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mass spectrometry

Nov 18, 2014

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MASS SPECTROMETRYINTRODUCTIONMass Spectrometer (MS) is a kind of machine which uses an analytical technique to measure the mass-to charge ratio of ions. This analytical technique is also known as Mass spectrometry. And an ion is an atom or group of atoms which have lost or gained one or more electrons, making them negatively or positively charged. Mass spectrometry is an important emerging method for the characterization of proteins. The two primary methods for ionization of whole proteins are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). As it is an important tool in proteomics, it is essential to understand not only the results, but also the principles of Mass Spectrometer. This report is devoting to provide a simple but clear explanation to the principles of Mass Spectrometer. DEFINITION: Mass spectrometry (MS) is an analytical technique for the determination of the elemental composition of a sample or molecule. It is also used for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measurement of their mass-to-charge ratios

PRINCIPLE:In a typical MS procedure: 1. a sample is loaded onto the MS instrument, and 2. the components of the sample are ionized by one of a variety of methods (e.g., by impacting them with an electron beam), which results in the formation of charged particles (ions) 3. directing the ions into an electric and/or magnetic field 4. computation of the mass-to-charge ratio of the particles based on the details of motion of the ions as they transit through electromagnetic fields, and 5. Detection of the ions, which in step 4 were sorted according to m/z.

INSTRUMENTATION:A mass spectrometer consists of following basic components 1. The inlet system (or) sample handling system 2. The ion source or ionisation chamber 3. The ion separator 4. The ion collector (the detector and readout system) 5. The vacuum system

THE MASS SPECTROMETER

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THE INLET SYSTEM: Direct Vapour Inlet. Gas Chromatography. Liquid Chromatography Direct Insertion Probe Direct Ionization of Sample

IONIZATION SOURCES Chemical Ionisation (CI) Atmospheric Pressure CI!(APCI) Electron Impact!(EI) Electrospray Ionization!(ESI) Matrix Assisted Laser Desorption Ionisation!(MALDI) Field Desorption/Field Ionisation (FD/FI) Fast Atom Bombardment (FAB) Thermo spray Ionisation (TI)

ANALYZERS quadruples Time-of-Flight (TOF) ion trap analyzer Fourier transform analyzer Orbitrap analyzer

DETECTORS electron multiplier detector Faraday cup detector Array detector

THE INLET SYSTEM/ SAMPLE HANDLING SYSTEM:The selection of a sample inlet depends upon the sample and the sample matrix. Most ionization techniques are designed for gas phase molecules so the inlet must transfer the analyte into the source as a gas phase molecule. If the analyte is sufficiently volatile and thermally stable, a variety of inlets are available. Gases and samples with high vapor pressure are introduced directly into the source region. Liquids and solids are usually heated to increase the vapor pressure for analysis. If the analyte 3

is thermally labile (it decomposes at high temperatures) or if it does not have a sufficient vapor pressure, the sample must be directly ionized from the condensed phase. These direct ionization techniques require special instrumentation and are more difficult to use. However, they greatly extend the range of compounds that may be analyzed by mass spectrometry. Commercial instruments are available that use direct ionization techniques to routinely analyze proteins and polymers with molecular weights greater than 100,000 Dalton. DIRECT VAPOR INLET. The simplest sample introduction method is a direct vapor inlet. The gas phase analyte is introduced directly into the source region of the mass spectrometer through a needle valve. Pump out lines are usually included to remove air from the sample. This inlet works well for gases, liquids, or solids with a high vapor pressure. Samples with low vapour pressure are heated to increase the vapor pressure. Since this inlet is limited to stable compounds and modest temperatures, it only works for some samples. GAS CHROMATOGRAPHY. Gas chromatography is probably the most common technique for introducing samples into a mass spectrometer. Complex mixtures are routinely separated by gas chromatography and mass spectrometry is used to identify and quantitate the individual components. Several different interface designs are used to connect these two instruments. The most significant characteristics of the inlets are the amount of GC carrier gas that enters the mass spectrometer and the amount of analyte that enters the mass spectrometer. If a large flow of GC carrier gas enters the mass spectrometer it will increase the pressure in the source region. Maintaining the required source pressure will require larger and more expensive vacuum pumps. The amount of analyte that enters the mass spectrometer is important for improving the detection limits of the instrument. Ideally all the analyte and none of the GC carrier gas would enter the source region. The most common GC/MS interface now uses a capillary GC column. Since the carrier Gas flow rate is very small for these columns, the end of the capillary is inserted directly into the source region of the mass spectrometer. The entire flow from the GC enters the mass spectrometer. Since capillary columns are now very common, this inlet is widely used. LIQUID CHROMATOGRAPHY. Liquid chromatography inlets are used to introduce thermally labile compounds not easily separated by gas chromatography. These inlets have undergone considerable development and are now 4

fairly routine. Because these inlets are used for temperature sensitive compounds, the sample is ionized directly from the condensed phase. DIRECT INSERTION PROBE. The Direct Insertion Probe (DIP) is widely used to introduce low vapor pressure liquids and solids into the mass spectrometer. The sample is loaded into a short capillary tube at the end of a heated sleeve. This sleeve is then inserted through a vacuum lock so the sample is inside the source region. After the probe is positioned, the temperature of the capillary tube is increased to vaporize the sample. This probe is used at higher temperatures than are possible with a direct vapor inlet. In addition, the sample is under vacuum and located close to the source so that lower temperatures are required for analysis. This is important for analyzing temperature sensitive compounds. Although the direct insertion probe is more cumbersome than the direct vapor inlet, it is useful for a wider range of samples. DIRECT IONIZATION OF SAMPLE Unfortunately, some compounds either decompose when heated or have no significant vapor pressure. These samples may be introduced to the mass spectrometer by direct ionization from the condensed phase. These direct ionization techniques are used for liquid chromatography/mass spectrometry, glow discharge mass spectrometry, fast atom bombardment and laser ablation. The development of new ionization techniques is an active research area and these techniques are rapidly evolving. Direct ionization is discussed in greater detail in the next sectio

IONISATION SOURCE: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. Techniques for ionization have been key to determining what types of samples can be analyzed by mass spectrometry. Electron ionization and chemical ionization are used for gases and vapors.

1. CHEMICAL IONIZATIOChemical ionization (CI) is an ionization technique used in mass spectrometry. Chemical ionization is a lower energy process than electron ionization. The lower energy yields less fragmentation, and usually a simpler spectrum. A typical CI spectrum has an easily identifiable molecular ion. Mechanism In a CI experiment, ions are produced through the collision of the analyte with ions of a reagent gas that are present in the ion source. Some common reagent gases include: methane, 5

ammonia, and isobutane. Inside the ion source, the reagent gas is present in large excess compared to the analyte. Electrons entering the source will preferentially ionize the reagent gas. The resultant collisions with other reagent gas molecules will create ionization plasma. Positive and negative ions of the analyte are formed by reactions with this plasma. Primary Ion Formation

Secondary Reagent Ions

Product Ion Formation (Protonation) (H abstraction) (Adduct formation) (Charge exchange) Self chemical ionization occurs when the reagent ion is an ionized form of the analyte. Negative chemical ionization (NCI) Chemical ionization for gas phase analysis is either positive or negative. Almost all neutral analytes can form positive ions through the reactions described above. In order to see a response by negative chemical ionization, the analyte must be capable of producing a negative ion (stabilize a negative charge) for example by electron capture ionization. Because not all analytes can do this, using NCI provides a certain degree of selectivity that is not available with other, more universal ionization techniques (EI, PCI). NCI can be used for the analysis of compounds containing acidic groups or electronegative elements (especially halogens). Because of the high electronegativity of halogen atoms, NCI is a common choice for their analysis. This includes many groups of compounds, such as PCBs, pesticides, and fire retardants. Most of these compounds are environmental contaminants, thus much of the NCI analysis that takes place is done under the auspice