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1 Mass Spectrometry & Applications Ashraf M. Mahmoud, Associate Professor Contents I. Introduction: (Basics of Mass Spectrometry) II. 1. 2. 3. 4. 5. Ionization step: Electron Ionization Chemical Ionization Photoionization Matrix-assisted Laser Desorption/Ionization Electrospray Ionization III. 1. 2. 3. 4. Mass Analysis (Separation): Magnetic Sector Quadrupole Ion Trap Time-of-flight IV. Molecular fragmentation pattern V. Major Applications of Mass Spectrometry Theory of Mass Spectrometry Schematic of Mass Spectrometry Ionizer ↓ Mass-to-charge ratio Analyzer ↓ Detector channeltron, electromultiplier and microchannel plates. Theory of Mass Spectrometry 2 In a mass spectrometer, molecules in the gaseous state under low pressure (high vacuum) are bombarded with a beam of high energy electrons (70 eV) This bombardment can first dislodge one of the electrons of the molecule and produce a positively charged ion called “The molecular ion”→ ionization step M + Molecule e’ high-energy → M+• + 2 e’ molecular ion The molecular ion contains also an odd number of electrons, thus it is a Free radical, or generally “Radical Cation” An electron beam with an energy of about 70 eV not only produces molecular ions but also give to the molecular ions considerable amount of surplus energy which is quite sufficient to break any covalent bond in the molecular ion (4-7 eV) Thus soon after they are formed, most molecular ions undergo fragmentation. Fragmentation can take place in a variety of ways depending on the nature of the molecular ion. Ionization step Different types of systems are commonly used to ionize the Molecule: 1. Electron Ionization 2. Chemical Ionization 3. Photoionization 4. Matrix-assisted Laser Desorption/Ionization 5. Electrospray Ionization Ion separator systems Different types of systems are commonly used to separate ions on the basis of their m/z ratio → Ion separation step such as: 1. Magnetic sector instrument Advantages : a. High accuracy c. Resolve ions to a level of 0.0001 amu or more Disadvantage : a. Its Lower sensitivity b. Narrow range of kinetic energy 2. Quadrupole instrument It uses two electric fields applied at right angles to each other, rather than a magnetic field, to separate ions according to their m/z ratios, one of them is oscillating to create a resonance frequency for each m/z value. The ions which resonate at the frequency of the quadrupole are able to pass through it and be detected Ion separator systems (cont.) Advantage : a. Its higher sensitivity b. Wider range of kinetic energy c. More cheaper than magnetic sector Disadvantages : a. Low accuracy b. It can not resolve ions not more than 0.1 amu 3. Ion trap instrument 4. Time-of-flight instrument MS can give us highly useful information about the structure of a complex molecule. MS, then sorts the produced cations on the basis of their m/z ratios. Since for all the practical purposes, the charge on all of the ions = +1, thus the sorting of them actually on the basis of their masses. Diagram for Mass spectrometer electrons generator that give the high energy electrons beam Ionization region Heater to generate the gas beam of molecules Detector Mass Spectrum Mass Spectrum: 1. The mass spectrum—is a graph of relative abundance of each fragment (% Intensity) plotted against its m/z value. 2. Because the charge (z) = +1 for all fragments that reach the collector plate so the m/z value express molecular mass (m) of fragments. 3. Remember that only positively charged species reach the collector. 4. The mass spectrum is usually published either as a bar graph or in table form Note that: 1. Peak with the highest m/z value in the spectrum is due to the fragment that results when an electron is knocked out of a molecule of the injected sample. i.e. represents the molecular ion (M)+ of the studied compound. Thus m/z value of molecular ion gives the molecular mass of compound. Molecular, Base & Fragment ion peaks 2. Since it is not known what bond loses the electron, the molecular ion is put in brackets and the positive charge and unpaired electron are assigned to the entire structure. 3. Peaks with smaller m/z values—called fragment ion peaks— represent positively charged fragments of the molecule. 4. “Base peak” is the peak with the greatest intensity, due to its having the greatest relative abundance. The base peak is assigned a relative intensity of 100%, and the relative intensity of each of the other peaks is reported as a percentage of the base peak. 5. The mass spectrum gives us structural information about the compound because the m/z values and the relative abundances of the fragments depend on the strength of the molecular ion’s bonds and the stability of the fragments. Example Mass spectrum of pentane, shown as a bar graph and in tabular form. Base peak represents fragment that appears in greatest abundance. Value of molecular ion gives molecular mass of the compound. Note that: The way by which the molecular ion to be fragmented depends on the strength of its bonds and stability of the fragments. Note that: 1- In some cases the base peak is the molecular ion peak, but in most cases it is different from that of the molecular ion (according to the relative stability of ions during fragmentation). 2- Peaks are commonly observed at m/z values one and two units less than the m/z values of the carbocations because the carbocations can undergo further fragmentation by losing one or two hydrogen atoms. 3- Small peak that occurs at m/z 73 (0.52%) is known as M+1 peak to indicate that “It is one mass unit greater than the molecular ion(M+.), The M+1 peak appear because most of elements have more than one naturally occurring isotope. Relative abundances of different isotopes Principal stable isotops of common elements: From the table : •For C, H, N and Si the principal heavier isotope is M+1. •For O, Si, S, Cl and Br , the principal heavier isotope is the M+2 •For F and I, there is no heavier isotopes (not affect M+1 or M+2) Some guides to determine molecular formula of organic compounds: Nitrogen rule: If molecular ion (M+) is even number, compound must contain an even number of N atoms (zero is considered as even number) • Number of Carbon atoms: Relative abundance of M+1 peak can be used for determination of number of carbons assuming that silicon and large number of nitrogens (more than 3 N) are not present. No of Carbons = Relative abundance of M+1/ 1.10 • Relative abundance of M+2 peak indicates the presence (or absence) of Oxygen (0.2), Silicon (3.35),Sulfur (4.4), Chlorine (32.5) & Bromine (98.0) • Molecular formula after this can be established by adding the suitable number of hydrogens and oxygens if necessary. • The index of hydrogen deficiency (Due to double and triple bonds and cyclization) can be calculated for the compounds containing C, H, N, O, S & halogens from the formula: • Index = Carbons - Hydrogens/2 – Halogens/2 + Nitrogens/2 +1 •Divalent atoms like O and S are not counted in formula. The index mainly used to indicate the number of double bonds. Examples: Determine the molecular formula for the following compounds M/Z % 12 Note M/Z % 2.6 16 13 8.6 14 Note M/Z % 0.9 14 17 21.1 17.1 18 100 15 85.6 19 16 100 M+ 20 17 1.15 M+1 Note M/Z % Note 17.2 73 0.56 M+1 15 100 72 18.56 M+ M+ 19 2.0 71 4.32 0.06 M+1 31 10.4 57 11.20 0.2 M+2 33 89.4 43 100.0 34 95.4 M+ 29 26.65 35 1.1 M+1 15 4.22 GC-MS 1. Constructed By interfacing the GC to the MS by using jet separator 2. Different types of ionization were used: a. Electron impact b. Positive ion chemical ionization (reagent gas is used, methan, isobutane, or ammonia; gas interacts with electrons to produce positive ions which can either associate with the analyte or transfer a proton to the analyte c. Negative ion chemical ionization LC-MS 1. Constructed By interfacing the LC to the MS by using different types of interfaces a. Particle beam b. Thermospray (reagent gas is used, methan, isobutane, or ammonia; gas interacts with electrons to produce positive ions which can either associate with the analyte or transfer a proton to the analyte c. Fast atom bombardment d. Electrospray ionization e. Atmospheric pressure ionization Major Applications of Mass Spectrometry 1. MS provides a highly specific method for determining or confirming the structure or the identity of drugs and raw materials. 2. MS in conjunction with either gas chromatography (GC-MS) or liquid chromatography (LC-MS) provides a method for characterizing the impurities. 3. GC-MS and LC-MS provides a highly sensitive and specific method for determining drugs and their metabolites in biological fluids and tissues. 4. MS has become an important tool in proteomics, which is currently the major tool in drug discovery 5. MS using electrospray ionization and time of flight separation will be of major use in quality control of therapeutic antibodies and protein. 6. It is the best method for getting rapid identification of trace impurities. Limitations of Mass Spectroscopy 1. It is not currently used in routine quality control due to complexity of instrumentation 2. Expensive instrumentation 3. requires a high degree of skill 4. Regular maintenance Examples The IR and mass spectra for a compound is shown in the following Figure, Identify the compound If you know that M+ at m/e 100 (20%), M+1 at 101(1.32%) and M+2 at 102 (0.04%) . Operation of Mass Spectrometry •This operation begins by producing the gas beam of molecules, bombardment with a beam of high energy electrons and accelerating the ions beam through its pass through the curved tube. •This curved tube (Analyzer tube) passes through a variable magnetic field and the magnetic field exerts an influence on the moving ions deflecting the positively charged fragments in a curved path. •At a given magnetic field strength, the degree to which the path is curved depends on the mass-to-charge ratio (m/z) of the fragment. •Path of a fragment with a smaller m/z value will bend more than that of a heavier fragment. In this way, the particles with the same m/z values can be separated from all the others. •If a fragment’s path matches the curvature of analyzer tube, the fragment will pass through the tube and out the ion exit slit (These ions are said to be in register). Operation of Mass Spectrometry (cont.) •A collector records the relative number of fragments with a particular m/z passing through the slit. The more stable the fragment, the more likely it will make it to the collector. •Strength of magnetic field is gradually increased, so fragments with progressively larger m/z values are guided through tube and out the exit slit. Note that: •Actual sorting of ions takes place first in the gradually increased magnetic field •Ion-sorting can also be done with “Electrical Focusing”, in this technique the magnetic field is held constant and the accelerating voltage is varied. It gives the same sorting results. •In the high-resolution mass spectrometers both techniques are used to increase the system sensitivity. Example: If the molecule under bombardment is a molecule of ammonia, the following reaction will take place: H:N:H H H:N:H H + H:N:H + H Molecular ion of ammonia e' H:N:H + H H:N: 2 e' + H :N: + H Molecular fragmentation pattern 1. Homolytic and heterolytic α-cleavage - under electron impact ionization condition, the analyte developes a +ve charge through loss of one electron. - If there is an electronegative atom such as oxygen, sulphur or nitrogen, this +ve charge will be located on the electronegative atom. - If electronegative atom is absent, the charge is more difficult to locate with certainity. a. Homolytic α-cleavage - is promoted by the presence of hetero-atom - it often gives rise to the most abundant ion peak (base peak) - The hetero-atom becomes +vely charged - it favors the loss of the largest possible radical - This type of fragmentation dominates the mass spectra of many drugs (major pathway) b. Heterolytic α-cleavage - is promoted by the presence of hetero-atom - The hetero-atom is not the+vely charged ion - it does not favor the loss of the largest possible radical - This type of fragmentation is a minor pathway 2. Cleavage with proton transfer - is also common pathway in the mass spectra Molecular fragmentation pattern (cont.) 3. McLafferty rearrangement - is not particularly common pathway in mass spectra of drugs - occurs in carboxylic acids, esters, ketones and amides which have side chain of at least 3 carbon atom - associated with elimination of small, stable, neutral molecules such as carbon monoxide, olefines, water, ammonia, - it characterise the long chain lipid molecules -To undergo a McLafferty rearrangement, a molecule must possess an appropriately located heteroatom , a p-system (usually a double bond), and an abstractable hydrogen atom g to the C = O system. Examples The IR and mass spectra for a compound is shown in the following Figure, Identify the compound If you know that M+ at m/e 162 (100%), M+1 at 163(6.62%) and M+2 at 164 (98.0%) . Examples The IR and mass spectra for a compound is shown in the following Figure, Identify the compound If you know that M+ at m/e 72 (50%), M+1 at 73(2.3%) and M+2 at 74 (0.1%) . Ashraf M Mahmoud