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ion exchange and gel permetion chromatography


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ion exchange and gel permetion chromatography

  2. 2. Chromatography G.SHRAVANI 2
  3. 3. 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. G.SHRAVANI 3
  4. 4. 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. G.SHRAVANI 4
  5. 5. PROPERTIES OF ION EXCHANGE RESIN Color Amount or cross linking Porosity Capacity Surface area Density Mechanical strength Size G.SHRAVANI 5
  6. 6. Flow rates should be controlled due to the difference in rate of exchange Flow rate -0.5 to 5ml/minutes Capacity of ion exchange depends on the no. of sites available Cation resins(strong acid) are stable at 150degress & anions at 70 G.SHRAVANI 6
  7. 7. 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. 1. 2. G.SHRAVANI 7
  8. 8. Ion Exchangers G.SHRAVANI 8
  9. 9. Ion exchangers – Functional groups Anion exchanger Aminoethyl (AE-) Diethylaminoethyl (DEAE-) Quaternary aminoethyl (QAE-) Cation exchanger Carboxymethyl (CM-) Phospho Sulphopropyl G.SHRAVANI (SP-) 9
  10. 10. 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 G.SHRAVANI 10
  11. 11. Anion exchange chromatography Anion exchange chromatography retains anions using positively charged functional group: + + _ + + R-X A +M B R-X B + M + A G.SHRAVANI 11
  12. 12. Procedure 1. A sample is introduced, either manually or with an autosampler, into a sample loop of known volume. 2. The mobile phase (buffered aqueous solution) carries the sample from the loop onto a column that contains some form of stationary phase material. 3. Stationary phase material is a resin or gel matrix consisting of agarose or cellulose beads with covalently bonded charged functional groups. G.SHRAVANI 12
  13. 13. Procedure 4. 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. G.SHRAVANI 13
  14. 14. Procedure 5. The analytes of interest must then be detected by some means, typically by conductivity or UV/Visible light absorbance. 6. A chromatography data system (CDS) is usually needed to control an IC. G.SHRAVANI 14
  15. 15. Procedure G.SHRAVANI 15
  16. 16. 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. G.SHRAVANI 16
  17. 17. 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. G.SHRAVANI 17
  18. 18. 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* G.SHRAVANI 18
  19. 19. Choosing your ion-exchanger: know your proteins 1.   Stability of proteins stable below pI value, use cation-exchanger stable above pI value, use anion-exchanger 2.   Molecular size of proteins <10,000 mw, use matrix of small pore size 10,000-100,000 mw, use Sepharose equivalent grade G.SHRAVANI 19
  20. 20.  Important to consider the stability of proteins in choice of ion exchangers. Isoelectric focusing can be used to identify suitable ion-exchanger type G.SHRAVANI 20
  21. 21. Applications Determination of sodium and potasium in the mixture: column eluted with 0.1M HCL flow rate -0.6ml/sqmin Radiochemistry G.SHRAVANI 21
  22. 22. Gel Permeation GEL FILTRATION Chromatography G.SHRAVANI 22
  23. 23. DEFINATION Gel chromatography is a technique in which fractionation is based upon the molecular size & shape of the species in the sample. Gel chromatography is also called as gel permeation or exclusion or molecular sieve chromatography. G.SHRAVANI 23
  24. 24. TECHNIQUES IN GEL CHROMATOGRAPHY Gel chromatography is performed on a column by the elution method. The degree of retardation, depends upon the extent to which the solute molecules or ions can penetrate that part of the solution phase which is held within the pores or the highly porous gel like packing material. A series of resins with different pore sizes can be obtained by changing the amount of Epichlorohydrin.The resulting gel is called as SEPHADEX. G.SHRAVANI 24
  25. 25. In gel chromatography the granulated or beded gel material is called as packing material. The solutes which are distributed through the entire gel phase is called as stationary phase and the liquid flowing through the bed is called as mobile phase. The cross linked dextran (sephadex) & xerogels of the polyacrylamide (Bio-gel) originally used as stationary phases in gel permeation chromatography are semi rigid gels. G.SHRAVANI 25
  26. 26. These are unable to withstand the highpressure used in HPLC. Hence modern stationary phases consist of micro particles of stryene-divinyl benzene copolymers (ultrastyragel) silica or porous glass. G.SHRAVANI 26
  27. 27. GEL PERMEATION CHROMATOGRAPHY A chromatographic method in which particles are separated based on their size, or in more technical terms, their hydrodynamic volume.  Organic solvent as the mobile phase.  The stationary phase consists of beads of porous polymeric material. G.SHRAVANI 27
  28. 28. OBJECTIVE Analysis of synthetic and biologic polymers Purification of polymers Polymer characterization  Study properties like:  Molecular weight  Polydispersity Index  Viscosity  Conformation  Folding G.SHRAVANI 28
  29. 29.  Aggregation  Branching  Copolymer composition  Molecular size G.SHRAVANI 29
  31. 31. PRINCIPLE Different sizes will elute (filter) through at different rates.   Column 1. Consists of a hollow tube tightly packed with extremely small porous polymer beads designed to have pores of different sizes. 2. Pores may be depressions on the surface or channels through the bead. 3. Smaller particles enter into the pores, larger particles don't. G.SHRAVANI 31
  32. 32. The larger the particles, the less overall volume to transverse over the length of the column G.SHRAVANI 32
  33. 33. FEATURES Solvent 1. Should be kept dry 2. Should be degassed in some applications 3. The samples should be made from the same solvent 4. For GPC/light scattering the solvent should be filtered before it ever hits the pump 5. Common solvents for tetrahydrofuran (THF) & toluene. G.SHRAVANI 33
  34. 34. G.SHRAVANI 34
  35. 35. Pump &Filters  a. b. c.  • Pump: Designed to deliver very constant, accurate flow rates. At microprocessor-controlled rate.   Designed not to produce any pressure pulses. Filters: Prevent major junk from getting into the columns G.SHRAVANI 35
  36. 36. Injector Loop Injector • • • • Loop Allows you to load the sample loop which is a piece of tubing precut for a precise volume. the output of the pump flushes through the loop   Carries the sample to the columns. sends a signal to the detector to indicate that the sample has been loaded. G.SHRAVANI 36
  37. 37. Columns Columns  Contain the beads through which the sample is allowed to pass.  Reference column is also present  Very expensive    Never change the pumping rate by a large amount  They are very delicate G.SHRAVANI 37
  38. 38. Detectors Detectors o Viscosity o Light Scattering o Ultraviolet detectors o Differential Refractive Index detector placed at the end to reduce pressure on it G.SHRAVANI 38
  39. 39. Analysis Spectroscopic Techniques 1. Refractive Index 2. Light Scattering 3.   Ultraviolet Spectroscopy   Viscometry Techniques 1) Viscosity 2)   Flow rate G.SHRAVANI 39
  40. 40. Conventional GPC Analysis Molecules separated according to their hydrodynamic volume. • Molecular weights (MW) and molecular weight distribution can be determined from the Measured retention volume (RV) • A calibration curve (log MW against RV), using known standards RI signal = KRI . dn /dc . C • G.SHRAVANI 40
  41. 41. KRI = apparatus-specific sensitivity constant dn /dc = the refractive index increment C = concentration.  Limitation  Their signals depend solely on concentration, not on molecular weight or polymer size.  Not very reliable G.SHRAVANI 41
  42. 42. Molecular Mass Sensitive Detectors Detectors sensitive to molecular weight used to overcome limitations of Conventional GPC E.g., light scattering and viscosity detectors Advantages over Conventional GPC I. True molecular weight distributions can be obtained II. Structural information III.Size distribution molecular weight can be directly determined without a calibration curve G.SHRAVANI 42
  43. 43. Characterization Light Scattering LS signal = KLS . (dn/dc)2 . MW .  KLS = sensitivity constant      dn/dc = refractive index increment   MW = molecular weight   C = concentration dn/dc depends on the Polymer Solvent combination and if it is low, then proper analysis cannot be done. G.SHRAVANI 43
  44. 44. Applications          Polymer characterization Molecular weight Polydispersity Index   Viscosity   Folding   Aggregation   Branching   Copolymer composition   Molecular size G.SHRAVANI 44
  45. 45. Proteomics  Purification    Conformation  Hydrodynamic volume  G.SHRAVANI 45
  46. 46. Advantages Can be used to find shape also   Rapid, routine analysis   Identify high mass components even in low concentration   Can analyze polydisperse samples   Branching studies can be done   Absolute molecular weights can be obtained G.SHRAVANI 46
  47. 47. Drawbacks There is a size window   Bad response for very small molecular weights   Standards are needed.   Sensitive for flow rate variation. Internal standard should be used whenever possible.   High Investment cost G.SHRAVANI 47
  48. 48. G.SHRAVANI 48
  49. 49. REFERENCES Instrumental methods of chemical analysis .B.K Sharma p.g C123-170 www.gel permeation chromatography www.ion exchange chromatography. G.SHRAVANI 49
  50. 50. G.SHRAVANI 50