Identification of Biochemicals from Biological Materials
Category Test Name Procedure Observation Inference Basis of Test
Heat original solution (OS) in a No ppt → carbohydrate; Carbohydrate/protein Proteins coagulate upon heating;
Preliminary Heating Test
test tube White ppt → protein identified carbs don’t
Drop OS on filter paper → Let
Spot Test for Lipids Greasy transparent spot Lipid present Lipids make paper translucent
it dry
Iodine Test 2 ml starch solution + few Blue-black (starch), Red Starch/glycogen Forms polyiodide complex with
Carbohydrates
(for starch/glycogen) drops of iodine solution (I₂KI) (glycogen) present starch/glycogen
2 ml sugar solution + 2 ml
Green → Yellow → Reducing sugar converts Cu²⁺ →
Benedict’s Test Benedict’s → Boil for 2 Reducing sugar present
Orange → Red ppt Cu₂O (brick-red ppt)
minutes
2 ml sugar sol. + 1 ml Fehling
Fehling’s Test Same as Benedict’s Reducing sugar present Cu²⁺ reduced to Cu₂O (brick-red)
A + 1 ml Fehling B → Boil
2 ml protein sol. + equal vol.
Peptide bonds form purple complex
Proteins Biuret Test 5% KOH → Add 2 drops 1% Violet/purple color Proteins present
with Cu²⁺ in alkaline solution
CuSO₄
2 ml protein sol. + 1 ml White ppt turns Protein (tyrosine) Phenolic group of tyrosine forms red
Millon’s Test
Millon’s reagent → Heat red/salmon pink present complex with mercury nitrate
2 ml oil + 2 ml water → Add Red-stained oil layer Sudan III stains fat globules
Lipids Sudan III Test Lipid present
few drops Sudan III → Shake floats (which float on water)
Water causes lipid-alcohol mix to
2 ml lipid + 2 ml ethanol → Cloudy white
Emulsion Test Lipid present form emulsion
Shake → Add 4 ml cold water suspension (emulsion)
(light-scattering effect)
IDENTIFICATION OF BIOCHEMICALS FROM BIOLOGICAL MATERIALS
MATERIALS NEEDED
Solutions: 1% Iodine (I₂KI), Benedict’s, Fehling’s A & B, Millon’s, 5% KOH, 10% CuSO₄, Trichloroacetic acid, Diphenyl amine
Samples: Glucose, Maltose, Lactose, Egg albumin, DNA
Equipment: Test tubes, test tube stand, filter paper, spirit lamp
PRELIMINARY TESTS
1. Heating Test (for Carbohydrates and Proteins) 2. Spot Test (for Lipids)
Method: Heat a little of the original sample (OS) in a test tube. Method: Put a drop of sample on filter paper and let it dry.
Observation: Observation:
o No precipitate → Carbohydrate present o Greasy, clear spot → Lipid present
o White precipitate → Protein present
TESTS FOR CARBOHYDRATES
1. Iodine Test (for Starch & Glycogen) 2. Benedict’s Test (for Reducing Sugars like 3. Fehling’s Test (Also for Reducing Sugars)
glucose, maltose, etc.)
Method: Add 2 ml starch solution + few Method:
drops of iodine (I₂KI) Method: o 2 ml sugar solution + 1 ml
Observation: o 2 ml sugar solution + 2 ml Fehling’s A + 1 ml Fehling’s B
o Blue-black → Starch present Benedict’s o Heat
o Red → Glycogen present o Heat for 2 minutes Observation:
Observation: o Blue → Green → Yellow →
Why? Iodine forms a complex with starch o Blue → Green → Yellow → Brick-red
molecules. Brick-red (with increasing sugar
levels) Why? Same as Benedict’s — copper ions are
reduced.
Why? Sugars reduce blue Cu²⁺ to red Cu₂O
(copper oxide).
TESTS FOR PROTEINS
1. Biuret Test 2. Millon’s Test
Method: Method:
o 2 ml protein solution (egg white) o 2 ml protein solution + 1 ml Millon’s reagent
o Add equal amount of 5% KOH o Heat
o Add 2 drops of 1% CuSO₄ and shake Observation:
Observation: o White precipitate turns red → Proteins present
o Purple color → Proteins present
Why? Tyrosine in proteins reacts with mercury nitrate to give red color.
Why? Peptide bonds react with copper in alkaline solution.
TESTS FOR LIPIDS
1. Sudan III Test 2. Emulsion Test
Method: Method:
o Mix 2 ml oil + 2 ml water in a tube o Mix 2 ml fat/oil with 2 ml ethanol and shake
o Add a few drops of Sudan III and shake o Add 4 ml cold water
Observation: Observation:
o Red-stained oil layer floats on water o Cloudy white suspension forms → Lipid present
Why? Sudan stains fat globules (which are lighter than water). Why? Water causes lipid droplets to form an emulsion, scattering light.
