Investigation using thin layer of chromatography to separate photosynthesis pigment

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Investigation using thin layer of chromatography to separate photosynthesis pigment


Chromatography is a technique used for separating components or solutes of a mixture between two phases the mobile phase and the stationary phase. The stationary phase is a sheet of chromatography paper in paper chromatography. The mobile phase may either be a water-based liquid or a carbon-base organic solvent. For each chemical in the sample, there is a changing phase of balance between the stationary phase and the mobile phase.

A paper chromatogram can be produced in a number of ways, for example if you have three green pens and you’re trying to identify which one was used to write a letter, samples of each ink are spotted onto a pencil line that’s drawn on a sheet of chromatography paper, some of the ink from the letter is then dissolved in a small amount of suitable solvent and is then spotted onto the same line. The paper is then inserted into a container with a deep layer of a suitable solvent in it and it’s important that the solvent level is below the line with the spots and the container is then covered to make sure the atmosphere in the beaker is saturated with the solvent vapour as this stops the solvent from evaporating. As the solvent travels up slowly up the chromatography paper, the different components of the ink mixtures are separated into different colours spot due to the ink travelling at a different rate.

Different factors affect the chromatography movement such as temperature; solvent; mixture and polarity. Temperature affects the movement of chromatography as the temperature of the solvent can dissolve better in a chemical that’s been transported at higher temperature. The solvent can affect the chromatography movement as a solvent with a strong interaction for a specific chemical will more easily overcome any affinity for the absorbent layer. The mixture also affects chromatograph movement as mixtures of solvents can also have different effects depending on the amount of each solvent. As the polarity of a substance is increase all the components of the mixture will move faster during the chromatography experiment

To make the chromatography technique more scientific, Rf value was introduced (Retention Value). Every compound has a specific Rf value for every specific solvent and solvent concentration, Rf value is used to identify the rf value for the unknown sample with the Rf value of the known compounds. The Rf value is described as the ratio of the distance moved by the solute and the distance moved by the solvent.

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The overall separation depends upon how strongly attracted the chemicals are to the mobile and          the stationary phases. This produces a chromatogram where different samples can be compared to find       the pigment group.

Thin layer chromatography is also like paper chromatography, but the stationary phase is a glass plate with a uniform layer of silica gel instead of paper. TlC chromatography has some advantages over paper chromatography such as the moving phase can move more quickly through the stationary phase, TLC also tend to produce more chromatograms by showing a bigger separation ...

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