Introduction

Organic molecules consist of compounds that contain carbon, hydrogen and oxygen.
Examples of organic compounds in living organisms are carbohydrates, proteins, lipids and
nucleic acids. Each of the macromolecules or biomolecules (polymer) is produced from the
small subunit called monomers. Polymers produced by the removal of one molecule of water
(condensation) and require energy which will form covalent bonds between monomers. The
covalent bond then can be broken down by hydrolysis process which need an additional of
water molecule produced energy. Each of these biomolecules has different structures and
properties.
The presence of biomolecule in a sample can be traced by using a simple technique. Usually,
the experiments are conducted on a sample that the contents are unknown. Then, the results
are compared to the controlled sample. During the experiment, observed the reaction of the
unknown sample with the reaction of the controlled sample by using the same procedures.
In this practical, students are exposed to the technique to trace biomolecules contains in
samples. This analysis is a qualitative analysis, which can only show the presence of
substance, not the quantity of the substance contain in the sample.

Objective

1. To trace biomolecules carbohydrates, proteins and lipids qualitatively.

2. To recognize specific biomolecules such as monosaccharides, disaccharides,
polysaccharides, reducing sugars, amino acids, polypeptides and lipids.
3. To understanding the process of condensation and hydrolysis, peptide bond and the
glycosidic bond.

Carbohydrate
Carbohydrates are the main energy source for the human body. Chemically, carbohydrates
are organic molecules in which carbon, hydrogen, and oxygen bond together in the ratio:
CnH2nOn or (CH2O)n. The carbon bond in this compound produced high energy when it is
broken down.
Carbohydrates present in a formed of monosaccharide (a monomer of carbohydrate or a
simple unit of a simple sugar that consist of aldehyde group, -CHO or ketones group, C=O).
Glucose, fructose and galactose are the monosaccharide.
Carbohydrates also present in formed of oligosaccharide (sugar that consist of the
combination between two to 20 units of monosaccharide through the condensation process
such as sucrose, maltose and lactose) and polysaccharide (non-sugar that consist of the long
chain of monosaccharide, as example starch, glycogen, cellulose, inuline and dextrin).

The common chemical test conducted in tracing carbohydrates group are Benedict Test and
Iodine Test.

Practical 1: Carbohydrate Test

 A. Benedict Test
These tests are conducted to trace all the monosaccaharide and disaccharide that
contains free aldehyde and ketones functional group. The aldehyde and ketone group
consist in polysaccharide can react with the Benedict reagent but it is hard to trace.
Benedict test also can be conducted to trace the presence of reducing sugar by the
changing in colour from blue to red. The changes in colour are resulted from the
reducing of cuprum ion (Cu 2+) to cuprum oxide (Cu+). The final colour after this test
is differs according to the quantity of the cuprum oxide. As example, green colour
solution shows the small amount of reducing sugar , red colour solution shows the
large amount of reducing sugar while non-reducing sugar produce blue colour
solution.

B. Iodine Test
These tests are conducted to trace the polysaccharide in helix forms such as starch and
glycogen. Iodine will formed a blue colour chain of helix polysaccharide complex. At
least eight units of monosaccharide are needed in the carbohydrate chain to form a
helix complex that can be coloured by iodine. The chains that consist of more branch
will reduced the blue colour of the complex because of the loose helix formed.

Practical 2: Acid Hydrolysis of Carbohydrate

Protein

Proteins are complex molecules that consist of amino acid units. Amino acid contains amino
group (-NH2) and carboxyl group (-COOH) that attach to the same carbon.

N–C-C
There are at least 20 amino acids that had been recognized. Although each amino acid
contains the same amino and carboxyl group, the R group is different. The R group will
determined the physical and chemical characteristic of the amino acids and the proteins.
Peptides are formed when an amino acid combine with the other amino acid through peptide
bond during condensation process.
So, amino acids are bonded through the peptide bond. These bonds can be broken down by
enzymatic hydrolysis, a process that needs water molecules. Polypeptide chains consist of
two or more amino acids. So, proteins are polypeptide chains that contain certain molecular
weight, unique conformation and specific sequence of amino acids. Each protein has a
specific structure and function and plays a special role in organism.
The objective of this experiment is to trace the presence of protein and certain amino acid in
samples provided through Biuret test, Ninhindrin test, Xanthoprotein test, Millon test and
Hopkins-Cole test.

Practical 3:
 A. Biuret Test
This test is conducted to trace the presence of protein in sample through the formation
of purple colour. The purple colour are produced when cuprum ion (Cu2+) formed are
complex with peptide bond in protein. Free amino acids and short peptide chain not
showing the positive result because Cu2+ only react with at least four to six peptide
bond to produce colour.

B. Ninhidrin Test
Amino acids contain amino group and d free carboxyl group that can react with
Ninhidrin and producing blue- purple product. The colour was produced from the
nitrogen element in the α-amino group (bond to α-carbon) of amino acid structure.
Amino acids that contain the secondary amino group also can react with Ninhidrin
and producing yellow colour solution. Proline (Pro) is one of the amino acid that
producing yellow colour solution.

C. Xanthoprotein test
Some of amino acids contain aromatic group that originated from benzene. This
aromatic group can react just like the reaction of benzene. One of the reactions is the
titration of benzene ring and nitric acid. This reaction is called Xanthoprotein test and
producing yellow colour solution. Amino acid that produced yellow colour solution in
this test is Tyrosine (Tyr), Tryptophan (Trp) and Phenylalanine (Phe).

D. Millon Test
This test is specifically for Tyrosine (Tyr). Tyrosine is the only amino acid that
contains of Phenol group. Phenols are initially nitrated with nitric
acid and then forming a complex with the mercury ion in the Millon solution. In this
experiment, the presence of Tyr in protein produces white precipitate that changes red
when it is heated. Sometimes, the sample straightly becomes red. Both are positive
result for Tyrosine.

E. Hopkins- Cole Test

This test is specifically for Tryptophan (Trp). Trp are the only amino acids that
contain indole group. Indole ring react with glyocsilic acid in high concentrated acid
solution to form purple ring. Protein was initially hydrolyzed by high concentration
acid. Free Trp are then will react with glyocsilic acid.

Lipid
The lipids are a large and diverse group of naturally occurring organic compounds that are
related by their solubility in nonpolar organic solvents (e.g. ether, chloroform, acetone &
benzene) and general insolubility in water. The main component of lipids is fatty acids. Fatty
acid composed of chains of carbon atom that ends with a carboxyl group (-COOH). Fatty
acids are usually stored and transported as triglyceride, three fatty acids bonded to a molecule
of glycerol. The bond formed are called ester bond and produced by hydrolysis process. In
this experiment, Sudan IV Test and Osmic Test are conducted to trace lipid in samples.

QUESTION
Carbohydrates:
From Table 1 and Table 2
1. List of reducing sugar
Fructose, glucose, lactose, maltose, galactose.
2. List oligosaccharide sugars that do not have a free aldehyde or ketone group.
Sucrose
3. List polisakarida.Polisakarida not seem to diverge, why?
Liver extract, as branched polysaccharides would yield a blue solution is less clear in the
iodine test, liver extract gave a bright blue color in this test.
4. Samples of fruit or vegetables is polysaccharides have saved? sample is a reducing sugar?
Potato extract containing polisakarida.Ekstrak potato, ripe banana extract, banana extract and
young garlic extracts containing reducing sugars.
5. What type of saccharide found in each sample solution after the hydrolysis of table 2?
Can not be ascertained because of an error in the experiment, but the samples should contain
the monosaccharides glucose and fructose.

6. What is the mechanism by acid hydrolysis of carbohydrates?

Hydrolysis is a chemical process in which some molecules are divided into two parts by the
addition of a molecule air.Satu senggat will get a pair of molecular hydrogen ions (H +) of
additional water molecules will have one more tersebut.Kumpulan hydroxyl group (HO +).
Hydrochloric acid, HCl used as a catalyst.
Protein:
From Table 3
1. Samples of fruit or vegetable is can you eat to get protein?
Potato is the only vegetable protein that can give us the experimental ini.Pisang also contain
protein but low in quantity.
2. Samples of fruit or vegetables which contain
a) Prolina
Onion and potato
b) phenylalanine
banana
c) Tyrosine
Potato and liver
3. Are free amino acids containing peptide bond? Explain why.
No, because the peptide bond is formed when four or more amino acid is attached through a
condensation process of removing a water molecule in each peptide bond is formed.
Lipids:

From Table 4
1. Samples of fruit or vegetables which contain lipids?
Olive oil, coconut oil and onions.
2. What can you conclude about the level of lipid solubility in organic solvents?
Oils and fats are the basic unit lipid.Kedua them soluble in organic solvents because their
molecules are polar molecules which are attracted to organic solvents.
3. What can you conclude about the level of lipid solubility in water?
Lipids are basically fats and oils are not soluble in air.Molekul they are not attracted to water
molecules.
DISCUSSION
Condensation process is the removal of one molecule of water and energy needed to form a
covalent bond between the monomer-monomer.Proses hydrolysis is the process of adding a
water molecule and release tenaga.Proses hydrolysis can break covalent bonds between
monomer-monomer.
Some of the results of the experiment is probably due to an error salah.Ini in the control
experiments, such as contamination in a sample using the pipette, placing the sample in one
particular test tube and shake the test tubes after mixing the solution.

CONCLUSION
Biomolecular carbohydrates, proteins and lipids can be detected qualitatively using
techniques such as Benedict's test, iodine test, Biuret test, test Ninhidrin, Xanthoprotein test,
Millon test, test test Hopskin-Cole and Sudan IV.