Molecular Mass Spectrometry

Molecular Mass Spectra

• Relative Abundance vs m/z (mass/charge):
  • Example: Ethyl benzene with a molecular mass of 106. The spectrum shows peaks at m/z 106 and 91, along with other peaks. The question is posed: Why?

How Sample is Analyzed

1. Vapor Phase Start:
   • Begin with ethyl benzene in the vapor phase.
2. Electron Bombardment:
   • Hit the vapor with a stream of electrons.
   • This process knocks off an electron from the molecule, creating a molecular ion.
   • CH3CH2C6H5 + e^- \rightarrow CH3CH2C6H5^{*+} + 2e^-
   • The resulting ion, denoted as M^{*+} (M star plus), is a radical ion (indicated by the dot).
3. Fragmentation:
   • The collision with the electron excites the molecule.
   • To release energy, the excited ion often fragments into smaller pieces.
   • In the case of ethyl benzene, a common fragmentation is the loss of CH_3.
   • This results in the formation of C6H5CH_2^+, which has a molecular weight (MW) of 91.
   • Other smaller, positively charged ions are also formed during this process.

Mass Spectrometer Operation

• Ion Acceleration: All positive ions are attracted to a negatively charged lens, which accelerates the ions into the mass spectrometer.
• Mass/Charge Sorting: The mass spectrometer sorts the ions based on their mass-to-charge ratio (m/z).
• Display: The instrument displays the relative abundance or amounts of each ion found at a particular m/z value.
• Base Peak:
  • The largest peak in the spectrum is called the base peak.
  • It is arbitrarily assigned a value of 100.
  • The intensities of all other peaks are scaled relative to the base peak intensity.
  • This scaling and analysis are typically done automatically by the computer software that runs the instrument.

Mass Spectrometer Components

• Sample Inlet System: Introduces the sample into the instrument. Operates under vacuum conditions (10^{-5} to 10^{-8} torr).
• Ion Source: Generates gaseous ions from the sample.
• Mass Analyzer: Separates ions according to their mass-to-charge ratio (m/z).
• Detector: Detects the separated ions and measures their abundance.
• Vacuum System: Maintains a high vacuum inside the mass spectrometer.
• Signal Processor: Processes the signals from the detector.
• Readout: Displays the mass spectrum.

Ion Sources

• The appearance of the mass spectrum (relative sizes of all the peaks) depends on the method used to generate ions.

• Table 20-1 (not provided) likely lists different methods for creating gaseous ions.

• Two Major Categories:

  • Gas Phase Sources: Start with the molecule already in the gas phase.
  • Desorption Sources: Handle samples in liquid or solid form, requiring conversion to the gas phase.

• Commercial mass spectrometers often have interchangeable units for different ionization methods.

• Common Sources:

  • Electron Impact (EI)
  • Chemical Ionization (CI)

• Gas-Phase Sources: Typically used for stable compounds with boiling points (BP) less than 500°C and molecular weights (MW) usually less than 1000 Da.

• Desorption Sources: Can handle molecules with molecular weights up to 10,000 Da.

• Hard vs. Soft Sources:

  • Hard Sources (e.g., EI): Impart enough energy to the molecular ion to cause significant fragmentation, resulting in many fragments with m/z ratios less than the molecular ion.
  • Soft Sources (e.g., CI): Cause little fragmentation.

Hard vs Soft Ionization

• (a) Hard ionization (Electron Impact): Causes significant fragmentation.
• (b) Soft Ionization (Chemical Ionization): Results in less fragmentation.
• EI is considered a hard ionization method, while CI is a soft ionization method.

Electron Impact Ionization (EI)

• Components: Includes a filament, electron slit, repeller, ionization region, ion accelerating region, anode, and mass analyzer.

• Process:

  • A molecular leak introduces the sample.
  • Electrons are emitted from a filament and accelerated into the ionization region, where they collide with sample molecules.
  • Ions are accelerated through a series of slits into the mass analyzer.

Advantages and Disadvantages of EI Sources

• Advantages:
  • Easy to use.
  • Gives a comparatively large number of ions.
  • Extensive fragmentation provides a pattern useful for identifying compounds.
• Disadvantages:
  • Extensive fragmentation may make it difficult to find the molecular ion.
  • The sample must be a stable gas.
  • Thermal decomposition can occur before ionization in some cases.
  • Limited to molecular weights in the 10^3 range.

Chemical Ionization (CI)

• Usually an interchangeable unit with EI.
• Ions are formed by colliding the molecule with ions of an excess reagent gas.
• Can be both positive and negative mode, but positive CI is most common.
• Second most common ion source.
• Similar setup to EI with two key changes:
  • The entire setup is enclosed in a vacuum chamber.
  • The analyzer is filled with a reagent gas, resulting in a much higher concentration of gas molecules (about 10^3 to 10^4 times more) than target molecules.
• The reagent gas is preferentially ionized due to its higher concentration.
• Typical reagent gas: Methane (CH_4).

Chemical Ionization Ion Formation

• Primary Ions:
  • Several ions are formed from methane, including CH4^+, CH3^+, and CH_2^+.
  • The first two (CH4^+ and CH3^+) constitute about 90% of the ions.
• Secondary Reactions: These ions undergo further collisions and reactions:
  • CH4^+ + CH4 \rightarrow CH5^+ + CH3
  • CH3^+ + CH4 \rightarrow C2H5^+ + H_2
• Reactive Ions: The last two ions (CH5^+ and C2H_5^+) are highly reactive with the target molecule (MH).
• Reactions with Target Molecule:
  • Proton transfer:
    • CH5^+ + MH \rightarrow MH2^+ + CH_4
    • C2H5^+ + MH \rightarrow MH2^+ + C2H_4
  • Hydride transfer:
    • C2H5^+ + MH \rightarrow M^+ + C2H6
• The proton transfer reactions give you (M+1)^+ ions.
• The hydride transfer gives you (M-1)^+ ions.
• Adduct Formation: Sometimes, you see an (M+29)^+ ion when C2H5^+ sticks to the molecule.
• In summary, chemical ionization typically results in the formation of the molecular ion plus or minus 1 mass unit, with little additional fragmentation.

Field Ionization Source

• Ions are formed under the influence of a large electric field (10^8 V/cm).
• Uses emitters with numerous fine tips having diameters less than 1 μm.
• Field ionization (FI) involves the generation of M^{+} ions by removing electrons from gas sample molecules using a high electric field.
• This generally occurs at a sharp edge or tip biased to a high electrical potential.

Ion Sources Summary

• Gas Phase Ionization:
  • Electron Impact Ionization (EI)
  • Chemical Ionization (CI)
  • Field Ionization (FI)
• Desorption Ionization:
  • Matrix Assisted Laser Desorption Ionization (MALDI)
  • Electrospray Ionization (ESI)

Matrix-Assisted Laser Desorption Ionization (MALDI)

• Great for biopolymers in the 1,000 - 100,000's range.
• Developed independently in 1988 by Franz Hillenkamp and Michael Karas (Germans) and Koichi Tanaka (Japanese).
• Sample Preparation:
  • Mix the sample in an aqueous or alcohol solution.
  • The solution contains a large excess of a radiation-absorbing matrix material (examples are likely listed in Table 20-4).
  • Dry the mixture onto a metal target.
• Analysis:
  • Place the target into the instrument under vacuum.
  • Blast tiny spots on the surface with a laser pulse.
  • The laser is tuned to the absorption of the matrix.
  • Everything in the spot is instantly sublimed.
  • A mass spectrum is acquired.
  • The process is repeated with another laser pulse on a different spot.

MALDI Process

• A laser beam is directed onto a sample plate containing the analyte mixed with a matrix.
• The laser energy desorbs and ionizes the matrix and analyte molecules.
• Cations are often involved in the ionization process.
• Ions are then directed into a Time-of-Flight (TOF) mass analyzer via an extraction grid.

MALDI-TOF Process in the MassARRAY Analyzer 4

• The UV laser triggers a clock to start timing the ion flight.
• The detector measures ion arrival times.
• Lighter ions arrive at the detector first, while heavier ions arrive last.
• The software captures data and prepares it for analysis.

Electro Spray Ionization (ESI)

• Operates at ambient pressure and temperature.
• The sample solution is pumped through a needle.
• This technique is well-suited for coupling with HPLC/MS (High-Performance Liquid Chromatography/Mass Spectrometry).
• The needle is held at several thousand volts above a cylindrical electrode around the needle.
• This generates fine droplets with charges on them.
• Droplets pass through a desolvating capillary.
• As the droplets shrink, the charge density on the droplet increases, leading to the desorption of ions into the gas phase.
• Very little fragmentation occurs.
• Most molecules are multiply charged, which lowers the m/z ratio.
• This allows for the determination of the parent molecular weight, even if the true m/z of the molecular ion is not directly observed.

Electrospray Ionization Source

• Liquid sample is introduced through a needle into a cylindrical electrode.
• The process involves electrostatic lenses, a skimmer, a drying gas, and multiple pumping stages.

Electrospray Ionization Process

• A sample in solution flows through a capillary.
• A Taylor cone forms at the tip of the capillary, emitting charged droplets due to the high voltage (HV).
• Solvent evaporation occurs until the Rayleigh limit is reached, leading to Coulomb explosions.
• Further evaporation and Coulomb explosions continue until the solvent is completely evaporated, leaving charged particles.
• Nitrogen gas (N_2) is often used to aid in evaporation.

Resolution of Mass Spectrometer

• HPLC Resolution?
  • R = \frac{2(t2 - t1)}{W2 + W1}, where t1 and t2 are the retention times of two peaks, and W1 and W2 are the peak widths at the base.
• Mass Spectrometry Resolution?
  • R = \frac{M}{\Delta M}, where M is the mass of the ion and \Delta M is the difference in mass between two resolvable peaks.

Mass Resolution Example

• What resolution is needed to separate the ions C2H4^+ and CH_2N^+, with masses of 28.0313 and 28.0187?

Mass Resolution Problem

• What mass differences can just be resolved at an m/z value of 1500 if the mass spectrometer has a resolution of 3000?

Mass Analyzers

• Magnetic Sector Mass Analyzer
• Quadrupole Mass Analyzer
• Time of Flight (TOF) Mass Analyzer
• Ion Trap Mass Analyzer
• Orbitrap Mass Analyzer

Magnetic Sector Mass Analyzers

• A gaseous sample is ionized by an electron source (hot filament) in an ionization chamber.
• Ions are accelerated through a slit and pass through a magnetic field.
• The magnetic field deflects the ions based on their mass-to-charge ratio.
• Lighter ions are deflected more than heavier ions.
• Ions then pass through an exit slit and are collected by an ion collector.

Double Focusing Mass Spectrometers

• Uses both a magnetic analyzer and an electrostatic analyzer (ESA) to achieve higher resolution.
• Ions from the ion source pass through a source exit slit and then enter the ESA.
• The ESA focuses the ions based on their energy.
• Ions then enter the magnetic analyzer, which focuses them based on their momentum.
• The point of double focus is where ions are focused in both direction and energy.

Time-of-Flight (TOF) Mass Analyzers

• Ions are formed in the ionization region and accelerated into a field-free separation region.
• Ions are separated based on their time of flight through the field-free region.
• Lighter ions travel faster and arrive at the detector sooner than heavier ions.
• The detector consists of an ion cathode, dynode strip, and anode.
• Electron multiplication occurs as ions strike the dynode strip.

Ion Trap Mass Analyzers

• Ions are trapped using a combination of RF and DC fields.
• Consists of a ring electrode and two end caps.
• Ions are ejected from the trap based on their m/z ratio.

Orbitrap Mass Analyzers

• Ions are injected off-axis into the Orbitrap.
• The Orbitrap consists of a shaped inner electrode and a split outer electrode.
• Injected ions orbit and oscillate in complex paths.
• The induced transient signal is sent to a computer for analysis.

Tandem in Space

• Uses multiple mass analyzers in series to perform MS/MS experiments.
• Components include an ion source, quadrupole mass analyzers (Q1, Q2, Q3), a collision cell, and an electron multiplier detector.
• First-stage mass separation selects a precursor ion.
• The precursor ion is fragmented in the collision cell.
• Second-stage mass separation analyzes the fragment ions.

Tandem Mass Spectrometry

• Components include a linear trap (Q3), a quadrupole (Q1), a curved collision cell (Q2), and an ion guide.
• Collisional focusing and ion trapping (Q0) enhance sensitivity.

Tandem in Space Cont.

• Original ions from the sample are selected as a precursor ion by Mass analyzer 1.
• The precursor ion is fragmented into product ions in an interaction cell.
• Mass analyzer 2 then analyzes the product ions, which are detected by a detector.

Tandem in Time

• Uses an ion trap to perform MS/MS experiments in time.
• The ion trap consists of a ring electrode and two end caps.
• Ions are stored using RF and DC fields.
• Ions of a specific m/z can be ejected by scanning the field.
• Advantages include the ability to perform MS/MS/MS… experiments and high sensitivity full scan MS/MS.

Isotope Abundance

• Consider the relationship between the molecular ion peak (M+) and isotopes such as (M+1)+, (M+2)+, (M+4)+.
• Examples can be found in the textbook.

TABLE 20-3 Natural Abundance of Isotopes of Some Common Elements

• Lists the most abundant isotope for various elements and the abundance of other isotopes relative to 100 parts of the most abundant isotope.
• Elements listed include Hydrogen, Carbon, Nitrogen, Oxygen, Sulfur, Chlorine, Bromine, and Silicon.
• Fluorine (^{19}F), phosphorus (^{31}P), sodium (^{23}Na), and iodine (^{127}I) have no additional naturally occurring isotopes.

Exact Masses Unknown Identification

• TABLE 20-6 Isotopic Abundance Percentages and Molecular Masses for Various Combinations of C, H, O, and N
• Lists the isotopic abundance percentages and molecular masses for various combinations of carbon, hydrogen, oxygen, and nitrogen.
• Provides a means for identifying unknown compounds based on their isotopic abundance patterns.

Applications of Molecular Mass Spectrometry (TABLE 20-5)

1. Elucidation of the structure of organic and biological molecules.
2. Determination of the molecular mass of peptides, proteins, and oligonucleotides.
3. Identification of components in thin-layer and paper chromatograms.
4. Determination of amino acid sequences in samples of polypeptides and proteins.
5. Detection and identification of species separated by chromatography and capillary electrophoresis.
6. Identification of drugs of abuse and metabolites of drugs of abuse in blood, urine, and saliva.
7. Monitoring gases in a patient's breath during surgery.
8. Testing for the presence of drugs in blood in racehorses and Olympic athletes.
9. Dating archaeological specimens.
10. Analyses of aerosol particles.
11. Determination of pesticide residues in food.
12. Monitoring volatile organic species in water supplies.