bluebettafish
My chem notes:
Chromatography:
Chromatography is used to separate and analyse complex mixtures. All methods involve:
- A mobile phase - which is the solvent containing the sample (often a liquid)
- A stationary phase - which is the surface the mobile phase flows over/through.
The retention time (time taken for a component to pass through) is dependent on the ability for different substances to interact with each phase. This includes how well the stationary phase adsorbs to the sample - and how well the mobile phase dissolved the sample (desorption). It is often influenced by the polarity of the molecules.
High performance liquid chromatography (HPLC) uses a solid stationary phase consisting of tiny particles. The mobile phase is pumped in at high pressure and the eluent stream (mixture of sample + mobile phase) is monitored by a UV detector. This is presented on a chromatogram.
Each peak on a chromatogram represents a component of the sample. If a set of standards is used, with known concentrations, a regression line can be calculated providing a relationship between the area under a peak and the concentration.
Factors affecting retention time include:
- The polarity of the stationary/mobile phase
- The length of the column
- The temperature of the column
- The mobile phase flow rate
- The surface of the stationary phase
Explain how HPLC works:
- HPLC relies on the repeated transfer of components from the stationary and mobile phase
- Components that are more strongly adsorbed to the stationary phase will have higher retention times as they travel slower than components that more strongly desorb to the mobile phase.
- The different retention times lead to separation.
An increase in temperature will mean that:
- the components in the HPLC column are less adsorbed to the stationary phase and more strongly desorbed in the mobile phase
- as the components are more soluble with an increased temperature.
- This leads to a decreased retention time.