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Activity 7

The document outlines an educational activity focused on detecting proteins, fats, and separating plant pigments using chromatography. It details specific tests such as the Biuret and Xanthoproteic tests for proteins, solubility tests for fats, and a procedure for extracting and analyzing chloroplast pigments. Students are expected to record observations and answer questions related to the experiments conducted.

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12 views5 pages

Activity 7

The document outlines an educational activity focused on detecting proteins, fats, and separating plant pigments using chromatography. It details specific tests such as the Biuret and Xanthoproteic tests for proteins, solubility tests for fats, and a procedure for extracting and analyzing chloroplast pigments. Students are expected to record observations and answer questions related to the experiments conducted.

Uploaded by

go.donque
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Name: Score:

Course & Section: Date:

Activity #7: The Amino Acids


Objectives
After completing the task, students should be able to:
 to detect the presence of proteins;
 to detect the presence of fats (lipid) in different plants and animal materials; and
 Separation of plant pigments (chloroplast pigments) by paper chromatography.

Introduction
Proteins respond to some color reactions due to the presence of one or more radicals or
groups of the complex protein in the molecule. All proteins do not contain the same amino acids,
and hence do not respond to all color reactions. Nitrogen atoms in the peptide chain form a
complex with copper ions in the Biuret test. Xanthoproteic test is specific for protein containing
aromatic amino acids. The benzene ring in the amino acids is nitrated by heating with nitric acid
and forms yellow nitro-compounds which turn to orange color with alkali.
Lipids (fats) are usually insoluble in water but soluble in the organic solvent. Sudan III
reagent gives a specific red color with fats.
The chloroplasts contain photosynthetic pigments Chlorophyll a, Chlorophyll b,
Carotenes, and Xanthophyll’s. Pigments absorb solar radiation at different wavelengths of the
visible spectrum for photosynthesis. These pigments differ in their physicochemical properties,
such as molecular weight, solubility in the solvent system, on a fine quality cellulose paper
(Whatman No.1 chromatography paper). Movement of pigments on the chromatography paper is
governed by the principle of adsorption and capillary action. The solvent system components
vary in density and thus move different rates due to wick action through the chromatography
paper. Lighter components move faster than the heavier components. Differential solubility of
pigments in the solvent system and the differential rates of mobility of solvent system
components is used from separation of pigments.
Materials: Test tube, spirit lamp, test tube holder, test tube stand, boiling tube with split rubeer
cork, chromatography chamber or wide mouth test tube, 100 mL to 10 mL measuring cylinders,
50 mL beaker, capillary tube, mortar and pestle,
What to bring?
Gram Seed, mustard oil, ghee, or butter, mucilage-free leaves of locally available herbaceous
plants, acetone, petroleum, ruler, muslin cloth, sand, pins, and ordinary blotting sheet, and paper
tape
Test No 1. Biuret and Xanthoproteic Test
Procedure
(a) Biuret test
1. Take 2 mL of protein solution (milk, albumin of egg, or gram seed extract) in a
test tube.
2. Add 1 mL of 40% NaOH solution and or 2 drops of 1% CuSO4
Record Observation/s:

(b) Xanthoproteic test


1. Add carefully 1 mL of concentrated HNO 3 to 2 mL of protein solution (albumin
of egg, milk, or gram seed extract
2. Boil the solution.
3. Cool the test tube and add 2 mL of 20% NaOH (or ammonia solution) to make it
alkaline.
Record Observation/s:

Questions
1. Why does the skin turn yellow when it inadvertently comes in contact with HNO 3?
2. Why are only a few drops of CuSO4 solution added during the biuret test?

Test No. 2. Test for Solunility and Sudan III test


(c) Solubility test
1. Arrange five dry test tubes in a test tube stand
2. Add 2 mL of water, ether, chloroform, benzene, and ethyl alcohol to each test
tube followed by one drop of mustard oil, ghee, or butter.
3. Shake thoroughly.
Record Observation/s:

(d) Sudan III test


1. Take 2 mL of Mustard oil in a test tube.
2. Add equal volume of water to it and shake.
3. Add a pinch of sudan III and shake again.
Record Observation/s:

Questions
1. What is the simplest form of fat?
2. Will fat dissolved in organic solvent give a positive result with sudan III?

Procedure
 Select tender, herbaceous, mucilage free leaves for the experiment. Remove the petioke
and mid rib from 20-30 mature leaves.
 Cut the leaves into smaller bits and transfer them into a mortar. Add a pinch of sand, a
few mL of acetone and grind thoroughly until the leaf extract becomes dark green in
color.
 Filter extract through a muslin cloth and collect the filtrate in 50 mL beaker.
 Get a petroleum ether and acetone. Transfer 3-5 ml of this mixture into the
chromatography chamber or wide mouthed test tube. Close the chamber of the test-tube
with the split cork tightly as the solvents are volatile and keep it in standing position.
 Cut a strip of Whatmann No.1 chromatography paper (6” x 1/2 “), which can fit into the
chamber or the test –tube.
 Using scissors make one end of the chromatography paper into a triangular shape with a
pointed end.
 Using the test tube, load the pigment extract on the lower side of the chromatography
paper in the spot till the spot is not more than 3-5 mm. Let it dry by blowing warm air on
it. Never use ink/ball pen for making on chromatography paper.
 Suspend the loaded chromatography into the boiling tube with the help of a split rubber
cork or in chromatography chamber or wide mouth test tube.
 Make sure that the loaded spot is clearly above the solvent.
 Keep the apparatus straight and leave the set up in a test tube stand undisturbed for 40
minutes. Note the rise of solvent and pigments
 Cover the chromatogram with black paper to prevent photo oxidation.

Record Observation/s:
Questions
1. Which photosynthetic pigment moves farthest and why?

2. Leaves in general appear green although they possess yellow and orange pigments.
Comment.

3. Why do we add sand while preparing the pigment extract?

4. Among the two solvent system components in chromatography, which moves farthest
and why?

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