ion exchange chromatography

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TOPIC

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INTRODUCTION

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PRINCIPLE

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CLASSIFICATION & PROPERTIES

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TYPICAL ION EXCHANGE EXPERIMENT

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EXPERIMENTAL TECHNIQUE

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SEPARATION FACTOR

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FACTORS AFFECTING SEPARATION FACTOR

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TECHNIQUES

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DETECTION IN ION CHROMATOGRAPHY

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APPLICATIONS

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INTRODUCTION Ion-exchange can be described as the process of the reversible, stoichiometric exchange of ions of same charge between a mobile liquid phase and an insoluble solid (stationary phase).An ion exchanger is an insoluble material liberating the counter ions (mobile ions) by electrolytic dissociation. Amongest all the chromatographic techniques, ion exchange chromatography are considered to be a very versatile method and is very useful in the separation of ions of similar properties. It is treated as column technique because the ion exchange material is normally packed in the column. Many natural substances like clays and zeolites (aluminosilicates) have ion-exchange properties. They were used to purify water since 19th century. However, from the standard point of analytical chemistry, it was not until 1935 that ion exchange became important to analytical chemist. At that time, a series of polymeric ion exchangers were first synthesized by Holmes. These exchangers had the advantage of high exchange capacities .they were readily penetrated by solvents, possessed sufficient stability and provided reproducible response .Now a days, most of the ion-exchange materials used are synthetic resins.

PRINCIPLE 2

Ion Exchange Chromatography relies on charge-charge interactions between the protiens in your sample and the charges immobilized on the resin of your choice.Ion exchange chromatography can be sub divided into cation exchange chromatography,in which postively charged ions bind to a negatively charged resin;and anion exchange chromatography,in which the binding ions are negative,and the immobilized functional group is postive.Once the solutes are bound,the column is washed to equilibriate it in your starting buffer,which should of low ionic strength,and then the bound molecules are eluted off using a gradient of a second buffer,which steadily increases the ionic strength of the eluent solution.Alternatively,the PH of the eluent buffer can be modified as to give your protien or the matrix a charge at which they will not interact and your molecule of interest elutes from the resin.If you know the PH ,you want to run at and need to decide what type of ion exchange to use paste your protien sequence into the titration curve generator.If it is negatively charged at the PH you wish,use an anion exchanger.Ofcourse this means that your protien will be bindind under the condition you choose.In many cases,it may be more advantageous to actually select conditions at which your protien will flow through while the conatminates will bind.This mode of binding is referred to as “flow through mode”.This is a particularly good mode to use this type of mode to bind up endotoxins or other highly negatively charged substances well at the same time relatively simply flowing your protien through the matrix.

Ion Exchange Properties

Resins

:

Classification

and

3

Ion exchange resins are highly ionic, covalently cross linked, insoluble polyelectrolytes supplied as beads. The beads have either have a dense internal structure with no discrete pores or a porous multichanneled structure. They are commonly prepared from styrene a various level of cross linking agent divinyl benzene, which control the porosity of the particles. Porous beads can also be made by adding homopolystrene, which is soluble in precursor beads are post functionalized to yield the finished resin Acrylic based; ion exchange resins are also available. These ionic polymers contain two types of ions, those which are bound within the structure and the oppositely charged counter ions which are free. The property of ion exchange is a consequence of Donnan exclusion-when the resins is immersed in a solution in which it is soluble, the counter ions are mobile and can be exchanged for other counter ions from the surrounding medium; ions of the same type of charge as the bound ions do not have free movement into and out of the polymer. Ion exchange resins have been classified based on the charge on the exchangeable counter ion and the ionic strength of the bound ion. Thus there are four primary types of ion exchange resins: 1. Strong cation exchange resins: containing sulfonic acid group or

the corresponding salts. 2. Weak cation exchange resins: containing carbolic acid groups or

the corresponding salts. 3. Strong anion exchange resins: containing quaternary ammonium

groups. Of the se there are two types : Type 1: resins contain triakylammmonium chloride or hydroxide Type 2 : resin contains dialkyl 2-hydroxyethyl ammonium chloride or hydroxide. 4. Weak anion exchange resins: containing ammonium chloride or

hydroxide. Additional type of ion exchange resins include blends of cation and anion exchange resins, called as MIXED BED RESIN.A resin which contains both an anion and a cation as bound ion are called as AMPHOLYTIC.

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Some ion exchange resins are prepared from chelating properties making them highly selective towards certain ions. In addition to there use in ion exchange, organic polymer supports, many of which are based on PS-DVB resins, are being used as polymer catalysts in the expanding research area involving heterogenization of homogenous catalysts and as a polymeric supports and reagents in combinatorial chemistry. The internal structure of the resin beads, i.e whether micro porous or macroporous,is important in the selection of an ion exchanger.Macroporous resins with their high effective surface area, facilitate the ion exchange process. Also they give access to the exchange sites for larger ions, can be used with almost any solvent, irrespective of whether it is a good solvent for the uncrossed linked polymer, and take up the solvent with little or no change in volume. They make more rigid beads, facilitating ease of removal from reaction system. In the case of micro porous resins since they have no discrete pores, solute ions diffuse through the particle to interact with exchange sites. Despite diffusional limitations on reaction rates, these resins offer certain advantages: they are less fragile, requiring less care handling, react faster in funtionalization and application reactions, and possess loading capacities. In addition to being functions of bead morphology, the kinetics of exchange depend on the particle size distribution of resin. It is enhanced by monodisperse resin. BASIC REQUIREMENTS OF USEFUL RESINS : •

The resin must be sufficiently crossed linked to have only negligible solubility.

•

In order to permit diffusion of ions through the structure at constant and finite rate, the resin must be sufficiently hydrophilie.

•

The swollen resin must be denser than water.

•

The must be chemically stable.

•

It must contain sufficient number of accessible ionic exchange groups.

CLASSIFICATION OF ION EXCHANGERS :-

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According to their origin both, organic and inorganic ion exchangers may be natural or sythetic.For chromatographic purpose organic synthetic ion exchangers, ion exchangers and ion exchange derivatives of cellulose, polydetran and agarose are of the greatest importances. There are four basic types of resins which are commonly used in the ion exchange chromatography. Strongly acidic cation exchange resins – Sulphonate polystyrene resins belong to this class. They are useful between the ph ranges 1 – 1.4.These are used mainly in the fractionation of cations, inorganic separation, lanthanides, vitamins, peptides and amino acids. Weakly acidic cation exchange resins – Carboxylic polymethacrylate is an example of such type of resins. They are effective between ph 5 – 14.These is useful in functionation of cations, biochemical separation, transistion elements, amino acids, antibiotics and organic base. Strongly basic anion exchange resins – Quaternary ammonium polystrene belongs to this class and it is effective between ph 0 and 12.This type of resins are useful in fractionation of anions,halogens,alkaloids,vitamin b complex, fatty acids etc. Strongly basic anion exchange resins – phenol formaldehyde and polyamine polystrene resins belong to this class. They are effective in the ph range 0-9 and can be used for the fractionation of the anionic complexes of metals, anions of different valancies,vitamins and amino acids.

CATION EXCHANGE RESINS : Resins in which liable ions are cations are known as cation exchange resin. Thus a cation exchange resin is a high molecular weight, cross linked polymer having acid mobile ions or functional groups such as sulphonic, carboxylic, phenolic etc.In this cation exchanger the hydrogen ions are mobile and exchangeable with other cations.The anions remain attached to the resin network a widely used cation exchange resin is obtained by copolymerization of styrene with little of divinyl benzene followed by sulphonation. 6

Cation exchange may be sub divided into: •

Strongly acidic.

•

Weakly acidic cation exchangers.

A) Strongly acidic cation exchanger behaves like a strong acid

and is almost completely ionized over a wide range of pH;such ion exchanger can exchange ion rapidly. These resins are useful for the chromatography of amino acids, rare earths and other substances. An example is the exchanger containing the sulphonic solution of NaCl is passed through the resin; the H+ will be replaced by Na+ ion because the sulphonate group has less attraction for the hydrogen ion than for the sodium ion. The equilibrium can be represented as :

7

B) A weakly acidic cation exchanger mainly contain carboxylic

group or phenolic.Such as exchanger ionizes in alkaline media only and should therefore be used at pH greater than 7.The rate of exchange has found to increase with increase in pH.

ANION EXCHANGE RESINS : -

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Resins in which ions are anions are known as anion exchange resins. The anion exchange resin is a high molecular weight, cross linked polymer containing basic functional group such as amino groups, quaternary ammonium group, halides group etc.A widely used anion exchange resin prepared by co-polymerization of styrene and a little divinyl benzene followed by chromethylation and interaction with base such as trimethyl amine. Anion exchanger may be sub divided into: A) Strongly basic anion exchanger: - With positively charged

quaternary ammonium groups attached to cross linked polystrene framework belongs to this class. These resins ionize over the wide range of pH.They are useful in fractionation of anions, halogens, alkaloids, vitamin B complex etc.

B) Weakly basic anion exchanger: Ionize in acid media only and

should therefore be used at pH less than 7.

AMPHOTERIC:-

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Amphoteric ion exchanger contains both cation exchanging and anion exchanging groups in their matrix. These ion exchangers are capable of forming internal salts which dissociates in contact with electrolytes and bind both its components. However they can be easily regenerated with water dipolar ion exchangers; are a special kind of atmospheric ion exchangers. On the matrix are bound amino acids which dipoles in aqueous solution. These types of ion exchangers are suitable for the chromatography of biopolymer with which the dipole interacts selectively.

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Resin type

Chemical constitution

Usual form of purchase

Common trade name Rohm DOWEX & chemical Hass

Selectivity

Strongly acidic cation exchangers

Sulphonic acid groups attached to the styrene and divinylbenzene copolymer

Aryl-SO

Amberlite IR-120

Dowex 50w

Ag+>Rb+> Cs+>R+ NH4+>H+> Li+>Zn2+> >Cu+>Ni2 +>Co2+

Weakly acidic cation exchangers

Carboxylic acid groups attached to acrylic & divinylbenzene copolymer

R-COO-Na-

Amberlite IR 50

---------

H+>>Ag+> K+>Na+>> Fe+>Ba2+ Ca2+> Mg2+

Strongly basic anion exchanger

Quaternary ammonium groups attached to styrene & divinylbenzene copolymer

ArylCH2N(CH3)3 +Cl-

Amberlite IRA-400

Dowex1

I>Phenol Ic>HSO4>CIO3>NO3-> Ba->CN-> HSO3->NO2>Cl>HCO3-> HCOO-> Acetate> OH->F-

Wealky basic anion exchanger

Polyalkylamine groups attached to styrene & divinylbenzene co polymer

Aryl-NH(R)2-Cl-

Amberlite IR-45

Dowex 3

Aryl-SO 3h>Citri c>CrO3> HSO4>ta Taric>ox Alic>H3P O4>HNO**> HBr> HCL>H

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PROPERTIES OF ION EXCHANGE RESINS:Ion exchange resin of different origin, structure and components will have different properties. The most important properties of ion exchange resin are color, density, mechanical strength, particle strength andcapacity,selectivity,cross linking,swelling,porosity,surface area and chemical resistant. •

Cross linking is very important in ion exchange resin, because it effects swelling and strength of the resin. As the cross linking in the resin decreases, the resin swelling increases.Divinyl benzene is the most common employed cross linking joining the chains together at various position.

•

Swelling increases as the cross in resin decreases. Almost all the ion exchange resin swells when placed in the water. This probably is due to the hydration of their ions. When the resin is in the swollen condition, small ions can diffuse in and out. It is very difficult to maintain electrical neutrality under these conditions. Thus if Ca2 ion enter inside the resin, and then it is most essential that the H+ ions must leave the place so that electrical neutrality is maintained.

•

Particle size is also the important factor on which the effective separation is based Particle size ranges of 50-100 mesh or 100-200 mesh are mostly commonly employed for effective separation since equilibrium constant.

•

Since the equilibrium constant on which the separation depends is influenced by the temperature changes, the later also affects the effective separation.

•

The degree of ionization at a given pH and the nature and concentration of the ion in solution are also important. The effect that the pH of the medium has on the exchange capacity of all material is significant, when pH increases the exchange capacity of cation exchanger increases, and that of anion exchangers decreases and vice-versa.

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TYPICAL ION EXCHANGE EXPERIMENT The simplest apparatus for ion exchange analysis consist of a burette provided with a glass- wool plug or sintered glass disc at the lower end .A glass- wool pad may be placed at the top of the bed of resin and the eluting agent is added on top of the bed of resin should be of small particle size so as to provide a large surface of contact. In all cases, the diameter of the resin bed should be less than onetenth of that of the column. The resin should be stirred with water in an open beaker for several minutes, any fine particle if present, is removed by decantation. The resin slurry is then transferred portionwise to the column previously filled with water. To ensure the removal of trapped air bubbles or any particles or an uneven distribution of resin granules, the column is backwashed before use. A stream of distilled water or deionised water is run up through the bed from the bottom at a sufficient flow rate to loosen and suspend the exchanger granules. The excess of water is drained off. However, the water level must never fall below the surface of resin throughout the operation. Procedure:The solution containing a mixture of solutes is first passed through a column packed with ion exchange resin. The cations in solution undergo exchange with the hydrogen or any other cation that may be present in the ion exchanger. The cation which has the maximum capacity to undergo the exchange process is held further down the column in the order of their decreasing capacities to undergo the exchange reaction .Another solution containing a stronger exchanger such as H+ ions’ is then used as an eluent .HCl is a good eluent in many cases. It is allowed to percolate slowly through the column. H+ ions displace the other cations held on the resin. The eluate i.e. the liquid coming out from the bottom of the column, contains first the components (cation) which is least firmly held on the resin. It is followed by other components (cation) in the order of increasing strength with which they are held by the

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resin.Thus, the elute coming out last from the bottom of the column is that which was most firmly held by the resin.

The process of removing absorbed ions is known as elution, the solution employed for elution is term as eluent, and the solution resulting from elution is called the eluate. If the ion exchange column is loaded with several ions of similar charge and if the concentration of components in successive portions of the eluate is plotted against the volume of the eluate, an elution curve is obtained as

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EXPERIMENTAL TECHNIQUE There are three ways of performing an ion exchange separation: by column chromatography, by batch methods, and by expended bed absorption. This section will mostly deal with column chromatography. Column chromatography: COLUMN:Column should be so designed that there should be no hindrance in the flow of liquid. The liquid is poured on the top of the column and all operations are carried out in down flow direction. The liquid moves down the column and comes in contact with the ion exchange resin and as a result exchange takes place. As the liquid moves, more the ions are exchanged and finally all the ions in the eluted solvent are completely exchanged with the resin. It should be noted that there should be no air bubble in column and the column is not allowed to drain out. The column geometry is dependent entirely on the separation factor. The latter can be improved by increasing the length of the column. It should be noted that the length of the column can be increased only upto to certain height, called critical length, beyond which it cannot be further increased. The diameter of the column depends upon the material to be separated. Generally the ratio of 10:1 or 100:1 between height and diameter is maintained in most of the experiments. Columns should b neither too wide nor too narrow in sizes, otherwise uneven flow of liquid is expected to occur. PACKING OF THE COLUMN :The column is held in a vertical position and the slurry of resin is then poured into the column. It should be noted that their should be no air bubble in the column. More over swelling effect are avoided; otherwisethey will interfere with the flow rate and packing of the column.The slurry is added in several parts and the reason is allowed

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to settle down in between each addition. It is also advisable to bring the resin in equilibrium with solvent before packing the column. When the packing is complete, the eluent is allowed to pass through the column, for certain time in order to ensure uniform rate flow over the whole cross section of the column. Then the level of the liquid is so adjusted that it remains over the top of the resin bed. Now the ion exchange resin column is ready for the experiment and the sample solution to be separated is now poured on top of the resin in the column by making use of a micropipette or micro syringe. It has been observed that a high range of exchange has been affected with a resin of low cross linking, and small particle size. The rate of exchange has also been the solution. The column in ion exchange chromatography may be operated, by elution, frontal analysis and displacement analysis.

COLUMN OPERATION WITH AN ION EXCHANGE RESIN The column operation is quite better and efficient method than batch operation .the resin is placed on top of the glass wool plug in a vertical tube (for most analytical purpose, a burette has been employed).By passing a sufficiently concentrated solution of the desired cation through the resin column (or resin bed), a strong acidic cation exchange resin can easily be converted completely into the desired ionic form. It should be noted that the liquid level must always be above the top of the resin bed, otherwise air bubbles become entrapped and the active surface available for the ion otherwise air bubbles become entrapped and the active surface available for the ion exchange is reduced .Thus it is desirable to place the column at a constant rate(e.g. 3-5 ml per minute per sq.cm of the resin bed cross section).A complete conversion of sodium chloride to hydrochloric acid can therefore be achieved by column operation method, because the solution as it passes through the column will counter fresh, uniequilibrated layers of resin and conversion to HCl will be complete. It should however be noted that the exchange capacity of the bed must be greater than the amount of sodium ion to be exchanged .Moreover, the solution of sodium chloride must be sufficiently 16

dilute, otherwise a concentrated solution of HCl will obtained and equilibrium would not favor the complete exchange of sodium for hydrogen. The column becomes exhausted, when it is used for a prolonged time. The column can be regenerated by passing a concentrated solution of an appropriate ion.

SAMPLE PREPARATION

Sample concentration:The amount of sample which can be applied to column depends on the dynamic capacity of the ion exchanger and the degree of the resolution required .For the best resolution it is not usually advisable to use more than 10-20% of this capacity (23).Information on the available capacities for the different exchangers is given in the relevant product sections. Methods for determining available and dynamic capacities are given later in this chapter.

Sample composition:The ionic composition should be the same as that of the starting buffer. If it is not, it can be changed by gel filtration on sephadex G-25 using e.g.Amersham Biosciences Disposable PD-10, fast Desalting Column hr 10/10 or Hitrap Desalting Columns, dialysis, diafiltration or possibly by addition of concentrated start buffer.

Sample volume:If the ion exchanger is to be developed with the starting buffer (isocratic elution), the sample volume is important and should be limited to between 1 and 5%of the bed volume. If however, the ion exchanger is to be developed with a gradient, starting conditions are 17

normally chosen so that all important substances are adsorbed at the top of the bed. In this case, the sample mass applied is of far greater importance than the sample volume. This means that large volumes of dilute solutions, such as pooled fractions from a preceding gel filtration step or a cell culture supernatant can be applied directly to the ion exchanger without prior concentration. Ion exchange thus serves as a useful means of concentration a sample in addition to fractionating it. If contaminants are to be adsorbed, and the component of interest is allowed to pass straight through, then the sample volume is less important than the amount of contaminant which is present .Under these conditions there will be no concentration of the purified component, rather some degree of dilution due to diffusion.

Sample viscosity :The viscosity may limit the quantity of sample that can be applied to column .a high sample viscosity causes instability of the zone and an irregular flow pattern. The critical variable is the viscosity of the sample relative to the eluent. A rule of thumb is to use 4 cp approximately 5% .Approximate relative viscosities can be quickly estimated by comparing emptying times from a pipette .if the sample is too viscous, due to high solute concentration, it can be diluted with start buffer. High viscosity due to nucleic acid contaminants can be alleviated by precipitation with a poly-cationic macromolecule such as polyethylene mine or protamine sulphate.Nucleic acid viscosity can also be reduced by digestion with endo nuclease. Such additives may however be less attractive in an industrial process since they will have to be proven absent from the final product.

Sample preparation:In all forms of chromatography, good resolution and long column life time depend on the sample being free from particulate matter. It is important that “dirty” samples are cleaned by filtration 18

or centrifugation before being applied to the column. This requirement is particularly crucial when working with small particle matrices, such as Minibeads(3µm), MonoBeads(10µm),SOURCE(15 and 30µm) and sepharose high performance (34µm).The “grade” of filter required for sample preparation depends on the particle size of the ion exchange matrix which will be used. Samples which are to be separated on a 90 µm medium can be filtered using a 1µm filter. For 3,10,15,30 and 34µm media, samples should be filtered through a 0.45µm filter. When sterile filtration or extract clean samples are required, a 0.22µm filter is appropriate. Samples should be clear after filtration and free from visible contamination by lipids. If turbid solutions are injected onto the column, the column lifetime, resolution and capacity can be reduced. Centrifugation at 10000g for 15 minutes can also be used to prepare samples. This is not the ideal method of sample preparation but may be appropriate if samples are of very small volume or adsorb nonspecifically to filters.

SEPARATION FACTOR:Ion exchange chromatography is based on the exchange of ions between a solid ion exchanger and ions present in the solution. The exchange of ions obey the law of mass action. For example, the equilibrium between the ion exchanger and the solution can be represented as,

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The conditional ion exchange equilibrium constant in the first approximation can be expressed as

[B-] / [A-] = KAB [B+] / [A+]

Where [B-] and [A-] are the concentrations of the ions in the solid phase and [B+] and [A+] are the concentrations of ions in the liquid phase. KAB is conditional ion exchange equilibrium constant. It should be noted that the sorption of ions depend on the following factors:

1) 2) 3)

Nature and structure of the ion exchanger. Nature of analyzed substances. Experimental conditions such as pH, temperature, etc.

It should also be noted that sorption is physiochemical process resulting in the absorption of gases, vapors, radiations and solutes by a substance form the surrounding medium.

Sorption also includes, i.e., sorption on the interface, absorption i.e. sorption by the bulk of the sorbent and other process.

For dilute solutions, the activities may be replaced by the concentration of exchanging ions in solution, because activity coefficients are equal to unity. The activities of ions in an ion exchanger may also be replaced by the concentrations if the ratio of activities of ions to powers corresponding to the reciprocal of the ionic charge is assumed to be a constant quantity. Thus if ion exchange proceeds according to the equation,

Z2M1AZ1 + Z1M2Z2+ Where

Z1M2AZ1 + Z2M1Z1+

Z1 and Z2 = Charges of exchanging ions. 20

M1 and M2 = First and second exchanging ions of metals A

= Anion of resin

Applying law of mass action K = [M2AZ2]

Z1

[M1Z1+]

Z2

[M1AZ1]Z2 [M2Z2+] Z1 OR

[M2AZ2]1/Z2

= Kc C21/ Z2

[M1AZ1]1/Z1

C11/ Z1

.…(5)

Where C1 and C2 are the concentrations of exchanging ion in solution and KC is concentration exchange constant which depends on temperature and the chemical nature (the charge, and size of ions and their hydration) of exchanging ions and ions exchanger. It characterizes the strength of incorporation of a given ion in the lattice of an ion is the change of the cation, and it increases with atomic number of the element involved.

Equations (5) is known as Nikolsky equation and is valid if the volume of the ion exchanger does not change in the course of an experiment, the ions in the solution do not react with the ion exchange radical, molecular and ionic sorption is absent, and the ions to be separated freely permeate into an exchanger.

Static exchange capacity may be defined as the number of mg equivalent of the ion adsorbed per a certain time interval by 1g of the dry ion exchanger. The thermodynamic exchange capacity may be defined as the number of ions adsorbed by a layer of ion exchanger 20cm high and 1 sq. cm in the cross section at the rate of migration of 0.5 dm2 per hour. 21

The adsorptive effect of an ion is characterized by the DISTRIBUTION COEFFICIENTS.

D = Cr.m Cs.V

Where Cr and Cs are the equilibrium concentrations of ions in the respective phases and m is the mass of the exchanger in grams. V is the volume of aqueous phase in cm3.

The SEPARATION FACTOR α is the ratio of the distribution coefficients of the two components (for ions of like charge).Thus

α = DA DB Where DA and DB are the distribution coefficients of two components respectively.

FACTORS AFFECTING SEPARATION FACTOR :Ion exchange is a physio-chemical process. Hence both chemical and purely physical variable influence the separation factor. Chemical variable includes: •

pH

•

Nature of ions being separated

•

Concentration of ions in solution 22

•

Tendency of ion to hydrate

•

Chemical composition of the exchanger etc.

The pH effect on ion exchanging process depends upon the chemical composition of the exchanger as well as on the type of exchange. The exchange capacity of a cation exchanger generally increases in pH of the solution, while that of an anion exchanger is decreased. In case an ion exchanger contains weak acid groups such as – COOH,OH etc, an increase in pH cause an increase in the sorption capacity. In the case of an ion exchanger who contains strong acidic groups such as –SO3 H the sorption capacity remains constant over a wide range of pH.Sorbability of ions increase in their charges, atomic masses and ionic radii. The physical variables include: •

Rate flow of solution in the column

•

Size of exchanger grains

•

The height of the column

•

Temperature of the solution etc.

Optimum separation is effected by selecting an appropriate amount of an ion exchanger. It is possible to calculate the value of the ratio of the exchanger mass to the volume of the analyzed solution in cm3 i.e. m/V provided the distribution coefficient D and the capacity alpha of the ion exchanger is known. Ion exchange constant vary with the cations involved and are markedly different from unity. The rates of the movements of the ions through a layer of an ion exchanger are inversely proportional to the values of ion exchange constant. In this way, a number of mixtures of a complex composition can be separated into their components. In displacement elution, the extraction of the ions by an ion exchange is affected with the help of a solution containing ions of a higher ion exchange constant. These ions displace the ions absorbed by the resin which pass into the solution. Instead of ions with a higher exchange constant, it is better to use ions with a lower

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constant but present in a high concentration. Example of such ion is H+ ions. For this purpose HCL solution are commonly employed. It should be noted that efficient separation of ion can be achieved only when their is a considerable difference in ion exchange constants. Hydrated ions with a similar radius have a greater charge have higher distribution coefficient, and therefore similar elements are eluted from the column in the following sequence. Li

Na

Mg Ca Lu

Yb

Tu

Er

Ho

Dy

k

Rb

Sr

Ba Tb

Gb

Cs

Eu

Sm

Pm A considerable more complete separation can be achieved in complex forming elution. In this case,desorption can be accomplished with the help of a solution of a compound which together with the cation absorbed by the resin, form a complex ion bearing a charge of opposite sign. For example in the desorption of the cations of rare earth elements by a citrate solution, the cation pass onto solution as the complex ion. Better separation takes place if there is equilibrium between the ions in the solution and on the ion exchange resin. An increase in the surface area of the ion exchanger also improves the condition for attainment of an equilibrium and separation. For good separation to be ensured, the solution must be passed slowly through the ion exchange resin. The rate of attainment of equilibrium is also affected by the temperature. A rise in temperature in many cases improves the result of separation. The temperature, however, changes also the equilibrium constant of the exchange, which may either improve of the impair the separation condition. The separation is also influenced by the composition of cation exchange resin. Resins with high content of divinyl benzene have a high swelling ability and ion exchange capacity. The length of column also plays an important role in separation process. For the separation to proceed more rapidly, the columns are expected to be short. Short columns are used for separation of

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anions and cations.Columns separation of cations.

of

greater

length

is

needed

for

CHOICE OF BUFFER : As with the choice of an ion exchanger, there are a number of variables which have to be considered. These include: 1. The choice of buffer pH and ionic strength. 2. The choice of buffering substances. 3. The price of the buffer if it is to be used in production process.

BUFFERS : Choice of buffer pH and ionic strength The choice of the buffer pH has been discussed in the previous section. It should be pointed out, however that in many application the optimum separation may be achieved by choosing conditions so that major and troublesome contaminants are bound to be exchanger while the substance of the interest is eluted during the wash phase. This procedure is sometimes referred to as “starting state elution”. The highest ionic strength which permits binding of the selected substances and the lowest ionic strength that causes their elution should normally be used as starting and final ionic strengths in subsequent column experiments. A third and higher ionic strength buffer ionic strength buffer is frequently employed as a wash step before column regeneration and re use. The required concentration of the start buffer will vary depending on the nature of the buffering substances. In the majority of cases a

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starting ionic strength of at least 10mM is required to ensure adequate buffering capacity. Salt also play a role in stabilizing protein structures in solution and so it is important that the ionic strength should not be so low that protein denaturation or precipitation occur. A major advantage of using Amersham Bioscience ion exchangers is that they have excellent capacities and so the initial ionic strength of the buffer can be quiet high with out significant affecting capacity for sample 77 In case of prepacked ion packed ion exchangers and columns, which can be run conveniently quickly, trial experiments using salt gradients with allow the determination of an optimal starting ionic strength In the case of sephadex based exchanges for batch application or where column running times are prohibitively long a simple test tube technique is recommendation as a test for a suitable ionic strength. Choice of buffer substances If the buffering ions carry a charge opposite to that of the functional groups of the ion exchanger they will take part in the ion exchange process and cause local disturbances in pH, it is preferable, therefore to use buffering ions with the same charge sign as the substituent groups on the ion exchanger. There are of course exceptions to this rule as illustrated by the frequency with which phosphate buffers are cited in the literature in connection with anion exchangers. In those instances when a buffering ion which interacts with the ionic groups on the matrix is used, extra care must be taken to ensure that the system has come to equilibrium before application of sample.

TECHNIQUES Recent advances in ion chromatography are mostly related to highly selective separation and to extremely low detection limits. Through the choice of stationary phase and the variation of eluent composition, higher separation selectively can be obatined.Analyzing the samples in which analytes are presented at low concentration in the presence of high ionic strength or extreme pH matrix, the major

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matrix ions would displace the adsorbed analyte ions on the stationary phase. The result would be band broadening and poor separation because the extreme pH of sample causes disturbance to the equilibria in the eluent.Therefore, technique relating to sample preparation and separation need to make progress. Both suppressed and non-suppressed detection modes have been applied to the analysis of samples.However, the most common mode of detection for ion chromatography is suppressed conductivity in which a suppressor is used to reduce the background conductivity of the eluent and to increase the detection signal of analyte ions. The suppressor is a cation or anion- exchanger, installed after the separation column to replace the counter ions with H+ and OH-,to change the highly ionized eluent ions into weakly ionized species. Progress in the area of continous electrochemically regenerated devices associated with suppressed conductivity Recent detection has also been made.

ION CHROMATOGRAPHY BASED ON SUPPRESSORS :The first used suppressor was an ion exchange column in the form of H+ or OH-,2 but this technique is restricted by the limited capacity of the suppressor, which must be regenerated periodically off-line. This problem has been overcome with the introduction of the hollowfiber membrane suppressor.However; a problem of this kind of suppressor is the low surface area of the fiber, resulting in low ion exchange and suppression capacity. The appearance of the micro membrane suppressor, in which two flats of membrane were inserted into three ion exchange screens as a sandwich, solved this problem.

Electrolytic membrane suppressors The suppressor mentioned above employs diffusion alone for ion transfer. More recently, an electric membrane based suppressor.This,a “Self Regenerating Suppressor” (SRS) has a design similar to that of the micro membrane supressor

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were used to allow the generation of H+ or OH- necessary from water. In this device, no external regenerate is needed. In analysis, an anion SRS is used and H+ ions produced from anode migrate across the cation-exchange membrane to transfer OH- ions into water and meanwhile counter-cations in the eluent are driven by electric field to cathode. Gas waste from electrolysis reaction and liquid waste will move out of the regenerant chamber.Cation analysis will operate in the same way except that OH- instead of H+ will migrate across the anion exchange membrane. SRS can be performed in a couple of ways: “recycle mode” and “external water mode ”.The “recycle mode” is operated in the manner in which effluent from the conductivity detector servers as the source of the water required for electrolysis. While an external source of water is supplied to the regenerant chamber of electrolysis in an “external water mode”. The performance difference in between them comes from the fact that higher flow rate of external water employed in “external water mode” would result in a lower noise and better sensitivity.Upto now the “recycle mode” is the most commonly used operated mode for most samples because of its ease to operate.Recently,it was found that the problem of low sensitivity 28

could be circumvented if an inert gas was used to supplement the effluent flow from detector. This mode called “gas assisted recycle mode” speeds up the removal of eluent counter-ions and electrolysis products, hence leads to a performance comparable to “external water mode”.

“Packed column mini suppressor ” “Solid phase chemical suppression” employs a valve configuration and two suppressor catridges.The devices offers no significant improvements because the cat ridge needs to be regenerated offline from time to time. A modified device,” Electrochemically Regenerated Ion Suppression”(ERIS) was developed in which two solid-phase electrochemical suppressor cells replaced the catridges.Suppressor cells are composed of cation exchange resins or anion exchange resins with electrodes being applied. At the start, eluent from the separation column enter into one cell, and normal suppression reaction occurs in this cell, and the effluent from the detector cell will flow into the second cell, where regeneration of ion exchange resins take place as water is electrolyzed. The performance of this device is equivalent to that of self regenerated suppressor. It has an advantage over membrane-based suppressors in that it can withstand high back-pressures.

“Continuously regenerated packed-column suppressor” A newer design of packed column suppressors has been developed, which combines the benefits of both electrolytic SRS suppressor and packed column mini-suppressors. The mechanism of this device is based on the principles of “ion reflux”, in which electrically polarized resins are used with water as an eluent to regenerate the required H+ ions. In anion analysis. the effluent from the separation column is introduced in the opposite flow direction of H+ ions generated at the anode, hence the counter cation in the eluent are displaced by the continous H+ ions and driven towards the cathode, where they combine with OH- ions generated at the cathode. Because the H+ ions would not be depleted as long as the electrolysis reaction 29

occurs, the suppression capacity of this device is higher and background.

SINGLE COLUMN ION CHROMATOGRAPHY :

Recently, commercial ion chromatography instrumentation that requires no suppressor column has become available. This approach depends on the small difference in conductivity between sample ions and the prevailing eluent ions. To amplify these differences, low capacity exchangers are used that permit elution with solution with low electrolyte concentration. The typical eluent used in nonsuppressed IC are phthalic acid and p-hydroxybenzoic acid for the determination of anions and the methanesulphonic acid for the determination of cations.The equivalent conductance values of chloride, sulphate and other common anions significantly greater than that of eluent and their for a positive peak is detected as the 30

anions are carried through the detector. The equivalent conductive value of sodium, potassium, calcium, magnesium and other common cations are significantly lower than that of the cations (H-) in the eluent.In this instance a negative peak is detected as the cations are carried through the detected. Non suppressed IC is easier to perform, and it is a technique for determining ions of weak acids such as cyanide and sulfide, which are non conducting after chemical suppression but show a higher baseline noise pharmaceutical analyses can be performed in the non suppressed mode because the quantification limits are usually in the upper mg per L to low percentage levels.

DETECTION IN ION CHROMATOGRAPHY The following detection methods are available with ion-exchange chromatography: 1. Conductivity detection. 2. Electrochemical detection 3. Potentiometric detection. 4. Spectroscopic detection. 5. Post column reaction detection.

 Conductivity detection :-

Conductivity detection has two major advantages for inorganic ion analysis. First all the ions are electrically conducting, so that the detector should be universal in response, and second, the detectors are relatively simple to construct and operate. Conductivity detection will be discussed here in terms of the 31

principle of operation and performance characteristics, modes of detection, cell design post column signal enhancement i.e. suppression and applications.

Principle of operation: The mobile phase eluting through the detector is in fact a conducting electrolyte. It flows through two electrodes across which potential is applied. The more current conducted by the solution, the higher is the electrical conductivity. The conductance of a solution is determined by several factors, including the ionic strength and the type of species in the solution, as well as the temperature. The specific conductance depends on the cross sectional area (cm2) of the electrodes inserted into the solution, and L(cm) is the distance between them, and will vary with concentration. The conductance is increased for cells in which the electrodes are large in surface area and are close together. The equivalent conductance is subject to activity effects such as ion-ion interactions, therefore the relationship between G and C becomes nonlinear at high ionic strength. Since the conductance of the solution results from both the anions and cations of the electrolyte, conductance is calculated for individual anions and cations in the solution. Most of the common anions and cations have limiting equivalent ionic conductance of 30-100.The most conducting cation is the hydronium ion and the most conducting anion is the hydroxyl ions; their values are 350 and 198 respectively. The conductance of an ion increases with its charge density and decreases with its viscosity. Therefore when stroelutropic multiply charged ions are needed in the mobile phase they can exert high background, therefore, large ions such as phthalate, citrate, or trimesate are used in such cases. Sensitivity detection can result as long as there is a considerable difference in the ionic conductance of the solute and the mobile phase ion’s. This difference can be positive or negative, depending on whether the eluent ions are strongly or weakly conducting. If the ionic conductance of the eluent

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ions is low, then an increase in conductance occurs when the solute enters the detections cell, due to higher conductance. In general this detection mode is referred to as direct. On the other hand, when the mobile phase ions are highly conducting, a decrease in conductance occurs when the solute enters the detection cell, due to lower conductance. This mode of detection is referred to as indirect. Direct conductivity detection is used for most IC methods involving the separation of anions.Eluent for non-suppressed IC formed from the salts such as potassium hydrogen phthalate or sodium benzoate contain competing anions with moderately low conductance.Similarly,direct conductivity detection is possible with eluents containing with organic bases. Indirect conductivity detection can be applied to anions using hydroxide eluents and to cation using mineral acid eluents.

Electrochemical detection :The term “electrochemical detection” is applied loosely to describe a range of detection technique involving the application of electric oxidation-reduction potential via suitable electrodes to sample solution, containing oxidizable or reducible solutes. The resulting current is measured as function of time. Electrochemical detection has been applied in situation where extreme sensitivity is required. Most commonly the electrochemical detector has been operated in tandem with a conductivity detector, which acts as a universal detector that gives a more general sample analysis.

Voltametery:33

Voltametery is a well-established technique in which a changing potential is applied to a working electrode with respect to a reference electrode. The current resulting from the reaction of analyzed species at the working electrode is measured. The key factor is that the applied potential is varied over the course of the measurement.

Amperometry and coulometry : The term amperometry describes the technique in which a fixed potential is applied to a working electrode with respect to a reference electrode. The working electrode is located in the flow-cell through which the mobile phase passes and the current resulting from the oxidation –reduction reactions occurring at the working electrode is measured. The analyte to be detected undergoes a Faradaic reaction if the applied potential has appropriate polarity and magnitude.When the reaction is incomplete, causing only a fraction of the total analyte to react, the detection mode is termed amperometry,while when the working electrode has larger surface area and the reaction is complete the mod is called coulometry.

Spectroscopic Methods :Spectroscopic methods of detection are very common in ion chromatography and are second in their abundance. This mode of detection can be divided to two major categories: molecular and atomic spectroscopy. Molecular spectroscopy includes methods such as UV-IS absorption, refractive index, fluorescence and phosphorescence. Atomic spectroscopy includes flame atomic absorption, flame atomic emission and plasma atomic emission.

Molecular Spectroscopy :-

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a)

UV-VIS Absorption :

Many inorganic cations and anions do not have significant absorption in the UV-VIS range of the spectrum, therefore the direct detection cannot be used typically. However their are areas where ions can be detected directly by their UV or visible detection in the 185-220 nm range. Detection of non absorbing ion can be achieved using indirect photometric mode, similarly to the indirect conductivity mode of detection. In the indirect mode highly absorbing ionic species are used as the eluents with high background, and the detector response is zeroed on them. The non absorbing solutes are detected as negative peaks, since the detector measures the difference of absorption between the high background and the nonabsorbing species. The polarity of the detector can be then reversed and the peak appear positive .Benzenepolycarboxylic acid salts, such as phthalate,benzoate,phenylphosphonate,p-toluenesulfonate or trimellitate are used as chromophoric eluent anions that enable the sensitive detection of union UV-VIS absorbing ion. b)

Fluorescence:

Fluorescence detection is well known for its sensitivity. Since most of the ionic species analyzed by the ion chromatography do not exhibit fluorescence, direct mode of detection has only a limited scope. Usually the mobile phase includes a chelate or an ion pair reagent that forms a species with the ions that produces a signal in the fluorescence detector. It is more likely to find works that utilize the indirect mode of fluorescence detection. c)

Refractive index(RI):

Most of the solutes for ion chromatography is used normally are not detectable directly by refractive index detectors. The general exceptions are carboxylic acids, large species such as polyphosphonates or sulphonium ions and some inorganic ions. In cases where the ions cannot be detected directly by the RI detector, an indirect mode of detection was used.

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Atomic spectroscopy :- The combination of HPLC separation with various forms of atomic spectrometry gives a method of great sensitivity as well as a time resolved detection of species. a. Flame atomic absorption (AA) and atomic emission (AE).

Direct coupling of atomic absorption spectrometer to an HLPC system requires means to match the flow rates of the two techniques. The output of the IC system needs to be relatively high to accommodate the atomic absorption instrument; therefore, pure water is added some times as a “make up” solvent. b. Inductively coupled plasma(ICP) ICP with emission spectroscopy or with mass spectroscopy have emerged as a replacement to emission spectrometers and act as detectors for ion chromatography in recent years. The introduction of HPLC coupled directly to ICP MS led to the used of these properties in speciation analysis. The coupling of ion chromatography(IC) with ICP MS made possible the elimination of gram amounts of matrix in cases where it could be converted into an anion form, so that ultra-trace amounts of the cationic impurities could be determined. In the semiconductor field, such analyses have been carried out on matrices of Mo, W, Re, As and P.

Post column reaction Detection by post-column reaction (PCR) involves the chemical reaction of the solutes as they elute from the column on the fly, prior to their introduction to the detector. The main goal of such a procedure is to enhance selectivity and specificity to solutes of small quantities in the present of large quantities of interferences in the sample matrix. Some of the post column reagents are ammonium molybdate,4(2-pyridylazo) resorcinol, pyridine-2, 6-dicarboxylic acid,phenylfluorone,2-(5-bromo-2-pyridylazo)-5-(diethyl amino)phenol. 36

APPLICATIONS OF ION EXCHANGE CHROMATOGRAPHY :Ion exchange chromatography has proved to be excellent tool for solving many complicated problems in the field of biology, organic and inorganic chemistry. In biological field, it has especially been used in the separation of hydrolyze products of nucleic acid. In organic chemistry, a number of separations are based on ion exchange. Among these are the separations of acids, amino acids peptides and nucleotides etc.In organic chemistry, it used in the separation of rare earth etc.Some important applications of ion exchange chromatography are briefly described below: 1) Demineralization of water:Ion exchange resins have been used for the complete demineralization of water for chemical use. Water is first allowed to pass through a cation exchanger, which removes the anion replacing them with the exact amount of OH- ions necessary to neutralize the acid from the first operation. Thus if water is passed through two column, the first containing cation exchange resin in the hydrogen form and the second anion exchange resin in the hydroxide form (M+) and anions(X-),the reactions are :

The resulting water is as pure as distilled water and is far les expensive. Colloidal material of course not recovered. Water purification by this method in comparison to distillation has two main advantages, The ion exchange procedure as cheaper as well as faster than distillation in the laboratory.

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2) Determination of sodium and potassium in the mixture :According to beulankamp and Reimen,the separation of potassium and sodium ions in a mixture can be done by introducing the sample at the top of a containing a sulphonic acid resin saturated with H+ ions. The column is then eluted with a solution of 0.7M HCl at a flow rate of 0.6mL/sq.min.Sodium ions, being the less strongly held, move down the column more rapidly and can then be collected before potassium ions appear in the effluent. The solutions are evaporated to dryness and the alkali chlorides weighed. Divalent ions, if present do not come out of the cation exchange resin until all the potassium ions have appeared in the effluent.Partion of the sodium ions in each theoretical plate column involves,

The potassium ion behaves in a similar way but is partition coefficient is numerically larger. The alkali chlorides are then redissolved and analyzed by titration of the Cl- ion by the Mohr method. 3) Separation of transition metals: - Kraus and Moore have reported the separation of transition metals by introducing the sample at the top of the column containing a strongly basic anion exchange resin in the chloride form. In this process stepwise rather than continuous change in the eluent was effected. The column was initially filed with 12M HCl and the sample was introduced at the

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top.Elutionwas carried out by successively more dilute solution of HCl.The Ni was not retained at all even in the presence of concentrated HCl.When the HCl was diluted to 6M,the acid caused the elution of Mn;Co came out at 4M,Cu at 2.5M,and Fe at 0.5M and Zn at 0.0005M HCl.This type of elution indicates the relative stabilities of the complex chloride anions and varying affinity of the resin and of water for these ions as chloride ions. 4) Separation of interfering ions of opposite charge: - Ion exchange resin have also been used for the removal of interfering ions, particularly where the ions have a charge opposite to the species being determined. For example,Fe,AI and other cations cause interference in the determination of sulphate,because of their tendency to co precipitate with the barium sulphate.The solution to be analyzed is passed through a column containing a cation exchange resin as a result of which all cation are retained and corresponding number of protons(H+) are liberated. The sulphate ion passes unhindered through the column and its analysis can be performed on the effluent. Similarly phosphate ion, which interferes in the analysis of Ba++ or Ca++,can be removed by passing the sample solution through a column containing an anion exchange resin. Ion exchange chromatography has also beer used to remove PO4 from the solution in which cations are being determined by the acid-alkali scheme of analysis or M3+ is being determined quantitatively in the form of hydroxide; to remove trivalent metal from a solution where sulphate sulphur is determined by gravimetric technique technique, and so on. In the first case, when the solution is present through the cation exchanger, the cations are sorbed,while the phosphate ion remains in the solution. For the subsequent removal of ion exchange to the left, the cation exchanger again passes over into H+ form and the cations enter the solution containing no PO4 ions. While determining SO4 ions of trivalent metals are separated by passing the solution through strongly acidic cation exchanger H2SO4 remains in the solution, where it is determined. (5) Total conten5t of cation in a solution: - T he stoichiometric nature of ion exchange resin allows us to find the total content of cations in a solution by titrating an amount of protons equivalent to them and obtained by reaction of the cation exchanger and the solution being analyzed. 39

(6)Concentration of traces of an electrolyte:- Concentration of traces of an ion form a very dilute solution can also be carried by making use of ion exchanger resins. For example, traces of metallic elements can be collected from larger volumes of natural waters by making use of cation exchange resins. The resin is first treated with HCl to liberate the ions .As a result the solution becomes more concentrated for further analysis. (7)Conversion of salts to acids or bases:- The total salt content of a sample can also be determined by using ion exchanger resins. When the sample solution is passed through a cation exchange resin in the acid form, the cations present in the sample are absorbed or retained by the exchanger and an equivalent quantity of H+ ions is released .These can be collected in the washing from the column and then titrated. Similarly, a standard solution of an acid can be prepared from a salt. For example, acid form of a cation exchange resin can be titrated against a weighed quantity of solution of chloride. The salt liberates an equivalent quantity of HCl, which is collected in washings and diluted to a known volume .In an analogous manner; the OH- ions are liberated when the salt is treated with an anion exchange resin. In addition to above applications, ion exchange chromatography is an extremely valuable tool in the separation of complex mixture of compounds of biological interest .For example, Scott and his coworkers have resolved peaks corresponding to more than 100 constituents of urine. They used gradient elution with parallel column of anion and cation exchange resins. Forty six amino acids have been separated using ion exchange chromatography. The peaks have been observed with a photometric detector. (8) Separation of amphoteric metals from non-amphoteric metals:- Ion exchange chromatography has successfully been used in the separation of constituents of a mixture, separation of cations from anions ,isolation of cations ,isolation of anions etc.In these cases amphoterism, complex formation and regulation of acidity of solutions are frequently used. For example, when non-amphoteric metals such as Fe, Cu etc are separated from amphoteric metals such as Zn,Sn,Al etc,the mixture is passed through a column packed with a cation exchanger and then washed with an alkali solution. The non-amphoteric metals are adsorbed by the cation exchanger as

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hydroxides while the amphoteric metals are eluted as AlO 2- ,Zn2- ions etc.

(9) Separation of metals, alloys and high alloy steels :-Pure metals, alloys and high alloy steels can be analyzed using complexation processes. The method is based on different stability of the complexes formed at a definite pH value. For example, addition of HCl to a mixture of Cu2+,Zn2+,Cd2+,PB2+and Bi3+ ions produces the chloride complexes [CuCl4], [CdCl4], [PbCl3] - and [BiCl4]-.Their stability increases from copper to bismuth. The solutions obtained are percolated through a column packed with an anion exchanger, which adsorbs all the complexes. The metals are then eluted successively with dilute HCl, water and nitric acid,2N HCl elutes copper,6NHCl elutes zinc,0.3N HCl elutes cadmium, lead is eluted with water and bismuth id eluted with HNO3.

(10) Separation of substances processing related properties:Ion exchange chromatography has also been used for the separation of substances processing related properties such as cations of alkali and alkaline earth metals, rare earth and transuranium elements, twin elements such as zirconium and hafnium, cis-trans isomeric complexes of cobalt and platinum. It is only this method that is applied to the quantitative separation of copper from Cu-Fe alloys with the iron content below 50%. (11)Analysis of natural and industrial waters:- Ion exchange chromatography is an efficient concentration technique often used in the analysis of natural and industrial waters for their content of heavy metals. (12) Separation of complex mixtures of biochemical compounds:- Ion exchange chromatography is an extremely valuable tool in the separation of complex mixtures of compounds of biochemical interest. Scott and coworkers have resolved peaks corresponding to over 100 constituents of urine using parallel columns of anion and cation exchanger, with the gradient elution. Peaks in an ion exchange chromatogram of mixed amino acids, observed with a photometric detector. Complete special purpose 41

chromatographs for the analysis of amino acid mixtures are also available commercially. (13) Production of analytical concentrates:- The concentrations of substances can be increased 200-500 fold by percolating large volumes of dilute solutions through an ion exchanger layer and subsequent extraction of the adsorbed substances with a small amount of solvent .This method is generally used in the separation of non –ferrous metals in the production of rare elements,uranium,radioactive isotopes etc . (14) Identification of ions: - The selectivity of colorless ion exchangers can be used to detect colored ions in a mixture. Depending on their sorpitivity, the ions are distributed in the cation exchanger by zones, namely, the lower the sorptivity of a cation, the lower in the column will its zone be. The separation of a mixture into different colored zones.

REFERENCES Instrumental methods of chemical analysis : By B.K.Sharma. Pathways of analytical chemistry :- By Kelkar,Mishra. Principles of instrumental analysis :- By Skoog. Basic concepts of analytical chemistry :- By Khopkar

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