BIOCHEMISTRY LABORATORY C.

Electronic Determination (pH meter)

SUBSTANCE TESTED pH or (H+)

A. Wide range pH paper (pH= 1-14) Test Strips

SUBSTANCE COLOR CHANGE pH RANGE Fresh Milk 6.4 – 6.8

TESTED
Tea 6.0 – 6.8
Fresh Milk Gold to light 6 Pure Water 7.0
gold
Tea Gold to light 6 Vinegar 2.4 – 3.4
yellow Coke 2.5 – 4.2
Pure Water Gold to olive 7
Chlorox 11.0 – 11.9
green
Vinegar Gold to light 2 Soap Solution 8.0 – 9.0
red Calamansi 2.5 – 3.0
Coke Gold to orange 3
Rubbing Alcohol 5.0 – 5.5
Chlorox Gold to blue 11
Soap Solution Gold to light 8 Shampoo 5.0 – 8.0
green
Calamansi Gold to orange 3
QUESTIONS: pH
Rubbing Gold to yellow 5
Alcohol 1. How does the pH of your assigned sample as
Shampoo Gold to yellow 5 obtained from the pH paper compared with
the pH obtained from the pH meter?
- pH obtained in pH paper is only limited
B. Narrow range pH paper (Litmus Paper) in acquiring pH range (wide range) pH
SUBSTANCE COLOR CHANGE pH paper and in classifying the samples as
TESTED acid or base only (narrow range) pH in
meter but corresponds to the obtained in
Fresh Milk Blue to red acid pH meter is specific for it shows the (H+)
Tea Blue to red acid 2. Blood has a (H+) = 5x10-8. Calculate the pH
Pure Water No change neutral - pH= -log (H+)
- pH= -log (5x10 to the 8 power)
Vinegar Blue to red acid
- pH= 7.301
Coke Blue to red acid 3. Calculate the pH of 0.15 M acetic acid. Ka=
Chlorox Red to blue base 2x10-5
Soap Solution Red to blue base - (HA)= solution concentration
- (H+)= hydrogen ion concentration
Calamansi Blue to red acid
- (A-)= antelog concentration
Rubbing Alcohol Blue to red acid - Given: (HA) = 0.15 (molarity)
Shampoo Red to blue base Ka = 2x10-5
- Formula: Ka = (H+)(A)/(HA) = (H+)2/(HA)
(H+) = square root of Ka (HA)
(H+) = square root of (0.15)(2x10-5)
= 1.732050807 x 10-3
pH = -log (1.732050807 x 10-3)
pH = 2.761

QUESTIONS: BUFFER

1. Which is the best at resisting pH change when

acid is added: water, acetic acid, acetate ion
or buffer?
 - Buffer is the best at resisting pH change
when acid is added. It is a solution that
resists change in pH following the
addition of an acid or base.
2. Which is the best at resisting pH change when
base is added: water, acetic acid, acetate ion
or buffer?
- Buffer is the best at resisting pH change
when base is added.
3. Why would it be useful to “buffer” an Aspirin
tablet? (Aspirin is acetylsalicylic acid)
- The pH of buffer aspirin was a lot of
higher than others. Keeping the
acetylsalicylic acid in ionic form or
preventing it from dissolving until it
reached the small intestine would prevent
it from causing bleeding.
4. Explain the significance of buffer.
- Buffers are vital in health and biological
sciences for virtually all chemical
reactions in living systems take place in
buffered solution. This includes reaction
in intracellular and extracellular fluid as
well as in the blood.
QUESTIONS: OSMOSIS
OSMOSIS
- Is a process by which a solvent or some
species of a solution passes through a
membrane, which is not permeable to
other permeable to the other species of
the solution.
 At equilibrium, the chemical potentials of the
components that can penetrates the
membrane are equal on the sides of
membrane.
 When the phrases in contact with membrane
are at the same temperature but differ in
concentration, equilibrium can be achieved by
imposing pressure on one of that component
which penetrates the membrane to the
chemical potential of that component which
penetrates the membrane to the chemical
potential of the pure component at a fixed
temperature is the osmotic solution.
 The osmotic pressure equalizes the chemical
potentials on both sides of the membrane and
thus establishes equilibrium with respect to
the permeating species. Without a difference
in osmotic pressure, this species would pass
spontaneously through the membrane from
the phase of high chemical potential to the
solution where its chemical potential is lower.
 Osmotic pressure is important in living cells
because they are surrounded by
semipermeable cell membrane through which
they communicate with their environment.
Cells are designed to function with a certain
internal salt concentration. If they are put in a
concentrated salt solution, they loss water
through a membrane and shrivel. If they are
placed in a distilled water, they take up more
water and swell. If the osmotic pressure inside
becomes too great for the membrane
strength, the cell ruptures.
a. Why do cells tend to shrivel when placed in a
conecentrated salt solution?
- Cells tend to shrivel when placed in a
concentrated salt solution because the
osmosis of high concentration to low
concentration causes the cells to shrivel
or shrinks from lack of water balance.
b. What will happen when cells are in contact
with 0.9% NaCl solution (NSS)?
- A solution of 0.9% NaCl is isotonic to the
cytoplasm of cells. There will be equal
movement of water by osmosis. The cells
would maintain their size and shape.
2. What is osmotic pressure? Explain its
significance in biochemistry.
- Osmotic pressure which needs to be
applied to a solute=ion to prevent the
inward flow of water across a semi-
permeable membrane. In the molecular
level, storage cells maintain osmotic
pressure by staring energy in forms of
macromolecules such as polysaccharides,
rather than micro molecules, such as
glucose.
 EXPERIMENT: CARBOHYDRATE 8a REDUCTION TEST
 Objectives: identify the presence of SUGAR USING NaOH Ba(OH)2
different carbohydrates. SOLUTION
 To identify some properties of sugar. Glucose Dark Caramel Colorless Oderless
GENERAL TEST FOR CARBOHYDRATES Yellow

1. MOLISCH TEST Fructose Orange Odorless Colorless Odorless

- A sensitive chemical test for the
presence of carbohydrates based on
Galactose Light Pungent Colorless Odorless
the dehydration of the carbohydrates Yellow
by sulfuric acid to produce an Orange
aldehyde, which condenses with two COMPARISON
molecules of phenol through other
phenols. Only NaOH
 Composition: alpha-naphthol in 95% presents change
ethanol.
Only NaOH
EXPECTED RESULT: PURPLE RING/ DEEP RED presents change

SUGAR ACTUAL REMARK Only NaOH

SOLUTION RESULT presents change
Glucose Purple Ring Positive
Sucrose Purple Ring Positive 1. FEHLING’S TEST
- A chemical test to differentiate water
Xylose Purple Ring Positive
soluble aldehydes and ketone
Lactose Purple Ring Positive functional groups and test for
Starch Purple Ring Positive monosaccharides.
 Composition:
2. ANTHRONE TEST
o Fehling’s 1- (7g hydrated
- Determines both reducing and non
copper sulfate dissolved in 100
reducing sugars because of the
ml of distilled water.
presence of the strongly oxidizing
o Fehling’s 2-(35g of potassium
agent sulfuric acid.
sodium tartrate and 10g
 Composition: 2g of anthrone dissolved in
sodium hydroxide in 100ml
a mixture of 75ml of water and 950 ml of
distilled water)
sulfuric acid.
EXPECTED RESULT: BRICK-RED PRECIPITATE
EXPECTED RESULT: BLUE GREEN/BLUISH-GREEN
COLOR SUGAR ACTUAL REMARK
SOLUTION RESULT
SUGAR ACTUAL REMARK
Glucose Brick-red Positive
SOLUTION RESULT
precipitate
Glucose Green Color Positive
Fructose Brick-red Positive
Sucrose Green Color Positive precipitate
Fructose Green Color Positive Xylose Brick-red Positive
precipitate
Lactose Green Color Positive
Lactose Brick-red Positive
Starch Green Color Positive precipitate
Maltose Brick-red Positive
precipitate
3. ACTION OF ALKALI
 Sucrose Black Negative Starch No reaction Negative
Starch Dark blue Negative
4. NYLANDER'S TEST
2. BENEDICT'S TEST - A test for reducing sugar
- Test for reducing sugars. o-test to - It is a chemical test used for detecting
determine whether or not the the presence of reducing sugars.
carbohydrates contains a free - When Nylander's reagent, which
aldehyde or ketone group. consists of bismuth nitrate potassium
 Composition: sodium bicarbonate, sodium tartrate and potassium
sodium citrate, and copper sulfate. hydroxide is added to a solution with
reducing sugars, a black precipitate of
EXPECTED RESULT: BRICK-RED PRECIPITATE metallic bismuth is formed.
SUGAR ACTUAL REMARK  Composition: Rochelle salt potassium
SOLUTION RESULT sodium tartrate). potassium or sodium
Glucose Brick-red Positive hydroxide and bismuth subnitrate in
precipitate water.
Fructose Brick-red Positive
EXPECTED RESULT: BLACK PRECIPITATE
precipitate
Xylose Brick-red Positive SUGAR ACTUAL REMARK
precipitate SOLUTION RESULT
Lactose Brick-red Positive Fructose Black Positive
precipitate precipitate
Maltose Brick-red Positive Xylose Black Positive
precipitate precipitate
Sucrose Blue Negative Lactose Black Positive
Starch Blue Negative precipitate

5. PICRIC TEST
3. BARFOED'S TEST
- Test for detection of reducing sugars.
- Test used to distinguish reducing
monosaccharides from disaccharides EXPECTED RESULT: MAHOGANY RED SOLUTION
and polysaccharides.
 Composition: dissolve 0.66g cupric SUGAR ACTUAL REMARK
SOLUTION RESULT
acetate (monohydrate) and 0.18ml glacial
Glucose Mahogany Positive
acetic acid in 10ml of distilled water.
Red Solution
EXPECTED RESULT: RED PRECIPITATE Fructose Mahogany Positive
Red Solution
SUGAR ACTUAL REMARK Xylose Mahogany Positive
SOLUTION RESULT Red Solution
Glucose Red Positive Lactose Mahogany Negative
precipitate Red Solution
Fructose Red Positive Maltose Mahogany Negative
precipitate Red Solution
Xylose Red Positive Sucrose Blue Negative
precipitate
Starch Blue Negative
Lactose No reaction Negative
Maltose No reaction Negative
Sucrose No reaction Negative 6. TOLLENS TEST
 - Specific test for reducing sugars.
- Test used to distinguish aldehydes and
ketone
 Composition: Ammoniacal nitrate
EXPECTED RESULT: SILVER MIRROR PRECIPITATE
SUGAR ACTUAL REMARK
SOLUTION RESULT
Glucose Silver Mirror Positive
Precipitate
QUESTIONS:
1. Classify all the given carbohydrates based
on their number of sugar units.
 Monosaccharides
- simplest sugar which contains one
sugar unit
- water soluble crystalline compound.
They are aliphatic aldehydes or
ketones which contain one carbonyl
group or more hydroxyl group.
- Examples: glucose, fructose, xylose,
galactose, ribose
 Disaccharides
- contains two sugar unit
- formed when two monosaccharides
are joined together by glycosidic bond
and a molecule of water is removed
- Examples: lactose, sucrose, maltose
 Polysaccharides
- are long carbohydrates molecules of
repeated monomer units joined
together by glycosidic bonds.
- Examples:
o Storage polysaccharides: starch
and glycogen
o Structural polysaccharides:
Cellulose and Chitin
2. Molisch's test: Did all the given
carbohydrates in procedure A give a
positive result to Molisch, test? Why?
- Answer: Yes, because Molisch's test is
an identification test for
carbohydrates. All the solution of
Molisch reagent, those carbohydrates
changed in color or become violet
which is an indication of positive
result.
3. What are reducing sugars?
- Answer: Sugars that can be oxidized
by mild oxidizing agent because the
oxidizing agent is reduced in the
reaction.
o Reducing monosaccharides:
glucose, fructose, glyceraldehyde,
and galactose
o Reducing disaccharides: Lactose
and maltose
o Reducing polysaccharide: Starch
4. Which sugars are non-reducing? Account
for their non-reducing property?
- Answer: Sugars which are unable to
reduce oxidizing agents
o Non-reducing disaccharide:
Sucrose
CARBOHYDRATES 8b
 Objective: To detect the presence of
different carbohydrates.
Test For a Ketose sugar
1. SELIWANOFF'S TEST
- Chemical test which distinguishes
between ketone hexose and aldehyde
hexose.
- A ketone hexose also called
ketohexose and aldehyde hexose is
aldohexose.
- To differentiate between aldose and
ketose sugars
 Seliwanoff' reagent → consist of
resorcinol and conc. HCI
 Ketose → sugar containing a ketone
group; if the mixture turns red
 Aldose → sugar containing an aldehyde
group: mixture turns pink
 Composition: a solution or resorcinol in
concentrated HCI
EXPECTED RESULT: RED COLOR/CHERRY RED - Answer: Monosaccharide reacts most
rapidly to seliwanoff's test specifically
SUGAR ACTUAL REMARKS
those sugar that contains only one
SOLUTION RESULT
aldehyde group per module called
Fructose Red Positive
aldoses.
Glucose Light Pink Negative
Sucrose Red Positive Bial's Orcinol Test:
Maltose Light Pink Negative
1. What type of carbohydrates reacts most
rapidly to this test?
Test for Pentose Sugar - Answer: Pentoses

1. BIAL'S ORCINOL TEST Mucic acid Test:

- A chemical test to distinguish pentose
1. What is the function of nitric acid to this
from hexose sugar.
test?
- in the presence of pentose, the
- Answer: Nitric acid is able to oxidize
pentose will be dehydrated to form
the terminal groups of aldoses, but
furfural.
leaves the secondary hydroxyl groups
- Pentoses are converted to furfural by
unchanged.
Bial's reagent (orcinol/HCI)
2. What compound has been formed in the
- Green color
reaction?
 Composition: dissolve 0.3g reagent grade
- Answer: Galactaric acid/ crystals
orcinal and 0.05g ferric chloride in 100 ml
of concentrated HCI.
EXPECTED RESULT: BLUE COLOR/ BLUISH-GREEN

SUGAR ACTUAL REMARKS

SOLUTION RESULT
Xylose Bluish Green Positive
Glucose No change Negative
Arabinose Bluish Green Positive
Sucrose Green Negative

Test for Galactose

1. MUCIC ACID TEST
- Test used to detect galactose a-
oxidation of galactose and hydrolysis
of lactose will yield insoluble mucic
acid.
- a mucic acid crystals also known as
galactaric acid.
 Composition: sugar and nitric acid
QUESTIONS:
Seliwanoff's Test:
1. What type of carbohydrates reacts most
rapidly to this test?