# Practical: Investigating the Rate of Diffusion ## Overview - Diffusion rates can be investigated experimentally using different apparatus - Two main approaches can be used: - **Visking tubing** - to demonstrate selective permeability and molecular size effects - **Agar cubes** - to investigate surface area to volume ratio effects on diffusion - Both methods allow investigation of how various factors affect the rate at which substances diffuse --- ## Practical 1: Using Visking Tubing ### What is Visking tubing? - Visking tubing (also called dialysis tubing) is a **non-living partially permeable membrane** made from cellulose - The pores in this membrane are small enough to: - **Prevent passage** of large molecules (e.g. starch, sucrose) - **Allow passage** of smaller molecules (e.g. glucose) by diffusion - This selective permeability mimics biological membranes

Visking tubing is a partially permeable membrane that allows small molecules through but blocks larger ones

### Apparatus Required - Visking tubing - Starch solution - Glucose solution - Boiling tubes - Distilled water - Stopwatch - Test reagents (iodine for starch, Benedict's reagent for glucose) - Water bath (optional) - Measuring cylinder ### Method 1. **Prepare the tubing** - Cut a section of Visking tubing (approximately 15-20 cm long) - Tie a secure knot at one end to seal it - Fill the tubing with a mixture of starch solution and glucose solution - Tie off the other end, ensuring no air bubbles are trapped inside 2. **Set up the experiment** - Rinse the outside of the filled tubing under running water - This removes any starch or glucose that may have contaminated the outside during filling - Place the tubing into a boiling tube containing a known volume of distilled water (e.g. 20 cm³) 3. **Allow diffusion to occur** - Leave the tubing suspended in the water for a set time period - A minimum of 30 minutes is recommended to allow sufficient diffusion - The tube can be placed in a water bath at a specific temperature for more controlled conditions 4. **Test for diffusion** - At regular time intervals (e.g. every 10 minutes), take small samples of the water outside the tubing - Test each sample for the presence of: - **Starch** using iodine solution (turns blue-black if starch present) - **Glucose** using Benedict's reagent (turns orange-red when heated if glucose present) ### Expected Results | Molecule | Will it diffuse out? | Reason | |----------|---------------------|---------| | Glucose | ✓ Yes | Small molecule - can pass through pores in Visking tubing | | Starch | ✗ No | Large molecule - too big to pass through pores | - **Glucose**: Benedict's test on the water outside the tubing will show a positive result (colour change from blue to orange-red when heated), indicating glucose has diffused out - **Starch**: Iodine test on the water outside will remain brown/orange (no blue-black colour), indicating starch has NOT diffused out ### Analysis - This demonstrates that **molecular size** affects diffusion through partially permeable membranes - Only molecules small enough to fit through the membrane pores can diffuse across - This models how cell membranes selectively control which substances can pass through ### Investigating concentration gradient effects

You can make this investigation quantitative by using Benedict's reagent with colour standards or a colorimeter to measure glucose concentration at different time points

To investigate how concentration gradient affects diffusion rate: 1. Set up multiple boiling tubes with Visking tubing containing **different concentrations** of glucose (e.g. 0.1 M, 0.2 M, 0.5 M, 1.0 M) 2. Suspend each in the same volume of distilled water for the same time period 3. Use the **semi-quantitative Benedict's test** to estimate glucose concentration: - Perform Benedict's test on samples from each tube - Compare the colour intensity against a set of colour standards (produced using known glucose concentrations) - **OR** use a colorimeter for more quantitative results 4. Plot a graph showing: - **X-axis**: Initial glucose concentration inside tubing - **Y-axis**: Rate of diffusion (estimated from final glucose concentration outside tubing) **Expected pattern**: The greater the concentration gradient, the faster the rate of diffusion. The graph below shows the typical relationship you would expect to observe:

--- ## Practical 2: Using Agar Cubes ### Purpose - This practical investigates how **surface area to volume ratio** affects the rate of diffusion - It uses the diffusion of acid through agar blocks to model how substances diffuse into cells ### Why agar? - Agar can be made containing pH indicators (like Universal Indicator or phenolphthalein) - When acid diffuses into the agar, it causes a visible colour change - This allows us to **see** and **measure** how far diffusion has occurred ### Apparatus Required - Agar jelly containing pH indicator - Knife or scalpel - Ruler - White tile - Dilute hydrochloric acid (e.g. 0.1 M) - Boiling tubes or beakers - Stopwatch - Paper towels ### Method #### Preparing the agar - Prepare coloured agar by mixing agar powder with: - Very dilute sodium hydroxide solution (to make it alkaline) - Universal Indicator (produces purple/pink agar in alkaline conditions) - Allow the agar to set in a shallow tray - **Alternative**: Use phenolphthalein instead of Universal Indicator (pink in alkaline, colourless in acid)

Dilute hydrochloric acid is corrosive - handle with care and wear eye protection

#### Cutting the cubes 1. Cut the coloured agar into cubes of **different sizes** with equal-length sides 2. Suggested dimensions: - 0.5 cm × 0.5 cm × 0.5 cm - 1.0 cm × 1.0 cm × 1.0 cm - 1.5 cm × 1.5 cm × 1.5 cm - 2.0 cm × 2.0 cm × 2.0 cm 3. Measure each cube carefully using a ruler to ensure accuracy 4. Calculate the **surface area to volume ratio** for each cube size | Cube side length (cm) | Surface area (cm²) | Volume (cm³) | SA:V ratio | |----------------------|-------------------|--------------|------------| | 0.5 | 1.5 | 0.125 | 12:1 | | 1.0 | 6.0 | 1.0 | 6:1 | | 1.5 | 13.5 | 3.375 | 4:1 | | 2.0 | 24.0 | 8.0 | 3:1 | The chart below illustrates how surface area to volume ratio decreases as cube size increases:

#### Running the experiment 1. Pour the same volume of dilute hydrochloric acid into several boiling tubes or beakers 2. The acid must be at a **higher molarity** than the sodium hydroxide in the agar 3. Add one agar cube to each tube 4. Start the stopwatch 5. **Option A - Fixed time method**: - Leave all cubes for the same fixed time (e.g. 10 minutes) - Remove cubes and blot dry with paper towels - Cut each cube in half - Measure the distance the acid has diffused into the cube (shown by colour change) - The coloured area remains alkaline (hasn't been reached by acid) - The colourless/different coloured area is where acid has diffused to 6. **Option B - Time to completion method**: - Record the time taken for the indicator colour to completely change throughout each cube - This indicates acid has diffused throughout the entire volume ### Expected Results **Pattern observed**: - The **smallest cube** (highest SA:V ratio) will: - Show the greatest depth of acid penetration in a fixed time - OR have acid diffuse completely through it in the shortest time - The **largest cube** (lowest SA:V ratio) will: - Show the least depth of acid penetration in a fixed time - OR take the longest time for acid to diffuse completely through **Why does this happen?** - As cube size increases: - **Volume** increases faster than **surface area** (volume is cubed, surface area is squared) - This gives a **smaller surface area to volume ratio** - There is proportionally **less surface area** for molecules to diffuse across relative to the volume - Therefore diffusion takes longer and is less effective

The smaller the object, the larger its surface area to volume ratio, and the faster diffusion can occur throughout its entire volume

### Analysis 1. Calculate the **rate of diffusion** for each cube: - If using Option A: Rate = distance diffused ÷ time - If using Option B: Rate = 1 ÷ time taken for complete colour change 2. Plot a graph: - **X-axis**: Surface area to volume ratio - **Y-axis**: Rate of diffusion 3. Draw conclusions: - There is a **positive correlation** between SA:V ratio and diffusion rate - The greater the surface area to volume ratio, the faster the rate of diffusion - This explains why: - Single cells are typically very small - Large organisms have specialized exchange surfaces (like lungs, gills) - Substances can't simply diffuse into large multicellular organisms The chart below shows the expected relationship between SA:V ratio and diffusion rate:

### Biological significance This practical demonstrates why: - **Cells are microscopic** - their small size gives them a large SA:V ratio, allowing efficient exchange of materials - **Large organisms need specialized systems** - simple diffusion across the outer surface is too slow - **Exchange surfaces have adaptations** - features like folding increase surface area (e.g. villi in small intestine, alveoli in lungs) --- ## Common Limitations & Improvements ### For Visking Tubing Practical | Limitation | Improvement | |------------|-------------| | Contamination of tubing exterior with test substances during filling | Rinse thoroughly under running water before placing in test tube | | Different tubing pieces may vary slightly in permeability | Use tubing from the same batch; conduct multiple repeats | | Temperature fluctuations affect diffusion rate | Conduct in a water bath at constant temperature | | Difficult to get quantitative results with simple colour tests | Use colorimeter readings rather than visual comparison | ### For Agar Cube Practical | Limitation | Improvement | |------------|-------------| | Cubes may not be perfectly cut to exact dimensions | Use a sharp scalpel and ruler; measure carefully; calculate actual SA:V for each cube | | Different areas of agar may have different indicator concentrations | Mix indicator thoroughly before agar sets; conduct multiple repeats with cubes from different areas | | Acid concentration may change during experiment as it neutralizes the alkaline agar | Use large volumes of acid relative to agar size; replace acid between repeats | | Difficult to measure exact depth of colour change | Cut cube precisely in half; measure using a ruler to nearest 0.5 mm |

Always conduct risk assessments before practical work - acids are corrosive and scalpels are sharp

--- ## Key Conclusions From these investigations, we can conclude: 1. **Molecular size affects diffusion** through partially permeable membranes - Only molecules small enough to pass through membrane pores can diffuse across - This is why cells can selectively control what enters and leaves 2. **Concentration gradient affects rate** - The steeper the concentration gradient, the faster diffusion occurs - This is because there is a greater difference in concentration between the two areas 3. **Surface area to volume ratio is crucial** - A larger SA:V ratio leads to faster, more efficient diffusion - This explains why cells remain small and why large organisms need specialized exchange surfaces 4. **Diffusion is passive** - No energy input is required - Movement continues until equilibrium is reached (equal concentration on both sides) These principles apply to gas exchange, nutrient absorption, and waste removal in living organisms.