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ion exchange chromatography

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  • 1. P SE E B RE NT ND Y: K AM I .SH IL 1702-13-886-005 GUIDE B Dr.H D Y: RIDAY B RA E 1
  • 2. W hat Is Ion E xchange Chromatography? P rinciple Ion E xchangers P rocedure F actors Affecting Ion E xchange Chromatography Advantages & Disadvantages Applications References 2
  • 3. Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on their charge. Ion exchange chromatography is coined by SM L ST VE AL , E NS, B AUM ANN. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. 3
  • 4. Ion-E xchange chromatography retains analyte molecules on the column based on coulombic (ionic) interactions. T stationary phase surface displays ionic functional he groups (R-X) that interact with analyte ions of opposite charge. T type of chromatography is further subdivided his into: Cation exchange chromatography Anion exchange chromatography T ionic compound consisting of the cationic species he M and the anionic species B can be retained by the + 4 stationary phase.
  • 5. Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group Anion exchange chromatography retains anions using positively charged functional group 5
  • 6. Resins Gels Inorganic exchanger 6
  • 7. Resins are amorphous particles of organic materials Ion exchange resins are used for the separation of small molecules. Strongly acidic cation exchanger -sulphonic acid groups attached to styrene and di vinyl benzene copolymer. W eakly acidic cation exchanger-carboxylic acid groups attached to acrylic and divinyl benzene co-polymer Strongly basic anion exchanger-quaternary ammonium groups attached to styrene and divinyl benzene co-polymer W eakly basic anion exchanger-poly alkyl amine groups attached to styrene and divinyl benzene co-polymer 7
  • 8. Cellulose and dextran ion exchangers , which are polymers of the sugar glucose , posses larger pore sizes and lower charge densities. B ecause they are much softer than polystyrene resins , dextran and its relatives are called gels . 8
  • 9. Column : glass, stainless steel or polymers L ength: diameter ratio 20:100 to 100:1 Packing the column : W packing method et Application of the sample : After packing, sample is added to the top of the column, use syringe or pipette M obile phase : Acids, alkalis, buffers… E lution : Components of mixture separate & move down the column at different rates depending upon the affinity of the ion for ion exchanger. T elutes are collected at different stages he Analysis of the elute : spectrophotometric, flame photometry 9
  • 10. A sample is introduced, either manually or with an autosampler, into a sample loop of known volume. T mobile phase (buffered aqueous solution) carries the he 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. T target analytes (anions or cations) are retained on the he 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. 10
  • 11. F example, in cation exchange chromatography, or the positively charged analyte could be displaced by the addition of positively charged sodium ions. T analytes of interest must then be detected he by some means, typically by conductivity or UV/ Visible light absorbance. A chromatography data system (CDS) usually needed to control an IC. is 11
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  • 13. • • • • Nature & properties of ion exchange resins Cross linking & swelling is important If more cross linking , they are more rigid, but swelling is less swells less → separation of ions of different sizes is difficult Nature of exchanging ions valency of ions Size of ions P olarizability Concentration of solution 13
  • 14. 14
  • 15. Ion exchange chromatography is used to convert one salt to other. E we can prepare tetra propyl ammonium hydroxide from a g; tetra propyl salt of some other anion. Ion exchange is used to prepare de-ionized water Separation of similar ions A mixture of sodium, hydrogen and potassium can be separated using cation exchanger resin. A mixture of Chloride, bromide, and iodide can be separated using basic anion exchange resin. M T OD: M EH ixture of chloride, bromide & iodide is passed through basic anion exchanger using 0.5M sodium nitrate as eluant. Chloride will first elute. Raise the conc of Sodium Nitrate, B romide will elute, raise the conc of Sodium Nitrate further, 15
  • 16. Removal of interfering radicals: P hosphate ion is the interfering with the calcium & barium ions. Phosphate is removed using sulphonic acid cation exchanger. + Calcium & barium ions exchanged with H ions while phosphate ion pass through the column. Softening of hard water: H ardness of water due to cal, mg and other divalent ions. T water is passed through cation exchanger charged his with the sodium ions. Ca & M ions retained in the column while g sodium is exchanged. Complete demineralization of water: Removal of both cations & anions. Step A) H ard water is first passed through an acidic cation + exchanger- Ca, M & Na are exchanged by H ions. g Step B T ) his water is then passed thro a basic anion exchanger – Cl, NO2, SO4- are exchanged by OH ions of the exchanger. Separation of L anthanides- L Y, Ce, Rb etc a, 16
  • 17. P rinciples of instrumental analysis , skoog , latest edition, pno. 641-647 en.wikipedia.org/ wiki/ Ion_chrom atography www.separations.us.tosohbioscience.com .../ rinciplesofChrom / P at ography https:/www.gelifesciences.com .../ / / litdoc11000421_201310032330 16.pdf www.authorstream .com P / resentation/ shreekanta-1507297-ionexchange 17
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