Republic of Iraq

Ministry of Higher Education

And Scientific Research
Al-Nahrain University
College of Science
Department of Chemistry

Preparation and biological study of

some ionic liquids derived from
aluminum and calcium salts-amine
A Thesis
Submitted to the College of Science/Al-Nahrain University as a partial
fulfillment of the requirements for the Degree of Master of Science in
Chemistry

By:
Bassam Baqer Hasan
B.Sc. Chemistry / College of Science / University of Baghdad
(2000-2001)

Supervised by

Prof. Dr. Assist. Prof.Dr.

Hadi Mohammed Ali Abood Nadira S.Mohamed Al Gra’awy

2018 A.D 1439 A.H

 Supervisor Certification
We certify that this thesis entitled '' Preparation and biological study of some
ionic liquids derived from aluminum and calcium salts-amine'' under our
supervision at the College of Science/Al-Nahrain University as partial
fulfillment of the requirements for the Degree of Master of Science in
Chemistry.

Signature: Signature:
Name: Dr. Hadi M. A. Abood Name: Nadira S.Mohamed Al Gra’awy
Title: Prof. Dr. Title: Assist. Prof. Dr.
College of Science Forensic DNA Research and Training Center

University of Al-Nahrain University of Al-Nahrain

Date: / / 2018 Date: / / 2018

______________________________________________

In view of the available recommendation, I forward this thesis for

debate by the examining committee.

Signature:
Name: Dr. Mehdi S. Shihab
Scientific Degree: Prof. Dr.
Head of Chemistry Department
College of Science
Al-Nahrain University
Date: \ \ 2018
 Committee Certification

We, the examining committee certify that we read this thesis entitled
''Preparation and biological study of some ionic liquids derived from aluminum
and calcium salts-amine '' in its contents and that in our opinion, it is accepted
for the Degree of Master of Science in Chemistry.

Signature:
Name: Mahmoud Najim Abid Aljibouri
Scientific Degree: Prof. Dr.
Date: \ \ 2018
(Chairman)

Signature: Signature:
Name: Jwan Abdulmohsin Zainulabdeen Name: Ammar J. Alabdali
Scientific Degree: Assist. Prof. Dr. Scientific Degree: Instructor. Dr.
Date: \ \ 2018 Date: \ \ 2018
(Member) (Member)

Signature: Signature:
Name: Dr. Hadi M. A. Abood Name: Nadira S.Mohamed Al Gra’awy
Scientific Degree: Prof. Dr. Scientific Degree: Assist. Prof. Dr.
College of Science Forensic DNA Research and Training Center
University of Al-Nahrain University of Al-Nahrain
Date: / / 2018 Date: / / 2018
(Member/Supervised) (Member/Supervised)
__________________________________________________________

I, hereby certify upon the decision of the examining committee.

Signature:
Name: Hadi M. A. Abood
Scientific Degree: Prof. Dr.
Dean of the College of Sciences
Date: \ \ 2018
 ‫بِسْ ِمِ اهلل الرَّحْمنِ الرَّحِي ِمِ ِ‬

‫ِ‬
‫هلله العَظِي ِمْ ِ‬
‫صَدَقَِِا ِ‬

‫سورةِالنحل ِ‬

‫اآليةِ{‪ِ }78‬‬

‫ِ‬

‫ِ‬
 ‫اإلهداء‬
‫إلى من افتقده في مواجهة الصعاب ولم تمهله الدنيا ألرتوي من حنانه‪ ..‬أبي‬

‫الى من عانت الصعاب ألصل إلى ما أنا فيه وعندما تكسوني الهموم أسبح في بحر‬

‫حنانها ليخفف من آالمي ‪ ..‬أمي‬

‫إلى منهل العلم و النجاح والصبر استاذي ومشرفي الف اضل الدكتورهادي‬

‫الى اشراقة التف ائل وكوكب المعرفة استاذتي ومشرفتي الف اضلة الدكتورة نادرة‬

‫إلى من شجعني وساندني على مواصلة مسيرتي العلمية رفيقة دربي زوجتي‬

‫الغالية‬

‫إلى رياحين حياتي في الشدة والرخاء اوالدي االعزاء‬

‫الى عزتي واصراري أخي وأخواتي‬

‫وإلى كل من شجعني وساعدني على إتمام هذا العمل‬

‫أهدي ثمرة جهدي‬

‫بسام باقر حسن‬

 ACKNOWLEDGMENTS

First of all, Thanks to ALLAH for his blesses. The honor is mine to express
my sincere thanks and gratitude to my supervisors Dr. Hadi M. A. Abood and
Dr. Nadira S. Mohamed for suggestion this project, advice, continuous
encouragement, their valuable guidance and sustained efforts throughout my
study.
Sincere thanks are due to the Chemistry Department, Al-Nahrain University
and to Staff of the department, specially the head of the department Dr. Emad
Al Sarraj and Dr. Nasreen Raheem Jber .
A great thanks are gone to the staff of microbiology laboratories of Al Karama
Hospital in Baghdad for providing the bacteria strains used in this study, and
also to the staff of clinical chemistry laboratories of Al-Yarmouk Teaching
Hospital in Baghdad to examine the samples of serum.
My deepest thanks and appreciation to my lovely family, for their patience with
me and their moral support.
Finally, sincere thanks and deep respect go to all my doctors in chemistry
department for their support. Thanks to all my friends for their help and
support.

Bassam 2018
Abstract
The recent work includes attempts to prepare different types of ionic liquids
from direct reaction of some amines components like ethanolamine, urea,
acetamide, thiourea and thioacetmide, with aluminum salts like aluminum
chloride, aluminum nitrate, ammonium aluminum sulfate, potassium aluminum
sulfate and aluminum stearate. Some of these attempts produced ILs using
varying temperatures up to 80°C and other auxiliary factors (ccl4 and
chloroform). Some physical characteristics such as conductivity, melting point,
FT-IR and electronic spectra measurements have been measured to the ionic
liquids.

On the other hand deep eutectic ionic liquid was prepared by reacting
Calcium Chloride dehydrate with acetamide. The resulting clear colorless ionic
liquid showed good heat stability ranged between a lowest freezing temperature
(-7 ˚C) and highest decomposition temperature (286.7 ˚C) with (1:7) mole ratio
respectively having ionic conductivity of (0.3 mS/ cm). FT-IR and UV spectra
was used to establish its interaction between the components that formed the IL.
Other physical properties, such as viscosity, density and cyclic voltammetry
have also been measured for the IL.

In addition to the preparation of ILs, the biological activity of some

aluminum and calcium-amine ionic liquids were evaluated on human serum
(HS) and some gram negative (E.coli, K.peneumonice, Pseudomonas and
Proteus) and gram positive (Staph.aureus and Staph.epidermidis), by measuring
the inhibition zone (The Kirby-Bauer Disk Diffusion Test), beside that the
optical density was measured at 600 nm for bacterial broth incubated at 37 ºC
for 24hrs with different concentrations of ionic liquids (2, 5, 10, 20% (v/v)) .
Subsequently, the results of optical density were turned and calculated as colony
forming units (CFUs). This work showed a positive effect on inhibition bacteria
growth by several factors like the type, structures, kind of anion and cation of
ILs, water miscibility, concentration of ILs and the type of microorganism in
both solid and liquid media.

On the other hand, the concentration of ILs was calculated practically until
reached the optimal concentration of 2% (v/v) causing no variation on serum
parameters likes (sugar, lipid, protein, liver functions, kidney functions, and
electrolytes), by using Automatic biochemistry analyzers technique.
 List of Contents
Subject Page No.
Abstract. I
Contents. III
List of Figures. VIII
List of Tables. X
Abbreviations. XII

Page
Subject No.

Chapter one: Introduction

1:1 Ionic liquids 1

1.2 History of ionic liquid 3

1.3 Synthesis of ionic liquids 5

1.4 Deep Eutectic Solvents (DES) 9

1.5 Properties of ionic liquids 12

1.6 Classification of ionic liquids 14

1.7 Aluminum salts – amine ionic liquids 19

1.8 Biological activity of ionic liquids and toxicity 20

1.8.1 Organism-dependent biological activity of ionic liquids 22

1.8.2 The biological activity of ILs 23

1.9 Clinical biochemistry 26

1.10 Biochemical tests 27

1.10.1 Automated analyser 28

1.10.2 Automatic biochemistry analysers 29

1.11 Bacteria 30

1.12 Morphology and cellular structure 31

1.13 Bacterial culture media 32

1.14 Classification of bacterial culture media 33

1.15 Bacterial Enumeration 34

1.15.1 Standard plate count (colony-forming units) 34

1.15.2 Turbidimetric measurement 35

1.16 Antibiotic sensitivity testing: The Kirby-Bauer Disk

36
Diffusion Test

1.17 Aim of the work 38

Chapter Two: Materials and Methods

2.1 Chemicals 39
2.2 Instrumental and Techniques 40

2.3 Synthesis of Ionic Liquid 42

2.3.1 Preparing RTIL by reacting Lewis base with several 42

aluminum salts.
2.3.2 Preparation of calcium chloride dihydrate /acetamide new
43
room temperature ionic liquid

2.3.2.1 composition/ freezing point, phase diagram of calcium

44
chloride dihydrate /acetamide ionic liquid

2.3.2.2 Determination of Conductivity, Density and Viscosity 44

2.3.2.3 Determination of Cyclic Voltammetry (CV) 45

2.3.3 Preparation of aluminum nitrate nanohydrate and hydrated

45
ammonium alum with urea and acetamide (RTILs)

2.3.3.1 Ionic liquid preparation with acetamide: 46

2.3.3.2 Ionic liquid preparation with urea 46

2.4 Cultivation conditions 46

2.4.1 Liquid cultures 46

2.4.2 Solid cultures 47

2.5 Biochemistry Testes 47

Chapter Three: Results and Discussion

3.1 Preparing room temperature ionic liquids by reacting Lewis
48
base with several Aluminum salts.

3.1.1 Monoethanolamine with aluminum chloride. 48

 49
3.1.2 Monoethanolamine with aluminum chloride in CCl4.

50
3.1.3 Monoethanolamine with aluminum chloride in chloroform.
3.1.4 Monoethanolamine with aluminum nitrate, ammonium
51
aluminum sulfate and Potassium aluminum sulfate.

3.1.5 Monoethanolamine with aluminum stearate. 51

3.1.6 Thiourea with ammonium aluminum sulfate and potassium

56
aluminum sulfate.

3.1.7 Thiourea with aluminum nitrate. 59

3.1.8 Aluminum stearate with thiourea and with thioacetamide. 60

3.1.9 Thioacetamide with ammonium aluminum sulfate and

60
potassium aluminum sulfate.

3.1.10 Thioacetmide with aluminum Nitrate. 61

3.2 Synthesis of New Ionic Liquid from calcium chloride

62
dihydrate /acetamide

3.2.1 Phase diagram binary of calcium chloride dihydrate

62
/acetamide

3.2.2 Simultaneous thermal analysis STA (TG – DSC) 64

3.2.3 Interaction between calcium chloride dihydrate /acetamide 66

3.2.4 FT-IR spectra of calcium chloride dihydrate /acetamide

66
ionic liquid

3.2.5 Electronic spectrum of calcium chloride dihydrate 68

/acetamide ionic liquid
3.2.6 Some physical properties of calcium chloride dihydrate
70
/acetamide new ionic liquid of 1:7 mole ratio.

3.2.7 Cyclic voltammetry of calcium chloride dihydrate/

73
acetamide ionic liquid.

3.2.7.1 Cyclic voltammetry of monoethanolamine with aluminum

chloride in chloroform and without chloroform, and aluminum 75
nitrate with thiourea.

3.3 Biological activity of ionic liquids 77

3.3.1 The Kirby-Bauer Disk Diffusion Test 77

3.3.2 The optical density (OD) test and colony forming units count 82

3.3.3 Biochemistry testes 93

3.3.4 Inhibition mechanism 102

Conclusions 103

104
Suggestions for future work:

References 105
 List of Figures
Figure Page
Description
No. No.
1-1 Simplified model of an ionic liquid 2
1-2 σ-complex of chloroaluminate in the Friedel-Crafts react. 3
1-3 Synthesis path for the preparation of ionic liquids 6
Series of equilibria in the reaction between [emim]Cl and
1-4 7
AlCl3
Structures of some halide salts and hydrogen bond donors
1-5 11
used in the formation of deep eutectic solvents.

1-6 Types of cations 14

1-7 The structures of imidazolium and pyridinium triplets 15
1-8 Heterocyclic non-aromatic (a) and aromatic (b) cations 16
1-9 Sample structure AIL (a) and PIL (b) 17
1-10 Generations of ionic liquids 18
A conglomerate forming (aluminum chloride with
3-1 49
ethanolamine).
3-2 A white solid product from mixing drops of (aluminum 49
 chloride /ethanolamine) liquid with water
A conglomerate forming in mixture of (aluminum chloride
3-3 49
with ethanolamine in CCl4)
3-4 Mixture of ethanolamine and AlCl3 in chloroform 50
Liquid mixture of ethanolamine and AlCl3 in chloroform after
3-5 50
two days
A white solid product from mixing drops of (aluminum
3-6 50
chloride with ethanolamine in chloroform) with water
Schematic representation of a eutectic point on a two
3-7 52
component (ALST/MEA) phase diagram
FT-IR spectra of ALST, MEA and ionic liquid (1:10) mole
3-8 55
ratio
Schematic representation of a eutectic point on a two
3-9 57
component (ALUM/Thiourea) phase diagram
Schematic representation of a eutectic point on a two
3-10 58
component (ALUM(K)/Thiourea) phase diagram
3-11 Thiourea with Aluminum Nitrate (2:1) mole ratio. 60
Thioacetmide with aluminum nitrate(2:1,1:1 and 1:2) mole
3-12 61
ratio
Schematic representation the eutectic point of two component
3-13 63
(CaCl2.2H2O/Acetamide) IL.
A :( TGA) and B: (DSC) analysis of calcium chloride
3-14 65
dihydrate /acetamide IL.
FT-IR spectra for calcium chloride dihydrate /acetamide new
3-15 68
IL.
Electronic spectrum of calcium chloride dihydrate /acetamide
3-16 69
IL.
3-17 Density & conductivity versus temperature for the new IL 72
3-18 viscosity & conductivity versus temperature for the new IL 73
Cyclic voltammogram for Pt elctrode in CaCl2.2H2O/
3-19 74
acetamide, with 1:7 mole ratio mixture at room temperature
 Cyclic voltammogram for Pt electrode in monoethanolamine
3-20 with aluminum chloride in chloroform (Red) and without 76
chloroform (Blue) at room temperature.
3-21 Cyclic voltammogram for Pt electrode in aluminum nitrate 76
with thiourea at room temperature.
3-22 The Bacterial inhibition zone against ILs 78-79
The optical density value for bacteria growth with deferent
3-23 84-87
concentration of ILs
Influence of A. (AN-AC) B. (ALUM-UREA) on growth of
K.peneumoniae in liquid medium. a: control, b: blank and
3-24 87
numbers 1-4 corresponds to 2, 5, 10 and 20% (v/v) of IL,
respectively (as example) .
Influence of A. (AN-AC) and B. (ALUM-UREA) on growth
of St. Aureus in liquid medium. a: control, b: blank and
3-25 numbers 1-4 corresponds to 2, 5, 10 and 20% (v/v) of IL, 88
respectively (as example).

schemes for Colony forming units (CFUs)/ml for a. E.coli, b.

3-26 K.peneumoniae, c. Staph.aureus, d. Staph. Epidermidis, e. 89-92
Pseudomonas and f. Proteus bacteria growth.
Colony forming on the agar media for A- K.peneumoniae and
3-27 92
B- St.aureus for 2%, 5% of IL (as example).
Serum aggregation due to the direct addition of aluminum
3-28 94
salts ionic liquids.
A statistic was performed for each serum parameters with and
without ionic liquids, as shown in the graphs above. Where
… Q: patient’s serum without any additives, ALUM-UREA,
AN-UREA, ALUM-Ac, AN-Ac and ….
CaCl-Ac ionic liquids. A: Statistic of Glucose, B: Statistic of 96-
3-29 Urea, C: Statistic of ALT, D: Statistic of AST, E: Statistic of 101
Cholesterol, F: Statistic of Triglycerides, G: Statistic of Uric
acid, H: Statistic of Calcium, I: Statistic of Chloride, J:
Statistic of Potassium, K: Statistic of Sodium, L: Statistic of
Total Protein, M: Statistic of Total bilirubin, N: Statistic of
Alkaline Phosphatase, O: Statistic of Albumin, P: Statistic of
 Creatinine.

List of Tables
Table Page
Description
No. No.
The history of the discovery and use of quaternary ammonium
1-1 1-2
salts
1-2 Examples of ionic liquids prepared by anion metathesis. 8

1-3 General Formula for the Classification of DESs 10

1-4 Normal Common Blood Chemistries testes 27-28

2-1 Specification of the chemicals used in current work 39-40
2-2 Binary mixture of amines and aluminum salts. 43
Viscosity, Conductivity and Density at different temperatures
2-3 44-45
of Calcium Chloride dihydrate /Acetamide ionic liquid
Compositions and physical Measurements for Aluminum
3-1 52
stearate with Ethanolamine.
3-2 FT-IR absorption bands of starting materials and ionic liquid. 54
3-3 Compositions and melting point of ALUM(NH4)/Thiourea 56
3-4 Compositions and melting point of ALUM(K)/Thiourea 57
Compositions and freezing points of new ionic liquid
3-5 64
CaCl2.2H2O/AC
 FT-IR absorption bands of the starting materials and ionic
3-6 67
liquid (cm-1).
Comparison density, viscosity, acidity and conductivity for
3-7 71
different ILs.
3-8 Density & Conductivity versus temperature for the new IL. 71
3-9 viscosity & Conductivity versus temperature for the new IL 72
3-10 The Bacterial inhibition zone diameter (mm) against ILs. 78
The optical density (OD) value for bacteria growth culture
3-11 with deferent concentration of ILs (The OD value without 82-83
incubation has been subtracted from the results).
3-12 Colony forming units (CFUs)/ml after 24h incubation 88-89
Clinical biochemical testes for serum human (Patient ID: 1 /
Gender: Female / Age: 42 / Draw Time: Morning / Type of
3-13 95
test: Random) with 2% of ionic liquids.
 Symbols and Abbreviations
IL Ionic Liquids

RTILs Room Temperature Ionic Liquids

DESs Deep eutectic solvents

PILs Protic ionic liquids

AILs Aprotic ionic liquids

HILs Herbicidal ionic liquids

CaCl Calcium Chloride dihydrate

TA Thioacetamide

Ac Acetamide

ALST Aluminum stearate

AN Aluminum Nitrate nanohydrate

ALUM(K) Potassium aluminum sulfate dodecahydrate

ALUM Ammonium aluminum sulfate dodecahydrate

MEA Monoethanolamine

[EtNH3] Ethylammonium

[emim] 1-Ethyl-3-methylimidazolium

[PF6]- Hexaflourophosphate
[BF4] Tetraflouroborate
[(TFO)2N] Trifluoromethanesulfonylimide

OTF Trifluoromethanesulfonate (Triflate)

 [OAc] acetate

[TFA] Trifluoroacetic acid

CB11H12 Carba-closo-dodecaborate

[C2MIm]+ 1-ethyl-3-methylimidazolium

[C4MIm]+ 1-butyl-3-methyimidazolium
[NTf2]− bis(trifluoromethylsulfonyl)amide anion

[Cho][Cl] cholinium chloride

STA Simultaneous Thermal Analyzer

TGA Thermogravimetric Analysis

DSC Differntial Scanning Calorimetry

CV Cyclic Voltammetry

𝛿 Bending vibration

ν Stretching Vibration

NB Nutrient broth

NA Nutrient agar

MHA Mueller-Hinton agar

CFUs Colony-forming units

OD Optical density

(K-B) Kirby-Bauer

MIC Minimum inhibitory concentration

MBC Minimum bactericidal concentration

LD50 lethal dose fifteen

 Chapter
One
Introduction
 Chapter one
Introduction
1.1 Ionic liquids
Ionic liquids (ILs) are defined as chemical compounds of ion structure,,
[3,4]
which have the melting point below 100°C . This property is usually a
result of a large size difference, between an organic cation of expanded
asymmetric structure and a small, organic or inorganic, anion . This hinders
the formation of a uniform crystal lattice,, thus greatly reducing the
[5,6a]
solidification point of the compound Figure (1-1) presents a general
model of an irregular system of ions ,in the IL structure.

Fig (1-1): Simplified model of an ionic liquid.

One of the main directions of the global research , is the search for new
chemical compounds with special , properties. Ionic liquids are such
compounds Their application brings new , possibilities for modern chemical
technology . The ionic liquids, fit, well, in the assumptions of green chemistry
[1]
. In contrast to the previous approach ,,the green chemistry requires
design, ,development and implementation of new processes and chemicals
that allow the reduction or elimination of use and production of
 [1]
hazardous, materials . Twelve principles of green chemistry ,, formulated
[2]
in 1998 by Anastas and Warner ,, describe the methods of
implementation of these tasks . Ionic liquids meet at least three of these
principles,,, i.e. principle no. 5 (safer solvents),, no. 6 (provide energy
efficiency) and no. 9 (are used in catalytic reactions) . Ionic liquid precursors
are quaternary ammonium halides,, known from the 1890s , which were
widely used and tested in the 20 th century. The history of synthesis and
application of organic salts containing a quaternary nitrogen atom is
presented in Table (1-1).

Table (1-1): The history of the discovery and use of quaternary ammonium salts [25].
Year Practical use Authors

1890 Synthesis Menschutkin

1914 solvent Walden

1916 Bactericidal properties Jacobs

Surface active properties

1935 Disinfection Domagk

1960s Fabric softener

1960 Phase transfer catalysis Jarousse-Mąkosza

1970s Antielectrostatics

1977 Wood preservation Butcher et al.

1980s Asphalt modifiers

1990s Clay modifiers

1996 Ionic liquids Seddon, Rogers

From the beginning of the 20th century,, chemical abstracts have been used
two keywords to describe these compounds: room - temperature ionic liquids
(RTILs) and ionic liquids (ILs) that apply ,to liquid and solid salts,
respectively , at room temperature. Currently,, such keywords as molten salt,
liquid organic salt or fused salt are no longer used to describe, ionic
liquids. Generally accepted abbreviations are ILs ,and RTILs.

1.2: History of ionic liquid

To search for the origins of research on ionic liquids, one has to look
back to the ,mid-19th century, when a low-melting organic salt has been
observed for the first time . It was a by-product produced the reaction of
benzene alkylation in the Friedel - Crafts reaction, with aluminium chloride
as a catalyst . The “red oil”, as at the time the substance was called , had
remained unidentified for over 100 years. Only after the popularisation of
nuclear magnetic resonance technique, it was possible to identify its, structure
Fig (1-2) [7-8].

Fig (1-2): σ-complex of Chloroaluminate in the Friedel-Crafts react.

At the turn of the 20th century,, the first reports on low-melting salts were
published . In 1888, Gabriel and Weiner obtained , ethanolammonium nitrate
[8]
with the melting point of 52° . In 1914, an ethylammonium nitrate was
[9]
described (melting point 12°C) , considered, the first low-temperature ionic
liquid. Therefore, Walden is considered as a „father” of ILs., This is contested
by Everts [10]
who quotes Boeck on the, fact that the Walden’s paper indeed
discussed conductivity of ethylammonium nitrate,, but their author did not
recognize its potential application . Moreover,, Boeck states that six years
before Schall, Ostwald’s (Nobel laureate in 1909) ,student had published a
paper on organic salts with a low melting point,, which are nowadays
classified as ILs [11]. Nevertheless, this discovery was, not found interesting
in scientific circles. It may be assumed , that an important reason for that was
their poor solubility in water, which was at the time ,considered universal
solvent. The 1950s brought interest to organic liquid salts. In 1963 Yoke
,described a reaction of copper(I) chloride with triethylammonium
[12]
hydrochloride,, which gave a liquid product . The progress in IL studies
was particularly impacted, by the series of studies conducted in the 1960s
and 70s at the U.S. Air Force Academy by King and his co-workers – Wilkes
and Carlin. These studies aimed to find an electrolyte for the thermal
battery. Based on patents from 1948 [13],, a cell, was developed containing an
ionic liquid produced in ,the reaction of aluminium chloride with 1-
ethylpyridinium bromide as an electrolyte [14]
. On the other hand, Koch’s
research team used 1-ethylpyridinium chloroaluminate ,as a solvent in
[15]
catalytic reaction . 1-Butylpyridinium chloroaluminate to ,be more
effective, and melted at 40°C [16]. Even ,better results were obtained for the 1,3-
dialkylimidazolium cation.,Combining 1-ethyl- 3-methylimidazolium chloride
[17]
with aluminium chloride gave RTIL . In 1986, a research team under the
leadership of Wilkes used it, as a solvent in the Friedel-Crafts reaction, thus
[18]
proving its catalytic activity, at the same time . It was observed that
pyridinium, as well as imidazolium chloroaluminates, ,break down in the
presence of water with the exudation of gaseous hydrogen chloride and are
[18]
even sensitive to atmospheric moisture . As a consequence,, their
application was burdensome,, because their processing required the anhydrous
conditions. Replacing a chloroaluminate anion with another anion resulted in
obtaining ILs that were completely stable in contact with an aqueous
solution. 1 Ethyl-3-methylimidazolium ILs,, insensitive to water, were
synthesised using inorganic (BF4 -, NO3 -, SO4 2-) and organic (CH3COO-)
[19]
anions . The increased interest in, ILs in the last two decades was caused
by several factors. Ionic liquids quickly started to be considered a “green”
alternative for volatile, flammable and often toxic popular organic solvents.
The ILs have been recognized as more environmentally friendly, solvents and
catalysts, and at the same time their unique advantage, i.e. designability,, has
started to be appreciated. ILs can be designed by selecting a proper cation
and anion to obtain a compound with desired properties. In this way it is
[20]
possible to synthesise ILs for a specific application . The term was
coined in the literature for such type of compounds – task specific ionic
liquids (TSILs). The name, was approved by academic circles after Davis’
[21]
publication in 2004 . The number of possible combinations of a cation
and anion was, estimated to be at level of 1018 [22]
. Such a great diversity
makes it possible to design a structure that will provide optimum properties
for very ,specific purposes. This directly translates into a potential application
of ILs in various ,technological processes [3,23]. In 2002, BASF, as one of the
first companies, has implemented a technology based on ILs (BASIL ™ –
Biphasic Acid Scavenging Utilizing Ionic Liquids™) [24].

1.3 Synthesis of Ionic Liquids

As stated in previous section (1:2), the first room temperature ionic liquid
[9]
[EtNH3] [NO3] (m.p.12∘C) was discovered in 1914 , but interest did not
develop until the discovery of binary ionic liquids made from mixtures of
aluminum (III) chloride and N-alkylpyridinium or 1, 3-dialkylimidazolium
chloride [26, 27].
Ionic liquids usually come in two main categories, namely, simple salts (made
of a single anion and cation) and binary ionic liquids (salts where equilibrium is
involved). For example, [EtNH3][NO3] is a simple salt whereas mixtures of
aluminum(III) chloride and 1,3-dialkylimidazolium chlorides (a binary ionic
liquid system) contain several different ionic species, and their melting point
and properties depend upon the mole fractions of aluminum(III) chloride and
1,3- dialkylimidazolium chloride present.
The synthesis of ionic liquids can be described in two steps (Figure 1-3).

Fig (1-3): Synthesis path for the preparation of ionic liquids [28].

(1) The formation of the desired cation:

The desired cation can be synthesized either by the protonation of the amine by
an acid or through quaternization reactions of amine with a haloalkane and
heating the mixture.
(2) Anion Exchange:
Anion exchange reactions can be carried out by treatment of halide salts with
Lewis acids to form Lewis acid-based ionic liquids or by anion metathesis. The
most extensively studied and used Lewis acid based ionic liquids are AlCl 3
[29-31]
based salts . Such salts involve simple mixing of the Lewis acid and the
halide salt which results in the formation of more than one anionic species
depending upon the ratio of quaternary halide salt Q+X− and Lewis acid MX𝑛
as illustrated by the reaction between [emim]Cl and AlCl3 in Scheme (1-4).
However, AlCl3 – urea [177]
for an ionic liquid with organic active cation
[AlCl2.n urea]+ with AlCl4- instead of previous chlorinate ionic liquids which for
organic cation such as [emim]+ and AlCl4-. Latter number of inorganic cationic
ionic liquids were reported such as Al(NO3)3.9H2O – urea [85]
and
AlNH4(SO4)2.XH2O – Amide [83].

Scheme (1-4): Series of equilibria in the reaction between [emim]Cl and AlCl3.

When [emim]Cl is present in molar excess over AlCl3 the ionic liquid formed
is basic (1); however, the molar excess of AlCl3 leads to the formation of an
acidic ionic liquid (3).
When both [emim]Cl and AlCl3 are present in equimolar quantities, it results in
the formation of neutral ionic liquids. Apart from AlCl3, some other Lewis acids
[32] [33] [34] [35]
used are AlEtCl2 , BCl3 , CuCl , and InCl3 . Anion metathesis is the
methodology of choice for the preparation of water and air stable ionic liquids
based upon 1,3-dialkylimidazolium cations. This method involves the treatment
of the halide salt with the silver/sodium/potassium salts of NO2−, NO3−, BF4−,
SO42−, and CH3CO2− or with the free acid of the appropriate anion. Table (1-2)
gives examples of the few ionic liquids prepared by anion metathesis.
Table (1-2): Examples of ionic liquids prepared by anion metathesis [36].
Salt Anion source

[Cation][PF6] HPF6

[Cation][BF4] HBF4, NH4BF4, NaBF4

[Cation][(TFO)2N] Li[(TFO)2N]

[Cation][OTF] OTFCH3,NH4[OTF]

[Cation][OAc] Ag[OAc]

[Cation][TFA] Ag[TFA]

[Cation][CF3(CF3)3CO2] K[CF3(CF3)3CO2]

[Cation][NO3] AgNO3, NaNO3

[Cation][N(CN)2] Ag[N(CN)2]

[Cation][CB11H12] Ag[CB11H12]

[Cation][AuCl4] HAuCl4

It is clear from the above mentioned ILs that large number of ionic liquids can
be envisioned by simple combination of different cations and anions. The
estimated number of single ILs is 1018 which further increases if we include
binary and ternary ionic liquids. Because of their “tailor-made” nature the ionic
liquids find applications as storage media for toxic gases, catalysts/solvents in
organic syntheses, performance additives in pigments, and matrices [37-39].
Several new and improved methodologies using nonconventional techniques,
such as irradiation with microwaves (MW) and power ultrasound (US), whether
used alone or in combination, have considerably improved the synthesis of ILs,
[40-42]
cutting down reaction times and improving yields .The recent introduction
of efficient, solventless, one pot synthetic protocols should make ILs cheaper
and thus encourage a wider use of these neoteric solvents [43-45].
1.4 Deep Eutectic Solvents (DES)
Deep eutectic solvents (DESs) are now widely acknowledged as a new class
of ionic liquid (IL) analogues because they share many characteristics and
properties with ILs. DESs are systems formed from a eutectic mixture of Lewis
or Brønsted acids and bases which can contain a variety of anionic and/or
cationic species; in contrast, ILs are formed from systems composed primarily
of one type of discrete anion and cation. It was illustrated that although the
physical properties of DESs are similar to other ILs, their chemical properties
suggest application areas which are significantly different. DESs contain large,
nonsymmetric ions that have low lattice energy and hence low melting points.
They are usually obtained by the complexation of a quaternary ammonium salt
with a metal salt or hydrogen bond donor (HBD). The charge delocalization
occurring through hydrogen bonding between for example a halide ion and the
hydrogen-donor moiety is responsible for the decrease in the melting point of
[175-176]
the mixture relative to the melting points of the individual components .
In a 2001 study by Abbott et al. a range of quaternary ammonium salts were
heated with ZnCl2 and the freezing points of the resulting liquids measured. It
was found that the lowest melting point, 23−25 °C, was obtained when choline
[175]
chloride was used as the ammonium salt . This initial study has been
extended, and a range of liquids formed from eutectic mixtures of salts and
[179,180]
hydrogen bond donors have now been developed . These liquids were
termed deep eutectic solvents to differentiate them from ionic liquids which
contain only discrete anions. The term DES refers to liquids close to the eutectic
composition of the mixtures, i.e., the molar ratio of the components which gives
the lowest melting point.
Deep eutectic solvents can be described by the general formula Cat +X−zY
where Cat+ is in principle any ammonium, phosphonium, or sulfonium cation,
and X is a Lewis base, generally a halide anion. The complex anionic species
are formed between X− and either a Lewis or Brønsted acid Y (z refers to the
number of Y molecules that interact with the anion). The majority of studies
have focused on quaternary ammonium and imidazolium cations with particular
emphasis being placed on more practical systems using choline chloride, [ChCl,
HOC2H4N+(CH3)3Cl−]. DESs are largely classified depending on the nature of
the complexing agent used, see Table (1-3).

Table (1-3): General Formula for the Classification of DESs [179].

Type General formula Terms

type I Cat+X−zMClx M = Zn, Sn, Fe, Al, Ga, In

type II Cat+X−zMClx·yH2O M = Cr, Co, Cu, Ni, Fe

type III Cat+X−zRZ Z = CONH2, COOH, OH14

M = Al, Zn and Z =CONH2,

type IV MClx + RZ = MClx−1+·RZ
+MClx+1− OH

DESs formed from MClx and quaternary ammonium salts, type I, can be
considered to be of an analogous type to the metal halide/ imidazolium salt
systems. Examples of type I eutectics include the chloroaluminate/imidazolium
salt melts and less common ionic liquids formed with imidazolium salts and
various metal halides including FeCl2, AgCl, CuCl, LiCl, CdCl2, CuCl2, SnCl2,
ZnCl2, LaCl3, YCl3, and SnCl4. The range of nonhydrated metal halides which
have a suitably low melting point to form type I DESs is limited; however, the
scope of deep eutectic solvents can be increased by using hydrated metal halides
and choline chloride (type II DESs). The relatively low cost of many hydrated
metal salts coupled with their inherent air/moisture insensitivity makes their use
in large scale industrial processes viable. Type III eutectics, formed from
choline chloride and hydrogen bond donors, have been of interest due to their
ability to solvate a wide range of transition metal species, including chlorides
[175,179,180]
and oxides . A range of hydrogen bond donors have been studied to
date, with deep eutectic solvents formed using amides, carboxylic acids, and
alcohols (Figure 1-5). These liquids are simple to prepare, and relatively
unreactive with water; many are biodegradable and are relatively low cost.

Figure (1-5): Structures of some halide salts and hydrogen bond donors used in the formation
of deep eutectic solvents.

The wide range of hydrogen bond donors available means that this class of deep
eutectic solvents is particularly adaptable. The physical properties of the liquid
are dependent upon the hydrogen bond donor and can be easily tailored for
specific applications. Although the electrochemical windows are significantly
smaller than those for some of the imidazolium salt−discrete anion ionic liquids,
they are sufficiently wide to allow the deposition of metals such as Zn with high
current efficiencies. This class of deep eutectic solvents have been shown to be
particularly versatile, with a wide range of possible applications investigated
including the removal of glycerol from biodiesel, processing of metal oxides,
and the synthesis of cellulose derivatives. A majority of ionic liquids which are
fluid at ambient temperatures are formed using an organic cation, generally
based around ammonium, phosphonium, and sulfonium moieties. Inorganic
cations generally do not form low melting point eutectics due to their high
charge density; however, previous studies have shown that mixtures of metal
halides with urea can form eutectics with melting points <150 °C [175]. Abbott et
al. have built upon this work and shown that a range of transition metals can be
incorporated into ambient temperature eutectics, and these have now been
termed type IV DESs. It would be expected that these metal salts would not
normally ionize in nonaqueous media; however, ZnCl2 has been shown to form
eutectics with urea, acetamide, ethylene glycol, and 1,6- hexanediol.

1.5 Properties of ionic liquids

There is a number of unique properties related to the ionic structure of ILs,,
which in combination with the liquid state in a wide range of temperatures,,
determines their technological usefulness. Their, advantages include virtually
[46]
non-measurable vapor pressure under moderate conditions . This has a
positive,,,effect on their safety as solvents, and eliminates atmospheric, vapor
emission. No volatility, in combination with moderately low flammability of
ionic liquids, greatly, reduces fire or explosion risk,, as opposed to
traditional organic solvents. The majority, of ILs, exhibit high,, thermal
[47]
stability. The vast,, majority is inflammable . However, caution is ,advised
 [48]
as their , decomposition products may ignite . This property ,has been
used in the development of energetic ionic liquids [49]. ILs, are conductive –
they are applied as electrolytes. The conductivity ,is usually in a range of
0.1 to almost 20 mS/cm. High conductivity is typical for liquids with an
imidazolium cation and weakly coordinating,, anions, e.g. a tetrafluoroborate
[50]
anion . At the same time,, ILs are electrochemically stable in a wide
[51]
range,, usually of 4.5–5 V , which enables their use as electrolytes and
modifying agents for ,electrochemical processes. Recently, there was an
interest in ammonium ILs, containing a chlorine atom in an alkyl substituent
[52]
. Year 2002 brought a publication of a breakthrough, report on dissolving
cellulose in ILs, without any auxiliary substances [53]. Bio polymer solubility
in ILs opened new possibilities in biomass processing and contributed to an
[54-55]
intensified research . The studies conducted led to the conclusion that
cellulose is soluble in imidazolium and ammonium ILs, which mainly
contain acetate anions and short alkyl, substituents at a quaternary nitrogen
atom. The incorporation of the alkyl substituent with a higher, number of
carbon atoms into the cation structure results in visible reduction in
cellulose solubility,, up to non-solubility, with gained bactericidal and
fungicidal activity. A limiting substituent,, for which the IL stops dissolving
cellulose and gains biological activity, is octyl group [56]. Although ILs have
low volatility (nonmeasurable vapor pressure) at ambient ,temperature
certain ILs have characteristic smell, An unpleasant odor of the product
depends on impurity (usually of an original substrate). For ammonium ILs,
for example,, amine serves as a substrate, which has the odor threshold at a
very low level. Amine content is at a level of ppm. It can be completely
removed, but due to time required and profitability,, it is usually neglected or
intentionally disregarded. ILs have surprising functional properties. For
example, they can be used to preserve soft tissue [57],, as feeding deterrents [58]
[59]
or rubber accelerators , which motivates their further research. For
many, ionic liquids there are detailed knowledge about their availability,
price,, physicochemical and biological properties,, including toxicity and
Ecotoxicity. Biomass can, also serve as an ion source. ILs synthesized in this
manner are known as bioionic liquids [60].

1.6 Classification of ionic liquids

It is difficult to classify the previously described, ionic liquids, as there is
no single criterion allowing it. A partial solution is to classify them, based on
the physical differences of a cation and anion, or on their potential
applications. Liquid state of, ILs at relatively low temperatures is caused by
the presence of structures hindering crystallisation in their structure. These
include hydrogen bonds between a cation and anion [61-63] and a large size and
[64-66]
strong asymmetry of an organic cation .The ILs, containing a cation, in
which the positive charge is located on a nitrogen, phosphorus, sulfur or
oxygen atom. This is why they are divided into ammonium, phosphonium,
sulfonium and oxonium ionic liquids. So far, ammonium liquids are best
known, whereas the least known are oxonium ones, as a large, number of them
[67]
is metastable . The principle of the general classification based on the type
of an atom with a positive charge is presented in Figure (1-6).

1 1 1 1
R R R R
+ +
3 N 2 3 P 2 + +
R 4 R R 4 R 2 S 3 2 O 3
R R R R R R
Ammonium Phosphonium Sulfonium Oxonium cation
Fig (1-6): Some types of cations in ILs structures.

[68]
The cations may have one or more atoms with a positive charge . An
example of such, ILs are trigeminal tricationic ionic liquids, i.e. cations that
have three positively, charge nitrogen atoms. ILs with such structure (Fig 1-
7) can be obtained by chloromethylation of glycerol, followed by
quaternisation and substitution reactions [69].

Fig (1-7): The structures of imidazolium and pyridinium triplets[69].

The cation classification is also affected by more subtle differences, in the

structure. A positively charged atom may has separate, not interconnected
alkyl substituents, which determine great diversity of such cation
[70]
conformations and directly affects its, physicochemical properties . Such
cations are known as aliphatic. Heterocyclic cations are the opposite. In this
case,, each charged atom is a part of a cyclic or polycyclic group with
condensed rings. Among heterocyclic ammonium cations, two structure
subtypes can be distinguished depending on the hybridisation of a charged
nitrogen atom. By, means of this criterion, heterocyclic cations may be
divided into cations containing heteroatom of sp2 hybridisation (aromatic
heterocyclic cations) and sp3 hybridisation (non-aromatic heterocyclic cations).
Example structures ,are presented in Figure (1-8).

Fig (1-8): Heterocyclic (a) non-aromatic and (b) aromatic cations [69].

Regardless the element that is positively charged in the cation structure,, the
following classification criterion is distinguished. If the positively charged,
atom is chemically bonded with at least one hydrogen atom,, the salts of
such structures are known as protic ionic liquids (PILs) [71]. The presence, of
the hydrogen atom at a central cation atom leads to the formation of a
network of strong supramolecular hydrogen bonds,., which are characteristic
for PILs [72]. However, if the positively charged atom is not bonded with any
hydrogen atom, i.e. it has the, maximum order, such an ionic liquid is ,called
an aprotic ,ionic liquid (AIL) [73]. Examples of aprotic and protic ionic liquids
are presented in, Figure (1-9).
Fig (1-9): Sample structure (a)AIL and (b)PIL [69].

ILs differ greatly in terms of their physicochemical and biological,

properties, which results in even greater number of potential applications. A
general and useful criterion was created in 2007. Based on IL designability,
the existing ILs were divided into three generations (Fig 1-10) depending on
[74]
the type of properties provided by the structure of a cation and anion .
The generations, are ordered according to the “evolution” history and studies
of ILs. The 1st generation includes ILs in which the ,cation and anion
,structures were chosen to obtain a product of specific physical properties. The
effect of the, cation or anion, structures on conductivity is of special
[75]
importance as it, translated into their specific applications as electrolytes .
Ions ,of the 1st generation ILs ,were also selected to achieve , specific values
of melting point,, density, ,viscosity, .thermal stability,, hydrophilicity and
refractive index [74].
The usefulness of the 2nd generation ionic liquids is a result, of both suitable
physical parameters, and chemical properties. The latter includes their
reactivity, chirality [76], solvating ability and ability to ,extract various chemical
[22] [49]
substances . This group includes, TSILs, energetic, ILs and chemical
reaction ,catalysts [77].
 Melting Point
Density
Viscosity
Hydrophilicity

1st Generation

Solvation
Reactivity
Chirality
Wide electrochemical window
Blocking UV

2nd Generation

Antibacterial activity
Anti-inflammatory effect
Cytostatic effect
Analgesic effect
Herbicidal activity

3rd Generation

Fig (1-10): Generations of ionic liquids[69].

Dynamically growing knowledge of ILs resulted, in the creation of the 3rd
generation of ionic liquids in the early 21st century. It, includes compounds
that apart from selected physicochemical properties also, exhibit a specific
biological activity . One ,of the first, 3rd generation ILs were salts of a
[78]
strong bactericidal effect . The ions of pharmaceuticals used serve both as
cations and ,anions of the 3rd generation ILs, acting against pain and, anti-
[79]
inflammatory . This generation also includes plant protection, products,
including protic derivatives of triazoles showing fungicidal activity [80] and
herbicidal, ionic liquids (HILs) such as derivatives of phenoxyacids
[81]
,exhibiting a selective herbicidal activity against dicotyledonous plants .
The biological activity may be achieved, by both cations and anions, which
enables the synthesis of the multifunctional 3rd generation ILs [82]
. The
spectrum of IL applications is so extensive that new terms will certainly
appear.

1.7 Aluminum salts – amine ionic liquids

The recently synthesized ionic liquids were much stable than the old
halogenoaluminated systems. Ionic Liquid of alum such as (NH4Al
(SO4)2.XH2O, Al2(SO4)3.XH2O and AlK(SO4)2.XH2O) with urea are considered
useful as a green solvent because these have low vapor pressure and stable to air
or moisture such as alum sulfate (AlNH4(SO4)2.12H2O) instead of aluminum
[83]
chloride with urea salt . These deep eutectic ionic liquids are much stable
than chloroaluminate ionic liquids, offering relatively cheaper, easily prepared
ionic liquids with promising similar properties. As green ionic liquid it is
expected to be used in variable process such as metal coating as it offer good
media for some insoluble compounds in aqueous media to be dissolve easily in
[84]
this ionic liquid such as silver sulfate . Another room temperature ionic
 [85]
liquids based on hydrated aluminum nitrate Al(NO3)3.XH2O with urea or
acetamide were also published for its ease of handling ,cheap and possible
incorporating of these compounds in variable industrial applications such as
water purification.

1.8 Biological Activity of Ionic Liquids and Toxicity

An understanding of the toxicity of different ionic liquids towards, living
organisms is important both in terms of selecting appropriate ,ionic liquids
for biological catalyst based reactions, as wells as allowing us to understand,
the impact of the accidental release of these solvents into the environment.
As ionic liquids become more readily understood the number of
applications, in which they can be used increases. Currently ionic liquids
have been employed in a number of different industrial processes, and
products including a process for the dissolution of cellulose,, use as paint
[86]
additives and as pharmaceutical intermediaries . In addition, the possible
use of ionic liquids has been investigated in a number of diverse areas.
Some of these potential ,applications, Include use of some phosphonium and
ammonium, based ionic liquids in the treatment of cancer [87],, use as insect
[88] [89]
detergents ,, use as blood and tissue, preservatives , phenol degradation
[90] [91]
and use as wood preservatives . However, increasing the application of
ionic liquids increases the possibility of accidental release of these solvents into
the environment. This possibility, means that an understanding of the toxicity
of different classes of ionic liquids toward all taxa ,of life cannot be
overestimated, as it gives an understanding of how an accidental release of
solvent will affect the environment,, as well as giving information to enable
the synthesis of less toxic ionic liquid structures. In general toxicity and
biodegradation studies have focused on ,the most common ionic liquids
families, namely those containing imidazolium,,pyridinium, piperidinium and
quaternary ammonium cations . By examining this small sub set of possible
cation ,structures and testing them against a range of organisms, - including
gram positive ,and gram negative bacteria, fungi, algae, zebra fish and cell lines,
it became possible to elucidate a number of toxicity, to structure relationships.
Most widely, reported was the impact of increasing the length of the alkyl
chain associated with, the cation having a linear relationship with increasing,
toxicity of the ionic liquid, within a range of cation chain lengths from
[92]
approximately 1 to 12 carbons in, length . The elucidation ,of the lipophilic
nature of an ionic liquid correlating ,with its toxicity has led to an
understanding of the mechanism of toxicity for, ionic liquids. The lipophillic
nature of the ionic liquid allows it to permeate the phospholipid bilayer, of
the cell disrupting ,the membrane structure. The more lipophilic the ionic
liquid the further it is able to, permeate into the membrane structure [93]. The
effect of, the anion on the toxicity of the ionic liquid has ,also been
investigated. Although the choice of anion appears, to play a lesser role in
the determination of the overall toxicity, of an ionic liquid than does the
choice of cation it has been reported ,that the choice of anion, can impact
the overall toxicity of the ionic liquid,, with the anions [NTf2] and
[(CF3SO2)2N] reported as significantly increasing, the toxicity of the ionic
liquid [94]. The effects of altering the structure of the cation alkyl chain have
also been investigated, with the introduction of ester linkages, producing
[95]
more biocompatible cations . The work has reported so far on the
relationship, between structure and toxicity has been instrumental in
elucidating ‘design rules’,, allowing either for structures to be discarded or
included for consideration for use, in particular studies and to guide the
synthesis of new ionic liquids. However, the structures which have been so
far investigated ,represent only a small fraction of the potential number of
ionic liquids.
1.8.1 Organism-Dependent Biological Activity of Ionic
Liquids
Nowadays, it is clearly established that ILs have an impact on different
levels of life, from single proteins to higher multicellular organisms [96]. First, an
apparently high bioavailability of ILs should be taken into account. Although
the particular mechanism of biological activity of ILs may vary from one
organism to another, water is crucial for all living systems. Therefore, solubility
and interactions with water are one of the factors determining
environmental/biological activity of ILs. In the study on ILs with various
cations containing butyl or isobutyl side chains and the
bis(trifluoromethylsulfonyl)amide anion ([NTf2]−), the water solubility of ILs
decreased as follows, Imidazolium ILs > Pyrrolidinium ILs > Pyridinium ILs >
Piperidinium ILs, and was suggested to depend on the water cavitation
potential, which was influenced by the size of the IL cation and, to some degree,
[97]
by the IL aromaticity . A theoretical study on Imidazolium ILs with small
inorganic anions suggested that both the cation and the chloride ([Cl]−), bromide
([Br]−), and tetrafluoroborate ([BF4]−) anions interacted strongly with water
molecules, whereas the hydrophobic hexafluorophosphate ([PF6]−) anion formed
no stable interactions with water. According to this study, the ion pairs also
interacted with water, but these interactions were significantly weaker [98]. In the
case of aqueous 1-ethyl-3- methylimidazolium acetate ([C2MIM][OAc]), the
main interactions were hydrogen bonds between water molecules and the anion
[99]
. [C4MIM][OAc] demonstrated weakening of cation anion interactions due to
[100]
interactions between the anion and water in water solutions . Biological
activity of ILs was shown to depend on their hydration state, and a threshold
hydration number was established. About seven water molecules per ion pair
were suggested to be the boundary at which biological effects of IL− water
mixtures changed dramatically, independent of the ion nature. The first six
 [101]
molecules of water supposedly formed strong interactions with the ion pair .
Interestingly, when the ratio of water molecules to IL molecules did not exceed
one to three, the solution retained the polar ionic network. Upon subsequent
dilution, water molecules surrounded the IL network causing its stretching and,
[102]
finally, disruption . Corroborating the above-mentioned dependence of
biological activity of ILs on the presence of water (for possible structural
factors, simulations of interactions between water solutions of ILs and
phospholipid bilayers suggest that the behavior of the IL anion is governed by
its interactions with water molecules. Thus, small hydrophilic anions, such as
[Cl]−, stayed in the solution, whereas more hydrophobic, bulkier [PF6]− formed
a film at the lipid/water boundary. In the case of the hydrophobic [NTf2]−, the
anion followed the imidazolium cation into the lipid bilayer and was located in
the interjacent region between the hydrocarbon tails and polar heads of the
phospholipid [103]. The chloride anion reduced the cation insertion, as compared
[104]
to lactate . The analysis of the general profile of biological activity of ILs
shows that it is strongly dependent on the organism [105].

1.8.2 The biological activity of ILs.

Antimicrobial activity of ionic liquids has quickly attracted the attention of
researchers. When it became obvious that numerous types of ILs inhibited
growth of various bacterial and fungal species, the medical and industrial
potentials of ILs have manifested. Imidazolium, Pyridinium, Pyrrolidinium,
Piperidinium, ammonium, and other ILs were shown to inhibit growth of
[106]
pathogenic and nonpathogenic bacteria and fungi . These results have
presented both advantages and disadvantages. Thus, high activity toward
microorganisms may seriously hinder the application of ILs in biotechnology;
however, it may be used as a valuable property in medicine. From the
biotechnological point of view, toxicity of new IL solvents may become a
serious problem, and evaluation and selection of appropriate ILs for various
[107]
biotechnological processes have been actively investigated lately .
Imidazolium ILs were shown to be toxic toward the probiotic bacterium
Propionibacterium freudenreichii subsp. freudenreichii applied in the dairy
[108]
industry . whereas pyridinium ILs inhibited Clostridium sp. involved in
[109]
biosorption of uranium . Residual 1-ethyl-3-methylimidazolium acetate
([C2MIM][OAc]) in hydrolysates of pretreated lignocellulose biomass inhibited
growth and ethanol production of the model yeast Saccharomyces cerevisiae
[110]
. Addition of either 1-ethyl-3-methylimidazolium acetate or 1-ethyl-3-
methylimidazolium methylphosphonate in small quantities (≤5%) led to a
[110]
metabolic switch from respiration to fermentation (ethanol production) ; in
[111]
higher concentrations, both ILs inhibited the yeast growth . The
lignocellulolytic bacterium Enterobacter lignolyticus was tolerant to high
concentrations of [C2MIM][Cl], possibly due to changes in the cell membrane
composition, down-regulation of membrane porins, and up-regulation of drug
[112]
efflux pumps . Correspondingly, a method for tolerance enhancement via
[113]
heterologous expression of efflux pumps in bacteria was proposed , and a
technique for enrichment of IL-tolerant microorganisms in microbial
[114]
communities was suggested . Interestingly, a single mutation in a
[115]
transcriptional regulator made Escherichia coli tolerant to [C2MIM][OAc] .
[C4MIM]-[PF6] and [C4MIM][NTf2] were considered compatible with whole-
cell biotechnological applications.[116] Penicillium sp., the specimen of which
could produce valuable bioactive substances, demonstrated very high tolerance
to IL treatment; in some cases, even 50 mM of IL did not inhibit the growth of
the fungi.[117] Aspergillus isolates were tolerant to [C2MIM][OAc].[118]
Morpholinium ILs demonstrated low toxicity toward bacteria and fungi.[119]
Thus, a search for IL-tolerant valuable microbial communities is ongoing.[120]
From the medical point of view, ever-evolving resistance of microorganisms to
the existing drugs is an urgent issue, and a whole new class of possible
antimicrobial agents is a very timely finding. Targeted attempts to develop
antimicrobial ILs for medical purposes have been made. Thus, comparison of
activity of ampicillin-carrying ILs with that of sodium ampicillin clearly
showed possible advantages of using ILs as antimicrobial agents.[121] ILs have
demonstrated impressive inhibitory results even in the case of resistant and
biofilm-forming microorganisms. Low concentrations of [C16MIM][Cl] strongly
inhibited growth of multidrug-resistant Candida tropicalis,[122] whereas 1-alkyl-
3- methylimidazolium chlorides, bromides, and iodide were active toward both
planktonic and biofilm bacteria and fungi.[123] 1-Alkyl-3-methylimidazolium
fumarates possessed high antimicrobial potential and were suggested to be used
for medical purposes.[124] Ammonium ILs with azolate anions were found to be
powerful antibacterial and antifungal substances;[125] hydroxylammonium ILs
were highly active against such human pathogens as Staphylococcus aureus,
Salmonella typhi, and Vibrio cholera,[126] whereas diphosphohium ILs displayed
a broad spectrum of antimicrobial activity against ocular pathogens.[127] Of note,
triphenylamine phosphonium ILs, which self-assembled into nanostructures,
showed potent activity against Gram positive bacteria, including S. aureus.[128]
However, possible negative effects associated with broad application of ILs in
medicine should not be overlooked. While increased IL tolerance is a benefit in
biotechnologically relevant microorganisms, in pathogenic bacteria and fungi it
may become a significant difficulty. It seems that, similarly to conventional
antimicrobial agents, ILs also may provoke the appearance of resistant strains.
Thus, [C4MIM][PF6] was shown to induce the expression of antibiotic
resistance genes and to activate horizontal gene transfer in freshwater
bacteria.[129] Of note, cholinium chloride ([Cho][Cl]) and [C2MIM][Cl]
significantly influenced the expression of genes involved in primary and
secondary metabolism in Aspergillus nidulans; the authors suggested the
treatment with ILs to be employed for boosting the diversity of natural active
compounds produced by the fungus.[130] High antimicrobial activity of many ILs
may be possibly explained by interactions between IL and the cell
membrane.[131] Thus, alkyltributylphosphonium chlorides with long alkyl chains
provoked substantial damage in conidia of A. nidulans.[132] For imidazolium ILs
with short alkyl chains, the toxicity was found to depend on the chaotropicity of
the anion.[133] Antimicrobial properties of ILs were suggested to be employed in
the development of new IL fungicides,[134] and in the protection of natural
fabric,[135] paper,[136] and metal surfaces [137] against microbial growth. Similarly,
graft polymers of poly (ionic liquids) were proposed to be used in antibacterial
coatings.[138] Alkoxymethyl (2-decanoyloxyethyl) dimethylammonium bis-
(trifluoromethylsulfonyl) amides and 1-methyl-3-octyloxymethylimidazolium
tetrafluoroborate performed good in preservation and fixation of tissues.[139]
Attempts to create imidazoliumbased poly-IL membranes with improved
antibacterial activity have been made.[140]

1.9 Clinical biochemistry

Clinical biochemistry or chemical pathology is the study of chemical and
[141]
biochemical ,mechanisms of the body in relation to disease , mostly
through the analysis of body, fluids such as blood or urine. Many diseases
,show significant changes ,in the chemical composition of body fluids
such as the raised blood enzymes, due to their release from heart muscles
after a heart attack; ,or a raised blood sugar in diabetes, mellitus due to
lack of insulin. Biochemical tests are designed to detect these, changes
qualitatively or quantitatively compared to results, from healthy people.
Clinical biochemistry use a broad range of analytical techniques for
example,, molecular diagnostics, measurement of enzyme activities,,
spectrophotometry,, electrophoresis,, the separation of molecules based on
physical characteristics and immunoassays [142].
1.10 Biochemical tests
Biochemical tests are crucial to modern medicine. Most biochemical tests,
are carried out on blood using plasma or serum,, but urine, cerebrospinal
fluid (CSF), feces, kidney stones, pleural fluid, etc. are sometimes
required. Plasma is obtained by collecting blood into an anticoagulant and
,separating the fluid, plasma phase from the blood cells by, centrifugation.
Serum is the corresponding fluid phase when blood is allowed to clot. For
many (but not all) ,biochemical tests on blood, it makes little ,difference
whether plasma or serum is used. There are many hundreds of tests
available in clinical biochemistry, but a core of common tests makes up the
majority of tests requested in ,clinical biochemistry, [143].Table (1-4) describes
some of common blood chemistries .
Table (1-4). Normal Common Blood Chemistries testes [143].

Traditional Units
Parameter SI Units (Canada)
(USA)

Albumin 35 – 50 g/L 3.5 – 5.0 g/dL

Alkaline phosphatase
35 – 100 U/L 35 – 100 U/L
(ALP serum)

Aspartate
Aminotransferase 0 – 35 U/L 0 – 35 U/L
(AST)

Bilirubin serum /Total

< 26μmol/L < 1.5 mg/dL
Bilirubin, conjugated
< 7 μmol/L < 0.4 mg/dL
(direct)
Blood Urea Nitrogen 2.5 – 8.0 mmol/L 7– 22 mg/dL

Calcium serum 2.18 – 2.58 8.7 – 10.3 mg/dL

Cholesterol < 5.2 mmol/L < 200 mg/dL

Cholesterol, LDL < 2.0 mmol/L < 77.3 mg/dL

Cholesterol, HDL < 1.00 mmol/L < 40 mg/dL

Creatinine, serum
Male 70 – 120 μmol/L 0.8 – 1.4 mg/dL
Female 50 – 90 0.56 – 1.0

Glucose, fasting 3.3 – 5.8 mmol/L 59 – 105 mg/dL

Potassium 3.5 – 5.0 mmol/L 3.5 – 5.0 mEq/L

Protein, total 60 – 80 g/L 6.0 – 8.0 g/dL

Sodium 135 – 145 mmol/L 135 – 145 mEq/L

Triglyceride < 2.20 mmol/L < 195 mg/dL

Uric Acid 180 – 420 μmol/L 3.0 – 7.0 mg/dL

1.10.1 Automated Analyser

An automated analyser is a medical laboratory, instrument designed to
measure different chemicals and other characteristics, in a number of
biological samples quickly, with minimal ,human assistance. These
measured properties of blood and other fluids may be useful in ,the
diagnosis of disease. Many methods of introducing samples into the analyser,
have been invented. This can involve placing test tubes of sample into
racks, which ,can be moved along a track, or inserting tubes into circular
carousels that rotate ,to make the sample available. Some analysers require
samples to be transferred ,to sample cups. However, the effort to protect the
health and safety of laboratory staff, has prompted many manufacturers to
develop analysers that feature closed tube, sampling, preventing workers
from direct exposure to samples.[143] Samples, can be processed singly, in
batches, or continuously. The automation of laboratory ,testing does not
remove the need for human expertise, results must still be, evaluated by
medical technologists and other qualified clinical laboratory professionals,
but ,it does ease concerns about error reduction, staffing concerns, and
safety.

1.10.2 Automatic biochemistry analysers

These are machines that process a large, portion of the samples going
into a hospital or private medical laboratory. Automation ,of the testing
process has reduced testing time for many analytes, from days to minutes.
The history of discrete sample analysis for the clinical laboratory ,began with
the introduction of the "Robot Chemist" invented by Hans Baruch ,and
[144]
introduced commercially in 1959 . AutoAnalyzer is an automated
,analyzer using a special flow technique named "continuous flow analysis
(CFA)", ,invented in 1957 by Leonard Skeggs, PhD and first made by the
Technician ,Corporation. The first applications were for clinical (medical)
analysis. The Auto Analyzer profoundly changed the character of the
chemical testing laboratory by ,allowing significant increases in the numbers
of samples that could be processed., The design based on separating a
continuously flowing stream with air bubbles largely, reduced slow,
[145]
clumsy, and error prone manual methods of analysis .The types, of tests
required include enzyme levels (such as many of the liver function tests) ,
ion, levels (e.g. sodium and potassium, and other tell-tale chemicals (such as
glucose,, serum albumin, or creatinine). Simple ions are often measured
with ion selective ,electrodes, which let one type of ion through, and
measure voltage differences. enzymes, may be measured by the rate they
change one colored substance to another; in these , tests, the results for
enzymes are given as an activity, not as a concentration of, the enzyme.
Other tests use colorimetric changes to determine the concentration of, the
chemical in question. Turbidity may also be measured [145].

1.11 Bacteria
Originally called “animalcules” bacteria were studied, microscopically
by Antonie van Leeuwenhoek during the mid - 17th century [146]. They ,
were not known as bacteria until the 1830s when Christian Gottfried
Ehrenbug defined the genus based on his scientific observations as
“non -spore forming rod –shaped bacteria ” [146]
. However , bacteria are
defined as , single – celled , prokaryotic microorganisms [146]. Bacteria have
several, morphologies ( i.e. spheres , rods , and spirals ) and sizes
which range on the scale of microns . Most bacteria are spherical ,
named cocci , or rod – shaped , called bacilli [146]. Slight differences in
the morphology, from spheres or rods have led to different classes of
bacteria named by their shapes such as comma (Vibrio) ,, spiral
(Spirilla) , or coiled (Spirochaetes) . The shapes of bacteria are
[146]
determined by the cell wall and its cytoskeleton . Aside from
morphology ,, bacteria arrange in ordered arrays that add to their
diverse nomenclature . For example , Streptococci species form chains
whereas Staphylococci species form clusters .
1.12 Morphology and Cellular Structure
Bacteria are visually distinguishable using Gram – staining (named
after Christian Gram ,, Danish scientist and physician , 1853 – 1938 ) [146].
This bacteriological method separates , bacteria into two classes based on
their abilities to retain dyes and the physical properties , of their cellular
walls . Additionally , this method allows for their, morphologies and
arrangements to be visualized microscopically . The principle of , Gram –
staining is premised on the notion that Gram – positive, bacteria have a
thicker cellular wall than Gram – negative [146]
. Hence ,, Gram – positive
bacteria retain both the crystal violet and safranin stains while Gram –
negative, bacteria only show the pink safranin dye . Examples of Gram –
positive bacteria are Staphylococcus , and Enterococcus and examples of
Gram - negative bacteria are Escherichia and Salmonella . Bacteria are
structurally composed of two major parts : intracellular, and extracellular
components . The intracellular components are enclosed by a hydrophobic
cell membrane which envelopes its essential materials . Prokaryotes are
structurally simple as compared to eukaryotic cells since they do not
[146]
contain, various membrane bound organelles . Instead of
mitochondria ,, bacteria use micro – compartments that maintain cellular
metabolisms and membrane potentials which are generated via
[146]
biochemical reactions across cellular membranes . Bacteria have a
nucleoid that , stores its genetic material . The only common feature ,
amongst all cells is the presence of ribosomes which helps to generate
proteins and enzymes for routine function . The intracellular components
, are protected by a cellular wall composed of peptidoglycan ,, which is
also the lethal target of beta (β) – lactam antibiotics [146]. As previously
mentioned ,, Gram – staining reveals the structural differences in the
extracellular components of Gram - positive and Gram – negative, bacteria
. Gram – positive bacteria have a thicker cell wall that is composed of
both peptidoglycan and techoic acid layers ,, which helps to retain
Gram – stains [146]. However , Gram – negative bacteria have a thin layer
of a peptidoglycan cell wall with a thick secondary cell membrane
[146]
,composed of lipopolysaccharides and lipoproteins . Although the
conventional structure of bacteria contains a cell membrane and cell wall
, the thickness and assembly can vary affecting the bacterium’s ,
susceptibility
To many antimicrobial agents . Additional components on the
extracellular framework , can include flagella , fimbriae , and pilli as well
as the production of protective slime layers .

1.13 Bacterial Culture Media

When culturing bacteria , it is very important to provide similar
environmental and nutritional conditions that exist in its natural habitat
Most culture medium contains water , a source of carbon and energy ,,
source of nitrogen ,, trace elements and some growth factors . Besides
these, optimum pH, oxygen tension and osmolarity too have to be taken
into consideration . Some of the ingredients of culture media include:
While tap water is suitable for culture media, it must not be used if it
contains high amount of minerals . In such situations,, distilled or
demineralized water should be used. Peptone is a byproduct of ,protein
digestion . Proteins are often obtained from heart muscle ,, casein , fibrin or
soya flour and is digested using proteolytic enzymes such as pepsin ,,
trypsin or papain . The final product contains peptones , proteoses and
 amino acids besides a variety of inorganic salts including phosphates ,
potassium and magnesium . Casein hydrolysate is obtained from
hydrolysis of milk protein casein using HCl or trypsin . Meat extract
is obtained by hot water extraction of lean beef and then concentrated
by evaporation . Yeast extract is prepared from washed cells , of
bakers’ yeast and contains wide range of amino acids, growth factors
and inorganic salts .[147]

1.14 Classification of Bacterial culture media

Bacterial culture media can be classified in at least three ways ;

Based on consistency , based on nutritional component, and based on its
functional use .

i. Liquid media
In liquid medium , bacteria grow producing turbidity / surface,
pellicle ( Vibrio & Bacillus) / granular, deposits (Streptococci) . Culturing
bacteria in liquid media , has some drawbacks ., Properties of
bacteria are, not visible in liquid media and presence of more than ,
one type of bacteria cannot be detected. [147]

ii. Solid media

Any liquid medium can be rendered solid by , the addition of
certain solidifying agents. Agar (simply called agar) is the most
commonly , used solidifying agent. It is an unbranched polysaccharide
obtained from the cell membranes of some species of red algae such as
the genera Gelidium. Agar is composed of two long-chain polysaccharides
 (70% agarose and 30%, agarapectin). It melts at 95°C and solidifies at
42oC,, doesn’t contribute any nutritive property, it is not hydrolysed by
most, bacteria and is usually free from growth promoting or growth
retarding substances. Agar is available as ,powders. New Zealand agar and
Japanese, agar are, most commonly used at concentration of 2% and 4%
respectively to make a solid agarmedium . [147]

iii. Semi-solid media

Reducing the amount of agar to 0.2-0.5% renders ,a medium semi- solid
. Such media ,are fairly soft and are useful in, demonstrating bacterial
motility ,(U-tube and Cragie’s tube). Certain transport media , such as,
Stuart’s and Amies media are semi-solid in consistency. Hugh &
Leifson’s oxidation fermentation test medium as well as mannitol,
motility, medium are also semi-solid. [147]

1.15 Bacterial Enumeration

There are numerous occasions when it is necessary to either estimate
or determine the number of bacterial cells in, a broth culture or liquid
medium. Determination of cell numbers can be ,accomplished by a number of
direct or indirect methods. The methods include, standard plate counts,
turbidimetric measurements, visual comparison ,of turbidity with a known
standard, direct microscopic counts, cell mass, determination, and
measurement of cellular activity. Two methods of bacterial enumeration: the
standard plate count and turbidimetric, measurement.

1.15.1 Standard Plate Count (colony-forming units)

 A viable cell is defined as a cell which is, able to divide and form a
population (or colony) [147]. A viable cell count is usually , done by diluting
the original sample, plating aliquots of the dilutions onto, an appropriate
culture medium, then incubating the plates under proper conditions, so that
colonies are formed [147, 148]. After incubation, the colonies are counted and,
from ,a knowledge of the dilution used, the original number of viable cells
can ,be calculated. For accurate determination of the total number of viable
cells, it is critical ,that each colony comes from only one cell, so chains
and clumps of cells must be, broken apart. However, since one is never sure
that all such groups have been , broken apart, the total number of viable
cells is usually reported as, colony-forming units (CFUs) rather than cell
[146-148]
numbers . This method of enumeration is relatively easy to perform
and is much more sensitive , than turbidimetric measurement. A major
disadvantage, however, is the time ,necessary for dilutions, platings and
incubations, as well as the time needed, for media preparation.[148]

1.15.2 Turbidimetric Measurement

A quick and efficient method of estimating, the number of bacteria in a
liquid medium is to measure , the turbidity or cloudiness of a culture and
[149]
translate this measurement into ,cell numbers . This method of
enumeration is fast and is usually preferred when, a large number of cultures
are to be counted. Although measuring turbidity is, much faster than the
standard plate count, the measurements must be correlated initially, with cell
147-149]
number . This is achieved by determining the turbidity of different
concentrations of, a given species of microorganism in a particular medium
and then, utilizing the standard plate count to determine the number of
viable organisms ,per milliliter of sample. A standard curve can then be
drawn (e.g., this lab protocol section), in, which a specific turbidity or
optical density reading is matched to a specific ,number of viable organisms.
[147-150]
Subsequently, only turbidity needs to ,be measured . The number of
viable organisms may be read directly from the standard, curve, without
necessitating time-consuming standard counts. Turbidity can, be measured by
an instrument such as a colorimeter or spectrophotometer. These
,instruments contain a light source and a light detector (photocell) separated
by , the sample compartment. Turbid solutions such as cell cultures interfere
with ,light passage through the sample, so that less light hits the photocell
than would if, the cells were not there. Turbidimetric methods can be used
as long as each individual ,cell blocks or intercepts light; as soon as the
mass of cells becomes so large that ,some cells effectively shield other
[148]
cells from the light, the measurement is no longer ,accurate . Before
turbidimetric measurements can be made, the spectrophotometer, must be
adjusted to 100% transmittance (0% absorbance). This is done, using a
sample of uninoculated medium. Percent transmittance of various ,dilutions
of the bacterial culture is then measured and the values converted to
optical, density, based on the formula: Absorbance (O.D.) = 2 – log %
Transmittance. A wavelength , of 420 nm is used when the solution is clear,
540 nm when the solution is, light yellow, and 600-625 nm is used for
yellow to brown solutions.[147-149]

1.16 Antibiotic Sensitivity testing: The Kirby-Bauer Disk

Diffusion Test
Certain bacteria can display resistance, to one or more antibiotics.
Determining bacterial antibiotic ,resistance – whether a bacterium can
survive in the presence of an antibiotic - is , a critically important part of
the management of infectious diseases in patients. ,The Kirby-Bauer (K-
B) disk diffusion test is the most common method for antibiotic
,resistance / susceptibility testing. The results of such testing help
physicians in choosing which antibiotics to use when treating a sick
patient . The Kirby-Bauer (K-B) test utilizes small filter disks impregnated
with a known concentration of ,antibiotic (e.g. Rifampin , Trimethoprim,
Amikacin , Cefixime, ,Tetracycline ,Ampicillin , Cefotaxime ). The disks are
placed on a Mueller-Hinton ,agar plate that is inoculated with the test
microorganism. Upon incubation, antibiotic diffuses , from the disk into the
surrounding agar. If susceptible to, the antibiotic, the test organism will be
unable to grow in the area immediately surrounding, the disk, displaying a
zone of inhibition . The size of this zone is dependent on a number of
factors, including the sensitivity of the microbe to the antibiotic, the rate
of diffusion of the antibiotic through the agar, and the depth of the agar.
Microorganisms ,that are resistant to an antibiotic will not show a zone of
inhibition, (growing right up to the disk itself) or display a relatively small
zone.[150]
1.17 Aim of the work
1. The present work was aimed to prepare an ionic liquids from some
aluminum and calcium salts with amide salts.
2. Investigation the interaction and physiochemical properties for the new ionic
liquids by using FT-IR, UV-visible spectra, cyclic voltammetry as well as
conductivity, melting point, viscosity and density.
3. The new work was referred to study and evaluate the biological behavior of
ILs on several kind of bacteria and on some serum parameters.
4. Then ionic liquids are characterized by their water stable, room temperature
ionic liquids and containing water molecules in their structure which might
influence the mechanism of their activity with organism
Chapter Two
Materials
and
 Methods
Chapter Two
Materials and Methods

2.1 Chemicals:-
All chemicals were used in current work with highest purity available and
without further purification Table (2-1) shows the reagents, their purities and
suppliers:

Table (2-1): Specification of the chemicals used in current work.

Chemical Purity
NO. Formula Company
compounds %

1 Acetamide CH3CONH2 98 Fluka

Aluminum Nitrate
2 Al(NO3)3.9H2O 99.5 BDH
nanohydrate
Ammonium
3 aluminum sulfate NH4Al(SO4)2.12H2O 99.5 BDH
dodecahydrate

4 Urea CO(NH2)2 99 THOMAS BAKER

5 Potassium KAl(SO4)2.12H2O 99 BDH

aluminum sulfate
 dodecahydrate

6 Aluminum chloride AlCl3 98 Fluka

Aluminum
7 C54H105AlO6 99 H&W
tristearate

8 Monoethanolamine CH2(OH)CH2NH2 99 BDH

9 Thiourea CS(NH2)2 99 BDH

10 Thioacetamide CH3CSNH2 99 H&W

11 Chloroform CHCl3 99 Fluka

Carbon
12 CCl4 99.5 Fluka
tetrachloride
Calcium Chloride
13 CaCl2.2H2O 98 CHEM-SUPPLY
dihydrate

14 Nutrient Broth M002 - HIMEDIA

Mueller Hinton
15 M173 - HIMEDIA
Agar

16 Nutrient Agar M001 - HIMEDIA

2.2 Instrumental and Techniques:-

The instruments used in this work are describe as follows:

1. Electronic Absorption Spectra.

The electronic spectra of ILs were measured using UV-1650PC Shimadzu at
room temperature using quartz cells of 1.0cm path length in the wavelength
range of 200-1100 nm, at the laboratories of Chemistry Department, College of
Science, Al- Nahrain University. the optical density for bacteria growth
measured were done by using SHIMADZU UV spectrophotometer (UV – 1800
JAPAN) at 600 nm, instrument is in Al Karama Hospital-Baghdad.

2. Fourier Transform Infrared Spectrophotometer (FT-IR).

The infrared spectra were recorded on Fourier Transform Infrared
Spectrophotometer, ALPHA FTIR Spectrometer by Bruker Optik GmbH. The
spectra were recorded in the laboratories of Chemistry Department, College of
Science, Al- Nahrain University.
3. Automatic biochemistry analyzer.
The clinical biochemistry testes were measured by Automatic biochemistry
analyzers (ARCHITECT c4000 Abbott core laboratory made in USA). The
measurements were carried out in the laboratories of Chemistry of Al-Yarmouk
Teaching Hospital-Baghdad.

4. Automatic colony counter

The colony forming units plates are automatically counted by aCOLyte 3
device from Synbiosis made in United Kingdom the measurements were carried
out in the Laboratory department of Al-Doura Water Project – Baghdad.

5. Conductivity, Density, Viscosity, Melting and Freezing Point

Measurement.
The conductivity measurements were carried out using WTW inolab cond-
720(Germany) in mS/cm, the measurements were carried out in the Laboratory
department of Al-Doura Water Project – Baghdad. Viscosity measurement were
carried out manually by using Cannon Fenske Opaque Viscometer (Koehler-
USA), and the density measurement was done by using pycnometer (Germany)
size 5 ml, at room temperature. The melting point determined by Dynalon
DMP100 Digital Melting Point Device (USA). The Freezing point was obtained
from a thermometer immersed inside the tube containing 2 ml of ionic liquid
stored in a deep freezing refrigerator and the temperature was recorded when
the sample changed its state. All measurements were carried out in laboratory of
chemistry Department, College of Science/ Al-Nahrain University.

6. Simultaneous Thermal Analysis STA (TG – DSC).

Thermal behavior of ionic liquids was investigated using LINSEIS
Simultaneous thermal analyzer (STA PT1000), with Platinum Evaluation
V1.0.89 software. Ionic liquid samples were heated using alumina crucibles
under air atmosphere with heating rate of 10 °C/min and sample weight of
(23.7696 mg) from room temperature up to 600 °C, which was performed in
chemistry department of Al-Nahrain University.

7. Cyclic voltammogram by Digi-lvy-Dy2300 Bipotentiostat

A three-electrode system comprising a platinum electrode as the working
electrode (WE), a platinum plate as the counter electrode (CE) and a silver wire
as the reference electrode (RE) was used in the cycle voltammogram. The
platinum working electrode surface was freshly cleaned with alumina, polish
before each scan and rinsed with double-distilled water at each polishing step.
The instrument for cyclic voltammogram CV was carried out in the laboratories
of chemistry department of Al-Nahrain University by using Digi-lvy-Dy2300
Bipotentiostat.

2.3 Synthesis of Ionic Liquid.

2.3.1 Preparing RTIL by reacting Lewis base with several

aluminum salts.
The synthesis methods of variable ionic liquids by reacting aluminum salts
with different components were conducted for the binary components outlined
in Table (2-2). These reactions involved an acid-base reaction or deep eutectic
 [179-180]
solvent (DES) methods of attempted ionic liquid preparation. Each
reaction conditions and results will be covered in chapter (3.1), where their
outcomes are discussed separately in details.

Table (2-2): Binary mixture of amines and aluminum salts.

No. Binary mixture No. Binary mixture No. Binary mixture

MEA + AlCl3 Thiourea +

1 6 MEA + AN 11
AlST
MEA + AlCl3 TA +
2 7 MEA + ALST 12 Ammonium
(with CCl4)
alum
MEA + AlCl3
Thiourea + TA + potassium
3 8 13
(with CHCl3) Ammonium alum alum

MEA + Ammonium Thiourea +

4 9 potassium alum 14 TA + AN
alum

MEA + potassium Thiourea + AN

5 10 15 TA + ALST
alum

2.3.2 Preparation of calcium chloride dihydrate /acetamide new

room temperature ionic liquid.

Calcium chloride dihydrate and acetamide were used in this study to prepare
ionic liquids in different proportions. Solid salts of calcium chloride dihydrate
[CaCl2.2H2O] with m.p. 176°C and acetamide [CH3CONH2] with m.p. 81°C,
were mixed, then heated gradually to 80°C for half hour, with continuous
mechanical stirring until both salts melt together providing a colorless and
homogeneous liquid. The produced ionic liquid then cooled to room
temperature and stored in airtight container. The mixtures were investigated by
UV-visible and FT-IR spectroscopy, cyclic voltammogram, and other physical
properties were also studied. The mole ratios were in the range of 1:1 to 1:10
CaCl2.2H2O : acetamide incremented by 1 .

2.3.2.1 Composition/ freezing point, phase diagram of Calcium

Chloride dihydrate /Acetamide ionic liquid
The composition / freezing point phase diagram was obtained for the freezing
points of the mixtures of calcium chloride dihydrate with acetamide ionic liquid.
Different ionic liquid of variable mole ratios were prepared to determine the
freezing point of their mixtures, which were placed in an ice bath and the
temperature at which the melt frozen was visually recorded.

2.3.2.2 Determination of Conductivity, Density and Viscosity

The measurements of conductivity, viscosity and density were determined at 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 and 90 ℃ using the ratio of
(1:7). The plotting of these measurements for new ionic liquid Calcium
Chloride dihydrate /Acetamide are illustrated in Table (2-3).

Table (2-3): Viscosity, Conductivity and Density at different temperatures of Calcium

Chloride dihydrate /Acetamide ionic liquid for [1:7] mole ratio.

Temp. ℃ Viscosity (cp) Density Kg/m3 Conductivity mS/cm

20 497.33 1133 0.29

25 471.71 1131 0.31

 30 324.81 1127 0.33

35 242.64 1124 0.35

40 165.54 1119 0.40

45 141.32 1113 0.47

50 107.92 1109 0.57

55 88.53 1105 0.65

60 69.24 1098 0.76

65 55.17 1093 0.88

70 42.82 1085 0.98

75 34.90 1078 1.16

80 28.66 1074 1.63

85 24.93 1069 2.14

90 20.57 1061 2.53

2.3.2.3 Determination of Cyclic Voltammetry (CV)

The cyclic voltammetry (CV) at room temperature was carried out for the
calcium chloride dihydrate /acetamide ionic liquid at the mole ratio of (1:7). CV
was performed using platinum working electrode, platinum plate counter
electrode and Ag+/Ag electrode as a reference electrode with the scan rate of
0.05 V/sec.
2.3.3 Preparation of aluminum nitrate nanohydrate and hydrated
ammonium alum with urea and acetamide (RTILs):-
Ionic liquid was prepared by mixing a proper amount of aluminum nitrate
nanohydrate [Al(NO3)3.9H2O] or hydrated ammonium alum
[NH4Al(SO4)2.12H2O] either with urea [NH2CONH2] or acetamide
[CH3CONH2] were milled, mixed together and heated with gentle stirring until
clear colorless liquids were obtained which left to cool and kept sealed in
container for further use as follow:

2.3.3.1 Ionic liquid preparation with acetamide:

- Aluminum nitrate and acetamide were prepared in different mole ratios and
among them the 1:3 mole ratio was selected for the study due to their proper
stability. The mixture was heated to 80 °C for 1 hour [85].
- Ammonium alum and acetamide in the mole ratio of 1:12 (Ammonium alum -
Ac) IL heat gradually from 25 °C to 80 °C for 10 hours produced colorless
liquid [83].

2.3.3.2 Ionic liquid preparation with urea:

- Hydrated aluminum nitrate and urea salts in the mole ratio of (1:1.2) (AN-
Urea) IL were heated gradually from ambient temperature to 80 °C for 3 hours,
to obtain colorless liquid [85].
- Ammonium alum and urea in the mole ratio of (1:5) (Ammonium alum -Urea)
IL were also heated gradually from ambient temperature to 80 °C for 3 hours
producing colorless liquid [83].
2.4 Cultivation conditions:-
2.4.1 Liquid cultures
Six kinds of Bacteria E.coli, K.peneumonice, Pseudomonas and Proteus as
gram negative and Staph.aureus and Staph.epidermidisas gram positive were
prepared by inoculation 25µl of standard bacteria in sterile tube contained 5ml
NB. After incubation for 24hrs at 37ºC the suspension of bacteria was formed,
then 100 μl was taken from the culture and transferred to 5 mL of fresh media in
a new sterile tubes containing series dilutions of (AN-Ac), |(AN-UREA),
(Ammonium alum -UREA), (Ammonium alum -Ac), (CaCl2.2H2O-Ac) and
(ALST-MEA) ILs at concentrations of (2, 5, 10 and 20) % (v/v) incubated for
24hrs at 37ºC. Subsequently all cultures was measured by the optical density.
2.4.2 Solid cultures
Culture media of NA plates were used for colony counting to determine the
number of viable cells (only for 2% and 5% ILs concentration). The plates of 60
mm in diameter with final volume of 5 ml of agar were spread using sterile
glass spreader rod. Each number of colonies in solid plate were counted by
using automatic colony counter after 24h incubation at 37ºC and the device
converted into the colony forming units (CFUs). On the other hand, culture
media MHA was used for antimicrobial activity (The Kirby-Bauer Disk
Diffusion Test) containing the six kind of bacteria on agar surface using sterile
cotton swab and then holes were made in the agar plate filled up with 50μL of
ILs to measure the inhibition zone.

2.5 Biochemistry Testes

Many experiments were performed initially based on ILs without dilution,
which showed an extremely effective toward Serum leading to an
agglomeration. After that ILs was diluted with deionized water and added to
the serum. Dilutions was continued until (3 μl) of appropriate concentration 2%
v/v ILs added to 300μl Serum was obtained, which has maintained the stability
of the serum parameters level. A statistical analysis of serum parameters were
performed for 50 patients (that they have deferent diseases like diabetes
mellitus, renal diseases, liver diseases, high pressure and others) at Al-Yarmouk
Teaching Hospital to emphasis the appropriate ILs concentration. On the other
 ̅ ) and the standard deviation (SD) was calculated by using the
hand the mean (𝑿
following equation:
SD= standard deviation
̅) 𝟐
∑(𝑿−𝑿
SD=√ X= each value in the data set
𝑵−𝟏 ̅ = mean of all values in the data set (mean of X)
𝑿
N= number of value in the data set

Chapter Three
Results
 and
Discussion
Chapter Three
Results and Discussion

3.1 Preparing room temperature ionic liquids by reacting Lewis

base with several Aluminum salts.

The following attempts were conducted to prepare an ionic liquid suitable for
biological activity from different organic components such as ethanolamine,
urea, aetamide, thiourea and thioacetmide, with aluminum salt such as
aluminum chloride, aluminum nitrate, ammonium aluminum sulfate, potassium
aluminum sulfate and aluminum stearate. Only the latter salt showed a sign of
ionic liquid formation. Some physical characteristics such as conductivity and
melting point, FTIR electronic spectra have measured for the products of above
reactions. These reaction will be outline hereafter.
3.1.1 Monoethanolamine with aluminum chloride.
In open atmosphere at room temperature 1 ml of ethanolamine (pale yellow
liquid) was placed in a glass tube and aluminum chloride (white solid) gradually
added in small amounts with continuous stirring to make 1:2 mole ratio (MEA:
AlCl3). At each addition there was a vigorous exothermic reaction with the rise
of alkaline mist vapors (detected by litmus paper). At the end of addition a
white solid lump in the yellow liquid was formed. It turned to dark solution with
solid lump after a few days Figure (3.1). A drops of the liquid (before turned to
dark color) mixed with water forming a white solid product Figure (3.2). This is
considered a non-formation feature for ionic liquid to be used as biological
material, since it react with the aqueous media. Therefor no farther investigation
of the resulted product was performed.

Figure (3.1): A conglomerate forming Figure (3.2): A white solid product from mixing drops of
(Aluminum Chloride with Ethanolamine). (Aluminum Chloride /Ethanolamine) liquid with water.

3.1.3 Monoethanolamine with aluminum chloride in CCl4.

In an attempt to reduce the exothermic reaction behavior of MEA with
AlCl3, the reaction was carried out in CCl4, as this solvent is immiscible with
MEA and AlCl3 was insoluble in it. AlCl3 was added to a solution containing 1:1
volume ratio of MEA: CCl4, a vigorous reaction was observed while the
solution was under fast mechanical stirring forming a very small balls of CCl4 in
the MEA liquid. The reaction produced gradually a solution of dark color at the
end of experiment when a large lump of solid was formed surrounding the
magnetic stirrer Figure (3.3). A similar test with water as in section (3.2.1) was
performed during this reaction and similar conclusion was achieved.

Figure (3.3): A conglomerate forming in mixture of (Aluminum Chloride

……………...with
3.1.3 Monoethanolamine with
Ethanolamine in CCl4).aluminum chloride in chloroform.

Ethanolamine was found to be miscible with chloroform. A 2ml of a mixture

(1ml ethanolamine + 1ml chloroform) was taken to be a reaction media for the
ionic liquid formation attempt. An addition of AlCl3 to the mixture with
continuous mechanical stirring was carried out when a non-vigorous reaction
was observed. The final addition of AlCl3 (saturation solution) made a 3:1 mole
ratio with MEA. Then the final mixture was heated up to 80 °C in a water bath
to evaporate the chloroform. It started boiling and forming a stable colorless
liquid when evaporation ceased Figure (3.4). This liquid changed in to black
color after two days. Figure (3.5). A drops of the liquid (before turned to dark
color) mixed with water forming a white solid product Figure (3.6). This is
considered a non-suitable formation feature for ionic liquid to be used as
biological material, since it react with the aqueous media. Therefor no further
investigation of the resulted product was performed.
Figure (3.4) Mixture of Figure (3.5) Liquid Figure (3.6): A white solid product
ethanolamine and AlCl3 in mixture of ethanolamine from mixing drops of (Aluminum
chloroform and AlCl3 in chloroform Chloride with Ethanolamine in
after two days chloroform) with water.

3.1.4 Monoethanolamine with aluminum nitrate, ammonium

aluminum Sulfate and potassium aluminum sulfate.
The mixtures of monoethanolamine and aluminum nitrate, ammonium
aluminum sulfate or potassium aluminum sulfate were mixed and heated up to
80°C in an attempt to form a deep eutectic liquid. The mole ratios that were
tried in this method were 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4 and 1:5 of the
above aluminum salts to monoethanolamine. In addition increasing
monoethanolamine ratio to aluminum salts 10:1 did not show ionic liquid
formation.

3.1.6 Monoethanolamine with aluminum stearate.

The deep eutectic method was used to prepare an ionic liquid by mixing
aluminum stearate with monoethanolamine. The mole ratios and melting point
of the resulted ionic liquids from heating the mixture up to 80°C are outline in
Table (3.1). The resulted solid above room temperature are lipid crystal ionic
[152, 153]
liquid type as also described in literature for similar ionic liquid
formation as it give a soapy subsistence when mixed with water. However,
increasing the mole ratio of monoethanolamine more than 10:1 aluminum
Mole Ratio
No. of Melting point
Aluminum stearate : Ethanolamine

stearate form a liquid phase of aluminum stearate dissolution in

monoethanolamine and not of ionic liquid type. Therefore, the melting point
composition phase diagram of the above ratios is presented in Figure (3.7)
showing the minimum melting point of 1:10 of aluminum stearate:
monoethanolamine at 40°C. The latter composition showed a conductivity of
0.2 mS/cm. The conductivity of aluminum stearate dissolved in toluene was
found to be anon conductive solution and monoethanolamine has a negligible
conductivity about 0.01 mS/cm. This would give an indication to the formation
of ionic species from the interaction of monoethanolamine with aluminum
stearate. This value is considered to be one of the features of a typical ionic
liquid which is accepted to have a conductivity between 0.1 to 20 mS/cm [50].
Table (3.1): Compositions and physical Measurements for Aluminum stearate with
Ethanolamine.
 1 1:1 59

2 1:3 54

3 1:5 50

4 1:7 47

5 1:10 40

70 200

60
59 150
50 54
50
47 100
40
ALST

MEA
40
30
50

20
0
10

0 -50
1:01 1:03 1:05 1:07 1:10

Mole Ratio of ALST/MEA

Fig(3.7): Schematic representation of a eutectic point on a two component

(ALST/MEA) phase diagram

The mixtures of monoethanolamine (MEA) and aluminum stearate (ALST)

were investigated by FTIR to elucidate the possible interaction resulting from
forming different mole ratios of both salts. The resulted FTIR tests are presented
in Figure (3-8) and the main group vibrational frequencies are tabulated in table
(3-2) for starting materials and the 1:1 mole ratio ionic liquid formed by mixing
the salts. It be seen that the vibrational mode of both (-OH), (-NH2) and (-NH)
of the MEA had been effected in ionic liquid a large decrease in intensity and
small vibrational frequency shift were observed. Thus the vibrational mode (-
OH) and (-NH) groups at 3350 cm-1 and 3170,3285 cm-1 respectively were
shifted to broad 3235 cm-1 and broad 3400 cm-1 respectively, noting that both
suffer from reducing intensity from about 10% to about 70%. In addition, (-
NH) bending vibration at 1595 cm-1 and (-OH) bending vibration at 1355 cm-1
were also reduced intensity in ionic liquid. Both latter vibrational modes
increased their frequencies by 1628 cm-1 and 1405 cm-1 respectively.
Consequently, (-C-O) vibration of MEA of the (C-OH) is also effected by
decreasing intensity. However, vibration of carbonyl group 1583 cm-1 of ALST
suffered changes by decreased frequency 18 cm-1and only small changes in
intensity. There changes might be taken to indicate an interaction between
ALST and MEA in the ionic liquid of (1:1) mole ratio via interaction of
aluminum in the state salt with both hydroxyl and amine group of the MEA salt.
As a result of such interaction would also show vibrational band of Al-O and
Al-N which were assigned to 586 cm-1 and 528 cm-1 in ionic liquid.
In addition to that when the ratio of MEA were increased up to 10, the
vibrational bands of MEA appeared and increased in intensity with increasing
the amount of MEA. This indicated 1:1 mole ratio is sufficient for ALST to
interact with MEA while the extra MEA mainly effected the melting point of
the mixture see Table (3-1) and Figure (3-7).

Table (3-2): FT-IR absorption bands of starting materials and ionic liquid.

Functional group AL - ST MEA IL – 1:1

 primary amines
stretching υ(N-H)
- 3170 , 3285 S 3400 W
Asymmetric
symmetric

υ(O-H) group from

- 3350 S 3235 W
the hydroxyl amine

υ (C-H)
Asymmetric –
CH2–, symmetric –
2916 - 2849 2923 – 2855 S 2917 – 2850 S
CH3 and –CH2–
stretching
vibrations
For CO2 1731 W - -

υ (C=O) and υ (N-

1583 C=O 1595 N-H 1565, 1628
H) bending

υ (C-H2) bending 1466 1452 1464

υ(O-H) bending - 1355 1405

υ (C-H3) bending 1326 - 1322

υ (C-N) stretching - 1168 1092

1075, 1032
υ(C-O) 1095-1072 1031
Primary alcohol

(N-H) out of plane - 942, 865 877

More CH2 groups

719 W - 721 W
rocking

(C-H) out of plane 581, 541 W 517 W -

υ (Al-O) 664 586 W

υ (Al-N) - - 528
 SAMPL NAME / 1:1 ionic liquid
SAMPL NAME /AL-ST
SAMPL NAME /MEA

Figure (3.8): FT-IR spectra of ALST, MEA and ionic liquid (1:10) mole ratio.

It is fond that this kind of ionic liquid is soapy subsistence when mixed with
water, therefore it was not used in biological tests to evaluate the effect on
bacteria, because it gives a high degree of turbidity when added to the broth
culture media where identifying the inhibition activity by optical density
measurement would be difficult. The tests of biochemistry of ionic liquid
mixture with the serum was non-homogenous and the tests are chromatic which
would interfere with the absorption of the test sample to be measured.
3.1.6 Thiourea with ammonium aluminum sulfate and potassium
aluminum sulfate.
A binary mixture of deferent molar ratio of thiourea (182°C) with
ammonium alum (93.5°C) and potassium alum (95°C) were mixed and heated
normally up to 150°C or at which the substances became liquid. After cooling to
room temperature the mixtures were formed and become solids and the melting
point was measured for all mole ratios mixtures between thiourea and
Ammonium Aluminum Sulfate (Table (3.3) and Figure (3.9)) or thiourea with
potassium aluminum sulfate (Table (3.4) and Figure (3.10)).

Table (3.3): Compositions and melting points of Ammonium alum/Thiourea

No. Mole Ratio of Ammonium alum: Thiourea Melting point

1 1:8 133

2 1:5 116

3 1:3 102

4 1:2 97

5 1:1 91

6 2:1 86

7 3:1 90

8 4:1 94

9 5:1 96
 140 200

133
120
150
116
100
102
97 96
94 100

Thiourea
80 91 90
ALUM

86

60
50

40
0
20

0 -50
1:01 1:02 1:03 1:05 1:08 2:01 3:01 4:01 5:01

Mole Ratio of ALUM/Thiourea

Fig(3.9): Representation of a eutectic point on a two component
Ammonium ALUM and Thiourea phase diagram

Table (3.4): Compositions and melting point of Potassium alum and Thiourea

No. Mole Ratio of Potassium ALUM : Thiourea Melting point

1 1:5 153
2 1:4 148
3 1:3 133
4 1:2 126
5 1:1 110
6 2:1 106
7 3:1 104
8 4:1 99
9 5:1 100
 180 200

160 153

140 150
148
120 133
126 104 100
ALUM(K)

100

Thiourea
100 110 106
99
80
50
60

40 0
20

0 -50
1:01 1:02 1:03 1:04 1:05 2:01 3:01 4:01 5:01
Mole Ratio of ALUM(K)/Thiourea

Fig(3.10): Representation of a eutectic point on a two component Potassium

ALUM and Thiourea phase diagram

As indicated in table (3.3), depression of melting point of thiourea (182°C)

was obvious and the depression increased linearly with increasing the mole ratio
of ammonium alum (133°C m.p of 1:8 mole ratio of ammonium alum : thiourea
while 91°C m.p of 1:1 mole ratio of the mixture). The 1:8 mole ratio indicated a
formation of complex compound that on increasing the mole ratio of ammonium
alum to double the thiourea it formed a eutectic compound melting at 86°C.
However, increasing ammonium alum further increase the melting point of the
mixture, yet it was less than ammonium alum melting point of 93.5°C. Hence it
is also clear that thiourea also depressed melting point of ammonium alum
particularly when the mixture contain 2/3 ammonium alum and 1/3 thiourea (i.e.
2:1) mole ratio. The depression in melting point which was obvious on thiourea
indicated an interaction between ammonium alum and thiourea probably
through the interaction of aluminum metal cationic species with Sulphur
electron pair of thiourea in a similar behaver of aluminum chloride – urea type
ionic liquid [177] or aluminum chloride – thiourea ionic liquid. [178]
Again potassium alum mixture with thiourea also experienced a depression
melting point of thiourea as when the mole ratio of potassium alum – thiourea
was 1:5 the melting point of thiourea depressed from 182°C for thiourea alone
to 153°C in the mixture. Decreasing the ratio of thiourea (or increasing the ratio
of potassium alum) related in more depression until a 1:1 mole ratio registered a
melting point of 110°C. Again the 1:1 mole ratio, also showed distinguished
phase formation with highest melting point of 153°C. The latter melting point
lowered sharply to 106°C when the mole ratio was 2:1 for potassium alum:
thiourea. The rate of lowering melting point of the mixture went slowly when
the potassium alum fraction was increased until it reached around 99°C and
100°C at 4:1 and 5:1 potassium alum: thiourea mole ratios.
The interaction between thiourea and potassium alum could also arise from the
metal aluminum cation with the sulphur of thiourea as stated above with
ammonium alum.

3.1.7 Thiourea with Aluminum Nitrate.

Thiourea and aluminum nitrate were mixed in mole ratio of 5:1, 4:1, 3:1, 2:1,
1:1, 1:2, 1:3, 1:4 and 1:5. Most mole ratio showed no sign of ionic liquid
formation even on heating up to 80°C. However, the mole ratios of 1:1, 2:1 and
3:1 showed a vigorous exothermic reaction when temperature was increased
from 60°C to 80°C with an orange fumes evaporation. Figure (3.11). After
cooling the product of these mole ratios formed a colorless liquids at room
temperature and the product of 1:1 and 3:1 mole ratios changed to a solid
material within few minutes while the product of 2:1 mole ratio remained liquid
for 3 days at room temperature before changing into white solid material.
Therefore, due to no stable liquid formation of these products, this type wasn’t
investigated for biological tests.

Figure (3.11): Thiourea

with Aluminum Nitrate
(2:1) mole ratio.

3.1.9 Aluminum stearate with thiourea and with thioacetamide.

No reaction or interaction was observed when Aluminum stearate mixed
with thiourea or with thioacetamid separately in different mole ratios (i.e. 5:1,
4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4 and 1:5) respectively, even when lifting the heat
to above 100°C.

3.1.9 Thioacetamide with ammonium aluminum sulfate and

potassium aluminum sulfate.

Mixing and heating thioacetamide with ammonium aluminum sulfate or

potassium aluminum sulfate in different mole ratios (i.e. 5:1, 4:1, 3:1, 2:1, 1:1,
1:2, 1:3, 1:4 and 1:5) respectively also gave no reaction or interaction between
the materials to form ionic liquid, although lifting the heating temperature up to
100˚C.

3.1.10 Thioacetmide with Aluminum Nitrate.

The reaction mixture of Thioacetmide with Aluminum Nitrate was similar to
Thiourea - Aluminum Nitrate see section (3.1.7), when used the same mole ratio
and heating degree, where a vigorous with orange fumes was resulted for 1:1,
2:1 and 1:2 mole ratios of aluminum nitrate, thioacetmide respectively (Fig.
3.12), but eventually a yellow liquid was formed, which turned into solid yellow
material immediately when cooled to room temperature.

Figure (3.12): Thioacetmide

with Aluminum Nitrate (2:1,
1:1 and 1:2) mole ratio.
3.2 Synthesis of New Ionic Liquid from calcium chloride
dihydrate /acetamide
3.2.1 Phase diagram binary of calcium chloride dihydrate
/acetamide
A binary deep eutectic solvent was prepared by heating mixtures of calcium
chloride dihydrate and acetamide in different mole ratios (see chap.2.3.2). The
colorless homogeneous liquid obtained at 85 ℃ from each mixture was
maintained at this temperature for 2 hours to insure complete liquid state
formation then slowly cooled to room temperature. The novel binary deep
[179,180]
eutectic solvent (DES) systems, (CaCl/Ac) was found to have deferent
freezing points at deferent mole ratio, Figure (3-13), and Table (3-1). The phase
rule indicates that there is a single degree of freedom at the eutectic point
obtained from Gibb's phase rule as shown below [154]:

f´=1+C-P , when
f´= the number of degrees of freedom
C = number of components
P = number of phases.
f´=1+ 2 – 3 ⇒ F = 0

The lowest freezing point obtained for this ionic liquid of -7 ℃ for 1:7 mole
ratio of CaCl/Ac was found to increase if the mole ratio of acetamide was
altered. Hence at 1:8 and 1:9 mole ratio the freezing point of IL increased to -
3℃ and 1℃ respectively, while decreasing acetamide mole ratio to 1:6, 1:5 and
1:4 mole ratios resulting increased the freezing point to -2℃, 2℃ and 5℃
respectively. Ionic liquids were formed of Ac mole ratio ranged between 1:4
and 1:9 with freezing temperature less than 90℃. However, a solid phase was
formed if Acetamide exceeded mole ratio of 10 or lower than 4. Figure (3-13).
All liquids were found visually to be viscous compared to water.
It is worth noticed that increasing mole ratio of CaCl 2.2H2O over acetamide did
not form ionic liquid and only formed a solid lump.
This prepared ionic liquid was then taken for farther investigation.

200 200

176

150 150
Calcium chloride

100 100

Acetamide
90
82 81

50 61 50

42

0 0
5 2
-2 1
-3
-7

-50 -50
1:00 1:01 1:02 1:03 1:04 1:05 1:06 1:07 1:08 1:09 1:10 0:01

Mole Ratio of CaCl2.2H2O/acetamide

Fig(3-13): Schematic representation the eutectic point of two

component (CaCl2.2H2O/acetamide) IL
Table (3-5): Compositions and freezing points of new ionic liquid CaCl2.2H2O/AC

Mole ratio of Physical

CaCl2.2H2O Acetamide Melting Freezing
No. CaCl2.2H2O / state at
mole % mole % point ˚C point ˚C
Acetamide 26 ˚C

1 1:1 50 50 90 - solid

2 1:2 33.33 66.66 82 - solid

3 1:3 25 75 61 - solid

4 1:4 20 80 - 5 Liquid

5 1:5 16.66 83.33 - 2 Liquid

6 1:6 14.28 85.71 - -2 Liquid

7 1:7 12.5 87.5 - -7 Liquid

8 1:8 11.11 88.88 - -3 Liquid

9 1:9 10 90 - 1 Liquid

Liquid
10 1:10 9.09 90.9 42 -
+ solid

3.2.2 Simultaneous Thermal Analysis STA (TG – DSC).

The mole ratio of 1:7 with lowest freezing point was chosen to study its
thermal stability thus thermal analyses with differential scanning calorimetry
(DSC) and thermogravimetric analysis (TGA) were conducted on this IL. Figure
(3-14). The DSC graph indicated numbers of internal changes in the mixture
during heating. These changes were of endothermic nature as occurred at
148.1℃, 237.1℃ and 273.6℃ watch occur before decomposition of ionic liquid
started at onset of 286.8 ℃ as shown in the TGA graph. The third internal
changes at around 273.6℃ does not seems to be complete as the onset of the
thermal decomposition at 286.7℃ was very close to it. Probably resulted in
overlapping with each other giving a small internal heat changes while the
exothermal heat resulted from the thermal decomposition cover it. The
decomposition, which commenced at 286.7℃ was fast as its weight loss was
sharply inclined and reach completeness at around 344.4℃. However, at the
latter temperature it was followed by a small endothermic changes and farther
stage of decomposition. The product at this stage was not stable as a very slow
weight loss was recorded from about 325℃ until 600℃.

Fig (3-14): A: (TGA) and B: (DSC) analysis of calcium chloride dihydrate /acetamide ionic
pppppppp liquid.
3.2.3 Interaction between Calcium Chloride dihydrate /Acetamide

The ionic liquids obtained from mixing and heating the mixture of solid
CaCl2.2H2O (m.p 176°C) and Acetamide (m.p 81°C) which gave a liquid with
much lower melting point than the original solids. Such ionic liquid formation is
known as deep eutectic solvent (DES) [179, 180]. This type of liquid might will be
formed due to formation of large unsymmetrical species of negative and
positive charges which are weakly interacted. Such weak interaction would not
help to reform the original solid lattice of the reactant and therefore the ionic
[157]
species forming a liquid state. Hydrogen bond formation between these
species could be one of the interaction. The endothermic stages during the
course of heating cold be related to rearrangement of coordination of the anionic
and cationic species of the ionic liquids. To ensure an evaluation of the
interaction the following FT-IR analysis was performed.

3.2.4 FT-IR spectra of calcium chloride dihydrate /acetamide

Ionic Liquid

The vibration frequencies FT-IR spectrum are showen in Figure (3-15) of

ionic liquid at (1:7) mole ratio and its calcium chloride dihydrate and acetamide
salts. It can be seen that the initial vibration frequencies of both solid salts are
shifted to either higher or lower frequencies in the ionic liquid indicating an
interaction between their functional groups. Hence, the vibration frequency of
carbonyl group 𝛎(C=O) changed from 1667 cm-1 to 1649 cm-1 with a shift of 18
cm-1 to a lower frequency than in free Acetamide. The stretching frequencies of
ν(N-H) 3304 cm-1(asym) and 3154cm-1(sym) for Acetamide were also suffered
a blue shift by about 23 cm-1 in IL for the symmetrical vibration and about 14
cm-1 for the asymmetric vibration. The latter changes could probably resulted
due to H-bonding of O-H stretching of water molecule along with that of N-H
stretching [155, 156]. The changes in the bands of the vibrations of 𝝂 (CO) and (N-
H) in acetamide indicate a coordination or interaction when ionic liquid is
formed but the coordination or interaction with nitrogen of amide seems to be
stronger than the carbonyl group because of the relatively larger shift that
occurs in amide group. Other frequencies of stretching vibration bands are
shown in the Table (3-6).

Table (3-6): FT-IR absorption bands of the starting materials and ionic liquid (cm-1).

Compounds group CaCl2.2H2O H2NCOCH3 IL

υ(O-H) 3429 - -

υ(N-H) 3304 & 3154 3318 & 3177

υ(C-H) - 2814 2797

Over tone 2154 - -

υ(C=O) &(C=C)for
Acetamid tautomer - 1667 1649 & 1603
& (N-H) bending

υ(O-H) bending 1620 - -

υ(C-H) SP3 bending

- 1390 1390
and scissoring

υ(C-N) - 1143 1134

1007
υ(C-O) 1005

υ(C-H) SP2 bending 870 873

 a
b

a. Ionic Liquid

b. CaCl2.2H2O
c. Acetamide

Fig (3-15): FT-IR spectra for calcium chloride dihydrate /acetamide new IL.

3.2.5 Electronic spectrum of calcium chloride dihydrate

/acetamide Ionic Liquid
The electronic spectrum of the colorless ionic liquid at (1:7) mole ratio of
CaCl/Ac showed absorption bands in the ultraviolet region at (200 – 380 nm),
which was suggested to be due to and electronic transition
in amine and carbonyl groups, Figure (3-16). The electronic transitions between
 and normally occur at high-energy and involve very short
wavelength of ultraviolet light (< 200 nm). These transitions usually fall out of
the generally available measurable range of UV-visible spectrophotometers
(200-1000 nm) which requires vacuum UV spectrophotometer and this
instrument is unavailable. No absorption in the visible region was observed
because this ionic liquid was a colorless solution and such transition was not
expected to be observed.

Figure (3-16): Electronic spectrum of Calcium Chloride dihydrate /Acetamide Ionic

…………………Liquid.
3.2.6 Some Physical Properties of calcium chloride dihydrate
/acetamide new ionic liquid of 1:7 mole ratio.

The conductivity, density and viscosity were measured at 26℃ for the new
ionic liquid of 1:7 mole ratio of CaCl/Ac were found to be 0.31 mScm-1, 1.130 g
cm-3 and 442.63 cP respectively. The conductivities of other ionic liquids based
[85] [157]
on aluminum salts or choline chloride salt with urea showed a higher
conductivity than present ionic liquid. The conductivity for AN/Urea has 9.09
mScm-1, for ammonium alum /urea about 4.05 mScm-1, for AlCl3/Urea about
0.804 mScm-1 and ChCl/Urea 1.64 mScm-1. However, present ionic liquid has
higher viscosity than AN/Urea, AlCl3/Urea and ChCl/Urea ionic liquids with
20.579, 60 and 61.5 cP respectively. The density (Table (3-7)) of present ionic
liquid found to decrease with increasing the temperature, while the conductivity
was found to increase see Tables (3-8 and 3-9) and Figures (3-17 and 3-18). The
lower conductivity of the ionic liquid could be related to the high viscosity
value of CaCl/Ac compared to the other ionic liquids in table (3-7). The high
viscosity known to reduce charge movement within the viscous solution or
[158]
liquid . This is in agreement with the low viscosity and high conductivity
values of other ionic liquids such as AN/urea which has the highest conductivity
and lowest viscosity among other ionic liquids stated in table (3-7). On the other
hand the higher viscosity of CaCl-Ac compared with other ionic liquids was
related to “Hole Theory” [159]
, in which the viscosity of a range of ionic and
molecular fluids is shown to be proportional to the probability of finding an
adjacent hole of sufficient dimensions to permit movement [160].

One of the features of this ionic liquid is its miscibility with polar protic
solvents such as water, ethanol and propanol but was not miscible with non-
polar organic solvents like toluene and olefin oil or polar aprotic solvent like
acetone. This feature is normally useful in the application of separation
technique of the analytical chemistry.
This new ionic liquid could be considered useful as a green solvent because it
has a low vapor pressure and stable to air moisture and the properties of this
liquid are close to those of the most common ionic liquids [161].

Table (3-7): Comparison density, viscosity, acidity and conductivity for different ILs.

Density Viscosity Cond.

Ionic Liquid Ref.
(g. cm-3) (c P) (mScm-1)

In this
CaCl2.2H2O/Acetamide 1.130 442.63 0.31
work

Al(NO3)3.9H2O/Urea 1.6028 20.579 9.09 [85]

NH4Al(SO4)2.12H2O/Urea 1.5 - 4.050 [83]

AlCl3/Urea 1.4 60 0.804 [181]

ChCl/ Urea 1.21 61.5 1.640 [157]

Table (3-8): Density and Conductivity versus temperature for the new IL.

Temp Density Conductivity Temp Density Conductivity Temp Density Conductivity

˚C g/m3 mS/cm ˚C g/m3 mS/cm ˚C g/m3 mS/cm

20 1.133 0.29 45 1.113 0.47 70 1.085 0.98

25 1.131 0.31 50 1.109 0.57 75 1.078 1.16

30 1.127 0.33 55 1.105 0.65 80 1.074 1.63

35 1.124 0.35 60 1.098 0.76 85 1.069 2.14

40 1.119 0.4 65 1.093 0.88 90 1.061 2.53

 Fig (3-17) :Density and Conductivity versus temperature for
the new IL
1140 3

1133
1131
1127
1120 1124 2.5
2.53
1119
1113
1109

Conductivity mS/cm
1100 1105 2.14 2
Density Kg/m3

1098
1093
1.63
1080 1085 1.5
1078
1074
1069
1060 1.16 1
1061
0.98
0.88
0.76
1040 0.65 0.5
0.57
0.47
0.4
0.33 0.35
0.29 0.31

1020 0
20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
Temperature ˚C

Density Conductivity

Table (3-9): viscosity and Conductivity versus temperature for the new IL.

Temp Viscosity Conductivity Temp Viscosity Conductivity Temp Viscosity Conductivity

˚C cP mS/cm ˚C cp mS/cm ˚C cp mS/cm

20 497.33 0.29 45 141.32 0.47 70 42.82 0.98

25 471.71 0.31 50 107.92 0.57 75 34.90 1.16

30 324.81 0.33 55 88.53 0.65 80 28.66 1.63

35 242.64 0.35 60 69.24 0.76 85 24.93 2.14

40 165.54 0.4 65 55.17 0.88 90 20.57 2.53

 600 3

2.53

500 2.5
497.33
2.14
471.71

400 2

Conductivity mS/cm
Viscosity cp

1.63

300 324.81 1.5

1.16

0.98
242.64
200 0.88 1
0.76
141.326
0.65
165.54 0.57
0.47
0.4
100 0.31 0.33 0.35 0.5
0.29 107.92
88.53 34.9 28.66
69.24 24.93 20.57
55.17
42.82
0 0
20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
Temperature ˚C

Viscosity Conductivity

Fig (3-18): viscosity and Conductivity versus temperature for the new
IL

3.2.7 Cyclic Voltammetry of calcium chloride dehydrate/

acetamide ionic liquid.

The common method to determine the electrochemical window is the cyclic

voltammgrams that gives the behavior of redox active compounds in ionic
liquids. The difference between the reductive potential limit and the oxidation
[20]
potential limit is the electrochemical potential window of ionic liquids .
Figure (3-19 a) shows the scale of electrochemical window for the ionic liquid
CaCl/Ac, with a mole ratio of 1:7 with platinum as the working electrode. The
electrochemical window of this ionic liquid was found to be about (-0.2 to 1.2)
smaller with other ionic liquids as ChCl/ urea that was reported to has a
[157]
potential window of 2.4 V (-1.2 to +1.2) and [C2mim][NTf2] reported to
have 4.5V [157], and relatively higher than aqueous systems that possess about
[151]
1.23V (- 0.82 to + 0.41) . The reduction potential of the ionic liquid was
scanned to more negative value (3-19 b) in an attempt to determine the possible
reduction of calcium cation to calcium metal. This was not observed probably
due to the presence of water molecules which might prevent deposition of
calcium on platinum electrode by the reduced proton to hydrogen gas which
[151]
will make a barrier on the surface of platinum electrode . The study of
electrochemical window of ionic liquid is interesting for many applications,
such as batteries and fuel cells... etc. [162].

Fig. (3-19): Cyclic voltammogram for Pt elctrode in CaCl2.2H2O : Acetamide, with 1:7
mole ratio mixture at room temperature
3.2.7.1 Cyclic Voltammetry of monoethanolamine with aluminum
chloride in chloroform and without chloroform, and aluminum
nitrate with thiourea.

The ionic liquid formed from mixing MEA with aluminum chloride in
chloroform and after evaporation of chloroform (section 3.1.3) was investigated
by cyclovoltammogram to determine it electro chemical redox behavior, Figure
(3-20). The liquid showed a range of -0.6v to +1.1v determined reduction and
oxidation potential with electrochemical window of 1.7v. However when the
redaction potential was scanned to more negative potential (-2.0v), the only
differences was that the reduction was faster in chloroform than after
evaporation of chloroform. This would indicate the dilution effect to enhance
rate of reaction by increasing charge mobility. In the same manner the oxidation
of ionic liquid was enhanced by dilution with chloroform.

However, aluminum nitrate mixed with thiourea (3.1.7) which gave liquid
was also investigated by cyclovoltamatric method, Figure (3-21). Which gave a
redox potential of -0.8v to +1.6v with electrochemical window of 2.4v. The two
reduction waves were observed at -0.4v and -0.95v, both reduced species were
not shown in the cyclovoltammogram to be oxidize, which indicated either anon
oxidized species or species that reduced to gas form which would evaporated
from the liquid. In the same manner the oxidized species on the oxidation scale
after 1.6v did not reduced when the scan was reversed showing most probably
gas formation.
Fig. (3-20): Cyclic voltammogram for Pt electrode in monoethanolamine with aluminum
chloride in chloroform (Red) and without chloroform (Blue) at room temperature.

Fig. (3-21): Cyclic voltammogram for Pt electrode in aluminum nitrate with thiourea at
room temperature.
3.3 Biological activity of ionic liquids
Due to the large number of ionic liquids which are currently used, it is
desirable to have a fast and reliable method for initial toxicity screening against
a range of microorganisms. To this end, a simple and cost effective method for
ionic liquid toxicity testing using agar diffusion plates and the optical density
(OD) test, has been developed and employed to test the toxicity and biological
behaver of ionic liquids of the present work against a number of gram positive
and gram negative bacteria.

3.3.1 The Kirby-Bauer Disk Diffusion Test

Recently the Agar Diffusion Test (or Kirby-Bauer test) has been adopted to
[163]
measure ionic liquid toxicity . This method is widely used in clinical
[164]
laboratories to determine antibiotic susceptibility of microorganisms . The
method is simple, inexpensive, requires no specialized equipment and uses only
a small volume of ionic liquid.
One of the methods used in this test is the addition of ionic liquid to a sterile
filter paper disc, but studies [165] have shown that some ionic liquids can interact
with the cellulose filter paper and that will lead to a wrong results, so to avoid
this overlap, filter paper disc test was excluded and replaced by holes that was
made in agar culture media filled with ionic liquids.
The (AN-Ac), (AN-urea), (ammonium alum-urea), (ammonium alum-Ac) and
(CaCl-Ac) ILs that were used in this test found to decrease the activity of
(E.coli, K.peneumonice, Staph.aureus, Staph.epidermidis, Pseudomonas and
Proteus) bacteria growth, evidently through measuring the inhibition zone (in
millimeter) that appeared around the holes filled up with (50 μl) concentrated
ionic liquids as shown in Table (3-10), and Figure (3-22) below:
Table (3-10): The Bacterial inhibition zone diameter (mm) against ILs.

Ionic liquid E.coli K.peneumoniae Staph.aureus Staph.epidermidis Pseudomonas Proteus

AN-Ac 24 25 28 28 25 27

AN-urea 22 21 24 24 23 22

ammonium
alum -Ac
19 17 20 18 17 18

ammonium
alum -urea
17 17 22 18 18 16

CaCl-Ac 20 18 16 16 18 15
 Staph.epidermidis Pseudomonas Proteus

Fig. (3-22): The Bacterial inhibition zone against ILs.

A clear inhibition zone is formed around the holes, this indicates that the ionic
liquid is inhibitory. The size of the inhibition zone can be measured to give an
indication of the degree of toxicity. From the data in the Table (3-10) and figure
(3-22) it was noted that the inhibition zone of AN-Ac IL was higher than the
other ILs in all types of tested bacteria and that gave an initial impression that
AN-Ac IL's ability to inhibit and kill bacteria is higher than the other ILs. In
contrast the inhibition zone of CaCl2.2H2O-Ac IL had the lowest results in some
bacteria, this result may be due to high viscosity of CaCl-Ac IL which effect its
diffusion compared with the other less viscous ionic liquids (see Table (3-7)).
Since the effect of liquids used on different types of bacteria will be arranged as
follows:

(AN-Ac) > (AN-urea) > (ammonium alum-urea) = (ammonium alum -Ac) > (CaCl-Ac)
Note that the presence of Aluminum Nitrate in ionic liquids gave a higher effect
on the bacteria compared to ammonium alum and also the presence of
Acetamide in AN ionic liquids gave a slight increase compared to presence of
urea. However, urea showed better effect in ammonium alum ionic liquid which
may be related to the presence of two amide group while one group of amide in
Ac. The difference in AN ionic liquid of the presence of urea over Ac may be
related to the more effective nitrate group than amine group.
The advantages of this test is simple, cheap, requires little preparation and only
basic microbiology skills.

The agar diffusion method has previously been used to screen a range of ionic
[174]
liquids against both Gram positive and Gram negative bacteria . Although
the test can be used reliably to identify highly inhibitory ionic liquids, it is more
[174]
difficult to rank the least inhibitory structures . Therefore the method is
intended only as preliminary screening tool to identify ionic liquids suitable for
further in depth ecotoxicological testing. There are a number of potential
problems with the microbial toxicity tests and the agar diffusion method in
particular. These problems must be carefully considered when reviewing the
data generated from these tests.
Of most concern when assessing the results of any test for ionic liquid toxicity
is the lack of understanding of the way in which the ionic liquid interacts with
the culture media and any other test reagents. Ionic liquids are salts and may,
[166]
therefore react with the ionic components of the culture medium , formation
of precipitates. Such interactions would affect measurements of microbial
growth rates, and in extreme cases may affect the integrity of the tests.
Similarly, these interactions may affect the agar diffusion test, since it relies on
diffusion of water miscible ionic liquids through the medium to create a
concentration gradient. Interactions between medium components and the ionic
liquid may influence the diffusion rate or may affect the stability of the resulting
concentration gradient. The formation of a stable concentration gradient also
assumes a uniform diffusion through the agar for all ionic liquids, regardless of
size or other properties, this assumption may not be correct for all ionic liquids
[167]
. A further problem with screening ionic liquids in microbial systems is that
the ionic liquids themselves may be chemically reactive or may act catalytically
to accelerate changes in the composition of the culture medium or materials
used to prepare the assays. For example, the interaction between certain ionic
[165]
liquids and cellulose has been widely reported , and this may have an
influence on the outcome of the agar diffusion test, in which cellulose filter
papers are used. Due to these possible limitations no single test system should
be used in isolation to assess the toxicity of an ionic liquid. To get the most
accurate picture of an ionic liquid’s toxicity it is preferable to consider the data
collected from a range of different ecotoxicological tests. However the
combination of data from a disparate range of test organisms, using different
testing methods and presenting data in varying formats is challenging.

It is also worth mentioning that this rate of diffusion is governed by the physical
and chemical properties of ILs. Its relative hydrophobicity, aqueous solubility,
and molecular weight dictate the extent of diffusion through the agar. Hence the
major disadvantage of this method is that the bacteriostatic or bactericidal
[168]
concentration cannot be quantified effectively . Therefor this technique is
considered to give a qualitative susceptibility and a preliminary perception of
the effectiveness of ILs on the growth of bacteria. So this test was used in order
to establish the validity of the agar diffusion method as an initial screen for
ionic liquid toxicity.

It is worth mentioning that the problems listed for this test did not occur to the
ionic liquids that used in this work, perhaps only the effect of high viscosity of
CaCl/Ac IL on diffusion as previously mentioned.
3.3.2 The optical density (OD) test and Colony forming
units count.
The effects on the bacteria of exposure to different concentrations of
concerned ionic liquids has been investigated by means of optical density (OD).
This test is considered to be high accuracy with compared to the previous
method.

The optical density (OD) was measured by using spectrophotometer at 600nm.

This wavelength was chosen because it is a wavelength of light that is
minimally absorbed by material within cells and, therefore, can be more reliably
used in most common situations than other wavelengths to simply measure the
scattering of light due to turbidity (presence of particles in a solution) [169].

The OD measurement was made after 24h incubation at 37ºC for (E.coli,
K.peneumonice, Staph. Aureus, Staph.epidermidis, Pseudomonas and Proteus)
bacteria culture, contend 2%, 5%, 10% and 20% concentration (v/v ionic liquid
to distill deionized water) of (AN-Ac), (AN-urea), (ammonium alum -urea),
(ammonium alum -Ac) and (CaCl-Ac) ILs in broth liquid medium. The OD
value without incubation has been subtracted from the results which are shown
in Table (3-11), Figures (3-23), (3-24) and (3-25).

Table (3-11): The optical density (OD) value for bacteria growth culture with deferent
concentration of ILs (The OD value without incubation has been subtracted from the results).

ammonium ammonium
The type of bacteria Conc. v/v AN-Ac AN-urea CaCL-Ac
alum -Ac alum-urea
E.coli : 0.512(control
solution) 2% 0.074 0.091 0.110 0.117 0.410
Blank = (Broth+100µl
E.Coli without
incubation = 0.042)
5% 0.021 0.030 0.039 0.045 0.159
 10% 0.005 0.007 0.011 0.009 0.003

20% 0.001 0.003 0.004 0.003 0.000

2% 0.080 0.085 0.093 0.097 0.435

K.peneumonice : 0.706
(control solution)
5% 0.025 0.027 0.031 0.030 0.160
Blank = (Broth+100µl
K.peneumoniae without
incubation:0.042) 10% 0.009 0.011 0.013 0.012 0.003

20% 0.000 0.001 0.002 0.005 0.000

2% 0.039 0.047 0.055 0.057 0.370

Staph.aureus : 0.470
(control solution)
5% 0.014 0.020 0.026 0.027 0.127
Blank = (Broth+100µl
Staph.aureus without
incubation:0.025) 10% 0.002 0.002 0.005 0.006 0.002

20% 0.000 0.000 0.001 0.003 0.000

2% 0.049 0.066 0.078 0.075 0.475

Staph.epidermidis :
0.811(control solution)
5% 0.006 0.009 0.026 0.027 0.164
Blank = (Broth+100µl
Staph.epidermidis
without
incubation:0.043) 10% 0.000 0.004 0.006 0.008 0.003

20% 0.000 0.000 0.000 0.001 0.000

Pseudomonas :
0.355(control solution) 2% 0.052 0.058 0.058 0.060 0.286
Blank = (Broth+100µl
Psendomonas without
incubation:0.029)
5% 0.012 0.013 0.018 0.018 0.096
 10% 0.001 0.003 0.007 0.008 0.005

20% 0.000 0.001 0.001 0.002 0.000

2% 0.036 0.040 0.044 0.052 0.344

Proteus : 0.593(control
solution)
5% 0.010 0.015 0.022 0.025 0.148
Blank = (Broth+100µl
Proteus without
incubation:0.047) 10% 0.001 0.002 0.004 0.009 0.004

20% 0.000 0.000 0.001 0.002 0.000

a. E.coli
0.6
0.512

0.5
0.41

0.4
turbidity

0.3
0.159

0.2
0.117
0.11
0.091
0.074

0.045
0.039

0.1
0.021
0.03

0.011
0.009
0.007
0.005

0.004
0.003

0.003

0.003
0.001

0
control solution 2% 5% 10% 20%

concentration of IONIC LIQUID v/v

ALN-ACE ALN-UREA ALUM-ACE ALUM-UREA CaCl/AC

 b. K.peneumoniae

0.706
0.8

0.7

0.6

0.435
0.5
turbidity

0.4

0.3

0.16
0.2

0.097
0.093
0.085
0.08

0.031
0.027
0.025

0.013
0.012
0.011
0.03

0.009

0.005
0.003

0.002
0.001
0.1

0
0
control solution 2% 5% 10% 20%

concentration of IONIC LIQUID v/v

ALN-ACE ALN-UREA ALUM-ACE ALUM-UREA CaCl/AC

c. Staph.aureus
0.47

0.5

0.45
0.37

0.4

0.35

0.3
turbidity

0.25

0.2
0.127

0.15
0.057
0.055
0.047

0.1
0.039

0.027
0.026
0.014
0.02

0.006
0.005

0.003
0.002
0.002

0.002

0.001

0.05
0
0
0

0
control solution 2% 5% 10% 20%

concentration of IONIC LIQUID v/v

ALN-ACE ALN-UREA ALUM-ACE ALUM-UREA CaCl/AC

 d. Staph.epidermidis

0.811
0.9

0.8

0.7

0.6

0.475
turbidity

0.5

0.4

0.3

0.164
0.2 0.078
0.075
0.066
0.049

0.027
0.026
0.009

0.008
0.006

0.006
0.004

0.003

0.001
0.1

0
0
0

0
0
control solution 2% 5% 10% 20%

concentration of IONIC LIQUID v/v

ALN-ACE ALN-UREA ALUM-ACE ALUM-UREA CaCl/AC

e. Pseudomonas
0.4
0.355

0.35
0.286

0.3

0.25
turbidity

0.2

0.15
0.096
0.058
0.058

0.1
0.052

0.06

0.018
0.018
0.013
0.012

0.008
0.007

0.05
0.005
0.003

0.002
0.001

0.001
0.001

0
0

0
control solution 2% 5% 10% 20%
concentration of IONIC LIQUID v/v

ALN-ACE ALN-UREA ALUM-ACE ALUM-UREA CaCl/AC

 f. Proteus
0.7

0.593
0.6

0.5

0.344
0.4
turbidity

0.3

0.148
0.2

0.052
0.044
0.036

0.025
0.04

0.022
0.1

0.015

0.009
0.004

0.004
0.002

0.002
0.001

0.001
0.01

0
0

0
0
control solution 2% 5% 10% 20%

concentration of IONIC LIQUID v/v

ALN-ACE ALN-UREA ALUM-ACE ALUM-UREA CaCl/AC

Fig (3-23). The optical density value for a. E.coli, b. K.peneumoniae, c. Staph.aureus, d. Staph.
Epidermidis, e. Pseudomonas and f. Proteus bacteria growth with deferent concentration of ILs.

Fig. (3-24). Influence of A. (AN-AC) B. (ammonium alum-urea) on growth of K.peneumoniae in liquid

medium. a: control, b: blank and numbers 1-4 corresponds to 2, 5, 10 and 20% (v/v) of IL, respectively
(as example) .
Fig. (3-25). Influence of A. (AN-AC) and B. (ammonium alum -urea) on growth of St. Aureus
in liquid medium. a: control, b: blank and numbers 1-4 corresponds to 2, 5, 10 and 20% (v/v) of
IL, respectively (as example).

After that another cultures were carried out to calculate colony forming units.
The tests was only performed for 2% and 5% concentrations because they had
bacterial growth. To perform colony forming units, a 0.1ml from each tube was
serial diluted and spread on solid media. The data of this test shown in Table (3-
12) and Figures (3-26) and (3-27).

Table (3-12): Colony forming units (CFUs)/ml after 24h incubation

Conc. ammonium ammonium

The type of bacteria AN-Ac AN-urea CaCL-AC
v/v alum -Ac alum -urea

2% 86x105 215x106 192x107 187x107 112x109

E.coli
5% 52x104 92x104 213x104 195x104 86x107

2% 116x106 185x106 205x106 235x106 156x109

K.peneumoniae
5% 33x104 48x104 63x104 58x104 92x107
 2% 11x105 29x105 41x106 63x106 99x108
Staph.aureus
5% 16X102 36x103 116x104 12x105 27x107

2% 51x103 105x103 65x104 78x104 59x1010

Staph.epidermidis
5% 13x102 55x102 25x104 92x104 107x107

2% 47x106 33x107 36x107 117x107 152x108

Pseudomonas
5% 10x102 11x102 22x102 63x102 118x103

2% 114x105 47x106 97x106 126x106 104x108

Proteus
5% 152x102 21x103 56x103 105x103 61x105

a. E.coli
1.00E+12
1.12E+11
1.92E+09

1.87E+09

1.00E+11
2.15E+08

1.00E+10
8.60E+08
1.00E+09
8.60E+06

2.13E+06

1.95E+06

1.00E+08
9.20E+05
5.20E+05

1.00E+07
CFUs/ml

1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
2% 5%
concentration of IONIC LIQUID v/v

ALN-ACE ALN-UREA ALUM-ACE ALUM-UREA CaCl/AC

 b. K.peneumoniae
1.00E+12 1.56E+11
1.00E+11

2.35E+08
2.05E+08
1.85E+08
1.16E+08
1.00E+10
9.20E+08
1.00E+09

6.30E+05

5.80E+05
1.00E+08

4.80E+05
3.30E+05
1.00E+07
CFUs/ml

1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
2% 5%
concentration of IONIC LIQUID v/v

ALN-ACE ALN-UREA ALUM-ACE ALUM-UREA CaCL-AC

c. Staph.aureus
9.90E+09
1.00E+10
6.30E+07
4.10E+07

1.00E+09 2.70E+08
2.90E+06

1.00E+08
1.20E+06
1.16E+06
1.10E+06

1.00E+07
3.60E+04

1.00E+06
CFUs/ml

1.60E+03

1.00E+05

1.00E+04

1.00E+03

1.00E+02

1.00E+01

1.00E+00
2% 5%
concentration of IONIC LIQUID v/v

ALN-ACE ALN-UREA ALUM-ACE ALUM-UREA CaCL-AC

 d. Staph.epidermidis

5.90E+11
1.00E+12
1.00E+11
1.00E+10
1.07E+09
1.00E+09

9.20E+05
7.80E+05
6.50E+05
1.00E+08

2.50E+05
1.05E+05
1.00E+07
5.10E+04
CFUs/ml

1.00E+06

5.50E+03
1.30E+03
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
2% 5%
concentration of IONIC LIQUID v/v

ALN-ACE ALN-UREA ALUM-ACE ALUM-UREA CaCL-AC

e. Pseudomonas
1.00E+11
1.17E+09

1.52E+10
3.60E+08
3.30E+08

1.00E+10
4.70E+07

1.00E+09

1.00E+08

1.00E+07
CFUs/ml

1.00E+06
6.30E+03

1.18E+05
2.20E+03

1.00E+05
1.10E+03
1.00E+03

1.00E+04

1.00E+03

1.00E+02

1.00E+01

1.00E+00
2% 5%
concentration of IONIC LIQUID v/v

ALN-ACE ALN-UREA ALUM-ACE ALUM-UREA CaCL-AC

 f. Proteus
1.00E+11
1.04E+10

1.26E+08
9.70E+07
4.70E+07
1.00E+10

1.14E+07
1.00E+09
1.00E+08

1.05E+05
6.10E+06

5.60E+04
1.00E+07

2.10E+04
1.52E+04
CFUs/ml

1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
2% 5%
concentration of IONIC LIQUID v/v

ALN-ACE ALN-UREA ALUM-ACE ALUM-UREA CaCL-AC

Fig (3-26): schemes for Colony forming units (CFUs)/ml for a. E.coli, b. K.peneumoniae, c.
Staph.aureus, d. Staph. Epidermidis, e. Pseudomonas and f. Proteus bacteria growth.

Fig. (3-26): Colony forming on the agar media for A- K.peneumoniae and B- St.aureus
for 2%, 5% of IL (as example).
The OD Table (3-11) and CFUs Table (3-12) tests indicated that at an increase
in all related ILs concentrations it decreases the bacterial growth that used in
this work, compared to bacterial growth without adding the ILs and it showed
that the 2% (v/v) ILs concentration is considered to be the minimum inhibitory
concentration (MIC) and 10% (v/v) ILs concentration is the minimum of
bactericidal concentration (MBC) for all ionic liquids that used in this research.
One of the advantages of this method is reliability and it gives greater precision
in understanding the behavior of ionic fluids with the growth of bacteria by
calculate the exactly number of viable bacteria.
It is worth noting that the related ionic liquids used are colorless and this has
helped to prevent interference with the measured absorption of bacterial cells,
which gave better credibility in the results. In addition this method is dependent
on the degree of solubility of ionic liquids in water (the main component in the
preparation of the culture medium) and hence it was found that all applied ionic
liquids have a high solubility in water see chapter 3 (3.1.3). Therefore the
methods of Kirby-Bauer disk diffusion test, optical density (OD) test and
Colony forming units count gave a clear perception that the (AN-Ac), (AN-
urea), (ammonium alum -urea), (ammonium alum -Ac) and (CaCl-Ac) ILs
which different cation and anion species that used in this research had a
biological and toxic effects against (E.coli, K.peneumonice, Staph. Aureus,
Staph.epidermidis, Pseudomonas and Proteus) gram (positive and negative)
bacteria. These results correspond with a similar effect of other researchers [170].

3.3.3 Biochemistry tests.

The effect of ionic liquids on human serum was determined by examining
changes in clinical biochemistry testes and selecting the optimal concentration.
The ionic liquids of (AN-Ac), (AN-urea), (ammonium alum -urea), (ammonium
alum-Ac) and (CaCl/Ac) were added in different concentrations to human
serum. Which when added without dilution, an aggregation of the serum was
observed (figure 3-28) but no changes were observed when (CaCl/Ac) IL was
used, because some type of ionic liquids can alter the structure, dynamics,
[171]
function, and stability of proteins and fats . The ionic liquids were then
diluted until the optimal concentration without affecting the serum parameters
was reached. Where 1μl have been taken from the ideal concentration of 2% v/v
aluminum salts ILs and 5μl of 2% v/v CaCl-Ac IL/water added to 100μl serum
severally. It was found at the addition and repeated examination that the
(CaCl/Ac) IL has a lower effect on the serum parameters compared with the rest
of the related ionic liquids, so the concentration was increased until the reaching
the ideal concentration. Subsequently clinical biochemistry tests like (sugar,
lipids, protein, liver functions, kidney functions, and electrolytes) Figure (3-29)
were performed for 50 patient’s serum taken from al-yarmouk teaching hospital
(chemistry lab). The result of patient (1) were taken as an example shown in
Table (3-10) it also showing the mean and the standard deviation (SD).

Figure (3-28): Serum aggregation due to the direct addition of aluminum salts ionic liquids.
Table (3-13): Clinical biochemical testes for human serum with mean and SD results for Patient
ID: 1 / Gender: Female / Age: 42 / Draw Time: Morning / Type of test: Random with 2% of
ionic liquids (for example).

Results
Assay Mean SD ± Range Units
ammoniu

ammoniu
AN-urea

CaCl/Ac
m alum-

m alum-
Control

AN-Ac
urea

Glu 102 98 98 Ac
99 98 98 98.83 1.79 65-110 Mg/dl

Urea 28 43 47 60 27 23 40 14.5 20-45 Mg/dl

Crea 0.65 0.64 0.66 0.65 0.66 0.66 0.65 0.009 0.57-1.25 Mg/dl

Alb 4.4 4.4 4.4 4.4 4.4 4.4 4.4 0 3.5-5.0 g/dl

TP 7.7 7.6 7.6 7.6 7.6 7.6 7.61 0 6.4-8.3 g/dl

ALT 6 7 7 8 8 8 7.33 0.8 0-55 U/L

AST 17 16 16 17 16 17 16.5 0 5-34 U/L

AlkP 117 115 111 114 114 113 114 2 40-150 U/L

BiliT 0.8 0.7 0.7 0.7 0.7 0.7 0.71 0 0.2-1.2 Mg/dl

Chol 176 169 170 169 171 170 170.83 0 0-199 Mg/dl

TG 81 78 77 78 77 78 78.16 0 0-149 Mg/dl

Ca 9.5 9.6 9.9 9.6 9.8 10.4 9.8 0.32 8.4-10.2 Mg/dl

UA 2.92 2.94 2.94 2.90 2.93 2.87 2.91 0.03 3.50-7.20 Mg/dl

Cl 105 103 103 103 103 103 103.3 0 98-107 mmol/L

K 4.2 4.2 4.2 4.2 4.2 4.1 4.18 0 3.5-5.1 mmol/L

Na 140 138 140 139 138 137 138.83 1.22 136-145 mmol/L
 A: Statistic of Glucose
300

250

200
Result

150

100

50

0
p1 p3 p5 p7 p9 p11p13p15p17p19p21p23p25p27p29p31p33p35p37p39p41p43p45p47p49
patient ID

Q ALUM-UREA AN– UREA ALUM- AC AN-AC CaCl-AC

B: Statistic of Urea
160
140
120
100
Result

80
60
40
20
0
p1 p3 p5 p7 p9 p11p13p15p17p19p21p23p25p27p29p31p33p35p37p39p41p43p45p47p49

patient ID

Q ALUM-UREA AN-UREA ALUM-AC AN-AC CaCl-AC

C: Statistic of ALT
250
200
150
Result

100
50
0
p1 p3 p5 p7 p9 p11 p13 p15 p17 p19 p21 p23 p25 p27 p29 p31 p33 p35 p37 p39 p41 p43 p45 p47 p49

patient ID

Q ALUM-UREA AN-UREA ALUM-AC AN-AC CaCl-AC

 D: Statistic of AST
80
70
60
50
Result

40
30
20
10
0
p1 p3 p5 p7 p9 p11p13p15p17p19p21p23p25p27p29p31p33p35p37p39p41p43p45p47p49

patient ID

Q ALUM-UREA AN-UREA ALUM-AC AN-AC CaCl-AC

E: Statistic of Cholesterol
400
350
300
250
Result

200
150
100
50
0
p1 p3 p5 p7 p9 p11 p13 p15 p17 p19 p21 p23 p25 p27 p29 p31 p33 p35 p37 p39 p41 p43 p45 p47 p49

patient ID

Q ALUM-UREA AN-UREA ALUM-AC AN-AC CaCl-AC

F: Statistic of Triglycerides
350
300
250
Result

200
150
100
50
0
p1 p3 p5 p7 p9 p11 p13 p15 p17 p19 p21 p23 p25 p27 p29 p31 p33 p35 p37 p39 p41 p43 p45 p47 p49

patient ID

Q ALUM-UREA AN-UREA ALUM-AC AN-AC CaCl-AC

 G: Statistic of Uric acid
9
8
7
6
Result

5
4
3
2
1
0
p1 p3 p5 p7 p9 p11 p13 p15 p17 p19 p21 p23 p25 p27 p29 p31 p33 p35 p37 p39 p41 p43 p45 p47 p49
patient ID

Q ALUM-UREA AN-UREA ALUM-AC AN-AC CaCl-AC

H: Statistic of Calcium
12
10
8
Result

6
4
2
0
p1 p3 p5 p7 p9 p11 p13 p15 p17 p19 p21 p23 p25 p27 p29 p31 p33 p35 p37 p39 p41 p43 p45 p47 p49

patient ID

Q ALUM-UREA AN-UREA ALUM-AC AN-AC CaCl-AC

I: Statistic of Chloride
140
120
100
Result

80
60
40
20
0
p1 p3 p5 p7 p9 p11 p13 p15 p17 p19 p21 p23 p25 p27 p29 p31 p33 p35 p37 p39 p41 p43 p45 p47 p49

patient ID

Q ALUM-UREA AN-UREA ALUM-AC AN-AC CaCl-AC

 J: Statistic of Potassium
6
5
4
Result

3
2
1
0
p1 p3 p5 p7 p9 p11 p13 p15 p17 p19 p21 p23 p25 p27 p29 p31 p33 p35 p37 p39 p41 p43 p45 p47 p49

patient ID

Q ALUM-UREA AN-UREA ALUM-AC AN-AC CaCL-AC

K: Statistic of Sodium
160
140
120
100
Result

80
60
40
20
0
p1 p3 p5 p7 p9 p11 p13 p15 p17 p19 p21 p23 p25 p27 p29 p31 p33 p35 p37 p39 p41 p43 p45 p47 p49

patient ID

Q ALUM-UREA AN-UREA ALUM-AC AN-AC CaCl-AC

L: Statistic of Total Protein

9
8
7
6
Result

5
4
3
2
1
0
p1 p3 p5 p7 p9 p11 p13 p15 p17 p19 p21 p23 p25 p27 p29 p31 p33 p35 p37 p39 p41 p43 p45 p47 p49
patient ID

Q ALUM-UREA AN-UREA ALUM-AC AN-AC CaCl-AC

 M: Statistic of Total bilirubin
1
0.9
0.8
0.7
0.6
Result

0.5
0.4
0.3
0.2
0.1
0
p1 p3 p5 p7 p9 p11 p13 p15 p17 p19 p21 p23 p25 p27 p29 p31 p33 p35 p37 p39 p41 p43 p45 p47 p49
patient ID

Q ALUM-UREA AN-UREA ALUM-Ac AN-AC CaCl-Ac

N: Statistic of Alkaline Phosphatase

160
140
120
100
Result

80
60
40
20
0
p1 p3 p5 p7 p9 p11 p13 p15 p17 p19 p21 p23 p25 p27 p29 p31 p33 p35 p37 p39 p41 p43 p45 p47 p49

patient ID

Q ALUM-UREA AN-UREA ALUM-Ac AN-AC CaCl-Ac

O: Statistic of Albumin
6

4
Result

0
p1 p3 p5 p7 p9 p11 p13 p15 p17 p19 p21 p23 p25 p27 p29 p31 p33 p35 p37 p39 p41 p43 p45 p47 p49
patient ID

Q ALUM-UREA AN-UREA ALUM-Ac AN-Ac CaCl-Ac

 P: Statistic of Creatinine
10
9
8
7
6
Result

5
4
3
2
1
0
p1 p3 p5 p7 p9 p11 p13 p15 p17 p19 p21 p23 p25 p27 p29 p31 p33 p35 p37 p39 p41 p43 p45 p47 p49

patient ID

Q ALUM-UREA AN-UREA ALUM-Ac AN-Ac CaCl-Ac

Fig (3-29): A statistic was performed for each serum parameters with and without ionic liquids,
as shown in the graphs above. Where Q: patient’s serum without any additives,
…….ammonium alum-urea, AN-urea, ammonium alum -Ac, AN-Ac and
…….CaCl-Ac ionic liquids. A: Statistic of Glucose, B: Statistic of Urea, C: Statistic of ALT, D:
Statistic of AST, E: Statistic of Cholesterol, F: Statistic of Triglycerides, G: Statistic of Uric
acid, H: Statistic of Calcium, I: Statistic of Chloride, J: Statistic of Potassium, K: Statistic of
Sodium, L: Statistic of Total Protein, M: Statistic of Total bilirubin, N: Statistic of Alkaline
Phosphatase, O: Statistic of Albumin, P: Statistic of Creatinine.

It is noted that all serum parameters were stable in (2% v/v) concentration of
ILs as showed in mean and SD values, except serum urea test showed an
increase due to the presence of amides groups in ILs. This increase clearly
shows by presence of ammonium alum -urea, ammonium alum -Ac, AN-urea
and AN-Ac ILs because it contains two amide groups in urea and one amide
group in Acetamide, and also calcium test showed an increase due to the
presence of calcium ions in CaCl-Ac IL, where the increase varies between 0.5
to 0.7 mg/dl. In spite of this increase in the levels of serum urea and calcium,
but it is within the normal levels of urea (20-50 mg/dl) and calcium (8.4-10.2
mg/dl) that is allowed in the normal human body.
The concentration of (2% v/v) of related ILs can be used in the toxicity
experiments to find the amount of lethal dose fifteen (LD50), and to be used for
example as an antibiotic or to mix it with different pharmaceuticals to give
higher effect. Therefore, it is recommended to use this concentration to start in
several scientific biochemistry experiments for blood and serum.

3.3.4 Inhibition Mechanism

The toxic influence of ILs may be associated to a common cellular structure
or process. It is supposed that the toxicity mechanism of ILs was related to an
interaction with the cell wall and membrane, leading to a membrane
disorder.[172, 173]
ILs containing cation-anion pairs have similar behaver of
surfactants, pesticides and antibiotics that attack lipid structure, and stimulate
polar narcosis, and may cause membrane-bound protein disorder.[174] It should
be noted that there is a need for future studies to determine the precise
mechanism of the ionic liquids used in this research and to establish their
behavior with human or other living organisms.
 Conclusions
In conclusion, it be drawn that reaction of aluminum salts with organic salts
having a pair of free electrons such as sulfur, oxygen or nitrogen atoms attached
to it (i.e. amide, carbonyl or sulfonyl group) was found possible (CaCl-Ac,
AlSt-MEA, AN-thiourea etc). However, the reaction may be a deep eutectic
liquid formation or an acid-base reaction forming liquids. The latter liquid
found to require more passive condition to keep the liquid stable, i.e. preventing
reaction with moisture.

The stable liquids of aluminum salts and organic compounds and that of
CaCl-Ac liquid were found to have biological activity and can be put in the
order of increasing activity as:

(AN-Ac) > (AN-urea) > (ammonium alum-urea) = (ammonium alum -Ac) > (CaCl-Ac)

The minimum concentration was found to be about 2%, which did not affect
human serum for (AN-Ac), (AN-urea), (ammonium alum -urea), (ammonium
alum -Ac) and (CaCl-Ac) ionic liquids.
Suggestions for future work:
The researcher of the present study has suggested the following for future
research:

1. Prepare a new ionic liquids by mixing the other aluminum salts with
organic amides and conducting a full investigation considering biological
activity.
2. Study the status stereochemistry and geometric shape of ionic liquids
prepared in this research.
3. Measure the lethal dose 50 (LD50) of ionic liquids used in this research.
4. Study the possibility of using ionic liquids as an antibiotic or with other
pharmaceuticals.
5. The possibility of studying the effect of ionic fluids on hormones and
cancer tumors.
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‫البصرية ( ‪ )optical density‬عند ‪ 600‬نانومتر لجميع البكتريا في الوسط الزرعي السائل عند ‪37‬‬
‫درجة سيليزية ولمدة حضن ‪ 24‬ساعة مضاف لها تراكيز مختلفة من السوائل االيونية (‪%20،10،5،2‬‬
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‫تحوي وسط زرعي صلب لحساب كمية المستعمرات( )‪.) colony forming units (CFUs‬‬
‫من جهة أخرى تم حساب تركيز السوائل االيونية بشكل عملي لحين تم التوصل إلى التركيز المثالي‬
‫(‪ % 2‬حجم‪/‬حجم) الذي خلط مع مصل الدم دون التسبب في أي تباين في الفحوصات البايوكيميائية مثل‬
‫فحص (السكر‪ ،‬والدهون‪ ،‬والبروتين‪ ،‬وظائف الكبد‪ ،‬وظائف الكلى‪ ،‬والشوارد) بإستخدام تقنية التحليل‬
‫التلقائي للفحوصات البايوكيميائية‪.‬‬
 ‫الخالصة‬
‫لقد أجريت العديد من التجارب لتحضير سوائل أيونية بإستخدام أنواع مختلفة من مركبات االمين‬
‫كااليثانول أمين واليوريا واالسيتاميد والثايويوريا والثايوأسيتاميد‪ ،‬مع العديد من أمالح االلمنيوم ككلوريد‬
‫االلمنيوم ونترات االلمنيوم و شب االمونيوم و شب البوتاسيوم و ستيارات االلمنيوم‪ .‬بعض المحاوالت‬
‫اعطت نتائج بإستخدام درجات حرارة متفاوتة وصوال الى ‪ 80‬درجة سيليزية وبأستخدام عوامل مختلفة‪.‬‬
‫بعدها تم إستخدام تقنيات مختلفة للقياسات الفيزياوية مثل قياس التوصيلية ودرجة االنصهار و قياس‬
‫المطيافية تحت الحمراء واالشعة فوق البنفسجية للتحقق من النتائج المستحصلة‪.‬‬

‫ومن جهة أخرى تم تحضير سائل أيوني جديد بطريقة ‪( )DESs( Deep Eutectic solvents‬وهو‬
‫نظام مشابه للسوائل االيونية تتكون من ايونات غير متناظرة تمتلك طاقة شبكية بلورية و درجة أنصهار‬
‫واطئة) عن طريق تفاعل كلوريد الكالسيوم ثنائي الماء ‪ CaCl2.2H2O‬مع االسيتاميد ‪CH3CONH2‬‬
‫وبنسب مولية مختلفة حيث كان السائل االيوني المتكون عديم اللون‪ ،‬وأظهراستقرارا جيدا للحرارة ما بين‬
‫أدنى درجة حرارة للتجمد (‪˚7-‬م) وأعلى درجة حرارة للتفكك (‪˚ 286.7‬م) وبنسة مولية (‪ )7 :1‬على‬
‫التوالي ويمتلك توصيلية ايونية بمقدار (‪ 0.3‬مليسيمنز ‪ /‬سم)‪ .‬إستخدمت تقنية القياس باالشعة تحت‬
‫الحمراء(‪ )FTIR‬واالشعة فوق البنفسجية ( ‪ )UV spectra‬لمعرفة التداخل والتناسق بين المركبين‬
‫لتكوين السائل االيوني الجديد‪ .‬وقد تم قياس بعض الخصائص الفيزيائية األخرى‪ ،‬مثل اللزوجة والكثافة‬
‫ودرجة الفولتامترية الحلقية‪.‬‬

‫تم ايضا دراسة الفعالية الدوائية البيولوجية لعدة أنواع من السوائل االيونية على مصل الدم البشري‬
‫وعلى نمو البكتريا‪ .‬فقد تم إجراء عدة إختبارات على ستة أنواع من البكتريا سالبة الغرام ‪E.coli‬‬
‫وموجبة الغرام ‪ Staph.aureus‬و‬ ‫‪Proteus‬‬ ‫و‪ K.peneumonice‬و ‪ pseudomonas‬و‬
‫‪ Staph.epidermidis‬لخمسة انواع من السوائل االيونية التي تم تحضيرها والمحضرة سابقا وهي‬
‫كلوريد الكالسيوم ثنائي الماء‪-‬االسيتاميد ونترات االلمنيوم المائية –االسيتاميد ونترات االلمنيوم المائية –‬
‫يوريا وكبريتات االمونيوم االلمنيوم‪-‬االستمايد وكبريتات االمونيوم االلمنيوم‪-‬اليوريا‪ .‬أظهر هذا العمل أن‬
‫العامل الرئيسي المؤثر هو نوع وتركيب و قابلية االمتزاج وتركيز و صنف االيونات السالبة والموجبة‬
‫للسوائل االيونية وكذلك نوع البكتريا المتواجدة في االوساط الزرعية السائلة والصلبة‪ ،‬حيث أعطت نتائج‬
‫ايجابية في تثبيط و قتل البكتريا بحسب الكميات المضافة للسوائل االيونية بواسطة قياس منطقة التثبيط‬
‫بأستخدام طريقة ( ‪ ،) The Kirby-Bauer Disk Diffusion Test‬باالضافة الى ذلك تم قياس الكثافة‬
 ‫جمهورية العراق‬

‫وزارة التعليم العالي و البحث العلمي‬

‫جامعة النهرين ‪ /‬كلية العلوم‬

‫قسم الكيمياء‬

‫تحضير ودراسة بايولوجية لبعض السوائل األيونية‬

‫المشتقة من أمالح األلمنيوم و الكالسيوم – أمين‬
‫رسالة‬
‫مقدمة الى كلية العلوم ‪ /‬جامعة النهرين‬
‫وهي جزء من متطلبات نيل درجة الماجستير في الكيمياء‬
‫من قبل‬

‫بسام باقر حسن‬

‫بكالوريوس في علوم الكيمياء جامعة بغداد(‪)2001-2000‬‬

‫بإشراف‬

‫أستاذ مساعد الدكتورة‬ ‫األستاذ الدكتور‬

‫نادرة سلمان محمد الغراوي‬ ‫هادي محمد علي عبود‬

‫‪ 1439‬هجرية‬ ‫‪ 2018‬ميالدية‬