MS 1

Day MS1

  • Read Chapter 16 Sections 16.1 – 16.2

Mass Spectrometry

  • Overview of Mass Spectrometry (MS)

    • Considered one of the most powerful and versatile analytical tools.

    • Key Features:

      • Sensitivity: Capable of detecting ions at very low concentrations.

      • Versatility: Applicable to a wide range of sample states, including solids, liquids, and gases, as well as techniques like Liquid Chromatography (LC), Gas Chromatography (GC), Capillary Electrophoresis (CE), Supercritical Fluid Chromatography (SFC), and plasmas.

      • Resolution and Precision: Ability to know the mass-to-charge ratio (m/z or m/ze) of an ion accurately.

    • Determining Molecular Properties:

      • Molecular formula can be deduced from the mass/charge ratio of the molecular ion peak and the relative abundances of ions around this peak.

      • Molecular structure can be inferred from ion fragmentation patterns or collision-induced fragmentation patterns.

      • Molar mass of macromolecules determined from the m/z values of the molecular ion or quasi-molecular ion.

Instrumentation

  • Basic Instrument Components:

    • Block diagram illustrates components of a generic mass spectrometer.

    • Mass analyzer and ion detector typically operate under high vacuum.

    • The exception: Ion trap mass spectrometer, operates at a pressure of 0.1 - 1 Torr.

    • Ion source and/or inlet system can be under high vacuum or at atmospheric pressure.

  • Basic MS Process:

    1. Sample introduction.

    2. Sample conversion to gaseous state.

    3. Ionization of sample.

    4. Dispersion of ions according to m/z.

    5. Detection of ions.

    6. Counting and measuring the relative abundances of ions with specific m/z.

  • High Vacuum Considerations:

    • Defined as a pressure range of 10^-5 to 10^-8 Torr or mmHg

    • Conversion: 1.3 x 10^-6 to 1.3 x 10^-9 kPa or 1.3 x 10^-8 to 1.3 x 10^-11 bar.

    • Mean free path ranges from 7.25 m to 7250 m; at 1 atm mean free path is 9.34 x 10^-8 m.

Ions in Mass Spectrometry

  • Definition of Ions:

    • Ions are charged atoms or collections of atoms.

    • Charge types:

      • Positive (most common).

      • Negative.

    • Charge range typically from +1 to higher numbers, depending on size, such as +50 for large biological macromolecules.

  • Mass-to-Charge Ratio (m/z):

    • Defined as the mass of the ion (in atomic mass units) divided by the charge (z):m/z=mass(amu)charge(z)m/z = \frac{mass (amu)}{charge (z)}.

    • Example: Carbon atom masses of 12.0000 amu or 13.0034 amu instead of 12.01 or 12.011 amu.

Benzene Ion Example

  • Analysis of Benzene (C6H6):

    • Most probable mass calculated as:
      78.0470 amu=(6×12.0000)+(6×1.00783)78.0470 \text{ amu} = (6 \times 12.0000) + (6 \times 1.00783).

    • Natural abundance produces some variants: about 1% of benzene molecules have mass of:
      79.0504 amu=(5×12.0000)+(1×13.0034)+(6×1.00783)79.0504 \text{ amu} = (5 \times 12.0000) + (1 \times 13.0034) + (6 \times 1.00783).

    • For a benzene ion with a +1 charge, the most intense peak in the mass spectrum would be at 78.0470 m/z.

    • For a +2 charge ion, the peak would shift to 39.0235 m/z.

Sample Introduction Methods

  • Overview of Sample Introduction Techniques:

    • Options include GC, LC, and syringe pumps with capillary interfaces.

    • Batch Inlet System: Involves a small leak of gaseous sample into the ionization source.

    • Direct Probe Injector: Utilized for solid or liquid samples that do not decompose upon heating; features a hole of approximately 50 μm in diameter.

Ionization Sources

  • Types of Ionization Methods:

    • Electron Impact Ionization.

    • Chemical Ionization.

    • Fast Atom Bombardment (FAB).

    • Matrix Assisted Laser Desorption (MALDI).

    • Electrospray Ionization (ESI).

    • Atmospheric Pressure Ionization (API).

  • Key Distinction:

    • Hard Ionization: Leads to many fragment ions and neutral fragments. Often, no molecular ion observed.

    • Soft Ionization: Minimizes fragmentation, allowing better observation of molecular or quasi-molecular ions in the spectrum.

Ionization Methods Explained

  • Electron Impact Ionization:

    • A schematic of the Electron Impact (EI) chamber.

    • Often surrounded by a magnetic field to enhance ionization through collisions between electrons and molecules.

    • Kinetic Energy (KE) of electrons in EI set to around 70 eV, commonly used in library records.

  • Ionization Energy Values:

    • Argon (Ar): 15.759 eV.

    • Helium (He): 22.59 eV.

    • Neon (Ne): 21.56 eV.

    • Krypton (Kr): 14.00 eV.

    • Common energy perspectives:

      • 1 eV = 96.5 kJ/mole,

      • C-C bond: 346 kJ/mole (3.6 eV).

    • Typically, most molecules have ionization energies less than or equal to 10 eV.

Kinetic Energy and Ion Velocity

  • Exploring Kinetic Energy and Velocity of Ions Exiting the Ion Source:

    • The formula for kinetic energy:
      KE=z×e×VKE = z \times e \times V

    • Alternative representation using mass and velocity:
      KE=12mν2KE = \frac{1}{2} m \nu^2

    • Rearranging yields velocity as:
      ν=2×z×e×Vm\nu = \sqrt{\frac{2 \times z \times e \times V}{m}}

  • Example Calculation:

    • For a benzene molecular ion with a +1 charge accelerated to 200 V:

    • 1 Volt=1 Joule/Coulomb1 \text{ Volt} = 1 \text{ Joule/Coulomb},

    • 1J=1kgm2/s21 J = 1 kg m^2/s^2,

    • The calculated velocity yields ν=2.22×104m/s\nu = 2.22 \times 10^4 m/s.

Soft Ionization Overview

  • Soft Ionization Principles:

    • Uses reagent ions for proton transfer, hydride transfer, or charge transfer aimed at generating analyte ions.

    • Often results in molecular or quasi-molecular ions, common gases used include CH4, NH3, He, and Ar at approximately 1 mmHg.

Chemical Ionization Process

  • Mechanism of Chemical Ionization:

    • Utilizes ion molecule reactions to produce analyte ions:

      • Example Reaction:CH<em>5++MH→MH</em>2++CH4CH<em>5^+ + MH \rightarrow MH</em>2^+ + CH_4 (Enthalpy \Delta H < 0, proton transfer).

    • Other reactions include:

      • C<em>2H</em>5++MH→MH<em>2++C</em>2H4C<em>2H</em>5^+ + MH \rightarrow MH<em>2^+ + C</em>2H_4 (proton transfer).

      • NH<em>4++MH→MH</em>2++NH3NH<em>4^+ + MH \rightarrow MH</em>2^+ + NH_3 (proton transfer).

      • C<em>2H</em>5++MH→M++C<em>2H</em>6C<em>2H</em>5^+ + MH \rightarrow M^+ + C<em>2H</em>6 (hydride transfer).

      • Ar++MH→MH++ArAr^+ + MH \rightarrow MH^+ + Ar (charge transfer).

    • The enthalpy changes ΔH\Delta H for these reactions range from -1 to -50 kJ/mole, indicating exothermic nature.

Mass Spectra Analysis Example

  • Analysis of Methyl Ester of Decanoic Acid:

    • Chemical formula: C11H22O2 (molar mass: 186.3 g/mol).

    • Spectra obtained using:

      • (A) Electron Ionization (hard ionization source).

      • (B) Chemical Ionization (soft ionization source).

Fast Atom Bombardment (FAB)

  • Overview of FAB:

    • Uses fast atoms, typically Ar, Cs, or Xe as ionizing agents with kinetic energy (KE) from 8-35 kV.

    • Usually applied in a viscous liquid layer, facilitating soft ionization.

    • Capable of generating multiply charged ions and quasi-molecular ions including M•H+ and M•Na+.

Nobel Prize Contributions in MS

  • Notable Figures:

    • John B. Fenn (1917 - Virginia Commonwealth University, Richmond, Virginia)

      • Awarded Nobel Prize in Chemistry 2002 for developing soft desorption ionization methods for biological macromolecule analysis.

      • Notable Achievement: Electrospray Ionization of Biomolecules.

    • Koichi Tanaka (1959 - Shimadzu Corporation, Japan)

      • Also awarded Nobel Prize in Chemistry 2002 for similar contributions related to soft desorption ionization methods.

      • Notable Achievement: Matrix-Assisted Laser Desorption/Ionization (MALDI).

Matrix-Assisted Laser Desorption Ionization (MALDI)

  • Characteristics of MALDI:

    • A soft ionization technique that often produces multiply charged ions and quasi-molecular ions.

    • Can generate both positive and negative ions (analysis limited to one type at a time).

    • Utilizes short pulse UV, Vis, or NIR lasers (4-10 ns, a few mJ per pulse) for vaporing and ionizing without causing thermal degradation.

MALDI-TOF Spectrum Example

  • A MALDI-TOF mass spectrum displaying mixture of three proteins.

    • Note peaks corresponding to cytochrome c ions with varying charges.

Electrospray Ionization (ESI)

  • Schematic of ESI System:

    • Characterized as a soft ionization method designed for coupling with LC and syringe pumps.

    • Often yields multiply charged ions and quasi-molecular ions.

    • Generates both positive and negative ions (analysis confined to one type at a time).

ESI and Carbonic Anhydrase II Analysis

  • Example Analysis:

    • ESI-MS of the enzyme carbonic anhydrase II, producing peaks solely from the pure enzyme sample.

Atmospheric Pressure Ionization (API)

  • API Overview:

    • Soft ionization technique designed for coupling with LC and syringe pumps,

    • Frequently results in multiply charged ions and quasi-molecular ions,

    • Similar to ESI and serves as its main competitor.

Worst middle school of the summer, which I like more like when they were actual people that I could talk with like action figure like a second grader, but at some point, it gets frustrating like the same thing every time where is middle school