Osmosis and Diffusion Through Solids and

Across Natural and Artificial Membranes

Introduction


Diffusion, or the passive movement of molecules from high concentrations to lower
concentrations, is an important process for the maintenance of living organisms. More
specifically, osmosis, or the diffusion of water, is absolutely critical to the proper functioning of
nearly every living thing. Living cells that are placed into a hypertonic solution, a solution that
contains more solutes than the cell, will be expected to lose water and shrink if the cell does not
actively utilize energy to combat this natural gradient. This result if from the passive diffusion
of water out of the cell, where water is in higher concentration, and into the hypertonic solution,
where water is concentrated less. Both plant and animal cells in this environment, and under this
condition, are under considerable stress and do not function at their optimal rates. Cells placed
into a hypotonic solution, a solution that contains less solute than the cell, will absorb water from
the solution. Under this influx of water animal cells could lyse, or burst, under the increased
pressure inside the cell membrane, effectively killing the cell. Plant cells are surrounded by a
rigid cell wall which prevents lysis. Instead water will diffuse into the cell, increasing its
pressure and creating a turgid internal environment. Plant cells function at their optimum under
this turgid condition. Under isotonic conditions, or when the concentration of solutes is equal
both inside the cell and in the surrounding solution, water enters the cell and leaves the cell at the
same rate. These are ideal conditions for animal cells (Campbell et al 1999).

Considering that osmosis, or diffusion in general, is so important to the understanding of cellular
maintenance it is imperative that scientists have a complete understanding of these two
processes. This experiment has three primary goals: 1) To quantify the osmotic flux across a
natural membrane, 2) To quantify the osmotic flux across artificial membranes, and 3) To
quantify the effects of molecule size on diffusion rates in solids, where it is proposed that smaller
particles will diffuse more rapidly in a restrictive environment. Based on well understood
scientific principles, natural and artificial membranes should allow the expulsion of water when
placed into hypertonic solutions and absorb water when placed into hypotonic solutions.
Likewise, molecules other than water that are small enough to fit through the membrane should
also follow a concentration gradient.


Methods


Osmosis across natural membranes
A core was taken from a white potato using a stainless steel coring tool and placed into a bowl of
deionized water prior to experimentation to eliminate desiccation. The soaked core was
randomly selected from six available in the water bowl and was blotted dry. The cores initial
mass was measured using a digital scale fitted with a plastic try to protect the scale. All mass
data was measured to the nearest 0.1 g. The core was placed into a bowl filled with a 20% (w/v)
sodium chloride solution and was measured every ten minutes for 1 hour by removing it from the
bowl with a metal probe, blotting it dry and placing it on the digital scale. After measurements
the core was placed back into the salt solution. Data was analyzed graphically using Microsofts
Excel program.

Osmosis across artificial membranes
Dialysis tubing was measured to 10 cm lengths and submerged in deionized water to hydrate the
membrane. Six different osmometers were created by filling a bowl with different media and
placing dialysis bags containing different solutions into them. The six different osmometers
treatments are summarized in Table 1.

Table 1: Summary of osmometer treatments.
Treatment name Solution in bowl Solution in the dialysis bag
1.0 M sucrose dH
2
O 1.0 M sucrose
0.6 M sucrose dH
2
O 0.6 M sucrose
0.3 M sucrose dH
2
O 0.3 M sucrose
0.5 M MgSO
4
dH
2
O 0.5 M MgSO
4

Control dH
2
O dH
2
O
Reciprocal control 1.0 M sucrose dH
2
O

Dialysis bags were created by folding over approximately 1 cm of tubing along one of the open
ends of the dialysis tube and tying it tightly with a piece of string. The remaining open end was
filled with the proper solution (Table 1) and air bubbles were removed by gently squeezing the
sides of the bag while pinching the end closed. The open end of the bag was then folded over
and tied tightly with a piece of string. Extra string was shortened by removal with a pair of
scissors. The initial masses, to the nearest 0.1 g, of each osmometer bag were measured by
blotting them dry and using a digital scale. Similar mass data was gathered every 15 minutes for
1 hour. Mass measurements were converted to % weight loss to correct for differences in initial
weights by the following formula:

(weight after immersion initial weight) x 100 = % weight change
Initial weight

The corrected osmometer data was analyzed graphically using Microsofts Excel program.

Differential diffusion
A beaker was filled halfway with deionized water prior to experimentation. A small subsample
of this water was tested for the presence of chloride and starch by precipitation with silver nitrate
and iodine, respectively and served as the control. Silver nitrate, in the presence of chloride ions,
will form a white precipitate while iodine, in the presence of starch, will form blue precipitate.
Failure to achieve either of these precipitates will indicate a negative result for the presence of
those substances. A dialysis bag was filled with equal portions of a 1% sodium chloride solution
and a 1% starch solution and closed by the method described previously. The dialysis bag was
then placed into the beaker of deionized water and allowed to stand for 1 hour. After 1 hour he
bag was removed and its contents were emptied into a dry, clean beaker. The contents of the
original beaker and the bag contents were analyzed for the presence of chloride and starch as
described previously.

Diffusion through a solid
An agar plate, prepared with an 8% technical agar solution, was altered by cutting 4 small wells
into the plate with a straw. Each of the 4 wells was labeled with one of four chemicals according
to the following treatment summarized in Table 2.

Table 2: Summary of agar plate chemical treatments.
Treatment chemical Gram-molecular weight (g)
Methylene blue 374
Potassium ferricyanide 212
Silver nitrate 112
1N Hydrochloric acid 35

The wells were filled simultaneously with the indicated solution by using a dropper bottle.
Plates were incubated at room temperature for 30 minutes, after which time the distance, to the
nearest mm, of chemical diffusion was measured using a small ruler. The distance of diffusion
was measured from the wall of the well to the outer most point along the diffusion ring.

Results


Osmosis across natural membranes

The initial mass of the potato core was 10 g and after 1 hour of submergence in a 20% sodium
chloride solution the mass of the potato had dropped to 7 g which accounts for a 30% drop in
weight. The mass of the potato core had decreased by 25% within the first 30 minutes of
treatment indicating that a majority of weight loss took place during the first half of treatment
and that additional measurements after 30 minutes showed decreasingly less weight loss with
increasing time (Figure 1).
6
7
8
9
10
11
0 10 20 30 40 50 60
Time (minutes)
M
a
s
s

(
g
)

Figure 1: Mass of a potato core during submergence in a 20% sodium chloride solution
for 1 hour.


Osmosis across artificial membranes

Osmometer bags containing sucrose solutions or MgSO
4
gained appreciable mass when placed
into deionized water for one hour. The amount of mass that each bag gained decreased with
increasing time. The 1.0 M sucrose osmosmeter gained the most mass and was 30% heavier
after one hour. The 0.5 M magnesium sulfate osmometer gained slightly less mass and was
approximately 25% heavier at the end of one hour. The 0.6 M sucrose and 03 M sucrose
osmometers gained 20% and 10% more mass, respectively over the course of the one hour
treatment. The control osmometer displayed slight fluctuations in mass over the course of the
first few measurements but eventually leveled off at 0% weight change at 45 minutes and
remained that way until the end of the experiment. The reciprocal control, or water filled
osmometer bag floating in 1.0 M sucrose solution, lost 30% of its mass over the one hour
duration. A graphical summary of the osmometer masses over the one hour experimental period
is displayed in Figure 2.


-40%
-30%
-20%
-10%
0%
10%
20%
30%
40%
0 15 30 45 60
Time (minutes)
%

W
e
i
g
h
t

c
h
a
n
g
e
1 M sucrose 0.6 M sucrose 0.3 M sucrose 0.5 M MgSO4 Control Reciprocol



Differential diffusion

The control sample of deionized water tested negative for both the presence of chloride and
starch when tested with silver nitrate and iodine, respectively. After one hour of soaking in
deionized water the contents of the bag tested positive for both starch and chloride. The contents
of the beaker, in which the dialysis bag had been soaking, tested positive for chloride but
negative for starch indicating that starch was not present in the beaker (Table 3).

Table 3: Summary of results testing for the presence of chloride and starch. A plus sign
indicates that the molecule was present. A minus sign indicates that the molecule was
absent. Treatment was for 1 hour.
Control sample (beaker
contents before
treatment)
Bag contents Beaker contents after
treatment
Starch - + +
Chloride - - +


Diffusion through a solid

The hydrochloric acid diffused 12 mm through the agar plate in 30 minutes and went
approximately twice as far as the next fastest molecule, silver nitrate, which traveled only 6 mm
in 30 minutes. Potassium ferricyanide and methylene blue traveled 5 mm and 2 mm
respectively. A plot of the diffusion distances against molecular weight clearly indicates that as
molecular weight increases the speed of diffusion decreases (Figure 3).

R
2
= 0.973
0
50
100
150
200
250
300
350
400
0 2 4 6 8 10 12 14
Distance (mm)
M
o
l
e
c
u
l
a
r

w
e
i
g
h
t

(
g
)

Figure 3: Scatterplot of molecular weight against distance diffused through an agar plate. The
four data points represent the data gathered above. A best-fit line has been included as a
reference.


Discussion

Osmotic balance and diffusion rates are critically important to the maintenance of cellular
activity and for sustaining life. In hypertonic environments cells typically lose water by the
passive diffusion of water across its osmotic gradient. In hypotonic environments cells typically
gain water by passive diffusion of water across its osmotic gradient. Only under isotonic
conditions are cells expected to have an equal amount of water diffusing across the cell
membrane both into and out of the internal cell compartment. When placed into a highly
hypertonic solution the mass of a potato was observed to decrease as expected (Figure 1). This
decrease in mass is most likely attributed to the osmotic loss of water from the potatos cells and
interstitial space. The initial loss of mass was great and with increasing time of suspension in the
hypertonic environment the loss of mass decreased. There are at least three possible
explanations for this observation. First, as water diffused from the potato cells the osmotic
gradient across the cell membrane decreased as the solutes in the solution became more diluted.
Second, plant cells may exhibit some water retaining abilities when a threshold for water loss has
been reached. Third, the potato was reaching the pint where nearly all of the water had been lost
by diffusion and could not lose any more. The third explanation is unlikely as a dehydrated
potato will shrivel to a much smaller size than was observed during the experiment. Van
overbeek (1942) proposed that tomato plants were capable of active water uptake by
concentrating solutes within their tissues by means of cellular respiration. This change in ionic
gradient could reclaim an osmotic gradient favorable to the uptake of water under less than ideal
conditions.

Osmometers made of artificial membranes gained mass when placed into hypotonic solutions
and lost mass when placed into hypertonic solutions as was expected (Figure 2). The gain and
loss of mass is most likely attributable to the uptake and loss of water across an osmotic gradient.
As the artificial membranes had no way to actively move water across them against a
concentration gradient it is assumed that the decreasing change in mass with increasing time of
being held in solution must be attributable to attaining equilibrium with the outer environment.
The slight fluctuation in the control osmometer may have been a result of measurement error or
technique. It is possible that an unequal amount of towel drying had been applied during some
of the weigh periods. If this indeed is true it did not appear to have an effect on the overall
results.

The differential diffusion experiment indicated that the deionized water in The University of
Texas Arlington biology laboratories is free of starch and chloride as would be expected if the
deionization method utilized by The University was operating properly. The results also
indicated that the artificial membranes utilized during the experiments had pore sizes large
enough to allow the passage of chloride ions and water but not large enough to allow the passage
of starch molecule which may attain relatively large molecular sizes (Table 3). Artificially
prepared membranes can be purchased with different pore sizes.

It was proposed that molecules of smaller molecular weight would diffuse through a semi-solid
substance faster than molecules with large molecular weights. The results of the solid diffusion
experiment confirmed that this was, in fact, true. The high molecular weight molecules diffused
much shorter distances over the same time period. Graphical analysis was fitted with a power-
function trendline allowing determination of suspected diffusion distances of molecules of
untested molecular weight. Further experimentation would need to be conducted to determine if
this relationship would hold up in different conditions other than 8% technical agar plates. The
diffusion speed of large molecules compared to small molecules is important in field of genetics
as this mechanism is the basis behind gel electrophoresis. Sections of DNA with different
lengths will pass through the gel at different speeds and result in identifiable bands indicative of
the DNA length.
Literature Cited

Campbell NA, Reece JB, Mitchell LG. 1999. Biology: Fifth Edition. California: Benjamin
Cummings. 1175 p.

Van Overbeek J. 1942. Water uptake by excised root systems of the tomato due to nonosmotic
forces. American Journal of Botany 29(8):677-683.