a process that
separation of ions
on the charge
properties of the
solution to be injected is usually called a
sample, and the individually separated
components are called analytes
can be used for almost any kind of charged
molecule including large proteins, small
nucleotides and amino acids.
is often used in protein purification, water
PROPERTIES OF ION EXCHANGE
or cross linking
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
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
cation exchange chromatography
anion exchange chromatography.
Cation exchange chromatography
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
Anion exchange chromatography
exchange chromatography retains
anions using positively charged functional
R-X A +M B
R-X B + M + A
A sample is introduced, either manually or with
an autosampler, into a sample loop of known
The mobile phase (buffered aqueous solution)
carries the sample from the loop onto a column
that contains some form of stationary phase
Stationary phase material is a resin or gel
matrix consisting of agarose or cellulose beads
with covalently bonded charged functional groups.
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
The analytes of interest must then be
detected by some means, typically by
A chromatography data system
(CDS) is usually needed to control an
have numerous functional groups
that can have both positive and negative
exchange chromatography separates
proteins according to their net charge, which
is dependent on the composition of the
Affect of pH in the separation of
adjusting the pH or the ionic
concentration of the mobile phase,
various protein molecules can be
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
Effect of pH in the separation of
are charged molecules. At
specific pH, it can exist in anionic (-),
cationic (+) or zwitterion (no net
pI isoelectric point*
Choosing your ion-exchanger: know
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
to consider the stability of proteins in
choice of ion exchangers. Isoelectric focusing can be
used to identify suitable ion-exchanger type
of sodium and potasium in
column eluted with 0.1M HCL flow
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
TECHNIQUES IN 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
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
The cross linked dextran (sephadex) & xerogels
of the polyacrylamide (Bio-gel) originally used as
stationary phases in gel permeation
chromatography are semi rigid gels.
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.
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
of synthetic and biologic polymers
Purification of polymers
Study properties like:
sizes will elute (filter) through at
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
larger the particles, the less overall
volume to transverse over the length of
1. Should be kept dry
2. Should be degassed in some applications
3. The samples should be made from the same
4. For GPC/light scattering the solvent should
be filtered before it ever hits the pump
5. Common solvents for tetrahydrofuran
(THF) & toluene.
Designed to deliver very constant,
accurate flow rates.
At microprocessor-controlled rate.
Designed not to produce any pressure
Prevent major junk from getting into the
Allows you to load the sample loop
which is a piece of tubing precut for a
the output of the pump flushes through
Carries the sample to the columns.
sends a signal to the detector to indicate
that the sample has been loaded.
the beads through which the
sample is allowed to pass.
Reference column is also present
Never change the pumping rate by a
They are very delicate
o Ultraviolet detectors
o Differential Refractive Index detector
placed at the end to reduce pressure on
Molecules separated according to their
• 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
KRI = apparatus-specific sensitivity constant
dn /dc = the refractive index increment
C = concentration.
Their signals depend solely on concentration,
not on molecular weight or polymer size.
Not very reliable
Molecular Mass Sensitive 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
II. Structural information
molecular weight can be directly determined
without a calibration curve
LS signal = KLS . (dn/dc)2 . MW .
KLS = sensitivity constant
dn/dc = refractive index increment
MW = molecular weight
C = concentration
depends on the Polymer Solvent
combination and if it is low, then proper
analysis cannot be done.
be used to find shape also
Rapid, routine analysis
Identify high mass components even in
Can analyze polydisperse samples
Branching studies can be done
Absolute molecular weights can be
is a size window
Bad response for very small molecular
Standards are needed.
Sensitive for flow rate variation. Internal
standard should be used whenever possible.
High Investment cost