Diffusion and Osmosis LAB

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Mars Hill College *

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348

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Chemistry

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May 2, 2024

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1 Diffusion and Osmosis (Part II) Pre-lab questions: 1. What type of plant will be used to investigate osmosis in exercise #1? - Elodea Leaf 2. How much dye will be transferred to each agar tube in exercise #2? Convert the volume to milliliters (ml). - -0.1 ML 3. Dialysis tubing selectively regulates the passage of material based on what? - Small ions and molecules, including water and glucose, but not larger molecules such as starch and proteins. - Size 4. What chemical reagent is used to detect the presence of glucose in a solution? - Benedict Reaction - Lugol’s Iodine solution 5. Describe the importance of positive and negative controls? - With negative controls it shows any positive effects of the new treatment that there isn’t a result of placebo effect. The positive control defects any problems in the experiment and benchmark result against another medication. 6. What factors influence the rate of diffusion? - The temperature of the environment, the density and distance traveled. - Concentration of gradient, size, and temperature. 7. Describe the similarities and differences between simple diffusion and osmosis. - They both have passive transport processes which means they do nor require any input of extra energy to occur. Particles moves from an area of higher concentration to one of lower concentration. - Diffusion is movement of substances from higher concentrations to lower concentrations. Osmosis it’s a passive transport H20 diffuses from an area of higher free energy. - The similarity is that they both are moving substances from one concentration to another concentration.

2 Objectives: 1. Investigate the diffusion of molecules through a selectively permeable barrier. 2. Investigate how the size of the diffusing material influences the rate of diffusion. 3. Examine the process of osmosis and how the rate of osmosis is influenced by differences in concentration gradients across a selectively permeable barrier. 4. Design an experiment to test a hypothesis. (SLO) 5. Develop and apply appropriate controls during analysis and interpretation of experimental data. (SLO) 6. Graph, interpret, and form conclusions based on experimental data. (SLO) In this lab, you will complete experiments that focus on the principles of simple diffusion and osmosis. Exercise #1: Osmosis in Plant Cells Osmosis is the movement of H 2 O across a selectively permeable membrane. It’s a type of passive transport; H 2 O diffuses from an area of higher “free” H 2 O concentration (lower solute concentration) to an area of lower “free” H 2 O concentration (higher solute concentration) without the requirement of energy. In this exercise, you will design an experiment to investigate the effect of extracellular solute concentration on osmosis in plant cells. 1. Design an experiment to test the following hypothesis: “Cells of an elodea leaf will lose water when placed in a hypertonic environment.” Materials Available Slides and Coverslips Elodea plant (freshwater plant) H 2 O – dropper bottle 10% NaCl – dropper bottle Compound light microscope Watch this video (Exercise #1) on how to prepare a wet mount. Before observing the results on the next page, use the space below to design an experiment to test the hypothesis provided above. Your experimental protocol must include enough detail that another student can use it to repeat the experiment. Now, observe the results provided and discuss whether they support or reject the hypothesis? Use evidence to explain your answer. - 1. Elodea leaf cells will lose water when are placed in a hypertonic environment. - 2. Materials Available A. Slides and coverslips B. Elodea plant C. H2O – dropper bottle D. 10% NaCI – dropper bottle E. Compound light microscope 3. Control Group: Water Experimental Group: Sodium Chloride. a. A drop of water to control group at noon and wait for five minutes.

3 b. A drop of Nacl to experimental group at noon and Waite 5 minutes. 4. Before the experiment begins, review the elodea leaf under the microscope and then 5 minutes after the water and NaCl have been added on the leaf when the experiment ends for both groups control and experimental. 5. Compare the change on the leaves the hypothesis is supported because in the wet mount with water the cells lose water, and the cells in cells in NaCl clump together. Exercise #2: Molecular weight and rate of diffusion Diffusion is the random movement of substances from areas of higher concentration to areas of lower concentration, until an equilibrium (equal distribution throughout an area) is reached. The rate of diffusion is influenced by several factors, including size (molecular weight), concentration gradient (difference in the concentration between two areas), and temperature. In this exercise, you will examine how the rate of diffusion is influenced by the size of the diffusing molecule. You will compare the diffusion rate of 3 different dyes through a gel-like substance, called agar. The size (molecular weight) of each dye is provided in Table 1. Protocol: Watch this video (Exercise #2) to follow the experimental setup and view results. Use the predictions and data provided in Table 1 to answer the questions below. 1. Before starting the experiment, predict which dye will have the fastest and slowest rate of diffusion based on size (molecular weight) and record in Table 1. ( THIS HAS BEEN DONE FOR YOU)

4 2. Next, obtain and label 3 test tubes filled with agar. Make sure to discard any water on the surface of the agar by shaking it off into the sink. 3. Use a micropipette to transfer 100 µl of each dye directly onto the agar of the corresponding test tube. Be careful not to puncture the agar with the pipette tip. 4. Incubate the tubes for 60 minutes. Record the start and end times below: Start time _______________ End time _______________ 5. At the end of 60 minutes, discard any excess dye that has not diffused into the agar by shaking it off into the sink. This will make it easier to accurately measure the dye that has diffused into the agar. Using a metric ruler, measure the distance of diffusion for each dye in mm. Record the data collected as the rate of diffusion (distance diffused in mm/hr) in Table 1. Table 1. Rate of diffusion Dye Molecular Weight Prediction: fastest/slowest rate of diffusion Rate of diffusion (mm/hr) Observation: fastest/slowest rate of diffusion Potassium Permanganate (PP) 158 g/mole slowest 12 Fastest Methyl Orange (MO) 327 g/mole 7 Congo Red (CR) 696 g/mol fastest 5 Slowest Questions and analysis : 1. Based on analysis of the data, which dye had the fastest rate of diffusion? Which dye had the slowest rate of diffusion? Record the results in Table 1 under observation. - The CR had the slowest, at the end of its seating with the dye it still did not diffuse into any of the liquid. The PP had the fastest because after it had sat with the dye and liquid it was diffusing into liquid. 2. Did the predictions differ from the observed results? If yes, explain why. - Yes, the predictions ended up being the opposite of what was predicted on the PP and CR. - By prediction, the CR was the fastest and te PP was the slowest. The molecular weight of CR was 696 g/mol and the PP molecular weight was 158 g/mol.

5 3. Determine which type of graph will best present the experimental data. Construct the graph using the provided data (using google sheets) and copy and paste the graph into the space below. Analyze the results. Is there a correlation between size (molecular weight) and the rate of diffusion? Support your answer with evidence. - A column chart would be best because it shows the rate of diffusion and the different sizes which is the molecular weight. 4. Analyze the results. Is there a correlation between size (molecular weight) and the rate of diffusion? Support your answer with evidence. - Yes, there is a correlation. The lighter the molecular weight, the faster diffusion. Exercise #3: Diffusion across a selectively permeable membrane All living cells are surrounded by a selectively permeable plasma membrane that regulates the movement of substances into and out of the cell. In this experiment, you will set up a “model cell” to examine the selective permeability of a membrane. Dialysis tubing will be used to represent a simplified plasma membrane that separates the intracellular and extracellular fluids. The dialysis tubing has microscopic pores that can selectively regulate the passage of substances based on size. Some substances will be small enough to pass through the pores, while others will be too big. You will construct the model cell by filling a piece of dialysis tubing with intracellular fluid that contains protein, glucose and starch. The model cell will be surrounded by extracellular fluid, Lugol’s iodine solution. You will analyze experimental results to determine if water, glucose, protein, and/or starch can diffuse across the selectively permeable membrane of the “model cell”.

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