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Paper Chromatography

Investigatory Project and Viva Questions on Paper Chromatography


Introduction


Chromatography is a very efficient method for the separation and purification of different components present in a mixture. The method is based on the distribution of different components in a mixture between a fixed or stationary phase and a mobile or moving phase. There are a number of chromatographic techniques based on the nature of the stationary phase. The stationary phase may be an adsorbent column, a paper or a glass plate coated with a thin layer of an adsorbent, through which the mobile phase moves. The mobile phase may be a liquid or a gas. The separation takes place due to the differential migration of the components which in turn depends on the relative affinity of the components towards the stationary and mobile phases. The component which is less strongly held by a stationary phase moves faster in a mobile phase.


Aim


Separation of pigments present in leaves and flowers by paper chromatography and the determination of Rf values of the separated components.


Paper chromatography:


This method is used for the separation and identification of the components present in a mixture especially when they are present in small amounts. In paper chromatography the stationary and mobile phases are liquids. The stationary phase is water present in filter paper (cellulose) and the mobile phase is a pure solvent or a mixture of solvents. Therefore, the basic principle of separation in paper chromatography is partition of the components of a mixture in two liquids.


Some flowers or leaves are taken and they are ground well in a mortar using a pestle. About 3 mL of acetone is added to it and it is again ground. The coloured clean solution is decanted. A Whatman filter paper No. 1 is taken and is cut to get a size of approximately 5cm x 30cm (depending on the size of the jar used). A line is drawn using a pencil at a distance of about 2 cm away from one end of the paper. This line is known as the reference line. About 2 to 3 drops of the extract of the leaves or flowers are placed at the centre of the reference line. It is then dried using a drier.

About 40-50 mL of the mobile liquid (acetone or alcohol) is taken in a gas jar. The filter paper is hanged into the gas jar in such a way that filter paper tip touches the liquid and the reference line is above the surface of the mobile liquid. The gas jar is now closed with a lid which has a slit on it. The upper end of the filter paper strip passes through the slit. The filter paper is allowed to stand for about 2 hours.


The solvent moves up on the filter paper. Along with the solvent the different constituents also move with varying speeds according to their partition coefficients. The coloured components are finally separated from each other. When the solvent front traverses about 75% of the filter paper, the paper is removed and the solvent front is marked on the paper. The chromatogram is dried and the distances travelled by the solvent front and the different components from the reference line are measured. From these the Rf values of different components are calculated. Rf value (retention factor) is defined as the ratio of distance travelled by a particular component from the origin line to the distance travelled by the solvent from the origin line.


Rf = Distance travelled by a component from the origin line/Distance travelled by the solvent from the origin line


The Rf value depends on the nature of the substance, nature of the mobile and stationary phases and the temperature. Rf values can be used to identify the components in a mixture.


Calculation


Distance travelled by solvent = a cm

Distance travelled by component X = b cm

Distance travelled by component Y = c cm

Rf of component X = b/a

Rf of component Y = c/a


PAPER CHROMATOGRAPHY VIVA QUESTIONS WITH ANSWERS.


1. What is chromatography?


Ans: It is a technique for separating components of a mixture by their relative movements in a mobile phase against a stationary phase.


2. Why do different substances travel at different speeds in the mobile phase?


Ans: Different substances have different degree of attachments towards the stationery phase. Substances which are attached strongly will move slowly in the mobile phase. Those which are attached weakly will move faster in the mobile phase.


3. What is adsorption chromatography?


Ans: If a substance is held to the surface of another substance by surface forces it is called adsorption. Chromatography based on adsorption is called adsorption chromatography.


4. What is partition chromatography?


Ans: Here one substance penetrates into another substance. There is absorption rather that adsorption. For example a substance between two liquids. Chromatography based on absorption is called partition chromatography.


5. Why is paper chromatography considered as partition chromatography?


Ans: Here the substance concerned is partitioned between the mobile phase and the water present in the paper.


6. Mention two substances that are commonly used as stationary phase in adsorption chromatography. 


Ans: (1) Aluminium Oxide (2) Chalk


7. What is Rf factor?


Ans: It is the ratio of the distance travelled by a solute substance to the total distance travelled by the solvent front.


8. In paper chromatography Rf factors help to identify the substance. How?


Ans: If the Rf  factors of different substances concerned are known, then from the relative positions of various substances they can be identified.

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