1. ION EXCHANGE AND GEL PERMEATI
ON CHROMATOGRAPHY
BY
G.SHRAVANI
170213884010
G.SHRAVANI
1

2. Chromatography
G.SHRAVANI
2

3. 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.
G.SHRAVANI
3

4. Ion-exchange chromatography
The
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.
G.SHRAVANI
4

5. PROPERTIES OF ION EXCHANGE
RESIN
Color
Amount
or cross linking
Porosity
Capacity
Surface
area
Density
Mechanical
strength
Size
G.SHRAVANI
5

6. Flow
rates should be controlled due to
the difference in rate of exchange
Flow rate -0.5 to 5ml/minutes
Capacity of ion exchange depends on the
no. of sites available
Cation resins(strong acid) are stable at
150degress & anions at 70
G.SHRAVANI
6

7. 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.
1.
2.
G.SHRAVANI
7

8. Ion Exchangers
G.SHRAVANI
8

9. Ion exchangers – Functional groups
Anion
exchanger
Aminoethyl
(AE-)
Diethylaminoethyl
(DEAE-)
Quaternary
aminoethyl (QAE-)
Cation
exchanger
Carboxymethyl
(CM-)
Phospho
Sulphopropyl
G.SHRAVANI
(SP-)
9

10. 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
G.SHRAVANI
10

11. Anion exchange chromatography
Anion
exchange chromatography retains
anions using positively charged functional
group:
+
+ _
+ +
R-X A +M B
R-X B + M + A
G.SHRAVANI
11

12. Procedure
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.
G.SHRAVANI
12

13. Procedure
4.
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.
G.SHRAVANI
13

14. Procedure
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.
G.SHRAVANI
14

15. Procedure
G.SHRAVANI
15

16. 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.
G.SHRAVANI
16

17. 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.
G.SHRAVANI
17

18. 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.
cationic
pH =pI
anionic
pH increase
pI isoelectric point*
G.SHRAVANI
18

19. Choosing your ion-exchanger: know
your proteins
1.


Stability of proteins
stable below pI value, use cation-exchanger
stable above pI value, use anion-exchanger
2.


Molecular size of proteins
<10,000 mw, use matrix of small pore size
10,000-100,000 mw, use Sepharose equivalent grade
G.SHRAVANI
19

20.  Important
to consider the stability of proteins in
choice of ion exchangers. Isoelectric focusing can be
used to identify suitable ion-exchanger type
G.SHRAVANI
20

21. Applications
Determination
of sodium and potasium in
the mixture:
column eluted with 0.1M HCL flow
rate -0.6ml/sqmin
Radiochemistry
G.SHRAVANI
21

22. Gel Permeation
GEL
FILTRATION
Chromatography
G.SHRAVANI
22

23. DEFINATION
Gel
chromatography is a technique in which
fractionation is based upon the molecular size
& shape of the species in the sample.
Gel chromatography is also called as gel
permeation or exclusion or molecular sieve
chromatography.
G.SHRAVANI
23

24. TECHNIQUES IN GEL
CHROMATOGRAPHY
Gel
chromatography is performed on a
column by the elution method. The degree
of retardation, depends upon the extent to
which the solute molecules or ions can
penetrate that part of the solution phase
which is held within the pores or the highly
porous gel like packing material.
A series of resins with different pore sizes
can be obtained by changing the amount of
Epichlorohydrin.The resulting gel is called as
SEPHADEX.
G.SHRAVANI
24

25. In
gel chromatography the granulated or beded
gel material is called as packing material.
The solutes which are distributed through the
entire gel phase is called as stationary phase
and the liquid flowing through the bed is called as
mobile phase.
The cross linked dextran (sephadex) & xerogels
of the polyacrylamide (Bio-gel) originally used as
stationary phases in gel permeation
chromatography are semi rigid gels.
G.SHRAVANI
25

26. These
are unable to withstand the highpressure used in HPLC.
Hence modern stationary phases consist
of micro particles of stryene-divinyl
benzene copolymers (ultrastyragel)
silica or porous glass.
G.SHRAVANI
26

27. GEL PERMEATION
CHROMATOGRAPHY
A
chromatographic method in which particles are
separated based on their size, or in more technical
terms, their hydrodynamic volume.
 Organic solvent as the mobile phase.
 The stationary phase consists of beads of porous
polymeric material.
G.SHRAVANI
27

28. OBJECTIVE
Analysis
of synthetic and biologic polymers
Purification of polymers
Polymer characterization
 Study properties like:
 Molecular weight
 Polydispersity Index
 Viscosity
 Conformation
 Folding
G.SHRAVANI
28

29.  Aggregation
 Branching
 Copolymer
composition
 Molecular size
G.SHRAVANI
29

30. PRINCIPLE
G.SHRAVANI
30

31. PRINCIPLE
Different
sizes will elute (filter) through at
different rates.
 Column
1. Consists of a hollow tube tightly packed with
extremely small porous polymer beads designed
to have pores of different sizes.
2. Pores may be depressions on the surface or
channels through the bead.
3. Smaller particles enter into the pores, larger
particles don't.
G.SHRAVANI
31

32. The
larger the particles, the less overall
volume to transverse over the length of
the column
G.SHRAVANI
32

33. FEATURES
Solvent
1. Should be kept dry
2. Should be degassed in some applications
3. The samples should be made from the same
solvent
4. For GPC/light scattering the solvent should
be filtered before it ever hits the pump
5. Common solvents for tetrahydrofuran
(THF) & toluene.
G.SHRAVANI
33

34. G.SHRAVANI
34

35. Pump &Filters

a.
b.
c.

•
Pump:
Designed to deliver very constant,
accurate flow rates.
At microprocessor-controlled rate.
Designed not to produce any pressure
pulses.
Filters:
Prevent major junk from getting into the
columns
G.SHRAVANI
35

36. Injector Loop
Injector
•
•
•
•
Loop
Allows you to load the sample loop
which is a piece of tubing precut for a
precise volume.
the output of the pump flushes through
the loop
Carries the sample to the columns.
sends a signal to the detector to indicate
that the sample has been loaded.
G.SHRAVANI
36

37. Columns
Columns
 Contain
the beads through which the
sample is allowed to pass.
 Reference column is also present
 Very expensive
 Never change the pumping rate by a
large amount
 They are very delicate
G.SHRAVANI
37

38. Detectors
Detectors
o Viscosity
o Light
Scattering
o Ultraviolet detectors
o Differential Refractive Index detector
placed at the end to reduce pressure on
it
G.SHRAVANI
38

39. Analysis
Spectroscopic
Techniques
1. Refractive Index
2. Light Scattering
3. Ultraviolet Spectroscopy
 Viscometry Techniques
1) Viscosity
2) Flow rate
G.SHRAVANI
39

40. Conventional GPC
Analysis
Molecules separated according to their
hydrodynamic volume.
• Molecular weights (MW) and molecular
weight distribution can be determined from
the Measured retention volume (RV)
• A calibration curve (log MW against RV),
using known standards
RI signal = KRI . dn /dc . C
•
G.SHRAVANI
40

41. KRI = apparatus-specific sensitivity constant
dn /dc = the refractive index increment
C = concentration.
 Limitation
 Their signals depend solely on concentration,
not on molecular weight or polymer size.

Not very reliable
G.SHRAVANI
41

42. Molecular Mass Sensitive Detectors
Detectors
sensitive to molecular weight used to
overcome limitations of Conventional GPC
E.g., light scattering and viscosity detectors
Advantages over Conventional GPC
I. True molecular weight distributions can be
obtained
II. Structural information
III.Size distribution
molecular weight can be directly determined
without a calibration curve
G.SHRAVANI
42

43. Characterization
Light
Scattering
LS signal = KLS . (dn/dc)2 . MW .
 KLS = sensitivity constant



dn/dc = refractive index increment
MW = molecular weight
C = concentration
dn/dc
depends on the Polymer Solvent
combination and if it is low, then proper
analysis cannot be done.
G.SHRAVANI
43

44. Applications









Polymer characterization
Molecular weight
Polydispersity Index
Viscosity
Folding
Aggregation
Branching
Copolymer composition
Molecular size
G.SHRAVANI
44

45. Proteomics
 Purification
 Conformation
 Hydrodynamic volume

G.SHRAVANI
45

46. Advantages
Can
be used to find shape also
 Rapid, routine analysis
 Identify high mass components even in
low concentration
 Can analyze polydisperse samples
 Branching studies can be done
 Absolute molecular weights can be
obtained
G.SHRAVANI
46

47. Drawbacks
There
is a size window
 Bad response for very small molecular
weights
 Standards are needed.
 Sensitive for flow rate variation. Internal
standard should be used whenever possible.
 High Investment cost
G.SHRAVANI
47

48. G.SHRAVANI
48

49. REFERENCES
Instrumental
methods of chemical
analysis .B.K Sharma p.g C123-170
www.gel permeation chromatography
www.ion exchange chromatography.
G.SHRAVANI
49

50. G.SHRAVANI
50