Nursing Biochemistry (NurBio) 1

Laboratory Manual

Laboratory Procedure Activity

Biochemical Processes 2

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

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
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
Glass slide with cover slip 2 mL 25% trichloroacetic acid
Microscope 2 mL chloroform
1 mL 0.9% sodium chloride
Bring: 1 mL 0.1M sodium chloride
12”x12” cellophane 1 mL 1.0M sodium chloride
2 mL defribinated blood 1 mL Fehling’s A reagent
50 mL fresh milk 1 mL Fehling’s B reagent
10 mL bile

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.

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

2. Repeat the test 1 drop of red liquid food coloring instead of blue. Compare
the results.

B. Surface Tension

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.

C. Osmosis

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.

D. Dialysis (For Demonstration)

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 ) .

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).

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

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

trichloroacetic 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.

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

b. Test for Sugars

1. Mix 1 mL Fehling’s A and 1 mL Fehling’s B in a test tube and heat it

in a hot boiling water bath.
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.

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 and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Nursing Biochemistry (NurBio) 5
Laboratory Manual

Subject/Section:________________ Date Performed :___________

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

Data Sheet Activity

Biochemical Processes 2

A. Diffusion

Sample Reagent Added Time to Produce

Uniform Color
10% CuSO4
Distilled Water
10% Prussian blue

B. Surface Tension

Sample Reagent Added Observation

Chloroform
Distilled Water

Chloroform + soap
solution
Distilled Water

Sulfur powder
Distilled Water

Sulfur powder
Bile Solution

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Chemistry Laboratory 101: General and Inorganic Chemistry 6
Laboratory Manual

C. Osmosis

Sample Reagent Added Observation

0.9% NaCl solution

0.1M NaCl solution

Defribinated Blood
1.0M NaCl solution

“control”

D. Dialysis

Sample Reagent Added Observation

Dialysate + 25%
Test for Proteins Trichloroacetic Acid

Dialysate + Fehling’s
Test for Sugars reagents

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