Demonstration of osmosis

Transcription

1 Demonstration of osmosis We will try to carry out a classical experiment on demonstration of osmosis. The principle is shown in the figure. Water moves from the solution of lower osmolality, across the semipermeable membrane, into the solution of higher osmolality, and its level rises. In theory, the process should stop in time of balancing the osmotic pressure by hydrostatic pressure of the column of the solution inside a tube. π = h g h... difference in solution levels density of the solution g... gravitational acceleration 1. On a wider portion of the tube, fasten the cellophane, which will serve as a semipermeable membrane. 2. Dip the tube membrane down into a graduated cylinder with distilled water so that the water level in the cylinder reached the mark. 3. Into the tube, transfer carefully by pipetting saturated sucrose solution, so that liquid levels in the tube and in the cylinder are at the same level. For better visibility, sucrose solution was colored with blue food coloring. 4. Let the experiment run until the end of the class. Then, using a ruler, measure the difference between the two levels and make a conclusion. The beginning: "What time is it?" The end: "What time is it?" Time period: min Difference in solution levels (mm): Conclusion: 1

2 Monitoring the kinetics of dialysis This exercise demonstrates separation of high and low molecular weight substances (starch and NaCl) by dialysis. Moreover, we will monitor dialysis rate of chlorides. As a dialysis membrane, we will use a cellophane with the size of pores between 3 and 4 nm. These pores are permeable to small ions, but do not allow the passage of large molecules. Procedure: Preparing the dialysis experiment: 1. There is an empty dialysis container mounted the laboratory stand at your working place. The dialysis container is a glass tube with cellophane membrane on one side. 2. Using a graduated cylinder, put 150 ml of distilled water into the beaker (of a volume of 250 ml). Keep the dialysis container out of the beaker at this moment. Add a stirring bar into the beaker, place the beaker on a magnetic stirrer, switch on the stirring (not heating!) and adjust the speed of it to optimal. 3. Into the dialysis container, pipette 5 ml of starch solution and 10 ml of NaCl solution (c = 1.5 mol/l). Prepare a stopwatch so that it can be started immediately when you dip the dialysis container into the beaker (cellophane membrane must be below the level of the solution in the beaker). Before starting the experiment, ensure yourself you know what to do further (taking the samples as described below)! Let the experiment run for 90 minutes. Monitoring of dialysis rate: At points in time of 2, 4, 6, 8, 10, 15, 20 minutes after starting the experiment, and later in 10-minute intervals, take (using a pipette) from the beaker samples of a volume of 2.5 ml. Put the samples into titration flasks labelled with numbers corresponding to the time sample was taken. Time (min) V titration (ml) V dif (ml) c () n (mmol) Time (min) V titration (ml) V dif (ml) c () n (mmol) V titration V dif c n mercurimetric titration of the sample - consumption of the standard reagent volume of the solution in the beaker (It is getting smaller by taking samples!) concentration of Cl - in the solution in the beaker amount of Cl - in the solution in the beaker 2

3 Estimation of chloride concentration: Concentration of Cl - can be estimated by mercurimetric titration. Mercurimetry belongs to complexometric titration methods. Standard reagent is a solution of Hg(NO 3 ) 2, that forms with Cl - ions soluble non-dissociated complex [HgCl 2 ]: Hg Cl - [HgCl 2 ] f = n(cl - )/n(hg 2+ ) = 2/1 For indication of the end point of the titration, we will use diphenylcarbazone that forms with the excess of Hg 2+ ions blue-violet coloration. The solution of Hg(NO 3 ) 2 is toxic! Keep safety in mind! After finishing your work, put the waste into the extra bottle! To the sample taken from the beaker you put into a titration flask, add 2.5 ml of distilled water and 0.3 ml of indicator (diphenylcarbazone). Titrate with standard solution of Hg(NO 3 ) 2 (c standard reagent = 12.5 ) to a color change to blue-violet coloration. Calculate the concentration of chlorides in individual samples and the amount of chlorides passed through the dialysis membrane into the beaker. Fill in the values into the table. c c standardreagent V.V sample titration.f n c.v dif V sample = 2.5 ml V dif = 150 ml, ml, 145 ml,... (It is getting smaller by 2.5 ml every time the sample is taken.) Checking what has happened with the starch: After taking of the last sample (90 minutes), stop stirring and take up the dialysis container from the beaker. Take two test tubes and transfer a small volume (~ 1 ml) of the solution from the dialysis container into one and from the beaker into the other one. Add 3 drops of Lugol solution (solution of iodine) into both test tubes and compare the results. Lugol test - solution from the dialysis container: Lugol test - solution from the beaker: Conclusion: 3

4 Evaluation of the dialysis rate: Make a graphical evaluation. Plot a graph showing increase in time of the amount of chlorides in the solution in the beaker. First, plot the individual points according to the data in the table. For t o =0 the amount of chlorides in the beaker is n o =0.Then draw optimal interlaced curve. axis x... time (minutes) axis y... n = amount of chlorides in the beaker (mmol) Divide the scale on axis x into 5-minute intervals (time t 1, t 2, t 3,...) and from the curve read on axis y the corresponding amount of chlorides (n 1, n 2, n 3,...). Time (min) n x (mmol) v x (mmol Cl - /min) Time (min) n x (mmol) v x (mmol Cl - /min) 4

5 Dialysis velocity (v) is defined as the amount of Cl - passing through the semipermeable membrane per minute. v 1 = (n 1 - n 0 ) : 5 [mmol Cl - /min] v 2 = (n 2 - n 1 ) : 5 v 3 = (n 3 - n 2 ) : 5 etc. Plot a graph of dialysis velocity in time. axis x... time (minutes) axis y... v = calculated velocity (mmol Cl - /min) At the cross section of the curve with axis x, read the time point at which dialysis stopped due to loss of concentration gradient (balancing of Cl - concentrations on both sides of the membrane). By extrapolation of the curve to zero time, find out the initial velocity at the cross section of the curve with axis y. Time point at which dialysis stopped: Initial velocity: Conclusion: 5

6 Determination of osmolality using cryoscopy Preparing of 250 ml of Ringer's solution Physiological solution in form of 0.9% NaCl contains only Na + and Cl - ions. However, there are also other ions in the blood plasma. In clinical practice, there are more types of infusion solutions in use, some of them more similar in ionic composition to blood plasma. Ringer's solution is one of them. 1. Weigh stepwise using a plastic weighing boat and transfer weighed quantities into an Erlenmayer flask (of volume of 250 ml): sodium chloride (NaCl) potassium chloride (KCl) calcium chloride (CaCl 2 ) g g g 2. Into the same Erlenmayer flask, flush also unobservable remnants from the plastic weighing bottle using a squirt bottle with distilled water. 3. Into the same Erlenmayer flask, add distilled water to a volume about ml and by swirl mixing thoroughly dissolve the contents. 4. Using a funnel pour the content of Erlenmayer flask into a 250 ml volumetric flask. Rinse the Erlenmeyer flask at least 2x with a little of distilled water, pour everything into a volumetric flask. 5. Fill the volumetric flask exactly to the mark with distilled water (i.e. a volume of 250 ml). 6. Close the volumetric flask by a stopper and thoroughly mix the contents. What is the concentration of the individual ions in the solution prepared? M(NaCl) = g/mol M(KCl) = g/mol M(CaCl 2. 2H 2 O) = g/mol Result Na + K + Ca 2+ Cl - From the known composition, calculate the osmolality (osmolarity) of this solution: 6

7 Measurement of osmolality by modern osmometer There is a modern freezing point osmometer Osmomat 3000 in the laboratory. The total osmolality of aqueous solutions is determined by comparative measurements of the freezing points of pure water and of solutions. The instrument requires very small sample volumes (50 l), test time is short (60 s). Preparing samples: Distilled water it is available in the laboratory Ringer's solution you have already prepared (previous exercise) Ringer's solution + ethanol Using a graduated cylinder, measure 50 ml of Ringer's solution, pour it into a small beaker and add by pipetting 0,25 ml of 40% ethanol. Calculate what rise of osmolality (osmolarity) should the addition of ethanol result in: Ringer's solution + glucose Using a graduated cylinder, measure 50 ml of Ringer's solution and pour it into a small beaker. Weigh using a plastic weighing boat 270 mg of glucose and dissolve it in the solution in the beaker. Calculate what rise of osmolality (osmolarity) should the addition of glucose result in: Sample Distilled water Ringer's solution Ringer's solution + ethanol Ringer's solution + glucose Measured osmolality 7