*MASS SPECTROMETRY(MS)
• It is the most accurate method for determining the molecular mass of the compound and its elemental composition.
• This operates by converting analytes molecule into gaseous, charged forms by application of electrostatic potential.
• It is used to measure the mass of charged particle that is mass to charge ratio of ions.
• It uses high energy electrons to break a molecule into fragments.
• Separation and analysis of the fragments provide information about :
➢ Molecular weight
➢ Structure
*MASS SPECTROMETER
mass spectrometer is an instrument which generates a beam of positively charged ions from the sample.
The essential components:
1. An ionization source
2. A mass analyzer
3. An ion detector
•Produce ions from the sample in the lonization source.
•Separate these ions according to their mass-to-charge ratio in the mass analyzer by accelerating them & subjecting them to magnetic or electric field.
• Detect the ions emerging from the analyzer and measure their abundance with the detector that converts the ions into electrical signals
•Process the signals from the detector that are transmitted to the computer to provide a mass spectrum.
Mass Spectrum
• Mass spectrum is a graph obtained by performing mass spectrometry. It is a relation between the mass to charge ratio and ion signal.
*IONIZATION SOURCE
• lonization of the organic compound is the primary step in obtaining the mass spectrum.
• Molecules in a sample are vaporized (converted to the gas phase by heating). Then, an electron beam bombards the vapors, which converts the vapors to ions.
• The ions are then transported by magnetic or electric fields to the mass analyzer.
Eg: Matrix Assisted Laser Desorption/Ionization(MALDI)
Electrospary Ionization (ESI)
*MALDI
•Sample is ionized by bombarding sample with laser light
• Sample is mixed with a UV absorbantmatrix(sinapinic acid for proteins, 4-hydroxycinnaminic acid for peptides)
•The laser light excites and vapourisesthe matrix and convert analyte into gas phase ions.
*MASS ANALYZER
• It identifies and sorts the analyte ions based on their mass to charge ratio
• Different type of mass analysers are available
• One of the simplest analyzer is Time Of Flight(TOF)
*TIME OF FLIGHT
• In which ions are accelerated through an elongated chamber under a fixed electrostatic potential
• It gives two ions of identical net charge, smaller ions require less time to transverse the chamber than the large ion
• The mass of the ions can be determined by measuring the time required for each ions to pass through the chamber
*DECTECTORS
Ions of increasing mass eventually reaches the dectector one after another and then its over to the computer to provide spectrum
Eg: Faraday cup,Electron multiplier
*Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE)
• SDS PAGE is a reliable method for determining the molecular weight of an unknown protein
• Electrophoresis consists of the migration of a charged molecules under the influence of electric field
• A buffer solution is use to conduct electricity through the whole setup
•Polyacrylamide gels are used in SDS PAGE
•The molecule will migrate through the gel depending upon the size, MW and shape.
•A denaturing detergent sodium dodecyl sulfate is used in PAGE to separate protein subunits according to molecular weight
•Proteins do not have a specific charge. SDS has a negative charge and by dissolving the protein in it, the protein becomes negatively charged.
• Then protein will to run properly through the gel(from negative to positive).
*Isoelectric Focusing
• Isoelectric focusing is a method in which proteins are separated in a pH gradient according to their isoelectric points
• It is carried out on a gel that contain a gradient PH.
• The PH gradient is created across the supporting medium & electrophoresis of protein mixture carried out through the gradient.
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