EXPERIMENT 1: Carbohydrates and Related Compounds

Test Test description Reagents Test positive result Test negative result
Used

Molisch Qualitative test for 1% Reddish Violet / Purple Colorless

Test determining the alpha-napht colored ring at the
presence of hol in junction of the two liquids
carbohydrates in a alcohol
solution
1mL conc.
H2SO4

All carbohydrates yield a

positive result

Anthrone Both qualitative test 1mL Yellow - Blue Green Colorless

Test and quantitative test Anthrone
for carbohydrates Reagent
(Anthrone +
Same principle with conc
methyl furfurals- give H2SO4)
condensation
products

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 All carbohydrates yield a
positive result

Fehling’s Contains CU2+ ions

Test in an aqueous basic
solution

Formation of red Blue color

precipitate

Glucose, Lactose, and Sucrose yields

Fructose yields positive negative result
result

Benedict’s Quantitative test

Test
Contains Cu2+ ions

Red in color Light blue in color

Glucose, Lactose, and Sucrose yields

Fructose yields positive negative result
result

Barfoed’s Similar to Fehling’s

Test test

Different types of
sugars react at
different rates.

It’s reagent is milder

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 than Fehling’s Produces dark red No precipitate
precipitate formation

Reducing Reducing
Monosaccharides react Disaccharides react
quickly with its reagent very slowly or
nothing at all
Glucose and Fructose
(and all other Sucrose does not
monosaccharides give give positive result (it
positive result) is a non-reducing
sugar)

Lactose does not

give positive result
(because it is a
reducing
DISACCHARIDE)

Tollen’s Turns Ag+ to Ag

Test

Formation of silver mirror

Sucrose gives a
Glucose, Lactose, and negative result
Fructose yields positive
result

Nylander’s Turns Bi+3 to

Test metallic Bi

Black solution
Sucrose gives a
Glucose, Lactose, and negative result
Fructose yields positive
result

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Picric Acid Will give mahogany
red color

Dark brown solution Yellow solution

Glucose, Lactose, and Sucrose yields

Fructose yields positive negative result
result

Seliwanoff’ Resorcinol-HCl Test

s Test
Test for Ketose

Ketose + HCl and

Resorcinol =
dehydrated (cherry
red complex) Cherry red complex Sucrose, Glucose,
and Lactose yields a
Only Fructose yields a negative result
positive result because it because they are
is the only sugar (out of aldoses
the four tested) that is a
ketose

Bial’s Test Orcinol-HCl Test Formation of bluish Formation of all other

precipitate colours
Ribose (a pentosan)

Test to determine the Sucrose, Glucose

presence of and Lactose do not
pentoses (5C yield a positive result
sugars) because they do not
have a pentose
Test specific for
pentoses

Ribose (additional sugar

tested) and Fructose are
the only ones to yield a
positive result because
they are the only ones to
have a pentose sugar

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Mucic Acid Oxidation of Sugar
Test to Sugar Acids

A.K.A Galactaric
Acid
Or 1,6- dicarboxylic
Formation of crystals
acid (an “aldaric
acid”)
A test to determine
galactose
Highly specific for
galactose

Obtained by the
Hydrolysis of
Lactose

Galactose is
converted to
saccharic acid due
to oxidation of both
aldehyde and
primary alcohol

Moore’s Action of strong Turns yellow to orange Any other colour

Test alkalies and finally dark brown, formation
liberating the odor of
caramel Sucrose does not
yield a positive result
because it lacks a
free aldehyde or
ketone group and is
non-reducing

Positive: Glucose and

Fructose and Lactose

Because Glucose and

Lactose has a free
aldehyde group and
fructose has a free
ketone group

Or in other words, all are

positive because they

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 are reducing sugars

Phenylhydr The reaction Same as moore’s

azine between three moles test
Reaction/ of phenylhydrazine
Osazone and one mole of
Formation aldose produces a
crystalline product
known as
phenylosazone

Formation of crystals

Same as moore’s test

SUMMARY

● Molisch and Anthrone = all yield positive result

● Sucrose does not yield a positive result in any reducing test, moore’s test and
osazone formation because it is a non-reducing sugar
● Only Glucose and Fructose yields positive result in Barfoed’s because Barfoed’s test
only yields positive result for monosaccharides
● Only Sucrose and Fructose yields a positive result for Seliwanoff’s test because
Seliwanoff’s test only yields positive result on ketoses, or those with a ketone group
● Only ribose yields a positive result in Bial’s test because it is the only sugar that has a
pentose

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 EXPERIMENT 2: Non-Reducing Disaccharides & Polysaccharides

Test Test description Reagents Test positive Test

used result negative
result

Hydrolysis of Common test used 2 mL of Fehlings

Non Reducing to identify and conc. HCI
Disaccharides characterize
and Some carbohydrates 3mL
Polysaccharides Fehling’s
solution
● Fehlings
● Seliwanoff 3mL
Seliwanoff’
s reagent Formation of red
precipitate

Seliwanoff’s test

Cherry red
complex

Fermentation Presence of yeast Yeast Bubble formation No bubble

formation
Decomposition of Glucose
carbohydrates Sucrose Galactose
brought about by the Lactose
action of Starch

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 microorganisms

Products: ethanol
and carbon dioxide
gas

Dialysis Separating Orange with ring Orange

molecules, without ring
semipermeable Glucose and
measure Sucrose (visible Starch
ring)

Iodine reaction Most important test Inulin

on for polysaccharides -Yellow
Polysaccharides since they form
different colors with Dextrin
iodine solution. -Purple

Test for Glycogen

polysaccharides -Reddish-brown

Forms a coordinate Cellulose

complex between -yellow
the helically
polysaccharide chain Starch
and iodine centrally -blue-black
located within the
helix due to
absorption

Reaction of Ability to break down Fehling’s Fehlings

polysaccharides or modify reagent
with Oxidizing polysaccharides by
Agent intruding chemical
change

Formation of red
precipitate

Hydrolysis of Cleavage of Hydrolyze: Fehlings

Polysaccharide glycosidic bonds Conc. HCI
between
monosaccharide Dilute
units resulting in NaOH

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 polysaccharides into
its constituent sugar Fehling’s
molecules reagent

Seliwanoff’
s reagent

Formation of red
precipitate

Seliwanoff’s test

Cherry red
complex

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EXPERIMENT NO. 3: Lipids

Test Test Description Reagents Test Positive Test

Used Result Negative
Result

Miscibility To know if the ● 2 drops Miscible - Immiscible -

substances mix in all of olive capable of being cannot be
proportions forming a oil mixed mixed
homogeneous 5 ml each of
solution the ff: Hot alcohol Water
● Water Ether Cold
● Cold Chloroform Alcohol
alcohol Carbon
● Hot tetrachloride
alcohol
● Ether
● Chlorofo
rm
● Carbon
tetrachlo
ride

Spot Test Physical test for fats. Coconut oil Translucent Spot No
Glycerol translucent
The formation of a Ether Non-volatile spot
translucent spot is Alcohol
caused by the high Coconut oil Volatile
molecular weight Glycerol
fatty acids which are Ether
non-volatile. Alcohol

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Acrolein Test Test for the presence Potassium Pungent Odor
of glycerol bisulfate Burnt Fat Odor

NOTE: Fats and oils Light

when heated with Brown-Brown
some crystals of color
potassium bisulfite
KHSO4 in a test Olive oil
tube; a pungent Glycerol
irritating odor or smell
of acrolein confirms
the presence of fat or
oil.

Test for Rancidity is brought Blue & red Fresh coconut -

Rancidity about by hydrolysis litmus paper neutral
and oxidation. pH: 7 to 8

1st process:
hydrolysis of fat and Rancid coconut
oil which cause the oil - acidic
liberation of free fatty
acids;
Followed by
oxidation of these
fatty acids to volatile
aldehydes and
ketones components

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Emulsification used to detect the Turbid solution/ 2 layers;
of Fats presence of lipids. cloudy white separate

It is the process that Permanent Temporary

stabilizes the water Emulsion Emulsion
and oil emulsion by
using the emulsifying Oil + water + Oil + water
agents. The lipid or egg white
oil in water will
appear as the Oil + water +
supernatant. soap solution

Emulsion - a solution
where two liquids that
normally do not mix
are mixed together
using an emulsifier

Emulsifying agents
a chemical
compound that
permits the mixing of
two or more
immiscible liquids.
Lecithin, soap, garlic,
egg white

Test for Bromine Number - a Bromine Using Iodine Using

Unsaturation measure of degree of solution bromine
(Bromine unsaturation of a Coconut oil -
Number) substance orange Coconut oil
- orange
Unsaturated fatty Cottonseed - color
acids reacts with dark orange
iodine/bromine
Olive oil - light SATURATE
D - no
double
Using Bromine double
carbon
Sunflower oil - bonds
colorless

Peanut oil -
colorless

UNSATURATED
Presence of
carbon double

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 bonds

Test for Lecithin Ethyl alcohol

Lecithin (phosphatidylcholine) After adding
is present in the Conc. HCl ammonium
nerve, brain and molybdate
other tissues of Dilute NaOH
animals, plants. Egg -> Dark Green
yolk contains lecithin. Dilute HNO3 (Glycerophosph
Lecithin has a polar oric acid is
head (ionic) and 2 Ammonium present)
nonpolar molybdate
hydrocarbon tails
(fatty acid units) I2 in KI After adding
which make it an Iodine
excellent emulsifying -> Cloudy
agent. orange
(Choline
present)

Test for the basis for the CHCl3 Cherry Red layer Yellow
Cholesterol qualitative solution
determination of Conc.
Salkowski’s cholesterol H2SO4
Test

Test for the basis for the CHCl3 Blue green

Cholesterol quantitative
determination of Acetic
Liebermann-B cholesterol anhydride
urchard
Reaction for Conc.
Cholesterol H2SO4

Extraction of ether Mass of lipid

Lipids from extracted / mass
Foods of food x 100 =
% lipid extracted

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SUMMARY:
● Miscibility test
○ immiscible/ polar - water, cold alcohol, & hot alcohol
○ miscible/ non-polar - ether, chloroform, & carbon tetrachloride
● Spot test
○ translucent spot/ non-volatile - coconut oil & glycerol
○ no translucent spot/ volatile - ether & alcohol
● Acrolein test
○ black ppt & pungent odor - olive oil & glycerol
● Test for Rancidity
○ Fresh coconut oil - neutral
○ Rancid coconut oil - acidic
● Emulsification of fats
○ Temporary - oil & water
○ Permanent - oil, water, soap/ egg white
● Test for Unsaturated (Bromine number)
○ Coconut oil - orange
○ Cottonseed - dark orange
○ Olive oil - light
● Test for Lecithin
● Test for Cholesterol
○ Salkowski’s test - Reddish Brown solution/ Red solution with yellow glow
○ Liebermann-Burchard test - Blue-green

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 EXPERIMENT 4: Proteins

Test Test description Reagents Test positive Test

used result negative
result

Coagulation of The term 5mL egg

Albumin with denaturation is used albumin
Alcohol more frequently than sol’n
coagulation by
scientific 10 mL 95%
investigators. ethyl
alcohol

Characteristics of Formation of
protein when coagulum
coagulated
- Loss of Alcohol and Egg
solubility in albumin solution
water and do not mix with
dilute salt each other, there
solutions are visible layers
and then the
Addition of heat to coagulum
action of acids,
Coagulation of alkalies, salts, 5mL egg
Albumin by heat alcohol, mechanical albumin
agitation, radiation,
and ultrasonic Heating
vibrations may
denature the protein
and convert it from a
soluble into an
insoluble form

Similar to test
with alcohol,
there is also a
coagulum
present

Precipitation of Egg
Albumin by Albumin
solutions of salts
of heavy metals 10% of the
(Metal Positive following:
Ions)
-mercuric
chloride
sol’n

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 -silver
nitrate sol’n
-lead (II)
acetate
sol’n
-cupric
sulfate
sol’n

Heller’s Test Precipitation with 5mL conc.

Strong Mineral Acids HNO3

2mL egg
albumin
sol’n
Formation of
white ring formed
at the junction of
two liquids

Robert’s Test Precipitation with 1mL egg

Strong Mineral Acids albumin
sol’n

1mL
Robert’s Formation of
reagent white ring formed
at the junction of
two liquids

Tanret’s Test Precipitation with 1mL

Strong Mineral Acids albumin
sol’n

Tanrets’s
reagent

Formation of a
red precipitate

Precipitation with 2mL of

Alkaloidal Albumin
Reagents
5% of
Tannic acid Tannic acid -
and Picric brownish white
Acid color of

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 precipitate

Picric acid -
yellow precipitate

Testing solubility
of the substances

Both precipitates
are insoluble

Biuret’s Test General Test for 3mL egg

(Color Reactions proteins albumin
of Proteins) sol’n
Given by
polypeptides, 10% NaOH
proteoses, peptones,
or where there is the 0.5%
presence of a CuSO4
peptide linkage. Pink-blue-violet
color

In experiment:
egg albumin plus
copper sulfate
solution yielded a
purple lavender
color solution

Ninhydrin Test An excellent general Colorless

(Triketohydrinden test for proteins solution
e Hydrate which is dependent
Reaction) on free NH2 (alpha
amino) group or
-COOH (carboxyl)
group

Important notes:

Ninhydrin
Oxidizing agent which Positive: blue or
leads to the oxidative purple color
determination of
alpha-amino groups
Formation of
Very important for the yellow: presence
detection and the of proline
quantitative analysis of
amino acids

Reacts with primary

amines, however
formation of carbon

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 dioxide is quite diagnostic
for amino acids

Xanthoproteic Indicates the 2 mL egg

presence of amino albumin
acids containing
phenyl group like 5 drops of
tyrosine, tryptophan, conc.
and phenylalanine HNO3 Formation of
yellow to orange
NH4OH color

Yellow -
presence of
phenyl group

Orange color is
due to NH4OH
(alk. pH ionizes
the phenolic
group)

Adamkiewicz Indicates the 2 mL egg

Reaction presence of an albumin Formation
indole group of the of black
amino acid 5 drops of precipitate
tryptophan. glacial
acetic acid
Involves the nitration Formation of
of the benzene ring H2SO4 violet ring
nucleus solution

Reduced Sulfur Test for cysteine, 2 mL of

Test cystine and egg
methionine amino albumin
acids
2 mL of
40% NaOH
solution

1-2 drops Formation of

of lead II) black precipitate
acetate
solution

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Molisch Test Will yield positive 3 mL of
result if the protein egg
compound contains albumin
sugar solution

5 drops of
molisch
reagent
Formation of
H2SO4
purple/violet ring
at the junction
between the 2
solutions

Test for Proteins Biuret

in Various Solution
Substances

Bread Butter
Milk
Gelatin
Peanuts
Flour

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