Name of Test Importance of Composition of Reagent Color of Chemical Group

Responsible for
Test Positive Positive Test
Result

Biuret test Used to detect Copper (II) sulfate (CuSO4) Rose-pink Due to the
the amount of presence of
proteins present to violet peptide or amide
in urine linkage (CONH2)

Ninhhydrin test Used in the Trikethohydirindene hydrate Blue color Due to the
analysis of presence of alpha-
amino acid in amino group in a
proteins molecule except
for proline and
hydroxyproline

Xanthoproteic Detect the Conc. Nitric acid, Sodium Yellow to Due to the
test presence of hydroxide orange presence of
aromatic amino Benzene ring in
acids in a protein the structure of
such amino acid
such as Tyrosine,
Tyrptophan and
Phenylalanine

SOLUBILITY

Test the solubility of 1 ml of fresh raw egg white in 2 ml. of each of the solvents listed below. Indicate
whether solubleor insoluble.

a. Water -soluble

b. 0.1M NaOH- insoluble

c. 0.1M HCl-soluble

d. Chloroform-insolu
Name of Test Importance of Test Composition of Color of Group Responsible
Reagents
Positive

Results

Millon’s Test Used to detect the Mercurous nitrate Flesh to Due to phenol group
presence of soluble in Nitric acid red
proteins contained in
color Tyrosine

Hopkins-Cole Used to detect the Glycooxylic acid Violet ring Due to the presence
Test for presence of and conc. Sulfuric of Indole nucleus in
Tryptophan acid the
Tryptophan
Tryptophan
component

Sakaguchi Test Used in the Alkaline solution, a- Red color Presence of Arginine
for Arginine naphthol and
calorimetric
Sodium
estimation of
hypochlorite
Arginine

Lead acetate for It is selective for Lead acetate, Black Due to the presence
Labile Sulfur deposit of Sulfur in amino
detection of Sodium hydroxide acids
Hydrogen sulfide in a
reaction

Protein is a macronutrient that is essential to building muscle mass. It is commonly found in animal
products, though is also present in other sources such as nuts and legumes.
Chemically, protein is composed of amino acids, which are organic compounds made of carbon,
hydrogen, nitrogen, oxygen or sulfur. Amino acids are the building blocks of proteins, and proteins are
the building blocks of muscle mass, according to the National Institutes of Health (NIH).

2. What are the functions of proteins?

1.Growth and maintenance

2. Causes biochemical reactions
3. Acts as a messenger
4. Provides structure
5. Maintains proper pH
6. Balances fluids
7. Boost immune system
8. Transport and stores nutrients
9. Provides energy
3. What are the elements present in proteins?

Protein is made up of amino acids composed of five elements: carbon, hydrogen, oxygen, nitrogen and
sulfur

4. Identify the essential and non-essential amino acids

Essential amino acids – Isoleucine, Histidine, Leucine, Methionineo Lysine, Phenylalanine, Tryptophan,
Threonine, Valine

Non-essential amino acids – Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamic Acid,
Glutamine, Glycine, Proline, Serine, Tyrosine

5. What is protein folding and briefly explain the four levels of protein folding mechanism.

✔ Primary Structure - describes the unique order in which amino acids are linked together to form a
protein. Proteins are constructed from a set of 20 amino acids.

✔ Secondary Structure - refers to the coiling or folding of a polypeptide chain that gives the protein its
3-D shape. There are two types of secondary structures observed in proteins.

1. alpha helix

2. beta pleated sheet

✔ Tertiary Structure - refers to the comprehensive 3-D structure of the polypeptide chain of a protein.
There are several types of bonds and forces that hold a protein in its tertiary structure.

1. Hydrophobic interaction

2. Hydrogen bonding
3. Ionic bonding

4. disulfide bridge

5. Van Der Waals forces

✔ Quaternary Structure - refers to the structure of a protein macromolecule formed by interactions

between multiple polypeptide chains. Each polypeptide chain is referred to as a subunit. o Proteins with
quaternary structure may consist of more than one of the same type of protein subunit.

o They may also be composed of different subunits.

o Hemoglobin is an example of a protein with quaternary structure

o Hemoglobin, found in the blood, is an iron-containing protein that binds oxygen molecules. It contains
four subunits: two alpha subunits and two beta subunits.

6. What is Marasmus? Explain briefly the relationship of protein to this disease.

✔ Marasmus is a form of malnutrition. It happens when the intake of nutrients and energy is too low
for a person’s needs. It leads to wasting, or the loss of body fat and muscle. A child with marasmus may
not grow as children usually do.

✔ Marasmus is a severe form of protein-energy malnutrition that results when a person does not
consume enough protein and calories. Without these vital nutrients, energy levels become dangerously
low and vital functions begin to stop

✔ The symptoms of marasmus include:

• weight loss

• dehydration

• chronic diarrhea

• stomach shrinkage

7. What is Kwashiorkor? Explain briefly the relationship of protein to this disease. (10 points) ▪
Kwashiorkor occurs in people who have a severe protein deficiency. Children who develop kwashiorkor
are often older than children who develop marasmus. Having a diet that’s mainly carbohydrates can
lead to this condition.

▪ The symptoms of kwashiorkor include:

∙ edema, or puffy or swollen appearance due to fluid retention
∙ bulging of the abdomen
∙ an inability to grow or gain weight
8. Explain how proteins become denatured.
A protein becomes denatured when its normal shape gets deformed because some of the hydrogen
bonds are broken. Weak hydrogen bonds break when too much heat is applied or when they are
exposed to an acid (like citric acid from lemon juice). As proteins deform or unravel parts of structure
that were hidden away get exposed and form bonds with other protein molecules, so they coagulate
(stick together) and becomes insoluble in water.

9. What is meant by isoelectric point? What is its importance?

The isoelectric point is defined as the pH at which no net migration takes place in an electric field, while
the isoionic point is defined as the pH at which there is no net charge on the molecule in a deionized
solution. The isoelectric point is significant in protein purification because it represents the pH where
solubility is typically minimal. Here, the protein isoelectric point signifies where mobility in an electro-
focusing system is zero—and, in turn, the point where the protein will collect.

MODULE 6

1. What amino acid makes Keratin? CYSTEINE

2. What amino acid is responsible for causing Sickle cell anemia? VALINE

3. What is the simplest amino acid? GLYCINE

4. This part of the cell is responsible for protein synthesis. RIBOSOMES

5. Give three functional groups found in the structure of amino acids AMINE, CARBOXYLIC ACID,
HYDROXYL B.

SOLUBILITY TEST

Test the solubility of a pinch of sucrose in 2 ml of each of the following solvents listed below. Indicate
whether soluble or insoluble.

A. Water SOLUBLE

B. 0.1M NaOH SLIGHLTY SOLUBLE

C. 0.1M HCl SOLUBLE

D. Ethyl alcohol SLIGHTLY SOLUBLE

E. Chloroform INSOLUBLE__

B. GENERAL COLOR TEST.

Molisch Test -To 3 separate test tube containing 1 ml each of glucose, sucrose and starch, add 3 drops
of alpha-naphthol solution. Incline the test tube and slowly and carefully add about 1 ml. of conc.
sulfuric acid (CAUTION!) down the side of the tube to form a layer below the sugar solution. Note the
color formed at the junction of the two liquids.
A violet ring is formed at the junction of two layers

2. Anthrone test

To 3 separate tubes containing 1 ml each of glucose, sucrose and starch, add 1 ml of anthrone reagent.
Mix thoroughly by swirling and heat in a bowling water bath for 3 minutes. Cool and observe the color
formed. Blue-green color complex is formed which infers presence of carbohydrate

C. TESTS FOR REDUCING SUGARS

Name of Test Composition of Reagent Carbohydrate Substance or Result

Obtained

Benedict’s Sodium carbonate, Sodium citrate, and Glucose Red precipitate

Test Copper(II)

sulfate pentahydrate Fructose Red or yellow orange

precipitate

Sucrose Color remains blue

Starch Color remains blue

Nylanders Bismuth nitrate, potassium sodium Glucose Black precipitate

Test tartrate and

potassium hydroxide Fructose Black precipitates

Sucrose Clear solution

Starch Clear solution

Barfoed’s Copper acetate in a dilute solution of Glucose Red precipitate

Test acetic acid
Fructose Red precipitate

Sucrose No red color precipitate

 Starch No red color precipitate

CONCLUSION:

When reducing sugars are mixed with Benedict’s reagent, Barfoed’s and Nylander’s, a reduction reaction
causes these reagents to change color. Benedict’s solution produces red precipitate as a positive result
that indicates presence of reducing sugars such as Glucose and fructose. If Benedict’s reagent remains
blue in color, the carbohydrate is non-reducing such as the case for sucrose and starch solution. The
positive test result for Nylander’s reagent is the formation of black precipitate in the presence of
reducing sugar. Nonreducing sugar will not form black precipitate but rather remains a clear solution.
Barfoed’s test is quite similar with Benedict’s reagent, they form red precipitate in the presence of a
reducing sugar.

D. MUCIC ACID TEST FOR GALACTOSE AND LACTOSE

NOTE: Instructor will only assign 2 groups to perform this test.

1. To 1 ml of galactose and lactose solution in separate evaporating dishes, add 5 ml of conc. Nitric acid.
Evaporate on a steam bath under the hood to a volume of 2 ml.
2. Allow to cool cover the evaporating dishes with watch glasses and set aside in your locker until the
next laboratory period.
3. Add 5 ml of distilled water to each dish, mix well a stirring rod, and pour the contents of the dishes
into clean test tubes.
4. Examine the crystals under the microscope.
5. What does the formation of crystals on the tube indicates?
It indicates the presence of galactose and lactose.
6. What does the absence of such crystals indicate?
It means there is no presence of such type of sugar as galactose and lactose because Mucic test is
specific only for galactose and lactose.
7. Write the equation involved in this test.

CH2OH-(CHOH)4-CHO + HNO3 → CH2OH-(CHOH)4-CHO-NO3 + H+

SELIWANOFFs test for Ketone

Place 1 ml. each of solutions of fructose and sucrose in separate test tubes. Add 1 ml of Seliwanoff’s
reagent to each tube and place of time of formation of this color in each tube.

Results: Formation of the cherry red-colored complex.

What is the chemical composition of Seliwadoff’s reagent? The reagent of this test consists of
resorcinol and concentrated HCl.

F. TEST FOR PENTOSES

1. Aniline Acetate Test

Place 3 ml. of arabinose in a test tube. Add 2 ml. of conc. HCI and carefully boil the resulting solution for
about 1 minute. Discontinue heating and hold a small piece of filter paper moistened with aniline
acetate over the mouth of the tube. Do not allow the paper to rest on the mouth of the tube. Hold the
paper, otherwise false results may be obtained.

Results: Bright pink color is produced.

2. Bial’s Test

To 1 ml. of Bial’s reagent in a test tube, add 1 ml. of arabinose and heat the mixture gently until it just
begins to boil. Cool and add about 1 ml. of amyl alcohol.

Results: The presence of a blue-green complex indicates the presence of pentoses in the sample. What
is the chemical composition of Bial’s reagent? It is a solution of orcinol, HCl and ferric chloride.

What are Carbohydrates?

Referred to as “hydrates of carbon” because the general formula of these compounds was (CnH2n)n or
Cn(H2)n. These are compounds which are either polyhydroxy aldehydes or polyhydroxy ketone or a
compound yields either or both of this hydrolysis. These are also product of photosynthesis, where
inorganic carbon dioxide becomes organic carbon with the utilization of solar energy, accompanied by
the release of oxygen gas.

2. Summarize the general characteristics of Carbohydrates.

A carbohydrate is a simple sugar. Its basic structure is composed of the elements carbon, hydrogen and
oxygen, with generally twice the hydrogen as carbon and oxygen. In its simplest form, a carbohydrate is
a chain of sugar molecules called monosaccharides. When these simple sugars are combined, you get
disaccharides, oligosaccharides and polysaccharides

3. Why do many carbohydrates give positive result for Molisch’s test?

Molisch test is a general test for all carbohydrates, either free or bound to proteins or lipids. The
objective of this test is to detect the presence of carbohydrates in a given sample and to distinguish
carbohydrates from other biomolecules.

4. What is the chemical name and formula for the precipitate obtained as positive result in Benedict’s
and Barfoed’s Test?
∙ Barfoed’s test is a chemical test used for detecting the presence of monosaccharides. It is based on
the reduction of cupric (II) acetate to cuprous (I) oxide (Cu2O), which forms a brick-red precipitate.

∙ Benedict’s test is performed by heating the reducing sugar with Benedict‘s reagent. The presence of
the alkaline sodium carbonate converts the sugar into a strong reducing agent called enediols. During
the reduction reaction, the mixture will change its color from blue to brick-red precipitate due to the
formation of cuprous oxide (Cu2O).

MODULE 7. LIPIDS

GUESS THE IDENTITY OF THE SUGAR based on the following test results obtained.

Name of the sugar: SUCROSE

A. Upon addition of alpha-naphthol solution and H2SO4, the solution formed a violet ring at the junction
of two layers.
B. Addition of Nylander’s reagent resulted into turbid solution
C. Color of the solution remains blue with Benedict’s reagent
SOLUBILITY TEST

SOLVENT COTTONSEED OIL VEGETABLE OIL LINSEED OIL

a. Water INSOLUBLE INSOLUBLE INSOLUBLE

b. Dil. HCl INSOLUBLE INSOLUBLE INSOLUBLE

c. Dil. NaOH INSOLUBLE INSOLUBLE INSOLUBLE

d. Ethanol SOLUBLE SOLUBLE SOLUBLE

e. Chloroform SOLUBLE SOLUBLE SOLUBLE

f. Ether SOLUBLE SOLUBLE SOLUBLE

∙ In what solvents is the oil soluble? They are more soluble in organic solvents such as ethanol,
chloroform and ether compared to inorganic solvents like HCl, NaOH and water.
∙ What are these solvents called? These organic solvents can be referred to as NOPOLAR SOLVENTS OR
FAT SOLUBLE solvents.
CONCLUSION:
It is one of the preliminary test that detects the presence of all lipids. Solubility test detects lipid
solubility in various solvents to check whether it is miscible or immiscible in polar or non-polar solvents.
Lipids are very nonpolar and will most likely dissolve in nonpolar solvents such as ether and chloroform.
However, there is an exception when it comes to polar solvent like ethanol, because of its distinct
characteristic to induced polarity to lipids rendering them miscible with each other.
The principle of solubility called “like dissolves like” applies also to Lipids. Hence, lipid substances are
immiscible with polar solvents which usually includes the inorganic compounds. And they are very
miscible with other fat or nonpolar solvents which usually includes organic compounds.
B. TRANSLUSCENT SPOT TEST

SAMPLE OBSERVATION

Coconut oil TRANSLUSCENT

Vegetable oil TRANSLUSCENT

Linseed oil TRANSLUSCENT

CONCLUSION:
A translucent spot test is also a preliminary test for the lipids, which is characterized by a translucent
and greasy spot. The lipid will not wet the filter paper, unlike water. The lipids will form a greasy or
translucent spot due to their greasy texture, and penetrate the filter paper.
C. ACROLEIN TEST
SAMPLE OBSERVATION

Coconut oil A pungent irritating odor of acrolein is produced

Vegetable oil A pungent irritating odor of acrolein is produced

Linseed oil A pungent irritating odor of acrolein is produced

CONCLUSION:
Acrolein test is used to detect the presence of glycerol or fat. When fat is treated strongly in the
presence of a dehydrating agent like potassium bisulphate (KHSO4), the glycerol portion of the
molecule is dehydrated to form an unsaturated aldehyde, acrolein that has a pungent irritating odour.

D. TEST FOR UNSATURATION (HUBL’S TEST)

SAMPLE NO. OF DROPS OF HUBL’S OBSERVATION
SOLUTION

Cottonseed NO DEFINITVE ANSWER The violet color persists indicating that the given fat
oil or oil is SATURATED

Linseed oil NO DEFINITVE ANSWER The color fades indicating that the given fat or oil is
UNSATURATED

CONCLUSION:
This test is used to detect the degree of unsaturation in oil or fat. Huble’s reagent reacts with an
alcoholic solution of iodine that contains some mercuric chloride. During the reaction, the violet colour
of iodine fades away if the oil or fat is unsaturated. If the oil or fat is saturated, the violet colour of
iodine does not fade away.
GUIDE QUESTIONS:
1. What are the general characteristics of Lipids?
The members of the lipids family have characteristic solubility and properties in common
but are rather diverse in their chemical institution, In general lipids are compounds having the following
characteristics.
1. They are insoluble in water but soluble in one or more solvents of low polarity (nonpolar)
2. They are esters of fatty acids
3. They are utilizable by the living organisms
2. What is meant by Iodine number? Give its significance.
The iodine value or iodine number is the mass of iodine in grams that is consumed by 100 grams of a
chemical substance.
Iodine numbers are often used to determine the amount of unsaturation in fats, oils and waxes. In
fatty acids, unsaturation occurs mainly as double bonds which are very reactive towards halogens,
the iodine in this case. Thus, the higher the iodine value, the more unsaturations are present in the
fat.
It can be seen that coconut oil is very saturated, which means it is good for making soap. On the other
hand, linseed oil is highly unsaturated, which makes it a drying oil, well suited for making oil paints.
3. What is the importance of Lipids?
a. As a source of energy - The oxidation of fatty acids was shown to be a major means of metabolic
energy production, and it became clear that their storage in the form of triacylglycerol was more
efficient and quantitatively more important than the storage of carbohydrates as glycogen.
b. As essential component of cellular structure especially cell membranes
4. Define Saponification of fats.
The hydrolysis of fats and oils in the presence of a base is used to make soap and is called
saponification. Ordinary soap is a mixture of the sodium salts of various fatty acids, produced in one of
the oldest organic syntheses practiced by humans
All soap is made from fats and oils, mixed with alkaline (basic) solutions.
Chemical reaction: Fat + Lye → Soap + Glycerol