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Premium member Presentation Transcript AN PRESENTATION ON FAB & MALDI : AN PRESENTATION ON FAB & MALDI SUMITTED BY:- PRAVESH KUMAR M.PHARM (P’CEUTICS) Invertis university bareillyINTRODUCTION OF FAST ATOM BOMBARDMENT: INTRODUCTION OF FAST ATOM BOMBARDMENT FAB first introduce in 1981 by M.Barber It is an ionization technique The fast atom bombardment (FAB) was coined & prevailed The intact molecular or quasimolecular ions could be generated even in case of highly polar compounds that were definitely not candidates for electron ionization or chemical ionization Use of liquid matrix where the analyte was dissolved for analysis brought the awaited improvementsPowerPoint Presentation: The sample is dissolved in a liquid matrix such as glycerol , thioglycerol , m- nitrobenzyl alcohol , or diethanolamine and a small amount (about 1 microliter ) is placed on a target. The atoms are typically from an inert ges such as :- Argon or xenon The target is bombarded with a fast atom beam (for example, 6 keV xenon atoms ) that desorb molecular-like ions and fragments from the analyte. Matrix assisted fast atom bombardment :-the started its career in organic mass spectrometry & soon became a powerful competitor of field desorptionPowerPoint Presentation: The properties of the liquid matrix are the key importance for the resulting FAB spectra The liquid matrix are :- glycerol ,thioglycerol, n-nitro benzyl alcohol or diethanolamine It is used to analyze nonvolatile thermally unstable & highly polarity compounds The material to be analyzed is mixed with a non volatile chemical protection environment called a matrixPowerPoint Presentation: Bombardment under vacuum with high energy(4000 to 10000 e) The most significant difference between FAB and SIMS is the sample preparation. In FAB the analyte is dissolved in a liquid matrix. A drop of the sample/matrix mixture is placed at the end of an insertion probe and introduced to the source region The fast atom beam is focusedon this droplet to produce analyte ions. Glycerol or similar low vapor pressure liquids are typically used for matrixPowerPoint Presentation: principle ions (Cs+) neutrals( Ar , Xe……) sample IONS analysedBENEFITS OF FAB :-: BENEFITS OF FAB :- The sample introduction can be though direct insertion probe or LC\MS (continuous flow FAB) Benefits :- Rapid ,sample Relatively tolerant of variations in Sampling God for a large for a Large variety of compound Useful fragmentation Pattern Strong ion current– good For high resolution measure - mentsLIMITATIONS: LIMITATIONS High chemical background defines detection limits May be difficult to distinguish low molecular weight Compounds from chemical background Analyte must be soluble in liquid matrix No good for multiple charged compounds with more then two charges Require a high concentration of the organic liquid matrixPowerPoint Presentation: MALDI MATRIX ASSISTED LASER DESORPTION/IONIZATIONWhat is MALDI :-: What is MALDI :- Matrix-assisted laser desorption/ionization mass spectrometry Ionizes molecules via laser pulses Separates molecules according to mass to charge ratio Mainly used for detection of large biomoleculesDevelopment of MALDI-TOF: Development of MALDI-TOF Developed in 1988 by Professor Hillenkamp Designed to enhance mass-spec. by solving two main problems Thermal instability and low volatility Large and heavy biomolecules Prof. Franz HillenkampPowerPoint Presentation: In 1987, Michael Karas and Franz Hillenkamp :-  successfully demonstrated the use of a matrix (a small organic molecule) in LD to circumvent the mass limitation. The matrix had a strong absorbance at the laser wavelength and was highly sublimable . A low concentration of the analyte was mixed with this matrix onto a probe or metal plate (see fig. 1) and introduced into a pulsed laser beam. A substantial burst of ions was produced with each laser pulse. An unexpected side effect of the matrix was that it allowed for the laser incidence spot to be refreshed between each pulse,PowerPoint Presentation: thus greatly enhancing shot-to-shot reproducibility. This was the foundation of MALDI Later developments by Koichi Tanaka  demonstrated the application of MALDI to a whole range of biological macromoleculesThe mechanism of MALDI is believed to consist of three basic steps: -: 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 being completely isolated from each other. This eases the formation of the homogenous 'solid solution' required to produce a stable desorption of the analyte.PowerPoint Presentation: (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.PowerPoint Presentation: ( 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 spectroscopy for analysisFactors Affecting Quality of Output: Factors Affecting Quality of Output Size of crystals Energy of laser Detector voltage Accelerator voltage Extraction time Number of laser shots Evaporation rate Sample concentration Baseline calculations Scanning settingsUses of MALDI :-: Uses of MALDI :- Used to characterize and identify large molecules Used in pharmaceutical for QC, monitoring of enzyme reactions Used in DNA sequencing for forensics Used to identify different strains of viruses to help develop vaccinesFigures of Merit: Figures of Merit Mass Range 1-400,000 Da Detection Limit 10 -15 – 10 -18 moles Accuracy 0.1 – 0.01% Sensitivity 1 uL containing 100 fmol – 10 pmol Sample Consumption Less than 1 pmolAdvantages / Disadvantages: Advantages / Disadvantages Small sample needed Works well with large biomolecules Gives absolute mass measurements Finding matrices to work with samples Ions may collisionally relax Sensitive to contaminants, such as saltsWhat’s its future?: What’s its future? Will help revolutionize the medical world and will help lead to treatments for many diseases Will be useful for DNA sequencing, thus can be useful for forensic investigationsSources: Sources http://www.psrc.usm.edu/mauritz/maldi.html http://www.psrc.usm.edu/macrog/maldi.htm http://www.metabion.com/techinfo/z-maltof.html http://cbms.st-andrews.ac.uk/services/maldi-tof/maldi.html http://www.lsc.psu.edu/stf/imsc/MaldiTof.html http://www.babraham.com/new/maldi.htm http://www.humboldt-forum-recht.de/6-1997/Biographie.html http://www.biotech.iastate.edu/facilities/protein/maldi.html http://www.geocities.com/justinhettick/maldi.html http://www.itl.nist.gov/div898/pubs/ar/ar1998/node13.html http://cmgm.stanford.edu/pan/voyager.html http://www.laserscience.com/maldi_tof.htmPowerPoint Presentation: THANK U You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.