BYBYG.SHRAVANIG.SHRAVANI170213884010170213884010

ION EXCHANGE AND GEL PERMEATION CHROMATOGRAPHY

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ChChromromatoatogragraphphyy

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DefinitionDefinition

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.

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Ion-exchange Ion-exchange chromatographychromatography

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.

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PROPERTIESPROPERTIES OFOF IONION EXCHANGEEXCHANGE RESIN RESIN

ColorAmount or cross linkingPorosityCapacitySurface areaDensityMechanical strengthSize

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Flow rates should be controlled due to the difference in rate of exchange

Flow rate -0.5 to 5ml/minutesCapacity of ion exchange

depends on the no. of sites available

Cation resins(strong acid) are stable at 150degress & anions at 70

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PrinciplePrinciple 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.

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Ion ExchangersIon Exchangers

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Ion exchangers Ion exchangers –– Functional Functional groupsgroups

Anion exchanger

Aminoethyl (AE-)Diethylaminoeth

yl (DEAE-)Quaternary

aminoethyl (QAE-)

Cation exchanger

Carboxymethyl (CM-)

PhosphoSulphopropyl (SP-)

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Cation exchange Cation exchange chromatographychromatographyCation exchange

chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group

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R-X C +M B R-X M + C + B

R-X C +M B R-X M + C + B

-- ++ ++ -- __ ++ ++ --

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Anion exchange Anion exchange chromatographychromatography

Anion exchange chromatography retains anions using positively charged functional group:

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R-X A +M B R-X B + M + AR-X A +M B R-X B + M + A++ __ ++ -- ++ -- ++ --

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ProcedureProcedure1. 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.

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ProcedureProcedure4. 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.

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ProcedureProcedure5. 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.

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ProcedureProcedure

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Separating proteinsSeparating 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.

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Affect of pH in the separation Affect of pH in the separation of proteinsof 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.

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Effect of pH in the separation of Effect of pH in the separation of proteinsproteins

Proteins are charged molecules. At specific pH, it can exist in anionic (-), cationic (+) or zwitterion (no net charge) stage.

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cationiccationic pH =pIpH =pI anionicanionic

pH increasepH increase*pI isoelectric point*pI isoelectric point

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Choosing your ion-exchanger: Choosing your ion-exchanger: knowknowyour proteinsyour 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

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

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ApplicationsApplications

Determination of sodium and potasium in the mixture:

column eluted with 0.1M HCL flow rate -0.6ml/sqmin

Radiochemistry

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Gel Permeation \GEL FILTRATION

Chromatography

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DEFINATIONDEFINATION

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.

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TECHNIQUES IN GEL TECHNIQUES IN GEL CHROMATOGRAPHYCHROMATOGRAPHY

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.

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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.

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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.

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GEL PERMEATION GEL PERMEATION CHROMATOGRAPHYCHROMATOGRAPHY

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.

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OBJECTIVEOBJECTIVE

Analysis of synthetic and biologic polymers

Purification of polymers Polymer characterization Study properties like:Molecular weightPolydispersity IndexViscosityConformation Folding

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AggregationBranchingCopolymer compositionMolecular size

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PRINCIPLEPRINCIPLE

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PRINCIPLEPRINCIPLE

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.

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The larger the particles, the less overall volume to transverse over the length of the column

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FEATURESFEATURESSolvent 1.Should be kept dry2.Should be degassed in some

applications3.The samples should be made from

the same solvent4.For GPC/light scattering the solvent

should be filtered before it ever hits the pump

5.Common solvents for tetrahydrofuran (THF) & toluene.

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Pump &FiltersPump &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

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Injector LoopInjector LoopInjector 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.

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ColumnsColumnsColumns 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 amountThey are very delicate

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DetectorsDetectors

Detectorso Viscosityo Light Scattering o Ultraviolet detectorso Differential Refractive Index

detector placed at the end to reduce pressure on it

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AnalysisAnalysis

Spectroscopic Techniques1. Refractive Index 2. Light Scattering 3.  Ultraviolet Spectroscopy   Viscometry Techniques1) Viscosity2)  Flow rate

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Conventional GPC 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 . C40G.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

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Molecular Mass Sensitive Molecular Mass Sensitive DetectorsDetectors

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 obtainedII.Structural information III.Size distribution molecular weight can be directly

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Characterization Characterization

Light ScatteringLS 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.

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ApplicationsApplications

Polymer characterization Molecular weight Polydispersity Index   Viscosity   Folding   Aggregation   Branching   Copolymer composition   Molecular size

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Proteomics Purification   Conformation Hydrodynamic volume

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AdvantagesAdvantagesCan 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

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DrawbacksDrawbacksThere 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

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REFERENCESREFERENCESInstrumental methods of

chemical analysis .B.K Sharma p.g C123-170

www.gel permeation chromatography

www.ion exchange chromatography.

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