Reverse Phase chromatography separates proteins and peptides
based on the difference in hydrophobicity, Reversible interaction with the
hydrophobic chromatographic matrix/medium results in the separation. As the
sample is loaded, Binding to the chromatographic matrix takes place, altering
the conditions results in elution of the protein or peptide from the matrix.
The reverse phase nature of column matrix causes strong binding, usually harsh
conditions (organic solvents or ion pairing agents) are required to elute the
protein out from the matrix.
based on the difference in hydrophobicity, Reversible interaction with the
hydrophobic chromatographic matrix/medium results in the separation. As the
sample is loaded, Binding to the chromatographic matrix takes place, altering
the conditions results in elution of the protein or peptide from the matrix.
The reverse phase nature of column matrix causes strong binding, usually harsh
conditions (organic solvents or ion pairing agents) are required to elute the
protein out from the matrix.
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In Reverse Phase high purity can be achieved. Since harsh
conditions are used proteins and enzyme with final tertiary structure need to
be maintained may not suitable for purification using RPC since the protein or enzyme
activity may lose during the process.
In Reverse Phase high purity can be achieved. Since harsh
conditions are used proteins and enzyme with final tertiary structure need to
be maintained may not suitable for purification using RPC since the protein or enzyme
activity may lose during the process.
RPC used is often used in the final purification of
oligonucleotides, peptides and proteins. It is ideal for analytical separations
such as peptide mapping.
Reverse Phase HPLC separation: Overview
oligonucleotides, peptides and proteins. It is ideal for analytical separations
such as peptide mapping.
Reverse Phase HPLC separation: Overview
Select suitable
Hydrophobic Ligand
Hydrophobic Ligand
Ligand is the material on which proteins will interact with
and separate based on the hydrophobicity. Commonly used ligands in RPC are C4,
C8, and C18 n-alkyl hydrophobic ligands. Select suitable ones based on their
degree of hydrophobicity required for the separation.
and separate based on the hydrophobicity. Commonly used ligands in RPC are C4,
C8, and C18 n-alkyl hydrophobic ligands. Select suitable ones based on their
degree of hydrophobicity required for the separation.
Highly hydrophobic molecules bind tightly to strong
hydrophobic ligand like C18.
hydrophobic ligand like C18.
Suitable matrix which gives high resolution and purity need
to be selected, screening of RPC media can be done to achieve this, trying from
low hydrophobicity to high hydrophobicity screening is usually done.
to be selected, screening of RPC media can be done to achieve this, trying from
low hydrophobicity to high hydrophobicity screening is usually done.
Sample volume and
binding capacity
binding capacity
RPC is a binding technique which is independent on sample
volume. Total binding capacity depends on the matrix used and the conditions
provided for the binding. Matrix should be selected in such a way that one
should get high resolution with high sample loading volume.
volume. Total binding capacity depends on the matrix used and the conditions
provided for the binding. Matrix should be selected in such a way that one
should get high resolution with high sample loading volume.
Sample and column
Preparation
Preparation
As like any other column chromatographic separation process,
sample should be free of particulates, this can be achieved by filtering the
protein sample with no or low binding filters. Sample with particles can lead
to clogging and results in poor resolution and or damage to the system due to
back pressure generation and other factors.
sample should be free of particulates, this can be achieved by filtering the
protein sample with no or low binding filters. Sample with particles can lead
to clogging and results in poor resolution and or damage to the system due to
back pressure generation and other factors.
Columns of RPC need to be equilibrated with enough column
volumes of equilibration buffer so as to give perfect binding condition for the
proteins.
volumes of equilibration buffer so as to give perfect binding condition for the
proteins.
Storage
Column need to be stored as per the specifications of the
column supplier.
column supplier.
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