Lab 1:

Tests for Carbohydrates

Carbohydrates present in a solution can be easily identified by performing certain tests in
the laboratory. The important tests for carbohydrate detection are as follows

1. Molisch’s Test:

It is the general test for all carbohydrates.

Monosaccharides give a rapid positive test.

Disaccharides and polysaccharides react slower.

Principle:

The reaction is due to the formation of Furfural and it’s derivatives by the dehydrating
action of acid on

sugar in the event of Sugar being (poly saccharide or disaccharide) the acid first
hydrolyzes it into

monosaccharides and then acts as a dehydrating agent.

The furfurals further react with α-naphthol present in the test reagent to produce a purple
product.

■ Method:

• 1ml test solution + 2 drops of α-naphthol

• mix well

• Add conc. H2SO4 down the side of the tube to form the ring at the interface of the two
layers.

Lab results:

all carbohydrate display the purple ring

Fehling Test

One of the most popular tests used for the estimation or detection of reducing sugars and
non-reducing sugars is the

Fehling’s Solution

Fehling’s test consists of a solution that is usually prepared fresh in laboratories. Initially,
the solution exists in the form of two separate solutions which are labelled as Fehling’s
A and Fehling’s B.
Fehling’s A is a solution containing copper(II) sulphate, which is blue. Fehling’s B is a
clear liquid consisting of potassium sodium tartrate
(Rochelle salt) and a strong alkali, usually sodium hydroxide. During the test solutions A
and B are prepared individually and stored.
The two solutions are later mixed in equal volumes to get the final Fehling solution
2+
which is deep blue. The deep blue ingredient is the bis(tartrate) complex of Cu . The
tartrate tetra-anions serve as a chelating agent in the solution.
Procedure
The procedure can be conducted as follows
▪ Add the sample in a dry test tube.
▪ Distilled water should be kept in another tube as control.
▪ Fehling’s solution is to be added in the tubes.
▪ The tubes must be kept in water bath.
▪ Make observations and record if there is any development of
red precipitate.

the result is positive if there is a formation of reddish brown precipitate while the result is
negative if there is no indication of such change

Benedict’s Test
The Benedict’s test identifies reducing sugars
monosaccharide’s and some disaccharides, which have free ketone or aldehyde
functional groups.
Benedict’s solution can be used to test for the presence of glucose in urine.
Principle of Benedict’s Test

When Benedict’s solution and simple carbohydrates are heated, the solution changes
to orange red/ brick red.

Procedure of Benedict’s Test

 1. Approximately 1 ml of sample is placed into a clean test tube.

2. 2 ml (10 drops) of Benedict’s reagent (CuSO4) is placed in the test tube.

3. The solution is then heated in a boiling water bath for 3-5 minutes.

4. Observe for color change in the solution of test tubes or precipitate formation.

Positive Benedict’s Test: Formation of a reddish precipitate within three minutes.

Reducing sugars present. Example: Glucose
Negative Benedict’s Test: No color change (Remains Blue). Reducing sugars absent.
Example: Sucrose.
Some examples of substances that yield positive results for Benedict’s test are listed
below.
• Glucose

• Fructose

• Ribose

Barfoed’s Test

It is a differentiating test to distinguish between monosaccharides and disaccharides.

Barfoed’s test is also based on the reduced ability of sugar.

Monosaccharides give an early positive test while disaccharides give a late positive

Principle

Reducing sugar undergo tautomerization in a mildly acidic medium to form enediols.

These enediols reduce cupric ions to cuprous ions that form cuprous hydroxide. This
cuprous hydroxide is converted to cuprous oxide on heating and precipitates are formed.

Its principle is similar to Benedict’s test except for the acidic environment.
Monosaccharides being strong reducing agents give this test much early.
Apparatus

• Test tube
• Test tube holder
• Dropper
• Pipette
• Stand
• Spirit or gas lamp
• Solution to be tested
Reagents

Barfoed’s reagent is used that contains:

• Copper Acetate in Glacial Acetic Acid

Procedure

1. Take 2 ml of Barfoed’s reagent in a test tube

2. Add 2 ml of the test solution to the above test tube
3. Mix the solutions
4. Hold the test tube on flame and boil for minutes
5. Allow to cool at room temperature
6. Look for the precipitates
7. If no precipitates are formed, boil for an additional 10 minutes
8. Allow to cool and look for the precipitates
Observations

1. Red precipitates are formed after the first 5 minutes

2. Red precipitates are formed after additional boiling
Results

1. The formation of red precipitates after the initial first 5 minutes indicates
the presence of a monosaccharide
2. If precipitates are formed after 15 minutes, a disaccharide is present in the
test solution
Points to Remember

• Thistest helps to differentiate between monosaccharides and disaccharides

• When the heating period is increased, the disaccharides are hydrolyzed to
monosaccharides that give the positive test
Precautions

• Keep proper track of the boiling time

• Allow gradual cooling at room temperature
• If reheating is necessary, do it after the solution has become cold
Iodine Test
The iodine test is a chemical reaction-based identification test for starch. In this test,
iodine and starch form a distinct blue-black colored complex.

This test helps to identify the presence of starch in a sample. It also helps to
distinguish between mono– or disaccharides from polysaccharides

Procedure of Iodine Test

The steps of the Iodine Test:

1. Take two test tubes and label your test tubes as- test sample and control sample
2. Take a small sample (solid sample:500 mg -1000mg; liquid: 1ml) in a clean and
dried test tube labeled as a test sample.
3. Take 1ml of the purified water in the clean and dried test tube labeled as the
control sample.
4. Add 2-3 drops of Lugol’s iodine solution to both the test tubes and mix it
thoroughly on a vortex mixer.
5. Observe the color that develops in both the test tubes.

Result and Interpretation of Iodine Test

Based on the observation in the color change of the samples, the following may be
concluded as the iodine test for starch results:

When the iodine is added to the solution, the color of the solution changes. It
may give the following colors;

• Blue
• Reddish-brown
Result

If the color of the solution changes upon adding iodine, it represents that
polysaccharide is present in the solution. The nature of polysaccharides is
detected based on the color formed.

• If blue color appears, amylase or starch is present in the solution

• If reddish-brown color appears, glycogen is present
Points to Remember

Protein

1- Biuret Test :
The Biuret Test is often used
-To detect the protein in the given solution.
-To demonstrate the presence of the peptide bond.
procedure
Add 2 cm3 of the liquid food sample* to a clean, dry test tube
• Add 2 cm3 of Biuret Reagent.
• Repeat steps the steps above with de-ionized water to prepare a negative
control and with albumin (egg white) to prepare a positive control.
• Shake well and allow the mixture to stand for 5 minutes
• Observe any color change.
2-PROTEIN PRECIPITATION
•The solubility of proteins is affected by pH, temperature, salts, heavy metal salts ..etc
•The change of one of these factors will lead to protein precipitation and/ or
denaturation.
•Proteins will get denatured while using some factors that lead to precipitation.
•Is widely used in downstream processing of biological products in order to
concentrate proteins and purify them from various contaminants.

DENATURATION OF PROTEINS
• Denaturation is a process in which the proteins losing its quaternary
structure, tertiary structure and secondary structure, by application
of some external factor or compound such as a strong acid or base,
an organic solvent (e.g., alcohol or chloroform), or heat.
•Protein will become more viscous, decreased solubility and
aggregation, and protein become inactive

PRINCIPLE:
•This test depend on affecting solubility of the protein as a function of changes in pH in
highly acidic media, the protein will be positively changed, which is attracted to the acid
anions that cause them to precipitate.

Lipid

solubility test for lipids

Lipids are all insoluble in polar solvents like water but highly soluble in the non-polar
or weakly polar organic solvents, including ether, chloroform, benzene, and
acetone. In fact, these four solvents are often referred to as "lipid-solvents" or "fat-
solvents"