1. Lecture 14 LC-MS Ionization

2. GC Computer MS GC-MS

3. LC Computer MS LC-MS

4. MS Analysis MS Gas phase Ions LC?

5. Condensed Phase Ionization Electrospray Ionization (ESI) Atmospheric Pressure Chemical Ionization (APCI) Atmospheric Pressure Photoionization (APPI) Matrix Assisted Laser Desorption/Ionization (MALDI) Desorption Electrospray Ionization (DESI) Direct Analysis in Real Time (DART) Liquid phase (LC) Solid phase

6. PCBs Production started 1920s Env Detection 1968 PFOS Production started 1930s Env Detection 2001 Difference?

7. GC-MS and LC-MS ionization are very different GC – Producing ions from gas phase analytes Producing gas phase compounds from charged analytes LC – MS Analysis Necessary for certain important analytes: –Biological molecules: Proteins Amino acids/nucleic acids sugars –Strong acids without the ability to absorption UV-vis

8. Problem solved… John Fenn Electrospray Ionization (ESI)

9. Electrospray Ionization (ESI) Gas Nebulized Spray LC Heater ESI Key Features: - Nebulized flow through capillary - Heat applied to evaporate solvent - Capillary (LC) 90 o to Cone (MS) Sample Cone MS Capillary Ionization Chamber 760 torr 10 -6 torr - Atmospheric pressure ion source - Voltage difference applied between capillary and cone - + - Detect (+) ions  Cap (+), Cone (-) - Detect (-) ions  Cap (-), Cone (+) What happens in the droplet?

10. Electrospray Ionization (ESI) Interested in Positive Ions - Apply (+) Voltage to capillary - (+) ions repelled from capillary - (-) ions attracted to capillary - Results in droplets enriched in (+) ions P. Kebarle, J. Mass Spectrom. 2000, 35, 804–817. +++ +++

11. Electrospray Ionization (ESI) Summary –Ions are formed in condensed phase In mobile phase droplets –Ions move into the gas phase by two mechanisms: Partition into gas phase due to columbic repulsion in droplet Solvent in droplet evaporates leaving ion behind Works well for compounds that can be charged in the condensed phase Strong or weak Acids and Bases

12. Atmospheric Pressure Chemical Ionization (APCI) Gas Nebulized Spray LC Heater APCI Key Features: - Nebulized flow through capillary - Heat applied to evaporate solvent - Capillary (LC) 90 o to Cone (MS) MS Capillary Ionization Chamber 760 torr 10 -6 torr - Atmospheric pressure ion source - No voltage applied to capillary - droplets are not charged - - Voltage still applied to cone - Detect (-) ions  Cone (+) - Detect (+) ions  Cone (-) Sample Cone - Heat evaporates solvent, analytes partition into the gas-phase uncharged - Need to make ions! Corona discharge needle

13. Atmospheric Pressure Chemical Ionization (APCI) Summary –First step is solvent evaporation Compounds in gas-phase as neutrals –Ions produced in the gas-phase Corona discharge ionizes carrier gas and mobile phase Acid/base reactions in the gas phase with mobile phase Works well for compounds that are protic but neutral at a pH between 2 and 9 Weak Acids / Bases

14. Atmospheric Pressure Photoionization (APPI) Gas Nebulized Spray LC Heater APPI Key Features (same as APCI): - Nebulized flow through capillary - Heat applied to evaporate solvent - Capillary (LC) 90 o to Cone (MS) MS Capillary Ionization Chamber 760 torr 10 -6 torr - Atmospheric pressure ion source - No voltage applied to capillary - droplets are not charged - - Voltage still applied to cone - Detect (-) ions  Cone (+) - Detect (+) ions  Cone (-) Sample Cone - Heat evaporates solvent, analytes partition into the gas-phase uncharged - Need to make ions! UV lamp

16. Summary –First step is solvent evaporation Compounds in gas-phase as neutrals –Ions produced in the gas-phase UV lamp ionizes compound by photon absorption or Charge exchange with dopant or Dopant ionizes mobile phase, followed by; Acid/base reactions in the gas phase with mobile phase Works well for compounds that are absorb UV radiation or are protic but neutral at a pH between 2 and 9 Weak Acids / Bases Nonpolar compounds that are UV active Atmospheric Pressure Photoionization (APPI)

17. Summary LC-MS Ionization www.agilent.com m/z (MW)

18. Matrix-Assisted Laser Desorption/Ionization (MALDI) Matrix molecule Analyte molecule Adapted from Dass, C. Fundamentals of Contemporary Mass Spectrometry, Wiley-Interscience, 2007. Laser beam + + - MALDI Key Features: - Analyte deposited on surface with excess matrix compound - Shine laser on the surface - h aborbed by matrix molecules - Matrix molecules transfer energy to analyte molecules - Results in desorption/ionization of both matrix and analyte MS Vacuum

19. Matrix-Assisted Laser Desorption/Ionization (MALDI) Common dopants: Analytes of interest: –Proteins –Polymers –High MW, ionization not possible using ESI, APCI, APPI –High MW separates these compounds from the background signal of the matrix

20. Desorption Electrospray Ionization (DESI) Z. Takáts et al. Science 2004, 306, 471-473.

21. Direct Analysis in Real Time (DART)