Mass Spectroscopy

What do the following techniques give information about?

NMR (locations of atoms within a molecule), IR (functional groups within a molecule), UV-Vis (conjugation), X-ray crystallography (3D arrangement of molecule), MS (fragmentation pattern, structure of molecule) - techniques are complementary

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What is a mass spectrometer?

A technique which measures molecules in the gas phase (ionised form). Charged species are generated and then sorted based on their mass to charge ratio (m/z)

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What is MS used for in pharmaceutical analysis?

Characterisation of potential drugs, drug degradation product analysis, PK bioavailability studies

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MS is used for which biomolecules in characterisation?

Peptides, proteins and antibodies. Oligonucleotides and synthetic polymers

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What is MS used for in environmental analysis?

Pesticides in food, soil/groundwater contamination

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How is MS used in forensic/clinical analysis?

Criminology, DNA, fingerprints, blood analysis

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What are the components of a mass spectrometer?

Sample injected (from syringe, HPLC/GC/solid), ionisation (ion source), mass analyser (ions separated by m/z), ion detector - system takes place in a high vacuum

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What is mass spectrometry?

Generation/separation/characterisation of gas phase ions according to their relative mass as a function of charge (m/z). Qualitative/quantitative tool, analysis 10^-15 g and 10^-21 mol for proteins. Used in molecular biology, semiconductors, etc.

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Describe features of the mass spectrum (1)

Ion abundance vs m/z ratio. Base peak (most abundant ion, usually 100%). Peak intensities relative to base peak as percentage. Molecule loses e- during ionisation. Molecular ion observed (gives molecular weight), M+ peak

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Describe features of the mass spectrum (2)

When a molecule loses a valence electron, bonds are broken or ion formed quickly fragments to lower energy ions. Neutral ions are not recorded

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Describe features of mass resolution

The ability to distinguish between ions differing slightly in m/z ratio. Two methods. A m/z of lowest mass peak divided by difference in m/z of peaks. Or delta m/z is the full width of peak at half maximum height

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Compare the resolution when using a quadrupole or time of flight

Time of flight has a better resolution due to having separated and sharp peaks (quadrupole has broader peaks with overlapping signals)

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In the mass spectrum for acebutolol, which unusual peaks are observed and why?

Peak after M+ peak and a solvent peak e.g. H2O. Isotopes will contribute to the MS (e.g. M+1 peak) - C, H, O, N and S (abundance %).

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Describe features of the monoisotopic and nominal mass

Monoisotopic mass (sum of exact masses of atoms within a molecule). Nominal mass (sum of average masses/isotopes of atoms within the molecule) - both give different values, M+1 and M+2 peaks

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Describe features of the isotopic peaks for chlorine

Diagnostic, M+2 isotope is prevalent. 35Cl and 37Cl in a 3:1 ratio. M+2 peak would be ~24% the size of the M+ peak if Cl is present

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Describe features of the isotopic peaks for chlorine

Diagnostic, M+2 isotope is prevalent. 79Br and 81Br in a 1:1 ratio. M+2 peak would be about the size of the M+ peak if Br is present

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Describe the isotopic peaks in insulin

For very large molecules, M+1, M+2 and M+3 bands become very important. Molecular ion(s) of insulin (257 atoms). Molecules that are completely 12C are now rare. At least 3 Cs are 13C

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Summarise resolution, accuracy and range on the MS

Mass range (range which contains peak). Resolution (width of peak at half height). Measured mass (m/z at peak point). True mass (shift in peak). Accuracy (closeness of measured mass to true mass)

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What is the effect of ionisation on the MS?

MS uses magnetic/electric fields to control the path of a compound based on m/z ratio. More charge leads to a lower m/z ratio e.g. COOH and OH (138), COO- and OH at pH 3 (137), COO- and O- at pH 8 (68)

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Describe features of ionisation

Have to convert flow of liquid from syringe or liquid chromatography system into gas phase before MS analysis. Different forms of ionisation from liquid into gas. Electron Impact. Electrospray ionisation, Matrix Assisted Laser Desorption Ionisation

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Describe features of electron impact

Analyte vapour enters from insertion probe. Sample bombarded with electrons. Trap (collect anions). Filament at 70 eV. Cations passed through to analyser. Harsh technique. System involves magnets and takes place in a vacuum

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Describe features of magnetic sector instrument with electron impact

Sample vaporised into ionisation source. Bombarded by electron beam (70 eV) removing valence electron to produce cation-radical in sample. Separation in magnetic field by m/z ratio. z = 1 for most ions fo value of m/z is mass of ion

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State features of the peaks for the MS of benzene using electron impact

Fragmentation patterns, base peak and M+1 peak

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State features of the peaks for the MS of benzyl chloride using electron impact

M+ and M+2 peaks, base peak, fragmentation patterns

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Give examples of other molecules analysed using electron impact and MS

Aspirin, ketoprofen, propanolol, ramelteon - EI shatters chemical bonds

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Describe features of EI and proteins

EI shatters chemical bonds. Any given protein contains 20 different amino acids. EI would shatter the protein into not only into amino acids but also amino acid sub-fragments and even peptides of 2,3,4 amino acids. Many signals/difficult to analyse

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Describe features of electrospray ionisation

Convert flow of liquid from a LC column or a syringe into mist for ionisation to occur. Sample passed through inlet, tip charged at 5 kV. Particles in become in gas-phase ion formation due to molecules becoming smaller and ionised. Sent to MS

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Describe features of ESI as a soft ionisation technique

Needle held at 5 kV relative to counter electrode (entrance to MS). Solution contains electrolyte/needle forms part of API source, fine mist of droplets emerge with +/- charge (polarity of needle/liquid). Dissolved analytes to MS by voltage

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Describe features of MALDI

Convert solid into the gas phase. Sample in matrix. Laser applied to system. Matrix becomes ionised. Proton transfer from matrix to sample, producing cation radicals

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Which molecules can be used in a matrix for MALDI?

-a-cynao-4-hydroxycannamic acid, sinapinic acid, 2,5-dihydroxybenzoic acid, nicotinic acid, 3-hydroxpicolinic acid (e.g. sample - proteins)

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What are the types of mass analyser?

Magnetic sector (instrument with EI), single quadrupole, triple quadrupole, time of flight

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Describe features of a quadrupole (1)

Four metal rods parallel to each other. Each opposing rod pair connected electrically with RF applied between rod pairs. Direct voltage superimposed on RF voltage. Only ions in certain m/z move through at specific voltage

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Describe features of a quadrupole (2)

Allows one m/z to be scanned or a range of m/z by varying voltage. Other ions have unstable trajectories and will collide with rods. Mass scanning mode or single mass transmission mode

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Describe features of the MS for propranolol using ESI

No fragmentation patterns due to technique not shattering chemical bonds

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Why does MS require gas-phase ions?

MS uses magnetic/electric fields to control the path of a compound based on m/z ratio

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How can you calculate the mass?

M = n (Mn -1) - application to ESI lysozyme

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Describe feature of time of flight (1)

Sample in tube with ion source, laser used for ionisation. Matrix ionised/proton transfer to sample. Ions formed in pulses, drift region is field free, measures time for ions to reach detector, small ions reach detector first, high resolution

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Describe feature of time of flight (2)

m/z = 2Vt^2 / L^2 (mass of ion, charge of ions, voltage, drift length tube, time) - application for MALDI-TOF for substance P and insulin

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What does MS-MS mean?

Using 2 mass analysers (in one instrument) to select ion from mixture, generate fragments from it to give structural information. MS/MS scan mode (ion source, parent ion transmission, collision chamber, mass analysis, detector) - spectrum

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Describe features of SRM

Sample - ionisation - m/z separation (MS1) - collision photon surface - fragments - m/z (MS2) - detection - spectrum (application of MS to fluticasone propionate study)

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Mass Spectroscopy

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