1.Introduction type of interaction to be successfully employed for the separation of chromatoraphy is based on this interaction. otides,and other c arged biome es(1).Th sons for the succes ving power,it ch.The h dbook isillustrated with examples of different types ecules wh have been sep 9
1. Introduction Adsorption chromatography depends upon interactions of different types between solute molecules and ligands immobilized on a chromatography matrix. The first type of interaction to be successfully employed for the separation of macromolecules was that between charged solute molecules and oppositely charged moieties covalently linked to a chromatography matrix. The technique of ion exchange chromatography is based on this interaction. Ion exchange is probably the most frequently used chromatographic technique for the separation and purification of proteins, polypeptides, nucleic acids, polynucleotides, and other charged biomolecules (1). The reasons for the success of ion exchange are its widespread applicability, its high resolving power, its high capacity, and the simplicity and controllability of the method. This handbook is designed as an introduction to the principles of ion exchange chromatography and as a practical guide to the use of the media available from Pharmacia Biotech. The handbook is illustrated with examples of different types of biological molecules which have been separated using ion exchange chromatography and different ways the technique can be used. For information on specific separations, the reader is recommended to consult the original literature. 9
2.Ion exchange chromatography The theory of ion exchange Separation in ion exchange chroma osite 5. Ad ▲■▲ ●●● 000 ■▲■ 00。 ●●● 000 0 ● 0/■ 6 000 ▲▲▲ ■■■●●● 000 000 OStarting buffer oounter-ions Substances to be separated dnn Fig.1.The principle of ion exchange chromatography (salt gradient elution). The first stage isequilibration in whi <changer is b ought to a starting able counter-ions (usually simple anions or cations,such as chloride or sodium). The second stage is sa e displace counter In the third s su ncreasing the i ngth of the cluting buffer or changing its pH.n Figure n is a y the introduc rdccertra their strengths of binding,the most weakly bound substances beingeutedfrst. 10
2. Ion exchange chromatography The theory of ion exchange Separation in ion exchange chromatography depends upon the reversible adsorption of charged solute molecules to immobilized ion exchange groups of opposite charge. Most ion exchange experiments are performed in five main stages. These steps are illustrated schematically below. Fig. 1. The principle of ion exchange chromatography (salt gradient elution). The first stage is equilibration in which the ion exchanger is brought to a starting state, in terms of pH and ionic strength, which allows the binding of the desired solute molecules. The exchanger groups are associated at this time with exchangeable counter-ions (usually simple anions or cations, such as chloride or sodium). The second stage is sample application and adsorption, in which solute molecules carrying the appropriate charge displace counter-ions and bind reversibly to the gel. Unbound substances can be washed out from the exchanger bed using starting buffer. In the third stage, substances are removed from the column by changing to elution conditions unfavourable for ionic bonding of the solute molecules. This normally involves increasing the ionic strength of the eluting buffer or changing its pH. In Figure 1 desorption is achieved by the introduction of an increasing salt concentration gradient and solute molecules are released from the column in the order of their strengths of binding, the most weakly bound substances being eluted first. ?W&? ?*@? ?W26X? ?W26X? ?W&? ?N@? ?.MB1? ?.MB1? W&@? @? ?J5? ?J5? ?W.Y@? @? W.Y? ?*U? ?7Y?@? @? ?W.Y ?N1? ?@@@@? @?e@? ?7Y? ?/KC5? @? @?@?@? ?@?@?@ ?@@@@??@ @?@?@? ?V40Y??@ @?@?@? @??@hg @? 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Ttcoerndthtagesarctheoalfontelgmcttbanertg c-cq arting conditions for the next purification. racti ns can be controlled nces in r to bind the subs ws a greater degree of fractionation and est. tscusedindetail ur.These effects are small and are mainly due to van der als forces and non-polar interactions Ion exchange separations may be carried out in a column,by a batch procedure or on,ample ion. three methodo ogies are performed in the stages dsorption etc.descri prev The matrix m It is possible to have both positively and negatively charged exchangers(Fig.2). e negatively charged counter-ions(anions)avai ers have positively charged counter-ions (cations)and are termed cation exchan ers. The matrix may be based on inorganic compounds,synthetic resins or polysaccha cy,capacity and recovery as w
The fourth and fifth stages are the removal from the column of substances not eluted under the previous experimental conditions and re-equilibration at the starting conditions for the next purification. Separation is obtained since different substances have different degrees of interaction with the ion exchanger due to differences in their charges, charge densities and distribution of charge on their surfaces. These interactions can be controlled by varying conditions such as ionic strength and pH. The differences in charge properties of biological compounds are often considerable, and since ion exchange chromatography is capable of separating species with very minor differences in properties, e.g. two proteins differing by only one charged amino acid, it is a very powerful separation technique. In ion exchange chromatography one can choose whether to bind the substances of interest and allow the contaminants to pass through the column, or to bind the contaminants and allow the substance of interest to pass through. Generally, the first method is more useful since it allows a greater degree of fractionation and concentrates the substances of interest. The conditions under which substances are bound (or free) are discussed in detail in the sections dealing with choice of experimental conditions, Chapter 9. In addition to the ion exchange effect, other types of binding may occur. These effects are small and are mainly due to van der Waals forces and non-polar interactions. Ion exchange separations may be carried out in a column, by a batch procedure or by expanded bed adsorption. All three methodologies are performed in the stages of equilibration, sample adsorption etc. described previously. The matrix An ion exchanger consists of an insoluble matrix to which charged groups have been covalently bound. The charged groups are associated with mobile counterions. These counter-ions can be reversibly exchanged with other ions of the same charge 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) available for exchange and are called anion exchangers. Negatively charged exchangers have positively charged counter-ions (cations) and are termed cation exchangers. The matrix may be based on inorganic compounds, synthetic resins or polysaccharides. The characteristics of the matrix determine its chromatographic properties such as efficiency, capacity and recovery as well as its chemical stability, mechanical 11
o ⑧ ⊙ + ⊕ ++ 6a Fig.2.Ion exchanger types. t its beba L s designed fo exchangers onsist of hydrophobic polymer matrices highly substituted with i ng and the hydro- rix tends all mat ed for use with biolog e the cellu gersdeveloped by Peterson and Sober (2).Because of the hydrophi c nature of lose,th eexchangers had little ndency to dena protein cellulose became soluble in water)and had poor flow p gular shape. deran(Sephadex)d by those bascd on garose ss-linke llulose (DEAE Sephacel)were the first ion have enabled this m croporosit and arc pharose Big Beads,and the eads,and SOURCE.These moc dern media e hable fast,h for extremely high resolution micropreparative or analytical separations. 12
Fig. 2. Ion exchanger types. strength and flow properties. The nature of the matrix will also affect its behaviour towards biological substances and the maintenance of biological activity. The first ion exchangers were synthetic resins designed for applications such as demineralisation, water treatment, and recovery of ions from wastes. Such ion exchangers consist of hydrophobic polymer matrices highly substituted with ionic groups, and have very high capacities for small ions. Due to their low permeability these matrices have low capacities for proteins and other macromolecules. In addition, the extremely high charge density gives very strong binding and the hydrophobic matrix tends to denature labile biological materials. Thus despite their excellent flow properties and capacities for small ions, these types of ion exchanger are unsuitable for use with biological samples. The first ion exchangers designed for use with biological substances were the cellulose ion exchangers developed by Peterson and Sober (2). Because of the hydrophilic nature of cellulose, these exchangers had little tendency to denature proteins. Unfortunately, many cellulose ion exchangers had low capacities (otherwise the cellulose became soluble in water) and had poor flow properties due to their irregular shape. Ion exchangers based on dextran (Sephadex), followed by those based on agarose (Sepharose CL-6B) and cross-linked cellulose (DEAE Sephacel) were the first ion exchange matrices to combine a spherical form with high porosity, leading to improved flow properties and high capacities for macromolecules. Subsequently, developments in gel technology have enabled this macroporosity to be extended to the highly cross-linked agarose based media such as Sepharose High Performance, Sepharose Fast Flow and Sepharose Big Beads, and the synthetic polymer matrices, MonoBeads, and SOURCE. 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Charged groups have ben chosn for use in on ehnrm fer Table 1.Functional groups used on Anion exchangers Functional group O-CH>CHz-N+H(CH2CH2 Quaternary aminoethyl (QAE) -O-CH,CH-N(CH )-CH,-CHOH-CH. Quatemary ammonlum() O-CHz-CHOH-CHzO-CHz-CHOH-CHz-N*(CH Cation exchangers Functional group 0-CH2C00 Sulphopropyl (SP) -O-CH-CHOH -CH-O-CH-CH-CH-so- O-CHz-CHOH-CHz-O-CH2-CHOH-CHSO orm ns strong a gth of re city varie of charge from the ion exchanger. A very simple mechanism of interaction exists between the ion exchanger and periments are more controllable since the charge characteristics media d Chapter 9) Resolution in ion exchange chromatography r mor The result of an ion exchang xperiment,as with any other chromatographic separation,is often expressed as the resolution between the peaks of interest
Charged groups The 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; their total 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 group Diethylaminoethyl (DEAE) -O-CH2-CH2-N+H(CH2CH3)2 Quaternary aminoethyl (QAE) -O-CH2-CH2-N+(C2H5)2-CH2-CHOH-CH3 Quaternary ammonium (Q) -O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-N+(CH3)3 Cation exchangers Functional group Carboxymethyl (CM) -O-CH2-COOSulphopropyl (SP) -O-CH2-CHOH-CH2-O-CH2-CH2-CH2SO3 - Methyl sulphonate (S) -O-CH2-CHOH-CH2-O-CH2-CHOH-CH2SO3 - Sulphonic and quaternary amino groups are used to form strong ion exchangers; the other groups form weak ion exchangers. The terms strong and weak refer to the extent of variation of ionization with pH and not the strength of binding. Strong ion exchangers are completely ionized over a wide pH range (see titration curves on page 49) whereas with weak ion exchangers, the degree of dissociation and 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 to loss of charge from the ion exchanger. • A very simple mechanism of interaction exists between the ion exchanger and the solute. • Ion exchange experiments are more controllable since the charge characteristics of the media do not change with changes in pH. This makes strong exchangers ideal for working with data derived from electrophoretic titration curves. (see Chapter 9) Resolution in ion exchange chromatography This section discusses the main theoretical parameters which affect the separation in ion exchange chromatography. For more in-depth information the reader is referred to standard works on the subject (4, 5). The result of an ion exchange experiment, as with any other chromatographic separation, is often expressed as the resolution between the peaks of interest. 13