Mass Spectrometry Instrumentation and Applications Monzir S. Abdel-Latif Chemistry Department IUG

Analytical method to measure the molecular or atomic weight of samples Mass Spectrometry Analytical method to measure the molecular or atomic weight of samples

Different compounds can be uniquely identified by their masses Butorphanol L-dopa Ethanol N OH HO -CH2- -CH2CH-NH2 COOH HO CH3CH2OH MW = 327.1 MW = 197.2 MW = 46.1

MS History MS concept first put into practice by Francis Aston, a physicist working in Cambridge, England in 1919 Designed to measure masses of elements Aston was awarded Nobel Prize in 1922 1920s - Electron impact ionization and magnetic sector mass analyzer were introduced

MS History 1948-52 - Time of Flight (TOF) mass analyzers introduced 1955 - Quadrupole ion filters introduced by Wolfgang Paul, also invented the ion trap in 1983 (wins 1989 Nobel Prize) 1968 - Tandem mass spectrometers were presented Mass spectrometers are now one of the MOST POWERFUL ANALYTIC TOOLS IN CHEMISTRY

MS Principles Find a way to “charge” an atom or molecule (ionization) Place charged atom or molecule in a magnetic field or subject it to an electric field and measure its speed or radius of curvature (as current) relative to its mass-to-charge ratio (mass analyzer) Detect ions using microchannel plate or electron multiplier tube

Mass Spec Principles Sample + _ Ionizer Mass Analyzer Detector

Typical sample: isolated A Typical Mass Spectrum O C H 3 Mass Spectrometer H C C N 3 N C C H C C O N N H Typical sample: isolated compound (~1 nanogram) Mass Spectrum 194 67 109 Abundance 55 82 42 94 136 165 40 60 80 100 120 140 160 180 200 Mass (amu)

Resolution Width of peak indicates the resolution of the MS instrument The better the resolution or resolving power, the better the instrument and the better the mass accuracy Resolving power is defined as: M/DM M is the mass number of the observed average of two adjacent masses and DM is the difference between the two masses

Resolution If we have 5000 resolution for a mass spectrometer, we can separate m/z 49.995 from m/z 50.005, or separate m/z 99.990 from m/z 100.010, or separate m/z 999.900 from m/z 1000.100 or m/z 9999 from m/z 10001 (all down to a 10% valley between the two peaks).

monoisotopic mass 1296.68518 R = 1000 R = 5000 average mass 1297.50248 Mass (m/z) average mass 1297.50248 I R = 1000 R = 5000 monoisotopic mass 1296.68518 . I Mass (m/z) 12C62H90N17O14 12C6113C1H90N17O14 12C6013C2 H90N17O14 12C5913C3H90N17 O14

Mass Spectrometer Schematic Inlet Ion Source Mass Analyzer Detector Data System High Vacuum System Rough pumps Rotary pumps Turbo pumps Diffusion pumps Vapor HPLC GC Solids probe MALDI ESI FAB EI/CI TOF Quadrupole Ion Trap Mag. Sector FTMS Microch plate Electron Mult. PC’s UNIX Mac

Different Ionization Methods Electron Impact (EI - Hard method) small molecules, 1-1000 Daltons Chemical Ionization (CI) and Fast Atom Bombardment, polar high molecular weight species (FAB – Softer) peptides, sugars, up to 10000 Daltons Electrospray Ionization (ESI - Soft) peptides, proteins, up to 200,000 Daltons Matrix Assisted Laser Desorption (MALDI-Soft) peptides, proteins, DNA, up to 500 kD

Fragmentation  Break Of Covalent Bond Fragment ion  An electrically charged dissociation product of an ionic fragmentation. Such an ion may fragmentate further to produce other electrically charged molecular or atomic moieties of successively lower formula weight. Fragmentation  Break Of Covalent Bond „Hard“ Ionization „Soft“ EI CI,FAB MALDI ESI

Electron Impact Ionization Source Electron Collector (Trap) Positive Ions + Repeller Neutral Molecules Inlet _ _ + to Analyzer + + + + + + e- e- e- _ Electrons Filament Extraction Plate

Electron Impact Ionization Sample introduced into instrument by heating it until it evaporates Gas phase sample is bombarded with electrons coming from rhenium or tungsten filament (energy = 70 eV) Molecules are “shattered” into fragments (70 eV >> bond energy) Fragments travel to mass analyzer The primary weakness is the loss of the molecular ion peak

Why it isn’t Wise to Use EI For Analyzing large molecules like proteins EI shatters chemical bonds Any given protein contains many different amino acids EI would shatter the protein into peptides of 2,3,4… amino acids and amino acid sub-fragments Result is a huge number of different signals from a single protein -- too complex to analyze

Chemical Ionization (CI) Electron ionization leads to fragmentation of the molecular ion, which sometimes prevents its detection. Chemical ionization (CI): Thus this technique presents the advantage of yielding a spectrum with less fragmentation in which the molecular species is easily recognized. Consequently, chemical ionization is complementary to electron ionization.

Chemical Ionization (CI) Typical reagent gases (ex. CH4, isobutane, or NH3) are present in a millionfold excess with respect to the analyte. Analyte is ionized by ion-molecule chemical reactions: Primary Ion Formation: CH4 + e-  CH4+ + 2e- Secondary Reagent Ions: CH4 + CH4+  CH5+ + CH3 CH4 + CH3+  C2H5+ + H2 Product Ion Formation: M + CH5+  CH4 + [M + H] + (protonation) AH + CH3+  CH4 + A+ (H− abstraction) M + CH5+  [M+ CH5] + (adduct formation) A + CH4+  CH4 + A+ (charge exchange)

Fast Atom Bombardment (FAB) Material to be analyzed is mixed with a non-volatile chemical protection environment called a matrix This is bombarded under vacuum with a high energy (4 – 10 keV) beam of atoms. Atoms are typically an inert gas (Ar or Xe)

Soft Ionization Soft ionization techniques keep the molecule of interest fully intact Electro-spray ionization first conceived in 1960’s by Malcolm Dole but put into practice in 1980’s by John Fenn (Yale) MALDI first introduced in 1985 by Franz Hillenkamp and Michael Karas (Frankfurt) Made it possible to analyze large molecules via inexpensive mass analyzers such as quadrupole, ion trap and TOF

Soft Ionization Methods 337 nm UV laser Fluid (no salt) + _ cyano-hydroxy cinnamic acid Gold tip needle MALDI ESI

Electrospray Ionization Sample dissolved in polar, volatile buffer and pumped through a stainless steel capillary (70 - 150 mm) at a rate of 10-100 mL/min!!! Strong voltage (3-4 kV) applied at tip along with flow of nebulizing gas causes the droplets to pick charges and nebulize (aerosol) Aerosol is directed through regions of higher vacuum until droplets evaporate to near atomic size (still carrying charges)

Electrospray Ionization Can be modified to “nanospray” system with flow < 1 mL/min Very sensitive technique, requires less than a picomole of material Strongly affected by salts & detergents

Electrospray (ESI)

Matrix-Assisted Laser Desorption Ionization 337 nm UV laser cyano-hydroxy cinnamic acid

MALDI Sample is ionized by bombarding sample with laser light Sample is mixed with a UV absorbant matrix (like 4-hydroxycinnaminic acid) Light wavelength matching the absorbance maximum of absorbant matrix will be absorbed and the matrix transfers some of its energy to the analyte (leads to ion sputtering)

MALDI Generally tolerates salts and nonvolatile components, an advantage over ESI Easier to use and maintain Requires as low as 10 mL of 1 pmol/mL sample

Mass Spectrometer Schematic Rough pumps Rotary pumps Turbo pumps Diffusion pumps High Vacuum System Inlet Ion Source Mass Filter Detector Data System Vapor HPLC GC Solids probe MALDI ESI FAB EI/CI TOF Quadrupole Ion Trap Mag. Sector FTMS Microch plate Electron Mult. PC’s UNIX Mac

Different Mass Analyzers Magnetic Sector Analyzer + Double Focusing High resolution, exact mass, original MA Quadrupole Analyzer Low (1 amu) resolution, fast, cheap Time-of-Flight Analyzer No upper m/z limit, high throughput Tandem Mass Spectrometers (MS-MS) V. good resolution Ion Trap Mass Analyzer Good resolution, simple Ion Cyclotron Resonance (FT-ICR) Highest resolution, exact mass, costly

Magnetic Sector Mass Analyzer

Double focusing mass analyzer

Quadrupole Mass Analyzer A quadrupole mass filter consists of four parallel metal rods with different charges The applied voltages affect the trajectory of ions traveling down the flight path For given dc and ac voltages, only ions of a certain mass-to-charge ratio pass through the quadrupole filter and all other ions are thrown out of their original path

Quadrupole Ion Filter resonant ion non-resonant ion _ Detector + + _ Source DC and AC Voltages

TOF

Principle Of Reflector-TOF acceleration region Field free drift region sample target 1 2 reflector m = m E < E 12 m/z detector

Ion Trap Mass Analyzer Invented by Wolfgang Paul (Nobel Prize1989) Offer good mass resolving power The two end-cap electrodes are grounded while the doughnut shaped ring electrode is connected to a variable RF voltage source

FT-ICR Uses powerful magnet (5-10 Tesla) to create miniature cyclotron Originally developed in Canada (UBC) by A.G. Marshal in 1974 FT approach allows many ion masses to be determined simultaneously (efficient) Has higher mass resolution than any other MS analyzer available

FT-ICR-MS instrument general scheme

A circulating ion in a magnetic field is capable of absorbing energy from an ac electric field provided the frequency of the field matches the cyclotron frequency the cyclotron frequencyA circulating ion perpendicular to field has a frequency called

FTICR: New Dimensions of High Performance Mass Spectrometry High mass resolution > 3 000 000 Accuracy of mass determination < 0.1 ppm Sensitivity (ESI, Octapeptide) ca. 50 attomol Structure-specific fragmentation MS/MS , MSn

Mass Spectrometer Schematic Rough pumps Rotary pumps Turbo pumps Diffusion pumps High Vacuum System Inlet Ion Source Mass Filter Detector Data System Vapor HPLC GC Solids probe MALDI ESI FAB EI/CI TOF Quadrupole Ion Trap Mag. Sector FTMS Microch plate Electron Mult. PC’s UNIX Mac

Mass Detectors Electron Multiplier

Electron multipliers with discrete dynodes In this device, positive ions strike a conversion cathode liberating electrons which are then accelerated and multiplied’ via a series of up to twenty dynodes. This type of detector is extremely sensitive, having a gain of up to 108. Aluminium-based dynodes have improved performances of the traditional materials (Cu/Be alloys) which age rather badly in the residual atmosphere of the spectrometers, or during non working periods (returning to atmospheric pressure).

Continuous dynode multipliers with a channeltron® The ions are directed towards a collector whose entrance, in the form of a horn, is made of a lead doped glass with which acts as the conversion cathode. The ejected electrons are attracted towards a positive electrode and their collisions against the internal walls give rise to multiplication, as with the separated dynodes. The assembly is usually mounted off-axis to avoid the impact of neutral species as well as photons emitted by the filament, equally susceptible to the removal of the electrons.

Microchannel plate detectors(MCP) They consist of the union of a large number microchanneltrons arranged like honeycombs. This resembles an electronic version of a photographic plate. Each individual detector is formed from a portion of microtube (25mm diameter) whose interior is coated by a semiconductor material acting as a continuous dynode. This system preserves the spatial resolution of the input charged ions.

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