High-effective IONIC CHROMATOGRAPHY Principles and Basics

1. High-effective IONIC
CHROMATOGRAPHY
Eng. Mustafa Ahmed Khaldoun Termanini

2. The main titles:
Definition of High-effective Ionic Chromatography.
Separation principle.
Types of High-effective Ionic Chromatography.
Instrumentation of High-effective Ionic
Chromatography.
Steps of the process of using High-effective Ionic
Chromatography.
Applications of High-effective Ionic Chromatography.

3. Definition of High-effective Ionic Chromatography
- It is an analytical technical process by
which ionic compounds are separated
based on their charge and affinity with ion
exchangers.
Separation principle:
The reverse exchange between the
ionic compounds present in the
mobile phase (liquid) that contains the
sample and the stationary phase
(solid) which contains insoluble
substances and ionic sites.

4. TYPES OF High-effective
IONIC CHROMATOGRAPHY

5. A. Cation exchange chromatography:
- in it, the stationary phase (solid supports) have negatively charged functional groups
It is used to separate molecules that have a positive charge.
R-X−𝐂+ + 𝐌+B− ⇄ R-X−𝐌+ + C+ +B−
- Commonly used cation exchange resins are S-resin, sulfate derivatives, and CM resins,
carboxylate derived ions.

6. B. Anion exchange chromatography:
- in it, the stationary phase (solid supports) have positively charged functional group
It is used to separate molecules that have a negative charge.
R-X+𝐀− + M+𝐁− ⇄ R-X+𝐁− + M+ + 𝐀−
- Commonly used anion exchange resins are Q-resin, a Quaternary amine; and DEAE resin, Diethyl
Aminoethane.

7. Instrumentation of High-effective Ionic
Chromatography
Instrumentation includes:
1. Pump.
2. Injector.
3. Column.
4. Suppressor.
5. Oven(optional).
6. Detector.
7. Recorder or data system.

8. 1. PUMP:
• Is one of the most important components
in the system.
• Which must provide a continuous constant
flow of the eluent through the injector,
column, and detector.
• A high-pressure pump with pressure and
flow indicator, to deliver the eluent.
• The pressure required for most instruments
is at least 600 psi up to 4000 psi.

9. 2. INJECTOR:
- The sample is manually injected into the column by injection valve using a syringe.
- Or automatically by using an automatic sampler.

10. 3. COLUMN:
• Used to separate the sample mixture into the individual components.
• its diameter from about 2mm to 5 cm and in length from 3 cm to 50cm.
• is made of glass, stainless steel, titanium, or polymers.

11. The suppressor reduces the background conductivity of the chemicals used
to elute samples from the ion-exchange column which improves the
conductivity measurement of the ions being tested and enhancing the
sensitivity.
4. OVEN (optional).
5. SUPPRESSOR:

12. 6. DETECTORS:
Detection methods include:
• Mass spectrometry.
• Atomic spectroscopy.
• Fluorescence.
• Luminescence.
• UV-vis, potentiometric.
• Electrical conductivity detector is
commonly used.
- Conductivity detector is the measure of a
material’s ability to conduct electricity.
Since conductivity is proportional to the
number of ions in solution, it is the primary
method of detection for ion chromatography.

13. 7. RECORDER OR DATA SYSTEM:
- For collecting and organizing the
chromatograms and data such as
a data station or minicomputer.

14. Steps of the process of using
High-effective Ionic Chromatography:

15. Steps of the process of using High-effective Ionic Chromatography:
1. Eluent Loading
2. Sample injection
3. Separation of sample
4. Elution of analyte A
5. Elution of analyte B
: Eluent ion .
: Ion A .
: Ion B .

16. Steps of the process of using High-effective Ionic Chromatography:
1. Eluent Loading:
The eluent loaded onto the column displaces any
anions bonded to the resin and saturates the resin
surface with the eluent anion.
2. Sample injection:
A sample containing anion A and anion B are injected
in the column. This sample could contain many
different ions. : Eluent ion .
: Ion A .
: Ion B .

17. Steps of the process of using High-effective Ionic Chromatography:
3. Separation of sample:
After the sample has been injected, the continued
addition of eluent causes a flow through the column.
As the sample elutes (or moves through the column),
anion A and anion B adhere to the column surface
differently. The sample zones move through the column
as eluent gradually displaces the analytes.
: Eluent ion .
: Ion A .
: Ion B .

18. Steps of the process of using High-effective Ionic Chromatography:
4. Elution of analyte A:
As the eluent continues to be added, the
anion A moves through the column in a band
and ultimately is eluted first.
5. Elution of analyte B:
The eluent displaces anion B, and anion B is
eluted off the column. : Eluent ion .
: Ion A .
: Ion B .

19. Stationary phase:
usually made from inert organic matrix chemically derivative with ionizable functional
groups (fixed ions) which carry displaceable oppositely charged ion.
The main classes of substances used are:
• Inorganic salts , Zeolites.
• Metal oxides and cellulose.
• Modified organic polymer resins, modified silica gels, (The most used).
Mobile phase:
Acids, alkalis, Buffers. Such as:
HCl, NaOH, phosphate buffers, acetate buffers, borate buffers, phthalate buffers.

20. RESINS
- Are made of polystyrene sulfonate.
CLASSIFICATION OF RESINS:
According to the chemical nature they classified as:
1. Strong cation exchange resin – Sulphonic acid.
2. Weak cation exchange resin – Carboxy methyl
compound.
3. Strong anion exchange resin – Quaternary ammonium
compound.
4. Weak anion exchange resin - Diethyl aminoethyl
compound.
Regeneration of ion exchange resin:
The procedure is performed using strong acids and alkalis such as
hydrochloric acid and sodium hydroxide

21. Applications of
High-effective Ionic Chromatography

22. Applications of High-effective Ionic Chromatography:
1. Softening of hard water:
The hardness of water is due to the presence of
𝐂𝐚+𝟐, 𝐌𝐠+𝟐, and other divalent ions that may be
removed by passing the hard water through the
cation exchanger charged with 𝐍𝐚+ ions.
2. Complete demineralization of water:
The ions (cations and anions) are completely
removed by passing water through two cation and
anion exchangers.

23. Applications of Ion High-effective Ionic Chromatography:
3. Separation of amino acids and protein and
small nucleotides:
Each amino acids and protein have
Isoelectric Point PI:
It is a point at which protein charges are equal
to those of the surrounding medium, so that
proteins precipitate, for example casein
precipitates at a pH of 4.6, and is used in the
separation of proteins and amino acids.

24. Applications of High-effective Ionic Chromatography:
4. Separation of different ions.
5. Other applications:
• For the measurement of various active ingredients in medicinal formulations.
• 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 in food and beverages.

25. References

26. References:
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[8] P. Stanton, “3,” vol. 251.
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10.1007/BF02263061.
[10] S. Together, “Separation of proteins with anion exchange chromatography on Sepapure Q
and DEAE Separation of proteins with anion exchange chromatography on Sepapure Q and
DEAE,” pp. 1–4.
[11] P. Of and I. O. N. Exchange, “Instrumentation PRINIPLES OF ION EXCHANGE.”
[12] Mohammed and JS, “A brief review on Ion Exchange Chromatography,” PharmaTutor, vol. 5,
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review-on-ion-exchange-chromatography.
[13] O. Bahadir, “Ion-Exchange Chromatography and Its Applications,” Column Chromatogr.,
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27. THANK YOU
Eng. Mustafa Ahmed Khaldoun Termanini