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Chapter -I
Review of Literature
� Introduction
� Classification of Dyes
� Chemistry of Dye Molecules
� Isolation Techniques
� Characterization Spectroscopic
Methods
� References
Introduction
Dyes are colored chemical compounds containing chromophores
and auxochrome groups, which when applied to a substrate impart color to
the substrate. Natural dye is a mixture of colored chemicals, often
characteristic of a certain plant of plant species, have long been admired
for their rich beauty and unique, earthy colors. Dyeing using vegetable
materials on textile fibres consists of first extraction of the coloring matter
and the fibre to be dyed. Textile fibres of animal origin, such as wool and
silk take the coloring matter quite easily, cotton on the other hand needs a
complex series of pre-treatments before it absorbs any dye except indigo,
with which it bonds naturally.
The most commonly used plants in Indian dyeing are Katha (Acacia
catechu), the fruit and leaf galls of Terminalia chebula known as
myrobalan, rhizomes of turmeric (Curcuma longa) and kingore
(Berberis). While the dried seed pods, barks, stems etc. which have high
tannin contents, are used to pre-treat the yarn for the absorption of
coloring matter. Terminalia is a well known forest tree, found throughout
the forests of India. Like most other dye producing plants, it is an
important ingredient in indigenous medicine. Other forest plants
commonly used for dyeing, and coloring agents are Mallotus philippensis
and Caesalpinia sappan, both used in silk and wool dyeing, Punica
granatum, Acacia catechu, Rubia cordifolia, Woodfordia fruticosa and
Onosma echilides.
Classification of Dyes
Dyes are classified into following classes:
According to sources of their Origin
(A) Natural Dyes
Those dyestuffs which were derived from plants, insects and
mineral sources. Natural dyes are classified into three groups
(I) Direct or substantive dyes
These dyes are water soluble dyes used primarily for dyeing
cellulose and protein fibers. These dyes have the advantage of being
applied directly in a hot aqueous dye solution in presence of common
salts, to stabilize the rate of dying. Examples of direct dyes include
safflower, berberry, turmeric, annatto etc.
(II) Mordant dyes
These dyes require a mordant to combine with and fix the dye
stuff. When the metallic salt is added to the dye molecule, it forms
relatively insoluble dyestuff with improved wet and light fastness
properties. Examples of mordant dyes are kermes, cochineal, lac, mode,
henna, wood, saffron etc.
(III) Vat dyes
Vat dyes are a group of insoluble compound which are converted
into an alkali soluble leucosis derivative by means of sodium
hydrosulphite and caustic soda and then applied to the fibers. Vat dyes are
colourless but the colour appears on re-oxidation by exposure air. Vat
dyes are suitable today cottons and rayon’s and provides a wide range of
colours except for red or orange. Vat dyes are not only resistant to light
and acid but they are equally resistant to strong oxidizing bleaches.
Indigo, its derivatives and catechu are important examples of vat dyes.
Natural dyes are also classified according to their chemical structure
into following classes-
1. Indigoid dyes - Indigo and its derivatives
2. Anthraquinone dyes - Anthraquinone and its derivatives
3. Vegetable dyes -
These dyes obtained from plant sources and are further classified
into various categories depending upon the parts of plants.
(I) Flowers dyes - Kusum, Aparajita, Rhododendron, Erythrena, Saffron
etc.
(II)Fruits dyes - Kamla, Annar, Orange etc.
(III) Leaves dyes- Indigo, Kaphal, Phuli, Tea leaves, Ankhrot, Kunjja
etc.
(IV) Bark dyes - Berberin, Kaphal, Lodh, Banj, Telleng, Acer,
Mangifera etc.
(V) Seeds dyes - Annato, Beri, Black glycine etc.
(VI) Wood dyes - Pantang, Catecheu, Mimosa etc.
(VII) Roots dyes - Madder, Berberis, Banj etc
(VIII) Tubers dyes- Haldi, Chukender, Peeyaz etc.
(IX) Needles dyes - Ankhrot, Acer, Kunjja etc.
(X) Lichens and parasitic plants-Archil, Akashmatra etc.
4. Animals dyes
Those dyes which obtained from animal tissues and Lichen species
are known as animal dyes. These are -
(I) Cochineal
It is obtained from the dried bodies of female insect Coccus cacti,
containing carmines acid, dyes bright scarlet and crimson on wool with
tin and alum mordents. Purple with chromium and iron, Claret with
copper.
(II) Tyrian purple
Obtained from Purpurea Shell fish (Murax brandaries). Each
mollusk contains a few drops of glandular mucous, this fluid at first
appears white but on exposure to light charges to reddish purple.
5. Mineral dyes
Mineral dyes obtained from natural earth pigments, which showed
their tinctorial property to the oxides and hydrated oxides of manganese.
They are insoluble in water and other solvents. These are resistant toward
light and chemicals but provide deeper and richest shades on calcinations.
Iron buff, chrome yellow, dul calcite and Prussian blue are some
important mineral dyes.
(B) Synthetic Dyes
(I) According to chemical composition or constituents
Dyes may be classified into following categories according to their
chemical composition.
1. Azo dyes
They contain an azo (-N=N-) group as chromophore in between two
aromatic rings. The azo dyes form the largest group among the synthetic
dyes and give a wide variety of colours. They are further classified
according to the number of azo groups, into mono-azo, di-azo, tri-azo
dyes etc. The azo dyes are prepared by coupling a diazonimum salt with
an amine or phenol. They are also classified on the basis of the nature of
the auxochrome.
(i) Acidic azo dyes
Contain the acidic groups like –SO3H, -COOH, -OH etc. They help
in making the dye soluble and also in fixing the dye on the fibers.
Example- Orange II.
(ii) Basic azo dyes
Contain basic groups like –NH2,-NR2 example of this type of dyes
are Aniline Yellow.
N
N
S O3Na
O H
Orange II
N N NH2
Aniline Yellow
2. Diazo dyes
They contains two azo groups in their structure. These are of
following types:
(i) Direct or substantive diazo dyes
These are the azo dyes which can be directly applied to cellulosic
substrates. They do no need the help of the intermediates called mordents
for their application. The example of direct azo dyes is Congo red
(ii) Ingrain diazo dyes
These are water insoluble azo dyes which are formed on the fibre
itself. The fibre is dipped in the alkaline solution of a naphthol and then
treated with a solution of the diazotized amine, which in results in the
formation of the azo dye on the fibres. The example of this type of dyes is
Para red.
(iii) Mordant diazo dyes
These are azo dyes which require a metal as a mordant. Chromium
is commonly used as the salt sodium chromate, dichromate or chromium
fluoride. The chromium salts can be applied to the fibre before dyeing
(Chrome-mordant method), alone with the dye (metachrome method) or
after dyeing (after chrome method). The application of mordant helps to
increase the molecular surface, decrease in solubility. Eriochrome black-T
and Red-B and Diamond black-F are examples of this class.
N NH2N
HO
Para Red
(iv) Synthetic fibre diazo dyes
There are several azo dyes used for dyeing synthetic fibres like
rayon, nylon, terrylene etc. they may be acidic, basic or disperse dyes. The
example of this type of dyes are Orange red and Celleton scarlet-B.
3 Anthraquinone dyes
They contains basic anthraquinone skeleton and are derivatives of
anthraquinone which has quinonoid system as the chromophoes. They
contain electron donating groups such as OH2, NH2, NR2, NHAr etc as
axochrumes.
N NH2N NH2
Orange dye
N NO2N N
CH2CH2OH
CH2CH3
Celliton Scarlet B
O
O
A nthraquinone
They are further classified as follows-
(i) Anthraquinone mordant dyes
They certain groups such as hydroxyl or Carboxyl group which can
combine with metal to from insoluble compounds called lakes. Example
of this type dyes are Alizarine.
(ii) Anthraquinone vat dyes
These are very important class of vat dyes. They are derivatives of
anthraquinone containing heterocyclic or other poly cycle rings. They may
also contain the grouping - C(CH=CH)n- C=O- attached to heterocyclic
rings. They have high molecular weight and are insoluble in water. They
are available in various brilliant colors and shades of exceptional fastness.
They are mainly used for dyeing of cotton, wool, silk and nylon and also
for printing. The anthraquinone vat dyes are first reduced by Sodium
Hydrosulphite (Na2S2O4) in alkaline medium to get a soluble form called
vat. Then the fabric is immersed in the vat solution. On exposure to air the
dye is regenerated on the fabric by oxidation. Anthraquinone vat dyes are
of two types:
(a) Lipophilic dyes - Insoluble dyes
(b) Hydrophilic dyes- Soluble dyes
O
O
Alizarin
OHOH
(iii) Anthraquinone acid dyes
These are water soluble anthraquinone derivatives which have the
solubilizing sodium sulphonate groups (SO3Na). They are used for dyeing
wool, silk, nylon leather and paper. These dyes are particularly fast when
applied to wool in acidic medium. Example of these types of dyes are
Ultra blue-B.
(iv) Anthraquinone disperse dyes
These are soluble derivatives of anthraquinone mainly used for
dyeing synthetic fibres like acetate, polyester and polyamide. They have
good light fastness, however exposure to gas fumes causes fading and
change of shade. This difficulty is overcome by substituting the ring with
halogens, hydroxyl, alkoxy, nitro and groups e.g. Violet 6-B and Dispersal
blue.
O
O
N H - C 6 H 5
S O 3 HN H 2
U ltr a B lu e B
O
O
N H C H 3
N H 2
V i o l e t B
O
O
Dispersal Blue
NH2 C H3
NH C H2C H2 O H
4. Reactive dyes
Reactive dyes are those dyes which reacts with hydroxide groups
of cellulose to form reactive system. Hence they will have good washing
fastness when applied to cotton fibres. They may be derived from azo,
anthraquinone and other chromophoric systems, example of this type dyes
are Prussion blue-HB.
5. Disperse dyes
The semi synthetic and synthetic fibres like cellulose acetate, nylon,
polyester, rayon etc, are hydrophobic fibres. Hence they cannot be dyed
by aqueous solution of dyes. Disperse dyes are insoluble but they are
finely ground and dispersed in water using suitable dispersing agents.
These micro fine dispersions are applied to fibres by using organic carriers
or by using high temperature and pressure. The fine particles of the dye
diffuse into the material of the fabric and then held by adsorption. The
disperse dyes may be nitro, azo or anthraquinone dyes. These dyes must
have low molecular weights and should have groups like ethanol amine
(NH CH2CH2OH) which helps in the formation of dispersion. The
example of this type dyes are Celliton scarlet-B.
N NO2N N
CH2CH2OH
CH2CH3
Celliton Scarlet B
6. Basic dyes
They are derivatives of heterocyclic rings containing -NH2 and –
NR2 groups as auxochromes. The heterocyclic ring in a quinonoid system
acts as a chromophore. Their salts with acid are coloured (Cationic dye).
They can be directly applied to protein fibres like wool and silk. However
they have poor affinity for cottons.
7. Intermediates dyes
The fairly pure organic chemicals used as raw materials for the
manufacture of dyes are called primaries. The primaries are never used
directly in the synthesis of dyes. The primaries are first converted into
derivatives which are called dye-intermediates. Which are then used in the
preparation of dyes. Thus,
Primaries Dye-intermediates Dyes.
Examples of intermediates dye are-
(i) p-nitro phenol:
p-nitro phenol is used as intermediates for sulpher dyes. Nitro dyes
are the nitro derivatives of phenols having at least one nitro group in the
O- or P-position to the hydroxyl group. They have the nitro group as the
chromophore and the hydroxyl group as the auxochrome.
O
N
O H
O O
P - n it r o P h e n o l
(ii) Picric acid:
Picric acid was the first synthetic dye, it dyes silk and wool directly.
It is now bounded due to its toxic and explosive nature.
(iii) Naphthol AS
When BON acid (β-oxy napthoic acid) Chloride is condensed with
Aniline naphthol-AS is obtained.
(iv) Quinizarine (1,4-dihydroxy anthraquinone)
It is prepared by heating pathalic anhydride, P-Chlorophenol, Conc.
Sulphuric acid, and Boric acid, at 200 °C.
OH
NO2
NO2
NO2
Picric Acid
C
O
N H
O H
N apthol AS
O
O O H
O H
Quinizarine
(v) Benzanthrone
It is prepared by reaction of anthraquinanone and acrolein to forms
anthrone through cyclisation, which leads to the fused ring formation to
give anthracene structure.
8. Azonic Dyes
Azonic dyes are insoluble azo colours which are formed on
cellulose fibres from selected diazo and coupling components. These are
prepared on the cellulose or cotton fibres in two different stages.
1. The fibre is treated with an alkaline solution of coupling component.
2. This is then developed with a diazonium salt.
The examples of azonic dye are Direct Red 65.
9. Sulphur Dyes
These constitute a group of dyes unknown constitution which can
be applied to fibres when reduced with sodium sulphide. Most of them are
insoluble in water before reduction after reduction they are soluble and
O
H 2 CC
H2
OH
NaO3S NHCONH NHAC
Direct Red
Benzanthrone
can be absorbed by fibres and then oxidized to an insoluble form with air.
These dyes are popular because of their heavy shades, such as blue, green,
black, brown etc. of reasonable fastness to light and ordinary washing at a
low costs. Example of sulphur dye is Sulphur black-I.
10. Heterocyclic Dyes
These include dyes involving heterocyclic rings and are not grouped
under other specific groups. They can be further divided as follows-
Quinone imine dyes and Quinone dimine
Indophenols and indamines derived from the above two compounds
are employed as dyes.
O H
N O 2
O 2 N
Sulphur Black I
NHHN
Quinone dimine
N H
Quinone imine
O
Indamines
H2N+
N
NH2+Cl-
(I) Indophenols
The simplest member of this group of dyes is indophenols blue. It
is obtained by oxidizing a mixture of P-phenylene diamine and phenol
with an alkaline hypochlorite solution.
The indophenols are blue. These are very sensitive to acids and
these are therefore, not now used in textile coloration. However, these are
applied in colour photography and also serve as intermediates for sulphur
dyes.
(II) Indamines
The simplest member of this group of dyes is phenylene blue. It is
obtained by oxidising a mixture of p-phenylene diamine and aniline
potassium dichromate in acetic acid.
These dyes are blue or green like indophenols these are very
sensitive to acids and they are therefore, not used for textile coloration,
however, they serve intermediates for the synthesis of azines, thiazines
and oxazines.
H2N+
N
O
Indophenol
11. Xanthene dyes
These are derivatives of xanthene. This group gives rise to brilliant
fluorescent dyes having red to yellow colour. xanthene dyes obtained from
xanthene by the introduction of auxochromes such as amino or hydroxyl
group into positions 3 and 6, the pre-position with respect to the carbon
atom linking the two benzene nuclei. Some important members of group
of these dyes are -
(i) Fluorescein
It is xanthene derivative and is obtained by heating phthalic
anhydride (1 molecule) with resorcinol (2 molecules) at 200 °C with
anhydrous oxalic acid.
Fluorescein is red powder, it is insoluble in water. When fluorescein
is dissolve in alkalis, it gives a reddish brown solution which on dilution
gives a strong yellowish-green fluorescence. The structure of fluoresein
onion is used in tracing underground currents in sea and rivers as well as a
marker during accidents.
O O O
C O O -
F l u o r e s c e i n a n i o n
(ii) Eosin
It is tetrabromo fluorescein and is obtained by the action of bromine
and fluorescein in glacial acetic acid solution. As eosin is a red powder.
The alkaline solution of eosin shows a yellow-green fluorescence.
Eosin is used to dye wool and silk a pure red, which a yellow
fluorescence. It is also used for poster printing. Most red inks are dilute
solutions of eosin.
(iii) Mercurochrome
It is di sodium salt of di bromo hydroxy mercurifluorescein. It is
prepared by heating di bromoflurorescein with mercuric acetate or
mercuric oxide in acetic acid and sodium hydroxide. It forms green scales
or granules. It dissolves in water forming a cherry-red solution. It is used
as an antiseptic in 2-5 % solution for the skin and in 1 % solution for
mucous membranes.
It has also found as application as a biological stain.
H O O + O H
C O O -
E o s i n
BrBr
Br Br
ON a O O
C O O N a
M e r c u r o c h r o m e
H g O H
Br Br
12. Di phenyl methane and Tri phenyl methane dyes
The di phenyl methane dyes are characterized by the presence of di
phenyl methane nucleus. Example-
(i) Auramine-G
The condensation of N-mono methyl-o-toluidine with formaldehyde
yields the product which on heating with sulphur in a current of ammonia
followed by treatment with hydrochloric acid yield Auramine-G is
greenish yellow dye.
This group of dyes is one of the oldest known synthetic dyestuff
groups. They are of brilliant color due to resonance and cover a range of
shades from red to blue, including violet and green. However the color
fades rapidly in light and due to this reason they find less uses in textiles
but are used for coloring papers, type writer ribbons and others articles
where fastness to light is not of much significance.
(ii) Malachite Green
On a large scale it is prepared by condensation of 2 moles of
dimethylamine with one mole of benzaldehyde at 100 °C in the presence
of Zinc chloride or cone, sulphric acid. The leuco base produced is
oxidized with lead dioxide in a solution of acetic acid having hydrochloric
C
H
MeHN
NH2
CH3
N HMe }
Auramine G
Cl+
acid. The resulting colour base on acidification with excess of
hydrochloric acid gives malachite green.
Malachite green dyes, wool and silk directly and cotton mordanted
with tannin.
(iii) Aurin
It is prepared by diazotizing pararosaniline and boiling the product
with water. Kolbe and Schmidit prepared this dye by oxidising a mixture
of phenol and formaldehyde or oxalic acid.
Aurin crystallizes in yellowish-brown prisms which are soluble in
alkalis to intense red solution. It is used as an indicator and for dyeing
paper.
C O
Aurin
OH
HO
C N Me2 }
Malachite Green
NMe2
Cl+
(iv) Chrome violet
It is prepared by heating salicylic acid with formaldehyde in
presence of con. Sulphuric acid and oxidizing agents.
13. Thio indingos and Indigos
(i) Thio indingo
When one or both the NH groups of the indigo are replaced by
sulphur atoms, the unsymmetrical and symmetrical thio indingos are
obtained. Examples are, Thio indigo scarlet-T and Ciba scarlet-G.
(ii) Indigo
It is the known dye. It occurs in the plants at indigofera group in
the form of glycoside the Indican. Indigo is also known as indigotin, it is
prepared by the reduction of isatin chloride (obtained by the action of
phosphorus pentachloride on isatin) with zinc dust in glacial acetic acid
yield indoxyl which upon oxidation in air gives indigotin. It is also
synthesized from anthranilic acid. When this acid is made to react with
chloroacetic acid, it yields the phenylgycine-o-carboxylic acid which
undergoes ring closure and decarboxylation to indoxyl on fusion with a
C
H O
Na O O C
O HC O O Na
C O O Na
O
C hro m e Violet
mixture of potassium hydroxide and sodamide. Atmospheric oxidation of
indoxyl yield indigotin.
Indigotin is a dark blue coloured powder. It is insoluble in water. It
may be reduced with alkaline sodium hyposulhide to a colourless form the
indigotin-white, which is soluble alkali. It is the alkaline solution which is
applied to the fibres. On exposure to air, the original blue colour indigotin
is regenerated in the cloth.
O
NH
NH
C
O
C CC Reduction
Oxidation
ONa
NH
NH
C
ONa
C CC
Indigotin-WhiteIndigotin
(III) Indigosol-O
Indigotin white is not stable, therefore in ordinary indigotin-
white is first converted its disulphonic ester (I) by treatment with chloro-
sulphonic acid in the presence of pyridine. The alkaline solution of ester
(I) is called the Indigosol-O.
C
C
NH
C
OSO2ONa
NH
NaOO2SO
Indoigosol O
13. Oxazine dyes
Oxazine class of dyes have characteristic oxazine rings system. This class
of dyes includes basic, mordant and direct dyes as well as dioxazine
pigments. The shades obtained are generally blue. Oxazines are obtained
by the condensation of para nitroso-di alkyl aniline like para nitroso
dimethyl aniline with a suitable phenol in alcoholic solution in the
presence at zinc chloride. Thus Capri blue GN is obtained by the
condensation of meta diethyl aminophenol and para nitroso dimethyl
aniline. Capri blues are basic dyes. They posses brilliant shades, good
fastness to light and moderate fastness to washing.
Meldola’s blue and many other dyes of this class are obtained by
phenols. Dioxazine pigments and light fast direct dyes have dioxazinc ring
system which is generally built up by the condensation of an aryl amine or
heterocyclic amine with chloramin.
14. Cyanine Dyes
In cyanine dyes there are two nitrogen containing ring systems, in
one of these the nitrogen is quaternony while another, is tertiary. These
two nitrogen atoms have been linked by a conjugated chain of an uneven
number at carbon atoms, like-CH=and-CH=CH-CH= etc. or similar chains
having nitrogen atoms such as =CH-N=N- etc. The heterocyclic nitrogen
O
N
ClN (CH3)2
Meldola's Blue
O
N
ClN (CH3)2
Capri Bule GN
(CH3)2 N
++
having systems such as quinoline, pyridine, indole, benzothiazol etc. use
in their synthesis.
There are different classes of cyanine dyes which mainly depend
upon the position of heterocyclic systems attached to each other and the
number and type of linkages of a carbon chain or similar chain between
two nitrogen atoms.
(I) Krypto cyanine
It is derived from quinoline derivatives. It belongs to a
subclass carbocyanine because the two rings have been joined by the
linkage-CH=CH-CH=, and linked through their 4 positions of attachment
are 4, 4’ (the ring system are linked through their 4 positions).
Most of the cyanine dyes find use in photography as photographic
sensitisers and desensitizers,because they favour sensitization or
desensitization in a particular region of visible and infrared spectrum.
CH CH CH
Kryptocyanine
N C2 H5
I
H5C2 N+
-
(II) Astrazon pink-FG
NCH2CH2Cl
CH3
CH=CH
N
CH3
Cl
Astrozon Pink FG
+
Chemistry of different classes of dye molecules
Flavonoids
Flavonoids constitute the largest group of plant secondary
metabolites, widespread in nature and found in most of the plant families.
These are photosynthetic products, distributed either vacuoles dissolved
deep in the cell sap or located externally on the surface of stems and
leaves. These are water soluble yellow pigments.
Flavonoid occur as aglycones, glycosides, methylated, acetylated or
as fluoroglucinol derivatives [1-3]. Flavonoids are classified into different
subclasses according to their substitution pattern in their basic skeleton.
(Figure 1, a-i)
1. Flavone - Figure 1 a
2. Chalcones - Figure 1 b
3. Aurones - Figure 1 c
4. Flavonones - Figure 1 d
5. Dihydroflavonol - Figure 1 e
6. Flavonol - Figure 1 f
7. Isoflavone - Figure 1 g
Flavonoid aglycone may be mono,di, tri, tetra and penta substituted
in A ring, B ring or C ring. The substitution may be hydroxy, methoxy,
acetoxy, fluroglucinol, prenylated or glycosilated. Flavonoid glycosides
widely distributed in most of the plants and constitute important
chemotaxonomic marker by physicians in the treatment of various
diseases [4]. The most common glycosidic linkage is C-3 or C-7 in A ring.
On the basis of the nature of glycosidic linkage flavonoid glycoside
divided into two categories.
1. C- glycoside (Figure 1 h)
2. O-glycoside (Figure 1 i)
O
b
f
O
O
OH
O
O
CH
c
i
O O
O H
O H
O H
O
Glu O
d
O
O
g
O
O
h
O
O O H
O H Glu
a
O
O
e
O
O
OH
Figure-1
Flavonoid-O-glycosides are the condensation products of
hydroxylated aglycone and glycone. Chemically these are easily
hydrolysed by acids, alkali or enzymes furnished the hydroxylated
flavonoid and sugar moiety. These are more common glycoside occur in
plant families.
Flavonoid-C-glycoside form glycosidic linkage through carbon-
carbon bond. These are resistant to the hydrolytic procedure and hence
difficult to determine the number and nature of glycone part present in
them by usual hydrolytic methods. Their structure can be established by
the analytical methods.
Xanthones
Xanthones are yellow plant pigments structurally related to flavonoids.
These are the condensation product of 2, 2- dihydroxy benzophenone
having a 6 membered heterocyclic ring (Figure 2a). These are simple,
hydroxy or methoxy substituted dibenzo- γ- pyrone hydrocarbon (Figure
2b). Gentisin (Figure 2c) was first naturally occurring xanthone isolated
from the roots or Gentiana lutea which is responsible for the yellow color
of fermented gentian root [5]. Robert et al.[6] reported eleven xanthone
from Angiospermic plants. Xanthones isolated from different sources are
placed into the following groups.
1. Simple oxygenated xanthones
A. Mono-oxygenated xanthone - Figure 3a.
B. Di-oxygenated xanthone - Figure 3b.
C. Tri-oxygenated xanthone - Figure 3c.
D. Tetra-oxygenated xanthone - Figure 3d.
E. Penta-oxygenated xanthone - Figure 3e.
O
O
OH
OH
OMe
c
Figure 2 (a-c)
F. Hexa-oxygenated xanthone - Figure 3f.
2. Dimeric xanthone - Figure 4a
3. Xanthonolignoids - Figure 4b.
4. Prenylated xanthones - Figure 4c.
5. Xanthone glycosides - Figure 4d.
O
O
a
O
O
b
O
O OH
OCH3
CH3O
OCH3
OCH3
OCH3
e
O
O OH
OCH3
CH3O
OCH3
CH3O
OH
f
Figure 3 (a-f)
O
O
OCH3
OH
OCH3
c
O
O OH
OCH3
OH
OCH3
d
O
O
OH
a
O
O
OH
OCH3
b
O
O OH
OCH3CH3O
OH
OO
OH
OCH3
OCH3
OHO
a
O
O OH
OCH3
O
O
OH
OCH3
OH
b
O
O O H
OCH 3
CH 3 O
O H
O
O
O O H
O H
O
O H O H O H
C H 3
CH 2 OH
Figure 4 (a-d)
d
O
O OH
OH
.
OH
.
CH3O
.
c
O
HH
a
O
O
b Figure 5 (a-b)
Anthraquinones
Anthraquinones are the derivative of anthracene nucleus distributed
widely in plants either in free state or in the form of glycosides. These are
well known for their dyeing and medicinal properties. Mostly these are
purgative and include the well-known natural drugs senna, cascara,
rhuburb, aloes, buckthron, cassia pulp etc. The well known dyeing
properties of plants eg. maddar or cochineal have great importance in
dyeing are due to presence of anthraquinone. Anthraquinone-O-glycosides
are easily hydrolysed by usual hydrolysing reagent but anthraquinone-C-
glycosides are resistant towards hydrolysing reagents. These are mostly
distributed among dicotyledons. The fungal anthraquinone pigments are
nearly all crysophenol or emodin derivative. Naturally occurring
anthraquinones are of two types.
(i) Anthranol and anthrones
These are reduced derivatives, brownish yellow fluorescent
coloured constituent of crysarobin (Figure 5a) found in trunk cavities of
the tree Andira araroba.
O
O
c
OH
O-glu OH
d
(ii) Dianthrone
These are the compound formed by the mild oxidation of two
identical or different anthraquinones moieties (Figure 5b) are the
important aglycones of purgative plant Cassia, Senna, Rhus and Rhamnus.
Naphthquinone
Naphthquinone occur in a number of plant families commonly in
reduced or glycosidic forms. These have the hydroxylated or acetylated or
methylated naphthalene nucleus (Figure 5c). The 4β-D-glucoside of α-
hydrojuglone (Figure 5d) a compound of walnut leaves after atmospheric
oxidation converted to the coloured compound, naphthaquinone
responsible for red color. The red color of henna (Lawsonia) (Figure 5e) is
due to hydroxy naphthaquinone [7]. Naphthaquinone distributed among
the plant families–Rubiaceae, Verbenaceae, Bignoniaceae, Juglandeaceae
etc. Naphthquinone have also used in pharmaceutical and cosmetic
industries [8-10]. Shikonin, alkannin and alkannan isolated from
Lithospermum officinale is presently used in various pharmaceutical
industries. Naphthaquinone occur as monomer, dimmer or complex trimer
[11].
O
OOH
R
OH alkannan R = - CH 2 - CH 2 - CH 2 HC
C H 3
C H 3
alkannin R = C
O HH
. CH 2 CH C
CH 3
CH 3
CHOH
. CH 2 CH C
C H 3
C H 3
Shikonin R =
Figure 5 (c-e)
Anthocyanins
Anthocyanins comparises a group of glycosidic pigments
responsible for various colors particularly red, violet, blue in flowers,
stems, berries, leaves and roots etc. These have flavylium chloride (fig
6a), benzopyrylium chloride nucleus (Figure 6b). All the anthocyanins are
the derivatives of 3, 5, 7-trihydroxyflavylium chloride (Figure 6c) (parent
compound) differ in the number, nature and position of hydroxy, methoxy
or sugar residue. The anthocyanin aglycones are known as anthocyanidin
and the commonly occurring sugar in anthocyanin are glucose, galactose
and rhamnose. The sugar is generally attached to C-3 or rarely at C-5
position of aglycone. Some naturally occurring anthocyanins are
acetylated derivative of two common acids p-hydorxybenzoic acid and
malonic acids. A new 6-hydroxy anthocyanidin has been isolated from the
red flowers of Alstroemeria, which occur as 3-glycoside [12]. The
anthocyanin give different color in different medium viz. red color in
acidic medium, purple in neutral medium and blue in alkaline medium
[13], eg. in aqueous sodium acetate soloution (pH-8) the solution of
cyanin chloride (red salt) is violet due to formation of anhydrobase. On
standing this solution it becames colorless which when kept in alkaline
medium (pH-12) it again gave blue color due to formation of anion
O
O
OH
OH
e
OG
O+Cl
b
O+Cl
a
O O H
OG
O H
O H
O O H
OG
OG
O H
O AcONa
HCl
O O H
OG
OG
O
O O O H
OG
OG
O H
O H
Cyanidin chloride
NaOH HCl
anhydrobase anion Pseudobase
NaOH
Anhydrobase
on standing NaOH
d
Figure 6 (a-d)
anhydrobase (Figure 6d). Again this solution in acidic medium (pH-4),
turn to red due to regeneration of cyanidin chloride. The colour and shades
of anthocyanins also depend upon the concentration of anthocyanins,
position and number of hydroxy group and number and nature of sugar
moiety attached with the parent compound.
OHO
OH
OH
c
Figure 7 (a-c)
C H 3
C H 3
C H 3 C H 3
C H 3
HOOC
a
O
C H 3
C H 3 C H 3
C H 3
O
O
b
. . . .
. O H . .
O H
. .
c
Carotenoid
Carotenoid is a group of plant pigments responsible for variety of
characteristics colour shades, yellow, orange, purple, red etc. This group
was first derived from the orange pigments found in carrot. Carotenoid
possess a long aliphatic, polyene chain composed of isoprene units, eg.
Bixin (Figure 7a) obtained form Bixa orellana (annato) and crocin (Figure
7b) found in Crocus sativus (saffron), Nyctanthes arbortristris and
Gardenia jasminoides [14]. Carotenoids are fat soluble compound
produce deep blue colour with antimony chloride. These compounds
possess eight isoprene units in their structure, hence regarded as tetra
terpenes. Xanthophylls (Figure 7c) contain cyclic ring with long aliphatic
chain found in green plants and some green insects. Carotenoids may be
classified into following classes.
1. Hydrocarbon or carotene eg. Bixin Figure 7a
2. Oxygenated derivative eg. Xanthophyll Figure 7c
Some oxygenated carotenoid of plant origin are listed in Table –
Oxygenated carotenoids of plant origin
Carotenoids Formula Occurence
Luteuin C40H56O2 yellow blossoms
Bixin C25H30O4 annato (Bixa orellana)
Crocetin C20H24O4 saffron
Crocin C44H64O4 saffron
Fucoxanthin C40H60O4 brown algae
Zeaxanthin C46H56O2 Physalis
Capsanthin C40H58O3 Capsicum spp.
Capsorubin C40H60O4 Capsium
Tannins
The term ‘tannin’ was first introduced by Seguin in 1796, denote
substances present in plant extracts which were able to combine with
protein of animal hides, prevent their putrefaction and convert them into
leather. Tannins are bitter and astringent substances in plants often
occurring as excretions in the bark and other parts. Mostly, true tannins
have high molecular weights and many types of tannin are glycosides.
Modern authors [15] often treat tannins not as a specific phytochemical
group but as example of polyphenols illustrating particular aspects of
gallic acid flavan 3- ol. The characteristic properties of tannins derived
from the accumulation within a moderately sized molecule of a substantial
number (1-2 per 100 mol. wt.) of phenolic groups many of which are
associated with O- dihydroxy and O- trihydroxy orientation within a
phenyl ring. Tannins can be categorised in four different groups.
C
O
O
O H
O H
O H
O H
O H CO
O
O H
O
O
O O H
O H
O H
O C
O
O H
O H
O H
O C
O
O H
O H
O H
H 2 C
O C
O
O H
O H
O H
b
O
C
C
O
O H
O H
O H
O H
O
O
a
(i) Hydrolysable tannins
Acids or enzymes may easily hydrolyse these tannins. Hydrolysable
tannins are formed from several molecules of phenolic acids such as gallic
and hexahydroxydiphenic acids, which are attached by ester linkages to
the central glucose molecule. These are known as pyrogallol tannins e.g.
gallotannins (Figure 8a) and ellagitannins (Figure 8b) are of highly
medicinal importance. These tannins may be monomeric, dimeric or
oligomeric. Agromoniin, the first reported oligomeric tannin isolated from
Agromonia composed by four monomeric gallic acid units, whereas
geraniin (Figure 8c) and tellimagrandins (Figure 8d) are known to
composed by 20 such monomeric units.
R=OH Tellimagrandin R=β-OG Tellimagrandin
Figure 8 (a-c)
d
O H
O H O H
O H
O H O H
CO CO
O O
CH 2
O OG
O O
CO CO
O H
O H
O
H
O H O H
O H
O
c
C
COH
OH
OH
OH
OH
OH
O
O
O
O
O
R
O-G
CH2
O
G
OOH
OH
OH
OH
OH
a
OOH
OH
OH
OH
OH
b
O O H
O H
OH
O H
O H
O H
c
(ii) Condensed tannins (proanthocyanidins)
Condensed tannins are not readily hydrolysed to simpler molecules
by usual hydrolysing reagents and do not possess sugar moiety.
Condensed tannins are related to the flavonoid pigments and have
polymeric flavan-3-ol structures. Condensed tannins when treated with
acids or enzymes converted into red insoluble compounds known as
phlobaphenes, which is a coloring agent used in various drugs and food
industries. On dry distillation these tannins yield catechol and so these are
some times called catechol tannins (Figure 9a-9d).
O O H
O H
OH
O H
O H
O O H
O H
OH
O H
O H
d
Figure 9 (a-d)
(iii) Pseudotannins:
Pseudotannins are comparatively low molecular weight compound
than of tannins usually found in maximum quantity in dead or drying cells
[15]. These tannins do not respond Gold beater’s skin test of tannins. The
most common pseudotannins are obtained from a number of plants and
abundantly in rhubarb, ipecacuanha, mate, coffee (particularly unroasted),
nux vomica etc.
(iv) Complex tannins:
Complex tannins have not great significance to mainstream
pharmacognosy. Complex tannins have been isolated from the
Combretaceae, Fagaceae (Quercus castanea), Mystaceae, Polygonaceae,
(Rheum) and Theaceae (Camellia). These are biosynthetic product of
hydrolysable tannin and condensed tannin. The commonly known
hydrolysable tannin unit occur in comlex tannin is C–glucoside
ellagitannin. The chemical union occur through a C–C bond between C–1
of glycone and usually C-6 or C-8 of flavan–3–ol derivative. The most
common glycone is glucose found in the complex tannin. The monomer
also formed the oligomer.
Coumarins
Coumarins have been found in all parst of plants. These are
derivative of benzo-α- pyrone or the lactone of O- hydroxycinnamic acid.
Aesculatin, umbelliferone and scopoletin are common in plants both in the
free state and as glycosides. In ammonical solution these compounds gave
a blue, blue-green or violet fluorescence. Coumarin gave a characteristic
odour and occur in about 150 species belonging to over 30 different
families. The vast majority of coumarin carry an oxygen substituent at C-7
position.
The coumarin are classified into the following groups:
i. Simple coumarins
ii. Furano coumarins
iii. Pyrano coumarins
iv. Phenyl coumarins
v. Bis coumarins
vi. Tris coumarins
vii. Coumarin-lignoids or coumarin lignans
viii. Coumestane
(i) Simple coumarins
These are metabolic products of the plants, formed from
the corresponding substituted trans-cinnamic acid derivatives. 7–
Hydroxycoumarin is often regarded as the parent compound (Figure 10a).
(ii) Furano coumarins
Furano coumarins are linear or angular analgous with substituents at
one or both the benzenoid positions. These are compounds with a furan
ring fused with the benzene ring (Figure 10b) of coumarin [16].
(iii) Pyrano coumarins
Pyrano coumarin (ceylantin) (Figure 10c) have been isolated from
the heartwood of Atlantica ceylanica [17]. In pyrano coumarin, a pyran
(six membered) ring is present in place of furan ring as in furano
coumarin. These are categorised into different groups on the mode of
fusion of the ring. These are linear (xanthyletin) (Figure 10d), angular
(Figure 10e) and angular dihydropyrano coumarin (Figure 10f).
(iv) Phenyl coumarins
The phenyl coumarins have a benzopyrone nucleus. The 3-
phenylcoumarins may be regarded as substituted 4-hydroxycoumarins.
This group has varied type of structure as 3-phenyl coumarin and 4-phenyl
coumarin (Figure 10g)
(v) Bis coumarins
Bis coumarins are formed from two coumarin moieties and the
linkage may occur in a number of ways. Dicoumarol is formed at C-3 and
C-3` (Figure 10h) through a methylene group and the first isolated
compounds of this series. Some examples of this group are as daphnoretin,
O–dimethyl–3–8- bisiderin and aflavarin.
(vi) Tris coumarins
A coumarin moiety is attached through one or more C-C bond to
another structural entity [18]. The chirality was deduced to be S for the
triscoumarin [19]. The common examplesof this group are trimbelletin
and wikstorosin (Figure 10i).
(vii) Coumrin–lignoids or coumarin lignans
In most of coumarin lignoids a C6-C3 unit is linked with a coumarin
nucleus through a dioxane bridge. Common examples are cleomiscosin-,
hemidesmin – 1, hemidesmin – 2 and aquillochin (Figure10j).
(viii) Coumestane
The common compounds of this group are coumestan (Figure 10k)
4-methyl coumestan and coumesterol. 3-Aryl- 4-hydoxy coumarins
derived from 4-hydroxy coumarin and quinones may be oxidatively
cyclised to cumestane using potassium ferricyanide [20].
O OOH
a
O OO
b
O
O
OMeO
OMe
c
O OO.
.
d
O OO
e
O O
O
OH
.
.
f
O O
g
Figure 10 (a-k)
h
HO
O
O
O O
O
i
O
O
O
O
O O
O
j
O O
O
O
OH
OH
OMe
k
O
O
O
Isolation Techniques
1. Collection of plant materials
Flowers and fruit rind of the study plants were collected form
Ranichauri, Agrakhal-Kunjapuri of District Tehri Garhwal and Pasulok
(Rishikesh), Dehradun. Plant materials were washed, air dried in shade,
chopped into small pieces and make into fine powder before extraction
separately.
2. Extraction of plant materials
(a) Extraction with organic solvents:
The plant material was extracted first with light petroleum (60-80
°C) and then extracted with MeOH or EtOH, 2 or 3 times. Untill the
extractive became colourless. The extract was monitored on TLC
(CHCl3:MeOH:H2O) (65 : 35 : 10) and developed with 7 % H2SO4 to
show the appearance of some spots. The extract was then fractionated
through CC (Column Chromatography) using chloroform-methanol as
eluting solvent. The polarity of eluent was gradually increased by addition
of methanol to afford different fractions.
All these fractions are checked by HPLC by using Aceto nitrile :
Acetone : Methanol as mobile phase. The sharpness of peaks of different
fractions in HPLC gives an idea about the purity of compounds. Some
fractions gives large trailing in HPCL, this indicating the impurity of
samples, which could not be separated by column chromatography.
Chromatographic techniques:
The term chromatography refers to a group of methods in which
difference in the affinity of substance for an active or adsorbing material
are utilized by percolating the mixture through a fixed bed of the material.
This bed of adsorbing material is either packed into a glass tube (column
chromatography) or it takes in the form of porous surface. (Paper
Chromatography and Thin Layer Chromatography).
(i) Paper Chromatography
Paper chromatography is the oldest and most widely used method
of qualitative analysis of various compounds from crude extracts. A
number of phenolic compounds have been analysed by using the one-
dimensional and two dimensional paper chromatographic techniques. The
choice of the solvent used to run a chromatogram depends up on the
polarity of constituents present in extract.
Usually, non-polar solvents like benzene: acetone: water (7:4:5) are
the commonly used developing solvents for polar aglycone and
glycosides. Rf value and appearance of coloured spots is useful guide in
the preliminary identification of different compounds.
(ii) Thin Layer Chromatography
TLC is a powerful, less time consuming and simplest method for
demonstrating the homogeneity of known and unknown natural dyes and
frequently serves as a tool for identification. It may be considered a basic
method for qualitative analysis of different compounds. The efficiency of
this method has been demonstrated by Derquini et al. [21].
The detection of spots on thin layer plates is identified by their Rf
values and characteristic colour tests, fluorescence under short wave UV
illumination or with different reagents.
The most commonly used solvents for separation of carotenoids are
ether-benzene (50:50) and n-hexane-ether (30:70), for quinone groups,
most frequently used adsorbent are polyamide and silver nitrate, with the
developing solvents benzene, chloroform or acetone water.
The mixture of methanol: acetic acid: water is successively used for
the separation of glycosides and aglycones of aurones, chalcones,
flavanones, flavones, flavonols and isoflavones.
The spots may also visualized on thin layer plates under UV light
with or without the add of ammonia vapours. Sulphuric acid and iodine
are universally used visualizing reagent for the detection of all kinds of
compounds.
The quinone and its derivative turn brownish on treatment with
iodine vapours and violet with concentrated sulphuric acid at 110°C.
Whereas ubiquinone, plastoquinone and tocophenyl quinones turn blue
with sulphuric acid [22]. Sulphuric acid is less sensitive for the separation
of carotenoids but produce different colours on thin layer plates. Wender
and co-workers [23] separated a number of flavanone glycosides on
polymide TLC plates using solvent systems nitro methane: methanol (5.2
V/V).
The spots of some dye yielding material can be identified from their
fluorescence under UV radiation [24-26]. For example coumarins are
easily detected as yellow spots by viewing the chromatogram under long
wave UV light. The spot appearance in UV light alone and in the presence
of ammonia vapours for different flavonoids is presented in table.
Relationships between spot colour and flavonoid structure
Flavonoid Spot Color
UV/NH 3 Flavonoid Type
U.V. light
Deep purple
Yellow, yellow-
green or brown
a.Usually flavones with 5-OH and 4-OH or
3-OH substituted flavonols with 5-OH and
4-OH.
b. Some 5-OH flavones and 4-OH chalcones
lacking B-ring hydroxyl groups.
Little or no colour
change
a. Flavones or flavonols with 5-OH but with
the 4-OH absent or substituted.
b. Iso flavones, dihydro flavonols and some
flavanone with 5-OH.
c.Chalcones with 2` or 6`-OH but without a
free 2-or-4-OH.
Light blue, red or
orange
Some 5-OH flavanones, chalcones with a
free 2-and or 4-OH.
Fluorescent
light blue
Fluorescent yellow-
green or fluorescent
blue green
a. Flavones and flavanones lacking a free 5-
OH.
b. Flavonols lacking a free 5-OH but with
the 3-OH substituted.
Little or no colour
change Bright
fluorescent light
blue
Isoflavones lacking a free 5-OH
Isoflavones lacking a free 5-OH.
Invisible Fluorescent light
blue
Isoflavones lacking a free 5-OH.
Dull yellow
and yellow
orange
fluorescence
Little or no colour
change
Flavonols with a free 3-OH and with or
without a free 5-OH.
Fluorescent
yellow
Orange or red Aurones with a free 4`-OH and some 2-or 4-
OH chalcones.
Yellow-
green blue-
green or
green
Little or no colour
change
a. Aurones lacking a free 4-OH and flavones
lacking a free 5-OH.
b. Flavanols with a free 3-OH and with or
without a free 5-OH.
Pale yellow Light yellow purple Dihydroxy flavonols lacking a free 5-OH.
Column Chramatography
Column chromatography is the best technique for the quantitative
separation of complex mixture of compounds. A number of different
adsorbents like silica gel, cellulose powder, polyamide, charcoal, starch
etc. have been used for the column chromatographic separation. The
choice of solvent and adsorbents depends upon the nature of compounds
to be separated. The eleuotropic series of solvent used for specific
separation are: Petroleum ether <n-hexane<benzene <dichloromethane
<chloroform <diethyl ether <ethyl acctate <acetone <n-propanol <methyl
alcohol <ethyl alcohol <water. Silica gel is the most commonly used
adsorbents for the separation of all kinds of compounds and charcoal
packed columns used for the primary purification of mixture of phenolic
molecules from the crude extracts. A number of polar and non polar
coumarins have been successfully separated by using combination of n-
hexane-ether. [27-29] n-hexane ethyl acetate [30,31] and
dichloromethane-tetrachloromethane with increasing amount of ethyl
acetate [28,29]. For the best separation of all type of flavonoid, aglycone
and glycosides polyamide, starch and charcoal are the best used
chromatographic adsorbents [30]. Whereas mixture of methanol-water is
preferred as eluting solvent.
High Performance Liquid Chromatography (HPLC)
Liquid chromatography is the oldest and most powerful form of
chromatography. In late 1960’s more and more emphasis was laid down in
developing liquid chromatography as complimentary technique to gas
chromatography as it had attained a significant role in modern
instrumentation. In 1967, Huber and Hulseman discovered high speed
liquid chromatography also known as “High pressure” high performance
and modern liquid chromatography. In practice, liquid chromatography
was performed in large diameter glass coloumns under atmospheric
condition at that time. Advances in both instrumentation and column
packing occurred, so rapidly that it was difficult to maintain a state of art
expertise. Now a days liquid chromatography has emerged as the powerful
analytical technique being employed in every laboratory.
Droplet Counters Current Chromatography (DCCC)
It is a modern separation technique for the fractionation of extracts
of natural products. The phenolics, xanthones and related compound
bearing few hydroxyle groups in their structure can be separated by
DCCC by using less polar layer of mobile phase in descending mode. The
polar glycosides were separated by using n-butanol : acetic acid : water (4
: 1 : 5) solvent system. The DCCC separation of acetyl flavonoids was
reported by Tanaka et al. using CHCl3 : MeOH : H2 O solvent system
[31].
Characterisation
Spectroscopic techniques
1. Ultraviolet and Visible spectroscopy (UV-VIS)
UV and Visible spectroscopy is valuable tool, most frequently used
in the identification of some functional groups especially the chromophore
group and ethylenic bonds present in the molecule. A number of research
paper published regarding the UV interpretation of the dye molecule like
xanthones, flavonoids, anthraquinones, proanthocyanidins etc. The
chromophoric groups in a molecule is highly influenced by the nature of
parent nucleus. The absorption bands for the colorued compounds ranges
between 200-700 nm. In the measurement of UV spectroscopy the
compound is generally dissolved in methanol or ethanol in presence of
classical shift reagent like AlCl3, HCl etc [32] is a method of choice to
determine the structures.
The visible region or band-I associated with actual colour depends
on the number and positions of the hydroxyl or methoxyl attached with
the parent ring and when these groups are fixed the colour then depend on
pH and solvent used [33].
The UV spectra of the some dye yielding molecules e.g. Flavonoids
(flavones and flavonols) when acetylated with cinnamic acid showed wide
maxima at 310-330 nm, characteristics to phenolic acids [34]. The UV
spectra also used to differentiate chromones and coumarins. Chromones
generally exhibit strong absorprtion bands between 240-250 nm, whereas
coumarins usually have a maxima at this wave length [35] and showed
absorption maxima at 274-311 nm. The introduction of hydroxyl group in
coumarin nucleus cause a bathochromic shift in the principle absorption
band and the formation of new maxima depend on the conjugation of
hydroxyl group and chromophoric groups. Hydroxyl and alkoxy groups
intensify the long wave length band and the bathochromic shift, above
360nm being dominated by the number of α-hydroxy groups. The β-
hydroxy groups, relatively little influence unless adjacent to an α -hydroxy
group [36,37].
UV spectra of glycosides provide information about the position of
glycosilation and number of glycone attached to it. The principal
absorption bands of different dye yielding molecules is summarised in
table.
Principal absorption bands of different dye yielding molecules
S.
No
Parent compound Solvent Principal maxima
1. Chromones MeOH 240-250 (log e 3.8).
2. Anthracene EtOH 252 (5.29), 308 (3.15), 323 (3.47),
338 (3.75), 355 (3.86), 375 (3.87).
3. Phenolic acids MeOH 310-330.
4. Quinoline EtOH 226 (4.53), 230 (4.47), 281 (3.56),
301 (3.52), 308 (3.59).
5. Isoquinoline Hexane 216(4.91), 266(3.62), 306(3.35),
318(3.56).
6. Cyanidin MeOH-HCl λ max 535 nm.
7. Alkyl coumarin MeOH 274-311 (loge 4.03 and 3.72).
8. Pelargonidin MeOH-HCl Max 520 nm.
9. Kaempferol MeOH 253sh, 266, 294sh, 322sh, 367
10 Quercetin3-O-
glucoside
7-O-rhamnoside
NaOMe 244, 270, 396.
11. Apigenin-7-O-
glucoside
MeOH 268, 33.
12. Xanthotoxin MeOH 218, 249, 277.
13. Anthraquinone MeOH 260, 260-290, 320-330.
14. Proanthocyanidins MeOH 205, 230-240.
15. Coumarins MeOH 274-311.
16. Flavonols and
flavones
MeOH 240-280 band II,
300-380 band I.
2. Infra-Red Spectroscopy (IR)
Infra-red spectroscopy has great importance in the identification of
functional groups and stereochemistry of some positions of various
compounds. The characteristic bands observed in the spectra of dyes
depend on the nature and positions of functional groups and parent ring.
Generally the phenolic hydroxyls are absorbed strongly at 3650-3584 cm-1
[38], whereas the same bond of unsubstituted flavones appeared at 1650
cm-1 [39].
The C-H stretching frequencies appeared in the form of these bands
of medium or week intensity between 3025-3175 cm-1 in the spectra of
coumarins. These absorption bands appeared due to the C-H stretching
vibrations of pyrone, furane and benzene rings [40, 41]. Table represent
the type of stretching frequencies of the bands commonly occur in the
natural product.
S. N. Type of Bonds Absorption Regions
1 C-C, C-O, C-N 1300-800
2 C=C, C=O, C=N, N=O 1900-1500
3 C=C, C=N 2300-2000
4 C-H, O-H, N-H 3800-2700
The IR spectra of flavonoids displayed absorption in the
region1600-1700 cm-1 for α,β-unsaturated carbonyl function and the
aromatic absorption at 1500-1600 cm-1. Unsubstituted chalcone displayed
carbonyl stretching band at 1659 cm-1 and same band for un substituted
flavone at 1650 cm-1.
Mass Spectroscopy
A mass spectrometer is an instrument that produces ions and
separates them in the gas phase according to their mass-to-charge ratio
(m/z). Today, a wide variety of mass spectrometers is available, ranging
from bench top detectors for gas chromatography to warehouse sized
instruments such as accelerator mass spectrometers. All of these share the
capability to assign mass-to-charge values to ions, although the principles
of operation and the types of experiments that can be done on these
instruments differ greatly. Basically, a mass spectrometric analysis can be
envisioned to be made up of the following steps:
(1). Sample introduction
(2). Ionization
(3). Mass analysis
(4) Ion detection/Data analysis.
Samples may be introduced in gas, liquid or solid states. In the
latter two cases. Volatilization must be accomplished either prior to, or
accompanying ionization. Many ionization techniques are available to
produce charged molecules in the gas phase, ranging from simple Electron
(impact) Ionization (EI) and Chemical Ionization (CI) to a variety of
desorption ionization techniques with acronyms such as FAB, FD, ESI
and MALD. Mass spectrometers are operated at reduced pressure in order
to prevent collisions of ions with residual gas molecules in the analyzer
during the flight from the ion source to the detector. The vacuum should
be such that the mean free path length of an ion. i.e. the average distance
an ion travels before colliding with another gas molecules is longer than
the distance from the source to the detector. For example at a pressure of 5
x 10-5 torr for instance, the mean free path length of ion is approximately
one meter, i.e. about twice the length of a quadrupole instrument. Thus the
introduction of a sample into mass spectrometer usually requires crossing
of a rather large pressure drop, and several means have been devised to
accomplish this, gas sample may be directly connected to the instrument
and metered into the instrument via a needle valve. Liquid and solid
samples can introduced through a septum inlet or a vacuum-lock system.
1. Electron impact and chemical ionization
Volatile substances can be ionized by electron (impact) ionization
in a process involving the interaction of the gaseous sample with an
electron beam generated by a heated filament in the ion source. The
electron energy is defined by the potential difference between the filament
and the source housing and is usually set to 70 ev(~1.12x10-17J). A
magnetic field keeps the electron beam focused across the ion source and
onto a trap. Upon impact with a 70 ev electron, the gaseous molecule may
lose one of its electrons to becomes a positively charged radical ion, M+ e-,
M+ 2e- where M+ is termed the molecular ion. It carries an unpaired
electron and can occupy various excited electronic and vibration states. If
these excited states contain enough energy, bonds will break and fragment
ions and neutral particles will be formed. With electron energy of 70 ev.
enough energy is transferred to most molecules to cause extensive
fragmentation. All ions source by an electric field produced by the
potential difference applied to the ion source and a grounded electrode. A
‘repeller’ serves to define the field with in the ion source. Fragmentation
will either take place in the ion source-giving rise to stable fragment ions,
or on the way to the fragmentation occurs before the ion strikes the
detector, a signal for the detector, a signal for the molecular ion is
generated. The mass spectrum obtained form recording all of these ions
contains signal of varying m/z and intensities, depending on the number of
ions depend on the structure of the molecule. Such that similar structures
give similar mass spectra [42]. The instrumentation used for CI is very
similar to that used for EI. The major difference in the design of the
source is that it is more gastight so that the reactant gas is retained at
higher pressures in order to favour ion/molecular reactions. The pressures
inside the CI ion source is typically of the order of 0.1-1 tarr. A wide
variety of reagents have been used, some of which lead to fragmentation
hydrogen for which the reactant ion is H3O+. Other reagents give rises to
mass spectra are iso-butane and butane and n-pentane for which reactant
ions are C4H9+ and C5H11
+ [43].
2. Plasma Desorption Mass Spectroscopy (PDMS)
A break through in the analysis of bio-molecules came in 1974 with
the introduction of PDMS [44]. This technique uses 252 Cf fission
fragments to desorb large molecules from a target. The target is made of a
thin aluminum foil, often covered with a layer of nitro cellulose, to which
a droplet of the sample solution is applied. The adsorption of proteins to
nitro cellulose is believed to be washed off and chemical reactions to be
carried out on the target. Alternatively, the sample can be electro sprayed
directly onto Ni or Al foil, a technique that is move effective for smaller
peptides two atomic particles are produced by the 252 Cf fission reaction.
One causing adsorption of the analyte and the other providing the start
signal for the time-of-flight measurement. A time of-flight mass analyzer
is generally used for ion separations. PDMS has a reasonably good
sensitivity with peptides and small proteins and typically about 10
picomoles are needed for molecular mass determinations of such a
protein. Perhaps its major virtue is that it is simple method. Both easy to
use with easily interpretable spectra and well suited for the protein
chemists and others.
3. Fast Atom Bombardment (FABMS)
It is another soft ionization technique i.e. one that yields minimal
fragmentation that performs well for polar and thermally labile
compounds [45]. In a typical FAB analysis, the sample is usually
dissolved in an appropriate matrix, a viscous solvent, in order to keep the
sample in the liquid state. Some of the more common liquid matrices are
glycerol, 1-thioglycerol, a matrix of dithiothreitol and dithioerythritol, 3-
nitrobenzyl alcohol and tri ethanolamine. One major role of the matrix,
because of its low freezing point, is to keep the sample in a liquid state as
it enters the high vacuum ion source. This matrix also reduces damage to
the analyte caused by the high energy-bombarding particle. In FAB
ionization, the sample droplet is bombarded with energetic atoms (Ar, Xe)
of 8-10 Ke V kinetic energy. Ions (e.g. Cs+) can be used as the
bombarding particle in a similar technique termed Liquid Secondary Ion
Mass Spectrometry (LSIMS). The general process leading to the
formation of the molecular ion and involves several different mechanisms
including ejection of performed ions.
The major advantage of FAB is that it is easy and fast to operate,
the spectra are simple to interpret, and the source it self is easily retrofitted
on most mass spectrometers. Several mixtures have been reported for
mass calibration in FABMS.
4. Thermospray and Particle Beam
Thermospray ionization was introduced in 1983 for the coupling of
HPLC at conventional flow rates (0.5 to 1.5 ml/min) to a mass
spectrometer [46]. The effluent from the HPLC column is vaporized under
reduced pressure by heating a stainless steel tube of 0.10 to 0.15 mm inner
diameter. The resulting supersonic jet contains small droplets that
vaporize further due to the hot gas in this low pressure region of the ion
source. Complete evaporation of the solvent from the liquid droplets
produces gas phase ions from ionic compounds in the sample solution or
from gas phase chemical ionization, when an auxiliary filament or low
current discharged device is used. Ionization requires polar or charged
species and volatile buffers, the filament arrangement is used for semi
volatile samples, and the discharged device for highly aqueous effluents.
The temperature of the vaporizer is critically and has to be adjusted for a
given solvent compositions to give best results. Ions are drawn into the
analyzer by electric fields and enter through on orifice of about 0.5 mm
diameter. Thermospray is considered as a soft ionization technique and
induces only limited fragmentation of the analyte. The particle beam
interface, derived from the MAGIC interface (Monodisperse Aerosol
Generation Interface for Chromatography), has elements in common with
thermospray, but gives spectra with more fragment ions [47]. Again
formation of an aerosol is the initial step, followed by dispersion caused
by a gas stream (usually helium), and desolvation. In the original
interface, a momentum separator then separates the lighter dispersion gas
and vaporized solvent from the sample particles, which have higher
momentum, and finally ions enter the mass spectrometer ion source as a
beam of charged particles. PB mass spectrum however has a relatively
intense background at low mass due to the solvent and limited sensitivity.
PB is less sensitive than thermospray and is best suited for ionic
compounds.
5. Electrospray (ES)
ES ionization has a tremendous impact over the last few years on
the use of mass spectrometry in biological research. It was the first
method to extend the useful mass range of instruments to well over 50,000
Da. Although introduced in its present form in 1984, the technique goes
back to investigations of the electrically assisted dispersion of liquids at
the beginning of this century. This was done by spraying a sample
solution from a small tube into a strong electric field in the presence of a
flow of warm nitrogen to assist desolvation and then measuring the ions
mobility techniques. Further innovative experiments in this field led to the
introduction of the ES ionization source [48]. Since then, ES has
investigated a wide range of bio-molecules. The sample is usually
dissolved in a mixture of water and organic solvent, commonly methanol,
isopropanol or acetonitrile. It can be directly infused, or injected into a
continuous-flow of this mixture or be contained in the effluent of an
HPLC column or CE capillary. The ES source design is simple, with spray
formation occurring in a high voltage field. In one proposed mechanism,
ion formation is believed to result from an ion evaporation process, first
proposed in 1976 [49]. A spray of droplets is caused by electrostatic
dispersion from the liquid ejected from the capillary tip. Aided by the
heated bath gas (usually nitrogen), the droplets undergo declustering
losing solvent molecules in the process and eventually producing
individual ions. In another proposed mechanism, desolvation of the
droplet surface that will eventually cause a coulombic explosion that leads
to individual ions. Spray formation is the crucial part of the ES technique.
It is usually advisable to filter all the solvents and high concentrations of
electrolytes should be avoided because they can lead to electrical break
down and unstable operating conditions. Electrospray has been used in
conjunction with all common mass one advantage of ES over PD and
MALD is that as a consequence of the multi-charging phenomenon the
instrument can be calibrated in the low m/z range, using singly charged
celebrants with known exact masses. Recently electron spray techniques
has been used more successfully to study the non covalent interaction
among molecules. Probably attributed to its case of used with 100%
aqueous solutions.
6. Matrix-Assisted Laser Desorption (MALD)
A wide range of wavelength from UV to IR, have been used with
many different types of mass spectrometers for isotope measurements,
elemental composition analysis, and for pyrolysis of small organic and
inorganic molecules. A major breakthrough came in 1988 with the
introduction of MALD [50], a technique which now is able to detect
biomolecules over 3000.000 Da, in size. The technique involves
embedding the analyte in a solid matrix, which absorbs energy at the
wavelength of the laser. The actual mechanism of MALD, combinations
of desorption of matrix and embedded bio-molecules. The laser energy
absorbed by the matrix, typically on the order of 106 watts/cm2 leads to
intense heating and generation of a plume of ejected material that rapidly
expands and undergoes cooling. The phase transition (evaporation and
sublimation) is probably the rate determining step in ion formation. The
technique also generates single and multiple-charged clusters of the
analyte of low intensities, an undesirable situation in that these tend to
complicate the spectrum. MALD produces a relatively intense matrix
background generally below m/z 1000 that can be minimized
electronically. MALD is a very fast and sensitive technique implemented
on small, relatively inexpensive instruments that do not require extensive
expertise in mass spectrometry.
Nuclear Magnetic Resonance Spectroscopy (NMR)
NMR spectroscopy is the conventional method used for the
structure determination of natural products. The 1H and 13C-NMR spectra
reflects the distribution of electrons surrounding the hydrogen and carbon
nuclei and are sensitive techniques to provide evidence for configuration
and conformation characteristics.
The introduction of Fourier Transformation (FT) technique in the
pulsed NMR spectroscopy in 1966 by Ernst and Anderson [51] started a
new era in this branch of spectroscopy. In the last two decades,
tremendous studies were taken in the advancement of commercially
available instruments of several multiples NMR techniques, most notably
in two-dimensional transformation.
(a) One Dimensional NMR Spectroscopy
(I) 1H-NMR
All the protons in organic molecules produce NMR signals at
different field strength when definite radio frequency is applied. Thus,
very conclusive results have been obtained to determine the structure and
stereochemistry of organic compounds.
These spectra provide specific information in the C-methyl region
in steroidal sapogenins. 1H-NMR gives unambiguous information about
the presence of α or β linkages in the glycone part of the saponim [52-54].
The anomeric protons of various sugars give peaks in the region of δ 4.50-
6.30, β-linkage of D-sugar have large coupling constant (J=6-9) whereas a
rarely occurring α-linkage of D-sugars have low value (J=2-4Hz)
In 1H-NMR of penta cyclic tri terpenes sharp absorption are found
due to methyl ester and acetoxyl group. The absorptions due to the
presence of angular methyl groups are seen in the 0.82 to 1.13 ppm
region. Other functional groups such as olefine proton have low and
diffused absorption in the region 5.66-5.44 and 3.8-4.52 ppm respectively.
The chemical shift of highest (most shielded) methyl groups is partially
indicative of the position of carbomethoxy group in tri terpenes of ursane
or oleanene series.
(II) 13C-NMR Spectroscopy
13C-NMR spectroscopy with its wide chemical shift range about
220 ppm down field to TMS has proved to be distinctly advantageous in
structure elucidation of natural products [55] for such studies three types
of 13C-NMR spectra [56] are generated. In the Proton Noise Decoupled
(PND), non-equivalent carbons resonate as separate signal lines and
provide information about the number and nature of carbon atoms on the
basis of their chemical shifts.
The single frequency off-resonance decoupled (S fold) spectrum
gives the hydrogen substitution pattern where carbon signals are split
according to the number of attached hydrogen atoms.
The proton coupled spectrum, which gives JC-H coupling values
extending up to these bonds.
(III) Nuclear Overhauser Effect (NOE)
The NOE is probably the most powerful tool for finding out the
stereochemistry of molecules in the solution. The detail of its theory and
its chemical possibility is given by Noggle and Schemer [57], and Bell
and Saunders [58]. NOE is the change in intensity of resonance ‘A’ when
some other nucleus ‘X’ is irradiated. The techniques used for observing
NOE is gared decoupling in which the decoupler power is gated off just
before the Rf pulse so that the population effect can be observed without
any complication. The NOE is governed by relaxation processes are not
involve scalar spin-spin coupling between the nuclei A and X the cross
relaxation rate is proportional to the factor 1/r6 and depends therefore on
the distance(r) between the nuclei of interest Nuclear Overhauser Effect
(NOE) occupies a very special place in the application of modern NMR
methods. It is only technique that does not depend on the presence of the
scalar coupling for its operation instead the interaction involved is the
direct magnetic coupling between nuclei. The NOE provides an indirect
way to extract information about this dipolar coupling, which in turn can
be related to internuclear distance and molecular motion.
(IV) Insensitive Nuclei Enhancement by Polarization Transfer
(INEPT)
In this technique the polarization is transferred from a sensitive
nuclei (e.g. 1H) to intensitive nuclei (e.g.13C) i.e. to stay from a high
magnetogyric ratio to low magnatogyric ratio. In the specific pulse
sequence, the proton transition are put into antiphase, as in selective
polarization transfer, but using nonselective polarization transfer in a
manner independent of chemical shift. The important element of INEPT
sequence are the modulation of transfer magnetization of the sensitive
nucleus (A) by scalar coupling to the intensitive nucleus(X) and the
simultaneous application of two 180° pulse in the ‘A’ and ‘X’ region. The
most important aspect of the INEPT method is the fact that it allows a
much larger intensity for insensitive nuclei than in NOE. Further more,
negative t - factors have no disadvantage in INEPT than in NOE. For
example between 15N and 1H-NOE observed is negative whereas INEPT
experiments contribute to the population difference of the final signals of
the 15N nucleus [59].
(V) Distortionless Enhancement by Polarization Transfer (DEPT)
In practical application of polarization transfer experiments for
resonance assignment the DEPT sequence is usually preferred. The
experiments bring about polarization transfer in similar fashion to INEPT,
but with the important difference that all the signals of the insensitive
nucleus are in phase at the start of acquisition. The DEPT experiments
field multiples with uniform phase after three J/2 delays, with the
application of 1H-decoupling singlet signals for all types of 13C resonance.
The pulse angle Q of the last A pulse can be optimized for individual
groups in order to allow signal selection. The DEPT experiment with
angle 45° showed positive signals for all three multiples, with Q=90° only
methyl signals should appear and Q=135° methylene signals will appear
negative while methane and methyl remains positive. Indeed it is possible
in favorable cases to achieve a complete separation of the decoupled 13C-
NMR spectrum into CH, CH2 and CH3 sub-spectra by taking linear
combination of the DEPT or INEPT spectra with three different values for
Q-[60].
(b) Two Dimensional (2D) NMR Spectroscopy
The recently developed technique of two-dimensional 2D-NMR is
utilized for investigation of cross relaxation and chemical exchange
process.
Jeener first introduced the concept of 2D FT NMR in 1971 [61, 62],
which was analyzed in detail by Aye et al. [63]. The power of 2D-NMR
lies in its ability to resolve overlapping spectral lines, to enhance the
sensitivity and to provide information not available by 1D method.
The two frequency dimensions of 2D-NMR originate form the two
time intervals t1 and t2 during which the nuclei can be subjected to two
different sets of conditions. The amplitude of the signals detected during
time t2 is a function of what happened to the nuclei during the evolution
periods t1 i.e. S (t1, t2). If over ‘n’ experiments we increase each evolution
period t1 by constant time increment 0, t1 varying from zero to several
hundred millisecond, a set of spectra is obtained with the amplitude of the
resonance modulated with the frequencies that existed during the
evolution period t1. A Fourier-Transformation with respect to t2 yields a
conventional 2D-spectra, whose data points on time axis t1 define the
modulation frequency, which can be determined by a second Fourier-
Transformation with respect to t1.
In 2D-spectra a line detected t2 may have component lines in t1. It
therefore shows cross peak to those lines. The peaks along the diagonal in
this spectrum arise from magnetization components, which have the same
frequency during both t1 and t2 i.e. from the portion of magnetization that
was not transferred elsewhere during the second pulse. Thus, the diagonal
gives the essence of the normal 1D-spectrum where the off diagonal peaks
(cross-peaks) which are either a part of the same multiple or a part of
different multiples that have coupling since the magnetization is
transferred in both the directions between transitions, a cross peak V1 V2
have symmetrical partners at V1 and V2.
(i) 2D 1H-1H Cosy (Homonuclear Correlated Spectroscopy)
It is the homonulcear correcelation through J-coupling, the
information obtained from the spectrum are the scalar coupling
connectivity network pattern of the molecule concerned by the help of
cross peaks. The rows of the spectra luminated the nature of data, but it is
clustered and confusing as the spectral complexity increases. Therefore,
the clear representations of the spectrum can be obtained as counter and
choosing suitable experimental parameters [63] makes the cosy spectrum
an elegant approach for making connections through bonds.
(ii) 2D 1H- 13C-Cosy (Hetero Nuclear Correlated Spectroscopy)
Hetero nuclear correlated spectroscopy is one of the most powerful
2D-experiments, combing the excellent resolving power of decoupled 13C-
NMR with ease for interpretation of proton chemical shifts. It offers good
chemical information and allows the resolution of signal sites in all but the
intractable spin system. As in INPET and DEPT the usual experiment
relies on transferring 1H spin polarization to 13C through one-bond
coupling. The fundamental concept is to use the evolution period t1 for the
processional motion of the 1H spins, and 13C occurs in the mixing period,
which is introduced between the evolution and the detection time. The
coupled nuclei yield signals with the co-ordinates (A) and (X) [63].
(iii) Hetero nuclear Multiple Quantum Correlation ( HMQC)
The protonated carbon atoms can be detected by HMQC
experiments. In HMQC experiment a 180° pulse refocused chemical shift
evolution and the ∆1 delay is tuned for the heteronuclear coupling so that
at the end of this period a coupled ‘A’ magnetized is in anti-phase while
uncoupled magnetization of homonuclear ‘A’ coupling is neglected which
still points along the y-axis. The 90° Z pulse transforms this magnetization
to 2-direction. The delay ∆2 is tuned equal to ∆1. The HMQC experiments
thus provide one bond hetero nuclear correlation between two nuclei i.e.
the attached nuclei [58].
(iv) Hetero nuclear Multiple Bond Correlation (HMBC )
HMBC is long-range version of HQMC experiment and is the only
way to establish connectivity between proton and non-protonated 13C
sites. In this experiment the delays ∆1 and ∆2 are turned to one-bond and
long-range heteronuclear as X coupling respectively. The first two 90°
pulse separated by the delay ∆1=½1 J (AlX) eliminate one bond
correlations. The second 90° X pulse then creates after the appreciably
longer delay ∆2 C~60ms. The desired multiple quantum coherence based
on long-range couplings. This is the most effectively performed technique
using gradient spectroscopy. Which significantly improves the elimination
of t1 noise from the residual signals of molecules with non-magnetic X
nuclei [58].
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