Más contenido relacionado


Ion Exchange Chromatography and Column Chromatography

  1. CHROMATOGRAPHY Subject: Modern Pharmaceutical Analytical Technique Subject Code: MAT101T Topic: Ion Exchange Chromatography and Column Chromatography Prepared by: Garima Singh M. Pharma Ist SEM Department Of Pharmaceutics 1Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  2. Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi 2
  3. DEFINITION • Ion chromatography or ion-exchange chromatography) is a chromatography process that separates ions and polar molecules based on their affinity to the ion exchange 3Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  4. PRINCIPLE • Exchange of ions is the basic principle in this type of Chromatography. In this process two types of exchangers i.e., cationic and anionic exchangers can be used. • Cationic exchangers possess negatively charged group and these will attract positively charged cations. These exchangers are also called “Acidic ion exchange” materials, because their negative charges result from the ionization of acidic group. • Anionic exchangers have positively charged groups that will attract negatively charged anions. These are also called “Basic ion exchange” materials. • Anionic exchange Chromatography should be carried out with cationic buffers. • Cationic exchange Chromatography should be carried out with anionic buffers. • The pK of the buffer should be as near as possible to the pH at which the system is buffered. This result in high buffer capacity, which can with stand the local changes of pH in the column easily. 4Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  5. • BUFFERS PH range Ammonium acetate 4-6 Ammonium formate 3-5 Pyridiniumformate 3-6 Pyridinium acetate 4-6 Ammonium carbonate 8-10 5Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  6. ION EXCHANGE MATERIAL • •DEFINITION: A complex resin structure which is water insoluble & loaded with ions which can easily replace the desired ion from the solution. • •There are 3 types of ion exchange material A) Synthetic inorganic ion exchangers- • •These materials posses a relatively open three dimensional frame work structure with channel and interconnecting cavities • •E.g. alumina-silicates, TiO2, ThO2, zirconium oxide, phosphate etc. the hydrous oxide of tri and tetravalent metals are useful as cation exchanger. Besides phosphate, molybdate, tungsten etc. of some metal acts as cation exchanger materials. B) Natural organic ion exchangers- • Substance like coal, paper, cotton etc can be converted into cation exchange by reaction of sulphonation or phosphorylation. • They act as cation exchanger since they carry sulphonic acid or carboxylic group attached to them. • These materials are less uniform in structure and get readily affected by other chemicals. 6Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  7. C) Synthetic organic ion exchangers- • The synthetic organic ion exchanger resin are made of cross linked polymer network to which are attached various functional group. • The nature of functional group determines whether it is a • Cation exchanger • Anion exchanger • In cation exchanger materials the acid group are sulphonic acid, carboxylic acid or phenolic, while in anion exchanger resin the group are basic as amine, quaternary ammonium. • On the basis of strength of group, they are further divided into four categories:  Strongly acidic cation exchange resin: Sulphonic group  Weakly acidic cation exchange resin: Carboxylic group  Strongly basic anion exchange resin: Quaternary ammonium group  Weakly basic anion exchange resin: Polystyrene, formaldehyde 7Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  8. Requirements of ion exchange resin • It should have a sufficient degree of cross liking for use in chromatography. • It should be insoluble in common solvents. • It should be sufficiently hydrophilic to permit diffusion of ions through its structure at a constant and finite rate. • It should have desired particle size and shape. • It must be chemically stable. • It must contain sufficient number of ion exchanger group. • It must have the ability of regeneration and reuse. • The swollen resin must be denser than water. 8Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  9. METHODS OF ION CHROMATOGRAPHY • 1.Batch Method-It involves single step equilibrium. The resin and the solution are mixed in a vessel until equilibrium is attained. • The solution is filtered off, and further fresh portion of the resin is added to the solution. This process is known as batch operation, but is very little important in quantitative analysis. • Used for producing deionised and demineralised water and for softening of water. • 2.Column Method-It is better and efficient method. The resin is placed on the top of a glass or wool plug or sintered glass disc in a vertical tube or burette. • •By passing sufficient concentration of solution through the resin column, a strongly acidic cation exchange resin can be easily converted completely into the desired ionic form. 9Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  10. TECHNIQUES OF ION CHROMATOGRAPHY • Ion exchange chromatography is performed on column with adsorbents, exchangers and solution which is to be separated is poured on the column then elute from the column is investigate • [A] Column for Ion exchange chromatography • They are designed in such a way that no disturbance occurs in flow of liquid and all operation is carried out in downward direction. • As the liquid moves down, ions comes in contact with untreated resin and get completely exchanged with resin. • Column should not be too wide nor should be too narrow because it will produce uneven flow of liquid. • Ion exchange chromatography is based on exchange of ion between solid ion exchange and ions present in the solution. • • RA + B+ → RB + A+ • [B-] / [A-] = KA,B[B+] / [A] • • Where B-and A-are concentration of ion in a solid phase and B+ and A+ are concentration of ion in liquid phase and K is ion exchange equilibrium constant. 10Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  11. • [B] Packing of column • The resin is treated with the solvent and equilibriums are achieved before packing the column. • The slurry of the resin is poured in the column. • The solvent which is to be used as an eluent should be used for making the slurry. • The slurry is added in several portions allowing the resin to settle down. • When packing is complete the eluent is permitted to pass through the column for certain time so to ensure uniform rate of flow over whole cross section area of the column, and level of liquid is so adjusted that it remain below the top of the resin bed. • Now the column is ready for experiment and sample solution which is to be separated is introduced or poured on top of the resin in column by using micropipette. • [C]Operation of the column • There are three methods of operation-the basis of all of these operations is that the substance in solution has some affinity for the substrate over which it flows. The affinity is due to ion exchange properties of the column. 11Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  12. • Displacement Analysis: Most strongly absorbed material displaces less strongly absorbed material and each component in turn displaces less readily adsorbed component. • Frontal Analysis: If solution of mixture of ion having different affinity coefficient is passed through a column of ion exchange resin in sufficient quantity to exceed the exchange capacity of the resin then the least absorbed ion breaks first through the column. • Elution Analysis: Mixture to be separated is adsorbed in a narrow band at the top of the resin column and is desorbed by passing down the column of a solution of another ion which has a lower affinity coefficient then that of the component of mixture & which is already absorbed on a major portion of the column. • [D]Analysis of eluent • After flowing down the column the solution is passed through automatic fraction collector for continuous determination of PH, refractive index etc. • These reading are then plotted against the elute volume. Other methods are: • Spectrophotometric method: used for direct analysis. • Polarographic method: in this diffusion current under constant potential is recorded as function of time & the amount of solute present in solution can be calculated from area under the curve. • Conductometric method: Electrical conductance of elute from the column is recorded. 12Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  13. • [E] Regeneration of ion exchange resin: • Regeneration refers to the replacement of the exchangeable cations or anion present in the original resin. • Hence the regeneration of the cation exchange resin is done by the changing the column with strong acid like hydrochloric acid. • Regeneration of anion exchange resin is done by using strong alkali like sodium hydroxide.  Factor affecting ion exchange separation: • A)Nature & properties of ion exchange resins:cross linking & swelling is important factor which depends on the proportion of cross linking agent & polystyrene.When more cross linking agent is present they are more rigid but swell less.When swelling is less separation of ions of different size is difficult as they cannot pass through the pores present. B)Nature of exchanging ions: • Valency of ions: at low concentrations & temperature exchange increase with increase in valency. • Size of ions: exchange increase with decrease in particle size. • Concentration of solution: in dilute solution polyvalent anions are generally adsorbed preferentially. • Concentration & charge of ions: if the resin has higher positive charge & solution has lower positive charge exchange is occur at higher concentration 13Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  14. ION EXCHANGE CHROMATOGRAPHY Advantages • Useful to resolve very complex samples. • Useful for separation of mixtures of biological origin. • Useful for detection of many inorganic salts. • Also used for detection of organic ions with poor UV absorptive. Disadvantages • Column efficiency is less. • It is difficult to achieve control over selectivity & resolution. • Stability & reproducibility of the column is difficult. 14Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  15. APPLICATION OF ION CHROMATOGRAPHY • Softening of water: removal of monovalent & divalent ions likes sodium, potassium, calcium, magnesium. • Demineralisation of water: removal of different ions to get demineralised water. • Purification of solution • Separation of inorganic ions. • Biochemical separation: isolation of some drugs or metabolites from blood & urine. • Concentration of ionic solution: a cation or anion from a bulk of solution can be absorbed onto ion exchange resin. After absorption it can be eluted by using small volume of eluent. • As ion exchange column in HPLC. 15Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  16. 16Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  17. DEFINITION • Column chromatography is a separation technique in which the components of mixture is separated by using a glass column packed with S.P. & the liquid M.P. is continuously flowing through the column. • This is one of the most useful methods for the separation & purification of both solid and liquid. • This is a solid-liquid technique in which the a solid & M.P. is a liquid. PRINCIPLE • It is based on the adsorption principle. • Mixture of components dissolved in M.P. is introduced in to the column. • Components moves depending on their relative affinities. • The adsorbent is packed in a glass column & a solvent the M.P. is moves slowly through the packed column. • A compound attracted more strongly by the M.P. will move rapidly through the column dissolved in the M.P. • A compound more strongly attracted to the S.P. will move slowly through the column. 17Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  18. Requirement of column chromatography 1)Selection of S.P. • Success of chromatography depends upon proper selection of S.P. • It can depends on following  Removal of impurity  Number of components to be separated  Length of column used  Affinity difference between components  Quantity of adsorbent used 2)Selection of M.P. • They act as a solvent, developer& eluent. • The function of M.P. are oAs developing agent oTo introduce the mixture into the column oTo developing agent oTo remove pure components out of the column 3)Column characteristics • The main function of the entire column is to support the S.P. • The material of the column is of good quality so it should not affected by the solvents. • Better separation will be obtained with a long narrow column because number of plates will be more. 18Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  19. • The length of column is depends on  Number of components to be separated  Type of adsorbent used  Quantity of sample  Affinity of components towards adsorbent used 4)Preparation of the column • It consists of a glass tube with bottom portion of the column packed with glass wool/cotton above which the adsorbent is packed. • After the packing a paper disc kept on the top so that the adsorbent layer is not distributed during the introduction of sample or M.P. 19Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  20. 5)Packaging technique The column should be free from impurity before using. • It should be washed properly & dry it. • Before filling the column with S.P. cotton/glass wool is kept. • It should be uniformly filled. • There are 2 types of packaging • Dry packaging:adsorbent is packed in the column in the dry form.Fill the solvent till equilibrium is achieved. • Wet packaging: the material is slurred with solvent & generally added to the column.The S.P. is settled uniformly in the column & there is no entrapment of air bubbles.There will be not any crack in the column of the adsorbent. • 6)Introduction of the sample • The sample usually a mixture of components is dissolved in minimum quantity of the M.P. • The entire sample is introduced into the column at once & gets adsorbed on the top portion of the column. • From the zone individual sample can be separated by a process of elution. 20Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  21. • 7)Development technique • By elution technique the individual components are separated out from the column. • There are 2 techniques of elution • Isocratic elution technique: in this technique same solvent composition or solvent of same polarity is used throughout the process of separation. • Example: chloroform • Gradient elution technique: solvents of gradually increase polarity or increase elution strength are used during the process of separation. • Example: benzene – chloroform – ethyl acetate – chloroform • 8)Detection of components • If the compounds separated in a column chromatography procedure are coloured, the progress of the separation can simply be monitored visually. • If the compounds to be isolated from columnchromatography are colourless. • In this case small fractions of the eluents are collected subsequently in labelled tubes & the composition of each fraction is analyted by TLC. 21Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  22. • Factors affecting on column efficiency • Dimension of the column: column efficiency has been improved by increasing length/width of the column. • Particle size of the column packaging: separation is improved by decreasing the particle size of the adsorbent. • Activity of the adsorbent • Temperature of the column: the speed of the elution increases at higher temperature. • Packaging of the column • Quality of solvents: solvents having low viscosities are giving better results. 22Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  23. COLOUMN CHROMATOGRAPHY ADVANTAGES • Any type of mixture can be separated •Any quantity of mixture can be separated • Wider choice of M.P. • Automation is possible • In preparative type the sample can be separated & reused. DISADVANTAGES • Time consuming • More amount of M.P. are required • Automation makes the techniques more complicated & expensive 23Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  24. APPLICATION OF COLUMN CHROMATOGRAPHY • Isolation of active: from the plant extracts, from formulations or other crude extracts, active constituents or required constituents can be isolated. • Determination of primary & secondary glycosides in digitalis leaf. • Purification process • Estimation of drugs in formulation. • Isolation & purification of vitamins & hormones. • Examination of vegetable oils. • In the separation of components after organic synthesis to obtain desired molecule. • Removal of impurities or purification process: impurities present in a compound can be removed by using appropriate S.P. & M.P. • Isolation of metabolites from biological fluids: e.g. 17ketosteroids from urine, cortisol, other drugs etc from biological fluids like blood, urine & serum. • Separation of mixture components: column chromatography can be used for the separation of several classes of drugs & constituents like alkaloids, glycosides, amino acids, plant extracts, drugs & formulation. • Determination of phytomenadione in injection & tablets. • Separation of diastereomers. • Separation of inorganic ions like copper, cobalt, nickel etc. 24Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi
  25. REFERENCES: Ion Exchange Chromatography • Principles of Instrumental Analysis, Skoog, 6th edition , • Instrumental Method of Analysis by willard, dean,merit,settle,7th edition, • Instrumental Method of Chemical Analysis, Gurudeep R. Chatwal , Sham ,k.Anand, Column Chromatography • a.H.BECKETT & J.B. STENLAKE, Practical Pharmaceutical Chemistry, 4thedition, part II, • Ashutoshkar, Pharmaceutical Analysis-II, • Vogel's, Text book of quantitative analysis, Smt B.N.B Swaminarayan Pharmacy Collage Salvav Vapi 25