mass spectrometry

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IN 504 Analytical InstrumentsModule 5

1 Presented by; Anju Sunny CUSATReference Text: R S Khandpur Handbook of Analytical Instrumentation

Mass Spectrometer2

IntroductionMass spectrometers is an analytical instrument which can be used for all kind of chemical analysis like;To measure the elemental composition of matterTo analyze the structures of inorganic, organic and biological moleculesTo find the isotopic ratios of atoms in the sampleFor the detection of poisons in a sample

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General Block Diagram Mass Spectrometer

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Analogy between Optical Spectrophotometer & Mass Spectrometer

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Components of a Mass Spectrometer

IonisationIon DetectionIon Separation

Ion Source Mass Analyser DetectorElectron Ionisation (EI)Chemical Ionisation (CI)Fast Atom Bombardment (FAB) Electrospray Ionisation (ESI) Matrix-Assisted Laser DesorptionIonisation (MALDI)QuadrupoleMagnetic deflection typeElectrostatic type Time-Of-Flight (TOF) Ion TrapElectron MultiplierMultichannel plate Faraday Cup

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IONIZATION SOURCES1) Electron Ionization (EI)

- Commonly used for analysis of organic samples

- Electrons are emitted from a heated tungsten filament cathode.

- Electrons are accelerated towards the anode with a potential of about 50 100 V

- Electrons meet at right angles with the sample molecules

- Interaction with the high energy electrons causes ionization of sample molecules and fragmentation into smaller ions.

IONIZATION SOURCES2) Chemical Ionization (CI)

- A large excess of reagent gas (1000 10000 times) is introduced into the ionization region

- Pressures in source are typically higher than EI

- Electrons are allowed to bombard the gas-sample mixture

Examples of reagent gas- Methane, ammonia, isobutane

IONIZATION SOURCES3) Atmospheric Pressure Ionization (API) Sources

Two major typesElectrospray Ionization (ESI) - Atmospheric Pressure Chemical Ionization (APCI)

- Operate at atmospheric pressure

- Modified version of ESI is the Ion Spray Source

- Used for mixtures of nonvolatile high molecular weight compounds

IONIZATION SOURCES4) Desorption Ionization

- For direct ionization of solids

- Excellent tool for analysis of large molecules

- Solid samples are placed on a support and then bombarded with ions or photons

- Different types are available


a) Laser Desorption Ionization

- Uses pulsed laser - Provides selective ionization by choosing appropriate

- Laser is focused on a solid surface to ionize material

Examples of Lasers- IR laser: CO2 laser- UV laser: Nd:YAG (yttrium aluminum garnet)


b) Matrix-Assisted Laser Desorption Ionization (MALDI)

- Matrix disperses large amounts of energy absorbed by the laser

- Minimizes fragmentation of the molecule

- Used for study of polymers, proteins, peptides


c) Fast Atom Bombardment (FAB)

- Employs fast moving neutral inert gas atoms (Ar) to ionize large molecules

- Sample is dissolved in glycerol and spread in a thin layer on a metal probe

- Probe is then inserted into the mass spectrometer and a beam of fast moving atoms probe the surface

Types of Mass SpectrometersVarious types of Spectrometers are available in the means of separating the ions according to their mass-to-charge ratio or simply, on the basis of Mass Analyzers used. They are;1) Magnetic Deflection Mass Spectrometer2) Electrostatic Mass Spectrometer3) Time-of-Flight Mass Spectrometer4) Quadrupole Mass Spectrometer

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1) Magnetic Deflection Mass Spectrometer

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Magnetic Deflection Mass SpectrometerIn a mass spectrometer, the sample to be analyzed is first bombarded with an electron beam to produce ionic fragments of the original molecule.These ions are accelerated in a high vacuum into a magnetic field , which deflects them into circular paths.Since the deflection for light ions is greater than that for heavy ions, the ion stream separates into beams of different molecular weight.A suitably placed slit allows a beam of selected molecular weight to pass through to a collection electrode or detector.

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2) Electrostatic Mass SpectrometerElectrostatic sector analyzer consists of 2 curved plates of equal and opposite potential. As the ion travels through the electric field, it is deflected and the force on the ion due to the electric field is equal to the centripetal force on the ion. Here the ions of the same kinetic energy are focused, and ions of different kinetic energies are dispersed.Electrostatic and magnetic sector analyzers when employed individually are single focusing instruments. When both techniques are used together, it is called a double focusing instrument, because in this instrument both the energies and the angular dispersions are focused.

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3)Time of Flight (ToF) Mass Spectrometer In ToF Mass Spectrometer, ions of different mass/charge ratio are separated by the difference in time they take to travel over an identical path from the ion source to the collector at the detector. Here ions generated by bombardment of the sample with a brief pulse of electrons, secondary ions or laser-generated photons. Ions accelerated by electric field and they enter into field-free drift tube. Ions enter tube with same kinetic energy and ion velocity vary inversely with mass. Lighter particles arrive at detector before heavier particles and ions are separated in the drift tube according to their velocities (v)

3)Time of Flight (ToF) Mass Spectrometer

3)Time of Flight (ToF) Mass Spectrometer V = accelerating voltage

If L is the length of tube (typically 1-2 m) and t is the flight time of ion, then v = L/t

Implies mass-to-charge ratio and flight time can be found from

Advantages of ToFHigh speed operationAbility to record entire mass spectrum at one timeAccuracy only depend on electronic circuits

21Disadvantages of ToFPoor resolution due to display on an Oscilloscope screen

Applications of ToFQualitative AnalysisMolecular Weight DeterminationStructure DeterminationQuantitative AnalysisBiotechnologyAnalysis of proteins & peptidesPharmaceuticaldrug discovery

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4) Quadrupole Mass Spectrometer23

4) Quadrupole Mass Spectrometer Four parallel cylindrical rods serve as electrodes. Opposite rods are connected electricallyOne pair attached to positive side of variable dc sourceOne pair attached to negative side of variable dc sourceVariable radio-frequency ac potential (180o out of phase) applied to each pair of rods.The result is an ac potential superimposed on a dc potential and this creates an electric field. Ions accelerated through the electric field between rods.ac and dc voltages increased simultaneously with ratio being constantAll ions without specific m/e strike rods and become neutralOnly ions having a limited range of m/e reach detector.

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Features of Quadrupole Mass SpectrometerThe quadrupole acts as a filter so is often called the mass filter.Sample must be ionized and it should be in gas phase.Has smaller range and lower resolution than magnetic sector but faster.More compactless expensiveHigh scan rate25

DETECTORS1) Multichannel plate

- Detector which measures one m/e value at a time called single channel detectors.

- Multichannel plate detectors are used for the detection of multiple ion beams with different m/e value.

- High resolution magnetic sector instruments use multichannel detectors (called multicollectors)

DETECTORS2) Electron Multiplier (EM)

- The most common detector in Mass spectrometer for ions.

- Similar to PMT.

- Very sensitive and has fast response.

DETECTORS3) Faraday Cup

- A metal or carbon cup serves to capture ions and store the charge.

- Cup shape decreases loss of electrons.

- Least expensive detector for ions.

- Has long response time.

DETECTORS4) Array Detectors

- Used in ToF Mass spectometers.

- Employs a focal plane camera (FPC) consisting of an array of 31 Faraday Cup

- Up to 15 m/e values can be measured simultaneously.

- Exhibits improved precision compared with single channel detectors.

Molecular weight can be obtained from a very small sample.It does not involve the absorption or emission of light.Detection limits better by 3 orders of magnitude of other techniques. Remarkably simple spectra that are unique and easily interpreted.Ability to measure isotopic ratios.

Mass Spectrometry : AdvantagesMass Spectrometry : Disadvantages Instrument costs are 2 to 3 times higher than others. Instrument drift that can be as high as 5-10% per hour. Interference effects may occur.

Chromatography31

PrincipleIt is a separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium.Components:Mobile phase: a solvent that flows through the supporting medium.Stationary phase: a layer or coating on the supporting medium that interacts with the analyte.Supporting medium: a solid surface on which the stationary phase is bound or coated.

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Types of Chromatography

The primary division of chromatographic techniques is based on the type of mobile phase used in the system. Type of Chromatography Type of Mobile PhaseGas chromatography (GC) GasLiquid chromatography (LC) Liquid

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Types of Chromatography Further divisions can be made based on the type of stationary phase used in the system:Gas Chromatography Name of GC Method Type of Stationary PhaseGas-solid chromatography(GSC) solid supportGas-liquid chromatography(GLC) liquid-coated supportBonded-phase gas chromatography chemical support

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Types of ChromatographyLiquid Chromatography Name of LC Method Type of Stationary PhaseAdsorption chromatography - solid supportPartition chromatography - liquid-coated supportIon-exchange chromatography - support containing fixed chargesSize exclusion chromatography - porous supportAffinity chromatography - support with immobilized material

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Gas Chromatography (GC)36

Gas Chromatography- Equipments37Carrier source with pressure and flow regulators. (Eg: Ar, He, N2, H2 )Injector or sample application system.Chromatographic column with oven for temperature control. (Eg: 2-50 m, coiled stainless steel/glass/Teflon tube)Detector & computer or recorder. Eg: Thermal Conductivity Detector (TCD), Flame Ionization Detector (FID), Electron Capture Detector (ECD)

Gas Chromatography- Equipments38Mobile Phase or Carrier gasMain purpose of carrier gas in GC is to move the solutes along the column.Common carrier gas include He, Ar, H2, N2 etc.Carrier Gas or Mobile phase does not affect solute retention time. (Time required for the sample to travel from the injection port through the column to the detector). But does affect: Desired efficiency for the GC SystemStability of column and solutesResponse of the detector

Gas Chromatography- Equipments39Stationary PhaseIt is the main factor determining the selectivity and retention of solutes.There are three types of stationary phases used in GC:Solid adsorbents (GSC)Liquids coated on solid supports (GLC)Bonded-phase supports

How Does Chromatography Work?In all chromatographic separations, the sample is transported in a mobile phase. The mobile phase can be a gas or a liquid.The mobile phase is then forced through a stationary phase held in a column or on a solid surface. The stationary phase needs to be something that does not react with the mobile phase or the sample.The sample then has the opportunity to interact with the stationary phase as it moves past it. Samples that interact greatly, then appear to move more slowly. Samples that interact weakly, then appear to move more quickly. Because of this difference in rates, the samples can then be separated into their components.

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Chromatography is based on a physical equilibrium that results when a solute is transferred between the mobile and a stationary phase. K = distribution coefficient or partition ratio

K = C S

C M

Where CS is the molar concentration of the solute in the stationary phase and CM is the molar concentration in the mobile phase.

Gas-Solid Chromatography (GSC)Here same material is used as both the stationary phase and support materialCommon adsorbents include:Alumina Molecular sieves (crystalline aluminosilicates [zeolites] and clay)Silica Active carbon

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Gas-Liquid Chromatography (GLC)Here stationary phase is some liquid coated on a solid support.Over 400 liquid stationary phases available for GLC.Material range from polymers (polysiloxanes, polyesters, polyethylene glycols) to fluorocarbons, molten salts and liquid crystals .Disadvantage is the liquid may slowly bleed off with time.

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Bonded Phase Gas ChromatographyStationary phase is covalently attach to the solid support.So it avoids column bleeding in GLC.Bonded phases are prepared by reacting the desired phase with the surface of a silica-based support. Commonly recommended bonded-phases:DimethylpolysiloxaneMethyl(phenyl)polysiloxanePolyethylene glycol (Carbowax 20M)

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Liquid Chromatography (LC)Introduction:A chromatographic technique in which the mobile phase is a liquid.LC is a much older technique than GC, but was overshadowed by the rapid development of GC in the 1950s and 1960s.LC is currently the dominate type of chromatography and is even replacing GC in its more traditional applications.

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Advantages of LC compared to GC

Separation of any compound that is soluble in a liquid phase is possible. (Eg: biological compounds, synthetic or natural polymers, inorganic compounds etc.)Can be used at lower temperatures than GC.Retention of solutes in LC depend on their interaction with both the mobile phase and stationary phase, where GC retention based on volatility and interaction with stationary phase.Most LC detectors are non-destructive and most GC detectors are destructive.

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Classification of LC based on overall efficiency or performance Low-performance Liquid Chromatography (LPLC) High-performance Liquid Chromatography (HPLC)47

Low-performance liquid chromatography (LPLC)LC methods that use large, non-rigid support material.Poor system efficiency.Such systems have the following characteristics:broad peaks poor limits of detectionlong separation times columns can only tolerate low operating pressures48

Low-performance liquid chromatography (LPLC)Column chromatography an example of the equipment used in low-performance liquid chromatography .

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Solvent reservoirColumn headColumnColumn packingPorous glass plate

Sample is usually applied directly to the top of the column. Detection is by fraction collection with later analysis of each fraction

High-performance liquid chromatography (HPLC)

LC methods that use small, uniform, rigid support material.Good system efficiencies Such systems have the following characteristics:narrow peaks good limits of detectionshort separation times columns can tolerate high operating pressures and faster flow-rates

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High-performance liquid chromatography (HPLC)It requires higher operating pressures for mobile phase delivery and uses special pumps and other system components. Sample applied using closed system (i.e., injection valve) Detection uses a flow through detector.

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High-performance liquid chromatography (HPLC)Advantages:fast analysis timeease of automationgood limits of detectionpreferred choice for analytical applicationspopular for purification work

Disadvantages:greater expenselower sample capacities

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HPLC Vs LPLC

Types of Liquid Chromatography

Techniques in LC are classified according to the method of solute separation.1) Adsorption chromatography: Separates solutes based on their adsorption to the stationary phase.2) Partition chromatography: Separates solutes based on their partitioning between a liquid mobile phase and a liquid stationary phase coated on a solid support.3) Ion-exchange chromatography: Separates solutes by their adsorption onto a support containing fixed charges on its surface. 4) Affinity chromatography: Separates based on the use of immobilized biological molecules as the stationary phase. 5) Size-exclusion chromatography: separates molecules according to differences in their size.

Types of Liquid Chromatography

Chromatographic DetectorsAfter the components of a mixture are separated using chromatography, they must be detected as they exit the chromatographic column. Most commonly used detectors are:Thermal Conductivity Detector (TCD)Flame Ionization Detectors (FID) Electron Capture Detector (ECD)Atomic Emission Detector (AED)Mass spectrometer (MS)

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Thermal Conductivity Detector (TCD)

A TCD detector consists of an electrically-heated wire or thermistor.The temperature of the sensing element depends on the thermal conductivity of the gas flowing around it.Changes in thermal conductivity, such as when organic molecules displace some of the carrier gas, cause a temperature rise in the element which is sensed as a change in resistance. The TCD is not as sensitive as other detectors but it is non-specific and non-destructive.

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Thermal Conductivity Detector (TCD)58

Flame Ionization Detectors (FID) The effluent from the column is mixed with hydrogen and air and then ignited electrically. Most organic compounds, when pyrolyzed at the temperature of a hydrogen/air flame, produce ions and electrons that can conduct electricity through the flame.The ions are collected on a biased electrode and the resulting current (~10-12 A) is then measured. Extremely sensitive and have large dynamic range.Disadvantage of FID is that it is destructive of sample.

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Flame Ionization Detectors (FID) 60

Electron Capture Detectors(ECD)The ECD uses a radioactive source such as Ni63 which produces Beta particles, which react with the carrier gas producing free electrons. These electrons flow to the anode & producing an electrical signal . When electrophillic molecules are present, they capture the free electrons, lowering the signal. The amount of lowering is proportional to the amount of analyte present. It is widely used detectors for environmental samples, because it detects halogen containing compounds, such as pesticides and polychlorinated biphenyls.

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Electron Capture Detectors(ECD)62

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Atomic Emission Detector (AED)

One of the newest gas chromatography detector & it is quite expensive compared to other chromatographic detectors.The strength of the AED lies in the detector's ability to simultaneously determine elements. It uses microwave energy to excite helium molecules (carrier gas) which emit radiation & which breaks down molecules to atoms such as S, N, P, Hg, As, etc. These excited molecules emit distinctive wavelengths which can be separated by a grating and sent to the detector (typically a photodiode array) which produces the electrical signal.

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Atomic Emission Detector (AED)64

Mass Spectrometer (MS)Uses the difference in mass-to-charge ratio (m/e) of ionized atoms or molecules to separate them from each other.Molecules have distinctive fragmentation patterns that provide structural information to identify structural components.The general operation of a mass spectrometer is:Create gas-phase ionsSeparate the ions in space or time based on their mass to charge ratioMeasure the quantity of ions of each mass-to-charge ratio.

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Mass Spectrometer (MS)66

Centrifugal forceis the apparent force that draws a rotating body away from the centre of rotation.

Centripetal force is aforce that makes a body follow a curved path.67Extra reading

mass spectrometry

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