Benzene ring containing cationic gemini surfactants: Synthesis, surface properties and antibacterial activity

Article abstract of DOI:10.1016/j.molliq.2017.06.008

New quaternary ammonium gemini surfactants of the general formula C_nH_2n?+?1-Ph-NHCOCH₂N⁺(CH₃)₂-(CH₂)_s-N⁺(CH₃)₂CH₂CONH-Ph-C_nH_2n?+?1 (with n?=?8, 10, 12, 14, 16 and s?=?2, 4, 6) have been synthesized by an efficient synthetic pathway based on the quaternization of N,N,N′,N′-tetramethylalkylenediamine with 2-bromo-N-(4-(alkyloxy) phenyl) acetamides. Their surface properties were investigated by surface tension, electrical conductivity and dynamic light scattering (DLS) measurements. The study shows that the incorporation of a benzene ring in the hydrophobic tail prompts micelle formation which leads to a smaller cmc values in the range of 0.21–0.009?mM compared to analogous geminis bearing alkyl hydrophobic chains. The length of the spacer and the hydrophobic chain has a pronounced effect on the aggregation behavior of surfactants molecules. This was confirmed by the average surfaces occupied by these molecules at the water–air interface calculated from the Gibbs equation. The size of the aggregates was measured by employing dynamic light scattering technique. The antimicrobial activity of investigated surfactants was evaluated against three microorganisms: Staphylococcus aureus, Escherichia coli and Candida albicans.

Full text of DOI:10.1016/j.molliq.2017.06.008

Accepted Manuscript
Benzene ring containing cationic gemini surfactants: Synthesis,
surface properties and antibacterial activity
Khadidja Taleb, Mustapha Mohamed-Benkada, Nadjia
Benhamed, Salima Saidi-Besbes, Yves Grohens, Aicha Derdour
PII:
DOI:
Reference:
S0167-7322(17)31569-6
doi: 10.1016/j.molliq.2017.06.008
MOLLIQ 7453
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Journal of Molecular Liquids
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Revised date:
Accepted date:
12 April 2017
30 May 2017
1 June 2017
Please cite this article as: Khadidja Taleb, Mustapha Mohamed-Benkada, Nadjia
Benhamed, Salima Saidi-Besbes, Yves Grohens, Aicha Derdour , Benzene ring containing
cationic gemini surfactants: Synthesis, surface properties and antibacterial activity,
Journal of Molecular Liquids (2017), doi: 10.1016/j.molliq.2017.06.008
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ACCEPTED MANUSCRIPT
Benzene ring containing cationic gemini surfactants: Synthesis, surface
properties and antibacterial activity
Khadidja Taleb,1 Mustapha Mohamed-Benkada,2 Nadjia Benhamed,2 Salima Saidi-Besbes,1,*
Yves Grohens,3 Aicha Derdour1
1 Université Oran 1 Ahmed Ben Bella, Laboratoire de Synthèse Organique Appliquée
(LSOA), Département de chimie, Faculté des sciences exactes et appliquées, BP 1524 EL
Mnaouer, 31000 Oran, Algeria
2
Département de Biotechnologie, Faculté des Sciences de la Nature et de la Vie, Université
des Sciences et de la Technologie d'Oran-Mohamed Boudiaf (USTO-MB), Bir El Djir, Oran,
Algeria
3 IRDL-FRE CNRS 3744, Université de Bretagne Sud, Lorient 56100, France
*Corresponding author: saidi.salima@univ-oran.dz; salima_saidi@yahoo.fr,
Tel : +213 555529020
Abstract
New quaternary ammonium gemini surfactants of the general formula CnH2n+1-Ph-
NHCOCH2N+(CH3)2-(CH2)s-N+(CH3)2CH2CONH-Ph-CnH2n+1 (with n = 8, 10, 12, 14, 16 and
s = 2, 4, 6) have been synthesized by an efficient synthetic pathway based on the
quaternization of N,N,N′,N′-tetramethylalkylenediamine with 2-bromo-N-(4-(alkyloxy)
phenyl) acetamides. Their surface properties were investigated by surface tension, electrical
conductivity and dynamic light scattering (DLS) measurements. The study shows that the
incorporation of a benzene ring in the hydrophobic tail prompts micelle formation which leads
to a smaller cmc values in the range of 0.21-0.009 mM compared to analogous geminis
bearing alkyl hydrophobic chains. The length of the spacer and the hydrophobic chain has a
pronounced effect on the aggregation behavior of surfactants molecules. This was confirmed
by the average surfaces occupied by these molecules at the water–air interface calculated from
the Gibbs equation. The size of the aggregates was measured by employing dynamic light
scattering technique. The antimicrobial activity of investigated surfactants was evaluated
against three microorganisms: Staphylococcus aureus, Escherichia coli and Candida albicans.
Keywords: Gemini surfactant, Quaternary ammonium salt, Surface-active properties,
aggregates, antimicrobial activity
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1. Introduction
Gemini surfactants, also called dimeric surfactants, are composed of two monomeric
surfactant molecules linked covalently by a spacer group. Since the pioneer work of Bunton et
al. on bisquatarnary ammonium bromide gemini surfactants [1], this class of compounds is
gaining increasing attention due to their unique properties. In comparison with conventional
surfactants, gemini surfactants are known to be much more efficient in reducing the surface
tension of water and the interfacial tension of the oil-water interface [2-3]. Besides, they
exhibit extremely lower CMC values, better water solubility, unusual micelle structures and
aggregation behavior, and interesting rheological properties [4-6]. Thereby, they have find a
wide range of applications in advanced technologic domains such as the fabrication of high-
porosity materials [7], enhanced oil recovery process [8], genetic science and pharmaceutical
applications, detergents, cosmetics, skin and personal care products manufacturing [9-14].
In the last decade, a variety of geminis have been designed and synthesized in the aim to
highlight the effect of structural parameters on the physicochemical properties of related
surfactants [15-17]. One of the most studied gemini surfactants are bis(quaternary
alkylammonium bromide) surfactants [18]. The effect of the nature and the length of the
spacer group and the hydrophobic chain have been systemically investigated. Alkyl chains
have been usually used as hydrophobic tail of gemini surfactants whereas common spacers
used to control the separation between the head groups are apolar aliphatic or aromatic group
[19], polar polyether group [20], short (CH2)2 [21] or long (CH2)6-12 methylene group [22],
rigid (stilbene [23-24], benzene [25-27], adamantine [28-29]) or flexible (polymethylene)
groups [30].
It was shown that the length and the rigidity of the spacer group has a strong influence on
different surfactant properties such as cmc, surface tension, water solubility and foaming
ability [31]. For instance, Song and Rosen found that geminis with a flexible hydrophilic
spacer aggregates more readily than homologous surfactants with a rigid hydrophobic spacer
[32]. Similar conclusions were obtained by Wang et al. when comparing the aggregation
properties of dicationic quaternary ammonium gemini surfactants bearing diethyl ether, hexyl
or p-xylyl spacer group by using electrical conductivity and fluorescence measurements [33].
An opposite behavior was obtained for gemini quaternary ammonium salts bearing a rigid
adamantine spacer. These compounds exhibited lower surface tension values as compared to
conventional gemini quaternary ammonium salts and were able to pack more tightly at the air-
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water interface thereby promoting self-association in the bulk [34]. Zhang et al. showed that
the variation in the CMC values reported in the literature for geminis with spacers different in
rigidity is mainly caused by changes in the other properties of the spacer such as the
hydrophobicity, the chain length and the π–π stacking for phenyl spacers[35].
Besides these, only few studies have been devoted to the hydrophobic tail rigidity effect on
the micellization behavior.
In this context, we report in this work a new family of cationic gemini surfactants bearing 4-
alkylbenzene group on the hydrophobic chains, connected through an amide function, to the
ammonium headgoup, named as Gem n-s-n (n = 8, 10, 12, 14, 16 and s = 2, 4, 6). The amide
moiety is considered as a biodegradable linkage suitable for the preparation of eco-friendly
surfactants [36]. The aim of this study is to clarify the effect of benzene ring on the surface
properties of gemini surfactants and to highlight a structure-property relationship upon
variation in different structural parameters such as hydrocarbon chain and spacer lengths. The
surface and bulk properties of these surfactants such as critical micellar concentration (cmc),
surface tension (γcmc), surface area occupied by a molecule at the air-water interface (Amin),
degree of counterion binding (β) and standard free energy of micellization (∆Gm0 ic) have been
discussed on the basis of surface tension, conductivity and dynamic light scattering
measurements. The antibacterial activity against Gram-positve, Gram-negative and fungi
microorganisms was also evaluated.
2. Experimental part
2.1. Characterization
Confirmation of the structures of the intermediates and products was obtained by nuclear
1
magnetic resonance (NMR) and High resolution mass spectrometry (HRMS). H and 13C
NMR spectra were recorded on a Bruker 300 MHz spectrometer (Wissembourg, France).
Tetramethylsilane was used as an internal reference for chemical shifts. HRMS was carried
out using a Finnigan Matt TSQ 7000 mass spectrometer (EIS mode) coupled with liquid
chromatography interface.
Conductivity Measurements. Conductivities were determined using a digital conductimeter
apparatus (OHAUS conductivity STARTER 300C). The cmc was determined by adding
adequate quantity of a concentrated surfactant solution to water in order to change the
surfactant concentration from concentrations well below the cmc to up to at least 1 to 2 times
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the cmc. The solutions were thermostated in the cell at 25.0 ± 0.1 °C. The CMC values were
estimated from the break point on each curve of conductivity versus surfactant concentration.
Surface Tension Measurements. The cmc and surface tensions at the cmc values were
determined at 25°C, by the Wilhelmy plate method using a GIBERTINI K100 tensiometer at
25°C. The tensiometer was calibrated using Millipore water.
Dynamic light scattering (DLS) measurements. Particles size and polydispersity were
measured at 25°C using a Zetasizer Nano-S model (Malvern Instruments Ltd.) equipped with
a He–Ne laser ( = 633 nm, 4.0 mW). The aqueous solutions were prepared using
demineralized water and were filtered through a 0.45 μm filter. The time dependent
correlation function of the scattered light intensity was measured at a scattering angle of 173°
relative to the laser source (back-scattering detection). The Stokes radius (RS) of the particles
was estimated from their diffusion coefficient (D) using the Stokes–Einstein equation
D=kBT/6 πRS , where kB is Boltzmann’s constant, T is the absolute temperature, and  is the
viscosity of the solvent, All measurements were performed at 25.0 ± 0.1℃.
2.2. Materials
Chemicals used for the synthesis: 4-aminophenol, 1-bromooctane (99%), 1-bromodecane
(98%), 1-bromododecane (97%), 1-bromotetradecane (97%), 1-bromohexadecane (97%),
bromoacetyl chloride (95%) , N,N,N′,N′-Tetramethylethylenediamine (99%), N,N,N′,N′-
Tetramethyl-1,4-butanediamine (98%), N,N,N′,N′-Tetramethyl-1,6-hexanediamine (99%),
butanone (98%) were purchased from Sigma-Aldrich and used without further purification.
All other reagents employed were common laboratory materials. The solvents were of
commercial grade quality and were dried and distilled before use.
4-(alkyloxy)benzenamine (2a-e) were prepared using a three step procedure from 4-
aminophenol according to our previously described procedure [37]. Detailed characterizations
can be found in the Supporting information file.
2.2.1. Synthesis of 2-bromo-N-(4-(alkyloxy) phenyl) acetamide (3a-e)
2-bromo-N-(4-(Alkyloxy)phenyl) acetamide (3a-e) were synthesized via a slight
modifications of the literature procedures [38]. 13 mmol of 4-(alkyloxy)benzenamine (2a-e)
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were dissolved in 15 mL of dichloromethane then a solution of (2.76 g, 20 mmol) of
potassium carbonate K2CO3 dissolved in 15 mL of water was added. The solution was cooled
to 5 °C then (3.14 g, 20 mmol) of bromoacetyl chloride dissolved in 15 mL of
dichloromethane were added dropwise. The reaction mixture was stirred at room temperature
for about 3 h. The aqueous solution was separated and extracted two times with
dichloromethane. The organic phase was washed with water then dried over anhydrous
sodium sulphate and finally concentrated to yield a white product quantitatively. If required, it
can be further purified by flash column chromatography using dichloromethane as eluent.
2-bromo-N-(4-(Octyloxy)phenyl)acetamide (3a): white solid, yield (quantitative), m.p.102°C,
3
1H NMR (300 MHz, CDCl3):δ 0.88 (t, 3H, CH3, J = 6.88Hz), 1.28 (m, 8H, C2H5(CH2)4),
1.44 (m, 2H, CH3CH2-), 1.76 (m, 2H, CH2CH2O-), 3.93 (t, 2H, CH2O-, 3J = 6.55 Hz), 4 .02 (s,
3
2H ,COCH2Br), 6.86 (d, 2H, Ar-H , J = 8.97 Hz), 7.39 (d, 2H, Ar-H, 3J = 8.97Hz), 8.04 (s,
1H, NH).13C NMR (300 MHz, CHCl3): 14.14, 22.68, 26.04, 29.25, 31.83, 68.28, 114.83,
121.97, 129.67, 153.83, 163.15
1
2-bromo-N-(4-(decyloxy)phenyl)acetamide (3b): white solid, yield (99%), m.p.105°C, H
3
NMR (300 MHz, CDCl3): δ 0.87 (t, 3H, CH3, J = 6.88Hz), 1.26 (m, 12H, C2H5(CH2)6), 1.44
3
(m, 2H, CH3CH2-), 1.76 (m, 2H, CH2CH2O-), 3.93 (t, 2H, CH2O-, J = 6.55 Hz),4 .01(s, 2H,
3
3
COCH2Br), 6.86 (d, 2H, Ar-H, J = 8.97Hz),7.39 (d, 2H, Ar, J = 8.97Hz), 8.05 (s,1H,
NH).13C NMR (300 MHz, CDCl3): δ 14.16, 22.71,26.04, 29.25, 29.35, 29.58, 31.92,68.28,
114.83,121.97,129.66,156.65, 163.16.
2-bromo-N-(4-(dodecyloxy)phenyl)acetamide (3c): white solid, yield (95%), m.p.112°C,1H
NMR (300 MHz, CDCl3): δ 0.88 (t, 3H, CH3, 3J = 6.88Hz), 1.26 (m, 16H, C2H5(CH2)8),1.44
(m, 2H, CH3CH2-), 1.76 (m, 2H, CH2CH2O-), 3.93 (t, 2H, CH2O-,3J =6.55 Hz), 4.01(s, 2H
3
,COCH2Br), 6.86 (d, 2H, Ar-H , J = 9.00Hz),7.39 (d, 2H, Ar-H, 3J = 9.00 Hz), 8.05 (s, 1H,
NH).13C NMR (300 MHz, CDCl3): δ 14.15, 22.71, 26.03, 29.25, 29.41, 29.65, 31.93, 68.29,
114.84,121.96,129.69,156.66,163.16.
2-bromo-N-(4-(tetradecyloxy)phenyl)acetamide (3d): white solid, yield (85%),m.p.114°C.
3
1H NMR (300 MHz, CDCl3): δ 0.87 (t, 3H, CH3, J = 6.88Hz), 1.25 (m, 20H, C2H5(CH2)10),
1.42 (m, 2H, CH3CH2-), 1.76 (m, 2H, CH2CH2O-), 3.93 (t, 2H, CH2O-, 3J = 6.55 Hz), 4.01(s,
2H, COCH2Br), 6.86 (d, 2H, Ar-H, 3J = 9.0 Hz), 7.39 (d, 2H, Ar-H, 3J = 9.0Hz), 8.06 (s, 1H,
NH).13C NMR (300 MHz, CDCl3): δ 14.18, 22.73, 26.04, 29.25, 29.42, 29.69, 31.95, 68.27,
114.81,121.97,129.64,156.69,163.16.
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2-bromo-N-(4-(hexadecyloxy)phenyl)acetamide (3e): white solid, yield (80%),m.p.120°C.
3
1H NMR (300 MHz, CDCl3): δ 0.88 (t, 3H, CH3, J = 6.88Hz), 1.25 (m, 24H, C2H5(CH2)12),
1.39 (m, 4H, CH3CH2-), 1.76 (m, 2H, CH2CH2O-), 3.93 (t, 2H, CH2O-, 3J = 6.55 Hz), 4 .01(s,
2H, COCH2Br), 6.86 (d, 2H, Ar-H, 3J = 9.00Hz) ,7.39 (d, 2H, Ar-H, 3J = 9.00Hz), 8.02 (s, 1H,
NH).13C NMR (300 MHz,CDCl3): δ 14.10, 22.68, 26.02, 29.24, 29.49, 29.68, 31.92, 68.32,
114.88,121.94, 129.71,156.69,163.10.
2.2.2. Synthesis of Gemini surfactants (Gem n-s-n)
In a typical procedure, 20 mmol of 2-bromo-N-(4-(alkyloxy) phenyl) acetamide (3a-e) were
added to 10 mmol of N,N,N’,N’-tetramethylalkanediamine dissolved in 35 mL of diethyl
ether. The reaction mixture was stirred for 72 hours at 40°C. The progress of the reaction was
monitored by thin layer chromatography using a mixture of dichloromethane/ethyl acetate
(90:10) as eluent. The obtained solid was filtered, washed several times with diethyl ether
then recrystallized from pure ethanol to eliminate the residual conventional surfactant and
give the gemini surfactant (Gem n-s-n) with 85−99% yield.
Gem 8-2-8: white solid, yield (90%). 1H NMR (300 MHz, DMSO d6): δ 0.85 (t, 6H, CH3, 3J
= 6.90 Hz), 1.26 (m, 16H, C2H5(CH2)4), 1.39 (m, 4H, CH3CH2-), 1.68 (m, 4H, CH2CH2O-),
3.39 (s,12H, (CH3)2N+), 3.90 (t, 4H, CH2O-, 3J = 6.49Hz), 4.33 (s, 4H, N+(CH2)2N+), 4.42 (s,
4H, COCH2N+), 6.87 (d, 4H, Ar-H, 3J = 9.08Hz), 7.46 (d, 4H, Ar-H, 3J = 9.08 Hz), 10.58 (s,
13
2H, NH). C NMR (300 MHz, DMSO d6): δ 14.94, 22.06, 25.5, 28.66, 28.76, 31.22, 52.5,
55.61, 61.84, 67.50, 114.41,121.36, 130.37, 155.41, 160.91. HRMS (ESI ): m/z 320.2458
(calculated), 320.2466 (found), M2Br2+.
1
Gem 10-2-10: white solid, yield (87%). H NMR (300 MHz, DMSO d6): δ 0.85 (t, 6H, 3J =
6.76Hz, CH3), 1.24 (m, 24H, C2H5(CH2)6), 1.38 (m, 4H, CH3CH2-), 1.67 (m, 4H, CH2CH2O-
), 3.34(s, 12H, (CH3)2N+), 3.89 (t, 4H, CH2O-, 3J = 6.45 Hz), 4.31(s, 4H, N+(CH2)2N+), 4.39
(s, 4H, COCH2N+), 6.84 (d, 4H, Ar-H, 3J = 9.06 Hz), 7.42 (d, 4H, Ar-H, 3J = 9.04 Hz), 10.54
(s, 2H, NH). 13C NMR (300MHz, DMSO d6): 13.93, 22.08, 25.50, 28.69, 28.60, 28.96, 29.00,
31.27, 52.52, 55.58, 61.80, 67.49, 114.39, 121.34, 130.38, 155.40,160.90. HRMS (ESI ): m/z
348.2771 (calculated), 348.2776 (found), M2Br2+.
Gem12-2-12: white solid, yield (88%).1H NMR (300 MHz, DMSO d6): δ 0.85 (t, 6H, 3J =
6.87Hz, CH3), 1.24 (m, 32H, C2H5(CH2)8), 1.38 (m, 4H, CH3CH2-),1.68 (m, 4H, CH2CH2O-),
3.38(s, 12H, (CH3)2N+), 3.90 (t, 4H, CH2O-, 3J = 6.48Hz), 4.32(s, 4H, N+(CH2)2N+), 4.40 (s,
6

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4H, COCH2N+), 6.83 (d, 4H, Ar-H, 3J = 9.04 Hz), 7.42 (d, 4H, Ar-H, 3J = 9.03 Hz), 10.56 (s,
2H, NH). 13C NMR (300MHz, DMSO d6):14.42, 22.56, 25.99, 29.18, 29.28, 29.48, 31.76,
53.09, 55.98, 58.46, 68.12, 114.91, 121.91, 130.86, 155.93, 161.50.
1
Gem14-2-14: white solid, yield (81%), H NMR (300 MHz, DMSO d6):δ 0.86 (t, 6H, 3J =
6.82Hz, CH3), 1.26 (m, 40H, C2H5(CH2)10), 1.40 (m, 4H, CH3CH2-), 1.67 (m, 4H, CH2CH2O-
), 3.42 (s, 12H, (CH3)2N+), 3.93 (t, 4H, CH2O-, 3J = 6.49Hz), 4.35(s, 4H, N+(CH2)2N+), 4.45(s,
4H, COCH2N+), 6.86 (d, 4H, Ar-H, 3J =8.95Hz),7.46 (d, 4H, Ar-H, 3J =8.95Hz), 10.60 (s, 2H,
13
NH). C NMR (300MHz, DMSO d6):13.38, 21.57, 25.08, 28.18, 28.31, 28.50, 30.81, 52.29,
55.60, 62.29, 67.50, 114.32, 121.31, 130.04, 155.38, 164.47
Gem 16-2-16: white solid, yield (85%).1H NMR (300 MHz, DMSO d6): δ 0.86 (t, 6H, 3J =
6.50Hz, CH3), 1.25 (m, 48H, C2H5(CH2)12), 1.40 (m, 4H, CH3CH2-), 1.69 (m, 4H, CH2CH2O-
),3.41 (s, 12H, (CH3)2N+), 3.93 (t, 4H, CH2O-, 3J = 6.23Hz), 4.34 (s, 4H, N+(CH2)2N+), 4.43
(s, 4H, COCH2N+), 6.86 (d, 4H, Ar-H, 3J =8.57Hz), 7.45 (d, 4H, Ar-H, 3J = 8.57Hz), 10.48 (s,
13
2H, NH). C NMR (300MHz, DMSO d6): 14.23, 22.43, 25.95, 28.79, 29.04, 29.19, 29.39,
31.68, 53.18, 56.60, 68.45, 115.18, 122.18, 127.02,158.19, 166.17.
Gem 8-4-8: white solid, yield (quantitative).1H NMR (300 MHz, DMSO d6): δ 0.85 (t, 6H, 3J
= 6.96Hz, CH3), 1.26 (m, 16H, C2H5(CH2)4), 1.38 (m, 4H, CH3CH2-),1.68 (m, 4H,
CH2CH2O-), 1.80 (m, 4H, N+CH2(CH2)2CH2N+), 3.26 (s, 12H, (CH3)2N+), 3.61(s, 4H,
3
CH2N+), 3.91 (t, 4H, CH2O-, 3J =6.50Hz), 4.32 (s, 4H, COCH2N+), 6.90 (d, 4H, Ar-H, J =
9.04Hz), 7.50 (d, 4H, Ar-H, 3J =9.04Hz), 10.59 (s, 2H, NH). 13C NMR (300MHz, DMSO d6):
13.94, 19.24, 22.06, 25.48, 28.64, 28.72, 31.21, 51.32, 62.63, 63.47, 67.52, 114.51, 121.26,
130.51, 155.42, 161.24. HRMS (ESI): m/z 334.2615 (calculated), 334.2629 (found),
M2Br2+.
3
Gem10-4-10: white solid, yield (100%).1H NMR (300 MHz, DMSO d6): δ 0.86 (t, 6H, J =
6.34Hz, CH3), 1.26 (m, 24H, C2H5(CH2)6), 1.40 (m, 4H, CH3CH2-),1.69 (m, 4H, CH2CH2O-),
1.88 (m, 4H, N+CH2(CH2)2CH2N+), 3.30(s, 12H, (CH3)2N+), 3.66 (s, 4H, CH2N+), 3.93 (t, 4H,
CH2O-, 3J = 6 .41Hz), 4.38 (s, 4H, CH2N+), 6.90 (d, 4H, Ar-H, 3J =8.53Hz), 7.52 (d, 4H, Ar-
3
H, J =8.53Hz), 10.57 (s, 2H, NH).13C NMR (300MHz, DMSO d6): 13.88, 19.08, 21.57,
25.05, 28.17, 28.45, 30.81, 51.13, 62.51, 63.71, 67.50, 114.36, 121.67, 130.26, 155.32,
160.82.
7

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3
Gem12-4-12: white solid, yield (97%).1H NMR (300 MHz, DMSO d6): δ 0.84 (t, 6H, J =
6.86Hz, CH3), 1.23 (m, 32H, C2H5(CH2)8), 1.37 (m, 4H, CH3CH2), 1.67 (m, 4H, CH2CH2O-),
1.82 (m, 4H, N+CH2(CH2)2CH2N+), 3.27(s, 12H, (CH3)2N+), 3.63 (s, 4H, CH2N+), 3.90 (t, 4H,
CH2O-, 3J = 6.45Hz), 4.36 (s, 4H, CH2N+), 6.87 (d, 4H, Ar-H, 3J = 9.02Hz), 7.50 (d, 4H, Ar-
H, J=9.02Hz), 10.62 (s, 2H, NH).13C NMR (300MHz, DMSO d6): 13.92, 19.27, 22.09, 25.56,
28.67, 28.79, 31.29, 51.32, 62.36, 63.54, 67.52, 114.45, 121.24, 130.56, 155.41, 161.23.
3
Gem14-4-14: white solid, yield (90%).1H NMR (300 MHz, DMSO d6): δ 0.84 (t, 6H, J =
6.78Hz, CH3), 1.23 (m, 40H, C2H5(CH2)10), 1.37 (m, 4H, CH3CH2-),1.67 (m, 4H, CH2CH2O-
),1.81 (m, 4H, N+CH2(CH2)2CH2N+), 3.27 (s, 12H, (CH3)2N+), 3.62 (s, 4H, CH2N+), 3.90 (t,
3
4H, CH2O, 3J=6.43Hz), 4.33 (s, 4H, CH2N+), 6.88 (d, 4H, Ar-H, J = 8.96Hz), 7.50 (d, 4H,
3
Ar-H, J = 8.96Hz), 10.60 (s, 2H, NH).13C NMR (300MHz, DMSO d6): 13.87, 19.19, 22.01,
25.43, 28.64, 28.72, 28.92, 28.99, 31.21, 51.31, 61.12, 62.40, 63.49, 67.49, 114.45, 121.20,
130.50, 155.38, 161.18.
1
3
Gem16-4-16: white solid, yield (93%). H NMR (300 MHz, DMSO d6): δ 0.85 (t, 6H, J =
6.97Hz, CH3), 1.25 (m, 48H, C2H5(CH2)12), 1.40 (m, 4H, CH3CH2-),1.69 (m, 4H, CH2CH2O-
), 1.80 (m, 4H, N+CH2(CH2)2CH2N+), 3.28 (s, 12H, (CH3)2N+), 3.64(s, 4H, CH2N+), 3.93 (t,
3
4H, CH2O, 3J =6 .50Hz), 4.33 (s, 4H, COCH2N+), 6.90 (d, 4H, Ar-H, J = 9.03Hz), 7.48 (d,
3
4H, Ar-H, J =9.03Hz), 10.49 (s, 2H, NH).13C NMR (300MHz, DMSO d6):13.46, 19.08,
21.65, 25.13, 28.25, 28.36, 28.59, 30.88, 51.28, 62.53, 63.58, 67.55, 114.43, 121.28, 130.19,
155.41, 160.89.
3
Gem 8-6-8: white solid, yield (89%).1H NMR (300 MHz, DMSO d6): δ 0.85 (t, 6H, J =
6.89Hz, CH3), 1.25 (m, 16H, C2H5CH2)4), 1.34 (m, 4H, CH3CH2),1.34 (m, 4H,
N+C2H4(CH2)2C2H4N+), 1.67 (m, 4H, CH2CH2O), 1.80 (m, 4H, N+CH2CH2), 3.27 (s, 12H,
(CH3)2N+), 3.54 (s, 4H, N+CH2), 3.90 (t, 4H, CH2O, 3J = 6.49Hz), 4.36 (s, 4H, COCH2N+),
6.89 (d, 4H, Ar-H, 3J =9.06 Hz), 7.52 (d, 4H, Ar-H, 3J = 9.05Hz), 10.66 (s, 2H, NH).13C NMR
(300 MHz, DMSO d6): 14.40, 21.15, 22.54, 25.57, 25.97, 29.19, 29.21, 31.69, 51.77, 62.53,
64.99, 68.05, 115.01, 121.71, 131.10, 155.93, 161.79. HRMS (ESI): m/z 348.2771
(calculated), 348.2270 (found), M2Br2+.
3
Gem 10-6-10: white solid, yield (89%). 1H NMR (300 MHz, DMSO d6): δ 0.84 (t, 6H, J =
6.87Hz, CH3), 1.24 (m, 24H, C2H5CH2)6), 1.33 (m, 8H, CH3CH2 and N+C2H4(CH2)2C2H4N+),
1.67 (m, 4H, CH2CH2O), 1.79 (m, 4H, N+CH2CH2), 3.26 (s, 12H, (CH3)2N+), 3.54 (m, 4H,
8

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3
N+CH2), 3.90 (t, 4H, CH2O, 3J =6.45Hz), 4.34 (s, 4H, COCH2N+),6.88 (d, 4H, Ar-H, J =
9.02Hz), 7.48 (d, 4H, Ar-H, 3J = 9.02 Hz), 10.62 (s, 2H, NH).13C NMR (300MHz, DMSO d6):
13.88, 21.60, 22.02, 25.05, 25.43, 28.60, 28.71, 28.88, 28.93, 31.22, 51.23, 62.10, 64.30,
67.47, 114.96, 121.17, 130.49, 155.37, 161.25.
3
Gem12-6-12: white solid, yield (88%).1H NMR (300 MHz, DMSO d6): δ 0.84 (t, 6H, J =
6.86 Hz, CH3), 1.23 (m, 32H, C2H5(CH2)8), 1.33 (m, 8H, CH3CH2 and
N+C2H4(CH2)2C2H4N+), 1.67 (m, 4H, CH2CH2O), 1.78 (m, 4H, N+CH2CH2) , 3.25 (s, 12H,
(CH3)2N+), 3.54(m, 4H, N+CH2), 3.90 (t, 4H, CH2O, 3J = 6.46Hz), 4.30 (s, 4H, COCH2N+),
6.89 (d, 4H, Ar-H, 3J = 8.99Hz), 7.48 (d, 4H, Ar-H, 3J = 8.98Hz), 10.54 (s, 2H, NH).
3
Gem14-6-14 : white solid, yield (92%).1H NMR (300 MHz, DMSO d6): δ 0.85 (t, 6H, J =
6.70Hz, CH3), 1.25 (m, 40H, C2H5(CH2)10), 1.37 (m, 8H, CH3CH2 and
N+C2H4(CH2)2C2H4N+), 1.68 (m, 4H, CH2CH2O), 1.81 (s, 4H, N+CH2CH2), 3.26 (s, 12H,
(CH3)2N+), 3.55(s, 4H, N+CH2), 3.92 (t, 4H, CH2O, 3J =5.90 Hz), 4.32 (s, 4H, COCH2N+),
6.88 (d, 4H, Ar-H, 3J =8.29Hz), 7.49 (d, 4H, Ar-H, 3J =8.29Hz), 10.48 (s, 2H, NH). 13C NMR
(300 MHz, DMSO d6):14.24, 22.19, 22.43, 25.59, 25.91, 29.04, 29.15, 29.37, 31.67, 51.97,
63.09, 65.29, 68.33,115.21,121.99,130.05,156.16,161.76.
1
3
Gem16-6-16 : white solid, yield (94%). H NMR (300 MHz, DMSO d6): δ 0.86 (t, 6H, J =
6.73Hz, CH3), 1.25 (m, 48H, C2H5(CH2)12), 1.37 (m, 8H, CH3CH2 and
N+C2H4(CH2)2C2H4N+), 1.68 (m, 4H, CH2CH2O), 1.81 (s,4H, N+CH2CH2) , 3.26 (s, 12H,
3
(CH3)2N+), 3.54 (s, 4H, N+CH2), 3.92 (t, 4H, CH2O, J = 6.46Hz), 4.32 (s, 4H, COCH2N+),
3
6.89 (d, 4H, Ar-H, 3J =8.88Hz), 7.50 (d, 4H, Ar-H, J = 8.78Hz), 10.50 (s, 2H, NH).13C NMR
(300 MHz, DMSO d6): 14.20, 22.21, 22.40, 25.60, 25.90, 29.07, 29.14, 29.34, 31.64, 52.08,
63.19, 65.32, 68.43, 115.31, 122.1, 133.93, 156.09, 161.82
2.3. Antimicrobial activity.
Antibacterial and antifungal activities of Gem n-s-n surfactants were evaluated using
measurement of Minimal Inhibitory Concentration (MIC; lowest concentration of compound
at which in vitro tested microorganism do not show visible growth after incubation [39]) and
Minimal Lethal Concentrations (MLC; lowest concentration of antimicrobial agent that will in
vitro kill microorganism and consequently prevents it’s growth after subculture onto
antibiotic-free media). Bacterial and mycological nutrient broths where used for bacteria and
9

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fungi respectively. Gram-positive bacteria strain Staphylococcus aureus (ATCC 25923);
Gram-negative bacteria strain Escherichia coli (ATCC 2592 and fungal strain Candida
albicans (ATCC 10231) were used for this study. Standardized inoculum of bacteria and
fungal strains were prepared by incubating the microorganisms in nutrient bacterial (24 h at
37°C) and mycological broths (48h at 25°C) and then diluted to approximately 106 cfu/mL.
For MIC determination the studied compounds were dissolved in double distilled water and
sterilized (filtration, 0,45 µm, Millipore). 1 mL of each concentration (concentration range 2-
512 µg/mL) of the Gem n-s-n surfactant was added to 1 mL of sterilized nutrient broth in a
hemolysis tube then inoculated with 20 µL of standardized microbial inoculum. A negative
control sample was made up without surfactant. Triplicate experiments were carried out for
each concentration. The MLC were determined from aliquots taken from MIC tubes by
inoculating three PCA (plate count agar) plates with 20 µL of the concentrations above the
MIC. Plates were incubated for 24 h at 37°C for antibacterial test and for 72 h at 25°C for
antifungal test.
3. Results and discussion.
3.1 Synthesis
Gemini surfactants with different spacer length (s = 2, 4, 6) and hydrophobic chain length (n =
8, 10, 12, 14, 16) were prepared according to the straightforward synthetic procedure
illustrated in scheme 1.
The general procedure is based on the quaternization of commercially available N,N,N′,N′-
tetramethylethylenediamine,
N,N,N′,N′-tetramethyl-1,4-butanediamine
or
N,N,N′,N′-
tetramethyl-1,6-hexanediamine with 2-bromo-N-(4-(alkyloxy) phenyl) acetamides (3a-e)
under reflux of diethyl ether during 3 days. These latter derivatives were prepared in four-
stage process starting from 4-aminophenol. The initial step consists of the reaction of 4-
aminophenol with acetic anhydride followed by the etherification with the suitable alkyl
bromide compound in the presence of potassium carbonate. The amine function of resulted
compounds is then deprotected under reflux of a concentrated hydrochloric acid solution. The
reaction of 4-(alkyloxy)benzenamine (2a-e) with bromoacetyl chloride in the presence of an
aqueous solution of potassium carbonate at room temperature affords the 2-bromo-N-(4-
(alkyloxy) phenyl) acetamides intermediates (3a-e) with almost quantitative yields.
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After quaternization reaction, the gemini surfactants were recovered by filtration then
recrystallized from ethanol to give the final products in good yields and high purity. The
surfactants were characterized by 1H and 13C NMR and high resolution mass spectrometry.
Br
Br
NHCOCH3
NHCOCH2Br
NH2
NH2
N
N
(CH2)s
iv
i
ii
iii
O
O
HN
NH
OCnH2n+1
OCnH2n+1
OH
OCnH2n+1
3a-e
1a-e
2a-e
OCnH2n+1
OCnH2n+1
Gem n-s-n
n = 8,10,12,14,16
s = 2, 4, 6
Scheme 1. Synthetic pathway of gemini surfactants Gem n-s-n 1. (i) 1. Acetic anhydride,
H2O, 110°C, 2. CnH2n+1Br (n= 8, 10, 12, 14, 16), K2CO3, butanone, reflux; (ii) HCl, H2O,
reflux; (iii) BrCH2COCl, rt; (iv) (CH3)2N(CH2)sN(CH3)2 with s = 2, 4, 6, Et2O, reflux.
3.2. Surface activity
In the aim to investigate the surface activity of synthesized gemini surfactants and highlight
the effect of their molecular structure on the adsorption behavior, we determine the
equilibrium surface tensions as function of surfactant concentrations using the Wilhelmy plate
method. Surface tension (γ) versus the logarithm of the concentration of Gem n-2-n series
plots are shown, as an example, in figure 1. The breakpoint of the curves can be correlated to
critical micelle concentrations. The absence of a minimum around these points indicates the
purity of surfactants. The cmc and γcmc values of Gem n-s-n surfactants are collected in table1.
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Table 1.Surface activity parameters of gemini surfactants Gem n-s-n
cmc a
(mol/L)
γcmc
(mN/m)
cmcb
(mol/L)
cmc/C20
휞max
Amin
∆푮ퟎ풎풊풄
(kJ/mol)
Surfactant
β
pC20
(nm2)
(µmol/m2)
2.51 x 10-4
2.08 x 10-4
1.09 x 10-4
4.07 x 10-5
2.07 x 10-5
1.09 x 10-5
3.71 x 10-5
1.42 x 10-5
1.04 x 10-5
2.08 x 10-5
1.18 x 10-5
2.75 x 10-5
1.40 x 10-5
9.00 x 10-6
2.88 x 10-5
0.81
0.69
0.70
0.75
0.57
0.50
0.51
0.57
0.53
0.44
0.51
0.56
/
-39.94
-36.95
-38.85
-43.22
-37.22
-38.26
-35.76
-39.09
-40.26
-34.04
-36.52
-38.23
/
4.60
4.67
4.92
5.53
5.31
5.02
4.69
4.97
5.06
4.8
9.99
9.73
9.07
1.38
4.23
1.15
1.82
1.33
1.19
1.31
1.10
1.99
3.13
2.78
2.13
1.11
1.14
0.99
1.13
1.06
1.22
1.80
1.65
2.01
2.36
1.96
1.81
0.87
1.24
1.72
1.49
1.46
1.66
1.47
1.57
1.36
0.92
1.01
0.83
0.70
0.84
0.92
1.91
1.34
0.97
30.4
32.0
36.0
33.1
41.6
45.0
44.6
44.8
49.0
47.0
46.0
51.1
46.0
48.0
45.0
3.22 x 10-4
2.13 x 10-4
1.17 x 10-4
4.82 x 10-5
4.37 x 10-5
2.04 x 10-5
3.44 x 10-5
2.16 x 10-5
8.29 x 10-6
2.34 x10-5
2.51 x 10-5
2.50 x 10-5
/
Gem 8-2-8
Gem 8-4-8
Gem 8-6-8
Gem 10-2-10
Gem 10-4-10
Gem 10-6-10
Gem 12-2-12
Gem 12-4-12
Gem 12-6-12
Gem 14-2-14
Gem 14-4-14
Gem 14-6-14
Gem 16-2-16
Gem 16-4-16
Gem 16-6-16
4.97
4.86
5.35
5.49
4.87
1.98 x 10-5
2.34 x 10-5
0.47
0.47
-35.67
-35.42
a cmc from surface tension, b cmc from conductivity
As expected, all the investigated bisammonium surfactants displayed a micellization process
in water with a very low cmc values in the range of (0.21-0.009 mM) much lower than
conventional surfactants as dedecyltrimethyl ammonium bromide DTAB (0.846 mmol [40])
and dodecyl N-ethanamine N,N,N-trimethyl ammonium bromide C12H25-NHCOCH2N+(CH3)3
Br- (2.9 mM [38]). This indicates that gemini surfactants have a stronger tendency to self-
assemble in aqueous solution at lower concentrations than single chain monomeric surfactants
probably because of the increase of hydrophobic interactions. This trend is in good agreement
with those reported for many Gemini surfactants.
The positive effect of introduction of a rigid oxyphenyl group on the terminal hydrophobic
chain can be clearly highlighted by comparing the micellization data of previously reported
gemini ammonium surfactant analogous bearing amide functions and flexible dodecylalkyl
hydrophobic terminal chains C12H25-NHCOCH2N+(CH3)2(CH2)s-N+(CH3)2CH2CONHC12H25
(CMC = 0.30, 0.23, 0.19, 0.14, 0.11 mM for s = 2, 4, 6, 8 and 12 respectively [38]) with
newly synthesized Gemini surfactants Gem 12-2-12, Gem 12-4-12 and Gem 12-6-12 with 4-
dodedecylphenyl chains (0.0371, 0.0142, 0.0104 mM). The latter surfactants exhibit 8-10
times lower CMC values indicating their outstanding surface activity.
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These results suggest that the inclusion of a phenylene unit on the hydrophobic chain favored
micellization and can be explained from several factors. The benzene ring can acts as: a) a
hydrophobic unit since it is commonly admitted that a benzene group is roughly equivalent in
its effect on the cmc to three and a half methylene groups [41]; b) a rigid bulky spacer
connecting the terminal alkyl chain and the ionic head thereby affecting the orientation of the
hydrophobic tail and the self-association of surfactant molecules; c) it can also induce π-π
interactions among the adjacent molecules which should be a favorable factor for aggregation
of surfactant molecules.
This trend is in good agreement with that reported for conventional quaternary ammonium
surfactants containing a phenylene unit within a long alkyl hydrophobic chain. De et al.
showed that the formation and the structure of micelles are less hindered by a phenyl group
located near the head group than in a terminal position of the hydrophobic tail [42]. In this
latter case, smaller aggregates with significantly fewer surfactants were formed due the
coalescence of the alkylene segments. Similar results were obtained by Li et al. for
poly(oxyethylene) glycol alkyl ethers nonionic surfactants with a benzene ring in the
hydrophobic chains [43].
A progressive fall in cmc values of investigated gemini surfactants was noted in accordance
with the lengthening of the alkyl chain (n) and to lesser extent with the spacer chain (s) (figure
2). This is likely due to the enhancement of hydrophobicity with the number of methylene
group in the chains which makes the aggregation easier for surfactant molecules in the bulk
solution.
A distinct behavior was noticed for compounds Gem14-6-14 and Gem 16-6-16 bearing the
longer spacer chain (s = 6) and hydrophobic chain (n = 14 and 16) lengths. Higher cmc values
were reached by these compounds indicating a minimum stability of the surfactant in the
micellar state. Nevertheless, the difference between these two gemini surfactants with varying
lengths of the hydrophobic chain (tetradecyl and hexadecyl) was small.
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Fig.1 Surface tension curves of Gem n-2-n surfactants with n = 8, 10,12,14,16
0.25
0.20
s=2
s=4
s=6
0.15
0.10
0.04
0.03
0.02
0.01
0.00
8
10
12
n
14
16
Fig. 2 Variation of the cmc with the hydrocarbon carbon number n for Gem n-2-n, Gem n-4-n and
Gem n-6-n surfactants.
An important parameter to evaluate the surface activity of surfactants is the packing density of
surfactant molecules at the air-water interface. The maximum surface excess concentration
(Γmax) and the minimum surface area per molecule (Amin) at the air-water interface were
calculated using the Gibbs adsorption equations (1) and (2) [44].
ퟏ
흏휸
Гmax = − (풏×ퟐ.ퟑퟎ푹푻) (흏풍풐품푪
)
(1)
푻
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ퟏퟔ
ퟏퟎ
Amin=
(2)
Г
×푵
푨
풎풂풙
흏휸
where γ is the surface tension, (흏풍풐품푪)푻 : is the slope of the descending section of the surface
tension isotherm at the cmc, T is the absolute temperature in K, R is the gas constant (R =
8.314 J mol-1K-1), NA is Avogadro’s number, The prefactor n is related to the number of
species at the air-water interface. For gemini surfactant n is generally taken as 3 by
considering a divalent surfactant ion and two univalent counterions.
The values of Гmax and Amin are shown in table 1. It comes out that regardless the length of the
spacer, the Amin values of Gem n-s-n surfactants goes through a minimum for n= 12-14 then
increases abruptly for longer chain (figure 3). Most likely, the initial decrease of Amin reflects
that gemini surfactants with longer hydrophobic tails have higher packing densities at the air-
water interface. It is interesting to notice that the variation of the spacer length does not affect
the Amin to greater extent. This trend is different than that commonly observed for gemini
surfactants [45]. It seems that the presence of a rigid phenyl group on the hydrophobic chain
may assist the orientation of the hydrophobic chain toward the air-water interface leading to a
more tightly packing of surfactant molecules at the air-water interface. The existence of π-π
interaction among the adjacent phenyl groups of Gem n-s-n molecules may be the main
reason for the more compact aggregate structure [43].The presence of amide function that
links the ammonium headgroup with the alkylphenyl hydrophobic chain should probably also
facilities the intermolecular association among surfactant molecules through hydrogen
bonding interactions in self-organized assemblies [46].
For longer hydrophobic chain (n > 14 for s = 2, 4 and n >12 for s = 6), Amin increases abruptly
particularly for shorter spacer length (Amin (Gem 14-2-14) =0.70 nm2, Amin (Gem 16-2-16) =
1.91 nm2). This variation in minimum surface area per molecule values (Amin) indicates that
too long hydrophobic tail prevents the orientation of hydrophobic chains into the air. It is
noteworthy that the values of Amin increases only slightly for hexamethylene spacer s = 6
(Amin (Gem 14-6-14) = 0.92 nm2, Amin (Gem 16-6-16) = 0.97 nm2). This result suggests that
for hexedecyl spacer, a compact packing of surfactant molecules is maintained at the air-water
interface even for long hydrophobic tail. Such “scenario” can be achieved if the spacer adopts
a folder conformation toward the air side according to the conformation of surfactant
molecules represented in scheme 2.
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2.0
s=2
s=4
s=6
1.8
1.6
1.4
1.2
1.0
0.8
0.6
8
10
12
n
14
16
Fig. 3 Variation of Amin with the hydrocarbon carbon number n for Gem n-2-n, Gem n-4-n and Gem
n-6-n surfactants
Scheme 2. Plausible conformation of gemini surfactants: a) short spacer, n=8-12, b) short spacer, n=
14-16, c) long spacer (s = 6), n = 8-16
60
56
52
48
44
40
36
32
s=2
s=4
s=6
28
24
20
8
10
12
n
14
16
Fig. 4 Variation of cmc with the hydrocarbon carbon number n for Gem n-2-n, Gem n-4-n and Gem n-
6-n surfactants.
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10
s=2
s=4
s=6
8
6
4
2
0
8
10
12
n
14
16
Fig. 5 Variation of cmc/C20 ratios with the hydrocarbon carbon number n for Gem n-2-n, Gem n-4-n
and Gem n-6-n surfactants.
The performance of a surfactant in lowering the surface tension  of a solution was
investigated in terms of surface tension at cmc (cmc) and the adsorption efficiency (pC20) from
the surface tension isotherm. The pC20 corresponds to the logarithm of the surfactant
concentration C20 required to produce 20 mN.m-1 reduction in surface tension of water. The
pC20 values were evaluated by using equation 3 [47]:
pC20 = -log C20 (3)
The cmc values of gemini surfactants were found to increase with increasing length of the
hydrophobic chain (n) and tend to gentle at n = 12-14 (figure 4). The effect of the spacer is
less noticeable although with a slight increase of surface tension values with spacer chain
length (s).
The cmc/C20 ratio is considered as a convenient measure of the relative effects of structural
factors on the micellization and adsorption processes. The larger the cmc/C20 values, the more
efficiently the surfactant is adsorbed at the air-water interface at the expense of the
micellization process and the more efficiently it reduces surface tension. The cmc/C20 ratios
indicate that compounds with octyl alkyl chain have a greater preference for adsorption than
for micellization (figure 5).
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3.3. Conductivity measurements and thermodynamic of micellization
The critical micellar concentrations have also be evaluated by conductivity measurements.
Figure 6 shows an example of the variation in conductivity with concentration for Gem 8-4-8
surfactant. The cmc values were determined from the break points in the curves of specific
conductivity (K) versus gemini surfactant concentration (C) and are listed in table 1. One can
see that the cmc values obtained by electrical conductivity method are mostly slightly higher
than those determined by tensiometry. Nevertheless, similar decreasing trend of cmc values
with the elongation of the hydrophobic alkyl chain were obtained with distinct behavior of
compounds Gem14-6-14 and Gem 16-6-16 (see figure S1, supplementary file). Several
studies have shown that the cmc values may differ significantly when evaluated by different
techniques and was attributed to the formation of non-surface-active premicellar aggregates of
surfactant in aqueous solutions [44].
Fig. 6 Specific conductivity as a function of concentration for surfactant Gem 8-4-8
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Fig.7 Variation of β values with the hydrocarbon carbon number n for Gem n-2-n, Gem n-4-n and
Gem n-6-n surfactants
-34
-36
-38
-40
s=2
s=4
-42
s=6
-44
8
10
12
n
14
16
Fig.8 Variation of The Gibbs free energies of micellization (∆Gm0 ic) with the hydrocarbon carbon
number n for Gem n-2-n, Gem n-4-n and Gem n-6-n surfactants
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The degree of counterion binding to micelles (β) is important parameter that governs the
stability of micelles. It can be estimated according to the formula α+β = 1 where α is The
ionization coefficient obtained from the ratio of the slope values above and below the CMC
obtained from the electrical conductivity measurements (conductance vs. concentration). The
main approximation underlying this expression is that a micellized surfactant whose electrical
charge is not compensated by a bound counterion contributes to the conductivity of the
solution with the same amount as if it is free [48].
From table 1 and figure 7, one can be seen that β values decreases with the increasing of
hydrophobic chain length (n) particularly for the gemini surfactant series with a short spacer
length Gem n-2-n. The decrease in counterion binding indicates a lower surface charge
density at the micelle-solution interface for the compounds with longer hydrophobic tails.
This variation may be ascribed to a reduction in aggregation number of micelles or the
increase of micelle size and/or shape [48]. This trend was evidenced from DLS measurements
reported in the subsequent section.
The Gibbs free energy of micellization for gemini surfactants (∆Gm0 ic) is associated with the
transfer of one mole of surfactant from the aqueous phase to the micellar pseudophase. It was
calculated using Equation (4) [44]:
0
(
) (
)
∆Gmic = RT 0.5 + β ln 푋푐푚푐
(ퟒ)
Where R is the gaz constant (8.314 J mol-1 K-1); T is absolute temperature; β is the degree of
counterion binding; 푋푐푚푐 is the cmc molar fraction, 푋푐푚푐 = cmc/55.4 where cmc is in mol L-1
and 55.4 comes from 1L of water corresponding to 55.4 mol of water at 25°C.
The values of micellization free energy calculated for the studied gemini surfactants are
tabulated in table 1. It can be seen that all ∆Gm0 ic values are negative indicating that the
micellization is a thermodynamically spontaneous process. Compound Gem 10-2-10 exhibit
the lowest micellization free energy values reflecting it high tendency to form micelles.
The plot of ∆Gm0 ic versus the hydrophobic carbon number (n) shows two regimens (Figure 8).
∆Gm0 ic goes through a minimum in the range of n = 10-12 then increased gradually. This
variation in micellization free energies indicates that the increase of hydrophobic chain length
promotes the micellar growth for n  10-12 due to the strengthening on the hydrophobic
interaction whereas becomes unfavorable upon further increasing n.
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The length of the spacer length seems to affects also the micellization process. Interestingly,
Gemini surfactants with ethylene spacer (s=2) are more conducive to micellization for n  10.
The opposite trend was obtained for n = 12 to 16, i.e. the homologous surfactants with longer
spacer (s=6) exhibit the lowest ∆Gm0 ic values.
There are several factors that contribute on micellization process mainly electrostatic
repulsion between heads groups, attractive interactions between headgroup and bounded
counterion, steric hindrance, hydrophobic effect of chains and spacers and π-π interaction
among adjacent phenyl groups. For short spacer and hydrophobic chain, electrostatic
repulsion between heads groups and π-π interaction among adjacent phenyl groups must
dominate favoring micellization. As n increases, these interactions are weakened in favor of
steric hindrance whence the increases of Amin previously argued. As a result, the micellar
growth is hindered.
A different behavior is expected for long spacer. Because hexamethylene spacer (s=6) is more
flexible and hydrophobic in nature, it can adopts a folded conformation and become
incorporated into the hydrophobic micelle core; the hydrophobic effect becomes dominant
and favor the micellization process. It is noteworthy that for long alkyl chain (n > 12), the
orientation of alkyl chain toward water-air interface should be disturbed (Amin of Gem n-6-n
increases slightly for n varying from 12-16), as a consequence surfactant molecule has more
difficulty in aggregation. These findings go well in agreement with the CMC values.
3.4 Size of the aggregates
The size of the aggregates formed by investigated surfactants in aqueous solution has been
investigated above the cmc value by dynamic light scattering (DLS) measurements. Figure 9
shows the size distribution of gemini surfactants according to the spacer and hydrocarbon
chain lengths. One can be see that the varying terminal alkyl chain length or the spacer length
has a systematic effect on the size of studied samples.
Surfactants bearing C8H17, C10H21 and C12H25 hydrocarbon tail and a short to middle-length
spacer (s = 2, 4) provide mostly a bimodal distribution containing two peaks with an average
hydrodynamic diameter of 73.9-151 nm and 4- 19.8 nm, respectively. The aggregate sizes
increased with progressive increase of the alkyl chain length and spacer length.
The smallest population is expected to be related to micelle organization while the larger
aggregates suggest a spontaneous organization in vesicles. The coexistence of small micelles
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with large vesicles was already reported for other gemini surfactants [49]. The introduction of
an amide function on the molecular structure of surfactants is known to promote the formation
of bilayer vesicles due to the Intermolecular H-bonding between N-H and C=O functions
among neighboring molecules [50]. These H-bonding interactions are able to minimize the
repulsive interactions among cationic head groups leading to highly stable bilayer
organizations.
Compounds Gem 8-2-8 and Gem 10-2-10 show an additional population of bigger aggregates
in the range of 337 nm and 436 nm, respectively.
It is interesting to note that the aggregation profile of compounds evolves for longer spacer
length (s=6) towards a monomodal distribution with one obvious peak corresponding to an
average apparent hydrodynamic radius of about 186-330 nm.
Fig.9 Aggregates size distribution diameter of gemini surfactants from DLS analysis
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3.5 Antibacterial and antifungal activity
The antimicrobial activity of Gem n-s-n surfactants was tested against three microorganisms:
A Gram-positive bacteria (Staphylococcus aureus), a Gram-negative bacteria (Escherichia
coli) and a yeast (Candida albicans). The activity was evaluated by the minimum inhibitory
concentrations (MIC) and the minimal lethal concentrations (MLC). The MIC data reported in
table 2 show that Gem n-s-n are more active toward Gram-positive tested bacteria strain than
for Gram-negative one and the fungal strain. It is generally admitted that the quaternary
ammonium surfactants are membrane active agents and that their antimicrobial mechanism is
based on cytoplasmic membranes disturbance by combined hydrophobic and electrostatic
interactions [51]. In fact, the Gram-positive bacteria layer is composed of porous
peptidoglycan (complex polysaccharide) and a single underline phospholipid bilayer, while
Gram-negative bacteria have a double phospholipid bilayer sandwiching a thinner
peptidoglycan layer. Furthermore, their phospholipid outer bilayer membrane is connected
and surrounded by an extra hydrophilic lipopolysaccharide outer membrane which restricts
the entrance of lipophilic small molecules and biocides [52]. The cell wall of yeasts shows
intermediate Gram-positive and Gram-negative bacteria permeability. Therefore, the low
sensibility of Gram-negative and yeast microorganisms to gemini surfactants is related to the
composition and the organization of their outer layers.
The surfactants with tetradecyl and hexadecyl chain are inactive on the three tested
microorganisms up to the concentration of 512 μg/mL. Antibacterial activity depression may
be due to the poor water solubility of Gem 14-s-14 and Gem 16-s-14 derivatives and the
formation of viscous solutions that inhibit the transfer through the microbial cell membranes
[34,53].
MIC results show that increasing the hydrophobic chain length from 8 to 12 carbon atoms
promotes Staphylococcus aureus tested strain growth inhibition. This can be caused by the
surfactants higher hydrophobic nature, which stimulates interactions with the inner core of the
bacterial cell membrane [38, 54]. The role of the spacer on the activity of investigated gemini
surfactants is not so obvious. An optimum hydrophilic/hydrophobic balance should be a key
parameter that governs the bacterial activity of quaternary ammonium surfactants. Among all
of the investigated gemini surfactants, compound Gem 12-4-12 showed the maximal
antibacterial activity at 17 μM against Staphylococcus aureus. This value is significantly
lower than that reported for conventional dodecyltrimethylammonium bromide DTAB (MIC
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= 44.8 μM [55]) or for monomeric ammonium surfactant analogous containing an amide
function (MIC of dodecyl N-ethanamide N,N,N-trimethylammonium bromide is 43 μM [38]).
The presence of two positive charges and two hydrophobic chains in the dimeric surfactants
could exacerbate their adsorption on the interface of the negatively charged cell membrane
through electrostatic and hydrophobic interactions compared to monomeric quaternary
ammonium surfactants [34].
When comparing the MLC values of investigated gemini surfactant, one finds that compound
Gem 10-6-10 present the strongest lethal activity against both Gram-positive and Gram-
negative bacteria. The majority of here-investigated surfactants have a bacteriostatic activity
since they exhibit significant minimal bactericidal concentrations.
Table 2. Antibacterial activities of active gemini surfactants.
Surfactant
MIC (μmol/L)[MLC(μmol/L)]a
E. coli
S. aureus
159.8 [-]b
C. albicans
159.8 [319.7]
617.8 [-]b
639.4 [-]b
Gem 8-2-8
Gem 8-4-8
Gem 8-6-8
Gem 10-2-10
Gem 10-4-10
Gem 10-6-10
Gem 12-4-12
301.6 [-]b
301.6 [-]b
37.34 [597.5]
74.69 [597.5]
72.32 [144.6]
70.15 [140.3]
17.0 [544.1]
291.8 [-]b
149.4 [149.4]
74.7 [-]b
597.5 [-]b
144.6 [-]b
72.3 [-]b
561.2 [561.2]
544.1 [544.1]
140.3 [-]b
544.1 [-]b
a The MIC values of Gem 12-2-12, Gem 12-6-12, Gem 14-s-14 and Gem 16-s-16 with
s=2,4,6 are higher than 520 μg/L
b The MLC is above the detection limit
4. Conclusion
In summary, a novel series of gemini surfactants bearing 4-alkoxyphenyl hydrophobic chains,
amide connector groups linking these tails to the ammonium heads and different methylene
1
spacer lengths have been synthesized and characterized by H and 13C NMR and high
resolution mass spectrometry (HRMS). These surfactants were investigated with respect to
surface active properties and biological activities. The present work revealed the positive
contribution of the benzene ring, present in the hydrophobic chain, on the micellization
behavior of investigated surfactants. Lower cmc values were reached compared to
conventional gemini quaternary ammonium surfactants having alkyl hydrophobic chains and
similar spacer length. π-π interactions among benzene rings in hydrophobic chains should
assist favorable packing of surfactants molecules. The gemini surfactants with hexamethylene
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spacer were found to pack very tightly at the air-water interface. This behavior is most likely
due to the tendency of the spacer to adopt a folder conformation towards the hydrophobic
phase allowing to maintain the interaction of benzene rings and amide functions of two
neighboring molecules to the detriment of the electrostatic repulsion of the cationic head
groups. DLS measurements showed that the size of aggregates increases with progressive
lengthening of the hydrophobic and spacer chains for investigated amphiphiles. Concerning
the biocide properties, only the geminis with octyl, decyl and dodecyl chains (n= 8, 10, 12)
showed an activity towards the pathogenic E. coli, S aureus and C. albican microorganisms.
An optimum hydrophilic/hydrophobic balance and a good water solubility of surfactants are
key parameters that enable a better interaction with bacteria cell surface. Owing to their
superior surface properties and good antibacterial activity, such derivatives might be good
candidates for wastewater and surface treatments.
Acknowledgments. Authors are thankful to A. Berrabah, S.S. Daho and S. Houalef for help
with antibacterial tests.
Author contributions: SS-B designed the research, KT synthesized the compounds and
performed the charaterizations and the surface properties study, MMB and NB performed the
antibacterial study, DLS study was carried out at HG laboratory, SS-B and KT interpreted the
experiments and wrote the paper.
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