Theory & Procedure, Paper Chromatography | Additional Study Material for NEET PDF Download

Objective

Our objective is to;

1. Separate the coloured components present in the mixture of red and blue inks by ascending paper chromatography and find their Rf values.
2. Separate the coloured components present in the extract of spinach leaves by ascending paper chromatography and find their Rf values.

The Theory

Are all organic componds pure?

No, most organic compounds obtained from natural sources and synthesised in laboratories are not pure. Various methods are used for the purification of organic compounds that are based on the nature of the compound and the impurity present in it. Among the various separation techniques, chromatography is one of the most important separation technique extensively used to separate mixtures into their components and the purification of the compounds.

What is Chromatography?

The word chromatography originated from two greek words 'chroma' meaning 'colour' and 'graphine' meaning 'to write'. Chromatography means colour writing and it was first employed by a Russian scientist Mikhail Tsvet. This method was first used for the separation of coloured substances in plants.

In the chromatographic technique, the mixture of substances is applied onto a phase called the stationary phase. The stationary phase may be solid or liquid. A moving phase that can be a pure solvent or a mixture of solvents, or a gas is allowed to move slowly over the stationary phase. This moving phase is called the mobile phase. When the mobile phase is moved over the mixture on the stationary phase, the components of the mixture gradually separates from one another.

Classification of Chromatography

Depending on the basic principle involved in chromatography, it is mainly classified into two.

1. Adsorption chromatography

It is based on the differential adsorption of the components on the adsorbent (stationary phase). This means that different compounds are adsorbed on an adsorbent at different degrees. Following are the two main types of chromatographic techniques based on the principle of differential adsorption.

• Column Chromatography
• Thin layer Chromatography

Column chromatography

In column chromatography, the stationary phase or adsorbent is a solid and the mobile phase is a liquid. The most commonly used stationary phases are silica gel and alumina. The mobile phase or eluent is a pure solvent or a mixture of solvents. It involves the separation of a mixture over a column of adsorbent packed in a glass tube called chromatography column. Chromatography column is a glass tube with diameter from 5 mm to 50 mm with length 5 cm to 1 m with a stopcork at the bottom. The mixture adsorbed is placed on the top of the adsorbent column packed in the chromatography column. An appropriate eluent is allowed to run down the column slowly. Depending upon the degree to which the components are adsorbed, complete separation takes place. The most readily adsorbed components are retained near the top and others come down to various distances in the column.

Thin Layer Chromatography

Thin layer chromatography is another type of adsorption chromatography, which involves the separation of a mixture of substances over a thin layer of an adsorbent coated on a glass plate. In this case, the stationary phase is a glass plate of suitable size coated with a thin layer of stationary phase usually silica gel or alumina. This plate is known as thin layer chromatography plate (TLC plate) or chromaplate. The solution of mixture to be separated is applied as a small spot about 2 cm above one end of the TLC plate. The glass plate is then placed in a closed jar (chromatography chamber) containing the mobile phase. As the mobile phase rises up the plate by capillary action, the components of the mixture move up along with the solvent to different distances depending on their degree of adsorption and separation takes place. The relative adsorption of each component of the mixture is expressed in terms of its Retardation factor (Rf) (Retention factor).

What is Retention factor (Rf value)?

It is defined as the distance moved up or travelled by the component from the original line to the distance travelled by the solvent from the original line.

How do we detect the components on the TLC plate?

• The spots of coloured components are visible on the TLC plate due to their original colour. The spots of colourless components that are invisible to the eye but fluorescent in ultraviolet light can be detected by putting the plate under ultraviolet light.
• Another detection technique is to place the plate in a covered jar containing few crystals of iodine, spots of the components that absorb iodine, will seen as brown spots.
• Another method is to spray an appropriate reagent on the TLC plate. For example, amino acids may be detected by spraying the plate with ninhydrin solution.

2. Partition Chromatography

The basic principle of partition chromatography is the continuous differential partitioning of components of a mixture between the stationary phase and the mobile phase. An important partition chromatography is Paper Chromatography.

Paper Chromatography

In paper chromatography, the stationary phase is a special quality paper called chromatography paper. Mobile phase is a solvent or a mixture of solvents. A solution of the mixture is spotted on a line about 2 cm above from the bottom of the paper, called original line or base line and then suspended in a chromatography chamber containing suitable solvent. The solvent rises up the paper by capillary action and flows over the spot. The paper selectively retains different components according to their differing partition in the two phases. The paper strip so developed is called Chromatogram. The spots of the separated coloured compounds are visible at different heights from the position of initial spot on the chromatogram. The spots of the separated colourless components may be observed either under ultraviolet light or by the use of an appropriate spray reagent. The distance travelled by the solvent from the original line is called solvent front. The relative adsorption of each component of the mixture is expressed in terms of its Retardation factor (Rf) (Retention factor).

Ascending and Descending Paper Chromatography

The type of paper chromatography in which the solvent rises up is called Ascending paper chromatography. Alternatively, the solvent may be taken on the top in a container and be allowed to come down, in which case it is termed as Descending paper chromatography.  Below is shown the ascending paper chormatography.

Learning Outcomes

• Students understand the terms chromatography, adsorption chromatography, partition chromatography, column chromatography, TLC, paper chromatography, retardation factor etc.
• Students understand the principle of adsorption chromatography and partition chromatography.
• Students acquire the skill to perform the experiment by observing animation, simulator, video etc.
• Students understand the basic requirements for performing a paper chromatography.
• Students understand the different components present in the extract of spinach leaves.
• Students can identify the components based on their Rf value.

1. Separation of components from a mixture of red and blue inks

Materials Required

Real Lab Procedure

• Take a Whatman filter paper strip and using a pencil draw a horizontal line 4cm from one end of the paper. Then draw another line lengthwise (verticallly) from the centre of the paper. Name the point at which the two lines intersect as P.
• Using a fine capillary tube, put a drop of the mixture of red and blue inks at the point P. Let it dry in air.
• Put another drop on the same spot and dry again, so that the spot is rich in the mixture.
• Pour equal amounts of isopropyl alcohol and distilled water into a chromatographic chamber and mix it well using a glass rod. This is used as the solvent.
• Suspend the filter paper vertically in the chromatographic chamber containing the solvent in such a way that the pencil line remains about 2cm above the solvent level.
• Close the jar with its lid and keep it undisturbed.
• Notice the rising solvent along with the red and blue inks. After the solvent has risen about 15 cm you will notice two different spots of blue and red colors on the filter paper.
• Take the filter paper out of the jar and using a pencil mark the distance that the solvent has risen on the paper. This is called the solvent front.
• Dry the filter paper and put pencil marks at the centre of the red and blue ink spots.
• Measure the distance of the two spots from the original line and the distance of the solvent from the original line.
• Calculate the Rf values of the red and blue inks using the formula,

Observation

Observations can be recorded as shown.

Inference

• Rf value of red ink = ……………
• Rf value of blue ink = …………..
   

2. Separation of pigments from the extract of spinach leaves

Materials Required

Real Lab Procedure

• Take a Whatman filter paper strip and using a pencil draw a horizontal line 4cm from one end of the paper. Then draw another line lengthwise (verticallly) from the centre of the paper. Name the point at which the two lines intersect as P.
• Using a fine capillary tube, put a drop of the extract of spinach leaves at the point P. Let it dry in air. 
• Put another drop on the same spot and dry again, so that the spot is rich in the leaf extract.
• Pour equal amounts of isopropyl alcohol and distilled water into a chromatographic chamber and mix it well using a glass rod. This is used as the solvent.
• Suspend the filter paper vertically in the chromatographic chamber containing the solvent in such a way that the pencil line remains about 2cm above the solvent level.
• Close the jar with its lid and keep it undisturbed.
• Notice the rising solvent along with the coloured components of the leaf extract.
• After the solvent has risen to about 15 cm you will notice two different spots of coloured components on the filter paper.
• Take the filter paper out of the jar and using a pencil mark the distance that the solvent has risen on the paper. This is called the solvent front.
• Dry the filter paper and put pencil marks at the centre of each spot.
• Measure the distance of each spot from the original line and the distance of the solvent front from the original line.
• Calculate the Rf values of different components of leaf extract by using the formula,

Observation

Observations can be recorded as shown.

Inference

• Rf value of orange (Carotene) = ……………
• Rf value of Yellow (Xanthophyll) = …………..
• Rf value of Light green (Chlorophyll a) = …………..
• Rf value of Dark green (Xhlorophyll b) = …………..

Simulator Procedure (as performed through the Online Labs)

• You can select the mixture from ‘Select the mixture’ drop down list.
• To take the sample, drag the capillary tube to the watch glass and drag it back to the filter paper to drop the sample on the point ‘P’.
• To open the chromatographic chamber, click on its lid.
• Drag the filter paper to the chromatographic chamber containing solvent to suspend the filter paper in it.
• Click on the lid to close the chromatographic chamber.
• You can see that the components of the mixture rising along with the solvent.
• Wait till the solvent reaches its maximum height.
• Click on the filter paper to take it out from the chromatographic chamber.
• You can see the inference by clicking on the inference icon.
• You can measure the distance travelled by the solvent (solvent front) and the distance travelled by each component of the mixture from the original line using a scale by clicking on the ‘Show scale’ check box.
• You can see the zoomed view of the scale readings on the right side of the filter paper.
• You can verify the values by entering the values in the respective check boxes on the left side menu.
• You can calculate the Rf value of each component using the equation of Rf value and can verify your result by entering the values in the respective check boxes. (Round up the decimal values.  (E.g., 0.775 should be rounded up to 0.78).
• To redo the experiment, click on the ‘Reset’ button.

Note: Click on the ‘HELP’ button to see the instructions.

Precautions

• Use good quality pencil for drawing the reference line so that the mark does not dissolve in the solvent in which the chromatography is carried out.
• Always use a fine capillary tube.
• Keep the jar undisturbed and covered during the experiment.
• The spot should be small and rich in mixture.
• Allow the spot to dry before putting the strip in the jar.
• Keep the strip erect. Do not let it curl.
• Do not allow the spot to dip in the solvent.