Group 3

Group Members:

Mohd Hafiz bin Rashid Rosamemi Bt Jamal Nor Hafizah Bt Abd Raman Nurhasmah Mahatchakun Zaity Bt Ahmad Hani Nabila Bt Maamor Meriem Fekak Osman Fatin Nabila Abd Rashid Nur Amalina Bt Baharom Norzalina Noruldin

0717173 0811432 0811246 0717032 0813442 0734066 0738738 0814278 0816954 0733304

Derived from the Greek word Chroma meaning color. Chromatography provides a way to identify unknown compounds and separate mixture.

Definition Separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium. Components mobile phase: a solvent that flows through the supporting medium. stationary phase: a layer or coating on the supporting medium that interacts with the analytes. supporting medium: a solid surface on which the stationary phase is bound or coated.

Affinity Chromatography (AF) is a technique enabling purification of a biomolecule with respect to biological function or individual chemical structure. The molecule of interest will have a well known and defined property which can be separate from complex mixtures in single process. The target molecule becoming trapped on a solid or stationary phase or medium. The other molecule in solution will not become trapped as they do not possess this property. The solid medium can be removed from the mixture, washed and the target molecule released from the entrapment in a process known as elution.

 Liquid chromatography technique that separates biomolecules according to hydrophobicity.

Used to separate proteins with difference in hydrophobicity

Principle
Separation of substances is based on their varying strength of interaction with hydrophobic groups attached to an uncharged gel matrix. Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix.

How is this achieved?

General Concept:
o Salt -promoted adsorption.

Hydrophobic groups on gel matrix and soluble proteins are shielded by water molecules. To expose these hydrophobic regions, water must be removed, and this can be achieved by adding ammonium sulfate.

Exposed hydrophobic regions of proteins and ligand group will interact, leading to binding of protein to ligand.

To release the bound protein from the ligand, dissociation can be achieved by eluting bound protein with medium of low ionic strength

HIC media are composed of ligands containing alkyl or aryl groups. The medium is packed into a column to form a packed bed. The bed is then equilibrated with buffer that fills the pores of the matrix and the space in between the particles Interaction between the protein and the medium is promoted by moderately high salt concentrations The type of salt and the concentration required in the start buffer are selected

When sample loading is completed and the column has been washed so that all non-bound proteins have passed through (i.e., the UV signal has returned to baseline), conditions are altered to begin elution. Proteins are eluted by decreasing the salt concentration in the elution buffer. As the level of salt decreases those proteins with the lowest hydrophobicity begin to elute from the column. By controlling changes in salt concentration using gradients, proteins are eluted differentially in a purified, concentrated form.

Those proteins with the highest degree of hydrophobicity will be most strongly retained and will be eluted last. A wash step in a salt-free buffer removes most tightly bound proteins at the end of an elution. If the hydrophobicity of the medium and the proteins in the sample have been judged correctly, all proteins will be eluted by this stage. The column is then re-equilibrated in start buffer before applying more samples in the next run.

1 Starting Conditions

2 Adsorption of sample substances

3 Start of absorption

4 End of desorption

5 Regeneratio n

Starting buffer c0unter-ions

Substances to be separated

Gradient ion

IEC is a process that allows the separation of ions and polar molecule based on the charge properties of the molecules. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. The solution to be injected is usually called a sample, and the individually separated components are called analytes. It is often used in protein purification. Ion exchange chromatography retains analyte molecules based on (ionic) interactions. The stationary phase surface displays ionic functional groups that interact with analyte ions of opposite charge

The surface charge of the solutes (proteins, nucleic acids, endotoxin) which bind will be net negative. Commonly used anion exchange resins are Q-resin, a Quaternary amine; and DEAE resin, DiEthylAminoEthane (see figure below). AEC is often used as a primary chromatography.

The surface charge of the solutes (proteins, nucleic acids, endotoxin) which bind will be net positive. Commonly used cation exchange resins are S-resin, sulfate derivatives; and CM resins, carboxylate derived ions.

A sample is introduced, either manually or with an autosampler. A buffered aqueous solution known as the mobile phase carries the sample from the loop onto a column that contains some form of stationary phase material which is typically 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.

Affinity chromatography Fast separation technique

Ion exchange chromatography

Hydrophobic interaction chromatography

It is a non-denaturing Large volume of sample technique that can be used can be loaded at all stages and scales of purification It can serve as a Sample with high ionic concentrating step. A large strength can be used volume of dilute sample can be applied to a media, and the adsorbed protein subsequently eluted in a smaller volume It offers high selectivity, in that it can resolve molecules with small differences in charge Sample eluted with low salt

Simple and very effective purification tool

High selectivity

AFFINITY CHROMATOGRAPHY (AC)

HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC)

ION-EXCHANGE CHROMATOGRAPHY (IEC)

Expensive ligands Leakage of ligand

pH Salt used

May require high volume of eluent Usually a relatively slow process, but rapid selective elution processes are known Requires narrow pH control.

Degradation of the solid support

Buffer type

Limited lifetime-Resins seem to have finite lifetime, especially substrate analog resins. They lose effectiveness with time. Non-specific adsorption Relatively low productivity

Temperature

Each of these must be carefully controlled to achieve reproducible separation but also represent an opportunity for increased selectivity.

Widely application:

Affinity chromatography is widely used in the pharmaceutical industry to purify and extract molecules of interest from complex mixtures. Separation of native from denatured forms of proteins Removal of small amount of biomaterial from large amounts of contaminants Purify and concentrate a molecule from a mixture in solution, even at very low concentrations. Reduce the amount of a substance in a mixture. Purify and concentrate an enzyme solution.

 Softening

of water Demineralization of water Purification of solutions free from ionic impurities Separation of inorganic ions Separation of sugars, amino acids

Crude purification of human autotaxin. Purification of recombinant HIV reverse transcriptase

Purification of mammalian transcription factors

Micropurification of GTPase activating protein

Purication of recombinant nucleocapsid (NP)protein of Newcastle disease virus (NDV) from unclaried feedstock using expanded bed adsorption chromatography.
Source: Protein Expression and Purication journal (Tan et al., 2006)

NP protein is the major protein in Newcastle disease virus (NDV) and is highly immunogenic. It has successfully tested on ELISA for detecting the antibody (Timani, 2004). It can be used in immunodiagnostic kit for NDV detection in infected chicken serum.

To develop a simple and rapid technique to recover NP protein from unclaried E. coli using IMA-EBAC. ________________________________ **Immobilized metal affinity - expanded bed adsorption chromatography

IMAC-EBAC resulted in a 31% adsorption and 9.6% recovery of NP protein, improve compared to the 1.3% final yield obtained from the multistep conventional methods.

NP protein has been efficiently purified from unclarified feedstock by using IMA-EBAC without the need of any clarification and concentration steps. The purity of the NP protein recovered is reasonably high (about 70%)

Approximately, 38 mg or about 31% of bound NP protein has been eluted from the

adsorbent by elution buer with

high concentration (350 mM).

A two-step elution protocol was used to elute NP protein from the adsorbent containing 1. Elution buffer, (50 mM) can eliminate high amount of contaminating proteins. 2. Elution buffer, (350) efficient to elutes large amount of NP protein from the adsorbent.

IMA-EBAC has reduced the downstream processing time.