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Exercise :- 3 Determining Osmotic Potential by the Freezing
Point Depression Method
Osmotic potential (Ψs) represents the effect of solutes on
the energy state of water. Osmotic potential is related to other properties of the solution such as vapor pressure, boiling point, and freezing point. These properties, which are interrelated, are termed colligative properties, and are dependent on the mole fraction of solute. Since these properties are inter-related, one can be measured and used to calculate the others.
The freezing point of pure water is 0 C but the freezing point of aqueous solutions containing non-volatile dissolved solutes is less than zero. Thus, the addition of solutes depresses the freezing point of water. The extent to which the freezing point is depressed below O C is proportional to the concentration of dissolved solute particles; a 1 molal solution of an ideal non-ionized solute has an osmotic potential of -2.27 MPa and freezes at -1.86 C. Based on this relationship the osmotic potential of any unknown solution can be calculated: -2.27 MPa / -1.86 C = Ψs / Δf re-arranging: Ψs = (1.22 MPa deg-1 ) Δf
where
Δf = is the freezing point of the solution in oC.
Since this equation is for solutions at zero oC (273 K) we must correct the equation to yield our answer at room temperature by multiplying the equation by the ratio of the absolute temperatures (room temperature in K/273 K). Thus:
eqn. 1: Ψs = (1.22 MPa deg-1 ) Δf x (room temp in K/273 K)
Freezing point depression is generally determined on tissue sap. Sap
samples are prepared by freezing the tissue to rupture cells and then squeezing the frozen material by hand through cheesecloth or with a hydraulic press. Alternately, the tissue can be homogenized (without water) in a blender or with a mortar and pestle. In practice, the temperature of a sap sample is monitored with a thermocouple or sensitive thermometer as it is cooled. Data from a typical freezing point depression experiment is given below (Figure 1). Point A represents the extent of super-cooling of the solution. The line from A to B represents a very rapid freezing of the water in the solution after super-cooling. The increase is due to the release of the heat of fusion. Therefore, Point B is an estimate of the freezing point depression and is called the apparent freezing point (Δf'). The true freezing point (Δf) is obtained after correcting for super-
cooling according to the following equation (Reiss, 1996):
eqn. 2: Δf = Δf' - 0.0125 µ
where Δf = true freezing point Δf' = apparent freezing point µ = degrees of super cooling (negative in sign) 0.0125 = amount of water (1/80) that solidifies per degree of super cooling One disadvantage of this method is that the solute composition of the sap influences the accuracy of the measurement. The equation above is given for ideal solutions of glucose or mannitol. Sucrose, on the other hand has a freezing point depression of 2.06 oC, not 1.86oC. Nevertheless, the method still provides an estimate in reasonable agreement with those obtained by other methods.
Fig. 1. Typical freezing point curve Fig 2. Freezing point apparatus
Hypothesis:
Bland and Tanner (1985) report that the osmotic potential for a potato tube is in the
range of -0.58 to -1.53 MPa. Our tubers should fall within that range.
Protocol:
Set up the freezing point apparatus (Figure 2).
First, we will calibrate the thermometer by placing 3 mL of water in the sample test
tube.
Immerse this unit in a beaker (600 mL) containing salt and ice. The ice bath should
be between -5 and -10 oC.
Suspend a thermometer in the sample making sure that it doesn't touch the bottom
or sides of the tube.
Stir the sample continuously and watch the decrease in temperature. When the
temperature reaches 0oC, record the temperature every 10 seconds (Table 1). Continue readings as the sap cools. Note the temperature closely. Periodically tap
the thermometer gently to induce freezing. When freezing occurs there will be a rapid increase in temperature. Note this temperature which is the apparent freezing point (Δf') and continue to record temperatures until they become fairly constant. [In some cases, the sample may freeze without releasing a detectable heat of fusion. If this occurs, discard the sample and begin again. You can often determine that this has occurred if there is a plateau in the cooling rate of the sap.] The freezing point of the water should be zero. If the Δf' of the water is not 0oC, then correct the Δf' of the sap sample accordingly.
Repeat the experiment with a potato sap sample. To prepare the potato sap, obtain
frozen potato sections and carefully remove the peel . Homogenize the thawed potato in a blender. Transfer the homogenate to a centrifuge tube and spin at the highest setting for five minutes to recover the supernatant. Data: 1.Complete Tables 1 & 2. 2.Plot temperature vs. time for both sap and water. 3.Calculate the true freezing point (Δf) of the sap (Table 2).
4.Calculate the Ψs of the sap sample.
Table 1: Freezing response of water and potato sap
Temperature ('C) Time (sec) Water Sap Table 2: Summary Table - Freezing Point Depression room temperature (C) room temperature (K)
Δf' water (from graph, C)
Δf' sap (uncorrected; from graph, C)
Δf' sap, corrected for thermometer, C
sap - coldest temp during supercooling (from graph, C) sap - coldest temp during supercooling (corrected for thermometer; C) µ (degree of supercooling = Δf' sap - coldest temp, negative in sign)
