required practicals

• What does the rate of migration of individuals pigments depend on in chromatography?

  - solubility

  - mass

  - affinity to paper
• Describe how you would use chromatography to determine to solvent front and distance travelled by the solvent, in order to find the Rf value. (Give method.)

  - draw straight pencil line approximately 1cm above bottom of paper, draw an X in middle

  - cut section of leaf and grind with pestle and mortar to release pigments

  - use capillary tube to extract pigment and blot onto X

  - suspend paper in solvent (so that solvent doesn't live above pigment line

  - wait until pigment has up paper

  - remove paper from solvent

  draw a line where solvent line is and measure distance travelled by solvent from pencil line

  - measure distance travelled from pencil line to centre of pigment blob

  - calculate Rf value
• What does a higher Rf value mean?

  - pigment has run up further paper

  - so pigment has a lower affinity, and is more soluble
• Give the formula for Rf value, and how you would use that to find what pigments are in a sample.


  - check database to identify what pigment it is

  
• Why should you use a pencil to draw the line on the chromatography paper?


  - pen will obscure results by running up paper as well
• List the hazards of chromatography, risks associated with them, and any precautionary measures you should take.

  Hazard - solvent

  Risk - flammable, causes irritation to eyes, harmful if inhaled and can cause dizziness

  Precaution - avoid contact, wear goggles, keep away from naked flame, keep room well ventilated
• What is the role of dehydrogenase in the light dependent stage of photosynthesis?

  - enzyme

  - catalyses the reaction where NADP accepts electrons. (of hydrogen ions)
• What is DCPIP, and what does it do?

  - redox indicator dye

  - accepts electron instead of NADP
• What is the colour change when DCPIP is reduced? (and when is absorbance greater?)

  - blue > colourless when reduced

  - absorbance is greater when DCPIP is blue
• Give the method for investigating dehydrogenase activity in chloroplasts.

  - remove stalks from leaf samples and grind using pestle and mortar

  - place in chilled isotonic solution

  - use muslin cloth and filter into a beaker

  - keep beaker in ice to keep chilled

  - transfer to centrifuge tubes and centrifuge at high speeds for 10 minutes, to separate chloroplasts into a pellet

  - remove the supernatant and add to fresh cold isolation medium

  - place this 30cm away from lamp and add DCPIP

  - immediately take sample and place in cuvette, then measure absorbance on calorimeter

  - you should have zeroed the calorimeter earlier using distilled water and chloroplast extract

  - take sample and measure absorbance every 2 minutes for 10 minutes

  - repeat for different distances from light up to 100cm
• Why should the experiment be carried out in a dark room?

  - so that only source of light is lamp

  - light intensity of lamp is always controlled, only changing distance varies intensity on plant
• Why shouldn't the samples be placed close to the lamp?

  - temperatures may affect rate of photosynthesis

  - alternatively use LED lamp
• List the hazards of dehydrogenase experiment, risks associated with them, and any precautionary measures you should take.

  Hazard - DCPIP

  Risk - irritation to skin and eyes

  Precaution - wash hands after use and wear goggles
• Why should you remove the stalks from the leaf samples before grinding them?

  - don't contain chloroplasts
• Why should the solution be chilled and isotonic?

  - chilled to lower the activity of enzymes to prevent them from breaking down the chloroplasts

  - isotonic to prevent damage to chloroplasts by osmosis
• Why is the muslin cloth used?

  - to filter out debris (while letting chlorophyll through)
• What graph should you draw? What does it mean if the graph is steeper?

  - absorbance / time

  - steeper gradient means high rate of decrease in absorbance, so dehydrogenase is highly active
• What does it mean if DCPIP takes a longer time to change from blue to colourless?


  - rate of light dependent reaction is slower

  - lower light intensity means lower rate of photoionisation

  - so less electrons released from chlorophyll, and less electrons accepted by DCPIP

  - so DCPIP reduced at lower rate
• In what ways does yeast respire?

  - aerobically

  - anaerobically
• How can respiration be measured?

  - use redox indicator dye (e.g. methylene blue)

  - accepts electrons that have been transferred to synthesise ATP (instead of electron transfer chain accepting)

  - record time take for dye to turn colourless
• What is the colour change in methylene blue?

  blue > colourless
• How would you investigate respiration in a single celled organism? (yeast)


  - water bath at 35C

  - add 5cm^3 yeast and glucose solution to three test tubes

  - place in water baths and leave to equilibrate

  - add 2cm^3 methylene blue and start timer

  - shake for 10 seconds, then leave in bath

  - record how long it takes for each tube to turn colourless

  - repeat using different temperatures ( 40,50,50,70)

  - find mean at each temperature

  - calculate average rate of respiration
• Give the formula for rate of respiration.

  rate of respiration = 1 / mean time
• List the hazards of measuring respiration in yeast, risks associated with them, and any precautionary measures you should take.

  Risk - methylene blue / yeast / hot water

  Hazard - irritant to skin and eyes / allergies / scalding

  Precaution - goggles, wash skin / wash hands / use tongs and handle with care
• What graph should you plot? What can you tell from the peak?

  - rate of respiration / temperature

  - peak is where optimum temperature is
• What happens to the rate of respiration if temperature gets too high?

  - enzymes involved in respiration denature
• What does it mean if methylene takes a longer time to turn colourless?

  - lower rate of respiration

  - due to conditions being further from optimum
• Explain how woodlice use kinesis to remain in favourable conditions, to increase their chances of survival.

  orthokinesis - speed of movement

  klinokinesis - frequency of direction change

  
  

  - slower speed and more direction changes in favourable environment

  - faster speed and less direction changes in unfavourable environment
• What are the control variables in an experiment to investigate simple animal responses?

  - number of animals

  - environmental conditions available

  - condition of animals

  - time allowed for animals to move
• Describe how you could investigate simple animal responses.

  - set up choice chamber with quadrants:

  > dark + dry

  > dark + damp

  > light + dry

  > light + damp

  - use dark paper to keep conditions dark

  - use wet cotton wool to make areas damp

  - use drying agent to male areas dry

  - put 12 woodlice in middle of chamber (use spoon, not forceps as that harms them)

  - leave for 10 minutes

  - record how many woodlice are in each quadrant

  - repeat
• Why is it important that woodlice are all placed in middle of choice chamber at start of experiment?

  - so they are all equal distance away from each stimulus

  - no bias towards one quadrant
• List the hazards of investigating simple animal responses, risks associated with them, and any precautionary measures you should take.

  Risk - woodlice

  Hazard - contamination

  Precaution - wash hands after use
• In which quadrant of the choice chamber should you expect the most woodlice to be in?

  - dark + damp

  - damp conditions help them to breath (through gills)

  - dark conditions means more likely to be damp, and they are at less risk of drying out
• How can you determine if the number of woodlice in each quadrant is significant?

  - chi squared test

  - if value exceeds critical value, reject null hypothesis (that there is no significance)

  less than 5% down to chance
• Give the formula for the chi squared test.

  - expected values here is that there is an equal number in each environment

  
• How can an unknown concentration of glucose in urine be determined? What can this be used for?

  - quantitative Benedict's test

  - make a calibration curve of absorbance using known concentrations of glucose

  - used to see if someone has diabetes, is at risk of becoming diabetic, or had kidney failure
• What kind of sugar is glucose?

  - reducing sugar
• What happens to the Benedict's solution in the presence of glucose, and what colour change happens?

  - in presence of glucose, Cu2+ in Benedict's solution is reduced into insoluble copper (1) oxide

  - blue > brick red

  - extent of colour change depends of concentration of glucose
• Describe how you could measure the concentration of a urine sample by making and using a calibration curve.

  - create a dilution series of glucose using distilled water (2cm^3)

  - make concentrations 0-10mmoldm^-3

  - place 2cm^3 of each unknown concentration in tubes

  - add 2cm^3 of Benedict's solution to all tubes

  - place all tubes in water bath at 90C for 4 minutes

  - remove and leave to cool

  - zero calorimeter using distilled water (blue filter)

  - measure absorbance of each sample in a cuvette

  - make a calibration curve with known samples

  - read off what concentrations go with each absorbance to find glucose concentration of unknown urine samples
• What is a serial dilution? (dilution series)

  - dilution where successive concentrations increase or decrease in logarithmic fashion