Ion Exchange Chromatography Final Ppt

PRESENTATION ON ION EXCHANGE CHROMATOGRAPHY BY JITENDRA YADAV M.Pharm 1st year

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ION EXCHANGE CHROMATOGRAPHY Introduction:• Ion exchange chromatography is similar to partition chromatography in that it has a coated solid as the stationary phase. The coating is referred to as a resin, and has ions (either cations or anions, depending on the resin) covalently bonded to it and ions of the opposite charge are electrostatically bound to the surface. When the mobile phase (always a liquid) is eluted through the resin and the electrostatically bound ions are released as other ions are bonded preferentially. 3/04/2012

Contd.. Mobile phase

Sample containing ions

Resin with ion exchanger

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Principle of separation  Separation in ion exchange chromatography depends upon the reversible adsorption of charged solute molecules to immobilized ion exchange groups of opposite charge. Most ion exchange experiments are performed in five main stages. 1. The first stage is equilibration in which the ion exchanger is brought to a starting state, in terms of pH and ionic strength, which allows the binding of the desired solute molecules. The exchanger groups are associated at this time with exchangeable counter-ions (usually simple anions or cations, such as chloride or sodium) 3/04/2012

Contd..

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Contd.. 2. The second stage is sample application and adsorption, in which solute molecules carrying the appropriate charge displace counter-ions and bind reversibly to the resin surface. Unbound substances can be washed out from the exchanger bed using starting buffer. 3. In the third stage, substances are removed from the column by changing to elution conditions unfavorable for ionic bonding of the solute molecules. This normally involves increasing the ionic strength of the eluting buffer or changing its pH. 3/04/2012

Contd. 4. The fourth and fifth stages are the removal of substances from the column that are not eluted under the previous experimental conditions and reequilibration at the starting conditions for the next purification.

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Ion exchangers  An ion exchanger consists of an insoluble matrix to which charged groups have been covalently bound. The charged groups are associated with mobile counter ions. These counter-ions can be reversibly exchanged with other ions of the same charge without altering the matrix.  It is possible to have both positively and negatively charged exchangers. • Positively charged exchangers have negatively charged counter-ions (anions) available for exchange and are called anion exchangers. 3/04/2012

Contd.. • Negatively charged exchangers have positively charged counter-ions (cations) and are termed cation exchangers.

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Contd

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Contd..

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Contd..  Properties of ion exchangers • They are almost insoluble in water &organic solvent like benzene, ether etc. • They are complex in nature ,i.e. infact they are polymeric. • They have active or counter ions that will exchange reversibly with other similar ions in a surrounding solution without any substantial changes in the material. 3/04/2012

Practical Requirements 1. Column material & dimensions • Columns are generally made up of glass , high quality stainless steel or polymers which are resistant to strong acids & alkalis can be used. • The column length: diameter ratio of 20:1 to 100:1 can be used for high efficiency.

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Contd. 2. Ion exchange resin The selection of ion exchange resin depends upon the following properties, • Type of the ions to be separated – cations or anions. • Nature of the ions to be separated- strong or weak. • Efficiency of the resin. • Particle size of the resin-50-100 or 100-200 mesh is used. • Structural type of the resin-porous, pellicular etc. • Amount of cross linking agent present which decides swelling of the resin. 3/04/2012

Contd.. 3. Packing of the column Wet packing method is used in which resin is mixed with the mobile phase & packed in the column uniformly the sample to be separated is dissolved in the mobile phase &introduced all at once in to the column. 4. Mobile phase :- different strength of acids, alkalis & buffers are used as eluting solvents e.g. 0.1 N Hcl,1N NaoH, phosphate buffer, acetate buffer etc.& organic solvents are less useful. 3/04/2012

Contd.. 5. Development of the chromatogram & elution • After introduction of the sample, development of the chromatogram is done by using different mobile phases, there are two elution techniques i.e. a) isocratic elution( same solvent composition) b) gradient elution (different solvent composition)

Gradient elution is usually used for complex mixtures.

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Contd.. 6. Analysis of the elute Different fractions collected with respect to volume or time is analysed for their contents . Several methods of analysis can be used which depends upon the nature and quantity of the sample. They are spectrophotometric method , polarographic method conductometric method amperometric method, flam photometric method, radiochemical methods etc. after analysing similar fractions are mixed inorder to get pure ion or compound of each type. 3/04/2012

Contd.. 7. Regeneration of the ion exchange resin The ion exchange resin after separation may not be useful for next separation as exchangeable functional groups are lost. But due to the cost of the ion exchange resin, they cannot be disposed off. Hence regeneration of the rein is most important. Regeneration makes the used ion exchange resin to be as efficient as a vergin resin.

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Contd.. Regeneration refers to the replacement of the exchangeable cations or anions present in the original resin. Regeneration of; • Cation exchange resin is done by charging the column with strong acid like Hcl. • Anion exchange resin is done by using KOH, NaoH.

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Factors affecting ion exchange separations  There are two major factor that affect ion exchange separations are, 1. Nature & properties of ion exchange resins cross linking & swelling is important factor, when more cross linking agent is present they are more rigid but swells less & results separations of ions of different sizes is difficult as they can not pass through the pores present and it becomes selective to ions of different sizes. 3/04/2012

Contd.. When less cross linking agent is present they are less rigid but swells more & separation will not be efficient as exchange of functional groups dose not take place due to wide pores. Hence an optimum quantity of cross linking agent should be added to the polymeric ion exchange resins for the separation to be effective. 2. Nature of exchanging ions a. Valency of ions:- at low concentration & at ordinary temperature extent of exchanges increases with increase in valency. 3/04/2012

Contd.. b. Size of ions:-for similar charged ions exchange increases with decrease in the size of hydrated ion. Li ˂ H ˂ Na ˂ NH ˂ K ˂ Rb ˂ Cs c. Polarizability:- exchange is preferred for greater polarizable ions. I ˂ Br ˂ Cl ˂ F d. Concentration of solution:- in dilute solution polyvalent ions are generally absorbed preferentially. 3/04/2012

Resolution in ion exchange chromatography  The resolution is defined as the distance between peak maxima compared with the average base width of the two peaks. Elution volumes and peak widths should be measured with the same units to give a dimensionless value to the resolution(Rs).  If Rs = 1.0 then 98% purity has been achieved at 98% of peak recovery.  If Rs = 1.5 then purity of the peak is 100%.

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Contd.. adsorption

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Contd..  The resolution achievable in a system is proportional to the product of the selectivity, the efficiency and the capacity of the system, the three most important parameters to control in column chromatography. The analytical expression for Rs is:

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Capacity factor  The capacity or retention factor (k) is a measure of the retention of a component. The capacity factor is calculated for each individual peak,

(K) =

VR1 -Vt Vt

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Contd..

YR2 3/04/2012

Contd..  Hypothetical chromatogram. V0 = void volume, VR1 = elution volume for peak 1, VR2 = elution volume for peak 2, Vt = total volume, wb1 = peak width for peak 1, wb2 = peak width for peak 2.

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Efficiency  The column efficiency is related to the zone broadening which occurs on the column and can be calculated from the expression:

No. of Theoretical plate 3/04/2012

Contd.. • Column efficiency is expressed as the number of theoretical plates (N) for the column under specified experimental conditions. Efficiency is frequently stated as the number of theoretical plates per meter chromatographic bed, or expressed as H (height equivalent to a theoretical plate, HETP), which is the bed length (L) divided by the plate number. H = L/N 3/04/2012

Contd.. • In the case of ion exchange chromatography, efficiency is measured under isocratic conditions, using a substance which does not interact with the matrix, e.g. acetone.

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Selectivity  The selectivity (α) defines the ability of the system to separate peaks i.e. the distance between two peaks. The selectivity factor can be calculated from the chromatogram using the expression,

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Contd.. • Good selectivity is a more important factor than high efficiency in determining resolution. since Rs is linearly related to selectivity but quadratically related to efficiency. This means that a four fold increase in efficiency is required to double the resolution under isocratic conditions.

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Contd.. • Selectivity in ion exchange chromatography depends not only on the nature and number of the ionic groups on the matrix but also on the experimental conditions, such as pH and ionic strength. It is the ease and predictability with which these experimental conditions, and thus the selectivity, can be manipulated which gives ion exchange chromatography the potential of extremely high resolution.

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Application of ion exchange chromatography 1) Separation of similar ions from one another. eg. A mixture of Li+,Na+,K+ can be separated by passing their solution through cation exchanger, subsequently 0.1N Hcl has been used as eluent. 2) Removal of interfering radicals in quantitative analysis:In the estimation of Ca+2,Ba+2 ions by the oxalate or sulphate method phosphate ion is found to interfere which can be removed by passing the solution through H2So4. 3/04/2012

Contd.. 3) Softening of hard water:- removal of monovalent & divalent ions like Na, K, Ca, Mg etc. 4) Complete demineralization of water:- removal of different ions to get demineralised water. 5) Separation of lanthanides & actinides. 6) Purification of organic compound extracted in water:-many natural products extracted in water have been found to be contaminated with ions which can be removed by using ion exchange process 3/04/2012

Contd.. 7) Separation of amino acids:- the complex mixture of 18 amino acids obtained by the acid hydrolysis of proteins can be separated by ion exchange chromatography. 8) Preparation of pure reagents:-carbonate is invariably present in solution of sodium hydroxide used for volumetric determination which can be removed by ion exchange chromatography.

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Contd.. 9. Hydrometallurgy:-many metals are recovered and purified by ion exchange eg. Uranium, thorium, gold, silver etc.. 10. Biochemical separations:- like isolation of some drugs or metabolites from blood, urine etc.

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References 1. Gurdeep .R Chatwal, Sham K. Anand;instrumental method of chemical analysis(Analytical chemistry) Page. No.2.662-2.672. 2. Ion exchange chromatography amarshampharmacia biotech.

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