Principles of chromatography
Learning objectives:
1. Explain the principle that are common to all chromatographic methods.
2. Define and explain the significance of retention time, band width W, resolution Rs, number of theoretical plates N, linear
velocity of the mobile phase u.
3. Describe the account for the effects on peak sharpe of mobile phase velocity, stationary phase particle size and sample
loading
General principles
Molecules can be either in the stationary or mobile phase we say that they are in a partition
Substances present in a mixture are allowed to distribute (partition) themselves between 2 phases: the stationary phase
(fixed) and the mobile phase (moving).
The mobile phase contains the sample that’s being separated
As the mobile phase flows over the stationary phase, components of the mixture experience many transfers between the
2 phases
Depending on the property of the molecule to be separated and the properties of the mobile and stationary phase, the
molecule will distribute between the 2 phases, with a particular partition coefficient
Differential partitioning is the reason why can separate out components in a mixture
Depending on th affinity of the component to the molbile phase will depends on how long the component will bind.
Partition coefficient = [conc in stationaty phase]/[conc in mobile phase]
Components of the mixture that interact (bind) strongly with the stationary phase will travel more slowly down the
column
Column chromatography
Mixture is injected at one end of the column and the mobile phase is passed through at constant velocity (flow rate)
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 take least time to pass through the column and are eluted first i.e. they have the
shortest retention times (volume)
Retention time or retensio volume (whether a gas or liquid)
Good features of chromatography in general
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 need to be separated.
Most substances that can be dissolved are amenable to some form of chromatography.
These include biological molecules such as peptides and proteins that are often temerpature sensitive, water-solube and
not easy to purify by traditional chemical methods such as distillation, solvent extraction and recrystailsation
Typical chromatography set-up
Same for all chromatography but require different detector, stationary phase, mobile phase, pump….due to different
samples needed to be separated.
, Equipment
Pump: the flow of the molbile phase through the column is producedby : gravilty, pumps or in CG pressure is used
Column: the stationary phase might be packed in a glass (GC) or steel column ( for high pressure) and plastic for low
pressure
Detector: UV Absorption (most common), fluorescence, refractive index, electro chemical, combustion (GC-amount of
energy released is detected), or even NMR can be used
Example of a chromatogram
Here different proteins are separated, smaller proteins travel slower through this column
The grey peaks are standard
The 2 peaks in black show a monomer and dimer
Size-exclusion chromatography
Intensity at y axis
Elution volume (ml) on the x-axis
Abs 280nm aromatic side chains
Learning objectives:
1. Explain the principle that are common to all chromatographic methods.
2. Define and explain the significance of retention time, band width W, resolution Rs, number of theoretical plates N, linear
velocity of the mobile phase u.
3. Describe the account for the effects on peak sharpe of mobile phase velocity, stationary phase particle size and sample
loading
General principles
Molecules can be either in the stationary or mobile phase we say that they are in a partition
Substances present in a mixture are allowed to distribute (partition) themselves between 2 phases: the stationary phase
(fixed) and the mobile phase (moving).
The mobile phase contains the sample that’s being separated
As the mobile phase flows over the stationary phase, components of the mixture experience many transfers between the
2 phases
Depending on the property of the molecule to be separated and the properties of the mobile and stationary phase, the
molecule will distribute between the 2 phases, with a particular partition coefficient
Differential partitioning is the reason why can separate out components in a mixture
Depending on th affinity of the component to the molbile phase will depends on how long the component will bind.
Partition coefficient = [conc in stationaty phase]/[conc in mobile phase]
Components of the mixture that interact (bind) strongly with the stationary phase will travel more slowly down the
column
Column chromatography
Mixture is injected at one end of the column and the mobile phase is passed through at constant velocity (flow rate)
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 take least time to pass through the column and are eluted first i.e. they have the
shortest retention times (volume)
Retention time or retensio volume (whether a gas or liquid)
Good features of chromatography in general
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 need to be separated.
Most substances that can be dissolved are amenable to some form of chromatography.
These include biological molecules such as peptides and proteins that are often temerpature sensitive, water-solube and
not easy to purify by traditional chemical methods such as distillation, solvent extraction and recrystailsation
Typical chromatography set-up
Same for all chromatography but require different detector, stationary phase, mobile phase, pump….due to different
samples needed to be separated.
, Equipment
Pump: the flow of the molbile phase through the column is producedby : gravilty, pumps or in CG pressure is used
Column: the stationary phase might be packed in a glass (GC) or steel column ( for high pressure) and plastic for low
pressure
Detector: UV Absorption (most common), fluorescence, refractive index, electro chemical, combustion (GC-amount of
energy released is detected), or even NMR can be used
Example of a chromatogram
Here different proteins are separated, smaller proteins travel slower through this column
The grey peaks are standard
The 2 peaks in black show a monomer and dimer
Size-exclusion chromatography
Intensity at y axis
Elution volume (ml) on the x-axis
Abs 280nm aromatic side chains
