Mass Spectroscopy

In MS, compounds are ionized, ionized molecule fragments into smaller ions/radicals. The positively charged fragments produced are separated based on their nominal_mass/charge (m/z) ratio.

Mass Spectroscopy

ionization M+.

M

fragmentation M+1 + M+.2+..+N1+ N2. + …..

Parent ion

daughter ions, radicals, neutral

Most of the ions has z=+1; m/z = mass of the fragment. A plot of relative abundance vs m/z of all charged particles is presented as the MS spectrum.

Fragmentation:

Base peak Molecular ion M+.

M 1 + + N1 . M+.

M+

M1+

+N

M2+. + N2 M+. N. N Nominal mass

Radical ion (odd e) Neutral radical (odd e) Neutral (even e)

M+ (even e) would not break up into a radical ion….

Spectrum presented as a bar graph.

Isotope peaks: In mass spectroscopy mass of actual fragments generated are determined. Therefore fragments with different isotopes are distinguished, e.g. Lead metal.

Actual signal has peaks with a line width. For molecular fragments, the isotope peak abundance is dependent on the molecular constitution and the natural isotope abundance of the constituent elements.

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In mass spectroscopy the masses of individual ‘ions’ are measured. Mass and ‘abundance’ of each isotopic composition is measured!! – not the average molecular mass.

Isotope peaks Mass Spectrometer: Sample Introduction Create gas-phase ions of sample Separate ions in space or time basis based on m/z ratio accomplished by mass analyzers. Detect of the quantity of ions of each m/z ratio

Cl-CH2-CH2-S-CH2-CH2-Cl

Ionization: Electron Ionization (Electron Impact, EI) Electrospray Matrix Assisted Laser Desorption Ionization (MALDI) Atmospheric Pressure Chemical Ionization (APCI) Fast Atom Bombardment (FAB) Chemical Ionization (CI) Inductively Coupled Plasma (ICP)

Mass Analyzers: Magnetic Sector Mass Analyzer (Single/Double Focusing Tandem Mass Spectrometry Quadrupole Quadrupole Ion Trap Fourier-Transform Mass Spectrometry (FTMS) Time-of-flight (TOF)

Ion Detection: Faraday Cup Electron Multiplier Photomultiplier Conversion Dynode

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Electron impact ionization Magnetic sector separation single focusing

Electron impact ionization (EI)

Mass Spectrometer 70V

-

+

V Ion optics Ekin = zV = mv2/2

Evacuated System - 10-6 torr

70eV – high energy electrons, molecular ion - very energetic, low/no abundance.

Volatilized compound is ionized by electron impact. An electron beam is generated by a accelerating the electrons from a heated filament through an applied voltage. The electron energy is defined by the potential difference between the filament and the source housing and is usually set to 70 eV (~1.12x10-17J). A field keeps the electron beam focused across the ion source and onto a trap (collimating magnets). Upon impact with a 70 eV electron, the gaseous molecule may lose one of its electrons to become a positively charged radical ion, daughter ions, etc.

All ions are subsequently accelerated out of the ion source by an electric field produced by the potential difference applied to the ion source and a grounded Electrode, V. A 'repeller' serves to define the field within the ion source. Depending on the lifetime of the excited state, fragmentation will either take place in the ion source giving rise to stable fragment ions, or on the way to the detector, producing metastable ions.

Magnetic sector mass analyzer: Each m/z beam follows it’s own path (r) for a given B and V in the magnetic sector (60o/900). V

B

r

m/z = (eB2r2)/(2V) note: slits

Ion source accelerates ions to a KE KE = ½ mv2 = zeV In the magnet

For specific V and B ions of unique m/z pass thro’ the magnetic sector and reaches the stationary detector. Variations of V and/or B causes fragments of different m/z value to reach the detector.

F = mv2 /r = Bzev,

Upon rearrangement r = mv/zeB = (2Vm/ze)1/2/B

m/z = (eB2r2)/(2V)

Usually B is scanned to allow different m/z’s to reach the detector sequentially generating the complete mass spectrum.

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Magnetic Sector Mass Analyzer: Double Focusing (EB)

E

B

+ -

mv 2 mv 2 and zeV= r 2 2V m r= ; r independent of in E; focussing! z E zeE =

The m/z ratio of the ions that reach the detector can be varied by scanning either the magnetic field (B) or the applied voltage of the ion optics (V). i.e. by varying the voltage or magnetic field of the magnetic-sector analyzer, the individual ion beams are separable spatially, radius of curvature Is held constant.

The distribution of a given mass by way of energy distribution of kinetic energy refined.

Resolving power 1.

Actual signal has peaks with a line width. Imposes a limitation on the resolvability of consecutive peaks

10%

2.

State the method of calculation when expressing resolving power, and the position of the lower peak.

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Electrons in molecules occupy molecular orbitals and hence acquire the energy associated with such orbitals. To remove electrons from such orbitals and ionize the molecule energy is required.

Ionization:

The energy required depends on the orbital of electron occupation namely the HOMO. Thus the ease of ionization will depend on the “types of electrons” in the molecule.

The molecular ion (dominant) is formed by the removal of the least tightly bound electron.

Chemical ionization (CI): Interaction of the molecule M with a reactive ionized reagent species (gaseous Bronsted acids). E.g.., EI of methane, generates CH4+· which then reacts to give the Bronsted acid CH5+;

M+. nearly nonexistent.

CH4+· + CH4 → CH5+ + CH3· If M in the source has a higher proton affinity than CH4, the protonated species MH+ will be formed by the exothermic reaction. M + CH5+ → MH+ + CH4

Abundant M+.

CI is a softer ionization process.

Fast Atom Bombardment Ionization: The sample droplet is bombarded with energetic atoms (Ar, Xe) of 8-10 keV kinetic energy. Ions (e.g., Cs+) can be used as the bombarding particle in a similar technique termed liquid secondary ion mass spectrometry (LSIMS) Beam collides with the sample and matrix molecules, producing positive and negative sample-related ions that can be accelerated into the mass spectrometer.

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Quadrupole Mass Analyzer (spectrometer):

Fast Atom Bombardment Used for polar organic compounds, acidic and basic functional groups. Basic groups run well in positive ionization mode and acidic groups run well in negative ionization mode. FAB analytes: peptides, proteins, fatty acids, organometallics, surfactants, carbohydrates, antibiotics, and gangliosides.

4 parallel, polished metal rods +[U+Vcos(ωt)] - [U+Vcosωt]

y z x

Diagonal electrodes have potentials of the same sign U= DC voltage, V=AC voltage, ω= angular velocity of alternating voltage

Quadrupole Mass Analyzer (Spectrometer): Ions oscillate under the influence of the variable fields. Combined DC and RF potentials on the quadrupole rods passes only a selected m/z ratio (resonant ion) at a time. All other ions acquire unstable trajectories through the quadrupole mass analyzer. The mass spectrum is obtained by varying the voltages on the rods and monitoring which ions pass through the quadrupole rods.

The solution of equations of motion of ions traveling through a QM analyzer shows that for an ion with a particular m/z to pass through, certain combinations of U and V must be obtained. Varying rod voltages (scanning the spectrum): a. vary ω while holding U and V constant b. vary U and V but keep the ratio U/V fixed If U and V are scanned such that U/V = constant, then successive detection of ions of different m/z is achieved.

Quadrupole mass (QM) analyzer is a "mass filter".

Two functions a and q define a stable trajectory for which ions do not collide with the rods across a range of values of U and V.

−4 zeU a= 2 2 2 m r0 ω

−2 zeV q= 2 2 2 m r0 ω

Graphically, e.g. the three stability curves represent values of U and V for which the masses m1, m2 and m3 have stable trajectories through the quadrupole. Only those mass values above the operating line transmits.

In principle QM can be operated for a range of U and V values. (U)

a 2U = q V

(V)

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The resolution is determined by the magnitude of U/V ratio. Resolution of the mass analyzer can be increased by increasing the slope of the curve U/V = const., and that if U = 0 then ions of all m/z are transmitted. Because quadrupoles operate at lower voltages, they can be scanned at faster rates (~1000 a.m.u./s) than magnet based Spectrometers. QMs are better detectors for LC-MS and GC-MS implementation.

MS/MS

MS1

Time-of-Flight Mass Analyzers (spectrometer):

MS2 Dissociation region

TOF measures the mass-dependent time required for ions of different masses to move from the ion source to the detector. This requires that at the starting time t=0, (time ions leaves the ion source) to be well-defined.

QqQ Scan with MS1 (only) turned on – entire MS spectrum. Set MS1 to filter fragment of interest, dissociate further in q collisionally and mass analyze by scanning with MS2.

Ions are created by a pulsed method (MALDI), or by rapid electric field switching that serves as a 'gate' to release the ions from the ion source in a very short time.

L

KE = zeV =

mv 2 2

v=

L t

m 2Vt 2 = 2 ze L

t=L

1 2eV

m z

Reflection time-of-flight mass spectrometer

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Parent/Molecular Peak M:

High Resolution MS:

An molecular ion that has not lost/gained atoms. The nominal mass of which is calculated with the mass numbers of the predominant isotopes of atoms.

Using mass number for isotopes of atoms is approximate. Actual mass of a given isotope deviates this integer by a small but unique amount (∆E = ∆mc2). Relative to 12C at 12.0000000, the isotopic mass of 16O is 15.9949146 amu., etc.

Base peak: Base peak is the peak from the most abundant ion, which is often the most stable ion.

High resolution mass spectrometers that can determine m/z values accurately to four/more decimal places, making it possible to distinguish different molecular formulas having the same nominal mass.

m/z=74

Very short list.

MF Isotope

Accurate Mass

Unsaturation

C2H2O3

2.0

CH2N2O2

2.0

1-H 2-H

1.007825 2.014102

12-C 13-C

12.000000000 13.0033548

C2H3FN2

2.0

14-N 15-N

14.0030740 15.0001090

C3H6O2

1.0

16-O 17-O 18-O

15.9949146 16.9991306 17.9991594

C2H6N2O

1.0

C4H7F

1.0

C4H10O

0.0

C3H10N2

0.0

C6H2

6.0

C3H3FO

2.0

http://www.chem.queensu.ca/FACILITIES/NMR/nmr/mass-spec/mstable3.htm

m/z=74

MF

Unsaturation

Exact Mass

C2H2O3

2.0

74.00040

CH2N2O2

2.0

74.01163

C6H2

6.0

74.01565

C3H3FO

2.0

74.01679

C2H3FN2

2.0

74.02803

C3H6O2

1.0

74.03678

C2H6N2O

1.0

74.04801

C4H7F

1.0

74.05318

C4H10O

0.0

74.07316

C3H10N2

0.0

74.08440

MF finder

Isotope Peaks The peaks from the isotopes. The intensity ratios (relative intensities) in the isotope patterns are arising from the natural abundance of the isotopes, thus are valuable to ascertain the atomic composition of ions. M+1 peaks are primarily due the presence of 13C in the sample. M+2, M+4, .. indicative of presence of Br, Cl, S; (79Br:81Br = 1:1, 35Cl : 37Cl = 3:1)

8

Isotope peak abundance depends on the molecular constitution. Example, halogens Cl, Br.

100 0.801 4.52

Calculating isotope peak abundances (%) to confirm fragment and parent peaks.

X=M

Parent/Molecular Peak M: an ion that has not lost/gained atoms (odd electron fragment, U=integer; reverse not true). Parent Peak leads to molecular formula. Molecular formula leads to the structural (/partial) features of the possible molecular structure. Unsaturation can be calculated from the molecular formula of the parent ion.

U = R + DB + 2TB = c − h / 2 + n / 2 + 1 c = #C & Si E.g. %M+1=(0.012nH+1.08nC+0.369nN+0.038nO+5.08nSi+0.801nS)100

h = #H & halogens n = #N, P, As..

Rule of Thirteen: Used to generate possible formula for a given molecular mass. imax is the total number of different elements in the composition, Ni the number of atoms of element i, and Vi is the valence of atom i.

1. Generate a base formula; M r =n+ CnHn+r 13 13 2. Calculate the index of H deficiency, U for the base formula. U=

n−r+2 2

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3. If needs to find the formula with nO of O for the same M; New formula = Base formula + nO O – nOC – 4nOH which changes U to U+nO ; 4. If needs to find the formula with nN of N for the same M, New formula = Base formula + nN N – nN C – 2nN H and recalculate U; Fractional U’s – unlikely formula. U < 0 is an impossible combination, indicates likely presence of O and N.

Useful Links

http://www.colby.edu/chemistry/NMR/NMR.html http://www.colby.edu/chemistry/PChem/Fragment.html http://www.chemcalc.org/ Main Page MF finder

http://www.chem.uni-potsdam.de/tools/index.html

Example 1 (M+1)/M ratio is paticularly useful to estimate the #C in the species.

#C ≤

%M + 1 × 100 %M

(next slide)

m/z 64 65 66

only peaks Relative abundance(normalized) 100.0 0.9±0.2 5.0±0.5

Nitrogen Rule: Many peaks can be ruled out as impossible simply on the grounds of reasonable structure requirements. Molecule of even nominal mass must contain zero or even number of N atoms. An odd numbered nominal mass requires an odd number of N.

Example 2 m/z 58 59 60 43 44 45

http://www.colby.edu/chemistry/PChem/Fragment.html MF finder

Organic compound Relative abundance 12.0 0.5±0.2 0.0±0.2

Normalized 100.0 4.2±1.7 0.0±0.2

100.0 3.3±0.3 0.0±0.2

. . http://www.colby.edu/chemistry/PChem/Fragment.html MF finder

Aromatic parent ions – large abundance.

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Fragmentation: Fragmentation leads to smaller ions by the cleaving of parts of the molecule. Unreasonable losses from molecular ion:

Fragmentation pattern: From the fragment losses the parent peak may be predicted by working back.

M - [3-14] and M – [21-26] are unreasonable losses. (next slide)

n-decane

Reasonable losses from molecular ion Neutral fragments expelled by simple cleavage OE+• → EE+ + OE• Neutral fragments expelled by multi-centered fragments OE+• → OE+• + EE See handout

Notation:

+.

-29

two electron movement

57 29

cleavage here

Unique isotope peak patterns are useful for the analysis.

one electron movement

Unique isotope peak patterns are useful for the analysis.

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m/z=100

Fragmentation: Nominal mass of parent ion containing C, H, O, S, Si, P and halogens is even. odd e m/z ion (homolytic) cleavage R' R+. even m/z, no N

?

- neutral species, e.g. CO, water, .. even e m/z ion

Structural isomers differentiation

Fig. E3 M = odd, 17 M+1

0.4%

one N no C

Nominal mass 17 14 – 1N 3 3H

Consistent with 1N M M+1

use table

NH3 H

+N

H

H

Fig. E3

PE1

PE1

M = even M+1

M

1.1%

1C

Nominal mass 16 12 – 1C 4 4H CH4

M+1

12

PE2

PE2 1 O present

0.9 21.1

2 H present

100.0 0.06

M+1, M+2 peaks confirm H2O.

0.2

M+1

no C

Data:Contains 3 elements, one is F.

Nominal mass 34 12 – 1 C 22 19 - 1 F 3 3H

PE3

PE3

CH3F

H H

M+1

1C

Further 34-15=19

F H

15

H 3C

Fragment intensities depend on the stability of the ion and the probability of formation.

29 43

57 71

CH 3

note: reasonable losses

13

H3C

43 CH3

57

H3C

Electron repelling methyl group stabilizes the carbocation.

CH3

29 43

57 71

CH3

CH3 H3C

CH3

Four ways to form carbocation

CH3

Stabilization of carbocations: 1. Alkenes frequently undergo fragmentations that yield allylic cations

57

2. Carbon-carbon bonds next to an atom with an unshared electron pair usually break readily because the resulting cation is resonance stabilized.

Z =N, 0, or S; R may also be H.

3. Carbon-carbon bonds next to the carbonyl group of an aldehyde or ketone break readily because resonance-stabilized ions called acylium ions are produced.

4. Alkyl-substituted benzenes undergo loss of a hydrogen atom or methyl group to yield the relatively stable tropylium ion. This fragmentation gives a prominent peak (sometimes the base peak) at m/e 91.

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5. Substituted benzenes also lose their substituent and yield a phenyl cation at m/e 77.

Y =halogen,-NO2.-Keto group,-R. etc.

INTENSITY PE7 m/e (AS PERCENT OF BASE PEAK) 73 8.0 14 36 - 3C from M+1 38.6 15 37 16.3 18 39.7 1N odd M 1428 23.4 23 29 Recalculating 46.6 42 1O from M+2 16intensities to 10.7 43 normalize 7 7H 44

100.0 (base)

73

86.1 M+.

74

3.2

3.72

75

0.2

0.23

C3H7NO 1.0 100

C3H7NO Use this resource

Mass spectrum for Problem E. 7. FIG. E.8

INTENSITY m/e (AS PERCENT OF BASE PEAK) 14

8.0

15

38.6

18

16.3

28

39.7

29

23.4

42

46.6

43

10.7

44

100.0 (base)

73

86.1 M+.

74

3.2

3.72

75

0.2

0.23

C3H7NO No halogens Probably 1 O; (M+2) 4 C ; M+1

Confirming MF C3H7NO 3.81 0.25 73.0528 o 1 OH

H3C N 73-31

O

H3C

Nominal mass 72 48 – 4 C 24 16 - 1 O 8 8H

N

42 31

NH2 H3C

100

O

44 Calculation; C4H8O

Mass spectrum for Problem E. 7. FIG. E.8

4.56 0.28 72.0575 o 1

Use this resource

Cleavage of Two bonds: 1. Alcohols frequently show a prominent peak at M - 18. This corresponds to the loss of a molecule of water.

O H3C

H3C

CH3

O OH H3C

H3C OH

CH2

72-44=28 loss of 28 from M to form base peak

2. Cycloalkenes can undergo a retro-Diels-Alder reaction that produces an alkene and an alkadienyl radical cation.

No straight forward cleavage possible; probably a rearrangement occurs before cleavage.

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3. Carbonyl compounds with a hydrogen on their -/-carbon undergo a fragmentation called the McLafferty rearrangement.

The compound is butanal. m/z = 44 arises from the McLafferty rearrangement.

m/z=29 arises from acylium ion. Y may be R, H, OR, OH, etc. In addition to these reactions, we frequently find peaks in mass spectra that result from the elimination of other small stable neutral molecules, for example, N2, NH3, CO,HCN, H2S, alcohols, and alkenes.

M+=116 present MS of which compound? O

Isopropyl butyl ether

O

O

Propyl butyl ether

+.

+

O

O

73 101

101

73

O

+.

+

O

87 73

+

O

Chromatography – Mass Spectroscopy

+

87

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GC-MS, LC_MS are hyphenated analytical techniques. Two techniques are combined to form a single method for analyzing mixtures of chemicals. Chromatography separates the components and MS detects and characterizes each of the components. Combination of the techniques allows both qualitative and quantitative evaluation of analytical samples. The MS spectrometer can be highly selective for analyte of interest.

Chromatograph Output

Data Output

Inlet

Data System

Ion Source

Mass Analyzer

Electrospray API Vacuum Pumps

Each analyte’s MS produced

Ion Detector

QM All fragment ions/or single ion detected and (total) ion current determined

Total (reconstructed) ion current vs time

http://www.gmu.edu/departments/SRIF/tutorial/gcd/gc-ms2.htm

GC,LC/MS takes data in three dimensions simultaneously, recording the number of ions created along with their masses over time. This information is generally represented by examining the total ion chromatogram (TIC) and ‘slicing’ along the third dimension (m/z) of a given peak to look at the mass spectrum at a chosen time.

t

MS – analyte 1 MS – analyte 2 MS – analyte 3

t 3D data set

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MS requires high vacuum ~10-3 – 10-6 torr. The eluate from a chromatographic system has much more carrier (gas/eluent) than the analyte. If eluate from the chromatograph is allowed to enter directly into the MS it would overwhelm the vacuum system (especially in LC – liquid vaporizes into a large volume). To alleviate the strain on the system, different strategies are employed at the sample introduction/ionization stage.

Electrospray interface:

Coaxial flow

Strong electric field + nebulization; ions vaporize

For positive ion MS

Low collisional dissociation; minimal fragmentation. Positive ions accelerate and collide with N2 molecules; few fragments form. Changing skimmer voltage to larger –ve would produce more fragmentation.

Atmospheric Pressure Chemical Ionization API-CI

Coaxial flow Electrons formed at corona, injects into aerosol.

This technique produces single charged ions. Little fragmentation, less structural information.

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Selected ion monitoring possible with the quadrupole mass analyzer by appropriate tuning.

N 2 + e → N 2+. + 2e N 2+. + 2 N 2 → N 4+. + N 2 +. 4

N + H 2 O → 2 N 2 + H 2O

+.

Usually total ion current monitored ion current from all ions from an analyte is constructed.

H 2O +. + H 2O → OH . + H 3O + H 3O + + nH 2O → H 3O + ( H 2O )n H 3O + ( H 2O ) n + M → MH + + (n + 1) H 2O

Selected ion chromatogram QM filter tuned to select the ion

Selectivity and S/N ratio increased markedly with QqQ triple quad arrangement.

Quadrupole Ion Trap Mass Analyzer.

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MALDI

MALDI

L

KE = zeV =

mv 2 2

v=

L t

m 2Vt 2 = 2 ze L

t=L

1 2eV

m z

Lasers are used to deliver a focused high density of monochromatic radiation to a sample target, which is vaporized and ionized. The yield of ions is often increased by using a secondary ion source or a matrix.

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