Glossary

8.2 Ionization techniques (EI, CI, ESI, MALDI)

4 min read•august 14, 2024

Mass spectrometry is all about turning molecules into ions we can measure. Different ionization techniques help us analyze different types of compounds. Some methods are gentle, while others break molecules apart.

Electron ionization and chemical ionization work well for small, volatile molecules. Electrospray ionization and MALDI are better for big, fragile molecules like proteins. Picking the right method depends on what you're studying and what info you need.

Ionization Techniques in Mass Spectrometry

Principles and Applications

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  • Ionization techniques in mass spectrometry convert analyte molecules into gas-phase ions, enabling their separation and detection based on their (m/z)
  • The choice of ionization technique depends on the nature of the analyte (molecular weight, polarity, and stability) and the desired analytical information (molecular mass, structural elucidation, or quantification)

Hard vs. Soft Ionization Techniques

  • Hard ionization techniques (electron ionization, EI) cause extensive fragmentation of the analyte molecules, providing structural information but potentially losing molecular ion information
  • techniques (chemical ionization, CI; electrospray ionization, ESI; matrix-assisted laser /ionization, MALDI) result in less fragmentation and are more suitable for analyzing large, non-volatile, or thermally labile molecules
  • EI and CI are typically used for gas chromatography-mass spectrometry (GC-MS), while ESI and MALDI are commonly coupled with liquid chromatography-mass spectrometry (LC-MS) or used for direct sample analysis

EI and CI Principles and Applications

Electron Ionization (EI)

  • EI involves bombarding the analyte molecules with a beam of high-energy electrons (typically 70 eV), causing the ejection of an electron from the molecule and the formation of a radical cation (M•+)
    • The excess energy imparted to the molecular ion often leads to extensive fragmentation, generating characteristic fragment ions that provide structural information about the analyte
    • EI is considered a hard ionization technique and is widely used for the analysis of small, volatile, and thermally stable molecules (hydrocarbons, pesticides, drugs)
    • EI mass spectra are highly reproducible and can be compared to extensive libraries (NIST, Wiley) for compound identification

Chemical Ionization (CI)

  • CI involves the reaction of the analyte molecules with a reagent gas (methane, ammonia, or isobutane) that has been ionized by electron impact
    • The reagent gas ions transfer a proton or other charged species to the analyte molecules, forming quasi-molecular ions ([M+H]+, [M+NH4]+, or [M-H]-) with minimal fragmentation
    • CI is a softer ionization technique compared to EI and is useful for obtaining molecular weight information and analyzing compounds that are prone to extensive fragmentation under EI conditions (alcohols, amines, esters)
    • The choice of reagent gas can influence the type of quasi-molecular ions formed and the degree of fragmentation observed (positive ion CI with methane vs. negative ion CI with ammonia)

ESI and MALDI Mechanisms and Advantages

Electrospray Ionization (ESI)

  • ESI is a soft ionization technique that produces gas-phase ions from a liquid sample
    • The sample solution is passed through a capillary at a high voltage, creating a fine spray of charged droplets
    • As the droplets evaporate, the charge density on their surface increases until ions are ejected into the gas phase (ion evaporation model) or the droplets undergo Coulomb fission (charged residue model)
    • ESI is particularly useful for analyzing large, polar, and thermally labile molecules (proteins, peptides, oligonucleotides)
    • Multiple charging of analytes in ESI allows the analysis of high molecular weight compounds using mass analyzers with limited m/z range (quadrupoles, ion traps)

Matrix-Assisted Laser Desorption/Ionization (MALDI)

  • MALDI is a soft ionization technique that uses a laser to desorb and ionize the analyte molecules from a solid matrix
    • The sample is co-crystallized with an excess of a matrix compound that absorbs energy at the laser wavelength (α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid)
    • Upon laser irradiation, the matrix absorbs the energy, leading to rapid heating, desorption, and ionization of the analyte molecules
    • MALDI typically produces singly charged ions and is well-suited for the analysis of large, non-volatile, and thermally labile molecules (proteins, peptides, polymers)
    • The choice of matrix depends on the nature of the analyte and can influence the ionization efficiency and the formation of matrix-related ions (matrix clusters, matrix adducts)

Choosing Ionization Techniques for Samples

Factors to Consider

  • The selection of an ionization technique depends on the physicochemical properties of the analyte (molecular weight, polarity, and thermal stability) and the analytical goals (structural elucidation, molecular mass determination, or quantification)
  • For small, volatile, and thermally stable molecules (pesticides, drugs), EI is often the preferred ionization technique, as it provides extensive fragmentation and reproducible mass spectra for compound identification
  • CI is suitable for compounds that are prone to extensive fragmentation under EI conditions (alcohols, amines) and when molecular weight information is required

Techniques for Specific Analytes

  • ESI is the method of choice for large, polar, and thermally labile molecules (proteins, peptides, oligonucleotides), particularly when coupled with LC-MS
  • MALDI is well-suited for the analysis of large, non-volatile, and thermally labile molecules (proteins, peptides, polymers) and is often used for direct sample analysis or imaging applications
  • In some cases, a combination of ionization techniques may be employed to obtain complementary information or to analyze complex mixtures containing a wide range of analytes (using both EI and ESI for a drug metabolism study)

Key Terms to Review (17)

Charge Transfer: Charge transfer refers to the process where an electron is transferred between two chemical species, leading to the formation of ions. This phenomenon is crucial in various ionization techniques as it influences the efficiency and type of ions produced during the ionization process, impacting the overall analysis in mass spectrometry.
Chemical Ionization (CI): Chemical ionization is a soft ionization technique used in mass spectrometry that involves the ionization of analytes through their interaction with ions from a reagent gas. This process typically results in fewer fragment ions compared to other methods, providing a clearer molecular ion signal, which is essential for accurate mass determination. The technique can enhance sensitivity and specificity, making it a popular choice in various analytical applications.
Desorption: Desorption is the process by which a substance is released from or through a surface. This is particularly important in the context of separation techniques and mass spectrometry, where it influences how analytes interact with surfaces and their subsequent analysis. The efficiency of desorption can significantly affect the resolution and sensitivity of various analytical methods.
Direct vs. Indirect Ionization: Direct ionization involves the immediate ejection of an electron from a molecule or atom when it interacts with an incoming particle, usually resulting in a charged ion. In contrast, indirect ionization occurs when a neutral molecule is first excited, leading to a secondary process that ultimately results in ion formation. Understanding these two mechanisms is essential for grasping how various ionization techniques operate, including those that utilize energetic particles and photons.
Electron ionization (EI): Electron ionization (EI) is a technique used in mass spectrometry that involves bombarding gaseous molecules with high-energy electrons to produce positively charged ions. This method is pivotal for analyzing small, volatile compounds and typically results in fragmentation of the molecule, providing structural information that aids in identification. EI is a widely-used technique and serves as a fundamental method for various applications in analytical chemistry.
Electrospray Ionization (ESI): Electrospray ionization (ESI) is a soft ionization technique used in mass spectrometry that allows the generation of ions from liquid samples by applying a high voltage to a solution, creating a fine aerosol of charged droplets. This technique is particularly effective for analyzing biomolecules, such as proteins and nucleic acids, due to its ability to produce ions without extensive fragmentation, making it easier to identify and quantify large and complex molecules.
Fragmentation patterns: Fragmentation patterns refer to the specific way in which molecular ions break apart into smaller fragments during the ionization process, providing valuable information about the structure and composition of the original molecule. These patterns are essential in mass spectrometry, as they help identify compounds by revealing characteristic fragmentation pathways based on their chemical structure and bonding. Understanding these patterns can aid in interpreting mass spectra and determining the molecular formula of unknown substances.
Francois Barre-Sinoussi: Francois Barre-Sinoussi is a French virologist best known for her role in the discovery of the human immunodeficiency virus (HIV), which is responsible for AIDS. Her groundbreaking work has not only advanced our understanding of viral infections but also paved the way for the development of antiretroviral therapies that are crucial in managing HIV infections today.
Hard vs. soft ionization: Hard vs. soft ionization refers to two distinct approaches in mass spectrometry for generating ions from molecules, with hard ionization producing more fragmented ions and soft ionization preserving the molecular structure. Hard ionization techniques, like electron impact, tend to impart high energy that leads to extensive fragmentation, while soft ionization methods, such as electrospray ionization, allow for intact ions to be analyzed, making them suitable for larger and more fragile biomolecules.
High Sensitivity: High sensitivity refers to the ability of an analytical technique to detect even the smallest quantities of a substance in a sample. This feature is crucial for accurately identifying and quantifying compounds, especially in complex mixtures where target analytes may be present at trace levels. High sensitivity ensures reliable data collection, enabling better analysis and interpretation in various fields such as environmental monitoring, pharmaceuticals, and biochemistry.
Ion yield: Ion yield refers to the amount of ions produced during the ionization process in mass spectrometry. It is a critical parameter that affects the sensitivity and detection limits of various ionization techniques, highlighting the efficiency of transforming neutral molecules into ions for analysis.
John B. Fenn: John B. Fenn was an American chemist awarded the Nobel Prize in Chemistry in 2002 for his pioneering work in the development of electrospray ionization (ESI), a critical ionization technique that transforms biomolecules into ions for mass spectrometry analysis. His contributions significantly advanced the field of analytical chemistry, particularly in the analysis of large biomolecules like proteins and nucleic acids.
Mass spectrometer: A mass spectrometer is an analytical instrument used to measure the mass-to-charge ratio of ions, helping in the identification and quantification of chemical compounds. It works by ionizing chemical species and sorting the resulting ions based on their mass-to-charge ratio, allowing for precise analysis of complex mixtures and providing insights into molecular structures.
Mass-to-charge ratio: The mass-to-charge ratio, often abbreviated as m/z, is a critical parameter in mass spectrometry that describes the ratio of the mass of an ion to its charge. This ratio is essential for distinguishing between ions with different masses and charges during analysis, allowing scientists to identify and quantify molecules based on their unique m/z values. Understanding this concept is fundamental for interpreting mass spectra, optimizing ionization techniques, and effectively utilizing mass analyzers.
Matrix-assisted laser desorption/ionization (MALDI): Matrix-assisted laser desorption/ionization (MALDI) is an ionization technique used in mass spectrometry that allows for the analysis of large biomolecules, such as proteins and peptides, by converting them into ions using a laser. In MALDI, the sample is mixed with a matrix material that absorbs the laser energy, facilitating the desorption and ionization of the analyte into the gas phase. This method is particularly valued for its ability to analyze complex mixtures without extensive sample preparation.
Soft ionization: Soft ionization is a gentle ionization method that produces ions without causing significant fragmentation of the molecules. This technique is essential in mass spectrometry as it allows for the analysis of larger, more complex molecules while preserving their structural integrity, which is crucial for accurate mass determination and identification.
Time-of-flight mass spectrometry (tof-ms): Time-of-flight mass spectrometry (tof-ms) is a technique used to determine the mass-to-charge ratio of ions by measuring the time it takes for them to travel a specific distance. This method is crucial because it allows for the rapid analysis of various ionized molecules generated by different ionization techniques, such as electron ionization, chemical ionization, electrospray ionization, and matrix-assisted laser desorption/ionization, making it versatile for analyzing complex mixtures.
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