Chromatography and NMR
Binding curves:
CD can be used to monitor the binding if s substrate to a protein.
The substrate can give a very different CD spectrum when free in the solution relative to when bond in solution.
Outside of farUV: 180-240nm.
1. Near UV CD: 240n-320nm, Aromatic amino acids and disulphide bonds.
2. Visible CD: d-d transition in some metal protein complexes for eg Cu (II) prion.
Principles of Chromatography
Substances present in a mixture are allowed to distribute themselves between two phases: the stationary phase (fixed) and the mobile phase.
As the mobile phase flows over the stationary phase, components of the mixture experience many transfers between the two phases.
Depending on the properties of the molecule to be separated and that of the mobile phase and stationary phase, the molecule with distribute between the two phases with a particular partition coefficient.
Partition coefficient: [stationary phase]/ [mobile phase].
Components of the mixture then interact strongly with the station phase will travel more slowly down the column.
Column chromatography:
The mixture is injected at one end of the column and the mobile phase is passes through at constant velocity.
All components of the mixture must travel the full length of the column before they are eluted at the outlet of the column and are detected.
The fastest moving components takes less time to pass through and are eluted first and they have the shortest retention time.
Good features of chromatography:
1. There are many possible combinations of stationary and mobile phases. These can be selected taking into account the physical and chemical properties of the materials that are needed to be separated.
2. Most substances that can be dissolved are amenable to some from of chromatography. Like biological molecules such as peptides and proteins that are often temperature sensitive, water soluble and
CD can be used to monitor the binding if s substrate to a protein.
The substrate can give a very different CD spectrum when free in the solution relative to when bond in solution.
Outside of farUV: 180-240nm.
1. Near UV CD: 240n-320nm, Aromatic amino acids and disulphide bonds.
2. Visible CD: d-d transition in some metal protein complexes for eg Cu (II) prion.
Principles of Chromatography
Substances present in a mixture are allowed to distribute themselves between two phases: the stationary phase (fixed) and the mobile phase.
As the mobile phase flows over the stationary phase, components of the mixture experience many transfers between the two phases.
Depending on the properties of the molecule to be separated and that of the mobile phase and stationary phase, the molecule with distribute between the two phases with a particular partition coefficient.
Partition coefficient: [stationary phase]/ [mobile phase].
Components of the mixture then interact strongly with the station phase will travel more slowly down the column.
Column chromatography:
The mixture is injected at one end of the column and the mobile phase is passes through at constant velocity.
All components of the mixture must travel the full length of the column before they are eluted at the outlet of the column and are detected.
The fastest moving components takes less time to pass through and are eluted first and they have the shortest retention time.
Good features of chromatography:
1. There are many possible combinations of stationary and mobile phases. These can be selected taking into account the physical and chemical properties of the materials that are needed to be separated.
2. Most substances that can be dissolved are amenable to some from of chromatography. Like biological molecules such as peptides and proteins that are often temperature sensitive, water soluble and