Mass spectrometry

(about the theory and applications)

Lecture-3

By

Dr. Ahmed M. Metwaly

Objectives

Soft ionization techniques

• Chemical Ionization

• Fast Atomic Bombardment (FAB)

• Electrospray Ionization (ESI)

• Matrix Assisted Laser Desorption Ionisation (MALDI)

HR-MS

Chemical Ionization (CI)

• A “soft” ionization technique, which involves indirect ionization of

sample by ion-molecule reaction instead of by electron-impact.

• Chemical reagent gas added to modified EI ion source.

• Electron impact ionization of reagent gases such as ammonia or

methane. Sample molecules are bombarded with a reagent gas

ions which results in ionization of the sample.

• Ionization occurs through chemical reaction (gas-phase proton

transfer).

• Reagent gases can react with the sample molecules

to give

• [M+H]+,

• [M+NH3]+ or

• [M+CH4]+.

• These ions are more stable and called

pseudomolecular ions.

CI Reactions

• Typical reagent gas: CH4 (methane)

▫ Proton transfer occurs (Positive CI) - M+1 peak

▫ CH4 + e- CH4·+ + 2 e-

▫ CH4·+ + CH4 CH5

+ + CH3·

▫ CH5+ + M CH4 + MH+

▫ (Negative CI) (hydride extraction ) - M-1 peak

▫ CH4 + e- CH4·+ + 2 e-

▫ CH4·+ CH3

+ + H·

▫ CH3+ + CH4 C2H5

+ + H2

▫ C2H5+ + M M-H- + C2H6

Comparison of EI/CI

Comparison of EI/CI

CI EI

Has less fragmentation

More MW information

Less structural information

Has more fragmentation

Little/no MW information

Better fingerprint

Fast Atomic Bombardment (FAB)

• Works best for organic compounds with polarity and either acidic

and or basic functional groups.

• FAB-MS is suitable for thermo labile and non volatile liquids.

• Classes that use FAB are: peptides, proteins, fatty acids,

carbohydrates.

Method • Argon gas (neutral inert gas) is subjected to ionization to release Ar+

rapid.

• Fast moving beam containing Ar+ rapid is directed toward the sample.

• Sample is dissolved in a liquid matrix, which coats the target of the

beam.

• Beam collides with the sample and matrix molecules, producing

positive and negative sample-related ions (pseudomolecular ions)

that can be accelerated into the mass spectrometer.

FAB Matrix

1. Dissolve sample to be analyzed

2. Facilitate in the ionization of the sample

3. Be of low volatility

4. Should not undergo a chemical reaction with the sample

Common matrices- thioglycerol, glycerol, 3-nitrobenzyl alcohol.

Electrospray Ionization (ESI)

• Electrospray Ionization (ESI) is the most used technique for soft

ionization.

• Electrospray ionization is known as a "soft" ionization

method as the sample is ionized by the addition or removal

of a proton, with very little extra energy remaining (cannot

cause fragmentation of the sample ions).

• In ESI, samples (M) with molecular masses up to ca. 1200 Da give

rise to singly charged molecular-related ions, usually protonated

molecular ions of the formula (M+H)+ in positive ionization

mode, and deprotonated molecular ions of the formula (M-H)- in

negative ionization mode.

• In electrospray ionization, instead of the sample molecules being

ionized by the addition of a proton H+, some molecules have been

ionized by the addition of a sodium cation Na+.

• These can be identified as the sodium adduct ions, (M+Na)+.

• Other common adduct ions include K+ (+39) and NH4+ (+18) in

positive ionization mode and Cl- (+35) in negative ionization

mode.

• In positive ionization mode, a trace of formic acid is often added

to aid protonation of the sample molecules.

• In negative ionization mode a trace of ammonia solution or a

volatile amine is added to aid deprotonation of the sample

molecules.

• Proteins and peptides are usually analyzed under positive

ionization conditions.

• Saccharides and oligonucleotides under negative ionization

conditions.

• The sample is dissolved in a polar, volatile solvent

and pumped through a narrow, stainless steel

capillary tube. A high voltage is applied to the tip

(end) of the capillary (strong electric field), the

sample emerging from the tip as aerosol of highly

charged droplets, a process that is aided by a co-

axially introduced nebulizing gas flowing around the

outside of the capillary.

• This gas, usually nitrogen,

1. Helps to direct the spray emerging of the compound from the

capillary tip as aerosol.

2. Solvent evaporation assisted by a warm flow of nitrogen known

as the drying gas.

• The solvent evaporates, makes the droplets even smaller.

• Eventually the repulsion of charges in the droplets is so great that

the droplet explodes into smaller lesser charged droplets.

• The process is repeated (solvent evaporation, shrinking, and

explosion) until individually charged naked analyte ions are formed

and enter the analyzer.

Matrix Assisted Laser Desorption Ionisation

(MALDI) • It deals well with thermolabile, non-volatile organic compounds

especially those of high molecular mass and is used successfully in

biochemical areas for the analysis of proteins, peptides,

glycoproteins, oligosaccharides, and oligonucleotides.

• MALDI is also a "soft" ionization method.

• Fragmentation of the sample ions does not usually occur.

• In positive ionization mode the protonated molecular ions

(M+H+) are usually the dominant species, although they can be

accompanied by salt adducts (M+Na) or (M+K).

• In negative ionisation mode the deprotonated molecular ions (M-

H-) are usually the most abundant species, accompanied by some

salt adducts and possibly traces of dimeric or doubly charged

materials.

• MALDI is based on the bombardment of sample molecules

with a laser light to bring about sample ionization.

• Aqueous or alcoholic solution of sample is pre-mixed with a

highly radiation absorbing matrix.

• Sinapinic acid is a common one for protein analysis while

alpha-cyano-4-hydroxycinnamic acid is often used for

peptide analysis.

• Solution evaporated on metallic probe.

• The matrix exposed to pulsed laser beam.

• The matrix transforms the laser energy for the sample, which leads to

sputtering of analyte and matrix ions from the surface of the mixture.

• In this way energy transfer is efficient and also the analyte molecules

are spared excessive direct energy that may otherwise cause

decomposition.

• Matrix absorbs laser wavelength, sample must not (or else

fragmentation occurs)

Remember

Ion sources for molecular MS

Ionizing agent Mode Basic type

Energetic electrons EI Gas phase

Reagent gaseous ions CI

Energetic atomic beam FAB Desorption

High electrical field ESI

Laser beam MALDI

High Resolution Mass Spectrometry

(HRMS)

• HRMS is used for determination of exact mass and

a molecular formula of the compound and

differentiation between isotopes.

• In low resolution MS, a molecular weight of the

compound based on atomic weights that are the

average of weights of all natural isotopes of an

element.

• i.e. For normal calculation purposes, you tend to use

rounded-off relative isotopic masses. For example,

you are familiar with the numbers:

• H = 1

• C = 12

• N = 14

• O = 16

• But HRMS, determine the sum of the exact masses

of the most abundant isotope of each element

(Accurate isotopic masses).

• H = 1.0078

• C = 12.0000

• N = 14.0031

• O = 15.9949

Example-1

• A molecule with mass of 44 could be C3H8, C2H4O

or CO2 in low resolution mass.

• But in HRMS

• C3H8 44.0624

• C2H4O 44.0261

• CO2 43.9898

Example-2

• CO, N2 and C2H4, having a mass at m/z 28, in a low

resolution mass spectrometer.

• But in HRMS,

• 12C16O = 12.0000 + 15.9949 = 27.9949.

• 14N2 = 14.0031 + 14.0031 = 28. 0062.

• 12C2 1H2 = 24.000 + 4.0312 = 28.0312.

Formula C6H12 C5H8O C4H8N2

Mass 84.0939 84.0575 84.0688

Isotope patterns

• Mass spectrometers are capable of separating and

detecting individual ions even those that only differ

by a single atomic mass unit.

• We can use low resolution results and intensities of

isotope peaks to arrive to a possible molecular

formula.

• As a result molecules containing different isotopes

can be distinguished.

• Isotopes: present in their usual abundance.

• Hydrocarbons contain 1.1% C-13, so there will be a

small M+1 peak.

• If Br is present, M+2 is equal to M+.

• If Cl is present, M+2 is one-third of M+.

What is an M+1 peak?

• If you had a complete (rather than a simplified)

mass spectrum, you will find a small line 1 m/z unit

to the right of the main molecular ion peak. This

small peak is called the M+1 peak.

What causes the M+1 peak?

• The M+1 peak is caused by the presence of the 13C

isotope in the molecule.

• Carbon-13 makes up 1.11% of all carbon atoms.

This is most apparent when atoms such as bromine or

chlorine are present.

Peaks at "M" and "M+2" are obtained.

Bromine isotopes [79Br : 81Br] have the same

abundance intensity (having M and M+2 in ratio

1:1).

Chlorine [35Cl : 37Cl] have difference in the

abundance, so the intensity of M and M+2 is in ratio

3:1.

M+2 peak

• The intensity ratios in the isotope patterns are due

to the natural abundance of the isotopes.

• Therefore, differentiation between the mass spectra

of chlorine- and bromine-containing compounds is

possible.

Examples of haloalkanes with characteristic

isotope patterns

Mass Spectrum with Chlorine

2-chloropropane

• Note the isotope pattern at 78 and 80 that represent

the M and M+2 in a 3:1 ratio.

• Loss of 35Cl from 78 or 37Cl from 80 gives the base

peak a m/z = 43, corresponding to the secondary

propyl cation.

• Note that the peaks at m/z = 63 and 65 is due to

fragment ions also containing one chlorine atom -

which could either be 35Cl or 37Cl and therefore

also show the 3:1 isotope pattern.

• The fragmentation that produced those ions was:

So

• if you look at the molecular ion region, and find

two peaks separated by 2 m/z units and with a

ratio of 3 : 1 in the peak heights, that tells you that

the molecule contains 1 chlorine atom.

Mass Spectrum with Bromine

1-bromopropane

• Note the isotope pattern at 122 and 124 that

represent the M and M+2 in a 1:1 ratio.

• Loss of 79Br from 122 or 81Br from 124 gives the

base peak a m/z = 43, corresponding to the propyl

cation.

• Note that other peaks, such as those at m/z = 107

and 109 still contain Br and therefore also show the

1:1 isotope pattern.

So • If you have two lines in the molecular ion region

with a gap of 2 m/z units between them and with

almost equal heights, this shows the presence of a

bromine atom in the molecule.

M+4 peak Two chlorine atoms in a compound

• The lines in the molecular ion region (at m/z values

of 98, 100 ands 102 in the ratio of 9:6:1) arise

because of the various combinations of chlorine

isotopes that are possible.

• The carbons and hydrogens add up to 28 - so the

various possible molecular ions could be:

• 28 + 35 + 35 = 98

• 28 + 35 + 37 = 100

• 28 + 37 + 37 = 102

So

• If you have 3 lines in the molecular ion region

(M+, M+2 and M+4) with gaps of 2 m/z units

between them, and with peak heights in the ratio

of 9:6:1, the compound contains 2 chlorine atoms.

Common MS fragments of organic compounds

m/z lost Moiety Compounds exhibiting loss

1 H aldehydes

15 CH3 branched sites

16 O sulfoxides, nitro compounds

16 NH2 amides, aromatic amines

17 OH acids

18 H2O alcohols, aldehydes, ketones, ethers

m/z lost Moiety Compounds exhibiting loss

26 CN alkylcyanides

29 C2H5 or CHO alcohols

31 OCH3 or CH2OH methyl esters, alcohols

35 Cl halide-containing

45 OC2H5 or COOH ethyl esters or carboxylic

acids