BIOLAB Exp 1 To 15 Merged

1
Experiment 1 – Elementary Composition of 3. Mucic Acid Test

Proteins 4. Reduction Test
1. Carbon and Hydrogen a. Fehling’s Test
2. Nitrogen b. Benedict’s Test
3. Sulfur c. Barfoed’s Test
4. Phosphorus 5. Iodine test for Starch and Dextrin
6. Phenylhydrazine Reaction
Experiment 2 – Color Reactions of Proteins
1. Biuret Reaction _____________________________________________________________
2. Xanthoproteic Reaction
3. Millon’s Test Experiment 6 – Glycogen
4. Glyoxylic Acid Reaction A. Preparation of Glycogen
5. Heller’s Ring Test B. Reaction of Glycogen
6. Reduced Sulfur Test
7. Adamkiewicz Reaction Experiment 7 – Lipids
1. Solubility
Experiment 3 – Precipitation Reactions of 2. Reaction towards Indicators
Proteins 3. Formation of Translucent Spot
1. Precipitation by Heat Coagulation 4. Acrolein Formation
2. Precipitation by Organic Solvents 5. Emulsification
3. Precipitation by Salting Out 6. Test for Unsaturation
4. Precipitation by Heavy Metal Ions 7. Glycerol
5. Precipitation by Alkaloidal Reagents
Experiment 8 – Enzymes
Experiment 4 – Test for Carbohydrates I. Oxidases from Fruits
A. Physical Tests II. Catalase
1. Solubility A. Catalase from Potato
2. Dialysis III. Oxidases from Potato
B. Furfural Reactions of Carbohydrates IV. Peroxidase from Potato
1. Molisch Test V. Catalase from Liver
2. Thymol Test
3. Seliwanoff’s Reaction Experiment 9 – Test for Nutrients in Foods
4. Moore’s Test 1. Starch
5. Tollen’s Phloroglucin Reaction 2. Glucose
3. Fats
Experiment 5 – Special Test for Saccharides 4. Proteins
1. Nitro-Chromic Acid Test 5. Mineral Matter
2. Alcoholic Fermentation 6. Carr-Price Test for Vitamin A
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EXPERIMENT 1 ELEMENTARY COMPOSITION OF PROTEINS
PROTEINS o pH at which a particular molecule

-the most abundant macromolecules found within the carries no net electrical charge or is
living cells. electrically neutral in statistical mean
- organic compounds of high molecular weight. ↑ pH > casein = negative charge, stable
-made of alpha amino acids joined by means of peptide
linkage e.g. milk pH 6.6
-the most important of all biological substances. = casein is negative and stable
-the fundamental constituents of the protoplasm of the
cells *CHARRING of casein – presence of Carbon
Charring – to burn (to convert to charcoal)
- One of the building blocks (proteins, *MOISTURE of test tube – presence of Hydrogen
carbohydrates, lipids, nucleic acids) of life Moisture – liquid diffused or condensed in
- Catalysts, structural elements, lubricants, relatively small quantity
cellular communication agents
Charring is a chemical process of
- is known to contain elements carbon, hydrogen, incomplete
oxygen, nitrogen, sulfur, and phosphorus, together with combustion of certain solids when
traces of iron, copper, iodine, manganese, and zinc subjected to high heat.
- composed on hundreds or even thousands of amino
acids linked by peptide linkages (carboxyl group of one Heat distillation removes water
amino acid linked with the amine group of the other) vapor and volatile organic
compounds(syngas) from the matrix.
Most Abundant Elements:
Carbon, Hydrogen, Nitrogen, Oxygen The residual black carbon material
is char, as distinguished from the lighter
1. Carbon and Hydrogen colored ash.
Apparatus: test tube, Bunsen burner, thong
Materials: Casein [C38H57N9O9] By the action of heat, charring removes
Procedure: hydrogen and oxygen from the solid, so
Place a small amount of casein in a test tube. Heat it that the remaining char is composed
gently over a low flame. Observe the two substances primarily of carbon.
formed within the tube.
Charring – removes Hydrogen and
What does the charring of casein and the Oxygen from the solid, so the remaining
formation of moisture in the test tube indicate? char (residue) is composed of carbon
The charring of casein indicates the presence of carbon
and the formation of moisture in the test tube indicates CASEIN (Alpha-Casein) - C38H57N9O9
the presence of hydrogen. Arg-tyr-leu-gly-tyr-leu
CASEIN Chemical Equation: Casein + O2 → C + H2O

- Main protein in milk in
coagulated form SUMMARY
o solid/semisolid form Experiment Title:
- 80% in milk Elementary Composition of
- phosphoprotein of milk Proteins, Test for the Presence of
o protein that is post transitionally Carbon and Hydrogen
modified by the attachment of either a
Material:
single phosphate group, or a complex
Casein (heating it gently)
molecule such as 5’-phospho-DNA
through a phosphate group Positive result:
- Primary source of organic substance - charring of casein (carbon)
- Precipitated from milk by heating with an acid - formation of moisture in the test
- Hydrophobic tube (hydrogen)
- Isolectric point (pI): 4.6
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2. Nitrogen
Apparatus: mortar and pestle, test tube, red litmus
paper
Materials: soda lime [CaHNaO2], casein
[C38H57N9O9]
Procedure:
Mix 1 gram of soda lime and a piece of casein
(mongo bean size) in a mortar and mix. Transfer
the mixture to a dry test tube and heat slowly and
cautiously. Expose a piece of moist red litmus
paper to the vapors.
SODA LIME - CaHNaO2
Sodium Hydroxide (Na(OH) mixture with lime
What gas turned the moistened litmus paper to
blue? Ammonia (NH3), the urine-like odor signifies
the presence of nitrogen
Nitrogen’s presence can be tested in casein through

mixture with soda lime and heat. While heating, there
is a gas inside the test tube. The confirmatory product
is NH3 which turns red litmus paper to blue. The red
litmus paper turns to blue because NH3 is basic.
Casein
– primary source of organic substances
Chemical Equation: NH4(g) + OH2 ↔ NH4 + OH-
Soda Lime (turning red litmus to blue)
– to absorb mixture and gases
– mixture of calcium oxide and sodium hydroxide SUMMARY:
Soda lime, white or grayish white granular mixture of
calcium hydroxide with sodium hydroxide or Experiment Title:
potassium hydroxide. Soda lime absorbs carbon Elementary Composition of
dioxide and water vapour and deteriorates rapidly Proteins, Test for the presence of
unless kept in airtight containers. Medically, soda Nitrogen
lime is used to absorb carbon dioxide in basal
metabolism tests and in rebreathing anesthesia Material:
systems. In gas masks it is an absorbent for toxic Soda lime and Casein (mixing
gases. It is used in laboratories as a drying agent. A them in a mortar and then
highly corrosive poison, soda lime severely damages heating them)
the gastrointestinal tract if swallowed and may cause
death. Confirmatory Product:
Ammonia - NH3 (basic, is
CASEIN (Alpha-Casein) - C38H57N9O9 responsible for turning red
Arg-tyr-leu-gly-tyr-leu litmus paper to blue)
Positive Result:
Pungent, unpleasant, urine-like
odor due to the presence of
nitrogen
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Apparatus: porcelain crucible, pestle (anything to 3. Sulfur

mix them), tripod, wire gauze, Bunsen burner, Apparatus: test tube, tripod, wire gauze, Bunsen
thong, filter paper, funnel, test tubes burner, thong, dropper, blue litmus paper
Materials: Fusion mixture (Sodium Carbonate Materials: Fusion mixture (Sodium Carbonate
[Na2CO3] and potassium nitrate [KNO3]), water [Na2CO3] and potassium nitrate [KNO3]),
[H2O] Hydrogen Chloride / Hydrochloric Acid [HCl],
Procedure: Barium chloride [BaCl2]
FUSION: Perform the tests for sulfur and Procedure:
phosphorus on the fused mixture prepared as Add dilute HCl to the first portion of the above
follows: Place about 2 grams of solid fusion filtrate until acidic. Heat the solution to boiling and
mixture (2 parts Na2CO3: 1 part KNO3) in a add several drops of 0.1 N BaCl2 solutions.
porcelain crucible. Add a small amount of
powdered casein and mix thoroughly. Heat slowly Explain: The solution was cloudy and then as it
at the start and the strongly, until a clear mixture is rests it became clearer gradually.
formed. Cool and dissolve with a small amount of
warm water and filter. Divide the filtrate into two Write the equation involved:
parts BaCl2 + H2SO4 → BaSO4 + 2HCl
Fusion: The presence of sulfur was tested by using the filtrate

- The procedure that changes the compounds from the fusion method and HCl. It took 17 drops of HCl
into ions because ions are more readily active until it was made acidic. (Blue litmus paper turns red
when tested under acidic conditions.) It heated to boiling. BaCl2 was
- PURPOSE: Used to oxidize sulfur to sulfate then added. A white precipitate was then formed. This
and phosphorus to phosphate is the BaSO4 or barium sulfate.
- mix casein and fusion mixture, heat in a
porcelain crucible (substance turns black HCl BaCl2
then white) and cool -reagent for S test -for precipitation
- dissolve a small amount in hot water and -to acidify -reagent for S test
filter
Barium reacts with hydrochloric acid and form a
barium chloride:
Ba (s) + 2 HCl (aq) → BaCl2 (aq) + H2 (g)
BaCl2 + H2SO4 → BaSO4 + 2HCl

With H2SO4: Barium Chloride reacts with Sulfuric Acid
[H2SO4] to produce a white insoluble precipitate of
barium sulphate (BaSO4).
HCl acid does not play any active role here. Barium
2 parts Sodium Carbonate + 1 part Potassium Nitrate
chloride does not react with hydrochloric acid, since
both possess the same negative radical (Cl-). BaSO4 is
insoluble in HCl.
But why is HCl used in precipitating BaSO4 but not

H2SO4? Barium salts can react with water to make
Ba(OH)2, which is not highly soluble, and may remove
some of the Ba2+ ions before they are precipitated with
SO4. HCl can be added to prevent this by removing any
Ba(OH)2 and changing it back to BaCl2. The addition of an
excess of Na2SO4 will ensure complete precipitation of
Divided into 2 parts for the detection of Sulfur and BaSO4. The residual HCl can be evaporated off, whereas
Phosphorus H2SO4 would not evaporate.
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Solid Fusion Mixture ammonium phospo-molybdate. This precipitate is

Sodium Carbonate and Potassium Nitrate extremely insoluble in nitric acid.
Na2CO3 and KNO3
Hydrogen Chloride / Hydrochloric Acid - HCl HNO3 (NH4)2MoO4
0.1 N Barium chloride - BaCl2 -reagent for P test -for precipitation
-to acidify -reagent for P test
Barium Sulfate - BaSO4
When lipids containing phosphate groups in their
Chemical Equation: (not sure which one!) structures are added to a strong acid solution, the lipid
BaCl2 + H2SO4 → BaSO4 + 2HCl hydrolyses, producing free phosphate.
Ba2+ + SO42- → BaSO4
BaCl2 + SO42- → BaSO4 + 2 Cl- Purpose of test: To determine the presence of phosphate
group
Cl- and Na+ are spectator ions because
they do not participate in the reaction! Purpose of the reagents: To break the bonds in order to
reveal the free phosphate group
SUMMARY
Compound Responsible: Phosphate
Experiment Title:
The precipitation is usually used to identify phosphate.
Elementary Composition of Proteins,
The precipitation is the formation of yellow ammonium
Test for the presence of Sulfur
phosphomolybdate from ammonium molybdate in acidic
Materials: solution.
Reactant – Fusion mixture/filtrate
Reagents – Hydrochloric Acid (HCl) Solid Fusion Mixture
and Barium Chloride (BaCl2) Sodium Carbonate and Potassium Nitrate
Positive Result: Na2CO3 and KNO3
White precipitate – BaSO4 (Barium conc. Nitric acid - HNO3
Sulfate)
Ammonium Molybdate - (NH4)2MoO4
Ammonium Phosphomolybdate
(NH4)3PO4(MoO3)12
4. Phosphorus Chemical Equation:

Apparatus: test tube, tripod, wire gauze, Bunsen burner, PO43- + 3NH4 + 12MoO42- + 24H+ →
thong, dropper, blue litmus paper (NH4)3PO4(MoO3)12 + 12H2O
Materials: Fusion mixture (Sodium Carbonate [Na2CO3]
and potassium nitrate [KNO3]), conc. Nitric acid [HNO3], SUMMARY
ammonium molybdate [(NH4)2MoO4] Experiment Title:
Procedure: Elementary
Take a second portion of the filtrate and add Composition of
concentrated nitric acid drop wise until acidic. Add a Proteins, Test for
few drops of ammonium molybdate solution and heat the presence of
nearly to boiling. Phosphorus
Materials:
Q1. What is the precipitate formed? Ammonium Reactant – Fusion
phosphomolybdate (NH4)3PO4*12MoO3) mixture / filtrate
Reagents – conc.
Q2. Write the equation involved: Nitric acid (HNO3)
PO43- + 3NH4 + 12MoO42- + 24H+ → and Ammonium
(NH4)3PO4(MoO3)12 + 12H2O Molybdate
((NH4)2MoO4)
The presence of phosphorus was tested using the filtrate Positive Result:
from the fusion method and nitric acid. Nitric acid was Yellow crystalline ppt – ammonium
added until acidic. (Blue litmus paper turns red under acidic phosphomolybdate (NH4)3PO4(MoO3)12
conditions.) After that, ammonium molybdate was added
then heated. A yellow precipitate was formed. This is the
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EXPERIMENT 2 COLOR REACTION OF PROTEINS
Qualitative color reactions have been devised for the

detection of proteins. Due to the complexity of the protein
molecule and the difficulty of obtaining a single pure
protein compound, these tests are used for specific
chemical groups of the component amino acids. Since no
one test is absolutely specific for proteins, it is necessary to
apply several tests.
Proteins give various color reactions which is not specific

for protein molecules, but for certain groups of the
component amino acid.
The biuret test, also known as Piotrowski's test, is
1. Biuret Reaction a chemical test used for detecting the presence of peptide
bonds. In the presence of peptides,
Biuret test is a general test for proteins. A positive a copper(II) ion forms mauve-colored coordination
reaction is obtained with all native proteins and most of complexes in an alkaline solution. The copper(II) binds
the derived proteins. A violet color is obtained with long with nitrogens present in the peptides of proteins.
chain proteins while pink color is obtained with shorter
chain. This is due to the carrying amount of peptide The biuret reaction can be used to assess
linkage. Any compound containing two carbonyl groups the concentration of proteins because peptide bonds occur
(-CONH2) joined together through a single nitrogen or with the same frequency per amino acid in the peptide.
carbon atom will also give a positive test. The intensity of the color, and hence the absorption at
540 nm, is directly proportional to the protein
Alkaline solution of protein treated with CuSO 4 solution concentration, according to the Beer–Lambert law.
results in the production of a rose ink to violet
coloration. This is due to the presence of peptide linkage In alkaline medium copper
(-CONH). All substances therefore containing this from CuSO4 will form a
linkage respond positively to this test. coordination complex with the
peptide bond. A minimum of 3
The smaller peptides and amino acids show no peptides are required to
coloration. This test serves as a good index for answer this test. Biuret test is
determining the extend of protein hydrolysis. Their not performed on urine
hydrolytic products give colors ranging from violet to because of presence of peptide
pink depending upon the extent to which hydrolysis has like linkage.
progressed.
Apparatus: test tube, dropper SUMMARY

Materials: Experiment Title:
 2% albumin solution BIURET REACTION
 10% Sodium Hydroxide [NaOH] – Color Reactions of
 0.5% Copper Sulfate [CuSO4] Proteins
 2% peptone solution Materials:
Procedure: Reactants:
Mix 1 ml of 2% albumin solution and 1 ml of 10% NaOH. - albumin – dark violet
Add 0.5% CuSO4 drop by drop mixing thoroughly after - peptone – pink violet
each addition until a pink or violet color is obtained. Reagents:
Observation: Dark violet color - NaOH (Sodium
Hydroxide)
What chemical structure in the protein molecule is - CuSO4 (Copper Sulfate)
responsible for a positive test? Two peptide bond Positive Result:
structures. - dark violet / pink violet
Will simple amino acids give positive biuret test? No, solution color
because they don’t have any or enough peptide bonds. (↑ darker color = ↑ number
Repeat the test using 2 ml of 2% peptone solution of peptide linkages)
instead of albumin. What did you observe? It turned
into a light violet, pinkish color
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2. Xanthroproteic Reaction due to nitration of certain amino acids, most common

examples being tyrosine and tryptophan. This chemical
This reaction is due to the presence of phenyl group (- reaction is a qualitative test, determining the presence or
C6H5) in the protein molecules. It involves the nitration absence of proteins.
(chemical process for the introduction of a nitro group
into an organic chemical compound) of the phenyl rings In real life, when you drop nitric acid on your skin or nails,
present in the aromatic amino acids such as tyrosine, it turns yellow after some time, indicating the presence of
phenylalanine, and tryptophan, forming yellow nitro- protein. The finger nails show a bright yellow color (finger
substitution products that turn orange when alkali is nails are made up of keratin- a protein) which cannot be
added (salt formation). scraped off, unlike the yellow coloration on the skin which
can be peeled off.
Proteins, when treated with conc. HNO3 turn yellow and
change to orange when neutralized with an alkali This test is answered by aromatic amino acids like tyrosine
(NH4OH). This is due to the nitration of the benzene ring and tryptophan. Phenyl alanine gives a weak positive
(-C6H5) present in the structure of such amino acids reaction. When a preotein solution is heated with
such as tyrosine, tryptophan, and phenylalanine. (the concentrated nitric acid, the benzene ring under goes
yellow stain on the skin produced by HNO3 is due to this nitration to form yellow nitro derivatives. When treated
reaction) with NaOH the sodium salt formed is tense orange in color.
• Principle: Nitric acid gives color when heated

Apparatus: test tube, thong, Bunsen burner with proteins containing tyrosine (yellow color)
Materials: or tryptophan (orange color); the color is due to
 2% albumin nitration.
 conc. Nitric acid [HNO3] • Purpose: used to identify the presence of an
 ammonium hydroxide [NH4OH or H5NO] activated benzene ring.
Procedure:
Place 1 ml of 2% albumin in a test tube and add 0.5 ml
of conc. Nitric acid, and heat. Cool and add ammonium
hydroxide in excess.
Observation: Mixing 2% albumin with conc. HNO3
turned the solution into yellow. But when heated, cooled
and then NH4OH was added, it turned orange.
SUMMARY
Experiment Title:
Possible Questions: XANTHOPROTEIC REACTION
 What kind of amino acids will be detected? – Color Reaction of Proteins
AROMATIC AMINO ACIDS
 Process of introducing a nitro group into an Materials:
organic chemical compound? NITRATION Reactant:
 What alkali is used? AMMONIUM HYDROXIDE - albumin
Reagents:
- Conc. Nitric Acid
The xanthoproteic reaction is a method that can be used - ammonium hydroxide
to determine the amount of protein soluble in a solution,
using concentrated nitric acid. The test gives a positive Positive Result:
result in amino acids carrying aromatic groups, especially - yellow color
in the presence of tyrosine. If the test is positive the proof - when added with an alkali, it turns into
is neutralized with an alkali, turning dark yellow. The an orange color
yellow color is due to xanthoproteic acid which is formed
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3. Millon’s Test
• Principle: Millon's reagent (Hg/HNO3) gives
Millon’s reagent is prepared by mixing one part by positive results ( pink to dark-red color) with
weight of mercury with two parts of weight by conc. proteins containing the phenolic amino acid
Nitric acid and dilute the resulting solution with two “tyrosine”
volumes of water. • Purpose: To detect the amino acid that have
phenol group of tyrosin.
The Millon’s reagent is made by dissolving mercury in
HNO3; hence it consists of mercurous and mercuric
nitrates. When added to a protein solution, the protein
is precipitated as mercury salt. On heating, the
precipitate turns flesh to red color if proteins containing
tyrosine is present. This reaction is due to the phenol
group contained in tyrosine. Any substance therefore
containing this group will give a positive Millon’s test.
Apparatus: test tube, dropper, Bunsen burner, thong

Materials:
• 2% albumin solution
• Millon’s reagent
Procedure:
Place 1 ml of 2% albumin solution in a test tube and 1
drop of Millon’s reagent. Mix and heat
What did you observe? There is a flocculent red

precipitate
What is responsible for this change? Positive results

with proteins
SUMMARY
What amino acid gives a positive Millon’s test?
Experiment Title:
Tyrosine
MILLON’S TEST
– Color Reactions of Proteins
Write its formula. C9H11NO3
Materials:
Reactant:
Millon's reagent is an analytical reagent used to detect
- albumin
the presence of soluble proteins. A few drops of the
Reagent:
reagent are added to the test solution, which is then heated
- Millon’s Reagent – one part of
gently. A reddish-brown coloration or precipitate indicates
mercury with 2 parts of conc. Nitric
the presence of tyrosine residue which occur in nearly all
acid and diluting the resulting
proteins.
solution with 2 volumes of water.
Millon's test is not specific for proteins (it
Positive Result:
detects phenolic compounds), and so must be confirmed
Red precipitate (presence of
by other tests for proteins such as the biuret test and
tyrosine residue)
the ninhydrin reaction. The reagent is made by dissolving
metallic mercury in nitric acid and diluting with water. The
test was developed by the French chemist Auguste Nicolas
Eugene Millon (1812–1867).
Specific to phenolic group of tyrosine. The hydroxy

benzene group of tyrosine reacts with Millon's reagent to
form red colored complex.
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4. Glyoxylic acid reaction (Hopkins-Cole)
Hopkins-Cole reagent is prepared by mixing 10 grams of The indole group of tryptophan reacts with glyoxylic
powdered magnesium with sufficient water to cover it. acid in the presence of conc. H2SO4 to give a purple
Then add slowly 250 ml of cold water and shaking at the colored complex. Glyoxylic acid is prepared by reducing
same time. Filter and acidify the filtrate with acetic acid Oxalic acid with magnesium powder or sodium amalgam.
to prevent partial precipitation of magnesium and make Glacial acetic acid which has been exposed to the sunlight
the volume to one liter with distilled water. also contains glyoxylic acid and can thus be used for this
test.
When protein mixed with glyoxylic acid (CHOCOOH) is
treated with sulfuric acid, a violet ring is produced at • Objective: To detect the amino acid tryptophan
the point of contact of the two solutions. This is due to present in protein
the presence of an indole nucleus in the tryptophan • Principle: The indole ring reacts with glyoxylic
component. The indole group of tryptophan reacts with acid in the presence of a strong acid to form a
glyoxylic acid in the presence of conc. The tryptophan violet cyclic product.
condenses with the aldehyde to form colored • Purpose: The Hopkins-Cole test is specific for
compound. H2SO4 to give a purple colored complex. tryptophan, the only amino acid containing indole
group.
Glyoxylic acid – is made by the reduction of oxalic acid
with magnesium powder.
Apparatus: test tube, dropper

Materials:
• 2% albumin solution
• Hopkins-Cole reagent
• pure conc. Sulfuric acid [H2SO4)
Procedure:
Mix 1 ml of 2% albumin solution and 1 ml of Hopkins-
Cole reagent. Incline the tube and allow 1 ml of pure
conc. Sulfuric acid on the side of the tube to form a layer
beneath the protein mixture. If no color is formed, shake
gently.
What is the color produced at the point of contact of

the two liquids? Violet ring
SUMMARY
What is the cause of this color reaction? Hopkins-Cole
reagent gives positive results with proteins containing Experiment Title:
tryptophan, which can alsoindicates High Nutritional GLYOXYLIC ACID REACTION / HOPKINS-COLE
Value – Color Reactions of Proteins
Name the amino acid responsible for this test and Materials:
write its formula. Tryptophan C11H12N2O2 Reactants:
- albumin
Reagents:
Positive Hopkin’s-Cole test: purple color at the interface - Hopkins-Cole Reagent
(tryptophan and egg albumin) – prepared by mixing 10 grams of powdered
magnesium with sufficient water to cover it,
Negative Hopkin’s-Cole test: glycine then slowly adding 250 ml of cold water and
Also, nitrites, chlorates, nitrates and shaking at the same time. Filter and acidify the
excess chlorides prevent the reaction from occurring filtrate with acetic acid to prevent partial
precipitation of magnesium and make the
volume to one liter with distilled water
- pure conc. Sulfuric acid
Positive Result:
Violet ring/purple color at the interface
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5. Heller’s Ring Test

SUMMARY
This is used clinically to detect the presence of Experiment Title:
albumin in urine. HELLER’S RING TEST
– Color Reactions of Proteins
If nitric acid is introduced into a test tube as a
Materials:
lower layer beneath a solution of protein, a
Reactant:
white precipitate of coagulated protein
- conc. Nitric Acid
appears at the junction of the two layers.
Reagent:
- albumin solution
Apparatus: test tube Positive Result:
Materials: - a white ring is formed at the junction of two
solutions
 conc. Nitric acid [HNO3]
- this ring is made up of denatured protein
 2% albumin solution
- yellow color in the ring is due to nitro
Procedure:
compound of aromatic amino acids. It can be
Put 5 ml of conc. Nitric acid in a test tube,
obtained by treating the proteins with other
include the tube and allow 2% albumin
acids like HCl and H2SO4
solution to flow down the side of the tube
slowly.
What happens at the interface between the

two liquids? There is a formation of a white
ring or white precipitate
Heller's test is a chemical test that shows

that strong acids cause
the denaturation of precipitated
proteins. Concentrated nitric acid is
added to a protein solution from the
side of the test tube to form two
layers. A white ring appears
between the two layers if the test is
positive. Heller's test is commonly
used to test for the presence of
proteins in urine. This test was
invented by the Austrian
Chemist, Johann Florian Heller.
 Objective: to detect the properties of

proteins in a given solution and is also
a general test for proteins
 Principle: When proteins are treated
with strong mineral acid, they are
denatured
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6. Reduced Sulfur Test
Loosely combined sulfur after boiling with

sodium hydroxide forms sodium sulfide. A
positive test is given by amino acids which
contain the reduced sulfur group.
Solutions of protein containing cystine,

cysteine or methionine, when heated with
NaOH, splits up the sulfur to form sodium
sulfide [Na2S]. When treated with lead acetate
produced a black precipitate due to the
formation of lead sulfide.
SUMMARY
Apparatus: test tube, dropper, Bunsen burner Experiment Title:
Materials: REDUCED SULFUR TEST – Color Reaction of
• 2% albumin solution Proteins
• 40% Sodium Hydroxide [NaOH]
Materials:
• lead acetate solution
Reactant:
Procedure:
- albumin
To 2 ml of 2% albumin solution add 2 ml of
Reagents:
40% NaOH and 10 drops of 2% lead acetate
- NaOH
solution. Heat to boiling for about 2 minutes
- lead acetate
until distinct change is obtained.
Positive Result:
What did you observe? Mixing the albumin Black deposit of lead sulfide (PbS) in
solution with NaOH and lead acetate solution, albumin
gave a black precipitate and the lead gave black
deposits when heated.
• Principle: Proteins containing sulfur

(like: Methionine and cysteine) give a
black deposit of lead sulfide (PbS) when
heated with lead acetate in alkaline
medium.
• Purpose: To detect amino acid which
contain sulfur group.
Albumin + NaOH + Pb(Ac)2 = PbS (black ppt)

12
7. Adamkiewicz Reaction
Indole derivatives give a positive result with

this test.
The Adamkiewicz reaction is part of a

biochemical test used to detect the presence of
the amino acid tryptophan in proteins. When
concentrated sulfuric acid is combined with a
solution of protein and glyoxylic acid, a
red/purple colour is produced. It was named
after its discoverer, Albert Wojciech
Adamkiewicz.
Apparatus: test tube

Materials:
 glacial acetic acid [CH3COOH] SUMMARY
 conc. Sulfuric acid [H2SO4] Experiment Title:
Procedure: REDUCED SULFUR TEST – Color Reaction of
Add 3 drops of 2% albumin solution to 5 ml of Proteins
glacial acetic acid. Mix well. Pour conc. Sulfuric
Materials:
acid down the side of the tube and note the
Reactant:
color of the ring after it is made to stand for a
- albumin
few minutes.
Reagents:
- NaOH
Observation: Vinegar-like odor of glacial
- lead acetate
acetic acid; glacial acetic acid and albumin
solution separated from the sulfuric acid; Positive Result:
red/purple color in the ring; Black deposit of lead sulfide (PbS) in
albumin
Albumin + Glacial Acetic Acid + H2SO4 = violet ring

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EXPERIMENT 3 PRECIPITATION REACTIONS OF PROTEINS
Proteins from colloidal systems. Their solubility depends What is the principle of Heat Coagulation test? What is
on their electrical charge and the layer of hydration the purpose of adding dilute acetic acid to the
around them. Hence, proteins can be precipitated by solution?
dehydration and neutralization of electrical charge. Heat causes denaturation of proteins by increasing the
kinetic energy of the molecules and by breaking the weak
1. Precipitation by heat coagulation bonds like electrostatic interactions, hydrogen bonds and
hydrophobic interactions. The denatured protein becomes
Apparatus: test tubes, Bunsen burner, thong insoluble and precipitates out. Dilute acetic acid provides
Materials: an acidic medium and brings the pH close to Isoelectric pH.
 2% casein [C38H57N9O9] At Isoelectric pH the protein has a minimum solubility and
 2% albumin solution is thus precipitated. In urinalysis. upon heating urine in an
 1N acetic acid [CH3COOH] alkaline medium the phosphates produce the similar
 5N acetic acid [CH3COOH] turbidity as observed with proteins but the turbidity
Procedure: disappears upon addition of acid if the phosphates are
Place 5 ml of 2% casein in a test tube and heat to boiling there but it persists if it was due to proteins.
Observation: No coagulation with the casein proteins.

Casein proteins are denatured by a change in pH (will
coagulate at pH 4.6) CASEIN – did not coagulate, needs dehydrating
agent
In each of 3 test tubes place 1 ml of 2% of albumin
solution. To the first add 1 drop of 1N acetic acid, to the
second add 2 drops of 5N acetic acid. The third serves as
control. Boil the contents if these 3 tubes.
Which tube has the best coagulation? The test tube

with 2% albumin and 1 drop of 1N acetic has the best and
most coagulation.
Heat Coagulation - denatures proteins forming lumps

via disulfide chains N – Normality; is a measure of concentration equal to the
isoelectric point - the pH where the molecule has no gram equivalent weight per liter of solution. Normality is
net charge also known as the equivalent concentration of a solution.
Casein - milk protein, didn’t coagulate on heat
Acids and base - alters pH therefore changes the
isoelectric point SUMMARY
0.1 N Acetic acid - denatures albumin
0.5 N Acetic acid - denatures albumin but also dissolves Experiment Title:
the precipitate Precipitation by Heat
Coagulation
Heat coagulation and precipitation occur best near the Materials:

isoelectric point of the protein. Albumin coagulates - casein
when heated. Aqueous solutions of proteoses, peptones, Reactant:
gelatin, and casein are not coagulated by heat. - albumin
Reagent:
Denaturation - 1N acetic acid
Process where proteins or nucleic acid lose quaternary, - 5N acetic acid
tertiary and secondary structure because of the
presence of stress: acid, base, salt or heat. Positive Result:
Coagulation on test tube with
Acetic acid (CH3COOH), also called ethanoic acid, the 1N acetic acid
most important of the carboxylic acids.
14
2. Precipitation by Organic Solvents Why is alcohol used as an antiseptic?

Water miscible organic solvents, such as Acetone and
Apparatus: test tube, filter paper, funnel Ethanol are generally good protein precipitants
Materials: because their lower dielectric constants lower the
 ethyl alcohol [C2H5OH] solvating power of their aqueous solutions for
 2% albumin solution dissolved proteins. Hence the protein-protein
 water [H2O] interactions become stronger than protein solvent
Procedure: interactions and the proteins are precipitated out.
Add 4 ml of ethyl alcohol to 2 ml of 2% albumin Alcohol rubbed over the injured site acts by
solution. Remove a small amount of the precipitate precipitating membrane proteins of the micro-
and add water. organisms and hence the multiplication of the micro-
organisms is prevented at the site of tissue injury.
Does the precipitate dissolve in water? No, the Although alcohol cannot coagulate every single cell, it
precipitate does not dissolve in water functions well to inhibit the growth and reproduction
of many microorganisms, including bacteria, fungi,
Set aside the remainder of the precipitate in a protozoa. and viruses. Human skin cells are more
stoppered test tube until the next laboratory resistant to alcoholic coagulation than most
period. Again test the solubility of the precipitate microorganisms. This is why the human skin doesn't
in water. coagulate if it comes into contact with alcohol.
Observation: Coagulation after setting for a day
Filter the mixture and heat the filtrate using a

water bath to see if coagulation occurs. If no
coagulation occurs, it means that the protein has
been completely precipitated by alcohol.
How is this process applied in the fixing of

tissue for histological purposes? Alcohol disrupts
the intrahydrogen bonds of proteins causing
shrinkage and hardening of tissues for preservation Albumin + CH3CH2OH = white ppt (insoluble in
processes. water)
Organic solvents precipitate proteins, when made

to stand for sometimes they are denatured and SUMMARY
become insoluble in water.
Experiment Title:
Precipitation by Organic
Precipitation by dehydration Solvents
Organic solvents like ethanol and acetone also
precipitate proteins by reducing the amount of Materials:
water and by reducing the dielectric constant. Reactant:
Precipitation reaction can also be used under well- - albumin
defined conditions to separate particular protein Reagent:
from a mixture. Eg. precipitation of albumin from - ethyl alcohol
serum at full saturation with (NH4)2SO4
Positive Result:
Alcohol – alcohol owns its disinfecting power to Coagulation on test tube
this property, in the form of 50%-70% solution after setting it for a day
15
3. Precipitation by Salting Out – requires water molecules to surround them to remain

dissolved
Apparatus: test tube, filter paper, funnel, stirring rod
Materials: Explanation: in an aqueous solution with high ionic
 solid ammonium sulfate [(NH4)2SO4] strength, the water molecules sorround the ions and
 2% albumin proteins. At a certain ionic strength, the water
 Millon’s reagent molecules can’t support the charges of both ions and the
 10% commercial peptone proteins
 Biuret’s reagent
Procedure: Hoffmeister’s Series – series that induce selective
a. Add solid ammonium sulfate to 5 ml of 2% precipitation
albumin until it becomes saturated. Filter. Test
the precipitate by Millon’s reaction. Cations : NH4 > K > Na > Li> Mg > Ca
Observation: red precipitation (positive results Anions : F> SO4 > H2PO4 > H3COO> Cl > NO3 > Br > ClO3
with proteins) was formed > ClO
Make a biuret test on the filtrate. (NH4)2SO4 – ions produced are very high in the series
Observation: a violet (positive to proteins due
to peptide bonds) solution was formed Albumin
– positive on Millon’s reaction and negative on the
Conclusion: If there is a positive result, it means biuret reaction
that proteins haven’t been fully salted out by – has no peptide bonds but contains phenol
(NH4)2SO4 and separation is incomplete thus the
violet color. The color blue of the solution would Peptone
indicate that it is a negative with Biuret’s test – on biuret reaction
and thus, all proteins have been salted out. – has peptide bonds
b. Separation of peptones and proteoses.

Saturate 10 ml of 10% commercial peptone What is the difference between salting in and salting
with solid ammonium sulfate, stirring all the out?
time. Filter off the proteoses, peptone is found A protein's multiple acid base groups make its solubility
in the filtrate. Make a biuret test on the filtrate. properties dependent on the concentration of salts, the
Note: Peptone is pink with biuret reagent. polarity of the solvent, the p H and the temperature. The
solubility of a protein in aqueous solution is a sensitive
function of the concentration of the dissolved salts. The
Addition of salts and electrolytes leads to absorption of solubility of a protein increases upon addition of low
the solution envelope from the colloidal particles of a concentration of salt. This phenomenon is called salting in.
protein solution along with the neutralization of surface The explanation for salting in is that as the salt
charges leading the protein to precipitate. Depending concentration of the protein solution increases additional
upon the nature of the proteins, the amount of salt ions more effectively shield the protein molecules and thus
required varies. For example, albumin requires a higher promote protein water interactions, (protein -protein
salt concentration for precipitation than casein or interactions are minimized as the like charges repel each
gelatin. Albumin particles are smaller in size and hence other) resulting in increase in solubility of the proteins. At
have a large surface area. They also hold more water high concentration of salt. the solubility of the protein
around them. Hence a higher salt concentration is decreases. This effect is known as salting out and is
required to precipitate it. primarily a result of the competition between added salt
ions and protein molecules for the given solvent. At high
Neutral salts, like ammonium sulfate and magnesium salt concentration many of the added ions are solvated. so
sulfate, these substances are used to “Salt out” proteins that the solvent available becomes insufficient to dissolve
protein. Hence protein — protein interactions become
stronger than protein water interactions and the protein is
Salting out precipitate out. Salting out is the basis of one of the most
– purification method that utilizes the reduced common protein purification procedures. By adjusting the
solubility of molecules in a solution of high ionic amount of salt concentration in a solution containing a
strength mixture of proteins, the different proteins can be
– causes reduction of molecules solubility separated out.
Hyrodphilic groups/ charged amino acid
16
What is the difference between half saturation and full

saturation?
Half saturation and full saturation signify the amount of
salt required for precipitation of a protein. The amount of
salt required for precipitation depends upon the molecular
weight of a protein. There is generally an inverse
relationship between amount of salt required and the
molecular weight of the protein. The proteins with a large
molecular weight need less salt. Globulins are precipitated
by half saturation with ammonium sulphate, where as
albumin with a low molecular weight is precipitated at full
saturation. Fibrinogen is best precipitated by 1/5 th
saturation with ammonium sulphate. The commonly used
salt solutions are
i) Ammonium sulphate
ii) A mixture of sodium sulphate and sodium
sulphite.
Separation of Peptones & Proteoses

Positive Result:
Filtrate should be pink/light violet with biuret Test
SUMMARY
Experiment Title:
Precipitation by Salting Out (Millons)
Precipitation by Salting Out
Precipitate tested with Millons = flesh to red ppt
Materials:
a. Reactant: 2% albumin
Reagent: solid ammonium sulfate, tested by Millon’s
Reaction and the Biuret Test
b. Reactant: commercial peptone

Reagent: solid ammonium sulfate, tested by Biuret
Test
Positive Result:
a. blue color = no proteins
b. pink/light violet color = presence of peptones
(proteoses have been filtered)
Precipitation by Salting Out (Biuret)

Filtrate = blue color (negative, all proteins have been
salted out)
17
4. Precipitation by heavy metal ions The use of egg as an antidote for metallic poisoning is
based on the ability of egg white to be coagulated by the
Apparatus: test tubes, dropper metallic ion thus taking it our of the field of reaction.
Materials:
 lead (II) acetate [Pb(C2H3O2)2 or Pb(Ac)2] Why is milk or raw egg used as an antidote to heavy
 1% Silver Nitrate [AgNO3] metal poisoning?
 1% Copper Sulfate [CuSO4] Heavy metal salts act to denature proteins in much the
 0.1N Sodium Hydroxide [NaOH] same manner as acids and bases. Heavy metal salts usually
Procedure: contain Hg+2, Pb+2.Ag+1 TI+1, Cd+2 and other metals
Place 2 ml of 2% albumin solution in each of 3 tubes. To with high atomic weights. Since salts are ionic they disrupt
the first add 2 to 3 drops of Pb(Ac)2, to the second and 3 salt bridges in proteins.The reaction of a heavy metal salt
drops of 1% AgNO3 and to the third 1% CuSO4. with a protein usually leads to an insoluble metal protein
salt. This reaction is used for its disinfectant properties in
Observation: external applications. For example AgNO3 is used to
Lead (II) acetate prevent gonorrhea infections in the eyes of new-born
Albumin + Pb (Ac)2 ——> white ppt infants. Silver nitrate is also used in the treatment of nose
Albumin + AgNO3 ——> white ppt and throat infections, as well as to cauterize wounds.
Albumin + CuSO4 ——> light blue Mercury salts administered as Mercurochrome or
Merthiolate have similar properties in preventing
Repeat the test using fresh samples of albumin but add infections in wounds. This same reaction is used in reverse
few drops of 0.1N NaOH before adding the mental salts. in cases of acute heavy metal poisoning. In such a situation.
a person may have swallowed a significant quantity of a
NaOH heavy metal salt. As an antidote, a protein such as milk or
Albumin + NaOH + Pb(Ac)2 ———> white ppt egg whites may be administered to precipitate the
Albumin + NaOH + AgNO3 ———–> brown ppt poisonous salt. Then an emetic is given to induce vomiting
Albumin + NaOH + CuSO4 ————> bluish violet ppt so that the precipitated metal protein is discharged from
the body.
Which has more precipitate, the test with or without SUMMARY
NaOH? More ppt with NaOH
Egg albumin is used as an antidote for mercury or lead Experiment Title:
poisoning. Why? Egg albumin forms ppt with mercuric Precipitation by
salts hindering or slowing down the absorption of Heavy Metal Ions
mercury in the system
Materials:
Reactant: 2% albumin
At pH above their pI proteins are present as negatively Reagent: sodium
charged ions. The heavy metal cations neutralize the hydroxide,
negative charge on the surface and cause precipitations 3 heavy metals: lead
of proteins. Commonly salts like lead acetate, zinc acetate, silver nitrate,
sulphate and cupric sulphate are used as precipitation copper sulfate,
agents. (Egg albumin can used to precipitate lead and
mercury in the intestine using this principle.) Positive Result:
More precipitate with
Heavy metals – forms insoluble metal protein salt NaOH
Egg albumin forms precipitate with mercuric salts

hindering/slowing down the absorption of mercury in
the system
Salts of heavy metals, like mercuric chloride, silver

nitrate, forming the insoluble metallic proteinates. The
deficiency of disinfectants depends on this property of
coagulating the protein of the organism destroying its
life.
18
5. Precipitation by alkaloidal reagents positive charge on proteins and cause their

precipitation. Sulphasalicylic acid, pircic acid, and
Apparatus: test tubes, dropper tricholoro acetic acid are commonly used alkaloids.
Materials:
 2% albumin solution Alkaloid reagents are reagents that precipitate
 0.1N Hydrochloric Acid [HCl] alkaloids. Some examples are tannic acid, picric
 potassium ferrocyanide acid, iodine and mercuric chloride. Alkaloids are
[K4Fe(CN)6·3H2O] vegetable compounds that are similar to ammonia
 5% tannic acid [C76H52O46] and form salts by combining with acids. Both
 saturated picric acid [C6H3N3O7] alkaloids and their salts are bitter in taste.
Procedure:
Place 2 ml of 2% albumin solution in each of 3 Alkaloidal reagents, such as picric acid, tannic acid,
tubes and acidify with a few drops of 0.1N HCl. To phosphotungstic acid, phosphomolybdic acid.
the first tube add 3 drops of potassium Tannic acid is used to relieve diarrhea, to check
ferrocyanide, to the second 3 drops of 5% tannic secretions in the treatment of burns because it
acid, to the third 3 drops of saturated picric acid. produces an astringent effect on the tissues and
Why are they called alkaloidal reagents? They prevents absorption of toxins; phosphotungstic
prove the presence or absence of alkaloids acid is used to precipitate and remove proteins in
blood analysis.
Why is picric acid used in treatment for burns
and tannic acid for diarrhea? Picric acid has
antiseptic and antistringent properties and destroys
protens. Tannic acid have astringency property
which slows down the peristaltic movements of the
gastrointestinal tracts. It also has a natural
bateriastic agent to stop bacteria from reproducing.
Antistringent – contraction of skin cells

Antiseptic – prevent further wound formation from
around the wound thus encouraging healing
Write the general equation showing the ionization

of a protein in acid and basic medium.
SUMMARY
Experiment Title:
Precipitation by Alkaloidal Reagents
Materials:
Reactant: 2% albumin
Reagent: HCl, 3 alkaloidal reagents: potassium
ferrocyanide, tannic acid, saturated picric acid
Positive Result:
Potassium ferrocyanide – white precipitate/solution
At pH below their pI, proteins are present as Tannic acid – brown precipitate/solution
Picric acid – yellow precipitate/solution
positively charged ions. Alkaloids neutralize the
19
EXPERIMENT 4 TEST FOR CARBOHYDRATES
A. Physical Tests Water

– monosaccharides and disaccharides are soluble
Glucose – monosaccharide, aldose, hexose, because of -OH group
aldohexose – polar + polar = solubility
Fructose – monosaccharide, ketose, hexose, – polysaccharide’s polymer linkage prevents
ketohexose interaction with water
Arabinose – monosaccharide, aldose, pentose, – crowded polymerization causes steric hindrance
aldopentose that lessens ability of adjacent hydroxyl group to
Starch – polymer of D-glucose joined by glycosidic react with water
bond (covalent bond joining carbohydrate to other
groups) NaCl
Dextrin – same with starch but more complex – same with water but less solubility because of the
presence of another solute, NaCl. However, the NaCl
1. Solubility won’t react chemically.
Apparatus: test tubes
Materials: Alcohol
 arabinose [C5H10O5] – the sugars did not dissolve because it was not polar
 fructose [C6H12O6] enough to dissolve the solute.
 glucose [C6H12O6]
 sucrose [C12H22O11] 2. Dialysis
 starch [(C6H10O4)n] Apparatus: test tube, semi-permeable layer,
 dextrin [(C6H10O5)n] beakers,
 water [H2O] Materials:
 10% Sodium Chloride [NaCl]  0.1M glucose [C6H12O6]
 alcohol [C2H5OH]  0.1M sucrose [C12H22O11]
Procedure:  0.7% starch [(C6H10O4)n]
Test the solubility of arabinose, fructose, glucose,  water [H2O]
sucrose, starch, and dextrin in 2 ml of water, 10%  Molisch reagent
NaCl, and alcohol. Note: use only a pinch of solid Procedure:
carbohydrates. Tabulate your results. (Demonstration) To each of the three dialysis
tubes place 1 ml of 0.1M glucose, 0.1 M sucrose,
2 ml H2O 10% NaCl Alcohol and 0.7% starch. Immerse the ends covered with
Arabinose ✓ ✓ ✖ the semi-permeable membrane in 3 100-ml
✓ ✓ ✖
beakers with 50 ml water. Set aside for 15 minutes.
Fructose
Perform the Molisch test on the dialysate and
Glucose ✓ ✓ ✖ compare the intensity of the color produced.
Sucrose ✓ ✓ ✖ Tabulate your results and draw conclusions.
Starch ✖ ✖ ✖
Dextrin ✖ ✖ ✖ Glucose – light violet ring
✓ - soluble ✖ - insoluble Sucrose – lighter violet ring
Starch – lightest violet ring
Water – monosaccharides and disaccharides are
There was a formation of violet ring in glucose
soluble because of OH group
(monosaccharide) and sucrose (disaccharide) but
not in starch (polysaccharide), because the
The solubility to ordinary solvents is inversely
molecules of polysaccharides are not small enough
proportional to the complexity of their structures
to pass through the semi-permeable membrane.
both mono and disaccharides are readily dissolved in
Moreover, it shows that glucose has a more violet
water. Higher carbohydrates like starch dissolve only
ring because it has diffused more.
slightly, while form colloidal solutions which cannot
be dialyzed. Cellulose is practically insoluble.
20
Selectively permeable membrane - only allows SUMMARY

small sized molecules (water, glucose & amino acid)
but not the large sized molecules (protein & starch) Experiment Title:
to pass through. e.g. cell membrane of all living cells, 1. SOLUBILITY – Physical Test for Carbohydrates
the internal wall of the gut & visking (dialysis) tubing. 2. DIALYSIS – Physical Test for Carbohydraates
Materials:
1. Materials:
 arabinose [C5H10O5]
 fructose [C6H12O6]
 sucrose [C12H22O11]
 starch [(C6H10O4)n]
 dextrin [(C6H10O5)n]
 water [H2O]
 10% Sodium Chloride [NaCl]
 alcohol [C2H5OH]
2. Materials:
 0.1M glucose [C6H12O6]
 0.1M sucrose [C12H22O11]
 0.7% starch [(C6H10O4)n]
 water [H2O]
 Molisch reagent
Positive Results:
1.
2 ml H2O 10% NaCl Alcohol
Arabinose ✓ ✓ ✖
Fructose ✓ ✓ ✖
Glucose ✓ ✓ ✖
Sucrose ✓ ✓ ✖
Starch ✖ ✖ ✖
Dextrin ✖ ✖ ✖
✓ - soluble ✖ - insoluble
2. Glucose – light violet ring

Sucrose – lighter violet ring
Starch – lightest violet ring
21
B. Furfural Reactions of Carbohydrates

Molisch test: (General test for carbohydrates)
1. Molisch Test (Alpha-Naphthol Reaction) When sugar solution mixed with a - naphthol is
Apparatus: test tubes, dropper brought in contact with conc. H2SO4, a violet ring is
Materials: formed at the junction of the two liquids. The sulfuric
 Water acid acts as a dehydrating agent forming furfural
 0.02M glucose [C6H12O6] derivatives which interact with a - naphthol
 0.02M sucrose [C12H22O11] liberating a colored compound of unknown
 0.05% starch [(C6H10O4)n] constitution.
Procedure: Molisch’s test is based on the dehydration of the

This is a general test for carbohydrates. carbohydrate by sulfuric acid or hydrochloric acid to
produce an aldehyde, which condenses with two
Prepare 4 for test tubes with 1 ml water, 1 ml of molecules of a phenol (usually α-naphthol, though
0.02M glucose, 1 ml of 0.02 M Sucrose, and 1 ml of other phenols such as resorcinol and thymol also give
0.05% starch respectively. The first serves as a colored products), resulting in a red- or purple-
control. To each of the 4 solutions add 2 drops of coloured compound.
Molisch reagent (5% solution of alpha-naphthol in
alcohol). Mix thoroughly. Incline the tube and All carbohydrates – monosaccharides, disaccharides,
allow 1 ml of conc. H2SO4 to flow in the side of the and polysaccharides – should give a positive reaction,
tube. Note the color of the ring obtained after and nucleic acids and glycoproteins also give a
some time. The color of the ring is violet. positive reaction, as all these compounds are
Furfural derivative + alpha naphthol → violet ring eventually hydrolyzed to monosaccharides by strong
mineral acids. Pentoses are then dehydrated
What product from each of the sugar condenses to furfural, while hexoses are dehydrated to 5-
with alpha-naphthol? Furfural derivative (glucose hydroxymethylfurfural. Either of these aldehydes, if
+ H2SO4) present, will condense with two molecules of α-
naphthol to form a purple-colored product
Molisch test
—> general test for carbohydrates
—> a dehydration-condensation reaction with the
formation of furfural
Conc. H2SO4 alpha-naphthol

Carbohydrate furfural violet ring
—> test for carbohydrates or compounds which can

be hydrated to furfural or furfural derivatives with
the presence of H2SO4
H2SO4 –> dehydrates the sugar forming furfural and
3 water molecules SUMMARY
Experiment Title:
example: Molisch Test – Furfural Reaction of
glucose + H2SO4 ——> furfural derivative Carbohydrates
furfural derivative + alpha-napthol —> violet ring Materials:
This reaction is a complicated aldol condensation  Water
 sugar = (+)
Reagent: a-naphthol and conc. H2SO4  0.02M sucrose [C12H22O11]
Reaction: PURPLE RING bet. H2SO4 and sugar  0.05% starch [(C6H10O4)n]
Positive: Glucose, Galactose, Fructose, Maltose, Positive Result:
Lactose, Sucrose, Glycogen, Starch Water, Glucose, Sucrose, Starch = violet ring
22
2. Thymol Test
Apparatus: test tubes, thong, Bunsen burner
Materials:
 3% thymol [C10H14O] in – alcohol
 conc. Hydrochloric Acid [HCl]
Procedure:
Perform this test using the carbohydrates used
in Molisch test.
To about 0.5 ml (10 drops) of the solution of a

carbohydrate, add 3-6 drops of 3% thymol in –
alcohol. Add an excess (5-10 ml) of conc. HCl. SUMMARY
Boil gently for 2 minutes shaking the mixture Experiment Title:
at intervals. A carmine color develops (if Thymol Test – Furfural Reaction of
positive). Carbohydrates
Glucose – carmine red Materials:

Sucrose – darkest red (almost black)  glucose [C6H12O6]
Starch – lightest red  sucrose [C12H22O11]
Thymol Test  3% thymol [C10H14O] in – alcohol
—> same results in Molisch test but more stable.  conc. Hydrochloric Acid [HCl]
—> carmine red if positive
Glucose –> carmine red Positive Result:
Sucrose –> carmine red (darkest, almost black) Glucose –> carmine red
Starch –> carmine red (lightest) Sucrose –> carmine red (darkest, almost black)
Starch –> carmine red (lightest)
23
3. Seliwanoff’s test (Resorcin-HCl Test)

(Resorcinol Test) Seliwanoff’s test is a chemical test which
Apparatus: test tubes, beaker, wire gauze, tripod, distinguishes between aldose and ketose sugars.
Bunsen burner Ketoses are distinguished from aldoses via
Materials: their ketone/aldehyde functionality. If the sugar
 water contains a ketone group, it is a ketose. If a sugar
 Seliwanoff’s reagent contains an aldehyde group, it is an aldose. This test
 0.01M glucose [C6H12O6] relies on the principle that, when heated, ketoses are
 0.01M fructose [C6H12O6] more rapidly dehydrated than aldoses.
 0.01M arabinose [C5H10O5]
Procedure: Fructose and sucrose are two common sugars which
Place in each of 4 test tubes 5 ml of Seliwanoff’s give a positive test. Sucrose gives a positive test as it
reagent. To these tubes add respectively 1 ml is a disaccharide consisting of fructose and glucose.
water, 1 ml of 0.01M glucose, 1 ml of 0.01M
fructose, and 1 ml of 0.01M arabinose. Dip all the
four tubes in boiling water at the same time.
Observe the color changes in each tube during a 5-

minute period of heating.
Water – colorless Fructose – Mahogany red

Glucose – yellow Arabinose – Light yellow
What kind of sugar will give a positive reaction

with Seliwanoff’s reagent? Fructose
Seliwanoff’s test (Specific test for Fructose)

Fructose + Seliwanoff’s reagent -------- mahogany red
(Resocinol – (HCl) SUMMARY
Seliwanoff reagent –> Resorcinol + HCl Experiment Title

–>Resorcinol reacts with ketose Seliwanoff’s Test / Resorcin – HCl Test /
–> HCl hydrolysis complex sugar –>simple sugar –> Resorcinol Test
furfural
– Furfural Reaction of Carbohydrates
aldose–> colorless to faint pink
ketose–> red, deep cherry red, mahogany red
Materials:
dehydrated ketoses react with resorcinol to Reactants:
produce deep cherry red color  Water
aldoses react to produce faint pink
 Glucose
Reagent: Resorcinol and Conc. H2SO4
Reaction: DEEP CHERRY RED=Fructose and Sucrose  Fructose
FAINT PINK=Glucose, Galactose, Maltose, Lactose  Arabinose
Reagent: Seliwanoff’s Reagent
FRUCTOSE – KETOSE
Positive Result:
Ketose + Seliwanoff’s Reagent
→ mahogany red due to the reaction of hydroxy Mahogay Red coloration
methyl furfural with resorcinol
When added to a solution containing aldoses, a

slower forming light pink is observed instead.
24
4. Moore’s Test (action of conc. Alkali)

Apparatus: test tube, Bunsen burner, thong
Materials:
 5% glucose [C6H12O6]
 conc. Sodium hydroxide [NaOH]
Procedure:
Mix 1 ml of 5% glucose and 1 ml of conc.
NaOH. Boil. What is the color and odor of the
solution? Caramel-like odor with caramel-like
color
This test is based on the liberation of

aldehydes which polymerize to form a
resinous substance, caramel.
Moore’s Test
–> based on the liberation of aldehydes
subsequently polymerizes to form resinous
substance. “Caramel”
–> test for reducing sugar except sucrose SUMMARY
–> yellow/orange/dark brown
—> liberating caramel odor Experiment Title:
positive: glucose, galactose, maltose, fructose, Moore’s Test
lactose – Furfural Reaction of Carbohydrates
negative: sucrose, glycogen, starch
Materials:
When a solution of reducing sugar is heated
Reactant: Glucose
with an alkali (NaOH), it turns yellow to
orange, and finally dark brown, liberating the Reagent: Conc. NaOH
odor of caramel.
Positive Result:
Brown Caramel color with accompanying
sweet caramel-like odor
25
5. Tollen’s Phloroglucin Reaction

Apparatus: test tube, beaker, wire gauze,
tripod, Bunsen burner
Materials:
 water [H2O]
 0.01M fructose [C6H12O6]
Procedure:
Measure off 5-ml of phloroglucin solution into
each of four test tubes. To each add
respectively 1 ml water, 1 ml of 0.01M glucose,
1 ml of 0.01M fructose, and 1 ml of 0.01M
arabinose. Immerse all test tubes in boiling
H2O at the same time. Observe at the end of 1
hour. This reaction may be used to
differentiate pentoses from hexoses.
Observation:
H2O = very light yellow SUMMARY
Glucose (Hexose) = red-wine
Arabinose (Pentose) = betadine-like
Experiment Title:
color/reddish-brown
Fructose (Hexose) = black Tollen’s Phloroglucin Reaction
– Furfural Reaction of Carbohydrates
–> differentiates pentose from hexose
Phloroglucin –> behaves as ketone Materials:
water- very light yellow Reactant:
glucose – red wine (hexose)  water [H2O]
fructose- greenish brown (hexose)  0.01M glucose [C6H12O6]
arabinose- reddish brown  0.01M fructose [C6H12O6]
– betadine-like color (pentose
Positive test if the color is betadine like
Reagent: Phloroglucin Solution
Phloroglucinol is a reagent of the Tollens' test
for pentoses. This test relies on reaction of Positive Result:
the furfural with phloroglucinol to produce a Betadine-like coloration of solution
colored compound with high molar absorptivity.
This is based on the formation of similar
intermediate furfurals which condense with
phloroglucinol to form a red colored compound.
26
EXPERIMENT 5 SPECIAL TEST FOR SACCHARIDES
1. Nitro-Chromic Acid Test

Apparatus: test tube, stirring rod
Materials:
 0.01 M glucose [C6H12O6]
 potassium chromate [K2CrO4]
Procedure:
To 5 ml of 0.01 M glucose solution, add 3 ml of
conc. Nitric acid and 5 drops of 5% potassium
chromate solution. Mix well. A blue solution
appears after a minute if it contains sugar with
a concentration more than 1%. This test is due
to the free –CHOH groups in the sugar
molecule. Non-sugar compounds which
contain this group may also give a positive
reaction. Results?
Blue coloration – positive result of nitro-

chromic acid
HNO3 + KCrO4
SUMMARY
–> detection of primary and secondary alcohol
with special reference to saccharides Experiment Title:
2HNO3 + K2CrO4 —> H2CrO4 + 2KNO3 Nitro-Chromic Acid Test
H2CrO4 – acid +chromium salt – Special Test for Saccharides
– strong acid for oxidizing alchol ->ketones ->
carboxylic acid Materials:
– made through the reaction
Reactant: Glucose
primary alcohol oxidizes to carboxylic
Reagents: conc. Nitric Acid and potassium
secondary alcohol oxidizes to ketone
aldehyde oxidizes to carboxylic chromate
H2CrO4 + reducing sugars —> HCrO3
>reduced state Positive Result:
> color blue: This reaction is given by all Blue coloration of the solution
saccharides and other compounds containing a
primary or a secondary alcohol group
Chromium VI is reduced to Chromium IV

27
2. Alcoholic Fermentation The yeast used in the fermentation contains enzymes

Apparatus: test tube that catalyze the transformation of more complex
Materials: sugars into simple sugars, then to alcohol and CO2.
 baker’s yeast
 glucose [C6H12O6] C12H22O11 + H2O invertase 2C6H1206
 galactose [C6H12O6] C6H1206 zymase 2CH3CH2OH 2CO2
 phosphate buffer CO2 + Ca(OH)2  CaCO3 + H2O
Procedure:
Place in a test tube 5 ml of a 20% suspension of
baker’s yeast and add about 5 ml of sugar solution
and 5 ml of phosphate buffer (pH 6.4 to 6.8). allow
this to stand for one hour. Bubbles indicate
fermentation.
Use the following sugars: sucrose, glucose,

galactose
What are the bubbles due to? Carbon dioxide,
when complex sugars turn into simple sugars, then
to alcohol, then to CO2
Explain results. Yeast contain the enzymes

invertase into zymase. Invertase causes hydrolysis of
sugar solution into glucose and fructose. Zymase
brings about the fermentation into ethyl alcohol and
CO2
FERMENTATION
This is the decomposition of carbohydrates
brought about by the action of microorganisms such
as yeast, molds, bacteria, etc. When yeast is added to
a sugar solution, ethyl alcohol and carbon dioxide are
produced.
This process is utilized in the manufacture of
beverages and other valuable industrial products.
Yeasts ferments only the naturally occuring D - SUMMARY
glucose, D mannose, D fructose and with difficulty, D
– galactose (fermented by specially cultured yeast).
Experiment Title:
The synthetic L - forms remain unaltered. Maltose
and sucrose are fermented by yeast only after being Alcoholic Fermentation
split into their component monosaccharides. Some – Special Test for Saccharides
yeast do not ferment lactose and D- galactose at all.
This serves to differentiate from other sugars. Materials:
Aside from alcoholic milk sours due to the  baker’s yeast
conversion of lactose to lactic acid and sucrose
ferments to form citric acid.
Yeast alone can cause fermentation of sugars.  glucose [C6H12O6]
Yeast contains the enzymes invertase and zymase;  galactose [C6H12O6]
invertase causes the hydrolysis of sugar solution into
Positive Result:
glucose and fructose; zymase brings about the
fermentation into ethyl alcohol and CO2. Sometimes Bubbles
Pasteur's salt is added as food nutrients to the yeast.
28
3. Mucic Acid Test

Apparatus: test tube, beaker, wire gauze,
tripod, Bunsen burner
Materials:
 5% galactose [C6H12O6]
Procedure:
To 1 ml of 5% galactose add 1 ml of conc.
Nitric acid. Heat in a boiling water bath for 1 ½
hour. Allow to stand till the next laboratory
period. Examine the crystals under the
microscope and draw them. Mucic Acid Test
galactose + conc. HNO3 = white crystals
The mucic acid test takes advantage of the
relatively insolubulity of mucic acid (an isomer
of saccharide acid) which is formed by the SUMMARY
oxidation of galactose)
Experiment Title:
Galactose + conc. HNO3  white crystals Mucic Acid Test
– Special Test for Saccharides
–> test for identifying galactose
–> aldehyde and primary groups are oxidized to
carboxyl group in reaction of galactose with Materials:
HNO3 forming saccharic acid Reactant: galactose
–> saccharic acid is insoluble Reagent: conc. Nitric Acid
Mucic acid is another name of galactaric acid
which is an example of saccharic acid Positive Result:
Formation of white crystals
- test to identify galactose and lactose
- the nitric acid oxidizes galactose to an isomer of
tetrahydroxyadipic (tetrahydroxyhexanedioic)
acid that crystallizes out from water under the
conditions of the test.
- lactose is a disaccharide, galactosyl (1>4)
glucose. The nitric acid first catalyzes hydrolysis
of lactose to glucose and galactose. Then the
nitric acid oxidizes both of those sugars to the
tetrahydroxy acids. Mucic acid crystallizes out,
which is a positive test. The acid from glucose
remains in solution
- the test will give positive results if there is a
presence of crystal precipitate
29
(5) 0.03M glucose – positive

4. Reduction Test (6) 0.03 M lactose – negative
Procedure: (7) 0.03 M sucrose – negative
a. Fehling’s Test
Place 1 ml each of the following solutions into 5 Note the speed of reduction in different tubes
separate test tubes: 0.01M glucose, 0.01M fructose,
0.01M Sucrose, 0.7% Starch solution, water Fastest to slowest: fructose, glucose, lactose,
(control). To all 5 test tubes add 1 ml each of sucrose
Fehling’s solution A (CuSO4) and Fehling’s Solution
B (KOH and Rochelle Salt). Shake and immerse all Positive result: formation of Cu2O
tubes in boiling water and heat for 15 minutes. Glucose and Fructose produced tiny brick red
Observe the color produced. ppt though Fructose reacted faster than
glucose.
H2O – blue sol’n
Glucose – red ppt Barfoed’s test serves to detect reducing
Fructose – red ppt monosaccharides, fructose reacts faster than
Sucrose – blue sol’n glucose. Disaccharides also reduce this reagent if
Starch – blue sol’n the sugar concentration is high enough and the
time of heating is long.
What is the precipitate due to? Free
aldehyde/Ketone Group REDUCTION
Which carbohydrates have no reaction? Sucrose All sugars except sucrose undergo with the
and Starch absorption of energy and formation of products
Why? They are non-reducing sugars. convertible into fats. Reduction used in culture media
for the identification and differentiation of bacteria.
b. Benedict’s Test (qualitative test for Only one alcohol is formed by the reduction of an
glucose in urine) aldose. Reduction of the ketose sugar yields two
In 3 separate tubes place 5 ml of Benedict's alcohols because the carbon of the ketonic group
solution (CuSO4, Na2CO3, sodium citrate) and then becomes asymmetric and produces an isomer.
add 1 ml of 0.1 M, 0.01, and 0.002 M glucose
solutions. Place in a boiling water bath for 10 a. Fehling’s test
minutes. Observe the colors produced and Fehling's solution (sodium hydroxide, sodium
compare the intensities in each tube. potassium tartrate, and copper sulfate). The alkaline
solution of CuSO4 when heated with a reducing sugar
0.1M glucose – light brown – very dark brick red forms a yellow to red precipitate due to the reduction
ppt of cupric hydroxide into cuprous oxide.
0.01M glucose – brown – dark brick red ppt
0.002M glucose – dark brown – brick red ppt - aldoses are easily oxidized to yield carboxylic acids
- Cupric ion complexed with tartrate ion is reduced to
c. Barfoed’s Test Cuprous oxide (brick red ppt.)
Prepare 7 test tubes and label them with numbers -Tartrate ion complexing agent keeps copper ion
properly. Add to each 5 ml of Barfoed’s solution solution. If absent cupric hydroxide would precipitate
(Copper acetate in acetic acid). Then add to the from basic solution
corresponding tube 5 ml of each (1) 0.01M glucose, - Reduction of Cu2+> Cu+ by reducing sugar
(2) 0.01M fructose, (3) 0.01M lactose, (4) 0.01 M Reagent: Fehlings I and Fehlings II = deep blue
sucrose, (5) 0.03M glucose, (6) 0.03 M lactose, and solution
(7) 0.03 M sucrose. Place the seven tubes in a Reaction: ORANGE-RED PPT of Cu2O
boiling water bath and heat for ½ hour. Positive: Glucose, Galactose, Maltose, Fructose,
Lactose
(1) 0.01M glucose – positive Negative: Sucrose, Glycogen, Starch
(2) 0.01M fructose – positive
(3) 0.01M lactose – negative Fehling’s reagent–> CuSO4 + KOH + Rochelle salt
(4) 0.01 M sucrose – negative (sodium potassium tartrate)
30
–> differentiate reducing from non-reducing sugars b. Benedict’s Test

–> Cu2O red ppt
–>sucrose is a non-reducing sugar Benedict's Test:
–> anomeric carbon is not free because it is the one 2 Cu(OH)2 + R.CHO ------------- Cu2O + R:COOH 2HOH
used in bonding the glucose and fructose through the insoluble cuprous oxide
alpha-1,2-glyosidic bond
Sucrose and polysaccharide show little or no
reducing action because of bound condition between
reducing groups.
Benedict's and Fehling's solutions are the
reagents commonly used. Benedict's is a modification
of Fehling's solution both contain soluble cupric
hydroxide, (Cu(OH)2.
-used to detect presence of all monosaccharides and

generally all reducing sugars
Reagent: Sodium Citrate (Na3C6H5O7),Sodium
Carbonate, Sodium hydroxide and Copper sulfate
Reaction: GREEN, YELLOW, ORANGE,RED-BRICK
RED PPT
Positive: Glucose, Galactose, Maltose, Fructose,
Lactose
Negative: Sucrose, Glycogen, Starch
–> qualitative test for glucose in urine

–> forms Cu2O red ppt
–> Benedict’s reagent (CuSO4 + Na2CO3 + sodium
citrate)
c. Barfoed’s Test
31
Reducing monosaccharides also exert their actions

even in acid solution. Thus, solution of cupric acetate
in weak acetic acid (Barfoed’s reagent) is reduced by
monosaccharides but not by disaccharides. This
serves, therefore, to differentiate the two sugars.
- reducing monosaccharides
Reagent: Cupric Acetate (Cu(OAc)2) and Acetic acid
(CH3COOH), HCl
Reaction: REDDISH PPT. of copper (I) oxide
Positive: Glucose (Dextrose), Galactose, Fructose
(Levulose)
Negative: Maltose, Lactose, Surcose,Glycogen, Starch
–> Barfoed reagent (Copper acetate in acetic acid)

–> test to detect monosaccharide a reducing sugar in
solution
–> differentiate monosaccharide from disaccharide
–> reducing disaccharides undergo in a slower rate
–> used to distinguish monosaccharides from
disaccharides, continued boiling may cause it to react
with disaccharides
Positive result: formation of Cu2O

32
5. Iodine Test for Starch and Dextrin

Apparatus: test tubes, thong, Bunsen burner
Materials:
 0.7% starch [(C6H10O4)n]
 0.1% dextrin [(C6H10O5)n
 dil. Iodine [I2] solution
Procedure:
To 5ml portion each of 0.7% starch and 0.1%
dextrin add a few drops of dil. I2 solution until a
good color develops. Then warm each tube very
gently.
Observation:
Starch – blue-black
Dextrin – black-violet
What happens when the warm solution is cooled?

Starch – when heated turned colorless, then
turned back to its original color when cooled
Dextrin – yellow when heated, then to its original
color when cooled
Iodine test for starch and dextrin

–> red color indicates glycogen or erythrodextrin
–> blue color indicates starch
–> test for starch
–> dark blue color for positive results Iodine Test for Starch and Dextrin (heated then cooled)
–> test for the presence of polymer of sugar and how Starch = when heated turned colorless, then turned
complex it is back to its original color when cooled.
Dextrin = yellow(heated) to its original
The positive results are due to the presence of: color(cooled)
1. Amylose- straight chain helices of starch, long,

bonds with I2 SUMMARY
2. Amylopectin- branched chain helices of starch, Experiment Name:

short, doesn’t bond with I2
Iodine Test for Starch and Dextrin
Iodine reacts with the coil structure of
polysaccharides – Special Test for Saccharides
Starch- is a polysaccharide with more amylose. Materials:

Because of this, when it is heated, the bonds of Iodine Reactant: Starch and Dextrin
and amylose is not broken. What’s destroyed is the Reagent: Iodine solution
bonds of iodine and amylopectin.
Dextrin- polysaccharide with more amylopectin Positive Result:

which caused the decoloration when heated Starch – blue-black
Dextrin – black-violet
33
6. Phenylhydrazine Reaction Osazones formation test involves the reaction of

Apparatus: test tube, beaker, wire gauze, a reducing sugar (free carbonyl group) with
tripod, Bunsen burner excess of phenylhydrazine when kept at boiling
Materials: temperature. All reducing sugars form osazones.
 5% glucose [C6H12O6] Therefore, sucrose, for example, does not form
 fructose [C6H12O6] osazone crystals because it is a non reducing
 maltose [C12H22O11] sugar as it has no free carbonyl group.
 lactose [C12H22O11]
 sucrose [C12H22O11] The reaction can be used to
 phenylhydrazine mixture (prepared identify monosaccharides. It involves two
by mixing 2 parts of reactions. Firstly glucose with phenylhydrazine
phenylhydrazine hydrochloride and gives glucosephenylhydrazone by elimination of
3 parts of sodium acetate. The a water molecule from the functional group. The
mixture should be freshly prepared next step involves reaction of one equivalent of
glucosephenylhydrazone with two equivalents
Procedure:
To 0.5 g of phenylhydrazine mixture add 2ml of phenylhydrazine (excess).
of the sugar solution, shake well and heat in First phenylhydrazine is involved in oxidizing
boiling water bath for 30-45 minutes. Allow the alpha carbon to a carbonyl group, and the
the tube to cool (not under the tap) and second phenylhydrazine involves in removal of
examine the crystal under a microscope. one water molecule with the new-
formed carbonyl group of that oxidized carbon
and forming the similar carbon nitrogen
Sugar solutions to be used: 5% glucose,
fructose, maltose, lactose, sucrose bond. The alpha carbon is attacked here because
it is more reactive than the others.
Yellow ppt – result of phenylhydrazine test Osazones are highly coloured and crystalline
compounds and can be easily detected. Each
sugar has a characteristic crystal form of
Osazone Formation
osazones.
Reducing sugars form characteristic
 Maltose forms sunflower / petal-shaped
osazone crystals when heated with an excess of
crystals. Maltosazone, soluble
phenylhydrazine (C6H5NHNH2). This property is
 Lactose forms powder puff-shaped crystals.
attributed to the presence of aldehyde or ketone
Lactosazone, soluble
group in their molecules. The individual sugars
 Galactose forms needle-shaped/rhombic-
form typical yellow-orange crystals with definite
plate shaped crystals. Galactosazone,
melting points.
insoluble
Due to this property, sugars can be
 Glucose, fructose and mannose form
identified from a mixed sample. However, sugars
broomstick or needle-shaped crystals.
with the same configuration like glucose,
Glucosazone, Fructosazone,
mannose and fructose yield the same osazone
Mannosazone, all insoluble
crystal, in which case identification becomes
difficult.
Osazones are a class of carbohydrate derivatives

found in organic chemistry formed
when sugars are reacted with excess
of phenylhydrazine.
34
Reducing sugars (contain potentially free SUMMARY

aldehyde or ketone group) form osazones when
heated with an excess of phenyl hydrazine. Experiment Title:
Osazones are yellow crystalline compounds Phenylhydrazine Reaction
having characteristic forms and melting points. – Special Test for Saccharides
Osazones are useful in the identification of
sugars because they are easily prepared and the
Materials:
crystals can be identified through a microscope.
Reactant: sugar solution
Glucose, fructose, and mannose yield the same
osazone formation (glucosazone) because they Reagent: phenylhydrazine mixture
have identical configuration at carbons 3,4,5,6 in
which case identification becomes difficult. Positive Result:
Osazone formation (yellow ppt)
There are three stages in the chemical reactions
involved in osazone formation:
Reducing sugar → phenylhydrazine → osazone +

aniline + ammonia
35
EXPERIMENT 6 GLYCOGEN
A. Preparation of Glycogen the 1 and 5th position, or in other words

1. Mince one or two pieces of oyster meat (main glycosidic 1,4 bond. And when all of the
source of glycogen in this experiment) into carbohydrates bond with each other they
small pieces. form a compact compound called glycogen.
2. Mix with sand and grind in a mortar. (to really
grind the oyster meat) This is an efficient way of storing energy for
3. Transfer the mixture into an evaporating dish animals, because they are attached together
and add 40 ml of water. by a condensation reaction (removal of water)
4. Boil for 20 minutes. and some ATP (energy). So water takes up a
5. Replenish the water lost by evaporation. lot of weight, We get more energy per unit of
6. Filter. Wash the residue on the filter paper weight. So if you do the opposite hydrolysis
with two 10 ml portions of water, adding the (addition of water), water breaks the bonds
washings to the first filtrate. between the carbohydrates, making it less
7. Concentrate the filtrate to a small volume by compact, but easier to breakdown, since they
boiling. are in their elementary form.
8. Faintly acidify with acetic acid and filter off
any coagulated protein. (acetic acid = to So the products of hydrolysis of glycogen
coagulate the protein) should be carbohydrates or glucose.
9. Use the opalescent filtrate (glycogen) for the
following tests: And to be more specific these glucose
molecules are usually phosphorylated, that’s
B. Reaction of Glycogen how they are removed, so they can be either
1. To 5 ml of the glycogen solution add 3-5 drops glucose 1-P or glucose 6-P, where the
of NaCl and several drops of Iodine solution. numbers indicate the position of the
Note the color and compare it with the color phosphorous.
formed by starch with iodine.
Glycogen solution + NaCl = brown color Explain the results obtained:

Iodine + Starch = blue black
A1. Glycogen is made by joining carbohydrates at the 1-5th
2. Use 3 ml of glycogen solution and add 6 ml of portion or glyceride 1,4 bond, when all of the carbohydrate
95% alcohol. Note the precipitation of molecules bond with each other to form a compact
glycogen. compound called glycogen.
Denaturation by alcohol – precipitates the B1. When iodine solution is added to glycogen, a glycogen-
nucleic acid present in the solution (white iodine complex is formed. Iodo-starch complex is blue-
ppt) black while Glycogen-Iodine complex is blue-brown.
Glycogen is more branched than starch, therefore helices
3. Test the precipitate in no. 2 with Benedict’s of starch are longer than glycogen.
solution and boil for a short time.
2-3. The glycogen was not hydrolyzed in the presence of
Observation: It gave a negative result since the Benedict’s reagent (which is a test for reducing sugar)
glycogen is not a reducing sugar because it and didn’t form a red ppt or form the reduced copper
is a polysaccharide and therefore cannot because it is not a reducing sugar.
undergo oxidation.
4. Red ppt was formed because glycogen has undergone
4. Hydrolyze 5 ml of the glycogen solution with a saccharification and was hydrolyzed into glucose.
few drops of conc. HCl, boiling for 10 minutes.
Cool the solution, neutralize with NaOH and
test with Benedict’s solution.
What are the products of hydrolysis of

glycogen?
It should be glucose. Glycogen is made by

highly branching glucose molecules, or
putting together glucose molecules. Usually
glycogen is made by joining carbohydrates at
36
What is Glycogen?
Glycogen is the stored form of Glucose. It is Glycogen
another name for Animal Starch. It is a –> multibranched polysaccharide of glucose that serves as
polysaccharide (consisting of around 2,000-600,00 units of a form of energy storage in animals and fungi
glucose). It is similar to starch found in plants. In the –> animal starch
human body, when blood sugar level is high, the pancreas –> found in muscle cells and liver cells
releases insulin which stimulates the liver to produce –> constructed predominantly of alpha-1,4-glycosidic
enzymes(eg: glycogen synthase) that synthesize glycogen bonds
from excess glucose. –>has branches created through alpha-1,6-glycosidic
bonds
Preparation of Glycogen
Glycogen is prepared from oyster meat because Opalescent filtrate–> filtrate with varying colors
oyster meat contains around 1-10% glycogen which is high Oyster meat–> main source of glycogen
in comparison to other meat
Reaction of Glycogen
Reaction of Glycogen 1. Iodine Test
When iodine solution is added to glycogen, a Iodine
glycogen-iodine complex is formed. Iodo-starch complex is –> useful to distinguish starch and glycogen from other
blue-black while Glycogen-Iodine complex is blue-brown. polysaccharide
Glycogen is more branched than starch, therefore helices
of starch are longer than glycogen. This longer helix of Starch + I2——–> Blue/Black
starch (specifically in amylose) allows more iodine atoms Glycogen + I2———> brown
(in the form of triiodide ions) to bind, which makes a
stronger color change compared to glycogen Starch in the form of amylose and amylopectin has less
Water is very polar, it forms a dipole-dipole branches than glycogen. This means that the helices to
interaction with the substance and make a layer around it. form iodine complex is longer in starch than in glycogen,
This means that glycogen cannot interact with other therefore binding more iodine atoms
glycogen molecules. Ethanol is less polar than water
allowing glycogen to interact with itself and aggregate, Starch iodine complex=more bonds, more intense
eventually precipitating it. (See General Rule on Solubility) Glycogen iodine complex=less bonds, less intense
Benedict's Test is used to detect the presence of
HYDROLYZED GLYCOGEN, which is GLUCOSE. (refer to 2. Denaturation by alcohol
previous experiments) Alcohol
–> precipitates the nucliec acid present in the solution
–> results to formation of white precipitate
3. Benedict’s Test
It gave a negative result since glycogen is not a reducing
sugar because it is a polysaccharide and therefore, it
cannot undergo oxidation
4. Hydrolysis of glycogen
HCl
–> serves as the acid in the acid hydrolysis
Saccharification–> hydrolysis of carbohydrates to form
simple sugar
NaOH–> to neutralize
37
EXPERIMENT 7 LIPIDS
Lipids include both true fats and fat-like substances. True

fats contain glycerol and fatty acids in their molecules.
Compound fats like phospholipids have besides glycerol
and fatty acids, a nitrogenous base, and phosphoric acid.
The sterols are represented by cholesterol, ergosterol, and
7-dehydrocholesterol.
Lipids are any group of amphiphilic (water fearing and

fat loving) organic compounds that are soluble in non-
polar solvents. They do not have 1 common functional
group but share common physical properties.
1. Solubility
Test the solubility of one drop of coconut oil in 1 ml of the
following solvents: water, diluted HCl, diluted NaOH, cold
alcohol, hot alcohol, chloroform, ether, carbon
tetrachloride. Tabulate your results.
Dil. Cold
H2O Dil. HCl
NaOH alcohol
Coconut oil ✖ ✖ ✖ ✓
Hot alcohol CHCl3 Ether CCl4

Coconut oil ✓ ✓ ✓ ✓
✓ - soluble ✖ - insoluble Rancid oil is a kind of lipid that is hydrolyzed and/or

auto-oxidized when left exposed to air. The products of
COCONUT OIL – nonpolar and organic (because it contains oxidation are aldehydes, ketones, and alcohols that give
carbon) it a bad smell and taste. Volatile short chain fatty acids
are also produced, thus lowering the pH. When pH is
Nonpolar → organic = soluble lowered below 3.0, the Congo Red that was added to the
Nonpolar → inorganic = insoluble lipid will turn blue.
Lipids are non-polar, therefore, they are only soluble in 3. Formation of Translucent Spot:
non-polar solvents such as chloroform (though slightly Place 1 drop of coconut oil on a piece of paper. Allow to
soluble in hot ROH). Lipids which are non-polar are evaporate.
soluble to non-polar solvents because of London
dispersion forces, the weakest intermolecular force. Does the translucent spot disappear?
No, because it can’t evaporate without heat, so they
2. Reaction towards indicators leave translucent spots. Moreover, oil evaporates
Place in each of two test tubes, 1 ml of fresh coconut oil. To slowly because of the force of molecules that hold
the first add congo red, to the other red and blue litmus them together are stronger.
paper. Repeat the test on rancid oil.
Would essential oils leave translucent spot?
Differentiate the reaction of fresh coconut oil and rancid No, because they are not actual oils.
oil towards the indicators
Short explanation:
Translucent Spot will not disappear because oil evaporates
Fresh Coconut Rancid Oil very slowly.
Oil (neutral) (Acidic)
Congo Red Red/original Turns to blue (Too Long; Don't Read):
(neutral) black Paper is made from fibers/remains of trees.
Red Litmus Red (original) Red Paper is opaque because it has a very high index of refraction
Paper (Physics) compared to the air surrounding the individual,
thus, when light passes through it, light will be refracted very
Blue Litmus blue (original) Red
much such that the light won't be able to hit our eyes. When
Paper
38
you have 2 mediums that have very different indices of

refraction, light will be bent much more (Search Snell's Law Which tube or tubes formed a permanent emulsion? Test
for reference). tube 1
When you place a drop of water (i will explain oil
later, they have similar results) on a paper, it will turn
translucent because water will enter the spaces filled with air,
You will form a permanent emulsion when you add an
and water has a higher index of refraction than air, therefore, emulsifying agent because the polar substance (in this case,
the difference between the indices of refraction of water and water), would be attracted with the POLAR END of the
paper fiber is less compared to air and paper fiber. This lesser EMULSIFYING AGENT, while the non-polar substance (in
difference leads to a lesser bending of light, thereby allowing this case, oil), will be attracted to the NON-POLAR END of
light to enter our eyes since light since it is not obstructed by the EMULSIFYING AGENT, thereby forming a stable
bending when passing through the paper fibers. The emulsion.
transparent spot will then disappear because water
evaporates and the fibers will then become "re-immersed" in
6. Test for Unsaturation
air
When you put COCONUT OIL on the paper, the paper
Place 5 ml of oleic acid in chloroform in a test tube and add
will turn translucent, because oil has a high refractive index 5 Hubl’s Iodine solution drop by drop shaking between
(slightly higher than water). The TRANSPARENT SPOT DOES addition. Make a control by shaking in another tube a
NOT DISAPPEAR because oil evaporates VERY SLOWLY. mixture of chloroform and iodine with no oleic acid.
Explain Results.
4. Acrolein Formation
Place 0.5 g of KHSO4 in a clean dry test tube. Add a drop of Repeat the test using stearic acid, coconut oil, and linseed
coconut oil and heat. oil.
Describe the odor produced: piercing acrid odor / burnt Lightest to darkest:
fat odor Linseed oil > oleic acid > coconut oil > stearic acid
Write the equation involved: Which oil will absorb more iodine, oleic or stearic acid?
Process: dehydration Oleic
KHSO4 Stearic acid does not contain any double bonds

CH2OH-CH(OH)-CH2OH  CH2=CH-CHO (acrolein) + 2 H2O
Heat
CH3(CH2)16COOH therefore it does not absorb iodine.
Acrolein is the simplest unsaturated aldehyde caused by Coconut oil has an iodine value of 7-10
dehydration of glycerol (removal of 2 H2O). (Fats are Oleic acid has a value of 80-88
positive with this because they contain a glycerol group). Linseed oil has a value of 136-178
KHSO4 is used to as dehydrating agent
Which oil contains more unsaturated fatty acids, coconut
or linseed oil? Linseed oil
5. Emulsification
Prepare 4 test tubes with the following contents: Structurally, how does an unsaturated fat differ from a
saturated fat?
Tube 1 5 ml water Saturated fatty acids lack double bonds between the
Tube 2 5 ml of water + 3 ml soap solution individual carbon atoms, while in unsaturated fatty
Tube 3 5 ml water + 1 ml of 1% albumin solution acids there is at least one double bond in the fatty acid
Tube 4 5 ml water + 0.5 ml Na2CO3 Solution chain. Saturated fats are solid at room temperature,
while unsaturated fats are liquid.
To each test tube above, add 5 drops of coconut oil, shake,
and observe results.
#1 – presence of white ring, immiscible

#2 – miscible
#3 – miscible
#4 - miscible
What is emulsion? Emulsion is a dispersion of 2

immiscible liquids, though without and emulsifying
agent, the emulsion is temporary (because those liquids
repel each other, they won't mix).
39
What is the relationship of iodine no. and unsaturation of a

fat? 1. Solubility
Test for the solubility of coconut oil to the following solvents:
Iodine number is directly proportional to the unsaturation
of a lipid. If the iodine number of a carbohydrate is high, its A lipid a non-polar molecule and must require non-polar solvents
unsaturation of fat is also high. to dissolve
Water, HCl–> the coconut oil did not dissolve for these solvents
Hubl’s reagent is prepared by dissolving 26 grams of
are polar
iodine and 30 grams of HgI2 in 1 litre of 95% alcohol.
23 g I2 + 30 g HgI2 in 1 liter of 95% alcohol NaOH–> the coconut oil did not mix with coconut oil but instead
underwent saponification
Iodine number is defined as the amount of iodine in grams
that is consumed/absorbed by 100 grams of a chemical saponification–> the reaction that takes place when triglycerides
substance (in this case, oil). (coconut oil) is reacted to a base medium
In lipids, iodine is absorbed because of double bonds. Double cold alcohol–> it did not dissolve the coconut oil because of the
presence of hydrophilic end
bonds will break and allow iodine in the molecule
(halogenation). hot alcohol–> it did not dissolve coconut oil
GLYCEROL chloroform, ether, carbon tetrachloride- dissolves the lipids

because they are non-polar solvents
C3H8O3
2. Reaction with indicators
Fresh oil are neutral but when exposed to open environment it
undergoes oxidation called rancidity.
(-OH) in oils will turn to (COOH)

7. Glycerol
a) test the solubility of glycerol in water, alcohol, and ether Congo red–> an acid dye
b) make an acrolein test on glycerol
Coconut oil–> did not react with the congo red
result: ->blue litmus remained blue, red litmus remained red
a. Soluble in water and alcohol, in ether it is
Rancid oil–> after adding congo red, color became bluish violet
insoluble due to the lack of –OH group –> blue litmus turned red, red litmus remained red
b. Odor of burnt grease
c) fuse a drop of glycerol in a nichrome wire with

powdered borax. Note the green flame produced. This is
due to the glycerol ester of boric acid. 3. Formation of Translucent Spot
d) perform the Nitro-Chromic acid test on a 5% aqueous –> Lipids soak into the paper fibers but the lipid spot evaporates
solution of glycerol slowly
–> The forces that holds the molecules together are strong
–> It diffracts light because of the difference in refractive index
What is the color formed? Blue (translucent phenomenon)
–> The spot of grease do not receive enough heat to vaporize
This indicates the presence of what group in glycerol?
Hydroxyl group (-OH) terminal due to presence of 4. Acrolein Formation
polar ends –> to detect presence of fat or glycerin when heated strongly in
the presence of a dehydrating agent
Glycerol (C3H8O3) is polar because of the presence of polar KHSO4–> dehydrating agent
ends
(-OH terminals), therefore it is soluble in polar solvents such acrolein–> unsaturated aldehyde
as water and alcohol, but not in non-polar solvents and
coconut oil + heat——-> glycerol + lauric acid
solvents with very very low polarity, such as ether.
glycerol + heat + KHSO4——-> acrolein + water
40
5. Emulsification 7. Glycerol
–> to combine 2 liquids that does not usually mix together with Glycerol–> is a simple polyol (sugar alcohol)
the presence of emulsifying agent
a. Solubility
emulsifying agent–> needed to stabilize the emulsion Glycerol is soluble in water and alcohol due to the presence of (-
OH) group
Oil with the following: Glycerol is insoluble to ether because ether lacks -OH group
Water–> immiscible
Water and soap solution–> miscible b. Acrolein test
Water and albumin solution–> miscible formation of odor of burnt grease
water and Na2CO3–> miscible
c. Flame test
Borax is Na2B4O7*10H2O
6. Test for Unsaturation glycerol + borax—->Boro-glycerine
–> To indicate the presence of double bonds in lipid sample —> formation of green flame
Hubl’s iodine–> alcoholic solution of iodine containing some d. Nitrochromic acid test
mercuric iodide –> there was a formation of blue color due to the presence of
primary and secondary -OH group that reduces Cr VI into Cr IV
–> pink color due to the presence of iodine
–> fats that contain unsaturated fatty acids will have a positive
result
41
EXPERIMENT 8 ENZYMES
Enzymes are complex organic compounds with Boiling a solution containing enzymes
definite chemical structure, secreted by living cells. They  subjecting enzymes in high
have the property of initiation and hastening chemical temperatures denatures, and
reaction without themselves being affected in the process. therefore inactivates them
Enzymes activity is influenced by the concentration of the
enzymes, the concentration of the substrate the Potato  main source catalase
concentration of the products of the reaction, temperature,
pH, inorganic salts, and the presence of activators and H2O2  oxidizing agent and bleaching
inhibitors. All these factors therefore should be considered agent
while performing the following experiment.
Action:
Enzymes are organic substances secreted by living cells 1. Divide the filtrate into four parts and
that increase the rate of reaction without being consumed boil one part for one minute.
in the reaction. Most enzymes are generally proteins. 2. Place the 5 ml of the boiled filtrate in
one tube and 5 ml of the unboiled
Types of Enzymes: filtrate in another tube.
1. Oxidases - Enzymes that catalyze redox reactions. Uses 3. Add to each a few drops of 3%
oxygen as electron acceptor (oxidizing agent). hydrogen peroxide. Note what
2. Catalases - Enzymes that catalyze the decomposition of happens.
hydrogen peroxide to oxygen and water
3. Peroxidase - Enzymes that catalyze the oxidation Unboiled filtrate  there was a
(similar to oxidases) of a hydrogen donor (or electron formation of bubbles because of
donor) with the help of Peroxides (such as H2O2). more oxygen that was catalyzed by
the catalase
Boiled filtrate  there was less

I. Oxidases from Fruits formation of bubbles due to the
denaturation of the enzyme
Cut thin slices of apples, guava, chico, or catalase, that is why there is a
other available fruits and expose them to the formation of coagulants
air. Observe the darkening of the exposed
sliced slides. Explain the color change.
The exposed area turned brown due to the

formation of melanin. Oxidases are
enzymes that catalyzes redox reactions
and use oxygen as electron acceptors.
Fruits are oxidized because of the enzyme
tyrosinase, or monophenol oxidase that
reacts with oxygen and iron-containing
phenols that basically makes a layer of
rust on the surface of the fruit.
II. Catalase
A. Catalase from Potato
Preparation:
1. Pare a small potato and grate it to a
fine pulp.
2. Mix this pulp with 100 ml of water
and let it stand for 15 minutes and
stain this through a piece of cheese
cloth.
3. Filter the extract
42
Procedure:
III. Oxidase from Potato 1. Prepare 3 test tubes and place 5 drops
There are three oxidases: each of the following substances:
1. Monophenol oxidase (hyrosinase) – a. 1% phenol
responsible for oxidizing phenol to  Arene with 1 -OH group
cathecol, to O-quinone and finally forms Observation:
condensation brown compounds of Color changed from very light pink
unknown composition. to pink
Also called tyrosinase, it is responsible for b. 1% cathecol

oxidizing monophenols (phenols  Arene with 2 -OH groups
containing only one -OH group) to Observation:
diphenols (phenols containing 2 -OH Color changed from carmine red to
groups ). In the experiment, this enzyme red-orange
oxidizes phenol (C6H5OH) to
catechol(C6H4(OH)2) and then to O- c. Pyrogallol
quinone, also called benzoquinone (check  Arene with 3 -OH groups
wiki for formula) . Benzoquinone then Observation:
forms a brown condensation product. Color changed from tan to brown
2. Polyphenol oxidase (cathecol oxidise) – 2. Add 1 ml of potato extract (oxidase) to

facts on cathecol forming o-quinone, then each test tube and shake.
the unknown brown compounds. It acts 3. Allow them to stand until the end of the
on pyrogallol to form purpurogallin. laboratory period, observing and
recording the initial color change, then
It is responsible for oxidizing phenols the change at the end of the period.
containing more than one -OH group,
such as catechol. In this experiment, this Final results (lightest to darkest):
enzyme acts on catechol to form o- Phenol  light pink
quinone which then forms brown Catechol  light orange
condensation products(similar to Pyrogallol  brown
monopheno oxidase). It also acts on
pyrogallol to form pupurogallin.
3. Cytochrome oxidise acts in conjunction

with – cytochrome, oxidizing IV. Peroxidase from Potato
phenylediamine, which in the presence Peroxidases, unlike oxidases, may require
of alpha-naphthol forms indophenol. a co-factor, phenol-oxidase to complete their
action. They also require H2O2 as the source of
oxygen, and upon which the phenol-oxidases
act. Phenol oxidase is also found abundant in
potatoes.
Peroxidase  hemoprotein that catalyzes

the oxidation of H2O2
Procedure:
1. Place 3 test tubes and add 5 drops each of
the following substances:
a. 1% phenol
 Flesh-like (lighter than oxidase
action color)
b. 1% cathecol
 Deep red
c. Pyrogallol
 Dark brown
43
How do you know that the gas

liberated is oxygen?
2. Add 1 ml of potato extract (peroxidase) to
each. The flame in the splint continued
3. Mix well, then add 3 drops of 3% to glow due to the oxygen emitted
hydrogen peroxide to each. by the liver
Observe and record the color changes What does catalase do?
produced and compare them with the
results observed on the action of oxidases Catalase from liver catalyzes the
with the same reagents. decomposition of hydrogen
peroxide to water and oxygen gas
Phenol  light pink with bubbles (due at a rate of 40 million molecules of
to the presence of oxygen) hydrogen peroxide per second.
Catechol  light orange with bubbles The liver filters the body using
(due to the presence of enzymes as catalysts. It makes
oxygen) harmful substances less toxic like
hydrogen peroxide to water and
Pyrogallol  brown with bubbles (due oxygen, and alcohol to aldehyde.
to the presence of oxygen) Lack of catalase causes the graying
of hair in humans.
H2O2  H2O + O2
Enzymes–> are complex organic compounds with definite

chemical structure, secreted by living cells
B. Catalase from liver

1. Place 1 gram of liver, 4 ml of water,
and a little sand in a mortar and
grind.
2. Add 2 ml of 3% hydrogen peroxide.
3. Test the gas evolved using a glowing
splint.
44
1. Oxidase from Fruits 3. Oxidase from Potatoes
The exposed area turned brown due to the formation of a. Monophenol oxidase (tyrosinase) Cresolase activity:
melanin. monophenol + O2 —> diphenol catechol
Fruits contain a certain type of enzyme called monophenol b. Polyphenol oxidase (catechol oxidase) Catecholase
oxidase or tyrosinase that reacts with O2 and iron- activity: Diphenol catechol —> quinone
containing phenols that basically makes a layer of rust on
the surface of the fruit c. Cytochrome oxidase Berthelot’s Reaction:
phenylenediamene + alpha-napthtol ——> indophenol
Tyrosinase–> is a bifunctional copper containing oxidase
having both catecholase and cresolase activity Phenol- arene with 1-OH group
catechol- arene with 2 -OH group
Orthohydroxylation: —> addition of two -OH group in pyrogallol- arene with 3 -OH group
ortho-directing substitution
Phenols in fruits + O2 + 2H ion —–> catechols
after this:
Dehydrogenation: —> removal of hydrogen Catechols + 4. Peroxidase from Potato

O2—–> 2 orthoquinone —> melanin (brown pigment) Peroxidase –> is a hemoprotein that catalyzes the
oxidation of H2O2
2. Catalase from Potato Catalase—> speed up the phenol–> light pink with bubbles at the top due to
decomposition of hydrogen peroxide to water and oxygen presence of oxygen
catechol–> light orange with bubbles due to the presence
Potato —> main source catalase in the experiment of oxygen
pyrogallol–> brown with bubbles due to the presence of
H2O2—> colorless, viscous liquid used as an oxidizing oxygen
agent and bleaching agent
Results: 5. Catalase of liver

Unboiled filtrate—> there was a formation of bubbles Glowing splint–> test for an oxidizing gas (oxygen)
because of more oxygen that was catalyzed by catalase
Boiled filtrate—> there was less formation of bubbles due Liver–> filter of the body that uses enzymes as catalysts
to the denaturation of the enzyme catalase. That is why – -> makes harmful substances less toxic like:
there is a formation of coagulant H2O2–> water and oxygen
alcohol–> aldehyde
45
EXPERIMENT 9 TEST FOR NUTRIENTS IN FOOD
For each of the following tests, use the following food

samples: a. Microscopical examination: Examine the crystals
under a microscope. Sketch the crystals.
Fruit juice, potatoes, butter, green and ripe bananas,
carrots, cheese, cooked egg white, cooked egg yolk,
unsalted dried fish meat like dills, cooked rice ( mashed ),
chopped or ground peanuts ( uncooked ), ( margarine may
be used instead of butter).
1. Starch: Boil a small amount of the food sample in a test

tube with 7 ml of water. Cool by holding it under running
cold water, and add a drop of iodine solution. A deep blue
color indicates starch;
b. Acetic anhydride - H2SO4 test (Lieberman-Buchard
2. Glucose: Add a few drops of a fruit juice to 10 ml of test)
water in a test tube and then Benedict's solution sufficient Dissolve a few crystals of cholesterol in 2 ml of chloroform
to give a very light blue solution. Boil. A yellow to red, in a dry test tube. Add 10 drops of acetic anhydride and 2
precipitate indicates the presence of a simple sugar such as drops of conc. sulphuric acid, and mix. Allow to stand and
glucose or fructose. note the production of colors. This is the basis for the
quantitative determination of cholesterol. The solution
3. Fats: To examine foods for oil or fat, shake a small becomes red, then blue and finally bluish-green.
portion of the food with 3 ml of ether in a test tube for
several minutes, then decant to an evaporating dish or
watch glass and allow the ether to evaporate
spontaneously (without heating }. Place the residue on a
piece of white paper, warm. When held to the light it will
be translucent as an indication of a fat.
4. Proteins: Use 2 ml of the aqueous food solutions and

perform the Biuret test.
5. Mineral Matter: Bum a very small amount of cheese on

an evaporating dish until all of the black carbon is
oxidized. The formation of any white powdery residue
indicates the presence of mineral matter.
Negative and Positive Results.
6. Carr-Price Test for Vitamin A: To 1 ml chloroform add 2
drops or a pinch of the food sample. Cool in an ice bath and c. Sulphuric acid test (Salkowski's test): Dissolve a small
add 2 ml of a cold saturated solution of antimony portion cholesterol in a little amount of chloroform and
trichloride. Observe the changes in color. How long did the add an equal volume of conc. sulphuric acid layer. Note the
color persist? Perform this test on margarine or butter and color produced both in the chloroform and sulphuric acid
carrots only. layer. A play of colors from bluish-red to cherry red and
purple is noted in the chloroform layer while the acid layer
7. Preparation of Cholesterol: Evaporate the acetone- has a marked green fluorescence. Note: 1 ml of chloroform
ether filtrate from no.1 to about one-fifth its original and sulfuric acid will be sufficient.
volume of until crystals are formed. If it evaporates to
dryness, redissolve the solid in a very small amount of
acetone and then allow to cool. Use the solid for the
following tests:
46
Write the formula of Vitamin A and state its importance.
Vitamin A - Retinol
Molecular Formula : C20H30O
Vitamin A is important for normal vision, the immune

system, and reproduction. It also helps the heart, lungs,
kidneys, and other organs work properly.
Positive and Negative Results.
What are your conclusions regarding the general

constitution of your food samples? Tabulate your results in
a separate sheet of paper. Indicate by a positive sign (+)
the nutrients found in each food and the negative sign (-)
if absent.
Food Starch Glucose Protein Fats Mineral Matter Cholesterol Vitamin A

Ripe Banana
Green Banana
Carrots
Egg white (cooked)
Egg yolk (cooked)
Peanut Butter
Potato
Cheese
Fruit Juice
Dilis
Uncooked peanuts
Cooked Rice
Milk – Experiment 11
Milk 1. Reaction of Milk
- Most complete food
- Proteins, carbohydrates, vitamins, fats, inorganic pH - Milk is slightly acidic (fresh milk has a pH of
salts 6.7)
- is a complex dispersion of fat globules and
casein micelles in an aqueous suspension of 1 ml of milk – red & blue lithmus paper ; congo
whey proteins, soluble caseins, salts, and lactose. red ; phenolphthalein solution
-
Results:
Fresh Unboiled Milk  red & blue lithmus paper – stays the same
- Enzyme, protease, lipase, lactase, phospotase, - milk is neither acidic or alkaline
catalase, peroxidase  Congo red – red / pink color solution
- Redox reaction
Casein  Phenolphthalein – colorless
- Chief protein of milk -commonly used indicator for titration
- Can be precipitated by acid, and carrying with it
the milk fat 2. Determination of Specific Gravity
- is a phosphoprotein, a kind of conjugated - desimeter
protein. It is the chief protein found in milk.
- Fresh Milk - low specific gravity – contains milk fat
Fat Skimmed Milk – higher specific gravity – no milk fat
- Can be extracted by organic solvents
Milk Fat
Filtrate from casein and fat - Highest constituent of milk ‘
- Contains soluble constituents like lactose and - More  lighter
inorganic salts - Less  heavier
Proteins of milk 3.Film Formation

- Derived from amino acids of the blood, - 5 ml of diluted canned milk in a small beaker and boil
- The synthesis occurring in the mammary glands
Sour Milk – no film bec. the proteins are already
Milk Fat denatured by additional acetic acid
- Origin is in phospholipid of the blood Fresh Milk – forms film
Lactose of Milk Factors that affect Film Formation:

- Derived from glucose of the blood  Temperature
 Stirring/ Whisking
Derived from blood through the process of filtration  Presence/Absence of milk fat
- Inorganic salts: calcium, magnesium, sodium,  Type of milk
phosphates, citrates and chlorides
Film – milk skim or lactoderm is cause by denaturation
Bone of proteins such as beta-globulin
- Reserve supply of calcium - Soluble milk proteins denatured and coagulate
with milk fat
Human Milk differs from cow’s milk - whey proteins denature; at higher temperatures,
- Less casein and ash casein proteins also denature.
- More albumin and lactose
due to a transverse hydration gradient that drives
evaporation and induces stresses in the milk skin, which
Acidity of Milk is due to lactic acid. It produced by the are alleviated through wrinkling.
action of the lactic acid organisms on the milk sugar. It
is now generally believed to be due to the presence of The film, also called milk skin is formed by fat-protein
the carbon dioxide, acid phosphates and casein, all of interaction. When milk is boiled, the water at the surface
which are found in fresh milk and which have an acid.
evaporates, thereby exposing the fat and protein the isoelectric pH of casein, leading to increased
molecules, which bind and dry out. At high molecular repulsion and dissolution of casein.
temperatures, the proteins tend to clump up. As it is
heated even more, the soft layer dries up. Skin formation  NaCl – soluble
does not form in skim milk because of the absence of Non-Concentrated(10%?) NaCl Solution - Soluble.
fats; without fat, there is nothing that proteins would Low salt concentration increases the solubility of
bind to. Only surface fats and proteins contribute to the substances through the effect called "Salting-in"
film.
In sour milk, lactose from the milk is oxidized by  HCl – insoluble
bacteria into Lactic Acid, which acidifies the solution Dilute HCl Solution - Insoluble. Isoelectric pH
leading to the denaturation of the proteins. of casein is slightly acidic. The pH of this solution is
close to isoelectric pH of casein. Reduced molecular
4. Coagulation Test repulsion
- 1 ml of fresh milk in a tt and acidify with acetic acid.
Heat to boiling Millon’s test – formation of red ppt.
- formation of casein: involves two process: - Due to the denatured proteins that forms amino
 Rennin converts the caseinogen into paracasein acids tyrosine
or soluble casein
 The calcium salts present in the milk precipitate dilue acetic acid is added to reach the isoelectric point of
the paracasein as casein casein. Adding excess acid lowers pH beyond isoelectric
Is there coagulation? yes point of casein leading to dissolution.
-coagulation due to the presence of whey protein which The precipitated protein is positive with Millon's Test
is mainly composed of beta-lactoglobulin (red ppt formation)
Acidifaction of milk with acetic acid allows the proteins

to reach isoelectric point, lowering the intermolecular 7. 3-ml portions of whey
repulsion between proteins, leading to coagulation(or
increased precipitation) when heated. a. Coagulation by heat
- Formation of coagulation of the coagulum
5. Action of Hot Alkali (lactalalbumin & lactoglobulin)
- mix: 1 ml of milk with drops of 6 M NaOH Biuret test
- heat - Positive : violet
NaOH: oxidizing agent b.
Results: presence of brown solution and caramel odor  Phosphorus– yellow ppt ( ammonium
due to the reducing sugar glucose phosphomolybdate)
- Moore’s test – test with same principles Phosphate Ion + Ammonium molybdate =
ammonium phosphomolymdate
Moore's Test. Polymerization of aldehyde groups of
sugars (in milk, it is lactose).  Calcium – white ppt. (calcium oxelate)
6. Preparation of Casein Calcium Ion + ammonium oxalate =

-10 m of milk calcium oxelate + ammonium ion
- dilute acetic (1%) – drop by drop – flocculent ppt.
forms ; if excess dissolution may occur  Millon’s test – orange ppt
-supernatant fluid (whey)
-add ethyl alcohol : used to remove moisture
- ether – to remove milk fat  Benedict’s Test – Cuprous Oxide
- Red ppt. due to the presence of reducing sugar
Results:
 Water – soluble
Whey from milk contains all other substances found in
 NaOH – soluble ‘ milk such as lactalbumin and lactoglobin proteins
NaOH Solution - Soluble. Isoelectric pH of casein (Biuret test, violet sol'n), phosphates (yellow ppt,
is slightly acidic. The pH of this solution is far from Ammonium phosphomolybdate), calcium (white ppt,
Calcium oxalate) and lactose(Benedicts test, orange ppt,
Cuprous oxide) apart from the separated casein.
8. Milk Fat
- transfer filtrate from no. 6 in a evaporating dish
- place in a boiling bath to evaporate the ether
- presence of residue: MILK FAT
- touch residue with a piece of paper: formation of
translucent spot
Fat from milk is non-polar and dissolves in the non-polar

ether. When placed in a evaporating dish at room
temperature, ether evaporated due to its volatility and
left behind a residue - milk fat. Tested with Translucent
Spot Test
Salivary Digestion - Experiment 12
Saliva Proteins present in saliva:
- Moistens the mucous membrane of the mouth  Amylase
and helps in preventing tooth decay by cleansing  Defensins
the mouth of cariogenic carbohydrates and by  Cystatins
neutralizing lactic acid.  Histatins
- Contains 99% water and less than 1 % solid  Immunoglobulins
- Mucin: chief solid  Statherin
 Lactoperoxidase
- Secreted by 3 pairs of glands:  Lysozyme
 Parotid  Lactoferin
 Submaxillary
 Sublingual 1. Reaction
 Hundreds of buccal glands - Resting saliva: phenolphthalein, litmus and
congo red
- The flow is stimulated by: - Stimulated saliva (chewing paraffin for 5 mins)
 Psychic
 Chemical Resting Saliva
 Mechanical - Saliva found in mouth in the intervals of food
taking and mastication
- It contains a protein, mucin - Basic
- An enzyme, ptyalin Phenolphthalein Litmus Congo Red
- Inorganic salts
Inorganic Salts in saliva has 2 important functions: Violet Red  blue Red w/
 Phosphate acts as buffers that tend to Blue bubbles
maintain the reaction (pH) of the saliva
constant
 Chloride ions are essential as coenzymes for Stimulated Saliva
the salivary amylase. - Saliva secretions during stimulation
- more basic
The flow of saliva (about 1500 cc daily) is the result of - higher pH
stimulation of the salivary glands by the nervous system, Phenolphthalein Litmus Congo Red
as is evidence by the fact that actual contact with food is
unnecessary since sight, odor, or even though will cause
the salivary gland to secrete profusely. Violet Red  blue 2 layers:
Blue red (upper
part)
Salivary Amylase/Ptyalin clear (down)
- Begins the breakdown of starches, responsible
for the digestive function of the saliva
- Principal enzyme of saliva Paraffin – stimulates saliva secretions
- Capable of hydrolyzing cooked starch into The acidic pH is essential in dissolution of enamel to
dextrin and maltose produce dental carries
Starch  soluble starch  dextrin  maltose 2. Test for Mucin

- 3 ml of saliva
The maltose so produced is later hydrolyzed by the - 1-2 drops of dilute acetic acid
intestinal maltase into glucose.
Mucin – is a mucopolysaccharide or glycoprotein that is
chief constituent of mucus
Salivary Digestion - is formed when there is coagulation due to
- Hydrolysis of starch by salivary amylase heating and its function is to lubricate the mouth
(ptyalin) and prevent bacterial build up
- Takes place in buccal cavity
- produced by epithelial cells and used as food
lubricant Calcium
Result: - clear with moisture (once HCL is dropped)
- cloudy solution with white ppt (Mucic) - Positive
Function: - Name of ppt: Calcium Oxelate (CaC2O4)
Mucin can be a symptom to any abnormalities in the - Dominant metal ion present
body such as cancer
Sulfate
3. Inorganic Matter - Clear
- Acidify 10 ml of saliva with a drop or 2 of acetic - Negative
acid
- Heat to boiling NORMAL PH RANGE OF SALIVA: 5.6 – 7.9
- Filter to remove protein - Several factors influence the pH nature of the
saliva and varies depending to the food and
Test filtrate for CHLORIDES, PHOSPHATES, drinks intake of an individual
SULFATES AND CALCIUM
4. Digestion of Starch Paste
Chloride salts and phosphate salts – most abundant - 10 ml of 1% starch paste in a small beaker
inorganic matter in saliva - add 5 drops of saliva and stri
- iodine (after 5 mins)
Chlorides: AgNO3 + HCl - opalescence of the starch solution disappears due
Phosphates: HCl + (NH4)2MoO4 to the formation of soluble starch
Sulfates: BaCl2 + HCl
Calcium: HCl + K2C2O4 Maltose – responsible to reducing action
10-20 mins – it takes to completely transform starch to
Results: reducing sugar
Chlorides Turbid soln with white Reaction of iodine with Benedict’s Test: Blue (Positive)
ppt.
Phosphate Clear, colorless soln with 1. Boiled Starch + ptyalin = soluble starch
pale yellow ppt 2. Soluble starch + ptyalin = erythrodextrin +
Sulfates Slightly turbid soln maltose
Calcium Clear, colorless soln 3. Erythrodextrin + maltose + ptyalin =
achrodextrin + maltose
4. Achrodextrin + maltose + ptyalin = isomaltose +
Chlorides maltose
- WHITE ppt
- Positive The optimum pH for the action of Ptyalin is 6.7
- Name of ppt: Silver Chloride (AgCl) In a theoretical setup, if starch paste becomes negative in
- Major amylase activator the test with iodine, it is surely positive with the test for
- Chloride ions help in maintaining the osmotic sugar (because starch is completely converted to
balance in the mouth preventing any excess glucose).
inflow or out flow of water from water tissues
5.Influence of Acid
AgNO3 + Cl-  AgCl + NO3 - 2 ml of:
 0.25 % HCl
Phosphates:  0.1 % HCl
- Yellow ppt  0.05% HCl
- Positive  0.025% HCl
- Name of ppt: Ammonium Phosphomolybdate  0.0075% HCl – obtain the greatest digestion
(NH4)3PMo7O40 because it is the least acidic
- pH buffer - place in a water bath for 20 mins at 40 C
- functions: contributes to solubility product of
calcium phosphate, which is crucial in 6.Influence of Alkali
maintaining tooth structure, important as a - repeat test in #5 but use:
buffer and an essential nutrient for oral  2% NaOH
microflora for metabolic pathways  1% NaOH
 0.5% NaOH - Silver nitrate
 0.125% NaOH
 0.065% NaOH Reagent for phosphate test
- neutralize with acetic acid - Ammonium molybdate
Reagent for sulfate test

Acid – has the greatest inhibiting power because it - Barium chloride
disrupts the basicity of the saliva which is necessary for
buffering capacity Reagent for calcium test
- Ammonium oxalate
Gastric juice starts protein digestion while saliva starts
starch digestion What is responsible for reducing reaction in benedict’s test
- Maltose
It is inhibited more by acids, therefore ptyalin is slowly
inactivated by the acidic Gastric Juice in the stomach. Which has greater inhibiting power? Acid or alkali?
Salivary digestion continues in the stomach for 10 to 20 - Acid
minutes due to the slow penetration of acidic Gastric
juice into the bolus (mass of chewed food). After the 10 B.S Ptyalin
to 20 minute period, salivary digestion stops due to the
total inactivation of ptyalin.
Questions:
Saliva is secreted by 3 pairs of glands S.S
- Parotid
- Submaxillary
- Sublingual
Factors that stimulate salivary flow

- Psychic
- Chemical
- Mechanical
Protein in saliva
- Mucin
Enzyme in saliva
- Ptyalin
Aka salivary amylase

- Ptyalin
Hydrolyzes starch
- Salivary amylase
Where does salivary digestion occur

- Buccal cavity; fundic end of stomach
Why is saliva basic and not acidic

- If its acidic it will dissolve enamel producing dental
caries
Used to precipitate mucin out of saliva

- Dilute acetic acid
Function of mucin
- Lubricate mouth & prevent bacterial build up
Abundant inorganic salts found in saliva

- Chlorides & phosphates
Reagent for chloride test

Bile a. Gmelin’s Test
- Very complex and varies according to the Superimpose 1 ml of bile to 1 ml of conc. HNO3.
nutritional state of an individual Production of green, blue, violet, red and reddish-
- Secretion of the liver yellow layers of color at the point of contact. This color
- It is viscid and has alkaline reaction and its color formation is due to the oxidation of bile pigments by
is greenish brown HNO3.
- It’s important constituents:
 Bile acids b. Rosenbach's modification of Gmelin's Test
 Bile pigments bile is instead filtered and a drop of HNO3 is added at
 Inorganic salts (potassium, sodium, the dried cone. Formation of concentric succession of
bicarbonate) colors indicate presence of bile pigments.
 Cholesterol
4. Test for bile acids and bile salts
Organic Constituents: Bile acids - Primary bile acids for humans is Cholic
 Bile salts Acid and Chendeoxycholic acid.
 Bile acids
 Bile pigments There are two main groups of bile acids: taurocholic
acids and glycocholic acids.
Inorganic Constituents
 Chloride a. Pettenkofer's Test or Sucrose-H2SO4 test
 Sulphates Test for bile acids. One drop of sucrose sol'n is added to
 Phosphates 1 ml of dilute bile. H2SO4 is allowed to run down the
side of the tube. Red ring forms at the point of contact.
1. Reaction Shaking makes the whole solution red.
- Bile to litmus paper, congo red and
phenolphthalein b. Foam Test
1:1000 soln of furfural is added to 1 mL of bile solution.
Results Mixture is shaken to form foam. H2SO4 is added to the
Litmus Paper Blue foam. Foam produces a pink coloration. Color formation
Phenolpthalein Yellow indicates the presence of bile salts/acids
Congo Red Red
c. Hay's Test of Surface Tension
Normal pH value of bile: 7 – 8.4 (basic) Surface tension is the tendency of the surface of a liquid
to resist external force.
2. Inorganic Constituents Surface tension is due to the cohesion of water
- Evaporate 10 ml of bile to dryness molecules. Surface tension is inversely proportional to
- Fuse residue with fusion mixture (2 oarts of temperature that is why the water was cooled to 17 °C
sodium carbonate and 1 part potassium nitrate) in the experiment.
- Extract with 10 ml of water Bile salts are emulsifying agents. Emulsifying agents
- Acidify with HNO3 reduce the surface tension of liquids, for example,
Results water, allowing substances that float normally on water,
Chloride White Calcium chloride such as sulfur, to sink.
Sulphate Yellow Barium Chloride
Phosphate Turbid - 5. Cholesterol in Bile
Bile is evaporated to dryness. Ether was placed in the
3. Test for Bile Pigments residue to extract cholesterol since ether is non-polar.
Bile pigments: The ether extract is allowed to evaporate and the
 Bilirubin – gives the red color in the tests residue is tested with Liebermann-Buchard test on
 Biliverdin – gives the green color in the tests chloroform solution. A red ring formed which
 Bilicyanin – gives the blue color in tests progressed to form a green ring.
Normally, bile contains enough chemicals to dissolve a CONSTITUENTS OF BILE
normal amount of cholesterol. Excessive cholesterol in Secretion Components Excretion Components
bile forms crystals since cholesterol can no longer be  Bile Salts  Bile pigments
dissolved which leads to formation of Gallstones.  Sodium Hydrogen  Cholesterol
Gallstones can then lead to blockage of the path of bile. Carbonate
 Water
BILE
- A viscous, yellow to brown, bitter-tasting THE BILE SALTS OR BILE ACIDS
alkaline fluid
- Secreted 500 to 800 cc daily by the liver Bile
- Flows via the bile ducts into the duodenum - Contains no digestive enzymes
- Plays an important role in digestion and
Rate of secretion and of flow of bile absorption of fat and indirectly that of other
- Influenced by the nature of food undergoing foodstuffs
digestion - Alkaline
- The stimulation, probably being influenced by a - Bile, along with the pancreatic juice and
hormone: SECRETIN intestinal juice
o Neutralizes the acid chime from the
Epsom Salts stomach
- Salt that stimulates the flow of bile - Contains a type of compound called the bile
salts or the bile acids
Absence of food:
Most of bile is diverted to the gallbladder BILE SALTS (or bile acids)
(about 30 cc capacity) - The two most important of these substances
- Where it is stored and becomes concentrated to are:
one-tenth its previous volume by: 1. Sodium glycocholate
1. Lymphatic reabsorption of: 2. Sodium tyrocholate
o Water
o Salt - A most important property of the bile salts:
o Cholesterol o They act as wetting agents, lower
o Pigment from it surface tension and facilitate the
2. By the addition of mucin emulsification very little fact digestion
 A secretion from the wall of the by pancreatic lipase (streapsin) occurs
gallbladder in the intestine
Active digestion: - Play an important role in fat absorption by

The contents of the gallbladder are evacuated uniting with the insoluble fatty acids, liberated
into the duodenum and thus provide a from fats by lipase action, to form soluble
concentrated supply of bile compounds known as choleric acids
Bile Choleric acids

- May also be considered as excretion  Upon absorption, separate
o Since it eliminates as waste products of again and the fatty acids unite
cellular action with glycerol in the lymphatic
o Certain substances which it alone can vessels of the villi (lacteals) to
dissolve (e.g. cholesterol) as well as form fats which are transported
other end products by the lacteals to the thoracic
- Contains: duct and eventually to the
o Lipids bloodstream
o Mucin
o Bile pigments
o Certain salts
Liberated bile salts In this decomposition of hemoglobin into globin
 Are then available for further and hematin, the latter with loss of iron,
use by the liver changes into bile pigments which impart a
 Are resecreted into the bile yellow to brown color to bile.
 Thus undergo a type of
circulation: Bilirubin → has a reddish cast
BLOOD
↳ LIVER Biliverdin → oxidized product of bilirubin
↳ BILE → a green pigment
↳ INTESTINE
↳ BLOOD Normal: little or no biliverdin in the bile
- Possess the property of stimulating the Exposure of bile to air: it will turn green due to
secretion of bile oxidation of bilirubin to biliverdin
o As such, it can be regarded as
cholagogues Oxidation
 Substance stimulating the flow o Produces a series of colored
of bile compounds, including
bilicyanin
- Also aid peristalsis  A blue pigment
- Absence in the adequate bile flow: Series of colors on bruised skin:

o Extensive putrefaction of protein Undoubtedly due to the decomposition of
occurs in the lower portion of the hemoglobin liberated by injured red cells which
gastrointestinal tract exuded from the capillaries of the injured tissue
 Due in part of the decreased
peristalsis and in part to the Obstruction to the bile ducts
undigested fat
 Forming an oily film Disease and dysfunction of the liver or an abnormal
over protein particles destruction of the red blood cells
and retarding their - Will result in an increased amount of bilirubin in
digestion the blood
o This will diffuse into the skin and will
- Absence of bile give them a characteristic yellow color
o Causes serious disturbances of which known as jaundice or icterus
intestinal digestion and absorption
o Feces In the INTESTINES:
 Contain greatly increased - The reducing bacteria change bile pigments to
quantity of fat and may be clay stercobilin or urobilin
colored and greasy, and leave a o A brown pigment which accounts for
very foul odor the characteristic color of feces
o Constipation
 May result from decreased Urobilin
peristalsis - A part of the yellow color of urine is due to this
pigment
THE BILE PIGMENTS - Of secondary importance to another yellow
- Consists mainly of: pigment in urine called urochrome
o Bilirubin
o Biliverdin Diarrhea
- Mostly formed when the liver salvages “organic - Too frequent elimination
iron” from the blood pigments (hemoglobin) of - Does not allow for much reduction of bile
worn-out red blood cells pigments with the consequence that feces then
have a decided yellow instead of brown color,
the color depending somewhat on the cause of
diarrhea
CHOLESTEROL
- The remaining important excretory compound
of the bile
- Ordinarily soluble in bile
In the presence of foreign substances (such as

injured cells or bacteria):
- The cholesterol tend to crystallize, carrying with
it some bile salts and pigments to form
gallstones
- An alcohol (C27H45OH)
- Like glycerol (C3H5(OH)3), unites with fatty acids

to form esters but it differs in that its esters do
not easily saponify
Lanolin
- The fat of sheep’s wool
- Contains the stearic, palmitic, and oleic esters
of cholesterol
URINE Oxidation
- A filtrate from the blood - when allowed to stand without preservative,
- Serves as a medium for excretion of: urine becomes ammoniacal in odor and alkaline
o Water in pH
o Acids o because of the oxidation of urea to
o Bases ammonium carbonate
o Waste products of metabolism
o Other toxic materials Organic Constituents
- Helps in the maintenance of water balance, acid  Urea
base equilibrium  uric acid
- Serves as an important factor in the  creatinine
detoxification of the body
Inorganic Constituents
COLLECTION AND PRESERVATION OF URINE SAMPLE  Uric acid
24-hour specimen  Sodium urate
- Examined for the study of both the qualitative - precipitate in acidic conditions
and quantitative composition of urine o These precipitates are the primary
- Bladder is emptied at 8:00 AM (urine is components of kidney stones.
discarded)
o All the urine from this time up to Calcium phosphate
8:00 AM the next day is taken as sample - precipitates in alkaline urine
Toluene Specific Gravity Range of normal urine

- A thick layer of this is over-layed on the surface - range of 1.015 - 1.025
to preserve the urine
2. DETECTION OF CREATININE
1. GENERAL CHARACTERISTICS Creatinine
- breakdown product of creatine phosphate
1,000 – 1,500 mL
- Volume of the 24-hour urine
A. NITROPRUSSIDE TEST - Weyl
A. EXAMINE THE SPECIMEN AS TO COLOR, ODOR, 5 ml of urine
TRANSPARENCY AND REACTION + 3 drops SODIUM NITROPRUSSIDE
+ NaOH (to alkalinize)
Responsible for the normal color of urine: = ruby red color which turns yellow
- Urochrome/urobilin pigments (yellow) - indicative of the presence of creatinine
- Uroerythrin pigments (amber)
- Uses sodium nitroprusside
What happens when the urine is allowed to stand for - Red ppt indicates presence of creatinine
some time, exposed to air? - Color fades in normal urine if added with acid
- It smells of ammonia due to certain types of
bacteria present in the water that work on the
urea present in the urine and convert it back
into ammonia, thus the longer it sits the
stronger it gets
Urine pH of 6 when freshly voided due to:

- disodium phosphate - Na2HPO4
- monosodium phosphate - NaH2PO4
Freshly voided urine is clear and transparent.

If transparency is of cloudy flocculate, it might
be because of mucus and epithelial cells
B. PICRIC ACID REACTION - Jaffe 5. DETECTION OF CHLORIDE
5 ml urine 5 ml urine
+ aqueous sol’n of picric acid + acidify with 2 drops of HNO3
+ NaOH (to render the sol’n alkaline) + 2 drops of AgNO3
= red color = White ppt of AgCl formed
- Due to the formation of a red tautomer of
creatinine picrate + excess of NH4OH
- Turns yellow when the sol’n is acidified = Excess NH4OH dissolves AgCl2
- Glucose: gives a similar red color only upon
heating Normal amount of chlorides eliminated in 24 hours:
- 10-15 grams of chlorides are eliminated in 24
- Red ppt due to formation of tautomer hours.
of creatinine picrate o Usually eliminated in the form of NaCl.
- Normally turns yellow when acidified
- If it does not turn yellow, acetone bodies are 6. DETECTION OF PHOSPHATES
present. 10 ml urine
+ AMMONIUM HYDROXIDE (to alkalinize)
Jaffe reaction Warm.
- a colorimetric method used in clinical = Precipitation of earthly phosphates (Ca and Mg salts)
chemistry to determine creatinine levels in occur
blood and urine
- color change that occurred was directly → The earthy phosphates of Ca and Mg separate.
proportional to the concentration of creatinine → Filter off the earthy phosphates.
- however also noted is that several + small amount of MAGNESIA MIXTURE (to the filtrate)
other organic compounds induced similar → Warm the Solution.
reactions = Magnesia mixture precipitates alkali phosphates (Na
and K salts)
3. DETECTION OF PIGMENTS
→ Determine which form of phosphate is present in
AMMONIACAL ZINC CHLORIDE TEST larger amount.
2 ml urine
+ 1 ml of NH4OH = Alkali phosphates are larger in amount compared to
→ Let it stand for a while. Earthly phosphates with a ratio of 2:1
→ Filter.
+ 2 drops ZINC CHLORIDE SOLUTION (to the filtrate) PATHOLOGIC CONSTITUENTS
= greenish fluorescence
- indicates the presence of urobilin 7. ALBUMIN
other pigments normally present in urine: (? Not sure) A. COAGULATION TEST

- urochrome / urobilin 5 ml urine
- uroerythrin → Heat to boiling.
- indican → Filter if urine is not clear.
= if heated portion becomes cloudy, the turbidity may
INORGANIC PHYSIOLOGICAL CONSTITUENTS be due to phosphates
4. SULPHATES + 3-4 drops of VERY DILUTE ACETIC ACID

A. DETECTION IN INORGANIC SULPHURIC ACID → Warm.
5 ml urine = phosphates will dissolve
+ 5 drops of ACETIC ACID = a more flocculent ppt will be produced if only
+ BARIUM CHLORIDE sol’n albumin is present
= ppt of BARIUM SULPHATE
- White ppt of BaSO4 B. HELLER’S RING TEST
5 ml conc. Nitric acid
→ Slant the tube. Is bilirubin normally present in the urine?
→ Very carefully allow an equal amount of urine to Bilirubin is normally ABSENT in urine because
slowly run down the side of the tube. bilirubin is converted to urobilinogen in the intestines
= urine will float on the nitric acid and then absorbed in the bloodstream. The absorbed
= a white ring (precipitated protein) will appear at the pigment is then oxidized to urochrome or urobilin which
junction of the two liquids makes its way to the kidneys(responsible for urine
- This confirms the presence of albumin color).
Sometimes the white zone does not appear until What does its presence indicate?
allowed to stand for a few minutes. Presence of bilirubin indicates obstruction of
the flow of bile from the gall bladder so that the bile is
8. GLUCOSE absorbed in the blood and bilirubin goes to the kidneys.
BENEDICT’S TEST (semi-qualitative test) 11. ACETONE BODIES

5 ml of Benedict’s reagent
+ 5 drops of urine Ketone bodies
→ Boil vigorously for 2 mins - are brought about by increased lipid
→ Set aside to cool metabolism wherein the utilization of lipids is
incomplete. These bodies are:
Amount of ppt and its color (red, yellow, or green) o Acetoacetic acid (diacetic acid),
- Depend on the quantity of glucose present in o Acetone
the urine o Beta hydroxybutyric acid
- Presence of these substances in urine is called
Benedict's Test - Semi quantitative test. Color depends ketonuria
on the quantity of glucose.
 Red - conc. amt. of glucose There is no satisfactory, simple direct test for b-hydroxyl
 Yellow - glucose substantially present butyric acid in the urine.
 Green - little glucose present
 Blue - no color change; no glucose present Aceto-acetic acid
- Decomposes so rapidly with the formation of
9. BLOOD acetone, that the usual test for acetone are
(demonstration only) also given for aceto-acetic acid
BENZIDINE TEST Test for ketonuria

3 ml of urine - Are tests for either acetone or aceto-acetic acid
→ Heat to boiling. or both
→ Cool. - True for nitroprusside test
→ Treat with an equal volume of a saturated sol’n of
Benzidine in Glacial Acetic Acid NITROPRUSSIDE TEST – Legal’s
+ 1 ml of 3% H2O2 2 ml urine
= development of a blue or green color indicates the + 3 drops of 5% freshly prepared aqueous sol’n of
presence of blood sodium nitroprusside
+ NaOH (to alkalinize)
10. BILE = ruby red color indicates acetone
GMELIN’S TEST + 0.5 ml of acetic acid

1 ml of conc. Nitric acid = if red color persists, ketone bodies are present.
→ Superimpose 1 ml of urine.
→ Do not mix. If the test is made directly on urine, a red color is given
= At the point of contact, various color rings are noted: by creatinine which disappears on the addition of acetic
blue, green, violet, red, and reddish yellow, in the acid.
presence of bile pigments.
PAPER CHROMATOGRAPHY experiment is the Filter Paper (cellulose in the filter
- Separation of amino acid on the basis of the difference in paper is responsible for the degree of movement)
solubility of amino acid between 2 immiscible solvents 2. Mobile Phase - this is the solvent that flows through
- is an analytical method used to separate colored the stationary phase. This carries the components of
chemicals or substances the mixture. The mobile phase in the experiment is
- two phases: the developing solution
 Stationary Phase (a solid, or a liquid supported on a 3. Rƒ - this is the Retention Factor or the Retardation
solid) Factor. This is the ratio of the distance traveled by
 Mobile Phase (a liquid or gas) the substance to the distance traveled by the
- Type of chromatography: solvent.
 Liquid chromatography
 Gas chromatography A. Preparation of the Developing Chamber
 Ion exchange chromatography Pipette 8 mL of the solvent consisting of 4:1:5: (by volume)
 Affinity Chromatography mixture of butanol, acetic acid, and distilled water
- most effective for the identification of unknown respectively, and introduce into a dry 250 mL beaker.
substances when known samples are run on the same Avoids splashing the liquid on the sides of the beaker.
paper chromatograph with unknowns Cover with a piece of aluminum foil and let stand for 10
minutes for the atmosphere inside to become saturated
with the solvent vapor.
Term Definition
solvent moving through the column  The developing solution consists of 4:1:5 by volume
Mobile phase - the medium that accompanies the mixture of butanol, acetic acid and distilled water.
or carrier analyzed substance as it moves  The Developing Chamber is a 250 mL beaker covered
through the stationary phase with a piece of aluminum foil
substance that stays fixed inside the  The solvent is placed in the chamber and was
Stationary allowed to evaporate for 10 minutes to saturate the
column
phase or chamber with its vapor (since it is volatile). This step
- medium on which the separation
adsorbent is very important since saturating the atmosphere in
occurs
Eluent fluid entering the column the beaker with the solvent vapor stops the solvent
fluid exiting the column (that is from evaporating as it rises up the paper.
Eluate
collected in flasks)
B. Preparation of the Paper Chromatogram
the process of washing out a compound
Elution through a column using a suitable 1. With minimal handling (fingerprint can obscure the
solvent result) cut a piece of Whatmen filter paper no. 1, 16.50 cm
mixture whose individual components long and 8.0 cm wide. With a pencil, draw a line 6 mm
Analyte from the lengthwise edge of the paper and 1 cm from each
have to be separated and analyzed
crosswise edge, for handling.
- Phenomenon where the solution
Capillary (sometimes called the eluting
1CM
Action solvent) will begin to rise up the
paper
Principle of separation of different components: Differential

affinities (strength of adhesion) of the various components of
the analyte towards the stationary and mobile phase results
in the differential separation of the components. Affinity, in
turn, is dictated by two properties of the molecule:
6 MM GLY LYS
‘Adsorption’ and ‘Solubility’.
ASP UNKNOWN
Type of Chromatography
 Ascending Paper Chromatography was used in the 2. Mark lightly with pencil equidistant spots along the
experiment. Movement of the solvent is due to lengthwise line of the filter paper.
capillary action or capillarity
3. Gently and quickly touch the first mark with the point of
Definition of Terms a fine capillary tube (0.5 mm diameter) containing 0.5%
1. Stationary Phase - this is the structure that holds the glycine. Apply approximately 20 micrograms of the sample
on the mark, allowing the spot to dry before each
substance that is tested. The stationary phase in the
application. The wet area should not be more than 2 mm o Ninhydrin reacts with amino acids to form a
in diameter. blue-violet compound.
 NINHYDRIN TEST principles:
4. Repeat step no. 3 on the other marks using a different o Amines (including α-amino acids) react with
ninhydrin to give a coloured product.
amino acid for each mark ( 0.5% lysine, 0.5% aspartic acid,
o It can be used qualitatively (e.g. for
and 0.5% unknown solution) chromatographic visualisation) or quantitatively
(e.g. for peptide sequencing).
 Whatmen Filter Paper no.1 was used as the o The α-amino acids typically give a blue-purple
chromatogram product.
 Dimensions is 16.50 x 8.0 cm o Proline, a secondary amine, gives a yellow-
 With a pencil, draw a line 6 mm from the lengthwise orange product.
edge of the paper and 1 cm from each crosswise o The test is sensitive enough that ninhydrin can
be used for the visualisation of fingerprints.
edge, for handling
 The further the spot from the starting line, the higher the
 0.5% of glycine, lysine, aspartic acid and unknown affinity of the amino acid for the mobile phase and the
solution were used in the experiment faster its migration
D. Calculation of the Rƒ value

Why is the chromatogram developed in an essentially closed How to measure the Rf Value:
system?  Distance traveled by the amino acid: the first dot made
The chromatogram is kept in an essentially closed system in near the 6 mm line until the center of the colored
order to prevent it from acquiring contaminants and other substances (violet) spot
that can hinder in the separation of the organic pigments. It also  OVER
prevents the developing solvent used from evaporating since most  Distance traveled by the solvent: from 6 mm line until
solvents used in paper chromatography are highly volatile, and some the traced portion from the top.
can be flammable and toxic.
C. Development of the Spot

1. With careful handling at the crosswise edges, staple the paper
into a cylindrical form. Do not overlap the edges. Calculate the Rf value of each amino acid and identify the
unknown amino acid.
1. Distance traveled by substance in mm divided by

the distance traveled by the solvent. This measures
the attraction of the substance to both the stationary
phase and the mobile phase
2. If the substance is strongly attracted to the stationary
2. Put the cylindrical paper upright into the equilibrated beaker phase and not attracted to the mobile phase, then the
with the spotted edge at the bottom. The solvent should wet the substance does not move at all, so, the Rf is 0.
lower edge of the paper without reaching the spots. Put the 3. If the substance has no affinity to the stationary phase,
aluminum foil cover in place and let stand for 30 - 45 minutes or then the substance moves along the solvent front, so,
until the solvent front is 1 cm away from the upper edge. the Rf is 1.0.
3. Remove the paper from the beaker and open-up. Mark the In conclusion, the movement of the substance in relation
position of the solvent front before it dries up completely. to the solvent front (Rf) is dependent upon:
 Solubility of the substance to the mobile phase (solvent)
Trace the level of the wet portion in the upper part of the paper
o an amino acid that is highly soluble in the eluting
using a pencil.
solvent will have a higher affinity for the mobile
phase than an amino acid that is less soluble in
4. Spray the entire paper lightly with 0.2% ninhydrin solution and
the solvent.
dry it in the oven at 110° C. Heat if necessary for the color
forming reaction. The color should be readily visible after 20 - 30  Affinity of the substance to the stationary phase (how
minutes. much it sticks to the cellulose of the filter paper)
o If an amino acid has a higher affinity for the
Expected result: spot will be observed to have moved upward. It mobile phase than the stationary phase, it will
will travel and our purpose is to measure the separation or travel tend to travel with the solvent front and be
of the amino acid. relatively unimpeded by the filter paper.
o If the amino acid has a higher affinity for the
paper than the solvent, it will tend to “stick” to
 Solvent moves via capillary action
the paper and travel more slowly than the
 Solvent brings along the substances as it moves up solvent front.
 Ninhydrin (2,2-Dihydroxyindane-1,3-dione) was used to - these differences in the amino acid affinities that
react with the amino acid to form a colored compound lead to their separation on the paper.
(when heated at 110 °C).

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Experiment 1 – Elementary Composition of 3. Mucic Acid Test

EXPERIMENT 1 ELEMENTARY COMPOSITION OF PROTEINS

PROTEINS o pH at which a particular molecule

CASEIN Chemical Equation: Casein + O2 → C + H2O

EXPERIMENT 1 ELEMENTARY COMPOSITION OF PROTEINS

Nitrogen’s presence can be tested in casein through

EXPERIMENT 1 ELEMENTARY COMPOSITION OF PROTEINS

Apparatus: porcelain crucible, pestle (anything to 3. Sulfur

Fusion: The presence of sulfur was tested by using the filtrate

BaCl2 + H2SO4 → BaSO4 + 2HCl

But why is HCl used in precipitating BaSO4 but not

EXPERIMENT 1 ELEMENTARY COMPOSITION OF PROTEINS

Solid Fusion Mixture ammonium phospo-molybdate. This precipitate is

4. Phosphorus Chemical Equation:

EXPERIMENT 2 COLOR REACTION OF PROTEINS

Qualitative color reactions have been devised for the

Proteins give various color reactions which is not specific

Apparatus: test tube, dropper SUMMARY

EXPERIMENT 2 COLOR REACTION OF PROTEINS

2. Xanthroproteic Reaction due to nitration of certain amino acids, most common

• Principle: Nitric acid gives color when heated

EXPERIMENT 2 COLOR REACTION OF PROTEINS

Apparatus: test tube, dropper, Bunsen burner, thong

What did you observe? There is a flocculent red

What is responsible for this change? Positive results

Specific to phenolic group of tyrosine. The hydroxy

EXPERIMENT 2 COLOR REACTION OF PROTEINS

4. Glyoxylic acid reaction (Hopkins-Cole)

Apparatus: test tube, dropper

What is the color produced at the point of contact of

EXPERIMENT 2 COLOR REACTION OF PROTEINS

5. Heller’s Ring Test

What happens at the interface between the

Heller's test is a chemical test that shows

 Objective: to detect the properties of

EXPERIMENT 2 COLOR REACTION OF PROTEINS

6. Reduced Sulfur Test

Loosely combined sulfur after boiling with

Solutions of protein containing cystine,

• Principle: Proteins containing sulfur

Albumin + NaOH + Pb(Ac)2 = PbS (black ppt)

EXPERIMENT 2 COLOR REACTION OF PROTEINS

Indole derivatives give a positive result with

The Adamkiewicz reaction is part of a

Apparatus: test tube

Albumin + Glacial Acetic Acid + H2SO4 = violet ring

EXPERIMENT 3 PRECIPITATION REACTIONS OF PROTEINS

Observation: No coagulation with the casein proteins.

Which tube has the best coagulation? The test tube

Heat Coagulation - denatures proteins forming lumps

Heat coagulation and precipitation occur best near the Materials:

EXPERIMENT 3 PRECIPITATION REACTIONS OF PROTEINS

2. Precipitation by Organic Solvents Why is alcohol used as an antiseptic?

Observation: Coagulation after setting for a day

Filter the mixture and heat the filtrate using a

How is this process applied in the fixing of

Organic solvents precipitate proteins, when made

EXPERIMENT 3 PRECIPITATION REACTIONS OF PROTEINS