What are different about all plants?
Different photosynthetic pigments in their leaves
Each pigment absorb different wavelength of light
so more than one type of pigment increases range of wavelength of light the plant can absorb
Why do pigments play an essential role other than photosynthetic pigments?
Protecting leaves from excessive UV radiation
What do different species of plant contain?
Different proportion and mixture of pigments
How to experiment the different pigments presented in leaves?
(Thin layer chromatography)
What does TLC involve?
- Mobile phase
- Stationary phase
What is mobile phase?
Where molecules can move
in TLC this is a liquid solvent
What is stationary phase?
Where molecules move
TLC consist of solid (e.g glass plate)
plate with a thin layer of gel (e.g silica gel) on top
Why does TLC help?
A sample of pigments can be extracted from a plant and put onto the TLC plate
When a plate is placed vertically in solvent, solvent move upwards through the gel carrying the dissolved pigments through it
Some pigments travel faster or further through the gel than others making them separated
How to identify a certain pigment?
Calculating its R1 value and looking up in database
R1 is the distance a substance has moved through the gel in relation to solvent
Each pigment has a specific R1 value
How to investigate using TLC?
Compare pigments present in shade-tolerant plants and shade-intolerant plants
What are the safety factors in TLC investigation?
Wear lab coat, eye protection , gloves
Because of chemicals involed are toxic and highly flammable
What are the first step of TLC
Grind up several leaves from shade tolerant plant you are investigating with some andydrous sodium sulfate
Add few drops of propane
What are the second step of TLC?
Transfer liquid to a test tube add some petroleum ether and gently shake the tube
Two distinct layers will form in liquid
Top layer is the pigments mixed in with petroleum ether
How to do the step 2 efficiently and 5?
In a fume cupboard as chemicals are volatile
they evporate easily and vapours are hazardous
What is step 3?
Transfer some of the liquid from top layer into second test tube with some anhydrous sodium sulfate
What is the fourth step?
Draw a horizontal pencil line near the bottom of TLC plate
Build up a concentrated spot from liquid from step 3 on line by applying several drops and ensuring one is dry before adding another
This is point of origin
What is the fifth step?
- Once point of origin is dry
- Put plate in small glass containor with prepared solvent (point of origin should be above solvent)
- Put lid in containor and leave to develop
What happens as solvent spreads up plate?
Different pigments move with it at different rates so they separate
What is seventh step?
When solvent nearly reached the top, take plate out and mark the solvent front (furherest point the solvent has reached with pencil)
Leave the plate to dry in well-ventialted plate
What is the eighth step of TLC?
- Repeat process for shade-intolerant and compare pigments
How to calculate Rf value?
B/A = Distance travelled by spot/Distance travelled by solvent
What do you do after the Rf value?
Look up in a database and identify the pigments
How is the pigments of leaf in shade-tolerant plant different from shade intolerant plants?
- Adapt to light conditions by possessing different proportion of photosynthetic pigments allowing plant to make best use of light available
- Mixture of non-photosynthetic pigments are different
- e.g shade tolerant plants are adapted for photosynthesis in low light condition but really sensitive for higher levels of light
- These plants produce dark red and purple pigments called anthocyanins , protect chloropladts from brieg exposure to higher levels of light
What reaction is catalysed by dehyrogenase enzyme?
NADP acts as a electron accept and it is reduced
How to measure activity of dehyrogenase enzyme?
Adding redox indicator dye to extract of chloroplasts
Dye acts as an electron accept (e.g NADP) and gets reduced by dehydrogenase in chloroplasts
As dye gets reduced - see colour change
e.g DCPIP changes from blue to colourless being reduced
How to measure the rate of dehygrongenase activity?
Measuring the rate at which DCPIP loses its blue colour
Need a colorimeter
Measure how much light a solution absorbs when light source shoen directly through
A coloured solution absorb more than colourless solution
What things does investigating effect of light intensity on dehydrogenase activity in extract in chloroplasts use?
- A bench lamp as a light source and involve placing tubes of chloroplast extract mixed with DCPIP at range of different distance from light source
- Light intensity should decrease with increasing distance from lamp
What is the first step of effect of light intensity on dehydorgenase activity in extract of chloroplast?
Cut a few leaves (spinach) into pieces and remove any tough stalks
What is the second step of effect of light intensity on dehydorgenase activity in extract of chloroplast?
Use pestle and mortar , grind up the leaf pieces with chilled isolation solution (sucorse, potassium chloride and phosphate buffer at pH7)
Filter the liquid you make into beaker througha funnel lined with muslin cloth
What is the third step of effect of light intensity on dehydorgenase activity in extract of chloroplast?
Transfer liquid to centrifuge tubes and centrifuge them at high speed for 10 minutes
Make chloroplasts gather at bottom - pellet