Mass Spectrometry Introduction EI-MS and CI-MS Molecular mass & formulas Principles of fragmentation Fragmentation patterns Isotopic effects 1
Introduction to MS Mass spectrometry is the method of analysis that involves interaction of sample molecule with fast moving electrons. Structural information obtained usually based on fragmentation patterns of the molecule under study. The sample studied may be gas, liquid or solid. The information obtained is a molecular mass fragment which does not depend on EM radiations. There are two methods that are involved in molecule ionization; Electron ionization and Chemical Ionization. Other mass spectrometry techniques include fast atom bombardment (FAB), Matrix-assisted laser desorption ionization (MALDI), Electrospray ionization (ESI). 2
Introduction Molecules are subjected to bombardment by stream of high energy electrons (ca. 70eV) converting some of the molecule to ions. Accelerated ions are separated according to their mass charge ratio (m/e or m/z) in magnetic field or electric field. The ions with particular m/e that strike a detector are counted. The amplified output is recorded as mass spectrum. 3
rel. abundance Mass spectrum Mass spectrum is a graph of particles detected as function of m/e 100 M + M 0 m/e A Typical MS spectrum showing molecular ion peak (M. + ) and the base peak (M + ). 4
Mass spec. The base peak is the most abundant peak assigned an arbitrary intensity of 100. The relative abundance of all other ions is reported as a % of abundance of the base peak. Molecular ion peak is the peak with highest m/z which corresponds to mass of the compound 5
Schematic representation of Mass spectrometer 6
Electron Ionization mass spectrometry (EI-MS) Three distinct regions of MS machine where molecule under investigation Ionization chamber, Ion analyzer and Detector For EI-MS, Gas-phase molecules enter source through heated probe. 70 ev electrons bombard molecules forming M + ions that fragment in unique reproducible way to form a collection of fragment ions. i.e M + ē [M] + + 2ē A beam of high-energy electrons emitted from a very hot filament (thousand degrees Celcius) strike the stream of molecules and ionize it. 7
Representation of ionizing chamber 8
Note: The only detectable fragments are positively charged only. Neutral and unpaired electrons without positive charge will NOT be detected. A B + A + (positive charged fragment) + B (neutral/radical fragment) 9
Ion (mass) analyzer From ionization chamber the ions passes through the field freeregion and enter to the mass analyzer where the ions are separated according to their mass-to-charge ratios. Governed by the equation; m/e = H 2 r 2 /2V. This is the combination of two equations one expressing the kinetic energy of the accelerate electron mv 2 /2 = ev and another equation is due to the path of electron in the presence of magnetic field (H) with radius of curvature r = mv/eh. Where e is the charged ion and V is potential difference of ionaccelerating plate. 10
The detector A typical spectrometer consists of a counter that produces a current that is proportional to the number of ions that strike it. The current caused by just one ion striking the detector can be measured so accurately though the use of electron multiplier circuits. Thereafter the signal from the detector is fed to a recorder which produces the mass spectrum. Modern instruments produces data in different formats tabulated and bar graph of relative intensity versus m/z. Generally, a mass spectrometer is designed to do three things: Convert neutral atoms or molecules into a beam of positive (or rarely negative) ions. Separate the ions on the basis of their mass-to-charge (m/z) ratio. Measure the relative abundance of each ion. 11
Chemical Ionization (CI) CI is the ionization that occurs through a reaction between the sample molecule and the reagent gas (usually methane, isobutene or ammonia). Gas is reacted (i.e it is ionized) by a stream of electrons before it react with the sample to form positive charge ions that acts like Lewis acid. Only molecular ion peak is observed. The spectrum of CI-MS have m/z value corresponding to M+H a unit greater than M +. (molecular ion). 12
CH 3 OH EI- ionization e CH 3 OH m/z = 32 CH 3 + OH m/z = 15 Chemical ionization CH 4 + e reagent CH 4 + 2e CH 3 OH m/z = 31 + H primary ion CH 4 excess CHO + 2H m/z = 29 CH 5 secondary ion + CH3 Sample M M+H + CH 4 pseudo molecular ion 13
Difference between EI-MS and CI-MS EI -MS CI-MS Gas-phase molecules enter source through heated probe or GC column 70 ev electrons bombard molecules forming M. + ions that fragment in unique reproducible way to form a collection of fragment ions EI spectra can be matched to library standards No ions higher m/z than M. + Smaller M. + intensity Rich family of fragment ions Higher pressure of methane or amonia leaked into the source (mtorr) Reagent ions transfer proton to analyte, (a much lower energy process) Adduct ions higher m/z than MH +,,[M+C 2 H 5 ] +,[M+C 3 H 5 ] + [M+NH 4 ] + Large intensity of M. + Relatively few fragment ions 14
A partial mass spectrum of dopamine showing all peaks with intensity equal to or greater than 0.5% of the base peak 15
Determination of Molecular Mass and Formulas Molecular ion peaks Nitrogen rule Rule of thirteen 16
Determination of molecular mass& formulas The value of the mass of any ion accelerated in a mass spectrometer is its true mass and NOT its molecular weight obtained from the use of chemical atomic weights The masses that are observed for molecular ions are the masses of the molecules in which every atom is present as its most common isotopes An error of only one mass unity in the assignment off mass spectral peaks can lead to difficulty in determination of structure. Varying (lowering) the energy of the ionizing electron beam may confirm that particular molecular ion peak due to increased intensity as tendency of molecular ion to fragment lessens. 17
Nitrogen Rule The rule states that, If a compound has zero or an even number of nitrogen atoms, its molecular ion will have an even m/z value. An odd number of nitrogen atoms, its molecular ion will have an odd m/z value. Reason: N atom has an even mass, has an odd-numbered valence. Consequently, an extra hydrogen atom is included as a part of the molecule, giving it an odd mass. For example; Ethylamine C 2 H 5 NH 2 has one nitrogen atom, and its mass is an odd number, 45 whereas Ethylenediamine, H 2 NCH 2 CH 2 NH 2 has two nitrogen atoms and its mass is an even number, 60. A molecule free of nitrogen and gives an ion at m/z=201, then that peak cannot be the molecular ion. An odd molecular ion is an indication of the presence of nitrogen. Note: In the case of Chemical Ionization, where [M+H] + is observed, need to subtract 1, then apply nitrogen rule 18
Facts about molecular ion peaks The peaks must correspond to the ion of highest mass in the spectrum, excluding the isotopic peaks that occur even at higher masses. The ion must have an odd number of electrons (the charge of an ion is one) They must be able to form relatively stable fragments with high mass by loss of logical neutral fragments. Special attention in assigning M.+ must be taken when dealing with Chlorine and bromine due to isotopic effects Alcohols due to loss of water (m/z = 18) Conjugate acids of oxygen and nitrogen due its stability. 19
Determination of Molecular Formula (Precise mass determination) Resolution: Resolution is a measure of how well a mass spectrometer separates ions of different mass. i.e ability to distinguish the nearest peaks Low resolution: Refers to instruments capable of separating only ions that differ in nominal mass i.e ions that differ by at least 1 or more atomic mass units (amu). High resolution: Refers to instruments capable of separating ions that differ in mass by as little as 0.0001 amu 20
Examples C 3 H 6 O and C 3 H 8 O have nominal masses of 58 and 60, and can be distinguished by low-resolution MS. C 3 H 8 O and C 2 H 4 O 2 both have nominal masses of 60 can be distinguish by high-resolution MS, that is; C 3 H 8 O = 60.05754, whereas C 2 H 4 O 2 = 60.02112. R = 20,000 (which is 5 to 10 times Low -resolution MS) can distinguish these ions. Resolution, R is calculated using the formula bellow in considering; Two adjacent peaks of equal intensity The valley between them being < 10 % i.e R = Mn/ Mn-Mm Where Mn is higher mass number and Mm is low mass number. High-resolution MS provides molecular mass information from which the user can determine the exact molecular formula directly. 21
Rule of Thirteen Rule of Thirteen may be used to generate possible molecular formulas for a given mass when molar mass information is missing Rule of thirteen is expressed as; where M is molecular mass, n is a numerator and r is the remainder, thus the formula becomes C n H n+r. whose unsaturation index may be calculated from the formula U = (n - r + 2)/2. 22
For example You are given a molecular mass 94. Then, apply Rule of 13 U = (n - r + 2)/2. U = (7-3 + 2)/2= 3; Molecular formula C n H n+r. = C 7 H 10 The structure that fits this information could be: CH 3 23
Fragmentation of molecules For EI-MS the fragmentation occur and only stable cation or cation-radical will be detected The fragmentation involves bond cleaving, ether by homolytic cleavage or heterolytic cleavage. 24
Typical Homolytic and Heterolystic cleavage H Homolytic H 3 C C OCH 3 CH 3 H 2 C OCH cleavage 3 H cationic radical + H 2 C OCH 3 H 3 C H C Br Heterolytic cleavage H H 3 C C + Br CH 3 cationic radical CH 3 25
Fragmentation of saturated hydrocarbons Cleavage occur at the branched carbon atom to form a stable carbocation. Peak M-15 (loss of CH 3 ) usually observed, followed by loss of CH 2 units (i.e fourteen mass unit). Fragmentation of unsaturated hydrocarbons Terminal alkene tend to cleave at the second bond from the double bond to give an alkyl carbocation of m/z = 41 stabilized by delocalization to the double bond (i.e cleavage occur at a and b positions). 26
McLafferty rearrangement For the alkyl group with more carbon long a rearrangement called McLafferty rearrangement can occur. By definition McLafferty rearrangement is the electron impact induced cleavage of carbonyl compounds having hydrogen in the -position, to an enolic fragment and olefin e.g ketones and aldehydes. O H R' OH R' + R MacLafferty rearrangement R enol 27
Fragmentation patterns Alkanes, Alkenes, Cyclohexenes Alcohols and Cycloalcohols Carboxylic acids, aldehydes Esters, ethers Aromatic rings 28
Fragmentation patterns of alkanes Tends to occur in the middle of unbranched chains rather than at the ends. The difference in energy among allylic, benzylic, 3, 2, 1, and methyl cations is much greater than the difference among comparable radicals. Where alternative modes of fragmentation are possible, the more stable carbocation tends to form in preference to the more stable radical. Characteristically, strong M, loss of 14 units in series M-14, M-28, M-42, etc. 29
Fragmentation patterns of alkanes 30
cycloalkanes 31
cycloalkanes 32
Fragmentation patterns of alkenes Alkenes characteristically, show a strong molecular ion peak cleave to form resonancestabilized allylic cations. strong M, m/z = 41, M-15, M-29, M-43, M-57, etc. Resulting fragmentation ions have formula corresponding to C n H 2n+ or C n H n-1 + 33
Fragmentation patterns of alkenes CH 2 =CHCH 2 CH 2 CH 3 CH 2 =CHCH 2 + CH 3 CH 2 34
Fragmentation patterns of cyclohexenes Cyclohexenes give a 1,3-diene and an alkene, a process that is the reverse of a Diels-Alder reaction CH 3 CH 3 + H 3 C CH 2 H 3 C CH 2 Lemonene m/z = 136 A neutral diene A radical cation m/z = 68 m/z = 68 35
cyclohexene 36
Fragmentation patterns of alcohol One of the most common fragmentation patterns of alcohols is loss of H 2 O to give a peak which corresponds to M-18. Characteristically, weak or absent M, M-18 (loss of alkyl group) Cyclic alcohols may undergo dehydration in three different ways Dehydration may occur in two mechanisms for acyclic alcohols; 1,2-elimination (before the molecule it come in contact with ionizing electros) or 1,4-elimination (from the molecular ion) a cyclic mechanism. 37
1,4-Elimination H H 2 C H 2 C O C H 2 H CH R H 2 C H C dehydartion by 1,4-elimination R + H 2 O + H 2 C CH 2 1,2-Elimination RHC (CH 2 ) n H H-O CHR' (CH 2)n RHC CHR' + H 2 O n = 1 or 2 dehydartion by 1,2-elimination 38
Alcohol. Another common pattern is loss of an alkyl group from the carbon bearing the -OH group to give a resonance-stabilized oxonium ion and an alkyl radical. R R' C OH R' R + C OH R'' Loss of alkyl group R'' 39
2 o alcohol Loss of ethyl group 40
Cyclic alcohols 41
Cyclic alcohol 42
Ketones Similar to aldehydes except for cyclic ketones Characteristically: 1) strong M, 2) Aliphatic: M-15, M-29, M-43, m/z = 43, m/z = 58, 72 86, 1) Aromatic: m/e= 105, 120 43
Ketone cleavage 44
McLafferty rearrangement 45
Carboxylic acids Characteristic fragmentation patterns are a- cleavage on either side of a carbonyl to give the ion [CO 2 H] + with m/z 45 or loss of OH group McLafferty rearrangement is possible with acids containing -hydrogens. Characteristically: Aliphatic carboxylic acids weak M, M-17, M-45, m/z = 45, 60; Aromatic carboxylic acids strong M, M-17, M-45; M-18 46
Esters -cleavage and McLafferty rearrangement Characteristically: weak M ; Methyl esters: M-31, m/z = 59,74; Higher esters M-45, M-59, M-73; m/z= 73,87,101, m/z= 88, 102, 116;m/z= 61, 75, 86; m/z = 77, 105. 108; M-32, M-46, M-60 47
Ethers Aliphatic ethers have stronger molecular ion peaks compared to that of alcohols. There three principal modes of fragmentations; -cleavage 2) formation of carbocation fragments, and 3) loss of alkoxy group 48
Ethers... -cleavage (responsible for m/z = 87 of diisopropyl ether) R CH 2 OR H 2 C OR + R Formation of carbocation fragment (m/z = 43 for diisopropyl ether) H 3 C CH 3 H 3 C CH 3 CH O CH CH O + HC H 3 C CH 3 H 3 C CH 3 49
Ethers. Rearrangement reaction taking place on one of the fragmentations, rather than on the molecular ion. Particularly favoured when -carbon is branched (m/z= 45) H R CH O CH 2 CH 2 + R CH OH HC CH 2 R R 50
Phenols Loss of CO to form M-28 peak, and loss of HCO to from M-29 peak Example 2-methylphenol Characteristically: strong M, M-1, M-28, M-29 Aliphatic amines The most characteristic fragmentation pattern of 1, 2, and 3 aliphatic amines is b-cleavage. a- cleavage is also common to 1 amine and m/z = 30 51
Isotopic effect M+1, M+2 & M+4 peaks 52
Example of isotopes and their relative abundance Element Isotope/Relative abundance Hydrogen 1 H 100 2 H 0.018 Carbon 12 C 100 13 C 1.11 Nitrogen 14 N 100 15 N 0.38 Oxygen 16 O 100 17 O 0.04 Sulphur 32 S 100 33 S 0.78 Chlorine 35 Cl 100 37 Cl 32.5 bromine 79 Br 100 79 Br 98.0 53
M+1 Carbon in nature, 12 C (98.90%) and 13 C (1.10% ) Thus, there are 1.11 atoms of carbon-13 in nature for every 100 atoms of carbon-12 54
Question What is the maximum number of carbons of unknown substance can have if the relative abundance of the parent peak is 40 and the M+1 peak is 1.4? Solution 55
M+2 Chlorine and bromine are common elements that give rise to M+2 peaks In nature, chlorine 35 Cl is 75.77% and 37 Cl is 24.23%. A ratio of M to M+2 is approximately 3:1 indicates the presence of a single chlorine in a compoun Bromine in nature 79 Br is 50.7 % and 81 Br is 49.3%. A ratio of M to M+2 of approximately 1:1 indicates the presence of single bromine in compound. 56
One Chloride will contain a M+2 peak approximately 1/3 the intensity of the molecular ion peak because of the presence of 37 Cl. e.g chloroethane -35 57
One Bromide will contain a M+2 peak approximately equal the intensity of the molecular ion peak because of the presence of 81 Br. (e.g bromoethane) -79 58
Note for M+2 and M+4 of 1 or 2 isotopic elements One atom One Chloride will contain a M+2 peak approximately 1/3 the intensity of the molecular ion peak because of the presence of 37 Cl. Two atoms Two chlorides will contain a M+2 peak approximately 1/3 the intensity of the molecular ion peak and M+4 peak about 1/3 of the M+2 peak One Br will contain a M+2 peak approximately equal the intensity of the molecular ion peak because of the presence of 81 Br. Two Bromides will contain M+2 peak approximately twice intensity of the molecular ion peak and M+4 peak about equal to the molecular ion peak. 59