Chemistry - Option A : Modern Analytical Chemistry MASS SPECTROMETRY

Background and Overview of Mass Spectrometer

Determining the Structure of an Organic Compound The analysis of the outcome of a reaction requires that we know the full structure of the products as well as the reactants In the 19th and early 20th centuries, structures were determined by synthesis and chemical degradation that related compounds to each other Physical methods now permit structures to be determined directly. We will examine: mass spectrometry (MS) infrared (IR) spectroscopy nuclear magnetic resonance spectroscopy (NMR) ultraviolet-visible spectroscopy (VIS)

Major Types of Spectroscopy Infrared (IR) spectroscopy measures the bond vibration frequencies in a molecule and is used to determine the functional group. Mass spectrometry (MS) fragments the molecule and measures the masses. Nuclear magnetic resonance (NMR) spectroscopy detects signals from hydrogen atoms and can be used to distinguish isomers. Ultraviolet (UV) spectroscopy uses electron transitions to determine bonding patterns.

Mass Spectrometry (MS) Uses the interaction of electric and/or magnetic fields (i.e. eletromagnetic radiation) with matter to determine weight or mass Measures mass, not absorption or emission of electromagnetic radiation Measures molecular weight Sample vaporized and subjected to bombardment by electrons that remove an electron Creates a cation-radical Bonds in cation radicals begin to break (fragment) Charge to mass ratio is measured

MS History Concept first put into practice by Francis Aston, a physicist working in Cambridge England in 1919 Designed to measure mass of elements (esp. isotopes) Awarded Nobel Prize in 1922 Now one of the MOST POWERFUL ANALYTIC TOOLS IN CHEMISTRY

MS Principles Different compounds can be uniquely identified by their mass Butorphanol L-dopa Ethanol N OH HO -CH2- -CH2CH-NH2 COOH HO CH3CH2OH MW = 327.1 MW = 197.2 MW = 46.1

MS Principles Find a way to “charge” an atom or molecule (ionization) Place charged atom or molecule in a magnetic field or subject it to an electric field and measure its speed or radius of curvature relative to its mass-to-charge ratio (mass analyzer) Detect ions using microchannel plate

Mass Spec Principles Sample + _ Ionizer Mass Analyzer Detector

Typical Mass Spectrometer

Mass Spectrometer

The Mass Spectrum Plot mass of ions (m/z) (x-axis) versus the intensity of the signal (roughly corresponding to the number of ions) (y-axis) Tallest peak is base peak (100%) Other peaks listed as the % of that peak Peak that corresponds to the unfragmented radical cation is parent peak or molecular ion (M+)

Typical Mass Spectrum aspirin

Different Types of MS GC-MS - Gas Chromatography MS separates volatile compounds in gas column and ID’s by mass LC-MS - Liquid Chromatography MS separates delicate compounds in HPLC column and ID’s by mass MS-MS - Tandem Mass Spectrometry separates compound fragments by magnetic field and ID’s by mass

Gas Chromatography Sample (must contain stable, volatile compounds) is vaporized in a heated chamber Column is filled with silanized (silicon-coated) calcium silicate Column is kept hot (400 oC) in oven Sample is pushed through column using gas pressure (He or N2)

Electron Impact MS of CH3OH Molecular ion EI Breaks up Molecules in Predictable Ways

Electron Impact MS of CH3Br Isotopes can help in identifying compounds

The Mass Spectrum

Masses are graphed or tabulated according to their relative abundance. The Mass Spectrum Masses are graphed or tabulated according to their relative abundance.

MOLECULAR MASS DETERMINATION USING MASS SPECTROMETRY Mass spectrometry is used to identify unknown or new compounds. When a molecule is ionized it forms a MOLECULAR ION which can also undergo FRAGMENTATION or RE-ARRANGEMENT to produce particles of smaller mass. Only particles with a positive charge will be deflected and detected. The resulting spectrum has many peaks. The final peak (M+) shows the molecular ion (highest m/z value) and indicates the molecular mass. The rest of the spectrum provides information about the structure. IONIZATION MOLECULAR ION FRAGMENTION RE-ARRANGEMENT FRAGMENTION

highest m/z value usually corresponds to the molecular ion THE MASS SPECTRUM Spectra obtained for organic molecules have many peaks. Each peak is due to a particular fragment with a certain m/z value. highest m/z value usually corresponds to the molecular ion its position provides information about the molecular mass of a substance the tallest peaks come from the most stable species

differences between major peaks THE MASS SPECTRUM Interpretation of thousands of spectra has shown that many classes of organic compound show characteristic fragmentation patterns due to their functional groups. It is possible to identify the type of compound from its spectrum by looking at the ... position of peaks differences between major peaks

. THE MOLECULAR ION Abundance % m/z In the spectrum of octane, a signal occurs at 114 due to the species C8H18+ The species due to the final signal is known as the molecular ion and is usually corresponds to the molecular mass of the compound. 20 40 60 80 100 Abundance % molecular ion 114 . m/z 10 20 30 40 50 60 70 80 90 100 110 120 130 140

. THE MOLECULAR ION Abundance % m/z The small peak (M+1) at 115 due to the natural abundance (about 1%) of carbon-13. The height of this peak relative to that for the molecular ion depends on the number of carbon atoms in the molecule. The more carbons present, the larger the M+1 peak. 20 40 60 80 100 Abundance % 114 . m/z 10 20 30 40 50 60 70 80 90 100 110 120 130 140

. FRAGMENTATION Abundance % m/z The rest of the spectrum provides additional information of the molecule’s structure. Peaks appear due to characteristic fragments (e.g. 29 due to C2H5+) and differences between two peaks also indicates the loss of certain units (18 for H2O, 28 for CO). 43 20 40 60 80 100 Abundance % 29 57 71 85 114 . m/z 10 20 30 40 50 60 70 80 90 100 110 120 130 140

FRAGMENTATION PATTERNS ALKANES The mass spectra of simple hydrocarbons have peaks at m/z values corresponding to the ions produced by breaking C-C bonds. Peaks can occur at ... m/z 15 29 43 57 71 85 etc. CH3+ C2H5+ C3H7+ C4H9+ C5H11+ C6H13+ • the stability of the carbocation formed affects its abundance • the more stable the cation the higher the peak • the more alkyl groups attached to the carbocation the more stable it is most stable tertiary 3° > secondary 2° > primary 1° least stable alkyl groups are electron releasing and stabilize the cation

FRAGMENTATION PATTERNS HALOGENOALKANES Multiple peaks occur in the molecular ion region due to different halogen isotopes. There are two peaks for the molecular ion of C2H5Br, one for the molecule containing the isotope 79Br and the other for the one with the 81Br isotope. Because the two isotopes are of similar abundance, the peaks are of similar height. 10 20 30 40 50 60 70 80 90 100 110 120 130 140 m/z 20 40 60 80 100 Abundance % molecular ion contains...79Br 81Br

FRAGMENTATION PATTERNS Aldehydes and ketones The position of the carbonyl group influences the fragmentation pattern because the molecular ion fragments either side of the carbonyl group. O CH3 C C4H9 MOLECULAR ION has m/z = 100 • +

FRAGMENTATION PATTERNS Aldehydes and ketones The position of the carbonyl group influences the fragmentation pattern because the molecular ion fragments either side of the carbonyl group. O CH3 C C4H9 MOLECULAR ION has m/z = 100 • + O C4H9 C+ CH3• Breaking the bond between the methyl group and the carbonyl group produces two possible ions, depending on how the bond breaks. Two peaks at m/z values 15 and 85 will appear in the mass spectrum. m/z = 85 O C4H9 C• CH3+ m/z = 15

FRAGMENTATION PATTERNS Aldehydes and ketones The position of the carbonyl group influences the fragmentation pattern because the molecular ion fragments either side of the carbonyl group. O CH3 C C4H9 MOLECULAR ION has m/z = 100 • + O CH3 C+ Breaking the bond between the butyl group and the carbonyl group produces two further ions, depending on how the bond breaks. Two peaks at m/z values 43 and 57 will appear in the mass spectrum. C4H9• m/z = 43 O CH3 C• C4H9+ m/z = 57

FRAGMENTATION PATTERNS Aldehydes and ketones The position of the carbonyl group influences the fragmentation pattern because the molecular ion fragments either side of the carbonyl group. O CH3 C C4H9 Example; MOLECULAR ION has m/z = 100 • + O C4H9 C+ O CH3 C+ CH3• C4H9• m/z = 85 m/z = 43 O C4H9 C• O CH3 C• CH3+ C4H9+ m/z = 15 m/z = 57 A further peak occurs at m/z = 72 (100-28) due to loss of CO

Mass Spectra of Alkanes More stable carbocations will be more abundant.

Mass Spectra of Alkenes Resonance-stabilized cations favored.

Mass Spectra of Alcohols Alcohols usually lose a water molecule. M+ may not be visible.

Molecules with Heteroatoms Isotopes: present in their usual abundance. Hydrocarbons contain 1.1% C-13, so there will be a small M+1 peak. If Br is present, M+2 is equal to M+. If Cl is present, M+2 is one-third of M+. If iodine is present, peak at 127, large gap. If N is present, M+ will be an odd number. If S is present, M+2 will be 4% of M+.

Isotopic Abundance 81Br

Mass Spectrum with Sulfur =>

Mass Spectrum with Chlorine

Mass Spectrum with Bromine

MS Applications and Conclusions

Applications Determination or confirmation of chemical structure of drugs and drug metabolites (MS-MS) Detection/quantitation of impurities Detection/quantitation of drugs and their metabolites in biofluids and tissues High throughput drug screening Analysis of liquid mixtures (LC-MS)

MS vs NMR Absorbance aspirin EI-MS NMR

MS vs. NMR MS peaks are narrower than NMR peaks MS is much more (104 x) more sensitive than NMR (among most sensitive tools) MS generally allows one to analyze much larger molecules (>50 kD) than NMR MS samples are more difficult to prepare MS is not particularly quantitative MS instruments cost a little less than NMR