15.14
Elektrosprey İyonizasyon (ESI) Kütle Spektrometresi
Daha yüksek moleküler ağırlıklı biyomoleküller, geleneksel elektron darbe iyonizasyon yöntemleriyle kütle analizi sırasında iyonlaşmadan veya buharlaş…
Proteinler ve nükleik asitler gibi biyomoleküller, yüksek moleküler ağırlığa sahip düşük uçuculukta bileşiklerdir. Konvansiyonel elektron darbeli iyonizasyon kütle spektrometresi, ciddi moleküler parçalanma da dahil olmak üzere bu tür moleküllerle ilgili zorluklarla karşı karşıyadır.
Elektrosprey iyonizasyonu, bu tür biyomoleküllerin kütle spektrometrisi için radikal olmayan moleküler iyonlar üreten alternatif bir "yumuşak" iyonizasyon yöntemidir.
Bu yöntemde numune, ilave tuzlar olsun veya olmasın metanol, metanol-su veya asetonitril gibi polar bir çözücü ile karıştırılır.
Karışım, yüksek voltajlı bir kılcal damar yoluyla bir vakum odasına püskürtülür. Yüklü damlacıkların ince spreyinden çözücünün hızlı bir şekilde buharlaşması, sonuçta yüklü gaz halindeki numune molekülüne yol açar.
Bu yüklü numune molekülü, kütle spektrumunda, numune moleküler kütlesinin ve ilişkili iyonların kütlelerinin toplamı olarak tespit edilir.
Örneğin, iyonik çözeltide sodyum iyonları varsa, spektrum tipik olarak bir M+23 tepe noktasına sahiptir.
View the full transcript and gain access to JoVE Core videos
Q1: Why is electrospray ionization better than electron-impact ionization for biomolecules?
Electron-impact ionization causes severe molecular fragmentation of high molecular weight biomolecules like proteins and nucleic acids. Electrospray ionization is a soft ionization method that generates nonradical molecular ions, preserving the intact biomolecule structure. This allows mass spectrometry analysis of biomolecules without excessive decomposition.
Q2: How does electrospray ionization transfer ions from liquid to gas phase?
The sample is mixed with a polar solvent and sprayed through a high-voltage capillary into a vacuum chamber. Electrical energy applied between the capillary and analysis chamber entrance moves charged droplets toward the chamber. Rapid solvent evaporation from the fine aerosol leaves behind charged gaseous sample molecules ready for mass detection.
Q3: What role does the ionic liquid play in electrospray ionization?
The ionic liquid serves as the ionization source in the sample solution. It provides cations that associate with the analyte biomolecule during ionization. The type and mass of these cations directly affect the m/z values detected in the mass spectrum, determining peak positions.
Q4: What does an M+23 peak indicate in an ESI mass spectrum?
An M+23 peak indicates that sodium ions from the ionic liquid have associated with the analyte molecule during ionization. The m/z value equals the sample's molecular mass plus 23, the atomic mass of sodium. Different cations produce different mass shifts, allowing identification of which ions are present.
Q5: How does solvent composition affect electrospray ionization performance?
The sample is mixed with polar solvents such as methanol, methanol-water, or acetonitrile, with or without added salts. These solvents facilitate the formation of charged droplets and enable efficient solvent evaporation. The choice of solvent and salt composition influences ionization efficiency and the types of cations available for association.
Q6: How does ESI handle multiple cation associations with a single biomolecule?
A biomolecule can associate with multiple cations during electrospray ionization, resulting in variably protonated or ionized species. Each association produces a distinct m/z value in the mass spectrum. The sum of the molecular mass and all associated ion masses determines the detected m/z, allowing analysis of chemical ionization mass spectrometry patterns.
Q7: What is the relationship between capillary voltage and charged droplet movement in ESI?
The voltage difference applied between the capillary and the analysis chamber entrance creates an electric field that drives charged droplets toward the chamber. Higher voltage increases droplet acceleration and ionization efficiency. This electrical energy is fundamental to transferring ions from the liquid phase into the gaseous phase for mass analysis.