The chromatography technique can be widely used to separate, purify, and also identify the compounds. The substance balances in the chromatography between a stationary phase and a mobile phase.
To separate the pigments that are present in flowers and leaves by the paper chromatography technique and determine their Rf values.
Paper chromatography is mainly a partition chromatography. In this paper chromatography, the stationary phase is defined as paper. The paper contains 22 percent of water molecules absorbed on about 78 percent of cellulose.
The components mixture separation takes place by partitioning of the components between the mobile phase and stationary phase. The mobile phase travels via paper by the capillary action. Depending on the ways the solvent travels on the paper, there exist three types of chromatography, given as follows.
Ascending Paper Chromatography
Descending Paper Chromatography
Circular Paper Chromatography
The distribution takes place in a definite ratio that represents the solution's characteristic distribution coefficient.
The Rf Coefficient Ration is Given as Follows:
Rf=distance travelled by solute, distance travelled by the solvent
Various substances possess various Rf values. Rf depends on a number of factors; where a few of them are listed below.
If any compound is coloured, it can be easily located on a chromatographic paper. If the same substance is colourless, however, a reagent can be used to treat it, which produces a characteristic colour. The term developer is given to this reagent. And iodine is the most frequently used paper chromatography developer.
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Take the Whatman filter paper and then draw a line using a pencil above 4cm from one end.
Then, grind the flowers and leaves in a motor and transfer the paste into the test tube.
In the crushed material, add methanol or acetone, and shake well and filter the respective mixture.
The filtrate is collected in a test tube to perform experiments.
With the help of a capillary tube, add one drop of the filtrate on the filter paper and allow it to dry.
After that, hang the filter paper in a jar containing 20ml of chloroform and petroleum ether.
Keep the same jar till the mobile phase rises up to 2/3rd of the length of the paper.
Then, remove the filter paper from the jar and mark the solvent front.
Outline the spots using a pencil and allow the filter paper to dry.
Now, measure the distance between the centre of different spots and the solvent front in relation to the reference line as indicated
Then, determine the pigment number in the flowers and leaves extract.
Finally, calculate the Rf value of different spots using the expression.
Observations and Inference
The noticed Observations and Inferences can be recorded in the respective fields of the table, which is given below.
Results and Discussions
Always choose a fine capillary tube.
Do not allow the spots to spread while spotting the test solution available on the paper.
Do not disturb the jar once the experiment is arranged as long as the development of the chromatogram has been completed.
Use the capillary finely drawn to place the spot on the same paper.
Prior to developing the spots, allow the paper strip to become dry perfectly.
Handle the organic solvents carefully.
Finding the Number of Pigments in Leaves with the Help of Chromatography
If there exist more samples, it's spots are bigger on the TLC plate.
Like when we spill coffee on a white shirt- the more coffee, the larger the spot! In general, on a shirt, the water carries the solute, which is considered a poor example.
Remember that a sample is applied in a tiny spot that goes to thin-layer chromatography (TLC) glass or near the bottom of the paper or the plastic plate. Then, the plate is placed carefully in the appropriate solvent and moves in an upward direction uniformly. Solutes, which are otherwise called pigment molecules, dissolve. After that, move upward at various rates with the solvent while the paper or the TLC plate adsorbent wants to "hold on" to the molecules. This is the main reason why the molecules having different polarities are separated and hence generate an Rf value.