Baixe 01 Fundamental LC - MS Introduction e outras Notas de estudo em PDF para Química, somente na Docsity! electronic analytical reference library Fundamental LC-MS Introduction Learning Aims & Objectives Aims Introduce fundamental LC/MS concepts Explain the function of each major component of the LC/MS system Indicate the major advantages of LC/MS and the application areas in which it is used Objectives Atthe end of this Section you should be able to: Describe the function of the various elements that are present in a typical LC/MS system List and explain the two main considerations common to all interface types List the most common interfaces and be able to clearly describe the differences between them List the most common mass analyzer types there is interactive material that cannot be fully shown in this reference manual. [E] Wherever you see this symbol, it is important to access the on-line course as electronic analytical reference library Content Definitions Instrument Fundamentals Process Why and when to use LC/MS HPLC separations MS detection MS detection Ionisation Overview Atmospheric Pressure lonisation (API) Electrospray lonisation Atmospheric Pressure Chemical lonisation (APCI) Atmospheric Pressure Photo lonisation (APPI) Mass analysers Quadrupole Time-of-flight (TOF) Ion Trap Mass Analyser Tandem mass spectrometry (MS/MS) Detectors Point detectors Array detectors Applications References O Crawford Scientific SONNJNDDDOU A O electronic analytical Where: 1. lon source: The HPLC eluent is sprayed into the atmospheric pressure region 2. Skimmer Cone: A cone with a sampling orifice of reduced diameter to preferentially sample gas phase ions and reduce the gas load entering the vacuum system of the mass analyser device. 3, Quadrupole: Device that uses electric fields in order to separate ions according tot their mass to charge ratio (m/z) as they pass along the central axis of four parallel equidistant rods. 4.Collision Cell: lons emerging from the first mass analyser are accelerated using a potential difference and collide with neutral gas molecules such as Ho, N> or Ar, causing analyte fragmentation. 6. Detector: Once produced and separated, the ions need to be detected and transformed into a usable signal. Electron multiplier, Dynode, Photodiode, and Multi Channel Plate (MCP) ion detection systems are widely employed in most modern mass spectrometer systems. 7. Vacuum system: Mass analysers require high levels of vacuum in order to operate in a predictable and efficient way. The vacuum systems of most modern LC-MS systems consist of two or more differentially pumped vacuum chambers, separated by baffles or orifice plates of varying design depending upon the instrument manufacturer. Process There are several discrete stages in LC-MS analysis, typically these include: Separation of the sample components using an HPLC column where the analytes are differentially partition between the mobile phase (eluent) and the stationary phase (coated onto a support material and packed into the column). The mechanism of retention and separation will depend on the mode of chromatography but may include, Hydrophobic Interaction, lon Exchange, lon-Pair, Surface Localisation, etc. The separated sample species are then sprayed into an Atmospheric Pressure lon Source (API) where they are converted to ions in the gas phase and the majority of the eluent is pumped to waste. The mass analyser is used to sort ions according to their mass to charge ratio. Most popular analyser types include Quadrupole (shown opposite), Time of Flight, Ion Trap and Magnetic Sector. The mass analyser may be used to isolate ions of specific mass to charge ratio or to 'scan' over all ion m/z values present. The detector is used to 'count' the ions emergent from the mass analyser, and may also amplify the signal generated from each ion. Widely used detector types include: electron multiplier, dynode, photodiode and multi-channel plate. All mass analysis and detection is carried out under high vacuum established using a combination of foreline (roughing) and turbomolecular pumps. O Crawford Scientific 5 ef: pf) pa fec: M gca folia Teto ig From HPLC System HPLC Column [i] [E LC/MS system Why and when to use LC/MS The use of LC-MS in many application areas within analytical science continues to grow almost exponentially. Listed below are some pointers as to the applicability of both HPLC as a separative technique and MS as a means of detecting analyte species. HPLC separations e For HPLC analysis the analyte must be soluble in the mobile phase e HPLC can analyse samples over a wide polarity range including ionic samples * HPLC has no real upper molecular weight limit and large proteins of many thousands of Daltons may be analysed. Solubility in the mobile phase may preclude the analysis of very large molecules. The ESI LC/MS spectrum of the Locusta migratoria cuticular protein! (16512 Da) is presented below * HPLC samples are prepared in a solvent system that has the same or less organic solvent than the mobile phase and injection volumes of 1 to 50 ul are common (1-104ug of analyte per 1g packing material) + Relativa x» IM+H] Intansieçõe MH] 16512.5 a000"! B241.8 15256.6 ) 1 173527 À x ooo 1000 1200 1400 1800 1800 2006 miz ESI LC/MS spectrum of Locusta migratoria cuticular protein (16512 Da)? MS detection e Allows specific compound identification (structural elucidation via spectral interpretation combined with elemental composition from accurate mass analysers is possible) * Very sensitive (fempto-gram amounts have been detected by certain mass analyzer types) * Highly selective (certain analyzer and experiment combinations can lead to highly selective and sensitive analysis of a wide range of analytes) O Crawford Scientific 6 ic analytical reference ig ry Ionisation Overview lonisation is the process whereby electrons are either removed or added to atoms or molecules to produce ions. In LC-MS charge may also be applied to the molecule via association with other charged molecules —for example a proton (H”). Such ions are produced in LC/MS systems by the use of strong electric fields in the vapour or condensed phase. Interfaces whereby the sample is ionised or desolvated under atmospheric pressure conditions are termed Atmospheric Pressure lonisation (API). The most common ionisation methods in LC-MS include: * Electrospray lonisation (ESI) -ionisation in the condensed phase e Atmospheric Pressure Chemical lonisation (APCI) -ionisation in the gas phase e Atmospheric Pressure Photo lonisation (APP!) —ionisation in the gas phase Sample Introduction Lo D—— lon Source I Interface hlags Analyser Detector High Yacuum Data Syatem Mass Spectrometer « Yacuum System [O Where the “Flow rate” label denotes the effluent (analyte plus eluent and additives) coming from the HPLC system. O Crawford Scientific 7 ic analytical reference library Atmospheric Pressure Chemical lonisation (APCI) Atmospheric Pressure chemical lonisation uses analyte desolvation and charge transfer reactions in the vapour phase to produce vapour phase analyte ions. In APCI the eluent is introduced into the interface using a capillary of similar design to the ESI source. However, no potential is applied to the capillary but instead the liquid emerges from the capillary surrounded by a flow of inert, nebulising gas into a heated region. The combination of nebulising gas and heat forms an aerosol that begins to rapidly evaporate. A pin is placed within the heated region that has a high potential applied to it and produces an electrical discharge that ionizes eluent molecules, these ionized molecules impart charge to the analyte molecules via charge transfer reactions or molecular association. Both ESI and APCI are termed “soft” ionisation methods. This means that in the process of producing ions there is negligible energy transferred to the ion. As a consequence the ion formed does not fragment to small mass ions. The resultant mass spectrum therefore consists predominantly of pseudomolecular ions, either [M+H]* or [M-H]' or adduct ions like [M+Naj. Heated Vaporiser Tube Corona Electrode Pin APCI process The ionised form of the molecule: MS M* +e The M* is known as the molecular ion. Note that molecular ions do not typically occur in LC/MS O Crawford Scientific 10 Ion: | fo qa (or: asa ps gp ot aloe Pseudomolecular ion formation If the analyte (M) has a larger proton affinity than the solvent (S), then: M+[S+H] > [M+HJ +S If the analyte (M) has a lower proton affinity than the solvent (S), then: M+[S-H/ > [M-HV+S Atmospheric Pressure Photo lonisation (APPI) APPI, is a complement to ESl and APCI and has been developed to broaden the range of ionizable analytes at atmospheric pressure. APPlI is important in the analysis of certain compounds that are not easily ionisable by ESI or APCI like low- and non-polar compounds (APPI has been used in the analysis of polycyclic aromatic hydrocarbons). In APPI, the ionisation process is accomplished by exposing an aerosol of droplets to photoirradiation. A molecular radical ion is formed when the molecule absorbs a photon. This process is possible only when the irradiating Photon (of energy ) exceeds the ionization potential (IP) of the molecule. APPI, allows the formation of charged species in positive or negative ion mode, these two mechanisms will be explained in detail in a subsequent chapter. Heated Vaporiser Tube Lamp APPI process O Crawford Scientific 1 ic analytical reference library Mass analysers In its simplest form the process of mass analysis in LC/MS involves the separation or filtration of analyte ions or fragments of analyte ions created in the Atmospheric Pressure lonisation (API) interface or in the regions between the API interface and the high vacuum region of the mass analyser (products of collision-induced dissociation etc.). Sample Introduction Lo lon Source À Interface Mass Analyser Detector High Yacuum Data Syatem There are several very popular types of mass analyser associated with routine liquid chromatography mass-spectrometric analysis and all differ in the fundamental way in which they separate species on a mass-to-charge basis: Quadrupole and lon Trap Mass analysers: ions are filtered using electrostatic potentials applied to the elements of the mass analysers which are used to 'select ions according to their mass to charge ratio —non-selected ions are elected from the mass analysing device and are not detected. Time of Flight (TOF) mass analysers: use differences in flight times of accelerated ions through an extended flight path to separate ions. Magnetic Sector Mass Analysers: use magnetic fields to select ions by directing the beam of ions of interest towards the detector. The analyte and fragment ions are plotted in terms of their mass-to-charge ratio (m/z) against the abundance of each mass to yield a mass spectrum of the analyte as shown. Relative abundance m/z Mass spectrum O Crawford Scientific 12 electronic analytical reference library End-Cap Electrode Gate Control [i] Ring Elecirode Preamplifier lon Sigina Electron ! auR hultiplier Detector lon trap mass analyser Ion trap mass analyser Advantages Disadvantages * High sensitivity * Produces very unusual spectra if the ions are * Multiple Product lon scan stored in the trap too long. capability (MS)” e Easily saturated * High resolution * Poor for low mass work (below 100 Da) * Good for DDA analyses e Poor dynamic range (except the most modern devices) and hence may have limited quantitative use O Crawford Scientific 15 ef: pf) pa fec: M gca folia Teto ig Tandem mass spectrometry (MS/MS) MS/MS is the combination of two or more MS experiments. The aim is either to get structural information by fragmenting the ions isolated during the first experiment, and/or to achieve better selectivity and sensitivity for quantitative analysis by selecting representative ion transitions using both the first and second analysers. MS/MS analysis can be achieved either by coupling multiple analysers (of the same or different kind) or, with an ion trap and carrying out successive fragmentations of trapped ions. MS" (should read MS to the n) is an acronym that refers to multiple ion production and filtering within a single instrument. Most common instruments use a combination of quadrupoles (as shown below) with a collision cell (usually a multi-pole device) between the analyzing devices in which the emergent ions from the first analyzer are fragmented prior to secondary mass filtering. Other combinations of mass analysing devices such as quadrupoles and time of flight, or quadrupoles with magnetic sector instruments are possible. q2 . lon Bridge OM Static Colision cell og Scanning Tandem mass spectrometry Detectors Once the ions have passed the mass analyser they have to be detected and transformed into a usable signal.” The detector is an important element of the mass spectrometer that generates a signal from incident ions by either generating secondary electrons, which are further amplified, or by inducing a current (generated by moving charges). lon detector systems fall into two main classes: Point detectors: ions are not spatially resolved and sequentially impinge upon a detector situated at a single point within the spectrometer geometry. Array detectors: ions are spatially resolved and all ions arrive simultaneously (or near simultaneously) and are recorded along a plane using a bank of detectors. O Crawford Scientific 16 ic analytical reference libr: ry Sample Introduction lon Source | Interface [>] Mass Analyser | >] Detector High Vacuum Temporally resolved —— e o lons Point Detector Spatially resolved co array lons Detector Applications To give a full list with the applications of LC/MS is simply impossible, its flexibility makes it attractive in a lot of different fields. Some interesting applications are listed below. Proteomics:!” systematic analysis of proteins. +10 100 o! 1000 1200 1400 dio 1800 2000 2200 2400. Negative ESI-MS spectrum of holo-siderocalin (21195 + 1 Da) Although mass analyzing devices have a practical upper molecular weight limit of around 5KDA (5000Da), Electrospray MS is capable of imparting multiple charges onto the analyte ion. As analysing devices select ions based on mass to charge ratio (m/z), this protein is capable of holding 12 charges (12+), effectively lowering the “apparent molecular weight to 21195/12 = 1766 O Crawford Scientific 17 Yo ic analytical reference library Food analysis:!! protein characterization, and determination of organic food stuff (enzymes, flavonoids, etc). Total ion chromatograms of cocoa procyanidins obtained after postcolumn addition of 10mM ammonium acetate in both positive and negative electrospray ionization modes. Petrochemistry:!ºl analysis of oilderived molecules (like hydrocarbons) and contaminants. f & 10 12 14 Timeímin) Analysis of nitropyrene 247 Da present in urban soil. Negative ion chemical ionisation. 1-Nitropyrene and its isomers (environmental contaminants) are well known for its carcinogenic properties. Considerable amounts of these compounds have been detected in diesel engine exhausts. O Crawford Scientific 20 ic analytical reference library Cosmetics.!"! NH OH A 184 o 305 454 asa A N $124 é Bal a ata ê 5 & es E 34 23 ad paul 4 1 T T T T T T 200 250 300 350 400 450 so miz Piridoxamine is used in cosmetics to avoid proteins modification. The figure presents the chromatogram (direct injection positive ESI LCMS) of an aqueous solution of piridoxamine. Doping.! 100 RT3A0 cl NH Han UH % e Clenhuteral o ú 1 2 3 4 5 6 Time (min) Analysis of clenbuterol from a urine sample by (+) APCI In order to increase their performance, clenbuterol has been illegally used for some competitors. O Crawford Scientific 21 electronic analytical reference libra References 1. HPLC Channel of the Electronic Analytical Reference Library (EARL). 2. Dário Eluan Kalume, Sylvie Kieffer, Kate Rafn, Lene Skou, Svend Olav Andersen, Peter Roepstorff. “Sequence determination of three cuticular proteins and isoforms from the migratory locust, Locusta migratoria, using a combination of Edman degradation and mass spectrometric techniques.” Biochimica et Biophysica Acta 1645, (2003), 152- 163. 3. De Hoffmann, J. Charette, and V. Stroobant. “Mass Spectrometry. Principles and Applications.” John Wiley and Sons. Pp 91-97, 1996 4. Catalin E. Doneanu, Roland K. Strong, and William N. Howald. “Characterization of a Noncovalent Lipocalin Complex by Liquid Chromatography/Electrospray lonization Mass Spectrometry” Journal of Biomolecular Techniques, volume 15, issue 3, September 2004. Pp 208 - 212 5. Birgit Eiermann, Per Olof Edlund, Agneta Tjernberg, Per Dalén, Marja-Liisa Dahl, and Leif Bertilsson. “1- and 3-hydroxylations, in addition to 4-hydroxylation, of debrisoquine are catalyzed by cytochrome p450 2d6 in humans” drug metabolism and disposition. Copyright O 1998 by the American Society for Pharmacology and Experimental Therapeutics. Pp 1096 — 1101 6. William J. Griffiths, Andreas P. Jonsson, Suya Liu, Dilip K. RAI and Yugin Wang. “Electrospray and tandem mass spectrometry in biochemistry” Biochem. J. (2001) 355, 545+561 (Printed in Great Britain). 7. Mayumi Nishikawa, Munenhiro Katagi, Akihiro Miki, and Hitoshi Tsuchihashi. “Forensic Toxicological Determination of Surfactant by Liquid Chromatography/Electrospray lonisation Mass Spectrometry and Liquid Chromatography/Electrospray lonisation Tandem Mass Spectrometry” Journal of Health Science 49(2) 138 — 148 (2003). 8. A. Agúera and A.R. Fernández-Alba. “GC-MS and LC-MS evaluation of pesticide degradation products generated through advanced oxidation processes: An overview” EDP Sciences, Wiley-VCH. Analusis Magazine, 1998, 26, Nº6. Pp M123 — M130. 9. Yun-leong Hong, Diane M. Barrett, and Alyson E. Mitchell. “Liquid Chromatography/Mass Spectrometry Investigation of the Impact of Thermal Processing and Storage on Peach Procyanidins” Journal of Agricultural Food Chemistry 2004, 52, 2366-2971. 10. Hiroshi Moriwaki, Kunihiro Funasaka and Michiko Uebori. “Direct Determination of NO>-Substituted Pyrenes by Liquid Chromatography-Atmospheric Pressure Chemical lonization-Mass Spectrometry” Analytical Sciences December 2000, Vol 16, Pp 1247 - 1248. O The Japan Society for Analytical Chemistry. 11. Joelle M. Onorato, Alicia J. Jenkins, Suzanne R. Thorpe, and John W. Baynes. “Pyridoxamine, an Inhibitor of Advanced Glycation Reactions, Also Inhibits Advanced Lipoxidation Reactions. Mechanism of Action of Pyridoxamine” The Journal of Biological Chemistry. Vol. 275, No. 28, Issue of July 14, pp. 21177-21184, 2000. 12. Mark Churchill and Mark Harrison. “High Resolution Separation with Accurate Mass Determination of Two Co-Eluting Controlled Drugs of Abuse with Isobaric Mass” Chromatography and Mass Spectrometry, Application Note 329. Thermo Electron Corporation. O Crawford Scientific 22