9/7/2007

Applications of Chromatographic
Techniques
HPLC TYPE APPLICATIONS‐ Affinity , Ion Exchange, Gel Permeation

Affinity Chromatography

Ligand Spacer Matrix

Ligand : Site of Interaction

Spacer: what binds ligand to support
Matrix: Supporting Phase

1
 9/7/2007

Affinity Chromatography
• Ligand is an atom, ion, or molecule that generally
donates one or more of its electrons through a
coordinate covalent bond to, or shares its electrons
th
through
h a covalent
l t bond
b d with
ith one or more central
t l
atoms or ions .
• Two types of Ligands are brought into use:
– Specific
– General
• Specific Ligands : Binds only to one species
• Group Specific Ligands: Binds to specific groups on
target species.

Affinity Chromatography
Ligand Types

Samples Types of Ligands used

Enzymes Substrate, Inhibitor, Cofactor

Antibody Antigen, Virus, Cells

Lectin Polysaccharides, Glycoprotein,
Cell receptor
Nucleic Acid Complementary
p y base sequence,
q
histone, nucleic acid, polymerase
binding protein
Hormones Receptors, Carrier proteins

2
 9/7/2007

Affinity Chromatography
SPACER
• Carbon chain interposed between Ligand and Matrix.
• Used when active site is located deep within the
sample molecule
• If too long it can interact with sample species on its
own ( hydrophobic interactions)
• If too short the ligand is unable to reach the active
sample molecule.
molecule
• Commercial phases have spacers which are optimized
for specific separations.

Affinity Chromatography
• Should be a rigid, stable and high surface
area.
• It must be insoluble in solvents and
buffers employed in the process
• it mustt be
b easily
il coupled
l d to
t a ligand
li d or spacer
arm onto which the ligand can be attached
• must exhibit good flow properties and have a
relativley large surface area for attachment
• Agarose is the most popular however,
cellulose, dextrans and polyacrylamide has
also been used frequently.
• Sepharose is a bead form of agarose gel.
gel
• In general any matrix useful for ion
exchange or gel filtration is also good for
affinity chromatography.

3
 9/7/2007

Affinity Chromatography
FORMATION OF A PHASE
• Ligand should be bound to support to create a stable
phase‐ immobilization.
• Two Steps
–Activation of the support with a reactive
compound.
–Attachment of a ligand
• How the spacer arm is attached to the ligand is
important, as it should not interfere with ligand binding
to the protein?
• But usually the best way to attach a ligand has to be
worked out by trial and error, synthesizing small test
molecules with alkyl groups attached to the ligand in
various ways and determining which bind best to the
protein.

Affinity Chromatography
FORMATION OF A PHASE
• Ligand should be bound to support to create a stable
phase‐ immobilization.
• Two Steps
–Activation of the support with a reactive
compound.
–Attachment of a ligand
• How the spacer arm is attached to the ligand is
important, as it should not interfere with ligand binding
to the protein?
• But usually the best way to attach a ligand has to be
worked out by trial and error, synthesizing small test
molecules with groups attached to the ligand in various
ways and determining which bind best to the target
molecule.

4
 9/7/2007

Affinity Chromatography
FORMATION OF A PHASE
• Ligand should be bound to support to create a stable
phase‐ immobilization.
• Two Steps
–Activation of the support with a reactive
compound.
–Attachment of a ligand
• How the spacer arm is attached to the ligand is
important, as it should not interfere with ligand binding
to the protein?
• But usually the best way to attach a ligand has to be
worked out by trial and error, synthesizing small test
molecules with groups attached to the ligand in various
ways and determining which bind best to the target
molecule.

Affinity Chromatography
Specific Phases
Type Specificity
Protein A- Sepahrose Cl4B Fc region of IgG and related
molecules
Con A- Sepharose Terminal –D- glucopyranosyl ,
D- mannopyranosyl or similar
residues
Blue Sepharose- Cl6B Broad range of enzymes
which
hi h h
have nucleotide
l tid
cofactors, serum albumin etc.
Lysine- Sepharose 4B Plasminogen, ribosomal RNA

5
 9/7/2007

Affinity Chromatography

BASIC STEPS

• Sample Introduction
• Adsorption of the components of interest
• Removal of impurities
• Elution of components

Affinity Chromatography
BASIC STEPS
Sample Introduction

Ensure that the column has

adequate capacity

6
 9/7/2007

Affinity Chromatography
BASIC STEPS
Absorption
Use a slow
l fl
flow rate off the
h solvent
l
so that the SAMPLE is allowed to
move through the column.

Larger duration of stay of the

SAMPLE allows better interaction of
the sample with unreacted sites.

Affinity Chromatography
BASIC STEPS
Washing
Basically
i ll removing
i the
h impurities
i ii
out of the column by passing fresh
volumes of solvent in a repeated
fashion

7
 9/7/2007

Affinity Chromatography
BASIC STEPS
Elution
Removall off the
h target molecule
l l from
f
the matrix and its collection.

Washing buffer and eluting buffers are

different.

After elution the column generally gets

regenerated.

Affinity Chromatography
BASIC STEPS
Elution Methods‐ Biospecific
Inhibitor
hibi ( free
f li d) is
ligand) i added
dd d into
i the
h
eluting buffer ( solvent) where it
competes with the solute (TARGET
MOLECULES)

This will lead to the elution of the

TARGET MOLECULE

8
 9/7/2007

Affinity Chromatography
BASIC STEPS
Elution Methods‐ Non‐ specific
A reagent is
i added
dd d that
h denatures
d the
h
solute (TARGET MOLECULE)
(pH, KSCN, Urea, ionic strength etc.)

This will lead to the elution of the

TARGET MOLECULE as it will leave
LIGAND due to a conformational
change.

Affinity Chromatography
Affinity Chromatogram

9
 9/7/2007

Affinity Chromatography
Example 1

Affinity Chromatography
Example 2

10
 9/7/2007

Applications of Affinity Chromatography

Various techniques developed from Affinity Chromatography

Applications of Affinity Chromatography

Various techniques developed from Affinity Chromatography

11
 9/7/2007

Applications of Affinity Chromatography

• Boronate affinity chromatography (BAC) –
– Glycated haemoglobin analysis is used in the management of
diabetes, HbA1c provides an indication of the degree of
diabetic control over the preceding two‐
two to three‐month
three month
period. BAC is the definitive method for determination of
HbA1c as it is the only interference free method presently
available to the routine laboratory.
– At a pH above 8, most boronate derivatives form covalent
bonds with compounds that contain cis‐diol groups in their
structure
– sugars such as glucose possess cis‐diol groups, boronates are
valuable for resolving glycoproteins (e.g., glycohemoglobin)
from non‐glycoproteins (e.g., normal hemoglobin).

Applications of Affinity Chromatography

• Boronate affinity chromatography (BAC) –
– Also used in the purification of Catecholamines (Hormones) ,
nucleosides & Nucelotides.
• Compound which is a ligand in BAC is m m‐ aminophenyl
boronate forming a tetrahedral boronate ion under
alkaline conditions. This anion can bind to the 1,2‐ cis –
diol group and the interaction is enhanced in the
presence of Mg2+ ions and inhibited by amine containing
buffers.
• HEPES and Morpholine are the basic buffers used for
absorption while Tris and Sorbitol are used as desorption
( eluting
l ti ) Buffers.
B ff
• Lectin Affinity Chromatographyl‐ Lectins are non‐
immune system proteins that have the ability to
recognize and bind certain types of carbohydrate
residues

12
 9/7/2007

Applications of Affinity Chromatography

• Lectin Affinity Chromatography
– Concanavalin A, which binds to ‐D‐mannose and ‐D‐
glucose residues, and wheat germ agglutinin, which binds to
D‐N‐acetylglucosamine
– isolation of many carbohydrate‐containing compounds, such
as polysaccharides, glycoproteins, and glycolipids
– has been in the separation and analysis of isoenzymes like
immobilized wheat germ agglutinin was used to distinguish
between the liver
liver‐ and bone
bone‐derived
derived isoenzymes of alkaline
phosphatase in human serum
– Concanavalin A have been used to separate apolipoprotein
A‐ and apolipoprotein B‐containing lipoproteins in human
plasma

Applications of Affinity Chromatography

• Protein A/G Affinity Chromatography
– ligands that have been used in direct analyte detection by
affinity chromatography are antibody‐binding proteins such
as protein A and protein G
– protein A and protein G are bacterial cell wall proteins
produced by Staphylococcus aureus and group G
streptococci, respectively
– Protein A and protein G bind most strongly to
immunoglobulins at or near neutral pH, but readily
dissociate from these solutes when placed in a buffer with a
lower pH.
– The ability of protein A and protein G to bind to antibodies
make these good ligands for the analysis of
immunoglobulins, especially IgG‐class antibodies, in
humans.

13
 9/7/2007

Applications of Affinity Chromatography

• Immunoaffinity Chromatography
– The term "immunoaffinity chromatography" (IAC) is used
for an affinity chromatographic method in which the
stationaryy p
phase consists of an antibodyy or antibody‐related
y
reagent
– IAC have been developed for anti‐idiotypic antibodies ,
glucose‐containing tetrasaccharides , granulocyte colony‐
stimulating factor IgG , immunoglobulin E , interferon ,
tumor necrosis factor‐ , interleukins , ß2‐microglobulin and
transferrin
– determination of fibrinogen in human plasma
• the amount of fibrinogen in the retained peak was determined by
the measurement of its absorbance at 280 nm.
• The sample was a 20‐µL aliquot of plasma diluted 1:10

Applications of Affinity Chromatography

• an immobilized heparin column is used for the
determination of antithrombin III in human plasma
• Octylglutathione has been used as a ligand for the
p
separation and analysis
y of gglutathione S‐transferase
isoenzymes in human lung and liver samples.
• "Immobilized metal ion affinity chromatography
(MIAC)", also known as "metal chelate affinity
chromatography", is another method that has been
widely used in purification processes
– The affinity ligand is a metal ion that is complexed with an
i
immobilized
bili d chelating
h l i agent. Iminodiacetic
I i di i acid
id is
i the
h most
common chelating agent used, but carboxymethylaspartic
acid,tris‐carboxymethylethylenediamine,tris(2‐aminoethyl)
amine, or dipicolylamine sometimes are also used. The metal
ions placed within these chelating groups are Cu2+, Zn2+, Ni2+,
Co2+, or Fe3+.

14
 9/7/2007

Applications of Affinity Chromatography

• MIAC separates proteins and peptides on the basis of
interactions between certain amino acid residues (such as
histidine, tryptophan, or cysteine) and the metal ions
within the immobilized metal chelate and has been used
commerciali l purification
ifi i off severall peptides,
id proteins,
i andd
amino acids have been purified commercially since its
discovery
• The demand for pharmaceutical grade plasmid DNA
(pDNA) is expected to grow in the future, placing pressure
on industry to produce the required volumes of pDNA
• Affinity
ff chromatography
h h offers
ff a solution
l to the
h problem
bl
of purifying pDNA from E.coli for gene therapy and
vaccine applications without co‐ purification of
undesirbale products like C‐DNA, RNA and endotoxins.

Applications of Gel permeation Chromatography

• It has applications in PROTEOMICS wherein the protein in their
quaternary structures could be separated.
• It is also used to assess the size and polydispersity of the
synthesized
h d polymer.
l
• It is also used for characterization of food polysaccharides as
they are typically polydisperse compounds with wide
distribution in molecular weight, sequence and structure.
• food industry uses native and modified starches, dextrins,
dextrans, glucans, pullulans, modified celluloses, pectins,
carrageenans, and gums from both microbial and plant seed
sources. In foods and beverages, polysaccharides may find use
as thickening agents, emulsifiers, emulsion stabilizers, or to add
structure to solids.

15
 9/7/2007

Applications of Gel permeation

Chromatography
• They are also important for their ability to modify fat
and water‐holding
water holding properties,
properties and to control aroma
and/or flavor release
• GPC has also been employed for detection of
Pesticide contamination in lanolin ( a base material
for cosmetics & pharmaceutical application) – GLC
application.
application

IEC & Applications

• Ion‐exchange chromatography is a process that
allows the separation
p of ions and p
polar molecules
based on the charge properties of the molecules.
• Used for almost any kind of charged molecule
including large proteins, small nucleotides and
amino acids .

16
 9/7/2007

IEC
PRINCIPLE
ƒ Ion exchange chromatography retains analyte
molecules based on coulombic (ionic) interactions.
ƒ Stationary
S i phase
h surface
f di l
displays i i functional
ionic f i l
groups that interact with analyte ions of opposite
charge
TYPES
ƒ Cation exchange : positively charged cations because
the stationary phase displays a negatively charged
functional group such as a phosphonic acid ( NET
NEGATIVE CHARGE)
ƒ Anion exchange : negatively charged anions using
positively charged functional group such as a
quaternary ammonium cation ( NET POSITIVE CHARGE)

Column Chromatography
ƒ Net Charge on the Ion exchanger‐measure to retain
opposite charge ion clearly depends upon the pH of
the mobile phase with which it is contact.
ƒ Net charge is also a function of pKa of the ion group
involved.
ƒ (R+ X‐) + P‐ ( R+P‐) + X‐ ( Absorption)
ƒ ( R+P‐) + S‐ (R+S‐) + P ( Desorption)
ƒ Example of Anion Exchanger‐ Diethylaminoethyl
(DEAE) and Cation Exchangers is Carboxymethyl (CM).

17
 9/7/2007

Ion Exchange Chromatography

ƒ Wide range of materials have been used for forming
ion exchangers‐ cross linked polystyrene; cross linked
polydextrans, cellulose and silica.
ƒ Silica Gel based ion exchangers are not widely used as
they have poor stability in extreme pH conditions.
ƒ Branch of Ion exchange chromatography involving
separation of simple inorganic cations and anions is
known as Ion chromatography.

Ion Exchange Chromatography

FIVE BASIC STEPS OF ION EXCHANGE CHROMATOGRAPHY
Starting Adsorption Start of End of
Conditions Of Sample Desorption Desorption regeneration

+ ++ + ++
+ ++ + ++
+ ++ ++ ++
++ ++
++

Staring Buffer Counter ions

Gradient Ions
Substances to be separated

18
 9/7/2007

Ion exchange chromatography

• The first stage is equilibration in which the ion
exchanger is brought to a starting state, in terms of pH
and ionic strength, which allows the binding of the
desired solute molecules.
• The exchanger groups are associated at this time with
exchangeable counter‐ions (usually simple anions or
cations, such as chloride or sodium).
• The second stage is sample application and
adsorption, in which solute molecules carrying the
appropriate charge displace counter
counter‐ions
ions and bind
reversibly to the gel.
• Unbound substances can be washed out from the
exchanger bed using starting buffer.

Ion exchange chromatography

• Third stage: substances are removed from the column
by changing to elution conditions unfavourable for
ionic bonding of the solute molecules. This normally
involves increasing the ionic strength of the eluting
buffer or changing its pH.
• The fourth and fifth stages are the removal from the
column of substances not eluted under the previous
experimental conditions and re‐equilibration at the
starting conditions for the next purification.

19
 9/7/2007

Ion exchange chromatography

• Separation is obtained since different substances have
different degrees of interaction with the ion exchanger
due to differences in their charges, charge densities
and distribution of charge on their surfaces.
surfaces
• These interactions can be controlled by varying
conditions such as ionic strength and pH.
• In ion exchange chromatography one can choose
whether to bind the substances of interest and allow
the contaminants to pass through the column,
column or to
bind the contaminants and allow the substance of
interest to pass through.

Ion exchange chromatography

MATRIX OF ION EXCHANGE COLUMN CHROMATOGRAPH
• Ion exchanger consists of an insoluble matrix to which
charged groups have been covalently bound. The
charged groups are associated with mobile counter ions.
ions
• These counter‐ions can be reversibly exchanged with
other ions of the same charge without altering the
matrix.
• Positively charged exchangers have negatively charged
counter‐ions (anions) available for exchange and are
called anion exchangers.
• Negatively charged exchangers have positively charged
counter‐ions (cations) and are termed cation
exchangers.

20
 9/7/2007

Ion exchange chromatography

MATRIX OF ION EXCHANGE COLUMN CHROMATOGRAPH
• The matrix may be based on inorganic compounds,
synthetic resins or polysaccharides.
• The
Th characteristics
h t i ti off the
th matrix t i determine
d t i it
its
chromatographic properties such as efficiency, capacity
and recovery as well as its chemical stability,
mechanical strength and flow properties. The nature of
the matrix will also affect its behavior towards
biological substances and the maintenance of biological
activity.
i i
• The first ion exchangers designed for use with biological
substances were the cellulose ion exchangers
developed by Peterson and Sober

Ion exchange chromatography

MATRIX OF ION EXCHANGE COLUMN CHROMATOGRAPH
• Many cellulose ion exchangers had low capacities
(otherwise the cellulose became soluble in water) and
h d poor flow
had fl properties due
d to their
h irregularl shape.
h
• Ion exchangers based on dextran (Sephadex), followed
by those based on agarose (Sepharose CL‐6B) and
cross‐linked cellulose (DEAE Sephacel) were the first ion
exchange matrices to combine a spherical form with
high porosity, leading to improved flow properties and
high capacities for macromolecules.

21
 9/7/2007

Ion exchange chromatography

MATRIX OF ION EXCHANGE COLUMN CHROMATOGRAPH

Function Group Used on Ion Exchangers

Ion exchange chromatography

APPLICATIONS
• Purification of Biologically Active proteins‐ ENZYMES
like Creatine Kinase from Chicken Breast Muscle is
recovered by 90%.
• Purification of Immunoglobulins like IgG from Cell
culture preparations ( Eli Lilly, USA)
• Nucleic acid separations – Purification of Plasmid
HB101 (pBR322) has been separated using anion
exchange chromatography on Q Sepharose High
performance. Time required for preparation through
IEC – 1 hr. and traditional CsCl density gradient
centrifugation – 8 hours.

22
 9/7/2007

Ion exchange chromatography

APPLICATIONS
• Separation of peptides from Cyanogen bromide
fragments from Collagen.
• Peptide Mapping
• Separation of Oligonucleotides
• Cation exchange chromatography used for fermentative
production of enzyme β‐ galactosidase
• Purification of recombinant P. aeruginosa exotoxin A (
MW 55,000) expressed in E. coli for use as a vaccine.
Exotoxin was captured directly by 4 chromatography
steps using DEAE adsorbent.

23