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Ion exchange chromatography

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Ion exchange chromatography

  1. 1. ION EXCHANGE CHROMATOGRAPHY PRESENTED BY S.KAVIYA M.PHARM – FIRST YEAR
  2. 2. The Ion Exchange Chromatography (IEC) is first used as analytical technique for the softening of the hard water by the Gans by using the cation exchange resins. Later Adams and Holms proposed the method by using the modern exchange resins. This method involves the exchange of the ions by using the different types of exchange resins based on the nature of the ions to be separated. The procedure for the IEC is first developed by the Hamish Small and co-workers. PRINCIPLE : The main principle involved is charge–charge interactions. Here, the separation and identification of the compounds by the reversible adsorption of the charged molecules into the matrix which contains the opposite charge. It is mainly based on the interaction between the solute particles and the adsorbent particles on the matrix. The separation is influenced by the ionic strength and pH. The stationary phase surface contains the R–X functional group that interacts with the ions of opposite charge present in the sample.
  3. 3. Based on the principle of separation, the IEC is divided into the following : 1)‰ Cation exchange chromatography: * Retains positively charged cation by using the negatively charged functional group. (Or) The Cations to be separated are present in solution and exchanges for similar ions present in cation exchange resin , a solid matrix. SOLID ( R - X-) H+ + M+ (SOLUTION) ↔ SOLID( R - X- ) M+ + H+ (SOLUTION) * The cations retained by the solid matrix of ion exchange resin can be eluted by using buffers of different strength and hence separation of cations can be effected . 2) ‰ Anion exchange chromatography: * Retains negatively charged anion by using the positively charged functional group. (OR) The anions to be separated are present in solution and exchanges for similar ions present in anion exchange resin ,a solid matrix . solid (R-X) OH- + A- (SOLUTION) ↔ SOLID − A- + OH- (SOLUTION) * The anions retained by the solid matrix of ion exchange resin can be eluted by using buffers of different strength and hence separation of anions can be effected .
  4. 4. THEORY ; The theory of the IEC involves two main steps they are as follows: 1)‰ Binding the sample to a charged resin.
  5. 5. 2)Elution or displacement of the sample from the charged resin. This separation is mainly depends on the resin which is composed of the polystyrene and divinyl benzene.
  6. 6. PRACTICAL REQUIREMENTS : 1) RESINS AND PHYSICAL CHARACTERISTICS : The resins used in the IEC should posses the following requirements: *‰ It should be chemically stable. * It should be clinically stable . * ‰ It should posses the sufficient degree of cross-linking. * ‰ It should posses the sufficient number ion exchange group CLASSIFICATION OF RESINS : 1) ‰ Source (a) Natural exchangers: Cation: zeolytes and clays; anion: dolomite. (b) Synthetic exchangers: Inorganic and organic resins. 2)‰ Chemical nature (a) Strong cation exchange resins. Eg ; Polystyrene sulphonic acid resin. (b) Weak cation exchange resins. Eg : Polymethyl acrylic acid resins. (c) Strong anion exchange resins Eg: Polystyrene quaternary ammonium resins. (d) Weak anion exchange resins. Eg : Polystyrene tertiary amine resins.
  7. 7. Ion exchange resins consist of an insoluble matrix to which charged groups are covalently Bonded .
  8. 8. (1) Cation exchange resins: These are molecular weight, cross-linked polymer with sulphonic, carboxylic, phenolic, etc., groups. Cationic exchange resin is a polymeric anion to which the active cation is attached. Amberilite IR-120, dowex, zerolite, etc., are the commonly available commercial resins. a) STRONG CATION EXCHANGE RESIN The standard grade resin is treated with the strong acidic solution. The excess of the acid is drained and washed with distilled water. This activated resin is then used for the exchange of hydrogen ions with the cation present in the sample solution. Example: Polystyrene sulphonic acid resin
  9. 9. (b) Weak cationic exchange resin: These resins are in the form of hydrogen ions which are converted into sodium by the treatment with sodium hydroxide. Example: poly methyl acrylic acid resin. (2) Anion exchange resins: The anion exchange resin is a polymer with amine or quaternary ammonium groups in the resin and the outer part is with anions such as Cl−, SO4−. The commercially available exchangers are amberilite IRA-400, zerolit FF-IP, etc.
  10. 10. (a) Strong anion exchange resin: These resins are in the chloride form. The hydroxide ions are converted by the reaction with the strong sodium hydroxide. Eg: Polystyrene quaternary ammonium resins. (b) Weak anion exchange resins: These resins are in free base forms. The salt form is prepared by treating with the hydrochloric acid. Then rinse with the distilled water . Example: Polystyrene tertiary amine resins. EFFICIENCY OF THE RESIN : Measured by ion exchange capacity . Ion exchange capacity is the total ion exchange capacity in terms of the exchangeable functional groups expressed as milli equivalents per gram of the ion exchange resin . m.eq/ g = 1000 / eq.wt PARTICLE SIZE OF THE RESIN : 50 – 100 mesh or 100 – 200 mesh is used . STRUCTURAL TYPE OF THE RESIN ; Porous (5 -10mu size) ion exchange capacity is 0.5 -2 meq/g ,pellicular(30 -40mu size) ion exchange efficiency is 0.01-0.1 meq/g ,etc ., AMOUNT OF CROSS LINKING AGENT PRESENT : which decides swelling of the resin .
  11. 11. 2)Mobile phase: Commonly employed mobile phases in the IEC are acids, alkali and buffer solutions. Mostly the buffer solutions are used as mobile phases. For cation exchange resins, the following buffers are used for the separation. * Table for the functional groups attached to the cation exchange resins
  12. 12. * For anion exchange resins, the following buffers are used for the separation: Table for the functional groups attached to the anion exchange resins 3)Detectors: The commonly used detectors for the detection of the eluents are conductivity detector which measures the conductance difference by conductivity meter. The other detectors used for the detection are UV/vis detector and fluorescence detector.
  13. 13. 4) COLUMN MATERIAL AND DIMENSIONS : Columns used in the laboratories are made up of of glass . But those used in industries are made up of either high quality stainless steel or polymers which are resistant to strong acids and alkalies . The column dimensions are also important and a length : diameter ratio of 20: 1 to 100 : 1 for higher efficiency can be used . 5) PACKING OF THE COLUMN : Wet packing method is used. The resin is mixed with the mobile phase and packed in the column uniformly . The sample to be separated is dissolved in the mobile phase and introduced all at once in the column . 6) DEVELOPMENT OF THE CHROMATOGRAM AND ELUTION ; After introduction of the sample , development of the chromatogram is done by using different mobile phases . As mentioned earlier , organic solvents are less useful and only acids , alkalies and buffers of different p H are used .There are two elution techniques .
  14. 14. They are isocratic elution technique and gradient elution technique A) In isocratic elution technique , the same solvent composition is used . i.e. Same strength of acid or alkali or buffer . B) In gradient elution technique , initially less acidity or basicity character is used followed by increasing the acidity or basicity of the mobile phase . Gradient elution is usually used for complex mixtures . 7) ANALYSIS OF THE ELUTE ; * Different fractions collected with respect to volume or time is analysed for their contents . * several methods of analysis can be used which depends upon the nature and quantity of the sample . * They are spectrophotometric method , polarographic method , conductometric method ,amperometric method , flame photometric method ,radiochemical methods ( using Geiger Muller counter , ionisation , ionisation chamber method ) etc ., 8) REGENERATION OF THE ION EXCHANGE RESIN ; * The ion exchange resin after separation may not be useful for next separation as exchangeable fuctional groups are lost . * But due to the cost of the ion exchange resins , they cannot be disposed off . Hence like reactivation, regeneration of the resin is most important . * Regeneration makes the used ion exchange resin to be as efficient as a virgin resin .
  15. 15. * Regeneration refers to the replacement of the exchangeable cations or anions present in the original resin . * Hence regeneration of the cation exchange resin is done by the charging the column with strong acid like Hcl. * Regenaration of anion exchange resin is done by using strong alkali like sodium hydroxide or potassium hydroxide . FACTORS AFFECTING ION EXCHANGE SEPARATIONS ; A) NATURE OF ION EXCHANGE RESIN : * Crosslinking and swelling is important factor which depends on the proportion of cross linking agent (divinyl benzene ) and polystyrene . * When more crosslinking agent is present , they are more rigid , but swells less . When swelling is less, separation of ions of different sizes is difficult as they cannot pass through the pores present and it becomes selective to ions of diferent sizes . * When less cross linking agent is present , they are less rigid , but swell more . When swelling is more , separation will not be efficient as exchange of functional groups does not take place due to wide pore . * Hence an optimum quantity of cross linking agent should be added to the polymeric ion exchange resins for the separation to be effective .
  16. 16. B) NATURE OF IONS : 1) VALENCY OF IONS : At low concencentrations and at ordinary temperatures , extent of exchange increases with increase in valency . Na + < Ca2+ < Al3+ < Th4+ 2) SIZE OF IONS ; For similar charged ions , exchange increases with decreases in the size of hydrated ion . Li + < H + < NH 4+ < K+ < Rb + < CS + 3) POLARIZABILITY ; Exchange is preferred for greater polarizable ion . 4) CONCENTRATION OF SOLUTION : In dilute solution , polyvalent anions are generally adsorbed preferentially . 5) CONCENTRATION AND CHARGE OF IONS ; If resin has higher +ve charge and solution has lower +ve charge , exchange is favoured at higher concentration . If resin has lower +ve charge and solution has high + ve charge , then exchange is favoured at low concentration .
  17. 17. INSTRUMENTAL COMPONENTS (OR) INSTRUMENTATION ‰ a) Eluent: Sample solution is called as eluent solution. b)‰ Pump: Single or dual piston pumps are employed to employ the mobile phase. c) ‰ Separator column: This column is mainly used for the separation of the mixture into ions which contains the matrix bonded with ion exchange resins. d) ‰ Detector: The detector is mainly used for the detection of the separated charged ions concentration. e) ‰ Integrator: It is mainly used for the interpretation of the results to get the read out. f) Elution technique: Initially prepare the column based on the nature of the substance to be separated (cationic or anionic exchange resins). Then adjust the pH and initialise the counter ions by increasing the salt concentration that is by gradient method or isocratic method. Apply the sample which is in low ionic strength. Then wash the column with the suitable mobile phase followed by the elution with the mobile phase.
  18. 18. ION EXCHANGE RESINS EFFICIENCY PARAMETERS a) Distribution coefficient: The distribution coefficient is determined by the shaking the resin with the known concentration of the sample solution. In other terms, it is defined as the rate at which the two constituents are separated in the column.
  19. 19. Kd = amount of the solute ÷ weight of the resin in grams / amount of the solute in solution ÷ volume of solution in ml Separation factor: The separation factor is defined as the chromatographic separation in which the mixture is separated into the individual components. This is given by the following formula: a = Kd1/Kd2 where Kd1 is the distribution coefficient of the first constituent; Kd2 is the distribution coefficient of the second constituent. Volume of the eluent: The Vmax is the volume required to reach the maximum concentration of an eluted ion in the effluent. This is given by the following equation: Vmax = Kd V0 + V0 where V0 is the volume of the sample solution in resin beads. Volume distribution coefficient: Dv = Kd/b
  20. 20. ADVANTAGES OF IEC * ‰ High-speed separation * ‰ High sensitivity * ‰ High selectivity * ‰ Effective simultaneous detection APPLICATIONS * ‰ Used in the separation of similar ions from one another. Example: Li+, Na+ and K+ ions separation. * Used in the removal of interfering radicals. Example: Ca2+ and Ba2+ are removed from the water. *‰ Used in the softening of the hard water. Example: Softening the water by removing the chloride and calcium, Mg2+ ions.
  21. 21. Resin + Hard water  Retaining the calcium and magnesium into the resin + soft water *‰ Used in the separation of amino acids. Example: Alanine and phenyl alanine are separated from the mixture. * ‰ Used in the separation of sugars. Example: Biologically active sugars are separated. *‰ Used in the separation of the morphine from codeine. Example: Natural products seperation. *‰ Used in the separation of alkaloids from the crude extracts. Example: Reserpine ,morphine ,digitoxin etc. *‰ Used in the separation of lanthanides. Example: Rb, Sr, Cs and Zr are separated.
  22. 22. REFERENCE: * VOGEL'S TEXTBOOK OF QUANTITATIVE CHEMICAL ANALYSIS, G. H. JEFFERY,, J. BASSETT , J. MENDHAM, R C. DENNEY – pg.no. 186-215 * INSTRUMENTAL METHOD OF CHEMICAL ANALYSIS, Gurdeep R.Chatwal , Sham K. Anand – pg.no. 2.662 -2.672 * PHARMACEUTICAL ANALYSIS , P.D.Chaithanya Sudha -pg.no. 360- 366

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