characterization and biological evaluation of secondary
TRANSCRIPT
Characterization and Biological Evaluation of
Secondary Metabolites from Vernonia oligocephala,
Chemistry and Applications of Green Solvents
A Dissertation Submitted for
The Fulfillment of the Requirement for the
Award of Degree of Doctor of Philosophy
in Chemistry
By
Rizwana Mustafa
Department of Chemistry The Islamia University of Bahawalpur
Bahawalpur-63100, Pakistan
2016
Summary
Page # iv
SUMMARY
The present thesis consists of two parts, Part A deals with the isolation of
natural products. Plants are being used as medicine since the beginning of
human civilization, perhaps as early as origin of man. Healing powers are
reported to be present in plants and therefore it is assumed that they have
medicinal properties. The present Ph. D thesis deals with isolation of
bioactive constituents from medicinally important plant of Pakistan namely
Vernonia oligocephala. Part B deals with green solvents (ILs) and their
chemistry. Mixtures of ionic liquids, ILs, and molecular solvents are used
because of practical advantages. Solvation by mixture of solvents is,
however, complex because of preferential solvation.
Part A
Characterization and biological evaluation
of secondary metabolites from Vernonia
oligocephala
Part B
Chemistry and applications of green
solvents
Summary
Page # v
Part A: Characterization and biological evaluation of
secondary metabolites from Vernonia oligocephala
The genus Vernonia is the largest genus among the vernoniae tribe with up
to 1000 species. It is found mostly in tropical regions, mostly grow in marshy
and wet areas, tropical forest, tropical savannahs, desert, and even in dry
frosty regions. It consists of annuals, lianas, trees, shrubs and perennials.
The Genus Vernonia is important for medicinal, food and industrial uses e.g.
the leaves of V. amygdalina, and V. colrarta are eaten as food. The
methanolic extract Vernonia oligocephala results in isolation and structural
isolation of one new compound (142) and eight known compounds (43, 44,
126, 143-147) were isolated.
New Compounds isolated from V. oligocephala
1 Characterization of Oligocephlate (142)
O
O1
3 5 7
9
10
12
14 16
17
18
20
22
23 24
25 26
27
2829
30
142
R. Mustafa et al., J. Chem. Soc. Pak., 35, 972-975 (2013).
Compounds isolated for the first time from V. oligocephala
1. β-Sitosterol (126)
2. Oleanolic Acid (143)
Summary
Page # vi
3. 5,7,4'-Trihydroxyflavone (144)
4. Apigenin-7-p-Coumerate (145)
5. Kaemferol (44)
6. 1sorhamnetin (146)
7. β-Sitosterol 3-O-β-D-glucopyranoside (147)
8. Quercetin (43)
RO
HHHO
O
OH
H
H
H
O
OH
HO
O
OH
O O
OH O
OH
O
HO O
O
OH
HO
OH
OH
O
OH
OCH3
O
OH
HO
OH
HO
OH
O
OH
O
OH
OH
4a6
1
3 5 7
9
11 13
15
17
19 21
23 24
25 26
27
28
29 30
143
43
3
5
6
8
2'
5'
6'
4a
8a
5'
6'
2
3
5
6
8
9
10
2'
3'
144
1
2
3
5
6
8
9
10
2'
3'
5'
6'
1"
2"
3"5"
6"
8"
9''
145
1
126 R = H
147 R = Glucose
28
23
21
29
25
26
27
3
57
9
12
1416
17
18 20
10
19
28a
8
2'
3'
5'
6'
44
2
4a
5
6
8
2'
5'
6'
146
The structures of these compounds were elucidated by spectral studies
including UV, IR, EI-MS, HR-EI-MS, FAB-MS, HR-FAB-MS, NMR
techniques including 1D (1H, 13C) and 2D NMR (HMQC, HMBC, COSY,
NOESY) and chemical transformations. The new compound
Summary
Page # vii
oligocephalate (142) were tested against the enzyme α-glucosidase, which
displayed inhibitory activity against this enzyme.
Part B: Chemistry and applications of green solvents
Mixtures of ionic liquids, ILs, and molecular solvents are used because of
practical advantages. Solvation by mixture of solvents is, however, complex
because of preferential solvation. We probed this phenomenon by examining
the spectral response of a solvatochromic dye, 2,6-dichloro-4-(2,4,6-
triphenylpyridinium-1yl)phenolate (WB), in mixtures of the ILs 1-(1-butyl)-3-
methylimidazolium acetate, (1-methoxyethyl-3-methylimidazolium acetate,
with dimethyl sulfoxide, DMSO and water, W, over the entire mole fraction
() range, at 15, 25, 40, and 60 °C. The empirical polarity of the mixtures,
ET(WB) showed nonlinear dependence on DMSO and W due to dye
preferential solvation. We treated the solvatochromic data by a model that
includes the formation of the “mixed” solvents IL-DMSO, and IL-W; the
concentrations of these third components were calculated from density data.
Solvent exchange equilibrium constants in the solvation layer of WB (ϕ) were
calculated; their values showed that IL-DMSO and IL-W are the most
efficient solvent in each medium. Due to its hydrogen-bonding capacity, IL-W
is more efficient than IL-DMSO. We used the results of molecular dynamics
simulations to corroborate the conclusions drawn. Our solvatochromic
results are relevant to cellulose dissolution in IL-DMSO because the same
interaction mechanisms (solvophobic; hydrogen bonding) are determinant to
dye solvation and biolpolymer dissolution.
R. Mustafa et al., The Journal of Physical Chemistry B, (Submitted).
Chapter # 01 Introduction
Page # 1
CHAPTER# 1
INTRODUCTION
Natural products
Medicinal Importance of Natural Products and
Bioactive Secondary Metabolites
Chapter # 01 Introduction
Page # 2
1 The Importance of Medicinal Plants
With the beginning of life on earth, the association of human and animal with
the plants starts, because the supply of oxygen, shelter, food and medicine to
them by plants. With the passage of time, when human societies start
forming, they start to study plant according to the necessities of life and start
to categories it according to their uses. From the multiple uses of plants, their
ability to heal can be recorded from earliest of myths. With the passage of
time the coding of the plants continue according to the ability to ease pain
and to treat the diseases. This plant based medicine system start primarily
from the local area that in future lead to well develop medicinal system; the
Ayurvedic and Unani of the Indian subcontinent, the Chinese and Tibetan of
other parts of Asia, the Native American of North America, the Amazonian of
South America and several local systems within Africa. World Health
Organization (WHO) reported that about 70% world population use plants as
primary health remedy, 35,000 to 70,000 species has been used up to now
for medicine, from the 250,000 species of plants 14-28% occurred all around
the world (Farnsworth NR 1991; Akerele 1992; Fransworth 1992; Padulosi S
2002) and almost 35-70% species of all medicinal plants are being used
world-wide (Padula De 1999). Up to now more than 50 major medicines has
been formed from the tropical pants. From the 250,000 species of higher
plants from the whole world, only 17% has been properly investigated for
their active biological constituents (Fransworth 1992; Moerman 2009). Due to
this reason and of high chemical and biological diversity of plants there is a
Chapter # 01 Introduction
Page # 3
lot of renewable sources in the plant area that can help in the development of
pharmaceuticals (Moerman 2009).
Table 1. Important drugs produced by medicinal plants
Sr. No
Drug Structure Source Use Reference
1 Vinblastine
N
NH
N
H3CO
H
OH
N
H
OAc
OH
OCH3
O
H3CO
O
Catharanthus roseus Anticancer Bagg (2000)
2 Ajmalacine
NH
N
O
H3CO
O
H
H
H
Catharanthus roseus Anticancer,
Hypotensive
(Wink
1998)
3 Rescinna
mie NH
N
H3CO
HH
H
H3CO
OOCH3
O
O
OCH3
OCH3
OCH3
Rauvolfia serpentina Tranquilizer (Fife 1960)
4 Reserpine
NH
N
H3COOC
O
OCH3
H
H
H3CO
O
OCH3
OCH3
OCH3
Rauvolfia serpentina Tranquilizer (Baumeister
2003)
5 Quinine
N
N
OH
OCH3
Cinchona sp. Antimalarial, (Hanbury
1874)
6 Pilocarpine
O
H3C
O
H
N
N
CH3
H
Pilocarpus jaborandi Antiglucoma (Rosin 1991)
7 Cocaine N
O
H3C
O
OCH3
O
Erythroxylum coca Topical
Anaesthetic
(Aggrawal
1995)
Chapter # 01 Introduction
Page # 4
8 Morphine
N CH3
O
HO
H
HO
H
Papaver somniferum Painkiller (Smith 2007)
9 Codeine
N CH3
O
H3CO
H
HO
H
Papaver somniferum Anticough (Codeine
2011)
10 Atropine N
O
H3C
O
OH
Atropa belladonna Spasmolytic,
Cold
WHO
11 Cardiac
glycosides
O O
OAc
O
OH
OH
OH
HO
Digitalis sp. For congestive
heart failure
(Newman
2008)
12 Taxol
O NH
O
O
O
OH
H
O
O
AcO
OHOAcO
OH
Taxus baccata Breast and
ovary cancer
(Wani 1971)
13 Berberine
N+
O
O
H3CO
OCH3
Berberis Leishmaniasis (Exell 2007)
14 Pristimerin COOCH3
CH3
H3C
H CH3
CH3
H3C
O
HO
CH3
Celastrus paniculata Antimalarial (King 2009)
15 Quassinoids
O
OH
H
H
OH
HO
H
H
O
O
O
Ailanthus Antiprotozoal (Fiaschetti
2011)
Chapter # 01 Introduction
Page # 5
16
Plumbagin
O
O
OH
Plumbago indica
Antibacterial,
Antifungal
(van der Vijver
1972)
17 Diospyrin O OH
OHO
O
O
Diospyros Montana Antifungal (Ray 1998)
18 Gossypol
OH
OH
HO
HO
O
OHO
OH
Gossypium sp. Antispermato (Polsky
1989)
19 Allicin
S
S+
O-
Allium sativum Antifungal,
Amoebiasis
(Cavallito 1944)
20 Ricin
ON
N
N
N
HN
N
O
N
Ricinus communis Abundant
protein Source
(Lord 1994)
21 Emetine
N
HNOCH3
OCH3
H
H3CO
H3CO
H
H
Cephaelis ipecacuanha Amoebiasis (Wiegrebe
1984)
22 Glycyrrhizi
n
O
O
O
HOOC
HO
HOOH
O
O
H
COOH
H
H
HO
HO
Glycyrrhizia glabra
Antiulcer
(I. Kitagawa
2002)
24 Nimbidin
O
O
O
H3CO
O
OAc
H
H
O
OCH3
Azadirachta indica
Antiulcer (Santhaku
mari 1981)
Chapter # 01 Introduction
Page # 6
1.1 The Role of Herbal Medicines in Traditional Healing
By the use of herbs the pharmacological treatment of disease began long time
before (Schulz 2001). Herbs are the traditional source for this purpose. Below
are the some worldwide traditions to use herbs for the treatment purpose.
25 Catechin
OHO
OH
OH
OH
OH
Acacia catechu Antiulcer
(Zheng 2008)
26 Sophoradin
H3C
CH3 O
OH
H3C CH3
CH3
CH3
HO
OH
Sophora subprostrata
Antiulcer
(Kazuaki
1975)
27
Magnolol
OH
HO
Magnolia bark
Peptic ulcer
(Alice 1981)
28 Forskolin
O
OH
OAc
OH
H
OH
Coleus forskohlii Hypotensive,
Cardiotonic
(Bernard
1984)
29 Digitoxin,
Digoxin O
O
O
OH
O
O O
O
HO
OH
H
OH
H
OHH
H
Digitalis thevetia Cardio tonic (Belz 2001)
30 Indicine
N-oxide
N+
O
O
HO
OH
O-
OH
Heliotropium indicum Anticancer (Powis
1979)
31 Homoharr
ingtonine
N
O
O
O
O
O
O
O
HO
OH
H
Cephalotaxus Anticancer
(Kantarjia
and
Cancer.
2001)
Chapter # 01 Introduction
Page # 7
1.1.1 Traditional Chinese Medicine
Chinese people are using traditional medicine from ancient times. The major
source of Chinese medicine is botanical although they use animals and
minerals also. Among 12,000 healing medicines 500 are in use of common
man (Li 2000) ).
1.1.2 Japanese Traditional Medicine
In Japan the first classification of native herbs into traditional medicine was
done in ninth century. Before that Japanese were using the knowledge of
Chinese (Saito 2000).
1.1.3 Indian Traditional Medicine
Before 5000 years ago Ayurveda medical system, that includes diet and
herbal remedies, was first time used in India (Morgan 2002).
1.1.4 Traditional Medicine in Pakistan
In Pakistan traditional Unani medicine is being practiced among different
parts of the country. This Unani medicine was originated in Greece and first
time practiced among Muslims during their glorious time in history. Muslims
scholars introduce traditional medicine in Indo-Pak Subcontinent and here
used it for centuries (Hassan 2001).
QURAN is a great source of all knowledge's and it is not a new one, as great
Muslims scholars in the past have influenced by this view. We see references
to discover nature in more than 10% Quranic verses [Abu Hamid al-Ghazali].
The Holy QURAN claims that it covers every aspect of life and is full of
wisdom and knowledge. It speaks “We have neglected nothing in the Book”
Chapter # 01 Introduction
Page # 8
(Khan A S 1994). Keeping in mind the importance of medicinal natural
products in Islam, research workers are investigating on bioactive natural
products and creating awareness in all over the world about all the plant
species that are listed in Holy QURAN.
1.2 What are Natural Products?
A natural product is a substance or a chemical compound that usually has
pharmacological or biological activity and is synthesized by living organism.
That source may be plants, animals or microorganisms. These biological
active components are further used for drug designing. These natural
products are considered as such even if they are synthesized in the
laboratory. The popularity of natural products has been increased with the
passage of time because it is a source of novel drugs and leads towards the
knowledge of synthesis of non-natural drugs (Briskin 2000). From the
historical knowledge of ancient physician important clues can be provided for
the development of new drugs. After purifying the extracted compounds from
the natural products their structure elucidation, their chemistry, synthesis
and biosynthesis are important areas of organic chemistry. Naturally
occurring compounds are generally divided into three categories; First, those
types of compounds which are important part of the cells and play important
role in the process of metabolism and reproduction of the cells, and known as
primary metabolites (PM), second, this type consist of the compounds that are
with high molecular weight such as cellulose, lignin's and proteins, which
Chapter # 01 Introduction
Page # 9
take part in the structure formation of cell. Third type of the compounds is
present only in a limited species. This type is the secondary metabolites (SM).
They do not take part directly in body growth, but can function as
communications tools, defense mechanisms, or sensory devices. The major
difference between the PMs and SMs is that, the biological effect of PM retain
within the cell while in case of SM this biological effect have influence on
other organism also (Swerdlow 2011).
Throughout the development of chemistry the biological activities of all type
of natural products has been studied. Among the biological active
compounds, majority are of SMs. From the literature it has been estimated
that the 40% origin of the medicines is from these compounds. There is large
number of methods that are applies for the screening of these bioactive
compounds that leads towards new medicines, e.g Taxol is SM, used for the
treatment of various types of cancer.
1.3 Classification of Secondary Metabolites
From the natural process of building up structural characteristics in the
natural products, each class have some particular structure and have
number of compounds in it. Simple classification of SMs can be done by
dividing them into three major groups [Michael Wink];
1.3.1 Without Nitrogen (Maimone 2007)
a) Terpenes Consists of almost 15,500 species
Chapter # 01 Introduction
Page # 10
b) Phenolics Consists of almost 7,950 species
1.3.2 With Nitrogen Consists of almost 13,140 species
Table 2. Secondary Mrtabolites
Number of natural products
With Nitrogen
Sr. no
Type
Example
No. of
species
1 Alkaloids
N
N
12,000
2 Non-proteinamino acid
O
HN NH
NH2NH2
O
HO
700
3 Amines NH2
100
4 Cyanogenic glycosides
CH
Oglu
N
100
5 Alkamides
N
O
150
6 Glucosinoltes
N
S-Glu
O SO3-
100
Without Nitrogen
Chapter # 01 Introduction
Page # 11
7 Monoterpenes
OH
2500
8 Sesquiterpenes
O
OOH
O
5,000
9 Diterpenes O
O
O
O
O
O
OOH
OH
-O
2,500
10 Triterpenes ,
saponins, Steriods
COOH
RO
5,000
11 Teraterpenes
500
12 Phenylpropanoids,
Coumerins, Lignans O O
2,000
13 Flavonoids
O
OH
HO
HOOOH
HO
4,000
14 Polyacetylenes, Fatty
acids, Waxes HO
OH
1,000
Chapter # 01 Introduction
Page # 12
1.4 Secondary Metabolites with Nitrogen
SMs fix the atmospheric nitrogen in roots through their symbiotic Rihzobia,
so nitrogen is available readily in these SMs (protease inhibitors, alkaloids,
cyanogens, non-protein amino acids, lectins) (Wink 1993). Alkaloid is an
important class of SMs, contain one or more nitrogen in their structure
(Aniszewski 1994) and is synthesized by plants, animals, mushrooms, fungi
and bacteria (Aniszewski 2007). They show antimalarial, antineoplastic,
antiviral, antimicrobial and analgesic activities (Alarcon 1986; Caron 1988;
Gul 2005; Gupta 2005; Jagetia 2005; Kluza 2005). In medicine, the role of
alkaloids is tremendous. They are used usually in the form of salt. Important
alkaloids include caffeine (1), codeine (2), morphine (3), nicotine (4), quinine
(5), vinblastine (6) and ajmaline (7) are used as a cough medicine, analgesic,
stimulant, antipyretics, antitumor and antiarrhythemic drugs, respectively.
15 Polyketides O
O
OH
CH3
OH
H3C
750
16 Carbohydrates
OOH
OH
HOOH
O
O
OH
OH
OH
HO
≥200
Chapter # 01 Introduction
Page # 13
N
NHO OH
N
N
H
OH
NH
H3CO
N
H
OAcOH
OCH3O
H3COO
N
O
H3CO
H
HOCH3
H
N
O
H3CO
H
HOCH3
H
N
N
OCH3
OH
N
N
H
N
N N
N
O
O
2 3
5
41
76
Some alkaloid such as salt of nicotine and anabasine (8) which were used as
a insecticides before the development of synthetic pesticides with low toxicity.
However they are never been in use by human due to their high toxicity
(György Matolcsy 2002). Alkaloids have been used for long time as a
psychoactive substances, like cocaine (9) and cathinone (10) which are used
as a central nervous system stimulants (Veselovskaya 2000).
N
NH
O
O
NH3C
OO
CH3O
NH2
8 9 10
Non protein amino acids are those types of amino acids which do not take
part in the formation of genetic code and have 20 amino acids in coding
Chapter # 01 Introduction
Page # 14
except of 22. About 140 amino acids and thousand of more combinations are
known (Ambrogelly 2007). Important functions of the non proteinogenic
amino acids are i) intermediate in biosynthesis ii) natural pharmacological
compounds iii) part of meteorites and prebiotic experiments. From bioactivity
prospect orithine (11) and cirtrulline (12) are found in the cycle of urea and is
a part of catabolism they also take part in the formation of toxins in
secondary metabolites (Curis E 2005). Some of the non-protein amino acids
are neurotoxic by mimicking neurotransmitters amino acids e.g quisqualic
acid (13), canavanine (14), and azetidine-2-carboxylic acid (15) (Dasuri
2011).
H2N OH
O
NH2
NH
OH
O
NH2
H2N
O
NNH
HO
O
OO
NH2
N
O
OH
O
NH2
H2N
NH2
NH2+
CO
O
11 12
1314 15
Another important class of SMs with nitrogen are Amines, a class of organic
compounds which are the derivatives of ammonia in which substitution of
one or two hydrogen take place by alkyl or aryl group. Important examples of
amines include chloramine (16), amino acids (17), trimethylamine (18) and
aniline (19).
Chapter # 01 Introduction
Page # 15
N C C
H
H
H
R
OH
O
N
CH3
H3C CH3
NH2
Cl NH2
17 1819
16
Large number of Amines are found to be biologically active as many of
neurotransmitters are amines by nature like dopamine (20), serotonin (21),
histamine (22) and epinephrine (23) (Miguel 1998).
N
NH
s-Bu
NH
HO
NH2
HO
NH2HO
HO
OH
HN
OH
20
21 2223
Many natural compounds are also used in the pharmaceutical to
manufacture drugs like chlorpheniramine (24), antihistamine (25),
chlorpromazine (26), and ephedrine (27) used for the treatment of allergic
disorder, insect bites and stings, to relieve anxiety, restlessness and even to
treat mental disorders, and to used as decongestants, respectively.
Chapter # 01 Introduction
Page # 16
Cl
N
N
S
N
N
ClN
NH
CH2
H2N
CH3
HNCH3
OH
24 262527
Alkamides are wide and large group of the natural product that are
biologically active and found in at least 33 plant families. These have broad
structural variability despite the fact that they have simple molecular
structure e.g Achillea (28), Piper (29), Echinacea (30), Amaranthus (31),
Capsicum (32), Glycosmis (33). They show numerous biological activities like
antiviral, insecticides, larvicidal, antimicrobial, pungent, analgestic , and
antioxidant moreover they are being used in the potentiation of antibiotics
and RNA synthesis(Campos Cuevas 2008).
N
OO
O
O
NH
O
NH
HO
OOH
NH
O
OCH3
OHNS
H3C
O
2829 30
3132 33
Chapter # 01 Introduction
Page # 17
They are pungent/irritating in taste and used for the treatment of dental
disorders, to enhance the immune system of the body and used for the
treatment of influenza and respiratory infections. Additionally alkamides
exhibit a number of more bioactivities which make this family new and need
to be more investigated in the future.
1.5 Secondary Metabolites without Nitrogen
This class of SMs includes very important sub-classes that do not have
nitrogen in their basic skeleton. Its includes Terpenes a group of compounds
that are present in almost every natural food and are built up on the unit of
isoprene (Wagner KH 2003). These are isomeric hydrocarbons (C5H8)n present
in essential oils (especially from conifers and some insects) and in organic
chemistry mostly used as solvent during different synthesis. Within every
living thing, terpenes are the major building blocks e.g from the derivitization
triterpen Squalene (33), a compound lansterol (34) is obtained which is a
basic skeleton for all kind of steroids (Corey 1966).
HOH
33
34
From terpenes up to 2007, 55,000 types of metabolites have purified
(Maimone 2007). Essential oils which are obtained from many types of plants
and flowers also have terpenes and terpenoids as primary constituents of their
Chapter # 01 Introduction
Page # 18
structure. These essential oils are used in perfume industry, for giving natural
flavor to food and used in manufacturing of medicine field also. By considering
these C5 isoprene units as basic structural constituents of terpenes theses are
classified into seven major classes.
Hemiterpenoids (C5)
Monoterpenoids (C10)
Sesquiterpenoids (C15)
Diterpenoids (C20)
Sesterpenoids (C25)
Triterpenoids (C30)
Carotenoids (C40)
Examples are isovaleric acid (35), terpineol (36), abscisic acid (37), bietic acid
(38), cafestol (39), lanosterol (40) and β-carotenes (41), respectively (Bicchi
2011). Terpenoids have a numerous biological effects including antifungal,
cytotoxic, antiallergenic, used in the treatment of ulcer, anti cancer
(especially against breast and ovarian cancer) and anti malarial (Ajikumar
2008).
OH
CO2H
O
OH
H
HHO2C
HOH
O
H
H
H
OH
CH3
OH
OH
O
36
37
38
40
39
35
41
Chapter # 01 Introduction
Page # 19
The second major large subgroup of SMs is Flavonoids (42) belongs to the
phenolic group, found in high concentration in prokaryotes and plants
(Middleton 1998; Woo HH 2002; Carvalho 2006). Up to 2002, more than 6,500
types of flavonoids have been classified (Boumendjel A 2002). These are
derived from flavones that is commonly present in the young tissues of higher
plants (Kurian A 2007; Yoshida K 2009). In plants, the role of flavonoids is
very significant as they are detoxifying agent (Yamasaki H 1997; Jansen MAK
2001; Michalak 2006), stimulant for spores germination (Bagga S 2000;
Morandi D. 1992), coloration to the petals and flowers of plants, and as UV
filter (Vergas FD 2003; Lanot A 2005 ). In the body of human, flavonoids act
as antioxidant (Williams RJ 2004; Lotito SB 2006), and they are most common
group of human food obtained from plants. They have antiallergic, anti-
inflammatory (Amamoto 2001), anticancer (Sousa De RR 2007), anti-diarrheal
(Schuier M 2005), antiviral (González ME 1990) and anti microbial (Cushnie
TPT 2005; Cushnie TPT 2011) activities such as flavonoids; Kaempferol (44)
and quercetine (43) prevent carcinogenesis and mutagenesis in vivo and vitro
and increase the blood circulation in the body (Whalley 1990; Etherton 2002).
O
O
O
O
HO
OH
OH
OH
OH
O
OH O
OH
OH
HO
4342
44
Chapter # 01 Introduction
Page # 20
Another important class of SMs which is formed by the condensation of
acetate units is Polyketides which results in the formation of fatty acids.
Unsaturated fatty acids are preferably used in the food. Oleic acid (45) is a
major constituent of olive oil, linoleic (46) lenolenic (47) are used in paints and
varnishes after drying. Jasmonic acid (48) formed after oxidation of lenolenic
acid makes plants defense system and arachidonic acid (49) is used in
functioning of prostaglandin hormones.
CO2HMeCO2HMe
CO2HMe
CO2H
MeO
CO2H
Me
4546
47 4849
Moreover they are used as a good antibiotic and antifungal agents, and
important examples of these compounds showing these activities are
tetracycline (50), and wyerone (51), respectively.
OCO2H
Me
OO
NMe2
HH
Me
OH
OH
CONH2
OH
OOHOH
5150
Chapter # 02 Literature Survey
Page # 21
CHAPTER # 2
LITERATURE SURVEY
NATURAL PRODUCTS
Phytochemical Investigations of Family Asteraceae and Literature Study of Some Medicinally Important Species of Genus Vernoni
Chapter # 02 Literature Survey
Page # 22
2.1 The Family Asteraceae
The family Asteraceae is also called the Compositae family commonly known
as aster, sunflower and daisy family. It is the major family of Angiospermae
consists of 23,000 sepcies,1620 genera and 12 subfamilies (Badillo 1997)
which are different in shapes, growth and morphology depends on the
location and habitats of growth. More than 40 species of this family are
economically very important and are used as medicine (chamomile), oil
(sunflower and safflower), food (lettuce and artichopa) and as ornamental
shrubs (chrysanthemum) (Burkill 1985).
Asteraceae
Class Dicotyledoneae (Angiospermae)
Order Asterales
Common names
Sunflower family, Daisy family, Thistle family, Madeliefie-
family, Sonneblom-family
2.2 Medicinal Importance of the Family Asteraceae
Plants are the main source to maintain human life on earth by providing a
number of facilities economically and socially. Depending on the every plant
Chapter # 02 Literature Survey
Page # 23
habitat they have specific characteristics. Among the plants medicinally
important are those which have ability to treat various diseases and have
been used by the human being from long time (Rawat 1998). The family
Asteraceae is medicinally important family. Some important plants with their
medicinal used are given in Table 3.
Table 3. Important Medicinal Plants of Family Asteraceae
Sr. No Botanical Name Useful parts Major Use Reference
1 Aegaratum conyzoides Leaves Treatment of cut and sores,
Piles, Wound healing
(Patel 2012)
2 Anacyclus pyrethrum Roots, Flowers Dental pain, Tonsillitis, Diarrhoea, Sexual weakness
3 Bluemea lacera Leaves Bleeding control, Burning,
Diuretic
4 Eclipta prostrata Leaves Asthma, Hair shampoo,
Hair tonic, Anthelminti
5 Spilanthes aemella Roots, Flowers Tooth trouble, Inflammation
of jaw, Fever
6 Stevia rebaudiana Leaves, Stems Antimicrobial, Diuretic,
Diabetes, High Blood
Pressure, Cardiotonic
7 Tagetes erecta Leaves, Roots Insecticidal prosperity,
Muscular pain, Boil,
Stomachic, Scorpion bite
8 Arctium lappa Leaves, Seeds,
Roots, Stems
Blood purifier, skin
infections, boils acne, bites, rashes, ringworms, sore
throat, induce sweating.
(Chan Y.S.
2010)
9 Calendula officinalis Whole plant Skin diseases, pain,
antiseptic, used in
cosmetics
(M. Wegiera
2012)
10 Onopordon leptolepsis Whole plant Antioxidant, protecting
agent in medicine formation, antitumor
(Joudi L
2010)
11 Cichorium intybus Roots Essential oils, tonic,
gallstones, bruises, weight
loss, constipation
(Roberfroid
2002)
12 Sonchus oleraceus Leaves Food, asthma (Everitt
2007)
13 Tragopogon pratensis Shoots, Roots Diabetic salad, diaphoretic
property
(P. M.
Guarrera 2003)
14 Taraxacum officinale Flowers Dandelion wine, salads,
coffee, food, kidney disease,
Anti tumor
(G. Jan
2009)
Chapter # 02 Literature Survey
Page # 24
15 Chrysanthemum leucanthemum
Flowers Cure digestive problems.
food
(Gordon
1999 )
16 Senecio
chrysanthemoids
Leaves, Flowers Ulcer, Diabetes,
Neurodegenrative disease,
Antioxidant, astham
(Durre
Shahwar
2012)
17 Anaphalis triplinerus Leaves Cure wounds (Gul Jan
2009) 18 Artemisia trichophylla Leaves, Shoots Respiratory stimulant,
earache, burning,
construction roof
19 Senecio chrysanthemoides
Rhizome Used against asthma and
respiratory problems
20 Sonchus asper Shoots,
Flowers
Tonic, diuretic and
jaundice, constipation, food
2.3 The Genus Vernonia
The genus Vernonia is the largest genus among the “Vernoniae” tribe with up
to 1000 species (Keeley 1979). It is found mostly in tropical regions, mostly
grow in marshy and wet areas, tropical forest, tropical savannahs, desert,
and even in dry frosty regions (Gleason 1923; Keeley 1979). It consists of
annuals, lianas, trees, shrubs and perennials. The genus Vernonia is
important for medicinal, food and industrial uses e.g. the leaves of V.
amygdalina and V. colrarta are eaten as food (Burkill 1985.; Iwu 1993). V.
amygdalina is rich with amino acid, minerals, and vitamins (Alabi 2005;
Ejoh 2007; Eleyinmi 2008).
2.4 Medicinal Importance of Genus Vernonia
The Plants of the genus Vernonia have medicinal importance mostly in the
field of ethanomedicine, ethanoverterinary medicine and in
zoopharmacognosy by chimpanzees and gorillas (Chaturvedi 2011). 109
species of the genus Vernonia are used as folk medicine and showed
bioactivities. The plants belong to the genus Vernonia have a chemical
Chapter # 02 Literature Survey
Page # 25
diversity which leads towards the synthesis of different classes of
compounds particularly terpenes with majority of sesquiterpenes, flavoniods
(Igile 1994; Ku 2002), alkaloids (Eyong 2011). Some important species of
genus Vernonia are shown in Table 4.
Table 4. Some medicinally important species of genus Vernonia
Sr. No Vernonia Species Useful part Major Use Reference 1 V. adoensis Leaves, Roots Chronic, Cough, fever,
Stomach pain,
Digestive/ appetizer, TB, malaria, Snake bite, HIV/AIDS infections
(Hutchings 1996); (Burkill 1985)
2 V. aemulans Leaves TB, bacteria and viruses infection, febrifuge, gonorhoea
(Kisangau 2007a); (Vlietinck 1995)
3 V. ambigua Leaves, Roots Male/female sterility, impotence, postpartum pains, dysmenorrhea, cough and cold, malaria
(Focho 2009)
4 V. amygdalina Leaves, Roots Fever, malaria, measles, diarrhoea, diabetes, Pile, stomachic, worms, headache, mantural crmp, itching, Tosililis, Cough, laxative, infertility, dysentry, antisickling, sexully transmitted disease, dermatitis, hepatitis, ringworms, appetizer
(Mensah 2008); (Gbolade 2009)
5 V. anthelmintica Shoots, Whole plant
Intestinal disorder, skin ailments, asthma, ulcer, astrigent, worms, tonic, stomachic, febrifuge
(Rao 2010); (Joy P.P. 1998)
6 V. aristifera Roots Dysentery, hypermenorrhage
(Heinrich 1996)
7 V. auriculifera Leaves, Roots Toothache, sleeping skiness, placenta removal
(Freiburghaus 1996; Focho 2009)
8 V. Cinerea Whole plant Worms, asthma, mental disorder, skin infections, depression, malaria, cough, scapies, threadworms, kidney disease
(Moshi 2009); (Alagesaboopathi 2009)
9 V. colorata drake Leaves, Roots Malaria, tonic, boils, liver disorders, abdominal pains, diarhea, jundice, infectious disease
(Rabe 2002) (Gakuya 2012)
10 V. condensata Leaves Cough, pneumonia, digestive problems,
(Bandeira 2001); (Albuquerque
Chapter # 02 Literature Survey
Page # 26
hepatic problems, diarrhea, snake bite
2007)
11 V. conferta Leaves, Roots Laxative, stomachache, bronchitis,poison antidote, constipation,
absess, whooping cough, sores, worms
(Burkill 1985)
12 V. cumingiana Roots, Leaves Hepatitis, gastrointestinal disease, toxicosis, eye disease
(Zheng 2009)
13 V. galamensis Leaves Chest pain, externa injury/infection, wounds, diabetes, piscicide
(Teklehaymanot 2010)
14 V. glabra Leaves, Roots Diabetes, burns, gonorrhoea, diuretic,
dysentery, snake antidote
(Long 2005); (Burkill 1985)
15 V. guineensis Leaves, Roots Pain, toothache, sore, vomiting. spermatogenesis, prostatitis, urinary infection, prostate cancer, male infertility
(Noumi 2010)
16 V. hildebrandtii Leaves, Roots Mental disease, emetic, cough, diarrhea, relief
(Hedberg 1982)
17 V. nigritiana Leaves, Roots Vomiting, kidney, antidysentery, colic, blood purification, jaundice, fever, piles
(Diehl 2004)
18 V. oligocephala Leaves Malaria, stomach disorders, dysentry, diabetes, ulcerative colitis, colic, malaise
(Thring 2006); (De Wet 2010)
19 V. patula Mart. Whole plant Nose bleeding, vermfuge, inflammation, fever, colds, bacteria, fever, piles, respiratory tract disorders, impotency, oral infection
(Mollik 2010)
20 V. zeylanica Less Stems Inflammation (Ratnasooriya 2007)
2.5 Phytochemical Survey of Genus Vernonia
The genus Vernonia is very important because of its pharmacological
importance which makes its enforced for its phytochemical survey. Below are
described some important species of genus Vernonia.
2.5.1 Vernonia cinerea
Chapter # 02 Literature Survey
Page # 27
Vernonia cinerea is annual terrestrial erect herb with 80 cm height. It grows
in marshy area, like open waste water, at road side, dry grassy site and in
fields during plantation (Gani 2003). It is used to treat cancer and number of
gastrointestinal disorders (Yusuf M 1994). It shows anti-inflammatory,
antibacterial, antidiarrhoeal, cytotoxic (Kuo YH 2003), antifungal,
antioxidant and antiprotozoal bioactivities (Iwalewa 2003; Arivoli 2011;
Kumar 2011; Rizvi 2011) and also shows antidepression action (Munir
1981).
CH3
CH3
CH3
HH
CH3 H
CH3
HO
H3C
H3CO
OCH3
O
O
H
H
H3CO
OCH3
OH
HO
O
O
CH3
CH3
CH3
H3C
H
CH3
HCH3HO
OH
OH
HO
H
O
OH
OO
O
O
61 62
6463
Stigmasterol (61), (+)-lirioresinol B (62) and stigmasterol-3-O-β-D-
glucopyranoside (64) showed cytotoxic activity on PC-12, (Zhu HX 2008) and
vernolide A (63) have a cytotoxic affect on the cancer cells (Pratheesh kumar
Chapter # 02 Literature Survey
Page # 28
2009; Pratheesh kumar 2010a; Pratheesh kumar 2010b; Pratheesh kumar
2011a; Pratheesh kumar 2011b; Pratheesh kumar 2011c; Pratheesh kumar
2011d; Pratheesh kumar 2012a; Pratheesh kumar 2012b). 8α-
Hydroxyhirsutnolide (66), a sesquiterpenes lactone, its derivative 8α-
hydroxyl-1-O-methylhirsutinolide (67) and (65,68-72) have been isolated
from n-hexane fraction of V. cinerea showed TNF-α-induced NF-kB activities
(Ui Joung Youn a 2012).
O
O
O
OH
OHO
OH
O
OH
OHO
OH
O
OH
OH3CO
O
O
OAc
OHO
O
O
OAc
OH
O
O
OAc
OH3C
O
O
O
OAc
OH3C
O
O
O
OH
OH3C
6566 67
68
69 7071 72
2.5.2 Vernonia anthelmintica
Vernonia anthelmintica is 2-5 cm shrub or plant with 6 mm in diameter;
grows by the process of cutting as it does not have seeds. The leaves of this
plant are used as food to enhance the digestive system and to treat fever. In
Nigeria, V. anthelmintica is used as a source of beer. It is also used to treat
Chapter # 02 Literature Survey
Page # 29
gastrointestinal disease, showed antimicrobial and antiparasitic, (Argheore
E.M 1998) antimalarial, antidiabetic, antihelmitic activities, act as a laxative,
and to treat cancer. Vernodalol (73), vernodalin (75), butein (74) were
isolated from V. anthelmintica.
OO
OH3CO
O
HO
OHH
O
OH
OH
O
HO
OH
O
OO
O
O
O
O O
O
O
O
O
N
N
N
N
N
N
O
O
O
H
HO O
OH
HO
7374 75
76
4α-Methylvemosterol (77), a novel sterol has been isolated from the seed of
V. anthelmintica (Akihisa 1992). Vernolic acid (78) is obtained from the oil of
the seeds of V. anthelmintica which is used as heat stabilizer in plastic
sheets (George R. Riser 1962).
Chapter # 02 Literature Survey
Page # 30
CH3
CH3
CH3
CH3
HOH
CH3
H3C CH3CO2H
O
77 78
From the aerial part of V. anthelmintica, nine new stigmastane-type steroids
with oxygen, vernoanthelcins (79-88) and two new stigmastane-type
steroidal glycosides and vermoanthelosides (89-90) were isolated (Lei Hua a
2012).
O
HHO
O
R
H
HO
H
H
O
HO
HHO
O
R
H
HO
H
H
O
HO
HHO
O
R
H
HO
H
H
O
H
O
HHO
O
R
H
HO
H
H
O
HO
HHO
O
H
HO
H
H
O
H
HO
O
HHO
O
O
H
HO
H
H
O
H
88 89
79 R = OH
90 R = OGlc80 R = H, OH
81 R = O
82 R = O
83 R = OH, H
85 R = H
85 R = O
86 R = OH, H
87 R = OGlc, H
Chapter # 02 Literature Survey
Page # 31
2.5.3 Vernonia conferta
It is a 9m shrub or tree and commonly name as soap tree, because in Sierrea
Leone its branches are converted to ash after burning which is used to make
soap (Burkill 1985). It is distributed in all central Africa, in south Nigeria
and Fernando Po. In Ghana the bark of V. conferta is used to treat diarrhea
and constipation. It is used against convulsive cough, asthma, bronchitis,
wounds, sores, diuretic, opthalmais, laxatives, bronchitis, poisonantidote,
stomachache, jaundice, whooping cough, as a galactogogue, gonococcal
orcthitis and abscesses (Burkill 1985; Ajibesin 2008). It showed bioactivity
against filarial worms (loa-loa) (Mengome 2010); (Ayim 2007). A new
germacranlide, confertolide (91), deacetoxyconfertolide (92) and its dihydro
derivative (93) have been isolated from V. conferta (Toubiana 1974).
CH2OAc
OAcO
OAc
O
O
CH3
OAcO
OAc
O
O
OAcO
OAc
O
O
91 92 93
2.5.4 Vernonia galamensis
V. galamensis is annul herb with 1.30m in height and distributed
throughout Africa, East Africa, and in many parts of Ethiopia and more than
1000 species of V. galamensis are grown in the East Africa. It is useful
source of oil seed. This plant is toxic in nature and is used to build timber,
for the protection of palisades, oil used for the production of paints and to
Chapter # 02 Literature Survey
Page # 32
reduce smog pollution in PVC. It is important plant in the field of commerce
as compared to field of medicine (Baye 2001; McClory 2010). It is used to
treat diabetes and its tablets are available in market (Autamashih 2011). The
extract of the roots and leaves are used to increase the membrane stabilizing
property (Johri 1995). The most of phytochemical studies are based on the
seed oils of this plant (Ncube 1998). Phytochemical investigations of the
seeds of the V. galamensis results two derivatives of the vernolic acid: cis-
(12S,13R)-(3-methylpentyl)-vernolate (94) and cis-(12S,13R)-(2,3-
propanediol) vernolate (95) (A. Fiseha 2010). The seeds also contains linoleic
acid (96) 14%, oleic acid (97) 7%, and 2 to 3% for palmitic acid (98) and
stearic acid (99).
O
O
O
O
O
O
OH
OH
O
HO
OH
O
OH
O
OH
O
9594
96 97
98 99
2.5.5 Vernonia amygdalina
Chapter # 02 Literature Survey
Page # 33
V. amygdalina is a small tree or shrub with 2-5m in height. The leaves of
this plant are of bitter in taste and have specific odor. V. amygdalina has
been originated from Nigeria and is distributed in Africa. It consist of about
200 species (Bonsi 1995a). It is used to control almost 20 diseases described
in Table 4. The leaves V. amygdalina are used as food to enhance the
digestive process in body, and to treat fever. Medically, it is used to treat
leech, to get rid from parasitic attack in chimpanzees. It is used to make beer
in Nigeria. It is also use as a domestic plant and pot-herb. During
phytochemical analysis a number of important classes of compounds have
been isolated including flavoniods and terpenoids which showed cytotoxicity
against the cell lines of cancer (Jisaka 1992; Izevbigie 2003; Izevbigie 2004;
Erasto 2006; Opata 2006). The secondary metabolites present in V.
amygdalina used to treat breast cancer as this plant showed antimicrobial,
antioxidant, antiparasitic and anticancer activities. Xuan Luo isolated n-
hexadecanoic acid (100), stigmasterol (101), chondrillasterol (102), steroid
glucoside (103), succinic acid (104), vernodalinol (105), cynaroside (106),
stigmasterol (101), chondrillasterol (102), docosanoic acid (108) and uracil
(107).
Chapter # 02 Literature Survey
Page # 34
HOOH
O
O
O
O
O OH
O
OH
O
HOH
O
OH
HOHO OH
O O
OH
OH
OH O
HO
O
HO
H
H
H
HHO
H H
NH
NH
O
O
O
OH
HOHO OH
O OH
OH
O
104
105106
100 101102
107
103
108
Few sesquiterpene lactones vernodalin (109), vernodalol (110), vernolepin
(111), and flavonoids luteolin (112) and luteolin 7-O-β-glucoside (113) were
isolated and identified (I. Ijeh 2011).
Chapter # 02 Literature Survey
Page # 35
O
H3CO
O
OHH
O
O
HO
O
O
O
O
O
HO
OH
HO
HO
OH
OH
OH
OHO
OH
OH
OH
O
O
O
O
O
H2C
CH2
O
H2C
OH
O
O
OH
H
O
O
OCH3
H
113
111
112
109110
2.5.6 Vernonia scorpioides
V. scorpioides is a sub shrub up to 2.50 m tall, much branched (Lorenzi
2002; Buskuhl 2010), common in Brazil, found commonly in pastures
neotropical soils, defrosted and roadsides (Cabrera 1980). V. scorpioides is
used to treat ulcer, skin diseases and wounds (Buskuhl 2010). It shows
fungicidal, bactericidal, cytotoxicity against cancer cells and anti-
inflammatory properties. The first phytochemical investigation on V.
scorpioides has been performed in 1980 by Drew et al (Drew 1980). A
number of sesquiterpenes lactones have been isolated from this species
(Lopes 1991; Buskuhl 2010) which show antimicrobial, analgesic,
antifeedant and mulusscicide activities. Few sesquiterpene lactones (114-
119) isolated from the leaves of V. scorpioides.
Chapter # 02 Literature Survey
Page # 36
O
O
HO
HO
O
COOMeO
COOMe
O
O
HO COOMeO
COOMe
OOH
O
O
HO COOMeHO
COOMe
O
O
O
O
HO COOMe
COOMe
O O
HO HO
O
O
HO COOMe
COOMe
O
HO HO
O
O
HO OEt
COOMe
O
HO HO
114 115 116
117 118 119
Secondary metabolites polyacetylene lactone rel-4-dihydro-4β-hydroxy-5α-
octa-2,4,6-triynyl-furan-2-(5H)-one (120), ethyl 3,4-dihydroxy-6,8,10-triynyl-
dodecanoate (121), taraxasteryl acetate (122), lupeyl acetate (123), lupeol
(125), lupenone (124), β-sitosterol (126), stigmasterol (127) and luteolin
(128) from the n-hexane fraction of the ethanolic extract of the V. scorpioides
(Adalva Lopes 2013).
Chapter # 02 Literature Survey
Page # 37
AcO
H
H
HRO
H
H
H
H
H
H
H
H
HO
H
CCCCCCH3CO CH3
O
OH
OH
OH
OH
OOH
HO
O
H
H
H
H
O
CCCCCCH3C
O
OH
120
121
122 123 R = COCH3
125 R = H
124
126
5
5,22
127
128
2.5.7 Vernonia patula
V. patula is an annual plant with 2-7 cm length and 1-3 cm width found in
Tawain, and island of Melville (Chiu 1987). Medicinally, V. patula has been
used against hepatitis, inflammation, cold, antiviral and antipyretic. It is
used to treat headache, malaria, rehum and gstroenteritis (Compilation
Committee). In Bangladesh, it has been used on vast level for the production
of up to 20 folk medicines in the field of ethnomedicine (Saha 2012); (Ku
2002). From the whole plant extract of V. patula: a germacrane
sesquiterpenoid, incaspitolide D (129), along with (S)-N-
Chapter # 02 Literature Survey
Page # 38
benzoylphenylalanine-(S)-2-benzamido-3-phenyl- propyl ester (130), indole-
3-carboxylic acid (133), apigenin (132), diosmetin (133) and luteolin (134)
was isolated (Liang 2010).
O
O
O
O
O
O
OH
O
O
O
H3C
NH
O
NH
O
H
OHO
OH O
OH
O
OH
CH3
HO
OH O
O
OH
OH
HO
OH O
129 130 131
132 133 134
Bauerenyl acetate (135), friedelin (136), epifriedelanol (137), 20(30)-
taraxastene-3β,21α-diol (138) have been isolated from the whole plant
extract of V. patula (Liang QL 2003).
H
H
HH
O
H
H
HH
HO
H
H
OAc
H
137 138136
Chapter # 02 Literature Survey
Page # 39
2.5.8 Vernonia colorata
Vernonia colorata is a variable under shrub or tree with 8m height
distributed thought central and south tropical Africa, West Cameron and is
second most popular species after V. amygdalina. Medicinally, it is used in
pregnancy, blood disorders, emetics, general healing, kidneys, liver disease,
skin, food poisoning, vermifuges, laxatives, oral treatment, paralysis,
epilepsy, spasm, venereal diseases and to treat pulmonary troubles.
Vernodalin (139) isolated from V. colorata show antibacterial and
antiplasmodial activities in its pure form (Rabe 2002; Chukwujekwu 2009).
Other bioactive compounds isolated from V. colorata includes, vernolide
(140), dihydrovernolide, dihydrovernodalin (141) (Rabe 2002; Chukwujekwu
2009).
O
O
O
OH
O
O
O
O
O
O
O
OH
O
O
O
HO
O
O
HO
O
O
CH2
141140139
Chapter # 03 Results & Discussions
Page 40
CHAPTER # 3
RESULTS & DISCUSSION
NATURAL PRODUCTS
Chapter # 03 Results & Discussions
Page 41
3 Structure Elucidation of New Compound Isolated from Vernonia
oligocephala
3.1 Structure Elucidation of Oligocephlate (142)
O
O1
3 5 7
9
10
12
14 16
17
18
20
22
23 24
25 26
27
2829
30
142
Compound 142 was isolated as colorless amorphous solid. The high
resolution electron impact mass spectrometry (HR-EI-MS) determined the
molecular formula C32H52O2 through a molecular ion peak [M]+ at m/z
468.3980 (calcd. for C32H52O2, 468.3968) having seven double bond
equivalence (DBE). The IR spectrum of 142 showed the peaks for ester
carbonyl (1730 cm-1) and unsaturation (1640 cm-1), respectively.
The 1H NMR spectrum of compound 142 showed eight methyl signals
including six tertiary and two secondary methyls at δ 0.76, 0.83, 0.84, 0.93,
0.97, 1.05 (3H each, s) and 0.87 (3H, d, J = 6.4 Hz), 0.93 (3H, d, J = 6.4 Hz),
respectively. This observation indicated the presence of pentacyclic
triterpenoid skeleton (Jones 1951). A methyl singlet at δ 2.02 (3H, s) is
attributed to acetyl group in the molecule. An oxymethine proton was
Chapter # 03 Results & Discussions
Page 42
resonated at δ 4.50 (1H, dd, J = 11.6, 5.6 Hz) is assigned to H-3 and its
larger coupling constant value confirmed it axial and α in orientation
(Sharnma 1984).
The 13C NMR spectra (BB and DEPT) of 142 showed 32 carbon signals for
nine methyl, ten methylene, five methine and eight quaternary carbons. The
downfield carbon at δ 171.0 assigned to acetyl group and δ 141.0 and 131.0
to olefinic quaternary carbons. The positions of both acetyl group and double
bond was done by HMBC correlations in which H-3 showed correlation with
ester carbonyl at δ 171.0, CH3-27 (δ 1.05) with C-13 (δ 131.0) and CH3-28 (δ
0.76) with C-18 (δ 141.0) confirming the position of acetyl group at C-3 and
double bond between C-13 and C-18. The above data showed close
resemblance to the data reported for boehmeryl acetate (Son 1990).
The relative stereochemistry at C-17 was determined through 13C NMR
chemical shift of C-28 (δ 17.9) (Nakane 1999) confirmed CH3-28 as axial and
α and missing of its NOESY correlations with H-21 confirmed the orientation
of isopropyl group at C-21 as α.
Based on these evidences compound 142 was 3β-acetoxyneohop-13-ene
and named as oligocephlate (Riaz 2013).
Chapter # 03 Results & Discussions
Page 43
3.1.1 α-Glucosidase Inhibitory Activity of Oligocephalate (142)
Various concentrations of oligocephalate (142) were tested against enzyme α-
glucosidase, which displayed inhibitory activity against this enzyme. The IC50
values are depicted in Table 5.
Table 5. Inhibition of α-glucosidase by oligocephalate (142)
Compound IC50 ± S.E.Ma[µM]
142 18.51 ± 0.01
Acarboseb 38.25 ± 0.12
aStandard error of the mean of five assays
bStandard inhibitor of the α-glucosidase enzyme
Chapter # 03 Results & Discussions
Page 44
3.2 Structure Elucidation of Known Compounds Isolated from V.
oligocephala
3.2.1 Structure Elucidation of β-Sitosterol (126)
HO
HH
1
3
57
9
12
1416
17
18 20
10
19
126
28
23
21
29
25
26
27
The compound 126 was purified as colorless crystalline solid (m.p: 143-145
°C). It appeared pink on heating after spraying with ceric sulphate solution.
The IR absorptions were appeared at 3445, 2970, 2868, 1618, 1257, 1021,
985, 780 cm-1 characteristic for O-H, C=C and C-H bonds. The electron
impact mass spectrometry (EI-MS) spectrum of compound 126 showed the
molecular ion peak [M]+ at 414.38 and its molecular formula C29H50O was
determined by high resolution electron impact mass spectrometry (HR-EI-
MS) due to the molecular ion peak at m/z 414.3861.
The 1H-NMR spectrum of 126 displayed a signal at δ 3.17 (1H, m) for an
oxymethine and δ 5.23 (1H, br s) for an olefinic proton. Six methyl signals
showed their presence at δ 1.50, 1.45, 0.95, 0.85, 0.75 and 0.65 among
them two were angular which is characteristic in steroids.
Chapter # 03 Results & Discussions
Page 45
The 13C-NMR showed twenty nine carbon signals including six methyl (δ
30.2, 24.4, 18.7, 18.3, 13.3, and 11.1), eleven methylene (δ 41.5, 40.6,
36.72, 33.9, 32.1, 31.9, 27.5, 25.7, 23.2, 22.6, 20.4), nine methines (δ
122.3, 70.1, 56.6, 56.3, 49.9, 36.2, 35.2, 32.5, and 28.1) and three
quaternary carbons (δ 141.9, 42.9, 35.6). The position of both the double
bond and hydroxyl group was confirmed through HMBC correlations in
which Me-19 correlated with C-5 (δ 141.5) and the COSY correlation of
olefinic methine at δ 5.23 with H-7 (δ 2.16) confirm the position of double
bond between C-5 and C-6. The oxymethine was placed at position C-3 due
to its HMBC correlation with C-1 (δ 36.7) and C-5 (δ 141.5) and its COSY
correlation with H-2 (δ 1.73). Based on the above discussion the compound
126 have hydroxyl group at C-3 and olefinic bond between C-5 and C-6.
The above described data for 126 was found resembled to the data already
reported for β-sitoster (Kamboj 2011).
Chapter # 03 Results & Discussions
Page 46
3.2.2 Structure Elucidation of Oleanolic Acid (143)
HO
COOH1
3 5 7
9
11 13
15
17
19 21
23 24
25 26
27
28
29 30
143
H
H
H
Compound 143 was isolated as colorless amorphous solid (m.p: 271-273 °C).
Its IR spectrum showed the characteristic absorptions for O-H (3340 cm-1),
COOH (3124 cm-1), C-H (2930, 2880 cm-1) and C=C (1650 cm-1). Its
molecular formula C30H48O3 was established based on HR-EI-MS which
showed a molecular ion peak [M]+ at m/z 456.3593 indicating the presence
of six degree of unsaturation.
The 1H-NMR spectrum of the compond 143 showed signals for seven tertiary
methyls at δ 0.92 (3H, s, Me-25), 0.97 (3H, s, Me-29), 0.99 (3H, s, Me-30),
1.01 (3H, s, Me-26), 1.02 (3H, s, Me-24), 1.11 (3H, s, Me-23) and 1.12 (3H, s,
Me-27). A downfield triplet at δ 5.29 (1H, t, J = 6.5 Hz, H-12), indicating the
presence of double bond in a pentacyclic triterpene nucleus and a doublet of
doublet at δ 2.18 (1H, dd, J = 12.7, 4.3 Hz, H-18) together with an
oxygenated methine at δ 3.35 (1H, dd, J = 12.4, 5.0 Hz) indicated 143 a
triterpene of oleanane series (Hamzah 1998).
Chapter # 03 Results & Discussions
Page 47
The 13C-NMR spectrum (BB and DEPT) of 143 exhibited thirty carbon
signals for seven methyl at δ 32.9, 28.8, 25.9, 23.8, 17.7, 17.3 and 15.5, ten
methylene at δ 46.3, 39.0, 33.9, 33.3, 32.6, 28.6, 28.4, 24.5, 24.2, and 18.9,
five methine at δ 71.9, 55.4, 47.9, 41.8 and 121.6 and eight quaternary
carbon atoms at δ 180.0, 140.9, 46.4, 42.8, 38.9, 38.8, 37.1, 31.4. The
downfield signals at δ 180.0, 140.9, 121.6 and 71.9 were attributed to
saturated carboxylic acid, double bond and an oxygenated methine,
respectively. The double bond was fixed between C-12/13 by HR-EI-MS
fragmentation pattern showing peaks at m/z 248 (C16H24O2) and 203
(C15H23). Retero Diels-Alders (RDA) fragmentation through the cleavage of
ring C indicated the presence OH in ring A/B and carboxyl acid in ring D/E
on oleanene skeleton.
The position of all the substituents and the linkages at various positions
were confirmed by the long range HMBC correlations in which the triplet
appeared at δ 5.29 showed HMBC correlations with C-9 (δ 47.9), C-14 (δ
42.8) and C-18 (δ 41.8) supporting the position of double bond at C-12. The
oxygenated methine (δ 3.35) showed HMBC correlation with C-1 (δ 39.0) and
C-23 (δ 28.8) confirming its presence at position C-3. Its orientation was
deduced by 1H-NMR spectrum through larger coupling constant (12.4 Hz),
thus confirming it as axial and and corresponding OH as equatorial and β
in orientation.
Chapter # 03 Results & Discussions
Page 48
The above spectral evidences were in complete agreement with the data
reported for oleanolic acid (3β-hydroxyolean-12-en-28-oic acid (Bhatt 2011)
which was finally verified by its co-TLC with authentic sample.
Chapter # 03 Results & Discussions
Page 49
3.2.3 Structure Elucidation of 5,7,4'-Trihydroxyflavone (144)
O
OH
HO
O
OH
5'
3
5
7 9
10
1'
3'
144
1
Compound 144 was obtained as yellow needles from the n-hexane soluble
fraction by CC over silica gel eluting with n-hexane:EtOAc (5.5:4.5). The IR
spectrum showed absorption bands at 3455 cm-1 for hydroxyl group, 1555-
1493 cm-1 for carbon carbon double bond. UV band appeared at 318, 268,
259 nm indicated the presence of flavones skeleton (Dordevic 2000). The HR-
EI-MS of 144 showed the molecular ion peak at m/z 270.0475
corresponding to the molecular formula C15H10O5 (calcd. for C15H10O5,
270.1453).
The 1H-NMR spectrum of compound 144 showed A2B2 doublets at δ 7.79
(2H, d, J = 8.5 Hz), 6.90 (2H, d, J = 8.5 Hz), a singlet at δ 6.54 (1H, s), two
meta-coupled doublets at δ 6.41 (1H, d, J = 2.0 Hz), 6.22 (1H, d, J = 2.0 Hz)
and an olefinic singlet at δ 6.54 (IH, s) typical for apigenin nucleus (Pandey
2006).
The 13C-NMR (BB & DEPT) spectrum of compound 144 showed thirteen
carbon signals for fifteen carbons, five signals for seven methine carbons at δ
Chapter # 03 Results & Discussions
Page 50
129.2, 116.8, 103.4, 100.1 and 94.9, and for eight quaternary carbons at δ
183.3, 166.0, 165.8, 163.0, 161.6, 159.1, 122.9, 104.8. The signals at δ
161.6, 129.2, 122.9, 116.8 indicated the presence of p-substituted benzene
ring where the signals at δ 183.3, 166.0, 103.4 showed the presence of
oxygenated α,β-unsaturated ketone.
The substitutions and the linkages at various positions were confirmed by
2D-NMR spectroscopic techniques including COSY, HSQC and HMBC.
The above discussed spectral data when compared with the literature found
completely overlapped with the data reported for 5,7,4'-trihydroxyflavone
commonly known as apigenin (Mabry 1970).
Chapter # 03 Results & Discussions
Page 51
3.2.4 Structure Elucidation of Apigenin 7-p-Coumarate (145)
O
HO
O O
OH O
OH
5'
6'
2
3
5
6
8
9
10
2'
3'
1"
2"
3"5"
6"
8"
9''
145
Compound 145 was obtained as yellow amorphous powder (m.p = 267-268
˚C) from the n-hexane soluble fraction by CC over silica gel eluting with n-
hexane:EtOAc (5.5:4.5). The IR spectrum exhibited absorption band for
hydroxyl group at (3700-3050 cm-1), together with absorption bands at 1684,
1652, 1510, 1494, 1242, 1075, 830 cm-1 for Ar. C=C and C-O, respectively.
The UV band appeared at 320 and 268 indicated the presence of conjugated
system. The molecular formula of the compound 145 was established as
C24H16O7 by HR-EI-MS showing the molecular ion peak at m/z 416.0950
(calcd. for C24H16O7, 416.0945).
Its 1H-NMR spectrum of compound 145 showed the signals as for apigenin
nucleus same as for compound 145 with the additional signals for p-
coumaroyl moiety at δ 7.44 (2H, d, J = 8.4 Hz), 7.38 (1H, d, J = 16.0 Hz),
6.76 (2H, d, J = 8.4 Hz) and 6.39 (1H, d, J = 16.0 Hz), respectively.
Chapter # 03 Results & Discussions
Page 52
The 13C-NMR (BB & DEPT) spectrum of compound 145 showed twenty
carbon resonances for 24 carbons, nine signals for thirteen carbons at δ
144.0, 129.1, 128.6, 117.1, 116.8, 115.8, 103.9, 99.1, 94.6 and eleven
quaternary carbon atoms at δ 183.2, 169.5, 164.5, 163.2, 162.1, 162.0,
159.9, 156.4, 126.3, 121.9, 106.0. The signals disclosed the presence of
apeginin nucleus were appeared at δ 183.3, 166.0, 165.8, 163.0, 161.6,
159.1, 129.1, 122.9, 116.8, 104.8, 103.4, 100.1 and 94.9 where as the
signals for p-coumaroyl moiety were appeared at δ 172.0, 162.0, 146.2,
130.2, 127.5, 116.9 and 116.0.
The position of all the substituent were confirmed by 2D-NMR spectroscopic
techniques including COSY, HSQC especially HMBC.
This spectral data discussed for compound 145 was matched with the
literature values reported for apigenin 7-p-coumerate (Gabrieli 1990).
Chapter # 03 Results & Discussions
Page 53
3.2.5 Structure Elucidation of Kaemferol (44)
O
O
OH
HO
OH
OH
2
9
10
2'
3'
5'
6'
44
4
1
5
7
Compound 44 was isolated as a yellow needle with melting point 276-278 ˚C.
The IR spectrum displayed absorption bands at 3420, 2830, 1700, 1600,
1510, 1560 cm-1 for O-H, C-H, C=O and C=C functional groups, respectively.
The UV spectrum displayed absorption bands at 204, 265 and 365 nm
indicated the presence of substituted aromatic system. Its molecular formula
C15H10O6 was deduced by a molecular ion peak at [M]+ m/z 286.0477.
The 1H-NMR spectrum of compound 44 displayed two doublet at δ 8.98 (2H,
d, J = 8.4 Hz), 7.20 (2H, d, J = 8.4 Hz) splitted as A2B2 splitting pattern
indicated the presence of p-substituted benzene ring and two meta-coupled
doublet at δ 6.58 (1H, d, J = 2.0 Hz), and 6.29 (1H, d, J = 2.0 Hz) indicated
the presence of 1,2,3,5-tetra substituted benzene ring.
The 13C-NMR spectra (BB & DEPT) of 44 displayed total thirteen signals for
fifteen carbons including four signals for six methines at δ 130.9, 115.5,
98.4, 93.8, nine quaternary carbon signals at δ 175.9, 164.1, 160.8, 160.1,
156.3, 146.7, 135.7, 122.1, 103.8, respectively.
Chapter # 03 Results & Discussions
Page 54
The downfield signal at δ 175.9 was assigned to an unsaturated keteone,
especially in flavanols whereas the signals at δ 160.1, 130.9, 122.1, 115.5
indicated the presence of p-substituted benzene ring.
The structure was constituted by using HMQC and the COSY correlations
and the substitutions and the linkages at various positions were finally
confirmed through long range hetero nuclear multiple bond correlation
(HMBC) experiments.
The above discussed spectral data when searched in the literature found on
good concurrence with the spectral data reported for kaemferol (Marin 2009).
Chapter # 03 Results & Discussions
Page 55
3.2.6 Structure Elucidation of Isorhamnetin (146)
O
OH
OCH3
O
OH
HO
OH
2
4a
5
6
8
2'
5'
6'
146
Compound 146 was isolated as pale yellow amorphous powder (m. p. 307-
308 ˚C). The IR spectrum showed the peaks at 3416, 3174, 2923, 2854,
1710, 1420 cm-1 indicated the presence of O-H, C-H, C=O and C=C
functionalities. The UV spectrum displayed the absorption bands at 272,
336 nm typical for substituted aromatic system. Its molecular formula
C16H12O7 was deduced by HR-EI-MS through a molecular ion peak at [M]+
m/z 316.0583 with 11 double bond equivalences (DBE).
The 1H-NMR spectrum of compound 146 showed an ABX-splitting pattern at
δ 7.81 (1H, d, J = 1.5 Hz), 7.78 (1H, dd, J = 8.4, 1.5 Hz), 6.95 (1H, d, J = 8.4
Hz), together with two doublets at δ 6.58 (1H, d, J = 2.0 Hz), 6.23 (1H, d, J =
2.0 Hz) indicated the presence of a 1,3,4-trisubstituted and a 1,2,3-5-tetra-
substituted benzene ring, respectively. The signal for a methoxy group was
appeared at δ 3.28 (3H, s).
The 13C-NMR spectra (BB & DEPT) of 146 displayed total 16 carbon signals
including one methyl at δ 57.2, five methines at δ 131.6, 128.9, 122.9,
Chapter # 03 Results & Discussions
Page 56
113.8, 93.7 and ten quaternary carbons at δ 176.7, 165.2, 160.5, 157.8,
157.6, 150.4, 147.8, 134.9, 122.3, 115.2.
The greater number of aromatic quaternary carbons indicated the presence
of condensed aromatic class may be flavanol. The careful analysis of the
NMR data indicated that 146 be a flavanol with the tri-substution pattern in
ring C and tetra-substitution in ring A of a flavanol nucleus.
The position of all the substituents especially the position of methoxy group
was confirmed by HMBC correlations.
All the discussed when search in the literature found compatible with the
spectral data reported for isorhamnetin (Sikorska 2001).
Chapter # 03 Results & Discussions
Page 57
3.2.7 Structure Elucidation of β-Sitosterol 3-O-β-D-glucopyranoside
(147)
OO
HOHO
OH
OH
3
1
57
9
11 13
15
17
19
18
21
23
25
26
27
29
3'
1'5'
147
H H
H
Compound 148 was isolated as colorless amorphous powder. Its IR spectrum
showed absorption peaks at 3452, 3044, 1646, 1618, 1559, 1550 cm-1. Its
molecular formula C35H60O6 was based on HR-FAB-MS which showed
molecular ion peak [M+H]+ at m/z 577.4483 indicating the presence of six
degree of unsaturation.
The 1H-NMR spectrum of compound 147 displayed same pattern of splitting
as observed for β-sitosterol 147 indicating the presence of basic sterol
nucleus, with the additional signal for glucose moiety at δ 4.38 (1H, d, J =
6.8 Hz, H-1), 3.01 (m), 3.24 (m), 3.32 (m), 3.39 (m), oxygenated methylene δ
3.43 and 3.65 (m).
The 13C-NMR spectra (BB and DEPT) of 147 showed thirty five carbon
signals for six methyl at δ 19.8, 19.6, 19.0, 18.7, 12.1 and 11.9, twelve
methylene at δ 61.9, 42.8, 40.3, 36.9, 34.4,32.7 , 21.1, 31.4, 28.9, 25.5,
Chapter # 03 Results & Discussions
Page 58
23.2 and 32.2, fourteen methine at δ 121.8, 101.0, 79.2, 75.5, 55.9, 51.3,
48.9, 36.1, 32.0, 26.2, 73.4, 70.1, 70.0 and 56.6, and three quaternary
carbon at δ 140.1, 42.2, 36.6. The downfield signal at δ 140.1 and 121.8 are
attributable to double bond and the signals at δ 101.0 and 79.2 were due to
anomeric carbon and oxygenated methine, respectively. The signals at 101.0,
75.5, 73.4, 70.1, 70.0 and 61.9 were due to the presence of hexose moiety.
The attachment of sugar moiety was confirmed HMBC correlation in which
the anomeric proton (δ 4.38) showed HMBC correlations with C-3 (δ 79.2)
confirmed the attachment of glucose moiety at C-3. This was further
confirmed from the EI-MS spectrum which displayed base ion peak at m/z
414 (C29H49O) and loss of 164 due to (C6H11O5) fragment.
This above spectral data matched with the data reported for β-sitosterol 3-O-
β-D-glucopyranoside (Pouchert and Behnke 1992; Mizan ur Rahman, Mukta
et al. 2009) which was further confirmed by co-TLC with authentic sample.
Chapter # 03 Results & Discussions
Page 59
3.2.8 Structure Elucidation of Quercetin (43)
HO
OH
O
OH
O
OH
OH
43
3
5
6
8
2'
5'
6'
4a
8a
Compound 43 was isolated as pale yellow powder (M.P. 300-301 ˚C). The IR
spectrum displayed absorption bands at 3428-3369, 2985, 2871, 1708 cm-1
for O-H, C-H, C=O and C=C functional groups, respectively where as the UV
absorptions were observed at 314, 360 and 400 nm typical for substituted
aromatic system. The molecular formula C15H10O7 was deduced by HR-EI-
MS through a molecular ion peak [M]+ at m/z 302.0427 with 11 double bond
equivalences (DBE).
The 1H-NMR spectrum of 43 showed signals at δ 7.71 (1H, d, J = 2.1 Hz),
7.61 (1H, dd, J = 8.4, 2.1 Hz), 6.77 (1H, d, J = 8.4 Hz), 6.34 (1H, d, J = 2.1
Hz) and 6.27 (1H, d, J = 2.0 Hz), respectively, same as observed for
compound 146 indicated the presence of a 1,3,4-trisubstituted and a 1,2,3-
5-tetra-substituted benzene ring.
The 13C-NMR spectra (BB & DEPT) of 43 displayed altogether 15 carbon
signals including five methine signals at δ 124.1 , 120.8, 116.1, 99.1, 94.2
Chapter # 03 Results & Discussions
Page 60
and ten quaternary carbons δ 177.4, 165.7, 162.7, 158.6, 148.6, 148.2,
146.4, 137.3,104.7, respectively.
The substitution of this ring was confirmed by the combination of 2D NMR
by using HSQC, COSY and long range HMBC correlations.
The spectral data described above for compound 43 showed close
resemblance with the spectral data already reported quercetin (Razavi 2012).
Chapter # 04 Experimental
Page # 61
CHAPTER # 4
EXPERIMENTAL
NATURAL PRODUCTS
Chapter # 04 Experimental
Page # 62
4.1 General Procedure The chromatographic techniques were accomplished by applying commercial
grade solvents. The solvents were purified by the process of distillation at
their respective boiling points.
4.2 Spectroscopy
4.2.1 UV Spectra
UV-spectroscopy yields the data about the presence of conjugated double
bonds. For obtaining UV spectra, Schimazdu and UV -240U -3200 Hitachi
spectrophotometers were used.
4.2.2 IR Spectra
IR spectra assures us the presence of functional groups and Jasco-320-A
Infrared spectrometer was used for this intention.
4.2.3 Mass Spectrometry
All types of mass spectra were recorded by employing Finnigan MAT-112 and
MAT-113 spectrometers. Linked scan and peak matching experiment were
also executed on the same instruments.
4.2.4 Nuclear Magnetic Resonance (NMR) Spectroscopy
One and two dimensional NMR spectra were measured by using Bruker AM
400 and 500 MHz instruments in deutrated solvents. The chemical shift
values ( ) are depicted in ppm and the coupling constant (J) are in Hz.
Chapter # 04 Experimental
Page # 63
4.3 Techniques used for the Purification of Compound
Several techniques of isolation and purification were used to get pure
compounds from crude extract.
4.3.1 Column Chromatography (CC)
Silica gel of Merck, 70-230 mesh was utilized to accomplished column
chromatography. It was packed in the glass column and organic solvents
were pass across it as a mobile phase.
4.3.2 Thin Layer Chromatography (TLC)
Pre-coated aluminium TLC plates of GF254, Merck 0.25 mm utilized to foster
the purification of several fractions received from column chromatography.
4.4 Visualization of constituents on TLC plates
After the developing chromatograms, the colored compounds were initially
visualized with naked eye. The colorless compounds were observed under UV
lamp at 254 nm and 365 nm and spots were outlined with a lead pencil to
mark their positions.
4.4.1 Locating Reagents
After the development of chromatogram the position of separated compounds
can be visualized by applying locating reagent ceric sulphate and iodine
solution. The inactive compounds were visualized by applying ceric sulphate
10% as locating reagent.
Chapter # 04 Experimental
Page # 64
4.5 Collection and Identification of Plant Material
The whole plant material of Vernonia oligocephala (10 kg) was collected from
Lal Sohanra (District Bahawalpur) in April 2008 and identified by Dr.
Muhammad Arshad (Late), Plant Taxonomist, Cholistan Institute for Desert
Studies (CIDS), Baghdad-ul-Jadeed Campus, The Islamia University of
Bahawalpur, Bahawalpur, Pakistan, where a voucher specimen is deposited
(VO-CIDS/21/08).
4.6 Extraction and Isolation
The whole plant material of V. oligocephala (10 kg) was dried under shade,
crushed and soaked in Methanol and extracted thrice to get its extract. The
methanolic extract was concentrated under reduced pressure to get green
gummy mass (430 g). The methanolic extract (430 g) was dissolved in water
and extracted with n-hexane, ethylacetate and water soluble fractions. The
ethylacetate (EtOAc) soluble fraction (50 g) was subjected to silica gel column
chromatography (CC) and eluted with n-hexane, n-hexane:EtOAc, EtOAc,
EtOAc:methanol and methanol in increasing order of polarity to get ten sub-
fractions. These sub-fractions on further CC using gradient elusion resulted
into pure compounds by polarity orders to get oligocephalate (142, 35 mg) at
20 % EtOAc in n-hexane, β-sitosterol (126, 50 mg) at 25 % EtOAc in n-
hexane, oleanolic acid (143, 59 mg) at 40 % EtOAc in n-hexane, 5,7,4-
trihydroxyflavone (144, 24 mg) at 45 % EtOAc in n-hexane, apigenin 7-p-
coumarate (145, 30 mg) at 55 % EtOAc in n-hexane, kaempherol (44, 39 mg)
Chapter # 04 Experimental
Page # 65
at 80 % EtOAc in n-hexane, isorhamnetin (146, 16 mg) at 82 % EtOAc in n-
hexane, β-sitosterol 3-O-β-D-glucopyranoside (147, 70 mg) at 85 % DCM in
n-hexane and quercetin (43, 34 mg) at 100 % EtOAc, respectively.
Chapter # 04 Experimental
Page # 66
Methanolic extract
(430 g)
Powdered, extracted with methanol
and concentrated on rotary evaporater
Silica gel CC
EtOAc
FractionAqueous
fraction
Suspended in water
Fr. obtained
in n-hexane-EtOAc
(6:4)
Fr. obtained
in n-hexane-EtOAc
(5.5:4.5)
Further
purification
in
n-hex-EtOAc
(5.5:4.5)
144
Isolation scheme used for the purification of compounds from V. oligocephala
Fr. obtained
in n-hexane-EtOAc
(8:2)
Fr. obtained
in n-hexane-EtOAc
Fr. obtained
in 100 % EtOAc
(100 %)
Vernonia oligocephala
(10 kg)
Fr. obtained
in n-hexane-EtOAc
(4.5:5.5)
Further
purification
in
100 % EtOAc
43
Further
purification
in
n-hex-EtOAc
(1.5:8.5)
147
Further
purification
in
n-hex-EtOAc
(1.8:8.2)
146
Further
purification
in
n-hex-EtOAc
(2:8)
44
Further
purification
in
n-hex-EtOAc
(6:4)
143
Further
purification
in
n-hex-EtOAc
(8:2)
142, 126
Further
purification
in
n-hex-EtOAc
(4.5:5.5)
145
Extracted with EtOAc
Chapter # 04 Experimental
Page # 67
4.7 α-Glucosidase Inhibition Assay
The α-glucosidase inhibition assay was performed with slight modifications
as done by Pierre et al (Pierre 1978). Total volume of 100 µL reaction mixture
contained 70 µL 50 mM phosphate buffer, pH 6.8, 10 µL (0.5 mM) test
compound, followed by the addition of 10 µL (0.0234 units, Sigma Inc.)
enzyme. The contents were mixed, preincubated for 10 min at 37ºC and pre-
read at 400 nm. The reaction was initiated by the addition of 10 µL of 0.5
mM substrate (p-nitrophenyl glucopyranoside, Sigma Inc.). After 30 min of
incubation at 37ºC, absorbance of the yellow color produced due to the
formation of p nitrophenol was measured at 400 nm using Synergy HT
(BioTek, USA) using 96-well microplate reader. Acarbose was used as
positive control. The percent inhibition was calculated by the following
equation
Inhibition (%) = (abs of control – abs of test / abs of control) × 100
IC50 values were calculated using EZ-Fit Enzyme Kinetics Software.
Chapter # 04 Experimental
Page # 68
4.8. Characterization of New Compound Isolated from V. oligocephala
4.8.1 Characterization of Oligocephlate (142)
Colorless amorphous solid (35 mg)
IR (KBr) Vmax cm-1: 1730, 1640, 1390,
1380, 960, 840 cm-1.
O
O1
3 5 7
9
10
12
14 16
17
18
20
22
23 24
25 26
27
2829
30
142
1H-NMR (CDCl3, 300 MHz): 1.42 (1H, m, H-1), 1.66 (2H, m, H-2), 4.50 (1H,
dd, J = 11.6, 5.6 Hz, H-3), 1.37 (1H, m, H-5), 1.18 (1H, m, H-6), 1.27 (1H, m,
H-7), 1.55 (1H, m, H-9), 1.24 (1H, m, H-11), 2.17 (1H, m, H-12), 1.74 (1H, m,
H-15), 1.23 (H, m, H-16), 2.23 (H, m, H-19), 1.34 (H, m, H-20), 1.04 (H, m,
H-21), 1.52 (H, m, H-22), 0.84 (3H, s, H-23), 0.83 (H, s, H-24), 0.94 (3H, s,
H-25), 0.97 (3H, s, H-26), 1.05 (3H, s, H-27), 0.76 (3H, s, H-28), 0.93 (3H, d,
J = 6.4 Hz, H-29), 0.87 (3H, d, J = 6.4 Hz, H-30), 2.02 (3H, s, OAc).
13C-NMR (CDCl3, 100 MHz): δ 32.8 (C-1), 25.3 (C-2), 81.07 (C-3), 37 (C-4),
48.2 (C-5), 18.8 (C-6), 34.8 (C-7), 42.5 (C-8), 46.2 (C-9), 37.0 (C-10), 22.8 (C-
11), 26.3 (C-12), 131.0 (C-13), 42.7 (C-14), 30.3 (C-15), 37.4 (C-16), 43.0 (C-
17), 141.0 (C-18), 26.4 (C-19), 27.5 (C-20), 59.0 (C-21), 29.8 (C-22), 28.9 (C-
23), 17.1 (C-24), 22.8 (C-25), 25.6 (C-26), 26.7 (C-27), 17.9 (C-28), 22.9 (C-
29), 23.0 (C-30), 171.0, 21.3 (Ac).
HR-EI-MS: m/z 468.398 [M]+ (97.4 %).
HR-EI-MS: m/z 468.3980 (calcd. for C32H52O2, 468.3967).
Chapter # 04 Experimental
Page # 69
4.8.2 Characterization of Known Compounds Isolated from V.
oligocephala
4.8.2.1 Characterization of β-Sitosterol (126)
Colorless Crystalline Solid (50 mg).
M.p: 143-145 °C.
[α]D – 28.6 (c = 0.0015, MeOH).
IR (KBr) max cm-1: 3445, 2970,
2868, 1805, 1618, 1452, 1380,
1257, 1021, 985,780.
HO
HH
1
3
57
9
12
1416
17
18 20
10
19
126
28
23
21
29
25
26
27
1H-NMR (CD3OD, 500 MHz): δ 2.35 (2H, m, H-1), 1.73 (2H, m, H-2), 3.17
(1H, m, H-3), 2.27 (2H, m, H-4), 5.23 (1H, m, H-6), 2.16 (2H, m, H-7), 1.11
(1H, m, H-8), 2.11 (1H, m, H-9), 1.48 (2H, m, H-11), 1.77 (2H, m, H-12), 2.01
(1H, m, H-14), 1.75 (2H, m, H-15), 1.29 (2H, m, H-16), 1.85 (1H, m, H-17),
0.95 (3H, s, H-18), 1.50 (3H, s, H-19), 2.40 (1H, m, H-20), 1.45 (3H, d, J =
6.5 Hz, H-21), 1.79 (2H, m, H-22), 1.72 (2H, m, H-23), 1.95 (1H, m, H-24),
1.85 (1H, m, H-25), 0.75 (3H, d, J = 6.5 Hz, H-26), 0.65 (3H, d, J = 6.5 Hz, H-
27) 1.63 (2H, s, H-28), 0.85 (3H, t, J = 7.0 Hz, H-29).
13C-NMR (CD3OD, 100 MHz): δ 36.7 (C-1), 33.9 (C-2), 70.1 (C-3), 41.5 (C-4),
141.9 (C-5), 122.3 (C-6), 32.1 (C-7), 32.5 (C-8), 49.9 (C-9), 35.6 (C-10), 20.4
(C-11), 40.6 (C-12), 42.9 (C-13), 56.3 (C-14), 23.2 (C-15), 27.5 (C-16), 56.6
(C-17), 11.1 (C-18), 30.2 (C-19), 36.2 (C-20), 24.4 (C-21), 31.9 (C-22), 22.6
(C-23), 35.2 (C-24), 28.1 (C-25), 18.7 (C-26), 18.3 (C-27), 25.7 (C-28), 13.3
(C-29).
EI-MS m/z (rel. int.): 414.4 [M]+ C29H50O: 414 (4.2), 400 (2.9), 381 (2.8), 275
(2.8), 272 (2.0), 254 (5.9), 230 (4.0), 212 (6.0), 197 (3.7), 173 (5.1), 163 (5.5),
137 (7.9), 120 (9.8), 106 (15.2), 94 (19.9), 83 (16.0), 69 (42.0), 55 (52.0), 43
(100.0).
Chapter # 04 Experimental
Page # 70
HR-EI-MS m/z: 414.3861 [M]+ (calcd. for C29H50O), 414.3855.
4.8.2.2 Characterization of Oleanolic Acid (143)
White amorphous powder (59 mg).
M.P = 271-273 °C.
IR (KBr) max cm-1: 3340, 3124, 2930,
2880, 1650. HO
O
OH
1
3 5 7
9
11 13
15
17
19 21
23 24
25 26
27
28
29 30
143
H
H
H
1H-NMR (CDCl3, 500 MHz): δ 5.29 (1H, t, J = 6.5 Hz, H-12), 3.35 (1H, dd, J =
12.4, 5.0 Hz, H-3), 2.18 (1H, dd, J = 12.7, 4.3 Hz, H-18), 1.12 (3H, s, Me-27),
1.11 (3H, s, Me-23), 1.02 (3H, s, Me-24), 1.01 (3H, s, Me-26), 0.99 (3H, s,
Me-30), 0.97 (3H, s, Me-29) and 0.92 (3H, s, Me-25),
13C-NMR (CDCl3, 125 MHz): δ 180.0 (C-28), 140.9 (C-13), 121.6 (C-12), 71.9
(C-3), 55.4 (C-5), 47.9 (C-9), 46.4 (C-17), 46.3 (C-19), 42.8 (C-14), 41.8 (C-
18), 39.0 (C-1), 38.9 (C-8), 38.8 (C-4), 37.1 (C-10), 33.9 (C-21), 33.3 (C-22),
32.9 (C-29), 32.6 (C-7), 31.4 (C-20), 28.8 (C-23), 28.6 (C-15), 28.4 (C-2), 25.9
(C-27), 24.5(C-11), 24.2 (C-16), 23.8 (C-30), 18.9 (C-6), 17.7 (C-24), 17.3 (C-
26) and 15.5 (C-25).
HR-EI-MS: m/z 456.3593 (calcd. for C30H48O3, 456.3603).
Chapter # 04 Experimental
Page # 71
4.8.2.3 Characterization of 5,7,4'-Trihydroxyflavone (144)
Yellow needles (24 mg).
MP: 352 ˚C.
UV (MeOH) max (log ε) nm: 269 (4.27),
340 (4.32).
IR (KBr) max cm-1: 3455, 1555-1493.
O
OH
HO
O
OH
5'
6'
2
3
5
6
8
9
10
2'
3'
144
1
1H-NMR (CD3OD, 500 MHz): δ 7.79 (2H, d, J = 8.5 Hz, H-3,5), 6.90 (2H, d, J
= 8.5 Hz, H-2,6), 6.54 (1H, s, H-3), 6.41 (1H, d, J = 2.0 Hz, H-8), 6.22 (1H,
d, J = 2.0 Hz, H-6).
13C-NMR (CD3OD, 125 MHz): δ 183.3 (C-4), 166.0 (C-2), 165.8 (C-7), 163.0
(C-9), 161.6 (C-4), 159.1 (C-5), 129.2 (C-3,5), 122.9 (C-1), 116.8 (C-2,6),
104.8 (C-10), 103.4 (C-3), 100.1 (C-6), 94.9 (C-8).
HR-EI-MS m/z: 270.1475 (calcd. for C15H10O5, 270.1453).
Chapter # 04 Experimental
Page # 72
4.8.2.4 Characterization of Apigenin-7-p-Coumerate (145)
Yellow amorphous powder (30
mg). MP: 267 ˚C.
UV (MeOH) max (log ε) nm: 225
(4.0), 268 (3.92), 320 (3.75).
IR (KBr) max cm-1: 3700-3050,
1684, 1652, 1510, 1494, 1444,
1242,1180, 1075, 830.
O
HO
O O
OH O
OH
2
3
5
6
8
9
10
2'
3'
5'
6'
1"
2"
3"5"
6"
8"
9''
145
1H-MR (CD3OD, 500 MHz): δ 7.98 (2H, d, J = 8.4 Hz, H-2,6), 7.44 (2H, d, J
= 8.4 Hz, H-5,9), 7.38 (1H, d, J = 16.0 Hz, H-2), 7.10 (2H, d, J = 8.4 Hz,
H-3,5), 6.90 (1H, d, J = 2.0 Hz, H-8), 6.76 (2H, d, J = 8.5 Hz, H-6,8), 6.70
(1H, s, H-3), 6.58 (1H, d, J = 2.0 Hz, H-6), 6.39 (1H, d, J = 16.0 Hz, H-3).
13C-NMR (CD3OD, 125 MHz): δ 183.2 (C-4), 164.5 (C-7), 163.2 (C-2), 162.1
(C-5), 162.0 (C-4), 156.4 (C-9), 128.6 (C-2,4), 121.9 (C-1), 116.8 (C-3,5),
106.0 (C-10), 103.9 (C-3), 99.1 (C-6), 94.6 (C-8), 169.5 (C-1), 159.9 (C-7),
144.0 (C-2), 129.1 (C-5,9), 126.3 (C-4), 117.1 (C-2), 115.8 (C-6,8).
HR-EI-MS m/z: 416.0950 (calcd. for C24H16O7, 416.0945).
Chapter # 04 Experimental
Page # 73
4.8.2.5 Characterization of Kaemferol (44)
Yellow needles (39 mg)
UV λmax: 204, 265 and 365 nm.
M.p: 276-278 °C
IR (KBr) max cm-1: 3420, 2830, 1700, 1600,
1510 and 1560 cm-1.
O
O
OH
HO
OH
OH
2
4a
8a
6
8
2'
3'
5'
6'
44
1H-NMR (CD3OD 400 MHz): δ 6.29 (1H, d J = 2.0 Hz, H-6), 6.58 (1H, d, J =
2.0 Hz, H-8), 8.98 (1H, d, J = 8.4 Hz, H-2), 7.20 (1H, d, J = 8.4 Hz, H-3),
7.20 (1H, d, J = 8.4 Hz, H-5) and 8.98 (1H, d, J = 8.4 Hz, H-6).
13C-NMR (100 MHz, CD3OD): δ 146.7 (C-2), 135.7 (C-3), 175.9 (C-4), 103.8
(C-4a), 156.3 (C-5), 98.4 (C-6), 164.1 (C-7), 93.8 (C-8), 160 8 (C-8a), 122.1
(C-1), 130.9 (C-2), 115.5 (C-3), 160.1 (C-4), 115.5 (C-5) and 130.9 (C-6).
HR-EI-MS: m/z 286.0472 (calcd. for C15H10O6, 286.0476).
Chapter # 04 Experimental
Page # 74
4.8.2.5 Characterization of 1sorhamnetin (146)
Pale yellow amorphous powder (16 mg).
UV λmax: 272 and 336 nm.
M.p: 307 °C
IR (KBr) max cm-1: 3416, 3174, 2923, 2854,
1710 and 1420 cm-1.
O
OH
OCH3
O
OH
HO
OH
2
4a
5
6
8
2'
5'
6'
146
1H-NMR (CD3OD, 400 MHz): δ 6.23 (1H, d, J = 2.0 Hz, H-6), 6.58 (1H, d, J =
2.0 Hz, H-8), 7.81 (1H, d, J = 1.5 Hz, H-2), 6.96 (1H, d, J = 8.4 Hz, H-5),
7.78 (1H, dd, J = 8.4, 1.5 Hz, H-6) and 3.29 (3H, s, OMe).
13C-NMR (CD3OD, 100 MHz): δ 157.8 (C-2), 147.8 (C-3), 176.7 (C-4), 122.3
(C-4a), 157.6 (C-5), 122.9 (C-6), 165.2 (C-7), 93.7 (C-8), 160.5 (C-8a), 115.2
(C-1), 128.9 (C-2), 134.9 (C-3), 150.4 (C-4), 113.8 (C-5), 131.6 (C-6) and
57.2 (C-7).
HR-EI-MS: m/z 316.0578 (calcd. for C16H12O7, 316.0582).
Chapter # 04 Experimental
Page # 75
4.8.2.6 Characterization of β-Sitosterol 3-O-β-D-glucopyranoside (147)
Colorless amorphous
powder (70 mg).
[]D25 -14.5˚, (c = 0.003
MeOH).
IR (KBr) max cm-1:
3452. 3044, 1646,
1618, 1559, 1550.
OO
HOHO
OH
OHH H
3
1
57
9
11 13
15
17
19
18
21
23
25
26
27
29
3'
1'5'
147
1H-NMR (C5D5N, 400 MHZ): Δ 5.13 (1H, BR S, H-6), 4.38 (1H, D, J = 6.8 HZ,
H-1′), 3.65 3.43, (2H, BR S, H-6), 3.45 (1H, M, H-3), 3.39 (1H, M, H-5), 3.32
(1H, M, H-3), 3.24(1H, M, H-4), 3.01 (1H, M, H-2), 1.00 (3H, S, ME-19),
0.92 (3H, D, J = 6.2 HZ, ME-21), 0.86 (3H, T, J = 7.0 HZ, ME-29), 0.83 (3H, D,
J = 6.0 HZ, ME-26), 0.80 (3H, D, J = 6.0 HZ, ME-27) AND 0.68 (3H, S, ME-18).
13C-NMR (C5D5N, 100 MHZ):Δ140.1 (C-5), 121.8 (C-6), 101.0 (C-1), 79.2 (C-
3), 75.5 (C-5), 73.4 (C-2), 70.1 (C-3), 70.0 (C-4), 61.9 (C-6), 56.6 (C-14),
55.9 (C-17), 51.3 (C-9), 48.9 (C-24), 42.8 (C-4), 42.2 (C-13), 40.3 (C-12),
36.9 (C-1), 36.6 (C-10), 36.1 (C-20), 34.4 (C-22), 32.7 (C-7), 32.2 (C-16),
32.0 (C-8), 31.4 (C-2), 28.9 (C-23), 26.2 (C-25), 25.5 (C-15), 23.2 (C-28),
21.1 (C-11), 19.8 (C-27), 19.6 (C-19), 19.0 (C-21), 18.7 (C-26), 12.1 (C-29)
AND 11.9 (C-18).
HR-FAB-MS: [M+H]+ m/z 577.4483 (calcd. for C35H60O6, 577.4494).
Chapter # 04 Experimental
Page # 76
4.8.2.7 Characterization of Quercetin (43)
Pale yellow powder (34 mg).
UV λmax: 314, 360 and 400 nm.
M.p: 300 °C
IR (KBr) max cm-1: 3428, 2985, 2871 and
1708 cm-1
HO
OH
O
OH
O
OH
OH
43
3
5
6
8
2'
5'
6'
4a
8a
1H-NMR (CD3OD 400 MHz): δ 6.27 (1H, d, J = 2.0 Hz, H-6), 6.34 (1H, d, J =
2.0 Hz, H-8), 7.71 (1H, d, J = 2.1 Hz, H-2), 6.77 (1H, d, J = 8.4 Hz, H-5) and
7.61 (1H, dd, J = 2.1 Hz, H-6).
13C-NMR (CD3OD, 100 MHz): δ 148.2 (C-2), 137.3 (C-3), 177.4 (C-4), 104.7
(C-4a), 162.7 (C-5), 99.1 (C-6), 165 7 (C-7), 94.2 (C-8), 158.6 (C-8a), 124.1
(C-1), 116.0 (C-2), 146 4 (C-3), 148.6 (C-4), 116.1 (C-5) and 120 8(C-6).
HR-EI-MS: m/z 304.0578 (calcd. for C15H10O7, 304.0582).
Chapter # 05 Introduction
Page 77
CHAPTER # 05
INTRODUCTION
GREEN CHEMISTRY
Chemistry and Application of Green Solvents
Chapter # 05 Introduction
Page 78
5.1 Effect of Solvent in Chemistry
The need for understanding of the term “Solvation” is very obvious by the
fact that most of the reactions are carried out in liquid phase and here the
role of the solvent is not only as "spectator" despite of that it acts as a
transfer agent for heat and mass, and participate in the transfer of proton
(for acid/base catalyzed reactions) and also for the Solvation of dipolar and
ionic species. The effect of the solvent in chemistry has vital importance as it
has great effect on solvent reactivity including the effects on reaction rates,
stability and solubility. However this phenomenon is very complex because
of various numbers of solute and solvent interactions. To understand this
effect let's consider these three reactions.
1. (C2H5)3N + C2H5I ---> (C2H5)4N+ I- (1)
2. N2O5 ----> N2O3 + O2 (2)
3. (CH3CO)2O + C2H5OH -----> CH3COOC2H5 + CH3COOH (3)
Rate constant varied from 0.00018 in hexane to 1.33 in benzyl alcohol and
70.1 in nitrobenzene for 1st reaction. The rate constant of the 3rd reaction
was almost the reverse of the 1st reaction (0.0119 in hexane and 0.00245 in
nitrobenzene) while the rate constant of 2nd reaction was almost same in
different solvents. Solvent affects the reaction rates in three different ways.
5.1 Solvent Polarity
Chapter # 05 Introduction
Page 79
Polar solvents accelerate the reactions in which the products are more polar
then the reactants. In reaction, first the product is more polar as compared
to reactant because of being a salt, so that's why in the presence of polar
solvents like benzyl alcohol the reaction is accelerated, on the other hand the
polar solvent decrease the reaction rate if the reactants are more polar then
the products like in reaction (3) (Seoud 2007). Generally, the Polar solvents
favor the reaction in the direction of increasing polarity. Polarity of solvents
will have no influence on the rate of the reaction and the rate is independent
of the nature of the solvent which is what happened in reaction when both
the reactants and products are non polar.
5.2 Solvation Influence
The interaction of reactant, product or activated complex with solvent has
influence on the rate of reaction. After the interaction of the reactant with
solvent, and after getting solvated it causes to lower the potential energy of
the reactant, increasing activation energy and lowers the reaction rate. While
on the other hand the interaction of activated complex with solvent, after
solvated it lowers the potential energy, decrease in activation energy cause to
increase the rate of reaction. The influence of solvent on the rate may not be
considerable, if both the activated complex and as well as reactant is
solvated. The Solvation of the product in the solvent has no effect on the rate
of reaction unless it is reversible reaction.
5.3 Dielectric Constant of the Solvent
Chapter # 05 Introduction
Page 80
The dielectric constant (D) of the solvent plays a major role if the ionic
reaction is taking place in the presence of solvent. With increasing value of
D, ionization energy will also increase. This work is equal to the electrostatic
contribution to the increase in Gibbs free energy from initial to final state.
The work will be positive if the sign on the chargers are same, and will
negative, if they are different. With dielectric constant, the logarithm of rate
constant of ionic liquids varies inversely (D. S. Kemp 1975).
Example
The effect of the solvent on reactivity can be predominantly described by
considering the spontaneous decomposition of the 6-nitro-3-
carboxybenzisoxazole (Figure 1). The effect of changing the solvent on the
observed rate constant Kobs, can obviously recognized due to solvation
difference between the reaction state, here the negative charge is concerted
on the carboxylate anion and the transition state and the charge dispersed
over many atoms. The half-lives of this reaction in hexamethyl
phosphotriamide, acetonitrile and water are 0.001 second, 11.6 minutes,
and a day, respectively (Grate 1993).
O
N
O2N
OO
ON
O2N
OO
O2N O
CN
CO2
Chapter # 05 Introduction
Page 81
Figure 1 Schematic representation of the spontaneous decomposition of 6-
nitro-3-carboxybenzisoxasole
To understand these large differences in kobs with solvent properties a
correlation is shown in Figure 2 and Table 6 in which the subscript (S) refers
to solvent, r, ET(30), SA, SB refer to the solvent relative permittivity, its
empirical polarity, hydrogen-bond donation capacity or “acidity”, and
hydrogen-bond acceptance capacity or “basicity”, respectively.
Figure 2. Attempt at correlation of the rate constant of the reaction shown in
Figure 1 with a function of dielectric constant of the medium
Chapter # 05 Introduction
Page 82
Table 6. Correlations between log in different solvents
Solvent property Coefficients of the correlations between
log kobs and solvent propertyc
r = 2(r -1) / (2r +1) 0.0778
ET(30) 0.1572
ET(30) + r 0.7864
ET(30) + SA 0.7791
ET(30) + SB 0.5167
ET(30) + SA +SB 0.8928
ET(30) + r + SA 0.8916
ET(30) + r + SA + SB 0.9485
aValues of kobs were taken from reference 1; bThe solvent properties include
relative permittivity, r; empirical polarity, ET(WB); acidity, SA; and basicity,
SB; cThe correlation coefficients are (r) and (r2) for linear, and multiple
regression analysis, respectively
From Figure 2 and Table 6, it is clear that there is no relationship between
log kobs and any single solvent property. It is concluded form the data given
in Figure 2 and Table 1 that the effect of solvent on chemical reactivity and
most probably other phenomena such as chemical equilibrium and
spectroscopic values are very complex to understand and most commonly, it
is very accidental to obtain good correlation by using single descriptor.
It is clear that the solvents play a major role in chemical reaction therefore
the choice of good solvent is very important. The availability of large number
of solvents and the complexity of solute-solvent interaction make the choice
Chapter # 05 Introduction
Page 83
very difficult. It is then physico-chemical properties, such as melting and
boiling points, heat of vaporization, density, index of refraction, vapour
pressure, dipole moment, dielectric constant, specific conductivity,
polarization, viscosity, surface tension, etc, dictate the choice.
Characterization and classification of the solvent is commonly based on their
physico-chemical properties. Taking few of these properties into account,
mostly results poor classification of solvents. To consider many of these
properties with chemometric tools make it possible. To classify and select the
organic solvents multivariate statistical methods have been applied in recent
years ( Reichardt 2003).
The experimental evidence proves that in actual situation the solvent can be
characterized as a pure solvent in a simple and precise way and this made
possible by using the term the pure solvent dipolarity-polarizability (SDP),
solvent basicity (SB), and solvent acidity (SA) scales, which were established
from suitable probe/homomorph couples.
The position and intensity of absorption bands in UV/Vis/near-IR, IR, ESR,
and NMR spectroscopy as well as rates and equilibrium positions of chemical
reactions are solvent-dependent. The careful selection of an appropriate
solvent for a reaction or absorption under study is part of its craftsmen’s
skill and now-a-days, this is generally known to every chemist.
The dependence of multi parameters of chemical reaction on solvent is
because of many solute-solvent interactions and their effects on chemical
Chapter # 05 Introduction
Page 84
reaction. Both specific and non specific interactions come into account in
this case i.e london or dispersion interactions, hydrogen bonding and dipolar
interactions (ion-dipole, dipole-dipole, dipole-induced dipole). To calculate
the dependence of chemical reaction on the properties of solvent, the most
remarkably the (simplify) Taft-Kamlet-Abboued equation (Seoud 2009).
Effect of the medium = Constant + a SA + b SB + d/p SDP (4)
The effect of medium, in which there is a linear combination of two hydrogen
bond donating terms, that acts like hydrogen bond accepter (b SB), or
hydrogen bond donor (a SA), and dipolarity/polarizability (d/p SDP), because
of the determination by using solvatochromic probes (via IR) the parameters
SB, SA, SDP are known as solvatochromic parameters. Those substances
whose absorption or emission spectra are mainly sensitive to these specific
solvent properties (acidicity, basicity etc). Empirical polarity scale that gives
the information about the solvation of the probe in a series of solvent is
represented as ET(probe) and can be calculated as (Seoud 2009)
ET (probe), kcal/mol = 28591.5 / max (nm) 5)
This equation is used to convert electronic transition into the relative intra-
molecular charge transfer energy observed within the probe.
5.2 Solvatochromism
Solvatochromism is the ability of a chemical substance to change color due
to a change in solvent polarity (Marini 2010). Negative solvate`ochromism
Chapter # 05 Introduction
Page 85
(blue shift) will result, if the ground state molecule is better stabilized by
solvation than the molecule in the excited state, with increasing the solvent
polarity. The increase in solvent polarity, better stabilization of the molecule
in the first excited state relative to the ground state, will lead to positive
solvatochromism (red shift) (Reichardt 2003). The Figure 3 shows the
difference between two types of solvatochromic behaviors.
Positive Solvatochromisms Negative Solvatochromism
Increase in Solvent polarity Increase in Solvent polarity
Excited State
Ground State
Figure 3. Systematic representation of Positive and negative solvatochromism
The sign of the solvatochromism depends on the difference in dipole moment
between the ground and the excited states of the chromospheres.
Solvatochromism is results due to the difference in solvation of the light
absorbing molecules, of the ground and the first excited state
(Hadjmohammadi 2008). On the basis of the energy difference between two
states of the probe polarity scale has been developed as
ETmax = NA hc / λmax =28951.5/ λmax (kcal.mol-1) (6)
Chapter # 05 Introduction
Page 86
Where h = Plank's contant, C = Speed of light, λmax is the maximal
energy. The wave number related to λmax of most of the polar solvent was
subtracted from that of the most non polar solvent (and considered as ∆Ѵ), to
show positive or negative solvatochromism for each probe. The probe has red
or blue shift is indicated by positive and negative sign of ∆Ѵ, respectively.
Solvents can bring about a change in the position, intensity and shape of
absorption bands and it has long been known that UV/Vis/near-IR
absorption spectra of chemical compounds may be influenced by the
surrounding medium (Kundt 1878; Scheibe 1927; Reu 1942).
Hantzschlater was the pioneer of the term solvatochromism. However, now
the meaning of solvatochromism that introduced by Hantzsch is differ
generally from the accepted term of solvatochromism. In order to understand
that scopic probe molecules cannot only measure the polarity of liquid
environments but also that of solids, glasses, and surfaces. The value of ET
is the measure of difference interaction energies of solvent between ground
and excited state. So the greater the value of the ET, the larger will be the
polarity solvation shell of the probe. Therefore, some points should keep in
mind while using these probes
i) Every probe imparts a specific color to the solution during
solvatochromism. e.g. RB gave solution of red, purple, green and blue
in methanol, ethanol, acetone and anisole with 4% methanol.
Chapter # 05 Introduction
Page 87
Figure 4. Staining probe MePMBr2 in solvents, from left to right, Water ethanol,
acetone and dichloromethane, respectively.
ii) The difference of dipole moment between the ground and excited state
of probe is considerable as it shown in the figure below (D = 15mg; µe
= 6D). This is due to the intra-molecular charge transfer (Kososwer
1968).
Here some solvents with their dielectric constant (D) have been presented in
Table 7.
Chapter # 05 Introduction
Page 88
Table 7. Some commonly used solvents with their Dielectric constant values
(D)
Sr. No Solvent Dielectric Constant
1 Hexane 1.879
2 CCl4 2.209
3 p-Xylene 2.269
4 Benzene 2.275
5 Tolune 2.379
6 Diethylether 4.335
7 Chloroform 4.806
8 Ethylacetate 6.02
9 Acetic acid 6.15
10 THF 7.58
11 DCM 8.93
12 n-BuOH 17.51
13 i-PrOH 19.92
14 n-PrOH 20.33
15 Acetone 20.70
16 Ethanol 24.55
17 Methanol 32.70
18 DMF 36.71
19 Acetonitrile 37.50
20 DMSO 46.68
21 Water 78.39
iii) Polarity scale change with the change of probe because of the change
in PK, in hydrophobic/hydrophilic character and change in structure
Chapter # 05 Introduction
Page 89
(Reichardt 2003). In table some solvatochromic probes with their λmax
value in the polar and non-polar solvent.
Table 8. Selection of some solvatochromic compounds representative, their
respective values of λmax in polar and non-polar solvent and Dlmax
accordingly. (Reichard, 2010)
S. No. Probe λmax
(non-polar or
low polar
solvent)
λmax
(polar
solvent)
∆λmax
1 H3CN O
CH3
331,1 382.6 51.1
2 NO2(C2H5)2N
365 430.5 65.5
3
O
N
O(C2H5)2+N
3
521.1 620 98.9
4
(H3C)2+N
NO2
414.9 425.5 10.6
5 H3C O
CH3CH3
230.6 242.6 12
6
N+ O-
NO2C2H5
526.0 452.9 73.1
7
N+H3C
O-
620 442 178
8
N+
Cl
O-
Cl
690.6 407 283.3
Chapter # 05 Introduction
Page 90
9
N+
O- CH3
574.1 443.3 130.8
10
N+
SO3-Na+
O- CH3
539.5 440.5 89
iv) Solvatochromism is also effected by temperature which known as
thermosolvatochromism. Temperature has influence on the intramolecular
interaction between probe and solvent, and solvent-solvent interactions so
effect the value of ET. Hence with these types of the studies it is possible to
study the effect of temperature on pure solvent and on solvation and
calculation of the energy involved in this whole process.
5.2.1 Solvatochromic Probes
Empirical parameters of solvent polarity have been preferably determined by
means of solvatochromic compounds, because of their simplicity of
UV/Vis/near-IR spectroscopic measurements. It is assumed that a
particular solvent-influenced Whishear-IR absorption, a suitable
representative model for a large class of other solvent-dependent processes.
The absorption range of suitable solvatochromic reference compounds does
not only include the UV and visible region but also the near-IR region
(Reichardt 1994).
Chapter # 05 Introduction
Page 91
The study of the solvatochromism of the fluorescence and derivatives of
different hydrogen bond accepter (HBA), hydrogen bond donor (HBD) is
calculated by using acceptor number (AN) and donor number (DN) of their
UV-Vis spectra. Results showed that change of solvent changed the position,
intensity and shape of absorption bands. These changes can be rationalized
by solvatochromic parameters such as α, β, ET (WB), AN and DN using
multiple linear regression (MLR) technique. The Correlation coefficients of
obtained equations were 0.965-0.999.
Table 9. Molecular structures of zwitterionic solvatochromic indicators,
(Scheibe 1927; S. E. Reu 1942)
Sr. No. Name Pka Log P Polarity scale Structure
1
MePM
8.37
-1.94
ET(MePM)
N+
CH3
O-
2
MePMBr2
5.15
-0.16
ET(MePMBr2)
N+
CH3
O-Br Br
Chapter # 05 Introduction
Page 92
3
BUPMBr2
5.15
1.12
ET(BUPMBr2)
N+
C4H9
O-Br Br
4
HxPMBR2
5.15
1.86
ET(HxPMBR2)
N+
C6H13
O-Br Br
5
OcPMBR2
5.15
2.70
ET(OcPMBR2)
N+
C8H17
O-Br Br
6
BuQMBr2
4.89
2.51
ET(BuQMBr2)
N+
C4H9
O-Br Br
7
RB
8.32
Large
ET(RB)
O-
N+
Chapter # 05 Introduction
Page 93
Recently, for the measurement of solvatochromic parameters, Catalan has
introduced a short number of solvatochromic probes. He introduced an
equation in order to split SDP into its components. The solvation equation is
Effect of the medium = Constant + a SA + b SB + d SD + p SP (7)
By using the absorption frequency () of the probe or the molecules which is
similar in size and shape known as homomorphs and every property in the
equation is calculated. The names and molecular structures of these
solvatochromic probes are: SA 3,6-Diethyl-1,2,4,5-tetrazine or a pair of o-
tert-butylstilbazolium betaine, o,o-di-tert-butylstilbazolium betaine, SB by
the pair 5-nitroindoline/1-methyl-5-nitroindoline, SDP by the pair 2-
(dimethylamino)-7-nitrofluorene)/2-fluoro-7-nitrofluorene and SP by ttbP9
8
WB
4.78
1.79
ET(WB)
Cl Cl
O-
N+
9
QB
6.80
-063
ET(QB)
N+
O- CH3
10
PB
5.2
-
ET(PB)
N+
CH3
O-
11
QBS
5.7
-1.94
ET(QBS)
N+
O- CH3
SO3-Na+
Chapter # 05 Introduction
Page 94
(3,20-di-ter-butyl-2,2,21,21-tetramethyl-5,7,9,11,13,15,17,19-
docosanonaene) (Catalán 2009)
N
NN
N
H3C
CH3
O2NF
FNF
O2NN(CH3)2
DMANF
NH
O2N
N
O2N
CH3
NI
MNI
NH3C
O
C(CH3)3
TBSB
NH3C
O
C(CH3)3
C(CH3)3DTBSB
DETZ
ttbP9
CH3H3C
CH3
CH3 CH3
CH3 CH3
H3C
H3C CH3
CH3
CH3CH3
CH3H3CCH3
H3C CH3
CH3
H3C
CH3
H3C
b-Carotene
Figure 5. Solvatochromic probes for measuring solvent
dipolarity/polarizability, SDP, 2-(dimethylamino)-7-nitrofluorene/2-fluoro-7-
nitrofluorene; solvent basicity, SB, 5-nitroindoline/1-methyl-5-nitroindoline;
solvent acidity, SA, 3,6-Diethyl-1,2,4,5-tetrazine or o-tert-butylstilbazolium
betaine, o,o-di-tert-butylstilbazolium betaine and solvent polarizability, SP,
Chapter # 05 Introduction
Page 95
(3,20-di-tert-butyl-2,2,21,21-tetramethyl-5,7,9,11,13,15,17,19docosanonaene),
ttbP9. The last substance is the natural product -carotene.
Five or six membered ring fluorophores which have intramolecular hydrogen
bonding, were used for the determination of their solvatochromism property.
The fluorescence of the frequency shift of the molecules relates directly to the
polarity SP and polarizability scale SPP, acidity SA and basicity SB also. Due
to the intramolecular hydrogen bonding in the fluorophores, a good relation
is found between the ground state and first excited state of the dipole
moments. Fluorescence shifts towards the red, blue or no shift, depends on
the solvatochromism shift (Javier Catalán 1999).
5.3 Preferential Solvation
In chemical and biochemical practice solvent mixtures are used on a large
scale to enhance the molecular environment to modulate interesting
phenomena such as organic synthesis, chromatographic separation, reaction
kinetics, protein folding unfolding or color of chromophores. To make a
change in the physical properties such as density, viscosity or vapor
pressure solvents are mostly used in the form of mixtures. When the
substrate is present in a large amount in a pure solvent, the description of
the solvation is considered to be more difficult of a neutral or ionic solute in
a solvation mixture (Reichardt 1988). The result of solvation in a mixture of
solvents is believed to be not only the key solute-solvent interactions but
also due to the interaction of others different species present in the mixture.
Chapter # 05 Introduction
Page 96
As Raoult’s law expressed mathematically this leads, among others, to
significant deviations from the ideal behavior in the vapor pressure of a
mixture. The explanation for the above-mentioned deviations may be that
the solvent ratio around the solute and in the bulk solution may be
significantly different. By becoming more negative the effect of solute being
preferentially surrounded by one of the solvents would be the result of the
Gibbs energy of solvation (H. Schneider 1969; Reichardt 1988). This would
ultimately reflect a difference between the macroscopic ratio and the
composition of the solvent shell around the solute. This phenomenon is
called “preferential solvation”. When probe is dissolved in a solvent mixture
for solvatochromism it acts like a solute, and in the process of solvation
three types of solvation may occur
1. The probe is dissolved equally by the both solvents. This is called ideal
solvation.
2. If the probe is solvated by the hydrated or anhydrate solvent the
solvation is called is preferentially solvated by water or aprotic
solvation.
3. If the composition of the solvation shell is surrounded by organic
solvents of the binary mixture, it is called preferential solvation by
organic solvent.
The model representation of this phenomenon is shown in Figure 6.
Chapter # 05 Introduction
Page 97
Figure 6. Solvation possibilities of the solute in Binary mixtures (Silva 2009)
This term mostly used to describe the situation in the bulk solvent the solute
create a change in its environment (known as dielectric enrichment) or via
specific solute-solvent interaction (e.g. complex formation). To find out the
composition of reaction mixture is not easy because this solvation is due to
preferential solvation of binary mixture. There are various ways to
determined the solvation process including the measurement of conductance
or transfer process ( Schneider 1969), NMR Spectroscopy (NOESY) (Bagno
1997; Bagno 2002), solvatochromic measurement by using IR (Popov and
Ritchie 1976) or UV-Vis region (Dawber 1988). Preferential solvation is used
to study specific and non-specific solute-solvent interaction in binary
mixtures. In BMs, which have hydrogen bonding non-specific interaction is
observed and vice versa (Ghoneim 2001). BMs also consists of micro-
heterogeneous mixtures, consists of different constituents formed by these
BMs and found both in protic and aprotic type. These clusters of mixtures
are detected by using mass spectroscopy (Wakisaka 1995; Wakisaka 2001),
Chapter # 05 Introduction
Page 98
fluorescence (Zana 1993) and by the calculations of Kirkwood-Buff integrals
(Marcus 2001). From the above discussion the complexity of the BMs clear
and we concentrate on the solvation process of aprotic binary mixtures
consists of ionic Liquids (IL) and DMSO.
To describe the preferential solvation different models have been developed,
the first one developed by Bosh & Roses (Bosch 1992), according to which
the binary mixtures consist two components one is organic solvent and other
is DMSO/Water. This theory didn't tell about the third component of the
binary mixture formed by aggregation of two solvents. So according to this
theory the solvation in binary mixtures is ideal solvation. In this case the
overall polarity of the solution is because of contribution of each component
of the mixture.
(8)
In case of binary mixtures factors f1 and f2 are introduced in equation 9 that
relate to the solvation shell of the (
) probe in the
solution ).
f1=
(9)
f2=
(10)
Polarity of the binary mixture is described in the equation (8). To represent
the preferential solvation, solvation preference factors (f1/f2) are used.
Chapter # 05 Introduction
Page 99
(11)
This model is very simple and does not describe the behavior of binary
mixture in the satisfactory way. This model lead towards the inconsistent
results, for example the solubility of RB probe in the binary mixture of water
and 2-methyl-2-propanol in the mole fraction range of 0 to 0.6 is 2 × 10-6
mol/L (Novaki 1997). This model was modified later (Bosch 1995; Bosch
1996; Ortega 1996; Bosch 1997; Rafols 1997; Buhvestov 1998; Herodes
1999). According to that modified form, the binary mixture is consist of three
species i.e. organic solvent, DMSO/Water and the other solvent formed by
mixture of Organic and inorganic solvent DMSO-solvent. Equation 12-15
describe this phenomenon
Solv + DMSO Solv-DMSO (12)
Probe (Solv)m + m(DMSO) Probe (DMSO)m + m(Solv) (13)
probe(DMSO)m + m(Solv -DMSO) probe(Solv -DMSO)m + m(DMSO) (14)
probe(R Solv)m + m(Solv -DMSO) probe(Solv - DMSO)m + m(Solv) (15)
Here (m) is the number of exchange of the molecules in the solvation shell of
the probe. The equilibrium constants of equation from 12-15 explain about
the relationship composition of the bulk solvent and the shell of the probe
solvation. These called "fractionation factor" and their values are
(16)
Chapter # 05 Introduction
Page 100
(17)
=
(18)
DMSO substituiting the organic solvent
Mixture of solvents replacing organic solnvent.
Complex substituting the DMSO
In the above equations Bk stands for bulk mixture and for molar fraction.
From the value of the solvation of the probe can be understand. In the
equation 16 DMSO is replacing organic solvent and if the value of
dmso/solv 1 the solvation shell have DMSO in excess but if the value
dmso/solv <1 the solvation of the probe is done by organic solvent. The same
idea applies to the value soln-DMSO/solv (complex solvent substituting
solv) and solv-DMSO/DMSO (complex solvent substituting DMSO) in
equation 17 and 18. According to this idea if the value of solv-DMSO/solv
and solv-DMSO/DMSO is greater than 1 the probe is solvated by the
complex of mixture, but in case of ideal solvation the value of should be
unity and the value of m to be near of unity. In the case of ideal solvation the
composition of the solvation shell of the probe and the BM is same. In ideal
solvation the composition of bulk BM and probe solvation is same.
Equations 12-15 describe about the presence of polarities of the species
present in the solution,
,
, and,
, which is multiplied
Chapter # 05 Introduction
Page 101
by the mole fraction of the related probe in the Solvation shell
,
and
, respectively. This is based on the effective mole
fraction rather than on analytical concentration of (Solv) and (DMSO) in the
bulk mixture:
=
(19)
Becasue
In case of ideal solvation their composition is described above in equation
16-18. We have another eqaution to describe this phenomenone.
=
(
)
(
)
(
)
(
)
(
)
(
)
(20)
The model developed by the Bosch et al has a shortcoming that it only deals
with solutions that have preferential Solvation. In case of the solutions that
do not have preferential solvation the value of is 1 according to that the
composition is same of both solvation shell of probe and bulk of mixture
solution. The equation 18 is modified to equation 20 for the ideal solvation
process.
(
) (
) (
) (21)
Chapter # 05 Introduction
Page 102
With the help of solvatochromic data it is possible to calculate the values of
and . To calculate these
parameters it is required to calculate the effective concentration of these
species in the media when equlibrium is reached between DMSO and
organic solvent. So it was necessary to calclate the constant of assocoiation
(Kassoc) between solvent-dmso after the formation of complex. To determine
the Kassoc the model used is devloped by the Katz et al (Katz 1986; Katz
1989), according to that the calculation of Kassoc is based on the densities of
BMs when it is in the form of solv:DMSO 1:1 complex and it constant of
disociation (kdissoc) is explained in equation (22). In eqaution (22)
[Solv],[DMSO] and [solv-DMSO] are the effective molar concentrations in the
BMs. The value of the Kassoc and of kdissoc are inverse to each other. The 1:1
compostion of the BMs is supported by the 1H-NMR and FTIR spectrs from
the literature FT (Chen and Shiao 1994; Eblinger and Schneider, 1996; Max
et al. 2002).
kdissoc= [ ][ ]
[ ] (22)
In equation 23 the density of the BMs Solv-DMSO is described
d=[ ] [ ] [ ]
[ ] [ ] [ ] (23)
Here M and V represent the molecular weight and molar volume of the
crossponding species. The equation (24-26) give the values of [DMSO][Solv],
Chapter # 05 Introduction
Page 103
[Solv-DMSO]. Here in these equation fv tell about the volume fraction of
organic components.
[DSMO]=
(24)
[Solv]=
[ ] (25)
[Solv-DMSO]=
[ ] (26)
To find out the value of (b) and (c) in the equation (24) equation (27-28) are
used.
b= Kdissoc+
(28)
c= Kdissoc(
) (29)
The advantage of this model is that to determine the kdissoc it does not
required any third solvent and it has been used in the past (Tada 2003; Tada
2003a.; Tada 2005). However, there are some uncertanities in this model as
from the composition of BM and correlation of BM eith thier densities the
calculations of kassoc and molar volume V solv-DMSO done which both are
dependent parameters.
5.4 Introduction of Green Chemistry
Green Chemistry deals with study to designing and invention of the methods
and their applications which help to reduce or eliminate the use of
hazardous substances. The term "Green Chemistry" came with the need of
industrial progress to meet the expectations of the present without
Chapter # 05 Introduction
Page 104
compromising the ability of future generations to meet their own needs. On
the other hand, the chemical activity is often related directly or indirectly,
the majority of so-called "environmental disaster", although other human
activities also exert an important role in the degradation and environmental
pollution (Anastas 1998). In the early 90s, a new trend in how the issue of
chemical waste should be treated began to take shape. We must seek an
alternative that avoids or minimizes the generation of waste, rather than
exclusive focus on the treatment of waste at the end of the production line.
Basically, there are twelve topics that must be followed when trying to
implement green chemistry in industry or educational institution
5.4.1 Characteristics of green chemistry
5.4.1.1 Prevention
Avoid the generation of waste is best to treat it or clean it after its
generation.
5.4.1.2 Atom Economy
One must try to design synthetic methodologies that should increase the
conversion of all reactants to the final products.
5.4.1.3 Synthesis of Products Less Dangerous
Try to synthesize those substances and chemicals that are less dangerous to
the living things and environment.
5.4.1.4 Designing of Safer Chemicals
Chapter # 05 Introduction
Page 105
Chemicals should be designed so that they perform the desired function and
simultaneously, are not toxic.
5.4.1.5 Design for Energy Efficiency
Energy use by chemical processes need to be recognized for their
environmental and economic impacts, and should be minimized. If possible,
the chemical processes should be conducted at low temperature and
pressure.
5.4.1.6 Use of Renewable Feedstock
Whenever technically and economically feasible, the use of renewable raw
materials should be chosen over non-renewable resources.
5.4.1.7 Avoid the Formation of Derivatives
Unnecessary derivatization (use of blocking groups, protection/deprotection,
temporary modification of physical or chemical processes) should be
minimized or, if possible, avoided, because these steps require additional
reagents and can generate waste.
5.4.1.8 Catalysis
Catalytic reagents (as selective as possible) are preferable than
stoichiometric reagents.
5.4.1.9 Design for Degradation
Chapter # 05 Introduction
Page 106
Designing of the chemical products should be in this way that final product
should be divided into smaller particles that do not remain in the
environment.
5.4.1.10 Real Time Analysis for Pollution Prevention
To develop the process by using that it is possible to analyze the process and
to have control on the formation of dangerous substances.
5.4.1.11 Safer Chemistry for Prevention of Accidents
To make use of those kind of chemicals in chemistry which decrease the
ratio of chemical accidents includes fires, explosions and gas releases.
5.4.1.12 Safer Solvents and Auxiliaries
Auxiliary substances e.g. solvents and separation substances should not be
used commonly, if necessary use of appropriate solvent should be made,
those are environment friendly.
5.4.2 Ionic Liquids
To date, solvents are mostly used to carry out different type of chemical
reactions. A new class of solvents have been introduced recently; Ionic
liquids (ILs) (Wasserscheid 2003). These are basically ionic species and
liquids at room temperature. They have been vastly used in different
processes including electrochemical devices and electrolytes, in the different
process of organic and catalytic chemistry, in the synthesis process of new
Chapter # 05 Introduction
Page 107
compounds and in separation and extraction chemistry (Huddleston 1998).
Ionic liquids (ILs) may organic or inorganic compounds which are made from
cations and anions and have boiling point less than water. They are also
named as "green solvents" because of several properties they have like low
volatility, low boiling and melting point, chemically and thermally stable,
posses high thermal conductivity and large ectrochemical potential (Zhao
2006). As they consists of ions so in the form of solutions it contains only
ions which make it differ from the other ionic solutions which contain salts
and may be molecular solvents as shown in Figure 8 (Welton 2011). This
property used for the solubilization of cellulose in ILs. ILs are able to dissolve
carbohydrates and number of polar and non polar compounds which leads
towards the synthesis of new substances.
Figure 7. Difference between ionic solutions and ionic liquids
In Figure 8, cations and anions can combine together to form ILs. They can
combine together in unlimited ways and create the ILs according to use.
Chapter # 05 Introduction
Page 108
N NR1
R3
R2
N
R2
R1
N+
R1
CH3R2
CH3
P+
C6H5
R1 C6H5
C6H5
+ +
R1= Alkyl, alkenyl
R2= H, CH3
R3= CH3
Halides
SCN-
BF4
PF6
CF3COO-
C6H5COO
(CF3SO2)2N-
CH3SO3-
(CN)2N-
Figure 8. Various cations and anions that combine to form the ILs
N N R'RN
R
N+
R R'
R4P+
Imidazolium Pyridinium Pyrrolidinium Phophonium
+ +
Most commonly used anions
BF4-, PF6
-, CF3SO3
-, (CF3SO2)NO
-, Cl
-, Br
-, CH3C6H4SO3
-
Figure 9. Typical cationic and anionic components of ionic liquids
The present work deals with the solubilization of cellulose checked by using
imidazole based ILs having imidazolium cations, which need the detail
description of the reactivity of these ILs as shown in Figure 10.
Chapter # 05 Introduction
Page 109
5.4.2.1 Reaction with Electrophilic Reagents
On reactions with haloalkanes, imidazole goes nucleophilic substitution and
gave a salt of 1,3-dialkylimidazolium on reaction with second mole of
haloalkanes.
N-
NH
R1 X
N+
NH
R1
X-
N
N
R1
N
N
R1
R2
X--HX
++R
2-X
Figure 10. Schematic of the formation of ILs
5.4.2..2 Solvation of ILs
Solavtion process in imidazole based ILs depends on the nature of the anion,
the concentration of salt and the position of the H2, as it reactive as
compared to H4 and H5. That's why the nature of solvent has a great
influence on the chemical shift of the hydrogens.
5.5 Mechanism of Dissolution of Cellulose
Cellulose is the most widespread organic chemical on the earth and has
great importance as a recycle material. But only 5% have been used for
further processing from up to 40 tons produced naturally due to the lack of
appropriate solvent. ILs are considered best solvent to solubilize cellulose
(Richard P. Swatloski 2002). The dissolution of cellulose in ionic liquid was
first checked by Graenacher in 1934 in N-ethylpyridinium chloride, in which
Chapter # 05 Introduction
Page 110
the dissolution of cellulose occur in the presence of nitrogen containing
bases. After a long time in 2002 again solubilization of cellulose in ILs was
checked. Swatloski and coworkers used alquilimdizole ionic liquids and from
their studies they conclude that best ionic liquid for the solubilization of
cellulose is [BuMeIm][Cl] (Swatloski 2002). The reason for this solubility is
due to the hydrogen bonding between the chloride ion of ionic liquid and
hydrogen of hydroxyl group of the cellulose (Remsing 2006). In 2005, Zhang
stated that [AlMeIm][Cl] can dissolve cellulose without activation or
pretreatment (Zhang 2005). Later on it was observed that ILs with acetate
anion show more solubility due to lower melting point and lower viscosity
like [EtMeIm][CH3COOH-] (Cao 2009).
Mechanism of dissolution of cellulose in ionic liquid involves the formation of
electron-electron donor recipient complex. In this complex OH group of
cellulose act as electron donor and hydrogen atom as electron recipient.
Cations of the ILs act as electron acceptor and anion as electron donor.
These two centers (acceptor/donor) should be close enough to each other so
that the formation of complex take place easily. Polymer chain separated
after the formation of complex, resulting in the breaking of molecular chain
by breaking hydrogen bonding between it which leads towards the
dissolution of cellulose (Cao 2009).
Chapter # 05 Introduction
Page 111
Figure 11. Mechanism of dissolution of cellulose in RTILs
Solubilization of the cellulose depends on the type of the cation and anion
used. A number of different ionic liquids have been developed by changing
the alkyl chain and anions (Xu 2010).
Chapter # 06 Results & Discussion
Page 112
CHAPTER # 06
RESULTS & DISCUSSION
GREEN CHEMISTRY
Chapter # 06 Results & Discussion
Page 113
The objectives of the present studies is to understand the solvatochromic
properties of binary mixtures of imidazole-based ionic liquids that are
employed in cellulose dissolution with molecular solvents, e.g., DMSO and to
determine the correlation of these properties with the solubilization of
cellulose in these media and to measure the preferential solvation in binary
mixtures by using density data. By using solvatochromic probes allot of work
have been done in past on this field to get information about the medium
(pure solvent or binary mixtures). The studies was done by using only
limited number of probes which do not address the properties of binary
mixtures (acidity and basicity polarizability/dipolorazibility) and theoretical
studies to make comparison with experimental data.
So the main objective of the present studies is to emphasize on the detail
and dependent studies on the existing points instead of repeated work.
Thus result and discussion are organized as
1. Check the preferential solvation of mixture of ILs and molecular
solvent by spectral response of a solvatochromic dye, 2,6-dichloro-4-
(2,4,6-triphenylpyridinium-1yl)phenolate (WB), in mixtures of the IL 1-
(1-butyl)-3-methylimidazolium acetate with dimethyl sulfoxide, and
water, over the entire mole fraction () range, at 15, 25, 40, and 60 °C.
2. We treated the solvatochromic data by a model that includes the
formation of the “mixed” solvents IL-DMSO, and IL-W; the
Chapter # 06 Results & Discussion
Page 114
concentrations of these third components were calculated from density
data.
3. Our solvatochromic results are relevant to cellulose dissolution in IL-
DMSO because the same interaction mechanisms (solvophobic;
hydrogen bonding) are determinant to dye solvation and biolpolymer
dissolution.
4. We try to describe the overall polarity of the pure solvent and binary
mixtures of the of ILs/DMSO and discuss the difference between
behavior of two Ionic liquids i.e. 1-methyl-4-butyl-imidazolium acetate
and 1-methoxyethyl-3-methylimidazolium acetate
5. After that thesolvatochromic properties (acidity and basicity
polarizability/dipolorazibility) of the binary mixtures and pure solvents
are discussed.
6.1 Selection of Appropriate Ionic Liquid and Polarity Probe
The first step before start work is the selection of ionic liquids to make binary
mixtures. Imidazole based ionic liquids are getting importance among the
chemists with the passage of time due to their abilities to work as water
purification agent, electromechanical actuator membranes and diluents,
biphasic reaction catalysis, separation science membranes. An important
property of imidazole based ionic liquid is to tune the ability of the ionic
liquid which formed a combination anion and cation (imidazole) and change
in physical properties such as boiling point, melting point and viscosity
which also change with by changing the counter anion and substituents on
Chapter # 06 Results & Discussion
Page 115
imidazole ring. Finally the ability of the imidazolium ionic liquids to
coordinate with transition metals, and the ability of polymer synthesis due to
catalyze atom transfer radical polymerization and their ability of rapid
solubilization (Green 2009) as compared to other ionic liquids make these
ionic liquids more important to use (Headley 2006). Here in present study we
use two ionic liquids one without oxygen: 1-methyl-4-butyl-imidazolium
acetate (C4MeImAc) and the other with oxygen: (1-methoxyethyl-3-
methylimidazolium acetate (C3OMeImAc) to observe the effect oxygen of IL on
the solvatochromic probes, role in salvation in binary mixtures and effect in
dissolution of cellulose. The second step was the selection of appropriate
polarity probe. In the present study a less basic version of betaine dye, the
dichloro-substituted betaine dye 2,6-dichloro-4-(2,4,6-triphenyl-pyridinium-
1-yl)-phenolate ET(33) of most commonly used solvatochromic dye 2,6-
diphenyl-4-(2,4,6-triphenyl-pyridinium-1-yl)-phenolate ET(WB) is used in
which both of the phenyl group are replace with chloro group. However,
interestingly, the sensitivity shown by both indicators towards solvent
"acidity" is the same as calculated by Taft-Kamlet-Abboud equation (Kamlet
1981).
Chapter # 06 Results & Discussion
Page 116
Ph Ph
Ph
O-
Cl Cl
N+ N+Ph Ph
Ph
O-
Ph Ph
ET33 ET30
pKa = 4.78 8.32
- ∗ leads toward the
ET(33) scale (Reichardt 2003). The interaction between the molecule of the
probe and solvent leads towards the molar transition energy which can be
measured by applying following formula
ET(33) =
(36)
6.2 Determination of the Kassoc between ILs and Water
With the help of density data calculations of Kassoc was done according to
the method described in detail Scott (Scott 2000). In the present studies
we plot a graph between the molar fraction of DMSO and density of
binary mixtures ( to see the affect of different concentrations variation of
binary mixtures with 16 different concentrations mixtures and two
samples for pure ionic liquids and DMSO. All these density measurement
were made on four different temperatures i.e. 15, 25, 40 and 60 °C. This
behavior is illustrated in the figure 10.
Chapter # 06 Results & Discussion
Page 117
Figure 12. Variation of density of BMs of IL (C4MeImAc) & DMSO of system in
relation of the molar fraction of DMSO, showing the synergetistic effect. The
measurements were taken at 15, 25, 40 and 60 oC.
Chapter # 06 Results & Discussion
Page 118
Figure 13. Variation of density of BMs of IL (C3OMeImAc) & DMSO of system in
relation of the molar fraction of DMSO, showing the synergetistic effect. The
measurements were taken at 15, 25, 40 and 60 oC. .
The change in the behavior towards the density measurement for both ILs
one without oxygen (C4MeImAc) Figure 12 and second with oxygen
(C3OMeImAc) is obvious in Figure 13. Density of BMs of IL with oxygen
(C3OMeImAc) is more as compared to BMs of IL without oxygen (C4MeImAc)
and decreases with increase of temperature in both cases. In case of
(C4MeImAc) density increases as we move towards the more concentration of
DMSO and almost straight line graph obtained with a slight below the line
curve in the initial values and final values which shows that the density of
pure IL is greater than the BM and for pure DMSO is less than BMs. Where
as in case of (C3OMeImAc), the density of both pure IL & DMSO is less than
Chapter # 06 Results & Discussion
Page 119
the density of BM and a cured graph is obtained which show first increase in
the density value and then decrease with the increase concentration of
DMSO.
6.3 Calculation of Kdissoc from density data
By using the results obtained dissociation constant Kdissoc of BMs measured
by applying the equations (37) and (38)
[ ] [ ]
[ ] (37)
=[ ] [ ] [ ]
[ ] [ ] [ ] (38)
Here Bk; Effective is the effective concentration in bulk at equilibrium. M
and V are the molar mass and molar volume of the respective species. Curve
fitting of versus molar fraction of DMSO for the calculation of Kdissoc is
carried out by fixing some variables, and the interaction continued until the
value of chi2 become constant. The data of Kdissoc of binary mixtures with
both IL is listed in Table 10. The linear correlation obtained between the
lnKdissoc and 1/T as shown in Figure 14. From the data of the density
measurements of ILs & DMSO the calculation of Kassoc ( Kassoc =1/ Kdissoc) was
done at four different temperatures and listed in Table 11 by using the Van't
Hoff's equation. It is obvious from the results that the correlation between
lnKdissoc show linear relationship versus 1/T. shown in Figure 14.
Chapter # 06 Results & Discussion
Page 120
Table 10. The value of Kdissoc calculated by using density data
Sr. No Binary mixture Temperature °C Kdissoc, L mol-1
1
C4MeImAc/DMSO
15 0.016
25 0.027
40 0.042
60 0.06
2
C3OMeImAc/DMSO
15 0.02
25 0.03
40 0.042
60 0.064
Below are straight line graph between lnKdissoc and temperature show a
linear relationship between lnKdissoc and temperature of both BMs.
Figure 14. Plot of InKdissoc versus 1/T of BM C4MeImAc/DMSO
Chapter # 06 Results & Discussion
Page 121
Figure 15. Plot of InKdissoc versus 1/T of BM C3OMeImAc/DMSO
Table 11. Values of Kassoc calculated by using the Van't Hoff's equation
Sr.No Binary mixture Temperature °C Kassoc, L mol-1
1 C4MeImAc/DMSO 15°C 62.5000
25°C 37.0370
40°C 23.8095
60°C 16.6667
2 C3OMeImAc/DMSO 15°C 50.0000
25°C 33.3333
40°C 23.8095
60°C 15.6250
Chapter # 06 Results & Discussion
Page 122
Figure 16. Applications of Eq van't Hoff to Kassoc (=1/Kdissoc) of BM of
C4MeImAc/DMSO
Figure 17. Applications of van't Hoff Eq for calculation of Kassoc (=1/Kdissoc)
for BM of C3OMeImAc/DMSO
From the data listed in Table 10 & 11 for Kdissoc and Kassoc for both BMs it is
obvious that these two values are inverse of each other for each BM. The
Chapter # 06 Results & Discussion
Page 123
value of Kdissoc for BM C4MeImAc/DMSO is less than the value of
C3OMeImAc/DMSO and inverse relation obtained in case of Kassoc. Linear
graph obtained in both plots of lnKdissoc and lnKassoc versus 1/T (Kelvin).
6.4 Thermo-solvatochromism in Binary Mixtures of DMSO and ILs.
The dependence of ET (probe)obs on dmso (analytical) is described in Figure
(16) & (17) for both BMs (C4MeImAc/DMSO& C3OMeImAc/DMSO) at 15, 25,
40 and 60°C. The dependence of φ on the temperature, type of the IL and
BMs is described in the Table 12.
Chapter # 06 Results & Discussion
Page 124
Figure 18. Dependence of the empirical solvent polarity parameter ET(probe)
on the analytical mole fraction of DMSO, at 15, 25, 40 and 60 °C for mixtures
of IL(C4MeImAc) DMSO, respectively. The straight lines were plotted to guide
the eye; they represent ideal solvation of the dye by the mixture.
54
56
58
60
62
64
0 0 0 1 1 1
ET 15 oC
54
56
58
60
62
64
0 0 0 0 0 1 1 1 1 1 1
ET 25 oC
54
56
58
60
62
64
0 0 0 0 0 1 1 1 1 1 1
ET 40oC
54
56
58
60
62
64
0 0 0 1 1 1
ET 60oC
ET(3
3) (K
cal m
ol-
1)
///m
ol
DMSO
Chapter # 06 Results & Discussion
Page 125
Figure 19. Dependence of the empirical solvent polarity parameter ET(probe)
on the analytical mole fraction of DMSO, at 15, 25, 40 and 60 °C for mixtures
of IL (C3OMeImAc) DMSO, respectively. The straight lines were plotted to guide
the eye; they represent non-ideal solvation of the dye by the mixture.
Following results can be concluded from the obtained data
1. All plots are nonlinear in Figure 18 and 19 are above the line of
linearity. There are many factors for this behavior and/or may be due to the
mechanism of solute and solvent. This non-ideal behavior may be because of
55
57
59
61
63
65
67
0.0000 0.2000 0.4000 0.6000 0.8000 1.0000
ET 15oC
55
57
59
61
63
65
67
0 0 0 1 1 1
ET 25oC
54
56
58
60
62
64
66
0 0 0 1 1 1
ET 40oC
54
56
58
60
62
64
0 1 1
ET 60oC
ET(3
3) (K
cal
mol-
1)
DMSO Analytical
Chapter # 06 Results & Discussion
Page 126
dielectric enrichment which is the observation of enrichment Єr in the
solvation shell of solvent with relatively greater permittivity (Suppan 1997). It
is clear from the Figure 18 and 19, all the points lie above the line of
linearity, (the line which is connecting the polarities of pure DMSO and IL).
The second reason which causes this non-ideality is preferential solvation of
the probe by the component of solvent mixtures which are formed by the
specific or non-specific interactions (Hydrogen bonding, dipole-dipole
solvophobic interactions). To explain the interactions between BMs a large
number of calculations has been carried on like Kirkwood-Buff integral
functions which describe the interaction between the components of solvent
i.e. DSMO/W-IL, DMSO-DMSO/W-W, and IL-IL which explain that in the
solvent mixture different type of micro-domains are present like DMSO
surrounded by Organic solvent and organic solvent surrounded by DMSO.
The nature of these micro-domains depends on the nature of the solvent
used for studies. In our studies the possibility of preferential solvation of the
polarity probe is by polar moiety (IL-DMSO) as shown by graph line leading
towards the up the line deviation as shown in Figure 18 and 19. In
conclusion we can say that in case of solvation in BMs the non-ideal
behavior is not an unexpected trend and it may be above the line of linearity
or below the line, or both cases can be seen in the same graph.
Table 12. Analysis of Thermosolvatochromic Data in BMs of IL/DMSO
(C4MeImAc/DMSO& C3OMeImAc/DMSO)
Chapter # 06 Results & Discussion
Page 127
2. The best fit of the values in the solvation model is confirmed by the
values of r2 and chi2 and by the best fit of results of the experimental and
calculated of ET (WB) of IL, and ET (WB) of DMSO, respectively. Thus
assumption of IL-DMSO in 1:1 ratio is a general trend in solvation in case of
BMs solvation. The change in results is discussed on the base of structure of
IL and effect on temperature.
3. The calculated values of (m) are near to 1 and decreases with increase
of temperature in our present studies. These value of (m) should not be
confused with the total number of probe ET (WB) solvated by solvent but it is
the number of solvent molecules takes part in the intra-molecular charge
transfer between the +ive and -ive poles of the probe (WB).
Ionic
Liquid
T°
C
Φ(dmso/
Il)
Φ(il-
dmso/Il)
Φ(dms
o-
il/dmso)
ET(W
B)il
ET(WB)d
mso
ET(WB)D
mso-Il
r2 Chi2 M
15°C
0.116 (±0.03)
1.523 (±1.2)
13.129
59.84 (±0.03)
54.47 (±0.00)
56.7 (±0.51)
0.99837
0.00396
0.92
C4MeImA
c
25°C
0.132 (±0.02)
1.427 (±0.90
)
10.810
59.5
(±0.03)
54.24 (±0.00)
56.5 (±0.16)
0.99845
0.0379 0.91
40°C
0.144 (±0.06)
0.815 (±4.90
)
5.660 59.18 (±0.09)
54.09 (±0.07)
55.4 (±0.83)
0.99864
0.00317
0.85
60°C
0.168 (±0.00)
0.792 (±0.04
)
4.714 58.90 (±0.02)
53.69 (±0.01)
55.1 (±00)
0.99922
0.000184
0.80
C3OMeImAc
15°
C
0.115
(±0.01)
2.001
(±1.23)
15.00
2
62.36
(±0.10)
55.48
(±0.07)
62
(±0.27)
0.999
71
0.0036
5
1.0
1
25°C
0.128
(±0.00)
1.854 (±1.39
)
12.894
61.59 (±0.05)
55.04 (±0.05)
61.86 (±0.16)
0.99984
0.00278
1.00
40°C
0.139 (±0.03)
1.008 (±5.69
)
8.123 59.23 (±0.15)
54.65 (±0.04)
61.03 (±0.51)
0.99891
0.00184
0.99
60°C
0.148 (±0.00)
1.000 (±0.45
)
6.128 58.96 (±0.02)
54 (±0.01)
60.58 (±0.26)
0.99898
0.00255
0.98
Chapter # 06 Results & Discussion
Page 128
4. The value of DMSO/IL are less than unity in both BMs which
indicate that DMSO is not able to replace IL in the solvation sphere of probe.
This solvation preferably by IL is due to the acidic hydrogen of the
imidazolium ring and oxygen of the phenolate of the probe, the acetate
(CH3COO-) of the IL and positively charge nitrogen of the probe, and as well
as in the vide infrared reign during the solvatochromism.
5. All values of IL-DMSO/IL, IL-DMSO/DMSO are greater than one in
case of both BMs approximately as shown in Table 12, which shows that the
solubility of probe is more in IL-DMSO mixture as compared to pure
solvents. Moreover, all the values of IL-DMSO/DMSO are greater than IL-
DMSO/IL, which shows that IL-DMSO can more efficiently replace DMSO
than IL from the solvation shell of the probe. The method of solvation
involves dipole-dipole interactions and solvophobic interactions in both cases
of solvation i.e. with pure IL, DMSO, and IL-DMSO mixture.
6. The value DMSO replacing IL ( DMSO/IL) for BM of
(C3OMeImAc/DMSO) is lower than the BM of (C4MeImAc/DMSO), but the
value of Mixture of DMSO-IL replacing IL ( IL-DMSO/IL) and of IL-DMSO
replacing DMSO ( IL-DMSO/DSMO) are higher than the BM of
(C4MeImAc/DMSO), which shows that the C3OMeImAc is more efficient in
preferential solvation as compared to C4MeImAc. This difference in the
values is because of the difference in structure of ionic liquids.
Chapter # 06 Results & Discussion
Page 129
Figure 20. Species distribution at 15, 25, 40. and 60°C for BMs of
C4MeImAc/DMSO
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0 0.5 1
15 ºC
0 0.5 1
25 ºC
0 0.5 1
40 ºC
0 0.5 1
60 ºC
effecti
ve
DMSO Analytical
Chapter # 06 Results & Discussion
Page 130
Figure 21. Species distribution at 15, 25, 40 and 60°C for BMs of
C3OMeImAc/DMSO
The effective concentration of DMSO
, Ionic liquid
, and
1:1 IL-DMSO complex
present in the reaction mixture is
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.5 1
15ºC
0 0.5 1
25ºC
0 0.5 1
40 ºC
0 0.5 1
60 ºC
e
ffecti
ve
DMSO Analytical
Chapter # 06 Results & Discussion
Page 131
calculated by using equation 15, 16 and 17, and the result are represented
in figure no 18 & 19 at four different temperatures. It is obvious from the
graph that with the increase of temperature the maximum solubility curve
tends towards the DMSO in 1:1 DMSO-IL complex.
6.5 Determination of Basicity (SB) of BMs by Using Pair of Probes
The basicity of the binary mixture (SB) is calculated by the using the
equation 33 and it is always higher than the ideal value. The (SB) values of
for BMs of both ILs are given in Table 13 and 14 and their dependency on
the molar fraction of DMSO is given in Figures 22 & 23.
Table 13. SB scale based on the solvatochromism of the probe 5-
nitroindoline and its homomorph N-methyl-5-nitroindoline (SB) of BMs of
IL/DMSO (C4MeImAc/DMSO)
Sr. No SB (15°C) SB (25°C) SB (40°C) SB (60°C)
1 0.54519 0.54812 0.55630 0.55888
2 0.58137 0.59847 0.58809 0.61793
3 0.61211 0.62380 0.63239 0.65788
4 0.63847 0.64742 0.65468 0.66065
5 0.64304 0.64685 0.65351 0.65734
6 0.66938 0.67563 0.68140 0.69446
7 0.71039 0.71264 0.70318 0.70851
8 0.72027 0.73661 0.72408 0.72757
9 0.73464 0.74118 0.70644 0.70233
10 0.70043 0.66167 0.63148 0.59968
Chapter # 06 Results & Discussion
Page 132
Figure 22. Dependence of the of the overall solvent basicity parameter SB
(NI/MNI) on mole fraction of DMSO, at 15, 25, 40 and 60 °C for mixtures of
IL/DMSO (C4MeImAc/DMSO/), respectively.
The value of basicity of BMs of both ILs ranges from 0.54 to 0.70 at four
different temperatures. The SB scale is based on the solvatochromic data of
the probe 5-nitroindoline and its homomorphs N-methyl-5-nitroindoline. The
basicity rises with increasing concentration of DMSO and maximum value
for basicity obtained at the moles fraction of DMSO 0.9 and then it falls
down for pure DMSO. As a result a curved graph is obtained which shows
0.000000
0.200000
0.400000
0.600000
0.800000
0.000 0.200 0.400 0.600 0.800 1.000
SB 15 ºC
0.000000
0.200000
0.400000
0.600000
0.800000
0.000 0.500 1.000
SB 25 ºC
0.000000
0.200000
0.400000
0.600000
0.800000
0.000 0.500 1.000
SB 40 ºC
0.000000
0.200000
0.400000
0.600000
0.800000
0.000 0.500 1.000
SB 60 ºC
Basic
ity o
f B
Ms
Chapter # 06 Results & Discussion
Page 133
positive deviation from the ideal behavior, which is commonly obtained in
the case of basicity measurement of BMs.
Table 14. SB scale based on the solvatochromism of the probe 5-
nitroindoline and its homomorph N-methyly -5-nitroindoline (SB) of BMs of
IL/DMSO (C3OMeImAc/DMSO)
Sr. NO. SB (15°C) SB (25°C) SB (40°C) SB (60°C)
1 0.41445 0.45822 0.46185 0.47155
2 0.46933 0.47814 0.48934 0.52977
3 0.49372 0.52216 0.53504 0.59106
4 0.55744 0.55660 0.56581 0.61531
5 0.57741 0.58173 0.59358 0.63475
6 0.59224 0.59331 0.62295 0.64394
7 0.60974 0.63674 0.65173 0.68124
8 0.66940 0.68271 0.68437 0.70673
9 0.70836 0.72551 0.73797 0.74827
10 0.67438 0.67458 0.62596 0.72651
Chapter # 06 Results & Discussion
Page 134
Figure 23. Dependence of the of the overall solvent basicity parameter SB
(NI/MNI) on mole fraction of DMSO, at 15, 25, 40 and 60 °C for mixtures of
IL/DMSO (C3OMeImAc/DMSO), respectively.
Following conclusion can be drawn from the basicity results of the binary
mixtures of both ILs
1. The basicity value increases with increase in temperature and
concentration of DMSO in binary mixtures in case of both ILs but not in
the case of pure DMSO as shown in figure 22 & 23.
0.000000
0.200000
0.400000
0.600000
0.800000
0.00 0.20 0.40 0.60 0.80 1.00
SB 15 ºC
0.000000
0.200000
0.400000
0.600000
0.800000
0.00 0.20 0.40 0.60 0.80 1.00
SB 25 ºC
0.000000
0.200000
0.400000
0.600000
0.800000
0.00 0.20 0.40 0.60 0.80 1.00
SB 40 ºC
0.000000
0.200000
0.400000
0.600000
0.800000
0.00 0.20 0.40 0.60 0.80 1.00
SB 60 ºC
Basic
ity o
f B
Ms
Chapter # 06 Results & Discussion
Page 135
2. This continuous increase in the SB values with the increase in
temperature and then sudden decrease (as in case of pure DMSO) gave
a small positive deviation from linearity shown in Figure 22 & 23.
3. This non-ideal behavior towards basicity is same as in case of polarity
measurements (both are above the line of linearity), which is must be
the result of the molecular interactions in their bulk.
4. The difference in behavior of both ILs towards basicity is obvious from
their SB values listed in tables 13 & 14. The lowest and highest value of
SB observed in BM of C4MeImAc/DMSO is 0.545193 & 0.741182 and in
case of C3OMeImAc/DMSO 0.414455 & 0.748275.
5. The lowest value are recorded in case of both pure IL and the least value
is observed in case of IL with oxygen, which indicates that presence of
oxygen in IL make it less basic as compared to the IL without oxygen.
6.6 Determination of Acidity (SA) of BMs by using DETZ
Determination of acidity parameter was first tried to measure by using
homomorphs pair of TBSB/DTBSB, which shows good results with the ionic
liquids without oxygen (C4MeImAc/DMSO), but in case of IL with oxygen
(C3OMeImAc/DMSO) this homomorphs pair reacts and was not proceed
further. The reaction was most probably between the acidic hydrogen of the
imidazole ring of IL and oxygen of the TBSB/DTBSB. To solve this problem
another probe DETZ was used which was stable for acidic medium even can
be used for strong acids. The equation 34 & 35 are used to calculate the (SA)
from the spectroscopic data. The values of spectroscopic data is listed in
Chapter # 06 Results & Discussion
Page 136
Table 15 & 16 and their plot versus mole fraction of DMSO are given in
Figures 24 & 25.
Table 15. Δν scale based on the solvatochromism of the probe Synthesis of
3,6-diethyl-1,2,4,5-tetrazin for BMs of IL/DMSO (C4MeImAc/DMSO)
Sr. No Δν (15°C) Δν (25°C) Δν (40°C) Δν (60°C)
1 18756.68 18745.55 18738.76 18722.04
2 18698.70 18717.01 18737.24 18740.98
3 18693.45 18696.02 18726.83 18712.69
4 18635.63 18636.68 18681.24 18703.24
5 18613.19 18610.19 18640.84 18699.05
6 18584.48 18580.34 18628.68 18657.07
7 18571.60 18568.38 18604.88 18614.81
8 18549.78 18553.56 18581.04 18585.75
9 18546.80 18539.12 18572.30 18561.67
Chapter # 06 Results & Discussion
Page 137
Figure 24. Dependence of the of the overall solvent acidity parameter SA
(DETZ) on mole fraction of DMSO, at 15, 25, 40 and 60 °C for mixtures of
IL/DMSO (C4MeImAc/DMSO), respectively
18500
18550
18600
18650
18700
18750
18800
0 0.5 1
Δν 15 oC
18500
18550
18600
18650
18700
18750
18800
0 0.5 1
Δν 25 oC
18500
18550
18600
18650
18700
18750
0 0.2 0.4 0.6 0.8 1
Δν 40 oC
18500
18550
18600
18650
18700
18750
0 0.2 0.4 0.6 0.8 1
Δν 60 oC
ΔѴ f
or
DE
TZ
Chapter # 06 Results & Discussion
Page 138
Table 16. SA scale based on the solvatochromism of the probe synthesis
of 3,6-diethyl-1,2,4,5-tetrazin of BMs of IL/DMSO (C3OMeImAc/DMSO)
The following conclusion can be drawn from the results of ΔѴ of both BMs;
All the values of ΔѴ are obtained when interaction between the solvent
components is maximum.
Although the results of spectroscopic data are showing positive deviation
from the linearity, and are in sequence as excepted, but it was difficult for
our group to calculate the acidity values (SA) as it need the solvent polarity
polarizability (SPP) values of these BMs at the same temperatures. We tried
1st time ever to calculate these (SPP) of BMs but results do not look in
correlation with ΔѴ for acidity parameter.
Sr. No Δν (15°C) Δν (25°C) Δν (40°C) Δν (60°C)
1 18715.26 18783.82 18702.21 18744.27
2 18880.63 18856.66 18825.08 18851.33
3 18809.02 18781.39 18770.9 18802.65
4 18747.44 18736.53 18737.47 18757.28
5 18680.3 18670.87 18664.1 18675.53
6 18652.43 18629.72 18629.14 18652.9
7 18588.4 18598.31 18592.67 18611.94
8 18577.58 18578.96 18578.73 18593.82
9 18565.97 18548.87 18568.04 18590.93
10 18541.75 18525.19 18547.6 18543.48
Chapter # 06 Results & Discussion
Page 139
Figure 25. Dependence of the of the overall solvent acidity parameter SA
(DETZ) on mole fraction of DMSO, at 15, 25, 40 and 60 °C for mixtures of
IL/DMSO (C3OMeImAc/DMSO), respectively.
1. The thermosolvatochromic data of BMs for (SA) is showing positive
deviation from linearity and this deviation increases with increase in
temperature in case of both BMs.
2. IL with oxygen is more acidic than without oxygen, it has been proved
during the solvatochromic analysis with TBSB/DTBSB.
18500
18600
18700
18800
18900
0 0.5 1
Δν 15 oC
18500
18600
18700
18800
18900
0 0.2 0.4 0.6 0.8 1
Δν 25oC
18500
18600
18700
18800
18900
0 0.5 1
Δν 40oC
18500
18600
18700
18800
18900
0 0.5 1
Δν 60oC
ΔѴ f
or
DE
TZ
Chapter # 06 Results & Discussion
Page 140
6.7 Determination of Solvent Polarity (SP) of BMs by Using β-Carotene
The (SP) of the binary mixture is measured by using β-carotene, a natural
probe which was not easily soluble in BMs of IL/DMSO and take hour to
make a samples for solvatochromic studies. The equation 30 is used to
calculate (SP)
Table 17. SP scale based on the solvatochromism of the probe Beta-carotene
of BMs of IL/DMSO (C4MeImAc/DMSO)
Sr. No SP (15°C) SP (25°C) SP (40°C) SP (60°C)
1 -0.5108 -0.4495 -0.3593 -0.6468
2 -0.3597 -0.3569 -0.3249 -0.2952
3 0.2361 -0.2545 -0.2648 -0.0445
4 -0.1004 -0.3684 -0.2909 -0.1456
5 -0.3126 -0.3297 -0.3182 -0.2869
6 -0.4455 -0.3931 -0.4142 -0.4725
7 -0.2143 0.7816 -0.2462 -0.1492
8 0.0250 0.8208 0.7205 0.0659
9 0.7680 0.8322 0.7960 0.7432
10 0.8492 0.8484 0.8339 0.8192
Chapter # 06 Results & Discussion
Page 141
Figure 26. Dependence of the of the overall solvent polarity parameter SP
(Beta-carotene) on mole fraction of DMSO, at 15, 25, 40 and 60 °C for mixtures
of IL/DMSO (C4MeImAc/DMSO) respectively.
-1.0000
-0.5000
0.0000
0.5000
1.0000
0 1 1
SP 15°C
-1.0000
-0.5000
0.0000
0.5000
1.0000
0 1 1
SP 25°C
-0.5000
0.0000
0.5000
1.0000
0 1 1
SP 40°C
-1.0000
-0.5000
0.0000
0.5000
1.0000
0 1 1
SP 60°C
SP o
f B
MS
Chapter # 06 Results & Discussion
Page 142
Table 18. SP scale based on the solvatochromism of the probe Beta-carotene
of BMs of IL/DMSO (C3OMeImAc/DMSO)
From the Solvent polarity data of BMs calculated by using Beta-carotene
following observation can be discussed
1. The solvent polarity (SP) of the binary mixture of IL/DMSO is calculated
first time and the results are quite surprising, as the sample of BMs
with higher value of IL show negative value of (SP) and goes towards
positive value with increase in the DMSO amount.
2. From these values of SP it is clear that BMs of IL showing negative
deviation from the linearity.
3. These values of SP are higher in case of IL with oxygen, showing more
polarity of C3OMeImAc due to the presence of Oxygen.
Sr. No SP (15°C) SP (25°C) SP (40°C) SP (60°C)
1 -0.3936 -0.4357 -0.5061 -0.5504
2 -0.2026 -0.4187 -0.5642 -0.6581
3 -0.2663 -0.3114 -0.4491 -0.3972
4 -0.3224 -0.2770 -0.4034 -0.4565
5 -0.0456 -0.0379 -0.1660 -0.2948
6 -0.2742 0.0088 -0.1876 -0.2132
7 -0.2412 -0.2535 -0.3474 -0.3316
8 0.0403 0.0495 0.0344 0.0313
9 0.8339 0.8357 0.8235 0.8216
10 0.8530 0.8471 0.8326 0.8259
Chapter # 06 Results & Discussion
Page 143
Figure 27. Dependence of the of the overall solvent polarity parameter SP
(Beta-carotene) on mole fraction of DMSO, at 15, 25, 40 and 60 °C for mixtures
of IL/DMSO (C3OMeImAc/DMSO) respectively.
Maximum value of the SP in case of both BMs is obtained for pure DMSO,
and this value decreases as concentration of IL increases in the binary
mixtures.
6.8 Determination of Solvent Dipolarity (SD) of BMs by Using DMANF &
β-Carotene
-0.5000
0.0000
0.5000
1.0000
0.000 0.500 1.000
SP 15°C
-0.5000
0.0000
0.5000
1.0000
0.000 0.200 0.400 0.600 0.800 1.000
SP 25°C
-1.0000
-0.5000
0.0000
0.5000
1.0000
0.000 0.500 1.000
SP 40°C
-1.0000
-0.5000
0.0000
0.5000
1.0000
0.000 0.500 1.000
SP 60°C
SP o
f B
MS
Chapter # 06 Results & Discussion
Page 144
Determination of solvent dipolarity (SD) of BMs of ILs was done by using a
pair of probes i.e. DMANF and β-carotene and the conversion of the
solvatochromic data into SD value was done by using equation 31. The
DMANF is readily soluble in the BMs of IL/DMSO to make the samples for
solvatochromic studies. The values of SD for both BMs at four different
temperatures are given in Table 19 & 20 and their dependence on the molar
fraction of DMSO is given in Figure 28 & 29.
Table 19. SD scale based on the solvatochromism of the probe Beta-
carotene &DMANF of BMs of IL/DMSO (C4MeImAc/DMSO)
Sr. No SD (15°C) SD (25°C) SD (40°C) SD (60°C)
1 11.62515453 11.5911042 9.505475 10.23058839
2 10.43428577 10.8132806 9.244012 7.989365278
3 7.551742045 9.95671908 8.815442 6.403380122
4 8.391326208 10.8715704 9.01401 7.050522511
5 10.06319366 10.5900881 9.235801 7.963216052
6 11.11064037 11.137485 9.931709 9.147475941
7 9.288653935 1.52537589 8.738982 7.105709162
8 7.40262966 1.20048423 1.793183 5.744924819
9 1.547978494 1.13469532 1.269113 1.478217738
10 0.908249661 1 1 1
Chapter # 06 Results & Discussion
Page 145
Figure 28. Dependence of the of the overall solvent dipolarity parameter SD
(Beta-carotene & DMANF) on mole fraction of DMSO, at 15, 25, 40 and 60 °C
for mixtures of IL/DMSO (C3OMeImAc/DMSO) respectively
From the SD data of the BMs of IL/DMSO following conclusion can be drawn
The values of (SD) are inverse of the values of (SP) in BMs of ILs, which
indicates that if SP is increasing with increasing concentration of DMSO, SD
will decrease.
0
5
10
15
0 0 0 1 1 1
SD 15oC
-1
4
9
14
0 0 0 1 1 1
SD 25oC
0
5
10
15
0 0 0 1 1 1
SD 40oC
0
5
10
15
0 0 0 1 1 1
SD 60oC
SD
of
BM
s
Chapter # 06 Results & Discussion
Page 146
It is positive deviation from the line of linearity and trends in the values is
not constant as, they 1st decrease, then increase and again decrease with
the increasing concentration of DMSO in BMs.
Table 20. SD scale based on the solvatochromism of the probe Beta- carotene
&DMANF of BMs of IL/DMSO (C3OMeImAc/DMSO)
Maximum value of SD in both BMs is in the case of pure IL, but IL with
oxygen shows more dipolarity as compared to the IL without oxygen, due to
the presence of oxygen.
The value of SD decreases with the increase in temperature showing the
effect of temperature on the structure of the ILs.
Sr. No SD (15°C) SD (25°C) SD (40°C) SD (60°C)
1 12.21563 11.57704 10.31208 9.929203
2 10.44127 11.43781 10.72038 10.62694
3 10.79641 10.55908 9.911563 8.936861
4 11.37241 10.27732 9.589630 9.320929
5 9.02594 8.317733 7.920958 8.274053
6 11.07776 7.935058 8.072776 7.745124
7 10.74988 10.08416 9.195829 8.512019
8 8.24273 7.601397 6.511610 6.161958
9 1.097859 1.160215 0.963175 1.043095
10 1 1.066141 0.899043 1.01528
Chapter # 06 Results & Discussion
Page 147
Figure 29. Dependence of the of the overall solvent dipolarity parameter SD
(Beta-carotene & DMANF) on mole fraction of DMSO, at 15, 25, 40 and 60 °C
for mixtures of IL/DMSO (C3OMeImAc/DMSO)respectively.
-1
4
9
14
0.000 0.200 0.400 0.600 0.800 1.000
SD at 15
-1
4
9
14
0.000 0.500 1.000
SD at25
0
2
4
6
8
10
12
0.000 0.200 0.400 0.600 0.800 1.000
SD at 40
0
2
4
6
8
10
12
0.000 0.500 1.000
SD at 60
SD
of
BM
s
Chapter # 06 Results & Discussion
Page 148
6.9 Determination of Solvent dipolarity Polarizability (SDP) of BMs by
Using ClNF & DMANF
Solvent polarity values for DMSO/IL are given in Figure 30 & 31.
Determination of solvent dipolarity polarizability (SDP) of BMs of ILs was
done by using a pair of probes i.e. DMANF and ClNF and the conversion of
the solvatochromic data into SD value was done by using equation 32. The
DMAN & ClNF is readily soluble in the BMs of IL/DMSO to make the
samples for solvatochromic studies. The values of SDP for both BMs at four
different temperatures is given in Table 21 & 22 and their dependence on the
molar fraction of DMSO.
Table 21. SDP scale based on the solvatochromism of the probe ClNF &
DMANF of BMs of IL/DMSO (C4MeImAc/DMSO)
Sr. No SDP (15°C) SDP (25°C) SDP (40°C) SDP (60°C)
1 0.22573 -0.12752 -0.14688 -0.02118
2 0.17365 -0.35634 -0.14127 -0.22295
3 0.58630 -0.20148 -0.26731 -0.41393
4 0.73088 -0.24241 -0.23304 -0.24096
5 0.51720 0.102443 0.09327 0.082406
6 0.36439 0.37843 0.29567 0.29963
7 0.11394 0.19638 0.20571 0.19928
8 -0.0521 0.3961 0.36640 0.33289
9 0.29307 0.7271 0.73646 0.75521
10 1 1 1 1
Chapter # 06 Results & Discussion
Page 149
Figure 30. Dependence of the of the overall solvent dipolarity parameter SDP
(ClNF &DMANF) on mole fraction of DMSO, at 15, 25, 40 and 60 °C for
mixtures of IL/DMSO (C4MeImAc/DMSO)respectively
-0.5
0
0.5
1
1.5
0.00 0.50 1.00
SDP 15°C
-0.5
0
0.5
1
1.5
0.00 0.50 1.00
SDP 25°C
-0.5
0
0.5
1
1.5
0.00 0.20 0.40 0.60 0.80 1.00
SDP 40°C
-0.5
0
0.5
1
1.5
0.00 0.50 1.00
SDP 15°C
SD
P o
f B
Ms
Chapter # 06 Results & Discussion
Page 150
Table 22: SDP scale based on the solvatochromism of the probe ClNF &
DMANF of BMs of IL/DMSO (C3OMeImAc/DMSO)
Sr. No SDP (15°C) SDP (25°C) SDP (40°C) SDP (60°C)
1 -0.00222 0.07034 0.17418 -0.00552
2 0.01849 0.18011 0.15890 0.08015
3 0.36686 0.21445 0.17741 -0.06004
4 0.25598 0.17855 0.05781 -0.18076
5 -0.00497 -0.02696 -0.04666 -0.08143
6 0.01286 -0.16106 -0.24590 -0.23646
7 0.12179 -0.05925 -0.14453 -0.02858
8 0.07330 -0.01476 -0.01815 0.23017
9 0.38398 0.01955 0.01750 0.39299
10 1 1 1 1
Chapter # 06 Results & Discussion
Page 151
Figure 31. Dependence of the of the overall solvent dipolarity parameter SDP
(ClNF &DMANF) on mole fraction of DMSO, at 15, 25, 40 and 60 °C for
mixtures of IL/DMSO (C3OMeImAc/DMSO) respectively.
From the SDP data of the BMs of IL/DMSO following conclusion can be
drawn.
The values of (SDP) are inverse of the values of (SD) in BMs of ILs, and have
negative values in the data, which indicates the negative dipolarity
polarizability of the BMs.
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
0.00 0.20 0.40 0.60 0.80 1.00
SDP 25oC
-0.2
0
0.2
0.4
0.6
0.8
1
0.00 0.20 0.40 0.60 0.80 1.00
SDP 15oC
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
0.00 0.20 0.40 0.60 0.80 1.00
SDP 40oC
-0.5
0
0.5
1
0.00 0.20 0.40 0.60 0.80 1.00
SDP 60oC
SD
P o
f B
Ms
Chapter # 06 Results & Discussion
Page 152
It shows negative deviation from the line of linearity and trends in the values
is not constant as, first increase, then decrease and again increase in the
SDP values observed with the increasing concentration of DMSO in BMs.
Maximum value of SDP in both BMs is in the case of pure DMSO, but IL
without oxygen shows more SDP value as compared to the IL with oxygen.
The value of SDP increases with the increase in temperature showing the
effect of temperature on the structure of the ILs
6.10 Dependence of ET(WB) on Binary Mixture Composition
Parts A and B of Figure 32 show the dependence of ET(WB) on DMSO and
W, at 15, 25, 40, and 60 °C, respectively. All plots are not ideal, i.e., the
calculated ET(WB) are not linear function in S. We exemplify the ideal
behavior by the straight lines that we draw to connect the data of pure IL
and DMSO (at 60 °C), and pure IL and W (at 15 °C). Ideal behavior is
observed when the compositions of the probe solvation layer and bulk
solvent are the same. The salient feature in Figure 32 is the different
behavior of IL-DMSO (positive deviation from linearity) from that of IL-W
(negative deviation from linearity). All ET(WB) values at maximum deviation
lie, however, between ET(WB) of the two pure solvents. That is, there is no
“synergism”, where the empirical polarity at maximum deviation lies above
the value of the more polar solvent (for positive deviation) as was observed,
e.g., for mixtures of the IL 1-(1-butyl)3-methylimidazolium tetrafluoroborate
or hexafluorophosphate with some alcohols.
Chapter # 06 Results & Discussion
Page 153
Figure 32. Dependence of the calculated empirical polarity, ET(WB) on the
composition of binary solvent mixtures. Part A is for IL-DMSO, whereas part B
is for IL-W. The ideal behavior is depicted by the straight lines that we draw
between ET(WB) of the pure solvents. For simplicity, we draw these lines for
the data of a single temperature, 60 °C for IL-DMSO and 15 °C for IL-W.
Instead of reporting extensive lists of ET(WB) and binary mixture
compositions, we have calculated the (polynomial) dependence of the
empirical polarity on the analytical mole fraction of DMSO or W, and present
the data in Tables 23 and 24. The quality of the fit is indicated by values of
the regression coefficient, r2 and ΣQ2, the sum of the squares of the
residuals. The degree of polynomial employed is that which gave the best
data fit, as indicated by statistical criteria e.g., the IL-DMSO data at 25°C
could have been conveniently adjusted with a fifth, or a fourth power
polynomial, leading to r2 = 0.99196, 0.98662, and ΣQ2 = 0.23511, 0.42428,
respectively. The same observation applies to IL-W mixtures.
Chapter # 06 Results & Discussion
Page 154
ET(WB) = A + B(DMSO) + C(DMSO)2 + D(DMSO)3 + E(DMSO)4 + F(DMSO)5 +
G(DMSO)6
Table 23. Polynomial dependence of ET(WB) on the mole fraction of DMSO
(DMSO) at different temperatures
r2' and ΣQ2 refer to the nonlinear correlation coefficient and the sum of the
squares of the residuals, respectively.
ET(WB) = A + B(W) + C(W)2 + D(W)3 + E(W)4 + F(W)5 + G(W)6
Table 24. Polynomial dependence of ET(WB) on the mole fraction of water
(W) at different temperatures
Chapter # 06 Results & Discussion
Page 155
6.11 Rational for the Solvatochromic Data of WB in Binary Solvent
Mixtures
Non-ideal behavior may originate from the so-called “dielectric enrichment”,
i.e., enrichment of the probe solvation layer in the solvent of higher relative
permittivity, ε 43 The values of ε are 48.2 (20 °C), 78.5 (25 °C), and between
7 and 17.2 (25 °C) for DMSO, W, and a series of ILs with diverse cations
(including BuMeIm+) and anions (BF4-, PF6-, Cl-, methyl to n-octylsulfates,
etc.), respectively (Khirade 1999, ; Uematsu 2008). If this solvation
mechanism were operating, then the ET(WB) versus W plots should show
positive deviation, i.e., should lie above the straight line connecting ET(WB) of
IL and W; this is not the case. The curves for DMSO lie above the straight
line. There is no particular reason, however, that IL-DMSO mixture shows
dielectric enrichment, whereas water whose ε is 63% larger than that of
DMSO does not. Therefore, we seek another mechanism to explain the non-
ideal behavior.
The model given from equation 12 to 15 analyzes the solvatochromic data in
terms of the effective (not analytical) concentrations of IL, W, and a
“complex” solvent (IL-W). The latter is formed by the interaction of the two
solvents, e.g., via hydrogen bonding, dipolar, and hydrophobic interactions.
Equation 12 shows the association of the two solvents, whereas equations 13
to 15 describe the solvent exchange equilibria in the solvation layer of the
probe.
Chapter # 06 Results & Discussion
Page 156
The assumption we made in equation 12 (1:1 stoichiometry for IL-S) is a
practical and convenient one because it renders subsequent calculations
tractable, and has been previously employed to describe solvatochromism
(Bosch .E 1997; Buhvestov .U 1998). Additionally, hydrogen bond formation
between IL and DMSO; and IL and W was demonstrated by IR, NIR, NMR
and dielectric spectroscopy, (Rebelo 2004; Martins CT 2008; Bešter-Rogač
2011; Takamuku 2014; Radhi 2015; Zhu 2016) and predicted by theoretical
calculations (Wang 2006; Li 2007; He 2015).
Mixed solvent species with stoichiometry other than 1:1 may be treated, to a
good approximation, as mixtures of the 1:1 structure plus excess of a pure
solvent. We designate the equilibrium constants of equations 12 to 15 as
solvent “fractionation factors, ϕ”. These are defined on the mole fraction
scale, after rearrangement, as shown in equations 16 to 17 (examples shown
for IL-W).
We consider our solvatochromic results in conjunction with those of MD
simulations. The Gromacs program considers solvation by pure solvents
only, i.e., the IL-S species is not taken into account. MD simulations provide
the radial distribution function, g(r) that describes the probability to find an
atom in a layer at a distance (r) from another atom, chosen as a reference
point. Information about the interaction between the species present in the
simulation box is extracted from: the sharpness of the first g(r) peak (first
solvation layer), strong interaction leads to sharp peaks; the relevant
Chapter # 06 Results & Discussion
Page 157
distances between pairs of species, and the number of interacting elements
of the one specie (in relation to other one), calculated from the area under
the normalized g(r) curve. Some of these MD plots are shown in Figure 33,
34 and 35. The former Figure shows the number and mole fraction of the
solvent molecules within the solvation layer of WB, set at 0.5 nm. Figure 34
shows the g(r) plots for the interactions of WB with DMSO and W, whereas
Figure 35 shows the g(r) plots for the interactions of the IL with DMSO and
W. The curves of g(r) between WB and the solvent molecules (Figure 34)
show that the probe first solvation layers end at 0.545 nm (DMSO), and
0.458 nm (W); this being the reason for setting the solvation layer at 0.5 nm.
Table 25. Data analysis of solvation of WB in mixtures of IL-DMSO and
water in the temperature range 15 to 60 °Ca,b
a-Analysis according to Eqns. 16 to 18, see the Calculations section of SI.
b- For pure solvents, the values within parenthesis refer to the difference:
(Experimental ET(WB) - calculated ET(WB)).
Chapter # 06 Results & Discussion
Page 158
The values reported for the mixed solvent IL-S are the calculated ones.
Table 26. Results of MD simulations for the solvation of WB in mixtures of
IL-DMSO and IL-water
a- The term atom pair refers to the pair of interacting atoms. Thus, the
representation WB-O- S+OMe2 refers to the interaction of the phenolate anion
of WB with the sulphur atom of DMSO. The number data means that (on the
average) there as 3.3 molecules of DMSO interacting (inside a layer of 0.5
nm) with each phenolate anion of WB; these interacting atoms are at 0.478
nm apart.
b- H2 refers to the relatively acidic H2 of the imidazolium ring.
Chapter # 06 Results & Discussion
Page 159
Table 27. Results of MD simulations for the interactions of IL with DMSO
and water
IL-DMSO
IL-W
Atom Pair Number Distance, nm Atom Paira Number Distance, nm
AcO-..H2 2.6 0.328 AcO-..H2 3.1 0.243
AcO-..SOδ+(OMe)2 2.2 0.477 AcO-..H2 δ+O 7.2 0.164
Me2SOδ-..H2b 2.0 0.250 H2Oδ-..H2b 6.1 0.254
a- Number of the second species solvating the first one. E.g., the first entry
of the Table indicates that 2.6 acetate anions solvate, on the average, one
BuMeIm+ cation.
b- H2 refers to the relatively acidic H2 of the imidazolium ring.
Chapter # 06 Results & Discussion
Page 160
Figure 33. Calculated composition of WB first solvation layer (set at 0.5 nm),
expressed in number of specie (Part A) and in mole fraction (Part B). The
columns in red color refer to DMSO, those in blue color to water
Figure 34. Radial distribution functions g(r), showing the following
interactions of the probe with the two solvents: the phenolate moiety of WB
and the sulphur atom of DMSO, or hydrogen atom of water, part A; the
interactions of the quaternary nitrogen of WB and the oxygen of DMSO or the
oxygen of water, part B. The colors are red (DMSO) and blue (W).
Figure 35. Radial distribution functions {g(r)} for the two solvent systems. The
plots show the interactions between the oxygen of the acetate anion and “H2”
Chapter # 06 Results & Discussion
Page 161
of in ILDMSO and IL-W (Part A). Part B shows the interactions between oxygen
of the acetate anion and the positive pole of the second solvent (Sδ+ of DMSO,
and H2 δ+O of W). Part C shows the interactions between “H2” and the
negative pole of the second solvent (Oδ- of DMSO, and Oδ-H2 of W). The colors
are red (DMSO) and blue (W).
Regarding all previous data, the following is relevant
(i)- The quality of fit of the above-discussed solvation model to our data is
shown by values of (r2) and ΣQ2, and by the excellent agreement between
experimental and calculated ET(WB) in pure solvents at different
temperatures.
(ii)- The second column of Tables 25, the fifth and eleventh columns of Table
26 show that the values of (m) are not far from unity. That is, a small
number of solvent molecules perturb the intramolecular charge-transfer
between the phenolate oxygen and quaternary nitrogen of WB, leading to the
observed dependence of ET(WB) on S, see Figure 32.
(iii)- As shown in Table 25, all values of ϕ(DMSO/IL) and ϕ(W/IL) are less
than unity, showing that WB is more efficiently solvated by the IL than by
DMSO or W, in agreement with previous studies on solvation of WB and
merocyanine probes of different hydrophobic character by IL-W (Martins CT
2008; Sato 2012).
(iv) Table 25 shows that all ϕ(IL-S/IL) and ϕ(IL-S/S) are larger than unity.
That is, the most efficient solvent is the (IL-S) species that displaces both IL
Chapter # 06 Results & Discussion
Page 162
and DMSO or W in the probe solvation layer. The values of ϕ(IL-S/IL) and
ϕ(IL-S/S) decrease as a function of increasing temperature, showing that WB
is desolvated in the same direction. This probe desolvation agrees with the
known effect of temperature on the structure of molecular solvents (Marcus
2001), and ILs (Khupse 2011) due to less efficient hydrogen-bonding and
dipolar interactions at higher temperatures. Because the solvation of zwitter
ionic probes reflects essentially solvent stabilization of the their ground
states, a decrease in this stabilization (due to decreased solvent-probe
interactions) is expected to lead to a blue shift in λmax, i.e., a decrease in
ET(probe), see equation 14.
(iv)- All values of ϕ(IL-DMSO/IL) are < ϕ(IL-W/IL). Likewise all values of ϕ(IL-
DMSO/DMSO) are < ϕ(IL-W/w). That is, the mixed solvent IL-W is more
efficient in displacing IL and W than does IL-DMSO in displacing IL and
DMSO. To analyze these results we considered: (iv-a) The strength of
interactions of the IL with DMSO and W; (iv-b) The composition of the
solvation shell as reviled by Table 25 and Figure 31; (iv-c) The mechanism of
solvation by the two types of binary mixtures. Point (iv-a) is important
because the interactions between solvent components bears on the nature of
IL-S, hence on solvation of WB. Several pieces of evidence, including FTIR
and NMR spectroscopy (Takamuku 2014; He 2015; Radhi 2015; Chen. 2014)
isothermal titration calorimetry, (Rai 2014) and theoretical calculations (Ding
2012),indicate a strong association between ILs (including as acetates),
DMSO and W. As shown in Table 27, the average distances between the AcO-
Chapter # 06 Results & Discussion
Page 163
….H2; Me2SO…H2 in IL-DMSO, are practically the same as the AcO-….H2;
H2O…H2 in IL-W. The strength of these interactions is also corroborated by
the sharpness of the first g(r) peaks in part C of Figure 35. That is the
difference between WB solvation by IL-DMSO and IL-W is not due to a
massive difference in the interactions of IL with S. Concerning point iv-b,
Figure 33 and Table 27 show no regular trend regarding the concentrations
of solvent species in the solvation layer of WB. Whereas DMSO in IL-DMSO
is > W in IL-W (although the molecular volume of the former is larger 0.118
and 0.030 nm3/molecule, for DMSO and W, respectively), (Carmen Grande
2007) the inverse is true for IL in both media (IL in IL-W > IL in IL-
DMSO). Therefore, there is some compensation (due to differences in local
concentrations) between the interactions of WB with IL (essentially
Coulumbic) and DMSO or W (dipolar and hydrogen bonding). A corollary to
the previous statement is that the difference in probe solvation in IL-DMSO
and IL-W is not largely dependent on the differences in the concentrations of
IL and S in its solvation layer. Regarding (iv-c), the probe-solvent interactions
of concern are those with the phenolate oxygen. The reason is that Table 26
shows that the distances between WBN+ and solvent acceptor atoms are
either at the upper limit, or greater for efficient Coulumbic interactions.
Compare, e.g., the following MD-based distances (in nm): IL-DMSO, WB-N+⋅⋅-
OAc- (0.325), WB-N+⋅Oδ-SMe2 (0.478); IL-W, WB-N+⋅⋅-OAc- (0.635) and
WBN+⋅Oδ-H2 (0.342) with X-ray based intermolecular distance Nδ+…Oδ- in
methoxybearing thioureas (0.31).(Venkatachalam 2005) The reason for little
Chapter # 06 Results & Discussion
Page 164
interaction is steric crowding around the quaternary nitrogen of WB, as
indicated by theoretical calculations for the structurally similar RB probe
(2,6-Diphenyl-4-(2,4,6-triphenyl-1-pyridinio)phenolate) (Chiappe 2012). On
the other hand, the phenolate oxygen of RB forms efficient hydrogen bonds
with protic solvents, as indicated by NMR spectroscopy (Dawber 1986) and
X-ray of the complexes between several betaine solvatochromic dyes and
aliphatic alcohols (methanol, ethanol, 1-propanol, and 1-butanol; probe-O-
….HOR, ca. 0.19 nm). Likewise, the distances WB-O-⋅⋅⋅H2 in IL-DMSO and
IL-W (0.244 nm) are within the accepted range for hydrogen-bonds between
an aromatic hydrogen atom and the oxygen of a hydroxyl group of, e.g.,
1,3,5-tris(4-hydroxyphenyl)benzene (0.242 to 0.246 nm)(Thallapally 2002).
As shown in Table 25, IL-S is the most efficient solvent in the solvation layer
of WB. Consequently, the mechanisms of WB solvation by IL-DMSO and IL-
W are of prime importance. The results of several techniques (IR, NMR,
isothermal titration calorimetry) (Dawber 1986; He 2015) and theoretical
calculations (Chiappe 2012) indicate the importance of H2…OSMe; AcO---
H2O and H2…OH2 species. Whereas IL-DMSO solvates WB by solvophobic
and dipolar interactions, the corresponding species for IL-W have hydrogen-
bond donation as an additional important solvation mechanism. That is, the
species present in IL-W perturb more the intramolecular charge transfer of
WB than IL-DMSO, because of hydrogen bonding to the probe phenolate
anion. This conclusion is in agreement with the ϕ(IL-S/S) results of Table 25,
Chapter # 06 Results & Discussion
Page 165
and the probe free energies of solvation; |(ΔGSolv)IL-W| > |(ΔGSolv)IL-
DMSO.
In summary, the clear preferential solvation observed in Figure 32 and Table
25 is caused by a combination of differences in the effective S in the
solvation layer of WB, and an additional, efficient solvation mechanism open
for IL-W, but not for IL-DMSO. The positive and negative deviations observed
in Figure 32 may be a consequence of the interplay between these two
factors.
6.12 Relevance of the Solvatochromic Results to Cellulose Dissolution
in IL-DMSO
The requirement for cellulose dissolution is the disruption of the intra and
intermolecular hydrogen bonding present in the biopolymer chain. This
disruption occurs via hydrogen bonding between the hydroxyl group of the
anhydroglucose unit, the anion of the IL and the dipole of the molecular
solvent, as well as solvophobic interactions with the IL cation (El Seoud
2007; Hauru LKJ 2012; Medronho 2012). As shown above, these
interactions are operative in the solvation of WB. Therefore, we investigated
whether the solvatochromic data of WB can be exploited to explain the
dependence of cellulose dissolution in IL-DMSO on mixture composition. As
Figure 34 shows, the dependence of solubility of MCC and Mcotton on binary
mixture composition shows the same trend as ET(WB), namely a nonlinear
change in wt % dissolved cellulose with maximum deviation in the solubility
Chapter # 06 Results & Discussion
Page 166
curve at DMSO = 0.25. The difference between the wt% dissolved cellulose
of the two samples is essentially due to the higher molar mass and the
fibrous nature of Mcotton. This nonlinear solubility, and the DMSO at
maximum biopolymer dissolution (0.25) is similar to that observed for the
dissolution of cellulose samples in binary mixtures of 1-allyl-3-
methylimidazolium chloride (AlMeImCl) and DMSO at 60 °C. The position of
maximum cellulose dissolution is different from the position of maximum
deviation in part A of Figure 32 because unlike WB, cellulose is practically
insoluble in IL-DMSO at DMSO > ca. 0.6 (dissolved biopolymer < 1% wt%
for MCC and <0.1 wt% for M-cotton). This is certainly related to IL
dissociation as a function of its mole fraction, as shown especially by
conductivity data (Bešter-Rogač 2011; Lopes 2011; Bioni 2015; Radhi 2015).
This dissociation results in free anions that are required for cellulose
dissolution (Gericke 2012). That is, the x-axis of Figure 36 may be
considered as “displaced” to the left relative to that of Figure 32. The relevant
point, however, is that the solubility of cellulose shows non-ideal behavior
with positive deviation. This can be traced to cellulose interactions with both
solvents, akin to WB.
Chapter # 06 Results & Discussion
Page 167
Figure 36. Dependence of cellulose dissolution in IL-DMSO on the mole
fraction of DMSO, DMSO, at 80 °C. The parts refer to microcrystalline
cellulose and mercerized cotton.
Chapter # 06 Results & Discussion
Page 168
(Clear) (Turbid) (Clear) (Turbid)
(Clear) (Turbid) Clear) (Turbid)
Figure 37. Solubilization of cellulose in Binary mixtures
Chapter # 07 Experimental
Page # 169
CHAPTER # 7
EXPERIMENTAL
GREEN CHEMISTRY
Chapter # 07 Experimental
Page # 170
7.1 Chemicals
All the solvents and reagents were purchased from Alfa-Aeser, Aldrich or
Merck; were treated with appropriate drying agents, according to the
literature (Armarego 2003) and distilled at reduced or amdient pressure as
needed. Microcrystalline cellulose (MCC) was purchased from Fluka and
dried. Ethanol were refluxed with sodium metal and distilled. 1-chloro-2-
methoxyethane, 1-chloro-2-methoxyethane, N-methyl-imidazole, DMSO,
were stirred with CaH2 and distilled. Alcohols and 2-chloroethanol, 3-chloro-
1-propanol and K2CO3 were stirred with anhydrous MgSO4 and filtered, and
distilled in the presence of K2CO3. All solvents, except acetone were distilled
after packed with activated molecular sieve 4A, activation was done by
heating at 150 °C for three hours, cooling at reduced pressure and
immediate employment (This measure aimed at minimizing the water
absorption of the solvent). The purity of the solvents was checked by density
measurements and polarity ET (WB). The probe -carotene (Fluka, purity
97.0%) was employed either as received; the probe WB was available from a
previous study (Reichardt 2003). Commercially available ClNF gave
calculated values of elemental analysis. C 63.5, H 3.2, N 5.7; analyzed C
63.4, H 3.0, N 5.1.
7.2 Equipment
The melting points were determined with Electrothermal IA 6304 mp
apparatus (London). Elemental analyses were carried out at the central
Chapter # 07 Experimental
Page # 171
analytical facility of this Institute, using Perkin-Elmar Elemental Analyser
CHN 2400. All densities were measured by using DMA-40 resonating tube
digital densimeter (Anton Paar, Graz).1H and 13C NMR measurements were
recorded with Varian Innova-300 or Bruker DPX 300 NMR spectrometers
(both operating at 300 MHz for 1H, δ in ppm, J in Hz).
7.3 Synthesis and Purification of the Solvent Acidity Probe, SA (DTBSB)
(X. Q. Cheng 2008)
H3C-N
O
C(CH3)3
C(CH3)3
269-271 °C
Melting Point
Color
Sharp Green
It is a two steps synthesis. First step involves the synthesis of Picolinium
salt, leasds to the condensation reaction of 3,5-di-tert-butyl-4-
hydroxybenzaldehyde and of 1,4-methylpyridinium iodide (Picolinium salt)
for the formation of a (DTBSB).
7.3.1 Synthesis of 1,4-methylpyridinium iodide
2.48g (20 mol) of Idomethane having molecular mass 141.93 and boiling
point 42 ˚C was treated with picoline 1.77g (19 mol) in the presence of
acetonitrile (B.P 82 ˚C). The reaction mixture was reflux for 5 hours. The
obtained product was Picolinium salt having meting point 156-158 ˚C.
Chapter # 07 Experimental
Page # 172
RIN+
CH3
I-
R
CH3
N
+
Acetonitrile
Reflux 5 hours
Scheme 5 Synthesis of 1, 4-methylpyridinium iodide
7.3.2 Synthesis of O-di-tert-butylstilbazolium betaine (DTBSB)
N+
CH3
RI-
CH
t-Bu
OH
t-Bu
O
H3C-N
O
C(CH3)3
C(CH3)3
+
Pipredine
KOH Base
Scheme 6 Synthesis of o-di-tert-butylstilbazolium betaine (DTBSB):
DTBSB was prepared by taking 1.0 g of 1,4-dimethylpyridinum iodide (4.25),
1.03 g of 3,5-di-tert-butyle-4-hydroxybenzaldehyde (4.25) 3.6 g of piperidine
(4.25), dissolving them into anhydrous 7ml of EtOH. After a refluxing of 22
hours, on cooling at room temperature a solid residue formed, this was
filtered off and washed with 15 ml of EtOH, four times. Then in 25 ml of
0.2M KOH, the solid residue was dissolved, stirred and heated for 3 hours.
On cooling slowly at room temperature, this solution turns grey and new
solid obtained by filtration. Upon recrystallization from hot water, deep-
green, well-shaped crystals obtained whish have melting point (274 ˚C)
(Decomposed). Purification was checked by taking NMR, TLC, Melting Point,
and solvatochromism in 5 solvents. The Molecular structure, number of
hydrogens and 1H NMR data for (DTBSB) is given below.
Chapter # 07 Experimental
Page # 173
Table 28 1H NMR data of (DTBSB)
7.4 Synthesis of 2,6-dichloro-4-(2,4,6-triphenyl-N-pyridinio)-phenolate
ET (WB) (Reichardt 2003)
Synthesis of 2,6-dichloro-4(2-,4,6-triphenyl-N-pyridinio)-phenolate involves
first the formation 2,4,6-triphenyl-pyrylium hydrogen sulphate, and then its
reaction with 4-amino-2,6-dichlorophenol in the presence of ethanol and
sodium acetate leads to the formation of ET(WB).
7.4.1 Synthesis of 2,4,6-triphenylpyrylium Hydrogen sulphate
H3C-N
O
C(CH3)3
C(CH3)3 (DTBSB)
Sr. No δ/ppm No. of H
1 1.4 (s) 9H
2 4.0 (s) 3H
3 6.5 (d) 1H, HG
4 6.8 (d) 1H, HD
5 7.3 (d) 1H,HF
6 7.35 (s) 1H,HC
7 7.78 (2d) 3H,HE+HB
8 8.4 (d) 2H,HA
Chapter # 07 Experimental
Page # 174
O+
Ph
Ph Ph
HSO4-
Melting Point
Color: Light Yellow
269-271 °C
Procedure
In a 25 ml flask, chalcon (4.28g, 0.0206 mol), acetophenone (1.24g, 0.0103
mol) and conc. H2SO4 (3.02 ml) was heated on a steam bath for 3 hours. 20
ml of water was added after 3 hours reflux, precipitate formed that dissolve
on further heating. In the presence of heating dark brown color oil was
separated. It was then removed by gravity filtration. The filtrate was set aside
and yellow crystals obtained. The black oil was removed from the filter paper
with help of hot water and the filtrate was treated with 0.2 ml of conc.
H2SO4. Upon cooling the additional product of 2,4,6-tripheny-pyrylium
hydrogen sulphate was obtained. The purity of the 2,4,6-tripheny-pyrylium
hydrogen sulphate was checked by taking melting point and TLC.
O
O
CH3
O+
H2SO4
+
Conc.H2SO4
Scheme 7 Synthesis of 2, 4, 6-triphenyl-pyrylium hydrogen sulphate
Chapter # 07 Experimental
Page # 175
7.4.2 Synthesis of 2,6-dichloro-4-(2,4,6-triphenyl-N-pyridinio)-phenolate
ET(WB) by using 2,4,6-triphenyl-pyrylium hydrogen sulphate.
Ph Ph
Ph
O-
Cl Cl
N+
Melting Point : 210oC
Color : Dark blue
Synthesis of 2,6-dichloro-4-(2,4,6-triphenyl-N-pyridinio)-phenolate ET(WB)
was done by taking,4,6-tripheny-pyrylium hydrogen sulphate (6.16) and 4-
amino 2,6-dchlorophenol (4.40). Both are dissolved in 95% ethanol (70 ml).
After addition of anhydrous sodium acetate (4.0 g) the mixture was heated to
reflux for 3 hours. Then a 5% aqueous solution of sodium hydroxide (70 ml)
was added to the hot solution and ethanol removed in vacuum to yield deep
purple crystals, which were first washed with 1% sodium hydroxide solution
until the washing liquid become pale yellow. Finally these crystals were
washed with distilled water. During Drying over P2O5 at 120 ˚C and 1mbr the
color changing form purple, via orange to dark blue, because of the partial
loss of water molecules. Half of the molecules of water remain in the crystals
of the product during this drying process.
Chapter # 07 Experimental
Page # 176
H2SO4
Cl Cl
NH2
OH
N+
Cl Cl
O-
O+ +
1) NaOAc/EtOH
2) NaOH
The melting point of the final dried product is 210 ˚C (Decompose). The
product was confirmed by taking H-NMR in DMSO. Below is the data of
H-NMR.
Table 29 H-NMR data of ET(WB
N+
Cl Cl
O- ETWB
Sr. no δ/ppm No. of H
1 8.47 (s) 2H
2 8.26 (m) 2H
3 7.60 (m) 3H
4 7.40 (m) 10H
5 6.90 (s) 2H
Chapter # 07 Experimental
Page # 177
7.5 Synthesis of 1-Methyl-5-nitroindoline (MNI) (J. Catalan 2006)
N+
O-
O
N
CH3
Color: Orange
Melting Point: 113-114oC
5-nitrorindoline (NI) was purchased from Aldrich and was purified by using
by using dichloromethane/hexane (6:4) as eluent in silica gel column
chromatography.
To a solution of 6.0 g (0.037 mol) of 5-nitroindoline and 3.00 g (0.037 mol) of
sodium carbonate in a 20 ml of tetrahydrofurane, 2.9 ml (0.047) of
idomethane was added drop wise, with stirring and boiling and reflux for 24
hours. The reaction medium was made basic, after the refluxing completed
by the addition of sodium carbonate and extracted with chloroform. The
extracted was dried by using magnesium sulphate, filtered and solvent was
removed. The resulting brown residue was purified by silica gel column
chromatography (hexane/dichloromethane/ethylacetate; 5.5:3.0:1.5) yielding
of 1-methyl-5-nitroindoline as an orange yellow solid with melting point 113-
114 ˚C.
Chapter # 07 Experimental
Page # 178
N
H
O2N
CH3I N
CH3
O2N
+Sodium Carbonate
Tetrahydrofurane
Scheme 9 Synthesis of 1-Methyl-5-nitroindoline (MNI)
Table 30 The H-NMR data of MNI (CDCl3)
N
N+
O-
O
CH3
MNI
Sr. No δ/ppm No. of H
1 7.96 (dd) 1H,HZ,6-H
2 7.77 (d) 1H,HZ,7-H
3 6.20 (d) 1H, HZ,7-H
4 3.60 (t) 2H, HZ,2-H
5 3.00 (t) 2H, HZ,3-H
6 2.87 (s) 3H
7.6 Synthesis of 3,6-diethyl-1,2,4,5-tetrazin (W. Skorianetz 1970)
N
NN
NCH3
H3CColor
Red oil
3,6-Diethyl-1,2,4,5-tetrazine
Synthesis of 3,6-diethyl-1,2,4,5-tetrazine involves three steps
Chapter # 07 Experimental
Page # 179
7.6.1 Synthesis and Characterization of 3,6-diethylhexahydro-tetrazine
NN
NN
CH3
H3CColor
Yellow
3,6-Diethyl-Dihydrotetrazine
5.9 ml of propionaldehyde was mix with 2 ml of ethanol. 3.2 ml of hydrazine
hydrate was added drop wise by putting the reaction mixture in ice bath.
After complete addition of hydrazine hydrate the reaction mixture was put in
ice bath for one hour. White fogy product appears after some time. Filtered
the product when all reaction mixture converted into the crystals, wash it
with ethanol and ether and recrystallized with chloroform yield (69%)
product with melting point 129-132 ˚C.
H3C H
O
NH2-NH2 H2O
HN
NH
NH
HN
CH3H3C+
ETOH
Scheme 10: 3, 6-diethyl-hexahydro-tetrazine
Table 31: Result of IR analysis of Intermediate (3,6-diethyl-
hexahydrotetrazine).
Chapter # 07 Experimental
Page # 180
Table 32 Elemental analysis of (3, 6-diethyl-hexahydrotetrazine).
.
7.6.2 Oxidation of 3,6-diethyl hexahydrotetrazine into 3,6-diethyl-1,6-
dihydrotetrazine
7g (0.0372) of 3,6-diethylhexahydrotetrazine dissolved in 3.3% of 93 ml of
sodium hydroxide solution. Then added 100 mg of platinum oxide and the
reaction mixture was stirred in the presence oxygen for 11 hours at 16 ˚C.
After stirring the reaction mixture was saturated with NH4Cl and extracted
with ether. After the evaporation of ether and concentrating the reaction
mixture the yellow oil is obtained. The confirmation of the product formation
HN
NH
NH
HN
CH3
H3C
DETZ
Sr. No Type of gorup IR (KBr cm-1)
1 N-H 2970-2840
2 C-H 1625-1500
3 N-H 1544
4 C-H 1370-1385
Sr. No % Hydrogen % Nitrogen % carbon
1. 11.09 38.58 49.76
Chapter # 07 Experimental
Page # 181
was checked by taking UV-VIS spectra and observes the λmax for different
solvents. M.P 41-43 ˚C.
N
NN
HN
CH3H3C
HN
NH
NH
HN
CH3H3C
PO2/O2
Scheme 11 Oxidation of 3, 6-Diethyl hexahydro-tetrazine into 3, 6-Diethyl-
1,6 dihydro-tetrazine
7.6.3 Oxidation of 3,6-diethyl-1,6-dihydrotetrazine into 3,6-diethyl-
1,2,4,5-tetrazine
N
NN
N
CH3H3C Color
Red Oil
400 mg 3,6-dethyl-1,6-dihydrotetrazine was dissolved in 70 ml of water with
2.89 g of sodium nitrite and 2.44 ml of glacial acetic acid was added drop
wise in the reaction mixture. The reaction mixture is stirred at 0 ˚C for one
hour and at room temperature for 2 hours. After stirring the product was
extracted with ether until the reaction mixture become colorless and all
products was extracted, after evaporation the ether left red color oil product.
For purification the solution of product in dimethyl chloride filtered over 40 g
of silica. 354 g (90%) of red oil pure product was obtained. Confirmation of
the product was done by measuring λmax with different solvent and taking IR.
Chapter # 07 Experimental
Page # 182
N
NN
N
CH3H3C
N
NN
HN
CH3H3C
NaNO2/HNO3
Scheme 12 Oxidation of 3, 6-diethyl 1, 6-dihydrotetrazine into 3,6-diethyl-
1,2,4,5-tetrazine
Table 33 Elemental analysis of 3, 6-Diethyl-1,2,4,5-tetrazine
Table 34 UV.VIS results data of 3, 6-Diethyl-1,2,4,5-tetrazine
Sr. No Solvent Measured value λmax Literature Value λmax
1 EtOH 539 539
2 MeOH 535 536
3 Acetic acid 534 534
7.7 Synthesis of ILs (C4MeImCl) by Microwave Assistance
N NH3CH3C Cl N NH3C
R
Cl-
+ +
Scheme 13: 1-(1-butyl)-3-methylimidazole acetate
Sr. No % Hydrogen % Nitrogen % carbon
1 51.97 7.29 40.05
Chapter # 07 Experimental
Page # 183
In a glass jar with 100 ml of capacity, equipped with a reflux condenser was
added the 23.14 g (22.27 ml, 0.25 mol) of 1-chlorobutane, and 20.99 g
(19.94 ml, 0.25 mol) of N-methylimidazole. The flask containing the reaction
mixture was inserted into the microwave oven (Model DU-8316 Discover,
CEM, Matthews, temperature control device with infrared) in which reaction
undergone a power of 100 W, at temperature 100 ˚C for 1:40h. After
completion the reaction, the product was extracted four times with ethyl
acetate to remove the unreactive reactants and to neutralize the product.
The recorded melting point of the C4MeImCl was 41-42 ˚C.
Figure 36. 1H NMR spectrum in CDCl3 C4MeImCl
7.8 Synthesis of IL (C3OMeIm)Cl by Microwave Assistance
Chapter # 07 Experimental
Page # 184
N NH3C
OCH3
Color
Light Yellow
M.P 52-53oC
In a glass jar with 100 ml of capacity, equipped with a reflux condenser was
added the 25.12 g (24.27 ml, 0.25 mol) of 1-chloro-2-methoxyethane, and
18.48 g (17.94 ml, 0.22 mol) of N-methylimidazole. The flask containing the
reaction mixture was inserted into the microwave oven, T = 100 ˚C for 60
minutes. After completion the reaction, the product was extracted four times
with ethyl acetate to remove the nonreactive reactants and to neutralize the
product. The recorded melting point of the C3OMeImCl was 71-72 ˚C.
NN
H3CH3C
O ClNNH
H3CR
Cl -
+
Scheme 14 Synthesis of IL C3OMeImCl
Chapter # 07 Experimental
Page # 185
Figure 37. 1H NMR spectrum in CDCl3 C3OMeImCl
Table 35: 1H NMR spectrum in CDCl3 C3OMeImCl
7.9 Ion Exchange of Anions Cl- to CH3COO-
NNH3C
OCH3
Cl-
H1
H5 H7
H6
H7 H8
H2
C3OMeImCl
NNH3C
CH3
Cl-
H8
H7
H6
H1
H2
H5 H4
H9
C4MeImCl
δ/ppm J/HZ δ/ppm J/HZ
H2 10.448 (s) 10.548 (s)
H4 7.620 (s) 7.793 (s)
H5 7.617 (s) 7.639 (s)
H6 4.604 (t) J6-7=4,8 4.349 (t) J6-7=7.4
H7 3.777 (t) 1.913 (qt)
H8 3.368 (s) 1.377 (m)
H9 0.958 (t)
H10 4.132 (s)
H1 4.113 (s)
Chapter # 07 Experimental
Page # 186
For the purpose of verifying the influence of anions on the dissolution of IL
the pulp was performed changed, as described below.
NNH
H3CR
Cl-
CH3COO-
NNH
H3CR
CH3COO-
Ion Exchange
Scheme 14 Ion exchange of anions Cl- to CH3COO-
Given mass of said IL was dissolved to prepare a solution 0.1 mol/IL in
methanol. This solution was eluted on a column which was packed with 170
ml of purolite resin SGA-55-0OH (1.10 meq/ml) (Cl- →-OH) with methanol as
the solvent. The passage of the solution through the column was tested by
the absence of chloride ion, using an acid solution nitrate silver (AgNO3). The
elute was collected and neutralized with a solution methanolic acetic acid
(CH3COOH). The solvent was removed on a rotary evaporator (Buchi
Rotavapor Model R 110). The reaction mixture was placed in an ice bath (-30
°C) and to it was added 30ml of ethylacetate, under intense agitation. After
addition, the mixture was kept standing for 3 hours was placed in an ice
bath (-30 °C) and to it was added 30 ml of ethylacetate, under intense
agitation. After addition, the mixture was kept standing for 3 hours was
noted for phase separation. With the aid of a dropping funnel, the ILs were
separated. The ILs were subjected to 1 atm and temperature of 80 °C. The
final product obtained was in both cases a yellowish liquid.
Chapter # 07 Experimental
Page # 187
Table 36 1H NMR spectrum in CDCl3 C3OMeImCH3COO-
NNH3C
O CH3
CHCOO-
H8H7
H6
H1
H2
H5 H4
H9
C30MeImAc
H9NNH3C
CH3
CHCOO-
H8
H7
H6
H1
H2
H5 H4
H10
C4MeImAc
δ/ppm J/HZ δ/ppm J/HZ
H2 11.047 (s) 11.001 (s)
H4 7.437 (s) 7.426 (s)
H5 7.336 (s) 7.343 (s)
H6 4.540 (t) J6-7 = 4.8 4.248 (t) J6-7 = 7.4
H7 4.739 (t) 1.879 (qt)
H8 3.351 (s) 1.372 (m)
H9 1.965 (s) 0.945 (t)
H10 1.954 (s)
H1 4.032 (s) 4.050 (s)
Figure 38 1H NMR spectrum in CDCl3 C4MeImAc
Chapter # 07 Experimental
Page # 188
Figure 39. 1H NMR spectrum in CDCl3 C3OMeImAc
7.10 UV/Visible Spectroscopic Measurements of Dye Solvatochromism
Spectrophotometer measurements have been done on 15 0.1 ˚C, 25 0.1
˚C, 40 0.1 ˚C and 60 0.1 ˚C by using Shimadzu UV-2500
spectrophotometer, equipped with model 4029 digital thermometer (Control
Company, Friendsmood), with following experimental conditions; At 140
nm/min each spectrum was calculated three times; silt width 0.5 nm; with
sample interval 0.2 nm. To check the accuracy of peaks of λmax known peaks
of a helium oxide glass filter (model 666-F1, Hellma Analytics, Müllheim)
used routinely. The calculation of the λmax was done by taking first derivative
of the absorption spectra by using commercial software (GRAMS/32 version
5.10, Galactic Industries); the uncertainty in λmax is ± 0.2 nm. The
concentration of final probe was 2-5x 10-4 mol L -1. No change in the λ max or
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Page # 189
shape of the charge transfer band in the UV-visible spectra of the probe
solution was observed in the range of 1x10-4-1x10-3 mol L-1. From this
behavior it was clear that during the experimental conditions no
intermolecular interaction was occur. The determination of parameters takes
place by taking into account the value of μ (wave number) of the respective
probe for the corresponding calculations.
The determination of parameters takes place by taking into account the
value of µ (wave number) of the respective probe for the corresponding
calculations. Following correlation has been used;
*
The value of * is calculated by considering the values of of DMANF and
CLNF and of DMSO = 6862 by using the following correlation (30). It
includes first the calculations of solvent polarity (SP) and solvent dipolarity
(SD) of binary mixtures which can be calculated by using following equation:
SP = (gas phase - ∆ʋ solvent)/(gas phase - CS2) (30)
SD = (Vo
max;DMANF,solvent-Vmax;DMANF, solvent) / (Vomax;DMANF;DMSO –Vmax;DMANF;DMSO)
(31)
*= (solvent- difference)/( difference) /( DMSO) (32)
The value of was calculated by using the values of 1-methyl-5-nitroindoline
(MNI) and nitroindoline (NI) and the constant values of solvent 0 and solvent
202 from the list available in literature by using following equation: i.e.
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Δʋ Sol 0 =1570 and Δʋ solv 202= -165
(sol-Sol 0)/(sol 202-sol 0) (33)
The value of acidity was calculated by using 3,6-diethyl-1,2,4,5-tetrazin and
taking into account the values of SPP already calculated by using (31) and
(32).
DETZ = (1.0147±0.0579) SPP + (17.511±0.045) (34)
±0.069) DETZ + (0.339±0.024) (35)
7.11 Density measurement:
By using DMA 4500M resonating tube density meter (Anton Paar) the
density of the above mentioned liquids( pure solvent and binary mixtures)
has been measured at 15, 25,40 and 60C .
7.12 Preparation of binary Mixtures:
Preparation of samples of binary mixtures (16 per set, S = solvent, DMSO/W)
for solvatochromic studies and density measurement was done at 25 oC. The
range of the sample concentration was from 0(pure IL) to 1 (pure S) range of
or . For solvatochromic studies the addition of probe was done by
following method;
Preparation of stock solution in acetone of known concentration by
using I ml volumetric tube.
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From stock solution pipette 1 ml volume in a tube so that the final
concentration of the solution stay between 2 and 5x10-4 mol L-1, at the
end acetone is evaporated under reduce pressure in the presence of
P4O10. Finally the binary mixture of solvents is added in the probe and
the solubilization done with the help of pipes (Labquake, Lab
Industries 30min).
7.13 UV/Vis Spectroscopic measurements of ET(WB):
Spectrophotometer measurements have been done on 15 0.1C, 25 0.1C,
40 0.1C and 60 0.1C by using Shimadzu UV-2500 spectrophotometer,
equipped with model 4000A digital thermometer (Control Company,
Friendsmood), with following experimental conditions; At 140nm/min each
spectrum was calculated three times; silt width 0.5nm; with sample interval
0.2nm. To check the accuracy of peaks of λmax known peaks of a holium oxide
glass filter (model 666-F1, Hellma Analytics, Müllheim) used routinely. The
calculation of the λmax was done by taking first derivative of the absorption
spectra by using commercial software (GRAMS/32 version 5.10, Galactic
Industries); the uncertainty in λmax is ± 0.2 nm. The concentration of final
probe was 2-5x 10-4 mol L -1. No change in the λ max or shape of the charge
transfer band in the UV-visible spectra of the probe solution was observed in
the range of 1x10-4-1x10-3 mol L-1. From this behavior it was clear that during
the experimental conditions no intermolecular interaction was occur. The
determination of parameters takes place by taking into account the value of μ
(wave number) of the respective probe for the corresponding calculations.
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Page # 192
7.14 Measurement of density of pure and binary mixtures:
For the measurement of density digital denstimeter (DMA 40 digital
densimeter, Anton Paar) was used which is equipped with thermostated
enclosure and density of binary mixtures and pure solvents were measured
at 15, 25, 40 and 60C.
7.15 Solubilization of Cellulose in ILs-DMSO mixtures:
Cellulose samples (MCC or M-cotton) were stirred with Il-DSMO mixtures
(e.g;50 mg cellulose in 5g solvent mixtures) in closed vials at 80 oC for 30
min. Cellulose dissolution was judged visually, using a magnifying glass
provided with white led light (1x2 amplification), and then with aid of Nikon,
Eclipse 2000 microscope (x40; polarized light). If the cellulose was not
soluble, we heated the mixture for additional 90 min, and examine the
sample after each 30 minutes. We considered that cellulose is insoluble if its
hits fibers were still visible after each 2h of heating. The results of this
experiment is given in wt % dissolved cellulose=[ mass(cellulose)/mass(cellulose=IL-
DMSO)].
7.16 Theoretical calculations:
7.16.1 Molecular dynamics, MD simulations:
We used Gromacs 5.0 software package for MD simulation (van der Spoel
2005). Two systems were simulated, each containing the following numbers
of molecules: 20, WB; 330, IL; 670, DMSO, or 670 (SPC/E model) water.
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(Berendsen 1987) These compositions correspond to the following
concentrations (in mol L-1) = 0.176, 2.91, and 5.90 for WB, IL and DMSO,
respectively.
The corresponding concentrations for the water-containing box are: 0.256,
4.22, and 8.57 for WB, IL and W, respectively. These concentrations were
calculated based on box volumes of 188.616 nm3 for WB/IL-DMSO, and
129.798 nm3 for WB/IL-W.
We optimized the geometry of WB and the IL (gas phase) by using DFT
calculations, employing “good-opt” parameter, using the Orca 2.9 program
(Neese 2011). Partial charges on the atoms were calculated by using the
RESP (Restrained ElectroStatic Potential fit) approach (Bayly 1993) as
calculated by the RED (RESP ESP charge Derive) on-line server (Vanquelef
2006) The topologies files for GAFF (General Amber Force Field) were
generated using the Acpype (Wang 2004) and Antechamber 12 programs
(Martínez 2009) GAFF-optimized geometry and topology of (SPC/E) water
were taken from the Gromacs package; those for DMSO molecules were
taken from literature (Bennett 1976) The simulation boxes were generated by
using Packmol program.
We carried out an initial equilibration phase for both boxes, first using a NVT
ensemble, followed by using a NPT ensemble; each equilibration for 100 ps.
Subsequently, both systems were “annealed” as follows: the simulation
boxes were heated from 298 to 473K (DMSO) or 370K (W) in 2 ns under
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constant volume. They were kept at 473 (DMSO) or 370K (W) for 6 ns, and
then cooled to 298K in 2 ns. After pressure equilibration to 1 bar (during
0.25 ns), the simulation data were acquired for 40 ns under NTP conditions
and at 298K. We repeated this annealing procedure four more times, each
starting from the previously annealed and equilibrated system. We checked
the equilibration of the ensembles by monitoring the potential energy and
density (in g mL-1) as a function of simulation time. The former reached
equilibrium, i.e., remained essentially constant, after ca. 10 ns from the
start- until the end of simulation. The calculated averaged system densities,
ρ were = 1.1192 ± 0.0003 and 1.1113 ± 0.0002 g/mL for IL/DMSO and
IL/W, respectively. The experimental densities, at the same temperature,
298K were 1.07670 ± 0.00005 g/mL and 1.06310 ± 0.00005, for IL-DMSO
and IL-W, respectively; leading to 3.9% and 4.5% difference between the
calculated and experimental densities, respectively. We analyzed the results
of these five MD annealing cycles using the radial distribution function of
pairs, g (r). Based on this function, we calculated the numbers, and
distances between pair of species, which can be atoms, ions or molecules.
7.16.2 Free energy of solvation of WB (ΔGSolv):
The value of (ΔGSolv) was calculated from MD simulations using Bennett
Acceptance Ratio (BAR) free energy perturbation approach (van der Spoel
2005) as implanted in the Gromacs 5.0 software package. We did this as
follows: with the system thermally equilibrated, we manipulated the program
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to “turn off” the Coulumbic and van der Waals (Lennard-Jones) interactions
between WB and the components of binary mixture. This was done with the
help of the so-called coupling parameter, λ. We turned off each of these
interactions separately in 21 steps, each corresponding to 10% increment in
λ (the initial condition plus 10 steps for Coulumbic interactions, and 10
steps for van der Waals ones). This results in a free energy change of
solvation, ΔGSolv (= ΔGSolv,Coulumbic + ΔGSolv,van de Waals), calculated from the
derivative of the corresponding enthalpy with respect to λ (∂H/∂λ). The
calculated values of (∆GSolv) of WB in mixtures containing = 0.67 were -
176.02 ± 4.28 and -126.64 ± 1.90 kJ mol-1 for IL-W, and IL-DMSO,
respectively. At this the plots of ET (WB) versus showed maximum
deviation from linearity.