intensity Ion Intensity

CH CH4+.

0.1

0.5 ln(Source Pressure)

Source pressure(torr) (torr)

1.0

7

Chemical Ionization • There are 4 general pathways to form ions from a neutral analyte M in CI: M + BH+ M+B

MH+ + B [M-H]- + BH+

proton transfer

M + X+

MX+

electrophilic addition

M + X+

[M-A]+ + AX

anion abstraction

M + X+. M+. + X charge exchange M-. + X M + X-. And others eg anion attachment (Cl-)

8

Thermochemical Considerations • The tendency of a molecule M to accept a proton is quantitatively described by it’s proton affinity (PA): B + H+ BH+ -ΔHr0 = PAB eg CH4, NH3 It is the negative of the enthalpy change in the gas-phase association reaction between a proton and the neutral

9

Positive Ion Chemical Ionization Reagent Gas

Reagent Ion

Hydrogen, H2

H3+

101

CH5+

128

C2H5+

161

Water, H2O

H3O+

170

Methanol, CH3OH

CH3OH2+

182

Acetonitrile, CH3CN

CH3CNH+

187

Isobutane, i-C4H10

C4H9+

194

Ammonia, NH3

NH4+

202

Methylamine, CH3NH2

CH3NH3+

211

Methane, CH4

PA (kcal/mol)

10

Thermochemical Considerations • In the subsequent CI experiment where: M + BH+

MH+ + B

protonation will occur as long as the process is exothermic ie PAB < PAM This exothermicity can result in fragmentation of the MH+ ion: Eint(M+H)+ = PAM - PAB 11

Thermochemical Considerations • Protonation under CI conditions AB + H+ → ABH+ ΔHRx = -PA(AB) = ΔHf(ABH+) – ΔHf(AB) – ΔHf(H+) Rearranging yields: ΔHf (ABH+) = ΔHf (AB) + ΔHf (H+) – PA (Proton affinity)

12

Thermochemical Considerations • In positive chemical ionization the reactant gas and the analyte molecule compete for the proton • Only if the proton affinity of the analyte molecule is higher than that of the reactant gas protonation can occur • In an ideal case the difference in proton affinities is transferred as internal energy to the analyte molecule during protonation • By proper choice of the reactant gas the ionization can be tailored in such a way that a minimum of energy is transferred to the analyte • → can yield soft ionization with little fragmentation 13

Thermochemical Considerations Example: Protonation of ethylamine (PA = 208kcal/mol) •

With methane (PA = 128kcal/mol) excess energy = 208 – 128 = 80kcal/mol → strong fragmentation

•

With methylamine (PA= 211) excess energy = 211 – 208 = 3kcal/mol → little fragmentation

•

If ammonia is used for CI of analytes their PA‘s determine whether a [M+H ]+ or [M+NH4 ]+ is formed:

•

PA (NH3) ~ PA (analyte) → [M+NH4 ]+ and/or [M+H ]+

•

PA (NH3) < PA (analyte) → [M+H ]+

•

PA (NH3) > PA (analyte) → [M+NH4 ]+ or analyte not observed

14

Comparison of EI, CH4CI and NH3CI for mw=340 species EI no M+.

CH4CI

NH3CI

[M+H]+

[M+NH4]+

15

Charge Exchange Chemical Ionization • The interaction of a positive ion with a neutral can lead to charge exchange: X+. + M

M+. + X

ΔH = IE(M) – RE(X+.) • This reaction will occur if the recombination energy (RE) of the reactant ion (X+.) is greater than the ionization energy of the neutral M ie if the reaction is exothermic. • Results in the formation of a radical cation (M+.) where the excess internal energy can be controlled by selection of X!

16

Charge Exchange Chemical Ionization • Also results in some degree of selectivity as if the IE of M is higher than the RE of X, then no electron transfer will occur and M will not be observed • Reactions of the type: A-. + B

B-. + A

ie formation of a negative ion can also occur provided the electron affinity of B is greater than the electron affinity of A. • Rarely used in practice (+ve or –ve CECI)

17

Charge Exchange Chemical Ionization Ion

Recombination Energy (eV)

Ne+.

21.6

Ar+.

15.8

N2+.

15.3

Kr+.

14

CO+.

14

CO2+.

13.8

Xe+.

12.1

CS2+.

9.5-10

C6H6+.

9.3

18

Charge Exchange Chemical Ionization For example: •if the IE of M is 11eV, CECI with Ne+. will yield excess internal energy of ~+10eV extensive fragmentation •if the IE of M is 11eV, CECI with Xe+. will yield excess internal energy of ~+1eV little or no fragmentation •if the IE of M is 11eV, CECI with C6H6+. will yield excess internal energy of ~-1.5eV M will not be ionized

19

Negative Ion Chemical Ionization (NCI) • Reactant Ion Negative Chemical Ionization – Mixture of N2O and methane forms [OH]– M + [OH]- → [M – H]• that is, proton abstraction – CH2Cl2 or CHCl3 forms Cl- by dissocaitive electron capture • Proton abstraction [M-H]- from very acidic compounds • Cl- attachment ion [M+Cl]- formed with acidic compounds 20

Negative Ion Chemical Ionization (NCI) • Electron Capture Negative Chemical Ionization – 3 mechanisms of ion formation: – M + e-

M-.

Resonance electron capture (0-2eV)

– M + e-

[M-A]- + A.

Dissociative electron capture (>2eV)

– M + e-

[M-B]- + B+ + e- Ion pair formation (>10eV)

– Generation of thermalized electrons (0 to 2eV kinetic energy) by bombardment of reagent gas (methane or ....) – Very soft ionization – Negative ion yield increases with increasing electron affinity – Introduction of groups with high electron affinities by derivatization such as pentafluorobenzyl derivatives 21

Summary • CI – Analtye ions must be volatile (same as EI) • naturally, by sublimation or by derivatization (non-ionic) – Volatility demand leads to small (<1kDa) molecules • heating biopolymers destroys them • extensive derivatization cumbersome • Desorption techniques minimize any thermal input to the analyte (DCI) sample

CI source

CI source

sample heat Direct insertion

Desorption

22

Field Ionization (FI) • Ionization mechanism – If atoms or molecules are exposed to very high electric fields (107 –108 V/cm) near a metal surface, a valence electron of the atom or molecule may tunnel into the anode (+vely charged) – this is termed Field Ionization, FI (Inghram and Gomer, 1955; Beckey, 1959) – Such high electric fields are produced if a voltage of 3 –10 kV is applied to a fine metal tip (emitter) of < 1 µm radius of curvature opposite to a counter electrode – During ionization hardly any excess energy is tranferrred to the ion “soft ionization“ that is, little or no fragmentation – Usually forms M+. but also [M+H]+ possible and M-. 1

Field Ionization (FI) • Ionization – Much higher ion yields can be generated if a large number of tips are generated on a thin metal wire (3 – 10 µm i.d.) by creation of whiskers (dendritic microneedles) by polymerization of a suitable organic compound (benzonitrile), a process termed activation C whiskers – As in electron impact (EI) and chemical ionization (CI) the sample is introduced to the emitter via the gas phase – GC, direct insertion probe, heated inlet – Very polar, non volatile compounds can not easily be introduced

– Poor sensitivity due to low probablity of desorbed neutral coming close enough to the emitter to be ionized 2

Field Ionization: Activated Emitter

3 courtesy of Springer 2004

Field Desorption (FD) • In field desorption mass spectrometry (FD-MS) the sample is dissolved in a suitable solvent and applied directly to the emitter. By applying high voltage to the emitter (ca. 104 V/cm) and simultaneously heating the emitter even compounds of low volatility, such as organic salts, are transferred into the gas phase • Yields both M+. and [M+H]+ ions depending on sample polarity • field desorption was the first mass spectrometric technique which was suited for the analysis of non-volatile compounds, such as oligopeptides, oligosaccharides and organic salts. • Much better sensitivity than FI courtesy of Springer 2004

4

Field Ionization and Field Desorption

5 courtesy of Springer 2004

Field Desorption – Trityl Chloride

6 courtesy of Springer 2004

Particle Bombardment Three main techniques: • Fast Atom Bombardment (FAB) employing 5-10keV Xe atoms • Liquid Secondary Ion Mass Spectrometry (LSIMS) using 2030keV Cs+ ions • 252Cf Plasma Desorption – MeV particles created from radioactive decay of 252Cf • allowed the MS analysis of high mass (>1000), polar, involatile and/or thermally labile species – no direct heating of sample • rely on momentum transfer from fast moving species to sample suspended in a matrix (FAB/LSIMS) or on a foil (252Cf PD) 1

Fast Atom Bombardment (FAB) •

Fast atoms are produced in a FAB gun.

8KeV Xe+. +

Xeo

8KeV Xeo +

Xe+.

That is, a charge exchange process

•

Non-volatile molecules are transferred to the gas-phase by bombardment 2 with a beam of fast atoms (4-10 keV). (Barber et al.,1981)

FAB: Mechanism of Ion Formation

courtesy of Springer 2004

3

FAB: Mechanism of Ion Formation

courtesy of Springer 2004

4

FAB/LSIMS of Inorganic Salts •

Clusters of Cs+ with I- forms series of ions: – +ve ion MnXn-1 eg Cs5I4+ – -ve ion MnXn+1 eg Cs4I5-

• • •

+ve and –ve ion FAB of CsI ions observed up to m/z >12,000 used for mass calibration in FAB and LSIMS

5 courtesy of Micromass

FAB/LSIMS: Role of the matrix The matrix: • Absorbs primary energy • Helps to overcome intermolecular forces between analyte molecules • Helps to maintain a long lasting sample supply ie replenishes the damaged surface by diffusion • Important for ion formation: proton donator or acceptor or electron donor/acceptor • Sample must be soluble in the matrix • Must be a viscous liquid that can survive the conditions in the high vacuum source to allow extended analysis 6

FAB and LSIMS: Matrices

courtesy of Springer 2004

7

FAB mass spectrum of glycerol matrix

8 courtesy of Springer 2004

1981 – the 1st analytical use of FAB Peptide – 11mer

Michael Barber et al., J.C.S. CHEM. COMM., 1981

9

Cs+ LSIMS

These spectra represent state-of-the-art protein analysis in 1987

B.N. Green et al 35th ASMS conference - 1987

Note: not m/z!

10

Surface Analysis with SIMS • SIMS analyzes the secondary ions emitted when a surface is irradiated with an energetic primary ion beam • Ar+, O2+, Cs+, O- are formed in a source and accelerated to very high kinetic energies (keV) and causes the emission of secondary particles (+ve, -ve or neutral) from the surface • This beam is rastered across the targets surface • The secondary ions are subsequently accelerated and mass analyzed • This technique is mostly used with solids and is especially useful to study conducting surfaces. 11

Surface Analysis with SIMS • High resolution chemical maps ie pictures representing the chemical distribution of the surface can be produced by rastering a tightly focused ionizing beam across the surface. • Used for trace elemental analysis especially in the semiconductor and thin coating science • Excellent technique for the elemental analysis of solid inorganics

12

Ion Imaging

197Au

and 34S signal from a pyrite (FeS2) grain

13

SIMS Advantages: • only surface species are ionized • depth profiling is possible by “milling” away at the sample with an ion beam • ions of all elements can be produced • can be used for small sample quantities

14

SIMS Disadvantages: • elemental sensitivity varies (~104) between 10-4 and 10-8 mol L-1 • non-conducting samples will charge up leading to unstable signals • isobaric interferences ie. 56Fe and 28Si2 (m/z 55.9349 and 55.9539) •this requires a resolution in excess of 5000 to distinguish by high resolution/accurate mass MS •a double focusing sector instrument can have resolutions in the 500 to 10000 range remember, better resolution is offset by poorer sensitivity 15

Laser Desorption Ionization (LDI) • laser pulses yielding 106 – 1010 Wcm-2 are focused on a solid sample surface of about 10-3 – 10-4 cm2. • The laser pulse ablates material from the surface, creating a microplasma of ions and neutral molecules. eg.

CsI(s)

hυ

Cs(CsI)n+

• LD is used to study surfaces since you can good spatial resolution with such a small beam of photons • high sensitivity • large variation in ionization probability ie. different energy to vaporize and ionize different samples 1

LDI • large spread in ion kinetic and internal energies, normally only fragments of the sample are seen in the mass spectrum due to dumping so much energy into the molecules, therefore structure is destroyed • useful for metals, inorganic salts and some polymers • signals are very short, therefore LD is best used with a mass analyzer such as TOF

2

Matrix Assisted Laser Desorption Ionization (MALDI)

• the compound to be analyzed is mixed in a solvent containing an organic compound (the matrix) which strongly absorbs at the laser wavelength. • the solvent is evaporated off leaving the analyte embedded in the matrix material. • an intense laser pulse deposits large amounts of energy into the sample in a short period of time. • this ejects or ablates bulk portions of the solid which contain matrix and analyte molecules from the surface 3

MALDI

• both UV and IR lasers can be used but most often a N2 (UV@337nm) laser is used • little internal energy is transferred to the analyte due to expansion and evaporation of the matrix material from the analyte so there is little fragmentation • ionization reactions can occur at any point in the process but the origin of ions produced in MALDI is not fully understood with numerous possibilities: •desorption of preformed (M+H)+ and (M+Na)+ ions •gas-phase protonation 4 •direct photoionization M+. and M-.

MALDI • among the chemical and physical ionization pathways suggested for MALDI are: gas-phase photoionization, excited-state proton transfer, ion/molecule reactions, or desorption of preformed ions. • the most widely accepted mechanism involves gas-phase proton transfer in the expanding plume with photoionized or photoexcited matrix molecules. • since most matrix materials contain aromatic rings, they can act as energy gathering chromophores.

M + hυ M* + A

M* AH+ + (M-H)-

M* + M MH* + A

MH+ + (M-H)AH+ + M

M = Matrix A = Analyte 5

Laser Desorption and Ionization: Mechanism

6 courtesy of Micromass (Waters)

Laser Ionization (without matrix): ion emission as a function of λ

Absorption spectrum of matrix

Absorption spectrum of sample

7 courtesy of Micromass (Waters)

Laser Ionization (with matrix – MALDI): ion emission as a function of λ

8 courtesy of Micromass (Waters)

MALDI: Matrices

courtesy of Springer 2004

9

MALDI Target - Batch Introduction Process

Bruker

Micromass

Can be fully automated! courtesy of Springer 2004

10

MALDI

MALDI spectrum of an N-linked glycan courtesy of Springer 2004

11

MALDI–Tof of a monoclonal antibody (m/z ~ 150,000)

12

Protein Identification using MALDI • Large proteins (>100,000 Da) may be multiply charged (doubly or triply charged): [M+H]+, [M+2H]2+, [M+3H]3+ but no more - very unlike ESI! • Smaller proteins and peptides are always singly charged – again, this is not the case for ESI • MALDI typically exhibits better sensitivity than ESI because it has a higher tolerance for other non-peptide sample constituents attamole • MS/MS of [M+H]+ ions provides fewer structurally significant fragment ions than does [M+2H]2+ ions commonly seen in ESI • not as useful for peptide sequencing or MS/MS ion database searching • Difficult to interface to chromatography • Also used for synthetic polymer characterization and imaging

13

2D Gel Electrophoresis

14

Protein Identification using MALDI •

Separate proteins on a 2D polyacrylamide gel (2D-PAGE)

•

Excise individual spots

•

These spots, which may contain 1 or more proteins, are degraded into small peptides (enzymatically) and measured by MALDI (or ESI)

•

The resulting MALDI mass spectrum (primarily [M+H]+ ions) is converted into a table of molecular weights of the individual peptides present to yield a Peptide Mass Fingerprint

•

Peptide Mass Fingerprint (PMF) or peptide mapping is an ideal method to identify peptides derived from a protein which are already known and in a database

•

The quasimolecular ions of this mixture of smaller peptides provide a map or fingerprint which can be searched against protein databases to provide a protein identification 15

Peptide Mass Fingerprint using MALDI a .i.

1 50 0 0

1 00 0 0

5 00 0

0 1 50 0

2 0 00

2500

30 0 0

3 50 0

m /z

16

MALDI-Tof of Polystyrene (A) mwt~330,000

(B) mwt~600,000

(C) mwt~900,000

Reproduced from Schriemer & Li, 1996

17

MALDI Imaging

Courtesy of S. Khatib-Shahidi, M. Andersson, J. L. Herman, T. A. Gillespie, and R. M. Caprioli. Anal. Chem. 2006, 78, 18 6448-6456.

Inductively Coupled Plasma (ICP) MS •

Inductively coupled plasma used originally and still used today in emission spectroscopy

•

Plasma temperature may exceed 8000oC

•

Elements with IP < 10 eV fully ionized → mass spectrometry

•

Inorganic samples first digested (e.g with nitric acid), pneumatically nebulized and introduced as finely dispersed mist into an argon ICP at +1200 Watt

•

Coupled to quadrupole MS and to magnetic sector double focussing instruments (the latter have much better mass resolution)

•

Very sensitive, very specific

•

Can suffer from isobaric interferences

1

ICP-MS

2

ICP-MS • The Ar plasma is generated and maintained at the end of the glass torch located inside the loops of a water cooled copper load coil. • A radio frequency (RF) potential applied to the coil produces an electromagnetic field in the part of the torch located within its loops. • A short electric discharge from a wire inside the torch provides the electrons to ignite the plasma. • In the electromagnetic field of the load coil these electrons are accelerated and collide with Ar flowing through the torch producing Ar+. ions and free electrons.

3

ICP-MS • Further collisions cause an increasing number of Ar atoms to be ionized and result in the formation of plasma. • The plasma-forming process rapidly becomes self-sustaining and may be maintained as long as Ar gas continues to flow through the torch • These colliding species cause heating of the plasma to ~10,000 K. The high temperatures rapidly desolvate, vaporize, atomize and ionize the sample. Therefore the sample is turned into atomic ions which are then mass analyzed

4

ICP-MS • Each element shows up at its own m/z value, including isotopes • Intensities are directly proportional to the amount of element introduced to the torch • No structural information since complete atomization occurs • Different ionization efficiencies result in different sensitivity • Isobaric interferences: • ArO+ m/z 55.9573 / Fe+ m/z 55.9349 • Ar2O+ m/z 95.9197 / Mo+ m/z 95.9068 • Ar2O++ m/z 47.9599 / Ti+ m/z 47.9479

5

ICP-GCMS – Organotin standards 100.0

80.0

Sn isotopic distribution

60.0

40.0

20.0

0.0 110.00

120Sn+

115.00

120.00

monitored

12,000 ICP-MS Intensity

125.00

Detection Limit: 1. 2. 3. 4. 5. 6. 7. 8.

10,000 8,000 6,000

inorganic Sn – not reported MBT 4.4fg TPrT 5.3fg DBT 9.4fg MPhT 4.4fg TBT 9.9fg DPhT 10fg TPhT 11fg

4,000 2,000

0

5

10

1μL injection of 5ppb standard

15

Retention time (min)

6

Atmospheric Pressure Ionization (API) •

conventional ionization methods employ sources that are at high vacuum (EI, CI, FI/FD, FAB/LSIMS, MALDI) and/or temperature (EI, CI, FI/FD)

•

the introduction of API sources employing a number of different types of ionization has allowed very robust instruments to be developed for LC/MS

•

These “new” ionization techniques have greatly extended the range of analytes that can be studied by MS to compounds that are high molecular weight, thermally labile and polar.

•

While the sources are designed to operate at atmospheric pressure we must still maintain a high vacuum in the rest of the instrument if we want to perform mass spectrometry!! 1

API Source reduced pressure High vacuum

}

HPLC inlet

}

Atmospheric pressure Skimmers Vacuum Wall

Nebulizer gas inlet

Lenses

Octopole

Capillary

Nebulizer

+ +

+ + + + + +

+ +

+ +

+

+

+

+ +

+

Mass analyzer

heated N2

Spray is at right angles to entrance to MS - orthogonal Waste

Vacuum Pumps courtesy of Agilent

2

API Source •

High vacuum must be maintained in the mass analyzer and detector region even though the source is at atmospheric pressure

•

The region after the source is heavily pumped with rotary vacuum and turbomolecular pumps (usually)

•

Also, a series of skimmers and flow restrictors are placed between the source and the mass analyzer region

•

These skimmers allow ions to be efficiently transmitted to the high vacuum region while at the same time allow air, solvent vapours and other neutral volatile species to be pumped away

•

The exact design will depend on the specific instrument type and manufacturer 3

API Sources • Electrospray (ESI) • high flow rate (100μL/min – 1mL/min) • capillary flow rate (2μL/min - 100μL/min)

}

pneumatically assisted ESI

• low flow rate (<2μL/min) – nanospray (200-500nL/min) – ESI is most sensitive at these low flow rates

• Atmospheric Pressure Chemical Ionization (APCI) • Atmospheric Pressure PhotoIonization (APPI) • Atmospheric Pressure MALDI

4

Relative Applicability of API Techniques 100,000

Molecular Weight

ESI & APMALDI

10,000

1000

APCI & APPI

nonpolar

Analyte Polarity

ESI: Electrospray Ionization & APMALDI APCI: Atmospheric Pressure Chemical Ionization APPI: Atmospheric Pressure Photo Ionization EI, CI, GC-MS

very polar

5

Electrospray (ESI) • Based upon the electrostatic spraying of liquids where a solution is passed through a needle held at high voltage (kV) relative to a counter electrode (the entrance to the MS) • When the solution contains an electrolyte and the needle forms part of the API source then the fine mist of droplets that emerge from the needle tip possesses a net +ve or –ve charge determined by the polarity of the needle and the solution chemistry of the bulk liquid • These preformed and then sprayed ions, which are characteristic of the dissolved analytes, are attracted to the entrance of the MS by applying appropriate voltages 6

Electrospray (ESI) •

The formation of the spray must be aided by nebulization (pneumatically assisted) at liquid flow rates higher than a few μL/min

•

ions exist in solution, if not, electrospray doesn’t work, it is not an ion formation technique rather than a technique for extracting ions from the solution-phase into the gas-phase free of solvent for mass spectral analysis

•

The analyte must be an ion in solution either as a preformed ion such as or through modifying the solution chemistry to induce a charge N

•

This can be accomplished by changing solution pH or adding cations eg Li+, NH4+ etc or anions to form adducts eg Cl-, OAc- etc 7

Electrospray Ionization Charged Droplets containing ions in solution

Analyte Ions in the gas phase - both +ve and -ve

+ + + + + - + -

Nebulizer assisted >1μL/min - capillary 2-100μL/min - normal 0.1-1mL/min

+

+

+ +

“Classical” - nanospray < 1μL/min

Evaporation

+

+ + -- + + +

+

Solvent Ion Cluster

+

Analyte Ion (proton transfer and 8 adduct ions)

+

+ + + + +

+

+ + +

Rayleigh Limit Reached

++ + --- + - + +

Coulombic Explosions

The “Source” High voltage Power supply

electrons

-

+

-

+

-

+

+ + + +

+ +

+

+

+

+

++

+

+

+ + + + ++ + + + + + + + + + + + + + + + + + + + + +

-

-

+

+

-

to MS

+

cathode - reduction

Anode -oxidation

+

+

+

+

Taylor cone +

+

+

+

+

+

+

+

+

+

+ ++ + + + + + + + ++

+ +

+ + +

+ + + +

+ ++

9

Proposed Mechanisms: 1.

Charge Residue Model: where the droplet is completely evaporated leaving “bare’ analyte ions

2.

Ion Evaporation Model: field assisted ion desorption •

Requires ~ 107Vcm-1 and a final droplet diameter of 10nm

•

Fits well with the observed data

•

In either case it is required that the analyte be an ion in solution (+ve or –ve) or made to be charged by modifying the solution to cause the analyte to be ionized

•

This can be accomplished by changing pH, adding modifiers (Na+, Li+) 10

Electrospray Solution Chemistry • Mobile phase pH has a major effect for analytes that are ions in solution: – Basic pH for negative ions – Acidic pH for positive ions • Changing pH can enhance performance for analytes that are not normally ionized in solution Positive Ion Mode

R1 | :N - R2 + HA | R3

R1 | +HN - R 2 + A | R3

Base

Analyte Ion

Acid

O || R-C-OH + :B Acid

Base

Positive ion mode, [M+H]+

O || R-C-O- + H:B+ Negative ion mode, [M-H]Analyte Ion

11

Electrospray Solution Chemistry •

In the case of acid/base chemistry, ideally we want to be 2 pH units either side of pK in order to cause complete protonation (+ESI) or deprotonation (-ESI) to give maximum sensitivity

•

In the case of batch introduction (infusion) of sample this is easily accomplished however in the case when LC is employed it is the nature of the mobile phase that determines the ions we will observe and the sensitivity

•

For example, in a reversed phase (C18) separation of analytes, in order to achieve a good separation it is necessary for the analytes to be neutral in solution so that they may interact with the stationary phase and achieve a good separation. These neutral species will not yield the best sensitivity when ESI is used. 12

Electrospray Solution Chemistry •

Don’t forget, the ESI process is a competition for charge!

•

A neutral in solution will pick up charge in a variety of ways and while we can influence which process is favoured we can not eliminate all competing ion formation mechanisms

•

Not only do proton transfer reactions occur but adduct ion formation is commonly observed

•

Species such as [M+NH4]+, [M+Na]+ and [M+K]+ in positive ion and [M+OAc]- and [M+Cl]- in negative ion are often observed even though these modifiers may not have been deliberately added to the solution containing the analyte 13

+ESI of Nucleotide Homologue (mw=890) Sample in 1:1 CH3CN/H2O+0.2% formic acid [M+H]+ [M+Na]+

[M+K]+

[M+NH4]+

14

Electrospray Considerations Samples: • Ions in solution: catecholamines, sulfate conjugates, quaternary amines, carboxylates, phosphorylated compounds • Compounds that can have a charge induced: carbohydrates • Compounds containing heteroatoms: carbamates, benzodiazepines • Multiply charged in solution: proteins, peptides, oligonucleotides • A curious feature of ESI is the formation of multiply charged ions ie where z>>1 and sometimes as high as 100

15

Electrospray Considerations Solution Chemistry Parameters: • flow rate • sample pK, solution pH • solution conductivity Samples to Avoid: • extremely non-polar samples: PAHs, PCBs • Samples containing high levels of buffers/electrolytes as this will cause ion suppression Ion Suppression: • Competition and interference with analyte ionization by other endogenous matrix species resulting in decreased number of ions characteristic of the analyte(s)

16

Protein ESI-MS

• In this mass spectrum, each peak represents the quasi molecular ion of the protein with one more charge attached, usually, but not always, a proton (H+) eg m/z 942.6 is the [M+18H]18+ • Consequently, each peak can be used to calculate the mwt of the protein and the resulting values averaged across all charge states. • This results in mass accuracies for protein mwt determination of + 0.01% 17 or better depending on the type of mass spectrometer employed.

Protein ESI-MS • •

Let the unknown mass of the protein be M and the # on charges be n corresponding to the addition of (M+nH)+ For 2 adjacent measured masses m1 (high mass) and m2 (low mass) we can write 2 equations:

m1 = (M+n) (i) and m2 = (M+n+1) (ii) n (n+1) Solving for n: for the ion at m/z 998.0 (m1) = (M+n) 998n = M+n n for the ion at m/z 942.6 (m2) = (M+n+1) 942.6n+941.6 = M+n (n+1) Consequently:

998n = 942.6n+941.6

n =17 for m1 (m/z 998)

Substituting n=17 in (i) gives M = (m1n)-n = (998x17)-17 = 16,949 •

These laborious calculations can be performed for all ion in the distribution or a software deconvolution can be performed 18

+ESI of a ~39kDa Protein - Infusion@1μL/min [M+33H]33+

[M+32H]32+

100

%

100

%

0 1150

m/z 1160

1170

1180

1190

1200

1210

1220

1230

1240

1250

[M+50H]50+ [M+22H]22+ [M+18H]18+

0

m/z 200

400

600

800

1000 1200 1400 1600 1800 2000 2200 2400 2600 2800

19

Software Deconvolution Software manipulation of the full scan +ESI data to show protein mwt 39,643+1.3

%

100

0

mass 39300

39400

39500

39600

39700

39800

39900

40000

40100

20

pH=2.6

pH=3.0

pH=5.2

• the charge states of the gaseous ions generally represent the charge states in the condensed phase. These are sometimes modified by ion/molecule collisions. Ions such as large biomolecules are highly charged. • the transfer of ions to the gas phase is not an energetic process. Ions are cold, in fact the desolvation process further cools ions. • non-covalent interactions can be preserved when the species enters the gas phase. This is significant for the application of ESI to the study of biological molecules such as proteins. ESI mass spectra of bovine cytochrome c

21

Raffinose – trisaccharide, mwt=504 +ESI m/z 522 (M+NH4)+

in 1:1 MeCN/H2O+5mMNH4OAc

%

100

0 100

125

150

175

200

225

275

300

325

350

375

400

425

450

475

500

525

550

575

m/z 600

m/z 505 (M+H)+ m/z 522 (M+NH4)+

In 1:1 MeCN/H2O+0.2%FA

%

100

250

125

150

175

200

225

250

275

300

325

350

375

400

425

450

475

500

525

550

575

m/z 600

m/z 511 (M+Li)+ 100

+LiOAc %

0 100

22 0 100

125

150

175

200

225

250

275

300

325

350

375

400

425

450

475

500

525

550

575

m/z 600

Raffinose – mwt=504 +ESI vs -ESI In 1:1 MeCN/H2O+0.2%FA

m/z 505 (M+H)+

100

%

m/z 522 (M+NH4)+

0 100

125

150

175

200

225

250

275

300

325

350

375

400

425

450

475

500

525

550

575

m/z 600

m/z 503 (M-H)-

100

%

m/z 549 (M+HCOO)-

0 100

125

150

175

200

225

250

275

300

325

350

375

400

425

450

475

500

525

550

575

m/z 600

23

Not Always Protonated! decamethylferrocene

M+.

EI

Fe

100

M+.

%

+ESI in MeCN

0 100

+ESI in 1:1 MeCN/H2O+0.2%FA

m/z 120

140

180

200

220

240

260

280

300

320

340

360

380

400

420

M+.

%

100

0 80

160

m/z 100

120

140

160

180

200

220

240

260

280

300

320

340

360

380

400

420

440

ie no (M+H)+ observed! Electron transfer dominates Oxidation 24

440

ESI – a MS Revolution • Electrospray ionization (ESI) has allowed mass spectrometry to investigate a huge diversity of molecules that were very difficult or impossible to study by MS previously •proteins, DNA, RNA, oligonucleotides •polymers, non-volatile inorganic and organometallic molecules and salts • As a result it has completely revolutionized mass spectrometry. • It has also revolutionized the sales of mass spectrometers as the can be considered to be an analytical technique for biochemistry (big $$). • Also, it has spurred the growth of more sensitive and exotic types of MS and combinations of MS analyzers. 25

Atmospheric Pressure Chemical Ionization (APCI) •

gas phase chemical ionization (CI) process where the vapourized LC mobile phase acts as the CI reagent gas to ionize the sample

•

Mobile phase and analyte are first nebulized (N2) and vapourised by heating to 350-550oC

•

The resulting vapour is ionized using a corona discharge (source of electrons)

•

Subsequent ion/molecule reactions (CI) then cause ionization of the analyte

•

Unlike ESI, analyte ions do not need to exist in solution

•

Unlike ESI, best sensitivity is achieved at high liquid flow rates ie 200μL – 1mL/min therefore easily interfaced to conventional HPLC 26

•

Analytes must be thermally stable and “volatile”

APCI

27

APCI Process Analyte containing aerosol

Heat and N2 to aid volatalization Vapour

needle

+ ++ + + + ++ + + + + + + + + ++ + ++ ++

Charge transfer to analyte eg H+ transfer, charge exchange etc

Charged reagent gas formed

+ +

+ +

+

Analyte ions kV corona discharge - a robust source of e28

APCI Considerations Samples: • Compounds of intermediate mwt and polarity: PAHs, PCBs, fatty acids, steroids, phthalates. • Compounds that don’t contain acidic or basic sites (e.g. hydrocarbons, steroids, alcohols, aldehydes, ketones, and esters) • samples containing heteroatoms: ureas, benzodiazepines, carbamates • samples that exhibit a poor electrospray response, that is, APCI can be considered to be complimentary to ESI

29

APCI Considerations Solution Chemistry Parameters: • less sensitive to solution chemistry effects than ESI – ion suppression not so important • Best sensitivity at higher flow rates than ESI • accommodates some non-polar solvents not compatible with ESI (hexane, CH2Cl2 etc) Samples to Avoid: • thermally labile, polar and high mwt compounds due to the vaporization process

30

APCI Mechanism S + e- → S+. + 2e• Solvent molecules are ionized (S+.) • the solvent is usually a complex mixture of H2O, CH3CN/CH3OH and mobile phase modifiers

S+. + S → [S+H]+ + S[-H] • S+. abstracts a hydrogen atom ie a CI process

[S+H]+ + M → [M+H]+ + S • [S+H]+ ionizes analyte M by proton transfer or proton abstraction

S+. + M → M+. + S • charge transfer can also occur with solvents like CH2Cl2

31

Atmospheric Pressure Photo-Ionization (APPI) • Experimentally, you can view APPI as an APCI source where the corona discharge has been replaced with a Kr lamp • The 1st step is complete vapourization of the mobile phase used in the LC separation employing nebulization (N2) and heating to 350-550oC • gas phase photoionization process • where the vapourized mobile phase may be photoionized to form a CI plasma • or a modifier (dopant) is added to aid the photoionization process and formation of the CI plasma • or the analyte can be directly photoionized by photons from 32 the Kr lamp

Atmospheric Pressure Photo-Ionization (APPI) • It is ionized by high energy photons from a Kr lamp (usually) causing either direct or indirect (dopant) photoionization • Very useful for non-polar analytes that are difficult to ionize with ESI or APCI such as PAH’s • Unlike ESI, best sensitivity is achieved at liquid flow rates around 200mL/min therefore easily interfaced to conventional HPLC

33

APPI Process +

Analyte containing aerosol

+ +

+

+

Evaporation

Photon ionizes analyte - Direct

hυ

+ Vapour

hυ

+

+

+ + + + Dopant added

+ + + + + + + + + + + + + + + +

+ +

+ +

+

Analyte ions

Dopant is photoionized and acts as reagent gas – Indirect 34

APPI Mechanisms Direct APPI: M + hν → M+. + eAnalyte molecule M is ionized to molecular ion M+. – If analyte ionization potential is below Kr lamp photon energy

Subsequently: M+. + SH → [M+H]+ + S• Molecular ion M+. may abstract a hydrogen to form [M+H]+ ie a CI process

35

APPI Mechanisms Dopant APPI: D + hν → D+. + e• Photoionizable dopant D is in excess & yields many D+. ions D+. + M → → [M+H]+ + D • Analyte M ionized by proton transfer from dopant or solvent D+. + M → M+. + D • D+. ionizes analyte M by electron transfer ie charge transfer

36

Energetics for Photoionization PhotoMate™ lamp Krypton 10.0 eV, 10.6 eV Ionization Potentials (IP) Anthracene 7.4 eV Fluoranthene 7.8 eV Caffeine 8.0 eV 4-Nitrotoluene 9.5 eV 2,4,6-Trinitrotoluene 10.59 eV

Dopant Ionization Potentials Toluene

8.82 eV

Acetone

9.70 eV

Solvent Ionization Potentials Methanol

10.85 eV

Acetonitrile

12.19 eV

Water

12.61 eV

•

The photons from the Kr lamp can only photoionize compounds of lower IP

•

Common HPLC solvents like H2O, CH3OH and CH3CN are NOT ionized and therefore cannot aid ion formation

•

In this circumstance, only direct photoionization of the analyte can yield characteristic ions such as M+. (not very efficient) – Subsequent ion/molecule reactions can form [M+H]+

•

Dopants are used that will be ionized by the Kr lamp

37

Atmospheric Pressure Ionization Techniques Electrospray (ESI) • Volatility not required • Preferred technique for polar, high mwt, thermally labile analytes • Ions formed in solution • Can form multiply charged ions APCI/APPI • Some volatility required • Analyte must be thermally stable • Ions formed in gas phase • Forms singly charged ions only

38

Ionization of Analytes How do we choose which technique to use? – is the analyte volatile? – is the analyte thermally labile? – Does the analyte have heteroatoms that can accept (N > O) or lose (O >> N) a proton? – accepts a proton - use positive ion mode – loses a proton - use negative ion mode

Ion Suppression? – Dirty matrix would favour the use of APCI/APPI rather than ESI because they are more tolerant to matrix effects than ESI 39

Chromatographic Considerations ESI: • Concentration dependant – smaller i.d. column gives better sensitivity - nanospray at 200500nL/min • However also works well from 1µl/min to 1 ml/min • Post-column addition can be used to adjust ionization chemistry

APCI/APPI: • Mass flow dependant – column i.d. has little effect on sensitivity • Works well from 100 µl/min to 1.5 ml/min • Can be used with normal phase chromatography 40

General Mobile Phase Considerations • Metal ion buffers interfere with ionization • Surfactants/detergents interfere with evaporation • Ion pairing reagents can ionize and create a high background • Strong ion pairing with an analyte can prevent the analyte from ionizing • Some mobile phase additives will cause persistent background problems – TEA interferes in positive ion mode (m/z 102) – TFA interferes in negative ion mode (m/z 113)

41

Mobile Phase Considerations ESI: • Solution pH must be adjusted to create analyte ions – pH 2 units away from pK of analyte • Organic modifier (CH3OH/CH3CN) has little effect on ionization • Volatile buffer concentration should be <25mM • Non-volatile buffers should be avoided or their concentration should be very low <<5mM • Na+ and K+ adducts commonly occur

42

Mobile Phase Considerations APCI/APPI: • Organic solvent should be a good charge transfer reagent – use methanol instead of acetonitrile – proton affinity of CH3OH (182kcal/mol) vs CH3CN (187kcal/mol) • Chlorinated solvents can aid ionization in negative mode • Volatile buffer concentration should be <100 mM • Non-volatile buffer concentration should avoided or be very low <<5mM

• Ammonium adducts may occur with ammonium salt buffers • APPI may require a dopant (eg acetone) 43

Abundance

395.3

Mass Spectra of Prednisolone in Negative Mode APCI with CH2Cl2

Abundance

[M+Cl]-

no CH2Cl2

600000

600000

OH

500000

500000

HO

400000

400000

300000

300000

O OH

O

200000 421.3

100000

365.3 377.3

335.3

200000

100000

0

0 150

200

250

300

350

400

m/z

150

200

250

300

350

400

m/z

Prednisolone does not normally ionize in negative mode APCI. In the presence of CH2Cl2, a very intense [M+Cl]- ion is formed. 44

Mass Spectra of Curcumin in Negative Mode APCI with CHCl3

no CHCl3 [M-H]-

90000

367.0 90000

80000

80000

70000

70000

60000

60000

50000

50000

40000

40000

30000

30000

337.0

20000 10000 0 100

160.9

217.1 200

[M-H]-

10000 m/z

0 100

367.1

O O OH O HO

337.1

20000

307.1 300

O

191.1 200

307.1 300

m/z

Curcumin is an example of a phenolic compound that ionizes equally well in the presence of oxygen or CHCl3. 45

Caffeine

140000

Max: 13143

APPI 140000

Max: 71549

120000

120000

120000

100000

100000

100000 195.1

[M+H]+= 195

[M+H]+= 195

60000

60000

40000

40000

40000

0 200

400

600

800

m/z

80000

196.1

103.2 121.2

60000

195.1 217.1

80000

20000

20000

0 200

Max: 148840

[M+H]+= 195

80000

20000

195.1

140000

APCI

400

600

m/z

196.1

ESI

0 200

courtesy of Agilent

300

400

500

600 m/z

46

Methomyl

80000 70000

[M+H]+= 163

10000

348.1

163.1 186.1

20000

165.1

40000

70000

70000

400

600

800

[M+H]+= 163

m/z

Max: 95891

[M+H]+= 163

60000

50000

50000

40000

40000

30000

30000

20000

20000

10000

0 200

80000

60000

50000

90000

80000

163.1

60000

30000

Max: 3663

90000

10000

0

106.1 164.1

Max: 206617

APPI 163.1

APCI

347.1

90000

185.0

ESI

0 200

400

600

800

m/z

200

400

courtesy of Agilent

600

800

47

m/z

Budesonide

Max: 161681

120000

140000

Max: 78432

120000

60000

20000

60000

40000

20000

0

40000

323.2 341.2 395.2

40000

20000

0 200

400

600

800

m/z

[M+H]+= 431

80000

431.3

60000

100000

[M+H]+= 431

121.2

80000

431.3 413.2

80000

413.2

100000

[M+H]+= 431

Max: 140093

120000

103.2

100000

140000

413.2

140000

APPI 431.2

APCI

453.2

ESI

0 200

400

600

800

m/z

200

courtesy of Agilent

400

600

800

48

m/z

Sample Matrix Effects The MS hardware is robust and tolerates non-volatile components however… The ionization process is effected by the concentration and type of salt/buffer and results in “Ion Suppression” and is much more prevalent in ESI “Competition and interference with analyte ionization by other endogenous matrix species resulting in decreased number of ions characteristic of the analyte(s)” 49

Sample Matrix Effect in ESI Composition of HBSS: Component Sodium chloride Calcium chloride Potassium chloride Potassium phosphate monobasic Magnesium sulfate Sodium bicarbonate Sodium phosphate dibasic Glucose Phenol red

Sulfachloropyridazine (mwt=284) dissolved in water vs. Hanks Balanced Salt Solution (HBSS)

UV

r ate w in lfa u s

mAU 20 10

150000 100000 50000

4000

r ate w in lfa u s

r ate w in lfa u s

g/L

S HB n i lfa su

S

S HB n i lfa su

S

S HB n i lfa su

8 0.1 0.4 0.06 0.1 0.35 0.048 1 0.011

S

TIC Scan mode EIC, m/z 285

Signal suppression!

2000

0

1

2

3

4

courtesy of Agilent

min

50

Adapting Existing LC Methods to LC/API-MS • Replace non-volatile buffers with volatile buffers at a concentration of <10 mM for ES or <100 mM for APCI. • Substitute phosphates, sulfates, and borates with ammonium acetate or formate, trifluoroacetic acid (TFA), heptafluorobutyric acid (HFBA), tetrabutylammonium hydroxide (TBAH) • If a non-volatile buffer must be used, use a buffer where only the anionic or cationic part is non-volatile, i.e. ammonium phosphate, not sodium phosphate. • Keep the pH the same using volatile additives: Formic acid, acetic acid, TFA, ammonium hydroxide • Volatile ion pair reagents should be employed such as HFBA

51

Summary of Ionization Methods Compound volatile or semivolatile: • Electron impact (EI): • M+• and perhaps substantial fragmentation

• Chemical ionization (CI): • Positive chemical ionization, [M+H]+ (soft ionization - little fragmentation) • Negative chemical ionization (electron capture), [M]-. (soft ionization - little fragmentation, can be very sensitive)

• Field Ionization (FI): • M+•, (soft ionization - little fragmentation)

Compounds non-volatile, methods difficult to couple to HPLC: • Field Desorption (FD): • [M+H]+, [M+Na]+ (soft ionization - little fragmentation)

• Fast Atom Bombardment (FAB) and Liquid Secondary Ion Mass Spectrometry (LSIMS): • [M+H]+ , [M+Na]+, [M-H]- (soft ionization - quasimolecular ion and fragment ions) 52

Summary of Ionization Methods Compounds non-volatile, methods difficult to couple to HPLC: • MALDI: [M+H]+, [M+Na]+, [M-H]- some multiple charging observed (both soft and hard ionization, quasi molecular ion and fragment ions, biopolymer analysis)

Compounds non-volatile, methods can readily be coupled to HPLC • APCI: [M+H]+, [M+Na]+, [M+NH4]+, [M-H]- (soft ionization, low to medium molecular weight, medium to high polarity) • APPI: M+•, [M+H]+, [M-H]- (soft ionization, low to medium molecular weight, medium to high polarity) • ESI: [M+H]+, [M+nH]n+, [M+Na]+, [M+NH4]+, [M-H]-, [M-nH]n- (soft ionization, low to high molecular weight, medium to high polarity, biopolymers and organic salts) 53

Mass Separation • Magnetic sector instruments – Single focussing with magnetic sector (B) – Double focussing with a combination of magnetic and electric sectors (EB or BE)

• Linear quadrupoles (Q - mass filters) • Three dimensional quadrupoles (ion traps - IT) • Linear ion traps (2D) • Time of flight mass spectrometers (Tof) • Fourier transform ion cyclotron resonance (FTICR or FTMS) 1

Mass Separation and the Lorentz Force NOTE: •

All mass analyzers function on the basis of the Lorentz Force equation which describes the force exerted on a charged particle in an electromagnetic field. The particle will experience a force due to the electric field (qE), and due to the magnetic field (qvB). Combined they give the Lorentz force equation:

F = qE+qvB – – – –

F is the force (in newtons) q is the electric charge of the particle (in coulombs) = ze E is the electric field (in volts per meter) B is the magnetic field (in webers per square meter, or equivalently, teslas) – v is the instantaneous velocity of the particle (in m/s) q = ze therefore, F

= zeE+zevB

2

Mass Separation: Magnetic Fields (B) Deflection of ions in magnetic fields: an ion of mass m and charge z moving with velocity, v, that traverses a magnetic B at right angles to the direction of the field will follow a circular path of radius r that fulfills the condition of equilibrium of FL (Lorentz Force) and centripetal force FC (from the source accelerating voltage)

FL = qvB = mv2/r = FC

r = mv/qB

Right hand rule for a +ve charged particle moving through a magnetic field:

Fingers in the direction of B Thumb in the direction of v Palm in the direction of the experienced force 3

Mass Separation: Magnetic Fields This shows the working principle of a magnetic sector - the radius, r through which the ion will be deflected depends on the momentum (mv) of the ion ie the magnetic sector is a momentum analyzer not a direct mass analyzer! Since the initial kinetic energy of the ions is given by: zeV = mv2/2 where V is the accelerating potential And rearranging for v2: From:

v2 = (2zeV)/m (i)

zevB = mv2/r we can derive v2 = (zeBr)2/m2 (ii)

Substitute v2 from (i) in (ii) and rearrange:

m/z = eB2r2/2V 4

Mass Separation: Magnetic Fields m/z = eB2r2/2V • Therefore specific values of V and B allow ions unique in m/z to pass to the detector. Variations in V or B will cause ions to collide with the walls of the flight tube therefore at any unique value of V or B only one specific ion will be passed to the detector. In practice only B scans are preferrred when generating full scan data over a large (>50Da) mass range • One exception to this is when high resolution, accurate mass measurements are made where Vacc scanning is preferred as voltages can be controlled and measured much more accuartely than can B 5

Accurate Mass Determinations LR EI of mw=308 (Resn~950)

PFK

PFK

Resn~9200

308.1085 C16H20O4S1 1ppm error or 0.3mmu

PFK

6

Deflection of ions of different masses in a constant magnetic field

•This is how Aston’s original mass spectrograph operated! • In modern instruments, the magnetic field is scanned to bring ions of different m/z ratios successively to the detector 7

Directional (angular) focusing of a magnetic field

Divergent ions of the same m/z will be brought into focus by a magnetic field 8

Mass Separation: Magnetic Fields • One significant drawback with employing B scans is that the initially accelerated ions have a kinetic energy spread which exhibits itself as increased peak width ie low resolution. • To overcome this problem an electric sector (ESA) is combined with the magnetic sector to produce what is called a double focusing instrument.

9

Principle of the Electrostatic Sector (ESA) • Remember the Lorentz Force equation which describes the force exerted on a charged particle in an electric field F = qE • If a radial electrostatic field E is created between 2 curved plates held at oppositely charged potentials of +E and –E, an ion of charge z moving with velocity v will traverse this field when its electrostatic force equals the centripetal force: zeE = (mv2)/r And since the kinetic energy of an ion 1/2mv2 = zeV, then r = 2V/E Note: the trajectory is independent of m and z and so the ESA is not a mass analyzer but reduces kinetic energy dispersion which results in narrower peaks and increased mass 10 resolution

Double Focusing Mass Spectrometers • Many geometries have been tried however in general they can be categorized as: – Normal or Forward geometry where:

Source

ESA

Magnet

Detector

• and: – Reversed geometry where: Source

Magnet

ESA

Detector

11

Double Focusing (Nier Johnson): Reverse Geometry

12

Double Focusing (Mattauch-Herzog geometry) Double focusing in a plane → photo plate

13

Mass Resolution: Definition δm10%

δmFWHM

10% Valley

m

•

The 10% valley definition: δm = the mass difference between two peaks which are separated by a valley equal in height to 10% of the height of the smallest peak

•

The full width at half maximum (FWHM) definition: δm = the width of a peak at half-height 14

Mass Resolution • The FWHM definition is easier to apply (only need one peak), but gives a resolution about twice that of the 10% valley definition • Resolution for sector instruments is usually given as the 10% valley figure. • High resolution has some obvious advantages: -It allows one to resolve ions that are isobaric -The narrower a peak, the easier it is to measure its position accurately

15

Mass Resolution • Low resolution: <2,000. Suitable only for nominal mass measurement. • Medium resolution: 2,000-20,000. Suitable for accurate mass measurement. Resolve isotope clusters of high charge states. • High resolution: >20,000. Better than medium resolution. You can never have too much resolution! • In practice, there is a trade-off between resolution and sensitivity. The ions are not coming from a point source: they exit the source through a slit of finite dimensions, and cannot be perfectly focussed. Slits and lens help to compensate for this by cutting out ions from the centre of the beam and focussing. To get very high resolution, the slits have to be narrowed, which means that a lot of ions are lost. 16

How Many Possible Elemental Compositions? without isotope abundance information Mol mass

10ppm

5ppm

3ppm

1ppm

2% isotopic accuracy

5% isotopic accuracy

0.1ppm

3ppm

5ppm

150

2

1

1

1

1

1

1

200

3

2

2

1

1

1

1

300

24

11

7

2

1

1

6

400

78

37

23

7

1

2

13

500

266

115

64

21

2

3

33

600

505

257

155

50

5

4

36

700

1046

538

321

108

10

10

97

800

1964

973

599

200

20

13

111

900

3447

1712

1045

345

32

18

196

17

How Many Possible Elemental Compositions? • No difference between high mass accuracy in the low m/z range and 3ppm mass accuracy and 2% isotopic abundance accuracy • Magnetic sector – 1 to 5ppm • FTICRMS – 0.5 to 1ppm • Tof – 1-10ppm • Orbitrap – 1-5ppm

18

“New” Developments in Magnetic Sector Instruments • Large, high field magnets – Mass range up to 10,000 Da at full accelerating potential (10 kV) for analysis of large biopolymers – Example: bovine insulin (MW 5734) • Laminated magnets – To reduce magnetic hysteresis – Total cycle time < 1 sec, fast scanning

19

Linear Quadrupoles (2D - mass filters)

20

Linear Quadrupoles (2D - mass filters) • Four hyperbolic rods (cheap version: circular rods) – compromise! • Opposite pairs of rods are connected electrically but are of opposite polarity • Each pair of rods has a DC (U) + AC (V0 cosωt) Rf voltage applied: 1 pair of rods: -(U + V0 cosωt) and the opposite pair: +(U + V0 cosωt) where, ω = radial frequency = 2πf • During a mass scan, the DC and AC voltages are ramped but the ratio of DC/AC (ie U/V0)is kept constant • For a given DC and AC amplitude, only ions with a given m/z (or m/z range) have stable oscillations and are transmitted and can be detected 21

Quadrupole (end view)

Hyperbolic

Round

Equipotential Field Lines

22

Superposition of RF and DC Voltages Applied to Rods 4000

3000

2000 RF VOLTAGE 6000 V P/P AT 2500 u

1000 500 VOLTAGE (V)

dc VOLTAGE 500 V AT 2500 u

0 -500

-1000

-2000

ATOMIC MASS UNITS (u)

-3000

-4000

23

Ion Motion DC

DC

+

+ve ion

+ve ion

X

AC

AC

Z

+ AC+DC

-

+ve ion

Y

-

-

+ve ion

X

Z

Z

-

+

+ve ion

Y

Z

+

-

Y

-

Z

•This motion is very complex! •DC fields focus +ve ions in the +ve plane and defocus them in the –ve plane •The superimposed AC helps correct this defocusing effect 24

Linear Q: Equations of Motion From the electrical part of the Lorentz equation, we can derive the equation of motion (x and y directions) for a particle in a combination the Mathieu equation: of DC and AC Rf fields 2

d u + ( au − 2qu cos 2ξ )u = 0 2 dξ – u represents the x or y transverse displacement. We do not consider displacement in the z direction because the electric field is 0 along the asymptotes of the hyperbolic rods. – The 2 parameters characteristc of the field (a and q) are given by:

8 zeU a x = −a y = mr02ϖ 2

and

− 4 zeV q x = −q y = mr02ϖ 2 25

Linear Q: Equations of Motion •

Where the variable ξ is the time in radians of the applied field = ωt/2

•

U is the DC voltage and V is the AC Rf voltage of frequency ω

•

r0 is the radius of the instrument aperture

•

Plotting a against q gives the Mathieu stability diagram of

the linear

quadrupole field - a/q = 2U/V •

Typical values are: - U = DC voltage (~200 - 1000V) - V = AC voltage (~1000 - 6000V, 1-2MHz), - m = mass of ion, e = electonic charge, z = # of charges on ion - 2r0 = distance between the rods - 1-2 cm 26

Stability Diagram

27

aq Space • Note: – Both +ve and –ve abscissa with a values ranging up to 10 and q values ranging up to 20 – In practice we only operate in the +ve area of region I Why? – Because in order to have a and q values >1 we would require VERY high DC and AC voltages which is not practical 28

Stability Diagram L1, only 1 ion has a stable trajectory all others ions are lost therefore adjacent ions are resolved from each other

a 0.3 Y unstable

0.2

Operating lines a/q constant

. . . . . . .. ..

0.1

L1 L1 = L2 L2

L2, 3 ions have a stable trajectory at the same time therefore these 3 ions would not be resolved from each other

In practice, the ratio of a/q is changed by changing the DC voltage X unstable

X and Y Stable 0.4

0.8

What would happen if no DC voltage is applied? q

29

Ion Motion

30

Conceptualizing a Q scan a 0.3

Operating or scan line

stable region of m1 0.2

stable region of m2

m1 < m2 < m3

stable region of m3

0.1

0.4

0.8

q

31

Mass Range and Resolution • Depends on 5 parameters: • Rod length (L) – 50 to 250mm • Rod diameter (r) – 6 to 15mm aligned to μm accuracy • Maximum supply voltage (Vm) • AC (Rf) fequency (f) • Ion injection energy (Vz) - ~5 volts • From the theory of quadrupole operation the following relationship can be derived: Mmax = 7x106Vm/f2r2 Consequently, as r and f increase, Mmax decreases and as r and f decrease, Mmax increases 32

Mass Range and Resolution

• The resolution limit of a quadrupole is governed by the number of cycles of the Rf field to which the ions are exposed: 2 M/ΔM = 0.05 fL m/2eVz • Consequently, as both f and L increase so does resolution. If L in increased then f can be decreased and vice versa • Scanning speeds as high as 6,000 amu/sec and mass resolution of 10,000 is attainable 33

Linear Q Advantages: • Small and light weight ~1 foot long • Inexpensive • Simple to operate – complete computer control • Low accelerating voltage – handles high source pressures better • Full scan mass spectra and selected ion monitoring (SIM) for quantitation Disadvantages: • Unit mass resolution only and limited mass range • High mass discrimination • Rod contamination causes further imperfections in the quadrupole field – compromises resolution and sensitivity 34

Linear Q Other applications: • QQQ for MS/MS • Hybrid instruments eg BEQQ and QqTof • Ion lenses (hexapoles and octapoles) • Collision chambers for MS/MS ie QQQ and BEQQ etc • Prefilter – before mass resolving rods to reduce contamination

35

Quadrupole 3D Ion Trap (QIT) Ion trap consists of three electrodes: Cap Cap

r0 Ring Cap

• ring electrode (hyperbolic shape) • 2 hyperbolic electrodes - end caps • Orifice for ion injection • Orifice for ion ejection • Pulsed introduction of ions 36

Quadrupole 3D Ion Trap (QIT) • Ions accumulated in the trap ie “trapped” and then further experiments can be performed on the trapped population of ions eg MS or MS/MS or even MSn • During this time, ions are gated away from the IT and lost • employs: - AC (Rf) to trap and scan ions out to detector – no DC component - He buffer gas necessary for efficient trapping of ions directed into the ion trap z He present at all times therefore no delay between MS and MS/MS experiments 37

QIT (properties) •

Ion trap volume very small (7mm i.d.)

•

High sensitivity (10-18 mol) (scan mode)

•

High mass range : 6,000

•

Higher mass resolution than Q ~x2-3

•

High dynamic range: 106 depending on space charging

•

MSn capabilities

•

Low mass cut-off is a disadvantage

•

Helium is introduced intentionally into the ion trap (10–3 mbar) – Needed as a buffer to absorb kinetic energy of incoming ions without chemical interaction so they can feel the effect of the trapping field - dampening (cooling) of oscillations – collision partner for MS/MS and MSn

•

Ions are concentrated in center of ion trap

•

Better resolution and better sensitivity than Q 38

QIT (ion motion) • Between the three electrode a quadrupole field exists, which forces the ions to the center of the trap • The farther the ion is removed from center of trap the stronger is the exerted electric force • The ions oscillate within the trap, but with a rather complex sinusoidal motion • The ion motion can be described by Mathieu’s differential equations

39

Quadrupole 3D Ion Trap (QIT) • For the QIT, the electric field has to be considered in 3 dimensions. The electric field can be descibed by the expression: Φx,y,z = Φ (r2 - 2z2) r02 0

• The equations of ion motion in such a field are: d²z/dt² - (4e/mr0²) [(U - V cos2ωt)z = 0 d²r/dt² + (2e/mr0²) [(U - V cos2ωt)r = 0 • Solving these Mathieu type differential equations yields the parameters az and qz az = -2ar = 16eU/(mr0²ω²) and qz = -2qr = 8eV/(mr0²ω²) Where ω = 2πf, f = fundamental Rf frequency of the trap (~1MHz) 40

QIT (Ion stability diagram)

Endcap Ring Electrode q = 0.908

q < 0.908 Ring Electrode

Endcap 41 courtesy of Spektrum Akademischer Verlag

QIT (stability diagram)

• Ions are only stable both in r and z direction for certain defined values of a and q • Ions oscillate with so called “secular frequency”, f, which differs from the frequency of applied Rf field because of inertia (in addition oscillations of higher order) • Ions of different m/z are simultaneously trapped, V determines low mass cut-off at qz = 0.908, which increases with V

42

QIT (mass selective ion stability scan) • Mass scan is possible by increasing the amplitude of the voltage on the ring electrode (U = 0, az = 0 ie no DC voltage) • Scan line: While scanning along this line (a=0) ions become increasingly non stable and exit the stability diagram at qz = 0.908. • Trajectory of these ions in z- direction. • Ions exit from trap through holes in end cap. • Linear scan function 43

QIT (resonance ejection) • An additional Rf voltage with low amplitude is applied to end caps. • If frequency of this additional voltage is equal to frequency of oscillating ions, ions take up energy exponentially and become non stable: → Resonance ejection ( at q < 0.908) • Mass scan with resonance ejection leads to – Higher resolution – Higher sensitivity – Higher mass range – Faster scanning – up to 26,000 amu/sec – Improved reproducibility due to higher net ion sampling 44 rate

QIT: Mass Scan – resonance ejection

• 1: Clear Trap • 2: Accumulation Time • 3: Scan Delay Courtesy of Agilent

• 4: Mass Analysis

45

Space Charging ~ 300 Ions 524.3

Relative Abundance

100

~ 1500 Ions 100

524.4

~ 3000 Ions 100

524.5

~ 6000 Ions 100

80

80

80

80

60

60

60

60

40

40

524.8

525.7

40

40 525.3

20

20 526.3

0

0 522

530

m/z

Good resolution and mass accuracy

525.5

525.4

522

20 526.3 527.5 530

526.5 527.5

0 522

526.7

20

530

0 522

530

Poor resolution and mass accuracy

46

Courtesy of Agilent

QIT (space charge)

• With increasing number of ions trapped the space charge increases • Space charge distorts the electric field • Deterioration of resolution, sensitivity and mass accuracy Solution: Pre-scan or measure in real time to control the number of ions (or more correctly, the number of charges) in the trap (a maximum of ~103 - 104) 47

Linear (2D) traps • Similar idea to 3D traps with a “new” 2D geometry • Rf only quads with DC voltage end electrodes • Larger size than 3D IT – higher ion capacity (~x50) therefore fewer space charge problems • More than one design for this type of trapping instrument • Hybrids such as QQQ where Q3 can also be used as a linear trap and LT-FTICR

48

Trapping Forces in a Linear Ion Trap Radial Trapping RF Voltage

Axial Trapping DC Voltage

Axial Trapping

Exit Lens Radial Trapping RF Voltage Courtesy of Sciex

Resonance Excitation 49

Linear Ion Trap – 2nd design DC 1

DC 2

DC 3

• •

DC 2

RF -

RF -

z DC 1

y

RF +

y

DC 3

x

RF +

For Axial Trapping 3-130 V DC3>DC2>DC1 To Contain ions: DC1=DC3>DC2

Radial Quadrupolar Trapping 1.2 MHz 5KV0-P

y

GND AC -

AC+

x

GND

Courtesy of Thermo

Radial Dipolar Excitation 5-600 KHz 0-400 Vpp

50

Linear Ion Trap vs 3D Trap No low mass cut-off Trapping Efficiency:

>10

Detection Efficiency:

doubled

Overall Efficiency:

>10

Ion Capacity (Spectral):

>20

Scan Rate (amu/sec):

4x

Highly Efficient MSn:

5x over 3D IT

51

Time of Flight (Tof) Principle: Ions of different mass (accelerated by the same field, V) have different velocities and thus flight times. The larger the mass the slower the ion: zeV = mv²/2 Ion formation: Ions are introduced to the Tof in pulses (e.g. MALDI or orthogonal extraction from a continuous beam such as ESI) Ion detected by analogue or time to digital converter (GHz ADC or TDC) • Linear Tof (high mass range but low mass resolution) • Reflectron Tof (lower mass range but high mass resolution) 52

Mass Separation: Time of Flight (Tof) MS

acceleration region (drift region)

53

Basic Principles Since the initial kinetic energy of the ions is given by: zeV = mv²/2 (i) velocity: v = (2zeV/m)1/2 (ii) time of flight: t = L/v = L[m/(2zeV)]1/2 (iii) m/z = 2eVt2/L2 (iv) Example: For C6H5+. and C7H7+., (m/z 77 and 91), accelerated at 10kV, what are the velocities of these 2 ions and how long would it take them to traverse a 2m flight tube? using eqn (ii) v77 = (2x1x1.6022x10-19x10,000/m)1/2 m(kg) = 0.077/6.022x1023 = 1.279x10-25 v77 = 128,759m/s 15.53μs v91 = 118,457m/s 16.88μs similarly for v91 V is the extraction pulse potential (V) L is the length of field free drift zone (m) t is the measured time-of-flight of the ion (s) e = 1.6022x10-19C

54

Example cont • From eq (iii), difference in flight time: tA/tB = (mA/mB)1/2 • Consequently, this square root relationship causes Δt for a given Δm/z to decrease with increasing m/z • For example: Δt/amu is calculated to be 114ns at m/z 20 to be 36ns at m/z 200 to be 11ns at m/z 2000 • Tof mass analyzer depends on the ability to accurately measure these short time intervals to make it a useful MS 55

Linear Tof • Transmittance as high as 90% • Ions introduced into the flight tube have a temporal and kinetic energy distribution which yields relatively poor mass resolution. • Kinetic energy spread can be reduced by employing Delayed Ion Extraction Principle of Delayed Ion Extraction: • Ions are formed during a short pulse of a few nanoseconds • The acceleration (extraction) field is only applied after a delay of some hundreds of nanoseconds: • At the beginning of the extraction ions with high initial velocities have traveled further than slower ones. Therefore after the second extraction pulse they do not experience the full acceleration potential. • Thus the initially faster ions will be accelerated less than the initially 56 slower ions.

Reflectron Tof Same m/z but different kinetic energy

•

In a reflectron Tof, the ions traverse the drift tube and penetrate into an electric field (ion mirror) where their direction is reversed.

•

Faster ions (with higher kinetic energy) penetrate farther into the electric field than slower ions (with lower kinetic energy).

•

Thus faster ions have a longer flight path and therefore need approximately the same flight time as the slower ions which have a shorter flight path. 57

Tof: Advantages and Disadvantages •

Extreme mass accuracy – reflectron ~ 5-10ppm – limited with quadrupole MS, poor with ion traps and linear Tof

•

High mass resolution – reflectron ~5,000 to 20,000 – Quadrupole MS, ion traps and linear Tof operate closer to unit mass resolution at m/z ~ 103

•

Extreme mass range – linear >105 Da, reflectron <104 Da – Ion traps and quadrupoles are limited to ~6,000 Da

•

Acceptable linearity for linear and reflectron Tof – not as good as quadrupole MS, but similar to ion traps

•

Very good scan-to-scan reproducibility for linear and reflectron Tof – as good as quadrupole MS

58

Fourier Transform Ion Cyclotron Resonance (FTICRMS – FTMS) Principle: An ion of velocity v entering a uniform magnetic field B perpendicular to its direction will move on a circular path by action of the Lorentz force, the radius rm is given by: rm = mv/qB Upon substitution with v = rmω, the cyclotron angular frequency ωc becomes: ωc = qB/m • cyclotron angular frequency is independent of ions initial velocity but is a function of it’s mass, charge and the applied magnetic field • once trapped, the ions oscillate with a cyclotron frequency that is inversely related to their m/z ratio 59

FTICR MS •

Basic Construction: – a cell where ions are trapped by intense, constant magnetic field and applied voltage – The cell accepts ions in a “pulsed” mode from the continuous ion beam – Detection of the ions is based on the FT deconvolution of the image current the circulating ions induce in a pair of detector plates after excitation with a resonant Rf pulse.

60

FTICR MS cont. • MS/MS: Excitation of the ion is achieved using a variety of techniques. Namely: – Sustained off-resonance Irradiation Collision-Induced Dissociation (SORI-CID) – Infrared Multiphoton Dissociation (IRMPD) – Electron Capture Dissociation (ECD) – Blackbody infrared radiative dissociation (BIRD) •

High sensitivity and >105 mass resolution

•

MS/MSn

•

Cyclotron frequencies can be measured with very high accuracy and precision leading to ultra high resolving power and high accuracy mass measurements 61

Ion Trapping and FTICR MS

•

ions enter the cell (or are created internally) and they begin their cyclotron motion, orbiting around the centre of the magnetic field

•

since the magnetic field is quite high (typical minimum of 4.7T, but this is increasing) the ions are trapped in the radial (x,y) direction.

•

Resolving power and scan speed increase linearly with B

62

Ion Trapping and FTICR MS

•

by applying small, equal potentials to the two end or “trapping” electrodes, the ions are confined in the z or axial direction.

•

ions can be confined for very long periods of time such that ion/molecule reactions or even slow unimolecular dissociation processes can be observed and monitored. 63

FTICR MS Detection •

In FT detection, all ions, regardless of their mass are detected at the same time. Ions before excitation. They have their natural cyclotron radius within the magnetic field.

•

Once ions are trapped inside the ICR cell they are excited by a fast sweep of all the Rf frequencies, exciting the ions to cyclotron motion with a larger radius.

64

FTICR MS - Detection

Before excitation

• • • •

After excitation

All ions are resonantly excited for the same amount of time. Each ion retains its characteristic cyclotron frequency (depending on m/z) but their radii of orbit increase. After excitation all ions have the same radii of motion since they were irradiated with Rf of the same amplitude for the same amount of time. Once the Rf is turned off, each ion packet, consisting of ions of the same m/z value induces an image current on two sets of receiver plates which are part of the ion cell. 65

FTICR MS - Detection When a packet of ions (+ve) approaches an electrode, electrons are attracted from ground and accumulate in that electrode causing a temporary current. As the ions continue to orbit, the electrons accumulate in the other electrode. The flow of electrons in the external circuit represents an image current. The amplitude of the current is proportional to the number of ions in the packet. 66

FTICR - Detection Rf Excitation

Time

Detected time domain image current

Time

Fourier Transform

Resulting mass domain Spectrum

•

•

m/z

the frequency of the image current oscillation is the same as the frequency of the ion’s cyclotron motion which is related to mass. A small AC voltage is created across a resistor and is amplified and detected. using FT techniques all ion packets, each containing ions of the same mass, are detected. The decay of the image current (as the excited cyclotron orbit radius decays) is detected in time and transformed into a frequency domain signal by a 67 Fourier transform.

FTICRMS • Very high resolution is possible. The current record is 8x108, and routine values are 100,000 or so. • Long trapping times are possible, allowing for ion-molecule reactions. • Good sensitivity. • Like the ion trap, the FTICR cell works well with pulsed sources. • MSn capability • However, expensive because of the cost of superconducting magnets and the very high vacuum requirements. • Difficult to operate 68

FTICRMS RT: 100

Sample:

very complex crude extract from human blood platelets Amount: unknown, but very low conc. Flow: 200 nl/min Scan Cycle: 1 spectrum every 3.5 s

95 90 85 80

Relative Abundance

75 70 65 60 55

78.81

86.60

50 45

49.33

40

72.26

35 30

66.96

25 20 15

100.26

10 5 40

45

50

55

60

65

70

75

80

85

90

95

100

105

110

Time (min) HCT116_A_030523101055 # 2189 RT: 72.26 FTMS + p ESI Full ms [ 200.00-2000.00]

T:

AV: 1

NL:

T:

579.29291

100

HCT116_A_030523101055 # 2189 RT: 72.26 FTMS + p ESI Full ms [ 200.00-2000.00]

7.84E4

AV: 1

NL:

7.84E4

579.29291 R=315540

100 95

95

90

90

85

RP: 400,000 @ m/z 400

80

85 80

75

75

70

70

Expand

65

857.44525

55 50 45

60 55 50

40

35

35

30

30

25

279.15924

891.38403

574.33777 R=312472

595.26740 R=300047

25

20 15

557.31122 R=323474

45

557.31122

40

65 Relative Abundance

Relative Abundance

60

20

301.14130

654.25793

15

10

10

400.23349

1157.58093

5 0

5 0

300

400

500

600

700

800 m/z

900

1000

1100

1200

550

555

560

565

570

575

580

585

590

595

600

605

m/z

69 Courtesy of Thermo

FTICRMS Resolution

70 Courtesy of IonSpec

Hyphenation in Mass Spectrometry

• Gas Chromatography – Mass Spectrometry (GC-MS) • Liquid Chromatography – Mass Spectrometry (LC-MS) • MS-MS and MSn - Tandem Mass Spectrometry WHY? • the ability to interface an orthogonal separation technique to MS greatly increases the information content that can be derived from complex mixtures

1

Gas Chromatography – Mass Spectrometry (GC-MS) • Both are gas phase techniques although at somewhat different pressures: • Ion source normally at high vacuum (EI and CI) • GC operates at ~ 5-10psi

GC

MS

Quite a stable and “friendly” relationship – they just get along 2 as both are gas-phase processes

GC-MS • Coupling of capillary GC to MS simple, as low carrier gas flow rates (1-2 mL/min) easily tolerated by MS vacuum system. • Carrier gas always helium (low viscosity, low mass). • Fused silica capillary introduced directly into the ion source via a transfer line which must be heated. • Operation modes of a GC/MS: – Full scan → TIC/RIC – Selected Ion Monitoring (SIM) 3

Operating Modes •

Full scan – selective, sequential transmission of ions to the detector in a continuous fashion (BE, EB, Q, QQQ, 3DQIT) – qualitative information

•

Selected Ion Monitoring (SIM or SIR) – Mass analyzer jumps from one pre-selected m/z value to the next – Only the response from the selected ions is recorded – no mass spectra – Detector time for the selected ions is increased by a factor of 10 – 100 and so is the sensitivity – Used for quantitative analyses – target compound analysis

•

Example: – If a Q is scanning from m/z 100 – 1100 in 1sec then each ion is recorded for 1msec – If the same Q is set to jump between only 4 ions in a SIM experiment then each ion is recorded for 250msec 4

GC-MS: Full Scan

5

GC-MS: Selected Ion Monitoring (SIM)

6

Liquid Chromatography – Mass Spectrometry (LC-MS) Historical approaches: •

There is a basic compatability problem when an LC is interfaced to a MS: – LC: 25-50oC, 200nL/mim – 1mL/min liquid flow rate at 100-3000psi – GC: 50-300oC, 1mL/min He(gas) at 5-10psi – MS: 200oC, 1x10-6mbar (high vacuum) and 20mL/min gas flow

•

The vacuum problem – gas load: • 1mL/min of hexane(l) yields approx 172mL/min gas • 1mL/min of water(l) yields approx 1240mL/min gas • Conventional MS can pump ~20mL/min of gas and maintain high vacuum

• Atmospheric Pressure Ionization solves many of these problems

7

Liquid Chromatography – Mass Spectrometry (LC-MS) • In GC, the “mobile phase“ normally referred to as the carrier gas is He and is easily removed however in LC the mobile phase is a complex mixture of solvents which might contain buffers, pH modifiers and/or ion-pair reagents – much more difficult to volatalize and remove • LC is most commonly used for compounds that are not easily analyzed by GC because they are thermally unstable, involatile or high mwt • MS requires species in the gas phase therefore we must transfer these difficult compounds into the gas phase without chemical or thermal modification 8

LC-MS: a difficult courtship MS Clearly defined boundary (picture courtesy of P.J. Arpino)

LC • It is the function of the interface to “blur” the distinction between the gas and liquid phase as far as the MS is concerned • Q: does the fish fly or does the bird swim? • A: a good question – compromise! Perhaps the bird doesn’t realize it is swimming and the fish doesn’t realize it’s flying!

9

LC-MS Why LC-MS? • LC is capable of providing separation of compounds unsuitable for GC (even with derivatization) • Polar, ionic, involatile, high mwt and thermally labile analytes are readily chromatographed by LC • Sample clean-up can be more straight forward for LC • LC allows selection of stationary and mobile phase • Conventional detectors (UV, FL etc) exhibit good detection limits but have limited specificity • The MS is a universal (sometimes), sensitive and highly specific detector HOWEVER: • A GC column is much more efficient than an LC column ie 10 HETP

Solutions to the Problem: •

There are 2 basic approaches to the problem of interfacing an LC to a MS: – The “spray” type interfaces which introduce all or a portion (splitting) of

the LC eluent to the source : – Direct Liquid Introduction (DLI) – Thermospray (TSP) – Continuous Flow FAB (CF-FAB) – Atmospheric Pressure Ionization (ESI, APCI and APPI) – The “enrichment” type interfaces which preferentially remove the LC

solvent from the less volatile analytes before introduction to the source: – Moving Belt – Particle Beam

•

This is by no means an complete list but describes the most important11

Direct Liquid Introduction (DLI)

•

Liquid is introduced directly into the high vacuum source – 10 – 40μL/min of liquid – Sprayed through a 2-5μm laser drilled hole – prone to blockage – Desolvation chamber vapourizes analyte and liquid – Vapour enters CI ion source – Proton transfer and proton abstraction dominates – Only CI type spectra with mobile phase forming the CI plasma – Only volatile buffers can be used – Works well for reversed and normal phase solvent systems

12

Thermospray (TSP)

• • • • • • •

Capillary is heated to partially vapourize the mobile phase supersonic jet of vapour and liquid Mobile phase @ 1mL/min must contain a volatile buffer - normally NH4OAc Reversed phase only ie solubilize the NH4OAc Ionization occurs through ion/molecule reactions with either NH4+ or OAcResulting mass spectra are NH3CI like in +ve mode Much more robust than DLI but classical TSP limited to reversed phase separations This can be overcome to some extent by employing an alternative form of ionization such as a discharge voltage (APCI like) then termed Plasmaspray or a heated 13 filament as in a conventional EI/CI source (fragile)

Continuous Flow FAB

14

Continuous Flow FAB • Analyte introduced continuously to the probe tip at 5 – 10μL/min and normally contains a small amount of a matrix eg glycerol • Tip is irradiated with a beam of Xe0 or Cs+ which causes sputtering of ions into the gas phase • Good for polar, thermally labile species but sensitivity can be 3-6 orders of magnitude lower than ESI

15

Electrospray (ESI)

•Electrospray (nanoESI) •up to 1 µl/min •Concentration dependant •Most sensitive

•Pneumatically assisted electrospray • flow rate range 5µl/min – 1mL/min • sensitivity poorer than nanoESI • Concentration dependant • More robust 16

ESI In-line spraying - most modern sources are orthogonal ie at right angles to the entrance to the MS •

•

•

All API sources/interfaces work because while the source is at atmospheric pressure the MS analyzer is at high vacuum. A series of skimmers and vacuum pumps is placed between the source and the MS to reduce pressure from AP to HV Orthogonal spraying helps reduce the gas load into the MS

17

ESI – Remember! • Droplets are highly charged during the spraying process and Coulombic repulsion causes the spray to “explode” into smaller drops as the solvent evaporates • As the droplets become smaller the charged analyte ions will be desorbed into the gas phase (IEM) or as the droplet becomes completely dry (CRM) and then extracted into the MS • Orthogonal source designs now ie sprayer at right angles to the entrance to the MS 18

APCI

19

APCI • Advantages: – Entire mobile phase and sample vaporized into the gas phase (with heat), then ionized – Accommodates high LC flow from (0.2 - 2 mL/min) • Uses heat (400–500 °C) and nebulizer gas to vaporize HPLC eluent and transfer sample into the gas phase for APCI – Temperature setting is not critical – Sensitive • Disadvantages: – Thermally labile analytes may degrade when vaporized – Low molecular weights only – CI mass spectra only, little fragmentation typically

20

APPI

courtesy of Applied Biosystems

21

APPI Advantages: – Entire mobile phase and sample vaporized into the gas phase (with heat), then ionized • Uses dopant (Toluene/Acetone) to promote ionization – Accommodates high LC flow from (0.2 - 2 mL/min) • Uses heat (400–500 °C) & nebulizer gas to vaporize HPLC eluent and transfer sample into the gas phase for photoionization – Detection limits are very good • Improvements over ESI and APCI compound dependant – Wider application range (low to medium polarity compounds) Disadvantages: – Thermally labile analytes may degrade when vaporized – Low molecular weight compounds only (<1000)

22

Moving Belt 3

4 A

B

2

1

• LC eluent is mechanically transported from the column (A) to the high vacuum ion source (B) • Combination of differentially pumped regions (1,2,3) and heating (4) removes solvent • The dried sample is flash vapourized into the source (EI/CI) or 23 irradiated with Xe0 or Cs+ ions (FAB/LSIMS)

Moving Belt • The polyimide belt contributes to the chemical noise of the system • Mobile phases containing high H2O concentrations are difficult to handle • Latent heat of vapourization of H2O is very high (compared to organic solvents) • Can bead on the belt (high surface tension) – destroys chromatographic integrity • Mechanically very complex - poor reliability • One of the first LC/MS interfaces available (along with DLI)

24

Particle Beam

25

Particle Beam

• •

• • •

LC eluent introduced with He to generate an aerosol of solvent droplets Solvent evaporates and the resultant beam of dry particles, He and solvent vapour enters a momentum separator which preferentially removes low mwt species (He and solvent) Dry particles enter a conventional high vacuum EI/CI source 0.1 – 1mL/min flow rates accommodated Samples must be volatile and thermally labile and mwt<1000

26

Tandem Mass Spectrometry – MS/MS •

The combination of 2 or more stages of mass analysis in one experiment

•

These stages are decoupled from one another in one of 2 ways: – MS/MS in space, where parent ion selection, dissociation and subsequent mass analysis are physically performed in different regions of the MS eg BE/EB, QQQ, Tof-Tof etc – Hybrid instruments, where 2 different types of mass analyzer are put together eg QTof, QTrap etc – MS/MS in time, where parent ion selection, dissociation and subsequent mass analysis are performed in the same region of the MS but are decoupled in time ie they are performed as a series of timed events following one another eg 2D and 3D ion traps, FTICRMS

•

The dissociation step is typically achieved using Collision Induced Dissociation (CID) sometimes called Collisional Activation (CA) 27

Ion Stability • 3 “types” of ions based on the mass spectrometric time frame: – Unstable – dissociate quickly ie before leaving the source k > 106s-1 and are never observed – Metastable – dissociate after leaving the source but before detection 105s-1 < k < 106s-1 can be detected – Stable – arrive intact at the detector k < 105s-1 • In order to perform MS/MS we must somehow destabilize the “stable” ions and cause them to dissociate • There are a number of ways to accomplish this but the most common is Collision Induced Dissociation (CID) 28

Collision Induced Dissociation (CID) • An ion/neutral species interaction wherein the projectile ion is dissociated as a result of interaction with a target neutral species (N2, Ar, He). This is brought about by conversion of part of the translation energy of the ion into internal energy of the ion during collision. AB+ + Ncg

AB+*

A+ + B + Ncg

vibrationally and electronically excited

• The internal energy (IE) of AB+* is composed of IE prior to collision (usually low) and the amount Q, transferred during collision: EAB+* = EAB+ + Q

29

CID •

The absolute upper limit for Q is defined by the “centre of mass” collision energy, ECM)

ECM = ELAB

(

m mN+mAB

N __________

)

Where: ELAB user set collision energy mN is the mass of the target or collision gas mAB is the mass of the ion •

ELAB can be in the 1-10keV range for BE/EB and Tof-Tof instruments – single or double collisions, He employed with short collision cells

•

ELAB can be in the 1-200eV range for QQQ, QIT, Q-Tof instruments employing multiple collision events with Ar, N2 or He (for QIT) and longer collision cells

•

Scattering can be an issue

•

Very fast process – 10-15 s

•

Must not ionize the collision gas in the collision event

30

CID • For example: – an ion of m/z100 with a collision energy of 50eV colliding with He will gain a maximum of 50*(4/(100+4)) = 1.9eV. – consequently, in the 1-200eV collision regime, we usually employ N2 or Ar as the collision gas to maximize the energy transfer – the same ion with 5 keV, colliding with He could in principle pick up 190eV of internal energy, and no doubt some do, but very, very few are seen. – consequently, in the keV collision regime, we usually employ He as the collision gas to minimize ion scattering and prevent excessive fragmentation 31

CID •

In high energy CID in a sector instrument, we expect to see only the products of glancing collisions which do not knock the ions out of the beam, and because such experiments are carried out under single-collision conditions (each precursor ion undergoes a maximum of one collision), the total internal energy is relatively low, say 1 – 15 eV.

•

As a general rule, the bigger it is, the harder it is to break. A simple way of understanding this is to keep in mind that the energy deposited in the ion may not be localized, and in any case, rapidly distributes over the bonds of the ion. Bigger ions Æ more bonds Æ less energy/bond, and less chance that a given bond will have enough energy to break.

•

A CID type process can also occur in the source of an API instrument because ions are accelerated (focused) through a high pressure region before they enter the mass analyzer – this can cause “unstable” ions to collide with neutral gas molecules and dissociate in the source – called “in-source CID” 32

“In-source” Collision Induced Dissociation (CID) mwt=451 396.128

100

Ions moving quickly through high pressure region

“normal” cone voltage 165V %

[M+H]+

352.143 164.071 397.145

0 100

452.198 474.184

m/z 125

150

175

200

225

250

275

300

325

350

375

400

425

450

475

[M+H]+

452.175

100

500

“low” cone voltage 65V %

Ions moving slowly through high pressure region

396.128 453.198

0 100

m/z 125

150

175

200

225

250

275

300

325

350

375

400

425

450

475

500

33

Why Tandem Mass Spectrometry? • To separate the ionization step from the fragmentation step – for example, in an EI full scan mass spectrum of an analyte that fragments extensively in the source, it can be difficult to determine which fragment ion is formed from which precursor – MS/MS allows individual ions to be selected and their fragmentation behavior studied in isolation from any other processes

• Direct mixture analysis • Increase specificity, reduce “chemical noise” for quantitative studies (SRM/MRM)

34

Tandem Mass Spectrometry • In the simplest of these experiments, MS/MS, the 1st mass analyzer is used to transmit only one ion observed (m2) in the full scan mass spectrum into the dissociation region • In the dissociation region (collision cell), ions are “excited” energetically in a variety of fashions depending on the instrument type. This leads to the dissociation of m2 into fragment ions • The 2nd mass analyzer is scanned to pass in turn the products of the dissociation of m2 onto the detector • This is the simplest of the MS/MS experiments and is called a product ion scan • There are many others: precursor ion scan, neutral loss scan, selected reaction monitoring 35

Tandem MS - Product Ion Scan Schematically Full scan MS of analyte m2

m3

Mass select m2 and dissociate MS/MS spectra of m2

Mass select another ion (m3) from the MS/MS spectrum of m2 and dissociate MS/MS/MS spectra or MS3

36

Magnetic Sector Instruments •

Ion dissociation in a Field Free Region (FFR): – Metastable decomposition – Collision Induced Dissociation (CID) – Dissociation causes partitioning of ion kinetic energy and momentum between the 2 particles

•

Constant Linked scan with EB instruments: – To look at dissociations occurring in the 1st FFR – Must scan B/E together (linked) – Not true tandem MS because B and E are not operated separately – Poor precursor ion resolution

•

Mass analyzed Ion Kinetic Energy Spectra (MIKES): – To look at dissociations occurring in the 2nd FFR – Only on BE instruments – Poor fragment ion resolution

37

Reminder: Metastable Ion (from section 3 (EI) pg7) Fragment ions M+.

Metastable ion • M+. and fragment ions~0.3Da wide • Metastable ion~3Da wide! 38

Reminder: Metastable Ion (from section 3 (EI) pg7

• It’s position at m/z 200.3 can be used to determine which ion is fragmenting to give which product: For the reaction, M1+ M 2 + + M3 apparent mass of the metastable, M* = M22 ____

M1 • In this case, solving the equation: M1+ is m/z 255 and M2+ is m/z 226 • Therefore this metastable ion corresponds to the fragmentation of the M+. ion to yield the fragment at m/z 226 • the other metastable ion at m/z 146.6 corresponds to m/z 226 fragmenting to give 182 39

Magnetic Sector Instruments • To overcome the resolution issues other sector based instruments have been built eg BEBE, EBEB etc

40

Triple Quadrupole – MS/MS in Space • Basic construction – QQQ configuration: Q1 and Q3 are mass filtering with Q2 being the collision cell – Ar or N2 collision gas employed for MS/MS Ion source

Q1

Q2

Q3

Detector

Product Ion Scan Park Q1 to Allow Only Ions of a Single m/z ratio to pass into Q2 These ions collide with collision gas at a given CE and dissociate (CID) Q3 is Scanned yielding the Full Scan Product Ion Spectra

41

QQQ – Full Scan Mass Spectrum Q1

Q2

Transmits all ions

Transmits all ions

Q3 Scan

• Full scan mass spectrum of all source generated ions

Typical ESI Full Scan MS: Q3 scan, Q1 and Q2 Rf only mode [M+H]+ little or no fragmentation observed

42

QQQ – Product Ion Scan Q1

Q2

Selected Ion

• •

Q3 CID

Scan

Full scan MS/MS spectrum of mass selected Parent Ion Used for structural elucidation

Typical ESI Full Scan MS: Q3 scan, Q1 and Q2 Rf only mode [M+H]+

Typical ESI Product Ion Scan: Q1 selects (M+H)+, CID occurs in Q2 Q3 scanned

little or no fragmentation observed [M+H]+

43

Precursor Ion Scan Q1

Q2

Scan

Q3

CID

Selected Ion

• Q3 is set to transmit only 1 ion characteristic of a specific class of compounds eg M1 = m/z 79 (PO3)• Then as species are introduced into the source, Q1 is scanned and only those compounds that fragment to yield M1 will be detected • Used for target compound class detection usually with chromatography

44

Precursor Ion Scan

Intensity

Intensity

Q3 monitoring only m/z 79

Time

• β-Casein Digest Full scan • Complicated with many peptides

Courtesy of the MSCLS at the Univ. of Minn.

Time

• β-Casein Digest Precursor Ion Scan • Detection of only Phosphorylated peptides

45

Neutral Loss Scan Q1

Scan

Q2

Q3

CID

Scan at Q1-neutral mass

• Q1 and Q3 are both scanned however there is a mass offset (Mn) between Q1 and Q3 • Then as species are introduced into the source, only those compounds that loose the neutral mass, Mn, will be detected • Used for target compound class detection usually with chromatography

46

Selected (Multiple) Reaction Monitoring – SRM/MRM Q1

Selected Ion

Q2

Q3

CID

Selected Fragment

• From a complex mixture, Q1 is set to pass only the parent ion from a specific analyte, M1, and Q3 monitors only 1 of the unique fragment ions formed from M1 • Ideally this allows the unambiguous detection of only 1 (or more) analyte in a very complex mixture • Used for target compound quantitation usually with chromatography • The most sensitive and specific of all MS techniques for quantitation 47

3D Quadrupole Ion Trap – MS/MS in Time Remember: He buffer gas necessary for efficient trapping of ions directed into the ion trap z He present at all times therefore no delay between MS and MS/MS experiments Ring z

Endcap +

+

+

+

+ +

+

+

+ +

detector

+ +

Ions gated into trap Endcap

Courtesy of Agilent

48

QIT (ion isolation) • An additional Rf voltage is applied to the end caps. • All but ions with one mass become non-stable and are ejected from trap • Resonance excitation of the trapped ion with additional Rf voltage → oscillations of trapped ion increases • The trapped ion collides with helium atoms and become internally excited • Collision Induced Dissociation (CID) • New mass scan → MS/MS or (MS)3 spectrum • In analogy: (MS)3, (MS)4, …. 49

QIT: MS/MS Scan

• 1: Clear Trap • 2: Accumulation Time

**

**

• 3: Isolation Delay • 4: Isolation begin • 5: Fragmentation delay

*

**

*

* *

• 6: Fragmentation begin* • 7: Scan delay • 8: Mass Analysis**

Courtesy of Agilent

50

3D - QIT • Can only perform product ion scans – no precursor, neutral loss or SRM functionality • Most often used in qualitative analysis ie structural elucidation • In scanning mode (MS, MS/MS), much more sensitive than a Q or QQQ • MS/MSn

51

QQQ with Linear Ion Trap - QTrap Dipolar Aux AC

Skimmer

N2 CAD Gas

Q0

Q1

Q2

Q3

Exit

IQ1 IQ2 LINAC

IQ3

linear ion trap 3x10-5 Torr

52 courtesy of Applied Biosystems

Q-Trap •

All the functionality of a QQQ – Product ion Scan – Precursor Ion Scan – Neutral Loss Scan – SRM

•

with the additional advantage that Q3 can be switched (software) into trapping mode operation – Very high scanning sensitivity compared to Q operated with AC (Rf) and DC – Linear trap also has some advantages over 3D QIT

•

Can perform quantitation and structure elucidation (MS/MS3) sequentially in a chromatographic time frame 53

QQQ with Linear Ion Trap - QTrap Dipolar Aux AC

Skimmer

N2 CAD Gas

Q0

Q1

IQ1

Q2

IQ2 LINAC

Q3

IQ3

Exit

linear ion trap 3x10-5 Torr

MS3 - Implementation • • • • • •

Precursor ion selection in Q1. Fragmentation in Q2. Trap products in Q3. RF/DC isolation in Q3. Single frequency excitation in Q3. Mass scan.

54 courtesy of Applied Biosystems

Complementary MS/MS Approaches: •Tandem in Space: Triple Quads •Poor scanning sensitivity •Great for quantitation (SRM/MRM) •Very selective scans •No low mass cut-off •Tandem-in-Time: 3D Ion Traps •Very sensitive scanning •Only product ion scans - MSn •Only scanning •Low mass cut-off! •3D vs 2D (linear) Traps •Linear traps have higher charge capacity •No low mass cut-off •Linear traps restricted to MS3

55

Hybrid Quadrupole Time of Flight - QTof • a QQQ where Q3 has been replaced by a reflectron TOF MS • TOF used to acquire (accumulate) both MS and MS/MS spectra • TOF accepts ions in a “pulsed” mode from the continuous ion beam passed to the pusher by the optics

56 Courtesy of Waters

Q-Tof • Advantages: • High resolution (104) and accurate mass • good scan sensitivity • MS and Product Ion MS/MS • Disadvantages: • historically, TOF instruments suffer from a “poor” dynamic range compared to Q instruments – this is changing • no Precursor Ion, Neutral Loss or SRM/MRM capability

57

+ve ESI - Peptide MS Glu-Gly-Val-Asn-Asp-Asn-Glu-Glu-Gly-Phe-Phe-Ser-Ala-Arg E-G-V-N-D-N-E-E-G-F-F-S-A-R mwt = 1569.67

[M+2H] 2+

100

785.82 786.32

%

797.31

804.80

203.05 149.02

805.29 815.79 816.29

411.26

219.02 355.07

776.82

[M+H]+

816.79 834.77

1570.67

0

m/z 200

400

600

800

1000

1200

1400

1600

58

+ve ESI - Peptide MS/MS 100

175.13

MS/MS of [M+H]+ CE = 85eV Ar 1570.80 684.37 684.41

1570.59

%

1056.54

1571.87 1570.51

1056.60 685.40 316.18 333.20 497.24

684.29

1057.56

813.47

1056.36 1039.53

813.38 814.48

1571.76

1570.40

1057.63

942.44

1571.90 1572.81 1572.89 1573.89

0

m/z 200

400

600

800

1000

1200

1400

1600

684.38

100

787.86

MS/MS of [M+2H]2+ CE = 35eV Ar

787.83 788.35

%

187.08

813.45

333.21

480.29

240.15

685.39

337.18

497.22

382.20

498.21

685.43 627.36

1056.53 1056.57 1285.65 1171.57 1286.60 943.51 1172.56 1286.71 924.44 1058.54 1287.59 815.46

814.42 942.44

740.30

0

m/z 200

400

600

800

1000

1200

1400

1600

Doubly charged peptide ions yield more sequence ions than does the singly 59 charged counterpart!

How Do Peptides Fragment? x3 y3

H2N

R1

O

C

C

H

z3

x2 y2

R2

O

N

C

C

H

H

a1 b1 c1

z2 x1 y1 R3

O

N

C

C

H

H

z1

H+

R4 N

C

H

H

COOH

a2 b2 c2 a3 b3 c3

– If this charge is retained on the N terminal fragment, the ion is classed as either a, b or c – If the charge is retained on the C terminal, the ion type is either x, y or z – A subscript indicates the number of residues in the fragment 60

+ve ESI - Peptide MS/MS Sequencing y13

y12

y11

y10

y9

y8

y7

y6

y5

y4

y3

y2

y1

E--G--V--N--D--N--E--E--G--F--F--S--A--R b1

b2

b3

b4

b5

b7

b8

y6 b2 187.08

b9

b10

b11

b12

b13

MS/MS of [M+2H]2+ CE = 35eV Ar

684.38

100

%

b6

787.86 787.83 788.35 y

7

y3

y4

333.21

813.45

480.29

240.15

337.18

497.22

382.20

498.21

y5 627.36

y11 1056.53 1056.57 y101285.65 814.42 942.44 1171.57 1286.60 943.51 1172.56 1286.71 924.44 1058.54 1287.59 815.46 y8

685.39 685.43 740.30

y9

0

m/z 200

400

600

800

1000

1200

1400

1600

61

• Low Energy CID (10eV to 100eV) – collision induced dissociation in a QQQ, IT or QTof – a peptide carrying a positive charge fragments mainly along its backbone, generating predominantly a, b and y ions – In addition, ions which have lost ammonia (-17 Da) denoted a*, b* and y* and water (-18 Da) denoted a°, b° and y° are often observed – Satellite ions from side chain cleavage are not observed.

• High Energy CID (keV) – collision induced dissociation in a Tof-Tof or a magnetic deflection instrument – All of the ion series described above are observed in high energy collision spectra. Relative abundances are composition dependent. – Unlike low energy CID, ions do not readily lose ammonia or water. – In addition, side chain cleavages can be observed, so called d, v and w cleavages 62

Others? • Of course: • • • • •

Orbitrap (linear trap – new type of 3D IT) Tof-Tof Linear trap-FTICR Trap-Tof etc etc Ion mobility coupled to other types of mass spectrometer eg QTof where the collision cell has been changed to allow IMS to be performed as well as conventional MS/MS expts

63

Quantitation in GC-MS and LC-MS • Every ionization technique exhibits a compound-dependent response – that is, the same amount injected of different analytes will give a different MS response • In general, every aspect of the MS experiment can influence the response from the analyte • Ionization method • Type of MS • Chromatographic method • Detector system

• Therefore a careful calibration of the instruments response versus the sample concentration is a pre-requisite for reliable quantitation • Most MS quantitation methods employ chromatography

1

Important Definitions • Limit of detection (LOD) – How small an amount can you see? Usually this is defined as the amount that will give a S/N of about 3.

• Limit of quantitation (LOQ) – How small an amount can be quantitatively measured? Usually significantly higher than the LOD.

• Sensitivity – What’s the response per unit of analyte?

• Specificity – Is the instrument response due only to the analyte? How well does our analysis cope with interferences?

• Linearity – Over what range is the response linear? 2

Important Definitions • Calibration standards – Prepared samples of known concentration for which corresponding responses can be obtained – Peak Area is most often employed as the response for mass spectrometric assays however Peak Heights can also be used but introduce a source of error

• Standard or Calibration Curve – A series of standards covering the analyte concentration range expected in the samples – Mathematical function or equation describing the relationship between instrument/ionization method/detector response and concentration

• Principle – Mass spectrometric quantification can be done ONLY by reference to standards (External or Internal) 3

Precision and Accuracy B

A

.. . . .... .. C

.

. . .. . . . . .

A: Accurate and Imprecise B: Accurate and Precise C: Inaccurate and Imprecise D: Inaccurate and Precise

....... D

......

. Precision (reproducibility): GC-MS < 1% HPLC-MS (electrospray): Quadrupole ~ 1-2 % Ion trap ~ 8 %

4

Quantitation in GC-MS and LC-MS • Calibration – In general, the response of a mass spectrometer to a specific analyte will vary significantly with time – Some of this variability can be directly attributable to parameters directly related to the ionization process and use of the MS as the detector however there are other sources of error: • injection reproducibility when chromatography is employed • Sample handling and preparation

• Why? – over time, MS performance will decrease as a result of detector aging and source/ion optics becoming contaminated – sample components causing ion suppression or isobaric interferences – this causes systematic variation in response/unit of analyte injected 5

Quantitation in GC-MS and LC-MS • Solution: – Quantitation calibration must be performed in real time to overcome these issues – 3 types of Calibration Methodologies: • External Standard Calibration • Standard Addition • Internal Standard Calibration

6

Quantitation in GC-MS and LC-MS • External Standard: – Construct a calibration curve by injecting a series of analyte standards and plotting response against concentration – The samples are then analyzed and their response is compared to the standard analyte response to derive their concentration

• However: – Number of ions produced by a given analyte at a given concentration varies with time therefore calibration curve less stable – How closely does the standard matrix resemble the sample matrix? – Did the MS response change between the analysis of the standards and samples? – These issues as well as some preparation errors can be overcome by using an internal standard method 7

Quantitation in GC-MS and LC-MS • Standard Addition: – The unknown sample is divided in 2 portions and a known amount of the analyte is spiked into one portion – Samples measured both before and after addition – The spiked sample shows a larger response and the difference in response between the spiked and unspiked is due to the spike and provides a calibration point to determine the amount of analyte in the original sample – Calculation of analyte concentration requires analysis of multiple samples of each analyte ie with and without standard added

• However: – A linear response is assumed when a 2 point determination is made, that is, no calibration curve 8

Quantitation in GC-MS and LC-MS • Internal standards (ISTD): – A known amount of a reference compound (the internal standard) is added to every sample – If it is added before sample workup/extraction and if it has similar chemical properties to the analyte then it can be used to compensate for: • differences in recovery during sample preparation (extraction) • Ion suppression by residual matrix components • Instrumental variability (injection volume etc)

– Chemically as similar as possible but not the analyte itself (obviously) – no co-elution with matrix components – However, we must have analyte free (blank) sample matrix to prepare calibration curve and QC’s – Gives improved accuracy and precision because an internal reference is in every sample and is the method of choice for MS 9 quantitation

Quantitation in GC-MS and LC-MS •

Internal standard selection: – isotopically labeled species is best

(13C/15N>2H)>homologue>>analogue – Could use an isomer of the analyte but if the MS/MS behavior is similar to the analyte then it must be chromatographically resolved from the analyte –

13C

or 15N labeled standards preferred

– Usually deuterated (2H ) standards are used as they are more available than 13C or 15N – The label must not be able to be exchanged during the analysis ie 1H/2H exchange – we need stably labeled ISTD’s – several isotopes to avoid overlap with isotopic peaks of unlabelled analyte >3 labels is optimum – In multi-component assays, the ideal situation is to employ an internal standard for each analyte 10

Schematic Representation of a Typical Quantitative MS Procedure SAMPLE

1

CRUDE EXTRACT

2

ANALYTICAL SAMPLE

3

RATIO OF RESPONSES [ANALYTE(S) TO ISTD(S)] 4

1a. Add internal standard(s) 1b. Homogenize 1c. Extract analytes and ISTD’s

QUANTITY OF ANALYTE IN SAMPLE

2a. Purify (chromatography, further extraction etc) 2b. Concentrate if necessary 2c. Derivatize if necessary 3. Mass Spectrometric measurement employing SIM or SRM with GC/MS(MS) or LC/MS(MS) 4. Comparison with calibration curve

11

Quantitation: Sample Introdution •

•

Direct introduction: •

Fast and simple

•

Dirty samples will require extensive cleanup to minimize suppression and interference (keep in mind that choice of ionization methods differ in their susceptibility to these problems)

Chromatography/MS: •

Not so simple to develop/find a method and then you need to solve any compatibility issues eg involatile buffers, ion pair reagents

•

Takes longer

•

Many problematic impurities can be separated from the analyte, eg salts which interfere with ESI will elute in the void volume of a reverse phase column - less sample cleanup may be required

•

Keep in mind that LC columns are not infinitely tolerant of “junk”, and are not cheap!

•

We may want to compare to say, a HPLC run using UV detection

•

Adds the specificity of retention time to the analysis, essential if we are 12 using an internal standard of the same molecular weight

Which Ionization Technique to Chose? • Electrospray (ESI) – – – –

little or no heat applied excellent for thermally labile molecules polar (ionic) to relatively non-polar molecules LC flow: <1 to ~1000µL/min Most susceptable to matrix effects ie SUPPRESSION

• APCI & APPI – Heat applied to vapourize sample and mobile phase therefore not for thermally labile molecules! – medium to low polararity analytes – LC flow: 100 – 2000µL/min – Less prone to matrix effects

• Which technique gives the best response (sensitivity) for your analyte? 13

Methomyl

80000 70000

[M+H]+= 163

10000

348.1

163.1 186.1

20000

165.1

40000

70000

70000

400

600

800

[M+H]+= 163

m/z

Max: 95891

[M+H]+= 163

60000

50000

50000

40000

40000

30000

30000

20000

20000

10000

0 200

80000

60000

50000

90000

80000

163.1

60000

30000

Max: 3663

90000

10000

0

106.1 164.1

Max: 206617

APPI 163.1

APCI

347.1

90000

185.0

ESI

0 200

400

600

800

m/z

200

400

courtesy of Agilent

600

800

14

m/z

Quantitation in GC-MS and LC-MS • Full scan: – Extracted ion chromatogram – Specificity based on mass alone eg (M+H)+ – Poor sensitivity compared to SIM and SRM

• Selected Ion Monitoring (SIM) – Specificity based on mass alone – More sensitive than full scan as we are only monitoring a few ions rather than the full mass range

• Optimum: – Selected reaction monitoring (SRM, MRM) in MS/MS with QQQ – Specificity based on mass and unique fragmentation – MS/MS with ion trap/QTof – no SRM mode – Significant reduction of chemical noise (less sample preparation) – Best S/N (sensitivity) of all

15

Full Scan vs SIM vs SRM

Same sample injected in different MS modes - increased specificity and detectability!

16

Calibration Curve with Internal Std Peak Area Ratio

QC3

QC2

. QC1

. •

.

.

.

.

.

Peak area Ratio = area of analyte response area of ISTD response

Stds, samples,QC’s and blanks ran in same “batch”

. Analyte amount

The “batch”: – Blanks, std curve (S1-S8), 2QC samples (QC1 and QC2), ½ of the unknowns, 2QC samples (QC1 and QC3), ½ of the unknowns, 2QC samples (QC2 and QC3) 17

Calibration Curve with Internal Standard •

Blanks are very important: – matrix blank: where matrix containing none of the analytes is extracted using the same sample preparation scheme – Internal standard blank: to assess whether the ISTD causes a response in the analyte channel – Solvent blank: does the solvent used to re-dissolve the extracted sample interfere – in many cases interference determines limit of quantitation (LOQ) and not the absolute detectability (LOD)

•

QC’s – Samples of known concentration extracted along with unknowns to assess method performance 18

Calibration Curve – General form Absolute Signal Intensity or Peak Area ratio

Peak area Ratio = area of analyte response area of ISTD response

.

.

saturation

. . chemical bkgd or memory

.

.

.

Linear range

. adsorption

Noise level concentration or amount 19

Linear Dynamic Range • Some mass spectrometric methods are more linear than others: – EI has the greatest linearity, say 5 to 7 orders of magnitude – ESI is probably the poorest exhibiting ~ 3 orders of magnitude – APCI/APPI is a little better with ~ 4 orders of magnitude – Trapping instruments tend to have lower dynamic range than Q type instruments because they fill up ie space charging – TDC with MCP type detectors used in Tof instruments are usually limited to ~3 orders of magnitude, whereas electron multipliers are much better ~ 6 – Linear or Quadratic regression is used to “fit” the calibration curve

20

Ion Suppression • Suppression: – Competition and interference with analyte ionization resulting in decreased number of [M+H]+ or [M-H]- ions

• Caused by salts that form adducts and clusters with analyte: – Strong bases in positive mode eg Triethylamine (TEA) – Acids in negative mode eg Trifluoracetic acid (TFA) – Non-volatile buffers (phosphate) and ion pair reagents – Non-covalent dimers [2M+H]+, trimers.... – Metal ion complexes, e.g. [2M+Cu]+, Cu+ eg from LC equipment – Other conatminants in the system eg PEG, platicizers, residual matrix species – Coeluting analytes with different proton affinities that is, analytes that compete for protons 21

Ion Suppression: Solutions •

Dilute sample (1:10+) before or after sample preparation

•

Select ISTD to coelute with analyte: - to achieve similar matrix effect for analyte and ISTD (compensation) - ideally the stable isotopically labeled analyte (2H, 13C, 15N) - force chemical analogue into coelution with analyte - individual ISTD for each analyte, if analytes are chromatographically resolved

•

Modify LC mobile phase composition, change stationary phase or perform a more extensive sample clean-up

•

In general, the more complex the sample, the more likely it is that chromatography will improve your quantitative method

•

Try APCI (or APPI) instead of ESI (if analyte is thermally stable) 22

Hints on Method Development •

Some form of sample preparation is critically important for rugged and robust quantitative measurements: – Solvent (liquid/liquid) extraction – Solid phase extraction

•

Prepare (external) calibration standards in same matrix as samples

•

Internal Standard should match analyte structure as closely as possible - goal is coelution of ISTD and analyte

•

Check mass spectrometric interference with coeluting compounds – interferences from analyte isotope peaks (eg Cl, Br, S) – metabolites and their fragmentation to the analyte

•

perform as much chromatography as is necessary 23

Ion Focusing •

Confine ions from dispersive environments and focus them spatially, temporally and/or energetically at a point in space or on a relevant detector to improve MS response

•

We can focus ions because their energy and/or the momentum is changeable – remember charged particles in an electric field

•

Ion optics need to be in a vacuum (greatest effect)

•

Accomplished using electrostatic lenses (plates, orifices, grids) or multipoles placed in or close to the ion beam to cause ions to be deflected/focused

•

Lens stacks for fast moving ions – While a charged particle is in an electric field force acts upon it. The faster the particle the smaller the accumulated impulse (rate of change of momentum = mΔv) – These plates can be stacked with as many as 30 sets to effect efficient focusing – very complicated

•

Simple lenses for slow moving ions – Einzel lens

1

Electrostatic lenses • Basic types – Immersion/aperture lenses – lens stacks and grids (shown below) – Unipotential lenses – Einzel lens

Ion Beam

• •

All lenses are convergent Focal point is independent of m/z

2

Einzel lens

• used in Tof’s, quads, ion entrance optics • Focuses ions without changing the kinetic energy 3

Ion Focusing •

quadrupoles (also hexapoles and octopoles) in Rf only mode ie no DC voltage (a=0) act as wide band pass filters for ions - very efficient especially at higher pressures

•

Collisional cooling of ions traveling slowly through Rf only multipoles with some collision gas present reduces the axial motion of ions and therefore increases the mass resolution (used in QTof instruments)

Without collisional cooling

With collisional cooling

4

Ion Detection

• The Faraday Cup: • The ion beam impacts the FC and deposit their charge – the resulting current flowing away from the FC results in a voltage that can be measured • Used rarely except in isotope ratio MS ie very accurate but not very sensitive 5

Discrete Dynode Electron Multiplier •

When an energetic particle (a positive or negative ion) impinges on the surface of a metal or semiconductor a number of 20 electrons are emitted from the surface

•

The ease of such emission is determined by the electron work function (we) of the respective material eg BeCu alloy oxide (we = 2.4eV) and the velocity of the impacting particle

•

The higher the velocity of the impacting particle and the lower the we the larger the number of 20 electrons formed: – larger, slow moving ions will yield fewer 20 electrons than small, fast moving ions which leads to high mass discrimination – Can be solved by employing a post-acceleration conversion dynode (PACD)

6

Discrete Dynode Electron Multiplier - no PACD

•

If an electrode opposite to the location of 10 emission is held at a more positive potential, then all emitted electrons will be accelerated towards and hit the surface where they in turn cause the release of even more electrons

•

With 12 to 18 discrete dynode stages held at about 100V more positive 7 potential allows the ion signal to be detected by a sensitive preamplifier

Discrete Dynode Electron Multiplier – no PACD • Normally have a gain of 106 – 107 (when new) • Have a lifetime or around 1 – 2 years before having to be replaced • Must be kept at high vacuum as the emission layers can be harmed by oxygen • over time and with use the first dynode surface becomes damaged and is therefore less efficient at releasing electrons ie gain is reduced • This can be compensated for to some extent by increasing the voltage difference between the discrete dynodes however eventually the multiplier must be replaced 8

Electron Multiplier with Post-Acceleration Conversion Dynode (ion beam) 20 electrons

Dynodes

Electron collector

Further

Conversion Dynode

Amplification and recording

Potential gradient

• All ions are accelerated to high velocity by the Post-Acceleration Conversion Dynode • All ions release ~ the same number of 20 electrons therefore high mass discrimination greatly reduced! 9

Channeltron Multiplier – continnuous dynode

• • • • • •

The inner surface is composed of a layer of silicon dioxide over a conductive layer of lead oxide This surface has sufficiently high resistance to withstand the ~1.5 to 2.5kV placed across the multiplier The high voltage drops continuously from the entrance to the exit of the tube Gain of around 105 to 106 Ages in a similar fashion to a discrete electron multiplier An array of linear channeltron multipliers is called a microchannel plate (MCP) –10 each multiplier is of the order of a few micrometers in size

Microchannel plate (MCP) Ions

•

Channels are inclined by some degrees from the perpendicular so that ions strike the inner surface and cause 20 electron emission

•

Gain is only ~ 103 to 104 so sometimes 2 MCP’s are sandwiched together so that the small offset angle of the channels oppose each other to form what is called a chevron plate 11

Reflectron Tof Head

Tof Pusher

MCP

12 Courtesy of Waters/Micromass

Photomultiplier with Conversion Dynode

• • •

The 20 electrons from the conversion dynode strike a phosphor which emits photons The photomultiplier detector has a much longer lifetime than an electron multiplier as it is a sealed device (under vacuum) 13 107 gain

Photomultiplier with Conversion Dynode •

Advantages: –

•

Long lifetime and gain does not decrease with time (compared with an electron multiplier)

Disadvantages: –

–

dark current – when an intense signal ie many electrons strike the phosphor it can take some time for the phosphor to stop releasing photons even when no electrons are hitting it Causes a signal to be recorded when there is no signal

14

Ionization Technique Summary Ionization

Polarity

Thermally

Mwt

Examples

EI

Low

stable

<1,000

Non-ionic organics

CI

Low

stable

<1,000

Non-ionic organics

FI

Low to med

stable

<1,000

Non-ionic organics

FD

Low to med

labile

<2,000

Non-ionic organics, peptides, organometallics, carbohydrates

FAB/LSIMS

Med to high

labile

<20,000

Polar/ionic organics, peptides, biomolecules, organometallics

MALDI

Med to high

labile

<200,000

Peptides, proteins, RNA, DNA, polymers

ESI

Med to high

labile

<100,000

Polar/ionic organics, peptides, proteins, biomolecules, polymers, organometallics

APCI/APPI

Low to med

stable

<1,000

Non-ionic organics

1

MS Experiment Summary Experiment Ionization

Instrument Type

Comment

Batch Introduction

All

All

DIP (EI, CI, FAB/LSIMS and MALDI) Infusion (ESI and CF-FAB)

Low resn

all

Q, QQQ, EB/BE, linear and 3D IT, linear Tof

Mass spectrum (some database searching capability for EI)

High resn

EI, FAB/LSIMS, MALDI, ESI

EB/BE, reflectron Tof and FTICRMS

Accurate mass yields elemental composition

MS/MS

all

QQQ, linear and 3D IT, QTof, and FTICRMS (other hybrids)

Structural elucidation and quantitation (SRM/MRM)

GC/MS

EI, CI, FI

EB/BE, Q, QQQ, 3D-IT, Tof

Complex mixture analysis of semi-volatile species

LC/MS

ESI, APCI, APPI

Complex mixture analysis Q, QQQ, linear and 3D IT, Tof, QTof, FTICRMS of more polar species 2

Mass Analyzers Summary Analyzer

Ion Detection

Ionization Modes

Sector (BE/EB)

continuous

EI, CI, FAB/LSIMS

Quadrupole (Q and QQQ)

continuous

Ion Trap (linear and 3D)

Mass Range

Resolution

~15,000

Variable up to 100,000

EI, CI, ESI, APCI, APPI

4,000

Normally 3,000 but 10,000 possible

pulsed

EI, CI, ESI, APCI, APPI, AP-MALDI

4,000

~3,000

FTICRMS

pulsed

EI, CI, ESI, APCI, APPI, MALDI

106

105 to 106

TOF

pulsed

all

Unlimited (L) ~1,000 (L) 10,000 (R) Up to 20,000 (R)

L = Linear Tof and R = Reflectron Tof

3