Page 4.1 Lab 4: Osmosis and Diffusion Cells need to obtain water and other particles from the fluids that surround them. Water and other particles also move out of cells. Osmosis (for water) and diffusion (for other particles) are important processes that allow for transport across the plasma membrane of cells. In addition, diffusion is an important mechanism of movement of particles between and within many body fluid compartments. For example, diffusion is the mechanism by which particles like oxygen and glucose travel between interstitial fluid and intracellular fluid. Osmosis is important in movement of water into and out of the GI tract, the urinary system, and capillaries throughout the body. In this Lab, you will analyze the parameters that affect diffusion and osmosis, and you will use red blood cells to analyze water and alcohol transport across the plasma membrane. Objectives At the conclusion of this laboratory the student will understand and be able to describe: 1. diffusion, osmosis, osmotic pressure, osmolarity, osmolality, selectively permeable membrane, tonicity, crenation, and hemolysis. 2. characteristics that affect membrane permeability of a substance. 3. an isotonic solution for mammalian blood cells. 4. expected results when hypothetical situations similar to the experiments performed are presented. 5. the difference between osmosis and dialysis. 6. why osmosis and diffusion are essential in physiological systems. 7. how to calculate osmolarity of a solution if given the solute s molarity or if given the solute s percent concentration and molecular weight. VOCABULARY LIST molarity mole molecular osmolarity osmole osmolality weight tonicity hypotonic isotonic hypertonic impermeant permeant diffusion dialysis osmosis semipermeable crenate osmometer lipid solubility solutes selectively permeable solutes hemolyze This lab consists of 2 Activities: ACTIVITY 1. Selective Permeability and Osmosis Students will observe (A) osmosis, (B) dialysis through a semi-permeable membrane, and (C) the effect of solutions of varying tonicity on red blood cells. ACTIVITY 2. Effect of Molecular Weight and Lipid Solubility on Membrane Permeability Students will determine which has a larger effect on the diffusion rate of alcohols across a selectively permeable cell membrane, molecular weight (size) or lipid solubility.
Page 4.2 BACKGROUND AND REFERENCES You should be familiar with many of the terms and concepts of this Lab from your general biology course; however, you may review this information in your textbook. Making Solutions To make a (w/v) solution given the desired percent of solute, add to a container an amount of solute in grams equal to the desired percent. Then add water until the final volume is 100 ml. Note that you will be adding LESS than 100 ml of water. Example: How would you make a 2% solution of NaCl? Answer: Add 2 grams of NaCl to a container. Then add water until the final solution volume is 100 ml. --------------------------------------------------------------------------------------------------------- What is molarity? It s a measure of how much solute is in a solution. But the amount of solute is measured in moles, not grams. So first, we need to know how to convert between grams and moles: Mass (in grams) of one mole of a substance = the # of grams of a substance that is its molecular weight Remember that molecular weight can be determined with a periodic table. The molecular weight of NaCl is 58. Accordingly, 1 mole of NaCl = 58 grams Now, the equation for molarity is: molarity = # of moles of solute 1 liter of solution Example: What is the molarity of the 2% w/v solution of NaCl? Answer: First, recognize that our solution was 2 grams of NaCl in 100mL final volume. But how many grams would that be if we had one Liter? 2g x 1000 ml = 20 g/liter 100 ml l liter Now, use the equation for molarity: molarity = # of moles of solute 1 liter of solution
Page 4.3 Therefore, in our example, Molarity of our solution = # of moles of NaCl = 20g x 1 mole = 0.34 mole 1 liter of solution 1L 58 g L A 1 molar solution of NaCl = 58 grams NaCl dissolved in water to equal 1 liter of solution. A 2 molar solution of NaCl = 58 x 2 = 116 grams NaCl dissolved in water to equal 1 liter of solution. --------------------------------------------------------------------------------------------------------- osmolarity = # of osmoles of solute # of osmoles = the # of moles times the number of 1 liter of solution particles that the substance breaks into when put into solution. Example: NaCl breaks (ionizes) into Na + and Cl - (2 particles) when put into water; therefore, 58 grams (1 mole) of NaCl dissolved in 1 liter of solution creates a 2 osmolar solution. For substances such as sucrose, which do not break into particles in water (they do not dissociate or ionize), the molarity of their solutions equals the osmolarity of their solutions. Note that osmolarity is a measure of the solute particle concentration of a fluid. This is NOT a relative measure. Instead, it is an absolute measure of concentration. We can describe the osmolarity of any solution in the units of osmoles/liter (Osm/L). Although osmolarity is not itself a relative measure, we can compare the osmolarities of two solutions. If solution A has a higher osmolarity than solution B, solution A is said to be hyperosmotic to solution B. If solution A has a lower osmolarity than solution B, solution A is said to be hyposmotic to solution B. If solution A has the same osmolarity as solution B, solution A is said to be iso-osmotic to solution B. Checkpoint: 1. How would you make a 30% (w/v) solution of glycerol? 2. What would the molarity of the above solution be? (The chemical formula for glycerol is C 3H 8O 3.)
Page 4.4 Osmolality = # of osmoles of solute 1 Kg water When water is the solvent, then 1 kg = 1L and the equation for osmolality above can be written as Osmolality = # of osmoles of solute 1 L water For our purposes, the difference between osmolarity and osmolality does not become significant; however, you should be aware of the fact that there is a difference between the two terms and that the word osmolality is not a "typo" error. These two terms are often used interchangeably; however, when dealing with osmosis, osmolality is actually the correct term to use. For osmolarity, the total volume of solute and solvent = 1 liter. For osmolality, the volume of solvent only = 1 liter. ---------------------------------------------------------------------------------------------------------- Tonicity: Tonicity describes the ability of solutions to pull water across a membrane, and is determined by the concentration of impermeant solutes (solutes that do not cross the plasma membrane) in a solution. It is most often used to compare the concentration of impermeant solutes inside a cell to the concentration of impermeant solutes in a solution surrounding the cell. If cells are placed in a solution, knowing the tonicity of the solution tells you whether that solution will cause cells to shrink (hypertonic solutions), swell (hypotonic solutions), or not change their volume (isotonic solutions). Understanding Tonicity from the Perspective of Impermeant solutes Tonicity is a relative measure, comparing the concentration of impermeant solutes in the intracellular fluid (ICF) to the concentration of impermeant solutes in the fluid surrounding the cell, that is, the extracellular fluid (ECF). Hypotonic solutions have a lower concentration of impermeant solutes than the ICF and will cause water to move from the ECF to the ICF; that is, into the cell. Isotonic solutions have the same concentration of impermeant solutes as the ICF and will cause no net water movement between the ICF and the ECF. Hypertonic solutions have a higher concentration of impermeant solutes than the ICF and will cause water to move from the ICF to the ECF, that is, out of the cell. The measurements of osmolarity and osmolality depend on ALL solutes in a solution. So why does tonicity depend specifically on impermeant solute concentrations? When a cell is placed in a solution that has solute particles that cannot enter the cell, these impermeant solutes in the solution can balance the impermeant solutes inside the cell. Because they do not move in or out of the cell, impermeant solutes on either side of a membrane can continually balance the impermeant solutes on the other side. Along with the impermeant solutes described above, a solution can also contain permeant solutes. Permeant solutes (solutes that do cross the plasma membrane) may initially cause water flow
Page 4.5 into or out of the cell, but the concentrations of permeant solutes can equilibrate (because they are permeant). Once they equilibrate, their concentrations will be the same inside and outside the cell, so they will eventually be irrelevant for the distribution of water. Permeant solutes in a solution cannot balance the impermeant solutes inside the cell; therefore, permeant solutes in a solution will not have an effect on the eventual volume of the cell. This is why permeant solutes are not relevant for the measure of tonicity. Because osmolarity and tonicity are measuring different things (all solutes, as opposed to impermeant solutes only) it is possible for a solution to be hyperosmotic to the ICF of cells, but still isotonic, as long as the concentration of impermeant solutes of the solution matches that of cells. For example, a solution with 320 mosm NaCl (impermeant) and 100 mosm urea (permeant) would have a total osmolarity of 420 mosm, which is hyperosmotic to ICF (320 mosm). But this solution would be isotonic. Cells placed in such a solution would INITIALLY shrink, but eventually their final volume would be the same as their initial volume, as the permeant solutes and water equilibrated. Overall, the solution does not cause shrinking or swelling of the cell (it is isotonic). In contrast, a solution can be iso-osmotic to the ICF of cells, but be hypotonic if the concentration of impermeant solutes is lower than that of the ICF. For example, a solution with 200 mosm NaCl and 120 mosm urea has a total osmolarity of 320 mosm, which is equal to the osmolarity of ICF. In this case, water will flow into the cell (as it follows the permeant solute urea). That water flow occurs because the high concentration of impermeant solutes in the ICF (320 mosm) can NOT be balanced by the low concentration of impermeant solute (200 mosm NaCl) outside the cell. Overall, the solution causes water to flow into the cell, and the cell will swell (therefore, the solution is hypotonic). After equilibration of the urea, the concentration of the ICF is 380 mosm (i.e. 320 + 60) while that of the solution is 260 mosm (i.e. 200 +60). The solution is hypotonic to the cell. Checkpoint: Choose the correct lettered answer (A, B, C or D) from below to complete each sentence: Imagine you are placing cells in some solution. If the solution is isotonic compared to the intracellular fluid (ICF), it. If the solution is isosmotic compared to the intracellular fluid, it. If the solution is hypotonic compared to the intracellular fluid, it. If the solution is hyposmotic compared to the intracellular fluid, it. A: will cause the cell to swell B: contains the same concentration of total solute particles as the ICF C: contains the same concentration of impermeant solutes as the ICF D: contains a lower concentration of total solute particles than the ICF What is the osmolarity of a 20% NaCl solution? Assuming that these solutes are impermeant, is the 30% solution of glycerol hypertonic, hypotonic, or isotonic to the 20% solution of NaCl?
Page 4.6 Activity 1: Selective Permeability and Osmosis In part A of this Activity, you will observe the movement of water across a semi-permeable membrane by the process of osmosis. By recording and graphing the extent of osmosis in three different situations, you will be able to determine which solution is most concentrated (i.e. has the most impermeant solutes in it) and which is most dilute. In part B of this Activity, you will see how a semi-permeable membrane can allow diffusion of some substances, but block diffusion of others. In part C of this Activity, you will observe the effect of hypertonic, hypotonic, and isotonic solutions on red blood cells. Diffusion describes the movement of particles from regions of high concentration to regions of low concentration (the particles move down their concentration gradient). The diffusion of solutes across a semi-permeable membrane is known as dialysis, while the diffusion of the solvent, water, across a semi-permeable (or selectively permeable) membrane is known as osmosis. All the rules of diffusion apply to osmosis and dialysis. Furthermore, the rate at which osmosis occurs is determined by such factors as (1) the osmolality of the two solutions separated by the membrane, and (2) the permeability of the membrane to the solutes involved. In part A of this Activity, you will observe the interaction of solutions with different osmolalities that are separated by a semi-permeable membrane (dialysis tubing). The direction and rate of osmosis will allow you to determine if the solution in the thistle tube is hypotonic, isotonic, or hypertonic to the solution in the beaker. The dialysis tubing represents a cell membrane, which separates the intracellular and extracellular fluids; however, the tubing is semi-permeable (meaning the inanimate structure of the membrane dictates its permeability) while the cell membrane is selectively permeable. Selective permeability uses active biochemical transport processes in addition to osmosis and diffusion to determine what particles will traverse the membrane. Thus, the dialysis tubing is a good, but not perfect approximation of how a cell membrane behaves. In part B of this Activity, you will observe dialysis: the movement of a solute across the semipermeable membrane of a dialysis bag. One of the solutes will be able to cross the dialysis tubing while the other solute will not be able to cross the tubing. In part C of this Activity, you will observe what happens to red blood cells when placed in solutions with different tonicities. Red blood cells will hemolyze (swell and burst) if they are placed in a hypotonic solution, and they will crenate (shrink) if they are placed in a hypertonic solution. A 0.32 osmolar saline solution is both iso-osmotic and isotonic to red blood cells. Red blood cells act as osmometers; the hemolysis or crenation of the red blood cells will be used to determine whether different solutions are hypotonic, isotonic, or hypertonic to the cells.
Page 4.7 Activity 2: Effect of Molecular Weight and Lipid Solubility on Membrane Permeability In this Activity you will observe the rate at which hemolysis of red blood cells occurs when placed in two different iso-osmotic, hypotonic alcohol solutions. The difference in hemolysis rates will be used to compare how the molecular weights and lipid solubilities of these two alcohols affects their membrane permeability and rate of diffusion. Remember that molecular weight (size) and lipid solubility are two factors that affect the rate of diffusion of a substance across a membrane. As mentioned above, the cell membrane is selectively permeable. The rate of diffusion of particles across the cell membrane can be influenced by several factors, including the size (molecular weight) of the particles and particle lipid solubility. The smaller (lower molecular weight) a particle is, the faster it will diffuse across the cell membrane. As you know, the cell membrane is made mainly of phospholipids and proteins. A particle that is highly soluble in lipids (i.e. oxygen) will be able to cross the cell membrane more easily and faster than one that is not soluble in lipids (i.e. an ion). Some particles that are not soluble in lipids can still cross the membrane because of special proteins that form channels and pumps in the cell membrane. In this Activity, you will determine the effects of molecular weight (size) and lipid solubility on the membrane permeability of two alcohols. Alcohols do not use carriers or pumps because they are lipid soluble and are able to diffuse directly across the plasma membrane. Generally speaking, the larger an alcohol molecule is, the more lipid soluble it is. This experiment will allow you to determine whether size or lipid solubility is more important for movement of these alcohols across red blood cell membranes. At the beginning of this Activity, the red blood cells will be placed in a solution which contains 0.3 mol/l alcohol and 0.02 mol/l NaCl. Checkpoint: Answer the following questions and defend your answers. Is this solution iso-osmotic to the cells? Isotonic? The alcohol concentration inside the cells is zero at the beginning of the Activity; therefore, the alcohol will diffuse into the cell. The rate of diffusion is dependent on how easily the alcohol can cross the membrane. Once alcohol crosses into the cells, water will follow by the process of osmosis to equalize the water concentration inside and outside of the cells. The entrance of water into the cells will result in hemolysis, As the red blood cells lyse, the solution will become clear. You will determine the time needed for hemolysis to take place for the two different alcohols; this time is directly proportional to the diffusion time of each alcohol.
Page 4.8 EXPERIMENTAL PROCEDURES Activity 1. Selective Permeability and Osmosis A. Osmometer Demonstration Students will collect data from a demonstration. There will be three osmosis apparatuses, labeled A, B, and C. The three thistle tubes in the demonstration are filled with 15% sucrose, 30% sucrose, or distilled water. The three beakers are filled with distilled water. You will determine which tube is filled with which solution. The initial position of liquid in each tube will be marked. Subsequent measurements of the height of the liquid above the initial position (in mm) will be recorded on the board. Record measurements every 10 minutes for the next 90 minutes. Each group should take at least one measurement, but be sure that you have recorded all measurements and times on the data sheet that is provided later in this lab. Checkpoint: Record the results, and construct a graph in Excel, with time on the horizontal axis and distance moved for the three solutions on the vertical axis. Remember to label the columns in your Excel spreadsheet. Graph the results. TIME (min) 0 10 20 30 40 50 60 70 80 90 SOLUTION A SOLUTION B SOLUTION C DISTANCE (mm) DISTANCE (mm) DISTANCE (mm) The solutions in the thistle tubes were either 15% sucrose, 30% sucrose, or distilled water. Which is solution A? Which is solution B? Which is solution C? How do you know? Did your results produce a straight line for each solution? Why or why not?
Page 4.9 B. Dialysis across a Semi-Permeable Membrane Students will collect data with a partner. Prepare a small beaker about half full of warm water, and place about 10 drops of iodine in the beaker. Then tie one end of a dialysis bag with several knots, making sure it is very tight. Mix about a teaspoon of starch in a separate container of 50 ml of water, stir thoroughly, and partially fill the dialysis bag with this starch solution. Place the knotted end of the bag in the beaker that contains the iodine, with the other end of the bag hanging over the side. After several minutes, note any changes in the color of the solution in the dialysis bag and the color of the solution in the beaker outside the bag. Checkpoint: Record your observations here. Color of fluid inside bag: Color of fluid outside bag: A color change occurs when starch interacts with iodine. Given the location of the color change, which particles (starch, iodine, or both) must have moved across the dialysis membrane, and why? The atomic weight of iodine is 127 g/mole. Is the molecular weight of the starch molecules higher or lower than this? C. Red Blood Cells as Osmometers Students will collect data with a partner. 1. Make a stock red blood cell (RBC) solution by diluting 20 drops of blood in 10 ml isotonic saline. Make sure the solution is well mixed because the cells tend to settle to the bottom of the tube. Do not throw this stock solution away because you will use it again in Activity 2.
Page 4.10 2. Obtain and label three test tubes (A, B, and C) and place 2 ml of the following solutions into the different tubes: A. distilled water B. 0.9% NaCl C. 2.0% NaCl 3. Add 0.1 ml of the stock RBC solution (from step 1) to test tube A. Immediately place a piece of lined notebook paper with letters on it behind the tube, and based on the clarity of the letters, decide whether the solution is clear or cloudy. Record the results in the checkpoint below. Repeat this for test tubes B and C. 4. After 5 minutes, mix the tubes well. Again place a piece of lined notebook paper with letters on it behind the tube, and decide whether the solution is clear or cloudy. Record the results in the checkpoint below. 5. Transfer a drop of each solution (after the RBCs have been added) to labeled microscope slides, add a cover slip to each slide, and observe the appearance of the cells under the microscope. Record your observations in the checkpoint below. 6. Based on the appearance of the cells in the microscope, determine the tonicity of each solution to RBCs, and record it in the checkpoint below. Checkpoint: Record a summary of your results from the RBCs in the following table. Clarity of solution (time = 0 min) Tube A: Distilled Water Tube B: 0.9% NaCl Tube C: 2.0% NaCl Clarity of solution (time = 5 min) Microscopic appearance (after 5 min) Tonicity of each solution to RBCs Which cells underwent hemolysis? Crenation?
Page 4.11 In the interest of time, lab instructors will perform steps 7 thru 9 on the videoscope. If you have time, you can perform this test yourself. 7. Transfer a drop of solution from test tube B (after the RBCs have been added) to a microscope slide and add a coverslip. Place the slide on the microscope and focus on the cells. Put 1-3 drops of distilled water next to the coverslip. Place a Kimwipe next to the coverslip on the opposite side to act as a wick, drawing the distilled water under the coverslip. Observe the cells and record your results on the data sheet. If nothing happens, add more drops of distilled water and observe again. Record your observations here for later transfer to the data sheets. 8. Transfer a drop of solution from test tube B (after the RBCs have been added) to a microscope slide and add a coverslip. Place the slide on the microscope and focus on the cells. Put 1-3 drops of 10.0% NaCl next to the coverslip. Place a Kimwipe next to the coverslip on the opposite side to act as a wick, drawing the distilled water under the coverslip. Observe the cells and record your results on the data sheet. If nothing happens, add more drops of 10.0% NaCl and observe again. 9. Look under the microscope at red blood cells (from test tube B above after RBCs have been added) that have been placed into distilled water and into saline solutions with the following concentrations: 10% - record your observation: 3.5% - record your observation: 0.9% - record your observation: 0.45% - record your observation: 0.2% - record your observation: distilled water - record your observation: Checkpoint: Based on your observations, which of these is isotonic to intracellular fluid? Explain your reasoning.
Page 4.12 Activity 2. Effect Of Molecular Weight and Lipid Solubility on Membrane Permeability 1. Obtain and label 6 test tubes. The experiment will be done in triplicate using 2 different alcohol solutions: methanol (tubes 1-3) and butanol (tubes 4-6). Place 2 ml of the appropriate alcohol into each tube. 2. Mix your RBC stock solution (from Activity 1 above) and add 0.1 ml of it to one of the alcohol tubes. Observe and record the time needed for hemolysis to occur (the solution becomes clear). This will happen within seconds so you must begin timing immediately after adding the RBCs. Repeat this for each of the remaining 5 tubes. Record your observations on the data sheet. 3. Convert the hemolysis time into a rate ("per second") by dividing 1 by the hemolysis time. 1 divided by hemolysis time = hemolysis rate Checkpoint: Record your results in the following chart and calculate the expected diffusion rate based only on molecular weight with the formula: R (diffusion rate) = 1 divided by the square root of molecular weight OBSERVED CALCULATED ALCOHOL HEMOLYSIS TIME HEMOLYSIS RATE AVERAGE HEMOLYSIS DIFFUSION RATE expected mw; lipid solubility (sec) (1/sec) RATE (R) by Molecular Weight alone METHANOL 32g/mole; 80 g/l BUTANOL 74g/mole; 6000 g/l Which factor (molecular weight or lipid solubility) appears to be the most important in the diffusion of each alcohol across the membrane? Which alcohol diffuses more rapidly? Was either of these alcohol solutions isotonic to the RBCs? Iso-osmotic to RBCs? If so, which one(s)? The lysis of these cells occurs within a few seconds. We could slow this process down to allow for more accurate measurements by reducing the concentration of alcohol to 0.15 Osmol/L (and increasing the concentration of NaCl to 0.17 Osm/L to maintain the same osmolarity). Why would the reduction in alcohol concentration slow down the process?