hill reaction Flashcards
(18 cards)
which indicators can be used
DCPIP, methylene blue
what do the indicators do
accept electrons instead of NADP
when the indicators accept electrons they change colour
this can be used to study the rate of the hill reaction-rate at which indicator changes colour from its oxidised to reduced state (faster change=faster rate)
DCPIP: what colour before and after reduction?
oxidised=blue
reduced=colourless
colour of reduced solution may appear green due to presence of chlorophyll
methylene blue: colour before and after reduction
oxidised=blue
reduced=colourless
what is the hill reaction referring to
NADPH being produced by electrons from electron transport chain and H+ from photolysis of water
which part of photosynthesis can it provide quantitative data on?
light-dependent reaction
what from leaves to we need to obtain to carry out this experiment
chloroplasts
what does a colorimeter measure
how much light passes through or is absorbed by a solution
transmission=amount of light that passes through
absorbance=light absorbed by solution
select appropriate wave length filter, usually between 500&600 nm
why is isolation solution used
to maintain pH and prevent damage to chloroplasts during the grinding and separation
process
maintains osmotic pressure of chloroplasts preventing them from bursting
added during first step when grinding leaves
what is pellet in centrifuge tube
heaviest organelles will form a sediment at the bottom of the tube/settled particles
supernatant in centrifuge tube
liquid which remains above the pellet after centrifugation/liquid above settled particles
which side of cuvette should face the light source
the clear side
method
-grind up leaves with 20cm3 isolation solution in pestle and mortar to break apart tissues of the leaf and protect chloroplasts from damage from osmotic imbalance or extreme pH
-filter liquid into clean beaker using funnel and cloth to get rid of big leaf pieces
-transfer to centrifuge tube and centrifuge for 10 mins
-pellet of chloroplasts will form at the bottom
-discard supernatant and keep pellet
-make chloroplast extract by mixing 2cm3 fresh isolation solution with pellet, stir with glass rod to re-suspend chloroplasts in the liquid
-transfer chloroplasts to ice water bath to slow down chloroplast activity
-place a test tube containing 0.5cm3 chloroplast extract a set distance from a lamp
-beaker of water placed between lamp and rack to prevent a temp increase from heat lamp which may invalidate results
-add 5cm3 DCPIP to chloroplast extract & mix together using clean glass rod
-use pipette to immediately place a sample of DCPIP-chloroplast mixture into a cuvette
-place cuvette in colorimeter and take a reading of absorbance
-repeat steps 9-10 every minute for 10 mins (placing DCPIP chloroplast mixture into a cuvette & take reading of absorbance in colorimeter)
-repeat at least twice ensuring variables are controlled
results: what should happen to the absorbance reading of the colorimeter over the 10 min test period?
absorbance should decrease, because DCPIP is reduced
when DCPIP is oxidised and blue it absorbs light at approximately 600nm, when its colourless less light is absorbed by the solution
about pigment of DCPIP not chloroplasts/chlorophyll
a graph of what against time can be drawn to show rate of hill reaction
absorbance against time
changing variables such as the dif limiting factors on photosynthesis would allow the effect or different variables on rate of hill reaction to be studied
control variables
age and source of chloroplasta
ph of isolation solution
electron acceptor/indicator used
DV
time taken for DCPIP to decolourise
IV
can be changing variables such as dif limiting factors of photosynthesis like light intensity
if measuring LI place one tube in the dark during step before addition of DCPIP as a control
