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Evolving Role of Mass Spectrometry in Bioanalytical Analysis Daniel Pentek February 1, 2007 -UCONN Bioanalytical Chem 395
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Feb. 1, 2007 Topics to be covered: Quick Review- MS Market & Technologies Basic ionization methods Electrospray, APCI, APPI Interfaces Orifice, capillary MS Analyzers Performance Criteria Strengths & weaknesses for bioanalytical analyses Quadrupole (single &triple quads) Ion traps (3-D and 2-D) Time of flight (linear, reflectron, MALDI) Hybrids (Quad-TOF) and others… FTMS (ICRs and orbitraps)
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Bioanalytical Chem 395 Feb. 1, 2007 What is a mass spectrometer? An instrument that separates molecules or atoms (e.g., ICP/MS) based on their mass/charge ratio (m/z). In order to control them, you need to put a charge on them. In order to separate them, you need an “analyzer” of some type…
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Bioanalytical Chem 395 Feb. 1, 2007 LC/MS Historical Perspective Industry was desperate for a decent, rugged LC/MS interface. GC/MS required derivatization, etc. Not applicable to most biomolecules (MW, etc.) API ionization ALONG WITH new interface designs provided the solution. Now, all interfaces are differentially pumped. Pumping of interfaces was critical Orifice – skimmer (nozzle) designs Heated (or cold) capillary designs LC/MS is big business now!
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Bioanalytical Chem 395 Feb. 1, 2007 Analytical Instruments Technology Segments… SDI data on analytical instrument technology shows mass spectrometry is a $2B market in 2006, and predicts… …that it will have fastest growth rate (8.3%) of any analytical instrument technology through 2010
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Bioanalytical Chem 395 Feb. 1, 2007 Distribution of Mass Spectrometry Techniques* *SDI Global 9 th Ed. Sept. 2006 PKI has product offerings * $255M CGR 6.2% $101M CGR 2.3% $643M CGR 9.5% $312M CGR 9.4% $139M CGR 17.0% $136M CGR 9.7% $391M CGR 4.9% $241M CGR 7.2% $61M CGR 3.8% Avg. CGR >10% Growth Opportunities
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Bioanalytical Chem 395 Feb. 1, 2007 LC/MS(/MS) is a Marriage of Liquid Chromatography and Mass Spectrometry Marriage (like any other close relationship) requires: COMPROMISE! LC Person: “MS is just another detector” MS Person: “LC is just an inlet” What’s good for LC may not be good for MS and vice versa. LC was around a long time before they figured out how to interface it to an MS.
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Bioanalytical Chem 395 Feb. 1, 2007 LC/MS Instrument Basics: Single MS- Primarily used as a detector Ion SourceInterfaceMass AnalyzersDetection -ESI -APCI -APPI -Orifice – Sk -Capillary (Hot or cold) 1- Quadrupole 2- Ion Trap (4 types) 3- Time of Flight CEM Discrete dynode CCD Photomultiplier LC MS System – under vacuum
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Bioanalytical Chem 395 Feb. 1, 2007 Today’s LC/MS Ionization Methods All Done at Atmospheric Pressure On-line techniques: Electrospray (ESI) - Fenn @ Yale ~1984 Shared Nobel Prize in 2002 for this work with K. Tanaka (MALDI) and K. W ü thrich (NMR) Atmospheric Pressure Chemical Ionization (APCI) Irabarne & Thomson ~1979 Atmospheric Pressure Photo Ionization (APPI) Emerging, not as widely used yet. All of the above are done at atmospheric pressure Significant change from traditional ionization methods which were all done within the vacuum chamber.
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Bioanalytical Chem 395 Feb. 1, 2007 Ionization Techniques – Application range Electrospray APPI
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Bioanalytical Chem 395 Feb. 1, 2007 Electrospray Basics (Spraying a charged “mist”) Turbo Gas ~10,000,000 ions on column ~ 4,000,000 - 40,000 ions Operator Impact Area ~1000 ions (IonSpray is a AB-Sciex trademark name for nebulizer assisted electrospray.)
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Bioanalytical Chem 395 Feb. 1, 2007 Electrospray – Based on Ion Evaporation Theory Rayleigh Limit = 10 cm 2 /V -The key is to get rid of the solvent before the ion enters the MS. -The higher the mobile phase flow rate, the more gas and heat that is required.
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Bioanalytical Chem 395 Feb. 1, 2007 Electrospray Take home message: Electrospray is concentration dependent technique. Started out as very low flow technique, which wasn’t very compatible with LC. LC Person: “Use lower flows and narrower column”. MS Person: “Buy ESI probe that has higher gas flows and desolvates better”. Ease of use and higher LC flow compatibility drove source development.
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Bioanalytical Chem 395 Feb. 1, 2007 Example: High Flow Electrospray Source AB-Sciex TurboIonSpray Source
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Bioanalytical Chem 395 Feb. 1, 2007 Example: High Flow Electrospray Source AB-Sciex TurboIonSpray Source Electrospray Probe Heater Gas Probe High Voltage Connector Temperature and Source ID Connector
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Bioanalytical Chem 395 Feb. 1, 2007 Newer Ion Sources are have Orthogonal Design: 90º to Ion Entrance (Orifice) of MS.
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Bioanalytical Chem 395 Feb. 1, 2007 Electrospray- Tips Modifiers Organic acids (e.g. formic, acetic) promote ionization of basic compounds (sp 3 N- containing) Neutral compounds containing nucleophilic lone pairs (sp 2 N, sp 3 O) can be desorbed by cationization with alkali metal or ammonium ions. Ammonium formate or acetate are recommended as buffers ( 2-10 mM optimum, can see suppression effects over 20 mM)
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Bioanalytical Chem 395 Feb. 1, 2007 Electrospray- Tips Modifiers (cont.) Salts can interfere with ionization and can cluster to complicate spectrum (but also aid in identification) Strong bases or quaternary amines can interfere with positive mode analytes Sulfonic acids interfere with negative mode analytes DO NOT USE PHOSPHATE BUFFERS
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Bioanalytical Chem 395 Feb. 1, 2007 Atmospheric Pressure Chemical Ionization (APCI) (“Steam distill” LC eluent past a HV needle) Liquid flows up to 2 mL/min are handled by using 2 additional gas flows and heat. To MS To the MS…
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Bioanalytical Chem 395 Feb. 1, 2007 Heated Nebulizer – APCI Probe Designed to Deliver Mist to Needle
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Bioanalytical Chem 395 Feb. 1, 2007 Atmospheric Pressure Chemical Ionization (APCI) Basics (“Steam distill” LC eluent past a HV needle) APCI utilizes corona discharge APCI is a “three” step process: 1) Needle at high voltage ionizes nebulizing gas (air or nitrogen) forming primary ions 2) Primary ions react immediately with solvent molecules forming reagent ions 3) Reagent ions react (by proton transfer) with analyte molecules forming (M+H) + in positive ion mode or (M-H) - in negative ion mode
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Bioanalytical Chem 395 Feb. 1, 2007 Atmospheric Pressure Chemical Ionization (APCI) Corona discharge example - positive ion 1) EI on atmosphere cause e - removal from N 2, O 2 forming N 2 +,O 2 + 2) In a complex series of reactions N 2 +,O 2 + react with H 2 O, CH 3 OH forming H 3 O + and CH 3 OH 2 + as reagent ions for CI. 3) H 3 O +, CH 3 OH 2 + donate protons to analyte forming [M+H] +
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Bioanalytical Chem 395 Feb. 1, 2007 APCI- Tips Buffers: Buffers/modifiers not required for ionization Volatile buffers tolerated up to 50 mM Very polar modifiers may reduce sensitivity to less polar analyte
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Bioanalytical Chem 395 Feb. 1, 2007 APCI- Heated Nebulizer Summary HN is a high flow (0.5-2.0 mL/min.) inlet Suitable for polar, thermally stable cmpds Usually, MW < 1000 amu Probe is heated to facilitate vaporization Requires nebulizing and auxiliary gas Requires corona discharge needle to produce ionization (APCI)
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Bioanalytical Chem 395 Feb. 1, 2007 ESI or APCI? - Which is better? For some applications, the choice is obvious… For analytes <1000 Da, you had to try both and see which one yielded the best sensitivity. Now, vendors are starting to offer “dual mode” sources to speed up method development…
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Bioanalytical Chem 395 Feb. 1, 2007 Dual Mode Source…
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Bioanalytical Chem 395 Feb. 1, 2007 Atmospheric Pressure Photo Ionization (APPI) Emerging technique (about 5 years old) Uses typical 10 eV UV lamp (similar to Photo- ionization lamps for GC). Similar to APCI, but applicable to broader range of compounds.
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Bioanalytical Chem 395 Feb. 1, 2007 LC/MS Instrument Basics: Interfaces- 2 basic designs… Ion SourceInterfaceMass AnalyzersDetection -ESI -APCI -APPI -Orifice – Sk -Capillary (Hot or cold) Quadrupole Ion Trap Time of Flight Magnet CEM Discrete dynode CCD Photomultiplier LC MS System – under vacuum
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Bioanalytical Chem 395 Feb. 1, 2007 Example: Orifice-Skimmer Interface (AB-Sciex) Differentially pumped +
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Bioanalytical Chem 395 Feb. 1, 2007 Vacuum Interface: Curtain Gas & Differentially Pumped Interfaces
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Bioanalytical Chem 395 Feb. 1, 2007 Vacuum Interface: Curtain Gas &Differentially Pumped Interfaces UHP nitrogen curtain gas (CG) keeps non- ionized species out of the orifice and analyzer region CG aids in ion declustering (with CID potentials) Two stage transition from atmosphere to low pressure region of analyzer (1 x 10 -5 torr) Curtain gas and ions are drawn in due to; Pressure differential (both ions & CG) Electric field gradients (ions only)
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Bioanalytical Chem 395 Feb. 1, 2007 LC/MS Instrument Basics: Ion SourceInterfaceMass AnalyzersDetection -ESI -APCI -APPI -Orifice – Sk -Capillary (Hot or cold) Quadrupole Ion Trap Time of Flight Magnet CEM Discrete dynode CCD Photomultiplier LC MS System – under vacuum Mass analyzers- where most of the differences occur…
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Bioanalytical Chem 395 Feb. 1, 2007 Analyzer (and System) Criteria Analyzer Considerations: Resolution Mass accuracy Scan speed Dynamic range System Considerations: “Sensitivity” Sample thru-put – Fast LC? Primary application: quantitation, qualitative or… Software- application based Ease of use!!! Price…
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Bioanalytical Chem 395 Feb. 1, 2007 Single MS and MS/MS Systems: Single MS systems (quadrupole or TOF) Have the added dimension of mass vs. UV or diode array detectors. However, chemical noise is the limiting factor for sensitivity (S/N) and dynamic range. “MS/MS Systems” Many variations now… 2 quadrupole MS’s, separated by a collision cell Quadrupole front end, collision cell, TOF back end TOF-TOF, separated by collision cell “MS n ” analyzers- ion traps, FTMS’s and others… Purposes essentially the same (regardless of analyzer type), select one ion from all the others, fragment it, and study the fragment ions
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Bioanalytical Chem 395 Feb. 1, 2007 2003 SDI- Price vs. Resolution SDI MAP October 2003
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Bioanalytical Chem 395 Feb. 1, 2007 2003 SDI- Price vs. Mass Range SDI MAP October 2003 Mag Sector Q-TOF TQ QTrap IT Nth LC-TOF Gen Purpose
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Bioanalytical Chem 395 Feb. 1, 2007 2003 SDI- Mass Range vs. Resolution SDI MAP October 2003
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Bioanalytical Chem 395 Feb. 1, 2007 Resolution- What is it? Ability to separate (resolve) adjacent ions Typically defined as: M/∆M M: Mass∆M: Full Width at Half Max. Quadrupoles: scan at constant peak width 30/1=30, 300/1=300, 3000/1=3000… Resolution increases as you go to higher mass TOFs: scan at constant resolution 10k res: m/z 10.001, 100.01, 1000.1, 10,001 Peak width increases as you go to higher mass
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Bioanalytical Chem 395 Feb. 1, 2007 Analyzer Types: Quadrupoles (and hexapoles, octopoles, etc.) Fundamental parts of virtually all LC/MS systems Serve one of two purposes: Ion transmission devices (quad, hex, oct…) Capture and transmit ions from one place to another… Ion filtering devices (quadrupoles only…) Act as a mass filter (analogous to a magnet)
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Bioanalytical Chem 395 Feb. 1, 2007 Quadrupole Theory Quad. as a mass filter Separates ions based on m/z ratio Quad. made of 4 rods “A pole” - vertical rods; “B pole” - horiz. rods (by convention) DC, RF volt. imposed: U=(DC) A -(DC) B (FDC) V: RF volt. peak-peak (RF p-p ) V = 7.22 * M * r 0 2 * f 2 ; i.e., V ~ M
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Bioanalytical Chem 395 Feb. 1, 2007 Quadrupole Theory (cont.) Resolution (pk width) of quad. defined by: M/ M =0.126/[0.168-U/V] M:Mass M:Full Width at Half Height As slope approaches 0.168, resolution approaches infinity (no signal) Resolution at any mass depends on U/V ratio (∆DC / ∆RF p-p )
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Bioanalytical Chem 395 Feb. 1, 2007 Typical “Single quad” MS Ion Path: Basic, “single MS” analyzer
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Bioanalytical Chem 395 Feb. 1, 2007 API-3000 “Triple Quad” Ion Path
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Bioanalytical Chem 395 Feb. 1, 2007 API-3000 Mass Filter Rail- Collision Cell
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Bioanalytical Chem 395 Feb. 1, 2007 All Triple Quads- Collision Cells must Overcome “Crosstalk” Crosstalk: can occur when measuring common fragment from 2 different precursor ions It takes time for ions to exit a collision cell ABI-Sciex uses “Linac” (linear accelerator) Eliminates cross-talk and allows faster MS/MS scanning without sensitivity losses Q2 rods are tilted and separate DC potentials are applied to each pair of rods to create an axial electric field Waters (Micromass) uses “T-Wave” Agilent uses something (they have to…)
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Bioanalytical Chem 395 Feb. 1, 2007 API-3000 Collision Cell - Linac Q2 Linac (linear accelerator) eliminates cross-talk and allows faster MS/MS scanning without sensitivity losses Q2 rods are tilted and separate DC potentials are applied to each pair of rods to create an axial electric field
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Bioanalytical Chem 395 Feb. 1, 2007 AB-Sciex’s LINAC Technology
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Bioanalytical Chem 395 Feb. 1, 2007 Triple Quad Scanning Modes: MS/MS - Product Ion Scan Product ion scan- common MS/MS mode After identification, the precursor ion is sent into the collision cell and fragmented Q1 is fixed, Q3 sweeps a given mass range Used for structural information gathering and identification of product ions First step to developing quantitative method
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Bioanalytical Chem 395 Feb. 1, 2007 MS/MS - Product Ion Scan (cont.) Product Ion Scan m1+m1+ m2+m2+ m2+m2+ m2+m2+ Product ion spectrum of a particular compound m 1 + set m 2 + scan
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Bioanalytical Chem 395 Feb. 1, 2007 Triple Quad Scanning Modes: MS/MS - Precursor Ion Scan Precursor ion scan Q1 sweeps a given mass range, Q3 is fixed Used to determine the “origin” of particular product ion(s) created in the collision cell Frequently used for drug metabolite identification (common product ion observed in the metabolites)
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Bioanalytical Chem 395 Feb. 1, 2007 MS/MS - Precursor Ion Scan (cont.) Precursor Ion Scan m1+m1+ m2+m2+ m1+m1+ m1+m1+ A set of compounds with a common product ion m 1 + scan m 2 + set
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Bioanalytical Chem 395 Feb. 1, 2007 Triple Quad Scanning Modes: MS/MS Constant Neutral Loss Neutral loss scan Q1 & Q3 both scan a given mass range but with a constant difference between ranges scanned Spectrum indicates which ions lose a neutral species equal to Q1 - Q3 difference Complement to Precursor Ion Scan Neutral “gain” indicates a multiply charged precursor ion was fragmented
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Bioanalytical Chem 395 Feb. 1, 2007 MS/MS Constant Neutral Loss (cont.) Constant Neutral Loss Scan m1+m1+ m2+m2+ m2+m2+ m1+m1+ A set of compounds with a common neutral fragment m 1 + scan m 2 + scan mm -m-m -m-m
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Bioanalytical Chem 395 Feb. 1, 2007 Triple Quad Scanning Modes: Multiple reaction Monitoring (MRM) If Q1 and Q3 width=0, then MRM Many precursor to product ion pairs can be monitored (A-B, A’-B’, A”-B”, etc.) MRM analysis is the best way to maximize signal intensity of product ions MRM used primarily for quantitation studies
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Bioanalytical Chem 395 Feb. 1, 2007 MS/MS - Multiple Reaction Monitoring (MRM) Precursor ion set Product ion set Fragmentation (CAD)
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Bioanalytical Chem 395 Feb. 1, 2007 Triple Quad MS/MS Example…
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Bioanalytical Chem 395 Feb. 1, 2007 MS Analyzer Comparison- Mass Accuracy Quadrupoles
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Bioanalytical Chem 395 Feb. 1, 2007 Analyzer Types: Ion Traps - MS/MS n Systems 3D Ion Trap
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Bioanalytical Chem 395 Feb. 1, 2007 4 “Types” of Ion Traps…
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Bioanalytical Chem 395 Feb. 1, 2007
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Bioanalytical Chem 395 Feb. 1, 2007 3D Ion Trap- MS/MS Operation
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Bioanalytical Chem 395 Feb. 1, 2007 Strengths/Weaknesses of 3D Traps
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Bioanalytical Chem 395 Feb. 1, 2007 Triple Quads vs. Ion Traps Complementary MS/MS Approaches: Tandem in Space: Triple Quads Poor scanning sensitivity Great for quant (MRM) Very selective scans Tandem-in-Time: Ion Traps Very sensitive scanning Only product ion scans Only scanning
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Bioanalytical Chem 395 Feb. 1, 2007 Ion Traps -2D (2002) 3-D Traps “Ion bottles” for optical spectroscopy. Minimize fringing fields to maximize performance. Ion accumulation for enhanced ms sensitivity. High quality mass spectrometer: RCM, 2002, 16, 512- 526. Linear Traps
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Bioanalytical Chem 395 Feb. 1, 2007 AB-Sciex Q TRAP™ System Ion Path N 2 CAD Gas linear ion trap 3-4x10 -5 Torr Dipolar Aux AC Exit IQ3 IQ2 LINAC IQ1 Skimmer Orifice Q0Q1Q2Q3
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Bioanalytical Chem 395 Feb. 1, 2007 Trapping Forces in a Linear Ion Trap Axial Trapping Exit Lens Radial Trapping RF Voltage Axial Trapping DC Voltage Resonance Excitation
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Bioanalytical Chem 395 Feb. 1, 2007 Run Thermo 2D Ion Trap Simulation…
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Bioanalytical Chem 395 Feb. 1, 2007 Linear vs. 3-D Ion Traps: Linear Trap Trapping Efficiency No quadrupole field on center line. Longer flight path. Extraction Efficiency 18-20% (measured) Ion Capacity: 3-D Trap Trapping Efficiency Quadrupole field gives amplitude and phase dependent injection eff’s. ~1 cm to lose injection energy. Extraction Efficiency < 50% (Probably ~30%) Ion Capacity: Linear trap is ~10X better 3-D trap is ~2X better 5-inch linear trap: 45X greater capacity
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Bioanalytical Chem 395 Feb. 1, 2007 FTMS- Ion Cyclotrons (ICR)
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Bioanalytical Chem 395 Feb. 1, 2007 Thermo FTMS (ICR) with 2D Ion Trap Front End…
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Bioanalytical Chem 395 Feb. 1, 2007 FTMS (ICR)…
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Bioanalytical Chem 395 Feb. 1, 2007 FTMS Data Example…
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Bioanalytical Chem 395 Feb. 1, 2007 New Analyzer: FTMS Ion Trap- Orbitrap Finnigan LTQ Orbitrap FTMS
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Bioanalytical Chem 395 Feb. 1, 2007 Finnigan LTQ Orbitrap FTMS
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Bioanalytical Chem 395 Feb. 1, 2007 Orbitrap (Brochure) Data
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Bioanalytical Chem 395 Feb. 1, 2007 2003 SDI- Price vs. Resolution SDI MAP October 2003 Orbitrap
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Bioanalytical Chem 395 Feb. 1, 2007 TOF Analyzer Linear Mode Reflectron Mode Common Ionization Methods for TOF MS MALDI ESI Sample Application ‘Time-of-Flight’ Mass Spectrometry
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Bioanalytical Chem 395 Feb. 1, 2007 Simplified Schematic- TOF-MS The analyser, detector and ionisation source are under high vacuum to allow unhindered movement of ions Operation is under complete data system control analyzer Time-of-Flight (TOF) drift tube
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Bioanalytical Chem 395 Feb. 1, 2007 TOF Mass Analyzer – The ‘Drift Tube’ + - +++++++++ + ++++++++ + + time
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Bioanalytical Chem 395 Feb. 1, 2007 LINEAR MALDI TOF MS
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Bioanalytical Chem 395 Feb. 1, 2007 REFLECTRON MALDI TOF MS
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Bioanalytical Chem 395 Feb. 1, 2007 The Mechanism of MALDI (Matrix-Assisted Laser Desorption/Ionization): Ion Desorption The Formation of a ‘Solid Solution’ Matrix Excitation Analyte Ionization Ionization Methods —MALDI
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Bioanalytical Chem 395 Feb. 1, 2007 MALDI Mass Spectrometry MALDI = Matrix-Assisted Laser Desorption / Ionization Mass Spec
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Bioanalytical Chem 395 Feb. 1, 2007 MALDI TOF Mass Spectrometry
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Bioanalytical Chem 395 Feb. 1, 2007 SELDI MS (Surface Enhanced Laser Desorption/Ionization) 4. Detection by TOF-MS 2. Washing 3. Add matrix (‘EAM’) 1. Add sample gifs from http://www.bmskorea.co.kr/new01_21-1.htm EAM = Energy absorbing molecule
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Bioanalytical Chem 395 Feb. 1, 2007 Orthogonal MALDI TOF MS PerkinElmer pro-TOF
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Bioanalytical Chem 395 Feb. 1, 2007 MALDI-TOF Ex. Data
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Bioanalytical Chem 395 Feb. 1, 2007
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Bioanalytical Chem 395 Feb. 1, 2007 Sample Applications (TOF MS) Biomarker Discovery
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Bioanalytical Chem 395 Feb. 1, 2007 Quadrupole- TOF MS/MS Systems (QTOF) Introduced commercially by Micromass around 1995. Brilliant innovation, first commercial hybrid MS/MS. They charged “what the market would bear” $500-600k No competition! Great qualitative analyzer TOF analyzer provided: Incredibly fast scan rates Accurate mass capability (MW confirmation) Higher resolution (8k initially, now 10-12k w/ 1 reflectron)
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Bioanalytical Chem 395 Feb. 1, 2007 QSTAR® XL System—Schematics Conducting liner DC Quad 250 L/s 770 L/s 2.5 Torr Q1 Accelerator column 10 -2 Torr Field Free Drift region 7x10 -7 Torr Ion Mirror (reflector) Effective Flight Path = 2.5 m 10 m Torr Curtain Gas Sample Ions LINAC Q2Q0 eliminate cross-talk fast switching MS MS 2 4-anode detector broad dynamic range saturation correction
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Bioanalytical Chem 395 Feb. 1, 2007 Micromass QTOF Premier
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Bioanalytical Chem 395 Feb. 1, 2007 What can a QTOF do for you…?
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Bioanalytical Chem 395 Feb. 1, 2007 TOF/TOF System (2001) Gas in Source 1 Source 1 Grid Lens 1 Collision Cell Source 2 Detectors Mirror Beam XY Deflector 1 Beam XY Deflector 2 U U U U TIS TOF 1TOF 2 Source 2 Grid CID
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Bioanalytical Chem 395 Feb. 1, 2007 ABI 4700 TOF/TOF Automation - Sample Loader Fast Sample Stage Collision Cell 200 hz Laser Patented TOF-TOF™ Analyzer Integration – Oracle Database
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Bioanalytical Chem 395 Feb. 1, 2007 New Model: ABI-4800 TOF-TOF
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Bioanalytical Chem 395 Feb. 1, 2007 Ion Trap- TOF New Hybrid Design
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Bioanalytical Chem 395 Feb. 1, 2007 Summary Role and importance of MS in bioanalytical analysis continues to grow and evolve MS is typically no longer the bottleneck (sample handling and data acquisition/processing slow thru-put) Power of new MS technology providing new dimension of information on biomolecules There are 3 fundamental MS analyzer technologies, each with it’s advantages and disadvantages. ‘Hybrids’ can take advantage of best of 2 technologies LC/MS continues to evolve at a rapid rate Better, faster, cheaper…
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