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Definition of Mass Spectrometry

Mass spectrometry (MS) :

An analytical technique by using mass spectrometry for the determination of the composition of a sample or molecule and elucidation of the chemical structures of molecules, such as peptides and other chemical compounds.

Mass spectrometry has been described as the smallest scale in the world, not because of the mass spectrometer’s size but because of the size of what it weighs -- molecules.

• Molecular weight

• Molecular formula (HRMS)

• Structure (from

fragmentation fingerprint)

• Isotopic incorporation /

distribution

• Protein sequence (MS-MS)

What information can be determined?

Rule of ThirteenCalculating Mass

• The “Rule of Thirteen” can be used to identify possible molecular formulas for an unknown hydrocarbon, CnHm.

– Step 1: n = M+/13 (integer only, use remainder in step 2)

– Step 2: m = n + remainder from step 1

Rule of Thirteen• Example: The formula for a hydrocarbon with

M+ =106 can be found:

– Step 1: n = 106/13 = 8 (R = 2)

– Step 2: m = 8 + 2 = 10

– Formula: C8H10

Nitrogen Rule

• This rule states that if a compound has an even no.of N atoms (or no N atom) , its molecular ion will appear at an even mass value.

• On the other hand , a molecule with an odd no.of N atom will form a molecular ion with an odd mass.

• The N rule stems from the fact that N , although it has an even mass , has an odd numbered valence.

• Consequently , an extra hydrogen atom is included as a part of a molecule , giving it an odd mass.

Nitrogen Rule

• To picture this effect, consider Ehtylamine, C2H5NH2.

• This substance has one N atom, and its mass is an odd number (45), whereas EhtyleneDiamine H2N-CH2-CH2-NH2, has 2 N atoms, and its mass is an even number (60).

Basic principle of working

• Find a way to “charge” an atom or molecule (ionization)

• Place charged atom or molecule in a magnetic field or subject it to an electric field and measure its speed or radius of curvature relative to its mass-to-charge ratio (mass analyzer)

• Detect ions using microchannel plate

Mass Spectrometry

• An outline of what happens in a mass spectrometer

• Atoms can be deflected by magnetic fields - provided the atom is first turned into an ion. Electrically charged particles are affected by a magnetic field although electrically neutral ones aren't.

»The sequence is :

Mass SpectrometryIonization

Slightly +veTo repel +ve ions Usually carry +1 chrge

Difficult to remove another electron

Mass Spectrometry

Mass Spectrometry

Lightest m/z ratio

Bulkiest m/z ratio Optimum m/z ratio

Assume all streams having +1 charge

Mass SpectrometryDetection

Creating electron vacancyCausing current to flow which is measured

Gas Phase/Ionize

Detector

Separate Based on Mass/Charge

Sample

Elements to Mass Spectrometry(J.J. Thomson ~ 1910)

• Electron Impact (EI)• Chemical Ionization (CI)• Electrospray (ESI)• Atmospheric Pressure Chemical

Ionization (APCI)• Photo-ionization (APPI)• Matrix Assisted Laser Desorption

and Ionization (MALDI)

14

Detector quiet

m/z too small

Mass SpectrometryThe Mass Spectrometer

Fundamental operating principleDetermine mass by manipulating flight path of an ion in a magnetic field

sampleintroduction

Measure ionmass-to-charge ratio

(m/z)

Detector

Ionization

Electron gun

+ -

Acceleratorplates

Magnet

m/z just right

Detector fires

Ionization: X + e- X+. + 2 e-

m/z too large

Detector quiet

15

Isotopes

Aston mass spectrum of neon (1919)•Ne empirical atomic weight = 20.2 amu•Ne mass spectrum: predict single peak at m/z = 20.2

Results m/z relative intensity 20.2 no peak 20.0 90% 22.0 10%

Conclusions•Neon is a mixture of isotopes•Weighted average: (90% x 20.0 amu) + (10.0% x 22.0 amu) = 20.2 amu

Isotopes: atoms with same number of protons and same number of electrons but different numbers of neutrons

•Nobel Prize in Chemistry 1922 to Aston for discovery of stable element isotopes

16

The Mass SpectrumOrigin of Relative Ion Abundances

M contributors M+1 contributors M+2 contributors

IsotopeNatural

AbundanceIsotope

NaturalAbundance

IsotopeNatural

Abundance1H 99.9855% 2H 0.015% 3H ppm12C 98.893 13C 1.107 14C ppm14N 99.634 15N 0.36616O 99.759 17O 0.037 18O 0.20419F 100.032S 95.0 33S 0.76 34S 4.22

35Cl 75.77 37Cl 24.2379Br 50.69 81Br 49.31127I 100.0

Ionization Methods

• Electron bomb Ionization (EI)

• Chemical Ionization (CI)

• Field ionization (FI)

• Matrix Assisted Laser Desorption Ionization (MALDI)

• Fast atom bombardment (FAB)

• Electro Spray Ionization (ESI)

But when to use which ionization technique?

Ion Source Depends on Sample

Solid Sample Liquid Sample Gas Sample

EICIAPCIMALDI ESIAPPI

Make into Solution ?Make into Solid ? Turn into Gas?

Chemical Properties of analyte in gas phase ?

Chemical Properties of analyte in solution phase ?

Electron Impact

M

e-e- e-

M(g) + e- M+(g) + 2e-

This reaction creates the molecular ion so is very useful. However, the excess energy from the electron can cause the molecular ion to fall apart:

s0

s1

IP

s0

s1

IP2NeutralMolecule

Ionized Molecule

Excess Energy get redistributed throughout ion to cause fragmentation.

Electron Impact

A+M

e-e- e-

M(g) + e- M+(g) + 2e-

M+(g) A+

Fragment 1 (g) + BFragment 2 (g)•Electron energy is chosen by compromise. •Fragment Information is useful. It can help structural determination. However, many ions produce only fragments with no molecular ion remaining. Molecular ion are often very unstable.•70 eV “Classical Spectra” to be used for comparisons

BB

Hard ionization

Gas-phase molecules enter source through heated probe or

GC column

70 eV electrons bombard molecules forming M+* ions that fragment in unique reproducible way to form a collection of fragment ions

EI spectra can be matched to library stds CI (soft ionization)

Properties of EI

Chemical Ionization

• EI is not appropriate for some molecules (it causes too much fragmentation)

• Instead, ionize a reagent gas (by EI) then react it with a analyte molecules

• Typically use methane or ammonia for reagent gas

Advantages

Parent IonInterface to GC

Insoluble SamplesCI is lower energy process than EI

Disadvantages

No Fragment LibraryNeed Volatile Sample

Need Thermal StabilityLow Mass Compounds

(<1000 amu)

Properties of CI

CI: Form Reagent Ions First

• For Example - Methane CI1. electron ionization of CH4:

• CH4 + e- CH4+ + 2e-

– Fragmentation forms CH3+, CH2

+, CH+

2. ion-molecule reactions create stable reagent ions:• CH4

+ + CH4 CH3 + CH5+

• CH3+ + CH4 H2 + C2H5

+ – CH5

+ and C2H5+ are the dominant methane CI reagent ions

Methane CI Reagent Ions

– Ions at m/z 17, 29, and 41 are from methane; • H3O+ is also formed from water vapor in the vacuum

system

Field ionization (FI)In field ionization, a high-potential electric field is applied to an emitter with a sharp surface, such

as a razor blade, or more commonly, a filament from which tiny "whiskers" have formed. This results in a very high electric field which can result in ionization of gaseous molecules of the analyte. Mass spectra produced by FI have little or no fragmentation. They are dominated by

molecular radical cations M+. and less often, protonated molecules.

Probe+

++ +

+

++

+++

++

+

Ionizationd<1mm

Probe

Field ionization (FI)

• Application:• FD/FI being used for analysis of polar and nonvolatile analytes such as polymers and

biological molecules. • However, FD/FI remains one of the only ionization techniques that can produce

simple mass spectra with molecular information from hydrocarbons and other particular analytes.

• The most commonly encountered application of FD/FI at the present time is the analysis of complex mixtures of hydrocarbons such as that found in petroleum fractions.

• Difference• There are three practical differences between CI and FI: there is less fragmentation in

FI• There is no high-resolution FI, and FI is less sensitive. • Sensitivity is not an issue unless there is an extremely small amount of sample. FI can

be performed by direct probe and GC/MS.

Field ionization (FI)

Matrix Assisted Laser Desorption Ionization (MALDI)

MALDI is achieved in two steps. In the first step, the compound to be analyzed is dissolved in a solvent containing in solution small organic molecules, called the matrix. •The second step occurs under vacuum conditions inside the source of the mass spectrometer.•The use of a chemical matrix in the form of small, laser-absorbing organic molecules in large excess over the analyte is at the core of the MALDI principle.•One important feature is the way in which the matrix and analyte interact in the MALDI sample. In a typical UV-MALDI sample preparation small volumes of an analyte is mixed with amount 106 of matrix. Upon solvent evaporation, thematrix crystallizes to form a bed of small crystals that range in size from a few toa few hundred micrometers, depending on the matrix and the details of the preparation.

Good solubility

Vapour pressure must be sufficiently low to maintain vacuum conditions

Viscosity must allow diffusion of the analyte from the bulk to the surface

Lower PRACTICAL detection limits

Easier to interpret spectra (less multiple charges)

Quick and easy

Higher mass detection Higher Throughput (>1000 samples per hour)

Low levels of some salts, buffers, and detergents can be tolerated as well as less than 2% of glycerol.

Properties of MALDI

MALDI mass spectrometry has become a powerful analytical

tool for both synthetic polymers and biopolymers.

Principle of MALDI

Electrospray is abbreviated to ESI , sample is sprayed out of a narrow nozzle in a high potential field. Generates positive (M+nH)n

+ and negative (M - nH)n- ions and almost no

fragmentation. Generates multiple charged ions.It is especially useful in producing ions from macromolecules because itovercomes the propensity of these molecules to fragment whenionized.

ElectroSpray Ionization (ESI)

http://en.wikipedia.org/wiki/Macromolecule

2. Principle

Advantages

Electrospray Ionization can be easily interfaced to LC.Absolute signals from Electrospray are more easily reproduced, therefore, better quantitation.Mass Accuracy is considered better.Multiple charging is more common then MALDI.

Disadvantages

No FragmentationNeed Polar Sample

Need Solubility in Polar Solvent (MeOH, ACN, H2O,

Acetone are best)Sensitive to Salts

Suppression

Properties of ESI

36

The Mass Spectrum

Example: methane CH4 + e- CH4+. + 2 e-

mass-to-charge ratio (m/z)

Rel

ativ

e io

n ab

unda

nce

(%)

Base peak: most abundant ion

m/z = (1 x 12) + (4 x 1) = 16C H

Tandem Mass Spectrometry

Tandem mass spectrometry, also known as MS/MS, involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages.

Fragmentation Patterns

• The impact of the stream of high energy electrons often breaks the molecule into fragments, commonly a cation and a radical.– Bonds break to give the most stable cation.– Stability of the radical is less important.


• Alkanes– Fragmentation often splits off simple alkyl groups:

• Loss of methyl M+ - 15• Loss of ethyl M+ - 29• Loss of propyl M+ - 43• Loss of butyl M+ - 57

– Branched alkanes tend to fragment forming the most stable carbocations.

Fragmentation Patterns• Mass spectrum of 2-methylpentane

Fragmentation Patterns• Alkenes:

– Fragmentation typically forms resonance stabilized allylic carbocations

Fragmentation Patterns• Aromatics:

– Fragment at the benzylic carbon, forming a resonance stabilized benzylic carbocation (which rearranges to the tropylium ion)

M+

CH

H

CH Br

HC

H

H

or

Fragmentation Patterns• Alcohols

– Fragment easily resulting in very small or missing parent ion peak

– Commonly losses H2O or OH• M+ - 17 or M+ - 18

– Commonly lose an alkyl group attached to the carbinol carbon forming an oxonium ion.• 1o alcohol usually has prominent peak at m/z = 31

corresponding to H2C=OH+


• MS for 1-propanol

M+M+-18

CH3CH2CH2OH

H2C OH

SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/28/09)


• Amines– Odd M+ (assuming an odd number of nitrogens are

present)– a-cleavage dominates forming an iminium ion

CH3CH2 CH2 N

H

CH2 CH2CH2CH3 CH3CH2CH2N CH2

H

m/ z =72

iminium ion

Fragmentation Patterns86

CH3CH2 CH2 N

H

CH2 CH2CH2CH3

72

Fragmentation PatternsAromatics may also have a peak at m/z = 77 for the benzene ring.

NO2

77M+ = 123

77


• Aldehydes (RCHO)– Fragmentation may form acylium ion

– Common fragments:

• M+ - 1 for • M+ - 29 for

RC O

R (i.e. RCHO - CHO)

RC O

Fragmentation Patterns• MS for hydrocinnamaldehyde

M+ = 134C C C H

H

H

H

H

O

133

105

91105

91



• Ketones– Fragmentation leads to formation of acylium ion:

• Loss of R forming

• Loss of R’ forming

RC O

R'C O

RCR'

O

Fragmentation Patterns• MS for 2-pentanone

CH3CCH2CH2CH3

O

M+

CH3CH2CH2C O

CH3C O



• Esters (RCO2R’)– Common fragmentation patterns include:

• Loss of OR’ – peak at M+ - OR’

• Loss of R’ – peak at M+ - R’

Frgamentation Patterns

M+ = 136

C

O

O CH3

105

77 105

77


54

2. Rearrangement

McLafferty rearrangement

Pattern I

+A

BC

D

EH

A

BC

D

EH

A

BH2C

D

EH

+A

BC

D

EH

A

BC

D

EH

CD

EH

56

Retro Diels-Alder rearrangement

R- e

R R R+

R R+

CH3 CH3

+

Examples:

57

Loss of small molecules, such as H2O, CO, C2H4

H HOC6H13 H2O + C6H13

- CO

O

O

O

- CO

+O

HO

H

OH2O

HOH

CH3

H2C CHCH3 CH2=CH2H2O+ +

58

Four-member ring rearrangement

Other rearrangement

CH3 CH2 O CH2 CH3

- C2H4

CH3 CH2 O CH2

H2C

H2C O

H=

CH2

- CH3

HO CH2

XR R +

X

oConcluding remarksoSoft techniques produces Molecular ion peaks whereas hard techniques produces Fregmentation.oBy Soft techniques exact molecular ion peak can be obtained for large and small molecules.oBy Fregmentation connection pattern of a molecule can be concluded, ehich helps in structure elucidation.

bw mass spectrometry - zeeshan

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