Mass Spectroscopy
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Mass Spectrometry
Dr Nizam M. El-Ashgar 1
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Mass Spectrometry • Mass spectrum is obtained by converting components of a sample into rapidly moving gaseous ions and resolving them on the basis of their mass-to-charge ratios. • Most generally applicable of analytical tools since capable of qualitative and quantitative information about both atomic and molecular composition of inorganic and organic compounds.
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Molecular Mass Spectrometry • Provides information about: • 1- The elemental composition of samples of matter • 2- The structures of inorganic, organic and biological molecules. • The qualitative and quantitative composition of complex mixture • The structure and composition of solid surfaces • Isotope ratios of atoms in samples. 3
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Acuracy of MWt Mesurments • For large samples such as biomolecules, molecular masses can be measured to within an accuracy of 0.01% of the total molecular mass of the sample. • Example: an error within a 4 Daltons (Da) or atomic mass units (amu) for a sample of 40,000 Da. This is sufficient to allow minor mass changes to be detected, e.g. the substitution of one amino acid for another, or a post-translational modification. • For small organic molecules the molecular mass can be measured to within an accuracy of 5 ppm or less, which is often sufficient to confirm the molecular formula of a compound, and is also a standard requirement for publication in a chemical journal.
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Where are mass spectrometers used? Mass spectrometers are used in industry and academia for both routine and research purposes. The following list is just a brief summary of the major mass spectrometric applications: Biotechnology: the analysis of proteins, peptides, oligonucleotides Pharmaceutical: drug discovery, combinatorial chemistry, pharmacokinetics, drug metabolism Clinical:, haemoglobin analysis, drug testing Environmental: water quality, food contamination
Geological: oil composition
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Mass Spectra • When the electron beam ionizes the molecule, the species that is formed is called a radical cation, and symbolized as M+•. • The radical cation M+• is called the molecular ion or parent ion. • The mass of M+• represents the molecular weight of M. • Because M is unstable, it decomposes to form fragments of radicals and cations that have a lower molecular weight than M+•. • The mass spectrometer analyzes the masses of cations. • A mass spectrum is a plot of the amount of each cation (its relative abundance) versus its mass to charge ratio (m/z, where m is mass, and z is charge). • Since z is almost always +1, m/z actually measures the mass (m) of the individual ions.
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Mass Spectrometry
• The tallest peak in the mass spectrum is called the base peak. • The base peak is also the M peak, although this may not always be the case. • Though most C atoms have an atomic mass of 12, 1.1% have a mass of 13. Thus, 13CH4 is responsible for the peak at m/z = 17. This is called the M + 1 peak. 7
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• Since the molecular ion is unstable, it fragments into other cations and radical cations containing one, two, three, or four fewer hydrogen atoms than methane itself. • Thus, the peaks at m/z 15, 14, 13 and 12 are due to these lower molecular weight fragments.
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Molecular Mass Spectra (hexane)
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Explanation • The collisions between energetic electrons and analyte molecules (enough E) lead to excitation. • Relaxation leads to fragmentation to lower masses ions • Attraction of positive ions through the slit of a mass spectrometer and sorted according to m/z ratios and appear in the Mass spectrum. • Mass spectrum is a plot of relative intensity versus m/z • Base peak has the value of 100 (arbitrarily) • The remaining computed as % of the base-peak height • Modern MS programmed base-peak and normalize the remaining peaks relative to that peak 10
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Mass Spectrometer All Instruments Have:
1. Sample Inlet 2. Ion Source 3. Mass Analyzer 4. Detector 5. Data System
http://www.asms.org
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Ion sources Starting point: Formation of gaseous analyte Ions.
Methods of ion formation: Two major categories: 1- Gas-phase sources -The sample is first vaporized and then ionized. -Restricted to thermally stable compounds of BP < 500 0C. -Limited to Compounds of MWt’s <103dalton . 2- Desorption sources The sample in a solid or liquid state is converted directly into gaseous ions (not require volatilization of analyte molecules) Applicable to nonvolatile and thermally unstable samples Applicable to analytes having of 105 dalton or larger. 13
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Basic Type
Gas Phase
Desorption
Name
Ionizing agent
Electron Impact (E I)
Energetic electrons
Chemical Ionization (CI)
Reagent gaseous ions
Field ionization (FI)
High-potential electrode
Field desorption (FD)
High-potential electrode
Electrospray ionization (ESI)
High electric Field
Matrix-assisted desorption/ionization (MALDI)
Laser beam
Plasma desorption (PD)
Fission fragments from 252Cf
Fast atom bombardment (FAB)
Energetic atomic beam
Secondary ion mass spectrometry (SIMS) Energetic beam of ions 14
Thermospray ionization (TI)
High temperature م09:40 22/12/2012
Hard and Soft Sources (another classification) • Hard Sources: • Impart sufficient E to analyte molecules (become of high excited E state). • Relaxation involves rupture of bonds producing fragment ions with m/z < than that of molecular ion. • Provides useful information about kinds of functional groups and structure information. • Soft Sources: • Causes little fragmentation. • Mainly the spectrum consists of the molecular ion peak and few peaks • Supplies accurate information about MWt of analyte. Both are useful for analysis 15
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The Electron-Impact Sources (EI) The sample is brought to a temperature high enough to produce a molecular vapor, which is then ionized by bombarding the resulting molecules with a beam of energetic electrons. • Positive ions forced by small potential difference through accelerator plates to mass analyzer. M + e- M.+ + 2ewhere, M = analyte molecule, M.+ = molecular ion. Relaxation then usually takes place by extensive fragmentation, giving a large number of positive ions of various masses that are less than that of the molecular ion. These lower mass ions are called daughter ions.
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Gaseous sample bombarded with beam of energetic electrons. Electrons produced at heated W or Rh wire and accelerated to energy of about 70 eV. Typically one in every million molecules undergoes ionization.
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Electron Impact (EI)
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• The positive ions produced are attracted through the slit in the first accelerating plate by a small potential diff (5 V). • With magnetic sector instrument high potentials (103 to 104) are applied to the accelerator plates) KE = qV = zeV • KE of an ion is independent of its mass and depends only upon its charge and accelerating potential • Velocity of an ion depends on its mass KE = 1/2m2 or = (KE/m)1/2
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Electron Impact Advantages • Well-Established • Fragmentation Libraries • Insoluble Samples • Interface to GC • Non-Polar Samples • They are convenient and produce high ion currents. • Extensive fragmentation can lead to unambiguous identification of analytes • Disadvantages • Parent Identification • Need Volatile Sample • Need Thermal Stability • No Interface to LC • Low Mass Compounds (<1000 amu) • Solids Probe Requires Skilled Operator • The need to volatilize the sample limits this method since it excludes analysis of thermally unstable compounds. • Excessive fragmentation can lead to the disappearance of the molecular ion peak therefore preventing the molecular mass of the analyte to be determined. 21
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Electron-Impact Spectra
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EI-MS of Methylene Chloride (top) and 1-Pentanol (bottom)
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•
Types of MS Peaks:
•
Molecular (or Parent) Ion – ion having same mass as the analyte Daughter Ion – ion having lower mass Base Peak – biggest peak
• • •
Size of peaks depends on relative natural abundance of isotopes The base peak in electron-impact spectra arise from fragments rather than from molecular ion. The molecular ion peak provides the MWt of the unknown. In EI certain molecules yield no molecular ion peak. 24
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Isotope Peaks •
Peaks occur at masses that are greater than that of the molecular ion are attributable to ions containing isotopes. • Examples: 12C1H 35Cl : m = 84 2 2 13C1H 35Cl : m = 85 2 2 12C1H 35Cl37Cl: m = 86 2 13C1H 35Cl37Cl: m = 87 2 12C1H 37Cl : m = 88 2 2 The size of various peaks depends upon the relative natural abundance of the isotopes. Note: F, P, I, Na occur only as single isotopes. Isotope peaks some times provide useful means for determining the formula for compound.
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Collision product peaks • Ion/molecule collisions, can produce peaks at higher mass numbers than that of the molecular ion. • At ordinary sample pressures, however the only important reaction of this type: transferring of H atom to the ion to give a protonated molecular ion, enhanced (M +1 )+ peak results (second order reaction). • The amount of product depends strongly upon the reactant concentration. • The height of an (M+1)+ peak due to this reaction increases with increase in sample pressure than do the heights of other peaks (detected).
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Advantages of EI-MS Convenient to use Produce high ion currents (gives good sensitivities) Often unambiguous identification of compounds possible (extensive fragmentations)
Disadvantages of EI-MS Extensive fragmentation may not leave parent ion (Molecular peak ion). Sample must be volatilized (thermal degradation) of some analytes before ionization to be occur. Only applicable to samples with molecular weights < 103 amu
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Chemical ionization sources and spectra Second most common process for ms • In modern MS, EI ionization and chemical ionization are interchangeable. Gaseous atoms are ionized by collision with ions produced by electron bombardment of an excess of a reagent gas. Positive ions usually used. negative ion used with analyte contain very electronegative atoms.
Experimentally: Modification of the electron beam ionization area of EI by adding vacuum pump capacity and reducing width of the slit to the mass analyzer so P reduced to 1 torr (Ionization area) and 10-5 torr in the analyzer. Gaseous regent introduced in the ionization region reagent:sample = 103-104. So electron beam reacts exclusively with reagent molecules.
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Common Reagents Methane • • • • • • • • • •
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Reacts with high E electrons to give several ions (CH4+ and CH3+ (90 %), CH2+). These ions react rapidly with additional methane molecules: CH4+ + CH4 CH5+ + CH3 CH3+ + CH4 C2H5+ + H2 Reactive collisions between sample molecules MH and CH5+ or C2H5+ take place: (proton and hydride transfer). CH5+ + MH MH2+ + CH4 (proton transfer) gives (M +1)+ ion peak C2H5+ + MH MH2+ + C2H4 (proton transfer) gives (M +1)+ ion peak C2H5+ + MH M+ + C2H6 (hydride transfer) gives (M -1)+ ion peak C2H5+ + M M- C2H5+ gives (M + 29)+ ion peak Other reagents: propane, isobutane and ammonia that give different spectra with a given analyte.
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EI hard source
CI Soft source
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Field ionization Sources • •
Ions are formed under large electric field (108 V/cm). The field produced by applying high voltages (10 to 20KV) to emitters having diameter < 1 m. • Example: W fine wire ( 10 m diameter) emitter of many hundreds of carbon microtips projecting from the surface tungsten wire. • FI are mounted 0.5-2 mm from the cathode (often serves as slit). • Gaseous sample is allowed to diffuse into the high-field area around the microtips of the anode. • EF is concentrated at the emitter tips and ionization occurs via a quantum mechanical tunneling mechanism in which electrons from the analyte are extracted by microtips of the anode. • Little vib. or rot energy is imparted to the analyte thus little fragmentation occurs.
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FI Source
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MS of Glutamic Acid Electron Impact
Field Ionization
Field Desorption
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Desorption sources Previous methods: The ionizing agents act on gaseous samples. • Such methods are not applicable to nonvolatile or thermally unstable samples. • Desorption ionization methods: are develop to deal with this type of samples. (thermally unstable biochemical species or that of 100, 000 Da). • The method is: dispenses the volatilization followed by ionization of the gaseous analyte molecules. • Instead: E in various forms is introduced into solid or liquid sample large molecules to overcome intermolecular forces to cause direct formation of gaseous ions.
• provide gentle ionization. e.g. carbohydrates, peptides, nucleic acids, organic salts & organometallics. • • 35
Simple spectra consist of only the molecular ion or the protonated molecular ions. Exact mechanism is not understood. م09:40 22/12/2012
Field Desorption Sources
• Multitipped emitter similar to FI source is used. • The electrode is mounted on a probe that can removed from the sample compartment and coated with a solution of the sample. • The probe is reinserted into the sample compartment. • Ionization takes place by the application of a high potential to this electrode. • Some samples needed heating the emitter by passing a current through the wire. • Thermal degradation may occur before ionization is complete. • Example of spectra (glutamic acid previously). • Spectrum is simpler than that of CI or FI consists of only the protonated molecular ion peak at mass 148 and isotope peak at mass 149. 36
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Matrix-Assisted Laser Desorption/Ionization (MALDI) • MALDI: is a new ionization method: provides accurate MWt of polar biopolymers (MWt few thousands to several hundred thousands da). • First technique (German group): an aqeous/alcohol solution of sample was mixed with large excess of a radiation-absorbing matrix material. • The resulting solution was evaporated on the surface of a metallic probe that was used for introduction of the sample into mass spectrometer. • The solid mixture exposed to a pulsed laser beam so analyte ions sublime. • Entire spectrum recording between the period of laser pulses.
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MALDI: Matrix Assisted Laser Desorption Ionization Sample plate
Laser
h
MH+
1. Sample is mixed with matrix (X) and dried on plate.
2. Laser flash ionizes matrix molecules. 3. Sample molecules (M) are ionized by proton transfer: XH+ + M MH+ + X.
+/- 20 kV 39
Grid (0 V) م09:40 22/12/2012
Requirement for MALDI: • Matrix compound must absorb the laser radiation strongly. • Soluble in sample solvent and present in large excess in the solid matrix deposited on the probe. • The analyte should not absorb laser to prevent fragmentation. • Only few compounds are suitable as matrices for biopolymers (Table 20-4).
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6. MALDI • matrix has strong A at laser λ • 500 fmol of betalow mass, so can sublime • D-galactosidase can measure Mr of proteins and biomolecules •
mixed with nicotinic acid on a silver surface using NdYAG 266 nm excitation RMM 117130 Fragments are cluster ions [nM+H]+ and multiplycharged ions [nM+zH]z+
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Electrospray ionization • ESI/MS (1984) now it is an important technique for analyzing biomolecules (polypeptides, proteins and oligonucleotides having MWt 0f 100,000 Da or more). Also used to identify inorganic species and synthetic polymers). • Takes place under atmospheric pressures and temperatures. • A solution of the sample is pumped through a stainless steel capillary needle at a rate of few microliters per minute. • The needle is maintained at several kilovolts with respect to a cylindrical electrode surrounds the needle. • The resulting charged spray of fine droplets then passes through a dissolving capillary . • Solvent evaporate and analyte charged and charge density increases and ions desorbed into the ambient gas. • Little fragmentation of large thermally fragile biomolecules occurs. • The ions formed are multiply charged (small m/z) detected by quadrupole instruments (range of 1500 or less). • Is ready adapted to direct sample introduction from HPLC and 42 electrophoresis columns. م09:40 22/12/2012
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proteinase K
ESI ms in 50% CH3CN/46% H2O/4% acetic acid with source at 100 oC.
29 kDa
thermolysin 34 kDa
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Fast Atom Bombardment FAB • FAB is a major role in the production of ions for ms studies of polar high MWt. species. • Samples in a condensed state often in a glycerol solution matrix are ionized by bombardment with energetic (several KeV) xenon or argon atoms. • Both +ve and –ve analyte ions are sputtered from the surface of the sample in a desorption process. • Rapid sample heating by this process reduces sample fragmentation. • A beam of fast atoms is obtained by passing accelerated Ar or Xe ions from an ion source or gun through a chamber containing Ar or Xe atoms at P of about 10-5 torr. • The high velocity ions undergo exchange reactions with the atoms without loss of translational energy and a beam of energetic atoms is formed. • The lower E ions from the exchange are removed by an electrostatic deflector. • FAB of organic or biochemical compounds usually produces significant amounts of molecular ions (and ion fragments) even for high MWt and thermally unstable samples (over 10,000 da) and detailed structure information obtained for samples of (3000 da). 45 م09:40 22/12/2012
FAB – sputtering/desorption technique. Sample dissolved in low-volatility, viscous solvent and placed on insertion probe. Bombard with atoms or ions 4-10 keV energy.
Xe/Ar atoms
Atoms/ions penetrate several layers, distribute k.e. 46
No fragmentn
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up to 9600 amu
FAB
Sample monolayer sputtered in few s. Nonvolatile glycerol allows surface layer to be renewed. Ar G (Gn+H)+ (M+H)+ sample M (M-H) (M+G+H)+ 47
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Mass Spectrometer • • •
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Several Types. Only two will be described: Quadrupole spectrometer and the time-of-flight spectrometer. General description of instrument components:
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Inlet System: To introduce a very small amount of sample (mol or less) into the mass spectrometer that converted to gaseous ions. ( a mean for volatilizing solid or liquid samples is presents).
Ion sources: Convert the components of a sample into ions. (generally the inlet system and the ion source are combined into a single component). The output is a stream of +ve or –ve ions are accelerated into the mass analyzer.
Mass analyzer analogous to grating in an optical spectrometer. Dispersion is based upon the mass/charge ratios of the analyte ions rather than upon the wavelength of photons. (Different categories of MS according to mass analyzer).
Detectors: Convert the beam of ions into an electrical signal that can then be processed, stored in the memory of a computer and displayed or recorded in a variety ways. Vacuum System: To create low pressure (10-4 to 10-8 torr) in all the instrument components except the signal processor and readout. To prevent interaction of components with atmosphere so destroyed. 49
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Sample Inlet System • For permitting introduction of a representative sample into the ion source with minimal loss of vacuum. Various types of inlets equipped to accommodate different samples: (Batch inlets, direct probe inlets, chromatographic inlets and capillary electrophoretic inlets). Batch Inlet Systems: Classical and simplest type. Sample is volatilized externally and then allowed to leak into evacuated ionization region. The figure shown is for a one that applicable to gaseous and liquid samples having PB up to 5000C. Gaseous samples: A small measured volume of a gas is trapped between the two valves and then expanded into reservoir flask. 50
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•
• • •
51
For liquids a small quantity of sample is introduced into a reservoir usually with a micrometer syringe. In either case vacuum system is used to achieve sample P 10-4 – 10-5 torr. For samples with boiling points > 1500C T must be maintained at elevated T by oven and heating tapes of maximum T of about 3500C. Sample is now in the gas phase is leaked into the ionization area of the spectrometer via a metal or glass diaphragm containing one or more pinholes.
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MS – Direct Sample Introduction
External Sample Introduction System
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Direct Sample Probe م09:40 22/12/2012
The Direct Probe Inlet. •
Solids and non volatile liquids can be introduced into the ionization region by means of a sample holder probe which is inserted through a vacuum lock. The lock system: Designed to limit V of air pumped after probe insertion. Probes also used for limited quantity samples (few nanograms)(less wasted than batch system). Probe: Sample is held on the surface of a glass or Al capillary tube, a fine wire or a small cup. The probe is positioned within a few mm of the Ionization source and the slit leading to spectrometer. Vacuum used to maintain thermally unstable compounds for spectrum measurements before major decomposition occurs. And to elevate nonvolatile conc. in the ionization area (carbohydrates, steroids, metalorganic species and low molecular weight polymeric substances). Partial pressure attained is at least of 10-8 torr before onset decomposition. 53
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• Chromatographic and capillary electrophoretic inlet System. • MS are often coupled with GC and HPLC or capillary electrophoresis columns. • Separation and determination of the components of complex mixture is obtained. • Specialized inlet systems is needed.
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Mass Analyzers • Several devices are available for separating ions with different m/z ratios. Mass analyzer should be: - Capable of distinguishing between minute mass differences. - Allows passage of sufficient number of ions to yield measurable currents. Resolution of mass spectrometer: R
R = m/Dm Where, m : mass of the first peak (or mean mass of the two peaks) Dm: mass difference between two adjacent peaks Two peaks are considered to be separated if height of valley between them 10% of their height. R of 4000 would resolve peaks occurring at m/z values of 400.0 and 400.1 (or 40.00 and 40.01)
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• R needed in MS depends upon its application. Example 1: same nominal mass ions: C2H4+, CH2N+, N2+ and CO+ (All ions of nominal mass 28 Da). Exact masses: 28.0313, 28.0187, 28.0061 and 27.9949 Da respectively. These requires an instrument with a resolution of several thousands. Example 2: Low MWt ions with a unit mass difference or more: NH3+ (m = 17) and CH4+ (m = 16) R instrument of 50 or less is sufficient. Commercial MS are available with R range of 500-500,000. Example: What R is needed to separate C2H4+ and CH2N+, ions? Dm = 28.0313 - 28.0187 = 0.0126 R =m/ Dm = 28.025/0.0126 = 2.22x103 Where 28.025 is the mean mass for the two species
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Magnetic Sector Analyzers • Employ a permanent magnet or an electromagnet. • Cause beam from the ion source to travel in a circular path most commonly of (180, 90 or 60 deg) • Schematic of a 90-deg magnetic sector spectrometer
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• •
Operation: Ions formed by Electron impact are accelerated through slit B into the metal analyzer tube internal P 10-7 torr. • Ions of different mass can be scanned across the exit slit by varying the field strength of the magnet or the accelerating potential between slits A and B. • Ions passing through the exit slit fall on a collector electrode, ion current resulted that amplified and recorded. • Translation or KE of an ion of mass m bearing z exiting slit B is given by: KE = ZeV = ½ mv2 Where V is voltage between A and B V is velocity of the ion after acceleration e is the electronic charge = 1.60x10-19 C Note: all ions having the same number of charges are assumed to have the same KE after accelerating regardless of their mass. (approximately true). All ions leaving the slit have approximately same KE, the heavier ions must travel at lower velocities. The path in sector by ions of a given m/z represents a balance between two forces acting upon them. 59
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The magnetic force FM: FM =BzeV
Where B is MF strength The balancing centripetal force: Fc = mv2/r Where r is the radius of curvature of the magnetic sector. For ion to traverse the circular path to the collector, FM and FC must be equal FM =BzeV = Fc = mv2/r v = Bzer/m Substituting the previous equation in ZeV = ½ mv2 m/z = B2r2e/2V Mass Spectra: Varying one of three variables (B, V or r) while holding the other two constants. Modern MS ions are sorted by holding V and r constant while varying the current in the magnet and thus B. In sector MS (using photographic recording) B and V are constants, r is the variable. 60
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•
What accelerating potential will be required to direct a singly charged water molecule through the exit slit of a magnetic mass spectrometer if B = 0.240 T and r of curvature of the ion through the magnetic field is 12.7 cm? SI units: ez = 1.60x10-19 Cx 1 r = 0.127 m
m
18.02 g H 2O / mol 6.02 x1023 H 2O / mol
x103
m = 2.99x10-26kg B = 0.240 T = 0.240 W/m2 m/z = B2r2e/2V or = [0.240 W/m2]2[0.127m]2[1.60x10-19C] 2x2.99x10-26kg = 2.49 x103 W2C/m2kg or volts
kg g
V = B2r2ez/2m
(In SI base units, the dimensions of the weber are kg.m2/S2.A . In derived units, they are volt-seconds V.s , or joules per amp J/A. The weber is a large unit, equal to 1 T m2 = 108 maxwells. •
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Double focusing spectrometers Previous are single-focusing spectrometers. Limitation: Low precision because of: 1- directional distribution of ions 2- energy distribution of ions Same m/z ratio but with small diverging directional distribution are focused so limiting the resolution of magnetic sector instruments (R2000). This because of the translational E distribution of ions leaving a source (Boltzmann dist.) arises from energies of original molecules and source field inhomogeneities. Spread of KE causes a broadening of the beam reaching the transducer and a loss of resolution.
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Double Focusing Spectrometers: Correction for both the directional and E distribution of ions. Both directional and E aberrations of a population of ions are simultaneously minimized by use of carefully selected combinations of electrostatic and magnetic fields.
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• •
• • • • • •
64
The ion beam is first passed through an electrostatic analyzer (ESA). Consists of two smooth curved metallic plates across which a dc potential is applied which limiting the KE of the ions reaching the magnetic sector to a closely defined range. Ions with E greater than average strike the upper side of ESA slit and lost to the ground. Ions with E less than average strike the lower side of the ESA slit and are thus removed. Directional focusing occurs along the focal plane (d). Energy focusing takes place along the plane e. Only ions of one m/z are double focused at the intersection of d and e for any given V and B. The collector slit is located at this locus of double focus.
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Quadrupole Mass Spectrometer • • • • •
Comparing with magnetic sector instrument it is: Less expensive and more rugged. More compact Found in commercial bench top ms Low scan times (<100 ms) which is useful for chromatography. • Most common mass analyzers in use today. • Quadrupole mass analyzer is responsible for filtering the samples ions. • Consists of four parallel metal rods. • Each rod pair is connected together electrically. • A radio frequency voltage is applied between one pair of rods then the other. 65
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• Ions travel down the quadrupole in between the rods • Electric field separates ions • Ions are subjected to complex forces • Only Ions of a particular m/z reaches the detector Advantages • Inexpensive • Easily Interfaced to Many Ionization Methods Disadvantages • Low Resolution (< 4000) • Low Accuracy (>100 ppm) • MS/MS requires multiple analyzers • Low Mass Range (< 4000) • Slow Scanning 66
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Time-of-Flight MS (TOF) •
• •
• • •
67
The +ve ions are produced periodically by bombardment of the sample with brief pulses of electrons, secondary ions or laser generated photons. The ions are then accelerated into a field-free drift tube by an electric field pulse of 103 – 104 V Separation of ions on the basis of mass occurs during the transition of the ions to the detector located at the end of the tube. All ion have same KE but their velocities are inversely proportional to their masses Lighter particles arrived earlier. Typical flight times are 1 to 30 s.
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Time-of-Flight MS (TOF)
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Advantages • Simplicity and ruggedness. • Ease of accessibility of the ion source. • Extremely High Mass Range (>1 MDa) • Fast Scanning Disadvantages • Low Resolution (4000) • Low sensitivity • Low Accuracy (>200ppm) • MS/MS not possible
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Ion Trap Analyzers • • •
71
Gaseous anions or cations can be formed and confined for extended periods by electric and/or magnetic fields. Several types, A simpler type of ion trap that used for GC/MS Now used to obtain mass spectra of a variety of analytes.
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• • • •
•
• •
73
It consists of a central doughnut-shaped ring electrode and a pair of endcap electrodes. A variable RF voltage is applied to the ring electrode while the two end-cap electrodes are grounded. Ions of appropriate m/z value circulate in a stable orbit within the cavity surrounded the ring. By increasing RF voltage the orbit of heavier ions become stabilized , while lighter become destabilized causing them to collide with the wall of the ring electrode. By RF voltage scanning the trapped ions destabilized and leave the ring electrode cavity via openings in the lower end cap then emitted to a transducer. Rugged compact and less costly than sector or quadrupole instruments. Capable of resolving ions that differ in mass by unit in the mass range of 500-1000 Da
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Operation of an Ion Trap MS
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Fourier Transform (FT) instruments • FTMS provide: • improved signal to noise ratios • Greater speed. • Higher sensitivity. • Higher resolution. The heart of FTMS is an ion trap within which ions can circulate in a well-defined orbits for extended periods.
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The Ion Cyclotron Resonance (ICR) • Gaseous ion drifts into or formed in a strong MF. • The motion becomes circular in a plane that is to the direction of the field. • The angular frequency of this motion is called cyclotron frequency c. c= v/r = zeB/m • In fixed field the c depends only upon the inverse of the m/z value. • Increases the velocity of an ion will be accompanied by increase of rotation of the ion. • This circulated trapped ion in the MF is capable of absorbing E from an ac EF. • So the EF frequency matches the c. • The absorbed E increases v of the ion and r of travel without disturbing c 76
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MS – Fourier Transform Analyzer Ion Cyclotron Resonance
Magnetic Field 77
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• Inner solid line represents the original path to the ion. • Dashed line shows spiral path when switch is moved briefly to position 1. • Outer solid line is new circular path when switch again opened. • For ensemble ions of the same m/z ratio between the two plates; when ac signal is applied the cyclotron resonance frequency sets all the particles into coherent motion that is in phase with the field. • Ions of different m/z ratios (different c) are unaffected by the ac field. Measurement of the ICR signal: Coherent circular motion of resonant ions creates image current observed current after termination of the frequency sweep signal (from position 1 to 2). The current decreases exponentially with time. It is a capacitor current induced by circular movement of a packet of ions with the same m/z ratios. 78
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Example: +ve ions approaches the upper plate electrons attracted from circuit common to this plate causing a momentary current. This current reversed at the other plate as ions reach the other plate. An ac current produced depends on number of ions in the packet Frequency of ac current is characteristic of m/z value of the ions in the packet. This current measures conc. of ions. The induced image current decays with time (few tenth of s to several s) by losing energy with collisions between ions (ions reach the thermal equilibrium) (time domain signal).
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Diagram of the cell used in pulsed ICR & in FT-MS
B┴ to front and back plates of cell
Pulsed ICR uses a single-frequency rf excitation, whereas a scanned frequency is used in 80 م09:40 22/12/2012 FT-ms. A voltage 1-5 V is used to trap +ions. The grid is used for pulsing the ion beam.
Generally equipped with a trapped ion analyzer cell. Gaseous sample molecules are ionized in the center of the cell by electrons that are accelerated from the filament through the cell to a collector plate. A pulsed voltage applied at the grid serves as a gate to switch the electron beam on and off periodically. The ions are held in the cell by a 1 to 5 V potential applied to the trap plate. The ions are accelerated by a radio-frequency signal applied to a transmitter plate (exited plate). The receiver (collector) plate is connected to a preamplifier to amplify the image current. Ions could be stored for several minutes. The dimensions of the cell are not critical (usually a few cm) on a side.
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Fourier Transform M.S. Mass analysis performed by detecting cyclotron frequencies of ions (depend on m/z) in uniform B, in time domain. Then F.T. to
frequency domain (mass spectrum). Advantages: No slits or ion optic lenses to adjust.
All ions detected simultaneously for a single ionizing pulse. Can produce very high mass resolution using slow scan. e.g. m/m ~ 220000 for m/z = 84 at B = 1.2T Detection sensitivity independent of m. Resolution α1/m (α B).
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FT-ms sequence of operations 1. Sample (plus R for CI) admitted to chamber. 2. Short e pulse causes EI/CI of sample. 3. Ions trapped by strong magnetic field, inducing ion cyclotron motion. 4. Then RF sweep (1 ms) coherently excites all m/z to larger r. 5. Then decay of cyclotron motion induces image currents in receiver circuit in cell walls. Observed for ms to s. 6. Quench pulse expels ions. 7. Decay signal in 5 amplified, phase-detected, filtered, digitized, stored. FT from time to frequency domain to get mass spectrum.
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apply V (rf) for t s Radius expands under V(rf) to r = V(rf)t/(2dB)
Frequency ν = w/(2π) = Bz/(2πm) s-1 Cyclotron frequency depends on m/z for fixed B. Principle of ICR spectrometer. Ion A has proper m/z, with v same as rf. It absorbs energy and describes an orbit of increasing r while keeping the same v. Ion B has different m/z and does not absorb energy. Ions of same m/z have same v. 84
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what is B/z range? High vac. So not FAB
FT-ICR
Ion image current generation
85
Rapid scan ICR
few ms; v varied linearly
wide-range ms
Ions subjected to slower scan. When v matches cyclotron resonance frequency م09:40 22/12/2012 of ion it is excited and detected.
Applications of Molecular Mass Spectrometry: The applications are numerous and widespread
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Identification of pure compounds Several kinds of data obtained from the spectrum of a pure compound. 1- MWt of the compound. 2- Molecular formula of the compound. 3- presence or absence of various functional groups (from fragmentation). Molecular weights from mass spectra: By identification of the ion peak or (M+1)+ or the (M-1)+ peak. Caution: when electron-impact source is used the molecular ion peak may be absent or small relative to impurity peaks. Molecular formula from exact molecular weights: The molecular ion peak can provide the exact mass. Requires high resolution instrument which capable of detecting mass differences of a few thousands of mass unit. Example: Molecular ions m/z of : Purine (m =120.044), benzamidine C7H8N2 (m =120.069), ethyltoluene C9H12 (m = 120.096) and acetophenon C8H8O (m=120.058).
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If the measured mass of the molecular ion peak is 120.070 (0.005) the all but C7H8N2 are excluded as possible formula. (precision 40 ppm) which achieved by high resolution double focusing instruments. Molecular formulas from isotopic ratios Data from a low resolution instruments can yield useful information about the formula of a compound if the molecular ion peak is sufficiently intense that its height and the heights of the (m+1)+ and (M+2)+ isotope can be determined accurately. Example: Calculate the ratios of the (m+1)+ to M+ peak heights for the following two compounds: dinitrobenzene, C6H4N2O4 (m = 168) and olefin C12H24 (m =168). 13C isotope = 1.08% For every 100 12C there is 1.08 13C atoms. In nitrobenzene C carbon present so 6x1.08 = 6.48 molecules having one 13C for every 100 molecules having non. S (m+1)+ peak will be 6.48% of the M+ peak. For other isotopic elements: 88
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MS – Isotopes Abundance
Most • H1 • C12 • N14 • S32 • • Cl35 • Br79 • Si28 • 89
Other H2 C13 N15 S33 S34 Cl37 Br81 Si29 Si30
Percentage (%) 0.015 1.08 0.37 0.8 4.4 32.5 98.0 5.1 3.4 م09:40 22/12/2012
13C 2H
15N 17O
6x1.08 = 6.48% 4x0.015 = 0.060% 2x0.37 = 0.74% 24x0.04 = 0.16%
So (M+1)+/M+ = 7.44% For C12H24 13C
12x1.08 = 12.96% 2H 24x0.015 =0.36% So (M+1)+/M+ = 13.32% Thus measuring the heights of (M+1)+ and M+ the two compounds can be discriminated (same MWt.)
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•
Determination of Molecular Formulas and Molecular Weights The Molecular Ion and Isotopic Peaks • The presence of heavier isotopes one or two mass units above the common isotope yields small peaks at M+.+1 and M+.+2
• The intensity of the M+.+1 and M+.+2 peaks relative to the M peak can be used to confirm a molecular formula • Example: In the spectrum of methane one expects an M+.+1 peak of 1.17% based on a 1.11% natural abundance of 13C and a 0.016% natural abundance of 2H
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Structural information from fragmentation It is seldom possible or (desirable) to account for all the peaks in the spectrum. Instead characteristic patterns of fragmentation are sought.
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• High-Resolution Mass Spectrometry • Low-resolution mass spectrometers measure m/z values to the nearest whole number • High-resolution mass spectrometers measure m/z values to three or four decimal places • The high accuracy of the molecular weight calculation allows accurate determination of the molecular formula of a fragment • Example – One can accurately pick the molecular formula of a fragment with a nominal molecular weight of 32 using high-resolution MS
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The exact mass of certain nuclides is shown – below
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•
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Fragmentation • In EI mass spectrometry the molecular ion is highly energetic and can break apart (fragment) • Fragmentation pathways are predictable and can be used to determine the structure of a molecule • The processes that cause fragmentation are unimolecular • The relative ion abundance is extremely important in predicting structures of fragments – Fragmentation by Cleavage at a Single Bond • Cleavage of a radical cation occurs to give a radical and a cation but only the cation is observable by MS • In general the fragmentation proceeds to give mainly the most stable carbocation – In the spectrum of propane the peak at 29 is the base peak (most abundant) 100% and the peak at 15 is 5.6%
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– Fragmentation Equations • The M+. Ion is formed by loss of one of its most loosely held electrons – If nonbonding electron pairs or pi electrons are present, an electron from one of these locations is usually lost by electron impact to form M+. – Loosely held nonbonding electrons on nitrogen and oxygen, and p electrons in double bonds are common locations for an electron to be lost (i.e., where the remaining unshared electron in M+. resides)
• In molecules with only C-C and C-H bonds, the location of the lone electron cannot be predicted and the formula is written to reflect this using brackets
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Example: The spectrum of hexane •
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• Example: spectrum of neopentane – Fragmentation of neopentane shows the propensity of cleavage to occur at a branch point leading to a relatively stable carbocation – The formation of the 3o carbocation is so favored that almost no molecular ion is detected
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– Fragmentation by Cleavage of 2 Bonds • The products are a new radical cation and a neutral molecule • Alcohols usually show an M+.-18 peak from loss of water
• Cycloalkenes can undergo a retro-Diels Alder reaction (section 13.11) to yield an alkadienyl radical cation
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Compounds identification from comparison spectra 1- Determination of MWt. and isotopic distribution and fragmentation patterns. 2- Narrowing the possible structures. 3- Comparing the unknown spectrum with the available reference compounds. Procedure assumptions: 1-mass fragmentation patterns are unique. 2- Controlling experimental conditions to produce reproducible spectra. The EI ionization is the method of choice for spectral comparison. Limitations: Heights of mass spectral peaks dependent upon: E of electron beam. Location of the sample with respect to the beam. Sample pressure and temperature. General geometry of the mass spectrometer. Generally it is desirable to confirm the identity of a compound by comparison of its spectrum to the spectrum of an authentic (standard) compound obtained with the same instrument under identical conditions.
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Hyphenated Mass Spectral Methods Mass spectrometers are coupled with various efficient separation devices. Used to analyze mixtures. Examples: GC/MS LC/MC Capillary electrophoresis/MS Tandem mass spectrometry (MS/MS).
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Tandem Mass Spectrometry (MS/MS) Coupling one mass spectrometer to a second. The first one serves to isolate the molecular ions of various components of a mixture. The second one used to fragment each molecular ion one at a time to give a series of mass spectra. The first MS equipped with a soft ionization source (CI) the output is a large molecular ion or protonated molecular ion. These ions then pass into an ion source for the second spectrometer. Further fragmentation of the former occurs to give numerous daughter ions which scanned by the second MS. Consider a hypothetical mixture of isomers ABCD and BCDA and other molecules such as UKL and UMN. 1- Singly charged molecular ions obtained by the first MS. 2- Ions with an m/z value corresponding to ABCD+ and BCDA+ (with identical m/z) are transmitted to the second MS. 102
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• • • • • • •
103
So molecular ions of isomers are separated from other components of the mixture. In the second MS ionization chamber fragmentation takes place and different daughter ions produced: AB+, CD+, BC+, DA+. Each fragment has a unique m/z so identification is possible in the second MS analyzer. The following using MS/Ms : The analyte consists of two different compounds that have identical masses (278). The first spectrometer set to mass of the protonated parent ions. The two quite different daughter ion spectra obtained after further ionization by collision and passage through the second spectrometer.
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Example of Daughter Ion MS/Ms Spectra
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Parent –ion MS/Ms •
The first spectrometer is scanned while the second spectrometer is set to the mass of one of the daughter ions. • Used to measure the identity and concentration of members of a class of closely related compounds . Example: Determination of alkylphenols (HOC6H4CH2R) in solvent refined coal. The second spectrometer is set at m/z value of 107 which corresponds to the ion HOC6H4CH2+ . The sample is then scanned with the first spectrometer. All of the alkylphenols in the samples yield an ion of mass 107 regardless of the nature of R. (measured in a complex sample)
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Instrumentation for Tandem MS •
Made up of various combinations of : magnetic sectors, electrostatic sectors, and quadrupole filter separators. Consists of: First type Magnetic sector Then an electrostatic sector Another type: Two double focusing mass spectrometers each made up of an electrostatic and a magnetic sector. Most widely used tandem mass spectrometer has three quadruple filters. Sample introduced into CI soft source. The ions are then accelerated into first stage parent ion separator Q1 filter. The separated ions rapidly moved to Q2 which is a collision chamber so further ionization of parent ions occurs, dc potential is applied across the rods. The resulting daughter ions pass Q3 where they scanned and recorded in the usual way. 106
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Schematic of Tandem Quadrupole MS/MS
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
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Applications of Tandem • • •
Analysis of complex mixtures either organic or biological. Faster than GC/MS separation complete in milliseconds. No dilution with mobile phase is needed (interferences occurs) as that of GC/MS and LC/MS. • So tandem MS is more sensitive than GC/MS or LC/MS (smaller noises). • Used for quant. And qualit. determination of the components of a wide variety of complex materials either in nature or industry. Examples: • identification and determination of drug metabolites, insect pheromones, alkaloids in plants, trace contaminants in air , alkaloids in plants, polymer sequences, petrochemicals, polychlorinated biphenyls, prostaglandins, diesel exhausts and odors in air. • It will find wider applications in future. Disadvantage: Greater cost. 108
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Quantitative Application • •
Fall into two categories: 1- Quantitative determination of molecular species in organic, biological, and inorganic samples. • 2- Determination of the concentration of elements in inorganic and less commonly organic and biological samples. Quantitative Determination of Molecular Species Performed by passage of the sample through a chromatographic or capillary electrophoretic column and into the spectrometer. Spectrometer set at suitable m/z value, the ion current is then recorded as a function of time (selected ion monitoring technique). Some techniques monitoring occurs at 3 or 4 m/z values. The plot of data consists of a series of peaks with each appearing at a time that is characteristic of one of the several components of the sample. Generally the Area to concentration of component. (MS detector).
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Second type: analyte conc. are obtained directly from the heights of the mass spectral peaks. For simple mixtures it is sometimes possible to find peaks at unique m/z values for each component. Calibration curves of peak heights versus concentration used for unkn. Analysis. Internal standard of fixed amount (for both sample and standard) is used to obtain more accurate results. (reduce prep. uncertainty) Plot of (peak In. of analyte/ peak In. of standard) versus analyte conc. Types of internal standards: 1- isotopically label analog تناظري. 2- Homolog of the analyte that yield intense peak of fragment similar to analyte fragment. Precision: 2-10 % relative. Accuracy depends upon complexity of the mixture: For gaseous hydrocarbon mixtures: (5-10 components) absolute error: 0.2-0.8 mol% appear. 110
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Applications: Mixture without sample heating: Natural gas: C3-C4 hydrocarbons, C6-C8 saturated hydrocarbons, C1-C4 chlorides and iodides, fluorocarbons, thiophenes, atmospheric pollutants exhaust gases,,,,,,. Employing higher temperatures: C16-C27 alcohols, aromatic acids and esters, steroids, fluorinated polyphenyls, aliphatic amides, halogenated aromatic derivatives and aromatic nitriles. High MWt. polymeric materials: The sample is first pyrolyzed The volatile products are then admitted into the spectrometer for examination. Or heating can be performed on the probe of a direct inlet system. Polymers yield single fragment: Isoprene (from natural rubber) Styrene (from polyester). Ethylene (from polyethylene). Polymers yield two products:
Depend on amount and kind of pyrolysis temperature. 111
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Dr Nizam M. El-Ashgar 1
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Mass Spectrometry • Mass spectrum is obtained by converting components of a sample into rapidly moving gaseous ions and resolving them on the basis of their mass-to-charge ratios. • Most generally applicable of analytical tools since capable of qualitative and quantitative information about both atomic and molecular composition of inorganic and organic compounds.
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Molecular Mass Spectrometry • Provides information about: • 1- The elemental composition of samples of matter • 2- The structures of inorganic, organic and biological molecules. • The qualitative and quantitative composition of complex mixture • The structure and composition of solid surfaces • Isotope ratios of atoms in samples. 3
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Acuracy of MWt Mesurments • For large samples such as biomolecules, molecular masses can be measured to within an accuracy of 0.01% of the total molecular mass of the sample. • Example: an error within a 4 Daltons (Da) or atomic mass units (amu) for a sample of 40,000 Da. This is sufficient to allow minor mass changes to be detected, e.g. the substitution of one amino acid for another, or a post-translational modification. • For small organic molecules the molecular mass can be measured to within an accuracy of 5 ppm or less, which is often sufficient to confirm the molecular formula of a compound, and is also a standard requirement for publication in a chemical journal.
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Where are mass spectrometers used? Mass spectrometers are used in industry and academia for both routine and research purposes. The following list is just a brief summary of the major mass spectrometric applications: Biotechnology: the analysis of proteins, peptides, oligonucleotides Pharmaceutical: drug discovery, combinatorial chemistry, pharmacokinetics, drug metabolism Clinical:, haemoglobin analysis, drug testing Environmental: water quality, food contamination
Geological: oil composition
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Mass Spectra • When the electron beam ionizes the molecule, the species that is formed is called a radical cation, and symbolized as M+•. • The radical cation M+• is called the molecular ion or parent ion. • The mass of M+• represents the molecular weight of M. • Because M is unstable, it decomposes to form fragments of radicals and cations that have a lower molecular weight than M+•. • The mass spectrometer analyzes the masses of cations. • A mass spectrum is a plot of the amount of each cation (its relative abundance) versus its mass to charge ratio (m/z, where m is mass, and z is charge). • Since z is almost always +1, m/z actually measures the mass (m) of the individual ions.
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Mass Spectrometry
• The tallest peak in the mass spectrum is called the base peak. • The base peak is also the M peak, although this may not always be the case. • Though most C atoms have an atomic mass of 12, 1.1% have a mass of 13. Thus, 13CH4 is responsible for the peak at m/z = 17. This is called the M + 1 peak. 7
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• Since the molecular ion is unstable, it fragments into other cations and radical cations containing one, two, three, or four fewer hydrogen atoms than methane itself. • Thus, the peaks at m/z 15, 14, 13 and 12 are due to these lower molecular weight fragments.
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Molecular Mass Spectra (hexane)
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Explanation • The collisions between energetic electrons and analyte molecules (enough E) lead to excitation. • Relaxation leads to fragmentation to lower masses ions • Attraction of positive ions through the slit of a mass spectrometer and sorted according to m/z ratios and appear in the Mass spectrum. • Mass spectrum is a plot of relative intensity versus m/z • Base peak has the value of 100 (arbitrarily) • The remaining computed as % of the base-peak height • Modern MS programmed base-peak and normalize the remaining peaks relative to that peak 10
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11
Mass Spectrometer All Instruments Have:
1. Sample Inlet 2. Ion Source 3. Mass Analyzer 4. Detector 5. Data System
http://www.asms.org
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Ion sources Starting point: Formation of gaseous analyte Ions.
Methods of ion formation: Two major categories: 1- Gas-phase sources -The sample is first vaporized and then ionized. -Restricted to thermally stable compounds of BP < 500 0C. -Limited to Compounds of MWt’s <103dalton . 2- Desorption sources The sample in a solid or liquid state is converted directly into gaseous ions (not require volatilization of analyte molecules) Applicable to nonvolatile and thermally unstable samples Applicable to analytes having of 105 dalton or larger. 13
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Basic Type
Gas Phase
Desorption
Name
Ionizing agent
Electron Impact (E I)
Energetic electrons
Chemical Ionization (CI)
Reagent gaseous ions
Field ionization (FI)
High-potential electrode
Field desorption (FD)
High-potential electrode
Electrospray ionization (ESI)
High electric Field
Matrix-assisted desorption/ionization (MALDI)
Laser beam
Plasma desorption (PD)
Fission fragments from 252Cf
Fast atom bombardment (FAB)
Energetic atomic beam
Secondary ion mass spectrometry (SIMS) Energetic beam of ions 14
Thermospray ionization (TI)
High temperature م09:40 22/12/2012
Hard and Soft Sources (another classification) • Hard Sources: • Impart sufficient E to analyte molecules (become of high excited E state). • Relaxation involves rupture of bonds producing fragment ions with m/z < than that of molecular ion. • Provides useful information about kinds of functional groups and structure information. • Soft Sources: • Causes little fragmentation. • Mainly the spectrum consists of the molecular ion peak and few peaks • Supplies accurate information about MWt of analyte. Both are useful for analysis 15
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The Electron-Impact Sources (EI) The sample is brought to a temperature high enough to produce a molecular vapor, which is then ionized by bombarding the resulting molecules with a beam of energetic electrons. • Positive ions forced by small potential difference through accelerator plates to mass analyzer. M + e- M.+ + 2ewhere, M = analyte molecule, M.+ = molecular ion. Relaxation then usually takes place by extensive fragmentation, giving a large number of positive ions of various masses that are less than that of the molecular ion. These lower mass ions are called daughter ions.
17
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Gaseous sample bombarded with beam of energetic electrons. Electrons produced at heated W or Rh wire and accelerated to energy of about 70 eV. Typically one in every million molecules undergoes ionization.
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Electron Impact (EI)
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• The positive ions produced are attracted through the slit in the first accelerating plate by a small potential diff (5 V). • With magnetic sector instrument high potentials (103 to 104) are applied to the accelerator plates) KE = qV = zeV • KE of an ion is independent of its mass and depends only upon its charge and accelerating potential • Velocity of an ion depends on its mass KE = 1/2m2 or = (KE/m)1/2
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Electron Impact Advantages • Well-Established • Fragmentation Libraries • Insoluble Samples • Interface to GC • Non-Polar Samples • They are convenient and produce high ion currents. • Extensive fragmentation can lead to unambiguous identification of analytes • Disadvantages • Parent Identification • Need Volatile Sample • Need Thermal Stability • No Interface to LC • Low Mass Compounds (<1000 amu) • Solids Probe Requires Skilled Operator • The need to volatilize the sample limits this method since it excludes analysis of thermally unstable compounds. • Excessive fragmentation can lead to the disappearance of the molecular ion peak therefore preventing the molecular mass of the analyte to be determined. 21
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Electron-Impact Spectra
22
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EI-MS of Methylene Chloride (top) and 1-Pentanol (bottom)
23
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•
Types of MS Peaks:
•
Molecular (or Parent) Ion – ion having same mass as the analyte Daughter Ion – ion having lower mass Base Peak – biggest peak
• • •
Size of peaks depends on relative natural abundance of isotopes The base peak in electron-impact spectra arise from fragments rather than from molecular ion. The molecular ion peak provides the MWt of the unknown. In EI certain molecules yield no molecular ion peak. 24
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Isotope Peaks •
Peaks occur at masses that are greater than that of the molecular ion are attributable to ions containing isotopes. • Examples: 12C1H 35Cl : m = 84 2 2 13C1H 35Cl : m = 85 2 2 12C1H 35Cl37Cl: m = 86 2 13C1H 35Cl37Cl: m = 87 2 12C1H 37Cl : m = 88 2 2 The size of various peaks depends upon the relative natural abundance of the isotopes. Note: F, P, I, Na occur only as single isotopes. Isotope peaks some times provide useful means for determining the formula for compound.
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Collision product peaks • Ion/molecule collisions, can produce peaks at higher mass numbers than that of the molecular ion. • At ordinary sample pressures, however the only important reaction of this type: transferring of H atom to the ion to give a protonated molecular ion, enhanced (M +1 )+ peak results (second order reaction). • The amount of product depends strongly upon the reactant concentration. • The height of an (M+1)+ peak due to this reaction increases with increase in sample pressure than do the heights of other peaks (detected).
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Advantages of EI-MS Convenient to use Produce high ion currents (gives good sensitivities) Often unambiguous identification of compounds possible (extensive fragmentations)
Disadvantages of EI-MS Extensive fragmentation may not leave parent ion (Molecular peak ion). Sample must be volatilized (thermal degradation) of some analytes before ionization to be occur. Only applicable to samples with molecular weights < 103 amu
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Chemical ionization sources and spectra Second most common process for ms • In modern MS, EI ionization and chemical ionization are interchangeable. Gaseous atoms are ionized by collision with ions produced by electron bombardment of an excess of a reagent gas. Positive ions usually used. negative ion used with analyte contain very electronegative atoms.
Experimentally: Modification of the electron beam ionization area of EI by adding vacuum pump capacity and reducing width of the slit to the mass analyzer so P reduced to 1 torr (Ionization area) and 10-5 torr in the analyzer. Gaseous regent introduced in the ionization region reagent:sample = 103-104. So electron beam reacts exclusively with reagent molecules.
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Common Reagents Methane • • • • • • • • • •
30
Reacts with high E electrons to give several ions (CH4+ and CH3+ (90 %), CH2+). These ions react rapidly with additional methane molecules: CH4+ + CH4 CH5+ + CH3 CH3+ + CH4 C2H5+ + H2 Reactive collisions between sample molecules MH and CH5+ or C2H5+ take place: (proton and hydride transfer). CH5+ + MH MH2+ + CH4 (proton transfer) gives (M +1)+ ion peak C2H5+ + MH MH2+ + C2H4 (proton transfer) gives (M +1)+ ion peak C2H5+ + MH M+ + C2H6 (hydride transfer) gives (M -1)+ ion peak C2H5+ + M M- C2H5+ gives (M + 29)+ ion peak Other reagents: propane, isobutane and ammonia that give different spectra with a given analyte.
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EI hard source
CI Soft source
31
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Field ionization Sources • •
Ions are formed under large electric field (108 V/cm). The field produced by applying high voltages (10 to 20KV) to emitters having diameter < 1 m. • Example: W fine wire ( 10 m diameter) emitter of many hundreds of carbon microtips projecting from the surface tungsten wire. • FI are mounted 0.5-2 mm from the cathode (often serves as slit). • Gaseous sample is allowed to diffuse into the high-field area around the microtips of the anode. • EF is concentrated at the emitter tips and ionization occurs via a quantum mechanical tunneling mechanism in which electrons from the analyte are extracted by microtips of the anode. • Little vib. or rot energy is imparted to the analyte thus little fragmentation occurs.
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FI Source
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MS of Glutamic Acid Electron Impact
Field Ionization
Field Desorption
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Desorption sources Previous methods: The ionizing agents act on gaseous samples. • Such methods are not applicable to nonvolatile or thermally unstable samples. • Desorption ionization methods: are develop to deal with this type of samples. (thermally unstable biochemical species or that of 100, 000 Da). • The method is: dispenses the volatilization followed by ionization of the gaseous analyte molecules. • Instead: E in various forms is introduced into solid or liquid sample large molecules to overcome intermolecular forces to cause direct formation of gaseous ions.
• provide gentle ionization. e.g. carbohydrates, peptides, nucleic acids, organic salts & organometallics. • • 35
Simple spectra consist of only the molecular ion or the protonated molecular ions. Exact mechanism is not understood. م09:40 22/12/2012
Field Desorption Sources
• Multitipped emitter similar to FI source is used. • The electrode is mounted on a probe that can removed from the sample compartment and coated with a solution of the sample. • The probe is reinserted into the sample compartment. • Ionization takes place by the application of a high potential to this electrode. • Some samples needed heating the emitter by passing a current through the wire. • Thermal degradation may occur before ionization is complete. • Example of spectra (glutamic acid previously). • Spectrum is simpler than that of CI or FI consists of only the protonated molecular ion peak at mass 148 and isotope peak at mass 149. 36
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Matrix-Assisted Laser Desorption/Ionization (MALDI) • MALDI: is a new ionization method: provides accurate MWt of polar biopolymers (MWt few thousands to several hundred thousands da). • First technique (German group): an aqeous/alcohol solution of sample was mixed with large excess of a radiation-absorbing matrix material. • The resulting solution was evaporated on the surface of a metallic probe that was used for introduction of the sample into mass spectrometer. • The solid mixture exposed to a pulsed laser beam so analyte ions sublime. • Entire spectrum recording between the period of laser pulses.
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MALDI: Matrix Assisted Laser Desorption Ionization Sample plate
Laser
h
MH+
1. Sample is mixed with matrix (X) and dried on plate.
2. Laser flash ionizes matrix molecules. 3. Sample molecules (M) are ionized by proton transfer: XH+ + M MH+ + X.
+/- 20 kV 39
Grid (0 V) م09:40 22/12/2012
Requirement for MALDI: • Matrix compound must absorb the laser radiation strongly. • Soluble in sample solvent and present in large excess in the solid matrix deposited on the probe. • The analyte should not absorb laser to prevent fragmentation. • Only few compounds are suitable as matrices for biopolymers (Table 20-4).
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6. MALDI • matrix has strong A at laser λ • 500 fmol of betalow mass, so can sublime • D-galactosidase can measure Mr of proteins and biomolecules •
mixed with nicotinic acid on a silver surface using NdYAG 266 nm excitation RMM 117130 Fragments are cluster ions [nM+H]+ and multiplycharged ions [nM+zH]z+
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Electrospray ionization • ESI/MS (1984) now it is an important technique for analyzing biomolecules (polypeptides, proteins and oligonucleotides having MWt 0f 100,000 Da or more). Also used to identify inorganic species and synthetic polymers). • Takes place under atmospheric pressures and temperatures. • A solution of the sample is pumped through a stainless steel capillary needle at a rate of few microliters per minute. • The needle is maintained at several kilovolts with respect to a cylindrical electrode surrounds the needle. • The resulting charged spray of fine droplets then passes through a dissolving capillary . • Solvent evaporate and analyte charged and charge density increases and ions desorbed into the ambient gas. • Little fragmentation of large thermally fragile biomolecules occurs. • The ions formed are multiply charged (small m/z) detected by quadrupole instruments (range of 1500 or less). • Is ready adapted to direct sample introduction from HPLC and 42 electrophoresis columns. م09:40 22/12/2012
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proteinase K
ESI ms in 50% CH3CN/46% H2O/4% acetic acid with source at 100 oC.
29 kDa
thermolysin 34 kDa
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Fast Atom Bombardment FAB • FAB is a major role in the production of ions for ms studies of polar high MWt. species. • Samples in a condensed state often in a glycerol solution matrix are ionized by bombardment with energetic (several KeV) xenon or argon atoms. • Both +ve and –ve analyte ions are sputtered from the surface of the sample in a desorption process. • Rapid sample heating by this process reduces sample fragmentation. • A beam of fast atoms is obtained by passing accelerated Ar or Xe ions from an ion source or gun through a chamber containing Ar or Xe atoms at P of about 10-5 torr. • The high velocity ions undergo exchange reactions with the atoms without loss of translational energy and a beam of energetic atoms is formed. • The lower E ions from the exchange are removed by an electrostatic deflector. • FAB of organic or biochemical compounds usually produces significant amounts of molecular ions (and ion fragments) even for high MWt and thermally unstable samples (over 10,000 da) and detailed structure information obtained for samples of (3000 da). 45 م09:40 22/12/2012
FAB – sputtering/desorption technique. Sample dissolved in low-volatility, viscous solvent and placed on insertion probe. Bombard with atoms or ions 4-10 keV energy.
Xe/Ar atoms
Atoms/ions penetrate several layers, distribute k.e. 46
No fragmentn
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up to 9600 amu
FAB
Sample monolayer sputtered in few s. Nonvolatile glycerol allows surface layer to be renewed. Ar G (Gn+H)+ (M+H)+ sample M (M-H) (M+G+H)+ 47
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Mass Spectrometer • • •
48
Several Types. Only two will be described: Quadrupole spectrometer and the time-of-flight spectrometer. General description of instrument components:
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Inlet System: To introduce a very small amount of sample (mol or less) into the mass spectrometer that converted to gaseous ions. ( a mean for volatilizing solid or liquid samples is presents).
Ion sources: Convert the components of a sample into ions. (generally the inlet system and the ion source are combined into a single component). The output is a stream of +ve or –ve ions are accelerated into the mass analyzer.
Mass analyzer analogous to grating in an optical spectrometer. Dispersion is based upon the mass/charge ratios of the analyte ions rather than upon the wavelength of photons. (Different categories of MS according to mass analyzer).
Detectors: Convert the beam of ions into an electrical signal that can then be processed, stored in the memory of a computer and displayed or recorded in a variety ways. Vacuum System: To create low pressure (10-4 to 10-8 torr) in all the instrument components except the signal processor and readout. To prevent interaction of components with atmosphere so destroyed. 49
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Sample Inlet System • For permitting introduction of a representative sample into the ion source with minimal loss of vacuum. Various types of inlets equipped to accommodate different samples: (Batch inlets, direct probe inlets, chromatographic inlets and capillary electrophoretic inlets). Batch Inlet Systems: Classical and simplest type. Sample is volatilized externally and then allowed to leak into evacuated ionization region. The figure shown is for a one that applicable to gaseous and liquid samples having PB up to 5000C. Gaseous samples: A small measured volume of a gas is trapped between the two valves and then expanded into reservoir flask. 50
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•
• • •
51
For liquids a small quantity of sample is introduced into a reservoir usually with a micrometer syringe. In either case vacuum system is used to achieve sample P 10-4 – 10-5 torr. For samples with boiling points > 1500C T must be maintained at elevated T by oven and heating tapes of maximum T of about 3500C. Sample is now in the gas phase is leaked into the ionization area of the spectrometer via a metal or glass diaphragm containing one or more pinholes.
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MS – Direct Sample Introduction
External Sample Introduction System
52
Direct Sample Probe م09:40 22/12/2012
The Direct Probe Inlet. •
Solids and non volatile liquids can be introduced into the ionization region by means of a sample holder probe which is inserted through a vacuum lock. The lock system: Designed to limit V of air pumped after probe insertion. Probes also used for limited quantity samples (few nanograms)(less wasted than batch system). Probe: Sample is held on the surface of a glass or Al capillary tube, a fine wire or a small cup. The probe is positioned within a few mm of the Ionization source and the slit leading to spectrometer. Vacuum used to maintain thermally unstable compounds for spectrum measurements before major decomposition occurs. And to elevate nonvolatile conc. in the ionization area (carbohydrates, steroids, metalorganic species and low molecular weight polymeric substances). Partial pressure attained is at least of 10-8 torr before onset decomposition. 53
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• Chromatographic and capillary electrophoretic inlet System. • MS are often coupled with GC and HPLC or capillary electrophoresis columns. • Separation and determination of the components of complex mixture is obtained. • Specialized inlet systems is needed.
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Mass Analyzers • Several devices are available for separating ions with different m/z ratios. Mass analyzer should be: - Capable of distinguishing between minute mass differences. - Allows passage of sufficient number of ions to yield measurable currents. Resolution of mass spectrometer: R
R = m/Dm Where, m : mass of the first peak (or mean mass of the two peaks) Dm: mass difference between two adjacent peaks Two peaks are considered to be separated if height of valley between them 10% of their height. R of 4000 would resolve peaks occurring at m/z values of 400.0 and 400.1 (or 40.00 and 40.01)
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• R needed in MS depends upon its application. Example 1: same nominal mass ions: C2H4+, CH2N+, N2+ and CO+ (All ions of nominal mass 28 Da). Exact masses: 28.0313, 28.0187, 28.0061 and 27.9949 Da respectively. These requires an instrument with a resolution of several thousands. Example 2: Low MWt ions with a unit mass difference or more: NH3+ (m = 17) and CH4+ (m = 16) R instrument of 50 or less is sufficient. Commercial MS are available with R range of 500-500,000. Example: What R is needed to separate C2H4+ and CH2N+, ions? Dm = 28.0313 - 28.0187 = 0.0126 R =m/ Dm = 28.025/0.0126 = 2.22x103 Where 28.025 is the mean mass for the two species
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Magnetic Sector Analyzers • Employ a permanent magnet or an electromagnet. • Cause beam from the ion source to travel in a circular path most commonly of (180, 90 or 60 deg) • Schematic of a 90-deg magnetic sector spectrometer
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• •
Operation: Ions formed by Electron impact are accelerated through slit B into the metal analyzer tube internal P 10-7 torr. • Ions of different mass can be scanned across the exit slit by varying the field strength of the magnet or the accelerating potential between slits A and B. • Ions passing through the exit slit fall on a collector electrode, ion current resulted that amplified and recorded. • Translation or KE of an ion of mass m bearing z exiting slit B is given by: KE = ZeV = ½ mv2 Where V is voltage between A and B V is velocity of the ion after acceleration e is the electronic charge = 1.60x10-19 C Note: all ions having the same number of charges are assumed to have the same KE after accelerating regardless of their mass. (approximately true). All ions leaving the slit have approximately same KE, the heavier ions must travel at lower velocities. The path in sector by ions of a given m/z represents a balance between two forces acting upon them. 59
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The magnetic force FM: FM =BzeV
Where B is MF strength The balancing centripetal force: Fc = mv2/r Where r is the radius of curvature of the magnetic sector. For ion to traverse the circular path to the collector, FM and FC must be equal FM =BzeV = Fc = mv2/r v = Bzer/m Substituting the previous equation in ZeV = ½ mv2 m/z = B2r2e/2V Mass Spectra: Varying one of three variables (B, V or r) while holding the other two constants. Modern MS ions are sorted by holding V and r constant while varying the current in the magnet and thus B. In sector MS (using photographic recording) B and V are constants, r is the variable. 60
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•
What accelerating potential will be required to direct a singly charged water molecule through the exit slit of a magnetic mass spectrometer if B = 0.240 T and r of curvature of the ion through the magnetic field is 12.7 cm? SI units: ez = 1.60x10-19 Cx 1 r = 0.127 m
m
18.02 g H 2O / mol 6.02 x1023 H 2O / mol
x103
m = 2.99x10-26kg B = 0.240 T = 0.240 W/m2 m/z = B2r2e/2V or = [0.240 W/m2]2[0.127m]2[1.60x10-19C] 2x2.99x10-26kg = 2.49 x103 W2C/m2kg or volts
kg g
V = B2r2ez/2m
(In SI base units, the dimensions of the weber are kg.m2/S2.A . In derived units, they are volt-seconds V.s , or joules per amp J/A. The weber is a large unit, equal to 1 T m2 = 108 maxwells. •
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Double focusing spectrometers Previous are single-focusing spectrometers. Limitation: Low precision because of: 1- directional distribution of ions 2- energy distribution of ions Same m/z ratio but with small diverging directional distribution are focused so limiting the resolution of magnetic sector instruments (R2000). This because of the translational E distribution of ions leaving a source (Boltzmann dist.) arises from energies of original molecules and source field inhomogeneities. Spread of KE causes a broadening of the beam reaching the transducer and a loss of resolution.
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Double Focusing Spectrometers: Correction for both the directional and E distribution of ions. Both directional and E aberrations of a population of ions are simultaneously minimized by use of carefully selected combinations of electrostatic and magnetic fields.
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• •
• • • • • •
64
The ion beam is first passed through an electrostatic analyzer (ESA). Consists of two smooth curved metallic plates across which a dc potential is applied which limiting the KE of the ions reaching the magnetic sector to a closely defined range. Ions with E greater than average strike the upper side of ESA slit and lost to the ground. Ions with E less than average strike the lower side of the ESA slit and are thus removed. Directional focusing occurs along the focal plane (d). Energy focusing takes place along the plane e. Only ions of one m/z are double focused at the intersection of d and e for any given V and B. The collector slit is located at this locus of double focus.
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Quadrupole Mass Spectrometer • • • • •
Comparing with magnetic sector instrument it is: Less expensive and more rugged. More compact Found in commercial bench top ms Low scan times (<100 ms) which is useful for chromatography. • Most common mass analyzers in use today. • Quadrupole mass analyzer is responsible for filtering the samples ions. • Consists of four parallel metal rods. • Each rod pair is connected together electrically. • A radio frequency voltage is applied between one pair of rods then the other. 65
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• Ions travel down the quadrupole in between the rods • Electric field separates ions • Ions are subjected to complex forces • Only Ions of a particular m/z reaches the detector Advantages • Inexpensive • Easily Interfaced to Many Ionization Methods Disadvantages • Low Resolution (< 4000) • Low Accuracy (>100 ppm) • MS/MS requires multiple analyzers • Low Mass Range (< 4000) • Slow Scanning 66
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Time-of-Flight MS (TOF) •
• •
• • •
67
The +ve ions are produced periodically by bombardment of the sample with brief pulses of electrons, secondary ions or laser generated photons. The ions are then accelerated into a field-free drift tube by an electric field pulse of 103 – 104 V Separation of ions on the basis of mass occurs during the transition of the ions to the detector located at the end of the tube. All ion have same KE but their velocities are inversely proportional to their masses Lighter particles arrived earlier. Typical flight times are 1 to 30 s.
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Time-of-Flight MS (TOF)
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Advantages • Simplicity and ruggedness. • Ease of accessibility of the ion source. • Extremely High Mass Range (>1 MDa) • Fast Scanning Disadvantages • Low Resolution (4000) • Low sensitivity • Low Accuracy (>200ppm) • MS/MS not possible
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Ion Trap Analyzers • • •
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Gaseous anions or cations can be formed and confined for extended periods by electric and/or magnetic fields. Several types, A simpler type of ion trap that used for GC/MS Now used to obtain mass spectra of a variety of analytes.
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• • • •
•
• •
73
It consists of a central doughnut-shaped ring electrode and a pair of endcap electrodes. A variable RF voltage is applied to the ring electrode while the two end-cap electrodes are grounded. Ions of appropriate m/z value circulate in a stable orbit within the cavity surrounded the ring. By increasing RF voltage the orbit of heavier ions become stabilized , while lighter become destabilized causing them to collide with the wall of the ring electrode. By RF voltage scanning the trapped ions destabilized and leave the ring electrode cavity via openings in the lower end cap then emitted to a transducer. Rugged compact and less costly than sector or quadrupole instruments. Capable of resolving ions that differ in mass by unit in the mass range of 500-1000 Da
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Operation of an Ion Trap MS
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Fourier Transform (FT) instruments • FTMS provide: • improved signal to noise ratios • Greater speed. • Higher sensitivity. • Higher resolution. The heart of FTMS is an ion trap within which ions can circulate in a well-defined orbits for extended periods.
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The Ion Cyclotron Resonance (ICR) • Gaseous ion drifts into or formed in a strong MF. • The motion becomes circular in a plane that is to the direction of the field. • The angular frequency of this motion is called cyclotron frequency c. c= v/r = zeB/m • In fixed field the c depends only upon the inverse of the m/z value. • Increases the velocity of an ion will be accompanied by increase of rotation of the ion. • This circulated trapped ion in the MF is capable of absorbing E from an ac EF. • So the EF frequency matches the c. • The absorbed E increases v of the ion and r of travel without disturbing c 76
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MS – Fourier Transform Analyzer Ion Cyclotron Resonance
Magnetic Field 77
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• Inner solid line represents the original path to the ion. • Dashed line shows spiral path when switch is moved briefly to position 1. • Outer solid line is new circular path when switch again opened. • For ensemble ions of the same m/z ratio between the two plates; when ac signal is applied the cyclotron resonance frequency sets all the particles into coherent motion that is in phase with the field. • Ions of different m/z ratios (different c) are unaffected by the ac field. Measurement of the ICR signal: Coherent circular motion of resonant ions creates image current observed current after termination of the frequency sweep signal (from position 1 to 2). The current decreases exponentially with time. It is a capacitor current induced by circular movement of a packet of ions with the same m/z ratios. 78
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Example: +ve ions approaches the upper plate electrons attracted from circuit common to this plate causing a momentary current. This current reversed at the other plate as ions reach the other plate. An ac current produced depends on number of ions in the packet Frequency of ac current is characteristic of m/z value of the ions in the packet. This current measures conc. of ions. The induced image current decays with time (few tenth of s to several s) by losing energy with collisions between ions (ions reach the thermal equilibrium) (time domain signal).
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Diagram of the cell used in pulsed ICR & in FT-MS
B┴ to front and back plates of cell
Pulsed ICR uses a single-frequency rf excitation, whereas a scanned frequency is used in 80 م09:40 22/12/2012 FT-ms. A voltage 1-5 V is used to trap +ions. The grid is used for pulsing the ion beam.
Generally equipped with a trapped ion analyzer cell. Gaseous sample molecules are ionized in the center of the cell by electrons that are accelerated from the filament through the cell to a collector plate. A pulsed voltage applied at the grid serves as a gate to switch the electron beam on and off periodically. The ions are held in the cell by a 1 to 5 V potential applied to the trap plate. The ions are accelerated by a radio-frequency signal applied to a transmitter plate (exited plate). The receiver (collector) plate is connected to a preamplifier to amplify the image current. Ions could be stored for several minutes. The dimensions of the cell are not critical (usually a few cm) on a side.
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Fourier Transform M.S. Mass analysis performed by detecting cyclotron frequencies of ions (depend on m/z) in uniform B, in time domain. Then F.T. to
frequency domain (mass spectrum). Advantages: No slits or ion optic lenses to adjust.
All ions detected simultaneously for a single ionizing pulse. Can produce very high mass resolution using slow scan. e.g. m/m ~ 220000 for m/z = 84 at B = 1.2T Detection sensitivity independent of m. Resolution α1/m (α B).
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FT-ms sequence of operations 1. Sample (plus R for CI) admitted to chamber. 2. Short e pulse causes EI/CI of sample. 3. Ions trapped by strong magnetic field, inducing ion cyclotron motion. 4. Then RF sweep (1 ms) coherently excites all m/z to larger r. 5. Then decay of cyclotron motion induces image currents in receiver circuit in cell walls. Observed for ms to s. 6. Quench pulse expels ions. 7. Decay signal in 5 amplified, phase-detected, filtered, digitized, stored. FT from time to frequency domain to get mass spectrum.
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apply V (rf) for t s Radius expands under V(rf) to r = V(rf)t/(2dB)
Frequency ν = w/(2π) = Bz/(2πm) s-1 Cyclotron frequency depends on m/z for fixed B. Principle of ICR spectrometer. Ion A has proper m/z, with v same as rf. It absorbs energy and describes an orbit of increasing r while keeping the same v. Ion B has different m/z and does not absorb energy. Ions of same m/z have same v. 84
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what is B/z range? High vac. So not FAB
FT-ICR
Ion image current generation
85
Rapid scan ICR
few ms; v varied linearly
wide-range ms
Ions subjected to slower scan. When v matches cyclotron resonance frequency م09:40 22/12/2012 of ion it is excited and detected.
Applications of Molecular Mass Spectrometry: The applications are numerous and widespread
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Identification of pure compounds Several kinds of data obtained from the spectrum of a pure compound. 1- MWt of the compound. 2- Molecular formula of the compound. 3- presence or absence of various functional groups (from fragmentation). Molecular weights from mass spectra: By identification of the ion peak or (M+1)+ or the (M-1)+ peak. Caution: when electron-impact source is used the molecular ion peak may be absent or small relative to impurity peaks. Molecular formula from exact molecular weights: The molecular ion peak can provide the exact mass. Requires high resolution instrument which capable of detecting mass differences of a few thousands of mass unit. Example: Molecular ions m/z of : Purine (m =120.044), benzamidine C7H8N2 (m =120.069), ethyltoluene C9H12 (m = 120.096) and acetophenon C8H8O (m=120.058).
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If the measured mass of the molecular ion peak is 120.070 (0.005) the all but C7H8N2 are excluded as possible formula. (precision 40 ppm) which achieved by high resolution double focusing instruments. Molecular formulas from isotopic ratios Data from a low resolution instruments can yield useful information about the formula of a compound if the molecular ion peak is sufficiently intense that its height and the heights of the (m+1)+ and (M+2)+ isotope can be determined accurately. Example: Calculate the ratios of the (m+1)+ to M+ peak heights for the following two compounds: dinitrobenzene, C6H4N2O4 (m = 168) and olefin C12H24 (m =168). 13C isotope = 1.08% For every 100 12C there is 1.08 13C atoms. In nitrobenzene C carbon present so 6x1.08 = 6.48 molecules having one 13C for every 100 molecules having non. S (m+1)+ peak will be 6.48% of the M+ peak. For other isotopic elements: 88
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MS – Isotopes Abundance
Most • H1 • C12 • N14 • S32 • • Cl35 • Br79 • Si28 • 89
Other H2 C13 N15 S33 S34 Cl37 Br81 Si29 Si30
Percentage (%) 0.015 1.08 0.37 0.8 4.4 32.5 98.0 5.1 3.4 م09:40 22/12/2012
13C 2H
15N 17O
6x1.08 = 6.48% 4x0.015 = 0.060% 2x0.37 = 0.74% 24x0.04 = 0.16%
So (M+1)+/M+ = 7.44% For C12H24 13C
12x1.08 = 12.96% 2H 24x0.015 =0.36% So (M+1)+/M+ = 13.32% Thus measuring the heights of (M+1)+ and M+ the two compounds can be discriminated (same MWt.)
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•
Determination of Molecular Formulas and Molecular Weights The Molecular Ion and Isotopic Peaks • The presence of heavier isotopes one or two mass units above the common isotope yields small peaks at M+.+1 and M+.+2
• The intensity of the M+.+1 and M+.+2 peaks relative to the M peak can be used to confirm a molecular formula • Example: In the spectrum of methane one expects an M+.+1 peak of 1.17% based on a 1.11% natural abundance of 13C and a 0.016% natural abundance of 2H
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Structural information from fragmentation It is seldom possible or (desirable) to account for all the peaks in the spectrum. Instead characteristic patterns of fragmentation are sought.
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• High-Resolution Mass Spectrometry • Low-resolution mass spectrometers measure m/z values to the nearest whole number • High-resolution mass spectrometers measure m/z values to three or four decimal places • The high accuracy of the molecular weight calculation allows accurate determination of the molecular formula of a fragment • Example – One can accurately pick the molecular formula of a fragment with a nominal molecular weight of 32 using high-resolution MS
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The exact mass of certain nuclides is shown – below
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•
95
Fragmentation • In EI mass spectrometry the molecular ion is highly energetic and can break apart (fragment) • Fragmentation pathways are predictable and can be used to determine the structure of a molecule • The processes that cause fragmentation are unimolecular • The relative ion abundance is extremely important in predicting structures of fragments – Fragmentation by Cleavage at a Single Bond • Cleavage of a radical cation occurs to give a radical and a cation but only the cation is observable by MS • In general the fragmentation proceeds to give mainly the most stable carbocation – In the spectrum of propane the peak at 29 is the base peak (most abundant) 100% and the peak at 15 is 5.6%
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– Fragmentation Equations • The M+. Ion is formed by loss of one of its most loosely held electrons – If nonbonding electron pairs or pi electrons are present, an electron from one of these locations is usually lost by electron impact to form M+. – Loosely held nonbonding electrons on nitrogen and oxygen, and p electrons in double bonds are common locations for an electron to be lost (i.e., where the remaining unshared electron in M+. resides)
• In molecules with only C-C and C-H bonds, the location of the lone electron cannot be predicted and the formula is written to reflect this using brackets
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Example: The spectrum of hexane •
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• Example: spectrum of neopentane – Fragmentation of neopentane shows the propensity of cleavage to occur at a branch point leading to a relatively stable carbocation – The formation of the 3o carbocation is so favored that almost no molecular ion is detected
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– Fragmentation by Cleavage of 2 Bonds • The products are a new radical cation and a neutral molecule • Alcohols usually show an M+.-18 peak from loss of water
• Cycloalkenes can undergo a retro-Diels Alder reaction (section 13.11) to yield an alkadienyl radical cation
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Compounds identification from comparison spectra 1- Determination of MWt. and isotopic distribution and fragmentation patterns. 2- Narrowing the possible structures. 3- Comparing the unknown spectrum with the available reference compounds. Procedure assumptions: 1-mass fragmentation patterns are unique. 2- Controlling experimental conditions to produce reproducible spectra. The EI ionization is the method of choice for spectral comparison. Limitations: Heights of mass spectral peaks dependent upon: E of electron beam. Location of the sample with respect to the beam. Sample pressure and temperature. General geometry of the mass spectrometer. Generally it is desirable to confirm the identity of a compound by comparison of its spectrum to the spectrum of an authentic (standard) compound obtained with the same instrument under identical conditions.
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Hyphenated Mass Spectral Methods Mass spectrometers are coupled with various efficient separation devices. Used to analyze mixtures. Examples: GC/MS LC/MC Capillary electrophoresis/MS Tandem mass spectrometry (MS/MS).
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Tandem Mass Spectrometry (MS/MS) Coupling one mass spectrometer to a second. The first one serves to isolate the molecular ions of various components of a mixture. The second one used to fragment each molecular ion one at a time to give a series of mass spectra. The first MS equipped with a soft ionization source (CI) the output is a large molecular ion or protonated molecular ion. These ions then pass into an ion source for the second spectrometer. Further fragmentation of the former occurs to give numerous daughter ions which scanned by the second MS. Consider a hypothetical mixture of isomers ABCD and BCDA and other molecules such as UKL and UMN. 1- Singly charged molecular ions obtained by the first MS. 2- Ions with an m/z value corresponding to ABCD+ and BCDA+ (with identical m/z) are transmitted to the second MS. 102
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• • • • • • •
103
So molecular ions of isomers are separated from other components of the mixture. In the second MS ionization chamber fragmentation takes place and different daughter ions produced: AB+, CD+, BC+, DA+. Each fragment has a unique m/z so identification is possible in the second MS analyzer. The following using MS/Ms : The analyte consists of two different compounds that have identical masses (278). The first spectrometer set to mass of the protonated parent ions. The two quite different daughter ion spectra obtained after further ionization by collision and passage through the second spectrometer.
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Example of Daughter Ion MS/Ms Spectra
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Parent –ion MS/Ms •
The first spectrometer is scanned while the second spectrometer is set to the mass of one of the daughter ions. • Used to measure the identity and concentration of members of a class of closely related compounds . Example: Determination of alkylphenols (HOC6H4CH2R) in solvent refined coal. The second spectrometer is set at m/z value of 107 which corresponds to the ion HOC6H4CH2+ . The sample is then scanned with the first spectrometer. All of the alkylphenols in the samples yield an ion of mass 107 regardless of the nature of R. (measured in a complex sample)
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Instrumentation for Tandem MS •
Made up of various combinations of : magnetic sectors, electrostatic sectors, and quadrupole filter separators. Consists of: First type Magnetic sector Then an electrostatic sector Another type: Two double focusing mass spectrometers each made up of an electrostatic and a magnetic sector. Most widely used tandem mass spectrometer has three quadruple filters. Sample introduced into CI soft source. The ions are then accelerated into first stage parent ion separator Q1 filter. The separated ions rapidly moved to Q2 which is a collision chamber so further ionization of parent ions occurs, dc potential is applied across the rods. The resulting daughter ions pass Q3 where they scanned and recorded in the usual way. 106
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Schematic of Tandem Quadrupole MS/MS
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
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Applications of Tandem • • •
Analysis of complex mixtures either organic or biological. Faster than GC/MS separation complete in milliseconds. No dilution with mobile phase is needed (interferences occurs) as that of GC/MS and LC/MS. • So tandem MS is more sensitive than GC/MS or LC/MS (smaller noises). • Used for quant. And qualit. determination of the components of a wide variety of complex materials either in nature or industry. Examples: • identification and determination of drug metabolites, insect pheromones, alkaloids in plants, trace contaminants in air , alkaloids in plants, polymer sequences, petrochemicals, polychlorinated biphenyls, prostaglandins, diesel exhausts and odors in air. • It will find wider applications in future. Disadvantage: Greater cost. 108
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Quantitative Application • •
Fall into two categories: 1- Quantitative determination of molecular species in organic, biological, and inorganic samples. • 2- Determination of the concentration of elements in inorganic and less commonly organic and biological samples. Quantitative Determination of Molecular Species Performed by passage of the sample through a chromatographic or capillary electrophoretic column and into the spectrometer. Spectrometer set at suitable m/z value, the ion current is then recorded as a function of time (selected ion monitoring technique). Some techniques monitoring occurs at 3 or 4 m/z values. The plot of data consists of a series of peaks with each appearing at a time that is characteristic of one of the several components of the sample. Generally the Area to concentration of component. (MS detector).
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Second type: analyte conc. are obtained directly from the heights of the mass spectral peaks. For simple mixtures it is sometimes possible to find peaks at unique m/z values for each component. Calibration curves of peak heights versus concentration used for unkn. Analysis. Internal standard of fixed amount (for both sample and standard) is used to obtain more accurate results. (reduce prep. uncertainty) Plot of (peak In. of analyte/ peak In. of standard) versus analyte conc. Types of internal standards: 1- isotopically label analog تناظري. 2- Homolog of the analyte that yield intense peak of fragment similar to analyte fragment. Precision: 2-10 % relative. Accuracy depends upon complexity of the mixture: For gaseous hydrocarbon mixtures: (5-10 components) absolute error: 0.2-0.8 mol% appear. 110
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Applications: Mixture without sample heating: Natural gas: C3-C4 hydrocarbons, C6-C8 saturated hydrocarbons, C1-C4 chlorides and iodides, fluorocarbons, thiophenes, atmospheric pollutants exhaust gases,,,,,,. Employing higher temperatures: C16-C27 alcohols, aromatic acids and esters, steroids, fluorinated polyphenyls, aliphatic amides, halogenated aromatic derivatives and aromatic nitriles. High MWt. polymeric materials: The sample is first pyrolyzed The volatile products are then admitted into the spectrometer for examination. Or heating can be performed on the probe of a direct inlet system. Polymers yield single fragment: Isoprene (from natural rubber) Styrene (from polyester). Ethylene (from polyethylene). Polymers yield two products:
Depend on amount and kind of pyrolysis temperature. 111
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