Apparatus and experiments (A)

Describe how pigments from a leaf of a plan can be isolated with paper chromatography (6 marks)

1. Crush leaves with solvent to extract pigments

2. Draw a pencil line on filter / chromatography paper, 1 cm above bottom

3. Add a drop of extract to line (point of origin)

4. Stand paper in boiling tube of (organic) solvent below point of origin

5. Add lid and leave to run (solvent moves up, carrying dissolved pigments)

6. Remove before solvent reaches top and mark solvent front with pencil.

Explain why the origin should be drawn in pencil rather than ink (in paper chromatography) (2 marks)

Ink is soluble in solvent. So ink would mix with pigments/line would move

Explain why the point of origin should be above the level of the solvent (in paper chromatography) (2 marks)

Pigments are soluble in solvent. So would run off paper/spots dissolve into solvent.

Explain why a pigment may not move up the chromatography paper in one solvent (in paper chromatography)

May be soluble in one solvent but insoluble in another.

Describe how pigments can be identified (2 marks)

Rf value= the distance moved by spot/solvent front. Compare Rf value to published value.

Explain why the solvent front should be marked quickly once chromatography paper is removed. (1 mark)

Once solvent evaporates, solvent front not visible.

Explain why the centre of each pigment spot should be measured (in paper chromatography). (1 mark)

Standardises readings as pigment is spread out, so allows comparisons to be made.

Explain why the obtained Rf values were similar but not identical, to the published values (1 mark)

Different solvent/paper which may affect Rf value.

Describe the role of enzyme dehydrogenase in photosynthesis (1 mark)

Catalyses the reduction of NADP in the light dependent reaction.

Describe how rate of dehydrogenase activity inn extracts of chloroplasts can be measured (4 marks)

1. Extract chloroplasts from a leaf sample using the method in ‘2.1.3 Methods of studying cells’

2. Set up test tubes as follows:

   1. Control 1 - set volume of DCPIP (redox indicator dye, electron acceptor), water and chloroplasts in isolation medium, covered in foil to block light

   2. Control 2 - set volume of DCPIP, water and isolation medium without chloroplasts

   3. Standard - set volume of water and chloroplasts in isolation medium, without DCPIP

   4. Experiment - set volume of DCPIP, water and chloroplasts in isolation medium

3. Shine light on test tubes and time how long to it takes for DCPIP to turn from blue (oxidised) to colourless (reduced) in tube D (tube A and B should show no change). Compare to a colour standard (tube C) to identify end point

4. Rate of dehydrogenase activity = 1 / time taken

Give examples of variables that could be controlled (when investigating the role of enzyme dehydrogenase in photosynthesis) (3 marks)

Source of chloroplasts. Volume of chloroplast suspension. Volume/concentration of DCPIP.

Explain the purpose of control 1 (with a set volume of DCPIP, water and chloroplasts, covered in foil to block light) (when investigating the role of enzyme dehydrogenase in photosynthesis) (2 marks)

Shows light is required for DCPIP to decolourise. Shows that chloroplasts alone do not cause DCPIP to decolourise.

Explain why DCPIP in control 1 (with a set volume of DCPIP, water and chloroplasts, covered in foil to block light) stays blue (when investigating the role of enzyme dehydrogenase in photosynthesis) (2 marks)

No light so no photoionisation of chlorophyll. So no electrons released to reduce DCPIP.

Explain the purpose of control 2 (with set volume of DCPIP, water without chloroplasts)(when investigating the role of enzyme dehydrogenase in photosynthesis) (2 marks)

Shows chloroplasts are requires for DCPIP to decolourise. Showing that light alone does not cause DCPIP to decolourise.

Explain why DCPIP changes from blue to colourless (2 marks)

DCPIP gets reduced by electrons. From the photoionisation of chlorophyll.

Suggest a limitation with the method and how the experiment could be modified to overcome this. (when investigating the role of enzyme dehydrogenase in photosynthesis) (2 marks)

End point (colour change) is subjective. Use a colorimeter instead. Measure the light absorbance of sample at set time intervals. Zero colorimeter using the colour standard.

Define gross primary production (1 mark)

Chemical energy store in plant biomass, in a given area or volume, in a given time.

Define net primary production (1 mark)

Chemical energy store in plant biomass after respiratory losses to environment taken into account.

State the formula for efficiency of energy transfer

Energy or biomass available after transfer / energy or biomass available before transfer

Formula for net primary production, and what does each component stand for?

NPP= GPP (gross primary production) - R (respiratory losses to the environment

Formula for net production of consumers, and what does each component stand for?

N= I - (F + R). Where I represents the chemical energy store in ingested food, F represents the chemical energy lost to the environment in faeces and urine and R represents the respiratory losses to the environment.

Describe how chemical energy stored in dry biomass can be estimated (3 marks)

Using calorimetry:

1. Known mass of dry biomass is fully combusted (burnt)

2. Heat energy released heats a known volume of water

3. Increase in temperature of water is used to calculate chemical energy of biomass

Explain how features of a calorimeter enable valid measurement of heat energy released (3 marks)

● Stirrer → evenly distributes heat energy (in water)

● Air / insulation → reduces heat loss & gain to & from surroundings

● Water → has a high specific heat capacity

How can biomass be measured?

Mass of carbon or dry mass of tissue per given area.

Describe how dry mass of tissue can be measured (3 marks)

Sample dried in oven at 100 degrees celcius (to avoid combustion). Sample is weighed and reheated at regular intervals. Until mass remains constant (all water has evaporated).

Explain why dry mass is more representative than fresh (wet) mass (1 mark)

Water volume in wet samples will vary but will not effect dry mass.

Describe how a respirometer can be used to measure the rate of aerobic respiration (by measuring oxygen uptake) (5 marks)

1. Add set mass of single-celled organism eg. yeast to set volume / conc. of substrate eg. glucose

2. Add a buffer to keep pH constant

3. Add a set volume / conc. of a chemical that absorbs CO2 eg. sodium hydroxide

4. Place in water bath at a set temperature and allow to equilibrate

5. Measure distance moved by coloured liquid in a set time

Explain why the liquid moves (when measuring rate of respiration) (4 marks)

Organisms aerobically respire to take in oxygen. Carbon dioxide given out but is absorbed by sodium hydroxide solution. So volume of gas and pressure in container decrease. Fluid in tube moves down the pressure gradient towards organism.

Explain why the respirometer apparatus is left open for 10 minutes. (3 marks)(when measuring rate of respiration)

Allow apparatus to equilibriate. Allow for overall pressure expansion throughout. Allow respiration of organism to stabilise.

Explain why the apparatus must be airtight (when measuring rate of respiration) (2 marks)

Prevent air entering or leaving, that would change volume and pressure, affecting movement of liquid.

Describe a more accurate way of measuring volume of gas (when measuring rate of respiration)(1 mark)

Use a gas syringe.

Suggest a suitable control experiment and explain why it is necessary.(when measuring rate of respiration)(2 marks)

No/dead/inert organisms and all other conditions/equipment kept the same. To show that respiring organisms are causing liquid to move.

Describe how a respirometer can be used to measure the rate of anaerobic respiration (by measuring carbon dioxide release) (2 marks)

Repeat experiment that measures aerobic respiration, but remove the chemical (e.g., sodium hydroxide) that absorbs carbon dioxide. Make conditions anaerobic, for example: layer of oil, add chemical that absorbs oxygen, leave for an hour to allow oxygen to be respired and used up.

When measuring anaerobic respiration using a respirometer, explain why the liquid moves (3 marks)

Yeast anaerobically respire so release carbon dioxide. So volume go gas and pressure in container increase. So fluid in capillary tube moves down a pressure gradient, away from the organism.

When measuring anaerobic respiration using a respirometer, explain why the apparatus is left for an hour after the culture has reached a constant temperature. (1 mark)

Allow time for oxygen to be used up/respired.

Describe how redox indicator dyes such as methylene blue can be used to measure rate of respiration (5 marks)

Add a set volume of organism (e.g., yeast) and a set volume of respiratory substrate (e.g., glucose) into tubes. Add buffer (keeping pH constant). Place in water bath at set temperature and allow to equilibrate for 5 mins. Add a set volume of methylene blue. Record time taken for colour to disappear in tube.

Give 5 examples of variables that should be controlled (when using a redox indicator to measure rate of respiration) (5 marks)

Volume of single celled organism. Volume of respiratory substrate. Temperature of water bath. pH. Volume of redox indicator.

Why leave tubes in the water bath for 5 minutes (when using a redox indicator to measure rate of respiration) (1 mark)

Allow for solutions to equilibrate and reach the same temperature as the water bath.

Suggest a suitable control experiment and explain why it is necessary (when using a redox indicator to measure rate of respiration) (3 marks)

Add methylene blue to boiled/inactive/dead yeast (denaturing enzymes). Keep all other conditions the same. To show change is due to respiration in organisms.

Suggest and explain why you must not shake tubes containing methylene blue (when using a redox indicator to measure rate of respiration) (3 marks)

Shaking would mix the solution with oxygen. Which would oxidise methylene blue (causing it to lose its electrons). So methylene blue would turn back to its original blue colour.

Suggest one source of error in using methylene blue. Explain how this can be reduced (when using a redox indicator to measure rate of respiration) (2 marks)

Subjective as to determination of colour change/end point. Compare results to a colour standard or use a colorimeter.

Describe how the effect of an environmental variable on the movement of an animal (e.g., woodlice) can be investigated using a choice chamber (5 marks)

Set up choice chamber (with different compartments) to create different environmental conditions. e.g., humidity- add a drying agent to one side and damp filter paper to the other. Control other environmental conditions. Use a teaspoon to place a set number of animals. After a set amount of time, record the number of animals in each section. Report after gently moving the woodlice back to the centre.

The woodlice were left for 15 minutes before their movement was recorded when investigating the effect of humidity. Explain why (When investigating an environmental variable on the movement of an animal) (3 marks)

Time to establish humidity. Woodlice no longer affected by handling. So that behaviour is typical of that humidity.

Explain how you would ensure the safe and ethical handling of animals (When investigating an environmental variable on the movement of an animal) (2 marks)

Safe: over open wounds/wash hands with soap before and after, to minimise risk of infection. Ethical: handle carefully/return to habitat ASAP.

Explain why a mesh platform is used when investigating the effect of humidity (When investigating an environmental variable on the movement of an animal) (1 mark)

To keep woodlice a safe distance from drying agent.

Describe how the effect of an environmental variable on the movement of an animal (eg. maggots) can be investigated using a maze (4 marks)

Change environment at one end of T shape, e.g., add a food source. Place animal at stem of T. Record whether animal moves towards or away from the food source Repeat with a large number of maggots. Wipe/clean the maze between trials. Repeat with the food on the other side of the T.

Explain why the same organism is not used more than once (When investigating an environmental variable on the movement of an animal) (2 marks)

Reduces stress on maggots. Prevents chance of learned behaviours.

Explain why a clean Petri dish/maze is used each time (When investigating an environmental variable on the movement of an animal) (2 marks0

Animals may leave chemicals/scents. Which influence the behaviour of other organisms.

Explain which statistical test should be used to analyse test results (When investigating an environmental variable on the movement of an animal) (3 marks)

Chi squared. As data is categorical and comparing frequencies. To see if theres a significant difference between the observes and expected frequencies.

Describe how a calibration curve could be produced for glucose (4 marks)

Use distilled water and a glucose solution (of known concentration) to produce a dilution series. Heat a set volume of each solution with a set volume of Benedicts. Measure absorbance of each solution using a colorimeter. Plot a graph of absorbance (y axis) against concentration of glucose concentration (on x axis) and draw a line of best fit.

Describe how the concentration of glucose in an unknown ‘urine’ sample can be identified using a calibration curve (3 marks)

Perform benedicts test on sample using same volumes of solutions. Measure absorbance using a colorimeter. Absorbance value read for urine sample read off calibration curve to find associated glucose concentration.

Give examples of variables that should be controlled (When investigating glucose concentration of a urine sample) (4 marks)

Volume of sample used. Volume of Benedicts solution. Temperature of water bath. Time samples were heated in water bath.

Explain why a high blood glucose concentration can cause glucose to be present in the urine of a diabetic person (2 marks)

Not all glucose is reabsorbed at proximal convoluted tubule. As glucose cotransporter proteins are saturated/working at maximum rate.

Describe how you could investigate the effect of an environmental factor on the distribution of a species in a habitat (random sampling in two areas) (5 marks)

Divide two areas into grids/squares (e.g., place 2 tape measures at right angles. Generate a pair of coordinates using a random number generator. Place a quadrate and hear and counter frequency of named species (standardise this e.g., only count if more than half is in the quadrat). Repeat a large number of times (10 or more) and calculate mean per quadrat for both areas. Measure environmental factor in each area.

Suggest why percentage core may be used rather than frequency (when investigating the effect of a named environmental factor on the distribution of a given species) (1 mark)

Too difficult to count individuals organisms/individual organism/individual organisms are too small.

Explain why random sampling is used (1 mark)

To avoid sampling bias.

Explain the importance of a large sample size (2 marks)

Minimises the effect of anomalies. Ensures the sample is representative of the population.

Describe how you could decide the number of quadrants that should be used in order to collect representative data (2 marks)

Calculate a running mean. When enough quadrants, the running mean shows little change. Enough to carry out a statistical test.

Describe how you could investigate the effect of a factor on the distribution of a species in a habitat (systematic sampling) (3 marks)

Place a transect line (tape measure) across an area with an environmental gradient (e.g., tree to full sun). Place quadrants at regular intervals and record the number of organisms and the named environmental factor. Repeat in other parallel areas and calculate mean number of each point along the transect.

Explain the limitations of using systematic sampling to estimate the population of a species in a field. (1 mark)

Not appropriate unless theres an environmental gradient. Transects only run in one direction.

Which statistical test should be used to determine the relationship between abundance and an environmental factor (1 mark)

Correlation coefficient. E.g., spearmans rank.

Describe when a chi-squared (X2) test can be used (2 marks)

When determining if observed results are statistically different from expected results (frequencies). When data is categorical (can be divided into groups e.g., phenotype).

Describe how a chi-squared value can be calculated (2 marks) Define each variable.

Sum of (O-E)squared / E. Where O is the frequencies observed. Where E is the frequencies expected (multiply total n with each expected ratio as a fraction).

Describe how a chi-squared value can be analysed (3 marks)

Number of degrees of freedom= number of categories-1. Determine a critical value at p=0.05 from a table. If X2 is greater than critical value at p<0.05, difference is significant so reject the null hypothesis, so there less than 5% probability that the difference is due to chance.

Explain how the mark release recapture equation can be derived

Number marked in sample 1 / Total population size = Number marked in sample 2 / Total number on sample 2.

What assumptions does the mark release recapture method make (4 marks)

Sufficient time has been left for marked individuals to mix/distribute evenly within the population. Marking not removed/not toxic so does not affect predication/chances of survival. Limited immigration/emigration. No/ few births/deaths/breeding.

Describe how DNA fragments can be produced using restriction enzymes (2 marks)

Restriction enzymes cut DNA at specific base ‘recognition sequences’ either sides of the desired gene (as they are complementary here). Many are cut in a staggered fashion forming “sticky ends”.

Describe how DNA fragments can be produced from mRNA (3 marks)

Isolate mRNA from a cell that readily synthesis the protein coded for by the desired gene. Mix mRNA with DNA nucleotides and reverse transcriptase to synthesise complementary DNA cDNA. DNA polymerase can form a second strand of DNA using cDNA as a template.

Describe how fragments of DNA can be produced using a gene machine (2 marks)

Synthesises fragments of DNA quickly and accurately from scratch. Which do not contain introns.

Explain how DNA fragments can be amplified by PCR (6 marks)

1. Mixture heated to 95oC	●  This separates DNA strands ●  Breaking hydrogen bonds between bases
2. Mixture cooled to 55oC	●  This allows primers to bind to DNA fragment template strand ●  By forming hydrogen bonds between complementary bases
3. Mixture heated to 72oC	●  Nucleotides align next to complementary exposed bases ●  DNA polymerase joins adjacent DNA nucleotides, forming phosphodiester bonds

Explain how gel electrophoresis can be used to separate DNA fragments (3 marks)

1. DNA samples loaded into wells in a porous gel and covered in buffer solution (which conducts electricity)

2. Electrical current passed through → DNA is negatively

   charged so moves towards positive electrode

3. Shorter DNA fragments travel faster so travel further

How can data showing results of gel electrophoresis be interpreted? (3 marks)

Run a standard with DNA fragments/proteins of known lengths under the same conditions. Compare to position of unknown DNA fragments/proteins to estimate their size. Shorter DNA fragments/proteins travel further/faster.

Explain how genetic fingerprinting can be used to analyse DNA fragments (6 marks)

1. Extract DNA from sample (eg. blood cells) and amplify by PCR

2. Cut DNA at specific base sequences / recognition sites (either side of VNTRs) using restriction enzymes

3. Separate VNTR fragments according to length using gel electrophoresis (shorter ones travel further)

4. Transfer to a nylon membrane and treat to form single strands with exposed bases

5. Add labelled DNA probes which hybridise / bind with complementary VNTRs (& wash to remove

unbound probe)

6. To show bound probe, expose membrane to UV light if a fluorescently labelled probe was used

OR use autoradiography (expose to X-ray film) if a radioactive probe was used

The student wanted to determine the rate of water loss per mm2 of surface area of the leaves of the shoot in a potometer.

Outline a method she could have used to find this rate. You should assume that all water loss from the shoot is from the leaves.

Method for measuring area; eg draw round (each) leaf on graph paper and count squares. Of both sides of (each) leaf. Divide rate (of water loss/uptake from potometer) by (total) surface area (of leaves).