| Separation on the basis of charge: ion-exchange chromatography
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| When a charged ion or molecule with one or more positive charges exchanges with another positively charged component bound to a negatively charged immobilized phase, the process is called cation exchange. The inverse process is called anion exchange. The cation exchanger, carboxymethyl-cellulose (-O-CH2-COO-), and anion exchanger, diethylaminoethyl (DEAE) cellulose (-O-C2H4-NH+[C2H5]2, are frequently used for the purification of proteins. Consider purifying a protein mixture containing albumin and immunoglobulin. At pH 7.5, albumin, with a pI of 4.8, is negatively charged; immunoglobulin with a pI ∼8 is positively charged. If the mixture is applied to a DEAE column, the albumin sticks to the positive-charged DEAE column whereas the immunoglobulin passes through the column. Figure 2.14 illustrates the principle of ion-exchange chromatography. As with gel permeation chromatography, proteins can be separated from one another, based on small differences in their pI.
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| Figure 2.12 Dialysis of proteins. Protein and low-molecular-mass compounds are separated by dialysis on the basis of size. (A) A protein solution with salts is placed in a dialysis tube in a beaker and dialyzed with stirring against an appropriate buffer. (B) The protein is retained in the tube whereas salts will pass through the membrane. |
| Figure 2.13 Fractionation of proteins by size: gel filtration chromatography of proteins. Proteins with different molecular sizes are separated by gel filtration based on their relative size. The smaller the protein, the more readily it exchanges into polymer beads, whereas larger proteins may be completely excluded. Larger molecules flow more rapidly through this column, leading to fractionation on the basis of molecular size. The chromatogram on the right shows a theoretical fractionation of three proteins, Pr1-Pr3 of decreasing molecular weight. |
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| Figure 2.14 Fractionation of proteins by charge: ion-exchange chromatography. Mixtures of proteins can be separated by ion-exchange chromatography according to their net charges. Beads that have positive-charge groups attached are called anion exchangers, whereas those having negative-charge groups are cation exchangers. This figure depicts an anion-exchange column. Negatively charged protein binds to positively charged beads, and positively charged protein flows through the column. |
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