BY G.SHRAVANI ION EXCHANGE AND GEL PERMEATI ON CHROMATOGRAPHY 1G.SHRAVANI

Chromatography 2G.SHRAVANI

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. 3G.SHRAVANI

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. 4G.SHRAVANI

PROPERTIES OF ION EXCHANGE RESIN Color Amount or cross linking Porosity Capacity Surface area Density Mechanical strength Size G.SHRAVANI5

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.SHRAVANI6

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: 1. cation exchange chromatography 2. anion exchange chromatography. 7G.SHRAVANI

Ion Exchangers 8G.SHRAVANI

Ion exchangers – Functional groups Anion exchanger Aminoethyl (AE-) Diethylaminoethyl (DEAE-) Quaternary aminoethyl (QAE-) Cation exchanger Carboxymethyl (CM-) Phospho Sulphopropyl (SP-) 9G.SHRAVANI

Cation exchange chromatography Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional groupcations 10 R-X C +M B R-X M + C + B _ _ G.SHRAVANI

Anion exchange chromatography Anion exchange chromatography retains anions using positively charged functional group: 11 R-X A +M B R-X B + M + A + + _ _ G.SHRAVANI

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.agarosecellulosecovalently 12G.SHRAVANI

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. 13G.SHRAVANI

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. 14G.SHRAVANI

Procedure 15

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. 16G.SHRAVANI

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. 17G.SHRAVANI

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. 18 cationic pH =pI anionic pH increase *pI isoelectric point G.SHRAVANI

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 mw, use Sepharose equivalent grade 19G.SHRAVANI

Important to consider the stability of proteins in choice of ion exchangers. Isoelectric focusing can be used to identify suitable ion-exchanger type 20G.SHRAVANI

Applications Determination of sodium and potasium in the mixture: column eluted with 0.1M HCL flow rate -0.6ml/sqmin Radiochemistry G.SHRAVANI21

Gel Permeation \GEL FILTRATION Chromatography 22G.SHRAVANI

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. 23G.SHRAVANI

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. 24G.SHRAVANI

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. 25G.SHRAVANI

These are unable to withstand the high- pressure used in HPLC. Hence modern stationary phases consist of micro particles of stryene-divinyl benzene copolymers (ultrastyragel) silica or porous glass. 26G.SHRAVANI

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. 27G.SHRAVANI

OBJECTIVE Analysis of synthetic and biologic polymers Purification of polymers Polymer characterization  Study properties like: Molecular weight Polydispersity Index Viscosity Conformation Folding 28G.SHRAVANI

Aggregation Branching Copolymer composition Molecular size 29G.SHRAVANI

PRINCIPLE 30G.SHRAVANI

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. 31G.SHRAVANI

 The larger the particles, the less overall volume to transverse over the length of the column 32G.SHRAVANI

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. 33G.SHRAVANI

34G.SHRAVANI

Pump &Filters  Pump: a. Designed to deliver very constant, accurate flow rates. b. At microprocessor-controlled rate. c. Designed not to produce any pressure pulses.  Filters: Prevent major junk from getting into the columns 35G.SHRAVANI

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. 36G.SHRAVANI

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 37G.SHRAVANI

Detectors Detectors o Viscosity o Light Scattering o Ultraviolet detectors o Differential Refractive Index detector placed at the end to reduce pressure on it 38G.SHRAVANI

Analysis  Spectroscopic Techniques 1. Refractive Index 2. Light Scattering 3. Ultraviolet Spectroscopy  Viscometry Techniques 1) Viscosity 2) Flow rate 39G.SHRAVANI

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 40G.SHRAVANI

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 41G.SHRAVANI

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 42G.SHRAVANI

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. 43G.SHRAVANI

Applications  Polymer characterization  Molecular weight  Polydispersity Index  Viscosity  Folding  Aggregation  Branching  Copolymer composition  Molecular size 44G.SHRAVANI

Proteomics Purification Conformation Hydrodynamic volume 45G.SHRAVANI

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 46G.SHRAVANI

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 47G.SHRAVANI

48

REFERENCES Instrumental methods of chemical analysis.B.K Sharma p.g C permeation chromatography exchange chromatography. G.SHRAVANI49

50G.SHRAVANI