Molecular Analytics Premaster L3 Mass Spectrometry PDF

Molecular Analytics Premaster L3 Mass spectrometry Jesper C. Ruiter Sectie BioAnalytische Chemie Vrije Universiteit Amsterdam [email protected] Friday 22 September 2023 Previously on MAP • What is a molecular vibration? • How do you determine the number of vibrational modes of a (non)-linear mo...

Molecular Analytics Premaster L3 Mass spectrometry Jesper C. Ruiter Sectie BioAnalytische Chemie Vrije Universiteit Amsterdam [email protected] Friday 22 September 2023 Previously on MAP • What is a molecular vibration? • How do you determine the number of vibrational modes of a (non)-linear molecule? • When do molecules absorb IR radiation? • How do you calculate the vibrational frequency of, for example, C=O? • Can you identify the groups discussed in this lecture in IR spectra? 2 Where are we? L1 L4 L5 L2 T2 T1 L3 3 Check-in 4 Learning objectives of today • Become familiar with terms such as: molecular mass, mass number, nominal mass and exact mass • Understanding what m/z is • Knowing what a molecular ion and base peak are • Understanding what mass resolution and mass accuracy are, what the relation between these two is and how to calculate both • Knowing what a mass spectrometer is • Knowing what a mass spectrum is • Understanding how we can ionise our analytes in EI • Understanding how heterolytic and homolytic cleavage reactions work • Understanding how McLafferty rearrangement works • Understanding what a mass analyzer is and how the TOF works • Understanding how we can detect the formed and separated ions 5 From the top: What is mass spectrometry? An analytical chemistry method that can identify the amount and type of chemicals present in a sample by measuring the m/z value and abundance of gas-phase ions Mass spectrometry (MS) is applied in all disciplines of science: -Chemistry -Physics -Biology -Medicine -Environmental science -Forensic science -And many more 6 MS: Chemistry • Assignment: synthesize paracetamol How sure are you that you have made paracetamol? • • MS can help with identifying your compound MS can also be used to elucidate the structures of new compounds 7 MS: Mass, mass and again mass (1/5) • • In MS we measure Molecular mass (atomic mass) • Molecular weight (MW) • The unit of atomic mass is Dalton (Da) 8 • Per definition: 1 Da = 1/12 of the mass of • Mass • 1 Da = 1 atomic mass unit (amu) = 1.660538921·10-27 kg 12C 12C O = 12.000000 Da 16O Mass number • Sum of the numbers of protons and neutrons of the elements in a compound • Mass number is unique for each isotope • How to write?: carbon-12 or atomic number = Number of protons mass number = Number of protons + Number of neutrons 12C 8 MS: Mass, mass and again mass (2/5) • Isotope: atoms of the same chemical element (so the same number of protons) where the number of neutrons in the nucleus are different mass number (8p + 8n) 16O mass number (8p + 9n) 17O mass number (8p + 10n) 18O Nominal mass of 16O = 16 Nominal mass of 17O = 17 Nominal mass of 18O = 18 Exact mass of 16O = 15.9949146 Da Exact mass of 17O = 16.99913 Da Exact mass of 18O = 17.99916 Da Natural abundance of 16O = 99.757% of total O Natural abundance of 17O = 0.038% of total O Natural abundance of 18O = 0.205% of total O • Average atomic mass = weighted average of the exact isotopic masses • Example: average mass of O = (0.99757·15.99491) + (0.00038·16.99913) + (0.00205·17.99916) = 15.99940 Da 9 MS: Isotopes Element Isotope (mass number) Exact mass (Da) Natural abundance (%) H 1 1.00782504 99.988 2 2.01410179 0.012 12 12.000000 98.93 13 13.003354 1.07 14 14.003074 99.632 15 15.00011 0.368 16 15.9949146 99.757 17 16.9991306 0.038 18 17.9991594 0.205 35 34.968854 75.78 37 36.965896 24.22 79 78.91834 50.69 81 80.91629 49.31 32 31.972074 94.93 33 32.97146 0.76 34 33.96787 4.29 36 35.96708 0.02 C N O Cl Br S Average mass 1.00794 12.0107 14.0067 15.9994 35.453 79.904 32.065 10 MS: Chemistry – structural analysis (1/4) • Determination of the elemental composition based upon accurate mass measurements and relative abundance of stable isotopes H, 2H C, 13C O, 18O S, 33S, 34S 35Cl, 37Cl Example: 1H 16O 2 The exact mass of water containing two 1H atoms and one 16O atom (1H216O) is (1.0078 + 1.0078 + 15.9949) Da = 18.0105 Da 11 MS: Chemistry – structural analysis (2/4) • Elemental composition (from exact mass measurements and stable isotopes) C10H15NO (exact mass: 165.115464 Da) • Prescence and position of functional groups -OH, SH, -NH2, -OR, -SR, NR2, COR, - CO2R, aryl groups, amino acids etc. Mass spectrometry: information about composition and functional groups 12 MS: Chemistry – structural analysis (3/4) • Bond connectivity in (bio)chemical compounds (exact mass, stable isotopes and fragmentation reactions): • Connectivity C10H15NO • Stereochemistry (cis-trans isomers) Mass spectrometry: information about connectivity and cis-trans isomers 13 MS: Chemistry – structural analysis (4/4) • Bond connectivity in (bio)chemical compounds (exact mass, stable isotopes and fragmentation reactions): • Enantiomers? Mass spectrometry: information about connectivity. MS is not a chiral method, meaning that enantiomers give the same mass spectra 14 MS: Mass, mass and again mass (3/5) • Nominal mass = sum of integer masses of the most abundant isotope of each element in the compound • For example: 12C has a natural abundance of 98.93% and 13C 1.07% → nominal mass of carbon is 12 Da • Exact mass = calculated mass of an ion or molecule with specified isotopic compositions • For example: the exact mass of water (H2O) consisting of twice 1H and one 16O, so 1.0078 + 1.0078 + 15.9949 = 18.0105 Da • The exact mass of D2O Consisting of twice 2H and one 16O, so 2.0141 + 2.0141 + 15.9949 = 20.0231 Da • When an exact mass value is given without specification of the isotopes? → refers to the most abundant isotope • MS measures exact molecular mass 15 Exact atomic mass ≠ nominal mass due to the mass defect MS: Mass, mass and again mass (4/5) • All together now: Compound Nominal mass Average mass Exact mass Most abundant isotope composition Carbon monoxide (CO) 28 28.0101 27.9949 12C16O Nitrogen (g) (N2) 28 28.0134 28.0061 14N Ethene (C2H4) 28 28.0532 28.0313 12C 1H 2 4 2 16 MS: Mass, mass and again mass (5/5) M+ Exact mass Mass defect = the difference between the nominal mass and the exact mass [M+1]+ m/z 17 Example: Insulin human insulin (C257H383N65O77S6) MS: Mass, mass and again mass (5/5) M+ Exact mass Mass defect = the difference between the nominal mass and the exact mass [M+1]+ m/z 19 m/z ratio In MS m/z is measured • Definition of m/z: • m is the number of amu’s (atomic mass unit) • z is the number of elemental charges (z = 1, 2, 3…) • m/z is a dimensionless quantity • Write m/z 100 (not m/z = 100) • 1 amu is the same as 1 Da = 1.660538921·10-27 kg 20 Molecular ion peak • Molecular ion: the radical cation formed by the loss of an electron from the neutral molecule M + e- → M•+ + 2e- • The molecular ion has a weight that is the actual molecular weight of the original molecule • The molecular ion is usually represented by M+ (of M•+) The molecular ion peak is the peak in the mass spectrum with the highest m/z-value 21 Molecular ion peak • Molecular ion: the radical cation formed by the loss of an electron from the neutral molecule M + e- → M•+ + 2e- • The molecular ion has a weight that is the actual molecular weight of the original molecule • The molecular ion is usually represented by M+ (of M•+) The molecular ion peak is the peak in the mass spectrum with the highest m/z-value 22 Isotopes! M+ m/z 194 12C 1H 14N 16O + 8 10 4 2 Are there other isotopes that could have caused this peak? Exact mass 12C 13C1H 14N 16O + 7 10 4 2 exact mass: 195.0837 [M+1]+ m/z 195 m/z 23 Isotopes! M+ m/z 194 12C 1H 14N 16O + 8 10 4 2 Possible ions Exact mass 12C 13 C1H 14N 16O + 7 10 4 2 12C 1H 14N 15N 16O + 8 10 3 4 2 12C 1H 2H14N 16O + 8 9 4 2 12C 1H 14N 16O17O+ 8 10 4 [M+1]+ m/z 195 m/z [M+2]+ m/z 196 Possibly 2x 13C of 1x 18O 24 Isotopes • Peaks caused by ions bearing those heavier isotopes (such as 13C) also appear in mass spectra • The relative abundances of such isotopic peaks are proportional to the abundances of the isotopes in nature • Next to the M+ peak you often see a M+1 peak (or more) 25 Relative abundance of several isotopes: C Mass spectrum of benzene 12C + 6H6 M+ m/z 78 100.0% 12C 13CH + 5 6 6.6% [M+1]+ m/z 79 Element Isotope Exact mass (Da) Natural abundance (%) C 12 12.000000 98.93 13 13.003354 1.07 26 Relative abundance of several isotopes: Cl Mass spectrum of CH3Cl CH335Cl+ 100% M+ m/z 50 CH337Cl+ 32% [M+1]+ m/z 51 [M+2]+ m/z 52 Element Isotope Exact mass (Da) Natural abundance (%) Cl 35 34.968854 75.78 37 36.965896 24.22 27 Relative abundance of several isotopes: Br M+ m/z 94 [M+2]+ m/z 96 CH379Br+ 100% CH381Br+ 98% Element Isotope Exact mass (Da) Natural abundance (%) Br 79 78.91834 50.69 81 80.91629 49.31 28 Mass resolution Resolving power, R The ability to separate two ions with different m/z The smaller the m/z difference (Δm) that can be distinguished, the higher the resolution The type (and quality) of the used mass analyzer determines the resolving power Mass analyzer • • • • • Quadrupole (Q) Ion trap (IT) Time-of-flight (TOF) Fourier transform ion cyclotron resonance (FTICR) Orbi trap Low R High R 29 Mass resolution: valley definition 10% valley: Δm is the difference between two peaks of the same height, separated by a valley which at its lowest point is equal to 10% of the peak height of either peak 𝑅= 𝑚 ∆𝑚 m = m/z of the first peak Δm = difference between the m/z of the two neighbouring peaks Δm = m2 ‒ m1 Example A mass analyzer with R = 1000 will separate two ions with a m/z of 500.0 and 500.5 with a 10% valley (Δm = 500/1000 = 0.5 m/z) height height m/z 30 Mass resolution: peak width definition Full Width at Half Maximum (FWHM): Δm is the peak width at 50% of the peak height Today the most used definition (because it is easier ☺) 𝑅= 𝑚 ∆𝑚 m = m/z of the first peak Δm = the width of the peak at a defined height: FWHM 𝑚 𝑅= 𝑊0.5 Example For an ion with m/z 500 a mass analyzer with R = 1000 will give a peak width at half height of 0.5 m/z (W0.5 = 500/1000 = 0.5 m/z) 31 Mass resolution: low and high m/z of an ion measured with a low resolving power W0.5 = 0.5 m/z of an ion measured with a high resolving power R = 570.3 / 0.5 = 1141 W0.5 = 0.01 R = 570.326 / 0.01 = 57033 Mass analyzer Quadrupole Ion trap m/z m/z Mass analyzer TOF FTICR Orbitrap The centroid can be estimated with an accuracy of about a tenth of the width at half maximum 32 Mass accuracy Mass accuracy The accuracy of the measured mass 𝑚𝑡𝑟𝑢𝑒 − 𝑚𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑚𝑎𝑠𝑠 𝑎𝑐𝑐𝑢𝑟𝑎𝑐𝑦 = ∙ 106 𝑝𝑝𝑚 𝑚𝑡𝑟𝑢𝑒 Example mmeasured = mtrue = 1000.200 1000.150 𝑚𝑎𝑠𝑠 𝑎𝑐𝑐𝑢𝑟𝑎𝑐𝑦 = (1000.200 − 1000.150) ∙ 106 𝑝𝑝𝑚 1000.150 = 5 ∙ 10−5 ∙ 106 𝑝𝑝𝑚 = 50 ppm An accuracy of 5 ppm or less is required for publication of chemical compounds High mass accuracy (small values) requires high resolution/resolving power 33 Intermezzo: What do we know so far • • We know: • Molecular mass (atomic mass): molecular weight • Mass number: the number of protons and neutrons in the nucleus • Nominal mass: sum of integer masses of the most abundant isotope of each element in the compound • Exact mass: calculated mass of an ion or molecule with specified isotopic compositions • m/z ratio • • Molecular ion: the radical cation formed by the loss of an electron from the neutral molecule • Mass accuracy: Mass resolution: The ability to separate two ions with different m/z 𝑚𝑎𝑠𝑠 𝑎𝑐𝑐𝑢𝑟𝑎𝑐𝑦 = 𝑚𝑡𝑟𝑢𝑒 − 𝑚𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 ∙ 106 𝑝𝑝𝑚 𝑚𝑡𝑟𝑢𝑒 What else do we need to know: • What is a mass spectrometer? • What is a mass spectrum? • How do we ionise our analytes? • How do we separate these ions? • How do we detect these ions? 34 Coffee time☺ 35 The mass spectrometer: an overview Mass spectrometer = an instrument that measures the m/z and the relative abundances of ions in the gas phase Vacuum Sample Inlet system Ion source Mass analyzer Detector Data system Massa spectrum 36 Massa spectrum: paracetamol • Massa spectrum = graph of the number of ions detected as a function of their m/z ratio • Base peak = the most abundant ion formed in the ionization chamber gives rise to the tallest peak in the mass spectrum, called the base peak Base peak 37 Inlet system Vacuum Sample Inlet system Ion source Mass analyzer • Before the ions can be formed, a stream of molecules must be introduced into the ion soure • The sample inlet system provides this stream of molecules • Detector Data system Massa spectrum The sample may be a gas, liquid or a solid. Depending on what phase your sample is in, the inlet system will be different 38 Ion source Vacuum Sample Inlet system Ion source Mass analyzer Detector • In the ion source the sample molecules are converted to charged particles • Many different ionisation methods: • Electron ionisation (EI) • Chemical ionisation (CI) • Matrix-assisted laser desorption ionisation (MALDI) • Electrospray ionisation (ESI) • And many more, in this course we focus on EI ☺ Data system Massa spectrum 39 Electron ionisation (EI) M Electron trap Molecules enter from the sample inlet system Sample Repeller plate Filament 40 Electron ionisation (EI) Electron trap Sample In EI-MS, a beam of high-energy electrons is emitted from a filament that is heated to several thousand degrees Celsius M Repeller plate e- e- e- Filament 41 Electron ionisation (EI) Electron trap These high-energy electrons strike the molecules in the ion source → leads to ionisation of the molecules Sample Ionization potential/energy = the energy required to remove an electron from an atom or molecule. Most organic compounds have ionization potentials between 8-15 eV M Repeller plate The beam of high-energy electrons have an ionization energy of 50-70 eV (70 eV is often used) e- e- e- Filament 42 Electron ionisation (EI) e- Electron trap A radical cation is formed due to the loss of an electron from a neutral molecule: M + e- → M•+ + 2e- Sample The repeller plate (positively charged) directs the newly created ions toward a series of accelerating plates and eventually to the mass analyzer Repeller plate e- e- eAccelerating plates Filament 43 Electron ionisation (EI) e- Electron trap Sample Repeller plate to mass analyser e- e- eAccelerating plates Filament 44 Electron ionisation (EI) • Ionisation & fragmentation • EI produces ions with a large distribution of internal energy • The excess electron energy spreads across the entire radical cation • This leads to fragmentation of your molecule (you see this a lot in EI-MS spectra) • Reflects the stability of molecules 45 Degree of fragmentation in EI • The relative intensity of the molecular ion depends on its stability • The stability of molecular ion depends on the compound class Decreasing stability of M+ ion Decreasing relative intensity of M+ ion (an increasing fragmentation) Hydrocarbons Hetero atom containing Aromatics S compounds Alkenes N compounds Alicyclic compounds O compounds Alkanes Branched alkanes 46 EI-MS van phenol 47 EI-MS van n-pentanol Molecular ion peak is not detected 48 Fragmentation reactions • Heterolytic cleavage • Homolytic cleavage • McLafferty rearrangement 49 Heterolytic cleavage Cleavage of a bond to a heteroatom with formation of a cation and a stable atom or radical • Common for halogen substituted molecules and sulfur compounds • Less important for alcohols, ethers and other oxygen containing molecules • Insignificant for amines 50 Heterolytic cleavage: examples (1/2) Alkyl halides can cleave heterolytically Alcohols can cleave heterolytically 51 Heterolytic cleavage: examples (2/2) Ethers can cleave heterolytically Option 2 Note that in the top a primary carbocation is formed, whereas at the bottom a secondary carbocation is formed, which is more stable and so this is more favourable 52 Heterolytic cleavage: relative stability Relative stability of simple organic carbenium ions • Size: • Type: • Hybridisation: 53 Heterolytic cleavage: relative stability Relative stability of simple organic carbenium ions • Delocalization: • Protonated benzene: 54 Homolytic cleavage Cleavage of the bond next to the α carbon Radical site initiated cleavage Note that we use single-headed curly arrows here 55 Homolytic cleavage: examples (1/4) Alkyl halides can cleave homolytically The carbon that is directly attached to the halogen, is the α carbon. Note that the bond next to the α carbon cleaves (α cleavage) 56 Homolytic cleavage: examples (2/4) Alcohols can cleave homolytically The carbon that is directly attached to the hydroxyl group, is the α carbon 57 Homolytic cleavage: examples (3/4) Ethers can cleave homolytically Option 2 58 The carbon that is directly connected to the oxygen, is the α carbon Homolytic cleavage: examples (4/4) Ketones can cleave homolytically These ions are called acylium ions. The triple Option 2 bond is responsible for the linear shape of the ion. Draw them this way! The carbon that is doubly bonded to the oxygen, is the α carbon 59 Homolytic cleavage: relative stability Relative stability of simple organic radicals • Size: • Type: • Hybridisation: 60 Homolytic cleavage: relative stability Relative stability of simple organic radicals • Delocalization: 61 McLafferty rearrangement Rearrangement reactions often lead to radical cations The process involves commonly hydrogen shifts followed by cleavage of a bond 62 McLafferty rearrangement: example McLafferty rearrangement is specific for ketones • The bond between the α and β is cleaved • A new π-bond forms between β and γ 63 Note that the labelling of the carbon atoms is different compared to homolytic cleavage What can we do with EI-MS? Determine the molecular mass Structure elucidation based on the fragment ions that are measured EI provides reproducable mass spectra: You can use spectral libraries to identify your compound 64 Mass analyzer Vacuum Sample Inlet system Ion source Mass analyzer Detector • Mass analyzer = the region of the mass spectrometer where the ions are separated according to their m/z ratios • There are many different mass analyzers: • Time-of-flight (TOF) • Quadrupole (Q) • Orbitrap • And again many more but we focus on the TOF ☺ Data system Massa spectrum 65 Time-of-flight (TOF) Principle Ions moving in the same direction and having the same constant kinetic energy, will have a velocity that is inversely proportional to √(𝑚Τ𝑧) Lighter will have a higher velocity L Detector Experiment • Acceleration of ions with a given Ekin • Ions enter the ‘’flight tube’’ • Flight time of the ions (time-of-flight) over L is detected • Time-of-flight is related to m/z • Repetition rate: ~1000 spectra per second • Resolving power: 1000-5000 for linear TOF instruments • Mass range: up to 106 Da (in principle unlimited) 66 Detector Vacuum Sample Inlet system Ion source Mass analyzer Detector • Detector = consists of a counter that produces a current that is proportional to the number of ions that strike it • Electron multiplier Data system Massa spectrum 67 Detector: electron multiplier Principle • Ion hits the surface of the electron multiplier • Two electrons are ejected from this surface • These electrons strike the surface again, each causing the ejection of two more electrons • This process repeats itself until the end of the electron multiplier is reached, and the electrical current is analyzed and recorded by the data system 68 Data system Vacuum Sample Inlet system Ion source Mass analyzer • The signal from the detector is fed to a recorder • Recorder: produces the mass spectrum Detector Data system Massa spectrum 69 Take home message You know the most important points of this lecture if you can answer the following questions: • What do the following terms mean: molecular mass, mass number, nominal mass, exact mass? • What is m/z? • What are the molecular ion and base peak? • What is mass resolution? What is mass accuracy and how do we calculate both? • What is a mass spectrometer? • What is a mass spectrum? • How does the ionisation method EI work? What is the general reaction equation of EI? • How do heterolytic and homolytic fragmentations look like? Can you draw the mechanisms for both for alkyl halides, alcohols, ethers and ketones? • How does McLafferty rearrangement look like? Can you draw the mechanism for a ketone compound? • What is a mass analyzer and how does the TOF mass analyzer work? • How are the formed and separated ions detected in MS? 70 Next lecture Friday 29 September at 13:30–15:15 ‘’NMR part 1’’ NU-4C07 71 End of today Questions? 72