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    Affinity Chromatography

    andIon Exchange Chromatography

    Shannon E. Spence

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    What on Earth did scientist do before

    Chromatography?

    -

    Extractionis based on the difference in solubility materialis grounded, placed with a solvent which

    dissolves soluble compounds. A second

    extract solvent . The mixture is placed in a

    separatory funnel

    -Crystallizationalso based on the difference of solubility. Thesolubility is solved in a fixed volume of solvent.

    The purified compound crystallizes as solution

    cools, evaporates or diffuses

    -Distillilation

    separates components based on their volatility

    typically via vaporization-condensationmethod

    Filtrationseparate components of a mixture based on

    their particle size. Used most often to

    separate a liquid from a solid

    www.chemguide.co.uk/.../idealfract.html

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    What entices the scientists to

    Chromatography?Just like the previous techniques

    chromatography is a way to separate

    two components based on a specific

    characteristic

    What makes chromatography so

    useful...The results are reproducible with

    better accuracy than the before

    mentioned separation techniques

    Chromatography can separate more

    complex mixtures than the previoustechniques

    Chromatography is less time

    consuming and cheaper

    http://www.residues.com/ion_chromatography.html

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    Brief History of Chromatography

    1903 Tswett, a Russian botanist

    coined the term chromatography. He

    passed plant tissue extracts through a

    chalk column to separate pigments by

    differential adsorption chromatogrpahy

    1915 R.M Willstatter, German Chemistwin Nobel Prize for similar

    experiement

    1922 L.S Palmer, American scientist

    used Tswetts techniques on various

    natural products

    1931 Richard Kuhn used

    chromatography to separate isomersoh polyene pigments; this is the first

    known acceptance of chromatographic

    methods

    http://www.chemgeo.uni -hd.de/texte/kuhn.html

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    History of the Main techniques

    1938 Thin Layer chromatography by

    Russian scientist N.A Izamailov and

    M.S Shraiber

    1941 Liquid-Liquid partition

    chromatography developed by Archer

    John, Porter Martin and RichardLaurence Millington Synge

    1944 Paper Chromatography one of

    the most important methods in the

    development of biotechnology

    1945 Gas Chromatography 1st

    analytical gas-solid (adsorption)

    chromatography developed by FritzPrior

    1950 Gas Liquid Chromatography by

    Martin and Anthony James; Martin

    won the Nobel Prize in 1952

    British chemist ArcherJohn Porter Martin, co-

    recipient, with Richard L. M. Synge, of the 1952

    Nobel Prize in chemistry, "for their invention ofpartition chromatography."

    http://www.chemistryexplained.com/Ma-Na/Martin-Archer-John-Porter.html

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    History of the Main Techniques

    1966 HPLC named by Csaba Horvath,

    but didnt become a popular method

    until 1970s

    1950s Ion-Exchange chromatography

    declassified this technique

    1970s Ion Chromatography wasdeveloped by Hamish Small and co-

    workers at the Dow Chemical

    company

    1930s Affinity Chromatography was

    developed for the study of enzymes

    and other proteins

    library.thinkquest.org

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    Chromatography

    Applies the principles of the fractional separation procedures

    Non-instrumental analysis which partitions components between two

    phases usually a mobile phase and a stationary phase, based onthe difference in the components physical properties

    Can separate complex mixtures composed of many very similar

    components.

    Chromatography is often coupled with analytical instruments to

    complete analysis. A single chromatographic analysis can isolate, identify , and

    quantitate multiple components of mixtures

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    Principles of Chromatography

    Chromatography is used when there is a difference in the retention times of

    different components

    Two types of phases

    1) Stationary phase

    2) mobile phases Properties of Chromatographic Properties

    1) immiscible stationary and mobile phases

    2) an arrangement where a mixture is depositied at one end of the

    stationary phase

    3) flow of the mobile phase towards the other end of the stationaryphase

    4) different rates of partitioning for each component

    5) means for visualizing the separation of each component

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    Techniques

    Ion Exchange Chromatography (IEC)separates biomolecules based on the their net surface charge

    Ion Chromatography (IC)more general form of IEC allows separation of ions and polar molecules

    based on the charge properties of the molecules

    Affinity Chromatography (AC)

    is the purification of a biomolecule with respect to the specific bindingof that biomolecule due to the chemical structure

    Gas Chromatography (GC)is a technique used to separate organic molecules that are volatile

    Gas-Liquid Chromatography (GLC)another name for GC

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    More Detailed History of

    IE Chromatography 1850 H. Thompson and J.T way treated various clas with ammonium sulfateor carbonate in solution to extract the ammonia and release the calcium

    1927 Zeolite (sodium aluminum silicates) mineral columns were used toremove interfering calcium and magnesium ions from solution to determinesulfate content of water

    1940s Modern Ion-Exchange Chromatography was developed during thewartime Manhattan Project

    - this technique was used to separate and concentrate the radioactiveelements needed to make an atomic bomb. The adsorbents would latchonto charged transuranium elements differentially eluting them

    1970s Hamish Small and co-workers ofDow Chemical Company developed

    ion-chromatography usable for automated analysis

    - IC uses weaker ionic resins for the stationary phase and a neutralizing

    stripper column to remove background eluent ions

    - used for determining low concentrations of ions in water and other

    environmental studies

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    Terminology

    Elution

    - washing of the mixture

    Eluent- additional solvents used for

    elution

    Effluent- exiting fluid stream

    Residency

    - time spent on column

    Stationary Phase

    -

    Mobile Phase

    - fluid carrying the mixture of

    analytes

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    Ion-Exchange Chromatography

    Usually employed with HPLC

    Ions are charged molecules

    - cation positively charged ion

    - anion negatively charged ion

    These ions do not separate smoothly under the traditional methods of theliquid and mobile phases of chromatography

    Requires alteration methods of either the mobile phase or stationary phase

    are required

    - mobile phase suppresses the ionic nature of the analyte

    - stationary phase incorporate ions of the opposite charge to attract and

    retain analyte

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    How do you get those columns

    to work There is a glass columncoated with a resin

    polymer

    The resin is eitherpositively charged (an

    acid) or negatively

    charged (a base)

    An analyte will have ions

    opposite of the resins

    charge eluting off the ion

    of interest

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    Resin

    Common resins are copolymerized styrenes

    vinylic and aromatic functional groups

    styrene derivative and divinylbenzene

    Creates better stability due to crosslinking of the benzene rings Creates a swelling within the polymer affecting the porosity while taking in

    the mobile phase liquid

    Aromatic substitution reaction makes these polymers ideal for charged

    functional groups

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    Cation Exchange resins

    The functional group in cation exchange resins are usually acids

    Sulfonic acids SO3H (strong acid resin) are added to the resin by

    sulfonation reactions

    Res-(SO3H) + M+

    Res-(SO3M) + H+

    Carboxylic acid COOH (weak acid resin)

    Res-COOH + M+ Res-COOM + H+

    With both the strong and the weak acid exchange sites an acidic Hydrogen

    is attached to a functional group chemically bound to the resin

    Cation exchange is good for removing metal ions from an aqueous solution

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    Anion Exchange Resins

    The functional groups added to the resin is similar to cation resins but are

    basic instead of acidic

    Quaternary ammonium a strong base -- CH2N(CH3)3+OH-

    CH2N(CH3)3+OH- + B- Res-CH2N(CH3)3

    +Cl- + OH-

    Polyalky amine a weak base -- NH(-R)2+OH-

    NH(-R)2+OH- + B- Res-NH(-R)2

    +B- +OH-

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    To Affinity and Beyond

    The rate of ion exchange is controlled by the law of

    mass action. At equal concentration the greater affinity

    molecule will control the cation exchange resins in the

    acid form.

    However if a much higher concentration of strong acid

    passed through the greater affinity molecule, such as

    sodium, will form the resin, reversing equilibrium and

    convert the resin back to an acidic form.

    Generally it is possible to return either ion exchangeresin column to a desired starting form by passing a

    large excess of the desired ion at very high

    concentration through the resin

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    What makes it unique

    Careful selection of the ionic

    composition of the eluent, and the

    gradual adjustment of its strength

    during elution using a controlled

    gradient

    The components of a mixture of

    ions can be induced to separate

    just as the components of a

    mixture separated by partitionion

    chromatography

    The parameters controlling the

    relative residence of the analyte or

    other eluent ions is the resin

    stationary phase or the ionic

    solution mobile phase

    1) both the relative selectivity of

    the resin for the ions and their

    relative concentrations in each

    phase

    2) In ion exchange, selectivity

    resides in relative ion-pairing

    interaction strengths only in the

    stationary phase

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    Relative Affinity of Ions

    The higher the charge the higher the

    affinityNa+ < Ca2+ < Al3+ and Cl- < SO4

    2-

    The Ion with the greatest size and charge

    has the highest affinity

    Li+ < Na+ < K+ < Cs+ < Be2+ < Mg2+ < Cu2+ and F- < Cl- < Br- < I-

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    Applications of IEC

    In cell and molecular biology, ion

    exchange chromatography is used to

    separate different proteins out of an

    eluant.

    areas of research such as the

    environment, industry, commercial

    products of organic molecules without UV-vis absorption

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    Ion Chromatography

    The analysis of ionic analytes by

    separation on ion exchange stationary

    phases with eluent suppression of excess

    eluent ions

    ex) when cations are being exchanged to effect aseparation, variable concentrations of HCl are used as

    an eluent passing through the analytical anion columnwithout being retained forming largely undissociated

    species such as water, carbonic acid and bicarbonate

    ions

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    AFFINITYCHROMATOGRAPHY

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    Affinity History

    1930s, first developed by Arne Wilhelm

    Tiselius, won the Nobel Prize in 1948

    Used to study enzymes and other proteins Relies on the affinity of various

    biochemical compounds with specific

    properties

    ex) enzymes for their substrates

    antibodies for their antigens

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    How do they get those iddy bitty

    molecules in there?

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    So now what.

    The Sample is injected into the equilibrated

    affinity chromatography column

    Only the substance with affinity for the ligand are

    retained on the column

    The substance with no affinity to the ligand will

    elute off

    The substances retained in the column can beeluted off by changing the pH of salt or organic

    solvent concentration of the eluent

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    Specificity of Affinity

    Chromatography Specificity is based on three aspect of affinity

    1) the matrix

    2) the ligand

    3) the attachment of the ligands to the matrix

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    Matrix

    The matrix simply provides a posre structure to increase

    the surface area to which the molecule can bind

    This has been what kept the Affinity Chromatographyfrom being developed earlier and useful to the scientific

    community

    The matrix must be activated for the ligand to bind to itbut still able to retain its own activation towards the

    target molecule

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    Matrix

    Amino, hydroxyl, carbonyl and thio groups located with

    the matrix serve as ligand binding sites

    Matrix are made up of agarose and otherpolysaccharides

    The matrix also must be able to withstand the

    decontamination process of rinsing with sodiumhydroxide or urea

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    Ligand

    The Ligand binds only to the desired molecule within the solution

    The ligand attaches to the matrix which is made up of an inert

    substance

    The ligand should only interact with the desired molecule and form a

    temporary bond

    The ligand/molecule complex will remain in the column, eluting

    everything else off

    The ligand/molecule complex dissociates by changing the pH

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    So many ligands so little time

    The chosen ligand must bind strongly to the molecule of

    interest

    If the ligand can bind to more than onel molecule in thesample a technique, negative affinity is performed

    - this is the removal of all ligands, leaving the

    molecule of interest in the column

    -done by adding different ligands to bind to theligands within the column

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    Applications

    Used in Genetic Engineering

    Production of Vaccines

    And Basic Metabolic Research

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    Gas Chromatography

    Gas-Liquid Chromatography

    AndGas-Solid Chromatography

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    History

    1945

    technique developed by Fritz Prior out of post-WWII

    Europe

    Fritz Prior was a only a graduate student at the time 1947

    Prior succeeded in separating O2 and CO2 on a

    charcoal column

    1950ArcherJ.P Martin and Anthony James developed Gas-

    Liquid Partition Chromatography (GLPC)

    this has become the method of choice

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    Gas Chromatography

    Gas- Solid Chromatography (GSC)

    is a process of repeated adsorption/desorption of

    sample from the carrier gas to the solid adsorbent

    Gas-Liquid Partioning Chromatography (GLPC)involves a sample being vaporized and injected onto the

    head of the chromatographic column. The sample is

    transported through the column by the flow of inert,

    gaseous mobile phase. The column itself contains a

    liquid stationary phase which is adsorbed onto the

    surface of an inert solid.

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    Instrumentation

    Carrier Gas

    Flow controller

    Injector port

    Column oven

    Column

    Detector

    Recorder

    http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/gaschrm.htm

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    Carrier Gas

    The carrier gas must be chemically inert.

    Commonly used gases include nitrogen, helium,

    argon, and carbon dioxide. The choice of carrier gas is often dependant

    upon the type of detector which is used.

    The carrier gas system also contains a

    molecular sieve to remove water and other

    impurities.

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    Sample injection port

    For optimum column efficiency, the sample should not be too large, and

    should be introduced onto the column as a "plug" of vapour

    - slow injection of large samples causes band broadening and loss of

    resolution.

    The most common injection method is where a microsyringe is used to

    inject sample through a rubber septum into a flash vapouriser port at the

    head of the column.

    The temperature of the sample port is usually about 50C higher than the

    boiling point of the least volatile component of the sample.

    For packed columns, sample size ranges from tenths of a microliter up to 20

    microliters. Capillary columns, on the other hand, need much less sample, typically

    around 10-3 mL. For capillary GC, split/splitless injection is used.

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    Sample injection port

    The injector can be used in one of

    two modes; split or split less.

    The injector contains a heated

    chamber containing a glass liner

    into which the sample is injected

    through the septum.

    The carrier gas enters the

    chamber and can leave by three

    routes (when the injector is in split

    mode).

    The sample vaporizes to form a

    mixture of carrier gas, vaporized

    solvent and vaporized solutes

    A proportion of this mixture passes

    onto the column, but most exits through

    the split outlet.

    The septum purge outlet prevents

    septum bleed components from

    entering the column

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    Columns

    There are two general types of column,

    1) packed

    contain a finely divided, inert, solid support material (commonly based on

    diatomaceous earth) coated with liquid stationary phase. Most packed columns are

    1.5 - 10m in length and have an internal diameter of 2 - 4mm.

    2) capillary(also known as open tubularCapillary columns have an internal diameter of a few tenths of a millimeter. They

    can be one of two types;

    a) wall-coated open tubular(WCOT)

    consist of a capillary tube whose walls are coated with liquid stationary

    phase

    b) support-coated open tubular(SCOT).the inner wall of the capillary is lined with a thin layer of support material

    such as diatomaceous earth, which the

    stationary phase has been adsorbed.

    SCOT columns are generally less efficient than WCOT columns. Both types of

    capillary column are more efficient than packed columns.

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    Column

    In 1979, a new type of WCOT column was devised - the Fused Silica Open

    Tubular(FSOT) column

    These have much thinner walls than the glass capillary columns, and are

    given strength by the polyimide coating. These columns are flexible and can

    be wound into coils. They have the advantages of physical strength,

    flexibility and low reactivity.

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    Column Temperature

    For precise work, column temperature must be controlled to within

    tenths of a degree.

    The optimum column temperature is dependant upon the boiling

    point of the sample.

    As a rule of thumb, a temperature slightly above the average boilingpoint of the sample results in an elution time of 2 - 30 minutes.

    Minimal temperatures give good resolution, but increase elution

    times.

    If a sample has a wide boiling range, then temperature programming

    can be useful. The column temperature is increased (either continuously or in

    steps) as separation proceeds

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    Detectors There are many detectors which can be used in gas chromatography. Different detectors will give different types of selectivity.

    A non-selective detector responds to all compounds except the carrier gas

    a selective detectorresponds to a range of compounds with a common

    physical or chemical property and a specific detectorresponds to a single

    chemical compound.

    Detectors can also be grouped into concentration dependant detectors and

    mass flow dependant detectors.

    The signal from a concentration dependant detector is related to the

    concentration of solute in the detector, and does not usually destroy the

    sample

    D

    ilution of with make-up gas will lower the detectors response.

    Mass flow dependant detectors usually destroy the sample, and the signal

    is related to the rate at which solute molecules enter the detector.

    The response of a mass flow dependant detector is unaffected by make-up

    gas.

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    Flame Ionization Detector (FID) The effluent from the column is mixed with

    hydrogen and air, and ignited.

    Organic compounds burning in the flame

    produce ions and electrons which can

    conduct electricity through the flame.

    A large electrical potential is applied at the

    burner tip, and a collector electrode is

    located above the flame.

    The current resulting from the pyrolysis ofany organic compounds is measured.

    FIDs are mass sensitive rather than

    concentration sensitive; this gives the

    advantage that changes in mobile phase

    flow rate do not affect the detector's

    response.

    The FID

    is a useful general detector for theanalysis of organic compounds; it has high

    sensitivity, a large linear response range,

    and low noise.

    It is also robust and easy to use, but

    unfortunately, it destroys the sample.

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    Applications

    Main purpose is to

    separate and analyze

    multiple component

    mixtures: Essential oils

    Hydrocarbons

    solvent

    Biomedical

    Biochemical

    Physics

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    Mass Spectrometry

    Creates ions from molecules

    It analyzes those ions, providing

    information about its molecular weight and

    chemical structure based on thefragmentation patterns

    http://www.chem.arizona.edu/massspec/intro_html/intro.html

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    Instrumentation

    Sample introduction/separation

    Ionization method

    - Electron Impact ionization Ion separation method

    - Low (unit) resolution 1 Dalton

    - High resolution 0.0001 Dalton Ion Detector

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    History

    1886 Eugene Goldstein

    observed rays which travelled through channels of a perforated

    cathode. These rays would travel towards an anode

    1899 William Wien

    discovered the rays could be deflected by either a strongelectrical field or a strong magnetic field

    constructed a device which could separate the positive rays by

    their mass to charge ratio (m/z)

    1918 and 1919 ArthurJeffrey Dempster and F.W Aston

    (respectively)created the modern day Mass spectrometer

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    History of Modern Day MS

    1929 WalkerBleakney

    developed Electron Impact mass spectrometry

    hard impact technique

    1987 Franz Hillenchamp and Michael Karas

    developed Matrix Assisted Laser

    Desorption/Ionization

    used in the identification of biomolecules

    2002 John Bennett Fenn

    developed ESI

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    Mass Spectrometry

    Mass spectrometry is an analytical tool used for measuring the molecular mass of a

    sample.

    For large samples such as macromolecules,molecular masses can be measured to

    within an accuracy of 0.01% of the total molecular mass of the sample

    within a 4 Daltons (Da) or atomic mass units (amu).

    This is sufficient to allow minor mass changes to be detectedthe 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 of5 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.

    Structural information can be generated using certain types of mass spectrometers,

    usually those with multiple analyzers which are known as tandem mass

    spectrometers. This is achieved by fragmenting the sample inside the instrument and

    analyzing the products generated.

    This procedure is useful for the structural elucidation of organic compounds and for

    peptide or oligonucleotide sequencing.

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    Applications

    Biotechnology: the analysis of proteins, peptides,

    oligonucleotides

    Pharmaceutical: drug discovery, combinatorial

    chemistry, pharmacokinetics, drug metabolism Clinical: neonatal screening, haemoglobin analysis,

    drug testing

    Environmental: PAHs, PCBs, water quality, food

    contamination Geological: oil composition

    Physics: identification of space particles

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    Great Scott, it can be used in

    Biochemistry too! Accurate molecular weight measurements:

    sample confirmation, to determine the purity of a sample, to verify amino

    acid substitutions, to detect post-translational modifications, to calculate the

    number of disulphide bridges

    Reaction monitoring:

    to monitor enzyme reactions, chemical modification, protein digestion Amino acid sequencing:

    sequence confirmation, de novo characterization of peptides, identification

    of proteins by database searching with a sequence "tag" from a proteolytic

    fragment

    Oligonucleotide sequencing:

    the characterization or quality control of oligonucleotides

    Protein structure:

    protein folding monitored byH/D exchange, protein-ligand complex

    formation under physiological conditions, macromolecular structure

    determination

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    Mass spectrometers can be

    divided into three fundamentalparts.

    Ionization source

    Analyzer

    Detector

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    Ionization Source

    The sample has to be introduced into the ionization source of the

    instrument.

    Once inside the ionization source, the sample molecules are

    ionized, because ions are easier to manipulate than neutral

    molecules. These ions are extracted into the analyzer region of the mass

    spectrometer where they are separated according to their mass (m)

    -to-charge (z) ratios (m/z) .

    The separated ions are detected and this signal sent to a data

    system where the m/z ratios are stored together with their relative

    abundance for presentation in the format of a m/z spectrum .

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    Analyzer

    The analyzer and detector of the mass spectrometer, and often the

    ionization source too, are maintained under high vacuum to give the ions a

    reasonable chance of travelling from one end of the instrument to the other

    without any hindrance from air molecules.

    The entire operation of the mass spectrometer, and often the sample

    introduction process also, is under complete data system control on modernmass spectrometers.

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    Sample Introduction

    The method of sample introduction to the ionization source often

    depends on the ionization method being used, as well as the type

    and complexity of the sample.

    The sample can be inserted directly into the ionization source, or

    can undergo some type of chromatography in route to the ionizationsource.

    This method of sample introduction usually involves the mass

    spectrometer being coupled directly to a high pressure liquid

    chromatography (HPLC), gas chromatography (GC) or capillary

    electrophoresis (CE) separation column.

    The sample is separated into a series of components which then

    enter the mass spectrometer sequentially for individual analysis.

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    Ionization Methods Many ionization methods are available and each has its own advantages and

    disadvantages

    The ionization method to be used should depend on the type of sample under

    investigation and the mass spectrometer available.

    Ionization methods include the following:

    1. Atmospheric Pressure Chemical Ionization (APCI)

    2. Chemical Ionization (CI)3. Electron Impact (EI)

    4. Electrospray Ionization (ESI)

    5, Fast Atom Bombardment (FAB)

    6. Field Desorption / Field Ionization (FD/FI)

    7. Matrix Assisted LaserDesorption Ionization (MALDI)

    8. Thermospray Ionization (TSP)

    The ionization methods used for the majority of biochemical analyses are

    Electrospray Ionization (ESI) and , and Matrix Assisted LaserDesorption Ionization

    With most ionization methods there is the possibility of creating both positively and

    negatively charged sample ions, depending on the proton affinity of the sample,

    therefore beginning an analysis, the user would need to determine if the ions are

    cations or anions.

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    Ionization MethodsIonization

    method

    Typical

    Analytes

    Sample

    Introduction

    Mass

    Range

    Method

    Highlights

    Electron Impact (EI) Relativelysmallvolatile

    GC orliquid/solidprobe

    to1,000Daltons

    Hard methodversatileprovidesstructure info

    Chemical Ionization(CI)

    Relativelysmall

    volatile

    GC orliquid/solid

    probe

    to1,000D

    altons

    Soft methodmolecular ion

    peak [M+H]+

    Electrospray (ESI) PeptidesProteinsnonvolatile

    LiquidChromatographyor syringe

    to200,000Daltons

    Soft methodions oftenmultiplycharged

    Fast AtomBombardment (FAB)

    CarbohydratesOrganometallicsPeptides

    nonvolatile

    Sample mixedin viscousmatrix

    to6,000Daltons

    Soft methodbut harderthan ESI or

    MALDI

    Matrix Assisted LaserDesorption

    (MALDI)

    PeptidesProteins

    Nucleotides

    Sample mixedin solid

    matrix

    to500,000Daltons

    Soft methodvery high

    mass

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    Electrospray Ionization (ESI)

    A high voltage of 3 or 4 kV is applied to the tip of the capillary,

    which is situated within the ionization source of the mass

    spectrometer, and as a consequence of this strong electric

    field, the sample emerging from the tip is dispersed into an

    aerosol of highly charged droplets, a process that is aided bya co-axially introduced nebulizing gas flowing around the

    outside of the capillary.

    This gas, usually nitrogen, helps to direct the spray emerging

    from the capillary tip towards the mass spectrometer. The

    charged droplets diminish in size by solvent evaporation,assisted by a warm flow of nitrogen known as the drying gas

    which passes across the front of the ionization source.

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    Electrospray Ionization (ESI)

    Eventually charged sample ions, free from solvent, are released from the

    droplets, some of which pass through a sampling cone or orifice into an

    intermediate vacuum region, and from there through a small aperture into

    the analyzer of the mass spectrometer, which is held under high vacuum.

    The lens voltages are optimized individually for each sample.

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    Electron Impact (EI)

    The gas molecules exiting the GC are

    bombarded by a high-energy electron beam

    (70eV)

    An electron will strike the molecule, supplyingenough energy to remove an electron from that

    molecule

    Will produce a singly charges ion containing one

    unpaired electron

    The instability on this molecule causes it to

    fragment into smaller pieces

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    Analyzers

    Analysis and Separation of Sample Ions

    The main function of the mass analyzer is to separate

    the ions formed in the ionization source of the mass

    spectrometer according to their mass-to-charge (m/z)

    ratios.

    There are a number of mass analyzers, the more

    common known mass analyzers are quadrupoles , time-of-flight (TOF) analyzers, magnetic sectors , Fourier

    transform and quadrupole ion traps .

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    Analyzers

    These mass analyzers have different features

    Including the m/z range that can be covered,

    the mass accuracy, and the achievable resolution.

    The compatibility of different analyzers with different ionization

    methods varies.

    For example, all of the analyzers listed above can be used in

    conjunction with electrospray ionization, whereas MALDI is not

    usually coupled to a quadrupole analyzer.

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    Analyzers

    Tandem (MS-MS) mass spectrometers are instruments that have

    more than one analyzer and so can be used for structural and

    sequencing studies.

    Two, three and four analyzers have all been incorporated intocommercially available tandem instruments, and the analyzers do

    not necessarily have to be of the same type, in which case the

    instrument is a hybrid one.

    More popular tandem mass spectrometers include those of thequadrupole-quadrupole, magnetic sector-quadrupole , and more

    recently, the quadrupole-time-of-flight geometries.

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    Detectors

    The detector monitors the ion current, amplifies it and

    the signal is then transmitted to the data system where it

    is recorded in the form of mass spectra .

    The m/z values of the ions are plotted against their

    intensities to show the number of components in the

    sample the molecular mass of each component, and the

    relative abundance of the various components in the

    sample.

    The type of detector is supplied to suit the type ofanalyzer; the more common ones are the

    photomultiplier, the electron multiplier and the micro-

    channel plate detectors.

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    Key Terminology

    Molecular Ion (M.+)is the charged molecule which remains intact, usually is the

    molecular weight of molecule

    Reference Spectramass spectral patterns which are reproducible

    Base peak

    100% abundance

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    Interpreting Spectra

    Ex) Methanol

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    Samples (M) with molecular masses up to 1200

    Da give rise to singly charged molecular-related

    ions, usually protonated molecular ions of the

    formula (M+H)+

    in positive ionization mode, anddeprotonated molecular ions of the formula (M-

    H)- in negative ionization mode.

    Protonated molecular ions are expected when

    the sample is analyzed under positive ionizationconditions.

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    References

    Robinson, Skelly Frame, Frame II, Undergraduate Instrumental Analysis, Chromatography pg,

    721-851

    Robinson, Skelly Frame, Frame II, 6th Edition, Undergraduate Instrumental Analysis. Mass

    Spectrometry, pg. 613-721

    Matthews, PhD, Fred, Organic Spectroscopy, Spring 2007 Lecture notes, Mass Spectroscopy and

    GLC

    B

    rennan, PhD

    , Carrie, Instrumental Analysis, Spring 2007 Lecture Notes, Chromatography Brennan, PhD, Carrie, Quantitative Analysis, Fall 2006 Lecture Notes, Chromatography

    Silberberg, Chemistry 3rd Edition, pg 75

    Silverstrin, Webster, Kiemle, Spectrometric Identification of Organic Compounds, 7th Edition,

    Chapter 1 Mass Spectrometry

    McMurry, Organic Chemistry, 6th Edition, Chapter 12

    http://pubs.acs.org/hotartcl/tcaw/98/sep/creat.html, accessed June 16, 2007

    www.chem.arizona.edu/massspec/inter_html/inter.html accessed July 3,2007 www.astbury.leeds.ac accessed July 3, 2007

    www.chemistry.wustl.edu accessed July 3, 2007