BIOCHEM LABORATORY QUIZ REVIEWER 2 beakers (250 mL)

EXPERIMENT #1: WATER AND ITS PROPERTIES Thistle tube

THEORY: 6 test tubes

-Water is important to all living systems. Test tube rack

-Serves as natural solvent for mineral ions and other String or rubber band
substances. PROCEDURE:
-the dispersion medium for colloidal cells like I. Water: A Universal Solvent
protoplasm.
a. Put about 0.5 grams of the following substances
-Serves as the medium for most biochemical
into six separate test tubes: NaCl, sugar, gelatin,
reactions, and is the most abundant component of
CuS04, lard, and ethanol.
cells.
Add 1 mL of water to each test tube and shake
-EXCEPT for bone tissues and enamel, water
vigorously to dissolve the substances.
constitutes about 70 percent of the human body.
To substances that did not dissolve, add another 1
OBJECTIVE:
mL of water and shake again. To the solids that still
-To determine the properties of water that make it a did not dissolve, add another 1 mL of water and
sustainable medium for sustaining life in biological shake.
systems. b. Repeat the solubility test using CCl4 instead of
III. MATERIALS water.

NaCl (Sodium Chloride) c. Describe the solubility in both solvents as soluble,

slightly soluble, and insoluble. Record observations in
Sugar the table.
Gelatin Substances Solubility in Solubility in
Water CCl4
CuSO4 (Copper Sulfate)
NaCl Soluble Insoluble
Lard
Sugar Soluble Insoluble
Ethanol (C2H6O) Gelatin Slightly Soluble Insoluble
CCl4 (Carbon Tetrachloride)
CuS04 Soluble Insoluble
Citric Acid Powder Lard Insoluble Soluble
1% NaCl in starch solution Ethanol Soluble Insoluble

0.1 M AgNO3 (Silver Nitrate)

Observation: Water dissolves more substances than
10% sucrose solution CCl4. Thus, water makes an excellent solvent, which
Deionized water means it can dissolve more substances.

Dialysis bag
Cellophane
II. Water: A Good Medium for Biochemical Reactions Cover the mouth of the thistle tube with a cellophane
membrane and keep it in place with a rubber band.
a. Mix 0.1 gram of dry, powdered citric acid, and
sodium bicarbonate (NaHCO3) Suspend the thistle tube in a beaker of water, making
sure that the levels of liquid inside and outside the
b. Add about 10 mL of water to the mixture and note tube are equal.
what happens.
Observe the difference in solution levels after 30
Observation: The presence of water, citric acid, and minutes. Did the sugar solution migrate inwards or
sodium bicarbonate mix to form carbon dioxide gas, outwards?
which disperses and produces bubbles in water.
Observation: As the tubing was submerged into the
III. Properties of Water Solutions water liquid arises, the sucrose solution is hypertonic
a. Dialysis. Obtain a dialysis bag about 20 to 25 cm to the water. So as the time passes by, we can
long and soak in clean water for about 10 minutes. observe that there is a movement of water molecules
from the water outside to the sucrose solution inside
Fill with 30 mL of 1% starch-NaCl mixture, tie the bag the tube. Thus, the solution should have risen.
and rinse thoroughly with water.
QUESTIONS:
Put the bag in a beaker containing deionized water.
Adjust the setup such that the levels of fluids inside 1. What are the different functions of water in living
and outside the bag are the same. systems?

After 1 hour, test 1 mL of dialysate (water iin the -Essential for the survival of all living organisms
beaker) with a few drops of 0.1 M AgNO3. Formation -Regulates body temperature
of a white precipitate of AgCl confirms the presence of
chloride ions in the dialysate. -Eliminate waste material and toxins

Observation: Dialysis is a process wherein the -Cushions the brain, spinal cord, and other sensitive
molecules separate through a semipermeable tissues
membrane.
-Maintains blood pressure
The solution inside the bag was slowly moving
-Breaks down food
outwards in the direction of the distilled water.
2. Based on the observations, how would you define
After an hour, the solution of starch and sodium
dialysis and osmosis?
chloride was separated on each side of the
semipermeable membrane. Dialysis is the transfer of solute through a semi
permeable membrane. It is used to separate different
The addition of 0.1 M AgNO3 dissolved in water
components or substances of a solution. The best-
formed chloride ions which diffused across the
known example of dialysis is the removal of waste
membrane.
and excess fluids from the blood when the kidney
Therefore, by replacing the solvent with a fresh does not work correctly.
solvent, you can eliminate unwanted substances.
On the other hand, osmosis occurs when fluid moves
b. Osmosis. Stopper the narrow end of a thistle tube form a high-water concentration to a lower
with a finger and fil it with sugar cane juice or a 10% concentration through the semi permeable
sucrose solution until the solution reaches up to the membrane. It is the absorption of water into a
base of the tube. concentrated solution. For instance, when plants
absorb water from the soil, the roots’ hair acts as a
semi permeable membrane, allowing water molecules
to move from high concentration (soil) to low A. Test Based on the Formation of Furfural and Its
concentration (roots). Derivatives

-Sugars are stable to hot dilute mineral acids. But hot

sulfuric acid will dehydrate them into furfural and its
EXPERIMENT #7: CARBOHYDRATES
derivatives.
THEORY:
-Pentoses, when heated with concentrated sulfuric
Carbohydrates include polyhydroxy aldehydes or acid, will form furfuraldehyde, while hexoses will form
ketones and their by-products. 5-hydroxymethyl furfural.

-They make up most of our diet and serve as the -In the presence of acid, phenolic compounds like a-
major source of energy. They play an important part naphtol, orcinol, and resorcinol will react with furfural
in metabolic processes by furnishing the carbon chain or hydroxymethyl furfural to form colored
for compound synthesis by living organisms. condensation products. The formation of these
colored constitutes a positive test for carbohydrates.
-Can be monosaccharide, disaccharide,
polysaccharide. 1. MOLISCH’S TEST

Monosaccharides are highly soluble in water, less -general test for carbohydrates
soluble in ethanol, and insoluble in ether. They cannot
-sugars are mixed with a-napthol
be hydrolyzed into simpler units.
-test tube is inclined, and concentrated H2S04
-can either be aldoses and ketoses depending on the
(sulfuric acid) is added along the side of the tube,
type of functional group present.
causing the formation of a lower layer of acid
-can be classified as tetroses, pentoses, or hexoses
-concentrated sulfuric acid will dehydrate the sugar
depending on the number of carbon atoms they
allowing it to react with the alcohol forming furfural or
possess.
hydroxymethyl-furfural
-exhibit mutarotation (can exist in a- (alpha) and b-
-Positive Result: formation of purple ring at the
(beta) forms.
interface of two liquids indicate the presence of
Disaccharides are formed by two molecules of carbohydrate
monosaccharides.
PROCEDURE:
-example is maltose, which are abundant in
a. (Control) Mix 4 mL of distilled water and two drops
germinating barley; sucrose (cane sugar or beet
of the Molisch’s Reagent in a test tube.
sugar); lactose (milk sugar) which does not taste very
sweet and is not fermented by yeast. b. (Glucose) Place a 4 mL of 3% solution of glucose
in a second test tube. Add two drops of the Molisch’s
Polysaccharides found in nature function either as
Reagent and mix the contents by gently shaking the
structural units (e.g., cellulose), or for storage such as
test tube.
starch, dextrin, glycogen, and insulin.
c. Incline the test tube and cautiously add about 5 mL
The CHEMICAL TEST used to detect the presence of
of concentrated sulfuric acid , allowing the acid to run
carbohydrates are based on the ability to: a. form
down the side of the tube. Sulfuric acid is denser than
furfural and its derivatives, and b. reduce and form
water and will form a lower layer.
characteristic compounds with reagents like Tollen’s
reagent and Benedicts’ solution. d. In the same manner of adding acid, add sulfuric
acid to the control tube.
 e. Repeat the above test with 3% solutions of 3. SELIWANOFF’S TEST
glucose, xylose, lactose, and starch.
-used to differentiate ketohexoses from aldohexoses
Substances tested Description of Visible Result
Control No visible purple ring -Ketohexoses react faster with the solution containing
Glucose Has visible purple ring hydrochloric acid and resorcinol than aldohexoses
Xylose Has visible purple ring -Dehydrated ketohexoses forms a bright cherry red
Lactose Has visible purple ring condensation product, while the aldohexoses yields
Starch Has visible purple ring
only a pale pink coloration, a negative result.
-In this test, prolonged heating of sample should be
Result: All carbohydrate solutions gave positive result.
avoided.
2. BIAL’S ORCINOL TEST
PROCEDURE:
-used to determine the presence of pentoses and a. Place 1 mL each of 3% xylose, glucose, fructose,
nucleotides that contain pentose sugars and sucrose in separately labeled test tubes.
-pentoses are treated with orcinol, furfurals are b. Add 4 mL each of the Seliwanoff’s test reagent to
formed and they will yield a blue-green compound in each test tube.
the presence of ferric ions.
c. Place the tubes in a water bath filled with boiling
-reaction is not specific for pentoses because other water and allow them to stay there for exactly 1
compounds like trioses, uronic acids, and certain minute.
heptoses will also give blue or green products
d. Note the changes and record which the test tube
-Hydroxymethyl furfural is formed from hexoses to gives a positive result in the shortest time.
give a yellow-brown condensation product
e. Continue heating and observe the color changes at
PROCEDURE: 1 minute.
a. Place 1 mL each of 3% solution of xylose, glucose, Substances Time Result Explanation
and starch in separately-labeled test tubes Xylose 7 mins (-) Dark Does not contain
Yellow either ketohexoses
b. Add 3 mL of Bial’s reagent to each test tube (Original
c. Carefully heat each tube over a Bunsen flame until Color of
reagent)
the solution begins to boil. Add one or two drops of
Glucose 7 mins (-) Dark Does not contain
10% FeCl3 (Iron Chloride) solution
yellow either ketohexoses
Substances Visible Result (Orignal
Control (water) Yellow brown color. color of
Negative result. Not a reagent)
pentose. Fructose 6 mins (+) Bright Contains
Xylose Blue green color. Cherry Ketohexoses
Positive result. A Red)
pentose. Sucrose 7 mins (-) Pale Contain
Glucose Yellow brown. Negative Pink aldohexoses
result. Not a pentose.
Starch Yellow brown. Negative
result. Not a pentose.

B. Tests Based on the Reducing Property of Sugars

Reducing sugars must have a free aldehyde or -its reagent contains cupric acetate in dilute acetic
ketone group. Therefore, all monosaccharides and acid and is used to distinguished between
some disaccharides can reduce oxidizing reagents monosaccharides, disaccharides, and
such as the cupric ion, dinitrosalicylic acid, and picric oligosaccharides.
acid.
-its reagent oxidizes monosaccharides but not
1. BENEDICT’S TEST oligosaccharides.

-very sensitive test done under mildly alkaline -disaccharides are less easily oxidized but are
conditions. oxidized if they undergo prolonged heating, causing
hydrolysis of the disaccharides into monosaccharides,
-reagent contains CuSO4 (Copper Sulfate), Na2C03 which will give a positive result.
(Sodium Carbonate), and sodium citrate
-concentration of sugar solution used in this test
-formation of a brick red precipitate of Cu2O (Copper should be approximately the same because the use of
oxide) is considered positive more concentrated disaccharide solution may give a
-most aldehydes have the ability to reduce Benedict’s faster reaction that that of a relatively more dilute
reagent monosaccharide solution

-other compounds like formic acid, hydrazobenzene, -unlike Benedict’s test, Barfoed’s is carried out under
phenols, phenylhydrazine, pyrogallol, and uric acid acidic rather than basic medium.
will also give a positive result. PROCEDURE:
PROCEDURE: a. Place 1 mL each of the 3% solutions of glucose,
a. Place 1 mL each of 3% solutions of glucose, xylose, fructose, lactose, and starch in separately-
xylose, fructose, lactose, and sucrose in separately- labeled test tubes.
labeled test tubes. Add 5 mL of Benedict’s reagent in b. Add 3 mL of Barfoed’s reagent in each test tube.
each test tube.
c. Prepare a control tube using distilled water instead
b. Place all test tubes in boiling water bath for 2 to 3 of sugar solution
minutes.
d. Place all test tubes in a boiling water bath for 10
c. Observe the color of the solution and note whether minutes.
a precipitate was formed. A change in color of the
solution is not considered a positive reaction Substances Result Explanation
Control (-) No change in
d. Avoid prolonged heating (distilled water) color
Glucose (+) Brick red color
Substances Result Explanation
precipitate.
Glucose (+)
Reducing
Xylose (+)
sugar
Fructose (+)
Xylose (+) Brick red color
Lactose (-) precipitate.
Sucrose (-) Reducing
sugar.
Fructose (+) Brick red color
precipitate.
2. BARFOEDS TEST Reducing
sugar.
Lactose (-) No change in
 color
Starch (-) No change in
color

Substances Result Explanation

3. PHENYLHYDRAZINE TEST Glucose Grey Glucose is an
aldehyde and
-when sugar is added to phenylhydrazine and NaAc manage to
(sodium acetate) and then heated, a yellow have a grey
precipitate is formed. colored
mixture.
-the precipitate formed may then be compared with Xylose Yellowish Grey Xylose is an
standards using difference in melting points to identify aldehyde and
the composition of the precipitate. created
yellowish grey
-Gluccosazones are fine yellow needles aggregated color.
like “bundles of hay”. Sucrose Silver Sucrose lacks
aldehyde
-Glucose, maltose, and mannose form the osazones which caused
-Lactosazone crystals are irregular cluster of fine it to have a
needles, and look like a “powder puff” silver color.

-Maltosazone crystals are “star shaped”

OBJECTIVE:
4. TOLLEN’S TEST
-To be able to identify the different types of
-sugars with aldehyde groups are capable of reducing carbohydrates using the different specific tests
Tollen’s reagent (an ammoniacal solution of Ag) to
form a gray to black precipitate.
-If the reaction vessel is clean and rate of deposition
is slow enough, the Ag deposits as a silver mirror.
PROCEDURE:
a. Place 5 drops of 3% solution of glucose, xylose,
and sucrose in separate test tubes.
b. Add 2 mL of Tollen’s reagent into each tube.
(Note: Prepare Tollen’s reagent by adding 1 drop of
NaOH (sodium hydroxide) solution to 6 mL of 5%
AgNO3. Add dilute NH4OH (Ammonium Hydroxide)
(1 mL concentrated NH4OH+5mL water) until the
brown precipitate of silver oxide that forms just
dissolves. This reagent mist be prepared fresh and
not stored since it decomposes when left standing
and yields an explosive decomposition product.
Discard all leftover materials.
c. Boil for about 5 minutes.