how will prepare solvent in chromatography separation of chlorophyll pigments in analytical chemistry ?
Answers
Explanation:
Aim
To distinguish and study the various pigments present in plants through the process of paper chromatography.
Theory
Plants carry out the process of photosynthesis, during which light energy from the sun is converted into chemical energy (food). The capturing of light energy is carried out by molecules known as pigments, which are present within the plant cells.
What are Pigments?
Pigments are chemical compounds, which are able to reflect only a particular range of wavelengths of visible light. Leaves of plants primarily contain different types of pigments within their tissues. The four different types of pigments are listed below in a tabular column along with their colours.ko
Pigment Colour
Chlorophyll A Dark green
Chlorophyll B Yellowish-green
Xanthophylls Yellow
Carotenoids Orange
In order to view and distinguish the primary four plant pigments, a simple technique known as chromatography can be used.
What is Chromatography?
It is a technique that is used to distinguish between different molecules. This differentiation is based on these attributes-shape, size, charge, mass, adsorption and solubility.
Types of chromatography:
Column chromatography
Paper chromatography
Partition chromatography
Thin-layer chromatography
Mechanism of Paper Chromatography
In this technique, the interaction between three components is involved – solid phase, separation of a mixture and a solvent
At first, the mixture is spotted onto the paper and is dried.
The solvent is made to flow through the capillary attraction.
While the solvent moves through the paper, the various components of the mixture differentiate into varied coloured spots.
Later the paper is allowed to dry and the position of various compounds is viewed.
The substance, which is the most soluble moves further on the paper as compared to the other substances that are less soluble.
Material Required
Chromatography chamber
Spinach leaves
Mortar and pestle
Scissors
Ether acetone solvent
Acetone
Capillary tube
Pencil
Spatula
Scale
Filter paper strips
Stapler
Thread
Watch glass
Procedure
In this experiment, spinach leaves are used to separate different pigments.
Pick a few fresh and green leaves of spinach and wash it.
Cut out small pieces of spinach using scissors. Add them to the mortar.
Accurately measure 5ml acetone using a measuring cylinder and add it into the mortar.
With the help of mortar and pestle, grind the spinach leaves into a smooth paste.
Shift the prepared paste of spinach into the watch glass with the help of a spatula.
Place a filter paper strip with a tapering notch towards one ending of the strip.
Horizontally trace a line with a scale and a pencil that is 2 to 3 cm apart from the notch’s tip.
Using a capillary tube, add 1 drop of the extract of the pigment in the midsection of the line.
Let the drop dry. Repeat the same process of adding a drop and allowing it to dry for 4-5 times.
In the chromatographic chamber, pour the ether acetone solvent.
Make sure to folded and stapled an end side of the paper.
Suspend the strip in the chamber.
The loading spot remains about 1 cm above the level of the solvent.
Let the chamber remain uninterrupted for a while.
We can notice that the solvent passes along the paper scattering various pigments of the blend to different distances.
Once the solvent reaches 3/4th of the strip, carefully take the strip off.
Allow the strip to dry.
Observation
The dried paper strip displays four different bands. Discrete pigments can be distinguished with the help of colours.
Conclusion
The Carotene pigment is observed at the topmost as an orange-yellow band of pigments distinctively.
Just below this band, a yellowish band appears which indicates the pigment xanthophyll.
The third band appearing dark green indicates chlorophyll-a pigment.
The yellowish-green band present at the bottom is the chlorophyll b pigment.
Precautions
The leaves that are selected should be green and fresh spinach leaves
From the tip of the notch, the loading spot needs to be 2 to 3 cm apart
While suspending the filter paper strips in the chamber, one need to ensure that the loading spot needs to be set up above 1 cm from the level of the solvent.