Osmosis Flow
Osmosis Flow
Osmosis Flow
The primary observations of osmosiswere originally filtration flow proportional to the pressure. Finally,
reported by Pfeffer in 1877 (5). Pfeffer made an he found that a closed cup containing a sucrose
artificial membrane in the walls of an unglazed solution would develop a pressure proportional to
porcelain cup by reacting copper salts with potas- the sucrose concentration. He defined the osmotic
sium ferrocyanide. He placed a sucrose solution pressure as the pressure applied to the solution side
inside the cup and water outside, and he found that that is necessary to stop the osmotic flow. This
water moved from the water side to the sucrose definition is restricted to osmosis across an ideal,
side. He then made three important observations. semipermeable membrane that allows water to pass
First, he found that the rate of flow was proportional but is impermeant to the solute. The pressure
to the sucrose concentration. Second, he observed defined in this way holds only at equilibrium, that is,
that a pressure applied inside the cup produced a when there is no net fluid movement. The magni-
tude of the pressure is then given by van? Hoff’s law across membranes. It is the flow that is physiologi-
(9) cally important and results in cell swelling or shrink-
ing. The swelling or shrinking requires fluid move-
T = RTC (1) ment, which occurs at some quantitative rate. These
where ?r is the osmotic pressure, R is the gas concepts are not easy to convey and are often
constant, T is the absolute temperature, and C is the omitted from the usual laboratory exercises. At-
concentration of impermeant solute. tempts to teach osmosis range from using animated
films (2) to computer simulations (7) and weighing
Pfeffer’s result, that the flow is linearly related to the potato cubes with time (1). A recent laboratory
concentration, allows us to write the equation manual describes a simple “osmometer,” consisting
of a one-hole stopper inserted into dialysis tubing
hydraulic conductivity and the reflection coefficient by simply tying it off, but then it would be difficult to
for particular solute-membrane pairs. produce a known area of membrane. Therefore the
membrane was sealed at both the top and bottom by
an 0 ring and clamp, where the 0 rings were seated
THE FLOW APPARATUS
in a machined groove. To save time in the labora-
The main problems in accurately measuring flow tory, the membranes were installed beforehand by
across membranes are to maintain a steady hydro- the instructor. Spectrapor membranes, 29 mm in
static pressure, expose a reproducible membrane diameter and of 3,500 and 1,000 mol wt cutoff
area to the solutions, and prevent leaks in the (MWCO), were used in all of the exercises. A hollow,
membrane. The device (Figs. l-3) surmounted truncated Delrin cone was used to assistin installing
these difficulties. Here a known area of dialysis the membranes and 0 rings (Fig. 2) and excess
magneticstirrer
A B
FIG. 1.
Apparatus for measuring osmotic flow. The horizontal flow tube was calibrated in centimeters and was supported
in a level position by the lip of the beaker and a clamp on a ring stand. The membranes used were Spectrapor 1,000
or 3,500 mol wt cutoff (MWCO). The membranes were sealed using an 0 ring and a metal circular clamp at the top
and bottom of the tube. The 0 rings were seated in grooves machined in both the clamp and the plastic supports,
thereby providing a well-defined surface area for the membrane. Leaks were tested (A) by applying a pressure
using a 60-ml syringe. When no leaks were detected, the syringe was removed and replaced with the horizontal
flow tube. The water solution in the inner compartment was removed and replaced with test solution (B). After
filling, the filling syringe was removed, and the position of the solution in the horizontal tube was recorded at
regular time intervals (C).
ASSEMBLER
$ DIA. 0
DIA.
FRONT
FIG. 2.
Dimensioned drawings of the body, ring clamps, and assembler. The ends of the body were
fabricated from methyl methacrylate plastic. A, inside the s/W-diameter hole was an 0 ring,
PRP-568-012,3/S” ID x l/2“ OD x l/16” cross section. B, semicircular groove for 0 ring was
l/64“ deep and l/16” wide; 0 ring was PRP-568-018, 3/4” ID x 7/S” OD x l/16“ cross
section. C, 30” relief angle. D, rods connecting the ends were l/S” stainless steel. Ring clamps
were made from aluminum. 0 ring shown with the ring clamp is the PRP-568-018 0 ring as
described in B. The groove in the ring clamp to receive the 0 ring was 0.095“ wide and 0.035”
deep. All edges on the ring clamp were rounded to avoid cutting the dialysis tubing. Screws for
ring clamp assembly were 4-40 socket head cap, l/2“ long. The assembler was constructed
from acetal plastic (Delrin). The 30” angle and dimensions of the assembler were designed to
fit over either end of the body to aid in placing the dialysis tubing over the body.
Luer lock plastic syringe was fitted to the opening moved and replaced with test solution, as shown in
for the flow tube (Fig. lA). This opening was sealed Fig. 1B. The test solution was added until it began to
with an 0 ring and fit the 60-ml syringe snugly. flow down the horizontal tube; the fill syringe was
Leaks were tested by adding a large pressure via the then removed and flow measurements were begun.
syringe. About one in four membranes leaked and During measurement of flow, the inlet aperture was
were replaced. In our experience, the leaks were left open to the atmosphere. Thus the pressure on
due not to unsuccessful sealsaround the 0 rings but the fluid in the horizontal calibrated tube was
to scratching the membrane and producing a hole maintained at atmospheric pressure while fluid
during assembly. After testing for leaks, the horizon- flowed down the tube. The tube was narrow enough
tal flow tube assembly was attached (Fig. IA and Fig. so that the moving water column had a vertical
3). The water in the inner compartment was re- meniscus that could easily be read on the scale.
TOP
-
I/ 8
Students were instructed to fill the dialysis sac until raised if desired (8). Stock solutions of 250 ml were
there was some fluid already in the tube, and to made, and - 55 ml filled the devices.
avoid air bubbles anywhere in the flow path because
these would interfere with the flow measurements.
DETERMINING FLOW RATES
Although flow began almost instantly, the students
were instructed to wait for up to 5 min before Students were assigned to one of four groups, each
beginning their measurements. of which had a corresponding set of test solutions
and a single test membrane. Each group was in-
THE SOLUTIONS structed to measure the position of the meniscus at
The solutes used in these experiments were urea, 5-min intervals and to record the result in tables
sucrose, and polyethylene glycol (PEG). Both urea provided in their handout. The students also wrote
and sucrose are readily available and solutions are their observations on the chalk board as they pro-
straightforward, but a note of caution is needed for gressed so that the data would be immediately
the PEG. PEG, with an average molecular weight of available to all students. Students plotted the dis-
3,350, was obtained from Sigma Chemical (cata- tance the meniscus traveled against the elapsed
logue no. P-3640). However, this is a weight-average time. The results of duplicate laboratory sessions
molecular weight. Osmotic experiments depend on performed in 1993 and 1994 for the l,OOO-MWCO
the number-average molecular weight, which is membrane are shown in Fig. 4. The flow was
smaller than the weight-averaged molecular weight routinely linear with time and was reproducible
for a polydisperse material. A number-average mo- from group to group and from year to year. Al-
lecular weight of 2,400 was used for this material. It though average numbers are used here, the data are
is probably unnecessary to explain this complication sufficiently reliable that single runs will provide
to students, but a discussion of the issue could be similarly quantitative and identically qualitative re-
TABLE 1
Osmosis across a model membrane: flow calculation
Membrane A: Membrane B:
n 18 1,000 Mwco 3,500 Mwco
E Solution
2 16 Ax/& Q”9 Ax/& Qvy
0.075M PEG cm/min cm3/min cm/min cm3/min
30 14
/ Sucrose
2 12 I 0.25 M 0.462 0.0416 0.400 0.0360
0.50 M 0.930 0.0837 0.832 0.0749
0.75 M 1.328 0.1195 1.224 0.1102
/ Urea, 1 M 0.024 0.0022 0.024
TIME (minutes)
FIG. 4. THINKING INTERLUDE
Position of meniscus in the flow tube related to
elapsed time. Position at time 0 was subtracted so that At this point, the students can be asked several
all flows would begin on the origin. Flow is shown for questions:
0.75 M sucrose (a), 0.50 M sucrose (A), 0.25 M sucrose
(0), 0.075 M polyethylene glycol (PEG, A), 0.0375 M
PEG (a), and 1 M urea (+). The membrane was 1) Is there a relationship between the osmotic flow
Spectrapor 1,000 MWCO. Values are means +: SE of 4 and the concentration of sucrose? If so, write an
separate determinations. equation for this relationship.
.13
.I2
r outlet tube was replaced
and the membrane
with a four-way
was mechanically
with stainless steel mesh. This allowed
stopcock,
reinforced
the develop-
ment of a significant hydrostatic pressure difference
.I1 across the membrane. Water was infused using a
Harvard syringe pump at a constant and known rate.
.I0 The pressure in the inside compartment rose, driv-
ing fluid out through the dialysis membrane. Eventu-
.09
ally a steady state was reached in which the rate of
infusion of water into the inner compartment was
.08
-n exactly equal to the rate of water exit through the
“0” ring
/
to pressure transducer
/
spring clamps -----+ membrane
\ /
FIG. 6.
Apparatus for measuring the area x filtration coefficient (A x Lp) for the
dialysis membranes. Apparatus was identical to flow device except that the
horizontal flow tube assembly was replaced with a four-way stopcock to
allow flow into the inner chamber via a syringe pump and simultaneous
measurement of pressure by a pressure transducer. Pressure increased
until a steady-state value was reached in which influx of water via the
syringe pump was exactly balanced by pressure-driven flow of water
across the dialysis membrane into the surrounding solution.
coefficient is viewed as being due to a steric hin- case, 0 < (T < 1.0, and the observed osmotic
drance of solute entry into a cylindrical pore ori- pressure is diminished. Various equations have been
ented perpendicular to the surface of the mem- derived to relate (Tto the geometric size and shape
brane. Because a solute molecule is larger than the of hypothetical pores within membranes (3,6, 10).
pore, then the solute cannot enter the pore, and all
collisions of the solute with the membrane result in
DEMONSTRATION OF THE MAGNITUDE OF
reflection of the solute molecule. The membrane is
OSMOTIC PRESSURE
impermeant, and u = 1 .O for this membrane. When
the solute is smaller than the pore, it can travel In a separate demonstration, the device shown in
across the membrane by going through the pore. Fig. 6 was filled with 1 M sucrose and placed in a
Because the solute molecules are larger than the beaker of pure water (Fig. 8). The outlet tube was
solvent water molecules, they are reflected back closed to the outside air but connected by a fluid-
from the membrane more often than water. In this filled cannula leading to a pressure transducer
TABLE 2
Osmosis across a model membrane: calculation of u
.04
1 ,ooo-Mwco 3,500~Mwco
Membrane Membrane
T = RTC,
Concentration
mmHg
c 03 Qv, u Qw u
‘c l
cm”/min cm3/min
.-
c9
Sucrose
E 0.25 M 4,636 0.0416 0.153 0.0360 0.072
-o_
to pressure
7
transducer
+----- beaker
FIG. 8.
Setup for measuring the pressure developed by osmotic flow. After the inner
compartment was filled with 1 M sucrose, the device was closed to air and
inserted in a beaker full of water. The hose connected the inner compart-
ment to a P23XL pressure transducer.
however, the pressure driving flow within the mem- semipermeable membrane, one which does not let
brane itself was established rapidly. solute pass at all, the observed pressure at equilib-
rium would be the osmotic pressure. Because the
KEY POINTS membranes used in this laboratory exercise are not
This laboratory exercise illustrated the following key ideal semipermeable membranes, the pressure ob-
points for the students. served at equilibrium would be the effective osmotic
pressure.
1) A membrane separating a solution from pure
water will result in fluid movement from the pure 3) Osmotic flow depends on the concentration of
water to the solution side. This is the principal solute (Figs. 4 and 5), because the effective osmotic
observation of osmosis, but it is not an explanation pressure across the membrane is proportional to
of it. concentration (the van? Hoff Law), and the greater
the osmotic pressure, the greater the flow.
2) This fluid movement can generate a pressure,
which in this exercise was > 1,000 mmHg. The 4) Osmotic flow also depends on the kind of solute
pressure that would be generated at equilibrium is (Fig. 5). This is due to the different values of u for
the observed osmotic pressure. For a perfectly each solute-membrane pair. Thus CTfor PEG was