Unit 3-Mass Spectrometry.docx

Mass Spectrometry- chapter 13, pg. 201 Tietz 8th edition Basic Concepts What is it? Mass spectrometry is useful for identifying and determining elemental composition and structure of compounds, as well as for performing quantitative analysis of components of samples- what it is and how much there i...

Mass Spectrometry- chapter 13, pg. 201 Tietz 8th edition Basic Concepts What is it? Mass spectrometry is useful for identifying and determining elemental composition and structure of compounds, as well as for performing quantitative analysis of components of samples- what it is and how much there is of it A mass spectrometer is an analytical instrument that ionizes molecules and then separates and measures the mass-to-charge ratio (m/z) of the ionized molecules and their fragments- the molecule that goes in does not have a charge will go in the instrument will give it a charge and turns it. Into a cation The sample in an MS is first volatilized and then ionized- turned into a gas A mass spectrum is represented by the relative abundance of ions plotted as a function of their m/z ratio When an ion has a single charge (z=1) the m/z ratio is equal to the mass of the ion- most melcules take on one charge so the m/z ratio is equal to the mass of the ion example carbon 1 charge = 12 mass to charge ratio is equal to their molecular weight The unfragmented ion is called the molecular ion- for example carbon dioxide Fragment ions are formed when a molecular ion breaks into smaller pieces The ion with the highest abundance in the mass spectrum is assigned a relative value of 100% and is called the base peak (may be the molecular ion or a fragment ion)- for example in the chart the base peak is carbon dioxide Molecules tend tofragment the exact same way, this is because of their molecular structure, we should get the same read out from whatever mass soec you are using therefore we have a huge library with documented sample identification so we can compare our sample to the library to find out the unknown Instrumentation All mass spectrometers include the following: Ion source- all MS techniques require an ionization step, in which an ion is produced from a neutral atom or molecule, or ions present in solution get transferred to the gas phase Vacuum system- required to prevent ions colliding with each other or other molecules during analysis( this whole process has to happen in a vacuum we don’t want them hitting other molecules) Mass analyzer Detector- nearly all mass spectrometers use electron multipliers for ion detection Computer Ion source Electron Ionization- the sample crosses a verticle path of electrons(using magnets), they are colliding with the sample and they are giving the sample a charge, because they are being bombarded by ions they are also getting a little bit fragmented as well In EI, gas phase molecules are bombarded by electrons emitted from a heated filament- process must occur in a vacuum- Electrons emitted by thermionic emission are directed to a flow of vaporized sample Electrons have enough kinetic energy to eject an electron from the analyte molecule, producing a radical cation The positive ions are drawn out of the ionization chamber by an electrical field and introduced into the mass analyzer In EI the mass spectral pattern in most cases is dominated by fragment ions and can be matched to entries in a mass spectral library Chemical Ionization-softer technique no fragments being made. This one uses the vertical electrons but there is a reagent gas being used as well, this gas is what is going to carry the electrons over to sample ions. This gas is normally methane and wont react with our sample. A “soft” ionization technique resulting in very little fragmentation during the process A proton is transferred from a gas phase analyte by a reagent gas molecule; commonly used are methane, ammonia, isobutane, and water An electron beam ionizes the reagent gas and produces reactive species followed by proton transfer to the analyte molecule CI results in a mass spectrum dominated by the unfragmented molecular ion Electrospray- not happening in a vacuum at this point, this is happening at atmospheric pressure, we have vapourized sample and spraying it with ionized solvent(mist) onto our molecules and fragments. They are in large solvent droplets as they move into the vaccum the solvent that was initially put on molecule starts to vapourized and we will end uop with just our charged molecules. ESI is a technique in which a sample is ionized at atmospheric pressure before it is introduced into the mass analyzer Effluent from a chromatographic column is passed through a capillary Electrostatic forces applied to the effluent result in formation of charged droplets A nebulizing gas helps to nebulize the liquid and direct the charged droplets toward a counter electrode While droplets evaporate and shrink, the field strength on the surface increases and solvated ions get expelled from the droplets In positive ion mode, the proton adducts of the molecule are “desolvated” to form “bare” ions, which then pass through an aperture inside the vacuum region of the mass analyzer Atmospheric pressure chemical ionization- happening at atmospheric pressure not in vacuum nitially. Instead of adding voltage to solvent fro electrospray. Here we have reagent gas that will have a chemical reaction with our sample this ionizes them(gives them a charge). We then have to separate the ions from the reaction so we use heat or another gas to get rid of what we don’t want before it goes into the spec. APCI is similar to ESI however, no voltage is applied to the inlet capillary Instead, a corona discharge needle is positioned on the path of the evaporated HPLC stream, to initiate a cascade of gas phase reactions that ionize compounds through a series of ion molecule reactions, similar to the reactions occurring in CI, but with molecules of the mobile phase constituents serving as reagent molecules Products of these secondary reactions typically contain clusters of solvent and analyte molecules, so a heated transfer tube or a countercurrent flow of an inert gas and potential differences are used to de-cluster the ions. Inductively couple plasma- considered hard, this fragments our molecules so much they turn to pure atoms at the atomic level. We like this when working wit elemental analysis for trace metal identification. This is for pure elements. We have to use acid digestion. We immerse it in acid and then we have to nebulize it or turn to vaoour and them it is introduced to hot plasma, it then gets its ionization from their, then it gets introduced to the analyzer Is a “hard” atmospheric pressure ionization method typically leading to complete atomization of the sample Its primary use is for elemental analysis ICP based instruments are extremely sensitive and are capable of a wide dynamic range The sample is typically prepared by acid digestion, and is introduced into the ion source via a nebulizer fed by a peristaltic pump The nebulized sample is transmitted into hot plasma maintained at atmospheric pressure by a high-power radio frequency (RF) generator A small orifice samples the plasma, and ions are transmitted to the vacuum region of a mass analyzer Matrix- Assisted Laser Desorption/Ionization (MALDI)- you take sample bacterial source and mix with a matrix gel, and you put on metal plate then let it dry, you put it into ypue sec and there is a lser that hits plate that breaks off analyte and matrix, from being in the matrix and broken off they have been analyzed and are now ready to go to the analyzer A “soft” ionization technique using a laser energy absorbing matrix to create ions The sample should be mixed with a solution of matrix , typically a low-molecular-weight UV-absorbing compound, placed on a target, and dried. As the liquid evaporates, the matrix crystallizes and incorporates analyte molecules in the crystals A pulsed laser irradiates the sample, triggering rapid vaporization and desorption of the sample and the matrix material; the analyte molecules are ionized in the plume of ablated gases, and are introduced into a mass analyzer Mass Analyzers Quadrupole- most common, has four rods, two pos across from each other and teo negatives. It is preset as to what ion we want to detect, what we want to detet will be allowed to pass through the rods and put through the analyzer, any component we don’t want will be thrown into the poles and will be destroyed The actual measuring of the m/z occurs when the gas phase ions pass into the mass analyzer Quadrupole- most common mass analyzer in use The electric field on the two sets of diagonally opposed rods allows only ions of a single selected m/z value to pass through to the detector. All other ions are deflected into the rods Ion Trap- similar to quadropole but this one is spere or square and traps the ionized molecules, they sit and wait after time period they will be ejected based on mass to charge ratio. We can ste what we are looking for specifically. They will sit and wait, once they are accumulated they will be sent out to the detector. This is more sensitized becase we can collect more fragments and more molecules. There is more time for those particles to reach the analyzer and be detected. Similar to a quadrupole analyzer A linear ion trap employs a stopping potential on the end electrodes to confine ions along the 2D axis of the quadrupoles In 3D, the four rods form a 3D sphere in which ions are “trapped” After a period of accumulation, the electric field adjusts to selectively destabilize the trapped ions, which are mass selectively ejected from the cavity to the detector based on their m/z. Tandem Mass Spectrometry- three quadropole analyzers in a row, in first, the sample that has been analyzed goes there at this pont we can select one ion of interest called the paents ion. It then goes to seconf where it collides with neutral gas and fragments even more called daughter ions. The third does all the analysis of wjat happede with the second quadropole or everything that happened. We could detect all ion products if we choose to. Analyzes all og the products, or it will only analyze what we set it to. Can be used for greater selectivity and lower detection limits A common form is to link three quadrupoles in series; such an instrument is referred to as a triple quad Following ionization, the first quadruple (Q1) is used to scan across a pre-set m/z range and select an ion of interest The second quadrupole (Q2) functions as a collision cell- ions are allowed to collide with neutral gas molecules to fragment the ions The third quadrupole (Q3) serves to analyze the ions generated in Q2. High Resolution- these types of instruments cn identity elements up to very small mass, they are very sensitive. They can figure out mass to 3-4 decimal places. We use this when we have an unknown analyte These instruments can measure large numbers of analytes simultaneously in complex biological matrices and have been particularly using for drug screening applications Traditional mass specs can determine masses to ~0.5 Da, whereas high-resolution instruments allow the exact mass of an unknown compound to be calculated to ~0.001 to 0.0001 Da. Detector- the make up of the detecter which is electron multiplier, instead of photons we have elctrones, they hit the dinodes and we have extrapolation of the ions. This produces a cascade or a chain and how much is multiplied goes to dectector and is converted to concentration The most common means of detecting ions employs an electron multiplier In this detector, a series of dynodes with increasing potentials are linked A cascade of electrons is formed by the end of the chain of dynodes, resulting in overall signal amplification on the order of 1 million or greater. Applications of MS in the Clinical Laboratory Small Molecule Analysis- we often couple mass specs with gas chromatography or liquid. Gc is to quantitate drugs of abuse, usually urine sample. The high resolution mass spec is for unknown toxicology an example their was a patient intoxicated they did usuall drug screen testing and they came back negative, they had to send out the sample they got results and the patient got drunk off of listerine, they were able to identify it Mass specs coupled to GC or LC can be used not only for the identification and quantitation of compounds but also for structural information and molecular weight determination GC/MS systems are widely used for measuring drugs of abuse in urine toxicology confirmations Drugs and metabolites must be extracted from body fluids and reacted with reagents to form compounds that are more volatile High-resolution MS has applications in unknown toxicology analysis- enable ID of drugs by identifying the compounds molecular weight to 3 or 4 decimal places LC/MS is used for measuring low-level and mixed polarity analytes such as vitamin D, testosterone, and immunosuppressant drugs In Proteomics and Pathogen ID- proteomics study of protein products, if we have gentic mutation we can use this to identify genetic diseases and diseases wih proteins. We compare samples to a librabry that we have and are looking for post translational modifications. Changes with our protein structure or if there are abnormality can be identified with mass spec Biomarkers for human diseases are found in the field of genomics (uses known sequences in entire human genome) and proteomics (protein products encoded by genes). A “shotgun” approach is often used in the discovery of new biochemical markers- proteins from samples from normal individuals are compared with those derived from patients with disease Complex mix of proteins are first separated using chromatographic techniques Proteins that only appear in either the normal or diseased specimens are further studied The proteins are then isolated and subjected to HPLC-MS/MS or to MALDI-TOF MS to identify the protein and possibly any posttranslational modifications that may have occurred MALDI-TOF- tof is time of flight. Take sample mix with matrix and let dry, use laser to get ions from it. Reason for tof is because the smaller ions will fly through faster than larger do id is based on time of flight. Used for the analysis of biomolecules, such as peptides and proteins Samples are mixed with an appropriate matrix solvent and spotted onto a stainless steel plate Solvent is dried and the plate is introduced into the vacuum system of the analyzer A laser irradiates the sample, causing desorption and ionization of both the matrix and sample Ions from the sample are focused into the mass spectrometer The time required for an ion with a given mass to reach the detector is a nonlinear function of the mass, with larger ions requiring more time than smaller ions The molecular weight of the proteins is used to determine the ID of the sample and any posttranslational modifications that may have occurred Pathogen ID- if there is bacteria we don’t know r fungus we can throw on maldi tof and can id based on known graphs. Sometimes there is weird bacteria Mainly used in the microbiology laboratory Isolated bacterial or fungal colonies can be directly spotted onto the MALDI plate and ionized, which results in a protein “fingerprint” of the species This “fingerprint” can be compared with a digital database of species to identify the pathogen