Department of Chemistry

G.C.W.U.S

Anum Naeem
Roll number: 13010203-012

Semester: 4th
Course code: CHEM-232
Course Title: BIOCHEMISTRY LABORATORY
Discipline: CHEMISTRY

Instructor Name: MISS AMNA KHALID

[SPRING 2015]
LAB # 1

Qualitative analysis of Carbohydrates, Proteins and Lipids

Theory:

Carbohydrates:
Molecules consist of carbon, hydrogen and oxygen atoms. A major food source and a key form
of energy for most organisms. When combined together to form polymers, carbohydrates can
function as long term food storage molecules, as protective membranes for organisms and cells,
and as the main structural support for plants and constituents of many cells and their contents.
Bread, pasta, beans, potatoes, bran, rice and cereals are carbohydrate-rich foods. Most
carbohydrate rich foods have a high starch content. Proteins and fats require more water for
digestion than carbohydrates. They are called carbohydrates because the carbon, oxygen and
hydrogen they contain are generally in proportion to form water with the general formula
Cn (H2O)

Saccharides, or carbohydrates, are sugars or starches. Saccharides consist of two basic

compounds: aldehydes (composed of double-bonded carbon and oxygen atoms, plus a
hydrogen atom), and ketones (composed of double-bonded carbon and oxygen atoms, plus two
additional carbon atoms).

There are various types of saccharides:

1-Monosaccharides
2-Disaccharides
3-Polysaccharides

Lipids:
Molecules consist of carbon, hydrogen, and oxygen atoms. The main constituents of all
membranes in all cells (cell walls), food storage molecules, intermediaries in signaling
pathways, Vitamins A, D, E and K, cholesterol. The lipids are the Organic compounds that are
insoluble in water but soluble in Organic solvents like Ether, chloroform, benzene and petroleum
Ether. They contain long chain of aliphatic hydrocarbons or benzene ring. One type of lipid, the
triglycerides, is sequestered as fat in adipose cells, which serve as the energy-storage depot for
organisms and also provide thermal insulation.
Lipids are molecules that can be extracted from plants and animals using nonpolar solvents
such as ether, chloroform and acetone. Fats (and the fatty acids from which they are made)
belong to this group as do other steroids, phospholipids forming cell membrane components
etc. Lipids as hydrophobic compounds are components of cellular membranes.
 Lipids that contain a functional group ester are hydrolysable in water. These include neutral
fats, waxes, phospholipids, and glycolipids. Non hydrolysable lipids lack such functional groups
and include steroids and fat-soluble vitamins (e.g. A, D, E, and K). Fats and oils are composed
of triacylglycerol or triglycerides. These are composed of glycerol (1, 2, 3-trihydroxypropane)
and 3 fatty acids to form a triester. Triglycerides are found in blood tests Lipids are also used to
store energy. Because of higher proportion of C-H bonds and low proportion of oxygen. Lipids
store double amount of energy as compared to same amount of any carbohydrates. Examples
of lipids include fats, oils, waxes, certain vitamins, hormones and most of the non-protein
membrane of cells.

Proteins:
Protein is found throughout the body—in muscle, bone, skin, hair, and virtually every other body
part or tissue. It makes up the enzymes that power many chemical reactions and the
hemoglobin that carries oxygen in your blood. They are present in all types of cells and in parts
of the cells.

Proteins are polymer chains made of amino acids linked together by peptide bonds. During
human digestion, proteins are broken down in the stomach to smaller polypeptide chains via
hydrochloric acid and protease actions. Most proteins consist of linear polymers built from series
of up to 20 different L-α-amino acids. All protein genic amino acids possess common structural
features, including an α-carbon to which an amino group, a carboxyl group, and a variable side
chain are bonded.

The number of amino acids varies from a few to 3000 or even more in different proteins.
The size of the proteins molecules are variable depending upon the no of amino acids present.
Generally the size of the protein is expressed in molecular weight that may range from 600 to
40,700,000.
Amino acids are found in animal sources such as meats, milk, fish, and eggs. They are also
found in plant sources such as soy, beans, legumes, nut butters, and some grains

Amino acids are classified into three groups:

 Essential
 Nonessential
 Conditional

 Essential amino acids cannot be made by the body, and must be supplied by

food. They do not need to be eaten at one meal. The balance over the whole day
is more important
 Nonessential amino acids are made by the body from essential amino acids or
in the normal breakdown of proteins.
 Conditional amino acids are needed in times of illness and stress.
 Procedure:

(a)Carbohydrates:

1-Molisch's Test:
It is a sensitive chemical test for the presence of Carbohydrates, based on the dehydration of

the carbohydrate by sulfuric acid to produce an aldehyde, which condenses with two molecules
of phenol (usually α-naphthol, resulting in a red- or purple-colored compound.

Procedure:
-Apply this test two different carbohydrate solutions of your own choice, preferably to one
monosaccharide and one polysaccharide. - Place 2 mL of a known carbohydrate solution in a
test tube, add 1 drop of Molisch’s reagent (10% α-naphthol in ethanol). - Pour 1-2 mL of conc.
H2SO4 down the side of the test tube, so that it forms a layer at the bottom of the tube. Observe
the color at the interface between two layers and compare your result with a control test. *A
brown color due to charring must be ignored and the test should be repeated with a more dilute
sugar solution.

2-Barfoed's Test:
It was invented by Danish chemist Christen Thomsen Barfoed It is a chemical test used for
detecting the presence of monosaccharides. It is based on the reduction of copper (II) acetate to
copper (I) oxide (Cu2O), which forms a brick-red precipitate RCHO + 2Cu2+ + 2H2O →
RCOOH + Cu2O↓ + 4H+ (Disaccharides may also react, but the reaction is much slower.) The
aldehyde group of the monosaccharide which normally forms a cyclic hemiacetal is oxidized to
the carboxylate. A number of other substances, including sodium chloride, may interfere.

Preparing Reagent:
Barfoed's reagent consists of a 0.33 molar solution of neutral copper acetate in 1% acetic acid
solution. The reagent does not keep well and it is therefore advisable to make it up when it is
actually required. Procedure: To 1-2 mL of Barfoed’s reagent, add an equal volume of sugar
solution. - Boil for 5 min. in a water bath and allow to stand. You will observe a brickred cuprous
oxide precipitate if reduction has taken place.

3-Seliwanoff's Test:
A biochemical test to identify the presence of ketonic sugars, such as fructose in solution. It
was devised by the Russian chemist F. F. Seliwanoff. A few drops of the reagent, consisting of
resorcinol crystals dissolved in equal amounts of water and hydrochloric acid, are heated with
the test solution and the formation of a red precipitate indicates a positive result. Procedure:
Heat 1 mL of sugar solution with 3 mL Seliwanoff’s reagent (0.5 g resorcinol per liter 10% HCl)
in boiling water in less than 30 seconds, a red color must appear for ketoses. 5- Iodine Test:
Iodine test is an indicator for the presence of starch. Iodine solution (iodine dissolved in an
aqueous solution of potassium iodide) reacts with starch producing a blue-black color. Apply this
test to all the polysaccharides provided. Procedure: To 2-3 mL of polysaccharide solution, add
1-2 drops of iodine solution.

(b)Proteins:
Biuret Test:
The biuret test is a chemical test used for detecting the presence of peptide bonds. In the

presence of peptides, a copper (II) ion forms violet-colored coordination complexes in an

alkaline solution. The biuret test uses an alkaline mixture, or reagent, composed of potassium
hydroxide and copper sulfate. The normal color of biuret reagent is blue. The reagent turns
violet in the presence of peptide bonds -- the chemical bonds that hold amino acids together.
The proteins detected must have at least three amino acids, which means that the protein must
have at least two peptide bonds. The reagent’s copper ions, with a charge of +2, are reduced to
a charge of +1 in the presence of peptide bonds causing color change.

(c)Lipids:

Sr# Sample Test Result Inference

1 Milk (A) Molisch Test Purple brown color Carbohydrates present

Barfoed Test Brick Red ppt Aldehydes present

Biurett Test Light purple color proteins present

Seliwanoff Test Red coloration Ketones present

Starch Test Bluish color Starch present
2 Glucose (B) Molisch Test Purple brown Carbohydrates present
Barfoad Test Brick red ppt Aldehydes present
Biurett Test No change Protein absent
Seliwanoff Test No change Ketones absent
Starch Test No change Starch absent
3 Egg yolk (C) Molisch Test Purple brown color Carbohydrates present
Barfoad Test Brick red ppt Aldehydes present
Biurett Test Violet pink color Protein present
Seliwanoff Test Red coloration Ketones present
Starch Test No change Starch absent
 4 Table sugar (D) Molisch Test Purple brown color Carbohydrates present
Barfoad Test Brick red ppt Aldehydes present
Biurett Test No change Protein absent
Seliwanoff Test Red coloration Ketones present
Starch Test No change Starch absent
5 Starch (E) Molisch Test No change Carbohydrates absent
Barfoad Test No change Aldehydes absent
Biurett Test No change Protein absent
Seliwanoff Test No change Ketones absent
Starch Test Bluish color Starch present

Result:
In sample A carbohydrates, protein, aldehyde, ketone and starch is present.

In sample B carbohydrates and aldehyde is present.

In sample C carbohydrates, aldehyde, ketone and protein is present.

In sample D carbohydrates, aldehyde and ketone is present.

In sample E starch is present.