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    PRINCIPLEBasic Principles of Ion Chromatography

    Basic process of ICThe basic process of chromatography using ion exchange can berepresented in 5 steps: eluent loading, sample injection, separation ofsample, elution of analyte A, and elution of analyte B, shown andexplained below. Elution is the process where the compound of interest

    is moved through the column. This happens because the eluent, thesolution used as the solvent in chromatography, is constantly pumpedthrough the column. The chemical reactions below are for an anionexchange process. (Eluent ion = , Ion A= , Ion B = )

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    Step 1: The eluent loaded onto the column displaces anyanions bonded

    to the resin and saturates the resin surface with the eluentanion.This process of the eluent ion (E-) displacing an anion (X-)bonded to the

    resin can be expressed by the following chemical reaction:Resin+-X- + E- Resin+-E- + XStep2: A sample containing anion A and anion B are injectedonto thecolumn. This sample could contain many different ions, butforsimplicity this example uses just two different ions ready tobe injectedonto the column.

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    STEP - 2

    (key: Eluent ion = , Ion A= , Ion B = )

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    Step 3: The continued addition of the eluent causes aflow through the

    column. As sample elutes, anion A and anion B adhere to thecolumnsurface differently. The sample zones move through thecolumn as

    eluent gradually displaces the analytes.In reality not every eluent ion is removed from the surface ofthecolumn. It depends on the amount of analyte loaded. A better

    representation of the column can be seen by just looking at aslice of thecolumn where the separation is occurring, as shown in thefigure below.

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    Step 4: As the eluent continues to be added, the anionA moves through

    the column in a band and ultimately is eluted first.This process can be represented by the chemical reactionshowing thedisplacement of the bound anion (A-) by the eluent anion

    (E-).Resin+-A- + E- Resin+-E- +

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    Step 5: The eluent displaces anion B,and anion B is eluted off thecolumn.

    Resin+-B- + E- Resin+-E- + B

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    The theory of ion exchangeSeparation in ion exchange chromatography

    depends upon the reversible adsorptionof charged solute molecules to immobilized ionexchange groups of oppositecharge. Most ion exchange experiments are

    performed in five main stages. Thesesteps are illustrated schematically below.

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    Fig. 1. The principle of ion exchange chromatography (salt gradient

    elution).The first stage is equilibration in which the ion exchanger is brought to astartingstate, in terms of pH and ionic strength, which allows the binding of thedesiredsolute molecules. The exchanger groups are associated at this time withexchangeablecounter-ions (usually simple anions or cations, such as chloride or sodium).The second stage is sample application and adsorption, in which solutemoleculescarrying the appropriate charge displace counter-ions and bind reversibly to

    thegel. Unbound substances can be washed out from the exchanger bed usingstartingbuffer.

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    In the third stage, substances are removed from the column

    by changing to elutionconditions unfavourable for ionic bonding of the solutemolecules. This normallyinvolves increasing the ionic strength of the eluting buffer orchanging its pH. InFigure 1 desorption is achieved by the introduction of anincreasing salt concentrationgradient and solute molecules are released from the columnin the order of

    their strengths of binding, the most weakly bound substancesbeing eluted first.

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    The fourth and fifth stages are the removal from the column of substancesnot elutedunder the previous experimental conditions and re-equilibration at thestartingconditions for the next purification.Separation is obtained since different substances have different degrees ofinteraction

    with the ion exchanger due to differences in their charges, charge densities

    and distribution of charge on their surfaces. These interactions can becontrolledby varying conditions such as ionic strength and pH. The differences inchargeproperties of biological compounds are often considerable, and since ion

    exchangechromatography is capable of separating species with very minordifferences inproperties, e.g. two proteins differing by only one charged amino acid, it isa very

    powerful separation technique.

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    In ion exchange chromatography one can choose whether to bindthe substancesof interest and allow the contaminants to pass through the column,

    or to bind thecontaminants and allow the substance of interest to pass through.Generally, thefirst method is more useful since it allows a greater degree of

    fractionation andconcentrates the substances of interest.The conditions under which substances are bound (or free) arediscussed in detailin the sections dealing with choice of experimental conditions,

    Chapter 9. In additionto the ion exchange effect, other types of binding may occur. Theseeffects aresmall and are mainly due to van der Waals forces and non-polar


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    Ion exchange separations may becarried out in a column, by a batchprocedure orby expanded bed adsorption. All threemethodologies are performed in thestages

    of equilibration, sample adsorptionetc. described previously.

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    The matrixAn ion exchanger consists of an insoluble matrix to which charged groups havebeen covalently bound. The charged groups are associated with mobile

    counterions.These counter-ions can be reversibly exchanged with other ions of the samecharge without altering the matrix.It is possible to have both positively and negatively charged exchangers (Fig. 2).Positively charged exchangers have negatively charged counter-ions (anions)availablefor exchange and are called anion exchangers. Negatively charged exchangershave positively charged counter-ions (cations) and are termed cation exchangers.The matrix may be based on inorganic compounds, synthetic resins orpolysaccharides.The characteristics of the matrix determine its chromatographic properties

    such as efficiency, capacity and recovery as well as its chemical stability,mechanical strength and flow properties. The nature of the matrix will also affect itsbehaviourtowards biological substances and the maintenance of biological activity.

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    The first ion exchangers were synthetic resins designed forapplications such asdemineralisation, water treatment, and recovery of ions from

    wastes. Such ionexchangers consist of hydrophobic polymer matrices highlysubstituted with ionicgroups, and have very high capacities for small ions. Due to their low

    permeabilitythese matrices have low capacities for proteins and othermacromolecules. In addition,the extremely high charge density gives very strong binding and the

    hydrophobicmatrix tends to denature labile biological materials. Thus despitetheirexcellent flow properties and capacities for small ions, these types ofion exchanger

    are unsuitable for use with biological samples.

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    The first ion exchangers designed for use with biological substances

    were the celluloseion exchangers developed by Peterson and Sober (2). Because of thehydrophilicnature of cellulose, these exchangers had little tendency to denatureproteins.

    Unfortunately, many cellulose ion exchangers had low capacities(otherwise thecellulose became soluble in water) and had poor flow properties dueto their irregular


    on exc angers ase on ex ran ep a ex o owe y ose ase on

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    on exc angers ase on ex ran ep a ex , o owe y ose ase ongaroseSepharose CL-6B) and cross-linked cellulose (DEAE Sephacel) were theirst ionxchange matrices to combine a spherical form with high porosity, leading

    omproved flow properties and high capacities for macromolecules.ubsequently,evelopments in gel technology have enabled this macroporosity to bextended tohe highly cross-linked agarose based media such as Sepharose Higherformance,epharose Fast Flow and Sepharose Big Beads, and the synthetic polymeratrices,

    onoBeads, and SOURCE. These modern media enable fast, high capacity,ighesolution ion exchange chromatography to be carried out at both analyticalndreparative scales. Non-porous polymer matrices, e.g. MiniBeads, are

    vailableor extremel hi h resolution micro re arative or anal tical se arations

    Ch d

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    Charged groupsThe presence of charged groups is a fundamental property of an ion exchanger.The type of group determines the type and strength of the ion exchanger; theirtotal number and availability determines the capacity. There is a variety of groups

    which have been chosen for use in ion exchangers (3); some of these are shown in

    Table 1.Table 1. Functional groups used on ion exchangers.Anion exchangers Functional groupDiethylaminoethyl (DEAE) -O-CH2-CH2-N+H(CH2CH3)2Quaternary aminoethyl (QAE) -O-CH2-CH2-N+(C2H5)2-CH2-CHOH-CH3

    Quaternary ammonium (Q) -O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-N+(CH3)3Cation exchangers Functional groupCarboxymethyl (CM) -O-CH2-COOSulphopropyl(SP) -O-CH2-CHOH-CH2-O-CH2-CH2-CH2SO3

    -Methyl sulphonate (S) -O-CH2-CHOH-CH2-O-CH2-CHOH-CH2SO3-

    S l h i d t i d t f t i

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    Sulphonic and quaternary amino groups are used to form strong ionexchangers;the other groups form weak ion exchangers. The terms strong and weak refertothe extent of variation of ionization with pH and not the strength of binding.

    Strong ion exchangers are completely ionized over a wide pH range (seetitrationcurves on page 49) whereas with weak ion exchangers, the degree ofdissociationand thus exchange capacity varies much more markedly with pH.

    Some properties of strong ion exchangers are: Sample loading capacity does not decrease at high or low pH values due tolossof charge from the ion exchanger. A very simple mechanism of interaction exists between the ion exchanger

    andthe solute. Ion exchange experiments are more controllable since the chargecharacteristicsof the media do not change with changes in pH. This makes strong exchangersideal for working with data derived from electrophoretic titration curves.

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    Components and principles of chromatography solute (i.e., molecule of interest) mobile phase (eg., solvent) stationary phase (eg., a solid matrix) the solute partitions between the two phases the interaction with the stationary phase determines how

    fast the solute migrates in themobile phase solutes that interact differently with the stationary phase canbe separated

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    stationary phase consists of a matrix (eg., cross-linkedpolymers) with fixed charges (i.e.,acids or bases)

    anion exchanger has fixed positive charges (i.e., bases) cation exchanger has fixed negative charges (i.e., acids) fixed charges can be 'strong' or 'weak' use strong exchanger for weaklyionizable groups and visa

    versa separation is based on charge differences betweendifferent solute molecules


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    COLUMNS OF ION EXCHANGE CHROMATOGRAPHYChromatography is the process use to separatemolecules based on SOME physical property ofthe molecule: Mass (i.e. size) ChargeAffinity for ligands or substrates Hydrophobic interactionsTwo phases in EVERY chromatographyexperiment:

    Stationary phase: a surface or resin that isinert Mobile phase: Comprised of the solvent andthe sample (eluant). Introduction of themobile phase can either be done by a gravity

    feed (siphoning) system or by a pumpingdevice (usually peristaltic). In most of thefigures for this chapter (see below), gravity feed systems are used.

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    Separation occurs due to VARYING degrees of

    interaction of sample with the stationary phase.Interaction of sample with stationary phase canbe modulated by changing the solventconditions (i.e. pH, ionic strength, competitiveligands, etc.).

    For column chromatography: stationary phaseis referred to as resin or gel or matrix.Three primary types of RESINS: Gel Filtration = Size Exclusion (SEC) =

    Molecular Sieve Ion Exchange (IEC)Affinity (ligands, substrates, or tags)

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    All column chromatographic methods

    attempt topurify a single protein from many otherproteinsbased on differential interactions. Rarely

    canthis be accomplished using one type ofcolumn!

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    Table 4. Volatile Buffer SystemsPh Substance Counterion2.0 Formic acid H+2.3-3.5 Pyridine/formic acid HCOO-

    3.0-5.0 Trimethylamine/formic acid HCOO-3.0-6.0 Pyridine/acetic acid CH3COO-4.0-6.0 Trimethylamine/acetic acid CH3COO-6.8-8.8 Trimethylamine/HCl Cl-7.0-8.5 Ammonia/formic acid HCOO-8.5-10.0 Ammonia/acetic acid CH3COO-

    7.0-12.0 Trimethylamine/CO2 CO3-27.0-12.0 Triethylamine/CO2 CO3-27.9 Ammonium bicarbonate HCO3-8.0-9.5 Ammonium carbonate/ammonia CO3-28.5-10.5 Ethanolamine/HCl Cl-8.9 Ammonium carbonate CO3

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    Important Factors in Optimizing an Ion Exchange orChromatofocusing Separation


    During ion exchange chromatography and chromatofocusing,control of the pH depends on buffer substances, salts, samplecomponents, organic solvents, other additives, and temperature.Each of these variables must be considered individually andin relation to each other for optimum separation of biomolecules.

    Titration curves provide an indication of the pH at which chargedifferences among sample components are the greatest. It isuseful to have an idea of the pKa values of the sample components,because changes in pH have the largest effect on analyteretention when the pH is near the pKa of the analyte. Changes in

    pH during an ion exchange experiment can make a greatdifference in the chromatogram. Many times, a sharp peakactually will consist of two or three components which have beencompressed into one peak due to a sudden change in pH.

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    BuffersIn all biomolecular work, the correct choice of buffer can becrucial to success. In ion exchange chromatography,maintenanceof pH is of primary concern, since ionic interactions vary

    with pH. The buffer components themselves are ionic andmaybe taking part in the ion exchange process. The buffer pKa


    should be within 0.5 pH units of the desired working pH. Ifconditions are used which cause the pH to change during anexperiment, adjust the pH of the buffer to the appropriatesideof its pKa to minimize the change.

    Since buffers can be prepared in different ways, detailed careintheir preparation is essential for reproduciblechromatography.

    A buffer consists of the salt of a weak acid/base and the

    corresponding acid/base, mixed to attain the desired pH.

    After adding sodium chloride or another nonbuffering salt to the

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    After adding sodium chloride or another nonbuffering salt to thebuffer, it might be necessary to adjust the pH of the buffer. If so,use an acid or base containing the counterion being used forelution (i.e., HCl/Cl- or LiOH/Li+). Likewise, use buffering ions that

    have the same charge as the ion exchange functional group: apositively charged buffering species with an anion exchangerand a negatively charged buffering species with a cation exchanger.This prevents fluctuations in pH and conductivity.

    To promote binding of the biomolecule, the buffer ionconcentrationnormally should be low to prevent aggregation andpreserve activity, but the minimum should be 10mM. In Tables1-4, concentrations between 20 and 50mM are recommended,depending on the pH and the buffer. The concentration of theinitial buffer for chromatofocusing is especially important, sincemicroenvironmental pH differences can be as much as 1 pH unit.

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    In ion exchange, the mobile phase is chosen to provide adequatesolubility for the various salts and buffers, to control sampleretention through solvent strength, and to provide separation

    selectivity. The buffers in Tables 1-3 describe optimized systemsfor most of the pH intervals listed. A blank run should always beperformed to determine the UV adsorption of the buffer. Highquality buffers and salts will minimize UV baseline rise due to

    batch-to-batch variations.Buffers for chromatofocusing are most often used at a dilution of1 in 10. For narrow pH intervals, dilutions up to 1 in 20 canprovide better resolution. Resolution can sometimes be improvedby adding small amounts of salt (10mM). If a collectedfraction is analyzed by reversed phase chromatography, theampholyte buffer may interact with pairing ions. If the pairing ionis very hydrophobic, the ampholyte will be retained on thecolumn and will have an absorbance maximum below 280nm.

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    ApplicationsIon exchange has proven to be one of the major methods of fractionation of labilebiological substances. From the introduction of the technique in the 1960s to thedevelopment of modern high performance media, ion exchange chromatography

    has played a major role in the separation and purification of biomolecules andcontributed significantly to our understanding of biological processes. Theexamplesgiven in the following section have been drawn from the published literatureas well as from work in our own laboratories.

    For detailed information on specific subjects the reader is referred to the originalwork.The design of a biochemical separationIon exchange chromatography, in common with other separation techniques inthe life sciences, is rarely sufficient as the sole purification stage in the separationor analysis of complex biological samples. Ion exchange is frequently combined

    with other techniques which separate according to other parameters such as size(gel filtration), hydrophobicity (hydrophobic interaction chromatography orRPC) or biological activity (affinity chromatography).

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    EnzymesIn the purification of biologically active proteins such as enzymes

    the recovery ofbiological activity is usually as important as the recovery of proteinmass or degreeof homogeneity. Ion exchange chromatography has played a role in

    the purificationof thousands of enzymes, and using modern matrices withoptimized separationconditions gives extremely high recoveries. This is exemplified bythe separationof enzymes from chicken breast muscle on Mono Q (Fig. 74). Therecovery ofcreatine kinase in this separation was 89%.

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    IsoenzymesNormally the isoforms of an enzyme have approximately the same molecularweight. This makes their separation impossible by gel filtration. However, thesmall differences in charge properties resulting from altered amino acid compositionenable the separation of isoenzymes using ion exchange chromatography.

    N-Acetyl b-D-glucosaminidases have beenwidely investigated in the diagnosis of haematologicalmalignancies. In the case ofcommon acute lymphoblastic leukaemia,an isoenzyme, referred to as Intermediate1 Form has been reported (29). Using high

    resolution ion exchange chromatography(Fig. 75) this previously single peak hasbeen resolved into a number of componentisoenzymes which had previously only beendetectable using isoelectric focusing.Fig. 75. Separation of Leukaemic cell N-Acetylb-D-glucosaminidase isoenzymes by anionexchange chromatography on Mono Q. NA-Gluactivities associated with distinct peaks (Ia-IXa)are indicated in relation to the NaCl gradients(29). (Reproduced by kind permission of the

    authors and publisher.)

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    Various other substances such as




    can also be subjected to the

    biochemical seperation.

    Purification of a recombinant

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    Pseudomonas aeruginosa exotoxin A, PE553DThis application shows a purification process for a genetically modified recombinantPseudomonas aeruginosa exotoxin A (MW 55 000) expressed in the periplasmofE. coli. The process was developed for large scale production of modified

    toxin, conjugation to a polysaccharide and use as a vaccine. The purificationstrategy used chromatography media differentiated to tackle the problems of capture,intermediate purification and polishing (for further information please referto Chapter 11.)The result was a highly purified exotoxin A from crude cell homogenate usingonly four chromatography steps, and taking less than half the time of a more conventional

    approach.Exotoxin A was captured directly from unclarified E. coli homogenate by expandedbed adsorption using STREAMLINE DEAE adsorbent in a STREAMLINE200 column (Fig. 88). The following intermediate purification step was hydrophobicinteraction chromatography (HIC) on Phenyl Sepharose 6 Fast Flow (highsub) packed in a BPG 200 column (Fig. 89). This step removed a substantial part

    of the UV absorbing material (including nucleic acids) that could interfere withthe following steps. The second intermediate purification step on SOURCE 30Q,packed in FineLINE 100 column, removed the majority of the remaining contaminants(Fig. 90). The polishing step was HIC on SOURCE 15PHE (Fig 91). Theprocess resulted in a pure protein, according to PAGE and RPC analysis (Fig 92and 93), and the overall recovery was 51 % (Table 24).

    C l STREAMLINE (i d )

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    Column: STREAMLINE 200 (i.d. 200 mm)Medium: STREAMLINE DEAE, 4.7 lSample: 4.7 kg of cells were subjected to osmotic shock and suspendedin a final volume of 180 litres 50 mM Tris buffer, pH 7.4 beforeapplication onto the expanded bed.Buffer A: 50 mM Tris buffer, pH 7.4

    Buffer B: 50 mM Tris, 0.5 M sodium chloride, pH 7.4Flow rate: 400 cm/h during sample application and wash100 cm/during elutionSystem: BioProcess Modular

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