Practical Techniques
A-Level Biology places significant emphasis on practical skills, data analysis, and mathematical competence. This lesson covers key practical techniques related to biological molecules, including serial dilutions, the use of buffers, calorimetry for measuring the energy content of food, and the statistical and mathematical skills needed to analyse experimental data and evaluate errors.
Serial Dilutions
A serial dilution is a stepwise dilution of a substance in solution. Each successive dilution reduces the concentration by a constant factor — most commonly by a factor of 10 (a tenfold serial dilution) or by a factor of 2 (a twofold serial dilution).
Method for a Tenfold Serial Dilution
- Start with a stock solution of known concentration (e.g., 1 mol dm⁻³ glucose).
- Transfer 1 cm³ of the stock solution into a test tube containing 9 cm³ of distilled water. Mix thoroughly (vortex or invert several times).
- This produces a solution with a concentration of 0.1 mol dm⁻³ (10⁻¹ of the original).
- Transfer 1 cm³ of this new solution into a second test tube containing 9 cm³ of distilled water. Mix thoroughly.
- This produces a concentration of 0.01 mol dm⁻³ (10⁻² of the original).
- Repeat the process for as many dilutions as needed.
Method for a Twofold Serial Dilution
- Transfer 5 cm³ of the stock solution into a test tube containing 5 cm³ of distilled water. Mix thoroughly.
- This halves the concentration. Repeat by transferring 5 cm³ from each tube into the next tube containing 5 cm³ of distilled water.
- Concentrations: stock, stock/2, stock/4, stock/8, stock/16, etc.
Why Serial Dilutions Are Important
- They allow you to create a range of concentrations quickly and accurately from a single stock solution.
- Essential for constructing calibration curves (e.g., for quantitative Benedict's test using a colorimeter).
- Used in microbiology to dilute bacterial cultures to countable numbers.
- Used in pharmacology to determine the effective concentration of a drug.
Exam Tip: In calculations, remember that adding 1 cm³ of solution to 9 cm³ of water gives a total volume of 10 cm³ — the dilution factor is 1/10, not 1/9. The dilution factor is calculated as: volume of sample / total volume after dilution.
Use of Buffers
Key Definition: A buffer is a solution that resists changes in pH when small amounts of acid or alkali are added.
Why Buffers Are Used in Biological Experiments
Many biological molecules and reactions are sensitive to pH changes:
- Enzymes have optimal pH values and are denatured by extreme pH.
- Protein structure depends on ionic bonds and hydrogen bonds between R groups, which are affected by pH.
- The ionisation state of amino acids and other biological molecules changes with pH, affecting their behaviour in chromatography and electrophoresis.
In practical work, buffers are used to:
- Maintain a constant pH during enzyme activity experiments, ensuring that any change in reaction rate is due to the independent variable (e.g., temperature or substrate concentration) and not to pH fluctuations.
- Standardise conditions in electrophoresis, ensuring consistent charge on molecules.
- Calibrate pH meters using standard buffer solutions of known pH.
How Buffers Work
A buffer typically contains a weak acid and its conjugate base (or a weak base and its conjugate acid):
- If H⁺ ions are added (pH would decrease), the conjugate base accepts them, minimising the pH change.
- If OH⁻ ions are added (pH would increase), the weak acid donates H⁺ ions to neutralise them, again minimising the pH change.
Common biological buffers include phosphate buffer (useful in the physiological pH range of 6.8–7.4) and Tris buffer (useful around pH 7.5–9.0).
Calorimetry — Measuring Energy in Food
Calorimetry is used to measure the energy content of food samples by burning them and measuring the temperature change in water.
Method
- Weigh a food sample accurately (e.g., 1.00 g of peanut) using a balance.
- Measure a known volume of water (e.g., 20 cm³ = 20 g, since the density of water is approximately 1 g cm⁻³) into a metal calorimeter (or a boiling tube clamped above the food).
- Record the initial temperature of the water using a thermometer.
- Set fire to the food sample using a Bunsen burner or match and immediately hold it under the calorimeter so the burning food heats the water.
- Stir the water gently to ensure even heating.
- When the food has completely burned (or has gone out), record the final temperature of the water.
- Calculate the energy released using the equation:
Energy (J) = mass of water (g) × specific heat capacity of water (4.18 J g⁻¹ °C⁻¹) × temperature change (°C)
Or: Q = m × c × ΔT
- Convert to energy per gram of food: divide the total energy released by the mass of food burned.
- Convert to kJ g⁻¹ by dividing by 1000.
Worked Example
A 0.50 g sample of cashew nut is burned and heats 25 cm³ of water from 21 °C to 52 °C.
Energy released = 25 × 4.18 × (52 − 21) = 25 × 4.18 × 31 = 3239.5 J
Energy per gram = 3239.5 / 0.50 = 6479 J g⁻¹ = 6.48 kJ g⁻¹