Reversed Phase Chromatography

Presented By,

Pooranachithra M,

Ist M.Tech Biotechnology.

Introduction:

It is an Adsorption technique.

The basic principle behind this is the molecular hydrophobicity.

The separation depends on the hydrophobic binding of the solute molecule from the mobile phase to the immobilized hydrophobic ligands attached to the stationary phase

Reason For Reversed Phase

In normal phase chromatography,

* Stationary phase – Polar mobile phase – Nonpolar

* for the separation of polar substances

In reversed phase chromatography,

* Stationary phase – Nonpolar mobile phase – Polar

* for the separation of nonpolar substances

Molecules that possess some degree of hydrophobic character, such as proteins, peptides and nucleic acids, can be separated

Theory Of RP Chromatography:

Hydrophobic Theory - the binding of mobile phase solute to an immobilized n-alkyl hydrocarbon or aromatic ligand occurs via hydrophobic interaction

Partition Theory - Analyte distributes between aqueous mobile phase and organic stationary phase

Adsorption Theory – Reversed phase chromatography is an adsorptive process by experimental design

The Matrix:

A reversed phase chromatography medium consists of hydrophobic ligands chemically grafted to a porous, insoluble beaded matrix.

The base matrix for the commercially available reversed phase media is generally composed of silica or polystyrene

Silanol groups on the silica surface of the silica gel is coupled to the hydrocarbon ligands using chlorotrialkylsilane reagents.

Silanol Groups

Ligands:

The selectivity is predominantly a function of the type of ligand grafted to the surface of the medium.

Linear hydrocarbon chains (n-alkyl groups) are the most popular ligands used in reversed phase applications.

A good rule of thumb is: The more hydrophobic the molecule to be purified, the less hydrophobic the ligand needs to be.

For example,

* C18 ligands - peptides and oligonucleotides

* C8 ligands - Proteins and recombinant peptides

Mixed Mode Retention

Mixed mode retention results from an

ion exchange interaction between

negatively charged silanol groups

exposed on the surface of the silica

and the positively charged amino

groups on the solute molecules.

The effect of mixed mode retention is

increased retention times with

significant peak broadening.

Properties Of The Matrix:

the chemical composition of the base matrix

particle size of the bead

the type of immobilized ligand

the ligand density on the surface of the bead

the capping chemistry used (if any)

the pore size of the bead.

Mobile phase

Term used for the mobile phases in reversed phase chromatography is “buffer”.

Organic solvent (modifier) is added to lower the polarity of the aqueous mobile phase.

The lower the polarity of the mobile phase, the greater its eluting strength in reversed phase chromatography.

Widely used organic solvents are - Acetonitrile, methanol, isopropanol.

Ion suppression

The major benefit of ion suppression in reversed phase

chromatography is the elimination of mixed mode retention

effects due to ionisable silanol groups remaining on the silica

gel surface

The retention of peptides and proteins in reversed phase

chromatography can be modified by mobile phase pH since

these particular solutes contain ionisable groups.

Ion Pairing Agents

The retention times of solutes such as proteins, peptides and oligonucleotides can be modified by adding ion pairing agents to the mobile phase.

The retention times of solutes such as proteins, peptides and oligonucleotides can be modified by adding ion pairing agents to the mobile phase.

Gradient Elution

The concentration of organic solvent is lower in the initial

mobile phase (mobile phase A) than it is in the final

mobile phase (mobile phase B).

The typical gradients for preparative reversed phase

chromatography of proteins and peptides are linear and

binary, i.e. involving two mobile phases

Resolution:

Resolution, Rs, is generally defined as the distance between the centres of two eluting peaks as measured by retention time or volume divided by the average width of the respective peaks.

The parameters that contribute to peak resolution are column selectivity, column efficiency and the column retention factor

Applications:

Preparative reversed phase chromatography has found applications ranging from

micro purification of protein fragments for sequencing (1)

To process scale purification of recombinant protein products (2).

Thank you