LABORATORY EXPERIMENTS FOR

CARBOHYDRATES
Background:
Carbohydrates are the most abundant organic molecules in nature.
Carbohydrates are polyhydroxy compounds that are either aldehydes or
ketones that contain only carbon, hydrogen, and oxygen. Carbohydrates
comprise about 60% of our daily caloric intake and are the major source of
metabolic energy. Carbohydrates are categorized as monosaccharaides,
disaccharides and polysaccharides.

Monosaccharaides:
Monosaccharaides (simple sugars) can be classified according to the number
of carbon atoms they contain. Examples of some monosaccharaides are
Glucose and Fructose. All digestible dietary carbohydrates in our diet are
eventually broken down into glucose, and therefore it is also commonly
known as blood sugar. Fructose is found in fruits and fruit juices and in
honey. All monosaccharaides are reducing sugars (i,e, they can be oxidized),
and either contain an aldehyde (aldoses) or a ketone (ketoses) group.

Disaccharides or Oligosaccharides:
Disaccharides contain two monosaccharide units are linked together via a
glycosidic bond. Maltose, lactose, and sucrose are three common
disaccharides. Lactose, also commonly known as milk sugar, is found in
milk and milk products. Lactose contains a glucose and galactose combined
via a glycosidic bond. Sucrose, common table sugar, is composed of glucose
and fructose. Unlike the monosaccharaides, not all disaccharides are
reducing sugars. Of the three common disaccharides Lactose and Maltose
are reducing sugars, while Sucrose is not.

1
Polysaccharides:
Polysaccharides are polymers composed of multiple monosaccharide units.
Amylose, Amylopectin, Glycogen, and Cellulose are all polysaccharides
containing glucose, and are different only in the manner in which glucose
units are attached to each other. Starch composed of the polysaccharides
amylose and amylopectin, is the major form of glucose storage in plants.
Amylose (~20% of starch) is a linear chain of glucose while amylopectin (~
80%) is a branched polymer of glucose. Glycogen is the major form of
glucose storage in animals, and is found in the liver and muscle. Glycogen,
like amylopectin in plants, is a branched polymer of glucose.

Tests for the identification of carbohydrates

1. General test for carbohydrates:

1.1. Molisch’s test:

Principle:
This test is useful for identifying any compound, which can be dehydrated to
furfural or hydroxyl-methyl furfural in the presence of H2SO4. Furfural is
derived from the dehydration of pentoses, while hydroxyl-methyl furfural is
produced from Hexoses. Oligosaccharides and polysaccharides are
hydrolyzed to yield their repeating monomers by the acid. The α-Naphthol
reacts with the cyclic aldehydes to form purple colored condensation
products. Although this test will detect compounds other than carbohydrates
(i.e. glycoproteins), a negative result indicates the absence of carbohydrates.
Molisch’s reagent is a solution of dissolves 10 g α-Naphthol in 100 ml 95%
ethanol.

Precautions:

2
 1- α-Naphthol solution is unstable and should be prepared fresh.
2- The conc. H2SO4 should be added carefully along the sides of the test
tube causing minimal disturbance to the contents of the tube.

Limitations:

In addition to carbohydrates, furfurals as such, some organic acids,

aldehydes and ketones also give this test. Secondly, a concentrated sugar
solution may give a red color instead of purple owing to charring action of
acid.

CHO

H C OH
O
H2SO4 COH
H C OH
-3H2O
H C OH

CH2OH
Furfural
Ribose

CHO

H OH
O
H2SO4 HOC CH2OH
HO H
-3H2O
H OH

H OH
5-Hydroxmethyfurfural
CH2OH

Glucose

3
 OH

O
HOC CH2OH
2
+

5-Hydroxmethyfurfural Alpha-Naphthol

-H2O H3O+

HOC OH
O HOC O
O
H3O+
+ -
-H , -2e

Purple-colored dye

Procedure:
1-To three separate test tubes, add 10 drops of 0.5% solutions of glucose,
sucrose and starch respectively.
2-Dilute each sugar solution with 2 ml of water.
3-Add 2 drops of α-Naphthol solution to each tube and mix.
4-Incline the test tube carefully pours drop wise 1ml conc. H2SO4, using a
dropper, on the inner wall of test tube.

4
 0.5% Glucose 0.5% Sucrose 0.5% Starch
10 drops 10 drops 10 drops
Water 2 ml 2 ml 2 ml

α-naphthol solution 2 drops 2 drops 2 drops

Mix
Con. H2SO4 1 ml 1 ml 1 ml

Observation:
A "violet colored ring or purple ring" appears at the interface between the
two layers indicates a positive test (Tube no. 2). A negative result by this
reaction is a good evidence of the absence of carbohydrates (Tube no. 1).
This reaction may give a green color under violet ring due to the reaction
more of α-Naphthol with Con. H2SO4 (Tube no. 3). Disregard a green color
if it appears.

Tube no. 2 Tube no. 3

Tube no. 1

5
1.2. Anthron test:

Principle:
It is another general test for carbohydrates. Its principle is same as that
Molisch’s test that furfurals and hydroxyl-methyl furfurals gives
condensation products with anthrone that are bluish green in color.

CHO

H OH O
HOC CH2OH
HO H Conc. H2SO4

H OH
5-Hydroxmethyfurfural
H OH

CH2OH O

Glucose

Anthrone

Bluish-green complex

Reagents:

Anthrone reagent: 0.2%(w/v) solution in conc. H2SO4.

6
Procedure:

Add about 2 ml of anthrone reagent to about 0.5-1ml of the test solution in a

test tube and mix thoroughly. Observe whether the color changes to bluish
green. If not, then examine the tubes again after keeping them in boiling
water bath for ten minutes. A blue green color indicates positive test.

2. Reactions of reducing sugars:

General principle:
A reducing sugar is sugar that has free or potentially free aldehyde or ketone
group. Sugars with free aldehyde groups can reduce either cupric ion in
alkaline medium (Fehling’s or Benedict’s reagents) or in acid medium
(Barfoed’s reagents) to produce red or orange colored precipitate of cuprous
ions. They can also reduce silver ions in alkaline medium (Tollen’s reagent)
to produce metallic silver giving a silver mirror. Heating in a boiling water
bath is necessary for these reactions.
If the alkaline copper solution is heated in the absence of reducing sugar, it
forms black precipitate of cupric oxide:
Heat
2Cu(OH)2 Cu2O + 2H2O
Black
In the presence of a reducing sugar, however, the alkaline solution of copper
is reduced to insoluble yellow or red cuprous oxide:
Heat
Reducing sugar + 2Cu(OH)2 Aldonic acid + CuO2 + 2H2O

All of the monosaccharaides are reducing sugar. A few of the disaccharides

including lactose and maltose are reducing sugars, but not sucrose because it
has not free aldehyde or ketone group. None of the polysaccharides, but
hydrolysis of polysaccharides and sucrose makes them reducing agents.

7
2.1. Reduction of Fehling’s test:

- Fehling A: (6.928g of CuSO4. 5H2O dissolved in 100 ml distilled water

containing 2 drops of dilute sulfuric acid, H2SO4).
- Fehling B: (35g sodium potassium tartarate and 12g of NaOH dissolved in
100 ml). Mix equal volumes of A&B Fehling solutions just before use
because mixing causes deterioration with time.

Procedure:

Add 1ml Fehling’s reagent (A&B) to 1ml of the test solution of

carbohydrate. Mix thoroughly and place the test tubes in boiling water bath.
Formation of yellow or red precipitates of cuprous oxide indicates the
presence of reducing sugar.

CuSO4 + 2NaOH Cu(OH)2 + Na2SO4

O CH COOK
OH CH COOK + H2O
Cu(OH)2 + Cu
O CH COONa
HO CH COONa
Deep blue colour (cupper, sodium,
Soudium potassium tartrate potassium tartrate complex

Heat
Cu(OH)2 + reducing sugar Cu2O + H2O

Note:
i) In case of mild reduction, leave the solution to stand for 10-15
minutes and then decant the supernatant. A small amount of red or
yellow precipitates may then be seen adhering to the inner side of
the tube.
ii) Fehling’s test is performed only alkaline solution.

8
 iii) Cuprous oxide is dissolved by ammonia. Hence it is not possible to
detect small quantities of reducing sugars in fluids saturated with
ammonium salts e.g. urine.

2.2. Reduction of Benedict’s test:

Benedict modified Fehling’s solution to produce an improved single reagent

which quite stable. Sodium citrate functions as a chelating agent. It is very
sensitive and even small quantities of reducing sugars (0.1%) yield enough
precipitates.

Reagents:
Dissolve 173g sodium citrate and 100g anhydrous sodium carbonate in 800
ml water by gently heating the contents. Then in a separate beaker dissolve
17.3g copper sulfate in about 100mL distilled water. Pour this solution
slowly, with constant stirring into the carbonate-citrate mixture and make up
to 1 L with distilled water.

Procedure:

Add 0.5-1ml of the test solution of carbohydrate to 2ml of Benedict’s

reagent. Keep the test tubes in boiling water bath for 3-5 minutes when the
content of the tube are cold, observe the formation of green, orange, yellow
or red precipitates which indicates the presence of reducing sugar in the
given solution. If the solution is the same color as the Benedict’s reagent in
water (the control), then there is no oxidation reaction. Record your
observations. Classify each as reducing or non-reducing sugar.

9
The concentration of the sugar affects the color of the test. Blue-sugar:
absent; Green: 0.5% sugar; Yellow: 1% sugar; Orange: 1.5% sugar; Brick
red; 2 % or more sugar.

10
 CuSO4 + Na2CO3 CuCO3 + Na2SO4

CH2 COONa
CH2 COONa
HO C COONa + CuCO3 Cu
HO C COONa + H2O
CH2 COONa
CH2 COONa
Sodium citrate
Complex compound with blue colour

CuCO3 + Reducing sugars Cu2O

Note:
i) This test is especially suitable for the detection of reducing sugar
in urine because it is more specific than Fehling test which also
positive for non-reducing substances such as urates present in
urine.

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2.3. Reduction of Barfoid’s test:

Barfoed’s test is a copper reduction test but unlike Benedict's test, it is

carried out in an acid (pH 4.6) rather than alkaline solution. The Barfoed’s
test contains copper (II) or cupric [Cu+2] ions in slightly acidic solution. The
Cu [II] ion is reduced to Cu [I] oxide, Cu2O. Reducing monosaccharaides
are oxidized by the copper ion in solution to form a carboxylic acid and a
reddish precipitate of copper (I) oxide within three minutes. The reaction
will be negative in the presence of reducing disaccharide sugars as they are
weaker reducing agents and react at a slower rate. A copious amount of
brick-red precipitate indicates a reducing monosaccharide. Some hydrolysis
of disaccharides may lead to trace precipitates (disaccharides generally don't
give any reaction even for ten minutes). The precipitate isn’t nearly as
voluminous as that seen with Benedict’s test and tends to adhere to the walls
of the test tube. So this test is used to detect the presence of reducing
monosaccharaides.

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Reaction mechanism:

CHO
O
H OH
CH3 C O
Cu +
H OH
CH3 C O
HO H
O
H OH
Cupric actate
CH2OH

Glucose
2H2O Heat

COOH

H OH

H OH + 4 CH3COOH + Cu2O

HO H Acetic acid Cuprous oxide

Reddish pecipitate
H OH

CH2OH

Gluconic acid

Reagents:

Dissolve 66.5 g of cupric acetate (acts as a source of Cu++) in 900 ml

distilled water. Boil and add 9 mL of glacial acetic acid (provides the low

13
pH). Cool and make the volume to 1 L with distilled water and filter if
necessary.

Procedure:

Take 2-3 ml of Barfoed’s reagent in a test tube and add 1 ml of the given test
solution of carbohydrate 0.5%(w/v). Keep the test tubes in boiling water
bath for 1-2 min only (over-heating should be avoided). Then allow the
tubes to cool down for a while. Thin red precipitates, at the bottom or sides
of the tube indicates the presence of a reducing monosaccharide.

Characteristics:

By use of the Barfoed’s reagent we can distinguish monosaccharaides from

disaccharides. This test is positive with solutions of all monosaccharaides of
con. 0.1% and above. Disaccharides do not produce any reduction unless
they are present in very high concentration.

2.4. Picric acid test:

Principle:

A number of polynitro derivatives can be reduced under the influence of

reducing sugars. For example, picric acid (2,4,6-trinitrophenol) is reduced to
picraminic acid (2-amino-4, 6-dinitrophenol). It is another test for the
detection of reducing sugars. The reducing sugars react with picric acid to
form a red coloured picraminic acid.

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 OH OH

O2N NO2 O2N NH2

Reduce

NO2 NO2

Picric acid Picramic acid

Reagents:

I. Saturated picric acid: Dissolve 1.3 g picric acid in 100 ml distilled

water, boil and cool.
II. Sodium carbonate (10% solution in distilled water or 1 M).

Procedure:

Add 1ml of the saturated picric acid to 1ml of the 1% carbohydrate solution
followed by 0.5 ml of 10% sodium carbonate. Mix thoroughly and put the
test tube in a boiling water bath. Appearance of red color indicates the
presence of reducing sugar in the solution.

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3. Specific tests for carbohydrates:

3.1. Iodine test for polysaccharides:

This test is performed to distinguish polysaccharides from mono- and

disaccharides.

Principle:

Iodine forms colored adsorption complexes with different polysaccharides.

These complexes are formed due to the adsorption of iodine on the
polysaccharide chains. The intensity of the color depends on the length of
the unbranched or linear chain available for the complex formation. Thus,
amylose, the unbranched helical component of starch gives a deep blue color
and amylopectin, the branched component gives red color because the chains
do not coil effectively. Glycogen, which is also highly branched, gives red
color with iodine. This test is conducted in acidic or neutral solutions.

Reagents:
Iodine solution:
Prepare 2%(w/v) solution of potassium iodide (KI) in water to which add a
few crystals of iodine until the solution assume a deep yellow color.

Starch solution:

Dissolve 1g starches in about 10-20 ml boiling water and make the volume
to 100 ml with saturated sodium chloride solution.

Procedure:

Take 2-3 ml of the test solution of carbohydrate in a test tube and add 1-2
drops of dil. HCl. Mix and then add 1-2 drops of iodine solution. Mix and
observe the color change. Iodine solution gives a blue color with starch that

16
disappears on heating and reappears on cooling. Iodine solution also gives a
brown color with glycogen.

3.2. Osazone formation test:

Principle:

Compounds containing the ketone and aldehyde (-CO, CHOH) group form
crystalline osazone compounds when heated with phenylhydrazine. Osazone
crystals have a characteristic shape under the light microscope and help in
the identification of the sugar type. The reaction is stepwise; first, phenyl
hydrazine reacts with carbonyl group of the sugar to form phenylhydrazone,
which then reacts with two further molecules of phenyl hydrazine to form
the osazone. The time of formation of the crystals, and whether the osazone
is precipitated from hot solution or after cooling, can help in the
identification of the type of carbohydrate. Phenylhydrazine reacts with
carbons #1 and #2 of reducing sugars to form derivatives called osazones.

17
The formation of these distinctive crystalline derivatives is useful for
comparing the structures of sugars. Glucose and fructose react as shown
below:
CHO

H OH

HO H
+ H2N NH
H OH

H OH
Phenylhydrazine
CH2OH
-H2O
Glucose

CH N NH

H OH

HO H
+ H2O
H OH

H OH

CH2OH

Glucose phenylhydrazone

18
As noted here the osazone reaction canceled the configuration of the –H and
–OH on carbon #1 and carbon #2 so, identical osazones are obtained from
D-glucose and D-fructose. This demonstrates that carbons #3 through #6 of
D-glucose and D-fructose molecules are identical. The same osazone is also
obtained from D-mannose. This indicates that carbons #3 through #6 of the

19
D-mannose molecule are the same as those of D-glucose and D-fructose
molecules. In fact, D-mannose differs from D-glucose only in the
configuration of the –H and –OH groups on carbon #2.

Reagents:

Dissolve 2 g of phenylhydrazine in 30 ml distilled water and mix well for

several minutes and filter if necessary. Add 3 g of sodium acetate and mix
thoroughly. Avoid getting phenylhydrazine solution on your hands. It is
absorbed in the skin and is poisonous.

Procedure:

I. Acidify 2 ml of sugar solution with 10 drops of glacial acetic acid.

II. Add 1 ml of phenylhydrazine solution and mix well.
III. Place the test tube in a boiling water bath for 30 minutes for each
shaking period.
IV. Identification of sugars by microscopic appearance of crystalline
osazones

Observation:

Osazones of monosaccharaides (glucose & fructose) are formed on hot after

about 15 minutes, and have the same crystal shape needles under the
microscope.
Osazones of reducing disaccharides (maltose & lactose) are formed after a
longer time (up to 30 minutes) and crystal appears slowly after cooling and
can be distinguished under the microscope as follows:
Lactose gives crystals in the form of a powder puff or tennis ball shaped.
Maltose gives crystals in the form of sunflower petals shaped.

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Carbohydrate Time of formation Solubility in hot
Crystalline structure
osazone (minutes) water
Fructosazone 2 Insoluble Needle shape
Glucosazone 5 Insoluble Needle shape
Galactosazone 20 Insoluble Thorny ball shape
Maltosazone 30-45 Soluble Sunflower shape
Lactosazone 30-45 Soluble Powder puff shape

Thorny ball shaped

Lactosazone

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4. Tests for individual sugars:

4.1. Bial’s test for pentoses

Principle:

Bial’s test is used to distinguish between pentoses and hexoses. When

pentoses are heated with concentrated HCl, furfural is formed which
condenses with orcinol, in the presence of ferric ion, to give a blue-green
color. The reaction is not absolutely specific for pentoses since prolonged of
some hexoses yields hydroxyl methyl furfural depends for boiling time,
which also reacts with orcinol, to give muddy brown-to-gray colored
complexes.

Muddy Brown-
Blue-green to-Gray
Glucose

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 CHO
HO OH
OH H
-3H2O
COH + 3H2O +
HO OH O
HCl
H OH Furfural
CH3
CH2OH
Orcinol

C OH
O

Blue-green colour
complex
H3C

Reagents:
Dissolve 3 g orcinol in 500 mL concentrated HCl, add 2.5 mL of a 10%
solution of ferric chloride hexahydrate (FeCl3), and dilute to one liter with
water; this is approximately 6 M HCl. The reagent is stable for months, but
its yellow color gradually darkens and some precipitate forms; this doesn't
seem to affect its reactivity. The “classical” Bial’s reagent is made with a
liter of concentrated HCl, undiluted with water. It gives a slightly stronger
reaction, and considerably faster (30-60 seconds), but is much less stable
than the recipe we've come up with, and the fumes are much more a problem
with concentrated than with 6 M HCl. The reaction even seems to work,
more slowly and with less intense color, if the final HCl concentration is
only 4 M.

23
Procedure:

I. Add about 2 ml of the sugar (test) solution to 5 ml of Bial reagent, in a

test tube in a boiling water bath, a positive test for pentoses is
formation of a green to blue color (not precipitate) in less than five
minutes.

4.2. Ketoses sugar test:

4.2.1. Seliwanoff’s test:

Principle:
Ketoses (fructose) are dehydrated more rapidly than aldoses (glucose and
lactose) to give furfural derivatives, which then condense with resorcinol, to
form a red complex. Thereby helping to distinguish between aldoses and
ketoses

Reagent:

Dissolve 1 g resorcinol in 330 ml concentrated HCl, dilute to one liter

(approx. 4 M HCl final). This reagent seems to be stable for more than a
year, though we usually make less than the recipe specifies.

Procedure:

I. Add 0.5 ml of the sugar (test) solution, to 2 ml of the Seliwanoff

reagent and mix well.
II. Heat in a boiling water bath is formation of an orange to red color (not
precipitate) within five minutes.

24
 HCl, resorcinol
Red colour
Ketohexoses and disaccharides Heat
composes of ketohexoses

25
 CH2OH CH2OH CH2OH CHO
O O
H OH H+
-3H2O
H H
HO H 5-Hydroxymethyl furfural
Furctose
3H2O

H+ OH

OH
Resocinol (dihydroxy benzene)

HO O O

O
CH2OH

Red-colou complex

4.3. Galactose and lactose sugars test:

4.3.1. Formation of mucic acid:

Principle:
This test is highly specific for galactose, which is either independently
present in solutions or obtained by the hydrolysis of lactose. Galactose is
converted to saccharic acid on heating with HNO3 (a strong oxidizing agent).

26
Mucic acid (galactaric acid), which is formed from galactose sugar due to
the oxidation of both aldehyde and primary alcoholic groups at C1 & C6. It
is the only saccharic acid, which is insoluble in cold water and thus helps in
the identification of galactose.

CHO COOH

H OH H OH

HO H HO H
+ HNO3
HO H HO H

H OH H OH

CH2OH COOH

Galactose Mucic acid

Reagents:
Conc. HNO3
Procedure:
I. Take about 1 g of galactose and 1 g of glucose separately in test tubes.
II. Add 1ml distilled water and 1ml conc. HNO3 to each tube and mix
well.
III. Heat the tubes in a boiling water bath for about 1.30 hr and let the
tubes to stand and cool slowly.
IV. Colorless needle like crystals will indicate the presence of galactose.
Note: Lactose will also give this test.

27
4.4. Test for sucrose:

Sucrose is classified as non-reducing sugar based on its ability to act as a

reducing agent during the Benedict’s test. A reducing agent donates
electrons during a redox reaction and is it oxidized. The aldehyde functional
group is the reducing agent in reducing sugars. Reducing sugars have either
an aldehyde functional group or have a ketone group in an open chain form,
which can be converted into an aldehyde. In fact, sucrose is the most
common non-reducing sugar because its constituent monosaccharaides
(glucose and fructose) linked by a glycosidic bond. Glycosidic bonds form
when the anomeric carbon of one sugar reacts with a hydroxyl group
belonging to a second sugar. Glucose and fructose can give a positive
Benedict’s test.

Reagents:
I. Sucrose solution 1%
II. Conc. HCl
III. Sodium carbonate solution 20%
IV. Litmus paper

Procedure:
I. Add 5 ml of 10% sucrose solution followed by 0.5 ml Conc. HCl and
mix well.
II. Place the tube in boiling water bath for five minutes and let the tube to
cool slowly.
V. Add sodium carbonate solution 20% into the tube to neutralize the
acid by using litmus paper.

28
VI. Do Benedict’s test to test for the presence of reducing
monosaccharaides in the tube test.

4.5. Pinoff’s test:

This test used to identify ketonic hexasaccharide i.e. fructose.

Add about (1ml) of 1% fructose solution in a test tube. Add an equal volume
of a freshly prepared (4% aqueous solution of ammonium molbdate) and
then (1drop) of glacial acetic acid. Heat the boiling the test tube in a boiling
water bath a greenish blue color with (3- 4 min) is obtained.

5. Identification of sugars by paper chromatograph methods:

Principle:

A method for the identification of sugars by differential migration along a

porous medium and the migration is caused by the flow of solvent. Paper
chromatography is an example of liquid-liquid chromatography. In this type
of chromatography separation is due to differential partition of solutes
between two liquid phases. One liquid phase is bound to the porous medium
for example; the water bound in the cellulose paper, this phase is referred to
as, the stationary phase. The other liquid phase, the mobile phase flows
along the porous medium. As the mobile phase flows over the solute
mixture, the individual solutes partition themselves between the aqueous
stationary phase and the organic mobile phase relative to their solubility in
the two phases. The more soluble a solute in the mobile phase, the faster it
will travel along the paper, and conversely, the mobile phase must be a
mixture in which the compounds to be separated are soluble or partially
soluble .In paper chromatography solute or solute mixture is spotted in

29
solution along a base line on a sheet of filter paper (whatman No. 1). The
mobile phase (solvent) is allowed to flow over the spots either ascending the
paper by capillary action or descending the paper by gravity. The separation
is measured in terms of a unit called Rf (relative rates of flow) with respect
to the solvent front. The figure below explains how to calculate this value.

The Rf value of a compound in a particular solvent system is constant under

identical conditions of the experiment, e.g. temperature, pH, etc. Because
most compounds are colorless the spots are visualized after separation by
specific reagent. Spraying the paper or rapidly dipping it in a solution of the
reagent in a volatile solvent applies the location reagent. Viewing under
ultraviolet light is also useful since some compounds, which absorb it
strongly, show up as dark spots against the florescent background of the
paper.

30
Reagents:

❖ Sugars solution (1%) in isopropanol alcohol.

❖ Aniline diphenylamine reagent: Mix 5 volumes of 1% aniline and 5
volumes of 1% diphenylamine in acetone with 1 volume of 85%
phosphoric acid. After spraying the dried chromatograms with this
solution the spots are visualized by heating the paper at 100C for a few
minutes (5-10 minutes).
❖ Sufficient solvent: isopropanol-water-acetic acid (4:1:5 v/v).

Procedure:
1. Place sufficient solvent into the bottom of the tank. Cover the led and
allow the tank to be saturated with the solvent.
2. Take a sheet of whattman NO. 1 chromatography paper (about 9 x 10
cm) and place it on a piece of clean paper on a bench.
3. Draw a fine line with a pencil along the width of the paper and about
1.5cm from the lower edge.
4. Along this line place four equality spaced (about 2cm apart) small
circles with a pencil.
5. Label the paper at the top with the name of each of the sugars and
label the last unknown.
6. Use a fine capillary or teeth pick to place the drops of the solutions of
the sugars, glucose, fructose, maltose, lactose and the mixture.
7. After spotting, dry the paper with hot air dryer for one minute, repeat
this step again.
8. Place the spotted paper in the chromatographic tank and make the
development by using the ascending technique.

31
9. Close the tank with lid, allow the solvent to flow for about 30-45
minutes.
10. Remove the paper and immediately mark the position of the solvent
front with a pencil.
11. After the chromatogram has dried, spray the paper with the locating
reagent.
12. You need to put the paper on the hot plate at low temperature or
expose it to the hot air dryer, until the colored spots appear. The
colors are stable for some weeks if kept in the dark and away from
acid vapors.
13. Circle the position of each spot with pencil.
14. Calculate the Rf value for each spot and also for the spots the mixture
contained
15. By comparing the Rf values of the mixture along with those for the
standards, state what sugars does this mixture contain?

32
 Scheme of unknown carbohydrates

Molisch's test

+Ve -Ve

Carbohydrates No carbohydrates
Iodine test

+Ve -Ve
Blue colour reduction experiments
unknown is starch

+Ve -Ve
reducing sugars non-reducing sugars
glucose, fructose, galactose, maltose and lactose unkown is sucrose
confirmatory test for
sucrose
Barfoed's test

+Ve -Ve
Monosaccharides Disaccharides
Ozazone test
Bial's test

Sunflower shape Powder puff

+Ve -Ve unknown is maltose shape
pentose Monosaccharides unknwon is
sugars glucose, fructose lactose
and galactose
Ozazone test

Needle shape Thorny ball shape

unknown is unknown is galactose
glucose or fructose

Selwanoff's test

+Ve -Ve
red color Unknwon is glucose
unknwon is fructose

33
 COLOR TESTS FOR PROTEINS AND AMINO ACIDS

1. General proteins color tests

1.1. Biuret test:

Principles:

It is a general test used for detecting the presence of proteins and peptides.
Biuret test is specific for proteins – to differentiate between proteins (+Ve)
and amino acids (-Ve). Protein sample treated with copper sulphate (CuSO4)
in an alkaline solution (NaOH) formed a pink-violet colored complex. This
color is due to a reaction between copper ions (Cu++) and peptide bonds
(CO-NH) in alkaline solution (at least two peptide bonds are required for a
positive test). Biuret compound (H2N-CO-NH-CO-NH2) react with CuSO4
in an alkaline solution and give the same color like protein, that is why the
test is called Biuret test. Proteins also contain amide groups. When an amino
group and a carboxyl group join to form a peptide bond, the amino group (-
NH2) becomes an amide group (-NH). Therefore, proteins will also complex
with copper ions at a basic pH. Because this reaction was first observed with
biuret, it is called the biuret reaction, and when this reaction is used to
measure protein concentrations, it is called the Biuret protein assay. Thus, in
this assay you will combine protein samples with Biuret reagent, which
contains copper ions in a basic solution.

34
 H 2N
O C
+ NH3
HN
O C

H 2N

Cu++ OH-

NH2 NH2
O C O C
HN NH
O C C
NH2 O NH2
Cu+2
NH2 O NH2
C
O C
HN NH
O C
O C
NH2 NH2
Biuret complex
pink or violet

Reagents:

I. CuSO4.5H2O solution 1%
II. NaOH solution 10%
III. Casein solution 0.5% (dissolve in 0.1M NaOH)
IV. Albumin solution 0.5% (dissolve in 0.1 M sodium chloride)

35
Procedure:

I. Add 2 ml of protein solution (albumin, casein, gelatin and peptone) in

tube test.
II. Add 5 drops of CuSO4 solution.
III. Add 2 ml of NaOH solution.
IV. Mix well; a violet color is obtained with albumin, casein & gelatin
and a pinkish violet color with peptone.

1.2. Ninhydrin test:

Principles:

Ninhydrin is specific for amino acids and proteins – to differentiate between

carbohydrates (-Ve) and amino acids & proteins (+Ve). Ninhydrin reacts
with α-amino acids (–NH2) in proteins giving a purple colored complex,
except proline and hydroxyl proline gives yellow color than purple color due
to lack of α-amino acids. Ninhydrin (triketohydrindene hydrate) degrades
amino acid into aldehyde (on pH range 4-8), ammonia and CO2 though a

36
series of reactions. Ninhydrin then condenses with ammonia and
hydrindantin to produce an intensely purple color.

37
 O
O
OH
+ H2N CH C OH
OH R Amino acid
O
Ninhydrin

O OH

R CH + NH3 + CO2 +
H
Hydrindantin O

OH

OH
2nd ninhydrin
O

O O

H
N + 3H2O

O O
Purple color

38
 O O

C OH
OH
+

OH HN

O
Ninhydrin Proline

+
N + CO2 + 2H2O

O-
Yellow color

Reagents:

I. Amino acid solution 1 % (tryptophan, tyrosine, glycine and proline).

II. 0.2% freshly ninhydrin solution in acetone.
III. Dilute ammonia solution (approximately 0.1 M).

Procedure:

I. Add 1 ml of amino acid solution in test tube and add 5 drops diluted
ammonia.
II. Add 1 ml of 0.1% aqueous ninhydrin to each tube.
III. Mix well, incubate in boiling water bath for 2-4 minutes, and observe
the colors after standing a few minutes.

39
IV. The color intensity produced is proportional to the amino acid
concentration.

Glycine Tryptophan Proline

1.3. Xanthobrotic’s test:

Principles:

This test is specific to the amino acids containing the benzene ring (aromatic
amino acids). Phenylalanine, tyrosine and tryptophan reacts with conc.
HNO3 at high temperature to form nitro-compounds, which are yellow in
color, it turns to orange color in an alkaline medium. Amino acids tyrosine
and tryptophan contain activated benzene rings, which are easily nitrated to
yellow colored compounds. The aromatic ring of phenylalanine dose not
react with nitric acid despite it contains a benzene ring, but it is not
activated, therefore it will not react.

40
 O O

H 2N CH C OH H2N CH C OH

CH2 CH2

Heat
+ Conc. HNO3

NO2

OH OH

Tyrosine

O
O
H 2N CH C OH
H2N CH C OH
CH2
CH2

Heat
+ Conc. HNO3
HN
HN
Tryptophan

O2N
O

H2N CH C OH

CH2

Heat
+ Conc. HNO3 NO reaction

Phenylalanine

41
Reagents:

I. Amino acid solution 1% (alanine, phenylalanine, and phenylalanine)

and 0.02% tryptophan.
II. Conce. HNO3.
III. 10% NaOH solution.

Procedure:

I. Add 1 ml of 1% alanine, 1% phenylalanine, 1% phenylalanine and 2

ml of 0.02% tryptophan to the separate test tubes.
II. Add 1 ml of concentrated HNO3 to each tube.
III. Incubate all the tubes in boiling water bath for 2 minutes.
IV. Cool. Describe the result in each test tubes, then, add carefully 10%
NaOH and see the deepens of yellow color to orange.

Phenylalanine Tryptophan Tyrosine

42
1.4. Millon’s test:

Principles:

This test is specific for tyrosine, the only amino acid containing a phenol
group, a hydroxyl group attached to benzene ring. The phenol group of
tyrosine is first nitrated by nitric acid in the test solution. Then the nitrated
tyrosine complexes mercury ions in the solution to form a brick-red solution
or precipitate of nitrated tyrosine, in all cases, appearance of red color is
positive test. The coloration is due to the mercury present in Millon’s
reagent.
OH

O C
H2
CH C OH + Conc. HNO3

NH2

Tyrosine -H 2O

OH
N O
O C
H2
CH C OH

NH2

Nitrated tyrosine

+2
Hg

N O
Hg O
O
R O
N R

Red coloured compound

43
Millon’s reagent:

• 15%w/v mercuric sulphate in 6N sulphuric acid.

• Sodium nitrite (NaNO2) solution (5%) [To be freshly prepared].
• 1% tyrosine solution.

Procedure:

I. Add 1 ml of 1% tyrosine solution in test tubes.

II. Add to the tube 2 ml Millon’s reagent and shake it well.
III. Place the test tube in the boiling bath with care, for 10 min.
IV. Cool the contents and add few drops of sodium nitrite solution.
V. A red – pink color appears.

1.5. Sakaguchi’s test:

Principles:

Sakaguchi’s test is a specific test for detection of amino acid containing

gauanidium group [R-NH-C= (NH2)2+-NH2]. In other words it’s a test for
guanidines, i.e arginine. In alkaline solution, arginine react with α-naphthol
and sodium hypobromite/chlorite as an oxidize agent, to form red complexes
as a positive result.

44
 O

H 2N CH C OH
OH
CH2

CH2
+ 2
CH2

NH
Alpha-naphthol
C NH

NH2
3 NaOBr
Arginine NaOH

Br

N O

C NH

(CH2)3

CH COOH
Br
NH2

Red coloured compound

45
Reagents:

• 1% arginine solution.
• 10% NaOH solution.
• 0.1% α-naphthol in 10% ethanol.
• 5% sodium hypobromate (NaOBr) solution.

Procedure:

I. Add 1 ml of the 1% arginine amino acid solution.

II. Add to the tube 2ml of NaOH solution. Mix well
III. Add to the tube 2ml of α-naphthol solution. Mix well
IV. Add to the tube 3 drops of sodium hypobromite solution.
V. Solutions in the test tube get a red color.

+ -

46
1.6. Lead acetate test:

Principles:

This is a specific test for sulfur-containing amino acids such as methionine

and cysteine. The protein solution upon adding a few drops of lead acetate
and heating in an alkaline medium becomes dark. The reaction takes place
also with free amino acids. By heating in an alkaline medium, they produce
sulfur and sodium sulfide:
O
O
H2N CH C OH
H2N CH C OH
CH2 CH2
+ 2NaOH Na2S + H2O
+
SH OH
Cysteine

Lead with soduim hydroxide produce Pb(ONa)2:

(CH3 COONa)3Pb + 2NaOH Pb(ONa)2 + 2CHCOOH

Sodium sulfide reacts with Pb(ONa)2 to form dark lead sulphide sediments:

Na2S + Pb(ONa)2 + H2O PbS + 4NaOH

Reagents:

• 1% cysteine and methionine solutions.

• 30% sodium hydroxide solution.
• 5% lead acetate solution.

Procedure:

I. Add 1 ml of amino acid solution in a test tube.

47
 II. Add 2 drops of 30% sodium hydroxide solution.
III. Add 2 drops of lead acetate.
IV. Mix well and put in a boiling water bath for few minutes.
V. A black deposit is formed with cysteine and methionine.

1.7. Nitroprusside test:

Principles:

It is specific for amino acids or proteins containing sulfur, -SH (in cysteine
& cystine) gives a red-purple color called “Mörner test”.

H2N CH C OH
Na2(CN)5Fe(NO). 2H2O + OH
-
CH2 Red complex
Nitroprusside SH
Cysteine

48
Reagents:

• 1% cysteine solution.
• 5% sodium nitroprusside solution.

Procedure:

I. Add 1 ml of amino acid solution in a test tube.

II. Add 3 drops of 5% sodium nitroprusside solution.
III. Mix well and add few drops of ammonia solution.
IV. A deep red-purple color appears.

1.8. Hopkin’s Cole test:

Principles:

This test is specific for tryptophan, the only amino acid containing an indole
group. The indole ring reacts with glyoxylic acid in the presence of a strong
acid to form a violet cyclic product.

H2N CH C OH
O O
CH2 H2SO4
+ H C CH Violet product

Glyoxylic acid
HN

Tryptophan

49
Preparation Hopkin’s Cole reagent:

Place 10 g of powdered magnesium in a large flask, and add enough

distilled water to cover the metal. Then add slowly 250 ml of a cold,
saturated solution of oxalic acid. The flask and contents must be cooled
under running water during the addition of the acid. When the reaction is
complete, filter off the insoluble magnesium oxalate and make the filtrate
slightly acid with acetic acid. Finally dilute the filtrate to 1 liter with distilled
water.

Procedure:

I. Place the 1% tryptophan solution in a test tube.

II. Add a little of the Hopkin’s Cole reagent (glyoxylic acid).
III. Then pour sulfuric acid down the side of the tube so that it
forms a layer at the bottom.
IV. A reddish-violet ring at the junction of the two liquids
indicates tryptophan or proteins containing this amino acid.

50
 Detection of amino acids
Ninhydrin test

-Ve +Ve
Carbohydrate Purple color
(amino acid/ protein

Biuret test

-Ve +Ve
amino aid Violet color
(Arg, Cys, Tyr,Try and Phe) (protein)

Millon's test

-Ve +Ve
(Arg, Cys, Try and Phe) Red Ppt. (Tyrosine)
Sakaguh's test

+Ve -Ve
Red color (Cys, Try and Phe)
(Arginine) Lead acetate test

+Ve -Ve
Red color (Cysteine) (Try and Phe)
Hopkin-Cole test

+Ve -Ve
Violet ring (Tryptophan) (Phenylalainie)

51
 2. PRECIPITATION REACTIONS OF PROTEINS

2.1. Precipitation by heavy metals

Principle:

Heavy metals (e.g. Hg2+, Pb2+, Cu2+) are high molecular weight cations.
The positive charge of these cations counteracts the negative charge of the
carboxylate group in proteins giving a precipitate.
Reagents:
• 0.5% egg albumin, casein, peptone and gelatin solutions.
• 10% copper sulfite and lead acetate solutions.

Procedure:

I. To 1 ml of protein solution in a test tube, add 1 drop of lead acetate; a

white precipitate is obtained.
II. To 1 ml of protein solution in a test tube, add 1 drop of 10% copper
sulfate; a blue precipitate is obtained.

2.2. Precipitation by alkaloids and acid reagents

Principle:

Alkaloids reagents (e.g. tannate and trichloroacetate) are high molecular

weight anions. The negative charge of these anions counteracts the positive
charge of the amino group in proteins giving a precipitate.
Reagents:
• 0.5% egg albumin, casein, peptone and gelatin solutions.
• 10% tannic acid
• 20% trichloroacetic acid.

52
Procedure:

I. To 1 ml of protein solution in a test tube, add tannic acid drop wise

until a buff precipitate is obtained.

II. − To 1 ml of protein solution in a test tube, add 1 ml of trichloroacetic

acid (TCA); a white precipitate is obtained.
N.B. Precipitation of proteins by heavy metals and alkaloids reagents
indicates the presence of both negative and positive charges and hence the
amphoteric nature of proteins.

OH OH
OH
OH
Protein
molecule
++++ +
O

C O
Positively charged Tannic acid

Protein
molecule
zero charge

Ppt.

53
2.3. Precipitation by denaturation

2.3.1. Denaturation by heat (heat coagulation test):

Principle:

Heat disrupts hydrogen bonds of secondary and tertiary protein structure

while the primary structure remains unaffected. The protein increases in size
due to denaturation and coagulation occurs.

Procedure:

• Put 2 ml of protein solution in a test tube, incline it and heat to

boiling.
• A permanent clotting and coagulation is obtained with albumin only.

2.3.2. Denaturation by acids (Heller’s test):

Principle:

Nitric acid causes denaturation of proteins with the formation of a white

precipitate (this differs from the nitration reaction in “xanthoproteic acid
test”).

Procedure:

• Put 2 ml of concentrated nitric acid in a test tube.

• Incline the tube and slowly add 1 ml protein solution drop wise to
form a layer above the nitric acid layer.
• A white ring is formed at the interface between the 2 layers.

2.4. Fractional precipitation by ammonium sulfate (salting out):

Principle:
Protein molecules contain both hydrophilic and hydrophobic amino acids.

54
In aqueous medium, hydrophobic amino acids form protected areas while
hydrophilic amino acids form hydrogen bonds with surrounding water
molecules (solvation layer). When proteins are present in salt solutions (e.g.
ammonium sulfate), some of the water molecules in the solvation layer are
attracted by salt ions. When salt concentration gradually increases, the
number of water molecules in the solvation layer gradually decreases until
protein molecules coagulate forming a precipitate; this is known as “salting
out”. As different proteins have different compositions of amino acids,
different proteins precipitate at different concentrations of salt solution.

Reagents:

• Saturated ammonium sulfate solution (77 g / 100 ml).

• Egg-white solution.
Procedure:

• To 2 ml of egg-white solution (containing both albumin & globulin),

add an equal volume of saturated ammonium sulfate solution;
globulin is precipitated in the resulting half saturated solution of
ammonium sulfate.
• Separate globulin by centrifugation and recover the clear supernatant.
• Add ammonium sulfate crystals gradually to the clear supernatant
until full saturation occurs; another precipitate (albumin) is obtained.
• Confirm this precipitate by protein tests.

55
N.B. The reason for the precipitation of globulin and albumin at different
ammonium sulfate concentration could be that the solvation layer around
globulin is looser and thinner than that around albumin. Therefore, globulin
needs only half-saturated ammonium sulfate to loose its solvation layer
while albumin looses its solvation layer in a fully saturated ammonium
sulfate solution.

H2O
H2O H2O
H2O H2O
H2O Protein H2O
molecule
H2O + (NH4)2 SO4
H2O
H2O +H2O
H2O H2O
H2O H2O
H2O H2O

Hydrated protein

-H2O

Protein
molecule
-H2O

Dehydrated protein

56
2.3. Picric acid test

To 3 ml of gelatin solution in a test tube, add 2 ml of saturated picric acid

solution, a yellow gelatinous precipitate is formed.

2.4. Separation of amino acids by paper chromatography

Principle:
Chromatography is a common technique for separating chemical substances.
In this experiment, very small volumes of solutions containing amino acids
will be applied (this process is sometimes called “spotting”) at the bottom of
a rectangular piece of filter paper. For ready comparison of each trial, it is
vital that each solution be applied on the same starting line. After the
solutions have been applied, the paper will be rolled into a cylinder and
placed in a beaker that contains a few milliliters of the liquid mobile phase.
For this separation, a solution containing n-propanol, water and acetic acid is
the optimum mobile phase. As soon as the paper is placed in the mobile
phase, the solution (sometimes called the eluting solvent) will begin to rise
up the paper. This phenomenon is called capillary action. A mixture of
unknown amino acids can be separated and identified by means of paper
chromatography. The positions of the amino acids in the chromatogram can
be detected by spraying with ninhydrin, which reacts with amino acids to
yield highly colored products.

Reagents:

• Whatman No. 1
• Solvent (n-propanol, glacial acetic acid and water) (20:5:5).
• Ninhydrin spray (2% solution of ninhydrin in ethanol).

57
 • Four separate test tubes containing respectively: 0.05 M glycine, 0.05
M tyrosine, 0.05 M leucine, and 0.05M aspartic acid in 1.5% HCl, an
unknown containing one to four of the above amino acids at a
concentration of about 0.05 M each in 1.5% HCl.
• Capillary tube, chromatography paper, (12 cm x 22 cm) and 500-ml
beaker.
Procedures:

1. Mix 20 ml of n-propanol solution with 5 ml of glacial acetic acid and

5 ml of water in a clean, 500 cm3 beaker, and cover tightly with a
piece of aluminum foil. This would be used as the solvent for the
experiment.
2. On a clean sheet of chromatography paper with size about 12 cm by
22 cm, mark a light pencil line parallel to the bottom and about 1.5 cm
away. Along this line mark ten light crosses ("x") at intervals of about
2 cm. Label each cross.
3. Using capillary tubes, place a small amount of each appropriate
solution on its two positions along the line on the chromatography
paper. Avoid getting the spot on the paper larger than about 3 mm in
diameter. Let the paper dry for a few minutes in air. Add a second
portion of the unknown to one of its two positions, to make certain
that sufficient quantities of each component of the unknown will be
present for good visual observation when the paper is developed.
4. Roll the paper into a cylindrical form. Staple the ends together in such
a fashion that they do not touch each other. Otherwise the solvent will
flow more rapidly at that point and form an uneven solvent front.

58
5. When the spots on the cylindrical paper are dry (it may be necessary
to place the paper in an oven at about 100Co for a short time), place it
carefully in the beaker of solvent, and cover carefully and tightly with
the aluminum foil. Make sure that the paper does not touch the wall of
the beaker.
6. Let the solvent rise up the paper for at least 1.5 hours. If the time is
shorter, the components may not be sufficiently separated for easy
identification. Remove the paper and place it upside down on the
desktop to dry. When most of the solvent has evaporated, open the
cylinder by tearing it apart where it was stapled and hang it in a fume
cupboard. Spray the paper lightly but completely with a solution of
ninhydrin, and leave the paper in the fume cupboard until the spray
solution is dry.
Hazard warning: The ninhydrin solution should be kept off the body
because it reacts with proteins in the skin to form a rather long-lasting
purple discoloration.
7. The teacher should ensure that student wear laboratory gowns, gloves
and safety spectacles in carrying the experiment.
8. Place the paper in an oven at 100-110Co for about 10 minutes, or until
all the spots have developed.
9. Circle each spot with a pencil, and measure the distance each spot
traveled (use the center of the for spot measurement). Measure the
distance the solvent traveled at each position, and calculate the Rf
values for each amino acid.
10. Determine the composition of the unknown by visual comparison of
spot colors and by comparing the Rf values.

59
 11. Rf = D1/D2; D1= distance traveled by the amino acid, D2= distance
traveled by the solvent.
2.5. Estimation of protein by Lowry’s method

Principle:

The principle behind the Lowry method of determining protein

concentrations lies in the reactivity of the peptide nitrogen[s] with the copper
[II] ions under alkaline conditions and the subsequent reduction of the
Folin-Ciocalteau phosphomolybdic phosphotungstic acid to
heteropolymolybdenum blue by the copper-catalyzed oxidation of aromatic
acids. The Lowry’s method is sensitive to pH changes and therefore the pH
of assay solution should be maintained at 10 - 10.5.

Reagents:

A. 2% Na2CO3 in 0.1 N NaOH

B. 1% NaK Tartrate in H2O

C. 0.5% CuSO4.5 H2O in H2O

D. Reagent I: 48 ml of A, 1 ml of B, 1 ml C

E. Reagent II: 5 ml of 2N Folin-Ciocalteu’s phenol reagent + 6 ml of H2O.

This solution light sensitive so keeps it an amber container. At the protocol
given by Gerhardt et al. (1994), the dilution ratio for the Folin-Ciocalteu’s
phenol reagent is 1:1 resulting in a 1N Folin-Ciocalteu’s phenol reagent.
BSA Standard: 0.2 mg/ ml. (weigh 0.05 g of BSA and add to a 500 ml
volumetric flask containing H2O. Stir well to dissolve and adjust the volume
to 500 ml with H2O. Final concentration of the stock is 100mg/L or
10mg/ml).

60
Procedure:
1. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 ml of BSA working standard in
8 test tubes and make up to 1ml using distilled water (20, 40, 60, 80,
100, 120, 140, 160 μg BSA/ml).
2. Although BSA is a water-soluble protein, it takes time to dissolve it
completely. So, prepare this stock solution and keep it mixed i.e., for
1 hour before starting the experiments.
3. The test tube with 1 ml distilled water serve as blank.
4. Add 4.5 ml of Reagent I and incubate for 10 minutes at room
temperature.
5. After incubation add 0.5 ml of Reagent II and incubate for 30
minutes at room temperature.
6. Measure the absorbance at 750 nm against blank and plot the standard
graph.
7. Estimate the amount of protein present in the given sample from the
standard graph.

Tube ml. of ml. of ml. of ml. of Con. O.D at

No. standard water Reagent I Reagent μg/ml 750 nm
solution added added II
Incubation for 10 min

Incubation for 30 min

added
1 0.1 0.9 4.5 0.5 20
2 0.2 0.8 4.5 0.5 40
3 0.3 0.7 4.5 0.5 60
4 0.4 0.6 4.5 0.5 80
5 0.5 0.5 4.5 0.5 100
6 0.6 0.4 4.5 0.5 120
7 0.7 0.3 4.5 0.5 140
8 0.8 0.2 4.5 0.5 160
9 0.0 1.0 4.5 0.5 0.0
Blank

61
 Detection of unknown protein

Biuret's test

-Ve +Ve
Carbohydrate or Violet color
amino acid solution is protein

Heat coagulation test

No cloudness at top of the Cloudness at

solution (Gasein- Gelatin-Peptone) top part of the
solution
(Albumin)
Reaction to litmus paper

Neutral Alkaline
(Peptone or Gelatin) solution is Gasein

Picric acid test

No yellow gelatinous Ppt. Yellow gelatinous Ppt.

solution is Peptone solution is Gelatin
(Peptone gives pink color with Biuret)

62
 3. LIPIDS

Background:

Lipids are a heterogeneous group of compounds synthesized by organisms

that are present in all biological tissues. These compounds are characterized
as being sparingly soluble in water, but highly soluble in nonpolar solvents
(e.g., chloroform, ether, hexane, etc.).

Classification of lipids:

I. Simple lipids:

They are esters of fatty acids with various alcohols. They include oils and
fats, which are esters of fatty acids with glycerol (i.e. triglycerides).

CH2 OH R1 COOH
+
CH OH
R2 COOH
+
CH2 OH
+
Glycerol R3 COOH
Fatty acids

CH3 (CH2)10 C O CH2

CH3 (CH2)10 C O CH Triglyceride lipid

CH3 (CH2)10 C O CH2

63
II. Complex lipids:

They contain (in addition to fatty acids and alcohols) additional groups as
phosphate (e.g. phospholipids), carbohydrates (e.g. glycolipids) and proteins
(e.g. lipoproteins).

III. Precursor and derived lipids:

They include fatty acids, cholesterol, steroid hormones, fat-soluble vitamins

(vitamin A, D, E & K) and eicosanoids, which include prostaglandins,
leukotrienes and thromboxanes.

H2 H2
H 3C C CH3
C
CH C HC
CH3 H2
CH3

CH3

HO Cholesterol

3.1. Test for solubility of lipids in polar and non-polar solvents:

• Add about 2 ml of the provided oil sample to 5 ml of water in a test

tube and try to mix oil with water.
• By shaking, oil and water mix initially; however, they gradually
separate out to form 2 layers.

64
 • Repeat the experiment using chloroform instead of water.
• Oil dissolves in the organic solvent “chloroform” but not in water
because oils contain long hydrocarbon tails which are hydrophobic in
nature.

3.2. Grease spot test:

In the test, some oil and some water are smeared onto a piece of paper. Some
time later, the water smear would become not translucent. But the smear of
oil would keep translucent for a long time. This is known as the grease spot
test.

65
3.3. Saponification of oils:

Saponification of oils means the hydrolysis of triglycerides in the oil in

presence of an alkaline medium (e.g. KOH) into glycerol and fatty acids
with the production of sodium salt of fatty acids or “soap”.
O

CH3 (CH2)10 C O CH2

O
+ KOH
CH3 (CH2)10 C O CH

CH3 (CH2)10 C O CH2

Triglyceride

CH2 OH R1 COOK
+
CH OH
R2 COOK
+
CH2 OH
+
Glycerol R3 COOK
Potassium soap

Procedure:

• Place 5 ml of the provided oil sample in a 250 ml beaker.

• Add 15 ml of alcoholic KOH solution.
• 0.5N alcoholic potassium hydroxide (alcoholic KOH) (prepared by
dissolving 30 g potassium hydroxide in 20 ml of water and make the
final volume to 1 L using 95 % ethanol. Leave the solution to stand
for 24 h before decanting and filtering the solution.

66
 • Cover the beaker with a watch glass and heat on a boiling.
• Stir the reaction mixture from time to time using a glass rod until a
semi-solid mass is formed.
• Put a small amount of this mass in a test tube containing about 5 ml of
water and shake well; a froth is obtained indicating the presence of
soap.

3.3. Test of cupric acetate for detecting fatty acids:

Principle:

This test is used for detecting saturated and unsaturated fatty acids, fatty acid
reacts with cupric acetate to form cupric salts of fatty acids. The cupric salts
of saturated fatty acids are not dissolved in water or petroleum ether;
therefore, it’s precipitated at the bottom of test tube. The cupric salts of
unsaturated fatty acids are dissolved in petroleum ether with blue-green
color.

Reagents:

• Saturated fatty acid (Stearic acid or Palmitic acid)

• Unsaturated fatty acid (Oleic acid)
• 10% aqueous cupric acetate
• Petroleum ether

67
Procedure:

1. Dissolve small mount of fatty acid in petroleum ether.

2. Add appropriate amount of aqueous cupric acetate solution.
Mix once by inversion (Don’t shake). Leave the tube until the emulsion will
separate into two layers. Observe green precipitate is appear at lower
aqueous layer (cupric acetate layer) where this is happened with saturated
fatty acids while at upper organic layer (petroleum ether layer), blue or green
color is appear when unsaturated fatty acid acetate layer) where this is
happened with saturated fatty acids while at upper organic layer (petroleum
ether layer), blue or green color is appear when unsaturated fatty acid.

3.4. Test for the degree of unsaturation of fatty acids:

Principle:

Fatty acids in animal fats are usually saturated, whereas those in vegetable
oils are generally unsaturated. Halogens (I, Br) will add across the double
bonds and thus the decolorization of an iodine or bromine solution will
indicate the presence of unsaturated fatty acids. Iodine test is used for

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distinguish between saturated and unsaturated fatty acids as well as between
oils and fats.

Procedure:

• Add one drop of Olive oil, one spatula-point of Butter and one
spatula-point of Lecithin to separate dry test tubes, and dissolve the
lipids in about 1 ml. of chloroform.
• Add 1 ml. of chloroform to other test tube to act as blank. By means
of Pasteur pipette add drop wise a solution of bromine in chloroform
until define yellow color is produced, note the number of drops in
each case, and comment on the results.

CH3 (CH2) 7 CH=CH- (CH2) 7 COOH + Br2

Oleic acid Bromine

H H

CH3 (CH2)7 C C (CH2)7 COOH

Br Br

Dibromo-Stearic acid

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3.5. Acrolein test

Principle:
When glycerol is heated with potassium bisulphate or concentrated H 2SO4,
dehydration occurs and aldehyde acrolein formed which has characteristic
odour. This test responds to glycerol free or linked as an ester.

CH2 – OH CH2
Heat
CH – OH KHSO4 or CH + 2H2O
Conc.H2SO4
CH2 – OH CHO

Glycerol Acrolein

Reagents:

1. Test compounds (Oil or fat, Oleic acid)

2. Potassium bisulphate or conc. H2SO4

Procedure:
1. Place 5 drops of test compound in a clean and dry test tube.
2. Add 1 ml of conc. H2SO4 carefully. Or 1.0 g of KHSO4
3. Heat the test tube directly.
4. Note the characteristic pungent odour of acrolein.

3.6. Test of triglycerides

Principle:

This test can be used to distinguish between triglycerides and fatty acids.
Triglycerides are chemically neutral while fatty acids are acidic due to their

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free carboxylic group and can decolorize the alkaline red color of
phenolphthalein.

Reagents:

• 20% Glycerin in chloroform

• 0.5% borax solution
• 1% phenolphthalein in 50% ethanol

Procedure:

• Place 5ml of borax solution (0.5%) in test tube.

• Add appropriate volume of phenolphthalein till red color is appearing.
• Add 20% glycerin drop by drop till red color is disappearing.
• Warm the test tube, the red color will appear again.
• Fatty acids are not responding to this test.

4.6. Tests of cholesterol

4.6.1. Liebermann–Burchard test

Principle:
This test is used in the estimation of blood cholesterol. Cholesterol produces
a characteristic green color when it is mixed with the Liebermann–Burchard
reagent, a mixture of acetic anhydride and sulfuric acid. The change in color
may be gradual, initially pink, then blue-purple, and finally deep green.

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 R
R

H2SO4
-H2O

HO

Cholesterol Deep green product

Reagents:
• 0.5 % cholesterol in chloroform
• Acetic anhydride
• Concentrated H2SO4

Procedure:

• Place 1ml of 0.5 % cholesterol in chloroform in a dry test tube.

• Add 5 drops of acetic anhydride.
• Add 1 drop of conc. H2SO4 and mix.
• Observe the appearance of pink color, which gradually turns into deep
green.

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4.6.2. Salkowski test
Reagents:

• 0.5 % cholesterol in chloroform

• Concentrated H2SO4

Procedure:

• Place 1ml of 0.5 % cholesterol in chloroform in a dry test tube.

• Add 1 ml of conc. H2SO4
• Mix carefully and allow standing and separating two layers.
• Observe the red color of the upper layer (chloroform) and green
fluorescence in the lower layer.

4.6.3. Zak test for cholesterol

This test is used for determination of cholesterol in blood.

Reagents:

• 0.2 g cholesterol in 1ml of conc. acetic acid.

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 • Ferric chloride
• Conc. Acetic acid
• Conc. Sulfuric acid

Procedure:

• Place 0.5 ml of prepared cholesterol solution in a dry test tube.

• Add 2 ml of colored solution (mixture of 10% ferric chloride, Conc.
CH3COOH and Conc. H2SO4)
• Observe appearance deep red which refers to existence of cholesterol

4.7. The Determination of the acid value of a fat

Principle:
During storage, fats may become rancid as a result of peroxide formation at
the double bonds by atmospheric oxygen and hydrolysis by microorganisms
with the liberation of free acid.
The amount of free acid present therefore gives an indication of the age and
quality of the fat. The acid value is the number of milligrams of KOH
required to neutralize the free fatty acid present in 1 g of fat.

Procedure:

• Weigh about 5 ml of fat in a flask.

• Add 25 ml of fat solvent to the flask.
• Add 1 ml of phenolphthalein 1% solution, mix well.
• Titrate with 0.01N KOH. End point is when the faint pink color
persists for 30 seconds.
• Note the volume (V) for KOH required. Calculate the acid value.

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Calculation:

• Molecular weight of KOH is 56

• 1 liter N KOH contains 56 g.
• 1 ml of N KOH contains 56 mg.
• 1 ml of 0.01N KOH contains 0.56 mg
• Acid value = (V X 0.56) / weight of fat used.

4.7. The iodine number of a fat

Principle:
Each carbon-to-carbon double bond of an unsaturated fat under suitable
conditions readily combines with two atoms of halogen (iodine, bromine and
chloride).
The number of grams of iodine absorbed by 100 g of fat, called the "iodine
number", therefore, is a measure of the degree of unsaturation.
Two methods are generally used for iodine number determination:
1) The Wijs method, which uses iodine chloride (ICL).
2) The Hanus method, which uses iodine bromide (IBr).

* The Hanus reagent is more stable, but the Wijs method gives results 2 to 5
percent higher and the iodine numbers are closer to the theoretical values.

Procedure:

1. Pipette 10 ml of each fat sample provided in separate flasks. Label

each flask.
2. Add exactly 25 ml of Hanus iodine solution from a burette to each
flask.

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 3. Set up a blank (separate flask) by adding 10 ml of chloroform to 25 ml
of Hanus iodine solution. Only one blank flask is enough for all the
students.
4. Close the flasks with glass stoppers, mix well by swirling and allow
standing at room temperature for 30 min. In a dark cabinet with
occasional swirling.
5. Add 10 ml of 15% potassium iodide solution to each flask and mix.
6. Add about 50 ml of water, washing down any iodine solution that may
be found on the wall of the flask and the stopper.
7. Titrate the iodine with 0.1N sodium thiosulphate from 50 ml burette
until the color of the solution is pale yellow.
8. Add 2 ml of 1% starch solution as indicator. The solution in the flask
turns blue.
9. Continue the titration until the blue color disappears, mixing well
during the final stages of titration.
10.To ensure complete removal of the iodine, stopper the flask and shake
vigorously. If the blue color returns, continue the titration.
11.Record the volume used for your sample and blank.

Calculation:

𝐁−𝐀 𝐱 𝐍 𝐱 𝟏𝟐𝟔.𝟗 𝐱 𝟏𝟎𝟎

Iodine number =
𝐖 𝐱 𝟏𝟎𝟎

Where A = ml of Na2S2O3 used for blank titration.

B = ml of Na2S2O3 used for sample titration.
N = normality of Na2S2O3 used.

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Where 126.9 is the millequivalent weight of iodine.
W = weight of sample fat used by gram.

Example for calculation:

mEq (1) of Na2S2O3 used for blank titration = ml. of Na2S2O3 X N of

Na2S2O3
mEq (2) of Na2S2O3 used for sample titration = ml. of Na2S2O3 X N of
Na2S2O3
mEq iodine was used in the reaction = mEq (1) – mEq (2) = C
Mg of reacted iodine = C x 126.9 = D
Number of reacted iodine by gram = D / 1000 = g of iodine
𝐠 𝐨𝐟 𝐢𝐨𝐝𝐢𝐧𝐞
Iodine number = x 100
𝐰𝐢𝐞𝐠𝐡𝐭 𝐨𝐟 𝐬𝐚𝐦𝐩𝐥𝐞

77
REFERENCE

Douglas, C. (1983). Selected exercises for the biochemistry laboratory,

Chapter 5. Oxford University Press, New York.
George, R. (1978). Synthetic peptides 1 pettit, New York: Academic Press.

Semishin, V. (1964). Laboratory exercises in general chemistry. Moscow:

Peace publishers.
Switzer, R. L., Clark, J. M. Jr. (1977). Experimental biochemistry (2nd. Ed.),
Freeman and company.
Wilson, K., and Walker, J. M. (1994). Principles and techniques of practical
biochemistry, Chapter 9. Cambridge, UK: Cambridge University Press.

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