Biochemistry (BTBIMOLAB) 1

Laboratory Manual

Laboratory Procedure
Biochemical Processes Activity
2

Natural Sciences Department, College of Science Information Technology and Engineering , Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 2
Laboratory Manual

Natural Sciences Department, College of Science Information Technology and Engineering , Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 3
Laboratory Manual

INTRODUCTION

Diffusion

The tendency of the solute to spread throughout the solution until the composition is
homogeneous is called diffusion. Small molecules and ions move with sufficient velocity
to distribute themselves throughout the solvent rapidly. On the other hand,
macromolecules move slowly because of their high molecular weight. The rate at which
a substance diffuses across a uniform cross-sectional area depends not only on the
molecular size and shape but also on the concentration gradient of the substance. In the
absence of any other influencing factor, particles of matter move spontaneously from a
region of high concentration toward one lower concentration.

Surface Tension

Surface tension is the tendency of a liquid surface to contract. The surface molecules
feel an unbalanced attraction and are pulled inward. This inward pull causes the
molecules at the surface to come closer together. The surface area tends to become
smaller giving rise to the membrane effect.

When two partially miscible liquids are placed in contact, each dissolves to certain extent
in the other causing marked changes in the surface tension. The addition of a third
component may lower the surface tension considerably. Soaps, detergents, and salts of
bile acids are especially effective in lowering surface tension.

Osmosis

Osmosis is the passage of a solvent through a semi-permeable membrane. Such a

membrane is permeable only to the solvent, not to the solute.

If two solutions of different concentrations are separated by a semi-permeable

membrane, the solvent will diffuse into the more concentrated solution. This flow of
solvent may be stopped or the direction of the flow reversed by the application of a
sufficient pressure to the more concentrated solution. The pressure applied to the
solution to prevent the flow of solvent through the membrane from the pure solvent is
called the osmotic pressure of the solution.

Osmotic pressure is important to living cells because of the enclosing semi-permeable

membranes of the cells through which they communicate with their environment. Cells
are designed to function with certain internal salt concentrations. If red blood cells are
placed in concentrated salt solution with an osmotic pressure greater than the normal
blood, water will pass out of the cells. As a result, the cells will shrink. On the other
hand, if red blood cells are placed in distilled water with osmotic pressure lower than
that of the normal blood, the cells will take up more water and swell. However, if the
osmotic pressure inside becomes too great for the membrane strength, the cell will

Natural Sciences Department, College of Science Information Technology and Engineering , Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 4
Laboratory Manual

burst. Thus, in preparation of solutions to be injected into the blood stream, the osmotic
pressure of the solution is adjusted until it is compatible with blood.

Dialysis

Dialysis is a common biochemical method of separation and purification by selective

passage of ions and small molecules through a semi-permeable membrane that will not
allow proteins to pass through. Some of the membranes used in dialysis are: visking
sausage casings, colloidion and cellophane. These membranes contain small pores that
allow the ions, but not the large colloidal particles to diffuse. The kidneys are made up
of a fine network of dialysis tubings. It excretes liquids, salts, and small waste molecules
but at the same time prevents the loss of protein from the body fluids.

The rate of dialysis depends on many factors: the area of the dialyzer, the size of the
pores, the temperature, the electric charges, and the relative concentration of solution
on the two sides of the membrane.

APPARATUS/MATERIALS CHEMICALS/REAGENTS

Borrow: Request:
Bunsen burner Blue liquid food coloring
Extension clamp with holder Red liquid food coloring
2 mL 25% trichloroacetic acid
1 mL Fehling’s A reagent
1 mL Fehling’s B reagent
0.5 ml soap solution
0.5 ml oil
Bring:
12”x12” cellophane 0.5 g sulfur
2 mL defribinated blood
10 mL bile
cooking oil
50 mL fresh milk

PROCEDURE:

A. Diffusion

1. Put 5 ml of distilled water in a test tube. Incline the test tube and carefully
add 1 drop of blue liquid food coloring. Note the time it takes for the entire
body of fluid to have the same color.
2. Repeat the test 1 drop of red liquid food coloring instead of blue. Compare
the results.
(NOTE: Please refer to page 4 for the Proper Waste Disposal of used samples)

Natural Sciences Department, College of Science Information Technology and Engineering , Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 5
Laboratory Manual

B. Surface Tension

SAFETY PRECAUTION:
When handling sulfur powder, safety glasses with side shields, mask,
and gloves are recommended to avoid excessive dust exposure.
Sulfur powder is flammable, make sure to ELIMINATE all ignition
sources (no flares, sparks, or flames) near your work area.

1. Prepare two test tubes labeled No. 1 and No. 2. To each test tube, place 1
mL distilled water and 5 drops of cooking oil. Add 5 drops of soap solution to
test tube No. 2. Share both test tubes, then let stand for a few minutes. Note
the time it takes for the drops to coalesce in each test tube.

2. Put 5 mL dilute bile solution in a dry evaporating dish. Sprinkle a pinch of

sulfur powder on the surface of the solution. Observe. Repeat the test using
distilled water instead of bile solution. Compare the results.
(NOTE: Please refer to page 4 for the Proper Waste Disposal of used samples)

C. Osmosis (THEORETICAL)
SAFETY PRECAUTION:
When working with blood samples, make sure to use protective barriers
such as gloves, laboratory coat, mask, and safety glasses with side.

1. Prepare 4 test tubes containing 5 drops each defribinated blood. Label test
Test tube no.4 as “control”.

2. To test tube no. 1 add 1ml 0.9% NaCl solution, to test tube no. 2 add 1 mL
of 0.1M NaCl solution, and to test tube no. 3 add 1 mL of 1.0M NaCl solution.

3. Examine the result of each test tube under the microscope. Record your
observation.
(NOTE: Please refer to page 5 for the Proper Waste Disposal of used samples)

D. Dialysis

1. Soak a piece of 12” x 12” cellophane in water for a few minutes to soften it.
Fold the soaked cellophane like filter paper to fit into a glass funnel and pour
50 mL of milk in it. Collect all the edges of the cellophane and tie with a
string to secure them. (NOTE: leave air space above the milk).

Natural Sciences Department, College of Science Information Technology and Engineering , Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 6
Laboratory Manual

2. Suspend the bag from an iron stand into a 500 mL beaker so that it almost
touches the bottom. Add distilled water to the beaker until it is level with the
surface of the milk. (Refer to the diagram below).

Iron Stand

Air space
Milk
Beaker with distilled water

3. Allow the milk to dialyze for 1 hour with constant stirring of the water
outside. Test the dialysate for proteins and sugars:

a. Test for Proteins

SAFETY PRECAUTION:
Trichloroacetic Acid (TCA) is a CORROSIVE CHEMICAL and
contact can severely irritate and burn the skin and eyes with
possible eye damage. Safety glasses with side shields,
laboratory coats, chemically resistant gloves, and closed toed
shoes should be worn when handling TCA.

1. Put 1 mL of the dialysate in a test tube. Add 1 mL 25%

trichloroacetic acid solution. If proteins are present, a white
precipitate or turbidity will appear.

2. To check the sensitivity of the test, dilute milk in a test tube. Add 1
mL 25% trichloroacetic acid solution.

b. Test for Sugars

SAFETY PRECAUTION:
While using water bath, use DISTILLED WATER instead of tap
water. DO NOT add too much water, so that the water will not
overflow when boiling. Hot water splash injuries can be
minimized by wearing goggles and lab coats.

1. Mix 10 drops Fehling’s A and 10 drops Fehling’s B in a

test tube and heat it in a hot boiling water bath.

Natural Sciences Department, College of Science Information Technology and Engineering , Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 7
Laboratory Manual

2. Add 1 mL of the dialysate and continue to boil for 1 to 2

minutes. A yellow or red precipitate indicates the presence of a
reducing sugar.

PROPER WASTE DISPOSAL

1. Dispose ALL USED SAMPLES into the sink under running water.
2. Place/pour ALL UNUSED EXCESS CHEMICALS in the containers on the
instructor’s table.
3. Dispose used tissue papers in BIODEGRADABLE trash bin and 12” x 12”
cellophane in NON-BIODEGRADABLE.

QUESTIONS:

1. What are the types of transport processes by which metabolites pass through
biomembrane?
2. What is the effect of the following on red blood cells?
a. Hypotonic salt solution
b. Hypertonic salt solution
c. Isotonic salt solution
3. How does the kidney maintain the body’s internal environment?
4. Explain how soap solution lowers the surface tension of fats.
5. What is the role of bile salts in fat digestion?

Natural Sciences Department, College of Science Information Technology and Engineering , Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 8
Laboratory Manual

Subject/Section:________________ Date Performed :___________

Name:________________________ Instructor’s Initials :___________
Course/year:___________________

Natural Sciences Department, College of Science Information Technology and Engineering , Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 9
Laboratory Manual

Data Sheet
Biochemical Processes Activity
2

A. Diffusion

Sample Reagent Added Time to Produce

Uniform Color

10% blue dye

Distilled Water
10% red dye

B. Surface Tension

Sample Reagent Added Observation

Distilled Water +
Cooking oil

Distilled Water +
soap solution
Cooking oil

Distilled Water sulfur powder

Bile Solution sulfur powder

C. Osmosis

Sample Reagent Added Observation

Defibrinated Blood 0.9% NaCl solution

Natural Sciences Department, College of Science Information Technology and Engineering , Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 10
Laboratory Manual

0.1M NaCl solution

1.0M NaCl solution

Control

D. Dialysis

Sample Reagent Added Observation

Dialysate
Test for Proteins
+ 25%
(Precipitation by
Trichloroacetic Acid
TCA)
Dilute Milk

Test for Sugars + Fehling’s reagents

Dialysate
(Fehling’s Test) 

Natural Sciences Department, College of Science Information Technology and Engineering , Ateneo de
Zamboanga University, Zamboanga City, Philippines.