Ion exchange chromatography

Principles of Ion -exchange chromatography, High performance liquid chromatography (HPLC) , chromatography generally stands for a technique which separates mixtures based on different dynamic sharing of their components between two distinct physio-chemical environments called mobile and stationary phase by repeated absorption/desorption steps. Ion chromatography (IC) is a member of large family of liquid phase
chromatographic methods (that is a mobile phase is a liquid and a stationary phase is a
solid).

1. Ion -exchange chromatography They could not drink of the water of Marah , for they were bitter...And he cried unto the LORD; and the LORD shewed him a tree , which when he had cast into the waters, the waters were made sweet... Exodus , Chapter 15, versus 23-25

2. Introduction to chromatography separates mixtures by repeated absorption /desorption steps components weakly held by the stationary phase move quicker down the column difference in migration rates through the column results cause distinct bands for sample components

3. Classification of Chromatography based on the mobile phase of the system (liquid or gas) and stationary phase (liquid or solid) Liquid-liquid eg . HPLC liquid-solid, eg . silica column chromatography, Ion chromatography (IC) gas-liquid , eg . GC gas-solid eg . Molecular sieves to purify gases

4. Classification of Chromatography based on the physical equipment used .   Column chromatography - flow through a packed column. Paper chromatography - selective migration of compounds across a plane of paper. Thin-layer chromatography - flow of a mobile phase through a small gap created by two planes of stationary material.

5.   subclasses of column chromatography based on column packing. Ion-exchange chromatography - ion-exchange resin as the adsorbent. Gel chromatography - controlled porosity gel as a packing. Affinity chromatography - certain proteins separated by protein-ligand interactions.

6. Classification of ionic samples based on ion chromatography Neutral sample- compounds that are neutral , eluted from the column without separation . Ionic samples - ( acids, bases, organic salts) ionic solute with organic molecule in protonization (base) or deprotonization (acid) in the pH range 1 – 14.

7. Principles of Ion -exchange chromatography Anions - separated on columns packed with an anion exchange resin. Cations - separated on columns with cation exchange resin Anion exchangers carry a positive charge (quaternary ammonium or amine groups) and separates anionic i.e. acidic compounds. Cation exchangers – ve charge ( sulfonate or carboxylate groups) and separates cations i.e . protonated bases .

8. Functional groups attached on ion exchangers Anion exchangers Functional groups diethyl amino ethyl (DEAE) quarternary amino ethyl (QEA) quarternary ammonium (Q)

9. Functional groups attached on ion exchangers Cation exchangers Functional groups carboxymethyl (CM) Sulphopropyl (SP) Methyl sulphonate (S)

10. Principles of Ion -exchange chromatography separation - reversible adsorption of charged sample molecules to an ion exchanger (matrix) of opposite charge. Adsorption organized by pH or ionic strength of the eluting buffer. four staged mechanism   Equilibration Sample Loading and Adsorption Desorption or Elution Regeneration

11. Equilibration- counter ions are ionically bonded to matrix A stationary phase of an exchange resin covered with counter ions from buffer with low ionic strength. equilibrating the ion exchanger with the starting buffer with desired pH and ionic strength. The pH for charged analyte , to bind resin

12. Sample adsorption – sample molecules displace counter ions the sample loaded into the column charged sample molecules displace the counter ions on the ion exchanger Chargeless unbound substances or the matrix charged (ion exchanger washed through the column.

13. Elution – sample molecules are displaced by ions from eluting buffer Attached solute molecules removed by changing the elution conditions ionic strength or pH of eluting buffer.

14. Elution – sample molecules are displaced by ions from eluting buffer Competition among salt ions (Na+ or Cl -) from the buffer compete with the bound compounds for the charged stationary phase. Retention of acidic or basic samples depends on the pH value . The higher the ionization, the higher retention will be. Lowering the pH will cause the analyte to be less negatively-charged and less likely to interact with the positively-charged anionic exchange resin.

15. Regeneration- matrix is regenerated with original counter ions All the bounded impurities are eluted (washed) from column the ion exchanger is regenerated with the original counter ions.

16. Applications of Ion-exchange chromatography 1. to isolate and purify protein samples For proteins, the charge is based on the protein's isoelectric point, where the protein is neutrally charged ( amphoteric), and measured as pI At a pH above its pI , the compound will be negatively charged, hence an anion exchange support is used, if stable at a pH above its pI , at a pH below its pI , the compound will be positively charged, hence an cation exchange support is used , Anionic exchange Chromatography is carried out with cationic buffers. Cationic exchange Chromatography is carried out with anionic buffers. The pK of the buffer is near to the pH at which the system is buffered.

17. Applications of Ion-exchange chromatography 1. to isolate and purify protein samples After sample adsorption, these can be eluted by Gradient Elution-if the sample components differ widely in retention or if the adsorption of sample mixture is strong, elution is done by selectively decreasing the affinity of the sample molecules for the charged groups on the ion exchanger. changing the composition, pH or ionic strength of the mobile phase over a period of time . elution by changing the pH towards isoelectric point (where no binding occurs) can desorb and elute the sample components from the column by increasing the ionic strength gradually reduces the availability of charged groups and elute the sample components from the column

18. Applications of Ion-exchange chromatography 1. to isolate and purify protein samples Common buffers used

19. Applications of Ion-exchange chromatography 2. water analysis Anion-exchange chromatography can be used to measure the concentration of anions, including sulfates, nitrates, nitrites, fluoride, and chloride. Cation -exchange chromatography is used to measure the concentration of cations such as sodium, potassium, calcium, and magnesium . magnesium and calcium ions in hard water can be removed from water using ion-exchange chromatography in water softeners by binding them to a resin, which releases bound sodium.

20. References Jackson, Peter; Haddad, Paul R. (1990). Ion chromatography: principles and applications. Amsterdam: Elsevier. Tatjana Weiss; Weiss, Joachim (2005). Handbook of Ion Chromatography. Weinheim : Wiley-VCH. https :// www.separations.us.tosohbioscience.com Lucy, C. A. (2003). "Evolution of ion-exchange: from Moses to the Manhattan Project to Modern Times". Journal of chromatography. A. 1000 (1–2): 711–24. Ion Exchange Chromatography Principles and Methods. General Electric Company. 2004. pp.  11–20 Jungbauer , Alois ; Hahn, Rainer (2009). "Chapter 22 Ion-Exchange Chromatography". Guide to Protein Purification, 2nd Edition. Methods in Enzymology. 463 . pp. 349–371. Jenke , D. (2011). "Application of Ion Chromatography in Pharmaceutical and Drug Analysis". Journal of Chromatographic Science. 49 (7): 524–39 . Gjerde , Douglas T.; Fritz, James S. (2000). Ion Chromatography. Weinheim : Wiley-VCH