PRACTICAL SKILLS 2

• Chromatography


  1. Draw a horizontal pencil line 1 cm above the bottom of the filter paper

  2. Add acetone and grind up the leaf sample to release pigments

  3. Transfer pigment onto the pencil line using a capillary tube

  4. Suspend the paper in the solvent until the liquid runs up near the top

  5. Remove paper, mark where the solvent moved up, and calculate the RF value for each spot
• The purpose of chromatography is to separate different components in a sample
• Factors affecting the rate of migration of different pigments

  • Solubility
  • Mass
  • Affinity to the paper
• RF value


  Formula: Distance moved by pigment/Distance moved by solvent
• The purpose of finding the RF value of a pigment is to compare the experimental RF value to a standard value in a database to identify the pigment
• Hazards and precautions in this practical: Solvents are irritant and flammable. Keep away from naked flames, wear eye protection, and avoid contact with skin. Leaf extract may be a biohazard. Wash hands after use
• Dehydrogenase Activity in Chloroplasts

  1. Function of dehydrogenase: Catalyses the acceptance of electrons by NADP in light-dependent reactions

  2. Purpose of DCPIP: Redox indicator dye, acts as an alternate electron acceptor instead of NADP, turns from blue to colorless when reduced

  3. Reason for chilling plant extract: To lower enzyme activity and prevent breakdown of chloroplasts

  4. Control set up: Fill a cuvette with chloroplast extract and distilled water

  5. Light intensity control: Adjust lamp distance, perform in a dark room with only lamp light

  6. Function of muslin cloth: Filter out debris in ground leaf mixture but allow chloroplasts to pass through

  7. Reason for removing stalks of leaves before grinding: Stalks contain fewer chloroplasts
• Investigating the effect of light intensity after chloroplast extract

  1. Set colorimeter to red filter, zero using cuvette with chloroplast extract and distilled water

  2. Place test tube 30 cm from light source, add DCPIP, measure absorbance

  3. Measure absorbance every 2 minutes for 10 minutes at different distances from lamp

  4. Repeat for distances up to 100 cm
• Respiration in Single-Celled Organisms

  1. Function of methylene blue: Acts as an alternate electron acceptor during ATP synthesis, turns from blue to colorless at endpoint

  2. Procedure to investigate effect of temperature on yeast respiration: Set up water bath at 35 degrees, add yeast and glucose to test tubes, equilibrate, add methylene blue, record time for decolourisation

  3. Use results to calculate rate of respiration at each temperature: Rate=1/time taken for methylene blue to decolourise

  4. Reason for buffering yeast solution: Maintain constant pH for optimal enzyme function

  5. Effect of temperature on respiration: Rate increases with temperature to an optimum, then decreases as enzymes denature
• Investigating Simple Animal Responses

  1. Use of choice chamber: Set up chambers with different environmental conditions, organisms move to preferred quadrant

  2. Factors to control when repeating experiment: Number of animals, environmental conditions, time allowed for choice

  3. Statistical test for analysis: Chi-squared test
• Factors to be controlled when repeating the experiment

  • Number of animals
  • Environmental conditions
  • Time allowed for animals to choose
• Chi squared test is used to analyse the results of this practical
• Chi squared test compares the expected and observed values, and tests if there is a significant difference
• If the calculated value is greater than the critical value in a Chi squared test
• Null hypothesis is rejected
• There is less than 5% probability that the difference is due to chance alone
• There is a statistically significant difference between the expected and observed values
• Animals move faster and change direction more frequently in unfavourable environments to increase their chances of survival
• Hazards and precautions in this practical

  • The live organisms used are a biohazard
  • Wash hands after handling
  • Benedict’s solution is an irritant
  • Wear eye protection
  • Avoid contact with skin
  • Handle the hot water bath with care
• Measuring Concentration of Glucose using a Calibration Curve

  1. Make a serial dilution of glucose ranging from 0 to 10 mmol dm-3

  2. Place 2 cm3 of each of the unknown samples in separate boiling tubes

  3. Add 2 cm3 of Benedict’s solution to all boiling tubes

  4. Place boiling tubes in a water bath at 90 degrees for four minutes

  5. Zero the colorimeter using a cuvette with distilled water and set to red filter

  6. Place known samples into cuvette and measure the absorbance of each using the colorimeter

  7. Make a calibration curve

  8. Measure the absorbance of the unknown samples using the colorimeter and use the calibration curve to determine glucose concentrations
• Serial dilution


  A dilution where successive concentrations increase/decrease in a logarithmic fashion
• Calibration curve


  A graph plotting absorbance against glucose concentration
• A high glucose concentration in urine may suggest diabetes
• Hazards and precautions of measuring glucose concentration

  • Benedict’s solution is an irritant
  • Wear eye protection
  • Avoid contact with skin
  • Handle the hot water bath with care
• To increase the accuracy of the estimate of the unknown glucose solution, increase the number of concentrations for the calibration curve within the range of concentrations that the unknown solution belongs in
• Abiotic factors


  • Light intensity
  • Humidity
  • Temperature
  • Wind speed
  • Water supply
  • Day length
  • Nutrient supply
  • Rainfall
• Biotic factors


  • Competition for resources
  • Predation
  • Disease
• Percentage cover is calculated by using a quadrat with squares, counting how many squares the required species is present in, and calculating the percentage of squares the species is present in
• Effect of Different Variables on Species Distribution

  1. Choose an area to take samples from

  2. Use a random number generator to generate 10 sets of random coordinates

  3. Use two tape measures to create a set of axes off which coordinates can be read

  4. Place the quadrat at each of the coordinates, placing the bottom left corner on the coordinate every time

  5. At each coordinate, a measure of the independent variable should be taken

  6. E.g. record light intensity using a photometer at each location
• The results can be used to determine the relationship between the chosen factor and the percentage cover by using a statistical test e.g. Pearson’s linear correlation, Spearman’s rank
• A random number generator should be used to avoid bias in random sampling
• Formula for the mark-release-recapture method
• Population size = no. in 1st sample x no. in 2nd sample/no. marked in 2nd sample
• Assumptions when using the mark-release-recapture method
• No births, deaths, or migrations
• Random mixing of marked individuals with the population
• Marking does not affect behaviour of individuals or make them more vulnerable to predators