Experiment Osmosis & Dialysis OBJECTIVES • • • Learn how to prepare a solution from a solid or by dilution Study the effects of different solution concentrations on cells Learn the differences between dialysis and osmosis BACKGROUND INFORMATION Osmolarity Osmosis is the spontaneous net movement (diffusion) of solvent molecules through a semi-permeable membrane into a region of higher solute concentration, thus equalizing the concentrations on each side of the membrane. In the body water, carbon dioxide, and oxygen can diffuse across cell membranes through osmosis. The direction of solvent flow during osmosis can be predicted by comparing the osmolarity of the solutions on either side of the membrane. Osmolarity is essentially the same as molarity except it accounts for the concentration of all solute particles in the solution, keeping in mind that some solute molecules (namely ionic compounds and strong acids) are known to dissociate when they dissolve. A 1 mol/L solution of the amino acid glycine, which is a covalently bonded molecule that does not dissociate, would also have an osmolarity of 1 Osmol/L. However, a 1 mol/L solution of potassium nitrate would have an osmolarity of 2 Osmol/L since KNO3 dissociates into two distinct particles when it dissolves: K+ and NO3-. The concept of osmolarity (which for most purposes is synonymous with tonicity) is extremely important biologically because cell membranes are semi-permeable, allowing small water molecules to pass through but blocking the transmission of larger ions such as Na+, K+ or Ca2+. These ions can only be transported in or out of the cell via specific membrane proteins known as ion channels. The fluid inside a cell, known as the cytosol, has a specific concentration of solute particles (mostly potassium ions and amino acids) equivalent to approximately 0.3 Osmol/L. Normal saline, which is a 0.9% or 0.15 M solution of NaCl, is designed to be isotonic with the cytosol. Dialysis Dialysis is the spontaneous net movement (diffusion) of solute molecules through a semi-permeable membrane. Dialysis selectively separates solute molecules. The kidneys utilize dialysis to remove specific toxic or waste molecules from the blood. Channels in the cell membrane allow the kidneys to separate molecules by size, shape, and charge. When a patient’s kidneys fail blood can be filtered using artificial membranes to prevent the build-up of waste and toxins. Cellulous membranes will form with gaps that act as pores for solvent and solute molecules to pass through. An example of the glucose polymer that makes up cellulose shown below.1 The size of the gaps control the size of molecule that can diffuse across the artificial membrane. 1 . The dialysis tubing used in this experiment is composed of cellulose with pores that can allow molecules with a molecular weight up to 12,000 g/mole pass through. LABORATORY TECHNIQUES Dialysis The semi-permeable nature of cell membranes will be simulated in this experiment using dialysis tubing, which is made out of a cellulose film engineered to contain a series of microscopic holes of a specific average diameter. The tubing used in this lab has holes that are large enough for water molecules to pass through but too small for the dissolved ionic solutes used in the experiment to pass through, just like a cell membrane. By enclosing a volume of aqueous solution (you will use potassium chloride since potassium is the most common ion in the cytosol) inside this dialysis tubing, we will be able to observe how different external concentrations affect a cell. In Part 3 of the expieriment you will test types of molecules that can pass through dialysis tubing by conducting a chemical reaction across the dialysis tubing. You will test Iodine, I2, glucose, KI, and starch. Dilution In this experiment, you will be required to prepare solutions of a specific concentration. For some solutions, it is more convenient to weigh a known amount of a solid solute and dissolve in a specific amount of solvent. For others, it is more convenient to start with a highly concentrated “stock” solution and dilute it by adding water to achieve the desired concentration. Since the amount of solute present is not changing during a dilution, only the volume, the following equation will always be true: M1V1 = M2V2 Where M1 and V1 are the molarity and volume respectively of the stock solution, and M2 and V2 are the molarity and volume of the diluted solution. In any situation where you need to calculate how to make a dilution, three of the four will be known and one will be unknown. In this experiment, you will need to calculate what volume of the stock solution (V1) is needed to make a specific volume and concentration by dilution. Therefore you will rearrange the above equation to solve for V1: 𝑉1 = 𝑀2 𝑉2 𝑀1 Student Name: Lab 6: Osmosis & Dialysis: Student Worksheet Please complete each section of this worksheet by watching the linked video for each section, filling out the data table, and answering any questions. You may use this as a template and modify the document so it contains your answers. Then you can convert this file to a pdf and submit it in canvas. All lab videos will be linked below, and they can be found at this youtube playlist: https://www.youtube.com/playlist?list=PLvetE9fWAB84m454EOB5zHIG-N7M-JcMK PART 1: Preparing Solutions Preparing an aqueous solution is a daily task in most labs. It can be accomplished by dissolving a solid solute in water to form a solution, or diluting a stock solution to a specific concentration. This portion of the lab will demonstrate this critical lab skill. In the following video an aqueous NaCl solution is made by dissolving solid NaCl in water. Record the measurements from in the video and then calculate the concentration in units of Molarity. Watch this video about preparing solutions: https://youtu.be/_ccG7asmHgs Table 1A: Data and calculations for the preparation of a NaCl aqueous solution Mass of NaCl (g) measured Calculated number of moles NaCl Volume of solution (mL) measured Calculated volume of solution (L) Calculated concentration of solution (M) 1) Given the concentration of the solution calculated in table 1A, do you expect it to be isotonic, hypotonic, or hypertonic with a simulated cell filled with 0.15 M NaCl? Explain the reasoning for your answer. Watch this video about preparing an aqueous NaCl solution by diluting a more concentrated stock solution: https://youtu.be/WBHCxd6O5E8 Table 1B: Data and calculations for the dilution of a NaCl aqueous solution Concentration of initial NaCl solution (M) This value is calculated in table 1A Volume of initial NaCl solution (mL) measured Calculated volume of initial NaCl solution (L) Volume of H2O (mL) measured Calculated Volume of H2O (L) Calculated total Volume of final Solution (L) Calculated concentration of final solution M) 2) Given the concentration of the solution calculated in table 1B, do you expect it to be isotonic, hypotonic, or hypertonic with a simulated cell filled with 0.15 M NaCl? Explain the reasoning for your answer. PART 2: Osmosis in Simulated Cells Dialysis tubing will be used to simulate a semi-permeable membrane. “Cells” will be created and filled with 0.15 M aqueous NaCl, a solution that is about isotonic with intracellular fluid. The mass of the cells will be observed before and after the cell is placed in a solution. Students will use the difference in mass to identify each solution as isotonic, hypotonic, or hypertonic with the simulated cell. Watch these videos of the simulated cell experiment and fill in the data on the next page with the observed measured values. Video 1: initial measurements: https://youtu.be/eYZ29o1DtXM Video 2: final measurements: https://youtu.be/G8k7JrCo-rk Table 2: Data and results for the change in mass in simulated cells in different concentration solutions. Identify the Identify Change in Solution as concentration Initial Mass (g) Final Mass (g) Mass (g) Isotonic, and solute in (final – initial) Hypotonic, or each beaker Hypertonic Beaker A Beaker B Beaker C Beaker D 3) Observations: What is the concentration of the solution inside the cells? _____________ How much time passed between the initial and final mass measurement? ____________ 4) Calculate and compare the osmolarity (Osmol/L) of beaker B, C, and D. Do the molarities or osmolarities of each solution better support the observed change in mass of each cell? Explain your answer. A picture of handwritten calculations can be inserted into the space below. 5) The solution diluted in part 1 (table 1B) of the lab was used in beaker B. Following up on your response to question 2, did your prediction match the observed experimental results in beaker B? If it did not, please suggest an experimental source of error. PART 3: Osmosis vs Dialysis: What will pass through dialysis tubing? This lab has used dialysis tubing to simulate a semipermeable membrane. This part of the lab will test the types of molecules that can pass through the holes in dialysis tubing. A mixture of potassium iodate, KIO3, and sodium bisulfite, NaHSO3, is combined in a beaker and undergoes a reaction to produce iodine, I2, and the brown color observed. A dialysis tubing cell is filled with a starch solution and glucose and placed inside the beaker. If the starch and iodine come in contact with each other (ie, if one of these molecules crosses the dialysis tubing) a bright blue color will form. Glucose testing strips will be used to test for the presence of glucose to see if it crosses the dialysis tubing barrier. Watch the following video from Bozeman Science that demonstrates the experiment described above. Record your observations and answer the following question. https://youtu.be/hxZFU2LBfgA?t=230 Observations Time Elapsed Inside the dialysis tubing cell Initial appearance of the two solutions (shown in video) Solution in beaker surrounding cell Appearance of the two solutions after 30 seconds (shown in picture to the right) Appearance of the two solutions after 10 minutes (shown in video) Inside the dialysis tubing cell Solution in beaker surrounding cell Inside the dialysis tubing cell Solution in beaker surrounding cell 6) Based on your observations in part 4, which of the following molecules can diffuse across the dialysis tubing? a. Iodine b. Starch c. Glucose 7) Which molecules that can diffuse across dialysis tubing would not be able to cross the semipermeable membrane of a cell wall? Explain your reasoning. 
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