Good Report On Practical 3: Diffusion Of Small Molecules Across Semi-Permeable Membranes Using Dialysis

Introduction

Body cells are surrounded by plasma membrane that controls the movement of substances into and out of the cells. Plasma membrane is a semi-permeable. Consequently, it can only allow certain substances to move across it into the cells or out of the cell. Three main processes are involved in the movement of substances across the plasma membrane: diffusion, active transport, and osmosis. Diffusion refers to the movement of molecules down a concentration gradient (Chiras, 1999). However, there are two main types of diffusion: Simple and facilitated diffusion. In simple diffusion, molecules pass through the semi permeable membrane without assistance. However, facilitated diffusion involves the passage of mostly relatively larger molecules across the plasma membrane through pores formed by integral proteins with the help of energy in the form of ATP. This process is passive.
Diffusion is very instrumental in the homeostatic functioning of the kidney. In this case, the kidney regulates the concentration of dissolved substances on both sides of the plasma membrane through diffusion. When a given substance is more concentrated on one side of the plasma membrane, its particles flow to the less concentrated side until a balance in concentration between the solutions on both sides of the membrane is achieved.

This experiment has two aims:

Methods
This experiment involved three main tasks:
setting up the calibration curve

Measuring diffusion across dialysis membrane

Measuring the effect of protein on dialysis
Setting up the Calibration Curve
This step involved first preparing 0.4% (v/v) solution of dye. In this case, 1mL of 100mM dyes stock solution to 249mL of water. Next, the following solutions were prepared from the 0.4% v/v: 0.2% v/v, 0.1%v/v, 0.05%v/v, 0.025%v/v, 0.0125%v/v, and 0.0063%v/v. In preparing the solutions, a series of two-fold dilution was made. 4mL of each solution was prepared. For example, in preparing 0.2%v/v solution, the following formula was applied:
C1V1=C2V2
Where; C1 refers to the concentration of 0.4%v/v (0.4%v/v)

V1 refers to the volume of 0.4%v/v that should be diluted to make 4mL solution

C2 is the concentration of the solution to be prepared, which is 0.2%v/v, in this case, (0.2%v/v).

V2 is the volume of the concentration to be prepared, in this case, 4mL.

A sample calculation for preparing 0.2% v/v solution is as shown below:
C1V1=C2V2
0.4*v1=0.2*4
V1=0.2*40.4
V1=2
Therefore, 2mL of 0.4%v/v was diluted with 2mL of water to make 4mL of 0.2%v/v.
In preparing 0.025%v/v, 2mL of water was added to 0.05%v/v solution. The method was applied for all the solutions. Next, 200µl of each standard was placed into the well of a 96well plate in triplicate and the absorbance read at 520nm. The concentration of each standard was then calculated and a calibration curve of concentration vs absorbance plotted.

Measuring diffusion

In this step, 1 ml of dye stock solution was put in a clean tube and diluted with 3mL of water. 250 mL of water was then added into a conical flask. The stirrer bar was then placed into the conical flask and the flask then placed on a stirrer. Then, the visking tubing was opened out in a beaker containing fresh water. A knot was then tied in one end of the tubing. The inside of the tubing was then rinsed out with water several times. Next, excess water was removed at the end of the rinsing process.
Next, 2.5ml of the diluted dye was placed into the visking tubing while the knot was being checked for any leaks. The other end of the tubing was then knotted, and a clip placed on both knotted ends. The visking tubing was then placed inside the flask. Then, 200µl of solution was removed from the flask and transferred to the wells at the following times: 5 min, 10min, 20 min, 30 min, 45 min, 1 hr, 1hr 15min, 1hr 30 min. The absorbance of each was measured at 520nm.
Measuring the effect of protein on dialysis
In this step, the dialysis membrane was first set up in the same way as had been done in the previous step (measuring diffusion). However, the dye solution used in this step was prepared by placing 1 ml of dye in a test tube with 3 ml of protein solution instead of water. The solutions were mixed and left to stand for sometimes while the tubing was being prepared. 250ml of water was then added into a conical flask. The stirrer bar was then placed into the flask and the flask placed on a stirrer. The visking tubing was then opened out in a beaker containing fresh water. A knot was then tied in one end of the tubing. The inside of the tubing was then rinsed out with water several times. Next, excess water was removed at the end of the rinsing process.
Next, 2.5ml of diluted dye was placed into the visking tubing while the knot was being checked for any leaks. The other end of the tubing was then knotted, and a clip placed on both knotted ends. The visking tubing was then placed inside the flask. Then, 200µl of solution was removed from the flask and transferred to the wells at the following times: 5 min, 10min, 20 min, 30 min, 45 min, 1 hr, 1hr 15min, 1hr 30 min. The absorbance of each was measured at 520nm.

Results

Figure 1 shown below shows the absorbance readings obtained from the standard solutions.
Figure 1: Table showing the various absorbance readings of the standard solutions used in the experiment
Figure 2: Table showing the various absorbance readings of the dye with distilled water and dye with protein used in the experiment
It was observed during the experiment that for both the dye with distilled water and the dye with protein, absorbance increased with time. However, the dye with distilled water recorded higher absorbance than the dye with protein at all time as shown in figure 3 below.
Figure 3: graph showing changes in absorbance of the solution removed from the flask for the solution with distilled water and that with protein.

Discussions

In the experiment, the diffusion of dye occurred faster when it was diluted with distilled water than when it was mixed with a protein solution. In other words, dye with distilled water diffuses faster than the dye mixed with protein. In addition, the magnitude of diffusion was higher in the dye diluted with distilled water than the dye diluted with a protein solution. The presence of protein molecules in the medium of diffusion increases the frequency of collision between the particles of the dye and protein molecules. Consequently, the rate of diffusion of particles of dye becomes slow. The diagram below illustrates the rate of diffusion between the visking with the dye mixed with water and that with the dye mixed with a protein solution.
Figure 4: Diagram showing that rate of diffusion of dye under different solutions
The amount of dye that can diffuse across the membrane of the visking tubing is affected by various factors. Such factors include the concentration gradient between the solution inside the visking tubing and the solution outside the tubing, temperature of the solution inside and outside the tubing, and the medium through which particles diffuse. Concentration gradient refers to the gradual difference in the concentration of a given substance between two regions separated by a semi-permeable membrane. The higher the difference in the concentration of a given substance between the two regions separated by a semipermeable membrane is, the higher the concentration gradient. The amount of dye that will diffuse from the visking tubing to the water in the flask will be higher if there will be higher concentration gradient between the solution in the tubing and the water in the flask. The temperature of both the solution in the visking tubing and the solution in the flask affects the amount of dye that would diffuse in the sense that the temperature increases the kinetic energy of molecules. High kinetic energy increases the speed of the molecules. Consequently, it leads to the diffusion of more particles. The medium of diffusion would also affect the amount of the dye that would diffuse from the visking tubing to the solution in the flask. The presence of many particles in the medium of diffusion increases the frequency of collision between the diffusing molecules and the particles in the medium. High frequency of collision between the diffusing molecules and the particles in the medium of diffusion leads to low rate of diffusion. This explains why there was less diffusion of the dye diluted with distilled water than the dye diluted with a protein solution.
Diffusion of water and molecules across the semi-permeable membrane is important in the physiology of kidney since it enables the kidney to purify the blood. Kidney nephron, the functional unit of kidney, performs the function of purifying blood. In this case, reabsorption of substances such as glucose occurs in the proximal convoluted tubule. This process takes place through diffusion.
The error in this experiment could have been caused by several factors. First, the instruments used may not be perfectly accurate. Consequently, the readings made for absorbance may have not been accurate. In addition, the continuous changes in the concentration of dye in the flask may have influenced the unstable readings recorded. In short, the two main sources of error in this experiment are the instruments used and the intrinsic factors to the solutions used.

Conclusions

This experiment found out that the presence of protein in the region of higher concentration of the dye leads to slow and low extent of diffusion of the dye. Therefore, the presence of many and larger molecules in the medium of diffusion leads to slow rate and low extent of diffusion of the semi-permeablediffusing particles. The experiment was successfully conducted since all the observations made are supported by scientific facts.

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