According to the IUPAC (International Union of Pure and Applied Chemistry), it is the branch of science dealing with all aspects of mass spectroscopes and results obtained with these instruments.
Mass Spectrometry Contents Brief History of Mass Spectrometry Nobel prize pioneers Mass spectrometer Structural analysis and Fragmentation Patterns interpretation of mass spectrum Applications of mass spectrometry
1897 1919 1934 1966 Mass Spectrometry Brief History of Mass Spectrometry J.J. Thomson. Discovered electrons by cathode rays experiment. Nobel prize in 1906. Francis Aston recognized 1st mass spectrometer and measure z/m of ionic compounds. First double focusing magnetic analyzer was invented by Johnson and Neil. Munson and Field described chemical ionization.
1968 1975 1985 1989 Mass Spectrometry Electrospray Ionization was invented by Dole, Mack and friends. Atmospheric Pressure Chemical Ionization (APCI) was developed by Carroll and others. F. Hillenkamp, M.Karas and co-workers describe and coin the term matrix assisted laser desorption ionization (MALDI). w. Paul discovered the ion trap technique.
Mass Spectrometry Understanding Mass Spectrometry In a mass spectrometer, the same thing is happening, except it's atoms and molecules that are being deflected, and it's electric or magnetic fields causing the deflection. It's also happening in a cabinet that can be as small as a microwave or as large as a chest freezer.
Mass Spectrometry Mass spectrometer is similar to a prism. In the prism, light is separated 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 in the mass analyzer, digitized and detected by an ion detector.
Mass Spectrometry Basic Components of Mass Spectrometer Four basic components
Mass spectrometer Sample Introduction Techniques Initial pressure of sample is 760 mmHg or ~10-6 torr Two techniques
Direct Insertion (commonly used in MALDI)
Direct infusion or injection (commonly used in ESI)
Mass spectrometer Direct Insertion sample introduction technique very simple technique Sample is placed on a prob and inserted into ionization source and then subjected to any number of desorption processes, such as laser desorption or direct heating, to facilitate vaporization and ionization.
Mass Spectrometry Direct infusion or injection sample introduction technique Frequently used due to high efficiently Used in coupling techniques like GC-MS and HPLC-MS
Mass Spectrometry Ionization Methods used in Mass spectrometry Commonly used
Mass Spectrometry Protonation Formation of positive ions by the addition of a proton Used for basic compounds like amines, peptides Used in MALDI, APCI and ESI
Mass Spectrometry Deprotonation Give net negative charge of 1- by removal of one proton Used for acidic species like phenols, carboxylic acid, sulfonic acid etc. Used in MALDI, APCI and ESI
Mass Spectrometry Cationization produces a charged complex by non-covalently adding a positively charged ion like alkali metal ion or ammonium ion to a neutral molecule. Used for Carbohydrates Used in MALDI, APCI and ESI
Mass Spectrometry Transfer of a charged molecule to the gas phase Cation from solution to gas Used in MALDI or ESI
Mass Spectrometry Electron ejection Electron is ejected to give positive ion Usually for non-polar compounds with low molecular weights like anthracene.
Mass Spectrometry Electron capture a net negative charge of 1- is achieved with the absorption or capture of an electron. Used for halogenated compounds
Mass Spectrometry Ionization Sources in mass spectrometer
Mass Spectrometry Types of Ionization Sources Hard ionization sources Soft ionization sources Little excess energy in molecule and produced unstable fragments which are again fragmented. leave excess energy in molecule and produced stable fragments which is not further fragarmented
Mass Spectrometry Electrospray Ionization (ESI) The sample solution is sprayed from a region of the strong electric field at the tip of a metal nozzle 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 charged droplets. Either dry gas, heat, or both are applied to the droplets at atmospheric pressure thus causing the solvent to evaporate from each droplet For example peptides, proteins, carbohydrates, small oligonucleotides, synthetic polymers, and lipids
Mass Spectrometry Nanoelectrospray Ionization (NanoESI) where the spray needle has been made very small and is positioned close to the entrance to the mass analyzer. The end result of this rather simple adjustment is increased efficiency, which includes a reduction in the amount of sample needed.
Mass Spectrometry Atmospheric Pressure Chemical Ionization (APCI) 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 source contains a heated vaporizer, which facilitates rapid desolvation/vaporization of the droplets. Vaporized sample molecules are carried through an ion-molecule reaction region at atmospheric pressure.
Mass Spectrometry Atmospheric pressure photoionization (APPI) it generates ions directly from solution with relatively low background and is capable of analyzing relatively nonpolar compounds. APPI vaporized sample passes through ultra-violet light. APPI is much more sensitive than ESI or APCI.
Mass Spectrometry Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) 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 a laser results in the vaporization of the matrix, which carries the analyte with it. The matrix plays a key role in this technique. The co-crystallized sample molecules also vaporize, but without having to directly absorb energy from the laser. Molecules sensitive to the laser light are therefore protected from direct UV laser excitation.
Mass Spectrometry Fast Atom Bombardment (FAB) Immobilized matrix is bombarded with a fast beam of Argon or Xenon atoms. Charged sample ions are ejected from the matrix and extracted into the mass analyzers Used for large compounds with low volatility (eg peptides, proteins, carbohydrates) Solid or liquid sample is mixed with a non-volatile matrix (eg glycerol, crown ethers, nitrobenzyl alcohol)
Mass Spectrometry Electron Ionization (EI) Energetic process a heated filament emits electrons which are accelerated by a potential difference of usually 70eV into the sample chamber. Ionization of the sample occurs by removal of an electron from the molecule thus generating a positively charged ion with one unpaired electron.
Mass Spectrometry Chemical Ionization (CI) process is initiated with a reagent gas such as methane, isobutane, or ammonia, which is ionized by electron impact. High gas pressure in the ionization source is required for the reaction between the reagent gas ions and reagent gas neutrals. possible mechanism Reagent (R) + e- -> R+ + 2 e- R+ + RH -> RH+ + R RH+ + Analyte (A) -> AH+ + R biologically important molecules (sugars, amino acids, lipids etc.).
Mass Spectrometry Thermal ionization (TI) Samples are deposited on rhenium or tantalum filament and then carefully evaporated and sent to mass analyzer. used to
quantify toxic trace elements in foods.
measurement of stable isotope ratio of inorganic elements.
Mass Spectrometry Mass Analyzer Properties of mass Analyzer Scan Speed Accuracy Mass Range Resolution The range over which a mass spectrometer analyzer can operate. is a measure of how close the value obtained is to the true value. The accuracy varies dramatically from analyzer to analyzer depending on the analyzer type and resolution. Analyzers are scanned with a regular cycle time from low to high m/z or vice versa. A measure of how well a mass spectrometer separates ions of different mass
Mass Spectrometry Quadrupoles -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 frequency Smalll and low cost Rmax~ 500 Harder to push heavy molecule - m/zmax < 2000
Mass Spectrometry Quadrupole Ion Trap The quadrupole ion trap typically consists of a ring electrode and two hyperbolic endcap electrodes. The motion of the ions induced by the electric field on these electrodes allows ions to be trapped or ejected from the ion trap. In the normal mode, the radio frequency is scanned to resonantly excite and therefore eject ions through 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.
Mass Spectrometry Linear Ion Trap The linear ion trap differs from the 3D ion trap as it confines ions along the axis of a quadrupole mass analyzer using a two-dimensional (2D) radio frequency (RF) field with potentials applied to end electrodes. The primary advantage to the linear trap over the 3D trap is the larger analyzer volume lends itself to a greater dynamic ranges and an improved range of quantitative analysis.
Mass Spectrometry Double-Focusing Magnetic Sector the ions are accelerated into a magnetic field using an electric field. A charged particle traveling through a magnetic field will travel in a circular motion with a radius that depends on the speed of the ion, the magnetic field strength, and the ion’s m/z. A mass spectrum is obtained by scanning the magnetic field and monitoring ions as they strike a fixed point detector.
Mass Spectrometry Quadrupole Time-of-Flight Tandem MS Time-of-flight analysis is based on accelerating a group of ions to a detector where all of the ions are given the same amount of energy through an accelerating potential. Because the ions have the same energy, but a different mass, the lighter ions reach the detector first because of their greater velocity, while the heavier ions take longer due to their heavier masses and lower velocity. Hence, the analyzer is called time-of-flight because the mass is determined from the ions’ time of arrival. Mass, charge, and kinetic energy of the ion all play a part in the arrival time at the detector.
Mass Spectrometry Quadrupole Time-of-Flight MS Quadrupole-TOF mass analyzers are typically coupled to electrospray ionization sources and more recently they have been successfully coupled to MALDI. It has high efficiency, sensitivity, and accuracy as compared to Quadrupole and TOF analyzer.
Mass Spectrometry Detectors used in mass spectrometer Faraday Cup Photomultiplier Conversion Dynode Array Detector Charge (or Inductive) Detector Electron Multiplier
Mass Spectrometry Faraday Cup A Faraday cup involves an ion striking the dynode (BeO, GaP, or CsSb) surface which causes secondary electrons to be ejected. This temporary electron emission induces a positive charge on the detector and therefore a current of electrons flowing toward the detector. not particularly sensitive offering limited amplification of signal is tolerant of relatively high pressure. – Ions are accelerated toward a grounded “collector electrode” – As ions strike the surface, electrons flow to neutralize charge, producing a small current that can be externally amplified. – Size of this current is related to # of ions in – No internal gain -> less sensitive
Mass Spectrometry Photomultiplier Conversion Dynode the secondary electrons strike a phosphorus screen instead of a dynode. The phosphorus screen releases photons which are detected by the photomultiplier. Photomultipliers also operate like the electron multiplier where the striking of the photon on scintillating surface results in the release of electrons that are then amplified using the cascading principle. is not as commonly Life limit is high as compared to others.
Mass Spectrometry Array Detector detects ions according to their different m/z, has been typically used on magnetic sector mass analyzers. The primary advantage of this approach is that, over a small mass range, scanning is not necessary and therefore sensitivity is improved.
Mass Spectrometry Charge (or Inductive) Detector Charge detectors simply recognize a moving charged particle (an ion) through the induction of a current on the plate as the ion moves past Detection is independent of ion size.
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 therefore more secondary electrons are generated.
Mass spectrometer Vacuum in the Mass Spectrometer All mass spectrometers need a vacuum to allow ions to reach the detector without colliding with other gaseous molecules or atoms. If such collisions did occur, the instrument would suffer from reduced resolution and sensitivity.
Mass spectrometer Structural analysis and Fragmentation Patterns Mass spectrum Graph of ion intensity (relative abundance) along x-axis versus mass-to-charge ratio (m/z) (units daltons, Da) along Y-axis
Mass spectrometer Molecular ion (Parent ion) the peak corresponding to the mol wt of the compound The peak of an ion formed from the original molecule by electron ionization, by the loss of an electron, or by addition or removal of an anion or cation and also known as parent peak, radical peak.
Mass spectrometer Fragmentation peaks The peaks observed by fragments of compounds. The molecular ions are energetically unstable, and some of them will break up into smaller pieces. The simplest case is that a molecular ion breaks into two parts - one of which is another positive ion, and the other is an uncharged free radical. The uncharged free radical won't produce a line on the mass spectrum. Only charged particles will be accelerated, deflected and detected by the mass spectrometer. These uncharged particles will simply get lost in the machine - eventually, they get removed by the vacuum pump.
Mass Spectrometry Base peak The most intense (tallest) peak in a mass spectrum, due to the most abundant ion. Not to be confused with molecular ion: base peaks are not always molecular ion and molecular ion are not always base peaks.
Mass Spectrometry Fragmentation Patterns By using fragmentation pattern we can easily study the structure of a compound.
Mass Spectrometry Stevenson’s Rule The most probable fragmentation is the one that leaves the positive charge on the fragment with the lowest ionization energy In other words, fragmentation processes that lead to the formation of more stable ions are favored over processes that lead to less-stable ions. Cleavages that lead to the formation of more stable carbocations are favored. When the loss of more than one possible radical is possible, a corollary to Stevenson’s Rule is that the largest alkyl radical to be lost preferentially.
Mass Spectrometry Homolytic bond cleavage A type of ion fragmentation in which a bond is broken by the transfer of one electron from the bond to the charged atom, the other electron remaining on its starting atom. The movement of one electron is signified by a fishhook arrow. The fragmentation of a ketone is shown in the figure.
Mass Spectrometry Heterolytic bond cleavage type of ion fragmentation in which a bond is broken by the transfer of a pair of electrons from the bond to the charged atom The movement of 2 electrons is signified by a double-barbed arrow and also referred to as charge-induced fragmentation.
Mass Spectrometry Alpha cleavage Alpha cleavage occurs on α-bonds adjacent to heteroatoms (N, O, and S). Charge is stabilized by heteroatom. Occurs only once in a fragmentation (cation formed is too stable to fragment further) For example in alcohols, aliphatic ethers, aromatic ethers, cyclic compounds and aromatic ketones etc.
Mass Spectrometry Beta-cleavage Fission of a bond two removed from a heteroatom or functional group, producing a radical and an ion. Also written as β-cleavage. For example allylic fragmentation.
Mass Spectrometry Inductive cleavage If an electron pair is completely transferred towards a centre of positive charge as a result of the inductive effect, shown schematically by the use of a double-headed arrow, then the ion will fragment by inductive cleavage. The figure illustrates this for a radical cation ether.
Mass Spectrometry Retro Diels-Alder Cleavage A multicentered ion fragmentation which is the reverse of the classical Diels-Alder reaction employed in organic synthesis that forms a cyclic alkene by the cycloaddition of a substituted diene and a conjugated diene. In the retro reaction, a cyclic alkene radical cation fragments to form 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 cation will dominate.
Mass Spectrometry McLafferty rearrangement An 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 a γ- hydrogen atom to the ionisation site through a ring system as shown here. The distonic radical cation so formed can break up by radical-site-induced (α), or charged site-induced fragmentation as shown in the figure. For example ketones, carboxylic acid and esters.
Mass Spectrometry Ortho effect The interaction between substituents oriented ortho, as opposed to para and meta, to each other on a ring system, can create specific fragmentation pathways. This permits the distinction between these isomeric species. The diagram shows a case in which only the ortho isomer can undergo the rearrangement.
Mass Spectrometry Onium Reaction Onium Ion: A hypervalent species containing a non-metallic element such as the methonium ion CH5+. It includes ions such as oxonium, phosphonium, and nitronium ions. Mostly observed in cationic fragments containing a heteroatom as charge carrier, e.g. oxonium, ammonium, phosphonium and sulphonium ions. The onium reaction is not limited to alkyl substituents acyl groups can also undergo the onium reaction
Mass Spectrometry CO Elimination Cyclic unsaturated carbonyl compounds and cationic carbonyl fragments which resulted from an a-cleavage tend to eliminate CO . If there is more than one CO group present sequential elimination of all CO groups is possible. From carbonyl compounds CO elimination reaction takes place like in aldehyde, ketones and phenols etc
Mass Spectrometry Rules for interpretation of mass spectrum
Mass Spectrometry DBR Calculations Double bond or ring calculations tell us about how many rings or double bonds are present in a compound. DBR= C-H/2+N/2+1 C= number of carbon atoms H= number of hydrogen atoms N= number of nitogen atoms
If a compound contains an even number of nitrogen atoms (or no nitrogen atoms), its molecular ion will appear at an even mass number.
• If, however, a compound contains an odd number of nitrogen atoms, then its molecular ion will appear at an odd mass value. • This rule is very useful for determining the nitrogen content of an unknown compound.
Mass Spectrometry Cycloalkanes Strong M+, strong base peak at M-28 (loss of ethene) A series of peaks: M-15, M-28, M-43 etc Methyl, ethyl, propyl with an additional hydrogen give peaks
Mass Spectrometry Alkenes Strong M+ Fragmentation ion has formula CnH2n+ and CnH2n-1 -Cleavage A series of peaks: M-15, M-29, M-43, M-57 etc
Mass Spectrometry Alkynes Strong M+ Strong base peak at M-1 peak due to the loss of terminal hydrogen Alpha cleavage
Mass Spectrometry Aromatic Hydrocarbons Strong M+ Loss of hydrogen gives base peak McLafferty rearrangement Formation of benzyl cation or tropylium ion
Mass Spectrometry Alcohols M+ weak or absent Loss of alkyl group via a-cleavage Dehydration (loss of water) gives peak at M-18
Mass Spectrometry Phenols Strong M+ M-1 due to hydrogen elimination M-28 due to loss of CO M-29 due to loss of HCO (formyl radical)
Mass Spectrometry Ethers M+ weak but observable Loss of alkyl radical due to a-cleavage B-cleavage( formation of carbocation fragments through loss of alkoxy radicals) C-O bond cleavage next to double bond Peaks at M-31, M-45, M-59 etc
Mass Spectrometry Aldehyde M+ weak, but observable (aliphatic) Aliphatic : M-29, M-43 etc McLafferty rearrangement is common gives the base peak A-cleavage B-cleavage
Mass Spectrometry Aldehyde M+ strong (aromatic) Aromatic: M-1 (loss of hydrogen) M-29 (loss of HCO) McLafferty rearrangement is common A-cleavage B-cleavage
Mass Spectrometry Ketones Strong M+ A series of peaks M-15, M-29, M-43 etc Loss of alkyl group attached to the carbonyl group by a-cleavage Formation of acylium ion (RCO+) McLafferty rearrangement
Mass Spectrometry Esters M+ weak but generally observable Loss of alkyl group attached to the carbonyl group by a-cleavage Formation of acylium ion (RCO+) McLafferty rearrangement Acyl portion of ester OR+ Methyl esters: M-31 due to loss of OCH3 Higher esters: M-32, M-45, M-46, M-59, M-60, M-73 etc
Mass Spectrometry Carboxylic acids Aliphatic carboxylic acids: M+ weak but observable A-cleavage on either side of C=O M-17 due to loss of OH M-45 due to loss of COOH McLafferty rearrangement gives base peak
Mass Spectrometry Aromatic carboxylic acids: M+ Strong A-cleavage on either side of C=O M-17 due to loss of OH M-18 due to loss of HOH M-45 due to loss of COOH McLafferty rearrangement gives base peak
Mass Spectrometry Amines M+ weak or absent Nitrogen rule obey A-cleavage
Mass Spectrometry Nitriles M+ weak but observable M-1 visible peak due to loss of termiminal hydrogen
Mass Spectrometry Nitro Compounds M+ seldom observed Loss of NO+ give visible peak Loss of NO2+ give peak
Mass Spectrometry Alkyl chloride and alkyl bromides Strong M+2 peak For Cl M/M+2 = 3:1 F or Br M/M+2 = 1:1 A-cleavage Loss of Cl or Br Loss of HCl or HBr
Mass Spectrometry Applications of Mass Spectrometry The technique has both quantitative and qualitative uses. These include identifying unknown compounds, determining the isotopic composition of elements in a molecule, and determining the structure of a compound by observing its fragmentation. Followings are the main applications
Mass Spectrometry Toxicity of Toothpastes DEG (diethylene glycol) which is a toxic chemical and usually present in Chinese toothpastes. Measuring nanoparticle size Mass spectrometry is used to measure nanoparticle size like platinum nanoparticles which is used as catalyst. Once size of a sphere is measured, its density is also calculated. 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.
Mass Spectrometry Protein characterization Mass spectrometry is an important emerging method for the characterization of proteins. The two primary methods for ionization of whole proteins are electrospray ionization (ESI) and (MALDI). Space exploration Mass spectrometers are also widely used in space missions to measure the composition of plasmas. For example, the Cassini spacecraft carries the Cassini Plasma Spectrometer (CAPS), which measures the mass of ions in Saturn's magnetosphere. Isotope dating and tracking Mass spectrometry is also used to determine the isotopic composition of elements within a sample. Differences in mass among isotopes of an element are very small, and the less abundant isotopes of an element are typically very rare, so a very sensitive instrument is required. These instruments, sometimes referred to as isotope ratio mass spectrometers (IR-MS).
Mass Spectrometry Molecular weight Molecular weight can be determined by mass spectrometry. Actual number of carbons, hydrogen, oxygen etc By using relative intensities(peak hight), we can easily calculated the actual numbers of C,H,O etc atoms. Bonding Bonding can be studied by fragmentation patterns for example, beta cleavage is possible only if double bonds or heteroatom present. Reaction mechanism Mass spectrometry is best technique to study reaction mechanism and intermediates produced in reaction, for example, in carboxylic acid and alcohols a peak at M-18 indicates that water is produced.
Mass Spectrometry Determination of Elements Bulk materials such as steel or refractory metals, elements are determined by low-resolution glow-discharge mass spectrometry. High-resolution GDMS has been used to study semiconductor materials. GDMS is considered virtually free of matrix effects. Detection limits in ICPMS as in Table
Mass Spectrometry Species Analysis Heavy metals in the environment are stored in complexes with humic acids, can be converted by microbes in different complexes, and can be transported in live animals and humans. This applies to many elements such as lead, mercury, arsenic, astatine, tin and platinum For example, tin and lead alkylates established in soil, water or muscle tissue by GC / MS after exhaustive alkylation or thermal spray, and ICP-LC/MS API methods.
Mass Spectrometry Environmental Chemistry the analysis of trace elements and compounds in environmental samples like air, water, soil etc because of its detection power, specificity and structural analysis functions Generally, sample preparation is at least one type of chromatography coupled with MS either offline or online like GCMS