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Slide 2 Evolving Role of Mass Spectrometry in Bioanalytical Analysis Daniel Pentek February 1, 2007 -UCONN Bioanalytical Chem 395 Slide 3 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) Slide 4 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 Slide 5 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! Slide 6 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 Slide 7 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 Slide 8 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 Whats 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. Slide 9 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 Slide 10 Bioanalytical Chem 395 Feb. 1, 2007 Todays 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 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. Slide 11 Bioanalytical Chem 395 Feb. 1, 2007 Ionization Techniques Application range Electrospray APPI Slide 12 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.) Slide 13 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. Slide 14 Bioanalytical Chem 395 Feb. 1, 2007 Electrospray Take home message: Electrospray is concentration dependent technique. Started out as very low flow technique, which wasnt 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. Slide 15 Bioanalytical Chem 395 Feb. 1, 2007 Example: High Flow Electrospray Source AB-Sciex TurboIonSpray Source Slide 16 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 Slide 17 Bioanalytical Chem 395 Feb. 1, 2007 Newer Ion Sources are have Orthogonal Design: 90 to Ion Entrance (Orifice) of MS. Slide 18 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) Slide 19 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 Slide 20 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 Slide 21 Bioanalytical Chem 395 Feb. 1, 2007 Heated Nebulizer APCI Probe Designed to Deliver Mist to Needle Slide 22 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 Slide 23 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] + Slide 24 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 Slide 25 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) Slide 26 Bioanalytical Chem 395 Feb. 1, 2007 ESI or APCI? - Which is better? For some applications, the choice is obvious For analytes