Explore the fascinating world of reverse phase chromatography in this comprehensive presentation. Reverse phase chromatography is a widely used technique in analytical chemistry and biochemistry for separating molecules based on their hydrophobicity and polarity.
In this presentation, we delve into the fundamental principles of reverse phase chromatography, discussing the mechanisms behind the separation process and the interaction between the stationary phase and mobile phase. We explore the role of various parameters such as column chemistry, mobile phase composition, and gradient elution in optimizing separation efficiency and resolution.
2. INTRODUCTION
◦Liquid chromatography:
Liquid chromatography is a seperation technique in
which the Mobile phase is liquid where sample ions or
molecules dissolved,the sample with mobile phase pass
through the column or plane which is packed with the
stationary phase Composed of irregular or spherical
shaped beads.
3. Contd.. introduction
◦Reversephase chromatography is a type of liquid
chromatography technique that involves the seperation of
molecules on the basis of hydrophobic interactions between the
solute molecules in the mobile phase and the ligands attached
to the stationary phase.
◦ It is column chromatography in which stationary phase is
packed in the column and the mobile phase containing solutes
will pass through it.
4. PRINCIPLE:
◦ The principle involved in reverse phase
chromatography is “adsorption”,as the seperations in
reverse phase chromatography relys upon reversible
absorption/ desorption of solutes with chainging levels
of hydrophobicity to hydrophobic stationary phase.
5. Components in reverse phase
chromatography :
◦Stationary phase:
◦Genrally composed of silica /synthetic
polystyrene.It should be physically and
chemically stable.
◦Mobile phase:
◦Organic solvents( modifiers)like
acetonitrile,methanol,isopropanol are used.
6. Contd…components
◦Ligands:
◦ Linear hydrocarbon chains (n-alkyl) groups are the most
popular ligands.
For eg: C18 ligands-for the seperation Of peptides
and nucleotides.
◦ C8 ligands –for the seperation of proteins and peptides.
7. Concept/mode of seperation in
RPC:
◦ Stationary phase- non polar
◦ Mobile phase- polar
◦ Column is packed with stationary
phase which is usually silica.
◦ Alkyl or aromatic ligands are
covalently bound to the silanol
groups on the surface of the silica to
provide a hydrophobic surface to
stationary phase.
8. Contd…concept:
◦ Mobile phase passing over the hydrophobic stationary
phase which is a polar solvent containing solutes.
9. Contd…concept:
◦ In this scenario,hydrophobic
solutes in the Mobile phase
tend to get bound to the
stationary phase ,via
hydrophobic interactions and
thus form the basis of
seperation.
10. Contd...concept
◦ The compounds which has
strong hydrophobic
interactions with the
stationary phase i.e nonpolr
will elute slowly.
◦ The compounds which has
less hydrophobic interactions
i.e high polar compounds
with stationary phase elute
fastly.
11. Elution type:
◦The type of elution in RPC is “ Gradient elution”.
◦Once the molecule of interest is bound to the
column matrix or stationary phase it is made to
dissociate from it by decreasing the polarity.
◦The polarity can be decreased by increasing the
conc of organic solvent in the Mobile phase.
◦This procesof varying the amount of organic solvent
in mobile phase to separate a molecule of interest is
called “gradient elution”.
12. Detectors:
◦The eluted compounds are sent to detectors
where they convert into electric signals ,these
electric signals are sent to recorder where peaks
of chromatogram are observed.
◦UV detectors should be used for effective reverse
phase seperation.
13. Advantages of reverse phase
chromatography:
◦Tested stability of the matrix under a variety of
mobile phase scenarios.
◦Reproducibility of seperations.
◦Excellent resolutions acheivable for molecules of
interest.
◦Appreciable recoveries and through output.
◦Ease of seperation provided by gradient elution.
14. Applications of Reverse phase
chromatography:
◦In the analysis and purification of peptides and
polypeptides.
◦In the seperation of proteins and recombinant
peptides.
◦Extraction of various contaminants in
environmental samples.
