Instrumental Analytics PDF

Instrumental Analytics Harald Hundsberger, Krems Wintersemester 23/24 Mass Spectroscopy Mass Spectroscopy What is mass spectroscopy ? Analytical Technology for determination of the molecular mass of ions in vacuum Mass Spectroscopy Components of Mass spectrometer Ion Source...

Instrumental Analytics Harald Hundsberger, Krems Wintersemester 23/24 Mass Spectroscopy Mass Spectroscopy What is mass spectroscopy ? Analytical Technology for determination of the molecular mass of ions in vacuum Mass Spectroscopy Components of Mass spectrometer Ion Source Electro spray ionization Matrix assisted laser disorption ionization Mass analyzer Combination of an electric and magnetic field Quadrupole (high frequency) Time of flight analyzer (TOF) Ion detector Faraday cup Mass Spectroscopy Various technologies for ionization of the sample- in this lecture focus on MALDI & ESI MALDI:Matrix assisted laser disorption/ionization ESI: Electrospray ionization Main progress was achieved by developing the moderate ionization techniques (MALDI and ESI) Large bio molecules could be analyzed without beeing destroyed during the ionization process ESI The analyte solution flow passes through the electrospray needle that has a high potential difference (with respect to the counter electrode) applied to it (typically in the range from 2.5 to 4 kV). This forces the spraying of charged droplets from the needle with a surface charge of the same polarity to the charge on the needle. The droplets are repelled from the needle towards the source sampling cone on the counter electrode (shown in blue). As the droplets traverse the space between the needle tip and the cone and solvent evaporation occurs. MALDI The mechanism of MALDI is believed to consist of three basic steps: (i) Formation of a 'Solid Solution': It is essential for the matrix to be in access thus leading to the analyte molecules bei ng completely isolated from each other. This eases the formation of the homogenous 'solid solution' required to produce a stable desorption of the analyte. (ii) Matrix Excitation: The laser beam is focussed onto the surface of the matrix-analyte solid solution. The chromaphore of the matrix couples with the laser frequency causing rapid vibrational excitation, bringing about localised disintegration of the solid solution. The clusters ejected from the surface consists of analyte molecules surrounded by matrix and salt ions. The matrix molecules evaporate away from the clusters to leave the free analyte in the gas-phase. (iii) Analyte Ionisation: The photo-excited matrix molecules are stabilised through proton transfer to the analyte. Cation attachment to the analyte is also encouraged during this process. It is in this way that the characteristic [M+X] + (X= H, Na, K etc.) analyte ions are formed. These ionisation reactions take place in the desorbed matrix-analyte cloud just above the surface. The ions are then extracted into the mass spectrometer for analysis. MALDI & ESI With the introduction of MALDI and ESI as ionization techniques, Mass spectroscopy became to the most important analytical methods in Proteinchemistry Mass of peptides and proteins could easily be determined and deviations in the theoretical mass of a proteins was due to specific postrtranslational modifications (glycosylation) MALDI-MS First attempts to analyze large biomolecules were done in the early seventies, Sample was applied via a thin layer and then was ionized by pulsed laser light Those spectra showed low intensities and fragmentation (protein degradation) 1987 Karas and Hillekamp showed when proteins are embedded in matrix of small organic molecules is beneficial Result: Matrix shows high absorption, higher intensities of the analyte molecules (proteins), much less fragmentation of the analyte Ionization Principle Mixing of analyte with matrix (1000-10000 fold molar excess) Next step is evaporation of the solvent which causes crystallization process where the analytes are part of the crystal In high vacuum the surface of the crystal is excited with short wavelength laser light (a few nanoseconds), this leads to evaporation and ionizatuion of analyte molecules Only intact proteins if energy of Laser is within a relative narrow bandwidth An electrostatic field is located next to the sample (100- several 1000V/mm) Dependent on the polarity of the field, positive or negative charged ions are accalerated to the mass analyzer Analysis of Mass with TOF-Technology TOF: means time of flight Determination of mass of the analyte is done via an electronic measurement from the ionization process till arrival to the detector The ionized analyte is accelerated by the electrostatic field to energies of more then keV After acceleration ions are drifting in a field free area In this area the analyte is seperated according to the mass/charge ratio Ions with different m/z values are brought to different velocities during acceleration With known flight distance and known acceleration voltage the measurement of the flight time can be used to calculate the mass/charge ratio of an analyte Calibration is done with reference molecules with known molecular weight Refelctron mode: doubling of the the fight distance Typical flight times are 1-100µsec. Resolution of Masses in MALDI TOF Key properties of mass spectrometer is the ability to resolve small differences in mass Modern MALDI can resolve differences in Isotope composition of peptides and proteins (