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Calculate Rf values from a chromatogram
Compare retention times and Rf values with standards to identify different substances.
Required practical 12: Separation of species by thin-layer chromatography.
Used to separate and identify the components in a mixture.
Types of chromatography include:
thin-layer chromatography (TLC) – a plate is coated with a solid and a solvent moves up the plate
column chromatography (CC) – a column is packed with a solid and a solvent moves down the column
gas chromatography (GC) – a column is packed with a solid or with a solid coated by a liquid, and a gas is passed through the column under pressure at high temperature.
Separation depends on the balance between solubility in the moving phase and retention by the stationary phase.
Retention times and Rf values are used to identify different substances.
The use of mass spectrometry to analyse the components separated by GC.
Chromatography is a technique used for separating mixtures. There are several types of chromatography, including:
• Thin-layer chromatography (TLC)
• Column chromatography (CC)
• Gas chromatography (GC), also known as gas-liquid chromatography (GLC)
The components in a mixture, when dissolved in a fluid (mobile phase), will flow through another material (stationary phase) at varying rates. The rate of separation depends on how the components in the mixture interact with the stationary phase (their retention) and how soluble they are in the mobile phase.
The retention factor (Rf value) is used to measure the degree of separation of components in a mixture. It is calculated by dividing the distance the compound moves by the distance moved by the solvent. Each component in a mixture will have a unique retention factor.
In gas-liquid chromatography (GLC), the term retention time is used. This refers to the time that elapses from the moment of injection to when a component exits the chromatography tube.
What affects Rf Values?
Since the solvent transports the chemical along the plate, as the mobile phase, the solvent has an impact on the chemical’s retention factor value. A solvent with a stronger interaction with a solute can easily overcome the solute’s affinity for the absorbent stationary phase and so can move the solute further in a given amount of time. Depending on the quantity of each solvent in a mixture, different effects can be achieved.
The temperature of the solvent and plate may make slight changes, since, the solvent can often better dissolve the chemicals it is transporting at higher temperatures. The retention factor might also be affected by human factors - too much sample can cause wide, diffuse bands of solute to move up the plate, making it difficult to estimate the distance the chemical has travelled.
Mobile Phase - fluid that carries the mixture through the stationary phase
Stationary Phase - material that the mobile phase flows through
Retention Factor (Rf value) - ratio of distance travelled by the compound to distance travelled by the solvent
Retention Time - time that elapses from the moment of injection to when a component exits the chromatography tube in GLC
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