LIPIDS - Experiment # 7

Fresh Coconut Oil and Congo Red - the

SOLUBILITY color is still red.

Lipids include both true fats and fat-like Rancid Coconut oil is acidic because it
substances. True fats contain glycerol and already underwent hydrolysis and oxidation
fatty acids in their molecules. Compound into shorter chain fatty acids which makes it
fats like phospholipids have besides acidic.
glycerol and fatty acids, a nitrogenous base, --------------------------------
and phosphoric acid. The sterols are FORMATION OF TRANSLUCENT SPOT
represented by cholesterol, ergosterol, and
7-dehydrocholesterol. Place 1 drop of coconut oil on a piece of
paper. Allow to evaporate. Does the
Water - immiscible translucent spot disappear?
Dil. HCL - immiscible
Dil. NaOH - immiscible When the light falls upon the paper a part of
Cold Alcohol - immiscible it is transmitted; a part TS scattered, d part
Hot Alcohol - slightly immiscible is absorbed, and a part is reflected The
CHCl3 - miscible degree of transparency / opaqueness of a
Ether - miscible medium depends on the ratio of light
CCl4 - miscible transmitted vs. light reflected or scattered.
The higher the light transmitted and lesser
Coconut oil is a non polar substance and it is reflected/scattered, the more will be
is immiscible in polar solvents like water, transparent. The amount of scattering
dilute NaOH, and Cold Alcohol, but they are depends on many things such as the size of
miscible in a non-polar solvent like the fibers and the difference in the index of
Chloroform, ether and Carbon the refraction between the particles & the
Tetrachloride. surrounding medium.

Solubility usually follows the rule, "Like Normally, when we look at the paper the
dissolves like". Hot Alcohol is slightly surrounding medium is air w/ an index of
immiscible. Although Alcohol is a polar refraction only slightly greater than 1.0. The
substance, but the high temperature paper fibers have much higher index of
increases the interactions of the solvent and refraction, probably much greater than 1.5.
the solute forming temporary emulsion. But
when it is cooled down, it becomes The fat also has a high index of refraction
immiscible . that it nearly marches the index of
-------------------------------- refractions of the paper. And reduces the
REACTION TOWARDS INDICATORS scattering significantly.

Fresh coconut oil is neutral The fat adhering to the cellulose fibers
Fresh Coconut oil and Litmus papers - So lowers the refraction of the cellulose & also
red litmus paper remains red and blue fills in air void so that visible light passes.
litmus paper remains blue.
through the bulb wic significantly lessens ● 4= 5 ml water + 0.5 mL Na2CO3
scattering solution (Sodium Carbonate)

The fat adhering to the cellulose fiber lowers To each test tube add 5 drops of coconut
the refraction of the cellulose & also fills in on, snake and observe results.
air void so that visible light passes. through
the bulb w/c significantly lessens scattering.
Test Tube 1 Formed temporary
emulsion right after mixing
We only see the light reflected from the
paper and much of the light that was Test Tube 2 Permanent emulsion
formerly scattered back to our eyes is now
Test Tube 3 Permanent emulsion
transmitted to the paper. The fat connects
the paper (fibers in the paper) w/ a liquid Test Tube 4 Temporary emulsion
w/c can transmit the refraction rather than.
scattering..
What is an emulsion?
- It is a stable dispersion of 2 or more
As a result, the paper, if thin enough, seems
immiscible liquids held in suspension
almost transparent. The translucent spot
by small percentage of substances
here will not evaporate easily because of its
called emulsifiers.
high boiling point.
--------------------------------
Which tube(s) formed a permanent
ACROLEIN FORMATION
emulsion?
- Test Tube 2 and Test Tube 3
Place 0.5g of KHSO4 in a dean dry test
tube. Add a drop of coconut oil & heat.
Both Soap & albumin are amphipathic
substances having both hydrophobic and
Describe the odor produced:
hydrophilic parts that could act as an
- burnt fat-like odor
emulsifying agent.
--------------------------------
Write the equation involved:
TEST FOR UNSATURATION

Coconut oil will undergo hydrolysis and

Place 5mL of oleic acid in chloroform in a
oxidation w/ the dehydrating agent.
rest tube and add Hubl's iodine solution
potassium bisulfate (KHS04) & produce
drop by drop shaking between addition.
acrolein (fatty acid & water)
Make a control by sharing in another tube a
--------------------------------
mixture of chloroform and iodine w/ no oleic
EMULSIFICATION
add

Prepare 4 test tubes w/ the ff. contents: tube

Explain results:
1 = 5 mL water
Iodine is soluble in chloroform & no
chemical reactions take place. oleic acid is
● 2 = 5mL water + 3mL soap solution
added to the mixture of iodine and
● 3 = 5 mL water + 1mL of 1% albumin
chloroform oleic acid absorbs and reacts w/
Solution
iodine forming a colorless halogenated fatty
acid: when an unsaturated fatty do NOT have carbon-carbon double
acid/unsaturated triglyceride absorbs and bonds in its triglyceride components.
reacts w/ iodine, the intensity of the color of
the solution becomes lesser / lighter than What is the relationship of Iodine no. &
the iodine+chloroform alone unsaturation of a fat?
- The more rodine is absorbed the
Repeat the test using stearic acid, coconut move unsaturated the fat / oil.
oil & linseed oil. (w/c will absorb more --------------------------------
Iodine) GLYCEROL

SAMPLE 1 A. Test the solubility of glycerol in

Stearic acid - with Hubl's Iodine water, alcohol, ether
- darker red than coconut, (dark B. Make acrolein test on glycerol"
orange to red)
- oleic acid (is yellow to ording), Results:
linseed (light orange) THE LIGHTER ● Water - Soluble
THE COLOR THE MORE IODINE IS ● Alcohol - Soluble
ABSORBED. ● Ether - Soluble
- Oleic is light yellow because it
absorbs more Todime than stearic Glycerol is a polar Substance and is
acid, since oleic acid is an soluble/ miscible m polar solvents-produces
unsaturated fatty acid and can burnt fat odor
absorb/react with the Iodine. Stearic
acid is saturated fatty acid and does C. Fuse a drop of glycerol in a
have unsaturation (carbon) that nichrome wire with powdered borax.
could react w/ lodine Note the green flame produced. This
is due to the glycerol ester of boric
Which oil contains more unsaturated fatty acid.
acids; coconut or linseed oil?
- Linseed oil contains more The solution turns to bluish-green
unsaturated fatty acids in its due to the release of chromic ion.
component triglycerides than
coconut oil, since linseed oil absorbs D. perform the nitro chromic acid test
more iodine than coconut oil. on 57. aqueous solution of glycerol.
What is the color formed?.
Which one will absorb more iodine, oleic or - blueish (bluish)-green with
stearic acid? potassium chromate in acidic
- Oleic. medium, the addition of nitric
acid.
Structurally, how does an unsaturated fat This indicates the presence of what
differ from a saturated fat? group in glycerol?
- Unsaturated fat is a mixture of - secondary alcohol group in
triglyceride that contains 2 carbon- glycerol.
carbon double bonds. Saturated fats --------------------------------
ENZYMES - EXPERIMENT # 8 ● Place 5mL of the boiled filtrate in
one tube and 5mL of the unboiled
Enzymes are complex organic compounds filtrate in another
w/ definite chemical structure, secreted by ● Add to each a few drops of 3%
living cells. They have the property of hydrogen peroxide. Note what
initiation and hastening chemical reaction happens.
w/out themselves being affected in the - unboiled filtrate - formation of
process Enzymes activity is influenced by bubbles because, more
the concentration of the products reaction, oxygen that was caralyzed
temperature, pH, inorganic salts of the and by the catalase (more)
the presence of activators & inhibitors. All - boiled filtrate - less formation
these factors should be considered while of bubbles due to the
performing the following experiments: denaturation of the enzyme
-------------------------------- catalase. There is a
OXIDASE FROM FRUITS formation of coagulants. (less
or none at all)
- When the fresh fruit (apple) are
peeled or cut open an enzyme called Boiling the potato extract reduces the
POLYPHENOL oxidase a.k.a catalytic. effectiveness of the enzymes
tyrosinase comprised of monophenol because heat can destroy the hydrogen
oxidase and catechol oxidase zing bonding it stabilizes the structural
enzymes when exposed to air/ conformation of protein enzymes.
oxygen will result in phenolic --------------------------------
compounds in the apple. OXIDASE FROM POTATO
- These tissues turn into quinones
(nave no color) but react w/ amino There are 3 oxidases:
acids & oxygen to produce melanin
and we get the brown color on ● Monophenol oxidase (tyrosinase)-
exposed sides of apple slices. responsible for oxidizing phenol to
-------------------------------- catechol to o-quinone and finally
CATALASE forms condensation browns
compounds of unknown
CATALASE FROM POTATO composition.
Preparation
● Prepare a small potato, and grate it ● Polyphenol oxidase (catechol
to a fine pulp oxidase) - facts on catechol forming
● Mix this pulp w/ 100 mL of water & o-quinone, then the unknown brown
let it stand for 15 minutes and strain compounds, Acts on pyrogallol to
this through a cheesecloth form purpurogallin.
● Filter the extract.
● Cytochrome oxidase - acts in
Action conjunction with-cytochrome,
● Divide the filtrate into four parts and oxidizing phenylenediamine, which
boil one part for one minute
 in the presence of alpha naphthol --------------------------------
forms indophenol. PEROXIDASE FROM POTATO

Procedure: Peroxidases, unlike oxidases, may require a

1. Prepare 3 test tubes and place 5 cofactor, phenol-oxidase to complete their
drops each of the following action. They also require H₂O₂ as the
substances: source of oxygen, and upon which the
a. 1% phenol phenol-oxidases act. Phenol oxidase is also
b. 1% catechol found abundant in potatoes.
c. pyrogallol
Procedure:
2. Add 1 ml of potato extract (oxidase)
to each test tube and shake. 1. Place 3 test tubes and add & drops
each of the following substances:
3. Allow them to stand until the end of
the laboratory period, observing and a. 1% phenol
recording the initial color change, b. 1% cathecol
then the change at the end of the c. pyrogallol
period. 2. Add 1 ml of potato extract
(peroxidase) to each.
3. Mix well, then add 3 drops of 3%
Initial Observations
hydrogen peroxide to each. Observe
1% Phenol Light brown and record the color changes
produced and compare them with
1% Catechol Brownish red the results observed on the action of
Pyrogallol red oxidases with the same reagents.

Initial Final
After 1 hour / end of lab period
1% Phenol Light brown Brownish
1% Phenol Brownish red red
1% Catechol Darker Brownish 1% Brownish Darker
red Catechol red Brownish
red
Pyrogallol red
Pyrogallol red red
Initially, phenol is converted first to catechol
to okinonce(?) Then forming a brown peroxidase catalyzes more pyrogallol to
substance (melanin) it takes time for phenol purpurogallin
to form quinone then catechol that shows --------------------------------
phenol is a lighter brown in color than
catechol. Pyrogallol is oxidized to purpu
rogallin w/ the presence of peroxidase in
potato.
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 glowing splint film continues to
glow due to the oxygen emitted by
the liver. Making it uneasy to put off.
Oxygen supports combustion

What does catalase do?

- catalase from the liver catalyzes the
decomposition of hydrogen peroxide
into oxygen gas and water
--------------------------------
TEST FOR NUTRIENTS IN FOODS -
EXPERIMENT # 9

STARCH GLUCOS PROTEIN FATS MINERAL CHOLEST VITAMIN

E MATTER EROL A

RIPE + + +- - - - +-
BANANA

GREEN +- + - +- + - +-
BANANA

CARROT + +- +- + - +
S

EGG - + + + - + -
WHITE
(COOKED
)

EGG - - + - - - -
YOLK
(COOKED

PEANUT + + + - - - -
BUTTER

POTATO + - + - + - -

CHEESE - + + + + - +-

FRUIT - + - - - - -
JUICE

DILIS - - + + + - -

UNCOOK + - + + + - -
ED
PEANUTS

COOKED + + + - + - -
RICE

Conclusion regarding the general constitutions of the food samples:

- The general composition and the amount of nutrients depends on the variety, source
and chemical reaction it underwent before the identification test. Most of the samples
contain 3 or more of these food nutrients but vary in Its concentration.
- Vitamin A or retinol is the immediate precursor to two important active metabolites.
Retinol plays a critical role in vision & retinoic acid w/c serves as an intracellular
messenger that affects the transcription of a number of genes
 - Vitamin A does not occur in plants but many plants contain carotenoids such as beta
carotene that can be converted to vitamin A within the intestine and other issues. The
observable results will depend on the traces amount of mese nutrients that can be
detected by this general rest quantitatively.

MILK - EXPERIMENT # 11 and lactose.

--------------------------------
Milk is the most complete food, it contains REACTION OF MILK
proteins, fats, carbohydrates, inorganic
Test 1mL of milk to both red & blue litmus
salts and vitamins. Fresh unboiled milk also paper, congo red and phenolphthalein
contains enzymes, protease, lactase, solution.

lipase, phosphatase, catalase, and What is the reaction of milk?

peroxidase. FRESH MILK
- Red Litmus paper remains red
The chief protein of milk is casein, which - Blue Litmus paper remains blue
can be precipitated by acid, carrying with it - congo red - red solution
the milk fat. The fat can be subsequently - phenolphthalein- colorless or low
extracted by organic solvents. The filtrate changing / no change in color
from the casein and fat contains the soluble
constituents like lactose and inorganic salts. SOUR MILK
- Blue Litmus paper turns red
The proteins of milk is derived from amino - Congo red - blue solution
acids of the blood, the synthesis occurring - phenolphtalein no change in color
in the mammary glands.
Fresh milk is slightly acidic to neutral, milk
Milk fat is believed to have its origin in the upon standing is acidic due to the oxidations
phospholipids of the blood. of lactose to lactic acid and its fats to
shorter carboxylic acids. Congo red with
The lactose of milk is derived from the sour milk is blue in color
glucose of the blood. --------------------------------
DETERMINATION OF SPECIFIC
The inorganic salts, like calcium, GRAVITY
magnesium, sodium, phosphates, citrates
and chlorides and other inorganic By The use of a desimeter determine the
constituents are likewise derived from the specific gravity of both the fresh milk and
blood, some by simple process of filtration. skimmed milk which has a greater specific
The bone acts as reserve supply of calcium. gravity? Why?
- Skim milk has a higher specific
Human milk differs from cow's milk in gravity because it has no or less far
having less casein and ash and more than fresh milk. Milk far is lighter in
albumin density than water and floats to the
surface of unhomogenized milk.
 When you skim off the surface some there will be no condensation of fats
of the fat, the denser portions remain and protein forming a film.
and the milk is denser. Explaining
why skim milk is denser. What factors facilitate the formation of
-------------------------------- surface film?
FILM FORMATION - heating & evaporation of the water
surface causing the surface fats &
Place 5 ml of diluted canned milk in a small proteins to condense or interact.
beaker and boil for a few minutes. Note the --------------------------------
formation of a film. What is the composition COAGULATION TEST
of the film?
- Milk forms a film on top when heated Place 1 ml of fresh milk in a test tube and
because of a chemical reaction that acidify with acetic the addition of acetic acid.
affects how proteins and far Heat to boiling.
molecules interact with each other.
When milk is heated rapidly, some of Is there any coagulation? Why?
the water in it evaporates from the - Yes, the supernatant whey becomes
surface; this exposes proteins and cloudy after heating because after
fat molecules w/c bind and dry out the casein is precipitated put the
as warming continues. solutions with acetic acid. There are
- The composition of the film (milk) still proteins that cannot form
are fats & proteins. insoluble precipitate with acetic acid,
these proteins / whey are soluble
Remove the film and heat again. Does the proteins and will coagulate when
film form again? heated. Heat will destroy the I
- Yes but lesser/ thinner than the first bonding of the protem molecules
and will make protein to coagulate.
Repeat the experiment using sour milk. Is --------------------------------
there any film formation? ACTION OF HOT ALKALI
- No. because it contains lesser fat
that could moract w/ the soluble Mix 1 ml of milk with drops of 6 M NaOH.
protein. Heat and describe the changes observed.
What is the cause of these changes?
What happened? Why? - The solution turns from yellow to
- Sour milk has lesser fat because brown due to the reactions of lactose
some fats are hydrolyzed and or the reducing sugar with a strong
oxidized into shorter chain carboxylic concentrated sodium hydroxide
acid w/c also contributes to the (NaOH).
souring of milk aside from lactic acid.
Because when water evaporates What test in the study of carbohydrates
from milk during heating, the milk involves the same principles?
proteins and fat molecules become - Moore’s test
more condensed on the surface, --------------------------------
since sour milk has lesser or no fat
PREPARATION OF CASEIN
FILTRATE
Take 10 ml of milk and dilute it with an
equal volume of water. Add dilute acetic Take two 3-ml portions of the why (filtrate
(1%) drop by drop until a flocculent ppt. from 6.) and make the ff tests:
forms. Avoid any excess as dissolution may
occur. Allow the ppt. to settle, decant the a. Coagulation by heating. Note the
supernatant fluid (whey) and reserve it for formation of coagulum. This consists
experiment 7. Filter off the ppt. Remove of lactalbumin & lactoglobulin. Make
excess moisture by washing the ppt. with a a biuret test on the coagulum.
few ml of ethyl alcohol. Transfer the casein - When the filtrate is heated,
to a dry test tube, cover it with ether and coagulations of protein is
heat on a water bath set at 50° C without observed due to the
flame for 5 to 10 minutes, shaking coagulation of lactalbumin
continuously. Filter and save the filtrate for lactoglobulin. These
no.8. Press the precipitate as dry as coagulated proteins are
possible between filter papers. Open the confirmed by blurrer test that
papers and allow the ether to evaporate shows purple colorations
spontaneously ( without heat). Grind the dry when heated w/ the millon's
ppt. to powder in a mortar and make the reagent.
following test:
b. Boil the second portion of the filtrate
a. solubility. Test the solubility of and remove every trace of
casein in water, dilute HCL, dilute coagulable protein. Filter Test the
NaOH and 10% NaCI. filtrare for the presence of P, Ca, &
reducing sugar.
- Benedict's test is used to
Water Slightly soluble
confirm the presence of
Dilute HCL insoluble reducing sugar. Ammonium
oxalate test for calcium and
Dilute NaOH soluble ammonium molybdate test
10% NaCl insoluble for phosphorus.

RESULTS:
b. Perform Millon's test on casein:
Result? There is a formation of a red
● Sugar - changes color to yellow,
ppt.
green, orange, and red of the
- casein positive for Millon's
solutions after heating w/ Benedicts.
test showing a flesh to red
This is due to the lactose which is
ppt due to the PHENOLIC
the most abundant carbohydrate in
GROUP OF TYROSINE IN
milk.
ITS PROTEIN CHAIN
--------------------------------
 ● Calcium sugar - there is a formation minutes. Do not use chewing gum for this
of white ppt (calcium oxalate) after purpose.
adding w/ ammonium oxalate.
What is the difference between the pH of
● Phosphorus - formation of yellow ppt the resting saliva and the stimulated saliva?
among phosphomolybdate after - Saliva is slightly acidic (pH of 6-7).
adding nitric acid ammonium Also contains buffer systems
molybdate responsible for maintaining proper
-------------------------------- acid base balance. Most important
MILK FAT role is played by the BICARBONATE
BUFFER; the buffers maintain the
Residue (milk fat?) she didn't say pH of the resting saliva between 5.7
& 6.2. While the pH of stimulated
Touch the residue w/ a piece of paper. saliva can reach to 8.
Observation: Formations of translucent spot
on the paper Is there any relation between the pH of the
-------------------------------- saliva and the susceptibility of dental
SALIVARY DIGESTION - EXPERIMENT # caries?
12 - Saliva also plays a very important
role in the inhibitions development of
Saliva is secreted by three pairs of glands, the caries lesions of the teeth.
the parotid, submaxillary and sublingual and Improving mineralizations of the
hundreds of small buccal glands. The flow is tooth enamel and preventing
stimulated by psychic, chemical and demineralization. When the pH of
mechanical factors. It contains a protein, this equation is w/ in 6-8-7.2, it
mucin and an enzyme, ptyalin end inorganic becomes a saturated solution of
salts. calcium phosphate which results in
quick and effective remineralization
By salivary digestion is meant the hydrolysis of the initial changes. However, if it
of starch by salivary amylase (ptyalin) which is likely acidified the environment,
is taking place in the buccal cavity and to a saliva becomes an unsaturated
certain extent in the fundic end of the solution and easily soluble calcium
stomach. hydrogen. phosphates are formed.
-------------------------------- Thus, the susceptibility of the teeth
REACTION caries increases.
Place a few drops of resting saliva in three --------------------------------
test tubes. Test the reaction with TEST FOR MUCIN
phenolphthalein, litmus and congo red.
From the color produced estimate the To 3 ml of saliva in a test tube add 1-2
approximate pH of resting saliva. drops of dilute acetic acid.

Repeat the experiment using stimulated What is the precipitate formed?

saliva. Stimulate the flow of the saliva by - After the addition of dilute acetic acid
chewing paraffin vigorously for at least 5 to the test tube containing saliva,
 coagulations of protein or the mucin - →when the benedict's test becomes
is observed. positive it means that the starch already
has hydrolyzed into reducing sugar
What is its function? (maltose). Maltose shows negative result
- mucin in saliva protects the surface in Iodine test because Iodine test is used
of oral mucous membranes from the to identify starch and (extruene)?
toxins and various types of irritants
contained in stimulants or Food. What is responsible for the reducing action?
Mucins are mainly responsible for - Maltose
the elasticity of saliva, it is also one
of the components of saliva that How long did it take for a complete
contributes to the lubricating transformation of the starch into reducing
properties of saliva. sugar?
-------------------------------- - The time to complete the hydrolysis
INORGANIC MATTER of starch into maltose in the salivary
digestion depends on how the starch
Acidify 10 ml of saliva with a drop or two of is exposed to the saliva and the
acetic acid, heat to boiling and filter to amount of saliva in this experiment.
remove the protein. Test the filtrate for - If the result in the iodine test is
chlorides, phosphates, sulfates and calcium. negative or no change in color
before & after addition of Iodine,
Which of the inorganic salt is found Benedicts should follow to confirm
abundant in the saliva? that the final result is maltose.
- chlorides and phosphate - If the result shows negative both
-------------------------------- iodine and Benedict the product of
DIGESTION OF STARCH PASTE hydrolysis of starch is not yet
maltose but it is achrodextrin and is
Place 10 ml of 1% starch paste in a small hydrolyzed further to become
beaker. Add 5 drops of saliva and stir positive w/ Benedict's test.
thoroughly. Add another 5 drops of saliva, if
blue color still forms with iodine after 5 Outline the different stages of starch
minutes. Gradually the opalescence of the digestion by ptyalin. How does this differ
starch solution disappears due to the from starch acid hydrolysis?
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 with
iodine test a portion with Benedict's test and
note the degree of reduction.

What is the reaction with iodine when the To differentiate it with starch acid hydrolysis,
Benedict's test becomes positive? the stages are the same but it doesn't end
with maltose. Maltose is further hydrolyzed catalytic activity. Although the
to glucose. normal pH range of caliva is
-------------------------------- between 6.8-7.2, the stimulated
INFLUENCE OF ACID paliva reaches to pH 8. Thus, an
acid has greater inhibiting power
Place 2 ml of one of the following strength because It. Lowers the pH of the
of acids in each of the 5 test tubes: 0.25%, saliva beyond its normal ranges and
0.1%, 0.05%, 0.025%, and 0.0075% HCI. reduces the catalytic effectiveness of
To each add 1 ml of 1% starch paste and 1 the enzyme
ml saliva, Shake well. Place in a water bath
at 40° C, for 20 minutes. At intervals of 5 What is the effect of gastric juice on the
minutes test a small portion with iodine and digestive section of saliva?
Benedict's test, until a positive result with - it starts protein digestion while sava
Benedict's and a negative result with iodine Starts starch digestion
is obtained. --------------------------------
BILE - EXPERIMENT # 14
In which tube did you obtain the greatest
digestion? Bile is a secretion of the liver. It is viscid and
- Test tube containing the lowest has an alkaline reaction and its color is
conc. of acid (0.025%. HCI). The greenish brown. Its important constituents
presence of high concentrations of are bile acids, bile pigments, inorganic salts
HCl in the saliva might denature the and cholesterol.
salivary amylase which is the --------------------------------
enzyme responsible for the REACTION
hydrolysis of starch Denaturations of
saliva results to a decrease of the Test the reaction of bile to litmus paper,
activity of the enzymes phenolphthalein, and red solutions. What is
-------------------------------- the reaction of bile?
INFLUENCE OF ALKALI
Red litmus paper Slightly blue
Repeat the above experiment using Instead
of HCI, NaOH with the following Blue litmus paper blue
concentrations: 2%, 1%, 0.5%, 0.125%, and
0.065%. phenolphthalein Slightly pink

Congo Red red

Neutralize the alkalinity before performing
the iodine test (use acetic acid to neutralize)
The results can be explained by The PH of
bile (slightly basic). Bile's normal pH value
Which has a greater inhibiting power, acid
is about 7-8 and is alkaline (slightly).
or alkali?
--------------------------------
- Salivary amylase works best at pH
INORGANIC CONSTITUENTS
level of saliva. Increasing or
decreasing a wave from the normal
Evaporate 10 ml of bile to dryness. Fuse the
pH range of saliva decreases its
residue with fusion mixture ( 2 parts of
sodium carbonate and 1 part of potassium into the top of the cone of the paper. Note
nitrate ) Cool and extract with 10 ml of the concentric succession of colors radiating
water. Acidify with HNO₂, until slightly from the tip of the cone.
acidic. Filter and test the filtrate for chloride, - different colors like green, blue,
sulphate, and phosphate. What inorganic violet, reddish yellow are observed
constituents are found in the bile? in the paper.
- Chloride, sulphate, phosphate --------------------------------
-------------------------------- TEST FOR BILE ACIDS AND BILE SALTS
TEST FOR BILE PIGMENTS
a. Pettenkofer's Test or Sucross-
a. Gmlin's test: Place 1 ml of conc. nitric Sulphuric acid test:
acid in a test tube. Superimpose 1 ml of
dilute bile, care being taken not to mix the Place 1 ml of dilute bile solution in a
two fluids. Note the production of different test tube. Add 1 drop of 5% sucrose
colors at the point of contact: green, blue, solution. Slowly run about 0.5 ml of
violet, red and reddish-yellow. conc. sulphuric acid down the side of
- depends on the fact that during the the tube. Observe the production of
stage OF oxidation the Bilirubin a red ring at the point of contact.
undergoes a series of changes in Shake and the whole solution
color which follows the sequence of assumes a red color. Place the tube
the familiar solar spectrum. Place a under running water to prevent the
drop of the fluid to be tested on a rise of temperature beyond 70° C.
white surface, capsule/ plate & allow
a drop of nitric acid. Yellow w/ The test is not reliable in the
nitrous dad fumes w/c make it more presence of proteins and other
oxidizing to run into it. As they chromogenic substance which give
mingle together, the rainbow-like color with sulphuric acid and
play of color appears. This when therefore, interfere with the reading
watched will be found to consist of a of the results.
series of changes to green, blue,
violet, red, and yellow. This can also b. Foam test:
be observed by allowing the acid to
trickle gently down the side of the Place 1 ml of dilute bile solution in a
test tube fixed in an inclined position test tube and add 1 drop of dilute
so that it cannot be shaken & the aqueous solution of furfural (1:1000).
play of color can be. seen Starting Shake and observe the production of
from the point of junction of the 2 a thick foam. Place a drop of conc.
fluids. H₂SO, on the foam. Note the
production of a dark pink color.
b. Rosenbach's modification of Gmlin's test: Formation of dark pink color is
Filter some very dilute bile (1:50) through a observed due to the condensations
small filter paper. When all has been of furfural & the bile acids and salts
drained through and the paper is still moist in dilute solution of bile.
throughout, place a drop of conc. nitric acid
 - The solution becomes red then due
c. Hay's Test of Surface Tension Test and finally bluish-green.
What is the danger of excessive amount of
This test is based upon the principle that cholesterol in the bile? Explain.
bile acids or bile salts have the property of - formation of gallstones: when bire is
reducing the surface tension of the fluid in oversaturated w/ excess cholesterol,
which they are contained. the excess crystalizes and forms
gallstones.
Cool about 5 ml of diluted bile in a test tube --------------------------------
to 17⁰ C or lower and sprinkle a little finely URINE - EXPERIMENT # 15
powdered sulfur on the surface of the fluid.
The presence of bile salts is indicated by Urine is a filtrate from the blood and serves
the sinking of sulfur, the rapidity depending as a medium for excretion of water, salts,
on the quantity of bile acids present. acids, bases, waste products of metabolism
and other toxic materials. As much, it helps
Repeat the experiment using water instead in the maintenance of water balance, acid-
of bile. Compare your results. base equilibrium and serves as an important
factor in the detoxification of the body.
- Test tube containing water and --------------------------------
sulfur powder. The sulfur powder COLLECTION AND PRESERVATION OF
floats at the surface of the water due URINE SAMPLE
to the surface tension between the
nonpolar sulfur and the polar water. For the study of both the qualitative and
- Test tubes containing dilute bile and quantitative composition of urine, it is better
sulfur powder. Sulfur powder sinks to examine the 24 hour specimen.
at the bottom of the test tube
because the bile and the bile salts The bladder is emptied at 8:00 a.m. and the
reduce to the surface tension urine is discarded. All the urine from this
between the water & the nonpolar time up to 8:00 a.m. the next day is taken as
Surface. a sample.
--------------------------------
CHOLESTEROL IN BILE To preserve the urine, a thick layer of
toluene is over-layed on its surface.
Evaporate to dryness 5 ml of undiluted bile.
Extract with a small amount of ether at a ● Qualitative - better to study the 24-
time. Evaporate the ether extract to dryness hour specimen.
on a hot water bath (no flame). Dissolve the
residue in 3 ml of dry chloroform. Perform ● Quantitative - bladder is emptied at
the Liebermann-Burchard test on the 8 am and urine is discarded. 8 am
chloroform solution. onwards until gam the next day urine
- Qualitative determination of is taken as sample.
cholesterol
- Involves Liebermann-Burchard test
on the chloroform solution.
To preserve Urine, a thick layer of toluene is - calcium phosphates, calcium
over layered on its surface. (don't use oxalates.
toluene in exps.) VELEZ OMITS TOLUENE
-------------------------------- What is the specific gravity of urine?
GENERAL CHARACTERISTICS - 1.015 1.025
--------------------------------
What is the volume of the 24-hour urine? DETECTION OF CREATININE
- 1 000 mL - 1 500 mL a) Nitroprusside test ( Wey!): To 5 ml of the
urine add 3 drops of sodium nitroprusside.
a) Examine the specimen as to color, odor, Alkalinize with NaOH.A ruby red color is
transparency and reaction. produced which turns yellow. This is
What substances are responsible for the indicative of the presence of creatinine.
normal color of urine? - Ruby red color produced that turns
- Urochrome and urobilin yellow. This is indicative of the
presence of creatinine
What happens when the urine is allowed to - RUBY RED to YELLOW
stand for sometime, exposed to air?
- bacteria causes urea to break down b) Picric acid reaction (Jaffe): Place 5 ml
into ammonia and therefore urine in a test tube, add an aqueous
increases pH. solution of picric acid and render the
solution alkaline with NaOH solution. A red
What is the normal reaction of urine when color is produced due to the formation of a
freshly voided? red tautomer of creatinine picrate. This tums
- acidic at around PH 6 yellow when the solution is acidified.
Glucose gives a similar red color, only upon
To what substance is the reaction due to? heating.
- presence of acid phosphates, - The solution turns red yellow when
organic acids acidified
--------------------------------
What happens when allowed to stand DETECTION OF PIGMENTS
without preservative? Why?
- urine develops foul odor and color Ammoniacal zinc chloride test.
becomes darker because of the
conversion of urea to ammonia and Place 2 ml of urine in a test tube and add 1
the decompositions or oxidations of ml of NH,OH stand for a while and filter. To
the organic substance by bacteria in the filtrate add 2 drops of zinc chloride
the urine. solution.

What constituents of the urine tend to - Ammoniacal zinc chloride test. The
precipitate when the reaction is addic? production of greenish fluorescence.
- urates (sodium urate) 4 uric acid The production of greenish
fluorescence indicates the presence
What substances precipitate in alkaline of urobilin.
urine?
What are the other pigments normally the remaining solutions may contain
present in the urine? alkali phosphate (sodium &
- Urochrome & uroerythrin potassium phosphate) which can be
-------------------------------- precipitated out from the solution by
INORGANIC PHYSIOLOGICAL adding magnesium mixture & will
CONSTITUENTS form. white gelatinous.ppt.
- Inorganic salt detection
Determine w/c form of the phosphate is
present in larger amounts.
SULPHATES - alkalí phosphates
--------------------------------
a) Detection in Inorganic sulphuric acid: PATHOLOGIC CONSTITUENTS
Place 5 ml of urine with 5 drops of acetic - healthy individual should have lower
acid. Add barium chloride solution. concentration of (Albumin, Glucose,
- formation of white ppt (barium Hellers ring).
sulphate)
-------------------------------- ALBUMIN
DETECTION OF CHLORIDES Coagulation test: Heat 5 ml of urine to
boiling in a test tube (filter if urine is not
What is produced? clear). If heated portion becomes cloudy the
- Formation of white ppt (silver turbidity may be due to phosphates. Add 3-4
chionde) drops of very dilute acetic acid and warm,
Add excess of NH40H. what the phosphates will dissolve, while if a more
happened in the ppt flocculent precipitate will be produced if only
- ppt will be dissolved & react w/ the albumin is present.
excess ammonium hydroxide - if tests are positive he/she may
(NH4OH) forming Soluble diamine have. Conditions that elevate the
silver-1-chloride concentration of the substances in
urine. Dier could also affect
What is the normal amount of chlorides
eliminated in 24 hours? b) Heller's Ring Test: Place 5 ml of conc.
- A common reference range for a nitric acid in a test tube, slant the tube, and
24hour urinary chloride is 110-250 very carefully allow an equal amount of
milli equivalence per day. Approx. urine to slowly run down the side of the
3.89-8.85g per day tube. The urine will float on the nitric acid,
and a white ring (precipitated protein) will
In what forms are they eliminated? appear at the junction of the two liquids.
- NaCl, KCl, MgCl₂, NH4Cl Sometimes the white zone does not appear
-------------------------------- until allowed to stand for a few minutes.
DETECTION OF PHOSPHATES. - if tests are positive he/she may
- formation of white gelatinous ppt the have. Conditions that elevate the
earthy phosphates (calcium & concentration of the substances in
magnésium phosphate) will urine. Dier could also affect
precipitate w/ ammonium hydroxide
c) Glucose Nitroprusside Test ( Legal 's): mix 2 ml of
Benedict's test. And 8 drops of urine to 5 ml urine and 3 drops of 5% freshly prepared
of Benedict's reagent and boil vigorously for aqueous solution of sodium nitroprusside.
2 minutes. Set aside to cool. The amount of Alkalinize with NaOH. Note the color
precipitate and its color (red, yellow or produced. A ruby red color indicates
green) depend on the quantity of glucose acetone. Add 0.5 ml of acetic acid and
present in the urine. make further observations.
- if tests are positive he/she may
have. Conditions that elevate the If the test is made directly on urine, a red
concentration of the substances in color is given by creatinine which
urine. Dier could also affect disappears on the addition of acetic acid.
--------------------------------
BLOOD
--------------------------------
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 bile?

- no

What does its presence indicate?

- possible jaundice, liver cirrhosis,
hepatitis and other Liver diseases.
--------------------------------
ACETONE BODIES

There is no satisfactory, simple direct test

for b-hydroxybutyric 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.