2. Introduction of Chromatography &
Reverse Phase Chromatography
Reverse Phase Chromatography is also known as
Adsorption chromatography.
It depends on the chemical interactions between solute
molecules and specifically designed ligands chemically
grafted to a chromatography matrix.
Reversed Phase Chromatography is a technique in which
the binding of mobile phase solute to an immobilized n-
alkyl hydrocarbon or aromatic ligand occurs via
hydrophobic interaction.
3. Application of Reverse
Phase Chromatography
Reversed phase Chromatography
has found both analytical and
preparative applications in the
area of biochemical separation
and purification.
Molecules that possess some
degree of hydrophobic character,
such as proteins, peptides and
nucleic acids, can be separated
by reversed phase
chromatography with excellent
recovery and resolution.
4. Mode of Separation
Reverse Phase Chromatography
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 a synthetic
organic polymer such as polystyrene.
The selectivity of the reversed phase medium is
predominantly a function of the type of ligand grafted to the
surface of the medium.
Generally speaking, linear hydrocarbon chains (n-alkyl
groups) are the most popular ligands used in reversed phase
applications.
Si
Si
O－Si－CH2(CH2)16CH3
CH3
CH3
O－Si－CH2(CH2)16CH3
CH3
CH3
O－Si－CH2(CH2)16CH3
CH3
CH3
CH3
CH3
O－Si－CH3
5. Reverse Phase Media
The reverse phase media has two parts.
Matrix Ligand
Synthetic organic polymers,
e.g. beaded polystyrene, are
also available as reversed
phase media.
6. Normal and Reverse Phase Chromatography
Normal Phase:
In the 1970s most liquid
chromatography was done
on non-modified silica or
alumina with a
hydrophilic surface
chemistry and a stronger
affinity for polar
compounds - hence it was
considered "Normal".
Reverse Phase:
The introduction of alkyl
chains bonded covalently
to the support surface
reversed the elution order.
Now polar compounds are
eluted first while non-
polar compounds are
retained (hydrophobic) -
hence "Reversed Phase".
7. Theory of Reverse Phase Chromatography
Step 1: Equilibrate the column packed with the RP medium under suitable initial
mobile phase conditions of pH, ionic strength and polarity (mobile phase
hydrophobicity). The polarity of the mobile phase is controlled by adding organic
modifiers acetonitrile.
Step 2: The sample containing the solutes is applied the chromatographic bed is
washed further with mobile phase A to remove unwanted and unbounded molecules.
Step 3: Bound solutes are next desorbed from the reversed phase medium by
adjusting the polarity of the mobile phase and elute from the column.
Step 4: Removal substances not previously desorbed from column by changing
mobile phase B.
Step 5: Re-equilibration of the chromatographic medium from 100%
mobile phase B back to the initial mobile phase conditions.
8. RP Mechanism (Simple)
Non-polar compounds in the mixture will tend to form attractions with the
hydrocarbon groups because of van der Waals dispersion forces.
Non Polar compounds are less soluble in the solvent because of the need to
break hydrogen bonds as they squeeze in between the water or methanol
molecules.
Therefore spend less time in solution in the solvent and spent more time way
through the column.
9. Resolution & Retention Time
Resolution is a combination of the degree of
separation between the peaks eluted from the
column (selectivity), the ability of the column to
produce narrow, symmetrical peaks (efficiency), the
amount (mass) of sample applied and the retention
time of the samples on the column.
Resolution (Rs) is defined as the distance between
peak maxima compared with the average base
width of the two peaks. Rs can be determined from
a chromatogram, as shown in Figure. The resolution
of two peaks is determined by three variables: the
retention factor (k), number of theoretical plates
(N), and the separation factor (α). The separation
factor (selectivity) α = k2/k1 (where k2>k1).
Retention Time: The retention time is defined as
the time between the sample introduction and the
elution from the column. The retention time can be
increased by adding more polar solvent or water
and decrease by adding organic solvent.
The resolution can be described by the
equation for two closely eluting compounds.
The resolution of two peaks can be
improved by increasing a, N, or k.
10. Some Terms to Understand
Retention Factor (K)
At any given time during the
migration through the
system, there is a
distribution of molecules of
each component between
the two phases.
K= ns/nm is called the
retention factor.
ns = Number of molecules in
the stationary.
nm= Number of molecules
mobile phases at a given
time.
Efficiency (N)
The efficiency of a packed
column is expressed by the
number of theoretical
Plates. N The greater the
number of theoretical plates
a column has, the greater its
efficiency and resolution.
The number of theoretical
plates, N, is given by
N = 5.54 (V1/W1/2)2
where V1 is the retention
volume of the peak and W1/2
is the peak width (volume)
at half peak height.
Selectivity
Good selectivity is the
degree of separation
between peaks.
It is an important factor in
determining resolution and
depends largely on the
nature of the RPC medium,
the nature and composition
of the eluent and the
gradient used for elution.
The selectivity, a, for two
peaks is given by
α = k2´ /k1´ = V2 - V0/V1 – V0
α = V2/V1
11. Critical Parameters in RP-chromatography
Column Length:
The resolution of high molecular weight biomolecules in reversed phase separations
is less sensitive to column length than is the resolution of small organic molecules.
Proteins, large peptides and nucleic acids may be purified effectively on short
columns and increasing column length does not improve resolution significantly.
Gradient elution:
Gradients in are usually performed either with increasing pressure (increasing
density) or with increasing temperature, or with increased concentration of polar
modifiers (such as methanol). It enhance the peak.
Elution mode is gradient elution by changing composition of the mobile phase.
pH:
Optimum pH is one of the most important parameters to establish. Selectivity can
be achieved changing the pH, for the separation.
Ion-pairing Agents:
A common way to increase the hydrophobicity of charged components, enhance
binding to the medium, and so alter retention time, is to add ion-pairing agents to
the eluent. Since most proteins and peptides are slightly basic, ion-pairing agents
are often acids such as trifluoroacetic acid (TFA) whereas a base such as
triethylamine is used for negatively charged molecules.
