Experimental evaluation of cationic-Schiff base surfactants based on 5-chloromethyl salicylaldehyde for improving crude oil recovery and bactericide

Article abstract of DOI:10.1016/j.molliq.2020.113862

In this study, a new family of cationic and cationic–Schiff Base surfactant was synthesized namely; N-(3-formyl-4-hydroxybenzyl)-N, N-dimethylhexadecan-1-aminium chloride (FHDHAC), (E)-N-(3-((butylimino)methyl)-4-hydroxybenzyl)-N,N-dimethylhexadecan-1-aminium chloride (4-IMHDAC), (E)-N-(3-((hexylimino)methyl)-4-hydroxybenzyl)-N,N-dimethylhexadecan-1-aminium chloride (6-IMHDAC) and (E)-N-(4-hydroxy-3-((octylimino) methyl)benzyl)-N,N-dimethylhexadecan-1-aminium chloride (8-IMHDAC) were prepared from 5-chloromethyl salicylaldehyde compound (5-CMS). The chemical structures of 5-CMS, 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC are characterized using FTIR, ¹H NMR instruments. The surface activity measurements of the prepared compounds showed a gradual increase in surface activity by increasing the alkyl chain length. Adsorption and micellization thermodynamic functions revealed the higher tendencies of the different surfactants towards adsorption at the organic/polar phase. The synthesized surfactants were evaluated in the enhanced oil recovery process and showed excellent efficiency (63.98%, 71.25%, and 75.72% for 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC, respectively) during the recovery process near their critical micelle concentrations. The prepared surfactants showed high antimicrobial efficacy against different bacteria and fungi.

Full text of DOI:10.1016/j.molliq.2020.113862

Journal Pre-proof
Experimental evaluation of cationic-Schiff base surfactants based
on 5-chloromethyl salicylaldehyde for improving crude oil
recovery and bactericide
Mohamed A. Betiha, Samy B. El-Henawy, Ahmed M. Al-Sabagh,
Nabel A. Negm, Tahany Mahmoud
PII:
S0167-7322(20)33662-X
DOI:
https://doi.org/10.1016/j.molliq.2020.113862
MOLLIQ 113862
Reference:
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
5 June 2020
13 July 2020
16 July 2020
Please cite this article as: M.A. Betiha, S.B. El-Henawy, A.M. Al-Sabagh, et al.,
Experimental evaluation of cationic-Schiff base surfactants based on 5-chloromethyl
salicylaldehyde for improving crude oil recovery and bactericide, Journal of Molecular
Liquids (2020), https://doi.org/10.1016/j.molliq.2020.113862
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Experimental Evaluation of Cationic-Schiff Base Surfactants Based on 5-Chloromethyl
Salicylaldehyde for Improving Crude Oil Recovery and Bactericide
Mohamed A. Betiha1,2, Samy B. El-Henawy1, Ahmed M. Al-Sabagh1, Nabel A. Negm1,2#
Tahany Mahmoud1
,
1- Egyptian Petroleum Research Institute, Nasr City, Cairo, Egypt
2- Egypt Nanotechnology Center, Cairo University, Sheikh Zayed, Egypt
Corresponding author: nabelnegm@hotmail.com
Abstract
In this study, a new family of cationic and cationic–Schiff Base surfactant was synthesized
namely;
N-(3-formyl-4-hydroxybenzyl)-N,N-dimethylhexadecan-1-aminium
chloride
(FHDHAC),
(E)-N-(3-((butylimino)methyl)-4-hydroxybenzyl)-N,N-dimethylhexadecan-1-
aminium chloride (4-IMHDAC), (E)-N-(3-((hexylimino)methyl)-4-hydroxybenzyl)-N,N-
dimethylhexadecan-1-aminium chloride (6-IMHDAC) and (E)-N-(4-hydroxy-3-((octylimino)
methyl)benzyl)-N,N-dimethylhexadecan-1-aminium chloride (8-IMHDAC) were prepared
from 5-chloromethyl salicylaldehyde compound (5-CMS). The chemical structures of 5-CMS,
4-IMHDAC, 6-IMHDAC, and 8-IMHDAC are characterized using FTIR, 1H-NMR
instruments. The surface activity measurements of the prepared compounds showed a gradual
increase in surface activity by increasing the alkyl chain length. Adsorption and micellization
thermodynamic functions revealed the higher tendencies of the different surfactants towards
adsorption at the organic/polar phase. The synthesized surfactants were evaluated in the
enhanced oil recovery process and showed excellent efficiency (63.98%, 71.25%, and 75.72%
for 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC, respectively) during the recovery process near
their critical micelle concentrations. The prepared surfactants showed high antimicrobial
efficacy against different bacteria and fungi.
Keywords: Wettability; cationic–Schiff Base surfactant, interfacial tension; EOR.
Introduction
Until now, with the entry of other sources of energy, such as solar energy, wind, etc., oil
maintains that it is the main source of energy, and the stock of oil is one of the factors that
evaluate the economies of some countries. The energies stored naturally inside the oil-bearing
formation (gas-drive, water-drive, or gravity drainage) is the main factor facilitating the
primary oil recovery. After the deterioration of the natural energies of the reservoirs, the
reservoir is injected with water or gas to enhance the production of the reservoir, which is
called secondary oil recovery. During this stage, the injected liquid is usually water or gas that
is mechanically injected through the injection wells. During this operation, the In general, the
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second stage does not include adding any chemicals or carrying out chemical reactions inside
the reservoir, as this method is considered as mechanical or physical operation in order to
enhance production from the oil reservoirs [1]. This stage reaches aging when the ratio of
water to the resulting oil is very high. It should be noted that the oil reservoir may maintain a
percentage of crude oil that may exceed two-thirds of the crude that has been predicted for the
reservoir [2]. The main reason behind the decrease in the production of crude oil from the
petroleum reservoir is the increased viscosity of crude oil, in addition to the high interfacial
tension (IFT) between the water and the remaining oil. These two factors lead to the
emergence of a large capillary force that traps the crude oil inside the capillaries of the
reservoir rocks [3]. Therefore, extracting other quantities of the remaining oil after the second
stage constitutes a challenge for the industry in the field of oil production, and this challenge
calls enhanced chemical recovery of oils.
Chemical floods are one of the most widely used and influencing methods for enhancing oil
production after the second stage [4]. Adding alkalis to surfactant causes emulsification of oil
droplets and regulates the phase behavior of emulsified oil by increasing the ionic strength to
achieve a very low IFT rate through carrying the saponification process [5]. One of the most
important advantages of using alkali is the cheapness in addition to reducing the adsorption of
surfactants on the solid instead of improving the interfacial activity of the added surfactants
[6].
Surface active agents are those that have the ability to form self-assembled molecules in
clusters (micelles) and can be adsorbed between various phases interface in the solution [7].
The amphiphilic nature of these molecules is due to they possess a hydrophilic
moiety/moieties and a lipophilic moiety/moieties [8]. The surface activities of the cationic and
nonionic surfactants were found to increase their interfacial applications especially during
emulsification and wettability [9, 10]. The process of increasing the oil extraction by
surfactant was classified based on the amount of surfactant concentration in the solution, so it
is called micellar flooding if the concentration of surfactant corresponds to critical micelle
concentration (CMC) values and if the concentration exceeds the CMC the operation is called
surfactant flooding [11]. By using surface flooding, the emulsification process
(microemulsions) of the crude oil droplets occurs, and as a result, the entrainment, and
coalescence of the crude oil drops with the water occurs as a result of a reduction in the
surface tension between the oil and the water. In addition to the previous action, the surfactant
had the ability to alter the rock wettability.
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Vatanparast et al.[12] discussed the phase behavior of cetyltrimethylammonium bromide
(CTAB) and concluded that CTAB improved oil production from a carbonate reservoir and
the mixing of CTAB with silica nanoparticle led to more oil recovery. The authors also
concluded that the increase in recovered oil was due to a decrease in IFT between oil and
water even at high temperatures due to the alteration rock surface by CTAB. Sharma and
Mohanty have studied the same compound again for the same purpose with changing
conditions of the petroleum reservoir, where they used a solution with a salinity estimated at
600000 ppm and a temperature of 100 °C. The authors reported that the CTAB was unable to
reduce the IFT at 100 °C despite its stability or alter the surface of carbonate rocks [13].
To some extent, the authors did not find any publications on the use of Schiff-base and cation
surfactant in the operation of improving oil recovery. Therefore, this manuscript is the first
report about the use of a single compound carrying a pair of an active group, cationic, and
Schiff base combinations. This study aims to prepare a 5-chloromethyl salicylaldehyde
compound using zinc chloric as a catalyst and get the benefit from the chloromethyl and
aldehydic groups to obtain the cationic and Schiff moieties and determine their surface
properties in order to maximize the benefit from them in the field of heavy oil production.
2. Experimental Section
2.1 Chemicals
Paraformaldehyde (≥99%), salicylaldehyde (≥99%), N,N-dimethyl hexadecyl amine (≥99%),
1-aminobutane (≥99%), 1-aminohexane (≥99%), 1-aminooctane (≥99%), hydrochloric acid
(36.5%), sodium chloride (90%) were obtained from Sigma Aldrich (Egypt). Diethyl ether
(≥99.0%), toluene (≥99.5%), methanol (anhydrous, 99.8%), and ethanol (96.0-97.2%) were
purchased from ADWIC Chemicals Company, Egypt. Prior to solvent use, the solvents were
distilled, followed by the drying over A4–molecular sieve. The crude oil was donated by
Egypt's Petroleum company, and its basic analysis of the crude oil and brine analysis was
given in Table 1.
2.2 Synthesis of cationic surfactants
2.2.1 Preparation of 5-Chloromethyl-2-hydroxybenzaldehyde (5-CMS)
Hydrochloric acid (150 mL, 37 wt.%) and 133.066 mmol of paraformaldehyde (10.8 mL, 37
wt.%) were charged into 250 mL-two neck flask. The solution was further purged with HCl
gas produced from the reaction between concentrated sulfuric acid and sodium chloride. After
30 min, 15 mL (141.39 mmol) of salicylaldehyde was added drop-wise. The solution was left
to stir for 24 h at the same condition until precipitation of a solid adduct. The solid was
dissolved in an appropriate volume of diethyl ether, and then water was added to obtain two
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separate layers. The organic layer was extracted, and diethyl ether was evaporated to obtain
the pure product of 5-chloromethyl-2-hydroxybenzaldehyde. The product was further washed
with (150 mL diethyl ether/200 mL water) several times till the pH of the medium remained
at 6.9. Finally, diethyl ether was vacuum evaporated to obtain crystallized 5-chloromethyl-2-
hydroxybenzaldehyde (Yield: 62.9 %, 15.24 g).
2.2.2
Preparation
of
3N-(3-formyl-4-hydroxybenzyl)-N,N-dimethylhexadecan-1-
aminium chloride (FHDHAC)
Equimolar amounts (10 mmol, 1.7 g) of 5–CMS and N, N-dimethyl hexadecyl amine (11
mmol, 2.96461 g) were dissolved in the appropriate volume of toluene, and the mixture was
stirred for 3 h at 75 °C. A rotary evaporator then removed the solvent, and the product was
washed three times with diethyl ether, followed by vacuum drying at 40 °C to obtain the
desired product (3-(3-formyl-4-hydroxybenzyl)-N,N,N-dimethyl hexadecyl ammonium
chloride, (FHDHAC).
2.2.3 Preparation of Schiff Base based on cation surfactants (4-IMHDAC, 6-IMHDAC,
and 8-IMHDAC)
FHDHAC (10 mmol), 1-aminobutane (0.8 mmol), trimethylamine (0.2 mL), and 30 mL of
dry ethanol were charged in a 100 mL flask, and the temperature of the mixture was raised the
to 70 °C for 6 h. The yellow product of the reaction was distilled to remove ethanol and
washed several times with ethanol to afford (E)-N-(3-((butylimino)methyl)-4-
hydroxybenzyl)-N,N-dimethylhexadecan-1-aminium chloride, (8.9 mmol, 4-IMHDAC). The
reaction was repeated by replacing 1-aminobutane by 1-aminohexane or 1-aminooctane to
obtain: (E)-N-(3-((hexylimino)methyl)-4-hydroxybenzyl)-N,N-dimethylhexadecan-1-aminium
chloride
(6-IMHDAC)
and
(E)-N-(4-hydroxy-3-((octylimino)methyl)benzyl)-N,N-
dimethylhexadecan-1-aminium chloride (8-IMHDAC), respectively (Scheme 1).
2.3. Measurements
To perform transform infrared spectroscopy (FTIR), 0.25ꢀmg of samples 5-CMS, FHDHAC,
4-IMHDAC, 6-IMHDAC, and 8-IMHDAC are well-dried and ground with KBR powder
(300ꢀmg) and analyzed on a Thermo iS50-FT-IRNICOLET 6700 instruments (resolution:
4ꢀcm–1 with 512 scans). To determine the structure of compounds, Bruker Nuclear Magnetic
Resonance Spectroscopy using DMSO-d6 as a solvent.
2.3.1 Surface tension (γ) and interfacial tension (γIFT) measurements
The surface tension values (γ) of the 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC surfactants at
serial concentrations started with initial concentration of 0.01 mM were measured using
Attension Theta High-pressure chamber (Biolin Company, Finland) (accuracy ±0.01 mN/m)
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using pendant drop technique at 25 and 50 oC. The surface was bound to the downward shape
according to Equation 1:
ΔρgR0
γ=
(1)
β
R0
β
surface tension, density, gravitational constant,
Where the symbols
γ
,
,
g
,
and

radius of the drop curvature, and factor based on the drop shape, respectively. β equals to 3
according the equation of Young-Laplace symbolized in Figure 1.
γ
The pendant drop method was applied for measuring the interfacial tension values ( IFT ) of
0.01g/mL surfactants solutions through a time range of 1 minute until the readings were
stabilized at 25 and 50 oC. Attention Theta has intended in a way that could be analyzed
γIFT
and contact angle using an online image capturing system, which enables the recording of
γIFT
data periodically as desired. In general, the
measurement was accomplished by injecting
a drop of surfactant solution from the hocked needle into a solid bulk phase under the required
pressure and temperature (ASTM ISO 19403-4).
2.3.2 Contact angle and Wettability measurement
Prior to performing analysis, the flat rocks were surface-cleaned using Soxhlet extractor filled
with toluene as a solvent, dried under vacuum at 112 °C for 12 h. Then, the flat rock is
immersed in brine for 12 h, and after that, the flat rock is submerged in the crude oil that will
be used in the flooding test to achieve the oil-wet rock samples. The contact angle between
the oil-wet rock and distilled water and different 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC
surfactants solutions were identified by Sessile Method using Attention Theta high-pressure
chamber ( (ASTM ISO 19403-5) at 25, 50 °C.
2.3.3 Kraft point
Kraft temperature (KT) was determined by heating surfactants solutions (4-IMHDAC, 6-
IMHDAC, and 8-IMHDAC) (1 wt.%) to obtain clear using the visual observation method,
and the temperature was obtained for an average of three readings [14].
2.3.4 Antimicrobial activities
The 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC surfactants were tested for their potency as
antimicrobial agents using the agar diffusion technique [15]. The 4-IMHDAC, 6-IMHDAC,
and 8-IMHDAC surfactants were assessed at 5000 ppm against Staphylococcus aureus ATCC
35556, and Bacillus subtilis ATCC 6633 as an example of a Gram-positive bacteria, and
Pseudomonas aeruginosa ATCC 10145, Escherichia coli ATCC 23282 as an example for
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Gram-negative bacteria. Candida albicans IMRU 3669 and Aspergillus niger ATCC 16404
are also considered as an example of yeast and filamentous fungus.
The different microorganisms were cultivated in their growing media (bacteria and yeast:
nutrient agar, fungus: Czapek's Dox agar). Erythromycin and Metronidazole (commercial
medicinal drugs) were used as controls bacteria and yeast, and fungus, respectively. All
examinations were executed in triplicates, and data were their averages.
Core flooding experiments
A distinction was made between the different 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC
surfactants on their ability to enhance oil recovery after the exhaust of secondary oil recovery
(water flooding), thereby increasing the oil proceeds tertiary recovery. An instrument has
been designed to simulate the oil production system from wells, consisting of the core holder
that contains silica as an oil-bearing material. A pump whose goal is to displace the crude oil
and surfactant solution at the desired rate or quantity, cylinders for the crude oil, brine and
surfactant solution, and cylinders to measure the volume of the resulting oil. The simulated
rock's core holder is characterized by a porous volume of 102.4 cm3, the total volume of the
reservoir is 496 cm3, and the crude oil residue after exhausting the second stage is 71 cm3.
While conducting the flooding process with the 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC
surfactants, it should focus on using the optimum concentration of the surfactants. To
optimize the concentration of the surfactant, three concentrations of best surfactant will be
used, 1 g/L, 2 g/L, and 4 g/L, respectively, at 50 °C. The injection of the surfactant rate into
the core holder was at 1 mL/min. The slug is allowed to remain in the core holder for two
hours to cause a change in the wetness of the rocks that can be studied.
3. Result and discussion
3.1.1 FTIR spectroscopy
The 5-CMS compound shows the absorption bands at 3200-3500 cm–1 assigned for the
starching vibration of hydroxyl groups, low intense bands at 2823 and 2920 cm–1 correspond
to sym- and asymmetrical vibration of C–H bonds, sharp 1730 cm–1 band for the two carbonyl
groups, 1350 cm-1 apportioned for C-Cl bond [16], 850-720 cm–1 bands for the phenyl group
[17, 18] (Figure 2). FGDHAC compound shows similar absorption bands as 5-CMS
compound. The disappearance of the 1350 cm–1 band was recorded due to the incorporation of
the C–Cl bond in the reaction. Also, with a minor decrease in the intensity of the 1730 cm–1
absorption band of the carbonyl group due to the incorporation of one carbonyl group in 5-
CMS in the reaction and only one group is remained. Otherwise, the main bands were
remained unchanged (Figure 2). The 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC compounds
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show similar absorption bands as FHDHAC compound with the diminished of the band at
1730 cm–1 and the appearance of the new absorption band at 1640 cm–1 due to mainly the
formation of imine group, –C=N–. Moreover, it is possible to see the aromatic stretching
band (=C-H at 3026 cm–1, and –C=C–at 1580 cm–1), and the aliphatic bending bands (-CH2 at
1377 cm–1, and CH3 at 1463 cm–1) in addition to C-N vibration at 1073 cm–1 (Figure 2).
3.1.2 1H-NMR spectroscopy
The 5-CMS compound displays the following signals at 4.64 ppm (s, 2H, Cl–CH2-Ar), 6.92-
7.64 ppm (dd, 3H, H–Ar), 10.24 ppm (s, 1H, O=CH-Ar) (Figure 3). The FGDHAC
compound shows the following signals at 0.96 ppm (t, 3H, –CH3), 1.29 ppm (m, 26 H, –CH2–
), 1.73 ppm (t, 2 H, –CH2-CH2 N+(CH3)2–), 3.24 ppm (t, 2 H, -CH2–N+(CH3)2–), 3.3 ppm (s,
+
6H, (CH3)2N+–), 4.5 ppm (s, 2H, N-CH2-Ar), 5.0 ppm (s, 1H, HO-Ar), 6.80-7.44 ppm (dd,
3H, H-Ar), 10.24 ppm (O=CH-Ar) (Figure 3). The 4-IMHDAC compound showed the
following signals at 0.86 ppm (t, 6H, -CH3), 1.26 ppm (m, 28 H, -CH2-), 1.72 ppm (m, 2 H, –
CH2–CH2N=C-), 1.85 ppm (-CH2–CH2N+(CH3)2–), 3.2 ppm (–CH2-N+(CH3)2–), 3.31 ppm (s,
6H, –N+(CH3)2–), 3.61 ppm (t, 2H, –CH2–N=C–), 4.5 ppm ((CH3)2N+–CH2-Ar), 4.9 ppm (s,
1H, HO-Ar), 6.74 ppm, 7.42 ppm, 7.65 ppm (d, 3H, H-Ar), 8.3 ppm (s, 1H, -N=CH) (Figure
3). The 6-IMHDAC compound showed the following signals at 0.86 ppm (t, 6H, –CH3), 1.26
ppm (m, 36H, –CH2–), 1.72 ppm (m, 2H, –CH2–CH2N=C-), 1.85 ppm (-CH2–CH2N+(CH3)2-
), 3.2 ppm (–CH2-N+(CH3)2–), 3.31 ppm (s, 6H, –N+(CH3)2–), 3.61 ppm (t, 2H, –CH2-N=C–),
4.5 ppm ((CH3)2N+–CH2-Ar), 4.9 ppm (s, 1H, HO–Ar), 6.74 ppm, 7.42 ppm, 7.65 ppm (d,
3H, H-Ar), 8.3 ppm (s, 1H, -N=CH-) (Figure 3). The 8-IMHDAC compound showed the
following signals at 0.86 ppm (t, 6H, –CH3), 1.26 ppm (m, 42H, –CH2–), 1.72 ppm (m, 2H, –
CH2–CH2N=C-), 1.85 ppm (–CH2–CH2N+(CH3)2–), 3.2 ppm (-CH2–N+(CH3)2–), 3.31 ppm (s,
6H, –N+(CH3)2–), 3.61 ppm (t, 2H, –CH2-N=C–), 4.5 ppm ((CH3)2N+–CH2–Ar), 4.9 ppm (s,
1H, HO–Ar), 6.74 ppm, 7.42 ppm, 7.65 ppm (d, 3H, H-Ar), 8.3 ppm (s, 1H, –N=CH–)
(Figure 3).
3.2 Surface active properties of the prepared surfactants
The surface properties of the 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC surfactants were
determined based on the surface tension data obtained from the measurements of the
surfactant's solutions surface tension at 25 °C and 50 °C in twice-distilled water. Figure 4
represents the variation in surface tension values obtained for 4-IMHDAC, 6-IMHDAC, and
8-IMHDAC surfactants at different temperatures. The concentrations of surfactants at
equilibrium, which associated with the stability of these values, were defined for CMC
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(critical micelle concentration). The CMC and γCMC values were established from the break-
point of the profiles, Table 2.
Obviously, there is a gradual decrease in CMC values by increasing the alkyl group chain
length attached to the azomethine group (Schiff Base group) of the used surfactants at 25 °C.
The lowest CMC value was obtained at 6.73 mM for octyl-chain of 8-IMHDAC surfactant. At
50 °C, the critical micelle concentration values were decreased compared to these obtained at
25 °C. That can be attributed to the increase in the repulsion between the hydrophobic chains
and the aqueous phase by the action of heat, which facilitates the reduction of micellization of
surfactant molecules. Generally, the surface tension at the micellization was decreased by
increasing both of the alkyl chain length and the temperature.
The effectiveness values (πCMC) are the difference between the surface tension values of the
bidistilled water and surfactant solution CMC and πCMC was determined for the prepared
surfactants using Equation 2 [19]:
πCMC = γW - γS (2)
γW
γS
are the water and surfactant surface tension at CMC. By calculating the
Where
and
π
average πCMC values, it was found that CMC of 8-IMHDAC was the highest among the
prepared surfactants.
The maximum surface excess concentration (Γmax) is described as the concentration per area
of surfactants molecules at the interface at the equilibrium, and can be achieved by applying
Equation 3:
1


MAX =-

(3)
RT lnC
is the excess surface concentration in (mol/cm2), T (absolute value), R
MAX
where

lnC
(universal gas constant, 8.314 J mol–1 K–1), and
is the slope of γ-ln C profile.
From the obtained data (Table 2), it was established that as the alkyl chains increase, the Γmax
values considerably decreased. The 8-IMHDAC surfactant had the lowest Γmax value
(6.54x10-11 mol/cm2) at 25 °C, and this value was decreased to 2.22x10-11 mol/cm2 by
increasing the temperature up to 50 °C. The average area occupied by surfactant molecules
(Amin) was determined for each 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC surfactants in the
solution at the equilibrium using Equation 4:
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where NA is equal 6.023×1023 molecule/mole (Avogadro's number).
Also, through Table 2, Amin values increases as both hydrophobic chain length and
temperature increase due to the decrease in Γmax values. The longest Amin value was 253.48
nm2 for 8-IMHDAC, whilst the 4-IMHDAC has the smallest area of 177.98 nm2 at the
interface at 25 °C. The increase in the temperature of the surfactant solution causes an
increase in the repulsion forces of the hydrophobic alkyl groups, and consequently, the
micellization increased at lower concentrations, and the area available for each molecule at
the interface was increased, as listed in Table 2.
3.3 Thermodynamic evaluation of the prepared surfactants
The surface tension data of the prepared cationic-Schiff Base surfactants were used to define
the free–energies of both micellization and the adsorption processes at the interfaces and the
bulk of the cationic-Schiff Base surfactants solutions at 25 °C and 50 °C. The reduction in
surface tension is in regard to the adsorption of the cationic-Schiff Base surfactant molecules
at the air-aqueous interface. The adsorption energy of the cationic-Schiff Base surfactant
molecules at the equilibrium can be calculated from Equation 5:
Furthermore, the free energy of the surfactant's molecules after micellization in the bulk of the
solution can be determined from Equation 6:
Gmic  RT lnCMC (6)
Gads
Where
ΔGads and ΔGmic are the molar Gibbs free energies of adsorption and
micellization processes in kJ/mol. The calculated values of ΔGmic and ΔGads for 4-IMHDAC,
6-IMHDAC, and 8-IMHDAC surfactants were listed in Table 3.
The data in Table 3 indicated that both ΔGads and ΔGmic values have negative signs,
suggesting the spontaneously of the two processes. The spontaneous behavior of these
processes comes from the repulsion that occurred between the hydrophobic chains of the
cationic-Schiff Base surfactant molecules and the polar aqueous phase. Also, the negativity of
the two values is increased by raising the temperature from 25 °C to 50 °C. Furthermore, it is
clear that ΔGads have more negative values than ΔGmic values, indicating the higher tendency
of the 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC surfactants towards adsorption at the
aqueous/air interface than the micellization in the solution bulk to form micelles [20]. The
preferability of the surfactants molecules towards adsorption and micellization was
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determined from ∆Gmic-∆Gads values. Obviously, the ∆Gmic-∆Gads values were increased by
increasing the temperature from 25 °C to 50 °C, indicating the increased adsorption tendency
of 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC surfactant molecules in their solutions at higher
temperatures.
3.4 Wettability alteration using 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC surfactants
The wettability of the surfaces is an important property which can be achieved by the
surfactants during several applications. The wettability tendencies of the prepared surfactants
were evaluated using interfacial tension and contact angle measurements at the crude
oil/surfactant solution interface. The alteration of the wettability of reservoir rocks of
petroleum wells is an essential requirement during enhanced oil recovery processes (EOR).
3.4.1 Interfacial tension (γIFT
)
The dynamic interfacial tension (γIFT) variations of the crude oil/surfactant solutions systems
at the CMC of the 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC surfactants at 25 °C and 50 °C
were represented in Figure 5. It is clear that the dynamic γIFT profiles of the crude
oil/surfactants solutions systems reach the equilibrium after 5 to 10 min. The variation in
dynamic γIFT involved different patterns; at the initial time (0-1 min), the γIFT values were
decreased sharply (i.e., the high slope of 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC is of
0.6514, 0.3714, 0.3514, respectively) indicating the fast adsorption of the cationic–Schiff
Base surfactant molecules at the interface. In the second region at (2-4 min), γIFT values were
decreased gradually (i.e., medium slope values of 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC
is of 0.05, 0.075, 0.07, respectively), indicating the surface equilibrium between adsorption
and desorption processes. At the longer time (4-10 min), γIFT values were almost stable (i.e.,
small slope values of 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC is of 0.0126, 0.0084, 0.0063,
respectively) indicating the achievement of equilibrium between the adsorption and
desorption of surfactants molecules to- and from the crude oil/solution interface. The main
reason for this response is the occurrence of adsorption and desorption of the cationic-Schiff
Base surfactant species at the interfacial crude oil/aqueous layer [21, 22]. The primary
decrease in γIFT could be attributed to the hydrophobic characters of the different cationic-
Schiff Base molecules, which forced the molecules to adsorb at the interface. The
adsorption/desorption equilibrium of the cationic-Schiff Base molecules at the interface is
started at the second time region while attaining the complete equilibrium has occurred at the
final time region after 4 minutes.
The equilibrium γIFT of the studied crude oil/surfactants solutions systems were varied
between 0.32 and 0.69 mN/m at 25 °C (Table 4). Rising the temperature to 50 °C was
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decreased the interfacial tension values of the different surfactants to much lower magnitude
(Table 4), which can be assigned to the increase of surfactant molecules pumping to the
interface as the increase of the repulsion between these molecules and the aqueous medium.
Decreasing the crude oil/surfactants solutions interfacial tension values by increasing the
temperature to 50 °C indicates the increase in surface wettability by the surfactant's solutions.
The high depression of the interfacial tension at the temperature of the crude oil reservoir
enables the use of 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC as additives for enhancing
crude oil production from exhausted wells.
3.4.2 Contact angle (θ)
During enhanced oil recovery processes, contact angle (θ) of the flooding solutions and the
rock surface saturated by crude oil plays an important role as that determines the interaction
between the rock and flooding solutions in petroleum reservoirs. Reservoir rocks can be
o
classified into three phases: water wet (θ ˂ 75 ), oil-wet (105 ˂ θ ˂180°) and moderate wet
(75 ˂ θ ˂105°) as shown in Figure 6.
The effect of the 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC surfactants at CMC
concentration on the contact angle at 25 and 50 °C was identified by measuring the contact
angle (θ) between saturated sand–rock saturated by crude oil and surfactants solutions (Table
4). The average contact angles of 4-IMHDAC, 6-IMHDAC and 8-IMHDAC surfactants were
observed at 30.9°, 29.2°, and 27.1° at 25 °C, and at 23.9°, 19.2°, and 13.5° at 50 °C,
respectively. The spreading of crude oil on the surface of the sand rock is increased by
increasing the alkyl group chain length of the surfactant, indicating wettability alteration of
the rock system from moderate or oil-wet to water-wet [23]. The change in the angle of
contact (θ) or the change in the wettability is the result of adsorption of the cationic–Schiff
Base molecules on the external and internal surface of the rock [24]. Moreover, increasing of
alky group chain length of cationic–Schiff Base surfactant favors the in-situ formation of the
surfactant at the crude oil/water interface due to the interaction between the long alkyl chain
and the crude oil.
After the formation of the in-situ surfactant, the surfactant moves to the aqueous phase as the
action of emulsification. In this case, some of the surfactant molecules are adsorbed on the
internal and external surface of rocks, resulting in wettability alteration, which facilitates the
desorption of the crude oil from the rocks. The results of the contact angle and interfacial
tension measurements of the 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC surfactants can
explain their mechanism during the enhanced oil recovery process. In the next section, the
efficiencies of the 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC surfactants during the enhanced
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oil recovery process of crude oil from exhausted wells will be measured in a simulated
flooding system containing similar wells conditions of salinity, temperature, and pressure
3.4.3 Enhanced oil recovery
The results of the flooding experiments of crude oil-saturated sandstone were represented in
Table 5. The obtained results from the use of 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC
surfactants at three different concentrations 1, 2, and 4 g/L, which represents the
concentration before, at, and higher than the critical micelle concentration values. The
cumulative oil recovery (%) obtained by using the 4-IMHDAC, 6-IMHDAC and 8-IMHDAC
surfactants at different concentrations showed their high comparative efficiency during the
enhanced oil recovery process (Table 5). At low concentration (1 g/L), the cumulative
recovery of 4-IMHDAC was 54.7%, while this value was increased to 68.9% in the case of 8-
IMHDAC. By increasing the concentration of the cationic-Schiff Base surfactants in the
flooding solutions up to 2 g/L, i.e., near the CMC of the used surfactants, a considerable
increase in the cumulative recovery was obtained. Additionally, a further increase in the
surfactant concentrations above their CMC values (4 g/L) showed an extreme increase in the
cumulative oil recovery (%) to reach 84.5% in the case of 8-IMHDAC flooding solution.
The results of the cumulative oil recovery from sandstone rock revealed several points. First,
at flooding solutions with concentrations lower than the CMC values, the efficiency of the
recovery was based on the interfacial properties of the different surfactants used during the
recovery process. Surfactants with lower interfacial tension values and contact angles were
more efficient in the recovery process. That can be attributed to the high ability of these
surfactants to be adsorbed at the sandstone/flooding solution interface, which facilitates their
diffusion to the rock pores, i.e., increase the wettability, and consequently repels the adhered
crude oil droplets from the rock bulk to the solution (Figure 7). As can be seen from Table 4,
the interfacial tension and contact angle values were decreased by increasing the hydrophobic
chain length from 4-IMHDAC to 8-IMHDAC, which is in good agreement with the
increasing variety of the recovery efficiency. Second, at flooding solutions with
concentrations at the CMC (2 g/L), the cumulative recovering efficiency was increased
considerably for the used surfactant flooding solutions to reach its maximum at 75.7% for 8-
IMHDAC surfactant (Table 5). Similar behavior was observed for 4-IMHDAC and 6-
IMHDAC, as well. Finally, the maximum cumulative enhanced recovery was obtained in the
case of flooding solutions containing surfactants at a concentration of 4 g/L (higher than CMC
values). It is essential to optimize the concentration of surfactants concentration in the
flooding solutions during the recovery process to avoid economic and production problems.
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At the three ranges of surfactant concentrations used during the recovery process, the
concentration near the CMC values was the most suitable concentration due to two main
points. The first is economical; the high concentration of surfactants above CMC results in
more adsorption of their molecules at the rock surface and leads to loss of the surfactants,
which increase the cost of the process. The second is related to separation efficiency, high
stable water/oil emulsions are formed at high surfactant concentrations, and that leads to a
complication of crude oil separation process from the obtained emulsion, and the problem of
emulsion breaker is raised. Hence, the optimized surfactant concentrations in the flooding
solutions were at their CMC values. The temperature is the third parameter, which should be
taken into consideration during the enhanced oil recovery process. The viscosity of
surfactant/crude oil emulsion was reduced by rising the temperature (Figure 8), which leads
to the increase in the mobility of the displaced fluid (flooding solution), and consequently,
higher sweep efficiency was occurred accompanied by the oil recovery process. The most
suitable temperature was found to be at 50 °C, which is also the average reservoir's
temperature.
3.4 Antimicrobial activity
The prepared cationic–Schiff Base surfactants, 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC,
were tested against several potent microorganisms, including Bacillus subtilis (B. subtilis),
Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Pseudomonas aeruginosa
candida Albicans (P. aeruginosa, C. Albicans), and Aspergillus niger (A. niger) by means of
inhibition zone diameter procedures, (Table 6). Inhibition zone diameter results indicated that
the cationic Schiff Base surfactants have good antimicrobial activities; furthermore, the
antimicrobial activities are depended mainly on the hydrophobicity of the chain length. The
obtained results were in agreement with reported findings [8], where the difference in the
optimum activity appears towards different types of bacteria resulting from the difference in
the chemical structure of the cationic surface. Thereby, the 8-IMHDAC, which has the most
extended chain length (8–carbon attached to the azomethine group), has the optimal activity.
Conclusion
In this study, three positively charged surface-active agents were synthesized, and their
chemical structures were elucidated by 1H-NMR and FTIR. The surface activity
measurements of the prepared compounds showed a gradual increase in surface activity by
increasing the alkyl chain length. Adsorption and micellization thermodynamic functions
revealed the higher tendencies of the different surfactants towards adsorption at the
organic/polar phase. The synthesized surfactants were evaluated in the enhanced oil recovery
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process and showed good efficiency (63.98%, 71.25%, and 75.72% for 4-IMHDAC, 6-
IMHDAC, and 8-IMHDAC, respectively) during the recovery process near their critical
micelle concentrations. The prepared surfactants showed high antimicrobial efficacy against
different bacteria and fungi.
References
[1] J. Niu, Q. Liu, J. Lv, B. Peng, Review on microbial enhanced oil recovery: Mechanisms,
modeling and field trials, Journal of Petroleum Science and Engineering, 192 (2020) 107350.
[2] N. Youssef, D.R. Simpson, K.E. Duncan, M.J. McInerney, M. Folmsbee, T. Fincher, R.M.
Knapp, In situ biosurfactant production by Bacillus strains injected into a limestone petroleum
reservoir, Applied and Environmental Microbiology, 73 (2007) 1239-1247.
[3] E.J. Gudiña, J.F.B. Pereira, R. Costa, J.A.P. Coutinho, J.A. Teixeira, L.R. Rodrigues,
Biosurfactant-producing and oil-degrading Bacillus subtilis strains enhance oil recovery in
laboratory sand-pack columns, Journal of Hazardous Materials, 261 (2013) 106-113.
[4] A. Mandal, Chemical flood enhanced oil recovery: A review, International Journal of Oil,
Gas and Coal Technology, 9 (2015) 241-264.
[5] A.A. Olajire, Review of ASP EOR (alkaline surfactant polymer enhanced oil recovery)
technology in the petroleum industry: Prospects and challenges, energy, 77 (2014) 963-982.
[6] G.J. Hirasaki, C.A. Miller, M. Puerto, Recent Advances in Surfactant EOR, (2008).
[7] I.A. Aiad, N.A. Negm, Some Schiff base surfactants as steel-corrosion inhibitors, Journal
of Surfactants and Detergents, 12 (2009) 313-319.
[8] N.A. Negm, S.M.I. Morsy, Corrosion inhibition of triethanolammonium bromide mono-
And dibenzoate as cationic inhibitors in an acidic medium, Journal of Surfactants and
Detergents 8 (2005) 283-287.
[9] N.A. Negm, N.G. Kandile, M.A. Mohamad, Synthesis, characterization and surface
activity of new eco‐friendly Schiff bases vanillin derived cationic surfactants, Journal of
Surfactants and Detergents 14 (2011) 325-331.
[10] N.A. Negm, A.F.M. El Farargy, D.E. Mohammed, H.N. Mohamad, Environmentally
friendly nonionic surfactants derived from tannic acid: synthesis, characterization and surface
activity, Journal of Surfactants and Detergents 15 (2012) 433-443.
[11] J.J. Sheng, Modern chemical enhanced oil recovery: theory and practice, Gulf
Professional Publishing 2010.
[12] H. Vatanparast, A. Alizadeh, A. Bahramian, H. Bazdar, Wettability alteration of low-
permeable carbonate reservoir rocks in presence of mixed ionic surfactants, Petroleum science
and technology, 29 (2011) 1873-1884.
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Journal Pre-proof
[13] G. Sharma, K. Mohanty, Wettability alteration in high-temperature and high-salinity
carbonate reservoirs, SPE Journal, 18 (2013) 646-655.
[14] L. Wang, Y. Zhang, L. Ding, J. Liu, B. Zhao, Q. Deng, T. Yan, Synthesis and
physiochemical properties of novel gemini surfactants with phenyl-1,4-bis(carbamoylmethyl)
spacer, RSC Advances, 5 (2015) 74764-74773.
[15] N.A. Negm, E.A. Badr, I.A. Aiad, M.F. Zaki, M.M. Said, investigation the inhibitory
action of novel diquaternary Schiff dibases on the acid dissolution of carbon steel in 1M
hydrochloric acid solution, Corrosion Science 65 (2012) 77-86.
[16] M.A. Betiha, Y.M. Moustafa, M.F. El-Shahat, E. Rafik, Polyvinylpyrrolidone-
Aminopropyl-SBA-15 schiff Base hybrid for efficient removal of divalent heavy metal
cations from wastewater, Journal of Hazardous Materials, 397 (2020) 122675.
[17] M.A. Betiha, N.A. Negm, E.M. El-Sayed, M.S. Mostafa, M.F. Menoufy, Capability of
synthesized sulfonated aromatic cross-linked polymer covalently bonded montmorillonite
framework in productivity process of biodiesel, Journal of Cleaner Production, 261 (2020)
120995.
[18] N.A. Negm, M.A. Betiha, M.S. Alhumaimess, H.M.A. Hassan, A.M. Rabie, Clean
transesterification process for biodiesel production using heterogeneous polymer-heteropoly
acid nanocatalyst, Journal of Cleaner Production, 238 (2019) 117854.
[19] A. Al-Sabagh, N. Nasser, M. El-Sukkary, A. Eissa, K.I. Kabel, T.M. Abd El-Hamid,
Surface Active and Thermodynamic Properties in Relation to the Demulsification Efficiency
for Some Ethoxylated Derivatives of Alkyldiamines and Polyalkylenepolyamines, Journal of
dispersion science and technology, 34 (2013) 1356-1367.
[20] M.J. Rosen, Z.H. Zhu, Synergism in binary mixtures of surfactants. 7. Synergism in
foaming and its relation to other types of synergism, Journal of the American Oil Chemists'
Society, 65 (1988) 663-668.
[21] A.M. Elfadly, A.M. Badawi, F.Z. Yehia, Y.A. Mohamed, M.A. Betiha, A.M. Rabie,
Selective nano alumina supported vanadium oxide catalysts for oxidative dehydrogenation of
ethylbenzene to styrene using CO2 as soft oxidant, Egyptian Journal of Petroleum, 22 (2013)
373-380.
[22] R. Borwankar, D. Wasan, Dynamic interfacial tensions in acidic crude oil/caustic
systems. Part I: A chemical diffusion‐kinetic model, AIChE journal, 32 (1986) 455-466.
[23] S. Kumar, A. Mandal, Studies on interfacial behavior and wettability change phenomena
by ionic and nonionic surfactants in presence of alkalis and salt for enhanced oil recovery,
Applied Surface Science, 372 (2016) 42-51.
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[24] H. Gong, Y. Li, M. Dong, S. Ma, W. Liu, Effect of wettability alteration on enhanced
heavy oil recovery by alkaline flooding, Colloids and Surfaces A: Physicochemical and
Engineering Aspects, 488 (2016) 28-35.
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CRediT author statement
Ahmed M. Al-Sabagh, Nabel A. Negm, Mohamed A. Betiha, Tahany Mahmoud:
Conceptualization, Methodology, Writing - Review & Editing.
Mohamed A. Betiha, Tahany Mahmoud: Conceptualization, Methodology, Writing.
Samy B. El-Henawy: Validation
Tahany Mahmoud: Investigation
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Authors of manuscripts confirm that there is no conflict of interest of this study with other studied.
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Scheme 1: Synthesis protocol of the compounds 5-CMS 4-IMHDA, 6-IMHDA and 8-
IMHDA.
Figure 1: Illustration of the pendant drop method based on optical drop-shape analysis.
Figure 2. FTIR of 5-CMS, 4-IMHDA, 6-IMHDA and 8-IMHDA compounds.
Figure 3: 1H-NMR of 5–CMS, FGDHAC, 4-IMHDAC, 6-IMHDAC and 8-IMHDAC
compounds.
Figure 4: Surface tension vs. concentration profile of prepared surfactants: A) at 25 oC,
and B) at 50 oC.
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Figure 5: Dynamic interfacial tension values at surfactants solutions-crude oil interface
at: A) 25 oC and B) 50 oC.
Figure 6: Different cases of contact angle at a solid liquid interface.
Figure 7: Predicted mechanism of enhanced oil recovery using: 4-IMHDAC, 6-
IMHDAC and 8-IMHDAC surfactants.
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Figure 8. Variation of viscosity for crudeoil and surfactant (1 g/L)/crude oil emulsion by
raising the temperature.
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Table 1: physicochemical properties of crude oil used
Experiment
Method
Result
ISO 3675
ASTM D-4052
ASTM D-4052
ASTM D-4052
IP-143
0.8983
0.8892
824.1
27.1
7.63
17.0
Density at 15.56 (°C)
Specific gravity (g/cm3)
Viscosity (cp)
API gravity at 60 (°F)
Asphaltene content (wt.%)
Wax content, wt. %
UOP-64
Table 2. Surface properties of 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC surfactants at
50 °C and 50 °C
Surfactant
T, oC
CMC
γCMC
πCMC
Γmax
Amin
(mmol/L) (mN/m) (mN/m) (mol/cm2)
(nm2)
25
50
25
50
25
50
8.40
4.21
7.91
3.97
6.73
3.38
31.8
23.1
27.8
22.8
26.9
19.9
40.8
49.5
44.8
49.8
45.7
52.7
9.32x10-11 177.98
3.77x10-11 440.26
9.09x10-11 182.64
2.81x10-11 589.08
6.54x10-11 253.48
2.22x10-11 746.88
4-IMHDAC
6-IMHDAC
8-IMHDAC
Table 3. Thermodynamic parameters of 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC
surfactants at 25 and 50 °C
Surfactant T (°C) ∆Gmic (KJ/mol)
∆Gads (KJ/mol) ∆Gmic-∆Gads
25
50
25
50
25
50
-11.84
-14.68
-11.99
-14.85
-12.39
-15.28
-16.21
-27.81
-16.91
-33.97
-19.36
-37.24
4.37
13.12
4.92
18.69
6.97
4-IMHDAC
6-IMHDAC
8-IMHDAC
22.39
Table 4. Interfacial tension and contact angle of crude oil/surfactants solutions at 25 °C,
and 50 oC
Interfacial tension (γIFT, mN/m) Contact angle (θ, o)
Surfactant
25 °C
0.72
0.46
0.32
50 °C
0.18
0.07
0.01
25 oC
30.9
29.2
27.6
50 oC
23.9
19.2
13.5
4-IMHDAC
6-IMHDAC
8-IMHDAC
Table 5. The cumulative oil recovery (%) obtained using 4-IMHDAC, 6-IMHDAC, and
8-IMHDAC surfactants at different concentrations at 50 oC
Conc. 1 g/L
Conc. 2 g/L
Recovery,%
Conc. 4 g/L
Surfactant
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54.67
62.93
68.85
63.98
71.25
75.72
81.28
83.55
84.46
4-IMHDAC
6-IMHDAC
8-IMHDAC
Table 6. Antimicrobial activity of 4-IMHDAC, 6-IMHDAC, and 8-IMHDAC
surfactants against pathogenic bacteria and fungi
Inhibition zone diameter (mm)
Surfactant
B.
subtilis
21
S.
aureus
28
E.
coli
13
18
14
28
---
P.
C.
albicans
26
A.
niger
26
29
20
aeruginosa
14
19
13
28
---
4-IMHDAC
6-IMHDAC
8-IMHDAC
Erythromycin
Metronidazole
19
18
32
---
30
24
33
---
30
25
---
29
---
30
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Three positively charged surface-active agents were synthesized.
Gradual increase in surface activity was increased by increase of chain length.
Adsorption thermodynamic functions showed high tendency towards adsorption.
The surfactants showed good efficiency in oil recovery.
The surfactants showed high antimicrobial efficacy against bacteria and fungi.
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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5