12th Biology Practical ~ (Part B) 2. separation of plant photosynthetic pigment by paper chromatography

 12th Biology practical book answers pdf Maharashtra board 

~separation of plant photosynthetic pigment by paper chromatography

Aim: Separation of plant (photosynthetic) pigments by paper chromatography

The plants show presence of many pigments. The different photosynthetic pigments present in the leaf cells are: chlorophylls, carotenes and xanthophylls. These photosynthetic pigments can be separated using the technique of paper chromatography.

 

Principle:

The separation of solutes (chloroplast pigments) is based on the liquid-liquid partitioning of pigments in paper chromatography. The partitioning takes place between the solvent (water) molecules (static phase) adsorbed to the cellulosic matter of the paper (capillary action) and organic (mobile) phase.

 

Requirements:

Chromatography chamber, chromatography paper (Whatman's filter paper no.1), pestle-mortar, capillary tube, muslin cloth and suitable solvent system, fresh spinach leaves, etc.

 

Procedure:

·        Prepare solvent system - Petroleum ether: acetone (90:10), pour it in the chromatography chamber and cover it tightly. Keep it undisturbed for 1 hour for saturation of the solvent system.

·        Take few spinach leaves (5gms), wash and keep them dry.

·        Grind these leaves in acetone (organic solvent) with a small amount of MgCl₂ (as a buffer)

·        Filter it through muslin cloth and collect the filtrate.

·        Cut suitable strip of chromatographic paper and mark the loading point with pencil. With the help of capillary tube, the extract or filtrate is repeatedly loaded at the loading point.

·        Allow it to dry.

·        Now place the strip in the solvent system in such a way that its lower tip gets dipped in the solvent.

·        Take care not to dip it too close to the loaded spot. Cover the chamber and keep it undisturbed to get the chromatogram run.

·        Let the solvent travel close to the other end of the chromatography paper. Different pigments carried along with the solvent up to different heights on the basis of their molecular weights.

·         Take out the paper, allow it to dry and observe the separation of photosynthetic pigments in the form of separate bands of different colours.

 

 

Observation:

On this trip we can observe the separation of photosynthetic pigments as bands of different colours in a particular sequence.  from loaded spot up to the top of sequence is chlorophyll B (yellowish green) chlorophyll a (bluish green) xanthophylls (yellow) and carotene (orange)

 

Questions

 

1.    What is the composition of solvent used for separation of photosynthetic pigments?

petroleum Ether and Acetone in 90:10 proportion used for separation of photosynthetic pigment Acetone is best because as It Breaks down a cell walls and is miscible with pigments.

 

2.    Which pigment reaches to the top of chromatography paper?

pigment carotene reaches to the top of chromatography paper

 

3.    Which photosynthetic pigment is bluish in colour?

Chlorophyll-a is bluish in colour

 

4.    Why Acetone and MgCl₂ are used while grinding of leaves?

Acetone and MgCl₂ are having polarity its slide polarity allows it to dissolve polar substances and the fact that it is a less polar than water which allows greater resolution between pigments on paper this reason allows Acetone to be great solvent for pigment chromatography.

 

 

 

 fin :)