mass spectrometry

MASS SPECTROMETRY

INTRODUCTION

Mass Spectrometer (MS) is a kind of machine which uses an analytical

technique to measure the mass-to charge ratio of ions. This analytical technique is also known as Mass

spectrometry. And an ion is an atom or group of atoms which have lost or gained one or more

electrons, making them negatively or positively charged. Mass spectrometry is an important emerging

method for the characterization of proteins. The two primary methods for ionization of whole proteins

are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). As it is an

important tool in proteomics, it is essential to understand not only the results, but also the principles of

Mass Spectrometer. This report is devoting to provide a simple but clear explanation to the principles

of Mass Spectrometer.

DEFINITION:

Mass spectrometry (MS) is an analytical technique for the determination of the

elemental composition of a sample or molecule. It is also used for elucidating the chemical structures

of molecules, such as peptides and other chemical compounds. The MS principle consists of ionizing

chemical compounds to generate charged molecules or molecule fragments and measurement of their

mass-to-charge ratios

PRINCIPLE:

In a typical MS procedure:

1. a sample is loaded onto the MS instrument, and

2. the components of the sample are ionized by one of a variety of methods (e.g., by impacting

them with an electron beam), which results in the formation of charged particles (ions)

3. directing the ions into an electric and/or magnetic field

4. computation of the mass-to-charge ratio of the particles based on the details of motion of the

ions as they transit through electromagnetic fields, and

5. Detection of the ions, which in step 4 were sorted according to m/z.

INSTRUMENTATION:

A mass spectrometer consists of following basic components

1. The inlet system (or) sample handling system

2. The ion source or ionisation chamber

3. The ion separator

4. The ion collector (the detector and readout system)

5. The vacuum system

THE MASS SPECTROMETER

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THE INLET SYSTEM:

Direct Vapour Inlet.

Gas Chromatography.

Liquid Chromatography

Direct Insertion Probe

Direct Ionization of Sample

IONIZATION SOURCES

Chemical Ionisation (CI)

Atmospheric Pressure CI!(APCI)

Electron Impact!(EI)

Electrospray Ionization!(ESI)

Matrix Assisted Laser Desorption Ionisation!(MALDI)

Field Desorption/Field Ionisation (FD/FI)

Fast Atom Bombardment (FAB)

Thermo spray Ionisation (TI)

ANALYZERS

quadruples

Time-of-Flight (TOF)

ion trap analyzer

Fourier transform analyzer

Orbitrap analyzer

DETECTORS

electron multiplier detector

Faraday cup detector

Array detector

THE INLET SYSTEM/ SAMPLE HANDLING SYSTEM:

The selection of a sample inlet depends upon the sample and the sample matrix. Most ionization

techniques are designed for gas phase molecules so the inlet must transfer the analyte into the source

as a gas phase molecule. If the analyte is sufficiently volatile and thermally stable, a variety of inlets

are available. Gases and samples with high vapor pressure are introduced directly into the source

region. Liquids and solids are usually heated to increase the vapor pressure for analysis. If the analyte

is thermally labile (it decomposes at high temperatures) or if it does not have a sufficient vapor

pressure, the sample must be directly ionized from the condensed phase. These direct ionization

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techniques require special instrumentation and are more difficult to use. However, they greatly extend

the range of compounds that may be analyzed by mass spectrometry. Commercial instruments are

available that use direct ionization techniques to routinely analyze proteins and polymers with

molecular weights greater than 100,000 Dalton.

DIRECT VAPOR INLET.

The simplest sample introduction method is a direct vapor inlet. The gas phase analyte is

introduced directly into the source region of the mass spectrometer through a needle valve. Pump

out lines are usually included to remove air from the sample. This inlet works well for gases,

liquids, or solids with a high vapor pressure. Samples with low vapour pressure are heated to

increase the vapor pressure. Since this inlet is limited to stable compounds and modest

temperatures, it only works for some samples.

GAS CHROMATOGRAPHY.

Gas chromatography is probably the most common technique for introducing samples into a

mass spectrometer. Complex mixtures are routinely separated by gas chromatography and mass

spectrometry is used to identify and quantitate the individual components. Several different interface

designs are used to connect these two instruments. The most significant characteristics of the inlets are

the amount of GC carrier gas that enters the mass spectrometer and the amount of analyte that enters

the mass spectrometer. If a large flow of GC carrier gas enters the mass spectrometer it will increase

the pressure in the source region. Maintaining the required source pressure will require larger and more

expensive vacuum pumps. The amount of analyte that enters the mass spectrometer is important for

improving the detection limits of the instrument. Ideally all the analyte and none of the GC carrier gas

would enter the source region.

The most common GC/MS interface now uses a capillary GC column. Since the carrier Gas flow rate

is very small for these columns, the end of the capillary is inserted directly into the source region of the

mass spectrometer. The entire flow from the GC enters the mass spectrometer. Since capillary columns

are now very common, this inlet is widely used.

LIQUID CHROMATOGRAPHY.

Liquid chromatography inlets are used to introduce thermally labile compounds not easily

separated by gas chromatography. These inlets have undergone considerable development and are now

fairly routine. Because these inlets are used for temperature sensitive compounds, the sample is ionized

directly from the condensed phase.

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DIRECT INSERTION PROBE.

The Direct Insertion Probe (DIP) is widely used to introduce low vapor pressure liquids and

solids into the mass spectrometer. The sample is loaded into a short capillary tube at the end of a

heated sleeve. This sleeve is then inserted through a vacuum lock so the sample is inside the source

region. After the probe is positioned, the temperature of the capillary tube is increased to vaporize the

sample. This probe is used at higher temperatures than are possible with a direct vapor inlet. In

addition, the sample is under vacuum and located close to the source so that lower temperatures are

required for analysis. This is important for analyzing temperature sensitive compounds. Although the

direct insertion probe is more cumbersome than the direct vapor inlet, it is useful for a wider range of

samples.

DIRECT IONIZATION OF SAMPLE

Unfortunately, some compounds either decompose when heated or have no significant

vapor pressure. These samples may be introduced to the mass spectrometer by direct ionization from

the condensed phase. These direct ionization techniques are used for liquid chromatography/mass

spectrometry, glow discharge mass spectrometry, fast atom bombardment and laser ablation. The

development of new ionization techniques is an active research area and these techniques are rapidly

evolving. Direct ionization is discussed in greater detail in the next sectio

IONISATION SOURCE:

The ion source is the part of the mass spectrometer that ionizes the material under analysis (the

analyte). The ions are then transported by magnetic or electric fields to the mass analyzer. Techniques

for ionization have been key to determining what types of samples can be analyzed by mass

spectrometry. Electron ionization and chemical ionization are used for gases and vapors.

1. CHEMICAL IONIZATIO

Chemical ionization (CI) is an ionization technique used in mass spectrometry. Chemical

ionization is a lower energy process than electron ionization. The lower energy yields less

fragmentation, and usually a simpler spectrum. A typical CI spectrum has an easily identifiable

molecular ion.

Mechanism

In a CI experiment, ions are produced through the collision of the analyte with ions of a

reagent gas that are present in the ion source. Some common reagent gases include: methane,

ammonia, and isobutane. Inside the ion source, the reagent gas is present in large excess compared to

the analyte. Electrons entering the source will preferentially ionize the reagent gas. The resultant

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collisions with other reagent gas molecules will create ionization plasma. Positive and negative ions of

the analyte are formed by reactions with this plasma.

Primary Ion Formation

Secondary Reagent Ions

Product Ion Formation

(Protonation)

(H − abstraction)

(Adduct formation)

(Charge exchange)

Self chemical ionization occurs when the reagent ion is an ionized form of the analyte.

Negative chemical ionization (NCI)

Chemical ionization for gas phase analysis is either positive or negative. Almost all neutral analytes

can form positive ions through the reactions described above.

In order to see a response by negative chemical ionization, the analyte must be capable of producing a

negative ion (stabilize a negative charge) for example by electron capture ionization. Because not all

analytes can do this, using NCI provides a certain degree of selectivity that is not available with other,

more universal ionization techniques (EI, PCI). NCI can be used for the analysis of compounds

containing acidic groups or electronegative elements (especially halogens).

Because of the high electronegativity of halogen atoms, NCI is a common choice for their analysis.

This includes many groups of compounds, such as PCBs, pesticides, and fire retardants. Most of these

compounds are environmental contaminants, thus much of the NCI analysis that takes place is done

under the auspices of environmental analysis.

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2. ATMOSPHERIC PRESSURE CHEMICAL IONIZATION (APCI)

Chemical ionization in an atmospheric pressure electric discharge is called atmospheric pressure

chemical ionization. The analyte is a gas or liquid spray and ionization is accomplished using an

atmospheric pressure corona discharge. This ionization method is often coupled with high performance

liquid chromatography where the mobile phase containing eluting analyte sprayed with high flow rates

of nitrogen and the aerosol spray is subjected to a corona discharge to create ions.

Atmospheric pressure chemical ionization (APCI) is an ionization method used in mass spectrometry.

It is a form of chemical ionization which takes place at atmospheric pressure.

How it works

APCI allows for the high flow rates typical of standard bore HPLC to be used directly, often without

diverting the larger fraction of volume to waste. Typically the mobile phase containing eluting analyte

is heated to relatively high temperatures (above 400 degrees Celsius), sprayed with high flow rates of

nitrogen and the entire aerosol cloud is subjected to a corona discharge that creates ions. Often APCI

can be performed in a modified ESI source. This is basically a gas phase ionisation, unlike ESI which

is a liquid phase ionisation process. Also, we can use nonpolar solvent for solution making instead of

polar solvent for supporting ions in solution as gaseous state conversion of solvent before reaching to

corona discharge pin is carried out here, which well supports the ions formed. Typically, APCI is a less

"soft" ionization technique than ESI, i.e. it generates more fragment ions relative to the parent ion.

Schematic diagram of atmospheric chemical ionisation

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3. ELECTRON IONIZATION

Electron ionization (EI, formerly known as electron impact) is an ionization method in which energetic

electrons interact with gas phase atoms or molecules to produce ions. This technique is widely used in

mass spectrometry, particularly for gases and volatile organic molecules.

Principle of operation

Diagram representing an electron ionization ion source

The following gas phase reaction describes the electron ionization process

where M is the analyte molecule being ionized, e- is the electron and M+• is the resulting ion.

In an EI ion source, electrons are produced through thermionic emission by heating a wire filament

that has electric current running through it. The electrons are accelerated to 70 eV in the region

between the filament and the entrance to the ion source block. The accelerated electrons are then

concentrated into a beam by being attracted to the trap electrode. The sample under investigation

which contains the neutral molecules is introduced to the ion source in a perpendicular direction to the

e- beam. Upon interaction with the e- beam, the analyte molecules ionize to radical cations which are

then directed towards the mass analyzer by a repeller electrode. Due to the high energy electrons and

the initial thermal distribution of the neutral molecules, the ionization process frequently causes

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cleavage reactions that give rise to fragment ions, which can convey structural information about the

analyte.

The ionization efficiency and production of fragment ions depends strongly on the chemistry of the

analyte and the energy of the electrons. At low energies (around 20 eV), the interactions between the

electrons and the analyte molecules do not transfer enough energy to cause ionization. At around 70

eV, the de Broglie wavelength of the electrons matches the length of typical bonds in organic

molecules (about 0.14 nm) and energy transfer to organic analyte molecules is maximized, leading to

the strongest possible ionization and fragmentation. Under these conditions, about 1 in 1000 analyte

molecules in the source are ionized. At higher energies, the de Broglie wavelength of the electrons

becomes smaller than the bond lengths in typical analytes; the molecules then become "transparent" to

the electrons and ionization efficiency decreases.

4. ELECTROSPRAY IONIZATION

Electrospray (nanoSpray) ionization source

Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions. It is

especially useful in producing ions from macromolecules because it overcomes the propensity of these

molecules to fragment when ionized. The development of electrospray ionization for the analysis of

biological macromolecules was rewarded with the attribution of the Nobel Prize in Chemistry to John

Bennett Fenn in 2002.

Mass spectrometry using ESI is called electrospray ionization mass spectrometry (ESI-MS) or, less

commonly, electrospray mass spectrometry (ES-MS)

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Fenn's first electrospray ionization source (top) coupled to a single quadrupole mass spectrometer

IONIZATION MECHANISM

The liquid containing the analyte(s) of interest is dispersed by electrospray into a fine aerosol. Because

the ion formation involves extensive solvent evaporation, the typical solvents for electrospray

ionization are prepared by mixing water with volatile organic compounds (e.g. methanol, acetonitrile).

To decrease the initial droplet size, compounds that increase the conductivity (e.g. acetic acid) are

customary added to the solution. Large-flow electrosprays can benefit from additional nebulization by

an inert gas such as nitrogen. The aerosol is sampled into the first vacuum stage of a mass

spectrometer through a capillary, which can be heated to aid further solvent evaporation from the

charged droplets. The solvent evaporates from a charged droplet until it becomes unstable upon

reaching its Rayleigh limit. At this point, the droplet deforms and emits charged jets in a process

known as Rayleigh fission. During the fission, the droplet loses a small percentage of its mass along

with a relatively large percentage of its charge

There are two major theories that explain the final production of gas-phase ions:

The Ion Evaporation Model (IEM) suggests that as the droplet reaches a certain radius the

field strength at the surface of the droplet becomes large enough to assist the field desorption of

solvated ions.

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The Charged Residue Model (CRM) suggests that electrospray droplets undergo evaporation

and fission cycles, eventually leading progeny droplets that contain on average one analyte ion

or less. The gas-phase ions form after the remaining solvent molecules evaporates, leaving the

analyte with the charges that the droplet carried.

While there is no definite scientific proof, a large body of indirect evidence suggests that small ions are

liberated into the gas phase through the ion evaporation mechanism, while larger ions form by charged

residue mechanism.

The ions observed by mass spectrometry may be quasimolecular ions created by the addition of a

proton (a hydrogen ion) and denoted [M + H]+, or of another cation such as sodium ion, [M + Na]+, or

the removal of a proton, [M − H]−. Multiply-charged ions such as [M + nH]n+ are often observed. For

large macromolecules, there can be many charge states, resulting in a characteristic charge state

envelope. All these are even-electron ion species: electrons (alone) are not added or removed, unlike in

some other ionization sources. The analytes are sometimes involved in electrochemical processes,

leading to shifts of the corresponding peaks in the mass spectrum.

APPLICATIONS

1.Liquid chromatography–mass spectrometry (LC-MS)

Electrospray ionization is the ion source of choice to couple liquid chromatography with mass

spectrometry. The analysis can be performed online, by feeding the liquid eluting from the LC column

directly to an electrospray, or offline, by collecting fractions to be later analyzed in a classical

nanoelectrospray-mass spectrometry setup.

2. Noncovalent gas phase interactions

Electrospray ionization is also ideal in studying noncovalent gas phase interactions. The electrospray

process is capable of transferring liquid-phase noncovalent complexes into the gas phase without

disrupting the noncovalent interaction. This means that a cluster of two molecules can be studied in the

gas phase by other mass spectrometry techniques. An interesting example of this is studying the

interactions between enzymes and drugs which are inhibitors of the enzyme. Because inhibitors

generally work by noncovalently binding to its target enzyme with reasonable affinity the noncovalent

complex can be studied in this way. Competition studies have been done in this way to screen for

potential new drug candidates

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5. MATRIX ASSISTED LASER DESORPTION IONISATION

Matrix Assisted Laser Desorption Ionisation (MALDI) (F. Hillenkamp, M. Karas, R. C. Beavis, B.

T. Chait, Anal. Chem., 1991, 63, 1193) 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. It is relatively

straightforward to use and reasonably tolerant to buffers and other additives. The mass accuracy

depends on the type and performance of the analyser of the mass spectrometer, but most modern

instruments should be capable of measuring masses to within 0.01% of the molecular mass of the

sample, at least up to ca. 40,000 Da.

MALDI is based on the bombardment of sample molecules with a laser light to bring about sample

ionisation. The sample is pre-mixed with a highly absorbing matrix compound for the most consistent

and reliable results and a low concentration of sample to matrix work best. The matrix transforms the

laser energy into excitation 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. Most

commercially available MALDI mass spectrometers now have a pulsed nitrogen laser of wavelength

337 nm.

Matrix assisted laser desorption ionisation (MALDI)

The sample to be analysed is dissolved in an appropriate volatile solvent, usually with a trace of

trifluoroacetic acid if positive ionisation is being used, at a concentration of ca. 10 pmol/µL and an

aliquot (1-2 µL) of this removed and mixed with an equal volume of a solution containing a vast

excess of a matrix. A range of compounds is suitable for use as matrices: sinapinic acid is a common

one for protein analysis while alpha-cyano-4-hydroxycinnamic acid is often used for peptide

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analysis. An aliquot (1-2 µL) of the final solution is applied to the sample target which is allowed to

dry prior to insertion into the high vacuum of the mass spectrometer. The laser is fired, the energy

arriving at the sample/matrix surface optimised, and data accumulated until a m/z spectrum of

reasonable intensity has been amassed. The time-of-flight analyser separates ions according to their

mass (m)-to-charge (z) (m/z) ratios by measuring the time it takes for ions to travel through a field

free region known as the flight, or drift, tube. The heavier ions are slower than the lighter ones.

The m/z scale of the mass spectrometer is calibrated with a known sample that can either be analysed

independently (external calibration) or pre-mixed with the sample and matrix (internal calibration).

Simplified schematic of MALDI-TOF mass spectrometry (linear mode)

Sample target for a Maldi Tof Spectrometer

MALDI is also a "soft" ionisation method and so results predominantly in the generation of singly

charged molecular-related ions regardless of the molecular mass, hence the spectra are relatively

easy to interpret. Fragmentation of the sample ions does not usually occur.

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In positive ionisation mode the protonated molecular ions (M+H+) are usually the dominant species,

although they can be accompanied by salt adducts, a trace of the doubly charged molecular ion at

approximately half the m/z value, and/or a trace of a dimeric species at approximately twice the m/z

value. Positive ionisation is used in general for protein and peptide analyses.

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.

Negative ionisation can be used for the analysis of oligonucleotides and oligosaccharides.

Positive ionisation MALDI m/z spectrum of a peptide mixture using alpha-cyano-4-

hydroxycinnamic acid as matrix

USES:-

In Biochemistry

In proteomics, MALDI is used for the identification of proteins isolated through gel electrophoresis:

SDS-PAGE, size exclusion chromatography, and two-dimensional gel electrophoresis. One method

used is peptide mass fingerprinting by MALDI-MS, or with post ionisation decay or collision-induced

dissociation (further use see mass spectrometry).

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In Organic Chemistry

Some synthetic macromolecules, such as catenanes and rotaxanes, dendrimers and hyperbranched

polymers, and other assemblies, have molecular weights extending into the thousands or tens of

thousands, where most ionization techniques have difficulty producing molecular ions. MALDI is a

simple and rapid analytical method that can allow chemists to analyze the results of such syntheses and

verify their results.

In polymer chemistry

In polymer chemistry MALDI can be used to determine the molar mass distribution. Polymers with

polydispersity greater than 1.2 are difficult to characterize with MALDI due to the signal intensity

discrimination against higher mass oligomers.

Reproducibility and performance

The sample preparation for MALDI is important for the result. Inorganic salts which are also part of

protein extracts interfere with the ionization process. The salts are removed by solid phase extraction

or washing the final target spots with water. Both methods can also remove other substances from the

sample. The matrix protein mixture is not homogenous because the polarity difference leads to a

separation of the two substances during crystallization. The spot diameter of the target is much larger

than that of the laser, which makes it necessary to do several laser shots at different places of the

target, to get the statistical average of the substance concentration within the target spot. The matrix

composition, the addition of trifluoroacetic acid and formic acid, delay between laser pulses, delay

time of the acceleration power, laser wavelength, energy density of the laser and the impact angle of

the laser on the target are among others the critical values for the quality and reproducibility of the

method

6. FIELD DESORPTION IONISATION:

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Schematic of field desorption ionization with emitter at left and mass spectrometer at right

Field desorption (FD)/field ionization (FI) refers to an ion source for mass spectrometry first reported

by Beckey in 1969. 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 .

Mechanism

In FD, the analyte is applied as a thin film directly to the emitter, or small crystals of solid materials

are placed onto the emitter. Slow heating of the emitter then begins, by passing a high current through

the emitter, which is maintained at a high potential (e.g. 5 kilovolts). As heating of the emitter

continues low-vapor-pressure materials get desorbed and ionized by alkali metal cation attachment.

Applications

Many earlier applications of FD/FI to analysis of polar and nonvolatile analytes such as polymers and

biological molecules have largely been supplanted by newer ionization techniques. 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.

Liquid injection

The recently developed liquid injection FD ionization (LIFDI) technique "presents a major

breakthrough for FD-MS of reactive analytes" : Transition metal complexes are neutral and due to

their reactivity, do not undergo protonation or ion attachment. They benefit from both: the soft FD

ionization and the safe and simple LIFDI transfer of air/moisture sensitive analyte solution. This

transfer occurs from the Schlenk flask to the FD emitter in the ion source through a fused silica

capillary without breaking the vacuum.

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7. FAST ATOM BOMBARDMENT IONISATION:

Fast atom bombardment (FAB) is an ionization technique used in mass spectrometry. The material to

be analyzed is mixed with a non-volatile chemical protection environment called a matrix and is

bombarded under vacuum with a high energy (4000 to 10,000 electron volts) beam of atoms. The

atoms are typically from an inert gas such as argon or xenon. Common matrices include glycerol,

thioglycerol, 3-nitrobenzyl alcohol (3-NBA), 18-Crown-6 ether, 2-nitrophenyloctyl ether, sulfolane,

diethanolamine, and triethanolamine. This technique is similar to secondary ion mass spectrometry and

plasma desorption mass spectrometry.

How it works

FAB is a relatively soft ionization technique and produces primarily intact protonated molecules

denoted as [M+H]+ and deprotonated molecules such as [M-H]-. The nature of its ionization products

places it close to electrospray and MALDI

The first example of the practical application of this technique was the elucidation of the amino acid

sequence of the oligopeptide efrapeptin D. This contained a variety of very unusual amino acid

residues.[6] The sequence was shown to be: N-acetyl-L-pip-AIB-L-pip-AIB-AIB-L-leu-beta-ala-gly-

AIB-AIB-L-pip-AIB-gly-L-leu-L-iva-AIB-X. PIP = pipecolic acid, AIB = alpha-amino-isobutyric

acid,leu = leucine, iva = isovaline, gly = glycine. This is a potent inhibitor of the mitochodrial ATPase

activity.

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8. THERMOSPRAY IONISATION:

Thermospray is a form of atmospheric pressure ionization in mass spectrometry. It transfers ions

from the liquid phase to the gas phase for analysis. It is particularly useful in liquid chromatography-

mass spectrometry.

Ionization mechanism

TSP ionization is achieved by passing a pressurized solution through a heated tube which partially

vaporizes the effluent to generate a spray prior to entering the ion source. Droplets from the spray

contain a statistical imbalance of charges originating from charged solutes present in the solution. The

droplets gradually decrease in size by evaporation of neutral solvent molecules until the droplet

reaches a size at which the charge repulsion forces overcome the cohesive forces of the droplet.

ANALYZERS

Mass analyzer technologies

Mass analyzers separate the ions according to their mass-to-charge ratio. The following two laws

govern the dynamics of charged particles in electric and magnetic fields in vacuum:

1.Lorentz force law

2.Newton's second law of motion in non-relativistic case, i.e. valid only at ion velocity much lower

than the speed of light)

Here F is the force applied to the ion, m is the mass of the ion, a is the acceleration, Q is the ion

charge, E is the electric field, and v x B is the vector cross product of the ion velocity and the magnetic

field

Equating the above expressions for the force applied to the ion yields:

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This differential equation is the classic equation of motion for charged particles. Together with the

particle's initial conditions, it completely determines the particle's motion in space and time in terms of

m/Q. Thus mass spectrometers could be thought of as "mass-to-charge spectrometers". When

presenting data, it is common to use the (officially) dimensionless m/z, where z is the number of

elementary charges (e) on the ion (z=Q/e). This quantity, although it is informally called the mass-to-

charge ratio, more accurately speaking represents the ratio of the mass number and the charge number,

z.

There are many types of mass analyzers, using either static or dynamic fields, and magnetic or electric

fields, but all operate according to the above differential equation. Each analyzer type has its strengths

and weaknesses. Many mass spectrometers use two or more mass analyzers for tandem mass

spectrometry (MS/MS). In addition to the more common mass analyzers listed below, there are others

designed for special situations.

1. QUADRUPOLE MASS ANALYZER

The quadrupole mass analyzer is one type of mass analyzer used in mass spectrometry. As the name

implies, it consists of 4 circular rods, set perfectly parallel to each other. In a quadrupole mass

spectrometer the quadrupole mass analyzer is the component of the instrument responsible for

filtering sample ions, based on their mass-to-charge ratio (m/z). Ions are separated in a quadrupole

based on the stability of their trajectories in the oscillating electric fields that are applied to the rods.

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How it works

Image from "Apparatus For Separating Charged Particles Of Different Specific Charges" Patent

number: 2939952

The quadrupole consists of four parallel metal rods. Each opposing rod pair is connected together

electrically and a radio frequency voltage is applied between one pair of rods and the other. A direct

current voltage is then superimposed on the R.F. voltage. Ions travel down the quadrupole between the

rods. Only ions of a certain m/z will reach the detector for a given ratio of voltages: other ions have

unstable trajectories and will collide with the rods. This permits selection of an ion with a particular

m/z or allows the operator to scan for a range of m/z-values by continuously varying the applied

voltage.

Applications

These mass spectrometers excel at applications where particular ions of interest are being studied

because they can stay tuned on a single ion for extended periods of time. One place where this is useful

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is in liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry where they

serve as exceptionally high specificity detectors. Quadrupole instruments are often reasonably priced

and make good multi-purpose instruments.

TRIPLE QUADRUPOLES

A linear series of three quadrupoles can be used; known as a triple quadrupole mass spectrometer. The

first (Q1) and third (Q3) quadrupoles act as mass filters, and the middle (q2) quadrupole is employed as

a collision cell. This collision cell is an RF only quadrupole (non-mass filtering) using Ar, He or N gas

(~10-3 Torr, ~30 eV) to induce collision induced dissociation of selected parent ion(s) from Q 1.

Subsequent fragments are passed through to Q3 where they may be filtered or scanned fully.

This process allows for the study of fragments (daughter ions) which are crucial in structural

elucidation. For example, the Q1 may be set to "filter" for a drug ion of a known mass, which is

fragmented in q2. The third quadrupole (Q3) can then be set to scan the entire m/z range, giving

information on the sizes of the fragments made. Thus, the structure of the original ion can be deduced.

The arrangement of three quadrupoles was first developed by Jim Morrison of LaTrobe University,

Australia for the purpose of studying the photodissociation of gas-phase ions. The first triple-

quadrupole mass spectrometer was developed at Michigan State University by Dr. Christie Enke and

graduate student Richard Yost in the late 1970's.

2. TIME-OF-FLIGHT (TOF) MASS ANALYSIS

Theory and History:

    Time-of-flight mass spectrometry (TOF-MS) is probably the simplest method of mass measurement

to conceptualise, although there are hidden complexities when it comes to higher resolution

applications. The first commercial TOF instrument was marketed by the Bendix Corporation in the late

1950's. Their design was based on the Wiley & MacLaren instrument that was published in 1955 [1].

TOF-MS has really come into its own in recent years as being an essential instrument for biological

analysis applications - this is especially the case with the coupling of TOF-MS to MALDI and ESI

ionisation methods and the development of high-resolution and hybrid instruments (for example Q-

TOF and TOF-TOF configurations). The inherent characteristics of TOF-MS are extreme sensitivity

(all ions are detected), almost unlimited mass range and speed of analysis (modern instruments can

obtain full spectra in seconds). This makes TOF-MS one of the most desirable methods of mass

analysis.

Fig. 1: A Schematic of a Time-of-Flight mass spectrometer operating in Reflectron Mode

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    The general set up of TOF is shown in Fig. 1. The ions are introduced either directly from the source

of the instrument or from a previous analyser (in the case of Q-TOF) as a pulse. This results in all the

ions receiving the same initial kinetic energy. As they then pass along the field free drift zone, they are

separated by their masses, lighter ions travel faster. This enables the instrument to record all ions as

they arrive at the detector and so accounts for the techniques high sensitivity. The equation governing

TOF separation is:

m/z is mass-to-charge ratio of the ion

E is the extraction pulse potential

s is the length of flight tube over which E is applied

d is the length of field free drift zone

t is the measured time-of-flight of the ion

Theoretically then, all the ions are given the same initial kinetic energy by the extraction pulse and

then drift along the field free drift zone where they will be separated so that all ions of the same m/z

arrive at the detector at the same time. In practice, the pulse is not felt by all ions to the same intensity

and so a kinetic energy distribution for each discrete m/z exists. This lowers the resolution by creating

a time-of-flight distribution for each m/z [2]. This is relatively easily corrected for by the application of

a reflectron at the end of the drift zone [3]. This consists of a series of electric fields which repulse the

ions back along the flight tube - usually at a slightly displaced angle (see figure) - resulting in a

refocusing of ions with the same m/z value on the reflectron detector.

22

3. ION TRAP ANALYSER:

An ion trap is a combination of electric or magnetic fields that captures ions in a region of a vacuum

system or tube. Ion traps have a number of scientific uses such as trapping ions while the ion's

quantum state is manipulated and mass spectrometery. The two most common types of ion traps are

the Penning trap and the Paul trap (quadrupole ion trap).

When using ion traps for scientific studies of quantum state manipulation, the Paul trap is most often

used. This work may lead to a trapped ion quantum computer and has already been used to create the

world’s most accurate atomic clocks.

An ion trap mass spectrometer may incorporate a Penning trap (Fourier transform ion cyclotron

resonance), Paul trap or the Kingdon trap. The Orbitrap, introduced in 2005, is based on the Kingdon

trap. Other types of mass spectrometers may also use a linear quadrupole ion trap as a selective mass

filter.

In an electron gun (a device emitting high-speed electrons, such as those in CRTs), an ion trap may be

implemented above the cathode (using an extra, positively-charged electrode between the cathode and

the extraction electrode) to prevent its degradation by positive ions accelerated backward by the fields

intended to pull electrons away from the cathode.

23

4. FOURIER TRANSFORM ION CYCLOTRON RESONANCE

ANALYZER:

Fourier transform mass spectrometry, or more precisely Fourier transform ion cyclotron resonance

MS, measures mass by detecting the image current produced by ions cyclotroning in the presence of a

magnetic field. Instead of measuring the deflection of ions with a detector such as an electron

multiplier, the ions are injected into a Penning trap (a static electric/magnetic ion trap) where they

effectively form part of a circuit. Detectors at fixed positions in space measure the electrical signal of

ions which pass near them over time, producing a periodic signal. Since the frequency of an ion's

cycling is determined by its mass to charge ratio, this can be deconvoluted by performing a Fourier

transform on the signal. FTMS has the advantage of high sensitivity (since each ion is "counted" more

than once) and much higher resolution and thus precision

Ion cyclotron resonance (ICR) is an older mass analysis technique similar to FTMS except that ions

are detected with a traditional detector. Ions trapped in a Penning trap are excited by an RF electric

field until they impact the wall of the trap, where the detector is located. Ions of different mass are

resolved according to impact time.

5. ORBITRAP ANALYZER:

24

An orbitrap is a type of mass spectrometer invented by Alexander Makarov. It consists of an outer

barrel-like electrode and a coaxial inner spindle-like electrode that form an electrostatic field with

quadro-logarithmic potential distribution.

PRINCIPLE OF OPERATION

Figure 3 from U.S. patent 6995364 indicating the orbitrap (130), central electrode (140), two outer

electrodes (160 and 170), amplifier (180) and field compensator (200).

In an orbitrap, ions are injected tangentially into the electric field between the electrodes and trapped

because their electrostatic attraction to the inner electrode is balanced by centrifugal forces. Thus, ions

cycle around the central electrode in rings. In addition, the ions also move back and forth along the

axis of the central electrode. Therefore, ions of a specific mass-to-charge ratio move in rings which

oscillate along the central spindle. The frequency of these harmonic oscillations is independent of the

ion velocity and is inversely proportional to the square root of the mass-to-charge ratio (m/z or m/q).

By sensing the ion oscillation similar as in the FTICR-MS, the trap can be used as a mass analyzer.

Orbitraps have a high mass accuracy (1–2 ppm), a high resolving power (up to 200,000) and a high

dynamic range (around 5000).

Ion trajectories in an Orbitrap mass spectrometer.

25

Like FTICR-MS the orbitrap resolving power is proportional to the number of harmonic oscillations of

the ions, as a result the resolving power is inversely proportional to the square root of m/z and

proportional to acquisition time. Scigelova and Makarov show the resolving power to be 7500 per 0.1

second transient at m/z 400. A transient being the duration that the time domain signal is acquired for.

The resolving power decreases further as the m/z value increases so that at 4 times the m/z value (1600)

the resolving power has halved to 3750 per 0.1 second. Approximately 0.1 seconds per transient is

required for data processing, thus a 0.1 second transient has a cycle time of 0.2 seconds. These effects

in combination result in a resolving power of 7500 at m/z 400 and 3750 at m/z 1600 when the orbitrap

is operating at 5 acquisitions per second.

The linear trap quadrupole (LTQ) can be used as a front end for the orbitrap.

DETECTOR

A continuous dynode particle multiplier detector.

The final element of the mass spectrometer is the detector. The detector records either the charge

induced or the current produced when an ion passes by or hits a surface. In a scanning instrument, the

signal produced in the detector during the course of the scan versus where the instrument is in the scan

(at what m/Q) will produce a mass spectrum, a record of ions as a function of m/Q

1) ELECTRON MULTIPLIER DETECTOR:

26

Continuous dynode electron multiplier

An electron multiplier (continuous dynode electron multiplier) is a vacuum-tube structure that

multiplies incident charges. In a process called secondary emission, a single electron can, when

bombarded on metal (or PbO coated surface) induce emission of roughly 1 to 3 electrons. If an electric

potential is applied between this metal plate and yet another, the emitted electrons will accelerate to

the next metal plate and induce secondary emission of still more electrons. This can be repeated a

number of times, resulting in a large shower of electrons all collected by a metal anode, all having

been triggered by just one.

Operation

The avalanche can be triggered by any charged particle hitting the starting electrode with sufficient

energy to cause secondary emission. Hence the electron multiplier is often used as an ion detector. It

could also be triggered by a photon causing vacuum photoemission of at least one electron. In a

photomultiplier tube, a photo-emissive surface is followed by an electron multiplier with several

sequential multiplying electrodes called dynodes. Because these electrodes are separate from each

other, this might be called a "discrete-dynode" multiplier. A voltage divider chain of resistors is

usually used to place each dynode at a potential 100-200v more positive than the previous one.

A "continuous-dynode" structure is feasible if the material of the electrodes has a high resistance, so

that the functions of secondary-emission and voltage-division are merged. This is often built as a

funnel of glass coated inside with a thin film of semi-conducting material, with negative high voltage

applied at the wider input end, and positive voltage near ground applied at the narrower output end.

Electrons emitted at any point are accelerated a modest distance down the funnel before impacting the

surface, perhaps on the opposite side of the funnel. At the destination end a separate electrode (anode)

remains necessary to collect the multiplied electrons. This structure is also known as (single) channel

electron multiplier (CEM), and one of the most common is sold under the trade name Channeltron.

Geometry of continuous-dynode electron multiplier is called the microchannel plate. It may be

considered a 2-dimensional parallel array of very small continuous-dynode electron multipliers, built

together and powered in parallel too. Each microchannel is generally parallel-walled, not tapered or

funnel-like

2) FARADAY CUP DETECTOR:27

A Faraday cup is a metal (conductive) cup designed to catch charged particles in vacuum. The

resulting current can be measured and used to determine the number of ions or electrons hitting the

cup.[1] The Faraday cup is named after Michael Faraday who first theorized ions around 1830.

How it works

When a beam or packet of Ions hits the metal it gains a small net charge while the ions are neutralized.

The metal can then be discharged to measure a small current equivalent to the number of impinging

ions. Essentially the faraday cup is part of a circuit where ions are the charge carriers in vacuum and

the faraday cup is the interface to the solid metal where electrons act as the charge carriers (as in most

circuits). By measuring the electrical current (the number of electrons flowing through the circuit per

second) in the metal part of the circuit the number of charges being carried by the ions in the vacuum

part of the circuit can be determined. For a continuous ion beam of ions (each with a single charge)

Faraday cup with an electron-supressor plate in front

Where N is the number of ions observed in a time t (in seconds), I is the measured current (in amperes)

and e is the elementary charge (about 1.60 × 10-19 C). Thus, a measured current of one nanoamp (10-9

A) corresponds to about 6 billion ions striking the faraday cup each second.

28

Similarly, a Faraday cup can act as a collector for electrons in a vacuum (for instance from an electron

beam). In this case electrons simply hit the metal plate/cup and a current is produced. Faraday cups are

not as sensitive as electron multiplier detectors, but are highly regarded for accuracy because of the

direct relation between the measured current and number of ions.

3. ARRAY DETECTORS

Array detectors can attain very high sensitivities by collecting all the ions of a given mass range

continuously.This contrasts with conventional scanning methods using point detectors (e.g. electron

multipliers) that collect only a small fraction of the ions in each mass channel as the spectrum is

scanned.

Photodiode arrays and similar devices were limited by their low resolving powers and narrow mass

ranges. However, recent improvements have extended the mass range by sequentially stepping

‘scanning arrays’ under computer control. Alternatively, specially designed ion-optical systems have

been developed for use with wide-angle array detectors. Both developments allow reasonable scan

rates for the total spectrum. Array detection can increase the apparent sensitivity by two orders of

magnitude.

THE VACUUM SYSTEM:

All mass spectrometers operate at very low pressure (high vacuum). This reduces the chance of ions

colliding with other molecules in the mass analyzer. Any collision can cause the ions to react,

neutralize, scatter, or fragment. All these processes will interfere with the mass spectrum. To minimize

collisions, experiments are conducted under high vacuum conditions, typically 10-2 to 10-5 Pa (10-4 to

10-7 torr) depending upon the geometry of the instrument. This high vacuum requires two pumping

stages. The first stage is a mechanical pump that provides rough vacuum down to 0.1 Pa (10-3 torr).

The second stage uses diffusion pumps or turbomolecular pumps to provide high vacuum. ICR

instruments have even higher vacuum requirements and often include a cryogenic pump for a third

pumping stage. The pumpingsystem is an important part of any mass spectrometer but a detailed

discussion is beyond thescope of this paper

RESOLUTION IN MASS SPECTROMETER

In mass spectrometry, resolution is a measure of the ability to distinguish between two peaks of

different mass-to-charge ratio, m/z, in a mass spectrum. There are several ways to define resolution in

29

mass spectrometry, therefore it is important to report the method used to determine mass resolution

when reporting its value.

Wher m and delta m are mass numbers of two neighbouring peaks

VALLEY DEFINITION: The valley definition defines Δm as the closest spacing of two peaks

of equal intensity with the valley (lowest value of signal) between them less than aspecifiedfraction of

the peak height. Typical values are 10% or 50%. The value obtained from a 5% peak width is roughly

equivalent to a 10% valley.

DATA AND ANALYSIS

Mass spectrum of a peptide showing the isotopic distribution

DATA REPRESENTATIONS

Mass spectrometry produces various types of data. The most common data representation is the mass

spectrum certain types of mass spectrometry data are best represented as a mass chromatogram. Types

of chromatograms include selected ion monitoring (SIM), total ion current (TIC), and selected reaction

monitoring chromatogram (SRM), among many others. Other types of mass spectrometry data are well

represented as a three dimensional contour map. In this form, the mass-to-charge, m/z is on the x-axis,

intensity the y-axis, and an additional experimental parameter, such as time, is recorded on the z-axis.

DATA ANALYSIS

Basics

30

Mass spectrometry data analysis is a complicated subject matter that is very specific to the type of

experiment producing the data. There are general subdivisions of data that are fundamental to

understand any data.

Many mass spectrometers work in either negative ion mode or positive ion mode. It is very important

to know whether the observed ions are negatively or positively charged. This is often important in

determining the neutral mass but it also indicates something about the nature of the molecules.

Different types of ion source result in different arrays of fragments produced from the original

molecules. An electron ionization source produces many fragments and mostly odd electron species

with one charge; whereas an electrospray source usually produces quasimolecular even electron

species that may be multiply charged. Tandem mass spectrometry purposely produces fragment ions

post-source and can drastically change the sort of data achieved by an experiment.

By understanding the origin of a sample, certain expectations can be assumed as to the component

molecules of the sample and their fragmentations. A sample from a synthesis/manufacturing process

will probably contain impurities chemically related to the target component. A relatively crudely

prepared biological sample will probably contain a certain amount of salt, which may form adducts

with the analyte molecules in certain analyses.

Results can also depend heavily on how the sample was prepared and how it was run/introduced. An

important example is the issue of which matrix is used for MALDI spotting, since much of the

energetics of the desorption/ionization event is controlled by the matrix rather than the laser power.

Sometimes samples are spiked with sodium or another ion-carrying species to produce adducts rather

than a protonated species.

The greatest source of trouble when non-mass spectrometrists try to conduct mass spectrometry on

their own or collaborate with a mass spectrometrist is inadequate definition of the research goal of the

experiment. Adequate definition of the experimental goal is a prerequisite for collecting the proper

data and successfully interpreting it. Among the determinations that can be achieved with mass

spectrometry are molecular mass, molecular structure, and sample purity. Each of these questions

requires a different experimental procedure. Simply asking for a "mass spec" will most likely not

answer the real question at hand.

INTERPRETATION OF MASS SPECTRA

Since the precise structure or peptide sequence of a molecule is deciphered through the set of fragment

masses, the interpretation of mass spectra requires combined use of various techniques. Usually the

31

first strategy for identifying an unknown compound is to compare its experimental mass spectrum

against a library of mass spectra. If the search comes up empty, then manual interpretation or software

assisted interpretation of mass spectra are performed. Computer simulation of ionization and

fragmentation processes occurring in mass spectrometer is the primary tool for assigning structure or

peptide sequence to a molecule. An a priori structural information is fragmented in silico and the

resulting pattern is compared with observed spectrum. Such simulation is often supported by a

fragmentation library that contains published patterns of known decomposition reactions. Software

taking advantage of this idea has been developed for both small molecules and proteins.

Another way of interpreting mass spectra involves spectra with accurate mass. A mass-to-charge ratio

value (m/z) with only integer precision can represent an immense number of theoretically possible ion

structures. More precise mass figures significantly reduce the number of candidate molecular formulas,

albeit each can still represent large number of structurally diverse compounds. A computer algorithm

called formula generator calculates all molecular formulas that theoretically fit a given mass with

specified tolerance.

A recent technique for structure elucidation in mass spectrometry, called precursor ion fingerprinting

identifies individual pieces of structural information by conducting a search of the tandem spectra of

the molecule under investigation against a library of the product-ion spectra of structurally

characterized

MASS SPECTRUM ANALYSIS

Mass spectrum analysis is an integral part of spectroscopy and mass spectrometry dealing with the

interpretation of mass spectra Organic chemists obtain mass spectra of chemical compounds as part of

structure elucidation and the analysis is part of every organic chemistry curriculum.

32

BASIC PEAKS

Mass spectra have several distinct sets of peaks or ions:

1. the molecular ion,

2. isotope peaks

3. fragmentation peaks

4. metastable peaks

5. rearrangement ion

6. Multiply charged ion

7. Negative ion

1. the molecular ion

Mass spectra first of all display the molecular ion (or parent ion) peak which is a radical cation M+. as

a result of removing one electron from the molecule. In the spectrum for toluene for example the

molecular ion peak is located at 92 m/e corresponding to its molecular mass. The molecular ion peak

does not always appear or can be weak. The height of the molecular ion peak diminishes with

branching and with increasing mass in a homologous series. Identifying the molecular ion can be

difficult. A useful aid is the nitrogen rule: if the mass is an even number, the compound contains no

nitrogen or an even number of nitrogen’s. Molecular ion peaks are also often preceded by a M-1 or M-

2 peak resulting from loss of a hydrogen radical or dihydrogen.

2. Isotope ion

More peaks are visible with m/e ratios larger than the molecular ion peak due to isotope distributions.

The value of 92 in the toluene example corresponds to the monoisotopic mass of a molecule of toluene

entirely composed of the most abundant isotopes (1H and 12C). The so-called M+1 peak corresponds to

a fraction of the molecules with one higher isotope incorporated ((2H or 13C) and the M+2 peak has two

higher isotopes. The natural abundance of the higher isotopes is low for frequently encountered

elements such as hydrogen, carbon and nitrogen and the intensity of isotope peaks subsequently low

and the intensity quickly diminishes with total mass. In halogens on the other hand higher isotopes

have a large abundance which results in a specific mass signature for halogen containing compounds.

3. Fragmentation ion

Peaks with mass less that the molecular ion are the result of fragmentation of the molecule. These

peaks are called daughter peaks. The peak with the highest ratio is called the base peak which is not

33

necessary the molecular ion. Many reaction pathways exist for fragmentation but only newly formed

cations will show up in the mass spectrum and not radical fragments or neutral fragments.

4. The metastable ion

Metastable peaks are broad peaks at non-integer mass values. These peaks result from molecular

fragments with lower kinetic energy because of fragmentations taking place ahead of the ionization

chamber. They are not of analytical value.

5. Rearrangement ion

Fragment ions formed by the intramolecular rearrangements involving migration of hydrogen atoms

from one part of the ion to other part are called rearrangement ions Rearrangement process are

common in unsaturated compounds and are fovoured even at low electron voltage

The most common example is “McLafferty Rearrangement”

6. Multiply charged ions

In mass spectrometer, the ions are carrying a single positive charge. How ever, sometime doubly or

even triply charged ions are found in mass spectrum. The doubly or triply charged ions are recorded at

a half or a third of the m/e value of the singly charged ions. The formation of these multiply charged

ions are more common in hetero atomic molecules

7. Negative ions: Though in ionisation process positive ions are produced, in few cases negative

ions do get formed these result due to the capture of electron by a molecule during collision of

molecules. Their formation is very rare but these can be produced in three ways

1 dissociatin resonance capture

2. Resonance capture

3. Ion pair production

34

The negative ions are focussed by reversing the field in the mass spectrometer.these are not very useful

in structural determinations

Ex: o-, oh-.c2h-

FRAGMENTATION

The fragmentation pattern not only allows the determination of the mass of an unknown compound but

also allows guessing the molecular structure especially in combination the calculation of the degree of

unsaturation from the molecular formula (when available). Neutral fragments frequently lost are

carbon monoxide, ethylene, water, ammonia, and hydrogen sulfide.

Fragmentations arise from:

Homolysis processes. An example is the cleavage of carbon-carbon bonds next to a heteroatom

In this depiction single-electron movements are indicated by a fishhook arrow.

Rearrangement reactions, for example a retro Diels-Alder reaction extruding neutral ethylene:

Or the McLafferty rearrangement. As it is not always obvious where a lone electron resides in a

radical cation a square bracket notation is often used.

MCLAFFERTY REARRANGEMENT

The McLafferty rearrangement is a reaction observed in mass spectrometry. It is sometimes found

that a molecule containing a keto-group undergoes β-cleavage, with the gain of the γ-hydrogen atom.

This rearrangement may take place by a radical or ionic mechanism.

35

The reaction

A description of the reaction was first published by the American chemist Fred McLafferty in 1959

ALPHA CLEAVAGE

Alpha cleavage, (α-cleavage) in organic chemistry, refers to the act of breaking the carbon-carbon

bond, [1] adjacent to the carbon bearing a specified functional group.

Generally this topic is discussed when covering mass spectrometry and occurs generally by the same

mechanisms.

For example of a mechanism of alpha cleavage, an electron is knocked off an atom (usually by

electron collision) to form a radical cation. Electron removal generally happens in the following order:

1) lone pair electrons, 2) pi bond electrons, 3) sigma bond electrons

One of the lone pair electrons moves down to form a pi bond with an electron from an adjacent (alpha)

bond.The other electron from the bond moves to an adjacent atom (not one adjacent to the lone pair

atom) creating a radical. This creates a double bond adjacent to the lone pair atom (oxygen is a good

example) and breaks/cleaves the bond from which the two electrons were removed.

Example C-C-(O::)-H > C-C-(O:.+)-H > C' + (C=O:+)-H where: is a lone pair + is a positive charge

and ' is a radical/free electron

In molecules containing carbonyl groups, often competes with McLafferty rearrangement.

SOME GENERAL RULES:

Cleavage occurs at alkyl substituted carbons reflecting the order generally observed in

carbocations.

Double bonds and arene fragments tend to resist fragmentation.

36

Allylic cations are stable and resist fragmentation.

the even-electron rule stipulates that even-electron species (cations but not radical ions) will

not fragment into two odd-electron species but rather to another cation and a neutral molecule

NITROGEN RULE

The nitrogen rule states that organic compounds containing exclusively hydrogen, carbon, nitrogen,

oxygen, silicon, phosphorus, sulfur, and the halogens either have 1) an odd nominal mass that indicates

an odd number of nitrogen atoms are present or 2) an even nominal mass that indicates an even number

of nitrogen atoms are present in the molecular ion The nitrogen rule is not a rule, per se, as much as a

general principle which may prove useful when attempting to solve organic mass spectrometry

structures.

Formulation of the rule

This rule is derived from the fact that, perhaps coincidentally, for the most common chemical elements

in neutral organic compounds (hydrogen, carbon, nitrogen, oxygen, silicon, phosphorus, sulfur, and the

halogens), elements with even numbered nominal masses form even numbers of covalent bonds, while

elements with odd numbered nominal masses form odd numbers of covalent bonds, with the exception

of nitrogen, which has a nominal (or integer) mass of 14, but has a valency of 3.

It should be noted that the nitrogen rule is only true for neutral structures in which all of the atoms in

the molecule have a number of covalent bonds equal to their standard valency (counting each sigma

bond and pi bond as a separate covalent bond for the purposes of the calculation). Therefore, the rule is

typically only applied to the molecular ion signal in the mass spectrum.

Mass spectrometry generally operates by measuring the mass of ions. If the measured ion is generated

by creating or breaking a single covalent bond (such as protonating an amine to form an ammonium

center or removing a hydride from a molecule to leave a positively charged ion) then the nitrogen rule

becomes reversed (odd numbered masses indicate even numbers of nitrogens and vice versa).

However, for each consecutive covalent bond that is broken or formed, the nitrogen rule again

reverses.

Therefore, a more rigorous definition of the nitrogen rule for organic compounds containing

exclusively hydrogen, carbon, nitrogen, oxygen, silicon, phosphorus, sulfur, and the halogens would

be as follows:

37

An even nominal mass indicates that a net even number of covalent bonds have been broken or formed

and an even number of nitrogen atoms are present, or that a net odd number of covalent bonds have

been broken or formed and an odd number of nitrogen atoms are present. An odd nominal mass

indicates that a net even number of covalent bonds have been broken or formed and an odd number of

nitrogen atoms are present, or that a net odd number of covalent bonds have been broken or formed

and an even number of nitrogen atoms are present.

Inorganic molecules do not necessarily follow the rule. For example the nitrogen oxides NO and NO2

have an odd number of nitrogens but even masses of 30 and 46

ISOTOPE EFFECTS

Isotope peaks within a spectrum can help in structure elucidation. Compounds containing halogens

(especially chlorine and bromine) produce very distinct isotope peaks. The mass spectrum of

methylbromide has two prominent peaks of equal intensity at 94 (M) and 96 (M+2) and then two more

at 79 and 81 belonging to the bromine fragment.

Even when compounds only contain elements with less intense isotope peaks (carbon or oxygen), the

distribution of these peaks can be used to assign the spectrum to the correct compound. For example,

two compounds with identical mass of 150, C8H12N3+ and C9H10O2

+, will have two different M+2

intensities which make it possible to distinguish between them.

TOLUENE EXAMPLE

38

The mass spectrum for toluene has around 30 signals. Several peaks can be rationalized in this

fragmentation pattern.

39

Natural abundance of some elements

The next table gives the isotope distributions for some elements. Some elements like phosphorus and

fluorine only exist as a single isotope, with a natural abundance of 100%.

Natural abundance of some elements [3]

Isotope  % nat. abundance atomic mass isotope  % nat. abundance atomic mass

1H 99.985 1.007825 12C 98.89 12 (definition)

2H 0.015 2.0140 13C 1.11 13.00335

16O 99.76 15.99491 14N 99.64 14.00307

17O 0.04 15N 0.36 15.00011

18O 0.2

28Si 92.23 27.97693 32S 95.0 31.97207

29Si 4.67 28.97649 33S 0.76 32.97146

30Si 3.10 29.97376 34S 4.22 33.96786

35Cl 75.77 34.96885 79Br 50.69 78.9183

37Cl 24.23 81Br 49.31 80.9163

40

CHROMATOGRAPHIC TECHNIQUES COMBINED WITH MASS

SPECTROMETRY

Gas chromatography

Liquid chromatography

Ion mobility

1. GAS CHROMATOGRAPHY MASS SPECTROMETRY (GC/MS)

Gas chromatography mass spectrometry (GC/MS) is an instrumental technique, comprising a gas

chromatograph (GC) coupled to a mass spectrometer (MS), by which complex mixtures of chemicals

may be separated, identfied and quantified. This makes it ideal for the analysis of the hundreds of

relatively low molecular weight compounds found in environmental materials. In order for a

compound to be analysed by GC/MS it must be sufficiently volatile and thermally stable. In addition,

functionalised compounds may require chemical modification (derivatization), prior to analysis, to

eliminate undesirable adsorption effects that would otherwise affect the quality of the data obtained.

Samples are usually analyzed as organic solutions consequently materials of interest (e.g. soils,

sediments, tissues etc.) need to be solvent extracted and the extract subjected to various 'wet chemical'

techniques before GC/MS analysis is possible.

The sample solution is injected into the GC inlet where it is vaporized and swept onto a

chromatographic column by the carrier gas (usually helium). The sample flows through the column

and the compounds comprising the mixture of interest are separated by virtue of their relative

interaction with the coating of the column (stationary phase) and the carrier gas (mobile phase). The

41

latter part of the column passes through a heated transfer line and ends at the entrance to ion source

(Fig. 1) where compounds eluting from the column are converted to ions.

Two potential methods exist for ion production. The most frequently used method is electron

ionisation (EI) and the occasionally used alternative is chemical ionisation (CI). For EI a beam of

electrons ionise the sample molecules resulting in the loss of one electron. A molecule with one

electron missing is called the molecular ion and is represented by M+. (a radical cation). When the

resulting peak from this ion is seen in a mass spectrum, it gives the molecular weight of the compound.

Due to the large amount of energy imparted to the molecular ion it usually fragments producing further

smaller ions with characteristic relative abundances that provide a 'fingerprint' for that molecular

structure. This information may be then used to identify compounds of interest and help elucidate the

structure of unknown components of mixtures. CI begins with the ionisation of methane (or another

suitable gas), creating a radical which in turn will ionise the sample molecule to produce [M+H]+

molecular ions. CI is a less energetic way of ionising a molecule hence less fragmentation occurs with

CI than with EI, hence CI yields less information about the detailed structure of the molecule, but does

yield the molecular ion; sometimes the molecular ion cannot be detected using EI, hence the two

methods complement one another. Once ionised a small positive is used to repel the ions out of the

ionisation chamber.

The next component is a mass analyser (filter), which separates the positively charged ions according

to various mass related properties depending upon the analyser used. Several types of analyser exist:

quadrupoles (Fig. 2), ion traps, magnetic sector, time-of-flight, radio frequency, cyclotron resonance

and focusing to name a few. The most common are quadrupoles and ion traps. After the ions are

separated they enter a detector the output from which is amplified to boost the signal. The detector

sends information to a computer that records all of the data produced, converts the electrical impulses

into visual displays and hard copy displays. In addtion, the computer also controls the operation of the

mass spectrometer.

42

2.LIQUID CHROMATOGRAPHY MASS SPECTROMETRY

Liquid chromatography-mass spectrometry (LC-MS, or alternatively HPLC-MS) is an analytical

chemistry technique that combines the physical separation capabilities of liquid chromatography (or

HPLC) with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used

for many applications which has very high sensitivity and specificity. Generally its application is

oriented towards the specific detection and potential identification of chemicals in the presence of

other chemicals (in a complex mixture).

LIQUID CHROMATOGRAPHY

Scale

A major difference between traditional HPLC and the chromatography used in LC-MS is that in the

latter case the scale is usually much smaller, both with respect to the internal diameter of the column

and even more so with respect to flow rate since it scales as the square of the diameter. For a long

time, 1 mm columns were typical for LC-MS work (as opposed to 4.6 mm for HPLC). More recently

300 µm and even 75 µm capillary columns have become more prevalent. At the low end of these

column diameters the flow rates approach 100 nL/min and are generally used with nanospray sources

Flow splitting

When standard bore (4.6 mm) columns are used the flow is often split ~10:1. This can be beneficial by

allowing the use of other techniques in tandem such as MS and UV. However splitting the flow to UV

will decrease the sensitivity of spectrophotometric detectors. The Mass Spec on the other hand will

give improved sensitivity at flow rates of 200 μL/min or less. This is because the analyte ions must be

vaporised (nebulized) in order to become charged.

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MASS SPECTROMETRY

Mass analyzer

There are a lot of mass analyzers that can be used in LC/MS. Single Quadrupole, Triple Quadrupole,

Ion Trap, TOF (time of Flight) and Quadrupole-time of flight (Q-TOF).

Interface

Understandably the interface between a liquid phase technique which continuously flows liquid, and a

gas phase technique carried out in a vacuum was difficult for a long time. The advent of electrospray

ionization changed this. The interface is most often an electrospray ion source or variant such as a

nanospray source; however fast atom bombardment, thermospray and atmospheric pressure chemical

ionization interfaces are also used. Various deposition and drying techniques have also been used such

as using moving belts; however the most common of these is off-line MALDI deposition.

Applications

Pharmacokinetics

LC-MS is very commonly used in pharmacokinetic studies of pharmaceuticals. These studies give

information about how quickly a drug will be cleared from the hepatic blood flow, and organs of the

body. MS is used for this due to high sensitivity and exceptional specificity compared to UV (as long

as the analyte can be suitably ionised), and short analysis time.

The major advantage MS has is the use of tandem MS-MS. The detector may be programmed to select

certain ions to fragment. The process is essentially a selection technique, but is in fact more complex.

The measured quantity is the sum of molecule fragments chosen by the operator. As long as there are

no interferences or ion suppression, the LC separation can be quite quick. It is common now to have

analysis times of 1 minute or less by MS-MS detection, compared to over 10 mins with UV detection.

Proteomics

LC-MS is also used in the study of proteomics where again components of a complex mixture must be

detected and identified in some manner. The bottom-up proteomics LC-MS approach to proteomics

generally involves protease digestion and denaturation (usually trypsin as a protease, urea to denature

tertiary structure and iodoacetamide to cap cysteine residues) followed by LC-MS with peptide mass

fingerprinting or LC-MS/MS (tandem MS) to derive sequence of individual peptides. LC-MS/MS is

most commonly used for proteomic analysis of complex samples where peptide masses may overlap

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even with a high-resolution mass spectrometer. Samples of complex biological fluids like human

serum may be run in a modern LC-MS/MS system and result in over 1000 proteins being identified,

provided that the sample was first separated on an SDS-PAGE gel or HPLC-SCX.

Drug development

LC-MS is frequently used in drug development at many different stages including Peptide Mapping,

Glycoprotein Mapping, Natural Products Dereplication, Bioaffinity Screening, In Vivo Drug

Screening, Metabolic Stability Screening, Metabolite Identification, Impurity Identification, Degradant

Identification, Quantitative Bioanalysis, and Quality Control.

3. ION MOBILITY SPECTROMETRY-MASS SPECTROMETRY

Ion mobility spectrometry-mass spectrometry (IMS-MS) is a method that combines the features of ion

mobility spectrometry and mass spectrometry to identify different substances within a test sample.

Ion mobility spectrometry/mass spectrometry (IMS/MS or IMMS) is a technique where ions are first

separated by drift time through some neutral gas under an applied electrical potential gradient before

being introduced into a mass spectrometer. Drift time is a measure of the radius relative to the charge

of the ion. The duty cycle of IMS (the time over which the experiment takes place) is longer than most

mass spectrometric techniques, such that the mass spectrometer can sample along the course of the

IMS separation. This produces data about the IMS separation and the mass-to-charge ratio of the ions

in a manner similar to LC/MS.

The duty cycle of IMS is short relative to liquid chromatography or gas chromatography separations

and can thus be coupled to such techniques, producing triple modalities such as LC/IMS/MS.

Data and analysis

On the one hand, ion mobility spectrometry was developed in the 70s and typically separates charged

particles on a millisecond scale. On the other hand, time-of-flight mass spectrometry was developed in

the 50s and typically separates charged particles on a microsecond scale. The combination of both

instruments was pioneered in the end of the 90s at Indiana University by David E. Clemmer and co-

workers

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Instrumentation

The IMS-MS is composed of two major building blocks: the ion mobility spectrometer and the mass

spectrometer. Whereas the ion mobility spectrometer is usually made of a drift region at atmospheric

pressure or lower, the mass spectrometer is under a high vacuum.

Applications: The IMS-MS technique can be used in proteomics, for analyzing complex mixtures

of peptides.

MASS SPECTRA-REPRESENTATION

Mass spectrum of a peptide showing the isotopic distribution

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APPLICATIONS OF MASS SPECTROMETRY

1.Isotope ratio MS: isotope dating and tracking

Mass spectrometer to determine the 16O/18O and 12C/13C isotope ratio on biogenous carbonate

Mass spectrometry is also used to determine the isotopic composition of elements within a sample.

Differences in mass among isotopes of an element are very small, and the less abundant isotopes of an

element are typically very rare, so a very sensitive instrument is required. These instruments,

sometimes referred to as isotope ratio mass spectrometers (IR-MS), usually use a single magnet to

bend a beam of ionized particles towards a series of Faraday cups which convert particle impacts to

electric current. A fast on-line analysis of deuterium content of water can be done using Flowing

afterglow mass spectrometry, FA-MS. Probably the most sensitive and accurate mass spectrometer for

this purpose is the accelerator mass spectrometer (AMS). Isotope ratios are important markers of a

variety of processes. Some isotope ratios are used to determine the age of materials for example as in

carbon dating. Labelling with stable isotopes is also used for protein quantification. (see Protein

quantitation below)

2.Trace gas analysis

Several techniques use ions created in a dedicated ion source injected into a flow tube or a drift tube:

selected ion flow tube (SIFT-MS), and proton transfer reaction (PTR-MS), are variants of chemical

ionization dedicated for trace gas analysis of air, breath or liquid headspace using well defined reaction

time allowing calculations of analyte concentrations from the known reaction kinetics without the need

for internal standard or calibration.

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3.Atom probe

An atom probe is an instrument that combines time-of-flight mass spectrometry and field ion

microscopy (FIM) to map the location of individual atoms.

4.Pharmacokinetics

Pharmacokinetics is often studied using mass spectrometry because of the complex nature of the

matrix (often blood or urine) and the need for high sensitivity to observe low dose and long time point

data. The most common instrumentation used in this application is LC-MS with a triple quadrupole

mass spectrometer. Tandem mass spectrometry is usually employed for added specificity. Standard

curves and internal standards are used for quantitation of usually a single pharmaceutical in the

samples. The samples represent different time points as a pharmaceutical is administered and then

metabolized or cleared from the body. Blank or t=0 samples taken before administration are important

in determining background and insuring data integrity with such complex sample matrices. Much

attention is paid to the linearity of the standard curve; however it is not uncommon to use curve fitting

with more complex functions such as quadratics since the response of most mass spectrometers is less

than linear across large concentration ranges.

There is currently considerable interest in the use of very high sensitivity mass spectrometry for

microdosing studies, which are seen as a promising alternative to animal experimentation.

5. Protein characterization

Mass spectrometry is an important emerging method for the characterization of proteins. The two

primary methods for ionization of whole proteins are electrospray ionization (ESI) and matrix-assisted

laser desorption/ionization (MALDI). In keeping with the performance and mass range of available

mass spectrometers, two approaches are used for characterizing proteins. In the first, intact proteins are

ionized by either of the two techniques described above, and then introduced to a mass analyser. This

approach is referred to as "top-down" strategy of protein analysis. In the second, proteins are

enzymatically digested into smaller peptides using proteases such as trypsin or pepsin, either in

solution or in gel after electrophoretic separation. Other proteolytic agents are also used. The collection

of peptide products are then introduced to the mass analyser. When the characteristic pattern of

peptides is used for the identification of the protein the method is called peptide mass fingerprinting

(PMF), if the identification is performed using the sequence data determined in tandem MS analysis it

is called de novo sequencing. These procedures of protein analysis are also referred to as the "bottom-

up" approach.

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6.Space exploration

As a standard method for analysis, mass spectrometers have reached other planets and moons. Two

were taken to Mars by the Viking program. In early 2005 the Cassini-Huygens mission delivered a

specialized GC-MS instrument aboard the Huygens probe through the atmosphere of Titan, the largest

moon of the planet Saturn. This instrument analyzed atmospheric samples along its descent trajectory

and was able to vaporize and analyze samples of Titan's frozen, hydrocarbon covered surface once the

probe had landed. These measurements compare the abundance of isotope(s) of each particle

comparatively to earth's natural abundance.. Also onboard the Cassini-Huygens spacecraft is an ion

and neutral mass spectrometer which has been taking measurements of Titan's atmospheric

composition as well as the composition of Enceladus' plumes.

Mass spectrometers are also widely used in space missions to measure the composition of plasmas. For

example, the Cassini spacecraft carries the Cassini Plasma Spectrometer (CAPS), which measures the

mass of ions in Saturn's magnetosphere.

7.Respired gas monitor

Mass spectrometers were used in hospitals for respiratory gas analysis beginning around 1975 through

the end of the century. Some are probably still in use but none are currently being manufactured.

Found mostly in the operating room, they were a part of a complex system in which respired gas

samples from patients undergoing anesthesia were drawn into the instrument through a valve

mechanism designed to sequentially connect up to 32 rooms to the mass spectrometer. A computer

directed all operations of the system. The data collected from the mass spectrometer was delivered to

the individual rooms for the anesthesiologist to use.

This magnetic sector mass spectrometer's uniqueness may have been the fact that a plane of detectors,

each purposely positioned to collect all of the ion species expected to be in the samples, allowed the

instrument to simultaneously report all of the patient respired gases. Although the mass range was

limited to slightly over 120 u, fragmentation of some of the heavier molecules negated the need for a

higher detection limit.

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