Date:

Title: Carbohydrates
Aim: To investigate the three classes of carbohydrates using the Reducing sugars, Non-
Reducing Sugars, and Starch tests
Background:

Carbohydrates, or carbs, are the sugars, starches, and dietary fibre that occur in certain foods.
The body breaks them down into glucose, which provides energy. Carbohydrates are
composed of carbon, hydrogen, and oxygen and have a general formula that approximates
CH2O. Carbohydrates play an important role in the human body. They act as an energy
source, help control blood glucose and insulin metabolism, participate in cholesterol and
triglyceride metabolism, and help with fermentation. There are three different types of
carbohydrates: starches (examples potatoes, and bread), sugars (examples molasses and
honey) and fibres (examples whole grains and legumes). The following are the tests to
identify the presence of carbohydrates.

1. Molisch’s test
2. Fehling’s test
3. Benedict’s test
4. Tollen’s test
5. Iodine test

(a) Molisch’s Test:

Molisch’s test is a general test for carbohydrates. This test is given by almost all the
carbohydrates. In this test, concentrated sulfuric acid converts the given carbohydrate into
furfural or its derivatives, which react with α-naphthol to form a purple-coloured product.

(b) Fehling’s Test:

This test is given by reducing sugars. To the aqueous solution of carbohydrate Fehling’s
solution is added and heated in water bath. The formation of red precipitate confirms the
presence of reducing sugars. The copper ions present in Fehling’s solution in +3 state is
reduced to +2 oxidation state and in alkaline medium it is precipitated as red cuprous oxide.

(c) Benedict’s Test:

This test is given by reducing sugars. in an alkaline medium, sodium carbonate converts
glucose to enediol and this enediol reduces cupric to cuprous forming cuprous hydroxide.
This solution is kept in sodium citrate and on boiling, red precipitate of cuprous oxide is
formed.
(d) Tollen’s Test:

This test is given by reducing sugars. Carbohydrates react with Tollens reagent and forms a
silver mirror on the inner walls of the test tube. This confirms the presence of reducing
sugars. Silver ions are reduced to metallic silver.

(e) Iodine Test:

This test is only given by starch. Starch reacts with iodine solution forms complex blue
colour solution. On heating the blue colour disappears and on cooling the blue colour
reappears.

Even thou starch sugars and fibres are all carbohydrates iodine will not change colors when it
gets exposed to sugar. This is because starch is made up of many, many sugar molecules
chained together. Only the long chains found in starch can interact with the iodine. Tests for
Carbohydrates are important because they allow us to identify and quantify the amount of
carbohydrates present in a sample.

Apparatus: Test tubes, benedict solution, water, glucose and sucrose, water bath, hydrochloric
acid, litmus paper, potato, petri dish, iodine potassium iodide
Method:
Part 1 Benedict’s Test for Reducing Sugars
1. Three test tubes were labelled as water, glucose, and sucrose.
2. In each of the test tube, 2 mL of the respective solutions were added.
3. Benedict’s solution (2mL) was added in equal volume to each test tube.
4. The contents were gently mixed ensuring thorough mixing of solutions.
5. The test tubes were placed in a water bath for about 5 minutes.
6. Each test tubes were observed and recorded.

Part 2 Non-Reducing Sugars Test

1. Two test tubes were labelled as ‘control’ and ‘test’.
2. Two millilitres of sucrose solution were added to both test tubes.
3. A few drops (3) of hydrochloric acid (HCL) were added to the ‘Test’ test tube.
4. The contents were gently mixed of the ‘Test” test tube ensuring thorough mixing of
the solutions.
5. Both test tubes were placed in a water bath for about 5 minutes
6. The ‘Test’ test tube was cooled and neutralized with diluted NaOH (using litmus
paper)
7. Both solutions were tested for the presence of reducing sugar with Benedict’s
solution.
8. The observation for each test tube was recorded.
Part 3 Test for starch
1. A small section of potato was cut and placed in a petri dish.
2. A drop of iodine potassium iodide (IKI) solution was placed on the surface of the
potato.
3. The observation was recorded.

Results
TABLE 1 BENEDICT’S TEST FOR REDUCING SUGARS
Solutions Observation Inference
Water solution + Benedict’s The solution remains blue No reaction
after the benedict’s test
Glucose solution + The solution turns from blue The hotter the final color of
Benedict’s to green to brick red. the reagent, the higher the
concentration of reducing
sugar
Sucrose solution + Bendict’s The solution remains blue Complex sugar remain
after the Benedict’s test.

TABLE 2 PART 2 NON-REDUCING SUGARS TEST

Solution Observation Inference
Sucrose + Benedict’s The solution remains blue No reaction
solution after the Benedict
Sucrose + HCl + Benedict’s The solution turns from blue Double bond broken with
solution to red HCL to form simple sugar

TABLE 3 PART 3 TEST FOR STARCH

Solution Observation Inference
Potato + IKI The iodine solution turns Potatoes are rich in starch
from yellow brown to dark and thus show a positive test
blue-black contact with the with iodine.
potato
Discussion
Glucose is a reducing sugar. The reactions involved in Benedict’s test is that the reducing
sugars are oxidized by the copper ion in solution to form a carboxylic acid and a reddish
precipitate of copper (I) oxide. The formula for benedict’s reaction is RCHO + 2 Cu2+ + 5
OH − → RCOO − + Cu 2O + 3 H 2OMaltose (malt sugar) is a reducing disaccharide while
sucrose is a non-reducing one because of the absence of free aldehyde or ketone group in
sucrose. In maltose, there are two glucose present. The glycosidic oxygen atom of one
glucose is alpha and bonded to C-4 atom of another glucose unit which is aglycone. The
image below shows the structures of the sugars.

The functional group that is responsible for the difference is the aldehyde group. Non-
reducing sugars lack free aldehyde group and cannot reduce other compounds. This is
because the anomeric carbon lacks an OH group. The function of each tube is to see the
separate reactions that took place when respective substances reaction with Benedict’s
solution. When glucose reacts with benedict’s solution, the mixture changed from blue to
brick red. This means that there was a positive test for glucose, however when sucrose
reacted with glucose, no reaction took place because the solution remained blue, likewise
when glucose reacted with water it was a negative control.
Glucose and fructose are the monomers that makes up sucrose molecules. The type of
reaction that took place that cause the conversion of the sucrose to its monomers is known as
hydrolysis. Hydrochloric acid was used in the reaction to hydrolyse the bonds between the
disaccharides. During the hydrolysis process, several chemical reactions take place. Glucose
and fructose are converted to glucose, HCl, and water. A water molecule helps to break the
acetal bond as shown in red in the diagram below. The acetal bond is broken, the H from the
water is added to the oxygen on the glucose. The -OH is then added to the carbon on the
fructose.
If starch is reacted with HCl it will hydrolyse the glycoside bond, reduced the molecular
weight of starch, decreased the amount of amylopectin, and formed glucose monomers.
A linear triiodide ion complex is formed when IKI is added to potato. The water molecule is
removed to make the connection between IKI and starch. Hydrolysis is the type of chemical
reaction that took place. Starch is a branched polymer chain, whereas cellulose is an
unbranched single chain polymer. Therefore the structure of starch can be used to make
cellulose by hydrolysis in condensation reactions.
Starch and glycogen are made up of 𝜶-glucose subunits, whereas cellulose is made up of 𝛃-
glucose subunits. Cellulose is unbranched and a straight-chain polymer of glucose, whereas
starch and glycogen are branched.
Limitations
It is impossible to determine the exact concentration of reducing sugar; only a
semiquantitative estimate may be given and further testing is required to identify the
carbohydrate.
Precautions

● The measurement should be accurate.

● Do not overheat the mixture. It is best to heat over a water bath slowly.

● Use a test-tube holder while heating the fluid.

Conclusion:
It can be concluded that the reducing sugar is glucose, sucrose is the nonreducing sugar and
potato contains starch.
Experiment 2
Date:
Title: Proteins
Aim: To carry out confirmative tests to determine the presence of proteins and fats in
provided food samples
Theory

Proteins are complex molecules and do most of the work in cells. They are important to the
structure, function, and regulation of the body. Proteins contain the elements carbon,
hydrogen, and oxygen. Proteins have multiple functions, including acting as enzymes and
hormones, maintaining proper fluid and acid-base balance, providing nutrient transport,
making antibodies, enabling wound healing and tissue regeneration, and providing energy
when carbohydrate and fat intake is inadequate. A protein's primary structure is defined as the
amino acid sequence of its polypeptide chain example haemoglobin; secondary structure is
the local spatial arrangement of a polypeptide's backbone (main chain) atoms (example
keratin made up of fingernail); tertiary structure refers to the three-dimensional structure of
an entire polypeptide chain (example myoglobin); and quaternary structure is the three-
dimensional arrangement of the sub-units in a multi-sub-unit protein (ribosomes are examples
of such). The following are the simple tests carried to find the presence of proteins in the
given food samples.

Biuret test
The compounds with peptide linkage undergo this test. Proteins are polypeptides of amino
acids linked together by peptide bonds. An alkaline solution of protein is treated with a drop
of aqueous copper sulphate and a bluish violet colour is obtained. Formation of violet
coloration confirms the presence of Proteins.

Xanthoproteic test
Proteins on treatment with nitric acid give a yellow or orange colour. Concentrated nitric
acid is used for nitration. On the treatment of nitric acid, proteins give yellow precipitate
which turns to orange colour on treatment with alkali. The appearance of a yellow colour
solution confirms the presence of proteins.

Millions test
Phenolic group of tyrosine of proteins reacts with mercuric sulphate in the presence of
sodium nitrite and sulfuric acid to give red colour. Millon’s test is given by proteins
containing phenolic amino acids. Gelatin does not give this test. First, a white precipitate is
formed when proteins are treated with millions reagent and then turns to brick-red colour on
boiling, this confirms the presence of proteins. The appearance of brick red colour solution
confirms the presence of proteins.

Ninhydrin test
Proteins react with pyridine solution of ninhydrin and change to a coloured solution from a
deep blue to violet-pink or sometimes even to a red colour. Ninhydrin solution is prepared by
dissolving 0.1gm of ninhydrin in about 100ml of distilled water. But this solution of
ninhydrin is unstable and can be kept for two days. The appearance of violet colour solution
confirms the presence of proteins.

Apparatus: Test tubes, biuret reagent, water, serum albumin, whole milk, skim milk, regular

Soda, diet soda, red bull, egg white, egg yolk, mashed potato, mashed beans, coffee
Method:
1. Twelve (12) test tubes were labelled.
2. Each sample (of 12 mL) was added to each test tube.
3. Biuret reagent (of 2mL) was added to each test tube.
4. The contents of each test tube were added.
5. Each tube was carefully examined, and the colour change was noted.
6. The observations was recorded in a table.

Results
TABLE SHOWING RESULTS OF THE BIURET TEST FOR PROTEINS
Tube number The colour of Initial colour at Final colour Is this a positive
Biuret Reagent mixing after 2 minutes test?
is blue at room
Are peptide
temperature
bonds present
(Yes or no?)
1 Negative Blue Blue Negative test
control: water No peptide
bond
2 Positive control Blue Purple Positive
Serum albumin Peptide bond
present
 3 Whole milk Light blue Purple Positive test
Peptide bond
present
4 Skim milk Light blue Purple Positive test
Peptide bond
present
5 Regular soda Blue Blue Negative test
No peptide
bond
6 Diet soda Black Black Negative test
No peptide
bond
7 Red bull Black Black Negative test
No peptide
bond
8 Egg white Blue Purple Positive test
Peptide bond
present
9 Egg Yolk Yellowish blue Purple Positive test
Peptide bond
present
10 Mashed up Light blue Light blue Negative test
potato
No peptide
bond present
11 Mashed up Dark brown Violet Positive test
beans
Peptide bond
present
12 Coffee Black Black Negative test
No peptide
bond present

Discussion
Proteins are one of the primary constituents of living matter. They consist of long chains of
amino acids, which are bonded together by peptide linkages and thus called polypeptides.
Five functions of proteins include: act as enzymes and hormones, maintain proper fluid and
acid-base balance, provide nutrient transport, make antibodies and enable wound healing and
tissue regeneration.
The general formula of an amino acid is R-CH(NH2)-COOH.

Amino acids consist of the amino (NH2) and carboxyl (COOH) functional groups. Peptide
bonds form between the carboxyl group of amino acid 1 and amino group of amino acid 2.
Water is removed as a byproduct. The resulting peptide bond is a C-N covalent bond that
now links the two amino acids together to form a dipeptide.
 The complete structure of a protein can be described at four different levels of
complexity: primary, secondary, tertiary, and quaternary structure.
Primary structure - sequence of amino acids that form peptide covalent bonds with each other
to form a polypeptide chain.

Secondary structure - hydrogen bonds form as the protein folds to form an alpha helix
(coiled/helical) or a beta-pleated sheet.

Tertiary structure - the specific 3D shape/active site if an enzyme - the amino acid R groups
interact and ionic/disulfide/hydrogen bonds form between them to form a specific 3D shape.

Quaternary structure - hydrogen bonds between multiple polypeptide chains, e.g. in collagen
which forms a triple helix.
An example of the primary structure of a protein is the first six amino acids in haemoglobin.
An example of the secondary structure of a protein is Interconnected alpha-helices are found
in fibrous proteins, like the keratin that makes up human fingernails and hair.

Myoglobin is a typical example of tertiary structure of protein.

Examples of proteins with quaternary structure is ribosomes.

The primary structure is comprised of a linear chain of amino acids. The secondary structure
contains regions of amino acid chains that are stabilized by hydrogen bonds from the
polypeptide backbone. These hydrogen bonds create alpha-helix and beta-pleated sheets of
the secondary structure. The three-dimensional shape of a protein, its tertiary structure, is
determined by the interactions of side chains from the polypeptide backbone. The quaternary
structure also influences the three-dimensional shape of the protein and is formed through the
side-chain interactions between two or more polypeptides.
The biuret test is a chemical test that can be used to check for the presence of peptide
bonds in each analyte.
The biuret test measures peptide bonds in a sample. Recall that proteins are made up of
amino acids connected with peptide bonds. So, the biuret test cannot test for individual amino
acids, because those aren't connected with a peptide bond.
Precautions
1. Use the proper amount of sample and reagent; generally, the 1:1 ratio gives a better
result.
2. Excessive use of reagent will form the mixture blue instead of purple giving a false
negative result.

Limitations
1. The number of proteins present in the sample cannot be quantified.
2. Food testing is limited in scope and can only identify specific contaminants that the
testing is designed to detect.
Conclusion
The biuret test was used to confirm it protein was present in a food sample but cannot be used
to determine amino acids. Food samples that contains proteins were serum albumin, whole
milk, skim milk, egg white, egg yolk and mashed up beans.

Experiment 3
Date:
Title: Lipids
Aim: To test for presence of lipids (fats) in various food items such as nuts, cheese, potatoes,
and meat using the emulsion test.
Theory:
Lipids are organic compounds that contain hydrogen, carbon, and oxygen atoms, which form
the framework for the structure and function of living cells. Lipids contain the same elements
as carbohydrates: carbon, hydrogen, and oxygen (C, H, and O). However, lipids are mainly
made of hydrocarbon chains (or rings) and contain fewer polar hydroxyl groups (-OH). Some
functions of lipids include help with moving and storing energy, absorbing vitamins, and
making hormones.
The four main groups of lipids include:

● Fatty acids (saturated and unsaturated)

● Glycerides (glycerol-containing lipids)
● Nonglyceride lipids (sphingolipids, steroids, waxes)
● Complex lipids (lipoproteins, glycolipids)
The Ethanol Emulsion Test is a food test which determines the presence of a broad group of
naturally occurring compounds known as lipids. Lipids consist of fats and oils. Other lipid
tests include the Grease Spot Test and the Sudan Stain Test. The Grease spot test is
performed on fats - lipids which are solid at room temperature. Sudan stain colours lipids red
but is a less common bench reagent than ethanol. The Ethanol Emulsion Test is the most
common test amongst the three.

Apparatus:
Test tubes (one for each food item), nuts, cheese, potato, meat, vegetable oil, distilled water,
ethanol (95%), test tube rack, pipettes, beaker, and test tube holder.

Method:
Solid samples:
1. The food samples were crushed and placed in a dry test tube.
2. 2 ml of 95% ethanol was added to the sample and shaken thoroughly.
3. The solid was allows to settle (for about 3 minutes) to allow the lipid to be extracted.
4. The ethanol was decanted into another test tube.
5. 2 ml of distilled water was added to the second test tube.
6. The observations were recorded.

Liquid Sample
1. A few drops of the liquid food sample was added to a dry test tube.
 2. 2cm3 of ethanol was added to the test tube and shaken thoroughly.
3. 2cm3 of distilled was added to the test.
4. The observations were recorded.

Results

TABLE SHOWING RESULTS FOR EMULSION TEST

Food Observation Inference
Vegetable oil Milky appearance Lipids present
Nuts Milky appearance Lipids present
Cheese Milky appearance Lipids present
Potato Colourless appearance No lipid present
Meat Milky appearance Lipids present

Discussion
Lipids are fatty, waxy, or oily compounds that are soluble in organic solvents and insoluble
in polar solvents such as water. Triglycerides are esters, made up of a glycerol molecule
bound to three fatty acid molecules. Glycerol is a simple sugar alcohol with three available
hydroxyl functional groups. Fatty acids are long carbon chains, typically containing between
12 and 24 carbons, bound to a carboxyl group. Fats are triglycerides with long chain fatty
acids. Van der Waals interactions between the long chains give rise to high melting points.
Hence, fats are solid at room temperature. Oils are triglycerides with shorter fatty acid chains.
As the Van der Waals interactions are not great, oils have a lower melting point and are liquid
at room temperature. Lipids perform functions both within the body and in food. Within the
body, lipids function as an energy reserve, regulate hormones, transmit nerve impulses,
cushion vital organs, and transport fat-soluble nutrients. Three sources of dietary lipids are
poultry, fish, and red meat. A fat free diet is unadvisable because persons who have a fat-
deficient diet are also deficient in fat-soluble vitamins. (A, D, E and K). Symptoms include
blurred vision, weak bones – movement of calcium into soft tissues, digestive tract problems
and numerous skin, hair, and nail problems. Obesity is a chronic health condition that
raises the risk for heart disease. Three diseases associated with obesity include heart disease,
diabetes, and high blood pressure. The significance of crushing the solid food sample and
leaving it to stand in ethanol for a few minutes is to break the cells exposes the lipids stored
inside so that they can be extracted by the ethanol solvent. Lipids are insoluble in water and
soluble in ethanol (an alcohol). After lipids have been dissolved in ethanol and then added to
H2O, they will form tiny, dispersed droplets in the water. This is called an emulsion. These
droplets scatter light as it passes through the water, so it appears white and cloudy. One
precaution is that the measurements should be precise to prevent errors that may arise in the
experiment. One limitation would be the storage and expiration date of the food sample.
Conclusion
It can be concluded that when the solution remain colourless lipids are not present and when
a layer of cloudy white suspensions forms at the top of the solution, lipids are present. The
food samples that contain lipids are vegetable oil, nuts, cheese and meat while potato did not
contain lipids.