PPT On Ion Exchange Chromatography

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Ion Exchange Chromatography Presentation Transcript:
1.Chromatography

2.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.

3.Ion-exchange chromatographyThe solution to be injected is usually called a sample, and the individually separated components are called analytes
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
It is often used in protein purification, water analysis.

4.Principle
Ion exchange chromatography retains analyte molecules based on ionic interactions.
The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge.
This type of chromatography is further subdivided into:
cation exchange chromatography
anion exchange chromatography.

5.Ion exchange mechanism
  Composed of five steps:
Diffusion of ion to the exchanger surface. 
     (occurs quickly in homogenous solution)
Diffusion of ion through the matrix structure to exchange site (depends on degree of cross-linkage and conc. of solution.

6.3. Exchange of ions at the exchange site. (instantaneous and is an equilibrium process)
4. Diffusion of the exchanged ion through the exchanger to the surface.
5. Selective desorption by the eluant and diffusion of the molecule into  external solution.(By changes in pH or ionic conc. or affinity elution).

7.Ion Exchangers

8.Cation exchange chromatography
Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group

9.Anion exchange chromatography retains anions using positively charged functional group:

10.Procedure
A sample is introduced, either manually or with an autosampler, into a sample loop of known volume.
The mobile phase (buffered aqueous solution) carries the sample from the loop onto a column that contains some form of stationary phase material.
Stationary phase material is a resin or gel matrix consisting of agarose or cellulose beads with covalently bonded charged functional groups.

11.The target analytes (anions or cations) are retained on the stationary phase but can be eluted by increasing the concentration of a similarly charged species that will displace the analyte ions from the stationary phase.        For example, in cation exchange chromatography, the positively charged analyte could be displaced by the addition of positively charged sodium ions.

12.The analytes of interest must then be detected by some means, typically by conductivity or UV/Visible light absorbance.
A chromatography data system (CDS) is usually needed to control an IC.

13.Separating proteins
Proteins have numerous functional groups that can have both positive and negative charges.
Ion exchange chromatography separates proteins according to their net charge, which is dependent on the composition of the mobile phase.

14.Affect 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, then it will bind to a column of negatively-charged beads, whereas a negatively charged protein would not.

15.Proteins are charged molecules. At specific pH, it can exist in anionic (-), cationic (+) or zwitterion (no net charge) stage.