ION EXCHANGE CHROMATOGRAPHY&

GEL PERMEATION CHROMATOGRAPHY Presented by: Under the Guidence of

G.Shekhar, Mr.Uttam Prasad,

M.Pharm (Ceutics) Dept. Of Pharmaceutical Analysis,

256213886011. Malla Reddy College Of Pharmacy.

ION EXCHANGE CHRoMATOGRAPHY

• INTRODUCTION• PRINCIPLE• MECHANISM• INSTRUMENTATION• ION EXCHANGERS• CLASSIFICATION OF ION EXCHANGE RESINS• EFFECT OF PH ON ION EXCHANGE• ADVANTAGES&DISADVANTAGES• APPLICATIONS

INTRODUCTIONThe most popular method for the purification of proteins and other charged

molecules is ion exchange chromatography. * In ion exchange chromatography, retention is based on the attraction b/w

the solute ions and charged sites bound to stationary phase * columns used for ion exchange are characterised by the presence of charged

groups covalently attached to the stationary phaseTYPES OF IEC: Two types1)anion exchangers, 2)cation exchangersCation exchange chromatography: positively charged molecules are attracted

to a negatively charged solid support. Commonly used cation exchange resins are S-resin,sulfate derivatives;&CM resins, carboxylate derived ions

Anion exchange chromatography:negatively charged molecules are attracted to a positively charged solid support

Examples:Q-resin, a quaternary amine, diethylaminoethane

PRINCIPLE

Principle : IEC retains analyte molecules on the column based on ionic interactions . The stationary phase surface display 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. The ionic compound consisting of the cationic species M+ & the ionic species B- can be retained by the stationary phase.

CEC retains positively charged cations because the stationary phase displays a negatively charged functional groups

AEC retains anions using positively charged functional groups

Mechanism Selectivity for ion exchange : in general,ion exchangers favour the binding of ions of

* Higher charge * Decreased hydrated radius *Increased polarizability• Mechanism

To optimize binding of all charged molecules, the mobile phase is generally a low to medium conductivity (i.e., low to medium salt concentration) solution. The adsorption of the molecules to the solid support is driven by the ionic interaction between the oppositely charged ionic groups in the sample molecule and in the functional ligand on the support. By increasing the salt concentration the molecules with the weakest ionic interactions start to elute from the column first. Molecules that have a stronger ionic interaction require a higher salt concentration and elute later in the gradient. The binding capacities of ion exchange resins are generally quite high. This is of major importance in process scale chromatography, but is not critical for analytical scale separations.

Ion exchange chromatography (anion exchange)

INSTRUMENTATION

Ion exchangers There are 3 classes of ion exchangers , these include1)Resins 2)Gels 3)Inorganic exchangers :separations involving harsh chemical conditions (high

temp,high radiation levels,strongly basic solutions /powerful oxidizing agents )employ inorganic ion exchangers.

*ion exchange resins are used for the separation of small of molecules *ion exchange gels are used for the separation of large molecules like proteins ,

nucleic acid.*Resins are amorphous particles of organic materials*polystyrene resins for ion exchange are made by co-polymerization of styrene

and divinyl benzene *divinyl benzene content is varied from 1-16 % to increase the extent of cross

linking

Classification of ion exchange resins

Strongly acidic cation exchanger : sulphonic acid groups attached to styrene & divinyl benzene copolymer

Weakly acidic cation exchanger:carboxylic acid groups attached to acrylic acid & divinyl benzene co-polymer

Strongly basic anion exchanger:quaternary ammonium groups attached to styrene & divinyl benzene co-polymer

Weakly basic anion exchanger:poly alkylamine groups attached to styrene & divinyl benzene co-polymer

* Sulphonate groups of strongly acidic resins remain ionized even in strongly acidic solutions , where as carboxyl groups are protonated near PH 4 & loose their cation exchange capacity

Ion exchange gels *Cellulose & dextran ion exchangers ,which are polymers of the sugar

glucose posses.larger pore sizes & lower charge densities .*Because they are much softer than poly styrene resins,dextran&its

relatives are called gelsEFFECT OF PH ON ION EXCHANG:*Varying PH is usually a preferred way to change selectivity in ion

exchange separations*An increase in the PH leads to greater sample ionization & retention in

Anion exchange HPLC*Exm: antibiotics containing COOH groups*decrease in PH favours retention of bases by cation exchange HPLC.Exm: local anesthetics containing NH2 groups*only the ionized from of acid /base will be retained significantly.

Effect of organic solvents *additional of an organic solvent to mobile phase results in decreased retention ,just as in the

case of reserved phase HPLC*solvents such as methonal /aceto nitrile are also often used in ion exchange to create changes

in selectivity .EFFECT OF BUFFERS : *In ion exchange, sometimes a particular salt is selected to provide stronger /weaker retention*a strong displacer reduces sample retention more than the same concentration of weak

displacer*in general , more highly charged displacers are stronger• As a rule, the pH of the mobile phase buffer must be between the pI (isoelectric point) or

pKa (acid dissociation constant) of the charged molecule and the pKa of the charged group on the solid support. For example, in cation exchange chromatography, using a functional group on the solid support with a pKa of 1.2, a sample molecule with a pI of 8.2 may be run in a mobile phase buffer of pH 6.0. In anion exchange chromatography a molecule with a pI of 6.8 may be run in a mobile phase buffer at pH 8.0 when the pKa of the solid support is 10.3.

Interpretation

Advantages &disadvantages

Advantages:Detectability :useful for the detection of many inorganic salts.Separations :* usually preferred ,because of the availability of

volatile buffers . Volatile buffers make the removal of mobile phase easier.

*useful for separation of mixtures of biological origin inorganic salts.Disadvantages*Column efficiency is less*it is difficult to achieve control over selectivity &resolution*stability& reproducibility of the columns become questionable after

repeated use

Applications

*IEC is used to convert one salt to other Example:we can prepare tetra propyl ammonium hydroxide from a

tetra propyl salt of some other anion *it is useful for pre concentration of trace components of a

solution to obtain enough for analysis*it is used to prepare deionized water Separation of similar ions:.A mixture of Na,H&K can be seperted using cation exchange resin a mixture of Cl,Br&iodide can be seperted using basic anion

exchange resin

Method : mixture of Cl,Br&iodide is passed through basic anion exchanger using 0.5M sodium nitrate as eluant . Cl will first eluate .Raise conc of sodium nitrate ,bromide will eluate .raise the conc of sodium nitrate further ,iodide ion will eluate.

Removal of interfering radicals: calcium & barium ions exchanged with H+ ions while phosphate ion pass through the column

Softening of hard water :water passed through cation exchanged charged with the sodium ions Ca&Mg ions retained in the column while sodium is exchanged

• Complete demineralization of water • Separation of lanthanides • Separation of sugars • Separation of amino aids • Other applications :• For the measurement of various active ingredients in

medicinal formulations • For the measurement of drugs& their metabolites in

serum & urine ,for residue analysis in food raw materials

GEL PERMEATION CHROMATOGRAPHYIntroduction

• GPC, also known as Size Exclusion Chromatography SEC or Gel Filtration Chromatography GFC, is a chromatographic technique that separates dissolved molecules on the basis of their size by pumping them through specialized columns containing a microporous packing material.

• As the sample is separated and eluted from the column, it can be characterized by a single concentration detector (Conventional Calibration) or series of detectors (Universal Calibration and Triple Detection). Gel Permeation Chromatography GPC is used to characterize natural and synthetic polymers, biopolymers, proteins or nanoparticles.

• When the GPC separation is coupled with light scattering, viscometer and concentration detectors together (triple detection), it will provide a distribution of absolute molecular weight, molecular size, and intrinsic viscosity as well as information on macromolecular structure, conformation aggregation and branching.

• It is a chromatographic method in which molecules in solution are separated by their size, not by molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography

Principle of separation • Principle of separation• It is a kind of chromatography technique based on the difference

of molecular weight and is one of the effective and mild methods extensively used to isolate and analyze the biomacromocular substances.

• The stationary phase consists of beads containing pores that span a relatively narrow size range.

• when the gel is packed into a column and percolated with a solvent, it permits the large molecular weight compounds to pass rapidly without penetration of the pores

• Smaller molecules spend more time inside the beads than larger molecules and therefore elute later (after a larger volume of mobile phase has passed through the column).

• In addition to providing the molecular weight distribution, GPC also separates a complex polymeric compound into its component parts - polymer, oligomer, monomer, and additives

Working • GPC separates molecules in solution by their "effective size in solution." To prepare

a sample for GPC analysis the resin is first dissolved in an appropriate solvent.• Inside the gel permeation chromatograph, the dissolved resin is injected into a

continually flowing stream of solvent (mobile phase). The mobile phase flows through millions of highly porous, rigid particles (stationary phase) tightly packed together in a column. The pore sizes of these particles are controlled and available in a range of sizes. The width of the individual peaks reflects the distribution of the size of molecules for a given resin and its components. The distribution curve is also known as the molecular weight distribution (MWD) curve.

• Taken together the peaks reflect the MWD of a sample. The broader the MWD, the broader the peaks become and vice versa. The higher the average molecular weight, the further along the molecular weight axis the curve shifts and vice versa.

• Taken together the peaks reflect the MWD of a sample. The broader the MWD, the broader the peaks become and vice versa. The higher the average molecular weight, the further along the molecular weight axis the curve shifts and vice versa.

• Cross sectional view of porous particle

Though they are subtle, differences such as those shown in the molecular-weight distributions to the left, could cause marked variations in the performance of the polymer.

The Size Separation Mechanism

• Molecules of various sizes elute from the column at different rates. The column retains low molecular weight material (small black dots) longer than the high molecular weight material (large black dots). The time it takes for a specific fraction to elute is called its "retention time".

Instrumentation Schematic of a basic gel permeation chromatograph

This diagram illustrates how the sample is injected into the mobile phase and the path the sample takes to the detector.

1. Pump• Pumps the polymer in solution through the system.• Different polymers produce solutions of different viscosities. To

compare data from one analysis to the next, the pump must deliver the same flow rates independent of viscosity differences. In addition, some detectors are very sensitive to the solvent flow rate precision. Such constant flow must be a critical feature of the instrument.

2.Introduces the polymer solution into the mobile phase. The injector must be capable of small volume injections and large

volume injections The injector should not disturb the continuous mobile phase flow. It should also be capable of automatic multiple sample injection when the sample volume is large.

• 3. Column Set• Efficiently separates sample components from one another. • High efficiency columns give maximum separating capability and rapid

analyses. Every column must provide reproducible information over extended periods for both analytical and fraction collecting purposes.

• 4. Detector• Monitors the separation and responds to components as they elute

from the column. • Detectors must be nondestructive to eluting components if they are to

be collected for further analysis.• In addition, the detectors must be sensitive and have a wide linear

range in order to respond to both trace amounts and large quantities of material if necessary.

Graph

Graph

Advantages

1.Good separation of large molecules from the small molecules with a minimal volume of eluate,[1] and that various solutions can be applied without interfering with the filtration process, all while preserving the biological activity of the particles to be separated.

2.The technique is generally combined with others that further separate molecules by other characteristics, such as acidity, basicity, charge, and affinity for certain compounds.

3. There are short and well-defined separation times and narrow bands, which lead to good sensitivity. There is also no sample loss because solutes do not interact with the stationary phase.

Disadvantages

• Disadvantages are, for example, that only a limited number of bands can be accommodated because the time scale of the chromatogram is short, and, in general, there has to be a 10% difference in molecular mass to have a good resolution

• Another Disadvantages of GPC for polymers is that filtrations must be performed befour using the instrument to prevent dust

Applications • The main application of gel-filtration chromatography is the

fractionation of proteins and other water-soluble polymers, while gel permeation chromatography is used to analyze the molecular weight distribution of organic-soluble polymers. Either technique should not be confused with gel electrophoresis, where an electric field is used to "pull" or "push" molecules through the gel depending on their electrical charges.

• SEC is a widely used technique for the purification and analysis of synthetic and biological polymers, such as proteins, polysaccharides and nucleic acids. Biologists and biochemists typically use a gel medium — usually polyacrylamide, dextran or agarose — and filter under low pressure. Polymer chemists typically use either a silica or crosslinked polystyrene medium under a higher pressure. These media are known as the stationary phase.

Thank You