PHYTOCHEMICAL

• Define the properties and classification of

phytochemicals.
• Compare the types of phytochemicals in
plants
 Introduction
• Plant metabolism
– Primary
• directly involved in normal growth, development, and reproduction of
plant species
• Eg. Amino acid, ethanol, water, lactic acid
– Secondary
• not directly involved in those processes, but usually has important
ecological functions like defences against predators, parasites and diseases,
for interspecies competition, and to facilitate the reproductive processes
• Eg. coloring agents, attractive smells, etc

Both: famous for their beneficial effects on human health

 What is phytochemical?
• Secondary metabolite
• Phyto = plant + chemical
– Natural substance in plant
– Bioactive compound
– Not vitamin

• phytochemicals are classes of compounds that are only found in plants that do
not also fall into the category of essential nutrients.
– phytochemicals are compounds that we ingest when we eat plants, but they are the parts
that aren’t absolutely needed by the body – in other words, they are not nutritionally
essential.

• Generally, the arils of fruit contain large amounts of organic acids, sugars, minerals,
and vitamins, but the peels contain higher amount of phenolic compounds than the
flesh.
Phytochemical classes
Class

Sub class
Classification are based on
• Biosynthetic origin
• Solubility properties
• Presence of certain key functional groups
Phenolic compound Terpenoids Nitrogen
•Recognized by their •Share lipid properties compounds
hydrophilic compounds •Biosysnthetic origin •Recognized by their
•Common origin from from isopentenyl positive responses to
the aromatic precursor phytophosphate either ninhydrin or the
shikimic acid Dragendorff reagent

Sugar and their Macromolecules

Organic acids, lipid •Easily separated from
and other classes of derivatives
•Water-soluble other constituents by their
compounds high molecular weights
carbohydrates
 Methods of extraction and isolation -The
plant material
• Ideally – fresh plant tissue and should be plunged into boiling alcohol
within a minutes of its collection
• Alternatively – may be dried before extraction
– Drying under controlled conditions to avoid chemical changes
– Dried as quickly as possible without using high temp, in a good air
draft
• Take note for the nature of the compounds eg:
– Essential oil – sensitive to temp change and decreased over time–
avoid drying
– Flavonoids and alkaloids – remarkably stable with time
– Tannin – better to extract from vacuum-dried fresh leaves rather than
air-dried
• Free of contamination and disease – not affected by viral, bacteria or
fungal infection
• Botanical identity of the plant – not to be mistakes – taxonomy expect
 Methods of extraction and isolation -The
extraction
• No precise mode of extraction – no right or wrong method of extraction
• In general – ‘kill’ the plant tissue
– Prevent enzymic oxidation or hydrolysis
– Plunging fresh leaf or flower tissue
– Suitably cut up where necessary
• Boiling ethanol/ alcohol – good all-purpose solvent for preliminary extraction
• Plant material can be macerated in a blender and filtered
• Extraction will be assumed completed when its completely free of colour –
repeat extraction
• Classical chemical procedure for dried material – Soxhlet apparatus
• The extract obtained is clarified by filtration and then concentrated in vacuo –
rotary evaporator
• For volatile compounds – needs special precaution and apparatus.
 Methods of separation
• Chromatography
– Paper chromatography (PC)
– Thin layer chromatography (TLC)
– Gas chromatography (GC)
– High performance liquid chromatography (HPLC)
• The choice of technique depends on the solubility properties and volatilities
of the compounds
– PC – applicable to water-soluble compounds
– TLC – separating lipid-soluble compounds
– GC – volatile compounds
– HPLC – less volatile compounds and polar compounds
• Capillary electrophoresis, liquid-liquid extraction, droplet counter-current
chromatograhy (DCCC), affinity chromatography, differential
ultracentrifugation
 Methods of identification
• Isolation and purification
• First to determine the class of compound
• Then to find out which particular substance it is
within that class
• Methods
– UV and visible spectroscopy
– Infrared spectroscopy (IR)
– Mass spectroscopy (MS)
– Nuclear magnetic resonance spectroscopy (NMR)
Class

Sub class
Phenolic compounds
 Introduction
• Wide range of plant substances which possess in common an aromatic
ring bearing one or more hydroxyl substituent
• Phenolic substances tend to be water-soluble, since they are usually
located in the vacuole.
• The flavonoids form the largest group but simple monocyclic phenols,
phenylpropanoids and phenolic quinones all exist in considerable
numbers.
• Several important groups of polymeric materials in plants – the
lignins, melanins and tannins – are polyphenolic
• Function of some classes of phenolic compound – eg. The lignins as
structural material the cell wall; the anthocyanins as flower pigments,
• Phenolic compounds are all aromatic, so that they all show intense
absorption in the UV region of the spectrum
 Phenols and phenolic acids
• The free phenols and phenolic acids are best considered
together since they are usually identified together during plant
analysis
• Acid hydrolysis of plant tissue releases a number of ether-
soluble phenolic acids
• These acids are either associated with lignin combined as ester
groups or present in the alcohol-soluble fraction of the leaf,
maybe present in the alcohol-soluble fraction bound as simple
glycosides.
• Free phenols are relatively rare in plants
• Simple phenols are included in here because their identification
is important to determining the structure of flavonoid.
 Phenylpropanoids
• Naturally occurring phenolic compounds which have an
aromatic ring to which a three-carbon side-chain is attached.
• Derived biosynthetically from the aromatic protein amino
acid phenylalanine and they may contain one or more C6-C3
residue.
• The most widespread -hydroxycinnamic acids – which are
important not only as providing the building blocks of lignin
but also in relation to growth regulation and to disease
resistance.
• Four hydroxycinnamic acids are common in plants – ferulic
acid, sinapic, caffeic and p-coumaric acids.
 Flavonoid pigments
• Derived from the parent substance flavone – more
general
• Classes of flavonoid
• Mainly water-soluble compounds
• Can be extracted with 70% ethanol and remain in
the aqueous layer, following partition of this
extract with petroleum ether.
 Properties of different flavonoid classes
Flavonoid class Distribution
Anthocyanins scarlet, red, mauve and blue
flower pigment; also in leaf
and other tissue
Proanthocyanidins mainly colourless, in
heartwoods and in leaves of
woody plants
Flavonols mainly colourless
copigments in both cyanic
and acyanic flowers;
widespread in leaves
Flavones as flavonols
Characteristic properties
water-soluble, visible max
515-545 nm, mobile in
BAW on paper
yield anthocyanins (colour
extractable into amyl
alcohol) when tissue is
heated for 0.5h in 2 M HCl
after acid hydrolysis, bright
yellow spots in UV light on
Forestal chromatogram;
spectral max 350-386nm
after acid hydrolysis, dull
absorbing brown spots on
Forestal chromatogram;
spectral max 330-350nm
 Properties of different flavonoid classes
Flavonoid class Distribution Characteristic properties
Glycoflavones as flavonols contains C-C linked
sugar; mobile in water
unlike normal flavones
Biflavonyls colourless; mainly confined to the on BAW chromatograms
gymnosperms dull absorbing spot of
very high Rf
Chalcones and yellow flower pigments; give red colours with
aurones occasionally present in other tissue ammonia (colour change
can be observed in situ),
visible max. 370-410
Flavanones colourless; in leaf and fruit give intense red colours
(especially in Citrus) with Mg/HCl;
occasionally an intense
bitter taste
Isoflavanons colourless; often in root; only mobile on paper in water;
common in one family, the no specific colour tests
Leguminosae available
 Flavonoid
• Flavonoid are phenolic - change in colour when treated with base or
ammonia.
• Flavonoid contain conjugated aromatic systems - show intense
absorption bands in the UV and visible regions of the spectrum.
• Flavonoid generally present in plants bound to sugar as glycosides
and any one flavonoid aglycones may occur in a single plant in
several glycosidic combinations.
• When analysing flavonoids, it is usually better to examine the
aglycones present in hydrolysed plant extracts before considering the
complexity of glycosides that may be present in the original extract.
• Flavonoids are present in plants as mixtures and it is very rare to find
only a single flavonoid component in a plant tissue.
• In addition, there are often mixtures of different flavonoid classes.
Quercetin 3-O-glycoside 5-O-
rhamonside @
Quercetin 3-rutinoside
 Anthocyanins
• Most important and widespread group of
coloring matters in plant
• These intensely coloured water-soluble pigments
are responsible for nearly all the pink, scarlet,
red, mauve, violet and blue colours in the petals,
leaves and fruits
• The anthocyanins are all based chemically on a
single aromatic structure, that of cyanidin, and all
are derived from this pigment by addition or
subtraction of hydroxyl group or by methylation
or by glycosylation
• 6 common anthocyanidins (anthocyanin aglycones
formed when anthocyanins are hydrolysed with acid)
– Magenta-colored (cyanidin)
– Orange-red colored (pelagornidin)
– Mauve, purple and blue colour (delphinidin)
– Peonidin
– Petunidin anthocyanin methyl esthers
– Malvidin
• Occurs with various sugars attached – glucose,
galactose, rhamnose, xylose or arabinose
• The number of sugar units (mono, di- or tri-
glycoside) and the position of attachment of
sugar(usually to the 3-hydroxyl, or to the 3-
and 5- hydroxyls.
• Anthocyanin occur acylated with either an organic acid such
as malonic or with aromatic acid such as p-coumaric acid
• Acylation is commonly through the sugar unit of the 3-
position and both type of acylation may be present in the
same molecule.
• Anthocyanin are unstable in neutral or alkaline solution

• Stable in acid solution but the colour may fade due to

exposure to light

• Anthocyanin must therefore be extracted from plants

with solvents containing acetic or hydrochloric acid and
solution should be stored in the dark and preferable
refrigerated.
 Flavonols and flavones
• Flavonols occur most frequently in glycosidic
combination (glycone/presence of sugar)
• 3 at common – kaempferol, quercetin, myricetin
• Flavones only differ from flavonols in lacking a 3-
hydroxyl substitution; this affects their UV absorption,
chromatographic mobility and colour reactions
• 2 common flavones – apigenin and luteolin,
corresponding in hydroxylation pattern to kaempferol
and quercetin.
 Minor flavonoids, xanthones and stilbenes

• The chalcones, aurones, flavanones,

dihydrochalcones and isoflavones are
designated ‘minor flavonoids’ because each of
these classes is of limited natural distribution.
• Chalcones and aurones together comprise the
‘anthochlors’, yellow pigments which can be
detected by the fact that a change to orange or
red colour is observed when a yellow petal is
fumed with the alkaline vapour of a cigar or
with a vial of ammonia.
 Tannins
• Occur widely in vascular plants – associated with woody tissue
• By definition – they have ability to react with protein, forming
stable water-insoluble co-polymers.
• Industrially – tannins are substances of plant origin which have
ability to cross-link with protein that capable of transforming
raw animal skin into leather.
• In plant cell, tannins are located separately from proteins and
enzymes of the cytoplasm but when tissue s damage, eg when
animal feed, the tanning reaction may occur, making the
protein less accessible to the digestive juices of the animal.
• Plant tissue high in tannins but largely avoided by most
feeders because of the astringent taste.
• Two types of tannins
– Condensed tannins – occur almost universally in
ferns and gymnosperms and widespread among
angiosperms especially woody species.
– Hydrolysable tanins – limited to dicotyledonous
plants

– Both can occur together in the same plant such

as in oak bark and leaf.
• Condensed tannins or flavolans – formed biosynthetically
by the condensation of single catechin (or gallocatechins)
to form dimers and then higher oligomers, with C-C
linking one flavan unit to the next by a 4 – 8 or 6 – 8 link.

• Most flavonols have between 2 and 20 flavan units

• Proanthocyanidin is used alternatively for condensed

tannins because on treatment with hot acid, some of the
carbon-carbon linking bonds are broken and
anthocyanidin monomers are released.

• Most proanthocyanidins are procyanidins, which means

that they yield cyanidin on acid treatment.
General structure of flavan-3-ol unit.
A) (+)catechin and (-)epicatechin monomers, B) procyanidin B2 dimer and C) procyanidin oligomers with C4-C8 linkage
(Contreras-Domínguez et al., 2006).
• Hydrolysable tannins
– Galloylglucose – glucose core is surrounded by 5
or more galloyl ester groups
– Ellagitannins – esters of hexahydroxydiphenic acid
with glucose attachment
Nomenclature Structure Molecular weight
range
Condensed tannins Oligomers of 1000 - 3000
- Proanthocyanindins catechins and
(or flavolans) flavans-3-4-dials

Hydrolysable tannins
- Gallotannins Esters of gallic acid 1000 – 1500
and glucose

- Ellagitannins esters of 1000 – 3000

hexahydroxydipheni
c acid and glucose

Prototannins
- Tannin precursors Catechins (and 200 – 600
gallocatechins)
flavan-3,4-diols
 Quinone pigments
• The natural quinone pigments range in colour from pale
yellow to almost to black.
• Relatively little contribution to colour in higher plants
• Frequently present in bark, heartwood or root or leaves
tissue where their colours are masked by other
pigments.
• Bacteria, fungi and lichen – pigmented by quinones
• Identification – divided into 4 groups: bezoquinines,
naphthalquinone, anthraquinones and isoprenoid
quinones
 Recap - flavonoid

• have the general structure of a 15-carbon skeleton

• which consists of two phenyl rings (A and B) and heterocyclic
ring (C)
• occurs with various sugars attached
Terpenoids
 Introduction
• Terpenoids – all based on the isoprene molecules
• CH2=C(CH3)-CH=CH2
• General formula (C5H8)n
• Its range from essential oil components, the volatile
mono- and sesquiterpenes (C10 and C15), less volatile
diterpenes (C20), involatile triterpenoids and sterols
(C30 )and carotenoid pigments (C40)
• Classification – based on n values
• Significant either in pant growth, metabolism or
ecology
 General formula : (C5H8)n

Value of n (number Number of Name of class Main type and occurrence

of isoprene units) carbon
atoms
1 5 Isoprene Detected in Hamamelis japonica
leaf
2 10 Monoterpenoids Monoterpene in plant essential oil -
menthol from mint
monoterpene lactones
tropolones
3 15 Sesquiterpenoids Sesquiterpenes in essential oils
sesquiterpenes lactones (common
in Compositae)
abscisins (abscisic acid)
4 20 Diterpenoids Diterpene acids in plant resins
gibberellins (gibberellic acid)
5 25 Sesterpenoids
6 30 Triterpenoids Sterol
Triterpenes
Saponins
8 40 Tetraterpenoids Carotenoids
>8 >40 Polyterpenoids rubber eg in Hevea brasiliensis
 sesquiterpenes

monoterpenes
• Chemically, terpenoids are generally lipid-
soluble
• Located in cytoplasm of the plant cell
– Essential oils sometimes occurs in special glandular
cells on the leaf surface
– Carotenoids – associated with chloroplasts in the leaf
and with chromoplasts in the petal
• Extraction – light petroleum, ether or
chloroform
• Separation – by chromatography on silica gel or
alumina using same solvent
• Difficult in detection on a microscale except carotenoids
– Because terpenoids are colourless
– No sensitive universal chromogenic reagent
• Isomerism is common: geraniol and nerol
• Mostly alicyclic compound because the cyclohexane ring usually
twisted in ‘chair’ form, different geometric conformations are
possible, depending on the substitution around the ring – often
difficult to determine
• Functions of plant terpenoids
– growth-regulating properties – abscisins (sesquiterppenoid) and
gibberellins (diterpenoid)
– Agents of communication and defense among insects
– Non-volatile terpenoids – sex hormones among fungi
– Carotenoids
• plant colour - pale yellow through bright orange to deep red
• Accessory pigments in photosynthesis
– Mono- and sesquiterpenes – distinctive smells and odours
 Essential oils
• Volatile steam-distillable – characteristic scent, odour or smell
• Commercially important – natural perfumes, spices and flavoring in food
industry
• Chemically, terpene essential oils divided into 2
– Monoterpenes (C10) – boiling point 140-180 °C
– Sesquiterpenes (C15) – bp >200 ° C
• Mono – divided into three group depending on whether they are acyclic,
monocyclic, or bicyclic.
• Within each group, mono-maybe simple unsaturated hydrocarbons
(limonene) or may have functional groups and be alcohols (menthol),
aldehydes or ketones (menthone, carvone).
• Simple mono- are widespread and tend to occurs as majority of essential
oils.
• Flower and seed oils tend to have more specialized mono- present
• Sesqui- group according to the basic carbon
skeleton (same as mono-)
• Either acyclic, monocyclic or bicyclic.
• But within group – there are too many
different compounds known
• 2 special sesqui- because of the growth-
regulating properties – abscisic acid (hormone
controlling domancy in seed) and xanthinin
(auxin-antagonist)
• Isolation from plant tissues – both mono- and
sesqui- separated by extraction into ether,
petroleum or acetone.
• Classical extraction procedure – steam
distillation
• Due to the volatility – ideal for separation by
GC
 Diterpenoids and gibberellins
• Comprises of a chemically heterogeneous group of
compounds
• All with C20 carbon skeleton - based on four isoprene units
• Very limited distribution compound
• Probably the only universally distributed diterpene is
acyclic parent compound of the series – phytol (present as
the ester attachment in chlorophyll)
• 3 classes of diterpenes – resin diterpenes, toxic
diterpenes and gibberellins
• Resin diterpenes
– Have protective function in nature
– Exuded from wood of trees or latex of herbaceous
plants
• Toxic diterpenes
– Poisonous

• Gibberellins
– Group of hormone – stimulate growth
– Gibberellic acid – the most popular gibberellin
 Triterpenoids and steroid
• Compounds with carbon skeleton based on 6 isoprene units
• Derived biosynthetically from the acyclic C 30 hydrocarbon
• Relatively complex cyclic structure
• Most either alcohols, aldehydes or carboxylic acids.
• Colourless, crystalline, often melting, optically active
substance which generally difficult to characterize because
lack of chemical reactivity
• Widely used test – Liebermann-Burchard reaction (acetic
anhydride-conc H2SO4) – produces a blue-green colour with
most triterpenes and sterols
• Divided into at least 4 groups of compounds:
– True triterpenoid
– Steroids
– Saponin
– Cardiac glycosides occur mainly as glycosides
• Functions
– occur especially in waxy coatings of leaves and on
fruits – protective function in repelling insect and
microbial attack
– Taste properties – bitterness
• eg. Limonin – the lipid-soluble bitter principle in Citrus
fruits
 Carotenoids
• C40 tetraterpenoids
• Extremely widely distributed group of lipid-
soluble pigment
• 2 principle function
– As colouring matters in flowers and fruits
– Accessory pigment in photosynthesis
• Eg in flowers – yellow colour (daffodil, marigold)
• Eg in fruits – orange or red (tomato, paprika,
palm oil)
• Over 600 known carotenoids but only a few are common in
higher plant
• Identification easy to resolve – by reference to the common
substance
• Split into two class –
– Purely hydrocarbon and contain no oxygen - based on lycopene
• Chemical structure of lycopene consists of a long chain of 8 isoprene units
joined head to tall, giving a completely conjugated system of alternate
double bond – which is the chromophore giving it colour
• Cyclization of lycopene at one end give γ-carotene
• Cyclization of lycopene at both end give β-carotene
• β-carotene isomers (α- and ε-carotene) differ in the position of the double
bond in the cyclic end units
– Oxygenated derivatives (contain oxygen) – xanthophylls
• Monohydroxycarotenes - lutein, rubixanthin
• Dihydroxy – zeaxanthin
• Dihydroxyepoxy – violaxanthin
• Glycosides – very rare in higher plant – the
best known is water-soluble crocin
• Combined forms of carotenoid
– xanthophylls esterified with fatty acid
– Eg. palmitic, oleic or linoleic acid
Nitrogen compound
 Introduction
• Nitrogen compounds – nitrogen element
• Nitrogen compounds are usually basic, thus form salts with
mineral acids
• Can be extracted from plant tissue using weak acidic solvents
• Can be precipitated from extracts by addition of ammonia
• Many nitrogen compounds are charge molecules, so
electrophoresis can be used for separation
• Detection technique
– spray reagent Ninhydrin – amino acids
– Dragendorff reagent - alkaloids
 Amino acid
• Plant amino acid conveniently divided into two groups (although the
division between the two groups is not entirely sharp and methods
of identifying and separating both groups are essentially the same)
– Protein amino acid
• Generally recognized to be twenty in number
• Found in plant and animal
• Glutamic, aspartic acids, glutamine and asparagine - present in larger amount and
represent a storage form of nitrogen
• Histidin, trytophan, cystein and methionine – low amount in plant tissue and cannot
be readily detected.
– Non-protein amino acid
• Only γ-aminobutyric acid regularly present in plants
• Their role in plant is not clear, although present in high concentration in seeds
• May be important as nitrogen storage material
• Amino acid are colourless ionic compounnds
• Solubility properties and high melting point –
zwitterions
• Water soluble
• Amino acid have difference charge properties,
amino acid mixture can be divided into
neutral, basic and acidic fractions by using
either electrophoresis or ion exchange
chromatograhy.
Amino acid Ninhydrin colour Charge properties
Glycine red-violet neutral
Alanine violet neutral
Serine neutral
Cysteine neutral
Threonine neutral
Valine neutral
Leucine neutral
Isoleucine neutral
Methionine neutral
Aspartic acid blue-violet acidic
Asparagines orange-brown neutral
Glutamic acid violet acidic
Glutamine neutral
Arginine basic
Lysine basic
Proline yellow neutral
Phenylalanine grey-violet neutral
Tyrosine neutral
Tryptophan neutral
Histidine basic
 Amines
• Considered simply as the products of decarboxylation of amino
acid, formed by the reaction:
RCH(NH2)CO2H  RCH2NH2 + CO2
• Divided into three groups
– aliphatic monoamines
• Volatile compound eg methylamine (CH3NH2), n-hexylamine (CH3(CH2)5NH2
• Have an unpleasant fish-like smell
• Function in flowers as insect attractants
– aliphatic polyamines
• Less volatile
• Still possess offensive odours
• Eg putrescine, agmatine, spermidine, spermine
• Function – growth-stimulating activity in relation to their effect on ribosomal
RNA
– aromatic amines
• Many of known aromatic amines are physiologically active and sometimes
classified as alkaloids
 Alkaloids
• Largest single class of secondary plant substance
• No one definition of term alkaloid which completely
satisfactory
• Alkaloid generally includes ‘ those basic substance which
contain one or more nitrogen atom, usually in combination as
part of a cyclic system’
• Often toxic to man and many have dramatic physiological
activities; hence wide use in medicine
• Usually colourless, often optically active substance, most are
crystalline but a few liquid at room temp (eg. nicotine)
• Simple test for alkaloid – bitter taste
• Many alkaloid are terpenoid in nature (eg. solanine)
and some are best considered as modified terpenoid
• Others are mainly aromatic compound (eg. colchine) –
containing their basic group as side-chain attachment
• Specific to one family or to a few related plants – name
of alkaloid types are often derived from plant source –
eg nicotine (Nicotina tabacum), atropine ( Atropa
belladonna)
• Mostly in angiosperm, generally absent or infrequent
in gymnosperms, ferns, mosses and lower plant.
• Function of alkaloid – still obscure / not clear
• Some reported to be involved as growth
regulation, insect repellents or attractants
• Theoretically – they act as a form of nitrogen
storage in plants
• Most alkaloids are weak bases, but some, such as
theobromine and theophylline, are amphoteric
• Many alkaloids dissolve poorly in water but
readily dissolve in organic solvents, such as diethyl
ether, chloroform or 1,2-dichloroethane.
• Caffeine, cocaine, codeine and nicotine are water
soluble (with a solubility of ≥1g/L), whereas
others, including morphine and yohimbine are
highly water soluble (0.1–1 g/L).
• Alkaloids and acids form salts of various strengths.
• These salts are usually soluble in water and
ethanol and poorly soluble in most organic
solvents.
• Extraction - no single method of the extraction
from natural raw materials.
• Most methods exploit the property of most
alkaloids to be soluble in organic solvents but
not in water, and the opposite tendency of
their salts.

Crystals of piperine extracted from black pepper

 Chlorophylls
• Essential catalysts of photosynthesis
• Occur as green pigment in all photosynthetic
plant tissue
• Occur abundantly in the chloroplast, often
bound loosely to protein but are readily
extracted into lipid solvent such as acetone or
ether
• Chemically, chlorophyll contain a porphyrin (tetrapyrrole)
nucleus with a chelated magnesium atom in the centre and
a long-chain hydrocarbon (phytyl) side chain attached
through a carboxylic acid group.
• Thus, the structure of chlorophyll b only differs from that of
a in having an aldehyde group instead of a methyl
substituent attached to the top righ-hand pyrrole ring
• Chlorophyll are relatively labile and during isolation it is
necessary to protect them from degradation eg.
– Active chlorophyllase enzyme removes the phytol side chain –
chlorophyllides
– Lost of central magnesium atom – protochlorophylls
• Determination of chlorophyll – better to extract fresh tissue
and make measurement immediately
• Alternatively – stored in the dark in acetone containing
trace of NA2CO3 at -20 to -30 °C
• General precaution rules – work in dim light to avoid
pigment losses
 Fatty acids and lipids
• Mainly occurs in bound form, esterified to glycerol as
fats or lipid
• Comprise up to 7% of the dry weight in leaves in
higher plants and are important as membrane
constituents in chloroplast and mitochondria.
• Abundantly in seeds or fruit to provide plants with a
storage form of energy to use during germination.
• Seed oil from plant – commercially used – olive,
palm, coconut etc
• Lipids are defined by their special solubility
properties and are extractable with alcohol or
ether
• 3 main classes – due to different fatty acid
residue
– Triglycerides
– Phospholipids
– Glycolipids
• Identification – requires the determination of
their fatty acid component
Case study