3 - Spring 2022 Chemistry_ Solubility, Diffusion, & Osmosis-1

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Feb 20, 2024

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LAB EXERCISE: Chemistry: Solubility, Diffusion, Osmosis Topic Summary Chemistry is the foundation of physiology. We need to have a good understanding of key chemical concepts to understand how the human body functions, how and why we run lab tests, and what the test results mean. We will learn about three main chemical concepts: determining solute concentration, tonicity, and solubility. The cells in our bodies are affected by the solutions that surround them. Cells have semipermeable membranes that selectively allow certain items to cross the membrane. In general diffusion is when a solute moves passively down its concentration gradient, whereas osmosis is a very specific type of diffusion. Osmosis requires a water gradient and a semipermeable membrane; however, diffusion only requires a gradient. Water will move passively down the water gradient through a semipermeable membrane. The tonicity of a solution describes how a solution affects the final shape of the cell. The solution is said to be hypotonic if the net water movement is into the cell and the cell swells. The solution is said to be isotonic if the net water movement is zero and the cell does not change shape. The solution is said to be hypertonic if the net water movement is out of the cell and the cell will crenate (shrink). Gradients (the difference in concentration between two sites) affect the rate of diffusion and osmosis. The general rule is that the steeper the gradient is, the faster the rate will be. In today’s lab you will determine the tonicity of solutions using dialysis bags filled with sucrose solution. You will also determine which one has a faster osmotic rate. Solubility (property of a molecule that determines if it will dissolve into a solvent) determines various properties of chemicals in the body. A general rule to remember about solubility is that “like dissolves like”; this means that polar molecules mix with polar substances while nonpolar molecules mix with nonpolar substances. The body uses solubility to function properly. For example, the solubility of a hormone can help us determine how it is carried around the body, where it will likely bind to a cell, how the cell will likely respond to the signal, and how long the hormone will remain in the body. Insoluble molecules will not dissolve in the solvent. Soluble molecules will dissolve in the solvent. We will also determine if molecules are hydrophobic (water fearing), hydrophilic (water loving), lipophobic (lipid fearing), or lipophilic (lipid loving). Lastly, we will determine how amphipathic molecules (molecules that contain both a polar and nonpolar region) allow us to process and handle hydrophobic materials in our body by creating micelles. Our digestive system uses bile to create micelles and help us mechanically break down fat; in today’s lab we will use dish soap. 1

Goals Physiology students will learn about diffusion, osmosis, tonicity, and solubility. Physiology students will work safely and learn to use a graduated cylinder, serological pipette, dialysis tubing, and scale. Physiology students will properly handle all waste materials, equipment, and chemicals. ● Understand the concepts of gradients, diffusion, osmosis, and tonicity (hypertonic, isotonic, hypotonic solution). ● Understand the concepts of solubility and amphipathic molecules. ● Learn how to use a graduated cylinder properly and will be able to demonstrate this skill to their instructor. ● Learn how to use a serological pipette properly and will be able to demonstrate this skill to their instructor. ● Learn how to use a dialysis tubing properly and will be able to demonstrate this skill to their instructor. ● Learn how to use a balance (scale) properly and will be able to demonstrate this skill to their instructor. ● Learn to use MS Excel to create a line graph. ● Learn to work safely in a lab and properly handle all waste materials, equipment, and chemicals. 2

Lab Exercises: Orientation to Lab Equipment: This may be your first time using a graduated cylinder, serological pipette, dialysis tubing, and/or balance (scale). Your instructor will show you how to use the equipment correctly. Ask for help if needed at any point during this lab. How to Use a Graduated Cylinder: 1. Use a graduated cylinder to accurately measure and transfer large amounts of liquid. Before attempting to use the graduated cylinder with liquid, take some time to get to know the instrument and how to use it. 2. Your instructor will show the class how to use the graduated cylinder properly. An overview is found below. a. Look at the graduated cylinder and find the scale. Identify the major scale division and minor scale division. Identify the total amount of volume this graduated cylinder can hold. b. Determine the volume you want to measure and find it on the scale. c. Place the graduated cylinder on a stable/level table or lab bench. d. Add liquid to the graduated cylinder until the meniscus reaches the volume you want to measure. e. Pour that volume into your container. 3

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How to Use a Serological Pipette with Bulb: 1. Use a serological pipette to accurately measure and transfer large amounts of liquid. Before attempting to use the serological pipette with liquid, take some time to get to know the instrument and how to use it. 2. Look at the bulb. Know the different parts and their function. 3. Place the bulb on the serological pipet. 4. Press the Air Valve (A) to open it and press the bulb. Release the Air Valve (A). The bulb should remain deflated. 5. Place the tip of the serological pipette into the liquid. 6. Press the Suction Valve (S) to open it and draw fluid into the serological pipette. 7. Release the Suction Valve (S) at or above the desired volume. Use the meniscus to measure the volume. 8. If you are above the desired volume, press the Empty Valve (E) to slowly release liquid into a waste container until you are at the desired volume. Release the Empty Valve (E) when you are at the desired volume. 9. Place the serological pipette tip into the new container and press the Empty Valve (E) to release all of the fluid into the new container. 4

How to Use Dialysis Tubing Cells: 1. Our lab technician created artificial cells by using dialysis tubing to contain a volume of sucrose solution. Dialysis tubing has microscopic pores that allow specific molecules to cross. We placed clips at each end to hold the sucrose solution inside the dialysis tubing. Before attempting to use the artificial cells, take some time to get to know this piece of equipment and how to use it. 2. Handle dialysis tubing with care because if it is stretched, then the microscopic pores will get larger. Be careful to not stretch or squeeze your artificial cell. 3. You will blot your artificial cells with a paper towel before you weigh them with a scale. Dialysis tubing is porous and keeping the artificial cells on a paper towel will pull the water out and decrease the weight. 4. Dialysis tubing will allow permeable solutes to diffuse through the membrane until dynamic equilibrium is reached. Water will move via osmosis until dynamic equilibrium is reached. 5. After your experiment you can remove the clips, dispose of the solution, and dispose of the dialysis tubing in the trash. Rinse the clips and place them back at the station. 5

HINT: Begin the Diffusion, Osmosis, & Tonicity and Solubility Lab Activities at the same time. Diffusion, Osmosis, & Tonicity Lab Activity (80 minutes) Equipment: ● Balance/scale ● 500 mL beakers ● Paper towel ● Tap water ● Waxpen ● 10% Sucrose Artificial Cell: Dialysis bag filled with 10% sucrose solution and clipped with dialysis tube clips ● 30% Sucrose Artificial Cell: Dialysis bag filled with 30% sucrose solution and clipped with dialysis tube clips Protocol: 1. Go to the dialysis tubing station. Obtain the 10% Sucrose Artificial Cell and the 30% Sucrose Artificial Cell. 2. Blot the 10% Sucrose Artificial Cell with a paper towel, then use the balance/scale to determine the initial weight of the 10% Sucrose Artificial Cell . 3. Blot the 30% Sucrose Artificial Cell with a paper towel, then use the balance/scale to determine the initial weight of the 30% Sucrose Artificial Cell . 4. Using a waxpen label a 500 mL beaker “10” which stands for 10% Sucrose Artificial Cell. Then add 300 mL of tap water to each beaker. 5. Using a waxpen label a 500 mL beaker “30” which stands for 30% Sucrose Artificial Cell. Then add 300 mL of tap water to each beaker. 6. Do the following two steps at the same time: a. Add the 10% Sucrose Artificial Cell into the beaker labeled “10”. Allow the 10% Sucrose Artificial Cell to sit for 15 min. When the time is up, remove the 30% Sucrose Artificial Cell and blot dry using a paper towel. b. Add the 30% Sucrose Artificial Cell into the beaker labeled “30”. Allow the 30% Sucrose Artificial Cell to sit for 15 min. When the time is up, remove the 30% Sucrose Artificial Cell and blot dry using a paper towel. 6

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7. Use the balance/scale to determine the weight in grams of each Sucrose Artificial Cell. 8. Repeat steps 6-7 three more times (total of 60 min). a. Enter the weight in grams at each time interval. i. Notice that each dialysis bag begins at a different weight. Time (min) Weight of 10% Sucrose Solution Artificial Cell (g) Weight of 30% Sucrose Solution Artificial Cell (g) 0 15 30 45 60 9. Normalize the data you collected to allow for comparisons between the two Sucrose Solution Artificial Cells. a. Since each dialysis bag begins at a different weight in grams, we cannot compare the data in grams because the amount of change may be affected by the initial size of the Sucrose Artificial Cell. We need to normalize the data in order to compare the bags to each other. Normalizing data means that we transform our data so that they are both on the same scale of measurement. In this experiment we will normalize the data so that we transform the Sucrose Artificial Cell weight from grams into percentage. When we do this, we will accurately be able to compare the percentage of weight gained or lost between the 10% Sucrose Artificial Cell and the 30% Sucrose Artificial Cell. b. Calculate the Normalized Weight using the formula below: Normalized Weight (%) = (weight in grams at time x / weight in grams at time 0 min) * 100% c. Normalize the weight for the 10% Sucrose Solution Artificial Cell. i. The normalized weight at 0 minutes is ______________________. 1. Show your work below: 7

ii. The normalized weight at 15 minutes is _____________________. 1. Show your work below: iii. The normalized weight at 30 minutes is _____________________. 1. Show your work below: iv. The normalized weight at 45 minutes is _____________________. 1. Show your work below: v. The normalized weight at 60 minutes is _____________________. 1. Show your work below: d. Normalize the weight for the 30% Sucrose Solution Artificial Cell. i. The normalized weight at 0 minutes is ______________________. 1. Show your work below: ii. The normalized weight at 15 minutes is _____________________. 1. Show your work below: iii. The normalized weight at 30 minutes is _____________________. 1. Show your work below: 8

iv. The normalized weight at 45 minutes is _____________________. 1. Show your work below: v. The normalized weight at 60 minutes is _____________________. 1. Show your work below: e. Enter the Normalized Weight in the table below. Time (min) Weight of 10% Sucrose Solution Artificial Cell (%) Weight of 30% Sucrose Solution Artificial Cell (%) 0 15 30 45 60 10.Use MS Excel to create a line graph showing Time (min) on the x axis and Normalized Weight (%) on the y axis. Show the 10% Sucrose Artificial Cell and the 30% Artificial Cell data on the same line graph. Connect your data points using straight lines. Add a title (for example: The Effect of a Sucrose Gradient on the Rate of Osmosis) and key. You will turn in your final line graph. 9

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11. Use your line graph to answer the following questions: a. There was a net _____________________ (influx / efflux) of water when the 10% Sucrose Solution Artificial Cell was placed in a beaker of water. b. There was a net _____________________ (influx / efflux) of water when the 30% Sucrose Solution Artificial Cell was placed in a beaker of water. c. The 10% Sucrose Solution Artificial Cell _______________ (gained / lost) weight as a result of being placed in a beaker of water. d. The 30% Sucrose Solution Artificial Cell _______________ (gained / lost) weight as a result of being placed in a beaker of water. e. The 10% Sucrose Solution Artificial Cell was placed in a _________________________ (hypotonic / isotonic / hypertonic) solution. f. The 30% Sucrose Solution Artificial Cell was placed in a _________________________ (hypotonic / isotonic / hypertonic) solution. g. The ________ (10% / 30%) Sucrose Solution Artificial Cell had a larger absolute change in Normalized Weight after 60 minutes in a beaker of water. h. The ________ (10% / 30%) Sucrose Solution Artificial Cell had a smaller absolute change in Normalized Weight after 60 minutes in a beaker of water. i. The larger gradient was found between the beaker and the _____________ (10% / 30%) Sucrose Solution Artificial Cell (hint: the gradient is the difference between the two locations). j. The smaller gradient was found between the beaker and the _____________ (10% / 30%) Sucrose Solution Artificial Cell (hint: the gradient is the difference between the two locations). k. The faster osmotic rate was found in the beaker that held the _____________ (10% / 30%) Sucrose Solution Artificial Cell (hint: the steeper the gradient the faster the rate). l. The slower osmotic rate was found in the beaker that held the _____________ (10% / 30%) Sucrose Solution Artificial Cell (hint: the steeper the gradient the faster the rate). 10

12.Let’s practice. Look at this graph and answer the following questions: a. Line(s) showing an artificial cell gaining water (net influx): _____________ b. Line(s) showing an artificial cell losing water (net efflux): ______________ c. Line(s) showing an artificial cell at dynamic equilibrium (net zero): ______ d. Line showing an osmotic rate of 0 %/min: __________________________ e. Line showing the fastest osmotic rate (%/min) (hint: steepest slope): ____ f. Line showing the slowest osmotic rate (%/min) (hint: gentlest slope): ____ g. The portion of the line labeled E represents the time that the artificial cell reached dynamic ___________________________________________. 13.Clean-up: a. Take your beakers and Sucrose Solution Artificial Cells to the sink. i. Beakers: 1. Empty the beakers in the sink. Wash, rinse, and dry the beakers. Return the clean beakers to the station. ii. Sucrose Solution Artificial Cells: 1. Open the clips in the sink. Allow the sucrose solution to go down the drain. Wash, rinse, and dry the clips. Return the clean clips to the station. Throw the dialysis tubing in the regular trash. iii. Rinse the sink. Wipe down excess water around the sink. b. Clean up any sucrose residue from the balance/scale and lab bench. i. Use a paper towel soaked with warm water to clean any sucrose residue on the balance/scale/lab bench. Sucrose residue typically looks shiny and is sticky or tacky. 11

Solubility Lab Activity (30 minutes) Equipment ● graduated cylinder ● serological pipet & bulb ● test tube ● parafilm ● fume hood ● DI water (H 2 O) ● hexane (C 6 H 14 ) (no not touch) ● potassium permanganate (KMnO 4 ; dissociates into K + & MnO 4 - ) ● vegetable oil (unsaturated fatty acids) ● detergent (soap; amphipathic molecule) Protocol: 1. Obtain one test tube. a. When thinking about solubility, remember that “like dissolves like”. i. Polar molecules dissolve ________________________ (polar / nonpolar) molecules. ii. Nonpolar molecules dissolve _________________________ (polar / nonpolar) molecules. 2. Using a graduated cylinder, add 2 mL of water to your test tube. a. What color is water? __________________________________________ b. Water is a _________________________ (nonpolar / polar) molecule. 3. Meet your instructor at the fume hood. Using a serological pipet, your instructor will add 1 mL of hexane. They will then mix vigorously. a. Look at your test tube. Answer the following questions: i. What color is hexane? ___________________________________ ii. Hexane _________________________ (does / does not) mix with water. iii. Hexane _________________________ (floats / sinks) when placed with water. iv. This result shows that hexane is _________________________ (hydrophobic / hydrophilic). v. This result shows that hexane is a _________________________ (polar / nonpolar) molecule. b. Your instructor will then add 1 more mL of hexane. You now have 2 mL of water and 2 mL of hexane in your test tube. Your instructor will cover the test tube with parafilm and mix vigorously. 12

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4. Add 1 small crystal of potassium permanganate (KMnO 4 ). Cover with parafilm and mix vigorously. a. Look at your test tube. Answer the following questions: i. Potassium permanganate (KMnO 4 ) dissociates into _________________________ (ions / lipids) when placed in water. ii. Potassium permanganate (KMnO 4 ) mixes with _________________________ (water / hexane). We know this because when they mixed the soluble layer turned purple. iii. This result shows that potassium permanganate (KMnO 4 ) is _________________________ (hydrophobic / hydrophilic). iv. This result shows that potassium permanganate (KMnO 4 ) is a _________________________ (polar / nonpolar) molecule. 5. Add 1 mL of vegetable oil. Cover with parafilm and mix vigorously. a. Look at your test tube. Answer the following questions: i. Vegetable oil is a type of _________________________ (carbohydrate / lipid / protein / nucleic acid). ii. Vegetable oil mixes with _________________________ (water / hexane). We know this because the soluble layer increased from 2 mL to 3 mL. iii. This result shows that vegetable oil is _________________________ (hydrophobic / hydrophilic). iv. This result shows that vegetable oil is a _________________________ (polar / nonpolar) molecule. 6. Add 1 mL of detergent (soapy water). Cover with parafilm and mix vigorously (alternatively transfer the solution back and forth between two test tubes to mix well). a. Look at your test tube. Answer the following questions: i. After adding detergent and mixing vigorously, our test tube now shows ______________ (1 / 3 / 5) layers. ii. Detergent mixes with _________________________ (water only/ hexane only/ water & hexane). iii. This result shows that Detergent is a(n) _________________________ (polar / nonpolar / amphipathic) molecule. iv. Explain how the following amphipathic molecules help your body: 1. Phospholipid in the cell membrane: 13

2. Bile in the GI: 3. Surfactant in the lungs: v. Explain how micelles help mix polar and nonpolar molecules. The figure below may be useful. Practice using the terms that you have learned (i.e. solubility, nonpolar, polar, amphipathic, micelles). 14

7. Clean-up: a. Dispose of all liquids in the Chemical Waste Container (found near a sink). Make sure to close the container when you are done. b. Place the empty test tubes in the Test Tube Collection Bucket. c. Place used parafilm in the regular trash. d. Used gloves in the regular trash. 15

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3 - Spring 2022 Chemistry_ Solubility, Diffusion, & Osmosis-1

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