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Mass spectrometry


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Mass spectrometry

  1. 1. Generated by Foxit PDF Creator © Foxit Software For evaluation only.MASS SPECTROMETRYBy Mussarat Jabeen
  2. 2. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMASS SPECTROMETRYMass spectrometry is a powerful analytical technique that is used to identify unknowncompounds, to quantify known compounds, and to elucidate the structure and chemicalproperties of molecules. It is the smallest scale in the world, not because of the massspectrometer’s size but because of the size what it weighs...molecules. According to the IUPAC(International Union of Pure and Applied Chemistry), it is the branch of science dealing with allaspects of mass spectroscopes and results obtained with these instruments. The informationgiven by mass spectrometry is sometimes sufficient, frequently necessary, and always useful foridentification of species.History of Mass Spectrometry  Mass Spectrometry was started by J.J. Thomson. Until 1897, scientists believed atoms were indivisible; the ultimate particles of matter, but Thomson proved them wrong when he discovered that atoms contained particles known as electrons. He concluded this by his experiments on cathode rays. He found that the rays could be deflected by an electric field (in addition to magnetic fields, which was already known). By comparing the deflection of a beam of cathode rays by electric and magnetic fields he was able to measure the particles mass. This showed that cathode rays were matter, but he found that the particles were about 2000 times lighter than the mass of the lightest atom, hydrogen. He concluded that the rays were composed of very light negatively charged particles which he called electron. He also concluded that neon is composed of two isotopes and them which was the first example of mass spectrometry. On his discovery he was awarded Nobel Prize in 1906. Page 2
  3. 3. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRY In 1919, Thomson, with the help of his student Francis Aston (who would go on to win his own Nobel Prize in Chemistry in 1922), built what later would be recognized as the first mass spectrometer to measure the masses of charged atoms. In their first mass spectrometer they measure the charge to mass ratio (z/m) for several ionic species. In the expression z/m, z is the charge number, i.e. the total charge on an ion divided by the elementary charge (e), and m is the nucleon number, i.e. the sum of the total number of protons and neutrons in an atom, molecule or ion. In modern mass spectrometry, the parameter measured is m/z, rather than z/m: the unit of m/z was recently designated the Thomson (Th).This instrument used gas discharge tubes to generate ions, which were then passed through parallel electric and magnetic fields. The ions were deflected into parabolic trajectories and then detected on a photographic plate. In 1934, First double focusing magnetic analyzer was invented by Johnson E.G., Nier A.O. In 1939, Accelerator Mass Spectrometry was developed by Lawrence E.O., Alvarez L.W., Brobeck W.M., Cooksey D., Corson D.R., McMillan E.M., Salisbury W.W., Thornton R.LAn important tool in trace biomolecule detection, still coming into its own. In 1946, Time-of-Flight Mass Spectrometry was invented by W.Stephens the significance of TOF mass analyzers has grown over the last 20 years especially in the biomedical applications of mass spectrometry. In 1947, Preparative Mass Spectrometry was used to purify radioactive 235U by Siuzdak G., Bothner B., Yeager M., Brugidou C., Fauquet C.M., Hoey K., Chang C.M., which was then used to construct the first nuclear weapon. It was recently demonstrated that viruses as well as other types of molecules could also be separated and collected using electrospray ionization mass spectrometry. In 1953, Pauls invention of the Quadrupole and Quadrupole ion trap earned him the Nobel Prize in Physics. These mass analyzers are the most widely used today and are still being developed for an even wider range of applications. In 1956, Golhke R.S., McLafferty F., Wiley B., Harrington D invented GC/MS instruments. A very powerful tool and still one of the most popular forms of doing mass spectrometry. In 1956, Beynon J.H first time Identify Organic Compounds with Mass Spectrometry. In 1966, Munson and Field described chemical ionization (CI). One of the first soft ionization techniques In 1968, Electrospray Ionization was invented by Dole M., Mack L.L., Hines R.L., Mobley R.C., Ferguson L.D., Alice M.B. In 1975, Atmospheric Pressure Chemical Ionization (APCI) was developed by Carroll D.I., Dzidic I., Stillwell R.N., Haegele K.D., Horning E.C. In 1976, Ronald MacFarlane and co-workers develop plasma desorption mass spectrometry. In 1980, Inductively Coupled Plasma MS By Reed T.B. A very powerful tool for elemental composition analysis In 1985, Franz Hillenkamp, Michael Karas and co-workers describe and coin the term matrix-assisted laser desorption ionization (MALDI). Page 3
  4. 4. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRY  In 1989, Wolfgang Paul receives the Nobel Prize in Physics "for the development of the ion trap technique"  In 2002, John Bennett Fenn and Koichi Tanaka are awarded one-quarter of the Nobel Prize in chemistry each "for the development of soft desorption ionisation methods ... for mass spectrometric analyses of biological macromolecules." The Five Mass Spectrometry Nobel Prize PioneersJoseph John Francis Wolfgang Paul John Bennet Koichi TanakaThomson William 1989 Nobel Fenn 2002 Nobel1906 Nobel Aston Prize for 2002 Nobel Prize Prize forPrize for 1922 Nobel Physics for Chemistry ChemistryPhysics Prize for (for the (for the (mass(theoretical Chemistry development of development of spectrometricand (mass the ion trap Soft Desorption analyses ofexperimental spectrograph, technique) ionization biologicalinvestigations of isotopes, in Method) macromolecule)on the a largeconduction of number ofelectricity by non-gases) radioactive elements) Page 4
  5. 5. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMASS SPECTROMETERAn instrument which measures the ratio of mass to the number of charges of ions producedfrom elements and compounds. It is also of value in performing fundamental studies of theproperties of gaseous ions. A mass spectrometer is similar to a prism. In the prism, light isseparated into its component wavelengths which are then detected with an optical receptor,such as visualization. Similarly, in a mass spectrometer the generated ions are separated inthe mass analyzer, digitized and detected by an ion detector.Understanding Mass SpectrometryTo understand the basic principles of mass spectrometry, consider a person standing at thetop of a tower on a windy day. The person picks up various balls and drops them, one by one,from the tower. As each ball falls, wind deflects it along a curved path. The masses of theballs affect how they fall. A bowling ball, for example, is much heavier than a basketball and Page 5
  6. 6. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYis therefore harder to move. As a result, a bowling ball follows a different path than abaseball.In a mass spectrometer, the same thing is happening, except its atoms and molecules that arebeing deflected, and its electric or magnetic fields causing the deflection. Its also happeningin a cabinet that can be as small as a microwave or as large as a chest freezer.Basic components of mass spectrometerFour basic components are, for the most part, standard in all mass spectrometers: a sampleinlet, an ionization source, a mass analyzer and an ion detector. Some instruments combinethe sample inlet and the ionization source, while others combine the mass analyzer and thedetector. However, all sample molecules undergo the same processes. Sample molecules areintroduced into the instrument through a sample inlet. Once inside the instrument, the samplemolecules are converted to ions in the ionization source, before being electrostaticallypropelled into the mass analyzer. Ions are then separated according to their m/z within themass analyzer. The detector converts the ion energy into electrical signals, which are thentransmitted to a computer. Page 6
  7. 7. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYSample Introduction TechniquesIn order to perform mass analysis on a sample, which is initially at atmospheric pressure (760mmHg), it must be introduced into the instrument in such a way that the vacuum inside theinstrument remains relatively unchanged (~10-6 torr). The most common methods of sampleintroduction are direct insertion with a probe or plate commonly used with MALDI-MS,direct infusion or injection into the ionization source such as ESI-MS.Direct Insertion:Using an insertion probe/plate is a very simple way to introduce a sample into an instrument.The sample is first placed onto a probe and then inserted into the ionization region of themass spectrometer, typically through a vacuum interlock. The sample is then subjected to anynumber of desorption processes, such as laser desorption or direct heating, to facilitatevaporization and ionizationDirect Infusion: A simple capillary or a capillary column is used to introduce a sample as a gas or in solution.Direct infusion is also useful because it can efficiently introduce small quantities of sampleinto a mass spectrometer without compromising the vacuum. Capillary columns are routinelyused to interface separation techniques with the ionization source of a mass spectrometer.These techniques, including gas chromatography (GC) and liquid chromatography (LC), alsoserve to separate a solution’s different components prior to mass analysis. In gaschromatography, separation of different components occurs within a glass capillary column.As the vaporized sample exits the gas chromatograph, it is directly introduced into the massspectrometer Page 7
  8. 8. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYIonization Methods Ionization method refers to the mechanism of ionization while the ionization source is themechanical device that allows ionization to occur. The different ionization methods,summarized here, work by either ionizing a neutral molecule through electron ejection,electron capture, protonation, cationization, or deprotonation, or by transferring a chargedmolecule from a condensed phase to the gas phase.  Protonation  Deprotonation  Cationization  Transfer of a charged molecule to the gas phase  Electron ejection  Electron captureProtonation (positive ions) Page 8
  9. 9. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYProtonation is a method of ionization by which a proton is added to a molecule, producing anet positive charge of 1+ for every proton added. Protonation is used for basic compoundssuch as amines, to form stable cations. Peptides are often ionized via protonation. Protonationcan be achieved via matrix-assisted laser desorption/-ionization (MALDI), electrosprayionization (ESI) and atmospheric pressure chemical ionization (APCI).Deprotonation (negative ions)Deprotonation is an ionization method by which the net negative charge of 1- is achievedthrough the removal of a proton from a molecule. This mechanism of ionization, commonlyachieved via MALDI, ESI, and APCI is very useful for acidic species including phenols,carboxylic acids, and sulfonic acids.Cationization (positive ions)M + Cation+ → MCation+Cationization is a method of ionization that produces a charged complex by non-covalentlyadding a positively charged ion to a neutral molecule. While protonation could fall under thissame definition, cationization is distinct for its addition of a cation adduct other than a proton(e.g. alkali, ammonium). Moreover, it is known to be useful with molecules unstable toProtonation. The binding of cations other than protons to a molecule is naturally less Page 9
  10. 10. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYcovalent, therefore, the charge remains localized on the cation. This minimizes delocalizationof the charge and fragmentation of the molecule. Cationization is commonly achieved viaMALDI, ESI, and APCI. Carbohydrates are excellent candidates for this ionizationmechanism, with Na+ a common cation adduct.Transfer of a charged molecule to the gas phase (positive or negative ions)The transfer of compounds already charged in solution is normally achieved through thedesorption or ejection of the charged species from the condensed phase into the gas phase.This transfer is commonly achieved via MALDI or ESI. The positive ion mass spectrum oftetraphenylphosphine.Electron ejection (positive ions)As its name implies, electron ejection achieves ionization through the ejection of an electronto produce a 1+ net positive charge, often forming radical cations. Observed most commonlywith electron ionization (EI) sources, electron ejection is usually performed on relativelynonpolar compounds with low molecular weights and it is also known to generate significantfragment ions. The mass spectrum resulting from electron ejection of anthracene.Electron capture (negative ions) Page 10
  11. 11. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYWith the electron capture ionization method, a net negative charge of 1- is achieved with theabsorption or capture of an electron. It is a mechanism of ion-ization primarily observed formolecules with a high electron affinity, such as halogenated compounds. The electron capturemass spectrum of hexachloro-benzene.Ionization Sources  Electrospray Ionization (ESI)  Nanoelectrospray Ionization (NanoESI)  Atmospheric Pressure Chemical Ionization (APCI)  Atmospheric pressure photoionization (APPI)  Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)  Fast Atom Bombardment (FAB)  Electron Ionization (EI)  Chemical Ionization (CI)  Thermal ionization (TI)Types of Ionization Sources  Hard ionization sources  Soft ionization sourcesHard ionization sourcesLeave excess energy in molecule and produced stable fragments which is not furtherfragarmented.Soft ionization sourcesLittle excess energy in molecule and produced unstable fragments which are againfragmented.Electrospray IonizationElectrospray ionization (ESI) is a method routinely used with peptides, proteins,carbohydrates, small oligonucleotides, synthetic polymers, and lipids. ESI produces gaseousionized molecules directly from a liquid solution. It operates by creating a fine spray ofhighly charged droplets in the presence of an electric field. (An illustration of the electrosprayionization process. Page 11
  12. 12. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYThe sample solution is sprayed from a region of the strong electric field at the tip of a metalnozzle maintained at a potential of anywhere from 700 V to 5000 V. The nozzle (or needle)to which the potential is applied serves to disperse the solution into a fine spray of chargeddroplets. Either dry gas, heat, or both are applied to the droplets at atmospheric pressure thuscausing the solvent to evaporate from each droplet. As the size of the charged dropletdecreases, the charge density on its surface increases. The mutual Coulombic repulsionbetween like charges on this surface becomes so great that it exceeds the forces of surfacetension, and ions are ejected from the droplet through a “Taylor cone”. Electrosprayionization is conducive to the formation of singly charged small molecules, but is also well-known for producing multiply charged species of larger molecules. This is an importantphenomenon because the mass spectrometer measures the mass-to-charge ratio (m/z) andtherefore multiple charging makes it possible to observe very large molecules with aninstrument having a relatively small mass range.Many solvents can be used in ESI and are chosen based on the solubility of the compound ofinterest, the volatility of the solvent and the solvent’s ability to donate a proton. Typically,protic primary solvents such as methanol, 50/50 methanol/water, or 50/50 acetonitrile/H2O Page 12
  13. 13. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYare used, while aprotic cosolvents, such as 10% DMSO in water, as well as isopropyl alcoholare used to improve solubility for some compounds. Although 100% water is used in ESI,water’s relatively low vapor pressure has a detrimental effect on sensitivity; better sensitivityis obtained when a volatile organic solvent is added. Some compounds require the use ofstraight chloroform with 0.1% formic acid added to facilitate ionization. This approach, whileless sensitive, can be effective for otherwise insoluble compounds. Buffers such as Na+, K+, phosphate, and salts present a problem for ESI by lowering thevapor pressure of the droplets resulting in reduced signal through an increase in dropletsurface tension resulting in a reduction of volatility (see Chapter 3 for quantitativeinformation on salt effects). Consequently, volatile buffers such as ammonium acetate can beused more effectively.Advantages  practical mass range of up to 70,000 Da  good sensitivity with femtomole to low picomole sensitivity typical  softest ionization method, capable of generating noncovalent complexes in the gas phase  easily adaptable to liquid chromatography  easily adaptable to tandem mass analyzers such as ion traps and triple quadrupole instruments  multiple charging allows for analysis of high mass ions with a relatively low m/z range instrument  no matrix interference Disadvantages  the presence of salts and ion-pairing agents like TFA can reduce sensitivity  complex mixtures can reduce sensitivity  simultaneous mixture analysis can be poor  multiple charging can be confusing especially in mixture analysis  sample purity is important  carryover from sample to sampleNanoelectrospray Ionization (NanoESI)Low flow electrospray, originally described by Wilm and Mann, has been callednanoelectrospray, nanospray, and micro-electrospray. This ionization source is a variation onESI, where the spray needle has been made very small and is positioned close to the entranceto the mass analyzer. The end result of this rather simple adjustment is increased efficiency,which includes a reduction in the amount of sample needed. Page 13
  14. 14. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRY The flow rates for nanoESI sources are on the order of tens to hundreds of nanoliters per minute. In order to obtain these low flow rates, nanoESI uses emitters of pulled and in some cases metallized glass or fused silica that have a small orifice (~5µ). The approximate size of droplet in nanoESI is 0.2 micron diameter which is very small as compared to normal ESI with droplet size 1 micron diameter. Advantages  Very sensitive  very low flow rates  applicable to LC/MS  has reasonable salt tolerance (low millimolar)  multiple charging useful  reasonable tolerance of mixtures  Soft ionization (little fragmentation observed). Disadvantages  low flow rates require specialized systems  significant suppression can occur with mixturesAtmospheric Pressure Chemical IonizationAPCI has also become an important ionization source because it generates ions directly fromsolution and it is capable of analyzing relatively nonpolar compounds. Similar to Page 14
  15. 15. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYelectrospray, the liquid effluent of APCI is introduced directly into the ionization source.However, the similarity stops there. The droplets are not charged and the APCI sourcecontains a heated vaporizer, which facilitates rapid desolvation/vaporization of the droplets.Vaporized sample molecules are carried through an ion-molecule reaction region atatmospheric pressure. Advantages  As the solvent ions are present at atmospheric pressure conditions, chemical ionization of analyte molecules is very efficient.  At atmospheric pressure analyte molecules collide with the reagent ions frequently.  Proton transfer (for protonation MH+ reactions) occurs in the positive mode  electron transfer or proton loss, ([M-H]-) in the negative mode.  Multiple charging is typically not observed presumably because the ionization process is more energetic than ESI.Atmospheric Pressure PhotoionizationAtmospheric pressure photoionization (APPI) has recently become an important ionizationsource because it generates ions directly from solution with relatively low background and iscapable of analyzing relatively nonpolar compounds. Similar to APCI, the liquid effluent ofAPPI is introduced directly into the ionization source. The primary difference between APCIand APPI is that the APPI vaporized sample passes through ultra-violet light (a typicalkrypton light source emits at 10.0 eV and 10.6 eV). Often, APPI is much more sensitive thanESI or APCI and has been shown to have higher signal-to-noise ratios because of lowerbackground ionization. Lower background signal is largely due to high ionization potential ofstandard solvents such as methanol and water (IP 10.85 and 12.62 eV, respectively) whichare not ionized by the krypton lamp. Page 15
  16. 16. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYIn APPI Protonation, Deprotonation, Cationization reaction takes place.Disadvantages  It can generate background ions from solvents  It requires vaporization temperatures ranging from 350-500° C, which can cause thermal degradation.Matrix-Assisted Laser Desorption/IonizationMatrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was firstintroduced in 1988 by Tanaka, Karas, and Hillenkamp. It has since become a widespreadanalytical tool for peptides, proteins, and most other biomolecules (oligonucleotides,carbohydrates, natural products, and lipids).While the exact desorption/ionization mechanism for MALDI is not known, it is generallybelieved that MALDI causes the ionization and transfer of a sample from the condensedphase to the gas phase via laser excitation and abalation of the sample matrix. In MALDI Page 16
  17. 17. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYanalysis, the analyte is first co-crystallized with a large molar excess of a matrix compound,usually a UV-absorbing weak organic acid. Irradiation of this analyte-matrix mixture by alaser results in the vaporization of the matrix, which carries the analyte with it. The matrixplays a key role in this technique. The co-crystallized sample molecules also vaporize, butwithout having to directly absorb energy from the laser. Molecules sensitive to the laser lightare therefore protected from direct UV laser excitation.MALDI matrix -- A nonvolatile solid material facilitates the desorption and ionizationprocess by absorbing the laser radiation. As a result, both the matrix and any sampleembedded in the matrix are vaporized. The matrix also serves to minimize sample damagefrom laser radiation by absorbing most of the incident energy.Advantages  practical mass range of up to 300,000 Da. Species of much greater mass have been observed using a high current detector;  typical sensitivity on the order of low femtomole to low picomole. Attomole sensitivity is possible;  soft ionization with little to no fragmentation observed;  tolerance of salts in millimolar concentrations;  suitable for the analysis of complex mixtures. Disadvantages  Matrix background, which can be a problem for compounds below a mass of 700 Da. This background interferences is highly dependent on the matrix material; Page 17
  18. 18. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRY  possibility of photo-degradation by laser desorption/ionization;  Acidic matrix used in MALDI my cause degradation on some compounds.Liquid SIMS (LSIMS) or Fast Atom Bombardment (FAB)Fast atom ion bombardment, or FAB, is an ionization source similar to MALDI in that it usesa matrix and a highly energetic beam of particles to desorb ions from a surface. It isimportant, however, to point out the differences between MALDI and FAB. For MALDI, theenergy beam is pulsed laser light, while FAB uses a continuous ion beam. With MALDI, thematrix is typically a solid crystalline, whereas FAB typically has a liquid matrix. It is alsoimportant to note that FAB is about 1000 times less sensitive than MALDI.Fast atom bombardment is a soft ionization source which requires the use of a direct insertionprobe for sample introduction, and a beam of Xe neutral atoms or Cs+ ions to sputter thesample and matrix from the direct insertion probe surface. It is common to detect matrix ionsin the FAB spectrum as well as the protonated or cationized (i.e. M + Na+) molecular ion ofthe analyte of interest.FAB matrix -- Facilitating the desorption and ionization process, the FAB matrix is anonvolatile liquid material that serves to constantly replenish the surface with new sample asit is bombarded by the incident ion beam. By absorbing most of the incident energy, thematrix also minimizes sample degradation from the high-energy particle beam. Two of the most common matrices used with FAB are m-nitrobenzyl alcohol and glycerol. m-nitrobenzyl alcohol (NBA) glycerol Page 18
  19. 19. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYThe fast atoms or ions impinge on or collide with the matrix causing the matrix and analyte tobe desorbed into the gas phase. The sample may already be charged and subsequentlytransferred into the gas phase by FAB, or it may become charged during FAB desorptionthrough reactions with surrounding molecules or ions. Once in the gas phase, the chargedmolecules can be propelled electrostatically to the mass analyzer.Electron IonizationElectron ionization is one of the most important ionization sources for the routine analysis ofsmall, hydrophobic, thermally stable molecules and is still widely used. Because EI usuallygenerates numerous fragment ions it is a “hard” ionization source. However, thefragmentation information can also be very useful. For example, by employing databasescontaining over 200,000 electron ionization mass spectra, it is possible to identify anunknown compound in seconds (provided it exists in the database). These databases,combined with current computer storage capacity and searching algorithms, allow for rapidcomparison with these databases (such as the NIST database), thus greatly facilitating theidentification of small molecules.Energetic process a heated filament emits electrons which are accelerated by a potentialdifference of usually 70eV into the sample chamber.Ionization of the sample occurs by removal of an electron from the molecule thus generatinga positively charged ion with one unpaired electron. Page 19
  20. 20. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYAdvantages  Widely used technique when coupled to GC  Suitable for volatile organic compounds – eg hydrocarbons, oils, flavours, fragrances  Not really coupled to LC today  Also called electron impact  Produces M+.radical cation giving molecular weight  Produces abundant fragment ionsChemical IonizationChemical Ionization (CI) is applied to samples similar to those analyzed by EI and isprimarily used to enhance the abundance of the molecular ion. Chemical ionization uses gasphase ion-molecule reactions within the vacuum of the mass spectrometer to produce ionsfrom the sample molecule. The chemical ionization process is initiated with a reagent gassuch as methane, isobutane, or ammonia, which is ionized by electron impact. High gaspressure in the ionization source results in ion-molecule reactions between the reagent gasions and reagent gas neutrals.A possible mechanism for ionization in CI occurs as follows:Reagent (R) + e- → R+ + 2 e-R+ + RH → RH+ + RRH+ + Analyte (A) → AH+ + RIn contrast to EI, an analyte is more likely to provide a molecular ion with reducedfragmentation using CI. However, similar to EI, samples must be thermally stable sincevaporization within the CI source occurs through heating. Page 20
  21. 21. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYNegative chemical ionization (NCI) typically requires an analyte that contains electron-capturing moieties (e.g., fluorine atoms or nitrobenzyl groups). Such moieties significantlyincrease the sensitivity of NICI, in some cases 100 to 1000 times greater than that of electronionization (EI). NCI is probably one of the most sensitive techniques and is used for a widevariety of small molecules with the caveat that the molecules are often chemically modifiedwith an electron-capturing moiety prior to analysis.While most compounds will not produce negative ions using EI or CI, many importantcompounds can produce negative ions and, in some cases, negative EI or CI massspectrometry is more sensitive and selective than positive ion analysis. In fact, compoundslike steroids are modified to enhance NCI.Advantages  Produces M+H+ ions or M - H-ions  Gives molecular weight  Softer ionization techniqueThermal ionizationThermal ionization is based upon the generation of atomic or molecular ions at the surface ofan electrically heated filament. Samples are deposited on specially treated filaments (usuallyrhenium or tantalum), then carefully dried. The filaments are heated slowly, leading toevaporation and vaporization of the sample. It is useful for determining the elements thatevaporate at low temperature but require high ionization temperatures (Ca, for example). TIis generally used for precise and accurate measurement of stable isotope ratio of inorganicelements. It is also used to quantify toxic trace elements in foods. Page 21
  22. 22. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMass AnalyzersOnce ions have been formed and introduced into the vacuum, they are subjected to electrical(DC and/or RF) or magnetic fields. Their motion under these conditions is a function of manyparameters but all include the mass-to-charge ratio. Ions can be ejected from the analyser onem/z at a time or can be detected and measured, while trapped in the analyser.Mass analyzer should have following propertiesAccuracyThe accuracy of a mass measurement or concentration from a quantitative determination is ameasure of how close the value obtained is to the true value. The accuracy variesdramatically from analyzer to analyzer depending on the analyzer type and resolution. Page 22
  23. 23. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMass RangeThe range over which a mass spectrometer analyzer can operate. Quadrupole, Paul and linearion traps tend to be limited to upper m/z values around 4000. Penning FTICR and TOFanalyzers have mass limits that can extend this to well over 200,000 but there is a resolvingpower trade-off at high m/z values.Resolution (Resolving Power)Resolution is a measure of the ability of the mass spectrometer analyzer to separate two ionsof different, but defined, m/z value. For two overlapping singly- charged peaks m1 and m2 ofequal height, the resolving power is defined as m1/Δm, where m1 is the m/z value of one ionand Δm is the mass difference between m1 and m2 such that the two peaks are resolved witha defined interpeak valley. In the diagram, if h is 10% of the peak heights, then the method iscalled the 10% valley method. Other definitions include the 50% valley method; where h are50%, and the FWHM method. For two adjacent peaks at m/z values of 200.00 and 200.05separated by a 10% valley, the resolving power is 200.00/0.05 = 4000.In simple words we can say that resolution is a measure of how well a mass spectrometerseparates ions of different mass.Low resolution - capable of distinguishing among ions of different nominal mass that is ionsthat differ by at least one or more mass unitsHigh resolution - capable of distinguishing among ions that differ in mass by as little as0.0001 mass unitsThe resolving power A is defined as: Page 23
  24. 24. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRY– To resolve two mass e.g. 950 and 951 then a resolving power A needs to be 950  Low resolution spectrometers A = 1-2000  High resolution spectrometers A >100000Scan SpeedAnalyzers are scanned with a regular cycle time from low to high m/z or vice versa.Quadrupole analyzers tend to be scanned linearly in mass while a magnetic analyzer isscanned exponentially to provide peaks of equal width throughout the mass range. Fast scanspeeds are needed when a mass spectrometer is linked to a fast chromatographic system andTOF analyzers are currently among the best for this.QuadrupolesQuadrupole mass analyzers have been used with EI sources since the 1950’s and are still themost common mass analyzers in existence today. Interestingly, quadrupole mass analyzershave found new utility in their capacity to interface with ESI and APCI.Four parallel metal rods of circular cross section are electronically connected in pairs and acombination of DC and RF applied. At a specific RF field, only ions of a specific m/z canpass through the quadrupole.In order to perform tandem mass analysis with a quadrupole instrument, it is necessary toplace three quadruples in series. Each quadrupole has a separate function: the first quadrupole Page 24
  25. 25. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRY(Q1) is used to scan across a preset m/z range and select an ion of interest. The secondquadrupole (Q2), also known as the collision cell, focuses and transmits the ions whileintroducing a collision gas (argon or helium) into the flight path of the selected ion. The thirdquadrupole (Q3) serves to analyze the fragment ions generated in the collision cell (Q2)-Ions travel parallel to four rods- Opposite pairs of rods have rapidly alternating potentials (AC)- Ions try to follow alternating field in helical trajectories- Stable path only for one m/z value for each field frequencySmall and low costRmax ~ 500Harder to push heavy molecule - m/zmax < 2000Quadrupole Ion TrapIt is similar to Quadrupole analyzer but in an ion trap, rather than passing through aquadrupole analyzer with a superimposed radio frequency field, the ions are trapped in aradio frequency quadrupole field. The quadrupole ion trap typically consists of a ringelectrode and two hyperbolic endcap electrodes. The motion of the ions induced by theelectric field on these electrodes allows ions to be trapped or ejected from the ion trap. In thenormal mode, the radio frequency is scanned to resonantly excite and therefore eject ionsthrough small holes in the endcap to a detector. As the RF is scanned to higher frequencies,higher m/z ions are excited, ejected, and detected. Page 25
  26. 26. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYLinear Ion TrapThe linear ion trap differs from the 3D ion trap as it confines ions along the axis of aquadrupole mass analyzer using a two-dimensional (2D) radio frequency (RF) field withpotentials applied to end electrodes. The primary advantage to the linear trap over the 3D trapis the larger analyzer volume lends itself to a greater dynamic ranges and an improved rangeof quantitative analysis.Double-Focusing Magnetic Sector The earliest mass analyzers separated ions with a magnetic field. In magnetic analysis, theions are accelerated into a magnetic field using an electric field. A charged particle travelingthrough a magnetic field will travel in a circular motion with a radius that depends on thespeed of the ion, the magnetic field strength, and the ion’s m/z. A mass spectrum is obtainedby scanning the magnetic field and monitoring ions as they strike a fixed point detector. Alimitation of magnetic analyzers is their relatively low resolution. In order to improve this, Page 26
  27. 27. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYmagnetic instruments were modified with the addition of an electrostatic analyzer to focus theions. These are called double-sector or two-sector instruments. The electric sector serves as akinetic energy focusing element allowing only ions of a particular kinetic energy to passthrough its field irrespective of their mass-to-charge ratio. Thus, the addition of an electricsector allows only ions of uniform kinetic energy to reach the detector, thereby decreasing thekinetic energy spread, which in turn increases resolution. It should be noted that thecorresponding increase in resolution does have its costs in terms of sensitivity. These double-focusing (Figure 2.7) mass analyzers are used with ESI, FAB and EI ionization, however theyare not widely used today primarily due to their large size and the success of time-of-flight,quadrupole and FTMS analyzers with ESI and MALDI.Quadrupole Time-of-Flight Tandem MSTime-of-flight analysis is based on accelerating a group of ions to a detector where all of theions are given the same amount of energy through an accelerating potential. Because the ionshave the same energy, but a different mass, the lighter ions reach the detector first because oftheir greater velocity, while the heavier ions take longer due to their heavier masses andlower velocity. Hence, the analyzer is called time-of-flight because the mass is determinedfrom the ions’ time of arrival. Mass, charge, and kinetic energy of the ion all play a part in thearrival time at the detector. Since the kinetic energy (KE) of the ion is equal to 1/2 mv2, theion’s velocity can be represented as v = d/t = (2KE/m)1/2. The ions will travel a givendistance d, within a time t, where t is dependent upon the mass-to-charge ratio (m/z). In thisequation, v = d/t = (2KE/m)1/2, assuming that z = 1. Another representation of this equationto more clearly present how mass is determined is m = 2t2 KE/d2 where KE is constant. Page 27
  28. 28. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYIt is now widely used for ESI, MALDI, and more recently for electron ionization in GC/MSapplications.It combines time-of-flight technology with an electrostatic mirror. The reflectron serves toincrease the amount of time (t) ions need to reach the detector while reducing their kineticenergy distribution, thereby reducing the temporal distribution Δt. Since resolution is definedby the mass of a peak divided by the width of a peak or m/Δm (or t/Δt since m is related to t),increasing t and decreasing Δt results in higher resolution. Therefore, the TOF reflectronoffers high resolution over a simple TOF instrument by increasing the path length and kineticenergy focusing through the reflectron.Quadrupole Time-of-Flight MSQuadrupole-TOF mass analyzers are typically coupled to electrospray ionization sources andmore recently they have been successfully coupled to MALDI. It has high efficiency,sensitivity, and accuracy as compared to Quadrupole and TOF analyzer. Page 28
  29. 29. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYDetectorsA device that detects the ions produced in the mass spectrometer and produces a measurablesignal, generally an electronic signal. In most detectors, this signal is amplified. Commontypes include the Faraday cup, the electron multiplier, the microchannel plate detector and theDaly photomultiplier detector.  Faraday Cup  Photomultiplier Conversion Dynode  Array Detector  Charge (or Inductive) Detector  Electron MultiplierFaraday Cup A Faraday cup involves an ion striking the dynode (BeO, GaP, or CsSb) surface whichcauses secondary electrons to be ejected. This temporary electron emission induces a positivecharge on the detector and therefore a current of electrons flowing toward the detector. Thisdetector is not particularly sensitive, offering limited amplification of signal, yet it is tolerantof relatively high pressure. Page 29
  30. 30. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRY– Ions are accelerated toward a grounded “collector electrode”– As ions strike the surface, electrons flow to neutralize charge, producing a small currentthat can be externally amplified.– Size of this current is related to # of ions in– No internal gain → less sensitivePhotomultiplier Conversion Dynode The photomultiplier conversion dynode detector is not as commonly used at the electronmultiplier yet it is similar in design where the secondary electrons strike a phosphorus screeninstead of a dynode. The phosphorus screen releases photons which are detected by thephotomultiplier. Photomultipliers also operate like the electron multiplier where the strikingof the photon on scintillating surface results in the release of electrons that are then amplifiedusing the cascading principle. One advantage of the conversion dynode is that thephotomultiplier tube is sealed in a vacuum, unexposed to the environment of the massspectrometer and thus the possibility of contamination is removed. This improves thelifetimes of these detectors over electron multipliers. A five-year or greater lifetime is typical,and they have a similar sensitivity to the electron multiplier.Array Detector An array detector is a group of individual detectors aligned in an array format. The arraydetector, which spatially detects ions according to their different m/z, has been typically usedon magnetic sector mass analyzers. Spatially differentiated ions can be detectedsimultaneously by an array detector. The primary advantage of this approach is that, over asmall mass range, scanning is not necessary and therefore sensitivity is improved. Page 30
  31. 31. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYCharge (or Inductive) Detector Charge detectors simply recognize a moving charged particle (an ion) through theinduction of a current on the plate as the ion moves past. A typical signal is shown in Figure.This type of detection is widely used in FTMS to generate an image current of an ion.Detection is independent of ion size and therefore has been used on particles such as wholeviruses.Electron Multiplier Perhaps the most common means of detecting ions involves an electron multiplier whichis made up of a series (12 to 24) of aluminum oxide (Al2O3) dynodes maintained at everincreasing potentials. Ions strike the first dynode surface causing an emission of electrons.These electrons are then attracted to the next dynode held at a higher potential and thereforemore secondary electrons are generated. Ultimately, as numerous dynodes are involved, acascade of electrons is formed that results in an overall current gain on the order of onemillion or higher. Page 31
  32. 32. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYVacuum in the Mass Spectrometer All mass spectrometers need a vacuum to allow ions to reach the detector withoutcolliding with other gaseous molecules or atoms. If such collisions did occur, the instrumentwould suffer from reduced resolution and sensitivity. Higher pressures may also cause highvoltages to discharge to ground which can damage the instrument, its electronics, and/or thecomputer system running the mass spectrometer. An extreme leak, basically an implosion,can seriously damage a mass spectrometer by destroying electrostatic lenses, coating theoptics with pump oil, and damaging the detector. In general, maintaining a good vacuum iscrucial to obtaining high quality spectra.STRUCTURAL ANALYSIS AND FRAGMENTATION PATTERNSWhen the vaporized organic sample passes into the ionization chamber of a massspectrometer, it is bombarded by a stream of electrons. These electrons have a high enoughenergy to knock an electron off an organic molecule to form a positive ion. This ion is calledthe molecular ion - or sometimes the parent ion. Page 32
  33. 33. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYThe molecular ion is often given the symbol M+ or - the dot in this second versionrepresents the fact that somewhere in the ion there will be a single unpaired electron. Thatsone half of what was originally a pair of electrons - the other half is the electron which wasremoved in the ionization process.FragmentationThe molecular ions are energetically unstable, and some of them will break up into smallerpieces. The simplest case is that a molecular ion breaks into two parts - one of which isanother positive ion, and the other is an uncharged free radical.The uncharged free radical wont produce a line on the mass spectrum. Only charged particleswill be accelerated, deflected and detected by the mass spectrometer. These unchargedparticles will simply get lost in the machine - eventually, they get removed by the vacuumpump.The ion, X+, will travel through the mass spectrometer just like any other positive ion - andwill produce a line on the stick diagram.All sorts of fragmentations of the original molecular ion are possible - and that means thatyou will get a whole host of lines in the mass spectrum. For example, the mass spectrum ofpentane looks like this:Its important to realize that the pattern of lines in the mass spectrum of an organic compoundtells you something quite different from the pattern of lines in the mass spectrum of anelement. With an element, each line represents a different isotope of that element. With acompound, each line represents a different fragment produced when the molecular ion breaksup.The tallest line in the stick diagram (in this case at m/z = 43) is called the base peak. This isusually given an arbitrary height of 100, and the height of everything else is measuredrelative to this. The base peak is the tallest peak because it represents the commonestfragment ion to be formed - either because there are several ways in which it could beproduced during fragmentation of the parent ion, or because it is a particularly stable ion. Page 33
  34. 34. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYFragmentation PatternsBy using fragmentation pattern we can easily study the structure of a compound.  Stevenson’s Rule  Homolytic bond cleavage  Heterolytic fragmentation  Alpha cleavage  Beta-cleavage  Inductive cleavage  Retro Diels-Alder Cleavage  McLafferty rearrangement  Ortho effect  Onimum Reaction  CO EliminationStevenson’s RuleWhen fragment ions form in the mass spectrometer, they almost always do so by means ofuni-molecular processes. The low pressure of the ionization chamber makes it unlikely asignificant number of bimolecular collisions could occur. The uni-molecular processes thatare energetically most favorable give rise to the most fragment ions. This is the idea behindStevenson’s Rule: The most probable fragmentation is the one that leaves the positive chargeon the fragment with the lowest ionization energy. In other words, fragmentation processesthat lead to the formation of more stable ions are favored over processes that lead to less-stable ions. This idea is grounded in the same concepts as Markovnikov’s Rule, which statesthat in the addition of a hydrogen halide to an alkene, the more stable carbocation forms thefastest and leads to the major product of the addition reaction. In fact, a great deal of thechemistry associated with ionic fragmentation can be explained in terms of what is knownabout carbocations in solution. For example, alkyl substitution stabilizes fragment ions (andpromotes their formation) in much the same way that it stabilizes carbocations. Other familiarconcepts will help one predict likely fragmentation processes: electronegativity,polarizability, resonance delocalization, the octet rule, and so on.Often, fragmentation involves the loss of an electrically neutral fragment. This fragment doesnot appear in the mass spectrum, but its existence can be deduced by noting the difference inmasses of the fragment ion and the original molecular ion. Again, processes that lead to theformation of a more stable neutral fragment are favored over those that lead to less-stableneutral fragments.An OE• + can fragment in two ways: cleavage of a bond to create an EE+ and a radical (R•) orcleavage of bonds to create another OE• + and a closed-shell neutral molecule (N). An EE+, onthe other hand, can only fragment in one way—cleavage of bonds to create another EE+ anda closed-shell neutral molecule (N). This is the so-called even-electron rule. The mostcommon mode of fragmentation involves the cleavage of one bond. In this process, the OE• +yields a radical (R•) and an EE+ fragment ion. Cleavages that lead to the formation of morestable carbocations are favored. When the loss of more than one possible radical is possible, acorollary to Stevenson’s Rule is that the largest alkyl radical to be lost preferentially. Thus,ease of fragmentation to form ions increases in the order Page 34
  35. 35. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYDescription of the fragmentation processes…Fragmentation of an odd electron molecular ion (M+.) may occur by hemolytic or heterolyticcleavage of sigma bond.Homolytic bond cleavageHomolytic bond cleavage is a type of ion fragmentation in which a bond is broken by thetransfer of one electron from the bond to the charged atom, the other electron remaining onits starting atom. The movement of one electron is signified by a fishhook arrow. Thefragmentation of a ketone is shown in the figure.Heterolytic bond cleavageHeterolytic bond cleavage is a type of ion fragmentation in which a bond is broken by thetransfer of a pair of electrons from the bond to the charged atom. In alpha cleavage, a bondalpha to the charged atom is broken and in beta cleavage, a bond two removed from thecharged atom is broken. The movement of 2 electrons is signified by a double-barbed arrowand also referred to as charge-induced fragmentation. The fragmentation of an even-electronion is shown in the figure. Page 35
  36. 36. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYAlpha cleavageAlpha cleavage occurs on α-bonds adjacent to heteroatoms (N, O, and S). Charge is stabilizedby heteroatom. Occurs only once in a fragmentation (cation formed is too stable to fragmentfurther) for example in alcohols, aliphatic ethers, aromatic ethers, cyclic compounds andaromatic ketones etc. Page 36
  37. 37. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYBeta-cleavageFission of a bond two removed from a heteroatom or functional group, producing a radicaland an ion is called beta cleavage and also written as β-cleavage for or example allylicfragmentation.Inductive cleavageIf an electron pair is completely transferred towards a centre of positive charge as a result ofthe inductive effect, shown schematically by the use of a double-headed arrow, then the ionwill fragment by inductive cleavage. The figure illustrates this for radical cation ether.Retro Diels-Alder CleavageA multicentered ion fragmentation which is the reverse of the classical Diels-Alder reactionemployed in organic synthesis that forms a cyclic alkene by the cycloaddition of a substituteddiene and a conjugated diene. In the retro reaction, a cyclic alkene radical cation fragments toform either a diene and an alkene radical cation or a diene radical cation and an alkene.Depending on the substituents present in the original molecule, the more stable radical cationwill dominate. Page 37
  38. 38. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMcLafferty rearrangementAn ion fragmentation characterised by a rearrangement within a six-membered ring system.The most usual configuration is for a radical cation formed by EI to undergo the transfer of aγ- hydrogen atom to the ionisation site through a ring system as shown here. The distonicradical cation so formed can break up by radical-site-induced (α), or charged site-inducedfragmentation as shown in the figure for example ketones, carboxylic acid and esters.Ortho effectThe interaction between substituents oriented ortho, as opposed to para and meta, to eachother on a ring system, can create specific fragmentation pathways. This permits thedistinction between these isomeric species. The diagram shows a case in which only the orthoisomer can undergo the rearrangement. Page 38
  39. 39. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYOnimum ReactionOnium ion, A hypervalent species containing a non-metallic element such as the methoniumion CH5+. It includes ions such as oxonium, phosphonium, and nitronium ions.The onium reaction is not limited to alkyl substituents acyl groups can also can undergo theonium reaction. An onium ion is a hypervalent species containing a non-metallic elementsuch as the methonium ion CH5+. It includes ions such as oxonium, phosphonium,sulphonium and nitronium ions.CO EliminationFrom carbonyl compounds CO elimination reaction from α-cleavage takes place like inaldehyde, ketones and phenols etc.If there is more than one CO group present sequential elimination of all CO groups ispossible. Page 39
  40. 40. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYINTERPRETATION OF MASS SPECTRUMMass spectrumIt is a simple graph between the abundance of an ion along Y-axis against its mass-to-chargeratio along X-axis. A mass spectrum contains a large number of peaks some are small andsome are large, these areMolecular ion peakThe peak of an ion formed from the original molecule by electron ionization, by the loss of anelectron, or by addition or removal of an anion or cation and also known as parent peak,radical peak.Fragmentation peaksThe peaks observed by fragments of compounds.Base peakThe most intense ion in a mass spectrum. The abundance of this ion is used as the base fromwhich to normalise the relative abundances of the remaining peaks in the spectrum and isgiven a nominal value of 100%.Isotopic peaksPeaks observed due to isotopes like in case of carbon with M+. M+1 peak also observed. Page 40
  41. 41. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYRules for interpretation of mass spectrumFollowing are the main rules for interpretationMolecular ion peakIf present it will be highest peak in all isotopic peaks.DBR CalculationsDouble bond or ring calculations tell us about how many rings or double bonds are present ina compound.DBR= C-H/2+N/2+1C= number of carbon atomsH= number of hydrogen atomsN= number of nitogen atomsNitrogen RuleThe nominal molecular weight of a compound will have an even-number value if there are nonitrogen atoms, or an even number of nitrogen atoms, present in the molecule. This holds forcompounds containing C, H, O, P, S, Si, or halogen atoms. Even-electron fragment ionscontaining an even number of nitrogen atoms occur at odd-number m/z values. Conversely, ifthere are an odd number of nitrogen atoms, the nominal molecular weight will be an oddnumber and even-electron ions containing an odd number of nitrogen atoms occur at even-number m/z values. Page 41
  42. 42. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYIsotopic effectMass spectrum can easily be drawn but there are some factors which make the spectracomplicated, one of these is isotopic effect.Mass spectra (examples)AlkanesWhen an alkane is bombarded by high energy electrons it will lose an electron to form aradical cation. This radical cation has the same mass as the parent compound and producesthe molecular ion (M+) peak. The type of radical formed follows the stability of radicals: 3o > 2o > 1o > methylThe alkane molecular ion can further fragment to form a homologous series of neutral alkylradicals usually beginning with the methyl radical. The methyl radical has a mass of 15 andthe next largest peak in the mass spectrum usually corresponds to the loss of methyl radical(M-15). Ethyl radical can also be lost (M-29) and so forth Page 42
  43. 43. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMechanism of fragmentation for butaneMass spectrum for butane Page 43
  44. 44. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYTypical fragments lost from straight chain alkanes Molecular Ion - Fragment Lost 1 H· 2 2 H· 15 CH3· 29 C2H5· 43 C3H7· 57 C4H9· 71 C5H11·Peaks in the mass spectra of straight chain alkanes will usually appear in groups of 14 massunit intervals (corresponding to one CH2 group). The most intense fragmentation peak isusually the 3 carbon fragment, with the intensities of the peaks decreasing with increasingmass. Often, the M-15 peak (loss of methyl radical) will be absent. Fragments to look for inthese spectra correspond to CnH2n+1+, CnH2n+, and CnH2n-1+.Branched alkanes tend to fragment very easily, due to the presence of 2o, 3o, and 4o carbonatoms in the structure. When branched alkanes fragment, stable secondary and tertiarycarbocations are formed. For this reason the molecular ion peak is much less intense than instraight chain alkanes. Figure 10 shows the mechanism of fragmentation for isobutane. Themass spectrum for isobutane is contained in Figure 11. Notice the reduced intensity of themolecular ion peak.Mechanism of fragmentation for isobutane Page 44
  45. 45. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMass spectrum for isobutaneSummaryStrong M+ (but intensity decreases with an increase of branches.Carbon-carbon bond cleavageLoss of CH units in series: M-14, M-28, M-42 etc Page 45
  46. 46. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYCycloalkanesThe fragmentation patterns of cycloalkanes may show mass clusters arranged in ahomologous series, as in the alkanes. However, the most significant mode of cleavage of thecycloalkanes involves the loss of ethene from the parent molecule or from intermediateradical-ions. Additionally, if the cycloalkane has a side chain, loss of that side chain is also afavorable mode of fragmentation. The mass spectrum of cyclopentane has an intense peak atm/e = 42 due to the loss of ethene. Page 46
  47. 47. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMechanism of fragmentation for cyclopentaneMass spectrum for cyclopentaneIn order for cycloalkanes to fragment, two carbon-carbon bonds must be broken. This processmay require a significant amount of time (relative to the amount of time it takes an ion toreach the detector in a mass spectrometer), therefore, a significant amount of the molecularion will reach the detector resulting in a large molecular ion peak. Page 47
  48. 48. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYCycloalkanes tend to cleave in CnH2n+, CnH2n-1+, and CnH2n-2+ fragments. The larger numberof even numbered mass fragments of cycloalkanes helps to distinguish this functional groupfrom the acyclic alkanes.SummaryStrong M+, strong base peak at M-28 (loss of ethene)A series of peaks: M-15, M-28, M-43 etcMethyl, ethyl, propyl with an additional hydrogen give peaksAlkenesThe mass spectra of most alkenes show distinct molecular ion peaks. This is probably due tothe loss of an electron in the p bond, leaving the carbon skeleton relatively undisturbed.Alkenes usually form fragments corresponding to CnH2n+1+, CnH2n+, and CnH2n-1+ (the lattertwo fragments are more intense). It is very difficult to locate the position of the double bondin an alkene because of its facile migration in the fragments. For this reason, the mass spectraof alkene isomers are nearly identical and almost impossible to discriminate between. Theonly exception would be terminal alkenes which fragment to form an allylic carbocation withm/e = 41 Page 48
  49. 49. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMechanism of fragmentation for 1-buteneMass spectrum for 1-buteneThe mass spectra of cycloalkenes show distinct molecular ion peaks. It may be impossible tolocate the position of a double bond due to migration. The mechanism of fragmentation forcyclic alkenes is virtually the same as for straight chain alkenes. One noteworthycharacteristic is the fragmentation of cyclohexenes which undergo a reverse Diels-Alderreaction. Page 49
  50. 50. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMechanism of fragmentation for cyclohexeneMass spectrum for cyclohexeneSummaryStrong M+Fragmentation ion has formula CnH2n+ and CnH2n-1α-CleavageA series of peaks: M-15, M-29, M-43, M-57 etc Page 50
  51. 51. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYAlkynesThe mass spectra of alkynes are virtually identical to those of alkenes. The molecular ionpeak is intense, and fragmentation parallels that of the alkenes.Two differences are worth mentioning: terminal alkynes fragment to form propargyl ions(m/e = 39), and can also lose the terminal hydrogen, yielding a strong M-1 peak.Mechanism of fragmentation for 1-butyne Page 51
  52. 52. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMass spectrum for 1-butyneSummaryStrong M+Strong base peak at M-1 peak due to the loss of terminal hydrogenAlpha cleavage Page 52
  53. 53. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYAromatic HydrocarbonsThe mass spectra of most aromatic compounds show distinct molecular ion peaks. This isprobably due to the loss of an electron in the p system, leaving the carbon skeleton relativelyundisturbed.When an alkyl side-chain is attached to the ring, fragmentation usually occurs at the benzylicposition, producing the tropylium ion (m/e = 91)Formation of tropylium ionHowever, fragmentation can also occur at the attachment point to the ring producing thephenyl cation (m/e = 77) as shown in figure 21.Formation of phenyl cationIf the side-chain is a propyl group or larger, then the McLafferty rearrangement is apossibility, producing a fragment of m/e = 92.Mechanism of McLafferty rearrangement Page 53
  54. 54. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYFormation of a substituted tropylium ion is typical for alkyl-substituted benzenes producing apeak at m/e = 105.Formation of substituted tropylium ionThe complete mass spectrum for propyl benzene is given in figure 24, which illustrates all ofthese points.Mass spectrum for propyl benzeneSummaryStrong M+Loss of hydrogen gives base peakMcLafferty rearrangement Page 54
  55. 55. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYFormation of benzyl cation or tropylium ionAlcoholsM+ weak or absentLoss of alkyl group via a-cleavageDehydration (loss of water) gives peak at M-18Loss of alkyl group in alcohol fragmentationA second common mode of fragmentation involves dehydration. The importance of thisfragmentation process increases with increasing chain length. Loss of water (M - 18) is veryindicative of an alcohol functionality (see figure 26). Page 55
  56. 56. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYDehydration of an alcoholPhenols can lose the elements of carbon monoxide to give strong peaks at M - 28.Phenols can also lose the elements of the formyl radical (HCO·) to give strong peaks at M -29.Mass spectrum for 2-pentanol Page 56
  57. 57. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYPhenolsStrong M+M-1 due to hydrogen eliminationM-28 due to loss of COM-29 due to loss of HCO (formyl radical) Page 57
  58. 58. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYEthersM+ weak but observableLoss of alkyl radical due to a-cleavageB-cleavage( formation of carbocation fragments through loss of alkoxy radicals)C-O bond cleavage next to double bondPeaks at M-31, M-45, M-59 etcCleavage of the C-C bond to the a-carbonA second common mode of fragmentation involves cleavage of the C-O bond (see figure 29).Cleavage of the C-O bond of etherHydride transfer from a b-carbon is an important rearrangement process in ethers as shown infigure 30.Rearrangement of an etherPeaks usually occur in CnH2nOH+ increments for ethers.. Page 58
  59. 59. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMass spectrum for n-butyl ether Page 59
  60. 60. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYAldehydesM+ weak, but observable (aliphatic)Aliphatic : M-29, M-43 etcMcLafferty rearrangement is common gives the base peakα-cleavageβ-cleavagea-cleavageA second common mode of fragmentation involves b-cleavageb-cleavageMcLafferty rearrangement can take place for aldehydes with at least 4 carbons. A fragmention of m/e = 44 is formed, and is considered quite characteristic of an aldehyde Page 60
  61. 61. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMcLafferty rearrangement of an aldehydeMass spectrum for hexanal Page 61
  62. 62. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYM+ strong (aromatic)Aromatic: M-1 (loss of hydrogen)M-29 (loss of HCO)McLafferty rearrangement is commonα-cleavageβ-cleavage Page 62
  63. 63. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYKetonesStrong M+A series of peaks M-15, M-29, M-43 etcLoss of alkyl group attached to the carbonyl group by a-cleavageFormation of acylium ion (RCO+)McLafferty rearrangementa-cleavageMcLafferty rearrangement can take place for ketones with at least 3 carbonsMcLafferty rearrangement of a ketoneFor aromatic ketones, a-cleavage usually occurs, which is followed by loss of carbonmonoxide as indicated in figure 38. Page 63
  64. 64. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYAromatic ketone fragmentationMass spectrum for 2-pentanone Page 64
  65. 65. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYEstersM+ weak but generally observableLoss of alkyl group attached to the carbonyl group by a-cleavageFormation of acylium ion (RCO+)McLafferty rearrangementAcyl portion of ester OR+Methyl esters: M-31 due to loss of OCH3Higher esters: M-32, M-45, M-46, M-59, M-60, M-73 etca-cleavage Page 65
  66. 66. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMcLafferty rearrangement can take place for esters in the alkyl portionMcLafferty rearrangement of an esterMass spectrum for ethyl acetate Page 66
  67. 67. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYCarboxylic AcidsAliphatic carboxylic acids:M+ weak but observableA-cleavage on either side of C=OM-17 due to loss of OHM-45 due to loss of COOHMcLafferty rearrangement gives base peaka-cleavage Page 67
  68. 68. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYLoss of the alkyl group as a free radical, leaving CO2H+, also occurs as shown in figure 44.Loss of alkyl radicalWith acids having g hydrogens, the principal pathway for rearrangement is the McLaffertyrearrangementMcLafferty rearrangement of an acidMass spectrum for pentanoic acid Page 68
  69. 69. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYAromatic carboxylic acids:M+ StrongA-cleavage on either side of C=OM-17 due to loss of OHM-18 due to loss of HOHM-45 due to loss of COOHMcLafferty rearrangement gives base peak Page 69
  70. 70. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYAminesM+ weak or absentNitrogen rule obeyα-cleavagea-cleavageLoss of hydrogen radical, is quite common in aminesLoss of hydrogen radical Page 70
  71. 71. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMass spectrum for diethyl amineNitrilesM+ weak but observableM-1 visible peak due to loss of termiminal hydrogen Page 71
  72. 72. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYLoss of hydrogen radicalMcLafferty rearrangement can take place for nitrilesMcLafferty rearrangement of a nitrileMass spectrum for pentanenitrile Page 72
  73. 73. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYAmidesThe molecular ion peak is usually observable, and will be a good indication of the presenceof an amide (nitrogen rule).a-cleavageMcLafferty rearrangement can take place for amides in the alkyl portion Page 73
  74. 74. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMcLafferty rearrangement of an amideMass spectrum for propanamideNitro CompoundsM+ seldom observedLoss of NO+ gives visible peakLoss of NO2+ gives peak Page 74
  75. 75. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYAlkyl Chlorides and Alkyl BromidesStrong M+ 2 peaksFor Cl M/M+2 = 3:1F or Br M/M+2 = 1:1A-cleavageLoss of Cl or BrLoss of HCl or HBr Page 75
  76. 76. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYAPPLICATIONS OF MASS SPECTROMETRYThe technique has both qualitative and quantitative uses. These include identifying unknowncompounds, determining the isotopic composition of elements in a molecule, and determiningthe structure of a compound by observing its fragmentation. Followings are the mainapplicationsToxicity of ToothpastesIn some Chinese toothpastes, a toxic compound known as DEG is sometimes used as asweetener. The compound is banned, but it is difficult to truly enforce the ban, sincetoothpaste is very difficult to test. It can be done, but it takes a lot of time. Until now, AChinese scientist, Huanwen Chen, has come up with a way of using mass spectrometry toquickly screed for toxins.DEG (diethylene glycol)Looking for pesticidesNutritional supplements are often touted as "natural" ways to boost health. However, the factof the matter is that pesticides can find themselves in supplements and food. Unfortunately,testing for multiple pesticides is difficult and is practically impossible without massspectrometry. Douglas Hayward and Jon Wong at the U.S. FDA have developed a massspectrometry method that can identify multiple compounds at once, hoping to reduce theamount of pesticides that enter the food supply. Page 76
  77. 77. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYMeasuring nanoparticle sizeUsually, nanoparticles are measured with the use of transmission electron microscopy or x-ray diffraction. Interestingly, it appears that you can use a MALDI-TOF spectrometer tomeasure the size of nanoparticles. Once size of a sphere is measured, its density is alsocalculated.Isotope Ratio Data a. Carbon, Hydrogen, Nitrogen, and OxygenWe must remember that molecular species exist that contain less abundant isotopes of carbon,hydrogen, nitrogen, and oxygen which will give rise to isotope peaks at M+1, M+2, etc.In above Figure, you will notice additional peaks at 122 (M+1) and 123 (M+2). These arepeaks due to the presence of isotopes of carbon, hydrogen, nitrogen, and oxygen inbenzamide, and are not to be confused with the molecular ion peak. Sometimes, the intensityof the M+1 and M+2 peaks can lead to valuable information about the molecular formula ofthe compound.To calculate the intensity of the M+1 peak (with respect to the M+ peak), use the followingequation: %(M+1) = (1.1 * #C atoms) + (0.016 * #H atoms) + (0.38 * #N atoms)To calculate the intensity of the M+2 peak, use the following equation: %(M+2) = [(1.1 * #C atoms)2 / 200] + [(0.016 * #H atoms)2 / 200] + (0.38 * #O atoms) Page 77
  78. 78. Generated by Foxit PDF Creator © Foxit Software For evaluation only. MASS SPECTROMETRYThe following example shows how the above equations can be used to help confirm theformula for a chemical compound.The above calculations will only approximate the sizes of the M+1 and M+2 peaks. Also,these formulas are really only useful if the molecular formula is already known, but theyprovide a good check on the validity of a proposed molecular formula. b. Bromine and ChlorineWhen bromine or chlorine is present in a compound, the M+2 peak becomes very significant.This is due to the fact that, for bromine, two isotopes (79Br and 81Br) are present in a 1:1 ratioin naturally occurring substances, and, for chlorine, two isotopes (35Cl and 37Cl) are present ina 3:1 ratio in naturally occurring substances. If a compound contains bromine, the M+ andM+2 peaks are present in equal intensities. Additionally, if a compound contains chlorine, theM+ and M+2 peaks will be present in a 3:1 ratio. The presence of these M+ and M+2 peaks isvery indicative of brominated and chlorinated compounds. Page 78