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Premium member Presentation Transcript Chromatography: Ch rom ato gra phy Ion Exchange Chromatography 1 SHREEKANTA RAMDefinition: Definition Ion-exchange chromatography (or ion chromatography ) is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules. 2Ion-exchange chromatography: Ion-exchange chromatography The solution to be injected is usually called a sample , and the individually separated components are called analytes It can be used for almost any kind of charged molecule including large proteins , small nucleotides and amino acids. It is often used in protein purification , water analysis. 3Principle: Principle Ion exchange chromatography retains analyte molecules based on ionic interactions. The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. This type of chromatography is further subdivided into: cation exchange chromatography anion exchange chromatography . 4Concept….: Concept…. Dr Gihan Gawish 5Ion Exchangers: Ion Exchangers 6Ion exchangers – Functional groups: Ion exchangers – Functional groups Anion exchanger Aminoethyl (AE-) Diethylaminoethyl (DEAE-) Quaternary aminoethyl (QAE-) Cation exchanger Carboxymethyl (CM-) Phospho Sulphopropyl (SP-) 7Cation exchange chromatography: Cation exchange chromatography Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group R-X C +M B R-X M + C + B - + + - _ + + - 8Example…: Example… 9Anion exchange chromatography: Anion exchange chromatography Anion exchange chromatography retains anions using positively charged functional group: R-X A +M B R-X B + M + A + _ + - + - + - 10Procedure: Procedure A sample is introduced, either manually or with an autosampler, into a sample loop of known volume. The mobile phase (buffered aqueous solution) carries the sample from the loop onto a column that contains some form of stationary phase material. Stationary phase material is a resin or gel matrix consisting of agarose or cellulose beads with covalently bonded charged functional groups. 11Procedure: Procedure The target analytes (anions or cations) are retained on the stationary phase but can be eluted by increasing the concentration of a similarly charged species that will displace the analyte ions from the stationary phase. For example, in cation exchange chromatography, the positively charged analyte could be displaced by the addition of positively charged sodium ions. 12Procedure: Procedure The analytes of interest must then be detected by some means, typically by conductivity or UV/Visible light absorbance. A chromatography data system (CDS) is usually needed to control an IC. 13Procedure: Procedure Dr Gihan Gawish 14Separating proteins : Separating proteins Proteins have numerous functional groups that can have both positive and negative charges. Ion exchange chromatography separates proteins according to their net charge, which is dependent on the composition of the mobile phase. 15Affect of pH in the separation of proteins: Affect of pH in the separation of proteins By adjusting the pH or the ionic concentration of the mobile phase, various protein molecules can be separated. For example, if a protein has a net positive charge at pH 7, then it will bind to a column of negatively-charged beads, whereas a negatively charged protein would not. 16Effect of pH in the separation of proteins: Effect of pH in the separation of proteins Proteins are charged molecules. At specific pH, it can exist in anionic (-), cationic (+) or zwitterion (no net charge) stage. cationic pH =pI anionic pH increase *pI isoelectric point 17Choosing your ion-exchanger: know your proteins: Choosing your ion-exchanger: know your proteins Stability of proteins stable below pI value, use cation -exchanger stable above pI value, use anion-exchanger Molecular size of proteins <10,000 mw, use matrix of small pore size 10,000-100,000 mw, use Sepharose equivalent grade 18Advantages of (IEC): Advantages of (IEC) versatility high resolving power high loading capacity multiple inlets column with large diameter straight forward basic principle . 19Applications….: Applications…. Separation of vitamins Separation of inorganic cations and anions Separation low molecular weight organic acids Analysis of serum Analysis of drugs 20Used for:: Used for: Antibody Purification Monoclonal antibodies produced in mouse ascites can be separated from other components of the ascites fluid. Immunoglobulins can be separated from albumin, transferrins and proteases. 21PowerPoint Presentation: Hemoglobin Separations Hemoglobins can be separated by high-performance cation -exchange chromatography using the Vydac strong cation -exchange column. Analysis of Glycosylated Hemoglobin 22 You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.