Benedict’s test for reducing sugars
Definition
Benedict’s test is a biochemical test performed to distinguish reducing sugars (monosaccharides
and some disaccharides) from non-reducing sugars.
Objectives
To detect the presence of simple carbohydrates in a solution.
To distinguish between reducing and non-reducing sugars.
Principle
The carbohydrates having a free or potentially free, aldehyde or ketone group can act as a
reducing agent.
In order to detect the reducing agent, Benedict’s reagent is used. It appears deep blue in
color and consists of copper sulfate mixed with sodium citrate and a weak alkali, sodium
carbonate.
When reducing sugars are heated in the presence of alkali, they get converted to enediols,
which are powerful reducing agents.
Enediols reduce the cupric ions (Cu2+) present in the benedict’s reagent to cuprous ions
(Cu+), which get precipitated as insoluble red-colored cuprous oxide.
The test is semi-quantitative since the color of the precipitate indicates an approximate
quantity of the sugar present in the sample.
For a sample containing reducing sugar the color of the sample during boiling progress
from blue (with no reducing sugar present), green, yellow, orange, red, and then brick red
or brown (with a high concentration of reducing sugars).
The citrate ions form a complex with cupric ions and prevent its precipitation with the
hydroxide ions as cupric hydroxide.
Reaction
Na2CO3 + 2H2 → 2NaOH + H2CO3
2NaOH + Cu(OH)2 → Na2SO4
Cu(OH)2 → CuO + H2O
D-glucose + 2CuO → D-gluconic acid + Cu2O (red ppt)
Requirements
Reagents
Benedict’s reagent: Benedict’s reagent is prepared by adding 17.3 gm of sodium citrate,
10 gm of sodium carbonate and 17.3 gm of sodium pentahydrate to 100 ml of water in a
beaker.
Test samples
Materials required
Test tube
Test tube stand
Pipettes
Equipment
Water bath
Procedure
About 1 ml of the test sample is added to a test tube along with 2 ml of benedict’s
reagent.
The test tubes are then placed in the test tube stand, which is kept in boiling water for 4-
10 minutes.
The color in the test tubes are observed and noted down.
Result and Interpretation
Figure: Observation (Results) of Benedict’s Test. Image Source: Chemistry Learner.
The appearance of a greenish precipitate indicates about 0.5 g% concentration; yellow
precipitate indicates 1 g% concentration; orange indicates 1.5 g% concentration and red
indicates 2 g% or higher concentration of reducing sugars.
The appearance of the blue color indicates the absence of reducing sugar and represents a
negative result.
Notes
Sucrose, starch, inositol gives a negative result, whereas lactose and maltose give a
positive result with benedict’s test.
Benedict modified the Fehling’s solution to make a single improved reagent, which is
quite stable. It is very sensitive to even small quantities of reducing sugars (0.1%) and
yields enough precipitate.
A false-positive reaction for urine sample may be obtained due to the presence of
reducing substances like uric acid, ascorbic acid or other drugs like levodopa.
Iodine test for starch
Definition
Iodine test, also known as a starch-iodine test, is a chemical test used to distinguish mono- or
disaccharides from specific polysaccharides like amylase, dextrin, and glycogen.
Objectives
To detect the presence of polysaccharide, primarily starch.
Principle
Iodine test is based on the fact that polyiodide ions form colored adsorption complex with
helical chains of glucose residue of amylase (blue-black), dextrin (black), or glycogen
(reddish-brown).
Monosaccharides, disaccharides, and branched polysaccharides like cellulose remain
colorless. Amylopectin produces an orange-yellow hue.
The reagent used in the iodine test is Lugol’s iodine, which is an aqueous solution of
elemental iodine and potassium iodide.
Iodine on its own is insoluble in water. Addition of potassium iodine results in a
reversible reaction of the iodine ion with iodine to form triiodide ion, which further reacts
with an iodine molecule to form pentaiodide ion.
Bench iodine solution appears brown, whereas, the iodide, triiodide, and pentaiodide ion
are colorless.
It is observed that the helix (coil or spring) structure of the glucose chain is the key to this
test.
Further, the resulting color depends on the length of the glucose chains.
The triiodide and pentaiodide ions formed are linear and slip inside the helix structure.
It is believed that transfer of charge between the helix and the polyiodide ions results in
changes in the spacing of the energy levels, which can absorb visible light, giving the
complex its color.
The intensity of the color decreases with the increase in temperature and the presence of
water-miscible organic compounds like ethanol.
On heating, the blue color amylase-iodine complex dissociates but is formed again on
cooling because the helical structure is disrupted; thereby amylose loses its iodine
binding capacity and the blue color.
The blue color reappears on cooling due to the recovery of iodine binding capacity due to
the regaining of the helical structure.
Requirements
Reagent
Lugol’s iodine: 5% elemental iodine is mixed with 10% potassium iodide to form the
Lugol’s iodine.
Test sample
Materials Required
Test tubes
Test tube stand
Equipment
Water bath
Procedure
1 ml of the test sample is taken in a test tube, to which 2-3 drops of Lugol’s reagent is
added.
The solution is then mixed in a vortex.
The color of the solution is observed. The test tubes are then placed in the boiling water
bath until the color disappears.
The tubes are then cooled, and the color of the solution is observed and noted down.
Result and Interpretation
Figure: Observation (Results) of Iodine test for starch. Source: Comprehensive Natural Science
(CNS).
The appearance of blue-black or purple color represents a positive test, indicating the
presence of starch.
If there is no change in color, the result is negative and indicates the absence of starch.
Notes
This test cannot be performed under acidic conditions as the starch hydrolyses under such
circumstances.
This test is a qualitative test and doesn’t signify the concentration of starch.
Emulsion test for Lipids
Definition
Emulsion test, also known as the Ethanol Emulsion test, is a general group test for the detection
of lipids.
Objectives
To determine the presence of lipids in a sample.
Principle
The presence of lipids is observed by the appearance of a cloudy white layer on top of the
reaction mixture.
This test is based on the fact that lipids dissolve in ethanol (due to hydrophobic
interaction), but on the addition of water, lipids spontaneously disperse to form micelles
(small droplets).
These droplets form the top layer as these are less dense than water and ethanol, and also
appear cloudy white as they diffract light.
The lipids come out of the solution because the overall strength of hydrogen bonding
interaction between ethanol and water is much higher than hydrophobic interactions
between lipids and ethanol.
Requirements
Reagents
Ethanol
Water
Materials required
Test tubes
Test tube stand
Pipettes
Procedure
Few drops of fat or lipid sample are added to a test tube. 2 ml of ethanol is added to the
same test tube.
To this solution, 2 ml of water is added, and the test tube is shaken well.
The appearance of cloudy suspension is observed.
Result and Interpretation
Figure: Positive Results of Emulsion test for Lipids. Source: Tinycards.
The appearance of cloudy suspension at the top layer of the solution indicates a positive
result. This represents the presence of lipids in the sample. Samples with high lipid
content will form a thicker cloudy suspension.
The absence of cloudy emulsion indicates a negative result and the absence of lipid.
Biuret test for proteins
Definition
Biuret test, also known as Piotrowski’s test, is a chemical test for the detection of peptide bonds
in a sample and can also be used for the quantification of proteins already in solution or easily
soluble in dilute alkali.
Objectives
To detect the presence of proteins or peptide bonds in a sample.
To determine the concentration of proteins present in a sample.
Principle
The biuret reagent contains sodium hydroxide, copper (II) sulfate, and potassium sodium
tartrate.
Under alkaline conditions of the biuret reaction (pH 14), deprotonation of the amide
nitrogen occurs which leads to high electron density at the nitrogen atom,
Further, copper (II) ion complexes with four peptide nitrogens to yield a tetradentate
violet colored coordination complex.
At high pH, Cu2+ bonding with OH– ion leads to an insoluble precipitate, which is
minimized by the addition of potassium sodium tartrate, which stabilizes the cupric ions.
Since peptide bonds occur with the same frequency per amino acid in proteins, the biuret
test can be used to assess the concentration of proteins.
Requirements
Reagent
Biuret reagent: 0.3 g of CuSO4 and 0.9 g of sodium-potassium tartrate are added to 50 ml
of 0.2N NaOH. To this, 0.5 g of KI added and the volume is made up to 100 ml by
adding 0.2N NaOH.
Sample
Materials required
Test tubes
Test tube stand
Pipettes
Equipment
UV spectrophotometer
Vortex
Procedure
1 ml of the sample is taken in a test tube to which few drops of Biuret reagent is added.
The test tube is then mixed well by shaking the test tube well.
The change in color of the solution is then observed and noted down.
Result and Interpretation
Figure: Results of the Biuret test for proteins.
The appearance of the purple color of the solution indicates a positive result. The purple
color present about 5-160 mg/ml concentration of proteins.
The absence of the purple color indicates a negative result and the absence of proteins in
the sample.
Notes
The color developed in this test is stable, but it is recommended to take the readings of
the sample within 10 minutes.
For significant measurable color, peptides with at least three amino acids are necessary.
Biuret is not a reagent in this test, but the test is so named because the reaction was first
discovered with peptide-like bonds in the biuret molecule. A more accurate name for the
reagent is the alkaline copper reagent (ACR) test.
