Class 12 Chemistry Notes Chapter 11 (Tests for Carbohydrates, Fats and Proteins) – Lab Manual (English) Book
Detailed Notes with MCQs of Chapter 11 from your Lab Manual: Qualitative Analysis of Biomolecules. This is important not just for your practical exams but also forms the basis for questions in various government competitive exams where general science or chemistry is a component. We'll look at the common tests used to identify carbohydrates, fats, and proteins in given food samples.
Understanding Biomolecules:
Before we dive into tests, remember:
- Carbohydrates: Primarily energy sources (e.g., glucose, fructose, sucrose, starch). Classified as monosaccharides, disaccharides, polysaccharides. Reducing sugars have a free aldehyde or ketone group.
- Fats (Lipids): Energy storage, insulation, components of cell membranes. Esters of fatty acids and glycerol. Generally insoluble in water but soluble in organic solvents.
- Proteins: Building blocks of the body, enzymes, hormones. Polymers of amino acids linked by peptide bonds.
I. Tests for Carbohydrates
These tests help identify the presence and type of carbohydrates.
-
Molisch's Test:
- Purpose: General test for all carbohydrates (mono-, di-, oligo-, poly-saccharides).
- Principle: Concentrated H₂SO₄ dehydrates carbohydrates to form furfural (from pentoses) or hydroxymethylfurfural (from hexoses). These aldehydes then condense with α-naphthol (present in Molisch's reagent) to form a reddish-violet or purple coloured complex.
- Reagent: Molisch's reagent (α-naphthol dissolved in ethanol) and concentrated H₂SO₄.
- Procedure: Add Molisch's reagent to the sample solution. Carefully add conc. H₂SO₄ along the sides of the test tube without mixing.
- Observation: Formation of a purple or reddish-violet ring at the junction of the two liquids indicates the presence of carbohydrates.
- Note: Proteins and lipids may sometimes give a faint greenish ring, but the distinct violet ring is characteristic of carbohydrates.
-
Fehling's Test:
- Purpose: Test for reducing sugars (all monosaccharides and disaccharides like lactose, maltose; not sucrose). Also positive for aldehydes.
- Principle: Reducing sugars reduce cupric ions (Cu²⁺) from Fehling's solution to cuprous ions (Cu⁺) in an alkaline medium upon heating. Cuprous ions precipitate as red cuprous oxide (Cu₂O). The alkaline medium is provided by NaOH, and sodium potassium tartrate (Rochelle salt) keeps the Cu²⁺ ions in solution, preventing precipitation as Cu(OH)₂.
- Reagent: Fehling's Solution A (aqueous CuSO₄) and Fehling's Solution B (alkaline solution of sodium potassium tartrate). Equal volumes are mixed just before the test.
- Procedure: Mix equal volumes of Fehling's A and B. Add the sample solution and heat in a water bath.
- Observation: Formation of a red or brown precipitate (Cu₂O) indicates the presence of reducing sugars. The initial blue colour of the solution disappears.
-
Benedict's Test:
- Purpose: Test for reducing sugars. More commonly used than Fehling's test in clinical labs.
- Principle: Similar to Fehling's test. Cupric ions (Cu²⁺) complexed with citrate ions in an alkaline medium (provided by Na₂CO₃) are reduced by the reducing sugar to red cuprous oxide (Cu₂O) upon heating. Sodium carbonate provides the alkaline medium, and sodium citrate acts as the complexing agent for Cu²⁺.
- Reagent: Benedict's reagent (contains CuSO₄, sodium carbonate, and sodium citrate dissolved in water). It's a single, stable solution.
- Procedure: Add Benedict's reagent to the sample solution and heat in a water bath.
- Observation: Formation of a red, yellow, or green precipitate indicates the presence of reducing sugars. The colour depends on the concentration of the sugar. The blue colour of the reagent diminishes or disappears.
-
Tollen's Test (Silver Mirror Test):
- Purpose: Test for reducing sugars (and aldehydes).
- Principle: Reducing sugars reduce ammoniacal silver nitrate solution (Tollen's reagent) to metallic silver (Ag). The silver deposits on the inner walls of the test tube, forming a mirror.
- Reagent: Tollen's reagent (freshly prepared by adding dilute ammonia solution to silver nitrate solution until the initially formed precipitate of Ag₂O just redissolves, forming the complex [Ag(NH₃)₂]⁺OH⁻).
- Procedure: Add Tollen's reagent to the sample solution and warm gently in a water bath. Caution: Tollen's reagent can form explosive silver fulminate upon standing, so prepare fresh and dispose of properly.
- Observation: Formation of a shiny silver mirror on the inner side of the test tube or a black precipitate of silver indicates the presence of reducing sugars.
-
Iodine Test:
- Purpose: Specific test for starch (a polysaccharide).
- Principle: Starch has a helical structure (amylose component). Iodine molecules (I₂ from the reagent) get trapped inside this helix, forming a charge-transfer complex which absorbs light, resulting in a characteristic blue-black colour. Simple sugars, disaccharides, and other polysaccharides like cellulose do not give this test. Glycogen gives a reddish-brown colour.
- Reagent: Iodine solution (Iodine dissolved in potassium iodide solution, I₂/KI).
- Procedure: Add a few drops of iodine solution to the sample solution (preferably neutral or slightly acidic and cold).
- Observation: Appearance of a deep blue or blue-black colour indicates the presence of starch. The colour disappears on heating and reappears on cooling.
II. Tests for Fats and Oils
These tests help identify lipids.
-
Solubility Test:
- Purpose: To check the characteristic solubility behaviour of fats.
- Principle: Fats are non-polar molecules ("like dissolves like"). They are insoluble in polar solvents like water but soluble in non-polar or weakly polar organic solvents.
- Reagent: Water, Organic solvents (e.g., ethanol, ether, chloroform, carbon tetrachloride).
- Procedure: Add a small amount of the sample to different solvents and observe solubility.
- Observation: Sample is insoluble in water but soluble in organic solvents like chloroform or ether. (Note: Sparingly soluble in ethanol).
-
Translucent Spot Test:
- Purpose: Simple physical test for fats/oils.
- Principle: Fats and oils are non-volatile and penetrate paper, making it translucent (allowing light to pass through diffusely). Volatile oils or other liquids will evaporate, and the spot will disappear.
- Procedure: Place a drop of the sample (or rub the foodstuff) on a piece of filter paper or plain paper. Let it dry.
- Observation: A permanent translucent spot on the paper indicates the presence of fat or oil.
-
Acrolein Test:
- Purpose: Test for the presence of glycerol, either free or bound in fat (triglycerides).
- Principle: When fat is heated strongly, especially in the presence of a dehydrating agent like potassium bisulphate (KHSO₄), the glycerol part of the lipid is dehydrated to form an unsaturated aldehyde called acrolein (prop-2-enal). Acrolein has a characteristic pungent, irritating odour.
- Reagent: Solid Potassium bisulphate (KHSO₄).
- Procedure: Heat a small amount of the sample with a few crystals of KHSO₄ in a dry test tube.
- Observation: Evolution of a pungent, irritating odour (smell of burnt grease) indicates the presence of glycerol, thus confirming fat/oil.
III. Tests for Proteins
These tests detect the presence of proteins or specific amino acids.
-
Biuret Test:
- Purpose: General test for proteins and peptides containing at least two peptide bonds. Not given by free amino acids or dipeptides (except Histidine).
- Principle: In an alkaline medium (provided by NaOH or KOH), Cupric ions (Cu²⁺) from copper sulphate solution react with the nitrogen atoms of the peptide bonds (-CO-NH-) to form a violet or purple coloured coordination complex. The intensity of the colour is proportional to the number of peptide bonds.
- Reagent: Biuret reagent (often added sequentially: 1% CuSO₄ solution and 40% NaOH solution, or a combined reagent).
- Procedure: Add strong alkali (e.g., NaOH) to the sample solution to make it alkaline. Then add a few drops of dilute copper sulphate solution and mix.
- Observation: Appearance of a violet or purple colour indicates the presence of proteins (or peptides with >2 peptide bonds). A pinkish colour may indicate smaller peptides.
-
Ninhydrin Test:
- Purpose: Test for α-amino acids (free or N-terminal amino acids in proteins/peptides).
- Principle: Ninhydrin (triketohydrindene hydrate) is a strong oxidizing agent that reacts with the α-amino group of amino acids between pH 4 and 8 upon heating. It results in oxidative deamination and decarboxylation of the amino acid, producing CO₂, NH₃, an aldehyde, and reduced ninhydrin. The released ammonia then reacts with another molecule of ninhydrin and the reduced ninhydrin to form a deep blue or purple coloured complex called Ruhemann's purple.
- Reagent: Ninhydrin solution (usually 0.1-0.2% in acetone or ethanol).
- Procedure: Add ninhydrin solution to the sample solution. Heat in a water bath for a few minutes.
- Observation: Appearance of a deep blue or violet colour indicates the presence of α-amino acids.
- Note: Proline and hydroxyproline, which are imino acids (secondary amines), react differently and yield a yellow colour. Ammonia and primary amines also give a positive test.
-
Xanthoproteic Test:
- Purpose: Test for proteins containing aromatic amino acids (specifically Tyrosine and Tryptophan; Phenylalanine reacts very slowly or not significantly under these conditions).
- Principle: Concentrated nitric acid (HNO₃) nitrates the benzene ring (phenyl group) present in tyrosine and tryptophan residues at high temperatures, forming yellow coloured nitro-derivatives. These derivatives turn orange upon addition of alkali (like NaOH or NH₄OH) due to the ionization of the phenolic group (in tyrosine) or the secondary amine formation.
- Reagent: Concentrated Nitric Acid (HNO₃) and a strong alkali (e.g., NaOH or NH₄OH solution).
- Procedure: Add concentrated HNO₃ to the sample solution and heat gently. Cool the solution and carefully add excess alkali.
- Observation: Formation of a yellow colour or precipitate on heating with HNO₃, which turns orange upon making the solution alkaline, indicates the presence of aromatic amino acids (tyrosine/tryptophan) in the protein.
-
Millon's Test:
- Purpose: Specific test for proteins containing the amino acid Tyrosine, which has a phenolic group.
- Principle: Millon's reagent contains mercury dissolved in nitric acid (Hg/HNO₃ mixture, forming mercurous and mercuric nitrates). The phenolic group of tyrosine reacts with Millon's reagent. Initially, a white precipitate may form (mercury complexed with protein), which upon heating turns brick red or pink, indicating the presence of tyrosine.
- Reagent: Millon's reagent (dissolve mercury in conc. nitric acid, then dilute with water). Caution: Mercury compounds are toxic.
- Procedure: Add Millon's reagent to the sample solution and heat gently in a water bath.
- Observation: Formation of a white precipitate that turns brick red upon heating indicates the presence of tyrosine (and thus, proteins containing tyrosine).
- Note: Chloride ions interfere with this test, so it may not work well with solutions containing NaCl (like urine). Any phenolic compound (not just tyrosine) will give a positive test.
Multiple Choice Questions (MCQs)
-
Molisch's test is a general test for which class of biomolecules?
a) Proteins
b) Lipids
c) Carbohydrates
d) Nucleic Acids -
A purple ring formed at the junction of two layers after adding conc. H₂SO₄ to a solution containing the sample and α-naphthol indicates a positive:
a) Biuret test
b) Molisch's test
c) Ninhydrin test
d) Xanthoproteic test -
Which of the following tests is used specifically to detect reducing sugars?
a) Iodine Test
b) Biuret Test
c) Benedict's Test
d) Acrolein Test -
Fehling's solution consists of two parts: Solution A (CuSO₄) and Solution B (Alkaline Rochelle Salt). What is the function of Rochelle salt (Sodium Potassium Tartrate)?
a) To provide an alkaline medium
b) To act as an oxidizing agent
c) To keep Cu²⁺ ions in solution as a complex
d) To form the red precipitate -
A student performs a test on a food sample and observes the formation of a shiny silver mirror inside the test tube. Which reagent was most likely used?
a) Benedict's reagent
b) Millon's reagent
c) Tollen's reagent
d) Fehling's solution -
The formation of a blue-black colour upon addition of iodine solution indicates the presence of:
a) Glucose
b) Starch
c) Protein
d) Fat -
Heating a substance with potassium bisulphate (KHSO₄) produces a pungent irritating odour. This indicates the presence of:
a) Peptide bonds
b) Aromatic amino acids
c) Starch
d) Glycerol (from fats/oils) -
The Biuret test detects the presence of:
a) Free amino acids
b) Peptide bonds (at least two)
c) Reducing sugars
d) Phenolic groups -
Which test gives a positive result (yellow colour turning orange with alkali) for proteins containing tyrosine and tryptophan?
a) Ninhydrin Test
b) Millon's Test
c) Xanthoproteic Test
d) Biuret Test -
Ninhydrin reacts with α-amino acids to produce a characteristic purple colour known as Ruhemann's purple. However, with which amino acid does it produce a yellow colour?
a) Glycine
b) Alanine
c) Proline
d) Tyrosine
Answer Key for MCQs:
- c) Carbohydrates
- b) Molisch's test
- c) Benedict's Test (Fehling's and Tollen's also detect reducing sugars, but Benedict's is a specific option given)
- c) To keep Cu²⁺ ions in solution as a complex
- c) Tollen's reagent
- b) Starch
- d) Glycerol (from fats/oils)
- b) Peptide bonds (at least two)
- c) Xanthoproteic Test
- c) Proline
Study these tests well, focusing on the principle, reagent, and the specific observation (colour change, precipitate formation, odour, etc.). Good luck with your preparation!