Chlorophyll

Overview of the pigment Chlorophyll, its sources, types, structure, photoreceptors, benefits, stability, degradation, preservation during food processing and technologies associated with it.

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Chlorophyll






Chandrima Shrivastava
13FET1001
S.Y. B. Tech (Food Engineering and Technology)
Institute of Chemical Technology, Mumbai

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I. INTRODUCTION
The green pigments of photosynthetic organisms are known collectively as the chlorophylls.
The term chlorophyll derives from the Greek words chloros, meaning "green" and phyllon,
meaning "leaf". The basis of chlorophyll is a macrocycle containing four pyrrole rings and
Mg
2+
ion in the centre. The side chains contain hydrocarbon radicals of various lengths and
saturations, and oxygen-containing functional groups.
In most green plants, chlorophyll a and b are in the ratio 3:1. These compounds are found in
the chloroplasts of photosynthetic tissues and are located in the thylakoids, the
photochemically active biomembranes.
Chlorophyll c is found together with chlorophyll a in marine algae, dinoflagellates, and
marine diatoms.1, 2 Chlorophyll d was discovered to be a minor constituent of red algae
(Rhodophyta) by Manning and Strain.
3

Chlorophyll f is a type form of chlorophyll that absorbs further in the red (infrared light) than
other chlorophylls. It is a relatively new discovery and chlorophyll f is made by unnamed
filamentous bacterium.
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II. FOOD SOURCES
Chlorophyll is present in all green plants, most of the algae and cyanobacteria. Chlorophyll is
abundant in leafy green vegetables and generally to a lesser extent in fruits. In spinach,
chlorophyll can be as high as 1% on a dry weight basis. Chlorophyll a is abundantly found in
Chlorella and Spirulina. Chlorella is called ‘Emerald food’ due to its extremely high
chlorophyll content, which is around 7% of the biomass. Chlorophyll is found in green
leaves, fresh herbs, blue-green algae, sprouts, wheatgrass, green vegetables & fruits, sea
vegetables - seaweeds.

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III. STRUCTURE
Both chlorophylls a and b are derivatives of dihydroporphyrin chelated with a centrally
located magnesium atom. Chlorophyll b differs from chlorophyll a only in one of the
functional groups bonded to the porphyrin (a -CHO group in place of a -CH3 group).
Chlorophyll c is closely related to chlorophylls a and b, and chlorophyll d is similar to
chlorophyll a except the vinyl group is replaced by a formyl group. The structures of
chlorophyll a and b are shown in Figure 1.

Figure 1: Structural formula of a and b type chlorophylls.
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Attached to the porphyrin is a long, C20 hydrophobic carbon-hydrogen chain which interacts
with the proteins of the thylakoids and serves to anchor the molecule in the internal
membranes of the chloroplast. Alternating single and double bonds, known as conjugated
bonds, such as those in the porphyrin ring of chlorophylls, are common among pigments, and
are responsible for the absorption of visible light by these substances. Both chlorophylls a
and b primarily absorb red and blue light, the colors most effective in photosynthesis.

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IV. CHEMISTRY
Chlorophyll contains a fully conjugated tetrapyrrole system (18 -electrons), and therefore
absorbs light in the visible range. It is the main structural unit of photosynthetic light-trapping
devices of green plants, which are nanosized supramolecular complexes containing up to
several hundred pigments from the protein environment. The main function of chlorophyll is
to absorb light, transform the light energy into electronic one and pass it to neighbouring
molecules by van-der Waals (dipole-dipole) interactions. The chain of chlorophyll transmits
electron energy to the photosynthetic reaction centre, where it is used for charge separation
and subsequent redox reactions. Chlorophylls are also contained in the reaction centres of
green plants, where they play the role of the primary electron donor.
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In purple and green bacteria, bacteriochlorophyll perform the functions of chlorophyll.
Unlike chlorophyll, they have one or two pyrrole rings partially hydrogenated, due to which
they absorb light of longer wavelength than chlorophylls.
Chlorophyll derivatives:
The central Mg atom is easily removed, particularly under acidic conditions, replacing it with
hydrogen and thus forming pheophytins. Hydrolysis of the phytyl group of pheophytin with
acid or alkali can proceed, forming the pheophorbides. Cleavage of the phytyl group without
removal of Mg atom, usually catalyzed enzymatically by chlorophyllase, produces the
chlorophyllides. Prolonged heating causes decarbomethoxylation at C-10 giving rise to pyro
derivatives.
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Figure 2 shows the relationship of chlorophyll with some of its derivatives.

Figure 2: Relationship of chlorophyll to some of its derivatives
9

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Chlorophyll as a photoreceptor:
Chlorophyll a and b are very effective photoreceptors because they contain a network of
alternating single and double bonds, and the orbitals can delocalise stabilising the structure.
Such delocalised polyenes have very strong absorption bands in the visible regions of the
spectrum, allowing the plant to absorb the energy from sunlight. Figure 3 depicts the
absorption spectra for Chlorophyll a and b.
The different side groups in the 2 chlorophylls 'tune' the absorption spectrum to slightly
different wavelengths, so that light that is not significantly absorbed by chlorophyll a, at, say,
460 nm, will instead be captured by chlorophyll b, which absorbs strongly at that wavelength.
Thus these two kinds of chlorophyll complement each other in absorbing sunlight. Plants can
obtain all their energy requirements from the blue and red parts of the spectrum, however,
there is still a large spectral region, between 500-600nm, where very little light is absorbed.
This light is in the green region of the spectrum, and since it is reflected, this is the reason
plants appear green. Chlorophyll absorbs so strongly that it can mask other less intense
colours. Some of these more delicate colours (from molecules such as carotene and quercetin)
are revealed when the chlorophyll molecule decays in the autumn, and the woodlands turn
red, orange, and golden brown. Chlorophyll can also be damaged when vegetation is cooked,
since the central Mg atom is replaced by hydrogen ions. This affects the energy levels within
the molecule, causing its absorbance spectrum to alter. Thus cooked leaves change colour -
often becoming a paler, insipid yellow-green.
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Figure 3: Absorption spectra of Chlorophyll a and b

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V. ALTERATIONS DURING FOOD PROCESSING
Colour is a major quality attribute of vegetable products. The most common alteration that
occurs in green vegetables is the conversion of chlorophylls to pheophytins, causing a
dramatic color change from bright green to olive brown.
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This conversion is enhanced
by extended heat treatment and is dependent upon the amount of acids formed during
processing and storage. Mild heat treatment, such as in vegetable blanching, induces the
formation of the C-10 epimers producing chlorophyll a’ and b’. During prolonged heat
treatment, such as in canning, almost all chlorophylls are converted to pheophytins and
pheophytin epimers.
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In some canned products, pyropheophytins were determined as the
predominant chlorophyll derivatives. The mechanism for chlorophyll decomposition during
canning is a two-step process: Chlorophyll  Pheophytin  Pyropheophytin
Generally, total destruction of chlorophylls results in the formation of pheophytins and
pyropheophytins. The pH of the product decreases with heat treatment, inducing further
pheophytinization. The extent of pheophytin formation was considered to be related to the
severity of the heat treatment.
In addition to the effects of pH, heat and metal complexes, other causes for the
decomposition of chlorophyll in foods have been suggested. In fermented products, such as
cucumbers (and wild rice), chlorophyllides and pheophorbides are produced in addition to the
pheophytins. The presence of pyropheophytin is attributed to the heat generated during
fermentation.
VI. STABILITY
Chlorophylls can be destabilised by high temperatures depending on the pH of the
environment. In an alkaline solution (pH 9), chlorophyll remains stable, but in an acidic
solution (pH 3) it is highly unstable. When plant material is heated, i.e. cooked, the plant cell
membranes break down and cause acid to be released, thus decreasing the pH of the
surrounding solution. The magnesium ion located at the centre of the porphin ring is normally
stable and difficult to remove, but at lower pH, the Mg
2+
ion is displaced by two H
+
ions,
resulting in formation of an olive brown pheophytin comlpex.
The heat induced cellular degradation can also predispose the chlorophyll pigments to photo-
degradation in which they chemically decompose in the presence of light.

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VII. DEGRADATION
Despite the importance of chlorophyll decomposition as a measure of ripening and quality,
the biochemistry of this process remains a biological enigma. Essentially, the process
involves release of chlorophyll from its protein complex followed by dephytlization and
possibly pheophytinization. Oxidation of the ring structure to chlorins occurs and ultimately
colorless end products form. Chlorophylls are degraded in the chloroplast by enzyme-
catalyzed process via pheophorbide a and the red chlorophyll catabolite (RCC) to give
primary fluorescent chlorophyll catabolites (pFCC, or its Cl-epimer, epipFCC). pFCCs are
modified further by unidentified hydroxylating enzymes. When carrying a free propionic acid
group, FCCs are transported into the vacuole where they isomerize by a spontaneous acid
catalyzed reaction to the corresponding nonfluorescent chlorophyll catabolites (NCCs).
EXAMPLES OF CHLOROPHYLL DEGRADARION
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Fruit maturation (on or off the plant):
 Ripening of tomatoes (chlorophyll  colorless compounds); induced by ethylene
 De-greening of bananas (chlorophyll  colorless compounds); induced by ethylene
 De-greening of lemons and oranges (chlorophyll  colorless compounds); induced
by ethylene
Processing:
 Canning of peas [chlorophyll  pheophytin (olive brown)]; induced by heat
 Brining of olives [chlorophyll  pheophytin (olive brown)  pheophorbide (olive
brown) and chlorophyll  chlorophyllide (blue green)  pheophorbide]; induced by
acid and chlorophyllase
 Blanching of snap beans, turnip greens and okra [chlorophyll  pheophytin (olive
brown)]; induced by heat
 Coleslaw processing [chlorophyll  pheophytin (olive brown)  pheophorbide
(olive brown)]; induced by acid and chlorophyllase
Senescence:
 Ready-to-eat salads (chlorophyll  colorless compounds); induced by ethylene
 Stored cabbage heads (chlorophyll  colorless compounds); induced by ethylene

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Figure 4: Possible chlorophyll degradation pathways in plant tissues or in processed foods.
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RCC = red chlorophyll catabolite
FCC = fluorescent chlorophyll catabolite
NCC = non-fluorescent chlorophyll catabolite

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A possible pathway for chlorophyll degradation is shown in Figure 4. The pathway of
chlorophyll breakdown includes the following steps:
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1) The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle
2) The hydrolysis of chlorophyll into chlorophyllide catalyzed by chlorophyllase
3) The removal of Mg
2+
to form pheophorbide catalyzed by Mg-dechelating agent
4) The oxidative cleavage of the tetrapyrrole ring by pheophorbide oxygenase
5) The reduction of a double bond to yield colourless but fluorescent chlorophyll catabolites
(FCCs) by the red chlorophyll catabolite reductase (RCCR)
6) The modification of FCCs by hydroxylation, glycosylation and esterification
7) The transport of the modified FCCs to the vacuole, where the fluorescing nature of the
catabolites (NCCs) is abolished by tautomerization.
VIII. PRESERVATION
Food ingredients and processing conditions greatly influence the rate and pathway of
chlorophyll degradation. Chlorophylls are extremely sensitive to low pH, high temperatures,
and length of heat treatment, in addition to the presence of salts, enzymes and surface-active
ions. Approaches for lengthening the shelf lives of fresh-packed vegetables are freezing,
cooling, application of modified atmosphere packaging, and gamma irradiation.
Low temperature storage preserves chlorophylls; however, cold stored products may develop
chilling injury symptoms. For example, green beans stored at 4
o
C maintained brighter green
color and better quality than those stored at 8
o
or 12
o
C, but developed latent chilling injuries
after 8 days of storage that became evident when the pods were transferred to 20
o
C.
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Blanching is a very common thermal treatment to inactivate enzymes that catalyze down-
grading reactions during storage, like chlorophyllase, magnesium dechelatase and oxidative
enzymes such as lipoxygenases, chlorophyll oxidase, and peroxidases that contribute to the
loss of green color and accumulation of oxidized chlorophyll catabolites.
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Although blanching produces a reduction of the concentrations of oxygen in plant tissues and
thus better retention of pigments initially, the acidic medium progressively promotes the
replacement of the centrally located magnesium ion from the chlorophyll molecule by two
atoms of hydrogen, producing pheophytins after prolonged storage. Additionally,
photoxidation and other oxidative reactions seem to be involved in later stages of the
degreening process by contact with oxidized lipids in the presence of oxygen.
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Irradiation is an effective method of reducing microbial contamination. Treatment with
gamma radiation in combination with storage at 8
o
or 10
o
C was efficient for bacterial
decontamination and elimination of potential pathogens without altering sensory attributes
like color. The advantage of irradiation is that it causes fewer changes to the sensory
attributes than blanching, application of fumigants, steam, microwaves, and other
treatments.
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The loss of chlorophylls to pheophytins can be retarded when the moisture content of
vegetables is reduced or the water activity is lowered. At lower activities, the chlorophylls are
bound in nonreactive compartments, or the water is not available for the reaction to form
pheophytins. The addition of agents like KMnO4, which absorbs ethylene, and sorbitol, which
reduces water activity may avoid water loss and diffusion of volatile off-flavors and odors
produced during storage under anaerobic conditions.
Generally, brighter-green vegetables are viewed as more appealing than darker coloured
ones; by reducing cooking time and boiling vegetables with the pan lid off to allow the
escape of volatile acids, the production of pheophytin and discoloration can be minimised.
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Adding a small amount of sodium bicarbonate (NaHCO3) to the water during boiling may
also help to keep the vegetables green as this raises the pH. This results in the formation of
chlorophyllin that has an unrealistic bright color and the texture of the vegetable is extremely
mushy. Mushiness can be prevented by the addition of calcium acetate or any other calcium
salt which will prevent breakdown of the hemicellulose in the alkaline medium
A patented process using Zn
2+
or Cu
2+
salts can retain the bright green color of vegetables.
These ions substitute for Mg
2+
atom in chlorophyll and form zinc or copper complexes of
chlorophyll derivatives.

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In canned peas, the acidity of the plant cells leads to loss of green color. Artificial colorants
such as tartrazine and green S (dyes) are added to restore the color. A derivative of
chlorophyll, sodium copper chlorophyllin gives an acceptable blue-green color to canned
peas. Though copper is toxic, the concentration level is too low to be a toxic hazard.
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In the process of pickling green vegetables like cucumbers, green mangoes, string beans,
ampalya, etc., lactic acid is produced by fermentation when the materials are soaked in weak
vinegar or acetic acid. Under this process, chlorophyll is broken up into phycophytin with the
alteration of the color from green to yellowish or brownish or greyish green, which tend to
lower the grade of the finished product. However, if these materials are soaked in a solution
of one part per million of copper salt before pickling, the green pigment is retained.
During heat treatment, chlorophyll degrades to pheophytin, which in turn decomposes to
other degradation products. Both chlorophyll and pheophytin conversion can be minimized
by the addition of maillard reaction products (MRPs) to improve the colour stability.
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Gibberellic acid (GA) is used to maintain fresh product activity in many agricultural
commodities. It increases fruit firmness, and enhances storage and shelf-life of fruits and
vegetables. Since chlorophyll degradation is delayed in GA-treated crops (Lers et al., 1998),
leafy vegetables such as parsley and celery maintain their green color longer in storage.
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IX. TECHNOLOGY AND ADVANCEMENTS
(A) Production of Chlorophyll Using Algae
The cyanobacterium Spirulina platensis is an alternative source of the pigment chlorophyll,
which is used as a natural color in food, cosmetic, and pharmaceutical products. This micro
alga presents one of the highest chlorophyll contents found in nature, corresponding to 1.15%
of its biomass.
Chlorophyll is generally produced by Spirulina using fermentation process. It has been shown
that the composition of the cultivation medium, cellular age, and light intensity are the main
factors influencing chlorophyll content in S platensis biomass. Research studies suggest an
inverse relationship between light intensity and chlorophyll content. Chlorophyll can be
extracted from the algal cells using dimethyl sulfoxide. Chlorophyll production from
Chlorella can be carried out in open ponds as well as fermenters.
25

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(B) Artificial Leaf:
The organic chemist responsible for this achievement was Robert Burns Woodward, from the
Converse Memorial Laboratory at Harvard University. “Artificial trees” replicate the
photosynthesis process to create hydrocarbon fuel directly from sunlight. This could help
offset the emission of CO2 from fossil fuels, and create an unlimited supply of fuel for
transport.
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Solar cells that mimic nature have been created at North Carolina State University where
water-gel-based artificial leaves containing chlorophyll produce electricity. They extracted
chlorophyll from leaves and trapped it in a transparent jelly. They put tiny electrodes made of
carbon nanotubes in this jelly. When sunlight falls on chlorophyll, it seizes a photon and
releases an electron. The electron is carried away by the carbon nanotubes to make electricity.
This electricity can drive a cell in which water is split to form hydrogen and oxygen.
(C) The Importance of Chlorophyll as a Water Quality Parameter
The measurement and distribution of the phytoplankton population enables researchers to
draw conclusions about a water body’s health, composition, and ecological status. Monitoring
chlorophyll levels is a direct way of tracking algal growth. Surface waters that have high
chlorophyll levels are typically high in nutrients, generally phosphorus and nitrogen. These
nutrients cause the algae bloom, leading to depletion in dissolved oxygen levels - a primary
cause of most fish kills. High levels of nitrogen and phosphorus indicate pollution from man-
made sources, such as septic system leakage, poorly functioning wastewater treatment plants,
or fertilizer runoff. There are various techniques to measure chlorophyll, including
spectrophotometry, high performance liquid chromatography (HPLC), and fluorometry.
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(D) Chlorophyll-based Phototransistor:
Chlorophyll is one of the most efficient light-absorbing materials known to science. Shao-Yu
Chen at the Institute of Atomic and Molecular Sciences in Taiwan has incorporated
chlorophyll into graphene transistors to make light-activated switches. The new
phototransistor design is relatively simple. It consists of two gold electrodes connected by a
sheet of graphene. The graphene is then covered by a layer of chlorophyll using a method
known as drop casting. This involves placing a drop of liquid containing chlorophyll on top

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of the graphene and letting it evaporate. When the chlorophyll is zapped by light of certain
frequencies, the current increases dramatically as the light causes chlorophyll to release
electrons into the graphene and this increases the current that flows.
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(E) Infrared chlorophyll to boost solar cells:
Stromatolites are among the most primitive of life forms. Their cyanobacteria contain a
newly discovered form of chlorophyll, the fifth known, which absorbs sunlight in the red and
infrared part of the spectrum. It could be harnessed to help solar cells convert more light into
electricity. Min Chen of the University of Sydney in Australia found a completely new type
of chlorophyll – chlorophyll f – made by an as-yet unnamed filamentous bacterium.
Because over half of the light from the sun comes in at infrared wavelengths, the makers of
photovoltaic panels have been working on ways to extend the section of the spectrum that
solar cells can absorb to beyond red.
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(F) Chlorophyll can help prevent cancer:
According to a recent study at Oregon State University, chlorophyll and its derivative,
chlorophyllin in green vegetables offers protection against cancer when tested against the
modest carcinogen exposure levels most likely to be found in the environment. It binds with
and sequesters carcinogens within the gastrointestinal tract until they are eliminated from the
body.
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