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Ion exchange chromatography
- 1. ION EXCHANGE CHROMATOGRAPHY SAKEENA ASMI MAHATMAGANDHI UNIVERSITY
- 2. • The firstdetailed description of chromatography is generally credited to Michael Tswett, a Russian biochemist, who separated chlorophyll from a mixture of plant pigments in 1906. • Because of the nature of the pigments in the sample, each band had a distinctive color. • Thus the name of the process was coined from the Greek words for color (chromo) and to write (graphy). • If the individual components of a mixture have widely dissimilar physical and chemical properties, it is very easy to separate one from another. CHROMATOGRAPHY
- 3. • But asthe individual components of a mixture get more and more similar in physical and chemical properties, it becomes increasingly difficult to separate them from one another. • However it can be readily achieved using chromatography. • The feature common to them all is that two mutually immiscible phases are brought into contact with each other. • One of these phases is stationary, while the other is mobile. • The mobile phase either moves over the surface or percolates through the interstices of the stationary phase.
- 4. PARTITION COEFFICIENT• Partition Coefficient(also known as distribution coefficient) is a definitive term normally used to describe the way in which a given compound distributes or partitions itself between two immiscible phases, the stationary and the mobile phase. K = Cs Cm where Cs and Cm are the concentrations of the compound in the stationary and the mobile phases respectively. • This concept of Partition Coefficient is the basic principle of all chromatographic methods.
- 5. TECHNIQUES OF CHROMATOGRAPHY • Thereare two basic techniques of chromatography : plane chromatography and column chromatography. PLANE CHROMATOGRAPHY • There are two variations of plane chromatography: paper chromatography and thin layer chromatography. • In paper chromatography the stationary phase is supported by cellulose fibres of the paper sheet. • In thin layer chromatography the stationary phase is coated onto a glass or plastic surface.
- 6. COLUMN CHROMATOGRAPHY • Incolumn chromatography the stationary phase is packed into a glass or metal column. • The stationary phase is either coated onto discrete small particles (the matrix) and packed into the column or applied as a thin film to the inside wall of the column. • As the eluent flows through the column the analytes separate on the basis of their distribution coefficients and emerge individually in the eluate as it leaves the column.
- 7. Avinash Upadhyay -Biophysical Chemistry-Principles & Techniques LIQUID
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- 9. INTRODUCTION • This techniquehas been developed since 19th century which was firstly used for purifying the drinking water. • Ion exchange chromatography is a distinct principle of chromatography performed in the column. • Ion exchange chromatography may be defined as the reversible exchange of ions in the solution with ions electrostatically bound to some sort of insoluble matrix or a stationary phase. • This technique is extremely useful in the separation of charge compound like proteins differing by only one charged amino acid.
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- 11. PRINCIPLE • Ion exchangechromatography relies on the attraction between oppositely charged stationary phase, known as an ion exchanger and analyte. • It is frequently chosen for the separation and purification of proteins, peptides, nucleic acids, polynucleotides and other charged molecules, mainly because of its high resolving power and high capacity. • There are two types of ion exchanger, namely cation and anion exchangers. • Cation exchangers possess negatively charged groups and these will attract positively charged cations. These exchangers are also called acidic ion exchangers.
- 12. PRINCIPLEContinued… • Anion exchangershave positively charged groups that will attract negatively charged anions. The term basic ion exchangers is also used to describe these exchangers. • The ion exchanger consists of an inert support medium coupled to positive (anion exchanger) or negative (cation exchanger) functional groups. • To these covalently bound functional groups the oppositely charged ions are bounded (mobile counter ion), which will be exchanged with like charge ions in the sample having charge magnitude more than the ions bounded to the matrix.
- 13. WORKING • Consider acolumn having E- Y+ cation exchanger in which E- is negative charged exchanger and Y+ is the mobile counter ion. • Let X+ be the cation in the sample having charge greater than Y+ . • The X+ ion can exchange sites with the counter ion Y+ with satisfying the following relationship : E- Y+ + X+ E- X + + Y+
- 14. Continued… Ion Exchange Chromatography(Cation Exchange)
- 15. Continued… • Bounded interestof ion (X +) can now be eluted by either of the two ways; 1. By adding the component Y+ having magnitude of charge more than that of X + so that Y+ will replace X + and X + will be eluting out. 2. By changing pH of the solvent (mobile phase) so that X + have no charge and is then unbounded from the matrix and can be eluted out.
- 16. Ion Exchange Chromatography(Anion Exchange)
- 17. ION EXCHANGERS • Anion exchanger consists of two basic components : 1. Ion exchange resin 2. Exchange medium (Cation exchanger and Anion exchanger) - Charged functional group. - Mobile counter ion.
- 18. ION EXCHANGE RESIN Two main groups of materials are used to prepare ion exchange resins : • Polystyrene • Cellulose Resin made from both of these materials differ in their flow properties, ion accessibility and chemical and mechanical stability. Selection of one or the other type of resin is done on the basis of compounds being separated.
- 19. 1. POLYSTYRENE • Polystyreneresins are prepared by polymerisation reaction of styrene and divinyl benzene. • Higher concentrations of divinyl benzene produces higher cross linkages. • Polystyrene resins are very useful for separating small molecular weight compounds, however, unsatisfactory for the separation of macromolecules.
- 20. 2. CELLULOSE • Celluloseis a high molecular weight compound which can be readily obtained in a high pure state. • Cellulose has much greater permeability to macromolecules.
- 21. EXCHANGE MEDIUM • Thechoice of ion exchangers depends upon the stability, molecular weight and ionic strength of the sample components. • The ion exchanger are packed in column having suitable buffer. • The ion exchangers are of two types : 1. Anion Exchangers 2. Cation Exchangers
- 22. 1. Anion Exchangers •The anion exchangers have positively charged exchanger with negatively charged mobile counter ion available for exchange. • If the basic functional groups are introduced, the resin becomes anion exchanger. • Tertiary amines Strong anion exchangers • Secondary amines Weak anion exchangers
- 23. Examples : AnionExchangers Source : Wilson K & Walker J (2010). Principles and Techniques of Biochemistry and Molecular biology.
- 24. 2. Cation Exchangers •The cation exchangers have negatively charged exchanger with positively charged mobile counter ion available for exchange. • If acidic functional group are introduced, then the resin becomes cation exchangers. • Sulphonic acid Strong cation exchangers • Carboxylic acid Weak cation exchangers
- 25. Examples : CationExchangers Source : Wilson K & Walker J (2010). Principles and Techniques of Biochemistry and Molecular biology.
- 26. PREPARATION OF ION EXCHANGERS •Preparation of the exchange medium is essential for satisfactory performance of ion exchange chromatography. • Apart from removing impurities, there are three major steps that are absolutely important in ion exchanger preparation. 1. SWELLING: For anion exchangers it is done by treating it first with an acid (0.5N HCl) and then with base (0.5N NaOH). Reverse is true for preparation of cation exchangers.
- 27. 2. REMOVAL OFFINE PARTICLES: Large number of such particle can result in decreased flow rate and improper resolution. For this the exchangers are repeatedly suspended in large volume of water. 3. ADDITION OF COUNTER IONS: Accomplished by washing the exchanger with suitable reagent depending upon which counter ion to be introduced. For e.g : NaOH (Na+), HCl (H+) etc…
- 28. CHOICE OF BUFFER •The choice of buffers which maintain the pH of the column is dictated by the compounds to be separated and the type of ion exchange being carried out (anionic or cationic). • Anion exchange chromatography should be carried out with cationic buffers. Reverse is true for cation exchange chromatography. • If anion exchange or cation exchange is carried out with anionic or cationic buffers respectively, the buffer ions will indulge in ion exchange and hamper sample component exchange. • The pH of the buffer should impart the same charge to the sample ions as is present on the counterion.
- 29. Some Volatile BuffersUsed in Ion-Exchange Chromatography Avinash Upadhyay - Biophysical Chemistry-Principles & Techniques
- 30. APPLICATIONS • Softening ofhard water. • Demineralization of water. • To analyze base composition of nucleic acid. • To concentrate the metal ions in the sample. • To measure the additives in food and drug sample. • To separate protein mixtures.
- 31. ADVANTAGES • Detectability: usefulfor the detection of many inorganic salts and also for the detection of organic ions with poor UV absorptivity like alkyl amines or sulfonates. • Preparative separations: usually preferred because of the availability of volatile buffers. Volatile buffers makes the removal of mobile phase easier. • Useful to resolve very complex samples, i.e in the case of multi step separation. • Useful for separation of mixtures of biological origin, in organic salts and some organo – metallics.
- 32. • Cost effective •Re-usable • Easily collectable • Low maintenance cost • Efficient technique • Quick separation
- 33. DISADVANTAGES • Column efficiencyis less. • It is difficult to achieve control over selectivity and resolution. • Stability and reproducibility of the columns become questionable after repeated use.
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