This document discusses reversed phase chromatography (RPC). RPC separates molecules based on differences in hydrophobicity using a hydrophobic stationary phase and polar mobile phase. Common stationary phases include silica or polystyrene beads chemically bonded to hydrophobic ligands like C18 chains. Mobile phase modifiers and ion pairing agents are used to control selectivity. RPC is useful for high resolution separation and analysis of proteins, peptides, and nucleic acids.

1. REVERSED PHASE
CHROMATOGRAPHY
PRESENTED BY:
VAISHALI JAIN

2. INTRODUCTION
• RPC has become increasingly important for high
resolution separation and analysis of proteins,
peptides and nucleic acids.
• This technique is ideal for applications such as
peptide mapping or purity checking and is often used
for final polishing of oligonucleotides and peptides.
• RPC is often combined with other chromatographic
methods to achieve identification and
characterisation of proteins and peptides.

3. PRINCIPLE
• RPC separates molecules according to differences in their
hydrophobicity.
• Stationary phase: hydrophobic
• Mobile phase: polar

5. MATRIX
• Medium consists of hydrophobic ligands chemically
grafted to a porous, insoluble beaded matrix.
• The matrix must be both chemically and mechanically
stable.
• The base matrix for the generally composed of silica or a
synthetic organic polymer such as polystyrene.

6. • Silica was the first polymer
used as the base matrix
• Produced as porous beads
which are chemically
stable at low pH and in the
organic solvents.
• The chemistry of the silica
gel allows simple
derivatisation with ligands
of various carbon chain
lengths.
• DISADVANTAGE:
Chemical instability in
aqueous solutions at high
pH. Not recommended for
prolonged exposure above
pH 7.5.

7. • Synthetic organic polymers, e.g. beaded polystyrene, are also
available as reversed phase media.
• ADVANTAGE: Stable at all pH values in the range of 1 to 12.

8. LIGANDS
• The selectivity of the reversed phase medium is
predominantly a function of the type of ligand grafted to the
surface of the medium.
• Generally linear hydrocarbon chains (n-alkyl groups) are the
most popular ligands used.
• The more hydrophobic the molecule to be purified, the less
hydrophobic the ligand needs to be.

9. END CAPPING
• Steric hindrance caused by C18 and C8 reagents prevents complete
derivatisation of all the available silanol groups. The residual silanol groups
are responsible for the deleterious mixed mode ion exchange effects.
• To prevent this, the residual silanol groups are reacted with smaller
alkylsilane reagents (chlorotrimethyl- and chlorotriethylsilanes).

10. MOBILE PHASE
ORGANIC MODIFIERS:
• The 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.
• Must be miscible in water.
• UV transparent
• Boiling point must be sufficiently low.

12. • ION PAIRING AGENTS: To
increase the hydrophobicity of
charged components, enhance
binding to the medium, and so
alter retention time.
• These agents bind via ionic
interactions with charged
groups and thereby suppress
their influence on overall
hydrophobicity.
• TFA for positively charged and
triethylamine for negatively
charged molecules.

13. Mixed mode retention &Ion
suppression
• Preventing mixed mode retention:
 End-capping
 Column ageing
 Addition of trifluroacetic acid
 Low pH environment

14. ELUTION
GRADIENT ISOCRATIC
Most frequently for
preparative and analytical,
high-resolution
separations of proteins
and peptides, in order to
minimize separation times.
Involve 2 mobile phases.
Mainly for high-
resolution analysis of
small organic molecules,
but can occasionally be
used to advantage as part
of an optimized
separation, also for
desalting sample.
Only one eluent is used.

15. GRADIENT ELUTION

17. ISOCRATIC GRADIENT
• DESALTING:

18. RESOLUTION
• Resolution (Rs) is defined as the distance between peak
maxima compared with the average base width of the two
peaks.

19. EFFICIENCY
• Column efficiency is the ability to elute narrow, symmetrical
peaks from a packed bed.
• It relates to the zone broadening that occurs on the column.
• Zone broadening can be minimized by minimizing distances
available for diffusion.
• A well packed column will contribute significantly for
resultion.

21. SELECTIVITY
• Selectivity is the degree of separation between peaks.

22. CRITICAL PARAMETERS
• Pore size
• Temperature
• Column length
• Flow rate

23. BINDING CAPACITY
• The binding capacity of an RPC medium is a quantitative
measurement of its ability to bind sample components under
defined static conditions.
• The dynamic binding capacity is a measure of the available
binding capacity at a specific flow rate.
• Porosity is a crucial factor in determining the binding capacity.