BLOCK I.

3
PRACTICAL SESSION OF BIOCHEMISTRY

MACRONUTRIENT:
CARBOHYDRATE, PROTEIN, AND LIPID

CONTRIBUTOR:
Prof. Dr.Dra. Sunarti, M.Kes
dr. Yogik Onky Silvana Wijaya, Ph.D
Dr. Dra. Prasetyastuti, Apt.,M.Kes
dr. Arta Farmawati, Ph.D.

Department of Biochemistry
Faculty of Medicine, Public Health, and Nursing
Universitas Gadjah Mada
Yogyakarta
2023

Student’s Book Block I.3 | 103

 Integrated Practical Session Guidelines

1. Students are asked to read Student’s Book Block I.2 which has been uploaded to Gamel
2. Students must do the mini quiz available in Gamel before the practical session according to
the schedule. The rules for doing a mini-quiz:
a. Choose TRUE or FALSE for each statement
b. If your score is 60 or above (≥60), you are allowed to take part in the practical session.
c. If your score is less than (<60), please repeat the mini-quiz until you get a minimum score
of 60 (unlimited repetition), As long as the link is still open, please work on it.
d. If students DO NOT do this Mini Quiz according to the existing schedule. The students
are allowed to follow the practical session according to the schedule and they must do the
assignment according to the on-going practical session topic.

MINI QUIZ
The link for the mini-quiz:
Regular class:
http://ugm.id/MinikuisBiokimReguler
International class:
http://ugm.id/MinikuisBiokimiaInternasional

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 CARBOHYDRATES

I. LEARNING OBJECTIVE
After this practical session, students are expected to be able to:
1. Explain the biomedical importance of carbohydrate
2. Explain the biomedical importance of protein
3. Explain the biomedical importance of lipid

II. INTRODUCTION
Carbohydrates are polyhidroxyaldehide, polyhydroxy ketone, or substances that yield this
compound on hydrolysis. They are widely distributed in plants and animals. In plants, glucose is
synthesized from carbon dioxide and water by photosynthesis and stored as starch or converted to
the cellulose of the plant framework. In animals, carbohydrates present as glucose and glycogen
which are important sources of energy. Many carbohydrates play a specific role in the cell,
including ribose in nucleotide, galactose in certain lipids, and lactose as an important component
of milk.
Functions of carbohydrate:
a. Source of energy for living beings, e.g. glucose
b. Storage form of energy, e.g. glycogen in animal tissue and starch in plants
c. Serve as a structural component, e.g. glycosaminoglycans in humans, cellulose in plants
and chitin in insects
d. Non-digestible carbohydrates like cellulose, serve as dietary fibers
e. Constituent of nucleic acids RNA and DNA, e.g. ribose and deoxyribose sugar
f. Carbohydrates are also involved in detoxification, e.g. glucuronic acid.

Carbohydrates are classified as follows (Figure 1):

a. Monosaccharides are carbohydrates that can’t be hydrolyzed into simpler carbohydrates
without losing their sugar properties.
b. Disaccharides yield two molecules of monosaccharides when hydrolyzed.
c. Oligosaccharides yield three to ten molecules of monosaccharides when hydrolyzed.
d. Polysaccharides yield more than ten molecules of monosaccharides when hydrolyzed.

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 Figure 1. Carbohydrate classification.

MONOSACCHARIDES
Monosaccharides are also called simple sugars. The term sugar is applied to carbohydrates that are
soluble in water and sweet to taste. They consist of a single polyhydroxy aldehyde or ketone unit,
and thus cannot be hydrolyzed into a simpler form. They may be subdivided into two groups as
follows:
a. Depending on the number of carbon atoms: trioses (3 Carbons), tetroses (4 carbons),
pentoses (5 Carbons), hexoses (6 Carbons), and heptoses (7 Carbons).
b. Depending on the functional aldehyde (CHO) or ketone (C=O) group: aldoses (for
aldehyde) and ketoses (for ketone).
Monosaccharides can form several types of isomerism (Figure 2). Here, we discuss a few of the
most biological importance of isomers:
a. Dextro- and Levo-isomerism. If the —OH group on the terminal alcohol carbon is on the
right, the sugar is in the d-isomer, and vice versa.
b. Pyran (a six-membered ring) and furan (a five-membered ring) ring structures. Almost all
glucose (99%) in the solution is in a pyranose form (pyran ring).
c. Epimer is a variation in the configuration of the —OH and —H on carbon atoms 2, 3, and
4 of glucose. The most important epimers of glucose are mannose (epimerized at carbon
2), and galactose (epimerized at carbon 4).

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Figure 2. Different types of Monosaccharides isomerism. (A) Dextro- and Levo-isomerism, (B)
pyran-furan rings, and (C) epimer.

Trioses (3 carbon atoms): Trioses are formed during the metabolic breakdown of hexoses in
muscle metabolism, for example, aldotriose (glyceraldehydes, glycerose) and ketotriose
(dihydroxy acetone). Fischer called the enantiomer with the OH group on the left side of the chiral
carbon the L (levo, left) and the one on the right side of the chiral carbon the D (dextro, right).
Tetroses (4 carbon atoms): One of the tetroses, erythrose, is an intermediate in the hexose
monophosphate shunt for the oxidation of glucose.
Pentoses (5 carbon atoms): The most important of pentoses are D-ribose and D-deoxyribose which
are found in ribonucleic acid (RNA) and deoxyribose nucleic acid (DNA), respectively. Ribose
also forms parts of ATP, coenzyme NAD, and NADP. The other aldo pentoses are D-arabinose
and D-xylose.
Hexoses (6 carbon atoms): The hexoses are the most common of all the carbohydrates. Of several
hexoses, the most important are glucose, galactose, mannose (aldohexose), and fructose
(ketohexose).
Glucose is known commonly as dextrose or grape sugar. It can be prepared by the hydrolysis of
amylum, sucrose, maltose, and lactose. Medically, when the term glucose is used, the D-isomer is
meant to be the biologically active isomer. Glucose contains four chiral centers (numbers 2,3,4,5)
and so has 24 or 16 optical isomers. Glucose is the most important monosaccharide, found in the
bloodstream and tissue fluids. It requires no digestion and can be given intravenously. The
presence of glucose in
Fructose, a ketohexose, is often called levulose or fruit sugar. It can be prepared by the hydrolysis
of sucrose (a disaccharide) and inulin (a polysaccharide). The chemical structure of fructose is
similar to glucose, carbon atom number 3 to 6 is the same. Therefore, osazones produced from
glucose and fructose are identical. Fructosemia is an inherited disease due to a deficiency of the

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enzyme fructose 1-phosphate aldolase. An infant who is suffering from this disease experiences
hypoglycemia, vomiting, and severe malnutrition.
Galactose differs from glucose only in the configuration of the H and OH about a single carbon
atom. Two sugars that differ only in the configuration of about a single atom are called epimers.
D-galactose can be converted to D-glucose in the liver by a specific enzyme called epimerase.
Galactose can be prepared by the hydrolysis of lactose and raffinose. Galactosemia results from
the inability of infants to metabolize galactose because of a deficiency of either the enzyme
galactose 1-phosphate uridyl transferase or the enzyme galactokinase. The galactose concentration
increases in the blood and presence in the urine (galactosuria).
Glycosides are molecules in which a sugar is bound to another hydroxyl group via a glycosidic
bond. The hydroxyl groups can be a non-sugar (aglycon) compound like a derivative of phenol or
an alcohol; or it can be another sugar compound as in cellulose, glycogen, or starch, which consists
of many glucose units. Glycosides are found in many drugs, including derivatives of digitalis and
strophantus such as ouabain, an inhibitor of the Na+-K+ ATPase of cell membranes.
Deoxy sugars are those in which a hydroxyl group attached to the ring structure has been replaced
by a hydrogen atom. Amino sugars are those that contain amino groups including D-glucosamine,
D-galactosamine, and several antibiotics (e.g., erythromycin).
Heptose (7 carbon atoms) includes sedoheptulose which is an intermediate compound of
carbohydrate metabolism.

BIOCHEMISTRY OF THE MONOSACCHARIDES

Reducing properties
Carbohydrates possessing a free or potentially free aldehyde or ketone group have the property of
readily reducing the ions of certain metals such as Cu, Bi, Fe, Hg, etc. The free carbonyl group of
aldehyde and ketone in the alkaline solution is converted to reactive enol and can be oxidized
easily (= reduce other compounds). Frequently used reagents are Benedict (contains CuSO4, Na-
citrate, and Na2CO3) and Fehling (contains CuSO4, K-Na-tartrate, and KOH). Na-citrate and K-
Na-tartrate are used to prevent the precipitation of C (OH)2 or CuCO3. Alkali is used to convert
sugar to enol and reduce Cu++ from the citrate or tartrate-complex compound to Cu+. The Cu+
and OH- ions form yellow-colored CuOH and are converted to red-colored Cu2O precipitation
with heating.
Reduction. The carbonyl group of carbohydrates can be reduced by a variety of reagents such as
Na-amalgam, H2, and Pt, to an alcohol group. Glucose is reduced to sorbitol, mannose to mannitol,
galactose to dulcitol, and fructose to a mixture of sorbitol and mannitol.
The effect of acids. If hexose is mixed with strong acids and heated, a hydroxymethylfurfural will
be produced. If pentose is mixed with strong acids and heated, a furfural will be produced. The
presence of furfural is a basic concept of several experiments such as the Molisch and the
Selliwanoff reaction. Purple colors appear in the Molisch reaction yielded from the condensation
of 4-hydroxymethylfurfural and alpha-naphthol. The Selliwanoff reaction is specific for levulose
such as fructose as well as other ketoses such as ketotrioses and ketopentoses. This reaction is also
positive for sucrose since sucrose is hydrolyzed to fructose and glucose by HCl presence in the
Selliwanoff’s reagent.

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 HC CH HC CH

H O H 2C – H C C - CHO HC C - CHO

O O
Hydroxymethylfurfural Furfural

Figure 3. The structure of hydroxymethylfurfural and furfural.

The effect of alkali. In the alkali solution, aldose and ketose will be converted to reactive enol.
Enol form of glucose, fructose, and mannose is similar.
Formation of osazones. When a solution of a reducing sugar is heated with phenylhydrazine,
yellow crystalline compounds called osazones are formed. Each individual sugar will give rise to
an osazones which is typical for that sugar. Glucose and fructose yield the same osazones because
of similarities of their molecule structures. Maltose and lactose yield maltosazone and lactosazone.
Non-reducing sugars like the disaccharide sucrose cannot form osazone due to the absence of a
free carbonyl (CHO or C = O) group in them.

DISACCHARIDES
Maltose contains 2 molecules of glucose with a C1-4 bond. It can be prepared by the hydrolysis
of amylum using the enzyme amylase/ptyalin or diastase. Hydrolysis of maltose using acids or
maltase yields 2 molecules of glucose which can reduce Benedict's solution since the free carbonyl
group is presence at the C1 atom. Maltose can form osazones and can be fermented.
Sucrose is also known as cane sugar. It is produced commercially from sugar cane and sugar beets.
When sucrose is hydrolyzed using acids or the enzyme sucrase (invertase), it forms a mixture of
glucose and fructose. This 50:50 mixture of glucose and sucrose is called inverted sugar because
it reverses the rotation of polarized light. Sucrose does not possess a free carbonyl group (non-
reducing sugar). The linkage in sucrose is 1,2 glycosidic linkage because it occurs between C1 of
the glucose molecule and C2 of the fructose. Sucrose produces osazones but cannot be fermented.
Lactose commonly known as milk sugar, is present in milk. Hydrolysis of lactose using acids or
enzyme lactase yields glucose and galactose. The linkage in lactose is 1,4 glycosidic linkages
between C1 of the glucose molecule and C4 of the galactose molecule. Lactose is a reducing sugar.
It forms osazones and can be fermented.
Trehalose yields 2 molecules of glucose when hydrolyzed. The linkage in trehalose is 1,1
glycosidic linkages between C1 of the two glucose molecules. Trehalose is a non-reducing sugar.
It cannot form osazones.

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OLIGOSACCHARIDES
Raffinose is a trisaccharide since it consists of 3 molecules of monosaccharides. Hydrolysis of
raffinose yields glucose, fructose, and galactose.

POLYSACCHARIDES

Polysaccharides are polymers of monosaccharides. Polysaccharides have high molecular mass, are
usually insoluble in water, and are tasteless. When starch is added to boiling water, a colloidal
dispersion is formed. Polysaccharides formed from pentoses are called pentosans, and those
formed from hexoses are called hexosans.
Amylum (starch) is a glucose. It is the most important source of carbohydrates and is found in
cereals, potatoes, legumes, and other vegetables. The 2 chief constituents are amylose (15-20%)
which has a non-branching helical structure and amylopectin (80-85%) which consists of a
branched-chain composed of 24-30 glucose residues united by 1,4 linkages in the chains and by
1,6 linkages at the branch points. Starch yields only glucose on hydrolysis by acids but yields
maltose on hydrolysis by the enzyme amylase. Starch gives a characteristic deep blue color with
iodine. Amylose gives blue color with iodine whereas amylopectin gives purple color. These
colors will disappear on heating but appear again on cooling. If alkali is added, the color will
disappear because alkali binds iodium. Amylum is a non-reducing sugar, it cannot form osazones.
When amylum is hydrolyzed, it forms dextrins (amylodextrin, erythrodextrin, achroodextrin), then
maltose, and finally glucose. Erythrodextrin turns red in the presence of iodine but both maltose
and glucose produce no color. Thus, it is possible to follow the hydrolysis of the starch by
observing the changing colors when iodine is added.
Dextran is produced by certain bacteria when they are grown on sucrose. Medically, dextrans are
used as blood extenders to hold water in the bloodstream and help prevent drops in blood volume
and blood pressure.
Dextrins are produced during the hydrolysis of starch. Amylodextrin, erythrodextrin and
achroodextrin give colors of blue, red, and colorless, respectively.
Glycogen is the storage of polysaccharides in the animal body. It is a more highly branched
structure than amylopectin. Glycogen is glucose in which chains of 10-18 glucose residues in 1,4
glycosidic linkage with branching by means of 1,6 glycosidic linkage. Hydrolysis of glycogen with
acid yields glucose, and hydrolysis with amylase yields maltose. Glycogen is a non-reducing sugar,
gives a red color with iodine, cannot form osazones, and can be precipitated by the addition of
saturated ammonium sulfate.
Cellulose consists of a large number of β-glucose units joined together in a chain by 1,4 glycosidic
linkages. It is the chief constituent of the framework of plants. Cellulose cannot be digested by
mammals including humans, since the absence of a hydrolase that attacks the β-linkage. In the gut
of ruminants and other herbivores, there are microorganisms that produce enzyme with the ability
to attack α-linkage, making cellulose available as a major source of calories for the animals.
Inulin is a fructose found in tuber a root of dahlia. In the medical field, it has been used to determine
the rate of glomerular filtration.

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Pentosan is a polymer of pentoses. Gum arabic, an important pentosan, may be hydrolyzed by
boiling with strong hydrochloric acid to yield arabinoses.
Chitin is an important structural polysaccharide and invertebrate. Structurally, it consists of N-
acetyl-D-glucosamine.
Glycoproteins and mucoproteins occur in the mucosal layer, as a fraction of plasma globulin, in
hormones (gonadotropin and thyrotrophin), and in isoagglutinin in the erythrocyte.
Galactans. One of the most important galactans is agar-agar, a product prepared from certain types
of seaweed. The hydrolysis products of agar-agar are D-galactose, L-galactose, and sulphuric
acids.

PROTEIN
Proteins are polymers of amino acids, which are linked to each other with peptide linkage. Amino
acids are the building blocks of proteins. There are only twenty kinds of amino acid as the building
blocks of protein molecules. They are glycine, alanine, valine, leucine, isoleucine, serine,
threonine, tyrosine, phenylalanine, tryptophan, aspartic acid, glutamic acid, lysine, arginine,
histidine, cysteine, methionine, asparagine, proline, and hydroxyproline.
Essential amino acids, also known as indispensable amino acids, are amino acids that humans and
other vertebrates cannot synthesize from metabolic intermediates. These amino acids must be
supplied from an exogenous diet because the human body lacks the metabolic pathways required
to synthesize these amino acids. In nutrition, amino acids are classified as either essential or non-
essential. These classifications resulted from early studies on human nutrition, which showed that
specific amino acids were required for growth or nitrogen balance even when there is an adequate
amount of alternative amino acids. Although variations are possible depending on the metabolic
state of an individual, the common thought is that there are nine essential amino acids, including
phenylalanine, valine, tryptophan, threonine, isoleucine, methionine, histidine, leucine, and lysine.

LIPID
Lipids are important dietary constituents not only because of their high energy value but also
because of the fat-soluble vitamins and the essential fatty acids contained in the fat of natural
foods. The term lipid is applied to a group of natural substances characterized by their insolubility
in water and their solubility in such fat solvents as ether, chloroform, boiling alcohol, and benzene.
They are limited to substances that are utilizable by the animal organism. Individual members of
this group show large individual variations in solubility, but as a class, the lipids are readily
distinguishable from the carbohydrates and the proteins, the other to great groups of naturally
occurring compounds. Chemically, the lipids are either esters of fatty acids or substances capable
of forming such esters. They are very widespread in nature, being found in all vegetable and animal
matter. Some members of this group, such as the phosphatides and sterols, are found in all living
cells where, with the proteins and carbohydrates, they form an essential part of a colloidal complex
of protoplasm. Complex lipids are also found in large quantities in brain and nervous tissues, thus
indicating the important role these substances must play in the living organism. Other lipids, such
as fats and oils, represent the chief form in which excess nutrients are stored in the animal body.
They arise from ingested lipids and from the metabolism of carbohydrates and proteins and are
stored in fat deposits, such as in the subcutaneous connective tissue, in the intermuscular
connective tissue, in the omentum, in the perirenal fat depots, and in the genital fat.

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Combinations of fat and protein (lipoprotein) are important cellular constituents, occurring both
in the cell membrane and in the mitochondria within the cytoplasm, and serving also as the means
of transporting lipids in the blood. Knowledge of lipid biochemistry is important in understanding
many current biomedical areas of interest, e.g., obesity, atherosclerosis, and the role of various
polyunsaturated fatty acids in nutrition and health.

CLASSIFICATION
Simple Lipids
The simple lipids are esters of fatty acids with certain alcohols. They are usually further classified
according to the nature of the alcohols, as follows:
1. Fats and Oils. Esters of fatty acids and glycerol. Oils are fats that are liquid at room
temperature.
2. Waxes. Esters of fatty acids with long-chain aliphatic alcohols or with cyclic alcohols. These
may be subdivided into (1) True waxes; (2) Cholesterol esters; (3) Vitamin A and its carotenol
esters; and (4) Vitamin D esters.
.
Derived Lipids
The derived lipids are substances formed in the hydrolysis of simple or compound lipids which
still retain the properties of this class of compound.
1. Fatty Acids. Saturated and unsaturated acids.
2. Alcohols. Compounds of high molecular eight but not glycerol. These may be classified as
follows:
a. Aliphatic Alcohols such as cetyl, stearyl, and myricyl.
b. Sterols that contain the phenanthrene nucleus (cholesterol, ergosterol, sitosterol,
stigmasterol).
c. Alcohols Containing the B-Ionone Ring. These include vitamin A.
3. Hydrocarbons. Compounds having no carboxyl or alcohol groups, and which cannot be
saponified.
a. Aliphatic Hydrocarbons
b. Carotenoids.
c. Squalene.
4. Lipid soluble vitamins (A, D, E, and K) and hormones

SATURATED FATTY ACIDS, CnH2nO2 or CnH2n+1COOH

The physical properties of the saturated fatty acids depend upon their molecular weights. Whereas
those fatty acids that contain ten carbon atoms or fewer in their molecules are liquids at room
temperature, the remainders are solids whose melting points rise with increasing molecular weight.
The liquid acids are also known as volatile fatty acids, since they may be distilled with steam,
whereas the others, the nonvolatile acids, are carried over by steam distillation only in traces or
not at all. Fatty acids with four carbon atoms or fewer are miscible with water in all proportions.
As the length of the carbon chain increases beyond this, however, the solubility rapidly diminishes
to zero. The common straight-chain saturated fatty acids found in nature as constituents of lipid
molecules are listed in the following table

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Table 1. List of saturated fatty acids.
Common Name Chemical Name Structure Occurrence
Butyric Butanoic CH3(CH2)2COOH Butter fat
Caproic Hexanoic CH3(CH2)4COOH Butter fat, coconut oil
Caprylic Octanoic CH3(CH2)6COOH Butter fat, coconut oil
Capric Decanoic CH3(CH2)8COOH Butter fat, coconut oil
Lauric Dodecanoic CH3(CH2)10COOH Laurel kernel oil, butter fat,
coconut oil
Myristic Tetradecanoic CH3(CH2)12COOH Nutmeg fat, butter oil,
vegetable fats
Palmitic Hexadecanoic CH3(CH2)14COOH Most vegetable and animal
fats
Stearic Octadecanoic CH3(CH2)16COOH Most vegetable and animal
fats
Arachidic Eicosanoic CH3(CH2)18COOH Peanut oil

Behenic Docosanoic CH3(CH2)20COOH Rapeseed oil, peanut oil

Lignoceric Tetracosanoic CH3(CH2)22COOH Cerebrosides, sphingomyelin,
peanut oil

UNSATURATED FATTY ACIDS

The unsaturated fatty acids are characterized by the presence of one or more double bonds in the
molecule. Because of the presence of the double bond, the unsaturated fatty acids are much more
reactive than the saturated fatty acids, the reactivity increasing with an increase in the number of
double bonds. The unsaturated fatty acids are capable of taking up one molecule of water,
oxygen, hydrogen, bromine, or iodine at each double bond, and the amount, of such substance
(e.g., iodine) absorbed by a given weight of acid is used to determine its degree of unsaturation.
It is obvious that a variety of isomerism is possible among the unsaturated fatty acids, depending
not only on the position of the double bond in the chain but also on cis-trans isomerism across a
double bond. Relatively few of the large number of possible isomers of the unsaturated fatty
acids are found in nature.
The chemical characteristics of these representative straight-chain unsaturated fatty acids are
provided in the table below.

Table 2. List of unsaturated fatty acids.

Number of
Common Name Empirical Formula Chemical Name
Double Bonds
Oleic Acid C18H34O2 1 9-Octadecenoic
Linoleic Acid C18H32O2 2 9,12-Octadecadienoic
Linolenic Acid C18H30O2 3 9,12,15-Octadecatrienoic
Arachidonic Acid C20H32O2 4 5,8,11,14-Eicosatetraenoic

BRANCH-CHAIN AND CYCLIC ACID

In addition to the straight-chain fatty acids so far described, a number of branched-chain and
cyclic fatty acids, both saturated and unsaturated, have been isolated from natural sources.

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Tuberculostearic acid (10-methyl stearic acid), a methylated branched chain C26 acid is possibly
3, 13, 19-trimethyltricosanoic acid, although this structure has been disputed. The latter appears
to be associated with some of the clinical manifestations of tuberculosis. It is interesting to note
that these and other fatty acids are found in tubercle wax as esters of the disaccharide trehalose.
In chaulmoogra oil the unsaturated cyclic fatty acids chaulmoogric acid acid and hydnocarpic
acid are found; these acids or their derivatives have been used in the treatment of leprosy.
Branch-chain and cyclic fatty acids
The fats. The fats are neutral esters of glycerol and fatty acids. An example is tristearin,
synthesized in living tissue from one molecule of glycerol and three molecules of stearic acid:
Alcohol + Fatty acid → Fat + water
Melting point
Tristearin, for example, melts containing saturated fatty acids, increase in molecular weight of
the component fatty acids results in a higher melting point; the change from a saturated to an
unsaturated fatty acid usually lowers the melting point.
Mixed glycerol containing a high proportion of unsaturated fatty acids are usually liquid at room
temperature, as indicated above, and are commonly called oils. Many commercial fats are
partially hydrogenated vegetable oils.

Rancid
When fat is allowed to stand for a sufficient length of time in contact with air and moisture,
particularly in the presence of light and heat, certain changes occur and it becomes rancid. Three
types of rancidity have been described; namely, oxidative, ketonic, and hydrolytic. The first is
the most important in the spoilage of fat. Rancid fats are unpalatable and appear to be slightly
toxic for some individuals and destructive to other factors in the food such as carotene and
vitamin A and vitamin E.
Emulsions
Emulsions when oil or liquid fat is shaken with water, it become finely divided and is dispersed
in the water to form what is known as an emulsion. Emulsification with water alone is of course
inefficient and transitory. In the presence of emulsifying agents dispersion of fat globules is more
complete and hence more permanent. Among the important emulsifying agents are soaps,
proteins, and bile salts.
Saponification
C3H5(OOCC5H31)3 + 3NaOH → C3H5(OH)3 + 3C15H31COONa

Formation of Acrolein
The glycerol of the fat is dehydrated and acrylic aldehyde or acrolein is produced. This is the
reaction which takes place:

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III. PRACTICAL STEPS AND PROCEDURES
A. PREPARATION AND PRECAUTIONS
Read the practical guidelines beforehand
Students must wear a laboratory coat
Please be careful when performing experiments with heat and fire.

B. LESSON PLAN

No Learning Activities Time

1. Briefing 10 minutes
Group will be divided into 2 subgroups consisting of 4-5 students. Each
subgroup has to perform all experiments.
The instructor guides and explains the experiment procedures.
2 Practical Session 60 minutes
Students perform the experiment according to the procedure in the
laboratory manual for about 60 minutes.
The instructor guides and supervises students during experiments.
Please discuss with your instructor if there were any difficulties.
3 Discussion 10 minutes
Students discuss the result of their experiment with their groups and
with the instructor. Discussion points may include interpretation of the
results or troubleshooting.
4 Feedback and Closing 10 minutes
Feedback and closing are carried out by the instructor.
5 Cleaning 10 minutes
Please clean your bench after the practical session is over. Ensure there
is no anything left behind before you leave your bench.
TOTAL 100 minutes

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C. PROCEDURE
EXPERIMENTS IN CARBOHYDRATES
Experiment 1 (Benedict test)
Monosaccharides can be oxidized by the effect of alkali in Benedict’s solution. Monosaccharide also
reduces Benedict’s solution. A deep-blue color of the solution appears, and a red cuprous oxide
precipitates.
Procedures

1. Add 5 mL of Benedict reagent and 8 drops of glucose solution in a test tube.

2. Heat using a Bunsen burner until boils.
3. Please try again using other solutions: fructose and pentose solutions.

Experiment 2 (Selliwanof test)

Monosaccharide (fructose) can be reduced by the effect of acid and resorcinol-HCL. It gives a
cherry-red color when there is ketose in the solution. In this test, you try the Selliwanof test for
sucrose and maltose. Which of them have positive or negative reactions? Explain your findings.
How to distinguish sucrose and maltose?
Procedures

1. Add 1 mL fructose solution and 5 mL of Selliwanof reagent in a test tube.

2. Heat using a Bunsen burner until boils.
3. What is the evidence of a positive test?
4. Please try again with glucose and pentose

Experiment 3 (Hydrolysis of gummi arabicum)

Gum Arab will be hydrolyzed by amylase to yield pentose. It has a positive reaction with
Tauber’s test or Benedict’s test.
Procedures
1. Add 1 mL of concentrated HCL and 4 mL solution of gum arab in a test tube.
2. Heat using a Bunsen burner until boils.
3. Cooling down with tap water.
4. After that, add sodium hydroxide to make an alkali (use indicator of litmus paper)
5. Carry out the Tauber’s test AND Benedict’s test.
6. What is the result of hydrolysis of gum Arab?

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Procedures of Tauber test
1. Add 2 mL of Tauber reagent and 2 drops of the solution from experiment 3 in a new test
tube.
2. Boil the solution directly in a few minutes and then cool it down with tap water. A cherry
red appearance will be present if there is pentose in the solution.
3. Please try again with glucose and fructose

EXPERIMENT IN PROTEIN
Experiment 1 (Color reaction for protein: Millon-Nasse Reaction)
Procedure
1. Add 2 mL of dilute protein solution and 1 mL of mercuric sulfate reagent (1% of HgSO4
in 10% of sulphuric acid).
2. Heat the tube until a yellow precipitate occurs
3. Cool down with running tap water.
4. Add 1 mL 1% NaNO2.
5. Heat again; the appearance of a deep red color indicates tyrosine or other 3,5-
unsubstituted phenol.

Experiment 2 (Coagulation of protein: Effect of strong acid and strong base)

Procedure
1. Place 3 mL of concentrated nitric acid in a test tube
2. Add 1 mL of diluted protein solution slowly from a pipette through the tube’s wall.
3. Allow the solution to run down the side of the tube and form a layer over the nitric acid.
Record the results! Note the appearance of a protein precipitate at the zone of contact
between the two fluids.
4. Now mix the contents of the tube thoroughly by careful shaking. Is protein precipitated by
concentrated nitric acid?
5. Repeat the above experiment using concentrated sodium hydroxide. How do these various
reagents differ in their action on proteins?
The formation of a protein precipitate by layering the solution over nitric acid as
described above is frequently used as a test for protein in urine and other fluids (Heller’s
test).

Student’s Book Block I.3 | 117

 Experiment 3 (The Appearance of the nitrogen gas)

Procedure
Place 3 mL of protein solution and a few drops of fresh sodium hypobromine solution, and mix
thoroughly. From this mixed solution, will nitrogen gas leave the solution?
Note: This reaction is also positive for urea and ammonium salts in any substance

EXPERIMENTS IN LIPIDS
Experiment 1 Unsaturated fatty acid (Iodine absorption test)
Procedure
1. Add 10 mL of chloroform in a test tube and 10 drops of Hubl’s iodine solution. The color of
the chloroform will turn pink, because of the free Iodine.
2. Distribute the solution into four test tubes. Label each tube.
3. To each tube different types of oils: coconut oil, peanut oil, sesame oil, or fat animal. Add drop
by drop and mix every time after adding oils into the tube.
4. Count how many drops of the oil are needed to eliminate the pink color. What is the result and
compare the degree of saturation between those oils and fat?

Experiment 2 H2SO4 test (Salkowski)

Procedure
1. Dissolve a few crystals of cholesterol in 2 mL chloroform
2. Add 2 mL of concentrated sulphuric acid. A play of colors from bluish-red to cherry-red
and purple will be formed.

Cholestadiena: cholesterol + chloroform reaction

Sulfonic acid: the cholestadiena + sulfuric acid reaction

Chloroform

Sulphuric acid

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DATA SHEET

Carbohydrate Results
Experiment 1

Experiment 2

Experiment 3

Protein
Experiment 1

Experiment 2

Experiment 3

Lipids
Experiment 1

Experiment 2

ASSESSMENT

1. Students must write a report and submit the report within one week after the practical session.
2. Students are required to take this practicum as a component of formative assessment and a
requirement to participate in a summative assessment at the end of the semester exam.
3. Summative assessment is integrated into the block cumulative exam and end-of-semester exam.

Student’s Book Block I.3 | 119

References
Naik, P. 2012. Essentials of Biochemistry. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd.
Hou Y, Wu G. 2018. Nutritionally Essential Amino Acids. Adv Nutr., 9(6): 849-851.
Hou Y, Yin Y, Wu G. 2015. Dietary essentiality of "nutritionally non-essential amino acids" for animals
and humans. Exp Biol Med (Maywood)., 240(8): 997-1007.
Reeds PJ. 2000. Dispensable and indispensable amino acids for humans. J Nutr., 130(7):1835S-40S.

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FEEDBACK
Practical Learning Feedback Sheet
Instructor Name :
Group Name :
Practicum Title :
Department :
Practicum Date :

-filled by student groups-

Well done aspect From Student: What was the best and most memorable
during the practicum?

From the Instructor: Describe the best and most memorable

observation results during the practicum?

Aspects that can be improve From Student: What should be improved regarding this
practicum?

From the Instructor: Explain what should be improved

regarding this practicum?

Student’s Book Block I.3 | 121

 Follow-up and commitment From Student: How to improve performance in the next
practicum?

From the Instructor: Give suggestions to improve

performance in the next practicum

-filled by the instructor-

Well done aspect Confirmation/clarification of student reflection:

Aspects that can be improve Confirmation/clarification of student reflection:

Follow-up and commitment Confirmation/clarification of student reflection:

122 | Faculty of Medicine, Public Health, and Nursing

Group performance in general Not Satisfactory/Borderline/Satisfactory/

Very Satisfactory*

Notes from the Practicum

Instructor

Student’s Book Block I.3 | 123

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