Organic Coloring materials

Color in organic compounds is associated with the presence of certain groups in the molecule.
The group that produces color is called chromophore and a molecule containing such a group is
known as chromogen. The most effective chromophores are nitroso, nitro, azo, o- and p-quinoid
groups. The presence of any one of these groups in a molecule is usually sufficient to produce color.
Thus nitrobenzene is pale-green, azobenzene is orange-red, o-quinones are orange or red, p-quinones
are yellow. Certain other unsaturated groups produce color only when several of them are present in a
O
O O
N=O ; N ; N=N ;
; C=C ; C=O ; C=N
O O
O
Nitroso Nitro Azo p-Quinoid o-Quinoid Ethylene Carbonyl Azomethine

O O O O O
CH3 C CH3 CH3 C C CH3 CH3 C CH2 C CH3

Acetone Biacetyl Acetylacetone

molecule in a conjugated form. These are ethylene, carbonyl and azomethine groups. Thus, although
acetone is colorless, biacetyl is yellow and acetylacetone in which the two carbonyl groups are not
conjugated is colorless. It is important to notice that certain groups those are not producing color
themselves, are able to intensify the color when present in a molecule together with a chromophore.
These are called auxochromes. The most effective auxochromes are: –OH, –NH2, –NHR, –NR2.
Thus the nitrophenols and nitroanilines are more intensify colored than nitrobenzene and are deep
yellow to orange.
Modern Theory of Color: For a better understanding of relationship of color and constitution,
one must consider the nature of light and its interaction with matter. The sensation of color is
produced when light having a wavelength within the visible region of electromagnetic spectrum
strikes the retina of eye. The visible region of the spectrum extends from 4,000 to 7500 Ǻ in
wave length (1 Ǻ= 10 -8 cm). The corresponding colors are those of the spectrum extending from
violet (shorter wavelengths, 4,000 to 4,300 Ǻ) to red (longer wavelength, 6,300 to 7,500 Ǻ).
Beyond the red end of the visible spectrum lies the infrared (7,500 to 5,000,000 Ǻ), and beyond the
violet end lies the ultraviolet (150 to 4,000 Ǻ). The energy of the visible light increases from the red
end to the violet end. Ultraviolet light has more energy than visible light; infrared has less.
Color and Absorption of Increasing Energy
High Low
light: When light passes through
matter or is reflected from it,
some of the light may be
Orange
Yellow
Violet

Green

Red
Blue

absorbed. The energy of light is Infrared

Visible
transformed into energy of Ultraviolet
motion of molecules, or electrons
in the molecules may be
4000 4500 5000 5500 6000 6500 7000 7500
promoted to higher energy levels. o

The energy is eventually Wave length (A)

transformed into heat. This implies, when matter absorbs light energy, its temperature is raised.
In rare cases, a part of light may be emitted as light of a longer wavelength, and this gives rise to
phenomenon of fluorescence. The most important source of color in organic compounds is by
absorption of light without subsequent emission.
If the color is absorbed from the infrared or

o
ultraviolet regions, no color will be seen; but if A
6 500
light is absorbed from the visible region, that

60
Orange

00
substance will appear to be colored. If substance Red o

A
absorbs all visible light except that corresponding o
Yellow o

5500 A
to yellow, for instance, it will transmit or reflect 7000 A
only yellow and will be seen as yellow. o
More commonly, however, light of only one color 4000 A Violet
Green
is absorbed, in which case the substance appears
45 Blue
to have the complementary color, as determined 00 o
by the use of the color wheel. Thus, if the light is

o
A 0 A
500
absorbed from the violet region of the spectrum,
the substance will be seen as yellow. If light is
absorbed from the red region, the substance will Color wheel of Munsell
appear green.
Color and conjugated system: When ultraviolet or visible light is absorbed by a molecule, an
electron is excited, that is, it is promoted to an orbital of higher energy. The molecule is said to be in
the excited state. The energy required to promote an electron varies with the environment of the
electron. The more stable the bond, the more difficult it is to promote an electron in that bond to the
excited state. A higher energy is required. Sigma ()-bond electrons are tightly held. Extremely high
energy (or short wavelengths) is necessary to promote electrons. The energy required to promote
-electrons is frequently high enough to break the -bond and form free radicals.
Pi ()-bond electrons are less tightly held and require less energy to be promoted. Electrons in
conjugated systems require even less energy. Conjugation and aromaticity help stabilize the excited
state by sharing and delocalizing the higher energy of the excited electron.
Organic compounds containing -bonds absorb light in the ultraviolet region of the spectrum,
while those containing only -bonds do not absorb light in this region. Conjugated -bonds require
less energy for electron promotion than isolated -bonds and absorb ultraviolet light at a longer
wavelength. As conjugation increases, the wavelength of light absorbed increases. When wavelengths
are long enough to be in the visible region, it shows color. This is illustrated by the following
examples.
Ethylene absorbs light of wavelength far into the ultraviolet part of the spectrum, at 1800 Ǻ,
Butadiene, with two conjugated double bonds, absorbs at 2170 Ǻ, a wavelength closer to the visible
region than that absorbed by ethylene. Hexatriene, with three conjugated double bonds, absorbs still
closer to the visible region, at 2580 Ǻ. All the three compounds are colorless. However, as the
number of conjugated double bonds increases, the position of absorption falls nearer the visible
region, and with enough
H C=CH2 ; CH2=CH CH=CH2 ; CH2=CH CH=CH CH=CH2
conjugation the molecules are 2
colored. For example, -carotene Ethylene 1,3-butadiene 1,3,5-hexatriene
Colorless Colorless Colorless
contains eleven conjugated double
bonds and is responsible for the
color of carrots. It absorbs at 4510 CH3 CH3 CH3 CH3 CH3 CH3
CH3 CH3
Ǻ, although the light absorbed is CH=CH-C=CH-CH=CH-C=CH-CH=CH-CH=C-CH=CH-CH=C-CH=CH

blue, carrots are, of course, orange.

CH3 -Carotene H3C
(Orange)
2
 Benzene absorbs light at 2550 Ǻ and is therefore, colorless, as are naphthalene and anthracene.
As the number of fused rings increases, the position of absorption approaches the visible region.
Thus naphthacene, with four linearly fused rings, is yellow (absorbs blue light) whereas Pentacene,
with five fused rings, is blue (absorbs orange light). Graphite is a sheet of benzene rings fused in all
directions; it is black, absorbing all colors almost completely.
Carbon-carbon double bonds and benzene rings are not the only groups, which produce color in
organic compounds. Other groups which contain easily excitable -electrons also form important
light absorbing (color producing) units. Thus it is significant to note that all chromophore have
multiple bonds, that is, they are -bonded systems. Intensification of color by auxochromes is due to
the formation of an extended, conjugated system (through delocalization) with the chromophore.

Benzene Naphthalene Anthracene Naphthacene Pentacene

(Colorless) (Colorless) (Colorless) (Yellow) (Blue)

Graphite
(Black)
Pigments: A pigment is a colored compound that is applied to the surface of the fiber.
Natural pigments are highly colored substances found in the living organisms, ie, either in plants or in
animals and are known as plant and animal pigments, respectively. However, some of them have
been isolated from both the sources, eg., carotenoids. In plants, carotenoids are present in colloidal
suspension in the form of esters or protein complex, whereas in animals these are present either
dissolved in fat or combined with proteins.
Classifications: Pigments may be classified in two ways according to their (I) Sources and
(II) Structural units:
(I) According to their sources: Classifying into i) Plastid pigments and ii) Sap pigments.
i) Plastid pigments: These pigments are found in plastids, which are specialized masses of
protoplasm lying in the cytoplasm. These are further classified into three types on the basis of colors;
(a) colorless plastids called leucoplasts. (b) green plastids called chloroplasts containing chlorophyll
and (c) chromoplasts containing coloring matter other than green, eg., carotenoids (yellow or orange).
ii) Sap pigments: The coloring matters found in the cell are known as sap pigments, viz.,
anthocyanins (bright red or blue) and anthoxanthins (pale-yellow).
(II) According to their structural units: Classifying into i) Polyene pigments, ii) Pyran or
pyrylium pigments, iii) Pyrone pigments and iv) Pyrrole pigments.

3
 i) Polyene pigments: This group contains a series of hydrocarbons, having a number of
conjugated double bonds, and their oxygenated derivatives, eg., carotenoids.
ii) Pyran or pyrylium pigments: Benzpyran is the structural unit of these types of pigments which
is present as 2-phenylbenzpyrylium chloride, eg., anthocyanins.
iii) Pyrone pigments: -pyrone is the basic structure of these types of pigments which is present as
benzo--pyrone, eg., flavones (anthoxanthins) and flavonols.
iv) Pyrrole pigments: Pyrrole is the structural unit of these types of pigments, eg., haemin and
chlorophyll.
Anthocyanins: These are natural plants pigments belongs to Pyran or pyrylium groups; these are
glycosides and their aglycons, ie., sugar-free pigments are known as the anthocyanidins. These are
responsible for various colors, particularly red, violet, blue, in flowers, fruits (berries), stems, leaves
and roots (beet root) of plants. They are soluble in water and generally
occur in the aqueous cell sap. Anthocyanins are amphoteric in nature; 8 + 2/ 3/
HO O Cl
their acid salts are red, alkali salts are blue and free anthocyanins 1 2 1
/ 4/
7 /
(or neutral) are violet. The different shades of the flowers are due to 3 6 5/
6
the presence of some anthocyanin in different media (acidic, alkaline 5 4 OH
or neutral). All the anthocyanins have flavylium chloride or OH
2-phenylbenzopyrylium chlorides as the parent compound.All the anthocyanins and anthocyanidins
are the derivatives of 3, 5, 7-trihydroxyflavylium chloride, the various pigments (anthocyanins and
anthocyanidins) differ in the number, nature and position of other hydroxyl groups, methoxy groups
and sugar residue. The anthocyanidins can be obtained by heating the pigment with 10% HCl.
Thus cyanin (anthocyanin) on heating with 10% HCl gives cyanidin (aglycon or anthocyanidin) and
two moles of glucose. The common sugars found in anthocyanins are glucose, galactose and
rhamnose and the more important of these is glucose. Some of the anthocyanins as well as
anthocyanidins are also acylated derivatives of the two common acids; p-hydroxybenzoic acid and
malonic acids. The acid radical may be attached either to the hydroxyl group in flavylium nucleus or
in sugar residue.
Geissman et al (1952) coined the term flavonoids to cover all O flavone
compounds whose structure is based on flavone:
Thus anthocyanins constitute as one group of flavonoid
O
compounds. The various groups of flavonoids give rise to
characteristic color reactions and thus they may be classified on the basis of their color reactions.

Class Color with aq. NaOH Color with conc. H2SO4 Color with Mg-HCl
Anthocyanins Blue to violet Yellow orange Red (fades to pink)
Flavones Yellow Yellow to orange Yellow to red
Flavonols Yellow to orange Yellow to orange Red to magenta
Flavanones (i) Yellow to orange (cold) Orange to crimson Red, magenta,
(ii) Red to purple (hot) violet, blue
Isoflavones Yellow Yellow Yellow
Leucoanthocyanin Yellow Crimson Pink

4
 * Isolation of anthocyanins:
The isolation of anthocyanins depends on the plant source. The earlier methods used solvent
extraction (ethanol, ether, acetone and light petroleum), but now-a-days chromatography is the main
method. With column chromatography (cellulose powder, silica gel, ion-exchange resin etc.) the
solvent used depends on the nature of adsorbent. Furthermore, since anthocyanins are colored, a
series of bands is produced on the column. The identity of the bands may then be determined by
specific color tests for the anthocyanins. A better way of identifying these compounds is to use paper
chromatography, together with known compounds for comparison. Moreover, paper chromatography
is particularly useful for micro-scale work. Other methods used for separation are paper
electrophoreses and counter-current distribution.
Anthocyanins are characterised by two absorption bands, band I, 475-560 nm (visible region) and
band II, 275-280 nm (ultraviolet region). The actual color (band I) depends on the number and
positions of the hydroxyl and methoxyl groups and when these are fixed, the color then depends upon
the pH and solvent.
* Determination of the structure of anthocyanins:
i) The anthocyanin on HCl
treated with hydrochloric acid C27H31O16Cl + 2H2O C15H11O6Cl + 2C6H12O6
gives anthocyanidin and sugar.
Cyanin Cyanidin Glucose
chloride chloride

ii) The carbohydrate is detected, estimated and its structure is determined by the usual methods;
various carbohydrates found in anthocyanins are glucose, galactose and rhamnose. In case two or
more molecules of monosaccharides are obtained from one molecule of anthocyanin, it becomes
necessary to know whether these are present as monosaccharides, disaccharides or trisaccharides in
the anthocyanin molecule.To do this anthocyanin is first methylated followed by hydrolysis with
suitable enzyme to give methylated sugar (ie., methylated di- or tri-saccharide).
iii) The next step is the determination of the structure of anthocyanidins (sugar free portion).
The most common and typical anthocyanidins with their corresponding anthocyanins are given here:

Anthocyanins Anthocyanidins
a) Cyanin chloride Cyanidin chloride or
3, 3/, 4/, 5, 7-pentahydroxy flavylium chloride
b) Pelargonin chloride Pelargonidin chloride or
3, 4/, 5, 7- tetrahydroxy flavylium chloride
c) Delphinin chloride Delphinidin chloride or
3, 3/, 4, 5, 5/, 7-hexahydroxy flavylium chloride
d) Peonin chloride Peonidin chloride or
3, 4/, 5, 7-tetrahydroxy -3/-methoxy flavylium chloride
e) Malvin chloride Malvidin chloride or
3, 4/, 5, 7-tetrahydroxy -3/ 5/-dimethoxyflavylium chloride
f) Hirsutin chloride Hirsutidin chloride or
3, 4/, 5-trihydroxy-3/, 5/, 7-trimethoxyflavylium chloride

5
  Determination of the structure of anthocyanidins:
i) The molecular formula is determined a usual, ii) The number of hydroxyl groups is determined
by acetylation, iii) Methoxy groups are estimated by Ziesel method.
iv) The anthocyanidin is fused with hot potassium hydroxide when phloroglucinol or methylated
phloroglucinol and a
/ /
phenolic acid are always HO 8 O+ Cl 2 3 4/ Conc. KOH HO
OH
7 1 2 1 /
OH + HOOC OH
formed; eg., On similar 3 6
/ 5/
boil
6
OH OH OH
fusion cyanidin chloride 5 4 OH
OH
produces phloroglucinol
and protocatechuic acid. Cyanidin chloride Phloroglucinol Protocatechuic acid

HO OH This method can be

+ HOOC OH successfully used for the
OH degradation of cyanidin,
OH pelargonidin and delphinidin
chloride; but can not be used
Protocatechuic acid
Phloroglucinol for the anthocyanidin
(-OCH3 group is hydrolysed to -OH group) containing methoxy groups,
(peonidin, malvidin and
Conc. KOH
boil hirsutidin chlorides) because
concentrated potassium
+Cl
HO O hydroxide solution will also
OH
hydrolyse – OCH3 groups to
OH OCH3 –OH and thus exact position
OH of the methoxyl groups can’t
be assigned. In such cases
Peonidin chloride the anthocyanidins are
treated with 9-10% solution
10% Ba(OH)2-H2 of barium hydroxide or
or 10% KOH-H2 sodium hydroxide in
presence of hydrogen.
Under these conditions, the
HO OH methoxyl group (s) remains
+ HOOC OH intact, eg; peonidin chloride.
OCH3 v) Now the last problem
OH
is to assign the position of
Phloroglucinol (-OCH3 group remains intact) sugar residues. There are
various methods for it:
a) The anthocyanin is methylated, hydrolysed with10% HCl to remove sugar residue, and the so
formed anthocyanidin is treated with barium hydroxide solution in an atmosphere of hydrogen; the
positions of the free hydroxyl groups indicate the points of attachment of sugar residues.

6
* Cyanin chloride gives the following products where G represents a glucose molecule:
These degradation does not +
HO O Cl (CH ) SO3 2 4 +
conclude which of the two OH H3CO O Cl
OCH3
OG OH
hydroxyl groups of monomethyl OG
OG OCH3
OG
phloroglucinol was originally Cyanin chloride (I)
HCl
attached to glucose (G)? Had the
structure of cyanin chloride been H3CO
+
O Cl
OCH3
(Ia) instead of (I), monomethyl
OH OCH3
phloroglucinol would still have OH
been obtained. Methylated cyanidin chloride

+ 10% Ba(OH)2-H2
GO O Cl
OH
OG OH H3CO OH
+ HOOC OCH3
OH OCH3
OH
Cyanin chloride (Ia)
Monomethyl phloroglucinol

ii) Anthocyanins are treated with 15% hydrogen peroxide

OH
HO
+ HOOC OCH3
in acetic acid, when pyrylium ring opens up between C2 and
H3CO
OCH3 C3 without removing the sugar residue.
Now, if the anthocyanin I has the sugar residue in position 3,
Monomethyl phloroglucinol the sugar can be removed from the degraded product II, by
dilute ammonia. On the other hand, if the sugar moiety in I is
in either 5 or 7-position, then this sugar molecule in degraded product II can be removed only by
heating with dilute hydrochloric acid. 8 + 2/ 3/
O Cl H2O2 HO OCO OH
Thus, the position 3 can be distinguished HO / 4/
/ OH
7 1 2 / 1
5 CH3COOH OH
3 6
from positions 5 and 7; but the latter two 6
5 4 OG OH OG
CH2COOG

positions (5 and 7) can’t be distinguished OG

from each other. Cyanin chloride I II

iii) The free nature of the hydroxyl

group of anthocyanins in position 3 is also provided by the rapid oxidation with FeCl3 solution during
which the anthocyanins are rapidly decolorized.
iv) Anthocyanins having a sugar molecule only in position 3 give a violet color with sodium
carbonate whereas anthocyanins which are 3,5-diglucosides always give blue color with sodium
carbonate.

v) The configuration of the anthocyanin, ie., weather the sugar is linked to anthocyanidin by  or
-linkage is ascertain by its hydrolysis, with the enzymes. Maltase attacks -linkage while emulsin
attacks the -linkage.
vi) The positions of the sugar residue are further confirmed by the synthesis of individual
anthocyanins. Generally, it is found that sugar moiety is linked either at position, 3 or 3 and 5; and
the linkage is usually .
Function of Anthocyanins: Anthocyanins are supposed to perform the following functions in
plants: a) Increase the osmotic pressure of the cell sap. b) As anthocyanins are beautifully colored
substances they help attracting the insects and thus assist cross-pollination. c) They also play some
important role in the process of photosynthesis and respiration. d) They act as light filters and thus
protect chlorophyll from decomposition against strong light.
7
 Flavones: The flavones, which are also known as anthoxanthins are widely distributed as yellow
plant pigments. These occur either in the free state or as glycosides associated with tannins.
The anthoxanthins also occur as colorless glycosides in the white corollas of several flowers, which
on treatment with ammonia vapor tyurn yellow (as the colorless precursors are converted into
flavones).
Isolation: The plant materials containing flavone is extracted with boiling water and the tannins
are removed as lead salts by means of lead acetates. The filtrate is diluted with water, acidified with
HCl and boiled few hours until the sugar free flavones are precipited out. These are extracted with
alcohols followed by purification through fractional crystallization of their acetates or by
recrystallization from some organic solvents like benzene, carbon disulphide, alcohol etc.
General properties: Flavones are crystalline compounds, soluble in water, dilute mineral acids,
alkalis, alcohol, etc., and are precipitated by means of lead acetate. It produces a dull green color or a
reddish brown color with ferric chloride. These are more highly colored in the acidic medium rather
than in alkali and from which they are derived. In acidic medium it forms oxonium salts, which
contribute the color, but this oxonium salt is
unstable and is hydrolysed back to free base O C6H5 O C6H5 O C6H5
(difference from anthocyanidins). The Cl
structure of flavone salts is not certain; these
OH OH OH
are probably best represented as resonating
structures. Flavones show two absorption
bands: Band I at 330-350 nm, and band II at 250-270 nm whereas anthocyanins shows bands I and II,
respectively at 475-560 and 275-280 nm. Thus flavones may be distinguished from anthocyanins on
the basis ob absorption bands and color reactions.

Basic structure of flavones and flavonols: The basic unit of flavones and flavonols is -pyrone
which is present as benzo--pyrone (chromone). The benzo- pyrone is substituted by a phenyl group
in position 2 to give the first members (flavone) of the class flavones. When hydrogen atom on C 3 in
the -pyrone ring of flavone is replaced by a hydroxyl group, 3-hydroxyflavone or flavonol is
produced which is the member of the class flavonols. The various flavones and flavonols are hydroxy
derivatives of the flavone (I) and flavonol (II), respectively. Mostly, the flavones and flavonols occur
as glycosides and on hydrolysis they O O 8 1
/
O 2 1
/
2 3 /
/
O
7 4
yield glucose or rhamnose and a 6 4 3 /
/
6 5 OH
sugar free portion (aglycon) known O O
5 O
O
as anthoxanthidin (flavone) or -pyrone benzo--pyrone 2-phenylbenzo--pyrone 3-hydroxyflavone
flavonol as the case may be. or flavone ,I or flavonol, II

Dyes: All color compounds are not dyes. Dyes are those colored compounds that can be firmly
fixed to the fabrics by chemical or physical bonding. A dye must show fastness to light, washing,
heat (sublimation) and bleaching.
Classification: Dyes may be classified in two ways. The organic chemists classifies them
according to a common different structure (Chemical classification. The dyer classifies them
according to the method of applications.
Xanthene dyes: Xanthene dyes can be identified by a common
O O
structural feature shown in the figure. These are obtained by condensing
phenols with phthalic anhydride in the presence of zinc chloride,
sulphuric acid or anhydrous oxalic acid, eg., Fluorescein, eosin and
rhodamine B. Common structure of dyes

8
 Fluorescein: It is HO
OH HO OH O OH
prepared by heating HO + HO O O
resorcinol (2molecules) H O H ZnCl2 NaOH
O
and phthalic anhydride C O -2 H2O C=O
COONa
(1 molecule) with zinc C=O
o
chloride at 190 C.
Fluorescein is of no value
Phthalic anhydride Fluorescein Uranine
as a dye. It is a red
powder, which is insoluble in water. A dilute solution of fluorescein in sodium hydroxide gives a
strong yellow-green fluorescene when exposed to light. It is used to trace pollution of water supplies
by sewrage, since if a small quantity of it is put in at the suspected source of pollution, the color will
be detectable at some distance from the source, even after extensive dilution. Fluorescein is also used
as a mild purgative. The sodium salt of fluorescein is called Uranine. It is used to dye wool and silk.
Eosin: It is the disodium salt of tetrabromofluorescein. Eosin is obtained by brominating
fluorescein in glacial acetic acid Br Br Br Br
HO OH NaO O
to give tetrabromofluorescein. HO O OH O O

Treatment of eosin with sodium Br2 Br Br NaOH Br Br

O O
hydroxide yields the dye. C=O CH3COOH C=O COONa
It is a red solid, which is soluble
in water. Alkaline solution of
eosin shows a yellow-green Fluorescein Tetrabromofluorescein Eosin
fluorescence. Eosin is used for dyeing wool, silk and paper; for making red ink and as the coloring
matter in lipsticks and nail polishes.

Rhodamine B: It is prepared by condensing phthalic anhydride with N,N-diethyl-m-aminophenol

In the presence of zinc chloride and treating the product with hydrochloric acid.It is used for dyeing
silk, wool and cotton.

(C2H5)2N OH HO N(C2H5)2 (C2H5)2N O N(C2H5)2 (C2H5)2N O N(C2H5)2

+
HCl
H O H ZnCl2 O Cl
C O -2 H2O C=O COOH
C=O

Phthalic anhydride Rhodamine B

Chlorophyll: It is the green coloring matter of leaves and green stems and its presence is
essential for photosynthesis. Photosynthesis is the process in which light energy is used by plants to
synthesize carbohydrates, proteins and fats. In green plants it is the chlorophyll, which absorbs the
light energy. Spectroscopic study shows that it is a mixture. When an ethereal solution of chlorophyll
is shaken with methanolic potassium hydroxide solution, various color changes occur.
With chlorophyll-a the green color immediately changes to yellow; with chlorophyll-b, from green to
carmine red; and with a mixture of the two chlorophyll, from green to yellowish brown. Then, after a
few moments, the green color reappears in the lower layer; the ether is colorless. This set of color
reactions is known as the phase test, and chlorophyll preparations that fail to give it are said to be
allomerised. The phase test involves the cyclopentanone ring in chlorophyll; the alkali forms traces of
some intermediate, which undergoes slow oxidation by oxygen (atmospheric).
When dried leaves are powdered and then digested with ethanol,’crystalline’ chlorophyll is
obtained after concentration of the solvent. If, however, ether or aqueous acetone is used instead of
9
ethanol, then the product is ‘amorphous’ chlorophyll. The extraction of chlorophyll is also
accompanied by the extraction of two other pigments, carotene and xanthophylls.
Crystalline chlorophyll was produced during the extraction of chlorophyll by means of ethanol, a
molecule of phytyl alcohol being replaced by ethanol under the influence of an enzyme,
chlorophyllase that’s present in leaves.
Initially the molecular formula of chlorophyll was assigned C55H72MgN4O6, butas it was obtained
from a wide variety of sources, so it was a mixture of two compounds, chlorophyll-=a and
chlorophyll-b. The separation was effected by shaking a light petrol solution of chlorophyll with
aqueous methanol; chlorophyll-a remains in the light petrol and chlorophyll-b passes into the aqueous
methanol.Chlorophyll-a is a bluish-black solid, giving a green solution in organic solvents;
chlorophyll-b is a dark green solid, also giving a green solution in organic solvents.
The two components occur in proportions of approximately 3 of a to 1 of b in natural chlorophyll.
The chlorophylls (and chlorophyll degradation products) are separated by chromatography.
Column chromatography (alumina, sugar, starch, etc.) may be used on a large scale, and partit ion,
paper and thin-layer chromatography are used for small-scale work, but the partition technique has
also been used on a preparative scale.
The molecular formulas of the two fractions of chlorophyll were assigned and these are
C55H72MgN4O4 and C55H70MgN4O6 for chlorophyll-a and chlorophyll b, respectively; these two
fractions have different absorption spectra: chlorophyll-a, 380, 418,428,510, 580, and 700 nm;
chlorophyll-b, 428, 464 and 675 nm. These characteristic absorption maxima have been used to
estimate the amounts of each chlorophyll in a mixture. Infrared spectroscopy has been used to detect
functional groups, from their examination of the infrared spectra of the chlorophylls, have shown that
these spectra provide a means of detecting trace amounts of chlorophyll-b in samples of
chlorophyll-a and, at the same time, offer a means of estimating the proportions of the two
components.
The hydrolysis of both chlorophylls with cold dilute potassium hydroxide solution gives one
molecule of phytol, C20H40O, one molecule of methanol and one molecule of chlorophyllide-a
(chlorophyllin–a) or chlorophyllide-b (chlorophyllin–b). Thus the chlorophylls are diesters.
When either chlorophyll is heated with an ethanolic solution of hydrated oxalic acid, the magnesium
atom is replaced by two hydrogen atoms to produce phytyl phaeophorbide-a or b; these phytyl
phaeophorbides are also known as phaeophytins a and b and ‘crystalline’ chlorophyll is ethyl
chlrophyllide. The foregoing reactions are shown in the figure.
Nomenclature of the COOH
KOH
chlorophyll degradation C32H30N4OMg + C20H40O + CH3OH
products: The derivatives COOCH3
COOH
of chlorophyll containing C32H30N4OMg Chlorophyllide-a
magnesium atom are COOC20H39
called chlorophyllides or COOCH3
Chlorophyll-a (COOH)2 C32H32N4O
chlorophyllins; whereas those
C2H5OH COOC20H39
in which magnesium atom is
Phytyl phaeophorbide-a
replaced by two hydrogen
atoms are known as phorbins, KOH
COOH
C32H28N4O2Mg + C20H40O + CH3OH
phorbides and phytins.
The symbol ‘a’ or ‘b’after COOCH3
COOH
C32H28N4O2Mg Chlorophyllide-b
the name of the product
indicates that the particular COOC20H39
COOCH3
product is obtained from Chlorophyll-b (COOH)2 C32H30N4O2
chlorophyll-a or chlorophyll C2H5OH COOC20H39
b, respectively. Phytyl phaeophorbide-b

10
 Finally, the structure of chlorophyll was confirmed by
synthesis and the structure was shown in the following
figure.
Chlorophyll contains Metallic magnesium in a complex
form and it is the haemin, which is responsible for color. The
intense green colour of chlorophyll is due to its strong
absorbencies in the red and blue regions of the spectrum,
shown in fig. Because of these absorbencies the light it
reflects and transmits appears green. Due to the green colour
of chlorophyll, it has many uses as dyes and pigments. It is
used in colouring soaps, oils, waxes and confectionary. The
uv/visible adsorption spectrum for chlorophyll is shown in
the figure.

Chlorophyll-a (C55H72MgN4O5, mol. wt.:

893.49). The methyl group marked with an
asterisk is replaced by an aldehyde in
chlorophyll-b (C55H70MgN4O6, mol. wt.: 906.51)

Haemoglobin: It is the red pigment of blood that is occurs almost in all vertebrates and also in
several invertebrates. It has also been found in certain strains of yeasts, moulds, etc. Chemically, it is
a chromoprotein (type of conjugated protein); the protein part is globin (94%) and the prosthetic
group is haem (6%). Globin consists of four polypeptide chains and further in the human globin the
chains are of two types, viz., -chains having valyl-leucyl as end-group and -chains having
valyl-histidyl-leucyl as end group. Normal adult haemoglobin contains two -chains having 141
amino acids in each chain and two -chains having 146 amino acids in each chain.
On the other hand, haem is an iron-protoporphyrin complex, If the iron atom present in the
ferrous state, the complex is called ferrous protoporphyrin, ferroprotoporphyrin, protohaem or haem,
and the molecule is electrically neutral. But when the iron atom is present in the ferric state, the
complex is called ferric protoporphyrin, ferriprotoporphyrin or haemin and the molecule carries a unit
positive charge and thus it must be associated with an anion. In the animal body, haemoglobin readily
combines with oxygen to form unstable oxyhaemoglobin. The oxyhaemoglobin loses its oxygen in its
way to various parts of the organism; thus haemoglobin acts as a carrier of oxygen from the organs of
respiration to the tissues where it (oxygen) is used in various metabolic reactions.
The poisonous character of carbon monoxide can be explained by the fact that haemoglobin forms a
compound with CO which is more stable than oxyhaemoglobin; so carbon monoxide displaces
oxygen in the blood as a result the tissues do not receive oxygen for their various metabolic reactions
(leading to death
of the organism). More stable compound CO
Haemoglobin O2
Oxyhaemoglobin
than oxyhaemoglobin

11
 Outside the organism, oxyhaemoglobin is changed into a stable compound, methaemoglobin, the
latter on treatment with acetic acid gives globin and a brownish-red pigment haematin
[C34H32N4O4Fe(III)]+OH. But if methaemoglobin is treated with acetic acid containing sodium
chloride, it is hydrolyzed into haemin and globin. It should be noted that iron free and iron containing
compounds are known as porphyrins and haems, respectively.
inside the body

O2 Oxyhaemoglobin
Haemoglobin

outside the body

Na2S2O4 CH3COOH
Haem Haemin Methaemoglobin
NaCl
C34H32N4O4Fe++ C34H32N4O4Fe+++Cl-
CH3COOH

Globin + Haematin
C34H32N4O4Fe+++OH-
Finally, the structure of haemoglobin was confirmed by synthesis and it is shown in the figure.
It was already mentioned that haemoglobin is a conjugated
protein consisting of globin and haem (Fe2+). Normally, six is the
covalence number of ferrous iron but in haem it is four.
So haem can easily add on substances capable of co-ordination,
eg., with pyridine it forms pyridinhaemochromogen.
Similarly, haemoglobin co-ordinates with the imidazole rings of
histidine, the main amino acid of globin.

Structure of Haemoglobin
Py
N N
2 Py
N Fe N N Fe N

N N Py
Py = Pyridine
Haem (Fe 2+) Pyridine-haemochromogen
(abbreviated formula)
Globin O2
N N N

N Fe N N Fe N N Fe N

N N Globin N Globin
Haem Haemoglobin Oxyhaemoglobin

12