Light which strikes a leaf may be : Reflected Transmitted pass straight through Absorbed

Light striking a leaf

Reflected

Absorbed

Transmitted
Only a small fraction of the light is absorbed by plant pigments In order to find out more about these plant pigments it is necessary to extract them and separate them using a technique called chromatography. Your teacher will explain the procedures carefully to you and you will be given an set of instructions as to how to write up your final report. This may be examined in the final exam so the following gives a summary of some of the important steps and why you have to design the experiment carefully. e

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COMPULSORY PRACTICAL Extraction and separation of leaf pigments The following table shows important design procedures Design feature Grind leaves with sand in acetone The mixture is filtered The pigment is spotted repeatedly into the chromatography paper Spotted paper is hung in chromatography solvent Solvent line marked with a pencil Lid kept on chromatography tank Paper not allowed to touch the side Solvent line marked immediately on completion Reason The sand ruptures the cell walls and the sand is used as a solvent for the plant pigments This separates the pigment solution from the debris This produces a concentrated spot so that the pigments will be visible even after they separate. This solvent is a mixture which helps maximum separation of pigments Prevents marked line dissolving in solvent This allows atmosphere to become saturated with solvents and helps separation of pigments Stops pigments running up the edge of the paper Allows Rf value to be calculated

Key ideas Pigment mix is dissolved in a solvent known as acetone Pigment mix is spotted onto chromatography paper Pigment mix is separated using chromatography solvent which is a mixture of acetone and petroleum ether Pigments are identified using their Rf value which is calculated by measuring the distance to each pigment and dividing it by the distance to the final solvent line. Your Rf valus can be compared against standard values to help you identify the pigments You must write this up as part of your course and hand in the finished lab report.

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After separation the pigment mix is shown to be made up of at least 4 different pigments

Carotene ( Orange ) Xanthophyll ( Yellow ) Chlorophyll a ( Blue-green) Chlorophyll b ( Dark green ) C

Solvent Front

Spot Line Concentrated Chlorophyll Spot

The visible spectrum Light can also be shown to be a complex mixture by shining it through a prism

Blue

Green

R Red

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The absorption spectrum If the light is passed through plant pigments before it gets to the prism the colours that are absorbed will disappear. This results in an abruption spectrum and shows that light is absorbed mainly at the blue and red ends of the spectrum.

Blue

Green

Red

The Action Spectrum An action spectrum can be produced by shining different colours of light onto a water plant and counting how quickly the bubbles of oxygen are given off. Comparing the light absorbed (absorption spectrum) and the light used for photosynthesis (action spectrum) shows that the two are closely linked. t

Blue

Green

R Red

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An action spectrum shows which wavelengths of light are used directly for photosynthesis.

Blue X

Green

Red

The pigments responsible for absorbing the light at the blue and red end of the spectrum are the chlorophylls which are known as the primary pigments. The carotenoids (xanthophylls and carotene) are known as accessory pigments and absorb light in slightly different regions see point X Accessory pigments pass the light energy to chlorophyll. A range a pigments are needed by a plant to increase the range of light wavelengths which can be absorbed by a plant for photosynthesis. Top Tips Learn the reason why a range of pigments are required as this is often asked. The fact that a pigment absorbs light does not necessarily mean that it uses it - my blue shirt absorbs red and green light!!

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Chemistry of photosynthesis The process of photosynthesis has two stages which take place inside organelles called chloroplasts.

Lamella part of inner membrane Stroma

Stage 1 Light dependant stage Takes place in grana Light energy is trapped by chlorophyll and used to split water photolysis. ATP and NADPH2 are produced

Granum contains chlorophyll Starch Grain

Stage 2 Light independent stage Takes place in stroma Involves cycle of enzyme reactions called the Krebs cycle Carbon dioxide is reduced to make glucose sugar this means it has hydrogen added to it by NADPH2 using energy supplied by ATP

Double Membrane

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CHEMISTRY OF PHOTOSYNTHESIS
Photosynthesis is the process by which organic compounds (e.g. glucose) are synthesised by the reduction of carbon dioxide.

Stage 1 Light dependent stage

The reduction of carbon dioxide requires energy in the form of ATP and a reducing agent (NADPH2) which are produced in the first stage of photosynthesis called the light dependant stage The light dependant stage takes place in the grana of the chloroplast and involves the trapping of light energy by chlorophyll and other accessory pigments During the light dependant stage light energy trapped by plant pigments is used to split water into hydrogen and oxygen. This process is called photolysis Energy released during photolysis is used to regenerate ATP from ADP and Pi. The ATP produced diffuses into the stroma of the chloroplast and is used as an energy source for a process called carbon fixation. The hydrogen produced during photolysis binds to a co-enzyme called NADP or nicotinamide adenine dinucleotide phosphate). The resulting NADPH is used as a reducing agent during carbon fixation Photolysis of water results in the release of oxygen as a by-product During the light dependant stage light energy is converted to stored

chemical energy (ATP)

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Summary of stage 1 Light Dependent Stage

ADP + Pi

Light Energy absorbed by chlorophyl l in

Water H 20
The splitting of water is known as photolysis

granum of chloropla st

O2- waste gas

H2
diffuses out of cell

ATP -to the

carbon fixation reactions found in the stroma of chloroplast

NADP

to the carbon fixation reactions found in the stroma of chloroplast

NADPH2

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Stage 2 Light independent stage

Stage 2 is called the light independent stage and involves the reduction of carbon dioxide to glucose The reduction of carbon dioxide is known as carbon fixation Carbon fixation involves a cycle of enzyme controlled reactions known as the Calvin Cycle and takes place in the Stroma of the chloroplast The series of reactions involved in the Calvin Cycle are affected by temperature and require ATP ( from the light dependent stage) and hydrogen (from photolysis) and carbon dioxide. The series of reactions involved in the Calvin Cycle can be summarised as follows The Calvin Cycle begins when carbon dioxide diffuses into stroma of the mitochondrion and is accepted by the 5-carbon compound ribulose 1,5 biosphosphate (RuBP) to form an unstable 6-carbon molecule see summary handout The unstable 6-Carbon molecule breaks down to a 3-carbon intermediate known as glycerate 3-phosphate (GP). Glycerate 3-phosphate (GP) is then reduced using hydrogen carried as NADPH from the photolysis reaction of the light dependent stage. This process requires energy supplied by the breakdown of ATP and produces 3-Carbon Triose sugar Triose sugars may be used to synthesis glucose, storage carbohydrates such as starch, structural carbohydrates such as cellulose or other major biological molecules such as proteins, fats and nucleic acids OR Triose sugars may be used to regenerate the original carbon fixing molecule in the Calvin Cycle (RuBP) so that the cycle of reactions can continue. This requires energy supplied by ATP Top Tip You do not need to name intermediate chemical compounds except GP (PGA) and RuBP (RuDP).

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Summary of stage 2 Light Independent Stage (also known as the carbon fixation stage or the Krebs Cycle CARBON DIOXIDE UNSTABLE COMPOUND (31C) (36C)
RuBP reacts with carbon dioxide from the air ( (35C) The 6C molecule is unstable and splits into two 3C molecules

RIBULOSE BIPHOSPHATE (35C)

CALVIN CYCLE

GLYCERATE-3PHOSPHATE (63C)
This is a reduction reaction with energy being supplied by the breakdown of ATP and the hydrogens being donated by NADPH2

The recycling of RuBP requires energy supplied by the breakdown of ATP

TRIOSE PHOSPHATE (63C)

GLUCOSE (6C)

STARCH

CELLULOSE

Starch is a storage carbohydrate as it allows the glucose made to be stored Cellulose is a structural carbohydrate as it is used to make plant cell walls
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LIMITING FACTORS

Limiting factors - Handout

The rate of photosynthesis is limited by the following environmental factors

Light intensity this affects the energy available for the light dependant reactions Carbon Dioxide concentration CO2 is the raw material required for the light independent reaction. Temperature this affects the rate of enzyme activity in the light independent reactions

Limiting factor graphs always have the same shape

7 6 5 4 3 2 Environmental factor X Prediction if Environmental X increases

Units of oxygen

Value 2 Part 2 Part 1

9 10 11

Value 1

General rules
Environmental factor X limits the rate of photosynthesis in part 1 of the graph A second requirement of photosynthesis limits the rate in part 2 of the graph

Describing - Mention both axis and value 1

Part 1 as factor X increases to value 1 units of oxygen evolved / minute increases Part 2 a further increase in factor X above value 1 gives no further increase in the units of oxygen evolved / minute

Explaining or accounting for trends in graph

Part 1 you must talk your way from factor X to oxygen evolved giving the importance of factor X in controlling the rate of photosynthesis

e.g. if factor X is light intensity you would say as light intensity increases to value 1 more energy is available for photosynthesis so the rate increases and the plant gives off more oxygen / minute

Part 2 you must talk your way from factor X to oxygen evolved mentioning the effect of a second limiting factor

e.g. if factor X is light intensity you would say as light intensity increases above value 1 the rate of photosynthesis is limited by a second factor such as CO2 concentration so the rate of photosynthesis cannot increase due to a lack of raw materials and the plant does not give off more oxygen / minute

Predicting trends in graph

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The prediction for increasing the concentration of a second environmental factor is always the same as that shown on the graph

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