BIOL110L Sautbayeva Z

Laboratory # 4
Study of diffusion of organic molecules across the
semipermeable membrane in a model membrane system
Introduction
Cells constantly exchange various molecules with their environment. Cells must
import such nutrients as amino acids, sugars and eliminate waste such as CO 2 and
regulate intracellular concentrations of inorganic ions. Plasma membranes are selectively
permeable, which means that only certain substances are allowed through the
membrane. Plasma membrane is represented by a lipid bilayer that is hydrophobic inside.
This chemical property of a membrane defines the ability of molecules to cross it.
Membrane contains lipids that are non-polar (hydrophobic) compounds. This means that
substances that are hydrophilic have difficulties crossing the membrane. On the contrary,
molecules with chemical structure that is similar to the cell membrane can pass through
much easier. In addition to solubility in the membrane, the ability of a molecule to cross
the membrane depends on other factors such as its size and charge.

Small hydrophobic molecules diffuse across the membrane very fast. The
examples of such molecules are oxygen, carbon dioxide and nitrogen. Very small polar
molecules such as water can also diffuse across the membrane. However, large polar
molecules and charged ions are not able to move across the membrane without the help
of special transporters that are embedded into a lipid bilayer.
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Substances can cross the membrane by passive and active transport. In the passive
transport solutes move down its concentration gradient which means that the molecules
spontaneously move from the region of high concentration to a region of low
concentration. No energy required for the molecules to move across the membrane by
passive transport. Passive transport includes simple and facilitated diffusion.

Movement of molecules across the membrane is possible due to the presence of

membrane transport proteins. The membrane transport proteins allow small hydrophilic
molecules to cross the membrane. Simple diffusion does not require the help of
membrane transport proteins. Facilitated diffusion requires either channel proteins or
transporters. Active transport requires energy in the form of ATP because solutes move
against its concentration gradient when moving across the membrane.

The movement of a solute through a semi-permeable membrane is defined as a

dialysis. In this experiment you will use dialysis tubing which is made of cellulose
membrane. A dialysis membrane is a semi-permeable with the pores that are placed
symmetrically throughout the membrane. Pores in the tubing allow the diffusion of
molecules only according to their size. Molecules larger than the pores cannot pass
through the membrane, but smaller molecules can do so freely. The pore size of a
dialysis membrane determines the molecular weight cut-off (MWCO) of the molecules
that can diffuse across it.
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In this laboratory you will use a sample that you will dialyze against a dialysate
(water). The amount of dialysate should be 200-500 times the volume of the sample. A
sample containing glucose, protein and starch will be placed inside the dialysis tube. You
will study the movement of organic molecules through the pores of the semipermeable
dialysis tubing by performing a dialysis. The movement of organic compounds will be
confirmed by performing special tests that will allow detection of glucose, protein and
starch.

Detection of Organic compounds

Living organisms composed of four classes of carbon-containing molecules:

carbohydrates, lipids, proteins, and nucleic acids. The cell composed of 50% protein,
15% nucleic acid, 15% carbohydrates, 10% lipids and 10% other substances. Organic
compounds can be detected by using colorimetric tests.

Glucose is a reducing monosaccharide that can be detected by using Benedict's

reagent. Benedict's reagent contains blue cupric copper which is reduced to red cupric
oxide in the presence of glucose. The blue color of Benedict’s solution will change
depending on concentrations of glucose in a solution going through green, yellow and red
as concentration of glucose increases.

Starch is polysaccharide that consists of two fractions, amylose and amylopectin. The
presence of starch can be detected by using Iodine/Potassium Iodide solution. A change
of color of Iodine solution to purple/black indicates the presence of starch in the solution.

Proteins can be detected by using Bradford reagent which contains Coomassie dye.
If present, protein binds to the Coomassie dye and as a result the color of Bradford
solution which is originally reddish/brown changes to blue.
BIOL110L Sautbayeva Z

Pre-lab questions:
1. This dialysis lab simulates a cell membrane. Explain why a cell membrane
allows a permeation of oxygen, water, CO2 and restricts the passage of
sucrose, proteins and starch.
2. What is the difference between osmosis and diffusion?
3. Why Benedict’s test cannot be used for sucrose detection, but is
widely used for glucose presence and quantification?

Materials:

1. Dialysis tubing (MWCO 14 kDa) (size for each piece is about 25-30 cm) (x3)
2. Large beakers (1 L), clean Eppendorf tubes (x15), tips, pipettes, tube racks,
binding clips
3. Distilled water
4. Iodine/Potassium Iodide solution
5. Benedict's Solution
6. Bradford reagent
7. Sample for dialysis (secret solutions):
✔ 30% Secrete solution 1
✔ 1% Secrete solution 2
✔ 4 mg/ml Secrete solution 3
11. Stirrer, stirring bars
12. Water bath set to 600C
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Laboratory procedure/protocol for each step

Laboratory Procedure 1: Confirmation of the presence of organic

molecules in the prepared solutions before the dialysis procedure

Pre-dialysis testing for glucose, protein and starch

1. Label nine clean Eppendorf tubes 1, 2, 3 (x3)

2. Add 1 mL of secret solution 1 into each Eppendorf tube 1 (x3)
3. Add 1 mL of secret solution 2 into each Eppendorf tube 2 (x3)
4. Add 1 mL of secret solution 3 into each Eppendorf tube 3 (x3)
Note: before adding the following reagents, resuspend them well by pipetting up and
down.
5. Add 200 µl of Benedict's solution to Eppendorf tubes 1, 2 and 3. Use a new tip
after each transfer to avoid cross-contamination. Heat the three tubes in a water bath
set at 600 C for 10-15 minutes.
6. Add 200 µl of Bradford solution to Eppendorf tubes 1, 2 and 3. Use a new tip
after each transfer to avoid cross-contamination. Wait for 10 minutes on the bench for
reaction to occur
7. Add 50 µl of Iodine solution to Eppendorf tube 1, 2 and 3. Use a new tip after
each transfer to avoid cross-contamination.

Tube number Benedict’s Bradford Iodine

solution solution solution

8. Record observed changes in Eppendorf tubes in the Tables 1 and 2

Table 1
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Table 2

Tube number Organic Test used Colour changes

Substances observed

3
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Laboratory procedure 2: Setting up the dialysis

1. Soak/pre-wet the dialysis tube in dH2O for 20 minutes and then form a bag
from the tubing by twisting and fixing one end with a binder clip.
2. Mix the content of the secret solution by turning the tube up and down. Open
the other end of the bag and pour 20mL of mixture solution into the bag. Twist
and fix the open end of the tubing. Make sure there are no leaks in the bag.
3. Place the bag in the beaker containing dH2O. The bag should be fully
immersed in the water. Place the beaker on a stirrer plate for 60 minutes at
room temperature at 250 rpm. Put a magnetic bead into the beaker. If needed,
add more water to immerse completely the tubing.
4. Repeat steps 1-3 for the remaining two secret solutions.

Laboratory procedure 3: Testing of solutions after the dialysis:

1. After 1 hour of dialysis at room temperature remove the dialysis bags from
the container and collect the contents from bags (1mL) into clean Eppendorf
tubes labelled 1i, 1o, 2i, 2o, 3i, 3o. (“i” stands for content taken from the inside
of the tubing and “o” stands for the content taken from the outside of the
tubing).
2. Collection: hold the bag over the beaker and open one binder clip and collect
1ml of the solution from the bags to correspondingly labelled Eppendorf
tubes.
3. Collect 1ml of the liquid that was left in the beakers after the removal of dialysis
bags (1mL) to correspondingly labelled Eppendorf tubes.
4. Test each Eppendorf tube for the presence of organic molecules by adding
appropriate developing reagent (Iodine, Bradford, Benedict’s solutions based
on your results obtained in Lab procedure 1) and record the results of testing
in the Table 3.

Table 3

Observations
Tube Organic Before
Test used After Dialysis
number Substance Dialysis
Control Inside Outside

3
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Post lab questions:

1. Provide your explanation for a color generation in the tube’s inside content
of all three secret solutions and absence of color change in the liquid taken
outside of the dialysis bags after 1 hour of stirring.
2. Design an experiment to show how you can use Benedict's solution to quantify
the presence of a carbohydrate by showing your calculations and results
interpretations.
3. Provide factors that affected a rate of diffusion in this lab’s experiment and justify
each of them.