Mass Spectrometry PDF

MASS SPECTROMETRY INTRODUCTION: ➔ Mass spectrometry is a powerful analytical technique used to identify unknown compounds within a sample, structure and chemical properties of different molecules. ➔ It is a microanalytical technique requiring only a few nanomoles of the sample to obtain char...

MASS SPECTROMETRY INTRODUCTION: ➔ Mass spectrometry is a powerful analytical technique used to identify unknown compounds within a sample, structure and chemical properties of different molecules. ➔ It is a microanalytical technique requiring only a few nanomoles of the sample to obtain characteristic information ( structure and molecular weight ). ➔ Now a day’s mass spectrometry is used in many areas including pharmaceutical, clinical, geological, biotechnology and environmental. 4  Main information given in MS analysis –Molecular weight –Number of specific elements (based on isotope peaks) –Molecular formula (with high resolution MS) –Reproducible fragment patterns (to get clues about functional groups and/or arrangement of components or to confirm compound identity) Roles of Mass Spectrometry ❖ Qualitative analysis (less useful than NMR for true unknowns, but can be applied to very small samples) ❖ Quantitative analysis (often used for quantitative analysis)  INTRODUCTION ➔It depends upon chemical reactions in the gas phase in which sample molecules are consumed during the formation of ionic and neutral species. ➔Only particles with a positive charge will be deflected and detected ➔ The mass spectrum of each compound is unique and can be used as a “chemical fingerprint” to characterize the sample. 5 It is an instrument in which the substances in gaseous or vapor state is bombarded with a beam of electrons, to form one unpaired electron and positively charged ions (Radical cations) which are further sorted according to their mass to charge ratio to record their masses and relative abundances.  PRINCIPLE ➔In mass spectrometry, organic molecules in gaseous state are bombarded with a beam of energetic electrons (70 eV) under pressure between 10-7 to 10-5 mm of Hg, using tungsten or rhenium filament. Molecules are broken up into cations and many other fragments ➔ These cations (molecular or parent ion) are formed due to loss of an electron usually from n or π orbital from a molecule, which can further break up into smaller ions (fragment ions or daughter ions). ➔ All these ions are accelerated by an electric field, sorted out according to their mass to charge ratio by deflection in variable magnetic field and recorded. The output is known as mass spectrum. ➔ Each line on the mass spectrum indicates the presence of atoms or molecules of a particular mass. ➔ The most intense peak in the spectrum is taken as the base peak. Its intensity is taken as 100 and other peaks are compared with it.  The final peak (M+) shows the molecular ion (highest m/z value) and indicates the molecular mass. The rest of the spectrum provides information about the structure ▪ Spectra obtained for organic molecules have many peaks. Each peak is due to a particular fragment with a certain m/z value. highest m/z value usually corresponds to the molecular ion its position provides information about the molecular mass of a substance The tallest peaks come from the most stable species BASIC PRINCIPLE OF MASS SPECTROMETRY 11  COMPONENTS OF A MASS SPECTROMETER: The essential components of a mass spectrometer consist of:  A sample inlet  An ionization source  A mass analyzer  An ion detector  Vacuum system 14 Components Of A Mass Spectrometer ION SOURCE  Ionization of the organic compound is the primary step in obtaining the mass spectrum. The minimum energy required to ionize the sample or organic molecule is called as its ionization potential.  The ion source is the part of the mass spectrometer that ionizes the material under analysis (the analyte).  The ions are then transported by magnetic or electric fields to the mass analyzer.  Molecular ions are formed when energy of the electron beam reaches to 10-15 eV.  Fragmentation of the ion reaches only at higher bombardment energies at 70 eV.  Function 1. Produces ion without mass discrimination of the sample. 2. Accelerates ions into the mass analyzer. 22 ESI (Electrospray Ionization)  It is a soft ionization technique.  It is typically used to determine the molecular weights of proteins, peptides and other biological macromolecules.  It provides a sensitive, robust and reliable tool for studying. Advantages a. Has ability to handle samples with large masses. b. One of the softest ionization methods available and has the ability to analyze biological samples with non-covalent interactions. c. Good sensitivity and therefore, useful in accurate quantitative and qualitative measurements. Disadvantages a.Cannot analyze mixtures very well & when forced to do so, results are unreliable. b.Apparatus is very difficult to clean and has a tendency to become overly contaminated with residues from previous experiments. c.The multiple charges that are attached to the molecular ions can make for confusing spectral data. 24 d.Prior separation by chromatography is required. ESI - Principle  ESI uses electrical energy to assist the transfer of ions from solution into the gaseous phase.  ESI works on the principle of Soft ionization.  Soft ionization is a useful technique when considering biological molecules of large molecular mass, because in this process macromolecule is ionized into small droplets, which are then desolvated into even smaller droplets, which creates molecules with attached protons. Applications a. Protein identification and characterization. b. Studying non covalent interaction. c. Probing molecular dynamics d. Monitoring chemical reactions and studying reactive intermediates. e. Chemical imaging. f. Identification and quantification of hemoglobin variants. g. Screening for inborn errors of metabolism.  Figure Representing ELECTROSPRAY IONIZATION MECHANISM MALDI (Matrix Assisted Laser Desorption Ionization)  MALDI is also based on “soft ionization” methods where ion formation does not lead to a significant loss of sample integrity.  Consequently, the high throughput and speed associated with complete automation has made MALDI-TOF mass spectrometer an obvious choice for proteomics work on large-scale. Advantages a. Gentle Ionization technique b. High molecular weight analyte can be ionized c. Molecule need not be volatile d. Wide array of matrices e. Produces singly charged ions thus interpretation becomes easy. f. Prior separation by chromatography is not required. Disadvantages a. MALDI matrix cluster ions obscure low m/z species (