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Introduction

Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles. It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and 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. For large samples such as biomolecules, molecular masses can be measured to within an accuracy of 0.01% of the total molecular mass of the sample i.e. within a 4 Daltons (Da) or atomic mass units (amu) error for a sample of 40,000 Da. This is sufficient to allow minor mass changes to be detected, e.g. the substitution of one amino acid for another or a post-translational modification. For small organic molecules the molecular mass can be measured to within an accuracy of 5 ppm or less, which is often sufficient to confirm the molecular formula of a compound, and is also a standard requirement for publication in a chemical journal. Structural information can be generated using certain types of mass spectrometers, usually those with multiple analyzers which are known as tandem mass spectrometers. This is achieved by fragmenting the sample inside the instrument and analyzing the products generated. This procedure is useful for the structural elucidation of organic compounds and for peptide or oligonucleotide sequencing.

In a typical MS procedure: 1. A sample is loaded onto the MS instrument, and undergoes vaporization 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. The ions are separated according to their mass-to-charge ratio in an analyzer by electromagnetic fields 4. The ions are detected, usually by a quantitative method 5. The ion signal is processed into mass spectra

MS instruments consist of three modules:

An ion source, which can convert gas phase sample molecules into ions (or, in the case of electrospray ionization, move ions that exist in solution into the gas phase) A mass analyzer, which sorts the ions by their masses by applying electromagnetic fields A detector, which measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present

Sample introduction
The method of sample introduction to the ionization source often depends on the ionization method being used, as well as the type and complexity of the sample. The sample can be inserted directly into the ionization source, or can undergo some type of chromatography en route to the ionization source. This latter method of sample introduction usually involves the mass spectrometer being coupled directly to a high pressure liquid chromatography (HPLC), gas chromatography (GC) or capillary electrophoresis (CE) separation column, and hence the sample is separated into a series of components which then enter the mass spectrometer sequentially for individual analysis.

Sample introduction:
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 thermallystable, a variety of inlets are available. Gases and samples with high vapor pressure areintroduced 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 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 vapor 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. However, wide bore capillaries and packed GC columns have higher flow rates. This significantly increases the pressure in the mass spectrometer. Several inlet designs are available to reduce the gas flow into the source. The simplest design splits the GC effluent so that only a small portion of the total flow enters the mass spectrometer. Although this inlet reduces the gas load on the vacuum system, it also reduces the amount of analyte. Effusive separators and membrane inlets are more selective and transport a higher fraction of the analyte into the source region. Each of these methods has efficiency and resolution drawbacks but they are necessary for some experiments.

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. These inlets are discussed in greater detail in the section on ionization techniques.

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 section.

Ionization Techniques
Glow discharge (GD) Electron impact ionization (EI) Chemical ionization (CI) Field ionization (FI) Inductively coupled plasma (ICP) Fast atom bombardment (FAB) Secondary ion mass spectrometry (SIMS) Thermospray (TSI) Ionspray (IS) Electrospray (ESI) Plasma Desorption (PD) Laser Desorption (LD) Matrix-assisted laser desorption/ionization (MALDI) A variety of ionization techniques are used for mass spectrometry. Most ionization Techniques excites the neutral analyte molecule which then ejects an electron to form a radicalcation (M+). Other ionization techniques involve ion molecule reactions that produce adductions (MH+) the most important considerations are the physical state of the analyte and the ionization energy. Electron ionization and chemical ionization are only suitable for gas phase ionization. Fast atom bombardment, secondary ion mass spectrometry, electrospray, and matrix assisted laser desorption are used to ionize condensed phase samples. The ionization energy insignificant because it controls the amount of fragmentation observed in the mass spectrum. .Although this fragmentation

complicates the mass spectrum; it provides structural information for the identification of unknown compounds. Some ionization techniques are very soft and only produce molecular ions,* other techniques are very energetic and cause ions to undergo extensive fragmentation. Although this fragmentation complicates the mass spectrum, it provides structural information for the identification of unknown compounds.

Electron Ionization.
Electron Ionization (EI) is the most common ionization technique used for mass spectrometry. EI works well for many gas phase molecules, but it does have some limitations. Although the mass spectra are very reproducible and are widely used for spectral libraries, EI causes extensive fragmentation so that the molecular ion is not observed for many compounds. Fragmentation is useful because it provides structural information for interpreting unknown spectra.

Fig: Electron Ionization Source. The electrons used for ionization are produced by passing a current through a wire Filament (Figure). The amount of current controls the number of electrons emitted by the filament. An electric field accelerates these electrons across the source region to produce a beam of high energy electrons. When an analyte molecule passes through this electron beam, a valence shell electron can be removed from the molecule to produce an ion.

Figure: electron ionization process. Ionization does not occur by electron capture, which is highly dependent upon molecular structure. Instead, EI produces positive ions by knocking a valence electron off the analyte molecule (Figure). As the electron passes close to the molecule the negative charge of the electron repels and distorts the electron cloud surrounding the molecule. This distortion transfers kinetic energy from the fast-moving electron to the electron cloud of the molecule. If enough energy is transferred by the process, the molecule will eject a valence electron and form a radicalcation (M+_).Since the ionization is produced by a single electron that is accelerated to 70 V, this is commonly referred to as 70 eV EI. This is enough energy to cause extensive fragmentation, and at this level small changes in the electron energy do not significantly affect the fragmentation patterns. The amount of energy transferred during this process depends upon how fast the electron is traveling and how close it passes to the molecule. In most 70 eV EI experiments, approximately 1400 kJ/mole (15 eV)* of energy is transferred during the ionization process. There is, however, a distribution of energy and as much as 2800 kJ/mole (30 eV) is transferred to some molecules. Since approximately 960 kJ/mole (10 eV) is required to ionize most organic compounds and typical chemical bond energy is 290 kJ/mole (3 eV), extensive fragmentation is often observed in 70 eV EI mass spectra. The distribution of energy transferred during ionization and the large number of fragmentation pathways results in a variety of products for a given analyte. Other electron voltages may be used to vary the amount of fragmentation produced during ionization. For most organic compounds the threshold energy for EI is about 10 eV.Because a mass spectrum is produced by ionizing many molecules, the spectrum is a distribution of the possible product ions. Intact molecular ions are observed from ions produced

Chemical Ionization
Chemical Ionization (CI) is a soft ionization technique that produces ions with little excess energy. As a result, less fragmentation is observed in the mass spectrum. Since this increases the abundance of the molecular ion, the technique is complimentary to 70 eV EI. CI is often used to verify the molecular mass of an unknown. Only slight modifications of an EI source region are required for CI experiments. In Chemical Ionization the source is enclosed in a small cell with openings for the electron beam, the reagent gas and the sample. The reagent gas is added to this cell at approximately 10 Pa (0.1 torr) pressure. This is higher than the 10-3 Pa (10-5 torr) pressure typical for a mass spectrometer source. At 10-3 Pa the mean free path between collisions is Approximately 2 meters and ion-molecule reactions are unlikely. In the CI source, however, the mean free path between collisions is only 10-4 meters and analyte

molecules undergo many collisions with the reagent gas. The reagent gas in the CI source is ionized with an electron beam to produce a cloud of ions. The reagent gas ions in this cloud react and produce adduct ions like CH (Figure 4), which are excellent proton donors.

When analyte molecules (M) are introduced to a source region with this cloud of ions, the reagent gas ions donate a proton to the analyte molecule and produce MH+ ions. The energetic of the proton transfer is controlled by using different reagent gases. The most common reagent gases are methane, isobutene and ammonia. Methane is the strongest proton donor commonly used with a proton affinity (PA) of 5.7 eV. For softer ionization, isobutene (PA 8.5 eV) and ammonia (PA 9.0 eV) are frequently used. Acid base chemistry is frequently used to describe the chemical ionization reactions. The reagent gas must be a strong enough Brnsted acid to transfer a proton to the analyte. Fragmentation is minimized in CI by reducing the amount of excess energy produced by the reaction. Because the adduct ions have little excess energy and are relatively stable, CI is very useful for molecular mass determination. Some typical reactions in a CI source are shown in Figure

Fast Atom Bombardment and Secondary Ion Mass Spectrometry.


Fast Atom Bombardment (FAB) and Secondary Ion Mass Spectrometry (SIMS) both use high energy atoms to sputter and ionize the sample in a single step. In these techniques, a beam of rare gas neutrals (FAB) or ions (SIMS) is focused on the liquid or solid sample. The impact of this high energy beam causes the analyte molecules to sputter into the gas phase and ionize in a single step (Figure). The exact mechanism of this process is not well understood, but these techniques work well for compounds with molecular weights up to a few thousand Dalton. Since no heating is required, sputtering techniques (especially FAB) are useful for studying thermally labile Compounds that decompose in conventional inlets

Mass analyzers:
Magnetic sectors Quadrupole (Q) Triple Quad (QQQ, QqQ or 3Q) Ion cyclotron with Fourier transformation (ICR-FT-MS) Ion trap (IT), linear trap (LT) Time-of-flight mass spectrometer (TOF) Hybrids (TOF-TOF, QTOF, etc)

Mass analyzers separate the charged species in the ionized sample according to their m/z ratios, thus permitting the mass and abundance of each species to be determined. Four commonly used analyzers are the magnetic sector; the quadrupole, the time off light, and the Fourier transform analyzers.

Magnetic Sector Analyzers


Ions generated in the ion source are collimated into a beam through the focusing action of a magnetic field and a slit assembly. After exiting the source, ions are subjected to a magnetic field perpendicular to the direction of the beam. Each ion experiences force at right angles to both its directional travel and the direction of the magnetic field, thereby deflecting the beam. The motion of each ion is given by m/z = H2r2/2V,

Where m is the mass in atomic mass units, z is the number of electronic charges, H is the magnetic field strength in gauss, r is the radius of the ion trajectory in centimeters, And V is the accelerating voltage. The mass spectrum is scanned by varying the strength of the magnetic field and detecting those ions passing through an exit slit as they come into focus. The magnetic sector mass spectrometer affords spatial resolution of ions, giving a unique trajectory at a given field strength for each value of m/z.

Quadrupole Analyzers
The instrument is based on four parallel rods in a square array. The ion beam is focused down the axis of the array and an electrical potential of fixed (DC) and radio Frequency (RF) components are applied to diagonally opposed rods. For a given combination of DC and RF components, ions of one specific m/z ratio have a stable path down the axis. All others are deflected to the sides and lost. Mass scanning is achieved by changing the DC and RF components of the voltage, while maintaining a fixed ratio. The quadrupole analyzer is a mass filter because it separates ions on the basis of their m/z ratio.

Ion-trap Analyzer
This quadrupole-type device is composed of a ring electrode placed between two ends cap electrodes. Depending upon the commercial version employed, the end caps are either held at ground potential or have an RF voltage applied to them, while an RF Voltage is placed on the ring electrode. As a result of these potentials, the hyperbolic surfaces of the three elements form a three dimensional quadrupole analyzer. Both ionization and mass analysis take place within the three-dimensional quadrupole field. In the ionization step, the RF voltage on the ring electrode is set low enough so that the ions within the mass range of interest are trapped within the device. Following ionization, mass analysis is accomplished through use of the mass selective instability mode of operation. That is, by raising the RF voltage on the ring electrode, ions of successively higher mass are ejected from the ion trap into an electron multiplier detector. In its most common application, the iontrap analyzer is used in conjunction with a gas chromatograph and covers the mass range of 10 to 560 Daltons. However, recent advances in ion-trap technology have extended the workable mass range to many thousands of Daltons.

Time-of-flight Analyzers
Ion separation is based on the principle that ions of different masses, possessing equal Kinetic energy, have different velocities. If there is a fixed distance for the ions to travel, the time of travel will vary with their mass, the lighter ions traveling faster and reaching the detector in a shorter period of time. The time-of-flight is given by tf = k m/z1 Where tf is the time-of-flight in seconds. Thus, the time-of-flight of the various ions is Simply proportional to the square root of the mass-to-charge ratio of ions. To measure

the time-of-flight, ions are introduced into the mass spectrometer in discrete packets so that a starting point for the timing process can be established. Ion packets are generated either through a pulsed ionization processor through a gating system in which ions are produced continuously, but areintroduced only at given times into the flight tube

Fourier Transform Analyzers


In a magnetic field of flux density B, ions move in circular orbits. The angular frequency, T of the orbital motion is given by T = (z/m)B In this type of mass spectrometer, the orbits are varied by subjecting the ions to a resonant alternating electric field. When the frequency of the alternating field matches the orbital frequency the ions are steadily accelerated to larger and larger orbits in coherent motion, developing a high level of kinetic energy. After the alternating electric field is turned off, the orbiting ion gives rise to an alternating image current on the electrodes. A frequency analysis of the signal yields the mass of the ions involved. Thus, the Fourier transform of the time domain transient signal yields the corresponding frequency spectrum form which the mass spectrum is computed. This is a high resolution technique, yielding m/z ratios accurate to about one thousandth of a Dalton.

Tandem Mass Spectrometry


Two mass spectrometers connected in series (MS/MS), tandem mass spectrometry, Refers to the use of two or more sequential mass analysis steps. In its simplest form MS/MS (Figure 2) consist of two mass spectrometers linked in such a way that ion pre selected by the first mass analyzer (MS1) are chemically or energetically modified and the results analyzed by the second mass analyzer (MS2).

Fig: Tandem mass spectrometry.

The basic concept of MS/MS involves the ability to determine the mass relationship Between a precursor ion in MS1 and a production in MS2. Different mass can be probed depending on how MS1 and MS2 are scanned. These include fragmentation of a precursor and measurement of all its fragments (a product scan), selection of multiple precursors and testing for a common fragment (a precursor scan), or scanning to see if a number of precursors all lose the same neutral species (a constant neutral loss scan).Fragmentation of the precursor ion can be induced by momentum transfer through collision with gas molecules and/or solid surfaces or by electronic excitation using lasers. These techniques are known as collision induced dissociation, surface-induced dissociation or laser-induced dissociation, respectively. Allowing the ion to fragment without additional activation is known as metastable decomposition. There are many applications of MS/MS to pharmaceutical problems. Product scans can be used to obtain qualitative information from precursor ions of drug substances, Impurities and contaminants. This can aid in the identification of unknowns. The method can also be used to determine the amino sequence of peptides and protein Fragments. MS/MS has advantage for mixture analysis. Even when then mass spectrometer is coupled to a separation device such as a liquid or gas chromatograph, the resulting signals may be a result of overlapping or unresolved components. MS/MS can be employed to select the precursor ion from one component and obtain structural information without interference with others. Selected reaction monitoring is used to reduce the interference encountered during quantitative analysis for low levels of drugs in biological matrices, as in pharmacokinetic studies. If analysis is for a drug specific ion, interfering signals from other compounds in the matrix can mask the desired signal. Interference is reduced if a drug-specific fragment is selected with MS1 and a structure-specific with MS2. The odds of another molecule producing the same mass relationship are diminishingly small. MS/MS can also be used in metabolism studies to search for molecules with common structural features such as metabolites related to the drug substance. All of the metabolites might contain the same functional group that is lost as a neutral fragment. In this case a constantneutral-loss scan will show all of these species. For instance, carboxyl acids will all lose neutral carbon dioxide. If the common functionality is lost as anionic fragment, then a precursor scan will show all of the molecules that produce that fragment ion DETECTORS: Detection of ions is based upon their charge or momentum. For large signals a faraday cup is used to collect ions and measure the current. Older instruments used photographic plates to measure the ion abundance at each mass to charge ratio. Most detectors currently used amplify the ion signal using a collector similar to a photomultiplier tube. These amplifying detectors include: electron multipliers, channel trons and multichannel plates. The gain is controlled by changing the high voltage applied to the detector. A detector is selected for its speed, dynamic range, gain, and geometry. Some detectors are sensitive enough to detect single ions.

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 (103 torr). The second stage uses diffusion pumps or turbo molecular pumps to provide high vacuum. ICR instruments have even higher vacuum requirements and often include a cryogenic pump for a third pumping stage. The pumping system is an important part of any mass spectrometer but a detailed discussion is beyond the scope of this paper. DATA ANALYSIS AND INTERPRETATION The mass spectral experiments yields information on the molecular weight of ions derived from the sample and the relative abundance of each of these ions. Spectra are Often complex, and not all of the ions maybe separated by the mass spectrometer. The ability of the instrument to separate ions is called the resolving power, commonly described by the 10 % valley definition. This states that the resolving power is the highest mass number at which two peaks differing by one molecular weight unit and of equal height have a valley between them that is equal to 10 % of the peak height. For low, medium, and high resolution mass spectrometers, this value is between 100 and 2000, 2000 and 10,000, and greater than 10,000, respectively. If one electron is removed or added to a neutral molecule, a molecular ion of essentially the same molecular weight as the parent molecule results. It is often possible to determine the mass of this ion with sufficient precision to enable the empirical formula of the compound to be calculated. Molecular masses may be determined accurately by using high resolution instruments or by peak-matching measurements using reference compounds. Fragment ions are those produced from the molecular ion by various bond cleavage processes. Numerous papers in the literature relate bond cleavage patterns (fragmentation patterns) to the molecular structure. In addition to measurements of the mass of a molecular ion and its dissociated fragmentations, mass spectrometers are also used to quantitative compounds with a high degree of selectivity, precision, and accuracy. Compounds are introduced into the mass spectrometer either via direct insertion probes, gas inlet, or, as is more common, via gas or liquid chromatographic interfaces, which provide sample purification. Ionization maybe by EI, CI, FAB, thermo spray, or electro spray and mass separation by magnetic sector, quadrupole, or quadrupole ion-trap mass spectrometers. Quantitative mass spectrometry involves measuring the abundance of a specific ion, or set of ions, and relating the response to a known standard. External or internal standards may be used, but the latter are preferred for greater accuracy. For mass spectrometry, internal standards may be either structural or stable isotope analogs. The former have the advantage of lower cost and availability while precision and accuracy are typically achieved by use of a stable isotope ( 2H, 13C, 15N) labeled analog of the analyte. The only requirements for labeling the analyte are that the ion monitored for the internal standard must retain an isotopic label after ionization and the label must not be exchangeable under the sampling, separation, or ionization conditions. Stable isotope internal standards are often required for acceptable quantization, particularly with FAB and LC/MS techniques such as thermo spray and electro spray. Relative abundances of the analyte and internal standard ions are typically determined by selected ion monitoring, by which one specific ions due to the analyte and the internal standard are monitored. This technique has the advantage

over scanning the full mass range in that more is spent integrating the ion current at a selected mass-to-charge ratio, thereby increasing sensitivity. Chromatographic peak or amount of analyte in a sample is calculated from the ratio of analyte to internal standard peak area (or height) and the regression parameters as determined by a calibration curve, using standard techniques.

Characteristics of Molecular Ions


1) The m/z value must correspond to a reasonable molecular formula with the proper isotope abundance. 2) Most compounds have an even molecular mass. The one common exception to this is the "Nitrogen Rule" discussed below. 3) The Nitrogen Rule: Any compound with an odd number of nitrogen atoms will have an odd molecular mass. Any compound with an even number of nitrogen atoms (including zero) will have an even molecular mass. This is because nitrogen is the only common atom where the most common isotope has an odd valence and an even mass. For example: the molecular ion for CH is m/z 16, the molecular ion for NH is m/z 17, and 43 the molecular ion for N H is m/z 32.24 4) If a peak is the molecular ion, the next highest mass fragment must correspond to the loss of a possible neutral fragment. For example, a peak that corresponds to loss of 5 u from the molecular ion is highly unlikely.

Molecular Ion.
The molecular ion provides the molecular mass of the analyte and is the first clue used to interpret a mass spectrum. The mass of the molecular ion is based upon the mass of the most abundant isotope for each element in the molecule. This is not the atomic weight from the periodic table. Since many mass spectrometers have unit mass resolution, the atomic mass is rounded to the nearest whole number; this is called the nominal mass. For example the molecular ion for CHBr is observed at m/z 250, not at the formula weight of 253.

Fragmentation.
Although the molecular ion is useful for identification, it does not provide any structural information about an unknown. The structural information is obtained from the fragmentation patterns of the mass spectrum. Identifying an unknown without analyzing the fragmentation patterns is like putting together a jigsaw puzzle without the picture. Fragmentation patterns are often complex, but they fit together like pieces of the puzzle to identify the structure of the molecule.

Figure: Fragmentation mechanisms.

McLafferty rearrangement:
The McLafferty rearrangement is a classic example of a rearrangement reaction. This rearrangement results in formation of an intact neutral molecule and a radical ion with an even mass to charge ratio. This reaction is significantly different from the cleavage reactions discussed previously. The McLafferty rearrangement is often observed for carbonyl compounds that contain a linear alkyl chain. If this alkyl chain is long enough, a six-membered

Figure: McLafferty rearrangement mechanism.

Ring forms from the carbonyl oxygen to the hydrogen on the fourth carbon. This spacing allows the hydrogen to transfer to the carbonyl oxygen via a six membered ring. The McLafferty rearrangement is energetically favorable because it results in loss of a neutral alkenes and formation of a resonance stabilized radical cation.

Isotope Abundance.
The existence of isotopes was first observed by Aston using a mass Spectrometer to study neon ions. When interpreting mass spectra it is important to remember that the atomic weight of an element is a weighted average of the naturally occurring isotopes. Mass spectrometers separate these isotopes and are even used to measure their relative abundance. Although this complicates the mass spectrum, it also provides useful information for identifying the elements in an ion. Abundance of Isotopes for Some Common Elements:

Exact Mass.
In most mass spectrometry experiments the nominal mass is used and the mass to charge ratio of an ion is rounded to the nearest whole number. High resolution instruments, including double focusing and FT-ICR mass spectrometers are capable of determining the "exact mass" of an ion. This is useful for interpretation because each element has a slightly different mass defect. This mass defect is the difference between the mass of the isotope and the nominal mass (which is equivalent to the number of protons and neutrons).Recall that the atomic mass scale is defined by carbon-12 with a mass of exactly 12.0000 u. The exact mass of a specific isotope is determined relative to 12C by high resolution mass spectrometry (see Table 3). High resolution mass spectrometry can distinguish compounds with the same nominal mass but different exact mass caused by different elemental composition. For example, C H , CHO, and NO all have a nominal mass of 30 u. Because they have 2 6 2 the same nominal mass, a mass spectrometer with unit mass resolution can not distinguish these three ions. However, the exact masses for C H (30.04695039), CH O (30.01056487) and NO 2 6 2 (29.99798882) are different and a high resolution mass spectrometer can distinguish these three compounds.

Exact mass of some common isotopes:

The Mass Spectrometer


How mass spectrometer works The basic principle
If something is moving and you subject it to a sideways force, instead of moving in a straight line, it will move in a curve - deflected out of its original path by the sideways force. Suppose you had a cannonball traveling past you and you wanted to deflect it as it went by you. All you've got is a jet of water from a hose-pipe that you can squirt at it. Frankly, its not going to make a lot of difference! Because the cannonball is so heavy, it will hardly be deflected at all from its original course. But suppose instead, you tried to deflect a table tennis ball traveling at the same speed as the cannonball using the same jet of water. Because this ball is so light, you will get a huge deflection. The amount of deflection you will get for a given sideways force depends on the mass of the ball. If you knew the speed of the ball and the size of the force, you could calculate the mass of the ball if you knew what sort of curved path it was deflected through. The less the deflection, the heavier the ball.

An outline of what happens in a mass spectrometer


Atoms can be deflected by magnetic fields - provided the atom is first turned into an ion. Electrically charged particles are affected by a magnetic field although electrically neutral ones arent. The sequence is :

Stage 1: Ionization
The atom is ionized by knocking one or more electrons off to give a positive ion. This is true even for things which you would normally expect to form negative ions (chlorine, for example) or never form ions at all (argon, for example). Mass spectrometers always work with positive ions.

Stage 2: Acceleration
The ions are accelerated so that they all have the same kinetic energy.

Stage 3: Deflection
The ions are then deflected by a magnetic field according to their masses. The lighter they are, the more they are deflected. The amount of deflection also depends on the number of positive charges on the ion - in other words, on how many electrons were knocked off in the first stage. The more the ion is charged, the more it gets deflected.

Stage 4: Detection
The beam of ions passing through the machine is detected electrically

Applications
1. Interpretation of Fragmentation Patterns (Mass Spectra) to Distinguish Positional Isomers

2. Drug Overdose: Use of GC/MS to Identify a Drug Metabolite

3. Verification that the Proper Derivative of the Compound of Interest Has Been Prepared

4. Use of a CI Mass Spectrum to Complement an EI Mass Spectrum

5. Use of Exact Mass Measurements to Identify Analytes According to Elemental Composition

6. Clinical Diagnostic Tests Based on Quantization of Stable Isotopes by Mass Spectrometry in Lieu of Radioactivity

7. Isotope ratio MS: isotope dating and tracking


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

8. 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.

9. 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

10. 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

11. Protein characterization


Mass spectrometry is an important emerging method for the characterization and sequencing of proteins.

12. Glycan analysis


Mass spectrometry (MS), with its low sample requirement and high sensitivity, has been the predominantly used in glycobiology for characterization and elucidation of glycan structures

13. 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.

14. 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

15. Compound Identification 16. Disinfection Byproducts Example: Mass spectrum of butane

Fig: Mass spectrum of butane

Conclusion
Mass spectrometry (MS) has undergone a profound change over the past decade. Instrumentation and techniques related to the automated analysis of bimolecular and new drugs now account for a large percentage of the research and publications in this field. In comparison, gas chromatography/mass spectrometry (GC/MS) and electron ionization (EI) mass spectra of small molecules play a less important role than they once did. But GC/MS is far from dead, and EIMS continues to be the ionization method of choice for many laboratories that routinely analyze volatilizable low molecular mass compounds such as drugs, flavor and odor components, pesticides, and petroleum products. This situation seems unlikely to change in the near future.

Reference

http://www.prenhall.com/settle/chapters/ch31.pdf http://www.asms.org/portals/0/WhatIsMSPoster.pdf http://www.forumsci.co.il/HPLC/usp-ms-2spaltet.pdf http://theory.caltech.edu/~politzer/supplements/mass-spectroscopy.pdf http://www.epa.gov/esd/factsheets/adv-mas-spec.pdf http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/MassSpe c/masspec1.htm http://en.wikipedia.org/wiki/Mass_spectrometry