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44 Cards in this Set
- Front
- Back
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Name four modern techniques for structure determination of molecules |
1. Ultraviolet Spectroscopy 2. Infrared Spectroscopy 3. Nuclear Magnetic Resonance 4. Mass Spectrometry |
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What is mass spectrometry?
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Mass spectrometry is the generation, separation and characterisation of gas phase ions according to their relative mass as a function of charge |
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What was a previous requirement of a sample for mass spectrometry that no longer exists? |
The sample had to be able to be vaporised |
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State four applications of mass spectrometry |
1. Pharmaceutical analysis 2. Biomolecule characterisation 3. Environmental analysis 4. Forensic analysis/clinical |
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What is the accuracy of mass spectrometry for small organic moleculs, and for large biomolecules? |
Small organic moleculs - MW within 5ppm or 0.0005% Large biomolecules - within accuracy of 0.01% |
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What are the three functions of a mass spectrometer? |
1. Creation of ions 2. Separation of ions 3. Detection of ions |
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What do all mass spectrometers have in common? |
Need a very high vacuum (10^-6 torr), while still allowing introduction of sample |
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How does a single focusing mass spectrometer generate ions? |
1. A small quantity of sample is injected and vaporised under high vacuum 2. Sample is then bombarded with electrons having 25-80eV of energy 3. A valence electron is 'punched' off the molecule, and an ion is formed |
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How does a single focusing mass spectrometer separate ions? |
1. Ions are accelerated using an anode towards the focusing magnet 2. Each ion has own kinetic energy 3. As ions enter magnetic field, their path is curved to different extent depending on mass/charge ratio 4. Only one mass has the correct radius path to pass through the magnet towards the detector 5. 'Incorrect' mass particles strike the magnet |
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What is the difference between a single focusing mass spectrometer and a double focusing mass spectrometer? |
In a double focusing mass spectrometer, the beam of sorted ions from the focusing magnet are focused again by an electrostatic analyser where the ions of identical mass are separated on the basis of differences in energy. More ions are 'lost' in the second focusing, so there is a decrease in sensitivity. |
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How does a quadrupole mass spectrometer work? |
Four magnets, hyperbolic in cross section are arranged with two positive rods opposite each other, and the two negative rods opposite each other. One pair has an applied direct current, the other an alternating current. Only a particular mass ion can resonate properly and reach the detector. |
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What is the advantage of a quadrupole mass spectrometer? |
Compact size of the instrument - each rod is about the size of a ballpoint pen. |
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What is the name of the tallest peak on a mass spectrum? |
The base peak |
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What are all other peak intensities relative to the base peak as? |
A percentage |
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What is the name of the peak produced by the molecule after it has lost only one electron? |
The M+ peak |
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What piece of information does the M+ give us? |
The molecular mass - assuming every atom is in its most abundant isotopic form |
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What three facts apply for a molecular ion peak? |
1. The peak must correspond to the highest mass ion on the spectrum excluding the isotopic peaks 2. The ion must have an odd number of electrons, usually a radical cation 3. The ion must be able to form the other fragments on the spectrum by loss of logical neutral fragments. |
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What is the nitrogen rule? |
If a molecule contains an even number of nitrogen atoms or no nitrogen atoms, the molecular ion will have an even mass value. If a molecule contains an odd number of nitrogen atoms, the molecular ion will have an odd mass value. |
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For molecules that contain Cl or Br, how are the isotopic peaks diagnostic?
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The M+2 peak is prevalent. 35Cl is ~75% and 37Cl is ~25% of naturally occuring chlorine atoms, therefore M+2 peak at ~25% intensity. 79Br is ~50% and 81Br is ~50% of naturally occuring bromine atoms,therefore M+2 peak at same intensity. |
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What indicates the resolution of the MS instrument? |
Peak width |
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What is resolving power defined as? |
∆M/M. M is the mass number of observed mass. ∆M is the difference between two masses that can be separated.
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State three different ionisation methods in MS |
1. Electron Impact 2. Electrospray Ionization 3. Matrix Assisted Laser Desorption |
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What molecules is electron impact ionisation useful for? |
Small molecules, 1-1000 Daltons |
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What molecules is electrospray ionisation useful for? |
small molecules, peptides, proteins, up to 200,000 Daltons |
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What molecules is MALDI ionisation useful for? |
peptides, proteins, DNA, >500 Da up to 500kD |
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Briefly outline electron impact ionisation |
1. Sample is vaporised onto ionisation source 2. Bombarded by electron beam (70 eV) dislodging valence electron of sample, producing cation-radical 3. Most cation-radicals fragment and are separated in magnetic field according to their m/z 4. Since z=1 for most ions the value of m/z is mass of ion |
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Why cannot you use electron impact ionisation to analyse proteins? |
- EI shatters chemical bonds - Any given protein contains 20 different amino acid - EI would shatter the protein into not only amino acids, but also amino acid sub-fragments and peptides - Result is 10,000s of differetn signals from a single protein - too complex to analyse |
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Name the two 'soft ionisation' techniques |
1. Electrospray Ionisation 2. Matrix Assisted Laser Desorption Ionisation (MALDI) |
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When was electrospray first conceived, and when was it first put into practice? |
Conceived - 1960s by Malcolm Dole Put into practice - 1980s by John Fenn (Yale) |
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When was MALDI first introduced? |
1985 by Franz Hillenkamp and Michael Karas (Frankfurt) |
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How does electrospray ionisation work? |
- Based on electrostatic spraying of liquids where a solution is passed through needle held at high voltage relative to a counter electrode (entrance to MS) - When the solution contains an electrolye and the needle forms part of the API source, then the fine mist of droplets that emerge from the needle tip possesses a net +ve or -ve charge determined by the polarity of the needle and the solution chemistry of the bulk liquid - These preformed and then sprayed ions, which are characteristic of the dissolved analytes, are attracted to the entrance of the MS by applying appropriate voltages |
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How does MALDI work? |
- Sample is mixed with a UV absorbant matrix (sinapinic acid for proteins, 4-hydroxycinnaminic acid for peptides) - Sample is ionised by bombarding sample with laser light - Light wavelength matches that of absorbance maximum of matrix so that matrix transfers some of its energy to the analyte (leads to ion sputtering) - Dissociation of matrix, phase change to super-compressed gas, charge transfer to analyte molecule - Expansion of matrix at supersonic velocity, analyte trapped in expanding matrix plume |
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What is the sample requirement of MALDI? |
Requires 10µL of 1pmol/µL sample |
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What are two advantages of MALDI? |
1. Easier to use and maintain 2. Capable of higher throughput |
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Name six applications of MALDI-TOF |
1. 2D gel separation of proteins 2. Comparative image analysis 3. Excision of regulated spots 4. Enzymatic digestion 5. MALDI-TOF-PMF 6. Protein ID by database search (MASCOT, Phenyx, etc.) |
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How is protein identification by MALDI-MS performed? |
1. Separate proteins on a 2D polyacrylamide gel 2. Excise individual spots 3. These spots may contain one or more proteins. Enzymatically digested with trypsin to generate small peptides. 4. Resulting MALDA-MS primarily (M+H)+ is converted into table of molecular weights of individual peptides present to yield a peptide mass fingerprint (PMF) 5. compare PMF with known proteins in genome database and identify protein |
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Name four types of mass analysers |
1. Magnetic Sector Analyser (MSA) 2. Quadrupole Analyser (Q) 3. Time-of-Flight Analyser (TOF) 4. Ion Trap Mass Analyser (QSTAR) |
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What are the three key specifications of mass analysers? |
1. Resolution 2. Mass measurement accuracy 3. Sensitivity |
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What types of mass analysers most commonly used for bioanalysis? |
- Quadrupole - Time-of-Flight - Ion Traps |
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How does a quadrupole mass analyser work? |
- Uses a combination of RF and DC voltages to operate as a mass filter. - Has four parallel metal rods - Lets one mass pass through at a time - Can scan through all masses or sit at one fixed mass |
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How does a time of flight mass analyser work? |
- Ions are formed in pulses - Drift region is field-free - Measures time for ions to reach detector - Small ions reach detector before large ones - High resolution technique |
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Name two types of ion detectors |
1. Faraday collector 2. Electron multiplier |
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What is MS/MS? |
MS/MS means using two mass analysers (combined in one instrument) to select an an analyte (ion) from a mixture, the ngenerate fragments from it to give structural information. |
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What is MRM? |
- Multiple reaction monitoring - Only daughter ion reaches detector - Sensitivity of MRM is a function of how much of the daughter ion is produced - Parent ion fragmentaation to daughter ion is commonly referred to as transition. |
