Ion exchange chromatography

Definition Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules.

History of IE Chromatography Ion Chromatography (IC) was introduced in 1975 by Small, Stevens, and Bauman as a new analytical method. In 1979, Fritz et al. described an alternative separation and detection scheme for inorganic anions, in which the separator column is directly coupled to the conductivity cell. At the end of the 1970s, ion chromatographic techniques were used to analyze organic ions for the first time. The 1980s witnessed the development of high efficiency separator columns with particle diameters between 5 µm and 8 µm, which resulted in a significant reduction of analysis time.   Since the beginning of the 1990s column development has aimed to provide stationary phases with special selectivity.

Types of Ion Chromatography 1. Ion-Exchange Chromatography (HPIC) (High Performance Ion Chromatography) 2. Ion-Exclusion Chromatography (HPICE) (High Performance Ion Chromatography Exclusion) 3. Ion-Pair Chromatography (MPIC) (Mobile Phase Ion Chromatography)

1. Ion-Exchange Chromatography (HPIC) (High Performance Ion Chromatography) This separation method is based on ion-exchange processes occurring between the mobile phase and ion-exchange groups bonded to the support material. Ion-exchange chromatography is used for the separation of both inorganic and organic anions and cations.

Ion-Exclusion Chromatography (HPICE) (High Performance Ion Chromatography Exclusion) Ion-exclusion chromatography is particularly useful for the separation of weak inorganic and organic acids from completely dissociated acids which elute as one peak within the void volume of the column. In combination with suitable detection systems, this separation method is also useful for determining amino acids, aldehydes, and alcohols.

Ion-Pair Chromatography (MPIC) (Mobile Phase Ion Chromatography) The dominating separation mechanism in ion-pair chromatography is adsorption. The stationary phase consists of a neutral porous divinylbenzene .resin of low polarity and high specific surface area Ion-pair chromatography is particularly suited for the separation of surface-active anions and cations, sulfur compounds, amines, and transition metal complexes.

The matrix 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. 1-Positively charged exchangers have negatively charged counter-ions (anions) available for exchange and are called anion exchangers. 2-Negatively charged exchangers have positively charged counter-ions (cations) and are termed cation exchangers.

CLASSIFICATION OF ION EXCHANGERS پلی ساکاریدی Anion exchanger دی اتیل آمینو سلولز (DEAE-cellulose) Cation exchanger کربوکسی متیل سلولز (CM. Cellulose) سنتتیک : وینیل بنزن، دی وینیل بنزن An almost unlimited variety of resins with different compositions and degrees of cross linking can be prepared. The resins consist of an elastic three-dimensional network of hydrocarbons which carry fixed ionic groups In a cation exchanger, the matrix carries ionic groups like − SO3−, − COO−, − PO3 and in an anion exchanger, it carries groups such as −NH3+, >NH2 , > N+

Ion Exchangers

Ion exchangers – Functional groups Anion exchanger Aminoethyl (AE-) Diethylaminoethyl (DEAE-) Quaternary aminoethyl (QAE-) amphoteric exchangers snake- cage Polyelectrolytes Cation exchanger Carboxymethyl cellulose (CM-cellulose) Sulphopropyl (SP-)

Cation exchange chromatography Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group - + + - _ - + + R-X C +M B R-X M + C + B

Anion exchange chromatography Anion exchange chromatography retains anions using positively charged functional group: _ - - - + + + + R-X A +M B R-X B + M + A

Important Points to Consider For Ion Exchange Chromatography Preparation of resin pH of buffers Buffer selection Elution PH change in ionic strength

procedure

Procedure Dr Gihan Gawish

Effect of pH in the separation of proteins By adjusting the pH or the ionic concentration of the mobile phase, various protein molecules can be separated. For example, if a protein has a net positive charge at pH 7.0, then it will bind to a column of negatively-charged beads, whereas a negatively charged protein would not.

Effect of pH in the separation of proteins Proteins are charged molecules. At specific pH, it can exist in anionic (-), cationic (+) or zwitterion (no net charge) stage. anionic cationic pH =pI pH increase *pI isoelectric point

Choosing your ion-exchanger: know your proteins Stability of proteins stable below pI value, use cation-exchanger stable above pI value, use anion-exchanger Molecular size of proteins <10,000 MW, use matrix of small pore size 10,000-100,000 MW, use Sepharose equivalent grade

Detection In Ion Chromatography 1. Conductivity detection  2. Electrochemical (amperometric or coulometric) detection  3. Potentiometric detection  4. Spectroscopic detection  5. Post-column reaction detection.

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 performance of any detector is evaluated according to the following criteria: • Sensitivity • Linearity • Resolution (detector cell volume) (• Noise (detection limit

Some advantage of strong ion exchangers are: • Sample loading capacity does not decrease at high or low pH values due to loss of charge from the ion exchanger . • A very simple mechanism of interaction exists between the ion exchanger and the solute. • Ion exchange experiments are more controllable since the charge characteristics of the media do not change with changes in pH. This makes strong exchangers ideal for working with data derived from electrophoretic titration curves. Ion-exchange chromatography separates proteins by charge under near physiological and non-denaturing conditions. its widespread applicability, its high resolving power

Some advantage of strong ion exchangers are: . Speed • Sensitivity • Selectivity • Simultaneous detection • Stability of the separator columns

Application i) Separation of metal ions and anions ii) Separation of organics iii) Separation of ionized from nonionized iv) Separation of actinide elements v) Miscellaneous applications.

Enzyme The recovery of creatine kinase in this separation was 89%.

isoenzyme

Immunoglobulins