Brief overview of ion chromatography and its applications

1. Ion
Exchange
Chromatography

2. CONTENTS
1. Brief history and introduction
2. Definition
3. Principle
4. Column Chemistry
5. Types of Ion exchange chromatography
6. General steps in IEC
7. Applications
 Separating proteins
8. Other applications
9. References

3. Brief history and introduction
 Old technique, separates by charge
-in Aristotle time , brakish water can be desalinated with sand
-The first report (1850) of research is by english agriculture
chemist , H.S. Thompson. He found that on passage of a
solution of ammonium sulfate through soil ,some or all the
ammonium was repalced by calcium ion. He called this ion
exchange
-An important advance was made in 1935 when Adams and
Holmes published the first paper on the synthesis of ion
exchange resins.
-AG resin introduced by BioRad in 1957, originally intended
for purification of TMV

4. 4
Definition
 Ion-exchange
chromatography (or
ion chromatography)
is a process that
separates ions and
polar molecules
based on their
affinity to the ion
exchanger.

5. 5
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.

6.  Beaded or Monolithic
 Hydrophilic
 Physically strong
Cellulose Agarose Polystyrene
Polymethacrylate Polyacrylamide
Stationary phase material
Column Chemistry

7. 7
Ion exchangers – Functional groups
Anion exchanger
 Quaternary aminoethyl (QAE-)
 Diethylaminopropyl (DEPE-)
 Diethylaminoethyl (DEAE-)
Cation exchanger
 Methylsulfonate(S-)
 Sulphopropyl (SP-)
 Carboxymethyl (CM-)

8. 8
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 + B + C
- + + - _
+ +-
1. Cation exchange chromatography
2. Anion exchange chromatography.
Types of Ion exchange chromatography

9. Anion exchange chromatography
 Anion exchange chromatography retains anions
using positively charged functional group:
R-X A +M B R-X B + M + A
+ - + - + - + -
9

10. General steps in IEC

11. 1
• A sample is introduced either manually or with an autosampler into a sample loop of
known volume.
2
• The mobile phase (buffered aqueous solution) carries the sample from the loop onto
a column that contains some form of stationary phase material.
3
• Stationary phase material is a resin or gel matrix consisting of agarose or cellulose
beads with covalently bonded charged functional groups.
4
• The target analytes (anions or cations) are retained on the stationary phase and can
be eluted by increasing the concentration of a similarly charged species that will
displace the analyte ions from the stationary phase.
5
• The analytes of interest must then be detected by some means, typically by
conductivity or UV/Visible light absorbance.
6
• A chromatography data system (CDS) is usually needed to control an IC. For
example EMPOWER for waters

12. APPLICATIONS
It can be used for almost any kind of charged
molecule including large proteins, small
nucleotides, nucleic acids and amino acids.
It is often used in protein purification, water
analysis.
Separation conditions are within physiological
range of salt and pH and in the most cases a
native protein can be obtained.
12

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.
 By adjusting the pH or the ionic concentration of
the mobile phase, various protein molecules can be
separated.
13

14. Electrostatic Potential Map of the Surface of a Protein

15. Mechanism of protein separation
 pH based separation:
 Salt based separation:

16. 16
Proteins are charged molecules. At specific pH, it can exist
in anionic (-), cationic (+) or zwitterion (no net charge)
stage.
[H+][OH-] [H+]
Protein becomes increasingly -veProtein becomes
increasingly +ve
[OH-]
pH ~3
At pH 3 the
protein will
be +ve
pH ~7
At pH 7
the protein
will be -ve anionic
cationic
pH =pI

17. 17
 The pH of buffers
should be
one unit below pI for cation exchangers and
one unit above pI anion exchangers
 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
Factors to be considered during protein
separation:

18.  EDTA – For Chelation
 Chaotropic Salts – Eg: Urea for
stabilization
 PEG – For Enhanced Selectivity
 DTT – To Prevent Oxidation
 Use Cationic Buffers in Anion Exchanger :
Eg: Alkyl Amines, Tris, Amino Ethyl Alcohol
 Use Anionic Buffers in Cation Exchanger:
Eg: Phosphate, Acetate,
Citrate, Barbiturate
Buffers Used in Ion Exchange Chromatography
Mobile Phase Modifiers

19. Factors affecting Ion Exchange
Chromatography
pH of mobile phase
Operating pH of Ion Exchange Chromatography (IEX) is mostly 2-9.
Ionic Strength
Mobile Phase Modifiers
Temperature

20. 20
Abstract
A great number of naturally occurring mononucleotides can be separated
and identified by poly(ethyleneimine)-cellulose thin-layer chromatography.
RF data for 33 compounds are given, and the factors are discussed which
influence the mobility under different elution conditions. The method is
compared with other present techniques for separating nucleotides.

21. Separation of Amino Acids:
The free amino acids in human CSF from eighteen subjects have been
determined.
Amino acids always found in readily detectable amounts were: taurine,
threonine, serine, glutamine, glutamic acid, citrulline, glycine, alanine, α-NH2-n-
butyric acid, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine,
ethanolamine, ornithine, lysine, histidine and arginine.

22. Abstract
High-speed liquid chromatography with superficially porous ion-exchange
column packings has been used to separate and quantitatively analyze the water
soluble vitamins nicotinic acid, thiamine, riboflavin, ascorbic acid, folic acid,
pyridoxine, pyridoxal and pyridoxamine. Chromatograms and chromatographic
conditions for these as well as the phosphoric acid esters of riboflavin and
pyridoxamine are shown. The reproducibility of retention time and peak area has
been demonstrated to be better than 1.5% while the lower limits of sensitivity for
the test vitamin compounds were better than 50 nanograms. Advantages of the
technique include fast analysis times and minimum of sample clean up.
Applications of the methods to vitamin analysis of food and pharmaceutical
products are discussed.
Analysis of Water-Soluble Vitamins by High-
Speed Ion-Exchange Chromatography
R
R.C. Williams
D.R.Baker
J.A.Schmit

23. 0ther applications
Ion exchange chromatography is used to convert
one salt to other.
Eg; we can prepare tetra propyl ammonium
hydroxide from a tetra propyl salt of some other anion.
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.

24.  For the measurement of drugs and their metabolites in serum
and urine, for residue analysis in food raw materials.
 For the measurement of additives such as vitamins and
preservatives in foods and beverages.
 Separation of similar ions
A mixture of sodium, hydrogen and potassium can be separated using
cation exchanger resin.
A mixture of Chloride, bromide, and iodide can be separated using basic
anion exchange resin.

25. References
 Ion Exchange in Analytical Chemistry: International
Series of Monographs in in analytical chemistry
volume 38 By William Rieman, Harold F. Walton.
 Himmelhoch, SR (1971) Chromatography of proteins
on ion-exchange adsorbents Meth. Enzymol 22:273-
286.
 Scopes, RK (1982) Ion exchangers-principles,
properties and uses. In “Protein Purification:
Principles and Practice” , pp75-101. Springer-Verlag,
New York.
 Practical HPLC method development,2nd Edition,
Lloyd r. snyder,pno.341-346
 Principles of instrumental analysis , skoog , latest
edition, pno. 641-647