BCHEM LAB KODIGS

EXP. 1: ELEMENTARY COMPOSITION OF boiling and add several drops of 0.1 N BaCl2
PROTEINS
I. PURPOSE
To determine the elementary
composition of different proteins and
understand their properties.
II. APPARATUS
Test tubes, test tube holder, test tube
rack, Bunsen burner, mortar and pestle,
porcelain crucible, stirring rad, filter paper,
beakers, red and blue litmus paper, pipettes,
wire gauze, tripod, tongs, funnel
III. MATERIALS
Casein (C81H125O39P) 1 g soda lime
(CaHNaO2), 2 g solid fusion mixture (2 parts
Na2CO3: 1 part KNO3), warm water (H2O),
dilute hydrochloric acid (HCl), several drops of
.1 N barium chloride (BaCl2) sol’ns, conc. nitric
acid (HNO3) [drop wise], few drops of
ammonium molybdate [(NH4)2MoO4)]
solutions.
Explain: There was a reaction with BaCl2 in
an acidic medium. The sol’n in the test tube
turned cloudy and a white precipitate named
barium sulfate (BaSO4) was formed. The
solution then became clearer as it rested
gradually.
Write the equation involved: BaCl2 +
H2SO4 → 2HCl + BaSO4/BaCl2 + Na2SO4 →
2NaCl + BaSO4 (white precipitate)
4. PHOSPHOROUS
Take a second portion of the filtrate and add
concentrated nitric acid drop wise until acidic.
Add a few drops of ammonium molybdate
solution and heat nearly to boiling.
What is the precipitate formed? Ammonium
phosphomolybdate [(NH4)3PO4 • 12MoO3)]
Write the equation involved: PO4^3- +
3NH4+ + 12MoO4²- + 24H+ → (NH4)3PO4 •
12MoO3 (yellow crystalline precipitate) +
12H₂O

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IV. PROCEDURE

1. CARBON AND HYDROGEN

Place a small amount of casein in a test tube.
Heat it gently over a low flame. Observe the two
substances formed within the tube.
What does the charring of casein and the
formation of moisture in the test tube indicate?
Charring of casein indicated the presence and
removal of carbon, while the formation of
moisture indicated the presence and removal of
hydrogen from the solid.

2. NITROGEN
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.
What gas turned the moistened litmus to
blue? Ammonia gas (NH3) turned the moistened
litmus to blue.

FUSION: Perform the tests for sulfur and

phosphorous on the fused mixture prepared as
follows: Place about 2 grams of solid fusion
mixture (2 parts Na2CO3: 1 part KNO3) in a
porcelain crucible. Add a small amount of
powdered casein and mix thoroughly. Heat
slowly at the start and then strongly, until a
clear mixture is formed. Cool and dissolve with
a small amount of warm water and filter. Divide
the filtrate into two parts.

3. SULFUR
Add dilute HCl to the first portion of the
above filtrate until acidic. Heat the solution to

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 BCHEM LAB KODIGS
EXP. 2: COLOR RXNS OF PROTEINS
I. PURPOSE
To determine the color reactions of
different proteins through various specific tests.
II. APPARATUS
Pipettes test tubes, beakers, test tube
holder, Bunsen burner, wire gauze, iron stand,
stirring rod
III. MATERIALS
2% albumin sol’n, 1 mL of 10% sodium
hydroxide (NaOH), .5% copper sulfate (CuSO4),
2 mL of 2% peptone sol’n, 5.5 mL conc. nitric
acid (HNO3), ammonium hydroxide (NH4OH)
[in excess], Millon’s rgt/HgN2O6 (1 part by wt.
of mercury/Hg: 2 parts by wt. of conc. nitric
acid/HNO3), 2 volumes of water (H2O), 10 g
powdered magnesium (Mg), 250 mL of cold
water (H2O), acetic (HAc), 1 mL of Hopkins-cole
rgt, pure conc. sulfuric acid (H2SO4), 2 mL of
40% sodium hydroxide (NaOH), 10 drops of 2%
lead acetate [Pb(C2H3O2)2]
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 test are used for specific
chemical groups of the component amino acids.
apply several tests. Since no one test is
absolutely specific for proteins, it is necessary to
apply several tests.
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IV. PROCEDURE
1. BIURET REACTION
Biuret test is a general test for proteins. A
positive reaction is obtained with all native
proteins and most of the derived proteins. A
violet color is obtained with long chain proteins
while pink color is obtained with shorter chain.
This is due to the varying amount of peptide
linkage. Any compound containing two carbonyl
groups (-CONH₂) joined together through a
single nitrogen or carbon atom will also give a
positive test.
Mix 1 mL of 2% albumin solution and 1 mL
of 10% NaOH. Add .5% CuSO4 drop by drop
mixing thoroughly after each addition until a
pink or violet color is obtained.
What chemical structure in the protein
molecule is responsible for a positive test? Two
peptide bonds/linkages in the protein molecule
Will simple amino acids give positive biuret
test? No, because biuret test will only react with
peptide linkages or a strong covalent bond that
will link two or more amino acids in polypeptide
or protein. [Simple] amino acids don’t have
peptide bonds.
Repeat the test using 2 mL of 2% peptone
solution instead of albumin. What did you
observe? The yellowish mixture of peptone
solution and NaOH turned a light violet color
upon addition of the bluish CuSO4 due to
peptone having less peptide bonds.
Albumin solution shows darker purple than
peptone, since albumin has a longer-chain
polypeptide than peptone (longer polypeptide
chain = darker solution).
2. XANTHOPROTEIC REACTION
This reaction is due to the presence of
phenyl group (-C6H5) in the protein molecules.
It involves the nitration of the phenyl rings
present in the aromatic amino acids such as
tyrosine, phenylalanine, tryptophan, forming
yellow nitro-substitution products that turn
orange when alkali is added (salt formation).
Place 1 ml of 2% albumin in a test tube, add
0.5 ml of conc. nitric acid, and heat. Cool and
add ammonium hydroxide in excess.
Observation? Before heating, the solution was
yellow. After it was heated and NH4OH was
added, the color of the solution changed to
orange. When the protein was treated with
conc. nitric acid, it turned yellow. And when it
was treated with an alkali (NH4OH), it turned
orange.
Yellow nitro-benzene substituted product or
precipitate is formed when conc. nitric acid
reacts with the aromatic amino acid in the
protein molecules. When NH4OH is added to the
precipitate, it turns to orange due to the
formation of ammonium-salt complex.
3. MILLON’S TEST
Millon’s reagent is prepared by mixing one
part by weight of mercury with two parts by
weight of conc. nitric acid and dilute the
resulting solution with two volumes of water.
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? A mercury salt
precipitate (which turned from flesh color to
red) formed at the bottom of the text tube.
What is responsible for this change?
Presence of a phenol group [of tyrosine] in the
protein
What amino acid gives a positive Millon’s
test? Tyrosine
Write its formula: C9H11NO3
4. GLYOXYLIC ACID REACTION (HOPKINS-
COLE)
Hopkins-Cole reagent is prepared by mixing
10 grams of powdered magnesium with
sufficient water to cover it. Then add slowly 250
ml of cold water and shaking at the same time.
Filter and acidify the filtrate with acetic acid to
prevent partial precipitation of magnesium and
make the volume to one liter with distilled
water.
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 BCHEM LAB KODIGS
Mix 1 mL of 2% albumin solution and 1 mL Formation of purple ring due to the indole
of Hopkins-Cole reagent. Incline the tube and group of tryptophan
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
What is the cause of this color reaction?
Presence of an indole nucleus in the tryptophan
component. The tryptophan condenses with the
aldehyde to form the colored compound.
Indole group of tryptophan in protein
molecule that reacts with the Hopkins-cole
reagent
Name the amino acid responsible for this test
and write its formula. Tryptophan
(C11H12N2O2)

5. HELLER’S RING TEST

This is used clinically to detect the presence
of albumin in urine.
Put 5 mL of concentrated nitric acid in a test
tube, incline the tube and allow 2% albumin
solution to flow down the side of the tube
slowly.
What happens at the interface between the
two liquids? A white precipitate of coagulated
protein appeared due to the change in pH
(specifically, denaturation) caused by the strong
mineral acid.
White/Heller’s ring is observed at the
junction of the solution due to the coagulation
in a strong concentrated acid. Usually, a yellow
precipitate is observed together with the white
ring due to the nitration of the aromatic amino
acid (xanthoproteic reaction).

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.
To 2 mL of 2% albumin solution add 2 mL of
40% NaOH and 10 drops of 2% lead acetate
solution. Heat to boiling for about 2 minutes or
until distinct change is obtained. What did you
observe? A black precipitate was formed (makes
the solution black in color) most likely due to the
formation of lead sulfide (PbS).
Reduced sulfur of cysteine and/or
methionine in protein chain into sulfide. When
lead acetate is added, the sulfide reacts with the
lead, to form PbS (black precipitate).

7. ADAMKIEWICZ REACTION
Indole derivatives give a positive result with
this test.
Add 3 drops of 2% albumin solution to 5 mL
of glacial acetic acid. Mix well. Pour conc.
sulfuric acid down the side of the tube and note
the color of the ring after it is made to stand for
a few minutes.
Light violet (lighter in color compared to the
one in Hopkin-Cole’s test)

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 BCHEM LAB KODIGS
EXP. 3: PRECIPITATION REACTIONS OF
PROTEINS
I. PURPOSE
To determine the components of proteins
and they precipitate in various chemical
reactions.
II. APPARATUS
Test tubes, beakers, wire gauze, iron
stand, Bunsen burner, test tube rack, pipettes,
stopper, filter paper, funnel, water bath, petri
dish, Erlenmeyer flask, test tube holder
III. MATERIALS
5 mL of 2% casein (C81H125O39P), 13
mL of 2% albumin solution, 1 drop of 1 N acetic
acid (HAc), 2 drops of 5 N acetic acid (HAc), 4
mL of ethyl alcohol (CH3CH2OH), water (H2O),
slid ammonium sulfate [(NH4)2SO4], Millon’s
reagent (HgN2O6), 1 mL of 10% sodium
hydroxide (NaOH), .5% copper sulfate (CuSO4),
10 mL of 10% commercial peptone, 2-3 drops
of lead (II) acetate [Pb(Ac)2], 3 drops of 1%
silver nitrate (AgNO3), 1% copper sulfate
(CuSO4), .1 N sodium hydroxide (NaOH), few
drops of .1 N hydrochloric acid, 3 drops of
potassium ferrocyanide {K4[Fe(CN)6] • 3H2O},
3 drops of 5% tannic acid (C76H52O46), 3 drops
of saturated picric acid [(O2N)3C6H2OH)
------------------------------------------------
IV. PROCEDURE
1. PRECIPITATION BY HEAT COAGULATION
Heat coagulation and precipitation occur
best near the isoelectric point of the protein.
Albumin coagulates when heated. Aqueous
solutions of proteases, peptones, gelatin, and
casein are not coagulated by heat.
Place 5 mL of 2% casein in a test tube and
heat to boiling.
Observation: The casein did not coagulate
because it needs a dehydrating agent and there
is a presence of surrounding water molecules.
No coagulation because of lesser intra-
molecular hydrogen bonding that stabilizes the
structure of casein. Disruption of these
hydrogen bonds that stabilize the secondary,
tertiary, and quaternary of protein results to
coagulation or denaturation of protein.
In each of 3 test tubes, place 1 mL of 2% of
albumin solution. To the first add 1 drop of 1 N
acetic acid, to the second add 2 drops of 5 N
acetic acid. The third serves as control. Boil the
contents of these 3 tubes.
Which tube has the best coagulation? Tube
1 (mixture of 2% albumin solution and 1 N
acetic acid)
When 1 N acetic acid is added to the albumin
solution, it makes the solution near the
isoelectric point of albumin (around 4.9). When
the solution is near the isoelectric point of the
protein, protein is less soluble or insoluble,
precipitating out of the solution. Heat can make
this less soluble protein coagulate.
(5 N = very acidic, more solid protein)
2. PRECIPITATION BY ORGANIC SOLVENTS
Add 4 ml of ethyl alcohol to 2 mL of 2%
albumin solution. Remove a small amount of the
precipitate and add water.
Does the precipitate dissolve in water? Yes,
since the denaturation is mild and reversible.
There’s little time of exposure of alcohol in
protein that makes the denaturation mild and
reversible when water is added, since the
alcohol is more soluble in water.
Set aside the remainder of the precipitate in
a stoppered test tube until the next laboratory
period. Again test the solubility of the precipitate
in water.
Observation: After setting the remainder of
the precipitate aside for a day, coagulation in
the test tube was observed.
Precipitate no longer soluble in water, since
the protein is exposed for a longer time in
alcohol that makes the denaturation irreversible.
When organic solvent is added in solution, water
molecules available for protein are reduced, and
precipitation occurs. Organic solvents reduce
the dielectric constants of the ---- of the major
protein precipitation (?).
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? With how
organic solvents disrupt hydrophobic and
hydrogen bonding, exposing internal
hydrophobic protein groups and causing
shrinkage and hardening of tissues for
preservation.
Preserves tissues
Organic solvents precipitate proteins. When
they are made to stand for sometimes, they are
denatured and become insoluble in water.
3. PRECIPITATION BY SALTING OUT
a. Add solid ammonium sulfate to 5 mL of 2%
albumin until it becomes saturated. Filter.
Test the precipitate by Millon’s reagent.
Observation: Flesh to red precipitate (protein
separated out from solution)
Make a biuret test on the filtrate.
Observation: The filtrate had a blue color
to it.
1 – solution can remain blue (nega) OR 2
– pink/purple (posi)
Conclusion: All the proteins were
precipitated from the addition of Millon’s
reagent and all proteins were precipitated from
the biuret test, since the blue color indicated a
negative result/absence of proteins.
b. Separation of peptones and proteoses.
Saturate 10 mL of 10% commercial peptone
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 BCHEM LAB KODIGS
with solid ammonium sulfate, stirring all the
time. Filter off the proteoses, and peptone is
found in the filtrate. Make a biuret test on
the filtrate. Note: peptone is pink with biuret
reagent. The filtrate should be pink/light
violet with the biuret best.
If the biuret test of the filtrate is positive, the
salting out is incomplete, since there’s still
protein molecules in the filtrate. If the biuret
test of the filtrate is negative, the salting out
is complete, since all the protein are
separated and precipitated out from the
solution.
4. PRECIPITATION BY HEAVY METAL IONS
Place 2 mL of 2% albumin solution in each
of 3 test tubes. To the first add 2 to 3 drops of
Pb(Ac)2, to the second and 3 drops of 1%
AgNO3 and to the third 1% CuSO4.
Observation: Addition of Pb(Ac)₂ = while
precipitate, addition of AgNO3 = white
precipitate, and addition of CuSO4 = [light] blue
precipitate
Repeat the test using fresh samples of
albumin but add few drops of 0.1 N NaOH
before adding the metal salts.
Which has more precipitate, the test with or
without NaOH? The test with NaOH
Protein (albumin) will precipitate with heavy
metal salts, forming
metal/proteinate/albuminate. Heavy metal ions
will interfere with the salt bridges, and the
disulfide bonds that stabilize the tertiary and
quaternary structure of albumin. Disruptions of
one or more of these bonds will denature the
protein, and form insoluble precipitate.
1 (with) – NaOH will make the solution basic
and very far from the isoelectric point of the
albumin, making it more soluble with larger
surface area, forming more insoluble metallic
albuminate/precipitate. OR 1 (without) – NaOH
will make the solution basic and very far from
the isoelectric point of the albumin, making it
more soluble. But, the excess NaOH tend to
dissolve the metallic albuminate when heavy
metal salts are added (appears lesser
precipitate).
Egg albumin is used as an antidote for
mercury or lead poisoning. Why? Egg albumin
will oxidize or “bind” with the organic mercury
or lead, thus inducing an individual to vomit the
egg albumin and mercury or lead from his
stomach. The egg albumin will form a
precipitate of mercuric or lead salts, slowing
down or hindering their absorption by the body.
Egg albumin will react first with the heavy
metals, forming insoluble metallic albuminate,
which is less toxic than the free heavy metals
that will absorb in the system.
5. PRECIPITATION BY ALKALOIDAL REAGENTS
Place 2 mL of 2% albumin solution in each
of 3 test tubes and acidify with a few drops of
0.1 N HCl. To the first tube add 3 drops of
potassium ferrocyanide, to the second 3 drops
of 5% tannic acid, to the third 3 drops of
saturated picric acid.
Why are they called alkaloidal reagents?
Because they precipitate complex amines.
Acidic reagents that react similarly as the
heavy metal, due in that acid will react with the
protein to form an insoluble salt. These acids
lower the pH of the medium when protein
carries net positive charges. These protein
cations combine with negatively charged ions to
form proteins ferrocyanide, tannate, and
picrate, and flocculent precipitate is formed.
Why is picric acid used in treatment for burns
and tannic acid for diarrhea? Picric acid is used
in treatment of burns because of its antiseptic
and astringent properties. Antiseptics help stop
the growth of microorganisms on the skin, while
astringents cause the contraction or shrinkage
of tissues, as well as drying up of secretions.
Tannic acid is used to treat diarrhea due to its
ability to improve epithelial barrier properties in
the intestine, reducing intestinal fluid secretion.
Picric acid can denature proteins by
disrupting salt bridges and hydrogen bonds that
stabilize the tertiary structure of protein. By
altering the state of either, these eventually
renders the hydrophobic end of protein in its
outer stratum, and the hydrophilic end at the
center. This precipitate clogs the pathway of any
pathogen, thus interstitial fluid to exit the
wound, or for the pathogen to enter.
Fibrinogens will fight against the progress of
infection, and the body will sustain good # of
monocytes in the infected area.
Tannic acid can improve epithelial barrier
functions (disfunction = intestinal diseases and
pathogenies of diarrheal diseases) and cause
constipation.
What is the general equation showing the
ionization of a protein in acid and basic medium?
Observation: All the alkaloidal reagents gave
positive results. Addition of potassium
ferrocyanides produced a white precipitate,
addition of tannic acid produced a fresh brown
precipitate, and addition of picric acid produced
a yellow flocculent precipitate.
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BCHEM LAB KODIGS

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 BCHEM LAB KODIGS
EXP. 4: TEST FOR CARBOHYDRATES
I. PURPOSE
To determine the presence of different
carbohydrates and observe their various
physical and chemical properties.
II. APPARATUS
Beakers, test tubes, test tube rack, droppers,
test tube holder, wire gauze, Bunsen burner,
clay stand/cover, dialysis tubes
III. MATERIALS
.01 M arabinose (C5H10O5), fructose
(C6H12O6), .1 M + .02 M + 5% glucose
(C6H12O6), .1 M + .02 M sucrose
(C12H22O11), .7% + .05% starch
[(C6H10O4)n], dextrin [(C6H10O5)n], water
(H₂O), 10% sodium chloride (NaCl), alcohol (-
OH), Molisch reagent (5% solution of alpha-
naphthol in alcohol), concentrated sulfuric and
(H2SO4), 3% thymol, concentrated hydrochloric
and (HCl), Seliwanoff’s reagent, concentrated
sodium hydroxide (NaOH), phloroglucin solution
------------------------------------------------
IV. PROCEDURE
A. PHYSICAL TESTS
1. Solubility
Test the solubility of arabinose, fructose,
glucose, sucrose, starch, and dextrin in 2 mL of
water, 10% NaCl, and alcohol. Note: use only a
pinch of the solid carbohydrates. Tabulate your
results.
Starch and dextrin = slightly soluble in water
and 10% NaCl (90% water, so can also form
hydrogen bonds in water that makes it soluble)
Lower membrane carbs = soluble in water
bec of more hydroxyl groups, which are polar,
that can form hydrogen bonds with [polar]
water. As polysaccharide chain increases,
solubility decreases.
When alcohol is added, which is less polar
than water, it lowers the solubility and potential
hydrogen bonds of the carbohydrates, because
the water is more attracted to the alcohol than
the carbohydrates, the carbohydrate is
dehydrated by the alcohol and form
precipitates.
2. Dialysis: (Demonstration) To each of the
three dialysis tubes place 1 ml of 0.1 M
glucose, 0.1 M sucrose and 0.7% starch.
Immerse the ends covered with the semi-
permeable membrane in 3 100 ml beakers
with 50 ml water. Set aside for 15 minutes.
Perform the Molisch test on the dialysate and
compare the intensity of the color produced.
Tabulate your results and draw conclusions.
B. FURFURAL REACTIONS OF
CARBOHYDRATES
1. Molisch Test (alpha-naphthol reaction)
This is a general test for carbohydrates.
Prepare 4 for test tubes with 1 mL water, 1
ml of 0.02 M glucose, 1 ml of 0.02 M sucrose,
and 1 mL of 0.05% starch, respectively. The first
serves as control. To each of the 4 solutions,
add 2 drops of Molisch reagent (5% solution of
alpha - naphthol in alcohol). Mix thoroughly.
Incline the tube and allow 1 mL of conc. H2SO4
to flow in the side of the tube. Note the color of
the ring obtained after sometime. Violet
What product from each of the sugar
condenses with alpha-naphthol?
Hydroxymethylfurfural from each of the sugar
condenses with alpha-naphthol. For glucose and
starch (which have hexoses), 5-
hydroxymethylfurfural condensed with alpha-
naphthol. For sucrose (a pentose), it was its
furfural molecule/s.
Carbs undergo dehydration upon intro of
conc. HCl/H2SO4, resulting in the formation of
an aldehyde or furural. This aldehyde undergoes
condensation, along with furfural-type
molecules such as alpha-naphthol, resulting in a
formation of a purple ring or reddish-purple-
colored complex.
2. Thymol Test
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. Boil gently for 2 minutes, shaking the
mixture at intervals. A carmine color develops.
There was no change of color in the test tube
with water. In the test tube with sucrose, a red
color appeared. And in the test tubes with
glucose and starch, a carmine red and a light
red (almost pink), respectively, were observed.
Carmine Test = alternative to Molisch
3. Seliwanoff’s Reaction (Resorcin - HCl test)
Place in each of 4 test tubes 5 mL of
Seliwanoff’s reagent. To these tubes, add
respectively 1 mL water, 1 mL of 0.01 M
glucose, 1 mL of 0.01 M fructose and 1 mL of
0.01 M 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. There were
no color changes in the test tubes with water
and 0.01 M arabinose. However, a light yellow
color was observed in the test tube with 0.01 M
glucose, and a deep red color was observed in
the test tube with 0.01 M fructose.
What kind of sugar will give a positive
reaction with Seliwanoff’s reagent?
Pentaketoses (such as fructose) because they
dehydrate faster and form 5-
hydroxymethylfurfural
Ketose (undergoes dehydration in the
presence of concentrated acid to yield 5-
hydroxymethylfurfural); reacts with 2 eqs. of
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 BCHEM LAB KODIGS
resorcinol in series of condensation reaction to
produce a complex called “xanthonoid” (deep
cherry red to mahogany red color)
4. Moore’s Test (action of conc. alkali)
Mix 1 mL of 5% glucose and 1 mL of conc.
NaOH. Boil. What is the color and odor of the
solution? Color = orangey brown, smell = sweet
(a lot like caramel)
This test is based on the liberation of
aldehydes which polymerize to form a resinous
substance: caramel.
When a solution of reducing sugar is heated
with an alkali, it turns yellow to orange then light
brown, liberating the odor of caramel (due to
the liberation of aldehyde, which subsequently
polymerizes to form a residue substance
caramel)
5. Tollen’s Phloroglucin reaction
Measure off 5 mL of phloroglucin solution
into each of four test tubes. To each add
respectively 1 ml water, 1 ml of 0.01 M glucose,
1 ml of 0.01 M fructose and 1 mL of 0.01 M
arabinose. Immerse all test tubes in boiling H2O
at the same time. Observe the color changes
every two minutes during the first 15 minutes of
heating, then observe at the end of 1 hour. This
reaction may be used to differentiate pentoses
from hexoses.
Observation: A change of color (light red-
orange) was observed first in the test tube with
fructose. Then, as it grew deeper, a light yellow
color was starting to be observed in the other
test tubes during the first 15 minutes of heating.
At the end of an hour: solution with arabinose
= dark brown black color, fructose = brown,
glucose = dark orange color, and water = light
yellow-orange color. As a pentose/pentaketose,
fructose indicated a positive result. And since
glucose and water are not
pentoses/pentaketoses, they indicated a
negative result.
Differentiates pentoses (reddish-brown
color) from hexoses (some may give reddish
color); observation may vary depending on
intensity and duration of heating

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 BCHEM LAB KODIGS
EXP. 5: SPECIAL TEST FOR SACCHARIDES
I. PURPOSE
To conduct special tests specific for
different saccharides and observe their various
results after.
II. APPARATUS
Droppers, test tubes, beakers, wire gauze,
Bunsen burner, day stand/cover, test tube rack,
test tube holder, microscope
III. MATERIALS
0.01, 0.1 M, 0.002 M, 0.03 M, and 5% glucose
(C6H12O6) solutions; conc. nitric acid (HNO3);
5% potassium chromate (K2CrO7); 20%
suspension of baker’s yeast (C19H14O2); 0.01
M, 0.03 M, and 5% sucrose (C12H2O11)
solution; galactose (C6H12O6); phosphate
buffer (pH 6.4-6.8); 0.7% starch [(C6H10O5)n]
solution; water (H2O); Fehling’s solution A
(CuSO4), Fehling’s solution B (KOH and Rochelle
salt); Benedict’s solution (CuSO4, Na2CO3,
Na3C6H5O7); Barfoed’s solution
[(ClCCH3COO)2 in HAC]; 0.01 M and 5%
fructose (C6H1206); 0.01 M, 0.03 M, and 5%
lactose (C12H22O11); 0.1% dextrin
[(C6H10O5)n], dil. iodine (I2) solution, 0.5 g of
phenylhydrazine solution (C6H8N2/2 parts
phenylhydrazine hydrochloride and 3 parts
sodium acetate), 5% maltose (C12H22O11)
------------------------------------------------
IV. PROCEDURE
1. NITRO-CHROMIC ACID TEST
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.
Result? The solution looked clear but was
actually light blue, indicating that it did contain
a sugar with a concentration more than 1%.
This reaction is given by all saccharides, as well
as other compounds with a primary or
secondary alcohol group.
The solution turned blue because of the
oxidation of the free aldehyde in glucose to a
carboxylic acid. This also reduced chromic avid
(H2CrO4) to HCrO3 (CrVI → CrIV), which is blue
(releases chromic ion, which is responsible for
bluish color of the solution).
2. ALCOHOLIC FERMENTATION
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
the (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? Production of
CO2 molecules by yeast
Explain results.
3. MUCIC ACID TEST
To 1 mL of 5 mL galactose, add 1 mL of conc.
nitric acid. Heat in a boiling water bath for 1½
hour. Allow to stand till the next laboratory
period. Examine the crystals under the
microscope and draw them.
The mucic acid test takes advantage of the
relative insolubility of mucic acid (an isomer of
saccharic acid) which is formed by the oxidation
of galactose.
Mucic/galactaric/aldaric acid = oxidized
product when the aldehyde group and primary
alcohol group of galactose are oxidized into
carboxylic acids with the presence of strong
nitric acid, serving as the strong oxidizing agent
4. REDUCTION TEST
A. FEHLING’S TEST
Place 1 mL each of the following solutions
into 5 separate test tubes: 0.01 M glucose, 0.01
M fructose, 0.01 M sucrose, 0.7% starch
solution and water (control). To all 5 test tubes,
add 1 mL each of Fehling’s solution A (CuSO4)
and Fehling's solution B (KOH and Rochelle
salt). Shake and immerse all tubes in boiling
water and heat for 15 minutes.
Observe the color produced. The mixture
with sucrose the mixture with water, and the
mixture with starch remained blue. Meanwhile,
the mixture with fructose turned a light blue, but
had a red precipitate. And, the mixture with
glucose turned red and had a red precipitate,
too.
What is the precipitate due to? Fructose and
glucose and reducing sugars, due to their free
aldehyde/ketone group.
Oxidization of reducing sugar (glucose and
fructose). Fehling’s = mild oxidizing agent that
oxidizes the aldehyde group of a reducing sugar
to aldonic acid, with the reagent being reduced
to cuprous oxide (brick red precipitate); only
reacts with reducing agent/sugar
Which carbohydrates have no reaction?
Sucrose and starch (non-reducing sugars)
9
 BCHEM LAB KODIGS
Why? Because these are non-reducing
sugars, which don’t have a free
aldehyde/ketone group
B. BENEDICT’S TEST (QUALITATIVE TEST FOR
GLUCOSE IN URINE)
In 3 separate tubes, place 5 mL of Benedict’s
solution (CuSO4, Na2CO3, sodium citrate) and
then add 1 mL of 0.01 M, 0.01 M, and 0.002 M
glucose solutions. Place in a boiling water bath
for 10 minutes.
Observe the colors produced and compare
the intensities in each tube. The test tube with
0.01 M glucose was observed to have some
brick red color precipitate at the bottom of its
mixture. The test tube with 0.1 M glucose was
observed to have a brick red color and
precipitate all throughout its mixture. The test
tube with 0.002 M glucose was observed to have
a bit of a brick red color precipitate at the
bottom of its mixture. The intensity in the
second test tube was the strongest, followed by
the first tube, then the third test tube.
This test is given by reducing sugars. in
alkaline medium, sodium carbonate converts
glucose to enediol and this enediol reduce cupric
to cuprous forming cuprous hydroxide. This
solution is kept in sodium citrate and on boiling,
[brick] red precipitate of cuprous oxide is
formed.
0.01 M has more glucose molecules that
could react with the Benedict’s reagent, forming
more cuprous oxide.
C. BARFOED’S TEST
Prepare 7 test tubes and label them with
numbers properly. Then add to each 5 mL of
Barfoed's solution (copper acetate in acetic
acid). Then add to corresponding tube 5 mL
each of (1) 0.01 M glucose, (2) 0.01 fructose,
(3) 0.01 M lactose, (4) 0.01 M sucrose. (5) 0.03
M glucose, (6) 0.03 M lactose, and (7) 0.03 M
sucrose. Place the seven tubes in a boiling water
bath the third and heat for ½ hour.
Note the speed of reduction in the different
tubes.
Barfoed’s test serves to detect reducing
monosaccharides; fructose reacts faster than
glucose. Disaccharides also reduce this reagent
if the sugar concentration is high enough and
the time of heating is long.
Mild oxidizing agent that can be reduced to
cuprous oxide; slower rate of reaction in
disaccharides
5. IODINE TEST FOR STARCH AND DEXTRIN
To 5 ml 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: The mixture in the test tube
with starch turned colorless, while the mixture
in the test tube with dextrin turned yellow-
orange.
What happens when the warm solution is
cooled? When the warm solution with starch
was cooled, a dark blue color was observed,
indicating a positive result for the presence of
polysaccharides (as dark blue was its original
color.). Meanwhile, when the warm solution
with dextrin was cooled, a dark blue-violet (near
black) color was observed, which was its original
color prior to heating. This also indicated a
positive result.
Starch and iodine before heating = blue-
colored solution; after = colorless
Dextrin and iodine before = purple; after =
colorless
Starch interacts with iodine to form inclusion
compounds called clathrates (?), in which the
molecules of one substance can enter the
molecular structure of another.
If you heat a test tube con-tain-ing a
so-lu-tion of starch, io-dine, and wa-ter over a
chemical burner for some time, the so-lu-tion
will turn white and trans-par-ent. This is
be-cause the com-pound of io-dine and starch
is unstable.
If you put the test tube containing starch and
iodine in cold wa-ter, a dark blue color solution
will form once more due to the partial hydrolysis
of starch in 2-dextrin*.
Dextrin might also turn back to its original
color, unless the heating makes it hydrolyze
further.
*When starch is heated to the boiling point, it
begins to break down and the amylose chains
will break, forming short chains of dextrin. So,
the color of starch will change.
6. PHENYLHYDRAZINE REACTION
To 0.5 g of phenylhydrazine mixture, add 2
mL of the indicated sugar solution, shake well,
and heat in a boiling water bath for 30 to 45
minutes. Allow the tube to cool (not under the
tap) and examine the crystal under art.
microscope.
Sugar solutions to be used: 5% glucose,
fructose, maltose, lactose and sucrose.
Glucosazone = needle-like, maltosazone =
sunflower petal, lactosazone = powderpuff (?)
hedgehog
If there are crystals observed in the test tube
containing sucrose, the crystals are
glucosazone/fructosazone.
Prolonged heating of sucrose with
phenylhydrazine = hydrolyze sucrose into
glucose and fructoes
10
 BCHEM LAB KODIGS
This test is useful in identifying sugars with
free aldehyde or ketone group. The shape of the
crystals and the time required for osazone
formation are important guides in identifying the
various sugars.
Compare results obtained with following
figures given by Mulliken:
Glucose – 4 to 5 minutes
Fructose – 2 minutes
Maltose – osazone soluble in hot water
Lactose – osazone soluble in hot water
Sucrose – 30 to 35 minutes (after hydrolysis)
Reagent: Phenylhydrazine mixture is
prepared by mixing 2 parts of phenylhydrazine
hydrochloride and 3 parts of sodium acetate.
The mixture should be freshly prepared.

11
 BCHEM LAB KODIGS
EXP. 6: GLYCOGEN
I. PURPOSE
To determine the reactive properties of
glycogen.
II. APPARATUS
Mortar and pestle, evaporating dish,
beakers, stirring rod, wire gauze, Bunsen
burner, clay stand/cover, funnel, filter paper,
Erlenmeyer flask, droppers, test tubes, test tube
rack, test tube holder
III. MATERIALS
Glycogen solution (oyster meat,
water/H2O, acetic acid/CH3COOH), sodium
chloride (NaCl), iodine (I2) solution, 95%
alcohol (-OH), Benedict’s solution, conc.
hydrochloric acid (HCl), sodium hydroxide
(NaOH), starch [(C6H10O5)n]
------------------------------------------------
IV. PROCEDURE
A. PREPARATION OF GLYCOGEN
1. Mince one or two pieces of oyster meat into
small pieces.
2. Mix with sand and grind in a mortar.
3. Transfer the mixture into an evaporating
dish and add 40 mL of water.
4. Boil for 20 minutes.
5. Replenish the water lost by evaporation.
6. Filter. Wash the residue on the filter paper
with two 10 mL portions of water, adding the
washings to the first filtrate.
7. Concentrate the filtrate to a small volume by
boiling.
8. Faintly acidify with acetic acid and filter off
any coagulated protein.
9. Use the opalescent filtrate (glycogen) for the
following tests:
B. REACTION OF GLYCOGEN
1. To 5 ml of the glycogen solution and 3-5
drops of NaCl and several drops of iodine
solution. Note the color and compare it with
the color formed by starch with iodine. When
iodine was added to the solution with
glycogen, a reddish brown ring formed at the
top of the mixture. Meanwhile, when iodine
was added to the solution with starch, the
mixture turned black, with a little bit of white
at the bottom.
Glycogen = reddish-brown-blue color,
starch = blue-black color
2. Use 3 mL of glycogen solution and add 6 mL
of 95% alcohol. Note the precipitation of
glycogen. The precipitation of glycogen
cloudy white and formed at the top of
glycogen. This is because glycogen is
insoluble in alcohol (specifically ethanol and
isopropanol). The precipitate traps target
nucleic acids.
Glycogen will form insoluble precipitate with
concentrated alcohol. Alcohol will interact
with the water more freely than the water
and glycogen; thus, glycogen will precipitate
out from the solution (formation of white
precipitate).
3. Test the precipitate in no. 2 with Benedict’s
solution and boil for a short time.
Observation: Before heating, the mixture
of the precipitate and Benedict’s solution
was a light blue. And after heating, it
remained the same or there was no chemical
change involved, indicating a negative
result. This is because the test done was a
test for reducing sugars.
Glycogen = nega in Benedict’s bec it’s a
non-reducing polysaccharide
4. Hydrolyze 5 mL of the glycogen solution with
a few drops of conc. HCl, boiling for 10
minutes. Cool the solution, neutralize with
NaOH and test with Benedict’s solution.
Before heating, the mixture was light blue.
During heating, it turned yellow. And after
heating/when the mixture was cooled, an
orange color was observed.
Brick red precipitate (cuprous oxide) =
confirmatory result
What are the products of hydrolysis of
glycogen? Glycogen is a glucose
polysaccharide, or a multi-branched polymer
of glucose. Through acid hydrolysis (in this
case, by HCl), glycogen can be broken down
into its D-glucose subunits; glucose
monomers are produced as glycosidic bonds
are broken in hydrolysis of glycogen.
Hydrolyzed product = glucose
Explain the results obtained:
A. The form in which carbohydrate is stored in
the body is called glycogen. Every glycogen
molecule is formed by the linkage in
branching chains of thousands of glucose
monomers. So, glycogen is a natural
polymer. It is a highly purified
polysaccharide that can be derived from
oysters, since it serves as a form of energy
storage in animals (including humans) and
fungi. Glycogen in oysters is stored as a
reserve food material and used in the
formation of gonad products; oysters can be
dried with no loss of glycogen or protein.
B.
1. This iodine test is useful to distinguish
glycogen and starch from other
polysaccharides. Glycogen reacts with iodine
to give a reddish brown color, while starch
reacts with iodine to give a blue-black color,
indicating a positive result (since this test is
primarily for the detection of the presence of
starch). Starch and glycogen form helical
coils, and iodine atoms can fit into the
helices to form a glycogen-iodine or a starch-
iodine complex.
12
 BCHEM LAB KODIGS
2. As a highly purified branched chain
carbohydrate, glycogen is insoluble in
ethanol and isopropanol and forms a
precipitate that traps nucleic acids, such as
DNA, rRNA, RNA, etc.
3. The Benedict’s solution is used to test the
presence of many simple carbohydrates,
turning from turquoise to yellow or orange
upon reaction with reducing sugars (simple
carbohydrates with unbound aldehyde or
ketone groups). Glycogen gave a negative
result because it is a complex carbohydrate
(polysaccharide), with each branch ending in
a nonreducing sugar residue. Glycogen is
considered a reducing sugar, but with only
one reducing end, which is visually
covalently linked to glycogenin and therefore
will not be reducing.
4. When glycogen was hydrolyzed, it
underwent saccharification (the process of
breaking a complex carbohydrate into its
monosaccharide components). Its glucose
units were removed one at a time from the
non-reducing ends, freeing its carbonyl
groups, and allowing for the presence of a
reducing sugar (glucose). Reducing sugars
then give positive result with Benedict’s
solution, as seen in the color change of the
mixture.

13
 BCHEM LAB KODIGS
EXP. 7: LIPIDS
I. PURPOSE
To determine the properties of lipids.
II. APPARATUS
Test tubes, test tube rack, droppers,
beakers, wire gauze, Bunsen burner, clay
stand/cover, ice [bath], blue and red
litmus paper, watch glasses, blank sheet
of paper (white), test tube holder
III. MATERIALS
Coconut oil (C33H62O6), water (H20),
dil. hydrochloric acid (HCl), dil. sodium
hydroxide (NaOH), cold alcohol
(CH3CH2OH) hot alcohol (CH3CH2OH),
chloroform (CHCl3), ether (R-O-R’),
congo red (C32H22N6NA2O6S2),
potassium bisulfate (KHSO4), soap
(C17H35COO-) solution, 1% albumin
solution, sodium bicarbonate (Na2CO3)
solution, oleic acid
[CH3(CH2)7CH=CH(CH2)7COOH),
Hubl’s iodine solution (26 grams of iodine
and 30 grams of Hgl2 in 1 L of 95%
alcohol), stearic acid
[CH3(CH2)16COOH], linseed oil
(C18H34O2), glycerol (C3H8O3),
powdered borax {Na2[B4O5(OH)4] •
8H2O}
------------------------------------------------
IV. PROCEDURE
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.
1. SOLUBILITY
Test the solubility of one drop of coconut oil
in 1 ml of the following solvents: water, dil. HCl,
dil. NaOH, cold alcohol, hot alcohol, chloroform,
ether, carbon tetrachloride. Tabulate your
results.
* back to our eyes is not transmitted thru the
Coconut oil is an non-polar substance and is
immiscible in polar solvents such as water, dil.
sodium hydroxide, and cold alcohol, but miscible
in non-polar solvents such as chloroform and
ether (+ carbon tetrachloride). Solubility usually
follows the rule “Like dissolves like.”
Hot alcohol is slight immiscible. Although
alcohol is a polar substance, the high
temperature increases the interactions of the
solute and solvent, forming temporary
emulsion. But when it is cooled down, it
becomes immiscible.
2. REACTION TOWARDS INDICATORS
Place in each of two test tubes, ml of fresh
coconut oil. To the first add congo red, to the
other red and blue litmus paper. Repeat the test
on rancid oil.
Differentiate the reaction of fresh coconut oil
and rancid oil towards the indicators. Fresh
coconut oil is neutral, thus the red litmus paper
remains red, and the blue litmus paper remains
blue. And towards congo red, it can be observed
that it remains red. Since rancid coconut oil is
acid (already underwent hydrolysis and
oxidation into shorter chain fatty acid, making it
acidic), there’s a change in coloration (reaction
towards the indicator).
3. FORMATION OF TRANSLUCENT SPOT
Place 1 drop of coconut oil on a piece of
paper. Allow to evaporate. Does the translucent
spot disappear? No, because oils evaporate very
slowly (as they have high boiling points as well).
Would essential oils leave translucent spot?
No, because essential oils are not real oils.
When light falls upon the paper, a part of it
is transmitted, a part is scattered, a part is
absorbed, and a part is reflected. The degree of
transparency and/or opaqueness of a medium
depends on the ratio of light transmitted vs.
light reflected or scattered. The higher the light
is transmitted and the lesser it is reflected or
scattered, the more will there be transparency.
The amt of scattering depends on many things,
such as the size of the fibers or fillers, and the
difference in the index of refraction b/w the
particles and the surrounding medium.
Normally, when you look at the paper, the
surrounding medium is air with an index of
refraction only slightly greater than 1.0. The
paper fibers have much higher index of
refraction, probably much greater than 1.5. The
fat also has high index of refraction, so that it
nearly matches that of the paper and produces
the scattering significantly. The fat adhering to
the cellulous fibers lowers the index of refraction
of the cellulose, and also fills in air void (?) so
that visible light passes thru the bag with
significantly less scattering. Normally, we only
see the light that’s reflected from the paper, and
much of the light that was formally scattered
paper. The fat connects the paper fibers with a
liquid, which can transmit the refraction rather
than scattering that falls upon it.
As a result, the paper – if thin enough –
seems almost transparent. The translucent spot
will not evaporate easily bec of its high boiling
pt.
4. ACROLEIN FORMATION
Place 0.5 g of KHSO4 in a clean dry test tube.
Add a drop of coconut oil and heat. Describe the
odor produced: Coconut oil will produce a
burned fat-like odor. It will undergo hydrolysis
14
 BCHEM LAB KODIGS
and oxidation with the dehydrating agent acid that has unsaturation that could react with
potassium bisulfate and produce acrolein, or the iodine
fatty acid and water. Which oil contains more unsaturated fatty
Write the equation involved: acids, coconut or linseed oil? Linseed oil
* contains more unsaturated fatty acids such as
triglycerides than coconut oil, since linseed oil
5. EMULSIFICATION absorbs more iodine than coconut oil.
Prepare 4 test tubes with the following Structurally, how does an unsaturated rat
contents: differ from a saturated fat? Unsaturated fat is a
Tube 1: 5 ml water mixture of triglyceride that contains two or more
Tube 2: 5 ml water + 3 ml soap solution carbon-carbon double bonds, while saturated
Tube 3: 5 ml water + 1 ml of 1% albumin fats don’t have carbon-carbon double bonds in
solution its triglyceride components.
Tube 4: 5 ml water + 0.5 ml Na₂CO3 solution What is the relationship of iodine no. and
To each test tube above add 5 drops of coconut unsaturation of a fat? The more iodine is
oil, shake and observe results. Test tube 1 & 4 absorbed, the more unsaturated the fat or oil.
= temporary emulsion [right after mixing], 2
and 3 = permanent emulsion. Both soap and 7. GLYCEROL
albumin are amphipathic substances of Test the solubility of glycerol in water,
hydrophobic and hydrophilic parts that can act alcohol and ether.
as an emulsifying agent. Make an acrolein test on glycerol. Result:
What is an emulsion? An emulsion is a stable Glycerol is soluble in water and alcohol, but
dispersion of two or more immiscible liquids held insoluble in ether. It’s a polar substance only
in suspensions by small %age of substance soluble and miscible in polar solvents. It
called emulsifiers. produces burned fat odor.
Which tube or tubes formed a permanent Fuse a drop of glycerol in a nichrome wire
emulsion? Test tubes 2 and 3 with powdered borax. Note the green flame
produced. This is due to the glycerol ester of
6. TEST FOR UNSATURATION boric acid.
Place 5 ml of oleic acid in chloroform in a test Perform the nitro-chromic acid test on a 5%
tube and add Hubl’s iodine solution drop by drop aqueous solution of glycerol.
shaking between addition. Make a control by What is the color formed? Solution turns
shaking in another tube a mixture of chloroform bluish-green bec of the release of chromic ion.
and iodine with no oleic acid. Solution turns bluish-green with potassium
Explain results: Iodine is soluble in chromate in acidic medium, or the addition of
chloroform and no chemical reaction takes nitric acid.
place. When oleic acid is added to the mixture This indicates the presence of what group in
of iodine and chloroform, oleic acid absorbs and glycerol? Secondary group in glycerol
reacts with iodine, forming a colorless,
halogenated fatty acid.
When an unsaturated fatty acid or
triglyceride absorbs and reacts with iodine, the
intensity of the color of the solution becomes
lesser or lighter than the iodine-chloroform
alone.
Repeat the test using stearic acid, coconut
oil and linseed oil. (This table assumes that the
initial color of all the oils is the same. Old stock
oils that maybe already underwent oxidation
have are darker in or have yellowish color.)
Result should be based on the change of the
intensity of the color before and after adding the
Hubl’s reagent.
Intensity of the color of stearic acid in Hubl’s
= darker red than coconut, coconut = dark
orange to red, oleic acid = yellow to
orange/light orange (?), linseed = light
orange/yellow (?). The lighter the color, the
more iodine is absorbed.
Which oil will absorb more iodine, oleic or
stearic acid? Oleic acid bec oleic acid is an
unsaturated fatty acid and can absorb or react
with the iodine. Stearic acid = saturated fatty

15
 BCHEM LAB KODIGS
EX. 8: ENZYMES
• Organic substances secreted by living cells
that increase the rate of reaction without
being consumed in the reaction
• Most = generally proteins
TYPES
1. Oxidases – catalyze redox reactions; uses
oxygen as electron acceptor (oxidizing
agent)
2. Catalases – catalyze the decomposition of
hydrogen peroxide to oxygen and water
3. Peroxidase – catalyze the oxidation (similar
to oxidases) of a hydrogen donor (or
electron donor) with the help of Peroxides
(such as H2O2).
OXIDASES FROM FRUITS
• The exposed area turned brown due to the
formation of melanin.
• Oxidases – catalyze redox reactions; uses
oxygen as electron acceptor (oxidizing
agent)
• 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.
o Tyrosinase – bifunctional copper
containing oxidase having both
catecholase and cresolase activity
o Orthohydroxylation – addition of two -OH
group in ortho-directing substitution
o Phenols in fruits + O2 + 2H ion →
catechols
o Dehydrogenation – removal of hydrogen
Catechols + O2 → 2 orthoquinone →
melanin (brown pigment)
* FROM POTATO
CATALASE
• Boiling a solution containing enzymes =
subjecting enzymes in high temperatures
denatures, and therefore inactivates them
o Potato – main source catalase
o H2O2 – oxidizing agent and bleaching
agent
o Boiled filtrate (potato extract with H2O2)
– there was less [or no] formation of
bubbles due to the denaturation of the
enzyme catalase, that is why there is a
formation of coagulants
o Unboiled filtrate = there was a formation
of bubbles because of more oxygen that
was catalyzed by the catalase
OXIDASE
• Final results (lightest to darkest):
o Phenol = light pink
o Catechol = light orange
o Pyrogallol = brown
• Monophenol oxidase (tyrosinase) Cresolase
activity: monophenol + O2 → diphenol
catechol
• Polyphenol oxidase (catechol oxidase)
Catecholase activity: Diphenol catechol →
quinone
• Cytochrome oxidase Berthelot’s Reaction:
phenylenediamene + alpha-napthtol →
indophenol Phenol- arene with 1-OH group
catechol- arene with 2 -OH group pyrogallol-
arene with 3 -OH group
PEROXIDASE
▪ Hemoprotein that catalyzes the oxidation of
H2O2
o Phenol = light pink with bubbles (due to
the presence of oxygen)
o Catechol = light orange with bubbles
(due to the presence of oxygen)
o Pyrogallol = brown with bubbles (due to
the presence of oxygen)
• Peroxidase – hemoprotein that catalyzes the
oxidation of H2O2
CATALASE OF LIVER
o Sand – increases friction for the [slimy]
liver to be grinded easily
o Glowing splint – test for an oxidizing gas
evolved (oxygen)
o Liver – filter of the body that uses
enzymes as catalysts → makes harmful
substances less toxic like: H2O2 → water
and oxygen + alcohol → aldehyde
• Split continues to glow bec of H2O2.
• Flame in the split continues to glow due to
the oxygen emitted by the liver. It is not
easily put off bec of the evolution of oxygen
from the decomposition of the H2O2 by the
enzyme catalase of the liver (oxygen
supports combustion)
• Catalase from liver catalyzes the
decomposition of H2O2 to water and oxygen
gas at a rate of 14M molecules H2O2 per
second.
• Liver – filter of the body that uses enzymes
as catalysts → makes harmful substances
less toxic like: H2O2 → water and oxygen +
alcohol → aldehyde
• Lack of catalyst → greying of hair in humans
------------------------------------------------
I. PURPOSE
To determine the presence and
properties of different enzymes.
II. APPARATUS
Knife, grater, test tubes, cheese cloth,
test tube rack, funnel, beakers, Bunsen
burner, wire gauze, flame shield, bamboo
splint, lighter, mortar and pestle
III. MATERIALS
Available fruits (apples, guava, or chico),
small potato, water (H2O), 3% hydrogen
peroxide (H2O2), 1% phenol (C6H5OH),
1% cathecol [C6H4(OH)2], pyrogallol
[C6H3(OH)3], liver, sand
16
 BCHEM LAB KODIGS
------------------------------------------------
IV. PROCEDURE
Enzymes are complex organic
compounds with definite chemical structure,
secreted by living cells. They have the property
of initiation and hastening chemical reaction
without themselves being affected in the
process. Enzymes activity is influenced by the
concentration of the enzymes, the concentration
of the substrate, the concentration of the
products of the reaction, temperature, pH,
inorganic salts and the presence of activators
and inhibitors. All these factors therefore should
be considered while performing the following
experiments.
I. OXIDASES FROM FRUITS
Cut thin slices of apples, guava, chico or
other available fruits and expose them to the air.
Observe the darkening of the exposed sliced
sides. Explain the color change.
The exposed sides of the apple slices
burned brown due to the formation of melanin.
A somewhat “layer of rust” can be made on the
surfaces of fruits when these fruits are oxidized
because of the enzyme tyrosinase or
monophenol oxidase reacting with oxygen and
iron-containing phenol. Basically, melanin was
formed due to the oxidation of iron-containing
phenols in the apple catalyzed by tyrosinase or
monophenol oxidase. The reaction can be
summarized as: tyrosinase + O2 → melanin
(brown pigment)
When fresh fruits (such as apples) are
peeled or cut open, an enzyme called
polyphenol oxidase/tyrosinase (comprised of
monophenol oxidase and catechol oxidase)
[when exposed to oxygen] will result in phenolic
compounds in the fresh fruits (such as apples).
These tissues turning into orthoquinones (no
color, but further react with amino acids and
oxygen to produce melanin (how we get the
brown color on the cut cells of the fruits).
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.
Action
1. Divide the filtrate into four parts and boil
one part for one minute.
2. Place 5 ml of the boiled filtrate in one
tube and 5 ml of the unboiled filtrate in
another tube.
3. Add to each a few drops of 3% hydrogen
peroxide. Note what happens.
In the boiled filtrate, the enzyme catalase
was denatured. So, there was less formation of
bubbles, but there was a formation of
coagulants, too. In the unboiled filtrate, more
oxygen vias catalyzed by the catalase. So, there
was more formation of bubbles. Subjecting
enzymes to high temperatures denatures and in
activates the enzymes. Likewise, when the
solution with enzyme catalase (which came from
the potato) was boiled, catalase was denatured
and inactivated.
Boiling the potato extract = reduces the
catalytic effectiveness of the enzymes bec heat
can destroy the hydrogen bonding that
stabilizes the structural conformation of the
protein enzymes.
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.
How do you know that the gas liberated is
oxygen? The gas liberated was oxygen because
what can make the flame in the splint continue
to glow is the oxygen emitted by the liver. A
glowing splint tests for oxidizing gases evolved,
and in this cave, it was oxygen. In this reaction,
the enzyme catalase of the live catalyzed the
decomposition of H2O2, causing the evolution
of oxygen and a flame not easily put off.
What does catalase do? Catalase
decomposes H2O2 into water and oxygen in the
procedure. In real life, catalase in the liver can
also turn alcohol to aldehyde. With catalase,
harmful substances can be made less toxic.
III. OXIDASES FROM POTATO
There are three oxidases:
1. monophenol oxidase (tyrosinase)
responsible for oxidizing phenol to
cathecol, to O-quinone and finally forms
condensation brown compounds of
unknown composition.
2. polyphenol oxidase (cathecol oxidase) -
facts on cathecol forming o-quinone,
then the unknown brown compounds. It
acts on pyrogallol to form purpurogallin
3. cytochrome oxidase acts in conjunction
with-cytochrome, oxidizing
phenylenediamine, which in the presence
of alpha napthol forms iodophenol
Procedure
1. Prepare 3 test tubes and place 5 drops
each of the following substances:
a. 1% phenol – light pink color was
observed; light brown to brownish red
b. 1% cathecol – light orange color was
observed; brownish red to darker
brownish red
c. pyrogallol – brown color was
observed; red to red
2. Add 1 ml of potato extract (oxidase) to
each test tube and shake.
17
 BCHEM LAB KODIGS
3. Allow them to stand until the end of the
laboratory period, observing and
recording the initial color change, then
the change at the end of the period.
Initially, phenol is first converted to
cathecol then o-quinone, forming a
brown substance (melanin). It takes time
for phenol to form o-quinone than
cathecol that shows phenol is lighter
brown in color than catechol. Pyrogallol
is oxidized to purpurogallin with the
presence of peroxidase in potato.

IV. PEROXIDASE FROM POTATO

Peroxidases, unlike oxidases, may
require 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.
Procedure
1. Place 3 test tubes and add 5 drops each
of the following substances:
a. 1% phenol – light brown to brownish
red
b. 1% cathecol – brownish red to darker
brownish red
c. pyrogallol – red to darker red
2. Add 1 ml of potato extract (peroxidase)
to each.
3. Mix well, then add 3 drops of 3%
hydrogen peroxide to each. Observe and
record the color changes produced and
compare them with the results observed
on the action of oxidases with the same
reagents.
A light pink color was observed in the test
tube with phenol, a light orange, was observed
in the test tube with cathecol, and a brown color
was observed in the test tube with pyrogallol. All
solutions in the test tubes bubbled as well, due
to the presence of oxygen, setting them apart
from the reactions with oxidases.
Peroxidases catalyzes more pyrogallol to
purpurogallin.

18
 BCHEM LAB KODIGS
EXP. 9: TEST FOR NUTRIENTS IN FOODS
I. PURPOSE
To test for or determine the various
nutrients in different food items.
II. APPARATUS
Test babes, test tube rack, beakers,
droppers, Bunsen burner, wire gauze,
flame shield, evaporating dishes or watch
glasses, white paper
III. MATERIALS
Food samples, water (H2O), iodine (I2)
solution, Benedict’s solution (CuSO4,
Na2CO3, sodium citrate), ether (R’-O-R),
chloroform (CHCl3), cold saturated
solution of antimony chloride (SbCl3),
sodium hydroxide (NaCl)
------------------------------------------------
IV. PROCEDURE
For each of the following tests, use the
following food samples:
Fruit juice, potatoes, butter, green and
ripe bananas, carrots, cheese, cooked egg
white, cooked egg yolk, unsalted dried fish meat
like dilis, 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.
2. Glucose: Add a few drops of a fruit juice
to 10 ml of water in a test tube and then
Benedict's solution sufficient to give a
very light blue solution. Boil. A yellow to
red, precipitate indicates the presence of
a simple sugar such as glucose or
fructose.
3. Fats: To examine foods for oil or fat,
shake a small portion of the food with 3
mi 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: Burn 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.
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 add
2 ml of a cold saturated solution of
antimony trichloride. Observe the
changes in color. How long did the color
persist? Perform this test on margarine or
butter and carrots only.
What are your conclusions regarding the
general constitution of your food samples?
nutrients found in each food and the negative
sign(-) if absent. (+) Only a few food samples
were complete with regards to general
constitution, such as peanut butter and un
animal products were ich in protein. Dilis tested
negative for most components of the general
constitution most likely because of its dryness.
The general constitution and the amount of
nutrients in the sample depends on the variety,
source, and chemical rxn it underwent before
the identi test. Most of the food samples contain
three or more of the food nutrients that vary in
each concentration.
Write the formula of Vitamin A and state its
importance. The formula of Vitamin A, a.k.a.
Retinol, is C20H30O. In general, it helps the
heart, lungs, kidneys, and other organs function
well. To be more specific, it is necessary for
good vision, immunity, and cell communication.
Vitamin A binds to retinoid receptors (which act
as transcription factors to alter for regulate gene
expression) and inhibit carcinogenesis (the
process by which normal cells die transformed
into cancer cells), inducing cell differentiation. It
makes up a portion of rhodopsin, a protein
important for light absorption in the retinas of
eyes.
Vit. A or retinol is the precursor to two active
metabolite: retinol (plays a critical role in vision)
and retinoic acid (intracellular messenger that
affects transcription of a number of genes). Vit.
A doesn’t occur in plants, but many plants
contain carotinoid, such as betacarotene that
can be converted to Vit. A w/in the intestine and
our tissues.
The observable result will depend on the
amount of nutrients that can be detected in
these general tests quantitatively.
19
 BCHEM LAB KODIGS
EXP. 11: MILK
I. PURPOSE
To determine the composition and
properties of milk.
II. APPARATUS
Beakers, watch glasses, stirring rods, test
tubes, test tube rack, droppers, Bunsen
burner, wire gauze, flame shield, test
tube holder, Erlenmeyer flask, funnel,
filter paper, crucible, desimeter
III. MATERIALS
Congo red (C32H22N6Na2O3S2);
phenolphthalein (C20H14O4) solution;
fresh milk; skimmed milk; dil. canned
milk; sour milk; acetic acid (HAc); 6 M,
dil., and 10% sodium hydroxide (NaOH);
ethyl alcohol (CH3CH20H); casein (C81H
125N22O39P); dil. hydrochloric and
(HCI); 10% sodium chloride (NaCl);
Millon’s reagent [Hg(NO3)2]; .5% copper
sulfate (CuSO4); red and blue litmus
paper; ammonium molybdate
[(NH4)2MoO4]; ammonium oxalate
[(NH)2C2O4]; Benedict’s solution
(CuSO4, Na2CO3, sodium citrate)
------------------------------------------------
IV. PROCEDURE
Milk is the most complete food, it
contains proteins, fats, carbohydrates, inorganic
salts and vitamins. Fresh unboiled milk also
contains enzymes, protease, lactase, lipase,
phosphatase, catalase, and peroxidase.
The chief protein of milk is casein, which
can be precipitated by acid, carrying with it the
milk fat. The fat can be subsequently extracted
by organic solvents. The filtrate from the casein
and fat contains the soluble constituents like
lactose and inorganic salts.
The proteins of milk is derived from
amino acids of the blood, the synthesis
occurring in the mammary glands.
Milk fat is believed to have its origin in
the phospholipids of the blood.
The lactose of milk is derived from the
glucose of the blood.
The inorganic salts, like calcium,
magnesium, sodium, phosphates, citrates and
chlorides and other inorganic constituents are
likewise derived from the blood, some by simple
process of filtration. The bone acts as reserve
supply of calcium.
Human milk differs from cow's milk in
having less casein and ash and more albumin
and lactose.
1. REACTION OF MILK
Test 1 ml of milk to both red and blue litmus
paper, congo red and phenolphthalein solution.
What is the reaction of milk? Fresh milk did
not react with both red and blue litmus paper,
congo red, and phenolphthalein solution.
However, sour milk turned the blue litmus paper
red, blue with congo red, and colorless with
phenolphthalein solution. All these were due to
sour milk being acidic (it became acidic because
of certain bacteria in the milk converting the
lactose to lactic acid).
Fresh milk + red litmus paper = red litmus
paper remains red, milk + blue litmus paper =
blue litmus paper remains blue, milk + congo
red = red sol’n, milk + phenolphthalein =
colorless/no change in color
Sour milk + red litmus paper = red litmus
paper remains red, milk + blue litmus paper =
blue litmus paper turns red, milk + congo red =
blue sol’n, milk + phenolphthalein =
colorless/no change in color
Fresh milk is slightly acidic to neutral. Milk
upon standing is acidic due to the oxidations of
lactose to lactic acid and its fats to shorter
carboxylic acid.
Congo red with sour milk is blue in color.
2. DETERMINATION OF SPECIFIC GRAVITY
By the use of a desimeter determine the
specific gravity of both fresh milk and
skimmed milk.
Which has a greater specific gravity?
Skimmed milk
Why? There is less fat or no fat in its
composition.
Milk fat is lighter in density than water
and floats on the surface on unhomogenized
milk. When you skim off the surface, some
of the fat in the denser portions remain and
the milk is denser. This explains why
skimmed milk is denser.
3. FILM FORMATION
Place 5 ml of diluted canned milk in a
small beaker and boil for a few minutes. Note
the formation of a film.
What is the composition of the film? The
composition of the film consists of surface
fats and proteins.
Milk forms a film on top when heated bec
of a chemical rxn that affects how protein
and fat molecules interact with each other.
When milk is heated rapidly, some of the
water in it evaporates from the surface,
exposing protein and fat molecules, which
bind and dry out as warming continues.
Remove the film and heat again. Does the
film form again? Yes, but lesser or thinner than
the first.
Repeat the experiment using sour milk. Is
there any film formation? No, bec it contains
lesser fat that can interact with the soluble
protein.
What happened? Why? The film formed
because of a fat-protein interaction that bonded
and dried out after the boiling of dilute canned
20
 BCHEM LAB KODIGS
milk. Sour milk, on the other hand, did not form
any film because its proteins were denatured by
lactic acid.
Sour milk has lesser fat bec some fats are
hydrolyzed and oxidized into shorter chain
carboxylic acid, which also contributes to
souring of milk aside from lactic acid. When
water evaporates from milk during heating, the
milk proteins and fat molecules become more
condensed on the surface. Since sour milk has
lesser or no fat, there will be no condensation of
fat and protein forming film.
What factors facilitate the formation of
surface film? Temperature, stirring process,
presence of fat, and type of milk
Heating and evaporation of the water’s
surface, causing the surface fats and proteins to
condense or interact
4. COAGULATION TEST
Place 1 ml of fresh milk in a test tube and
acidify with acetic acid. Heat to boiling.
Is there any coagulation? Why? Yes, there is
coagulation. This is because acetic acid brought
the proteins closer to their isoelectric points,
allowing them to clump up and bind easily.
Yes, the supernatant whey becomes cloudy
after heating. After the casein is precipitated,
put the solutions in acetic acid, there is still
proteins that cannot form insoluble precipitate
with acetic acid. These proteins/whey are
soluble proteins and will coagulate when
heated. Heat will destroy the H-bonding of the
protein molecules and will make the protein
coagulate.
5. ACTION OF HOT ALKALI
Mix 1 ml of milk with drops of 6 M NaOH.
Heat and describe the changes observed.
What is the cause of these changes? The
liberation of aldehyde groups in the lactose of
milk, which polymerized them to form an
aresinous substance
The sol’n turns from yellow to brown due to
the rxns of lactose or the reducing sugar with a
strong concentrating NaOH.
What test in the study of carbohydrates
involves the same principles? Moore’s Test
6. PREPARATION OF CASEIN
Take 10 ml of milk and dilute it with an equal
volume of water. Add dilute acetic (1%) drop by
drop until a flocculent ppt. forms. Avoid any
excess as dissolution may occur. Allow the ppt.
to settle, decant the supernatant fluid (whey)
and reserve it for experiment 7. Filter off the
ppt. Remove excess moisture by washing the
ppt. with a few ml of ethyl alcohol. Transfer the
casein to a dry test tube, cover it with ether and
heat on a water bath set at 50 °C without flame
for 5 to 10 minutes, shaking continuously. Filter
and save the filtrate for no. 8. Press the
precipitate as dry as possible between filter
papers. Open the papers and allow the ether to
evaporate spontaneously (without heat). Grind
the dry ppt. to powder in a mortar and make the
following test:
A. Solubility: Test the solubility of casein in
water, dilute HCl, dilute NaOH and 10%
NaCl. Casein is soluble in water (slightly)
and dilute NaOH, but insoluble in and
dilute HCl and 10% NaCl.
B. Perform Millon’s test on casein.
Result? Flesh to red precipitate due to the
phenolic group of tyrosine in its protein
chain
To what class of protein does casein
belong? Conjugated proteins, specifically
phosphoproteins
7. Take two 3-ml portions of the whey (filtrate
from no. 6) and make the following test:
a. Coagulation by heating. Note the
formation of a coagulum. This consists of
lactalbumin and lactoglobin. Make a
biuret test on the coagulum.
Observation? Violet color bec of the
presence of proteins
When the filtrate was heated,
coagulation of the protein was observed
due to the coagulation of lactalbumin and
lactoglobulin. These are confirmed by
biuret test that shows purple colorations
when heated with the Millon’s rgt.
b. Boil the second portion of the filtrate and
remove every trace of coagulable
protein. Filter. Test the filtrate for the
presence of P, Ca and reducing sugar.
Observations? The presence of
phosphate/phosphorous (P) was confirmed due
to the yellow precipitate present, the presence
of calcium (Ca) was confirmed due to the white
precipitate present, and the presence of the
reducing sugar/lactose was confirmed due to
the positive reaction with the Benedict’s test.
Draw your conclusions: Whey protein is
found in whey, which is the liquid part of milk
that results from the curdling and straining of
milk. It coagulates upon the addition of heat, as
well as takes on a violet color after the biuret
test because of the presence of albumins and
globulins. Phosphate was present because of
the formation of yellow precipitate with
(NH4)2MoO4, calcium was present because of
the formation of white precipitate with
(NH4)2C2O4, and reducing sugar was present
because of the positive Benedict’s test result.
Benedict’s test is used to determine the
presence of reducing sugar (changes the color
of the solutions to yellow, green, or red after
heating due to lactose, the most abundant carb
in milk), ammonium oxalate test for calcium
(white ppt – calcium oxalate), and ammonium
molybdate test for phosphorus (yellow ppt).
8. MILK FAT
Transfer the ether filtrate obtained in no. 6
into a dry evaporating dish and place it on a
21
 BCHEM LAB KODIGS
boiling water bath to evaporate the ether (turn
out all flames), leaving a small amount or
residue. What is the residue? Milk fat
Touch the residue with a piece of paper.
What do you observe? The piece of paper looks
translucent, which indicates a positive result for
fats.
Translucent spots on the paper

WARNING: Ether could cause severe headache

if smelled in larger quantities. Only the assigned
groups will be given ether. Tests on casein could
be done if addition of ether is omitted for those
groups not given ether.

22
 BCHEM LAB KODIGS
EXP. 12: SALIVARY DIGESTION
I. PURPOSE
To determine the components of saliva.
II. APPARATUS
Beakers, stirring rod, watch glasses, red
and blue litmus papers, test tubes, test
tube rack, test tube holder, Bunsen
burner, flame shield, wire gauze, put
plate/white tile
III. MATERIALS
Saliva, phenolphthalein (C20H14O4),
congo red (C32H22N6Na2O6S2), paraffin
(CnH2n+2), dil. acetic and (HAc), 1%
starch [(C6H10O5)n] paste, iodine (l2),
Benedict’s solution (CuSO4, Na2Co3,
sodium citrate), silver nitrate (AgNO3),
dil. hydrochloric acid (HCl), 0.5 barium
chloride (BaCl2), ammonium oxalate
[(NH4)2C2O4], HCl (0.1%., 0.25%,
0.05%, 0.025%, 0.0075%)
------------------------------------------------
IV. PROCEDURE
Saliva is secreted by three pairs of
glands, the parotid, submaxillary and sublingual
and hundreds of small buccal glands. The flow
is stimulated by psychic, chemical and
mechanical factors. It contains a protein, mucin
and an enzyme, ptyalin end inorganic salts.
By salivary digestion is meant the
hydrolysis of starch by salivary amylase (ptyalin)
which is taking place in the buccal cavity and to
a certain extent in the fundic end of the
stomach.
1. REACTION
Place a few drops of resting saliva in three
test tubes. Test the reaction with
phenolphthalein, litmus and congo red. From
the color produced estimate the approximate pH
of resting saliva.
Repeat the experiment using stimulated
saliva. Stimulate the flow of the saliva by
chewing paraffin vigorously for at least 5
minutes. Do not use chewing gum for this
purpose.
What is the difference between the pH of the
resting saliva and the stimulated saliva? The
difference between the pH of resting saliva and
stimulated saliva is that testing saliva is slightly
acidic (pH between 5.7 and 6.2), while
stimulated saliva is slightly basic (can reach pH
8).
Saliva is slightly acidic with a pH of 6-7 and
contains buffer systems responsible for
maintaining proper acid-base balance. The most
impt role is played by the bicarbonate buffer.
The buffers maintain the pH of resting saliva
b/w 5.7 and 6.2, while the pH of stimulated
saliva can reach 8.
Is there any relation between the pH of the
saliva and the susceptibility of dental caries?
Yes. The lower the pH of the saliva, the greater
the susceptibility to dental caries because of
enamel erosion.
Saliva also plays a v impt role in the
inhibitions and dev’t of the caries lesions (?) of
the teeth, improving remineralizations of the
tooth enamel and preventing demineralization.
When the pH of this secretion is within 6.8-7.2,
it becomes a saturated sol’n of calcium
phosphates, which results in quick and effective
remineralizations of the initial changes.
However, if we slightly acidify the envi, saliva
becomes an unsaturated sol’n and easily soluble
calcium hydrogen phosphates are formed. Thus,
the susceptibility of the teeth to caries
decreases.
2. TEST FOR MUCIN
To 3 ml of saliva in a test tube add 1-2 drops
of dilute acetic acid.
What is the precipitate formed? Mucin clot
Coagulation of protein/mucin
What is its function? Mucin is a gland
secretion that lubricates food, preventing it from
damaging any epithelial cells. Therefore, the
mucin clot test can help determine inflammatory
conditions wherein mucin is provided and
secreted (most frequently, it is in the joints).
Mucin also protects the surface of oral mucous
membranes from toxins and maintains the
avidity of saliva.
Mucin in saliva protects the surface of oral
mucous membranes from the toxins and various
types of irritants contained in stimulants or food.
Mucin is mainly responsible for the elasticity of
saliva and contributes lubricating properties of
saliva.
3. INORGANIC MATTER
Acidify 10 ml of saliva with a drop or two of
acetic acid, heat to boiling and filter to remove
the protein. Test the filtrate for chlorides,
phosphates, sulfates and calcium.
Which of the inorganic salt is found
abundant in the saliva? Chlorides and
phosphates
4. DIGESTION OF STARCH PASTE
Place 10 ml of 1% starch paste in a small
beaker. Add 5. drops of saliva and stir
thoroughly. Add another 5 drops of saliva, if
blue color still forms with iodine after 5 minutes.
Gradually the opalescence of the starch solution
disappears due to the formation of soluble
starch. In making the test with iodine, remove a
drop of the starch-saliva solution and transfer to
a spot plate or white tile at intervals of one
minute. Stir for sometime and test again with
iodine. When no more blue color is produced
23
 BCHEM LAB KODIGS
with iodine test a portion with Benedict's test
and note the degree of reduction.
What is the reaction with iodine when the
Benedict's test becomes positive? The reaction
is a negative one, as there is no longer any
starch. The iodine test can only identify starch
and dextrin.
When the Benedict’s test becomes positive,
this means that the starch already hydrolyzed
into reducing sugar maltose. Maltose shows
negative result in iodine test bec iodine test is
used to identify starch and dextrin.
What is responsible for the reducing action?
Maltose
How long did it take for a complete
transformation of the starch into reducing
sugar? It took 10-20 minutes to completely
transform starch into reducing sugar. However,
usually, the time to transform starch to a
reducing sugar (like maltose) depends on the
exposure of the starch to saliva and how much
saliva is present.
The time to complete the hydrolysis of starch
into maltose in the salivary digestion depends
on how the starch is exposed to the saliva and
the amt of saliva in the exp. If the result in the
iodine test is negative or there’s no change in
color before and after addition of iodine,
Benedict’s should follow to confirm if the final
result is maltose. If the results show negative in
both iodine and Benedict’s, the product of the
starch hydrolysis is not yet maltose, but aqua
dextrin and is needed to hydrolyze further for
the sol’n to be positive in Benedict’s test.
Outline the different stages of starch
digestion by ptyalin. How does this differ from
starch acid hydrolysis?
Starch Digestion by Ptyalin
Starch, to soluble starch, then amylodextrin
Starch to soluble starch – add iodine sol’n =
blue-black color
When iodine turns purple = amylodextrin stage
More saliva + iodine – red = erythrodextrin
Achrodextrin – yellow/colorless
Maltose – no color
Soluble starch – blue-black in color with iodine
Amylodextrin – purple
Erythrodextrin – red
Achrodextrin – yellow/colorless
Maltose – colorless
Starch acid Hydrolysis
Same stages as ^, but ends in glucose
5. INFLUENCE OF ACID
Place 2 ml of one of the following strength
of acids in each of the 5 test tubes: 0.25%,
0.1%, 0.05%, 0.025%, and 0.0075% HCI. To
each add 1 ml of 1% starch paste and 1 ml
saliva. Shake well. Place in a water bath at 40°
C, for 20 minutes. At intervals of 5 minutes test
a small portion with iodine and Benedict's test,
until a positive result with Benedict's and a
negative result with iodine is obtained.
In which tube did you obtain the greatest
digestion? The tube where the greatest
digestion was obtained was in the tube with
0.025%.
Test tube with lowest concs of acid = .025%
HCl. The presence of high concs of HCl in the
saliva might denature the salivary amylase,
which is the enzyme responsible for the
hydrolysis of starch. Denaturation of saliva
results to a decrease of the activity of the
enzymes.
6. INFLUENCE OF ALKALI
Repeat the above experiment using instead
of HCI, NaOH with the following concentrations:
2%, 1%, 0.5%, 0.125%, and 0.065%.
Neutralize the alkalinity before performing
the iodine test (use acetic acid to neutralize)
Which has a greater inhibiting power, acid or
alkali? Acid has a greater inhibiting power
(ptyalin is only slowly inactivated by acidic
gastric juice in the stomach). It disrupts the
saliva PH used in buffering; it lowers salivary pH
beyond normal and reduces the catalytic
effectiveness of an enzyme.
What is the effect of gastric juice on the
digestive section of saliva? Gastric juice slowly
penetrates into bolus (moss of chewed food),
continuing salivary digestion in the stomach.
Deactivating ptyalin, it lengthens the digestive
action of saliva in carbohydrates.
How long does salivary digestion continue in
the stomach? Salivary digestion can continue in
the stomach for as long as 10-20 minutes bec
gastric juice takes time to penetrate the bolus,
stopping after then due to the total inactivation
of ptyalin.
Salivary amylase works best at pH lvl of
saliva. Increasing or decreasing away from the
normal pH range of saliva decreases its catalytic
activity. Although the normal pH range of saliva
is b/w 6.8-7.2, the stimulated saliva reaches the
pH 8. So, acid bec it lowers the pH of the saliva
beyond its normal ranges and reduces the
catalytic effectiveness of the enzyme.
24
 BCHEM LAB KODIGS
EXP. 14: BILE
I. PURPOSE
To determine the properties of bile.
II. APPARATUS
Test tubes, test tube rack, droppers,
Erlenmeyer flask, funnel, filter paper,
beakers, test tube holder, evaporating
dish, Bunsen burner, wire gauze, flame
shield, litmus paper
III. MATERIALS
Bile, phenolphthalein (C20H14O4),
congo red (C32H22N6Na2O6S2), fusion
mixture (2 parts sodium
carbonate/NaCO3 and 1 part potassium
nitrate/KNO2), water (H2O), nitric acid
(HNO3), 5% sucrose (C12H22O11)
solution, sulfuric acid (H2SO4), furfural
(C4H3OCHO), finely powdered sulfur (S),
ether (R’-O-R), chloroform (CHCl3),
ammonium molybdate [(NH4)2MoO4],
silver nitrate (AgNO3), barium chloride
(BaCl2)
------------------------------------------------
IV. PROCEDURE
Bile is a secretion of the liver. It is viscid
and has an alkaline reaction and its color is
greenish brown. Its important constituents are
bile acids, bile pigments, inorganic salts and
cholesterol.
1. REACTION
Test the reaction of bile to litmus paper,
phenolphthalein, and congo red solutions. What
is the reaction of bile? Bile in red litmus paper =
slightly blue, bile in blue litmus paper = remains
blue, bile with phenolphthalein = slightly pink,
bile with congo red = remains red
Normally what is the pH value of bile?
Greater than 7 but less than 9
Slightly basic/alkaline (7-8)
2. INORGANIC CONSTITUENTS
Evaporate 10 ml of bile to dryness. Fuse the
residue with fusion mixture (2 parts of sodium
carbonate and 1 part of potassium nitrate) Cool
and extract with 10 ml of water. Acidify with
HNO3, until slightly acid. Filter and test the
filtrate for chloride, sulphate, and phosphate.
What inorganic constituents are found in the
bile? Chloride (Cl), sulphate (SO42-), and
phosphate (PO43-)
Test for chlorides = HNO3 + AgNO3; white ppt
of silver chloride (AgCl)
Test for sulfate = HCl + BaCl; white ppt of
barium sulfate (BaSO4)
Test for phosphate = NO3 + [(NH4)2MoO4];
yellow ppt of ammonium phosphomolybdate
3. TEST FOR BILE PIGMENTS
a. Gmlin's test: Place 1 ml of conc. nitric
acid in a test tube. Superimpose 1 ml of
dilute bile, care being taken not to mix
the two fluids. Note the production of
different colors at the point of contact:
green, blue, violet, red and reddish-
yellow. Based on the production of our
different colors at the point of contact,
bile pigment have been oxidized by conc.
HNO3.
Test depends on the fact that during the
stage of oxidation, bilirubin undergoes a
series of changes in color, which follow
the sequence of the familiar solar
spectrum. Place a drop of the fluids to be
tested on a white surface, capsule, or
plate, and allow a drop of HNO3 (yellow)
to fuse and make it more oxidizing, to run
into it. As they mingle together, the
rainbow-like color appears. This, when
washed, will be found to consist of a
series of changes from green, blue,
violet, red, and yellow. This can also be
observed by allowing the acid to trickle
down the side of the test tube fixed in an
inclined position for the play of color to
be seen, starting from the pt. of junction
of the two fluids.
b. Rosenbach's modification of Gmlin's test:
Filter some very dilute bile (1:50)
through a small filter paper. When all has
been drained through and the paper is
still moist throughout, place a drop of
conc. nitric acid into the top of the cone
of the paper. Note the concentric
succession of colors radiating from the tip
of the cone. The concentric succession of
colors radiating from the tip of the cone
indicated the presence of bile pigments.
The same diff colors are observed in the
paper.
4. TEST FOR BILE ACIDS AND BILE SALTS
a. Pettenkofer's Test or Sucrose-Sulphuric
acid test: Place 1 ml of dilute bile solution
in a test tube. Add 1 drop of 5% sucrose
solution. Slowly run about 0.5 ml of conc.
sulphuric acid down the side of the tube.
Observe the production of a red ring at
the point of contact. Shake and the whole
solution assumes a red color. Place the
tube under running water to prevent the
rise of temperature beyond 70° C. Bile
salt reacts with
hydroxymethylenefurfural (sucrose was
dehydrated by H2SO4).
To a fluid containing either or both bile
acids or any solns of cholic acids, add
some cane sugar, then slowly (drop by
drop) strong sulfuric acid, the soln turns
to cherry red then purple. Other
substances such as albuminous bodies
give under the tx a similar color. To make
25
 BCHEM LAB KODIGS
the rxns a trustworthy test for bile salts,
the two characteristic absorption bonds
given by the spectroscope should also be
observed.
The test is not reliable in the presence of
proteins and other chromogenic
substance which give color with sulphuric
acid and therefore, interfere with the
reading of the results.
b. Foam test: Place 1 ml of dilute bile
solution in a test tube and add 1 drop of
dilute aqueous solution of furfural
(1:1000). Shake and observe the
production of a thick foam. Place a drop
of conc. H2SO4 on the foam. Note the
production of a dark pink color. Color due
to the condensations of furfural and the
bile acids and salts in the dil. soln of bile
c. Hay's Test of Surface Tension Test: This
test is based upon the principle that bile
acids or bile salts have the property of
reducing the surface tension of the fluid
in which they are contained.
Cool about 5 ml of diluted bile in a test
tube to 17° C or lower and sprinkle a little
finely powdered sulphur on the surface of
the fluid. The presence of bile salts is
indicated by the sinking of sulphur, the
rapidity depending on the quantity of bile
acids present.
Repeat the experiment using water
instead of bile. Compare your results.
Sulfur powder floats at the surface of the
water due to the surface tensions b/w the
nonpolar sulfur and the polar water. Test
tubes containing dilute bile and sulfur
powder – sulfur powder sinks bec bile
and its salts reduce the surface tension
b/w the water and the nonpolar surface.

5. CHOLESTEROL IN BILE
Evaporate to dryness 5 ml of undiluted bile.
Extract with a small amount of ether at a time.
Evaporate the ether extract to dryness on a hot
water bath (no flame). Dissolve the residue in 3
ml of dry chloroform. Perform the Liebermann-
Burchard test on the chloroform solution.
Unsaturated steroids react with acetic anhydride
in chloroform in the presence of conc. H2SO4 to
produce a solution that is red, then tums blue,
and finally, becomes bluish-green. (quali
determination of chole)
What is the danger of excessive amount of
cholesterol in the bile? Explain. Formation of
gallstones. When bile is oversaturated with
excess chole, the excess crystallize and form
gallstones.

26
 BCHEM LAB KODIGS
EXP. 15: URINE
I. PURPOSE
To identify the properties of urine and
best for the different chemicals or
compounds found in urine.
II. APPARATUS
Test tubes, test tube rack, droppers,
beakers, Bunsen burner, wire gauze,
flame shield
III. MATERIALS
Urine, sodium nitroprusside
{(Na₂[Fe(CN)5No] • 2H2O}, sodium
hydroxide (NaOH), ammonium hydroxide
(NH4OH), zinc chloride (ZnCl2), acetic
and (HAc), barium chloride (BaCl₂), nitric
acid (HNO3), silver nitrate (AgNO3),
magnesia mix, Benedict’s reagent,
benzedrine (C18H28N2O4), aceto-acetic
acid (C4H6O3)
------------------------------------------------
IV. PROCEDURE
Urine is a filtrate from the blood and
serves as a medium for excretion of water, salts,
acids, bases, waste products of metabolism and
other toxic materials. As much, it helps in the
maintenance of water balance, acid-base
equilibrium and serves as an important factor in
the detoxication of the body.
Collection and Preservation of Urine Sample:
For the study of both the qualitative and
quantitative composition of urine, it is better to
examine the 24 hour specimen.
The bladder is emptied at 8:00 a.m. and
the urine is discarded. All the urine from this
time up to 8:00 a.m. the nest day is taken as
sample.
To preserve the urine, a thick layer of
toluene is over-layed on its surface.
1. GENERAL CHARACTERISTICS
What is the volume of the 24-hour
urine? 1K-1.5K mL
a. Examine the specimen as to color, odor,
transparency and reaction.
What substances are responsible for the
normal color of urine? Urochrome and
urobilin
What happens when the urine is allowed
to stand for sometime, exposed to air?
Bacteria causes urea to break down into
ammonia, therefore increasing pH.
What is the normal reaction of urine
when freshly voided? Acidic at around pH
6
To what substances is this reaction due
to? Presence of phosphates, organic
acids, and uric acids
What happens when allowed to stand
without a preservative? Why? Urine
develops foul odor and color becomes
darker. This is due to the conversion of
urea to ammonia and the decompositions
or oxidations of the organic substance by
bacteria in the urine.
What constituents of the urine tend to
precipitate when the reaction is acidic?
Urates or sodium urate and uric acid
What substances precipitate in alkaline
urine? Calcium phosphates, oxalates, and
What is the specific gravity range of
normal urine? 1.015-1.025
2. DETECTION OF CREATININE
a. Nitroprusside test (Weyl)
To 5 ml of the urine add 3 drops of
sodium nitroprusside. Alkalinize with
NaOH. A ruby red color is produced
which turns yellow. This is indicative
of the presence of creatinine. Sol’n
turns to ruby red then yellow
b. Picric acid reaction (Jaffe) : Place 5 ml
urine in a test tube, add an aqueous
solution of picric acid and render the
solution alkaline with NaOH solution.
A red color is produced due to the
formation of a red tautomer of
creatinine picrate. This turns yellow
when the solution is acidified. Glucose
gives a similar red color, only upon
heating. Sol’n turns red and yellow
when acidified
3. DETECTION OF PIGMENTS
Ammoniacal zinc chloride test.
Place 2 ml of urine in a test tube
and add 1 ml of NH,OH stand for a while
and filter. To the filtrate add 2 drops of
zinc chloride solution.
The production of greenish
fluorescence indicates the presence of
urobilin.
What are the other pigments
normally present in the urine? Urochrome
and uroerythrin
Inorganic Physiological Constituents
4. SULPHATES
a. Detection in inorganic sulphuric acid:
Place 5 ml of urine with 5 drops of
acetic acid. Add barium chloride
solution. White ppt (barium sulfate)
5. DETECTION OF CHLORIDE
Place 5 ml of urine in a test tube
and acidify with 2 drops of HNO3. Add 2
27
 BCHEM LAB KODIGS
drops of AgNO3. Note: What is
produced? White ppt (silver chloride)
Add excess of NH₂OH. What
happened with the precipitate? Ppt will
be dissolved and react with the excess
NH4OH, forming soluble diaminesilver-1-
chloride
What is the normal amount of
chlorides eliminated in 24 hours? 110-
250 mille eqs per day, approximately
3.89-8.85 g per day
In what forms are they
eliminated? NaCl, KCl, MgCl2, NH4Cl
6. DETECTION OF PHOSPHATES
Make 10 ml of urine alkaline with
ammonium hydroxide and warm. Result?
White gelatinous ppt
The earthy phosphates or
phosphates of Ca and Mg separate. Filter
off the earthy phosphates and small
amount of magnesia mixture to the
filtrate. Warm the solution. Result? White
gelatinous ppt
Earthy phosphate or Ca
phosphates and Mg phosphates will
precipitate with NH4OH. The remaining
solns may contain alkali phosphate,
sodium phosphate, and potassium
phosphate, which can be precipitated out
from the sol’n by adding Mg mixture and
will form white gelatinous ppt.
The alkali phosphates or the
phosphates of Na and K separate.
Determine which form of
phosphate is present in larger amount.
Alkali phosphates
PATHOLOGIC CONSTITUENTS
7. ALBUMIN
Healthy indi should have lower
concs of these or nega. If posi, might
have conditions that affect the concs, but
diet before test could also affect the
results (showing false-posi)
a. Coagulation test: Heat 5 ml of urine to
boiling in a test tube (filter if urine is not
clear). If heated portion becomes cloudy
the turbidity may be due to phosphates.
Add 3 - 4 drops of very dilute acetic acid
and warm, the phosphates will dissolve,
while if a more flocculent precipitate will
be produced if only albumin is present.
(no albumin in the sample)
b. Heller's Ring Test: Place 5 ml of conc.
nitric acid in a test tube, slant the tube,
and very carefully allow an equal amount
of urine to slowly run down the side of
the tube. The urine will float on the nitric
acid, and a white ring (precipitated
protein) will appear at the junction of the
two liquids. Sometimes the white zone
does not appear until allowed to stand for
a few minutes. (no formation of white
ring in the sample)
8. GLUCOSE
Benedict’s test. - And 8 drops of
urine to 5 ml of Benedict's reagent and
boil vigorously for 2 minutes. Set aside to
cool. The amount of precipitate and its
color (red, yellow or green) depend on
the quantity of glucose present in the
urine.
9. BLOOD (DEMONSTRATION)
Benzidine test: Heat 3 ml of urine
to boiling, cool and treat with an equal
volume of a saturated solution of
benzidine in glacial acetic acid. Add 1 ml
of 3% H2O2. The development of a blue
or green color indicates the presence of
blood.
10. BILE
Gmelin’s: Place 1 ml of conc. nitric
acid in a test tube and upon it
superimpose 1 ml of urine. Do not mix
the 2 fluids.
At the point of contact various
colored rings are noted: blue, green,
violet, red and reddish yellow, in the
presence of bile pigments. Is bilirubin
normally present in the urine? No
What does its presence indicate?
Possible jaundice, liver cirrhosis, hepa,
and other liver diseases
11. ACETONE BODIES
There is no satisfactory, simple
direct test for b-hydroxyl butyric acid in
the urine. Aceto-acetic acid on the other
hand decomposes so rapidly with the
formation of acetone, that the usual test
for acetone are also given for aceto-
acetic acid. The test for ketonuria
therefore, are tests for either acetone or
aceto-acetic acid or both. This is true of
the nitroprusside test.
Nitroprusside Test (Legal’s): mix 2
ml of urine and 3 drops of 5% freshly
prepared aqueous solution of sodium
nitroprusside. Alkalinize with NaOH. Note
the color produced. A ruby red color
indicates acetone. Add 0.5 ml of acetic
acid and make further observations.
If the test is made directly on
urine, a red color is given by creatinine
which disappears on the addition of
acetic acid.
28
 BCHEM LAB KODIGS
EXP. 17: [ASCENDING] PAPER IV. PROCEDURE
CHROMATOGRAPHY

Four types of chromatography: liquid, gas,

affinity, and exchange.
(Effective in identification of the unknown
substances [when known samples are run on
the paper chromatogram with the unknowns])
(Ascending) Movement of the solvent is due
to capillary action/*.

DEF’N OF TERMS

Stationary Phase – solid or liquid supported on

a solid; structure that holds the substance that’s
tested (ex. filter paper – presence of cellulose
responsible for the degree of movement)
Mobile " – refers to liquid or gas; solvent that
flows thru the stationary phase and carries the
components of the mixture (ex. developing
sol’n)
Rƒ or Retention/Retardation Factor – ratio of the
distance travelled by the substance to the
distance travelled by the solvent

I. PURPOSE
To separate amino acids based on
solubility between two immiscible
solvents [of amino acids] + analytical
method used to separate colored
chemicals or substances

II. APPARATUS
Droppers, beakers, aluminum foil,
Whatmen filter paper, capillary tube,
oven

III. MATERIALS
Butanol, acetic acid, distilled water
(H2O), 5% glycine, 5% lysine, 5%
aspartic acid, 5% unknown, .2%
ninhydrin sol’n

------------------------------------------------

29
 BCHEM LAB KODIGS
A. PREPARATION OF THE DEVELOPING
CHAMBER
Pipette 8 ml of the solvent
consisting of 4:1:5: (by volume) mixture
of butanol, acetic acid and distilled water
respectively, and introduce into a dry 250
ml beaker. Avoid splashing the liquid on
the sides of the beaker. Cover with a
piece of aluminum foil and let stand for
10 minutes for the atmosphere inside to
become saturated with the solvent vapor.
The developing solution consists
of 4:1:5 by volume mixture of butanol,
acetic acid and distilled water. The
Developing Chamber is a 250 mL beaker
covered with a piece of aluminum foil.
The solvent is placed in the chamber and
was allowed to evaporate for 10 minutes
to saturate the chamber with its vapor
(since it is volatile).
Saturation of the atmosphere in
the beaker with the solvent vapor = stops
the solvent from evaporating as it rises
up the paper
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.
2. Put the cylindrical paper upright into the
equilibrated beaker with the spotted
edge at the bottom. The solvent should
wet the lower edge of the paper without
reaching the spots. Put the aluminum foil
cover in place and let stand for 30 - 45
minutes or until the solvent front is 1 cm
away from the upper edge.
3. Remove the paper from the beaker and
open-up. Mark the position of the solvent
front before it dries up completely.
4. Spray the paper lightly with 0.2%
ninhydrin solution and dry it in the oven
at 110⁰ C. Heat if necessary for the color
forming reaction. The color should be
readily visible after 20-30 minutes.

B. PREPARATION OF THE PAPER

CHROMATOGRAM
1. With minimal handling (fingerprint can
obscure the result) cut a piece of
Whatmen filter paper no. 1, 16.50 cm
long and 8.0 cm wide. With a pencil,
draw a line 6 mm from the lengthwise
edge of the paper and 1 cm from each
crosswise edge, for handling.

2. Mark lightly with pencil equidistant spots

along the lengthwise line of the filter
paper.
D. CALCULATION OF THE Rf VALUE

Calculate the Rf value of each

amino acid and identify the unknown
amino acid/Calculate the Rf value of each
amino acid the unknown.
Unknown should have the same or
near the Rf found in one of the given
amino acids.
3. Gently and quickly touch the first mark
with the point of a fine capillary tube (0.5
mm diameter) containing 0.5% glycine.
Apply approximately 20 micrograms of
the sample on the mark, allowing the
spot to dry before each application. The
wet area should not be more than 2 mm
in diameter.
4. Repeat step no. 3 on the other marks
using a different amino acid for each
mark (0.5% lysine, 0.5% aspartic acid
and 0.5% unknown solution).

30