assignment ion exchange chromatography
- 1. ION EXCHANGE CHROMATOGRAPHY Introduction : Ion exchange chromatography is used to remove ions of one type from a mixture and replace them by ions of another type . The column is packed with porous beads of a resin that will exchange either cations or anions. There is one type of ion on the surface of the resin and these are released when other ions are bound in their place – e.g. a basic anion exchange resin might remove nitrate ions (NO3–) from a solution and replace them with hydroxide ions (OH–). Principles: • Separation is based on the attraction between oppositely charged particles. • Net charge exhibited by compound is dependent on their pKa and pH of the solution in accordance with HENDERSON HASSELBACH EQUATION. • Ion exchangers special type of polyelectrolyte and consist of to which are bonded a large number of electrically charged group. • Classification: Ion exchangers Based on the inorganic group Based on the nature of the source Based on the structure Anion exchangers: Anion exchangers contain bound positive groups, where as cation exchangers contain bound negative groups. Cation exchangers: Cationic exchangers are useful for separation of cations such as protonated bases and anion exchange columns are used for anions or acidic samples. Fig: Ion exchange chromatography
- 2. Resin: • 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 to 16 percent to increase the extent of cross linking. • Benzene groups are modified to produce cation exchange resin and anion exchange resin. Column & media preparation: Equilibrate column with 5–10 column volumes of start buffer or until the baseline, eluent pH and conductivity are stable. Using prepacked columns is highly recommended to ensure the best performance and reproducible results. An evenly packed column ensures that component peaks are not unnecessarily broadened as sample passes down the column so that the best resolution can be achieved. Sample applications: Desalt the sample and transfer to the buffer Adjust the sample to the chosen starting pH and ionic strength and apply to the column. Sample volume should be based on the capacity of ion exchange resins For protein samples maximum concentration of about 50- 70mg/ml can be used. Sample loading: Apply samples directly to the column via a chromatography system, a peristaltic pump or a syringe. The choice of equipment depends largely on the sample volume, the size of column, the type of IEX medium and the requirements for accuracy in gradient elution. Ensure that the top of the column bed is not disturbed during sample application Do not change buffer conditions until all unbound material has been washed through the column (monitored by UV absorbance) and until UV and conductivity values have returned. Re Equilibration: Wash with 5 column volumes of 1 M NaCl (100%B) to elute any remaining ionically-bound material. Include a wash step at the end of every run in order to remove any molecules that are still bound to the medium. Monitor UV absorbance so that the wash step can be shortened or prolonged, as necessary.
- 3. Re-equilibrate with 5–10 column volumes of start buffer or until eluent pH and conductivity reach the required values. A re-equilibration step after washing returns the column to start conditions before applying further samples. Whenever possible, monitor pH and conductivity to check when start conditions have been reached. The re-equilibration step can then be shortened or prolonged as necessary. Analysis of eluent: Spectrophotometric method. Flame photometry Conductometric methods Radio isotope method Advantages: • Detectability: useful for the detection of many in-organic 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- metallic Disadvantages: • Column efficiency is less • It is difficult to achieve control over selectivity and resolution • Stability and reproducibility of the columns become questionable after repeated use. Applications: • Ion exchange chromatography is used to convert one salt to other. • It is useful for pre concentration of trace components of a solution to obtain enough for analysis. • Ion exchange is used to prepare de-ionized water. • Water polishing equipment used in many laboratories uses several ion exchange cartridges.
- 4. Conclusion: Ion exchange chromatography has many advantages. This method is widely applicable to the analysis of a large number of molecules with high capacity. The technique is easily transferred to the manufacturing scales with low cost. High levels of purification of the desired molecule can be achieved by ion exchange step. Follow- up of the nonsolvent extractable natural products can be realized by this technique. References: Instrumental analysis of chemical analysis by Gurudeep R.Chatwal, Sham.K.Anand, Pg. 2.662- 2.672 Instrumental methods of chemical analysis by B.K.sharma, C-123 -150