Test for biological molecules

i) Benedict’s test for reducing sugar

Examples: All monosaccharides & some disaccharides eg. Lactose and maltose.
Principle:
 Benedict’s test makes use of ability of reducing sugars to reduce Cu2+ to Cu+.
 Reducing ability due to carbonyl group of the sugar, which reduces copper (II) sulphate
solution to insoluble copper (I) oxide.

Method:
 Add an equal amount of Benedict’s reagent to 2ml of the solution being tested.
 Mix well and place it in boiling water bath for 2 minutes.

Observations:
 Benedict’s test is partially quantitative as the colour of the reaction mixture indicates the
amount of reducing sugar present.
 Green colour indicates small amount of reducing sugar.
 Red colour indicates large amount of reducing sugar.
 Blue colour indicates the absence of reducing sugar.

ii) Test for non-reducing sugar

Example: Sucrose.
Method:
 Sucrose is heated with dilute hydrochloric acid to hydrolyse it to glucose and fructose.
 Cool the contents and neutralizing it with sodium hydrogencarbonate.
 Keep adding until no more bubbling observed.
 Carry out Benedict’s test for reducing sugar.

iii) Iodine solution test for starch

Principle:
 The iodine takes up a position in the centre of the amylase helix, forming a starch-iodine complex that
gives an intense blue-black colour.
Method:
 Add a drop of iodine in potassium iodide solution to 1ml of starch suspension.
Observations:
 A blue-black colour indicates the presence of starch.
 Brown colour of iodine remains in the absence of starch.
iv) Biuret test for proteins
Principle:
 The biuret test detects peptide bonds and thus all proteins give a positive result.

Method:
 Add 5% NaOH to 2ml of solution being tested.
 Shake it to mix the contents.
 Add 1% copper sulphate solution a drop at a time, shaking the tube after each drop.
 Note the colour of the contents in the tube.

Observations:
 A violet colour indicates the presence of protein.
 Blue colour indicates the absence of protein.

v) Ethanol emulsion test for lipids

Principle:
 Lipids are soluble in organic solvents such as ethanol, but not in water, with which they form emulsion
on vigorous shaking.

Method:
 Grind test item and pour it into a test tube.
 Use only two drops of test item.
 Add 2 ml of ethanol and mix well.
 Decant the ethanol into another test tube containing 2ml of water.
 Mix well and look for cloudiness.

Observations:
 If lipid is present, it dissolves in ethanol to form a homogeneous solution, which forms an emulsion with
water.
 If lipid is absent, a homogeneous solution is still formed with ethanol, which remains homogeneous
when water is added.
(vi) DCPIP test for Vitamin C (ascorbic acid)

Principle:
 DCPIP is a blue dye which is reduced to a colourless compound by ascorbic acid, a strong
reducing agent.

Method:
 Using 0.1% ascorbic acid solution as a standard
 Add 1cm3 of DCPIP solution to a test-tube. Fill a 1cm3 syringe with 0.1% ascorbic acid.
 Add the acid to the DCPIP drop by drop, stirring gently with the syringe needle. Do not shake.
 Add until the blue colour of the dye just disappears. Note the volume of the ascorbic acid.
Observations:
 Blue colour of the dye disappears to leave a colourless solution.