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Mass.pptx instrumentation, principle, theory
- 1. Mass spectrometry Presented by: Dr. Vijaya U. Barge (Vice Principal & Professor) Pune District Education Association’s Shankarrao Ursal College of Pharmaceutical Sciences & Research Centre.
- 2. LearningObjectives: • Mass spectrometryis sensitive analytical technique with potential applications in the field of analytical and bio-analytical chemistry. • This chapter covers all aspects of this versatile technique including instrumentationdetails and newer avenues being explored. • Soft ionization techniques are described, including chemical ionization Cl, electrospray ionization ESI, FAB, MALDI, etc. • Different types of ionic species are described, such as molecular ions, isotopic ions,metastable ions, rearrangement ions, etc. • Recognition of molecular ion peak is an important aspect of mass spectrometry and this has been described in detail along with application of nitrogen rule, index of unsaturation, ring rule, etc. • General rules for fragmentation are described including application of even electron rule. • Application of the technique for elucidating molecular formulae of the compounds is described. • Mass fragmentation pattern of different chemical classes of compounds is highlighted. • Common fragment ions and fragments lost from the molecular ion have been tabulated at the end of the chapter.
- 3. Mass spectrophotometry: Mass spectrometryis an analytical technique that is used to measure the mass-to-charge ratio of ions.The results are presented as a mass spectrum, a plot of intensity as a function of the mass-to-charge ratio mass spectrometers can be used to identify unknown compounds via molecular weight determination
- 4. Ionisation in massspectroscopy: The atom is ionised by knocking one or more electrons off to give a positive ion. (Mass spectrometers always work with positive ions). The particles in the sample (atoms or molecules) are bombarded with a stream of electrons to knock one or more electrons out of the sample particles to make positive ions.
- 5. • The choiceof ionization method depends on the nature of the sample and the type of information required from the analysis. So-called 'soft ionization' methods such as field desorption and elec- trospray ionization tend to produce mass spectra with little or no fragment-ion content.
- 6. Classification according tohard and soft source: Hard source: It imparts sufficiently energy to analyte molecule • Relaxation then involves rupture of bonds producing fragment ions that have mass to charge ratio less than that of molecular ion. Peaks in hard source provide useful information about the kind of functional groups and thus structural information about analytes. E.g. Chemical ionization
- 7. Soft source: Little fragmentation. •Supply accurate information about the molecular weight of the analyte molecules. E.g. 1)Matrix Assisted Laser Desorption Ionization (MALDI) 2) Electron impact ionization
- 9. • Electron Impact(El-Hard method) • - small molecules, 1-1000 Daltons, structure • Fast Atom Bombardment (FAB - Semi-hard) peptides, sugars, up to 6000 Daltons - • Electrospray lonization (ESI - Soft) peptides, proteins, up to 200,000 Daltons • Matrix Assisted Laser Desorption (MALDI-Soft) - peptides, proteins, DNA, up to 500 kD
- 10. • EI isdone by volatilizing a sample directly in the source that is contained in a vacuum system directly attached to the analyzer. • The gas phase molecules are bombarded by a beam of electrons formed by • heating a filament bias at a negative voltage compared to the source. The bias voltage is most commonly at -70 volts. • The electron beam ejects an ion from the gas phase molecule producing a radical ion. • This technique is considered a hard ionization technique, because it causes the • ion to fragment. EI is also the method that is most commonly used for GC-MS. Electron Impact ionization (EI) -
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- 13. Generally hydrogen (H2),methane (CH4), isobutane (iso- C4H10) and ammonia (NH3) are used as reagent gases in CI mass spectrometry; with all these CI gases the compounds form protonated molecule ion in their CI spectra. In CI mass spectrometry the molecules of a vaporized sample are ionized by a set of reagent ions (reagent plasma) in a series of ion-molecule reactions
- 14. • Electrospray reliesin part on chemistry to generate analyte ions in solution before the analyte reaches the mass spectrometer. • The LC eluent is sprayed (nebulized) into a chamber at atmospheric pressure in the presence of a strong electrostatic field and heated drying gas. The electrostatic field causes further dissociation of the analyte molecules. The heated drying gas causes the solvent in the droplets to evaporate. As the droplets shrink, the charge concentration in the droplets increases. Eventually, the repulsive force between ions with like charges exceeds the cohesive • forces and ions are ejected (desorbed) into the gas phase. • These ions are attracted to and pass through a capillary sampling orifice into themass analyzer
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- 16. Atmospheric pressure photoionization: •Atmospheric pressure photoionization (APPI) for LC/MS is a relatively new • technique. As in APCI, a vaporizer converts the LC eluent to the gas phase. • A discharge lamp generates photons in a narrow range of ionization energies. • The range of energies is carefully chosen to ionize as many analyte molecules • as possible while minimizing the ionization of solvent molecules. • The resulting ions pass through a capillary sampling orifice into the mass analyzer.
- 17. Fast Atom Bombardment(FAB) - • FAB is a technique that was popular in • the 80's to early 90's because it was the first technique that allowed • ionization of non-volatile compounds that could be done simply. • It was done by bombarding a sample in a vacuum with a beam of atoms, • typically Ar or Xe, accelerated to Kilovolt energies. The sample was • typically mixed in a matrix. The two most common matrixes were glycerol • and 3 Nitro-benzoic acid. • It was developed by Michael Barber at the University of Manchester in 1980.
- 18. Electrospray mass spectrometry(ESI-MS) • - Liquid containing analyte is forced through a steel capillary at high voltage to electrostatically disperse analyte. Charge imparted from rapidly evaporating liquid. If the sample has functional groups that readily accept H+ .(such as amide and amino groups found in peptides and proteins) then positive ion detection is used-PROTEINS • If a sample has functional groups that readily lose a proton (such as carboxylic acids and hydroxyls as found in nucleic acids and sugars) then negative ion detection is used-DNA
- 19. Matrix-assisted laser desorptionionization (MALDI) • - Analyte (protein) is mixed with large excess of matrix (small organic molecule) • Irradiated with short pulse of laser light. Wavelength of laser is the same as absorbance max of matrix. • Sample is ionized by bombarding sample with laser light • Sample is mixed with a UV absorbant matrix • Light wavelength matches that of absorbance maximum of matrix so that the matrix transfers some of its energy to the analyte (leads to ion sputtering)
- 20. Mass Analyzers Although intheory many type of mass analyzer could be used for LC/MS, four types are used most often •Quadrupole • Time-of-flight • Ion trap • Fourier transform-ion cyclotron resonance (FT-ICR or FT-MS)
- 21. Quadrupole • A quadrupolemass analyzer consists of four parallel rods arranged in a square. The analyte ions are directed down the center of the square • Voltages applied to the rods generate electromagnetic fields. These fields determine which mass-to-charge ratio of ions can pass through the filter at a given time • Quadrupoles tend to be the simplest and least expensive mass analyzers.
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- 23. Quadrupole mass analyzerscan operate in two modes: • Scanning (scan) mode • Selected ion monitoring (SIM) mode In scan mode, the mass analyzer monitors a range of mass-to-charge ratios. In SIM mode, the mass analyzer monitors only a few mass to-charge ratios.
- 24. SIM mode issignificantly more sensitive than scan mode but provides information about fewer ions. Scan mode is typically used for qualitative analyses or for quantitation when all analyte masses are not known in advance.
- 25. Time-of-flight • In atime-of-flight (TOF) mass analyzer, a uniform electromagnetic force is applied • to all ions at the same time, causing them to accelerate down a flight tube. • Lighter ions travel faster and arrive at the detector first, so the mass-to-charge • ratios of the ions are determined by their arrival times. Time-off light mass • analyzers have a wide mass range and can be very accurate in their mass measurements.
- 26. Metastable ion • Metastableion in mass spectrometry, An ion which is formed with sufficient excitation to dissociate spontaneously during its flight from the ion source to the detector. • Fragment of a parent ion will give rise to a new ion (daughter) plus either a neutral molecule or a radical. M1 + M2 + + non charged particle .An intermediate situation is possible; M1 + may decompose to M2 + while being accelerated. • The resultant daughter ion M2 + will not be recorded at either M1 or M2, but at a position M* as a rather broad, poorly focused peak. Such an ion is called a metastable ion.
- 27. Significance of Metastablelons: • Metastable ions are useful in helping to establish fragments routes. • Metastable ion peak can also be used to distinguish between fragmentation Processes, which occur in few microseconds
- 28. 1. Astronomy: analysisof astronomical components of the solar system 2. Electronics: analysis of microchips 3. Environmental: detection of toxic chemical, monitoring of nuclear facilities, analysis of petroleum products, etc. 4. Forensics: toxicology, trace metals, biological materials, etc. 5. Medical: drug abuse diagnosis, analysis of pharmaceuticals and products of genetic engineering 6. Military: mobile mass spectrometers are used to detect liquid chemica warfare agents Applications Many disciplines use mass spectroscopy for chemical identification.