Ion exchange chromatography and gec

1. ION EXCHANGE CHROMATOGRAPHY
&
GEL PERMEATION CHROMATOGRAPHY
Presented by: Under the Guidence of
G.Shekhar, Mr.Uttam Prasad,
M.Pharm (Ceutics) Dept. Of Pharmaceutical Analysis,
256213886011. Malla Reddy College Of Pharmacy.

2. ION EXCHANGE CHRoMATOGRAPHY
• INTRODUCTION
• PRINCIPLE
• MECHANISM
• INSTRUMENTATION
• ION EXCHANGERS
• CLASSIFICATION OF ION EXCHANGE RESINS
• EFFECT OF PH ON ION EXCHANGE
• ADVANTAGES&DISADVANTAGES
• APPLICATIONS

3. INTRODUCTION
The most popular method for the purification of proteins and other charged
molecules is ion exchange chromatography.
* In ion exchange chromatography, retention is based on the attraction b/w
the solute ions and charged sites bound to stationary phase
* columns used for ion exchange are characterised by the presence of
charged groups covalently attached to the stationary phase
TYPES OF IEC: Two types
1)anion exchangers, 2)cation exchangers
Cation exchange chromatography: positively charged molecules are attracted
to a negatively charged solid support. Commonly used cation exchange
resins are S-resin,sulfate derivatives;&CM resins, carboxylate derived ions
Anion exchange chromatography:negatively charged molecules are attracted
to a positively charged solid support
Examples:Q-resin, a quaternary amine, diethylaminoethane

4. PRINCIPLE
Principle : IEC retains analyte molecules on the column based
on ionic interactions . The stationary phase surface display
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. The ionic compound consisting
of the cationic species M+ & the ionic species B- can be
retained by the stationary phase.
CEC retains positively charged cations because the stationary
phase displays a negatively charged functional groups
AEC retains anions using positively charged functional groups

5. Mechanism
Selectivity for ion exchange : in general,ion exchangers favour the binding of
ions of * Higher charge
* Decreased hydrated radius *Increased polarizability
• Mechanism
To optimize binding of all charged molecules, the mobile phase is generally
a low to medium conductivity (i.e., low to medium salt concentration)
solution. The adsorption of the molecules to the solid support is driven by
the ionic interaction between the oppositely charged ionic groups in the
sample molecule and in the functional ligand on the support. By increasing
the salt concentration the molecules with the weakest ionic interactions
start to elute from the column first. Molecules that have a stronger ionic
interaction require a higher salt concentration and elute later in the
gradient. The binding capacities of ion exchange resins are generally quite
high. This is of major importance in process scale chromatography, but is
not critical for analytical scale separations.

6. Ion exchange chromatography (anion
exchange)

7. INSTRUMENTATION

8. Ion exchangers
There are 3 classes of ion exchangers , these include
1)Resins
2)Gels
3)Inorganic exchangers :separations involving harsh chemical
conditions (high temp,high radiation levels,strongly basic solutions
/powerful oxidizing agents )employ inorganic ion exchangers.
*ion exchange resins are used for the separation of small of molecules
*ion exchange gels are used for the separation of large molecules like
proteins , nucleic acid.
*Resins are amorphous particles of organic materials
*polystyrene resins for ion exchange are made by co-polymerization of
styrene and divinyl benzene
*divinyl benzene content is varied from 1-16 % to increase the extent
of cross linking

9. Classification of ion exchange resins
Strongly acidic cation exchanger : sulphonic acid groups
attached to styrene & divinyl benzene copolymer
Weakly acidic cation exchanger:carboxylic acid groups
attached to acrylic acid & divinyl benzene co-polymer
Strongly basic anion exchanger:quaternary ammonium
groups attached to styrene & divinyl benzene co-polymer
Weakly basic anion exchanger:poly alkylamine groups
attached to styrene & divinyl benzene co-polymer
* Sulphonate groups of strongly acidic resins remain ionized
even in strongly acidic solutions , where as carboxyl groups
are protonated near PH 4 & loose their cation exchange
capacity

10. Ion exchange gels
*Cellulose & dextran ion exchangers ,which are polymers of the sugar
glucose posses.larger pore sizes & lower charge densities .
*Because they are much softer than poly styrene resins,dextran&its
relatives are called gels
EFFECT OF PH ON ION EXCHANG:
*Varying PH is usually a preferred way to change selectivity in ion
exchange separations
*An increase in the PH leads to greater sample ionization & retention
in Anion exchange HPLC
*Exm: antibiotics containing COOH groups
*decrease in PH favours retention of bases by cation exchange HPLC.
Exm: local anesthetics containing NH2 groups
*only the ionized from of acid /base will be retained significantly.

11. Effect of organic solvents
*additional of an organic solvent to mobile phase results in decreased retention ,just
as in the case of reserved phase HPLC
*solvents such as methonal /aceto nitrile are also often used in ion exchange to create
changes in selectivity .
EFFECT OF BUFFERS :
*In ion exchange, sometimes a particular salt is selected to provide stronger /weaker
retention
*a strong displacer reduces sample retention more than the same concentration of
weak displacer
*in general , more highly charged displacers are stronger
• As a rule, the pH of the mobile phase buffer must be between the pI (isoelectric
point) or pKa (acid dissociation constant) of the charged molecule and the pKa of
the charged group on the solid support. For example, in cation exchange
chromatography, using a functional group on the solid support with a pKa of 1.2, a
sample molecule with a pI of 8.2 may be run in a mobile phase buffer of pH 6.0. In
anion exchange chromatography a molecule with a pI of 6.8 may be run in a
mobile phase buffer at pH 8.0 when the pKa of the solid support is 10.3.

12. Interpretation

13. Advantages &disadvantages
Advantages:
Detectability :useful for the detection of many
inorganic salts.
Separations :* usually preferred ,because of the availability of volatile
buffers . Volatile buffers make the removal of mobile phase easier.
*useful for separation of mixtures of biological origin inorganic salts.
Disadvantages
*Column efficiency is less
*it is difficult to achieve control over selectivity &resolution
*stability& reproducibility of the columns become questionable after
repeated use

14. Applications
*IEC is used to convert one salt to other
Example: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
*it is used to prepare deionized water
Separation of similar ions:
.A mixture of Na,H&K can be seperted using cation exchange
resin
a mixture of Cl,Br&iodide can be seperted using basic anion
exchange resin

15. Method : mixture of Cl,Br&iodide is passed through basic
anion exchanger using 0.5M sodium nitrate as eluant .
Cl will first eluate .Raise conc of sodium nitrate
,bromide will eluate .raise the conc of sodium nitrate
further ,iodide ion will eluate.
Removal of interfering radicals: calcium & barium ions
exchanged with H+ ions while phosphate ion pass
through the column
Softening of hard water :water passed through cation
exchanged charged with the sodium ions Ca&Mg ions
retained in the column while sodium is exchanged

16. • Complete demineralization of water
• Separation of lanthanides
• Separation of sugars
• Separation of amino aids
• Other applications :
• For the measurement of various active
ingredients in medicinal formulations
• For the measurement of drugs& their
metabolites in serum & urine ,for residue analysis
in food raw materials

17. GEL PERMEATION
CHROMATOGRAPHY
Introduction
• GPC, also known as Size Exclusion Chromatography SEC
or Gel Filtration Chromatography GFC, is a
chromatographic technique that separates dissolved
molecules on the basis of their size by pumping them
through specialized columns containing a microporous
packing material.
• As the sample is separated and eluted from the
column, it can be characterized by a single
concentration detector (Conventional Calibration) or
series of detectors (Universal Calibration and Triple
Detection). Gel Permeation Chromatography GPC is
used to characterize natural and synthetic polymers,
biopolymers, proteins or nanoparticles.

18. • When the GPC separation is coupled with light scattering,
viscometer and concentration detectors together (triple
detection), it will provide a distribution of absolute
molecular weight, molecular size, and intrinsic viscosity as
well as information on macromolecular structure,
conformation aggregation and branching.
• It is a chromatographic method in which molecules in
solution are separated by their size, not by molecular
weight. It is usually applied to large molecules or
macromolecular complexes such as proteins and industrial
polymers. Typically, when an aqueous solution is used to
transport the sample through the column, the technique is
known as gel-filtration chromatography

19. Principle of separation
• Principle of separation
• It is a kind of chromatography technique based on the difference
of molecular weight and is one of the effective and mild methods
extensively used to isolate and analyze the biomacromocular
substances.
• The stationary phase consists of beads containing pores that span
a relatively narrow size range.
• when the gel is packed into a column and percolated with a
solvent, it permits the large molecular weight compounds to pass
rapidly without penetration of the pores
• Smaller molecules spend more time inside the beads than larger
molecules and therefore elute later (after a larger volume of
mobile phase has passed through the column).

20. • In addition to providing the molecular weight
distribution, GPC also separates a complex
polymeric compound into its component parts
- polymer, oligomer, monomer, and additives

21. Working
• GPC separates molecules in solution by their "effective size in solution." To prepare
a sample for GPC analysis the resin is first dissolved in an appropriate solvent.
• Inside the gel permeation chromatograph, the dissolved resin is injected into a
continually flowing stream of solvent (mobile phase). The mobile phase flows
through millions of highly porous, rigid particles (stationary phase) tightly packed
together in a column. The pore sizes of these particles are controlled and available
in a range of sizes. The width of the individual peaks reflects the distribution of the
size of molecules for a given resin and its components. The distribution curve is
also known as the molecular weight distribution (MWD) curve.
• Taken together the peaks reflect the MWD of a sample. The broader the MWD,
the broader the peaks become and vice versa. The higher the average molecular
weight, the further along the molecular weight axis the curve shifts and vice versa.

22. • Taken together the peaks reflect the MWD of
a sample. The broader the MWD, the broader
the peaks become and vice versa. The higher
the average molecular weight, the further
along the molecular weight axis the curve
shifts and vice versa.
• Cross sectional view of porous particle

23. Though they are subtle, differences such as
those shown in the molecular-weight
distributions to the left, could cause marked
variations in the performance of the polymer.

24. The Size Separation Mechanism

25. • Molecules of various sizes elute from the
column at different rates. The column retains
low molecular weight material (small black
dots) longer than the high molecular weight
material (large black dots). The time it takes
for a specific fraction to elute is called its
"retention time".
•

26. Instrumentation
Schematic of a basic gel permeation chromatograph
This diagram illustrates how the sample is
injected into the mobile phase and the path
the sample takes to the detector.

28. 1. Pump
• Pumps the polymer in solution through the system.
• Different polymers produce solutions of different viscosities. To
compare data from one analysis to the next, the pump must deliver
the same flow rates independent of viscosity differences. In
addition, some detectors are very sensitive to the solvent flow rate
precision. Such constant flow must be a critical feature of the
instrument.
2.Introduces the polymer solution into the mobile phase.
The injector must be capable of small volume injections and large
volume injections The injector should not disturb the continuous
mobile phase flow. It should also be capable of automatic multiple
sample injection when the sample volume is large.

29. • 3. Column Set
• Efficiently separates sample components from one another.
• High efficiency columns give maximum separating capability and
rapid analyses. Every column must provide reproducible
information over extended periods for both analytical and fraction
collecting purposes.
• 4. Detector
• Monitors the separation and responds to components as they
elute from the column.
• Detectors must be nondestructive to eluting components if they are
to be collected for further analysis.
• In addition, the detectors must be sensitive and have a wide linear
range in order to respond to both trace amounts and large
quantities of material if necessary.

30. Graph

31. Graph

32. Advantages
1.Good separation of large molecules from the small
molecules with a minimal volume of eluate,[1] and that
various solutions can be applied without interfering with
the filtration process, all while preserving the biological
activity of the particles to be separated.
2.The technique is generally combined with others that
further separate molecules by other characteristics, such as
acidity, basicity, charge, and affinity for certain compounds.
3. There are short and well-defined separation times and
narrow bands, which lead to good sensitivity. There is also
no sample loss because solutes do not interact with the
stationary phase.

33. Disadvantages
• Disadvantages are, for example, that only a
limited number of bands can be
accommodated because the time scale of the
chromatogram is short, and, in general, there
has to be a 10% difference in molecular mass
to have a good resolution
• Another Disadvantages of GPC for polymers is
that filtrations must be performed befour
using the instrument to prevent dust

34. Applications
• The main application of gel-filtration chromatography is the
fractionation of proteins and other water-soluble polymers, while
gel permeation chromatography is used to analyze the molecular
weight distribution of organic-soluble polymers. Either technique
should not be confused with gel electrophoresis, where an electric
field is used to "pull" or "push" molecules through the gel
depending on their electrical charges.
• SEC is a widely used technique for the purification and analysis of
synthetic and biological polymers, such as proteins, polysaccharides
and nucleic acids. Biologists and biochemists typically use a gel
medium — usually polyacrylamide, dextran or agarose — and filter
under low pressure. Polymer chemists typically use either a silica or
crosslinked polystyrene medium under a higher pressure. These
media are known as the stationary phase.

35. Thank
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