303351 Instrumental Analysis

Mass Spectrometry Lecturer: !Somsak Sirichai

Mass SpectrometryWhat is Mass spectrometry (MS)? An analytic method that employs ionization and mass analysis of compounds in order to determine the mass, formula and structure of the compound being analyzed.

In general there are four steps associated with a mass spectroscopic experiment:• generate gas-phase molecules from analyse (solid, liquid, gas, etc)!• ionize those molecules!• separate the ions based on mass-to-charge ratio!• detect the ion beam

Molecular Mass Spectra

Figure 20-1 Mass spectrum of ethyl benzene

C6H 5CH 2CH 3 + e− → C6H 5CH 2CH 3i+ + 2e−

molecular ion

How are mass spectra produced?1. Ions are produced in the source and are transferred into the

mass analyser

2. They are separated according to their mass/charge ratio in the ! mass analyser (e.g. Quadrupole, Ion Trap, Time of Flight)

3. Ions of the various m/z values exit the analyser and are ! ‘counted’ by the detector

C6H 5CH 2CH 3 + e− → C6H 5CH 2CH 3i+ + 2e−

molecular ion

Mass Spectrometer

Figure 20-11 Components of a mass spectrometer

1. Sample Inlet Systems• to permit introduction of a representative sample into the ion

source with minimal loss of vacuum

• modern mass spectrometers are equipped with several types of inlets to accommodate various kinds of samples; these include batch inlets, direct probes inlets, chromatographic inlets, and capillary electrophoretic inlets.

Inlet system - three requires are:

• (1) that sample must be in the vapour phase prior to ionisation;!

• (2) that sample does not suffer thermal decomposition during

the vaporisation process;!

• (3) that during admission of the sample, the pressure inside the

mass spectrometer us kept as low as possible.

Batch Inlet Systems• the conventional (and simplest) inlet system

• the sample is volatilised externally and then allowed to leak into the evacuated ionisation region.

• Figure 20-12a is a schematic of a typical system that is applicable to gaseous and liquid samples having boiling point up to about 500o C

Figure 20-12 (a) Schematic of an external sample-introduction system

The Direct Probe Inlet• solid and nonvolatile liquid can be introduced into the ionisation

region by means of a sample holder, or probe, which is inserted through a vacuum lock (Figures 20-12b)

Figure 20-12(b) !a sample probe for inserting a sample directly into the ion source!!• temperature-programmable

under data system control!• vaporizes small amounts of

material into MS source!• requires pure sample for

meaningful spectra!• can “dirty” MS rapidly!• Labor-intersive: all-manual

operation

Chromatographic Inlet System

Figure 27-14 Schematic of a typically capillary GC/MS system

2. Ion Source• Once we have produced gas phase molecules, we can start to look at

the ionisation process

• the appearance of mass spectra for a given molecular species strongly depends on the method used for ion formation

• the methods fall into two major categories: (1) gas-phase sources and (2) desorption source (Table 20-1)

• gas-phase source: the sample is first vaporised and then ionised. ! generally restricted to ionisation of thermally stable compounds ! that have boiling point less than about 500oC

• desorption source: the solid- or liquid-state sample is converted directly into gaseous ions.!

advantages - they are applicable to nonvolatile and thermally ! unstable samples

Ion sources are also classified as being hard sources or soft sources.

Hard ionisation sources! impart enough energy to analyte molecules to leave them in a highly excited energy state. Relaxation then involves rupture of bonds, producing fragment ions that have mass-to-charge ratios less than of molecular ion.

Soft ionisation sources! cause little fragmentation. Thus, the mass spectrum from a soft ionisation source often consist of the molecular ion peak and only a few, if any, other peak.

Figure 20-2 Mass spectrum of 1-decanol from (a) a hard ionisation source (electron inpact) and (b) a soft ionisation source (chemical ionisation)

Hard- and soft- ionisation sources!!

both are useful for analysis. The many peaks in a hard-source spectrum provide useful information about the kinds of functional groups and thus structural information about analytes. !!

Soft-source spectra are useful because they supply accurate information about the molecular mass of the analyte molecule or molecules.

The Electron-Impact Source

ions from mass analysis were produced by electron impact. In this process, the sample is brought to a temperature high enough to produce a molecular vapour,, which is then ionised by bombarding the resulting molecules with a beam of energetic electrons.!!

Figure 20-3 is a schematic of a basic electron-impact ion source.

Figure 20-3 An electron-impact ion source

Figure 20-3 is a schematic of a basic electron-impact ion source. Electrons are emitted from a heated tungsten or rhenium filament and accelerated by applying approximately 70 V between filament and the anode. As shown in the figure, the paths of electrons and molecules are at the right angles and intersect near the center of the source., where collision and ionisation occur.

EI source: advantages vs. disadvantages• convenient to use and produce

high ion currents , thus giving good sensitivities. !

!• extensive fragmentation and

resulting large number of peaks is also advantage because it often makes unambiguous identification of analyte possible.

• it may be difficult to measure relative molecular masses for some molecules!

• it is difficult to distinguish between isomers!

• some compounds may undergo thermal decomposition prior to ionisation or be very prone to fragmentation after ionisation because of the temperature required for vaporisation !

• applicable only to analytes having molecular masses smaller than about 103 Da

Chemical Ionisation Source• most modern mass spectrometers are designed so that electron-

impact ionisation and chemical ionisation can be carried out interchangeably. Such sources are called EI-CI sources.

• In CI source, gaseous atoms of the sample (from either a batch inlet or a heated probe) are ionised by collision with ions produced by electron bombardment of an excess of a reagent gas (or buffer gas).

• Buffer gas often methane (CH4) is added in a large excess (about x100 fold) over the analyte. The electron beam ionises the methane, gas phase reactions produce molecular ions such as CH5+ which collide with analyte molecules (M) to gently produce the analyte ions (MH+)

Chemical Ionisation Source• can perform CI in same unit with EI except for much higher pressure!!• must provide additional pumping speed to move extra buffer gas

out of ioniser so as to not degrade vacuum in analyser

Methane reacts with high-energy electrons to give several ions such as CH4+, CH3+ and CH2+. The first two predominate and represent about 90% of the reaction products.

CH 4+ +CH 4 →CH 5

+ +CH 3

CH 3+ +CH 4 →C2H 5

+ +H 2

Step 1: Formation of Reagent Ions!• Primary step by electron impact

CH 4 + e− →CH 4

+• + 2e−

→CH 2

+• +H 2

→CH 3

+• +H •

• Secondary step by Ion-Molecule Reactions:

CH 4

+• +CH 4 →CH 5

+ +CH 3 i

CH 3

+• +CH 4 →C2H 5

+ +CH 2

C2H 5

+• +CH 4 →C3H 5

+ + 2H 2

Step 2: Ionisation of sample molecule!• Protonation/adduct formation

M +CH 5+ → MH⎡⎣ ⎤⎦

+

M +C2H 5+ → M •C2H 5⎡⎣ ⎤⎦

+

M +C3H 5+ → M •C3H 5⎡⎣ ⎤⎦

+

m/z = M + 1

m/z = M + 29

m/z = M + 41

• Hydride abstraction

M +CH 5+ → M −M⎡⎣ ⎤⎦

+i +CH 4 +H 2

Commom for large straight chain aliphatic molecules

Field Ionisation Sources• In field ionisation source, ions are

formed under the influence of a large electric field (108 V/cm)!

!• when a large potential difference

(10-20 kV) is applied across an object fashioned to come to an atomically sharp point, the electric field is HUGE (108 V/cm). These fields, across a single molecule, have enough potential to pull an electron directly from the molecule

Figure 20-5 Photomicrograph of a carbon microneedle emitter (Anode of FI source).

Field Ionisation Sources• Advantages:!! ! the field ionisation spectrum is relative simple (uncomplicated), ! with an easily distinguished (M+1)+

• Disadvantages:!! ! a limitation to field ionisation is its sensitivity, which is at least an order of magnitude less than that of EI sources; maximum currents are on the order of 10-11 A —- low sensitivity and resolution

Desorption sourcesThe ionisation methods discussed so far require that the ionisation agents act on gaseous samples. Such methods are not applicable to nonvolatile or thermally unstable samples.

A number of desorption ionisation methods have been developed for dealing with this type of sample. ! advantages: enable mass spectra to be obtained for thermally delicate biochemical species and species having molecular masses of greater than 100,000 Da.

Electrospray Ionisation (ESI)• one of the most important techniques for analysing biomolecule

such as polypeptide, proteins, and oligonucleotide, having molecular weights of 100,000 Da or more

• produce multiply charged ions based on ion evaporation process

Figure 20-9 Apparatus for electrospray ionisation

Fast Atom Bombardment Sources• FAB sources, aslo called liquid secondary-ion sources !!• concept: if a beam of fast moving neutral atoms are directed onto a

metal plate coated with a sample, then much of the high kinetic energy of the atoms is transferred to the sample molecules on impact. This energy ca be dissipated in various ways, some of which lead to ionisation of the sample. !

!• The bombarding atoms are usually rare gases, either xenon or

argon. In order for them to achieve a high kinetic energy, atoms of the gas are first ionised and these ions are then pass through an electric field.

3. Mass Analyzers• At the heart of a mass spectrometer is the mass analyser system.

The selection is always based upon a mass-to-charge ratio, rather than on absolute mass.!

!• ideally, the mass analyser should be capable of distinguishing minute

mass differences. In addition, the analyser should allow passage of a sufficient number of ions to yield readily measurable ion current!!

• The principal devices for mass selection are!! ! Magnetic sector Analyzer!! ! Double-Focusing Spectrometers!! ! Quadrupole Mass Spectrometers!! ! Time-of-Flight (TOF) Mass Analyzers!! ! Ion-Trap Analyzers

Example 20-3 !What resolution is needed to separate the ion C2H4+ and CH2N+, with masses of 28.0313 and 28.0187, respectively?

Resolution (R) of Mass Spectrometers R = mΔm

where is the mass difference between two adjacent peaks that are just resolved and is the nominal mass of the first peak (the mean mass of the two peaks is sometimes used instead)

Δmm

Magnetic SectorA charges particles (an ion) experiences a force that bends its path when moving through a magnetic field. The balance between magnetic force and centripetal force brings an ion of a particular m/z to entrance slit of a detector.

Magnetic Sector EquationsThe magnetic force Fm is given by

This is balanced by the centripetal force Fc

These are equated and solved for the velocity

Fm = Bzev

Fc =mv2

r

Bzev = mv2

r ⇒ v = Bzer

mSubstitute into expression for kinetic energy. Solve for m/z

Ek = zev = 12mv2 = 1

2m Bzer

m⎛⎝⎜

⎞⎠⎟2

⇒ mz

= B2r2e2V

Double-Focusing SpectrometersA single magnetic sector instrument’s resolution is limited by spread of translational energy of the ions coming from the source. A double focusing instrument uses an electrostatic field to narrow the energy spread before the ions enter the magnetic sector. Resolutions of 105 are achievable with these instruments.

Quadrupole Mass Spectrometers

Ion-Trap MS• This device is able to trap and

hold ions in a space charge region with their masses extending over a range of several thousand Da. Adjusting the end cap fields to a particular frequency can drive a narrow Da range into the detector. !

• has a high ion yield for species.!• dynamic concetration range is

low. Ions spends lots of time in the trap and ion-ion interaction can alter the fragmentation pattern.

Tandem Mass SpectrometryUsually referred to as MS/MS. Here one take a mass spectrum of a mass spectrum. A particular ion fragment isolated in the first stage is directed to the second phase for further fragmentation and dispersion. !!

There are three common types:!! EBEB: !! Double Double Focusing. Two double focusing ! ! !! ! ! ! magnetic sector stages.!! QQQ: !! A triple Quad. Three quadrupoles sections in series.!! Q-TOF: ! A quadrupole stage preceding a TOF analyzer

Figure 20-23 Schematic of a triple quadrupole mass spectrometer

TOFIons accelerated to same energy. Mass difference mean different velocities. There travel a long path (1-2 m) and arrive at the detector at different times. Time of arrival calculates m/z.

mz

= 2Vt2

L2 ⇒ t = L m

2zV