The reactions of photosynthesis

Photosynthesis is a very important process, involving the conversion of solar energy to chemical energy within molecules inside cells. It is carried out by all photoautotrophic organisms (green plants including algae and photosynthetic bacteria - cyanobacteria). This type of nutrition, based on the use of light energy to synthesise foods from carbon dioxide as the principal source of carbon, is sometimes called holophytic.

Obviously it is central to these green plants, as they directly use the resulting organic compounds as substrates for respiration (providing a constant energy supply for processes within their cells), and for the synthesis of more complex molecules (for growth). It also indirectly provides energy and organic compounds for the metabolism of animals, and also for fungi and bacteria. It is probably worth stating that the processes of photosynthesis and respiration are not 100% efficient in terms of energy transfer.

In an ecological context, photosynthesis effectively powers almost all living organisms on the planet. Green plants are producers, and animals are (primary, secondary, etc) consumers, obtaining organic food from plants. All the oxygen in the atmosphere is produced as a result - actually as a waste product - of photosynthesis, and it is essential for all aerobically respiring organisms. The chemical substrates for respiration (not just carbohydrates but potentially other organic molecules as well) are all produced by plants and passed to animals as they feed on other organisms within food chains. The transfer of this biomass and its stored chemical energy from one trophic level to another within a community is also very inefficient.

The processes within photosynthesis have a number of similarities with (aerobic) respiration. They occur inside a membrane-bound structure probably derived millions of years ago from another organism, and they involve transfer of electrons between a number of compounds embedded in the membrane, linked to movement of protons across the membrane, which sets up an electrochemical gradient. This results in the production of ATP using chemiosmotic energy as protons pass through the stalked enzyme ATP synthase. And there is a secondary cyclical section which uses ATP and reduced coenzymes. However respiration is a catabolic (breaking down) process and photosynthesis is anabolic (building up), and photosynthesis uses (reduced) NADP whereas respiration principally uses (reduced) NAD. And of course photosynthesis uses the energy of light to power the process, whereas respiration is all about the release of chemical potential energy.

Photosynthesis uses water and carbon dioxide as reactants, and carbohydrate and oxygen are its products. In higher plants, water is taken to the leaves via the xylem vessels leading up from the roots in the transpiration stream. Carbon dioxide gas enters leaves through stomata on the underside of leaves. The main photosynthetic region within leaves is known as mesophyll - which has 2 layers of cells: the spongy layer and the palisade layer.

But the reactants are actually delivered to organelles within the leaf cells: chloroplasts. Cells of water plants, algae and cyanobacteria are surrounded by water in which carbon dioxide is dissolved.

The photosynthesis process described here is essentially the same in green land plants, algae and cyanobacteria. It is called oxygenic photosynthesis, as it results in the production of oxygen as a waste product.

The simple overall equation for photosynthesis does not make allowance for the different processes detailed below.

carbon dioxide + water glucose + oxygen OK artificial light works as well as sunlight

It is now known that photosynthesis has 2 main stages -
the light-dependent reaction and the light-independent reaction.

The light-dependent reaction

(equations not 'balanced')
On the innermost membranes of the chloroplast, chlorophyll absorbs light energy, and as a result it is raised to a higher energy level so it gives off an electron, which is passed to other compounds (electron acceptors). The loss of the electron (a negatively charged particle - shown as e^-) causes chlorophyll to be photoionised (positively charged) for a short time.

As the electron is passed along an electron transfer chain in the membrane, it gives up its energy and some of this is used to move protons across the inner membrane, resulting in a buildup of protons in the space inside - the thylakoid lumen. At the end of the electron transfer chain, protons and electrons are used to reduce NADP.
Reduced NADP is used in the light-independent stage of photosynthesis.

The difference in concentration of protons on either side of the membrane establishes a chemiosmotic pressure, causing them to pass back across the membrane. Protons can only pass through ATP synthase molecules embedded in the chloroplast membrane, and their movement results in the production of ATP from ADP + Pi.
ATP is used in the light-independent stage of photosynthesis.

The charge lost from chlorophyll is repaid when water splits, in the related process of photolysis.
As a result, in the light water produces a steady supply of:

• electrons which are released and passed back to chlorophyll
• protons which build up and contribute to the proton gradient
• oxygen which is released as is not needed and it diffuses away to be used in aerobic respiration by the plant itself or by animals in the general environment.

Continue to the light-independent reaction

The Hill reaction

Robert Hill FRS (1899–1991) was a biochemist working at Cambridge who had a wide variety of research interests: haemoglobin, myoglobin and cytochromes, plant dyes and photosynthesis.

In 1939, he demonstrated that oxygen is produced during the light requiring stage of photosynthesis by measuring the evolution of oxygen by isolated chloroplasts exposed to light (in the complete absence of CO₂).
This showed that photosynthesis was not a simple (one-stage) process.

The Hill reaction is the photoreduction of an electron acceptor by hydrogen originating from water, with the evolution of oxygen.

In a plant cell the final electron acceptor is NADP, and it interacts with an enzyme called NADP reductase - which is not a dehydrogenase.

The rate of the Hill reaction in isolated chloroplasts may be measured using an artificial electron acceptor that changes color as it is reduced.

DCIP (2,6-dichlorophenolindophenol) is a suitable dye which is blue in its oxidized form and colourless in its reduced form.

The progress of the reaction can be followed using a spectophotometer to measure the change in absorbance (at 600nm).

This forms the basis of a number of practicals which are expected at this level.

The LDR in more detail

The reactions of photosynthesis are controlled by many molecules working together. A key feature of the first section is the dependence on the thylakoid membrane, the surface of which exposes pigments to light and this is folded to form enclosed shallow flattened spaces called the lumen. On the outside of this is a fluid filled space called the stroma.

There are different forms of the green pigment chlorophyll - 'a' and 'b' forms - as well as the yellow xanthophyll and carotenoids which are orange. All of these absorb light of different wavelengths, and they may pass on the energy to other compounds. Carotenoids act as anti-oxidants, preventing damage to the other pigments.
There is a section below on separating these pigments using chromatography.

A Biological Photocall

Embedded in the thylakoid membrane there are two 'photosystems' - protein complexes containing a number of subunits in specific positions next to one another, which controls their chemical interactions.
Distinct from these are a number of enzymes which function to produce ATP.
As they depend on light, they all have 'photo' in their names.

Photosystem II

The first component, Photosystem II, contains about 35 chlorophyll 'a' and 12 beta-carotene molecules, forming a light-harvesting or 'antenna complex' which absorbs light energy from photons and passes it to a reaction centre: one specific P680 chlorophyll molecule. This P680 is raised to a higher energy level - it may be called P680* - and it emits an electron, so it becomes (photo)ionised - oxidised: P680⁺.

The electron is passed to an electron transfer chain beginning with phaeophytin - the primary electron acceptor. Phaeophytin (not shown in the diagram above) is in fact a chlorophyll molecule lacking a magnesium ion. These energy-carrying electrons are passed (in pairs) to plastoquinone (PQ), which is able to move within the lipid bilayer.
As it leaves Photosystem II, plastoquinone also picks up (pairs of) protons from the stroma side (becoming plastoquinol - PQH₂ in the diagram) and carries them across the membrane to cytochrome b₆f, which then deposits the protons in the lumen. Electrons - by now at rather a low energy level - are picked up by plastocyanin and eventually transferred to Photosystem I (see below).

Also attached to Photosystem II (on the lumen side) is a group of 4 manganese and one calcium ions (a 'Mn₄O₅Ca cluster' with a curious cube-like structure) which make up the oxygen-evolving complex. This water-splitting enzyme is the site of the oxidation of water, passing through 4 states of oxidation/reduction involving Mn³⁺ and Mn⁴⁺.

This process - also called the photolysis of water - produces:
- electrons which are individually passed to P680⁺ to replace the lost electron emitted
- protons which remain within the thylakoid lumen at this stage
- molecular oxygen which is effectively a waste product of the process 2 H₂O → 4e^- + 4H⁺ + O₂

Photosystem I

Photosystem I does not have an equivalent of the oxygen-evolving complex. It has a larger antenna complex (90 chlorophyll 'a', and 22 beta-carotene molecules) which passes energy from a second photon of light to a pair of chlorophyll molecules P700. These emit an electron - effectively at a higher energy level than in PSII - which passes along a chain of electron carriers, all within PSI: These are another chlorophyll a molecule, then another quinone: phylloquinone (Vitamin K1), three 4Fe-S4 clusters , and a protein with a 2Fe-2S cluster. This protein, called ferredoxin, interacts with PSI on the stroma side.
An enzyme ferredoxin-NADP reductase (FDR) catalyses the final stage: conversion of NADP into reduced NADP.
NADP + 2 H⁺ + 2 e^- → Reduced NADP The electron coming from PSII via plastocyanin is used to repay the electron debt left by the photoionisation of the P700 molecules.

Photophosphorylation

As protons are deposited in the lumen without a balancing anion (counterion) the charge separation establishes an electrical (potential) gradient as well as a concentration gradient due to the difference in proton concentration on either side of the membrane. In fact the proton concentration in the lumen may be about 100 times that in the stroma, corresponding to a pH of 6 in the lumen as opposed to 8 in the stroma. Some estimates give a lower lumen pH (in the light), so there could be an even higher difference in proton concentration.

Together these factors provide a proton-motive force.

The accumulation of protons within the lumen thus provides a source of energy which can be used in the production of ATP from ADP + Pi.

Protons can pass through the cylindrical section of the ATP synthase enzyme which spans the membrane, and as they move from one section to the next ATP is produced (1 ATP molecule per 4 protons ?). Since they pass from a region with high proton concentration to a lower one, the comparison with water crossing a membrane by osmosis from high to low concentration means that this is called a chemiosmotic process.

This is comparable to the ATP synthase in mitochondria.

Cyclic photophosphorylation

Under some circumstances, photosystem I operates without the participation of photosystem II.

Photons excite chlorophyll P700 and this emits electrons which pass along the chain of electron acceptors but instead of involving NADP reductase, ferredoxin passes the electrons back to plastoquinone, thus shuttling protons across the membrane and into the lumen. The electrons pass on to cytochrome b₆f, and then to plastocyanin before returning to chlorophyll P700.
This process does not require the continuous input of an electron donor like water.
As a result :

• ATP is produced because of the proton motive force caused by the concentration of protons
• Water does not need to be broken down and no oxygen is released
• No reduced NADP is generated.

The light-independent reaction

Do not call this the dark reaction, or fall into the trap of assuming that it occurs later on at night.
It follows directly after the light-dependent reaction.

It is helpful to keep track of the number of carbon atoms (and phosphate groups) in the various compounds. The chemical conversions are all part of a cycle - usually called the Calvin cycle.

Carbon dioxide reacts with ribulose bisphosphate (RuBP) to form two molecules of glycerate 3-phosphate (GP). This reaction is catalysed by the enzyme RuBisCo.


Glycerate 3-phosphate is reduced to triose phosphate (3-C sugar with 1 phosphate), using ATP and reduced NADP from the light-dependent reaction.

Most of the triose phosphate (10 out of 12 molecules) is used in a multi-stage process to regenerate RuBP, so as to repeat the process of accepting carbon dioxide.

The rest of the triose phosphate is the product of the light-independent reaction, and 2 molecules of it are produced for every 6 molecules of CO₂ which enter the cycle.

This is converted to useful organic substances required by the plant, e.g. glucose (a 6-C sugar) which may be converted (by polymerisation) into other carbohydrates, e.g. cellulose for cell walls or stored as starch. It can also be converted into other classes of organic compound, e.g. lipids, amino acids and then proteins and nucleic acids.

The Calvin cycle

Melvin Calvin (1911–1997) was an American biochemist at the University of California, Berkeley.

Along with Andrew Benson and James Bassham, Calvin discovered the plant metabolic cycle which bears his name, sometimes together with the others.
He (alone) was awarded the 1961 Nobel Prize in Chemistry.

Calvin used this 'lollipop apparatus' in which he grew the green alga Chlorella in a liquid containing carbon dioxide labelled with radioactive ¹⁴C and he took extracts after illuminating it for different (short) periods of time.
Two-dimensional chromatograms were prepared and these were covered with photographic paper and left (in the dark) for some time before being processed in the normal way to develop a photographic image - an autoradiogram. Dark splodges showed the position of radioactive organic compounds resulting from the reaction, which were then identified. Reducing the time (and increasing the intensity of light) allowed the sequence of products to be determined.
It is interesting to note that in addition to carbohydrate products, amino acids have been formed in the later sample.
What is meant by a two-dimensional chromatogram?
> Chromatogram that is run in one direction, then dried, rotated through 90°, and run again, probably with a different solvent

What are its advantages?
> To separate overlapping compounds

The LIR in more detail

The following events take place within the stroma - a larger fluid-filled space outside the thylakoids which is surrounded by the two outer chloroplast membranes - each of which is itself a lipid bilayer, with embedded proteins. The lipid compositions of these membranes differ from one another and from most other cell membranes which are mostly phospholipid. In fact the outer membrane is about 48% phospholipids, 46% galactolipids and 6% sulpholipids, whereas the composition of the inner membrane is 16% phospholipids, 79% galactolipids and 5% sulpholipids. This is quite similar to composition of the thylakoid membrane.

The chemical reactions which take place in the stroma are a cycle of enzyme-controlled covalent conversions and do not rely directly on electron transfer which necessitates a membrane-bound chain of redox carriers.

Carbon dioxide fixation

Ribulose 1,5 bisphosphate (RuBP) is a double phosphate ester of the ketopentose sugar ribulose. This is similar to ribose - a 5-carbon sugar - but it has a >C=O keto group at carbon 2, and it is 'linear' not ring-shaped as it has two phosphate groups, one on each end of the molecule.

RuBP is carboxylated (it reacts with carbon dioxide) to form an unstable compound, 3-keto-2-carboxyarabinitol 1,5-bisphosphate.
The enzyme RuBisCO (Ribulose Bisphosphate CarbOxylase) which catalyses this reaction is said to be the most common protein in the world as it makes up 20-25% of the mass of protein in most leaves and it is produced at a rate of about 1000 kg/second on earth.

The diagrams above show molecules in zigzag skeletal format,
but the Carbon coming in from CO₂ is shown as a C
The unstable 6-carbon, 2 phosphate compound splits in the middle to form 2 molecules of glycerate-3 phosphate - GP - a 3-carbon, 1 phosphate compound similar to glycerol but with a carboxylic acid group - shown on the right as -COO^- (conjugate base of glyceric acid).

Reduction

GP is further phosphorylated using ATP to form glycerate 1,3 bisphosphate (G1,3BP) (1,3 phosphoglycerate).

The next stage is effectively the same as in glycolysis (steps 6&7), but in reverse.


G1,3BP is reduced by reacting with reduced NADP which provides protons and electrons.

The resulting products are glyceraldehyde 3-phosphate (G3P, also abbreviated to PGA, PGAL or GALP and others!) and inorganic phosphate. G3P is possibly best known as triose phosphate (TP), and some of this is converted into useful compounds for the plant. Typically 2 x G3P out of 12 molecules in the cycle can be converted into fructose 1,6 bisphosphate, fructose 6 phosphate, then glucose 6 phosphate, then glucose.

Regeneration of RuBp

The rest of the triose phosphate (10 out of 12 molecules) undergoes molecular rearrangement - in 8 stages - to form ribulose monophosphate - ribulose 5-phosphate, Ru5P. This is then phosphorylated to form ribulose 1,5 bisphosphate again, so the cycle can continue.

Both RuBP and ADP have two phosphate groups.
Why do we write RuBP as ribulose bisphosphate, but ADP as adenosine diphosphate?
> In ADP both phosphates are attached (in a row) to the same part of the molecule, in RuBP there are are 2 different parts - at opposite ends of the molecule

Photorespiration - the downside of RuBisCO

As well as catalysing the (carboxylation) reaction between RuBP and carbon dioxide, the enzyme RuBisCO can also catalyse the (oxygenation) reaction between RuBP and oxygen.
Presumably the molecules of CO₂ and O₂ are similar in size and shape so that both can fit into the active site of RuBisCO.

This reaction, known as photorespiration, is wasteful as it results in the production of 2-phosphoglycolate at well as glycerate 3-phosphate (2 molecules of which are normally produced). The 2-phosphoglycolate is processed via a different cycle before being recycled into RuBP to re-enter the Calvin cycle, and carbon dioxide is lost in the process.

This is a drawback to the normal ('C₃') photosynthesis, as described above. It is more marked in conditions of low carbon dioxide concentration - especially when stomata are closed due to drought - or higher temperature. Some plants use alternative methods of trapping carbon dioxide, using the enzyme phosphoenolpyruvate carboxylase, which produces 4-carbon compounds that are exported to another part of the leaf with a low oxygen concentration where CO₂ is released to be accepted by RuBisCO in the normal way, avoiding oxygenation.

Some of the world's most economically important (tropical, grasslike) crops - sweetcorn or maize (Zea mays), sugarcane (Saccharum officinarum), sorghum (Sorghum bicolor), and millets (several species) - use this C₄ photosynthesis.

Most succulent plants and cacti use another alternative: Crassulacean Acid Metabolism CAM in which CO₂ is captured at night when it is cooler and there is less water loss via open stomata, and it is converted into C4 compounds and malic acid which is stored in the vacuole. During daytime they release CO₂ from their reserves to perform photosynthesis when stomata are closed and water conservation mechanisms operate.

Evolution of photosynthesis

Early conditions on planet Earth were very different than today: Many environments were anaerobic and reducing, rather like hydrothermal vents today.
There was practically no oxygen in the atmosphere, but a higher amount of carbon dioxide
This meant that there was no ozone layer and much more ultraviolet light.
Carbon dioxide dissolved to produce hydrogencarbonate ions.

Originally there must have been bacteria with an established respiration process involving movement of electrons between a number of redox molecules like haem and cytochromes. Within one of these a single photosystem evolved, as magnesium replaced iron in the porphyin ring of haem and the pigment chlorophyll was formed, and this absorbed energy from light.

This early photosystem probably took electrons from hydrogen sulphide or iron, and using chlorophyll passed them into the respiratory cycles enabling organic compounds like sugars to be produced from CO₂.

Another version of the photosystem emerged which probably took excited electrons from chlorophyll and used them to move protons across the membrane and create ATP before returning the electrons to chlorophyll. This is similar to cyclic photophosphorylation. Both of these systems would differ somewhat and evolved to be more efficient in different environments.

It is thought that (possibly following gene exchange) some bacteria had both systems, but they had a mechanism which allowed them to use only one at a time, avoiding unnecessary synthesis of intermediates: the first provided organic compounds for growth when hydrogen sulphide was available, and the second gave ATP for maintenance when it was unavailable. This is called the "redox switch hypothesis".

Nowadays, several groups of bacteria perform photosynthesis but most have only photosystem I or photosystem II; only cyanobacteria have both systems available, and in some species they switch off PS II when performing nitrogen fixation, which is inhibited by oxygen.

Conceivably depletion of raw materials meant that these bacteria were subject to selection pressure. Manganese, which is quite common on the ocean floor today, absorbs ultraviolet radiation which could be destructive to organic molecules within cells, so it would act as a sort of antioxidant screen to this radiation. In doing so it emits electrons which would be absorbed by chlorophyll which has lost electrons as it absorbed light energy.

It is thought that cyanobacteria used manganese to split water, which is a much more widely available chemical resource. Furthermore it is thought that bypassing the redox switch enabled the two systems to work in synchrony. The resulting continuous electron flow could then be passed from one photosystem to the other, and used to produce both ATP and reduced NADP.

This double photosystem with the manganese-based oxygen-evolving complex became the standard combination when chloroplasts were formed by the coalescence of two cells, forming an early single-celled organism like an alga, from which many-celled plants presumably evolved. Cyanobacteria and higher plants have transformed Earth's atmosphere by adding oxygen obtained by splitting water, and this has allowed a number of aerobically respiring organisms to evolve.
See 'endosymbiont theory' (link below).

Most of the previous section dealt with the light-dependent reaction.
Several versions of the light-independent reaction (C₃, C₄, CAM) have been mentioned above, but earlier versions probably used respiration pathways (running in the opposite direction to 'normal').

For example, green sulphur bacteria are currently often found in deep waters, near to hydrothermal vents which emit a dull light as well as various mineral ions which provide reducing conditions resulting in an anoxic environment. Unsurprisingly, they are anaerobic.
They employ PS I for photosynthesis, using several bacteriochlorophylls which absorb light at 720-750 nm but the P840 chlorophylls at the reaction centre emit electrons which are passed to ferredoxin for reduction of NADP. Electrons are repaid following the oxidation of sulphide, resulting in the accumulation of elemental sulphur. The fixation of carbon dioxide is achieved by a reaction which is the complete reverse of the tricarboxylic acid (Krebs, citric acid) cycle, so they use a respiratory pathway.

Environmental factors that limit the rate of photosynthesis

Four factors interact to affect the rate at which photosynthsis proceeds.

Light intensity

Since light powers the light dependent reaction, the overall rate of photosynthesis is proportional to the intensity of light, provided that other factors are at acceptable levels.

Wavelength of light

Normal (white) light has a spectrum of different wavelengths - seen as different colours.
The action spectrum for photosynthesis (above) has peaks at either end of the spectrum, but it dips down in the central section -between 500 and 600 nm.

Chlorophyll absorbs light most strongly in the blue and red regions of the spectrum, as shown by the peaks at either end of the absorption spectrum shown above. Other pigments absorb light at slightly different wavelengths so the action spectrum for the photosynthesis process is not so sharply cut as the absorption spectrum here.

What happens to light in the central (lower) section, and what is the consequence of that to us and to the plant? (There is a clue there!)
> It is reflected off so we see the colour of chlorophyll and plant leaves as green
> Plants do not photosynthesise efficiently in green light

Temperature

The movement of molecules (especially in the light dependent reaction) is faster at higher temperatures and chemical conversions (especially in the light independent reaction) are under the control of enzymes which can be denatured at higher temperatures, so there is an optimum temperature for the overall photosynthesis process.

Different processes in the life cycle of plants are also affected by temperature so the overall growth of plants may have a slightly different optimum temperature.

Carbon dioxide concentration

Carbon dioxide is a reactant in the light independent reaction so it is a rate limiting factor. The normal concentration of carbon dioxide in the atmosphere is quite low: about 0.04%, and increases in the level of CO₂ around plants will raise the overall rate of photosynthesis. This will plateau if other factors such as light intensity or temperature are limiting.

Experiments to investigate the effect of environmental variables on the rate of photosynthesis

Some pointers to help you devise your own experiments
Aquatic plants like Elodea (canadensis) are available from shops specialising in tropical aquarium supplies.

When illuminated in a glass tube containing water or sodium hydrogencarbonate solution, sprigs of these plants give off fairly consistent steams of bubbles of gas (oxygen) from their cut surface so they are usually inserted upside-down, and counting the number (in a given time period) gives an estimate of rate of photosynthesis. The bubbles could be collected into a (very small) calibrated tube so the volume coild be measured.

The intensity of light may be varied by altering the distance between the light source (microscope light, in a darkened room) and the plant material, or by using an electronic dimmer, which may make the quality of the light change.
Colour of light may be varied using plastic sleeves of different colours.
The hydrogencarbonate ion (HCO₃^-) is the effective form of carbon dioxide in solution so different concentrations of sodium hydrogencarbonate solution may be used to show the effect of varying CO₂ concentration (much easier than varying gas concentration!)

Algal suspensions such as Scenedesmus quadricauda are quite popular for photosynthesis demonstrations and student experiments. Immobilised algal beads - algae mixed with sodium alginate solution, then dropped into calcium chloride solution to 'set' them - are an improvement for several reasons:
- they enable the amount of photosynthetic material to be standardised (count the balls!)
- they leave the liquid above them uncoloured and so chemical indicators can be used to determine the pH and hence the rate of consumption of CO₂ can be estimated, giving another way of calculating rate of reaction.

Leaf discs cut with (larger) cork-borers can be used for experiments, perhaps floating in liquids in Petri-dishes. They can be decolorised by boiling in alcohol and residual starch visualised with iodine solution.

Chloroplasts are fairly easily extracted from leaves. Spinach is most often used as a starting point, but Arabidopsis thaliana is sometimes used.

Chromatography of photosynthetic pigments

Biological molecules can be separated by using chromatographic techniques.

A sample of leaves are ripped into roughly centimetre-square pieces, placed in a small quantity of solvent such as propanone, together with a pinch of sand (or magnesium sulphate crystals), and ground using a pestle and mortar or small blender, resulting in a dark green liquid.

A piece of chromatography paper or TLC (thin-layer chromatography) paper is cut to fit the apparatus, and a pencil 'start line' is drawn across it about 1-2 cm from one end. Perhaps a point is cut in the end below this.

The liquid is applied to the start line - also called the origin - using a very fine capillary tube or a Pasteur pipette. After this has been left to dry it can be repeated (several times).

The paper is then trimmed or folded to fit and attached to the stopper which fits on top of the apparatus.

A small layer of solvent is placed into the bottom of the apparatus which is placed in position.

The paper is lowered in, checking that the origin line is above the (solvent) liquid level.

This is then left for some time as the liquid soaks into the paper and moves up gradually by capillarity.

When the liquid has nearly reached the top, the paper is removed and the solvent front is immediately marked, again with pencil.

The paper is normally taken away to dry, preferably in a fume cupboard.

What is the function of the sand?
> It helps break open cells and release pigments
What advantage do magnesium sulphate crystals give?
> They dissolve so there is no grit to block pipettes - will also draw liquid out by osmosis.
Why is the green liquid at room temperature, not ice-cold like in the DCPIP experiment?
> No enzymes are involved
Why is a pencil (not a ballpoint pen) used to draw lines?
> Ink would dissolve and move so starting position would be lost
Why is the start line annointed several times?
> To increase concentration of extract and make colours darker.
Why is the solvent front marked?
> All the Rf values depend on this: each component's distance moved is divided by the distance between the start line and this point.
And it will be invisible after drying.
What would happen if the liquid level at the start was higher than the origin line?
> Pigment would spread out into the liquid rather than move up the paper
The diagram below shows the resulting separation of several spinach leaf pigments by paper chromatography.


Consider the effects on the separation process of the following chemical and physical properties of the individual components (pigments) in the mixture of pigments from the leaf:

• solubility of component in the solvent used:
> more soluble substances move further/faster
• molecular weight/size of component:
>smaller ones move further/faster
• physicochemical interaction between solvent and component:
>similar hydrophobicity/H bonding causes more movement
• component's adhesion to paper:
>more adhesion causes less movement

Using the rule at the side, calculate the (approximate) Rf value for the following components:

• chlorophyll b
>1.1/7.5 = 0.13 to 1.4/7.5 = 0.19
• xanthophyll:
>3.5/7.5 = 0.47 to 3.8/7.5 = 0.51
• carotene
>6.8/7.5 = 0.91 to 7.3/7.5 = 0.97

Common agricultural and horticultural practices used to overcome the effect of these limiting factors

Some of these practices do not relate directly to photosynthesis but to other processes which contribute to overall crop productivity.

Agriculture

Growing crop plants in open fields, with no overhanging trees to block the light is so common that we see this as normal or natural in first world countries, but it is now seen as in opposition to biodiversity, especially when hedges are removed and field sizes are increased.

Clearing natural forests in tropical countries in order to maximise production of economically important species is often criticised: Large areas of Amazon rainforest have been destroyed in order to grow soybeans or create grassland prairies for cattle production. In south east Asia, especially Indonesia and Malaysia, much biodiverse rainforest has been cut down to permit Palm Oil production, with consequent habitat loss for many species including Orang-Utans and Sumatran tigers.

It is clear that much supermarket produce is grown in other parts of the world, which are generally sunnier and warmer. Production costs e.g. workers' pay may be lower, and the cost of transport must be economically viable, but the 'air miles' are another factor to be considered.

Horticulture

However much more control over all the limiting factors for photosynthesis is possible when crops are grown "under glass", i.e. in glasshouses (aka greenhouses).


Each factor adds to the rate of photosynthesis.
The main effect, noticeable in simple glasshouses, is increase in temperature.

Some plants like tomatoes are grown on support gantries which are winched to take growing tips to higher levels, exposing them to more light and heat.
In more high-tech horticultural enterprises, extra light may be provided by overhead electrical illumination, and this allows year-round production of tomatoes, cucumbers and peppers (see Thanet Earth).

Artificial illumination can also be used to extend the photoperiod, i.e the length of 'daylight time' within the 24-hour cycle, which may influence production of flowers, e.g. Poinsettia, and also crop plants which obviously flower before they produce fruit.


Modern LED systems can provide more light in the red and blue ends of the visible spectrum, which is more efficient in activating chlorophyll and increasing rate of photosynthesis.


This has even allowed plants to be grown in underground chambers where natural light does not enter.
This has opened up the possibility of growing salad vegetables for city restaurants in abandoned underground train stations, and growing cannabis in unexpected places like boarded-up domestic property, or nuclear bunkers.


Intensive producers heat their greenhouses by burning gas and they may release the gaseous products into the growing environment, raising the carbon dioxide concentration there. Similarly electrical generators powered by fossil fuels provide heat and carbon dioxide as well as electricity to power illumination and electrical equipment.
Elevated levels of carbon dioxide may cause drowsiness, headaches and other conditions in workers , and there is a workplace exposure limit of 5,000 ppm - 0.5% CO₂ - (as 8-hour TWA - time-weighted average).

In addition, if temperatures rise too high, ventilators can be opened or large sunscreens may be rolled out to provide shade, thus optimising the temperature.