3.5 RP

Question 1

Describe how pigments from a leaf of a plant can be isolated with paper chromatography

Answer 1

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

Question 2

1. Explain why the origin should be drawn in pencil rather than ink.
2. Explain why the point of origin should be above the level of the solvent.
3. Explain why a pigment may not move up the chromatography paper in one solvent.

Answer 2

1. ● Ink is soluble in solvent
   ● So ink would mix with pigments / line would move
2. ● Pigments are soluble in solvent
   ● So would run off paper / spots dissolve into solvent
3. May be soluble in one solvent but insoluble in another

Question 3

Describe how pigments can be identified

Answer 3

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

Question 4

Explain why the solvent front should be marked quickly once chromatography paper is removed.

Answer 4

● Once solvent evaporates, solvent front not visible

Question 5

Explain why the centre of each pigment spot should be measured.

Answer 5

● Standardises readings as pigment is spread out
● So allows comparisons to be made

Question 6

Explain why the obtained Rf values were similar, but not identical, to the published values.

Answer 6

● Different solvent / paper / running conditions may affect Rf value

Question 7

Explain why Rf values are used and not the distances moved by pigment spots.

Answer 7

● Solvent / pigment moves different distances
● Rf value is constant for same pigment / can becompared

Question 8

Describe the role of the enzyme dehydrogenase in photosynthesis

Answer 8

● Catalyses the reduction of NADP in the light-dependent reaction
- NADP accepts (gains) electrons from photoionisation of chlorophyll / photolysis of water

Question 9

Describe how rate of dehydrogenase activity in extracts of chloroplasts can
be measured

Answer 9

1. Set up test tubes as follows:
   A. Control 1- set volume of DCPIP, water and chloroplasts in isolation medium, covered in foil to block light
   B. Control 2 - set volume of DCPIP, water and isolation medium without chloroplasts
   C. Standard - set volume of water and chloroplasts in isolation medium, without DCPIP
   D. Experiment - set volume of DCPIP, water and chloroplasts in isolation medium
2. 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
3. Rate of dehydrogenase activity = 1 / time taken

Question 10

Give examples of variables that could be controlled.

Answer 10

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

Question 11

1. Explain the purpose of control 1 (tube A).
2. Explain why DCPIP in control 1 stays blue.
3. Explain the purpose of control 2 (tube B).

Answer 11

1. ● Shows light is required for DCPIP to decolourise
   ● Shows that chloroplasts alone do not cause DCPIP to decolourise
2. ● No light so no photoionisation of chlorophyll
   ● So no electrons released to reduce DCPIP
3. ● Shows chloroplasts are required for DCPIP to decolourise
   ● Shows that light alone does not cause DCPIP to decolourise

Question 12

Explain why DCPIP changes from blue to colourless.

Answer 12

● DCPIP is a redox indicator / DCPIP gets reduced by electrons
● From photoionisation of chlorophyll

Question 13

Suggest a limitation with the method and how the experiment could be modified to overcome this.

Answer 13

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

Question 14

Describe how a respirometer can be used to measure the rate of aerobic respiration

Answer 14

Measures O2 uptake:
1. Add a set mass of single-celled organism eg. yeast to a set volume / concentration of substrate eg. glucose
2. Add a buffer to keep pH constant
3. Add 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

Question 15

Explain why the liquid moves.

Answer 15

● Organisms aerobically respire → take in O2
● CO2 given out but absorbed by sodium hydroxide solution
● So volume of gas and pressure in container decrease
● So fluid in capillary tube moves down a pressure gradient towards
organism

Question 16

Explain why the respirometer apparatus is left open for 10 minutes.

Answer 16

● Allow apparatus to equilibrate
● Allow for overall pressure expansion/change throughout
● Allow respiration rate of organisms to stabilise

Question 17

Explain why the apparatus must be airtight.

Answer 17

● Prevent air entering or leaving
● Would change volume and pressure, affecting movement of liquid

Question 18

Describe a more accurate way to measure volume of gas.

Answer 18

● Use a gas syringe

Question 19

Describe how the rate of respiration can be calculated

Answer 19

1. Calculate volume of O2 / CO2 consumed / released (calculate area of a cylinder)
   a. Calculate cross-sectional area of capillary tube using πr²
   b. Multiply by distance liquid has moved
2. Divide by mass of organism and time taken
3. Units - unit for volume per unit time per unit mass eg. cm3 min-1 g-1

Question 20

Describe how a respirometer can be used to measure the rate of anaerobic respiration

Answer 20

Measures CO2 release:
● Repeat experiment as above but remove chemical that absorbs CO2
● Make conditions anaerobic, for example:
- Layer of oil / liquid paraffin above yeast → stop O2 diffusing in
- Add a chemical that absorbs O2
- Leave for an hour to allow O2 to be respired and used up

Question 21

Explain why the liquid moves

Answer 21

● Yeast anaerobically respire → release CO2
● So volume of gas and pressure in container increase
● So fluid in capillary tube moves down a pressure gradient away
from organism

Question 22

Explain why the apparatus is left for an hour after the culture has reached a constant temperature

Answer 22

● Allow time for oxygen to be used / respired

Question 23

Describe how redox indicator dyes such as Methylene blue can be used to measure rate of respiration

Answer 23

● Redox indicators (eg. methylene blue) change colour when they accept electrons becoming reduced
● Redox indicators take up hydrogens and get reduced instead of NAD / FAD → modelling their reactions

1. Add a set volume of organism eg. yeast and a set volume of respiratory substrate eg. glucose to tubes
2. Add a buffer to keep pH constant
3. Place in water bath at a set temperature and allow to equilibrate for 5 mins
4. Add a set volume of methylene blue, shake for a set time (do not shake again)
5. Record time taken for colour to disappear in tube
   Rate of respiration (s-1) = 1 / time (sec)

Question 24

Give examples of variables that could be controlled

Answer 24

● Volume of single-celled organism
● Volume / conc. / type of respiratory substrate
● Temperature (with a water bath)
● pH (with a buffer)
● Volume of redox indicator (only control)

Question 25

Why leave tubes in the water bath for 5 minutes?

Answer 25

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

Question 26

Describe a control experiment and why it would be done

Answer 26

• Add methylene blue to boiled / inactive / dead yeast (boiling denatures enzymes) • All other conditions the same • To show change is due to respiration in organisms

Question 27

Suggest and explain why you must not shake tubes containing methylene blue

Answer 27

• Shaking would mix solution with oxygen • Which would oxidise methylene blue / cause it to lose its electrons • So methylene blue would turn back to its original blue colour

Question 28

Suggest one source of error in using methylene blue. Explain how this can be reduced.

Answer 28

• Subjective as to determination of colour change / end point • Compare results to a colour standard (one that has already changed) • Or use a colorimeter for quantitative results