SOLUTION: molar masses/ molecular weight | My Assignment Tutor

OMED0104 – CHARACTERISATION OF AN UNKNOWN SAMPLE BY MASSSPECTROMETRY – Experiment 4 guidance notesThe aim of the experiment is to identify compounds A – D using:1. the molar masses/ molecular weight given to you of the 4 compounds(Aspirin, Ibuprofen, trans-Cinnamic Acid, Phenacetin);2. the Electrospray Ionisation (soft) mass spectra generated by LC-MS;3. the Electron Ionisation (hard) mass spectra generated by GC-MS;1. Using the molar masses/ molecular weight of the 4 compoundsa) You are given the structure, and molar mass (g/mol) for each compound:From the given molar mass for each compound we can determine themolecular weight, this is a simple unit change from g/mol to Daltons, thusgiving:Phenacetin = 179.2 DaAspirin = 180.2 DaTrans-cinnamic acid = 148.2 DaIbuprofen-Na salt = 228.3 DaNote:- although the ibuprofen salt is provided the ibuprofen free-base form ofthe drug is also present, and so the molecular weight must be re-calculated:C13H17O2Na – Na+ + H+ → C13H18O2 + Na+This gives us a molecular weight of 206.3 Da for Ibuprofen.b) Once we have the molecular weights of our four possible analytecompounds then we can predict their mass-to-charge (m/z) ratios for ionsgenerated by Electron Ionisation, and by Electrospray Ionisation.If you recall the ‘modes of ionisation’ from OMED0104 mass spectrometryLecture 1 (slide 23):The GC-MS instrument utilises (EI) electron ejection ionisation to generatepositive ions; as only a single electron is lost to generate a molecular radicalcation then the effect on the molecular weight of an analyte is negligible, andso the mass-to-charge ratio for each analyte is simply determined by(analyte molecular weight) / (z = +1)Phenacetin, m/z = 179.2 Da/zAspirin, m/z = 180.1 Da/zTrans-cinnamic acid, m/z = 148.2 Da/zIbuprofen, m/z = 206.2 Da/zThe LC-MS instrument utilises (ESI) electrospray ionisation to generate positiveions by protonation, and negative ions by deprotonation. As a proton (H+) isgained or lost during ionisation then we are generating a pseudomolecularcation, or anion, respectively. This, then, affects the mass-to-charge ratio ofthe observed pseudomolecular parent ion, with the effect on the molecularweight of an analyte determined by: +ve Ion Mode-ve Ion Mode(analyte molecular weight) + 1 / (z = +1)(analyte molecular weight) – 1 / (z = -1)Phenacetin, (+ve) m/z = 180.2 Da/zAspirin, (+ve) m/z = 181.2 Da/z(-ve) m/z = 178.1 Da/z(-ve) m/z = 179.1 Da/zTrans-cinnamic acid, (+ve) m/z = 149.2 Da/z(-ve) m/z = 147.1 Da/zIbuprofen, (+ve) m/z = 207.2 Da/z(-ve) m/z = 205.1 Da/z Note:- This is a key step as otherwise determination of the ion fragments andneutral losses resulting from ionisation of an analyte molecule becomesimpossible and will prevent you from correctly identifying the fragmentationmechanisms giving rise to each peak in the mass spectrum.Additionally, not all analytes generate an observable molecular, orpseudomolecular ion. Remember that we only observe the ions that have alifetime equal to, or greater than, the time taken for an ion to move from theionisation region of a mass spectrometer through to the detector! ANALYTEm/z (EI)m/z (ESI +ve)m/z (ESI -ve)PHENACETIN179.2180.2178.1ASPIRIN180.1181.2179.1TRANS-CINNAMIC ACID148.2149.2147.1PHENACETIN206.2207.2205.1 Once you have determined the expected molecular and pseudomolecular ionmass-to-charge ratios then you are ready to interpret the mass spectragenerated by the GC-MS and the LC-MS instruments.2. Interpretation of Electrospray Ionisation mass spectraElectrospray ionisation mass spectra are usually considered first owing to themethod being a ‘soft’ ionisation method, thus there is a reasonable probabilityof a pseudomolecular ion being observed in the mass spectrum of an analyte.This is very important as it provides confirmation of the molecular weight (m)of an analyte, which is then used in interpreting the Electron Ionisation massspectra correctly.Additionally, this can then be compared with the molecular weights for ouranalytes, and with the list of predicted pseudomolecular ions determined instep 1.b) above, thereby providing prima facie identification of our analytes.With the exception of aliphatic, acyclic, saturated n-hydrocarbons, then mostion fragmentation occurs around functional groups, with many neutralfragment losses resulting from the formation and subsequent loss of a stablemolecule, or ‘leaving group’.For example, consider an alcohol group on a molecule, either as a discreteentity or as a component of a carboxylic acid moiety, R – OH. When protonatedvia dative bonding between the oxygen-Lone Pairs and the free proton (H+)then the species R – OH2+ is formed, followed by charge migration and α-fragmentation, resulting in formation of a carbocation R+ and loss of theneutral water molecule.A second, common fragmentation observed is loss of a carboxylic acid group,most commonly observed for Negative Ion Mode ionisation duringElectrospray Ionisation, but also occasionally observed for Positive Ion Modeionsiation. Consider a carboxylic acid group on a molecule, R – CO2H. As ESIoccurs as a consequence of Acid – Base interactions, then during negativeionisation the acidic proton in the carboxylic acid group is stripped to revealthe carboxylate anion, R – CO2-, again charge migration can occur, followed byfragmentation and loss of the carboxylate group as carbon dioxide.To determine the molecular weight of a ‘neutral fragment’ loss to form aparticular ion peak in a mass spectrum then subtract the (m/z)-value of yourchosen peak from that of the pseudomolecular ion (m/z)-value,e.g. m/z (protonated Ibuprofen) = 206 Da/zm/z of unknown peak = 161 Da/zneutral fragment mass = 206 – 161 = 45 DaThere is only one functional group with a molecular weight of 45 Da and that iscarboxylic acid. Examination of the structure of Ibuprofen confirms that thereis indeed a carboxylic acid functional group present in Ibuprofen, and so loss ofCO2H would be consistent with Ibuprofen.3. Interpretation of Electron Ionisation mass spectraElectron Ionisation mass spectra are used in conjunction with the analytestructures given to you to provide structural confirmation of an analyte. AsElectron Ionisation results in substantial fragmentation, again generally aroundfunctional groups present, then each peak in the spectrum is representing astable fragment generated by loss of a neutral structural component. Byidentifying the ion fragment, and/ or the neutral fragment, resulting in eachpeak then these components can each be compared to the analyte structure todetermine if the proposed fragment is consistent with that analyte. This selfconsistency is key as you can only propose a fragment which is actually presentin an analyte structure.As an example, consider an ethene fragment, C2H4, and a carbonyl, CO, bothhave a nominal mass of 28 Da; if we are analysing 1,3-butadiene then wewould not expect to see loss of a carbonyl group!As with Electrospray Ionisation, to determine the molecular weight of a‘neutral fragment’ loss to form a particular ion peak in a mass spectrum thensubtract the (m/z)-value of your chosen peak from that of the molecular ion(m/z)-value. Remember – Electron Ionisation forms Molecular Ions,Electrospray Ionisation forms Pseudomolecular Ions.As Electron Ionisation does not involve protonation or deprotonation offunctional groups then this makes identification of neutral losses a little easier,for example -OH loss is observed at 17 Da rather than 18 Da (H2O) viaElectrospray Ionisation, although conversely this also means thatfragmentation is not just restricted to acidic or basic functional groups, but canalso occur around alkene, alkyl and aromatic skeletal functional groups, orbranched side-chains especially if a tertiary or secondary carbocation can beformed. It is also quite rare to see alcohol Molecular Ions as the alcohol protoncan be easily lost.Summary1. Determine your molecular weights (Da) for each analyte;2. Predict Molecular Ion, and Pseudomolecular Ion mass-to-charge ratios(Da/z) from your molecular weights;3. Look over your ESI and EI mass spectra and see if you can observe anexpected Molecular Ion, or Pseudomolecular Ion, to provide a primafacie identification of an analyte;4. Work through the EI and ESI mass spectra to obtain the key structuralevidence to support your identification of an analyte through its’structural components.5. When assigning structures to ion fragments or neutral losses thenconsider fragmentation mechanisms, for example α-cleave, σ-cleave,retro diels-alder, and especially McLafferty rearrangements for analytescontaining carbonyls.