Chromatography analysis: Thin layer chromatography
Chromatography is used to separate mixtures of substances in to components. Thin layer chromatography is done using a thin uniform layer of silica gel or alumina coated onto a piece of glass, metal or rigid plastic.
The silica gel is the stationary phase, TLC uses a thin uniform layer of silica gel or alumina coated onto a piece of glass, metal or rigid plastic. The stationary phase for thin layer chromatography also often contains a substance that fluoresces in UV light. Silica gel is a form of silicon dioxide (silica). The silicon atoms are joined via oxygen atoms in a giant covalent structure. The surface of the silica gel is very polar because the silicon atoms are attached to OH bonds that form hydrogen bonds with the compounds around them.
The mobile phase is the liquid solvent, it flows through the stationary phase and carries the components of the mixture with it. When setting up a chromatogram a pencil line is drawn near the bottom of the plate and a small drop of solution of dye mixture is placed on it. When the spot of mixture is dry, the plate is stood in a shallow layer of solvent in a beaker. As the solvent travels up the plate the different components of the dye mixture travels at different rates and the mixture is separated into different coloured spots.
For maximum separation the solvent should rise until it almost reaches the top of the solvent. The position of the solvent should be marked and the solvent allowed evaporating. If the components of the sample are coloured they can be observed directly. If they are not then they can be visualised by shining an ultraviolet light on the plate. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.
The measurement are then taken:
The Rf value of each die is then worked out using the formula
Rf = distance travelled by the component Distance travelled by solvent
For example if the red component travelled 1.5cm from the base line and the solvent line travelled 5cm then the Rf value for the red dye will be
Rf =1.5 5 =0.3
The way the compound is carried up the plate depends on how soluble the compound is in the solvent and how much the compound sticks to the silica gel. This depends on how much attraction there is between the molecule of the compound and the solvent
For best separation on the technique there is need to determine the best solvent to develop the TLC plate in, the polarity of the solvent can be varied in several trial runs. Then the ideal solvent that can be used is the one that gives maximum separation therefore use of polar solvent will result in rapid movement of a polar compound up the TLC plate
High performance liquid chromatography –HPLC
High performance liquid chromatography is a highly improved form of column chromatography, instead of the solvent allowed to drip through the column under gravity it will be forced under high pressure making it faster. The apparatus consists of a pumping system, an injector, a chromatographic column,…