Synthesis, surface adsorption, micellization behavior and antibacterial activity of novel gemini surfactants with morpholinium headgroup and benzene-based spacer

Article abstract of DOI:10.1016/j.molliq.2021.115781

Two series of novel gemini surfactants with morpholinium headgroup and benzene-based rigid spacers (abbreviated as (Mor)m-P-m and (Mor)m-BP-m, where m represents the carbon numbers of hydrophobic chains) were synthesized and characterized by ¹H NMR, ESI-MS, and FT-IR spectra. The effect of lengths of rigid spacer and hydrocarbon chain on their solution properties and antibacterial activities were systematically investigated. Due to the existence of morpholinium, they have a superior surface activity to the classic gemini surfactants. (Mor)m-BP-m possess lower CMC and γ_cmc than (Mor)m-P-m, which is assumed to be related to the conformation change of spacer. The possible arrangement models for the adsorptions of (Mor)m-P-m and (Mor)m-BP-m at the air-water interface were proposed by comparing their surface parameters with those of other similar surfactants. The pre-micellar associations occur in aqueous solutions of (Mor)16-P-16, (Mor)14-BP-14 and (Mor)16-BP-16. The thermodynamic parameters indicate the micellar processes are spontaneous, and (Mor)m-BP-m have stronger aggregation tendency than (Mor)m-P-m. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) show they spontaneously self-assemble into vesicle, and the aggregate size of (Mor)m-BP-m is larger than (Mor)m-P-m. These surfactants showed excellent antibacterial properties against Bacillus subtilis and Escherichia coli. Compared with (Mor)m-P-m, (Mor)m-BP-m exhibit a higher activity.

Full text of DOI:10.1016/j.molliq.2021.115781

Journal of Molecular Liquids 331 (2021) 115781
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Synthesis, surface adsorption, micellization behavior and antibacterial
activity of novel gemini surfactants with morpholinium headgroup and
benzene-based spacer
Le-Chi Zheng, Qing-Xiao Tong ⁎
Department of Chemistry and Key Laboratory for Preparation and Application of Ordered Structural Material of Guangdong Province, Shantou University, Guangdong 515063, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 October 2020
Received in revised form 18 January 2021
Accepted 24 February 2021
Available online 26 February 2021
Two series of novel gemini surfactants with morpholinium headgroup and benzene-based rigid spacers (abbre-
viated as (Mor)m-P-m and (Mor)m-BP-m, where m represents the carbon numbers of hydrophobic chains) were
synthesized and characterized by H NMR, ESI-MS, and FT-IR spectra. The effect of lengths of rigid spacer and hy-
1
drocarbon chain on their solution properties and antibacterial activities were systematically investigated. Due to
the existence of morpholinium, they have a superior surface activity to the classic gemini surfactants. (Mor)m-
BP-m possess lower CMC and γcmc than (Mor)m-P-m, which is assumed to be related to the conformation change
of spacer. The possible arrangement models for the adsorptions of (Mor)m-P-m and (Mor)m-BP-m at the air-
water interface were proposed by comparing their surface parameters with those of other similar surfactants.
The pre-micellar associations occur in aqueous solutions of (Mor)16-P-16, (Mor)14-BP-14 and (Mor)16-BP-16.
The thermodynamic parameters indicate the micellar processes are spontaneous, and (Mor)m-BP-m have stron-
ger aggregation tendency than (Mor)m-P-m. Dynamic light scattering (DLS) and transmission electron micros-
copy (TEM) show they spontaneously self-assemble into vesicle, and the aggregate size of (Mor)m-BP-m is
larger than (Mor)m-P-m. These surfactants showed excellent antibacterial properties against Bacillus subtilis
and Escherichia coli. Compared with (Mor)m-P-m, (Mor)m-BP-m exhibit a higher activity.
Keywords:
Gemini surfactant
Morpholinium headgroup
Rigid spacer
Aggregation behavior
Surface activity
Antibacterial property
©
2021 Elsevier B.V. All rights reserved.
1
. Introduction
spacer (p-benzenedio) showed rather normal aggregation behavior,
but for that with a long spacer (p-dibenzenediol), its hydrophobic
chains adopted an unusual conformation and unexpected network
structure aggregates were obtained in the solution.
Gemini surfactant is a new class of surfactant, which consists of two
hydrophilic headgroups and two hydrophobic chains connected cova-
lently by a space near, or at the level of the headgroups [1,2]. Compared
with traditional surfactants with only one hydrophobic chain and one
hydrophilic group, gemini surfactants have many advantages such as
higher surface activity, lower critical micelle concentration (CMC) and
stronger adsorption capacity at the interface, which are widely
employed in tertiary oil recovery, metal anticorrosion and sterilization
Biphenyl is a common rigid group. Antimicrobial preparations based
on biphenyl derivatives have a wide range of medicine and agriculture
applications, but their low water solubility hinders the wide practical
application [15]. Quaternary ammonium compounds are commonly
used as bactericides owing to their bactericidal activities. However,
after long-term use of the known antimicrobial agents, microbial strains
are prone to produce resistance, which necessitates the constant search
for new compounds with antibacterial properties. Gemini surfactants
containing biphenyl group, which have good water solubility and
thermo-stability, could be effective antimicrobial agents in agriculture
and industry.
Most studies about gemini surfactants have been focused on the cat-
ionic category alkanediyl-a-β-bis(alkyl dimethyl ammonium)
dibromide, denoted as m-s-m, where m and s stand for the number of
carbon atoms in the hydrophobic alkyl chain and methylene spacer re-
spectively [11]. To further improve the physicochemical properties of
m-s-m, researchers have made a lot of attempts and efforts, which
brings about some novel surfactants known as the heterocyclic (such
[3–6].
The softness and length of spacer have a profound impact on the so-
lution properties of gemini surfactants [7]. The previous reports show
that gemini surfactants with flexible spacers tend to form spherical or
elongated micelles [8–10]. But gemini surfactants with rigid spacers
are more likely to form pre-micelles and larger aggregations than mi-
celles, and their tails lie flat at the interface [11–13]. Zhao et al. [14] stud-
ied the aggregation behavior of gemini surfactants containing rigid
spacers of different lengths. They found the molecule with a short
⁎
Corresponding author.
E-mail address: qxtong@stu.edu.cn (Q.-X. Tong).
https://doi.org/10.1016/j.molliq.2021.115781
167-7322/© 2021 Elsevier B.V. All rights reserved.
0

L.-C. Zheng and Q.-X. Tong
Journal of Molecular Liquids 331 (2021) 115781
as pyridinium [16–20], pyrrolidinium [21–24], imidazolium [25–28]
and morpholinium [29,30]) gemini surfactants. Compared with classic
m-s-m type surfactants, these gemini surfactants have higher surface
activity and stronger aggregation ability due to the introduction of het-
erocycles [21,26,30,31]. Among the heterocycles, morpholine is distinc-
tive because of the existence of an oxygen atom [30]. For one thing, the
oxygen atom in morpholine can change the positive charge distribution
of the headgroup, which has a certain effect on the interaction between
the headgroups. For another, the presence of oxygen atom can result in
some weak interaction (such as hydrogen bond) between surfactant
and water. Morpholinium gemini surfactants with a flexible spacer
were reported [30,32], but there is no work about morpholinium gemini
surfactants with a rigid spacer.
In this work, we synthesized two series of novel morpholinium gem-
ini surfactants with phenylene (short) and biphenylene (long) rigid
spacer (Scheme 1). The effect of the lengths of spacer and hydrocarbon
chain on their surface activities, thermodynamic properties, aggregation
behaviors and antibacterial properties were investigated, which is con-
ducive to extend their potential applications such as wastewater treat-
ments [33], the synthesis of nanoparticles, nanorods and mesoporous
materials [11,34]. The surface parameters were also compared with
those of the similar gemini surfactants m-P-m [35] and (Mor)m-6-m
2.2. Surface tension measurement
The surface tension was carried out on an automatic A101 tensiom-
eter (Fangrui, China) by the Du Nouy ring method at 25 ± 0.1 °C. Before
the experiment, all the surfactant solutions were kept for 24 h to ensure
equilibrium status.
2.3. Electrical conductivity measurement
The electrical conductivities of the surfactant solutions were mea-
sured by a low-frequency conductivity analyzer (model DDS-307A,
Shanghai INESA Scientific Instrument Co., Ltd) at 15 ± 0.1 °C, 20 ±
0
.1 °C, 25 ± 0.1 °C, 30 ± 0.1 °C and 35 ± 0.1 °C respectively.
2.4. Steady-state fluorescence measurement
The fluorescence spectra of the surfactant solutions were carried out
by a Shimadzu RF-5300PC spectrofluorophotometer. The concentration
of fluorescence probe pyrene is 1 × 10 mol/L. The surfactant solutions
were excited at 335 nm wavelength and the emission spectra were
scanned from 350 to 450 nm wavelength. The excitation and emission
slit widths were 5 nm and 2 nm, respectively.
−6
[32] to illustrate the influence of the head group and spacer flexibility,
which is of fundamental importance in understanding the structure-
property relationship of gemini surfactants.
2.5. Dynamic light scattering (DLS) measurement
The DLS measurements were performed on a Malvern zeta sizer
Nano-zs 90 (Malvern, UK) with a scattering angle of 173° and He\\Ne
laser (λ = 632.8 nm) at room temperature. Every sample was measured
three times. The autocorrelation function was analyzed by the CONTIN
2
. Experimental
2.1. Materials
h
method to get a hydrodynamic radius (R ).
1
-bromodecane, 1-bromododecane, 1-bromotetradecane, 1-
bromohexadecane, morpholine, 1,4-bis(chloromethyl)-benzene, and
,4′-bis(chloromethyl)-1,1′-biphenyl were purchased from Energy
2.6. Transmission electron microscopy (TEM)
4
Chemical. Acetone, ethanol and ethyl acetate are all analytical grades.
Both the solvent and the reagents were used as received. Pyrene (En-
ergy Chemical 98%) was recrystallized from ethanol three times before
experiments.
The TEM micrographs were obtained using a JEM-F200 transmission
electron microscope (JEOL Ltd., Japan). All the samples were prepared
by the negative-staining method. Phosphotungstic acid (2%) was used
as the staining agent.
Scheme 1. Synthesis routes for (Mor)m-P-m and (Mor)m-BP-m (m = 10, 12, 14, 16)
2

L.-C. Zheng and Q.-X. Tong
Journal of Molecular Liquids 331 (2021) 115781
Fig. 1. The curves of the surface tension of (Mor)m-P-m (a) and (Mor)m-BP-m (b) at different concentrations.
Table 1
CMC values of the surfactants at 25 °C.
Surfactant
CMC (mM/L)
Surfactant
0-P-10
CMC (mM/L)
Surfactant
CMC (mM/L)
Surfactant
CMC (mM/L)
Surfactant
CMC (mM/L)
1
7.27
1.2
0.92
0.48
(Mor)10‐6-10
(Mor)12‐6-12
3.1
1.0
(Mor)10-P-10
(Mor)12-P-12
(Mor)14-P-14
(Mor)16-P-16
6.49
1.2
0.17
0.051
(Mor)10-BP-10
(Mor)12-BP-12
(Mor)14-BP-14
(Mor)16-BP-16
2.86
(
Mor)12
Mor)16
21.0
0.38
12-P-12
4-P-14
16-P-16
0.548
0.153
0.046
1
(
2.7. Thermal stability
The antibacterial rate η (%) was calculated from the following
Eq. [36]:
The thermaldynamic properties of the synthesized surfactants were
measured by thermal gravimetric analysis (TGA) under the nitrogen at-
mosphere at a heating rate of 10 °C/min.
A−B
A
ηð%Þ ¼
ꢀ 100
ð1Þ
where A and B were the average numbers of bacterial colonies without
and with surfactants respectively. Minimum inhibitory concentration
2.8. Antibacterial activity
(
MIC) was determined as the lowest concentration of the antibacterial
Gram-negative Escherichia coli (ATCC8739) and gram-positive Bacil-
agent with an antibacterial rate above 90% [36].
lus subtilis (ATCC6633) were used as the indicative bacteria to charac-
terize the antibacterial activity of the target surfactants by a plate
coating method.
3. Results and discussion
In a sterile environment, the bacterial strains were scraped into a
sterile liquid medium by an inoculation loop and the organisms were
shaken at 37 °C for 24 h to obtain the bacterial suspensions.
The synthesized surfactants were sterilized and dissolved in sterile
water to get the antibacterial solution. The antibacterial solution was
added to the bacterial suspensions and the mixed solution was incu-
bated by shaking at 37 °C for 1 h (the concentrations of bacteria were
3.1. Surface activity
The surface tension curves of (Mor)m-P-m and (Mor)m-BP-m
aqueous solutions are shown in Fig. 1. The CMC values of them and
7
7
approximately 1 × 10 –9 × 10 cfu/mL). The test organisms were coated
on sterile solid medium in the Petri dishes by 10 times dilution method
and cultured in an incubator at 37 °C for 24 h. Bacteria suspensions
without surfactants were also incubated for comparison. Two parallel
samples were measured for every organism. The average numbers of
bacterial colonies with and without surfactants were determined.
Table 2
Surface paraments of (Mor)m-P-m and (Mor)m-BP-m at 25 °C.
surfactant
CMC
mmol/L)
γ
cmc
pC20
Γ
Amin
(nm )
2
2
(
(mN/M)
(μmol/m )
(
(
(
(
(
(
(
(
Mor)10-P-10
Mor)12-P-12
Mor)14-P-14
Mor)16-P-16
Mor)10-BP-10
Mor)12-BP-12
Mor)14-BP-14
Mor)16-BP-16
6.487
1.312
0.171
0.051
2.859
0.548
0.153
0.046
39.90
38.24
42.45
49.06
35.99
35.40
41.67
35.96
2.56
3.35
4.18
4.49
3.30
4.29
4.53
5.70
1.63
1.31
1.29
0.94
1.32
0.99
0.86
0.68
1.02
1.26
1.29
1.77
1.26
1.68
1.93
2.44
Fig. 2. Values of Amin versus m of the gemini surfactants.
3

L.-C. Zheng and Q.-X. Tong
Journal of Molecular Liquids 331 (2021) 115781
the reference substances are listed in Table 1. (Mor)12-P-12 and
solution. However, that is not the case for (Mor)m-BP-m. In Table 1,
the CMC of (Mor)m-BP-m are smaller than (Mor)m-P-m and (Mor)m-
6-m, especially for m = 10 and 12, indicating the best aggregation abil-
ity of (Mor)m-BP-m in aqueous solution.
(
Mor)16-P-16 have lower CMC values than the corresponding
morpholinium surfactants with a single hydrophobic chain [37,38] de-
noted as (Mor)12 and (Mor)16, indicating the prominent aggregation
capacities of gemini surfactants. (Mor)m-P-m possess smaller CMC
than m-P-m, especially for m = 14 and 16, the CMC are about one
order of magnitude smaller, suggesting the surface activity was im-
proved by introducing morpholinium.
To better demonstrate the surface activities of (Mor)m-P-m and
(
Mor)m-BP-m, Amin and Γmax are calculated. Amin is defined as the
area occupied by a single molecule of surfactant at the air-water inter-
face, reflecting the stacking density of surfactant molecules. Amin can
We abbreviate the morpholinium gemini surfactant with n-
hexanediyl [32] as (Mor)m-6-m. The length of n-hexanediyl is almost
equal to that of phenylenediyl (5.5–6.0 methylene units [3]). The CMC
of (Mor)m-P-m are bigger than (Mor)m-6-m, which may be related to
the flexibility of the spacer. For the spacers with almost equivalent
length, the flexibility of n-hexanediyl makes the spacers easy to locate
at the aggregate-water interface, while the rigidity and steric hindrance
of phenylenediyl lead the spacers difficult to combine with the hydro-
philic outer layer of the micelle [13], resulting in the stronger aggrega-
tion tendency of (Mor)m-6-m. It seems that the rigid spacer is
unfavorable for the aggregation of gemini surfactants in aqueous
be obtained from the maximum surface excess concentration Γmax
which can be deduced from the Gibbs adsorption equation:
,
ꢀ
ꢁ
1
dγ
Γmax ¼ −
ð2Þ
ð3Þ
2:303nRT dlogC
T
1
24
Αmin ¼
ꢀ 10
N Γmax
A
where γ is the surface tension in mN/m; Γmax is the surface excess in
2
−1 −1
μmol/m ; R is the gas constant (8.314 J mol
K
); T is the absolute
Fig. 3. Schematic representations for the adsorption at the air-water interface (Mor)m-P-m (a) and (Mor)m-BP-m (b).
Fig. 4. The curves of specific conductivity versus concentration of (Mor)10-P-10 (a), (Mor)12-P-12 (b), (Mor)14-P-14 (c) and (Mor)16-P-16 (d) at different temperatures.
4

L.-C. Zheng and Q.-X. Tong
Journal of Molecular Liquids 331 (2021) 115781
temperature; C is the concentration of surfactant in mol/L; dγ/dlogC is
the slope of the linear part of the γ-logC curve where the concentration
of surfactant is lower than CMC. For gemini surfactant in aqueous solu-
(Mor)m-P-m orientate to the air, the chain density of (Mor)m-P-m
would be higher than (Mor)m-BP-m in the adsorption layer, and the
γ
CMC should be smaller. However, the results are contrary, which
might be ascribed to the structure of the spacer. We could infer the ar-
rangement of the hydrophobic chains of (Mor)m-P-m and (Mor)m-
BP-m at the air-water interface by comparing their Amin with those of
other similar gemini surfactants.
A
tions without addition counterion, n takes as 3. N is the Avogadro's
constant (6.02 × 10 mol ), and Amin is in nm .
2
3
−1
2
In Table 2, the CMC values of (Mor)m-P-m and (Mor)m-BP-m de-
crease obviously with m increasing from 10 to 16, indicating the stron-
ger aggregation ability for longer hydrophobic chains. Long alkyl chains
prefer to bend, leading to an increase of Amin and a decrease of Γmax. For
the same m, (Mor)m-BP-m have bigger Amin and smaller Γmax. The
length of biphenylene spacer is longer than phenylene spacer, which
leads to the further distance between two hydrophobic chains, bringing
Fig. 2 shows the effect of the carbon atoms number (m) in the hydro-
phobic chain on Amin (n = 3). The Amin of (Mor)10‐6-10 and (Mor)12‐
2
6
(
-12 are 0.593 and 0.611 nm respectively (black dots) [32]. The Amin of
2
Mor)10-P-10 and (Mor)12-P-12 are 1.02 and 1.26 nm (pink dots),
which are about double those of (Mor)10‐6-10 and (Mor)12‐6-12 re-
spectively. Such significant differences in the Amin among these gemini
surfactants having a similar spacer length proves that both tails of
about bigger Amin and smaller Γmax
.
From the γ-logC curve, we can also get two other parameters pC20
and γCMC. pC20 is defined as the negative logarithm of the surfactant
concentration required for reducing the surface tension value of water
by 20 mN/m. The larger the pC20 is, the higher the efficiency to reduce
surface tension will be. γCMC refers to the lowest surface tension of
water that the surfactant can reduce to. The smaller the γCMC are, the
stronger the ability to reduce the surface tension will be. The hydropho-
bicities of (Mor)m-P-m and (Mor)m-BP-m are enhanced with increas-
ing m, thus pC20 show an increasing trend. The γCMC of (Mor)m-BP-m
are smaller than (Mor)m-P-m, indicating the stronger ability of (Mor)
m-BP-m to reduce the surface tension, which may be related to the ar-
rangement of hydrophobic chains at the interface.
When the adsorption of surfactant at the air-water interface is satu-
rated, a nonpolar layer consists of hydrocarbon chains could be ob-
tained. A higher density of hydrophobic groups in the layer leads to
the smaller γCMC, and vice versa. Γmax of (Mor)m-P-m is bigger than
that of (Mor)m-BP-m, which means that the higher stacking density
of (Mor)m-P-m at the interface. If both the hydrophobic chains of
(
Mor)m-P-m are unlikely toward the air.
Ge et al. [13] reported a series of gemini surfactants with p-
xylylene spacer (denoted as m-PX-m, m = 10, 12, 14, 16) and the
min values of them are 1.28, 1.77, 2.29 and 3.4 nm2 respectively
red dots). Wang et al. [11] synthesized gemini surfactants containing
n-phenylacetamide in the spacer (regarded as m-PAM-m, m = 12, 14,
6) and their Amin values are 1.78, 2.03 and 2.56 nm2 respectively
blue dots). They found the presence of benzene increases the rigidity
A
(
1
(
of the spacer and hinders the change in the spacer conformation, mak-
ing the hydrophobic chains of m-PX-m and m-PAM-m lie at the inter-
face. Fig. 2 demonstrates that there is little diversity in Amin between
m-PX-m and m-PAM-m. Thus, Amin values of gemini surfactants
with a rigid spacer are relatively big, and their hydrophobic chains
tend to lie at the air-water interface. In Fig. 2, Amin of (Mor)m-P-m
(
(
pink dots) are far smaller than m-PX-m (red dots) and m-PAM-m
blue dots), indicating that both tails of (Mor)m-P-m are impossible
to lie at the interface.
Fig. 5. The curves of specific conductivity versus concentration of (Mor)10-BP-10 (a), (Mor)12-BP-12 (b), (Mor)14-BP-14 (c) and (Mor)16-BP-16 (d) at different temperatures.
5

L.-C. Zheng and Q.-X. Tong
Journal of Molecular Liquids 331 (2021) 115781
Based on the above analysis, for the saturated adsorption of gemini
surfactants at the interface, their Amin values are relatively small when
the tails are oriented to the air and relatively big when the tails lie at
the interface. From Fig. 2, the Amin of (Mor)m-P-m are between these
two cases. Therefore, the hydrophobic chains of (Mor)m-P-m are prob-
able to locate at the air-water interface adopting a mixed arrangement
of being orientated to the air and lying at the surface due to the rigidity
and steric effect of phenylene spacers (shown in Fig. 3a).
The spacer lengths of (Mor)m-BP-m (biphenylene) are longer than m-
PX-m and m-PAM-m. If the tails of (Mor)m-BP-m lie at the interface, Amin
of (Mor)m-BP-m will be bigger than m-PX-m and m-PAM-m. Neverthe-
less, the Amin values are even slightly smaller (green dots in Fig. 2), indi-
cating that the tails should not lie flat at the interface. The C\\C single
bond between two benzene rings of biphenylene is expected to enable
these benzene rings to rotate. What is more, the longer spacer can
weaken the interaction between two alkyl chains [7]. Therefore, both
the tails of (Mor)m-BP-m would be unfolded and orientated to the air
through the conformation change of spacer, leading to the higher density
of hydrophobic chains in the adsorption layer (as shown in Fig. 3b). As a
result, the γCMC of (Mor)m-BP-m is lower than (Mor)m-P-m.
turning point shows the CMC. The counterion binding (β) can be calcu-
lated through the following formula:
β ¼ 1−k2=k1
where k and k
ð4Þ
1
2
are the pre-micellar slope and post-micellar slope,
respectively.
The values of β and CMC obtained by the conductivity method are
listed in Table 3. The counterions are combined on the surface of mi-
celles by the electrostatic attraction. The electrostatic attraction is
weak at a high temperature, thus the β values decrease with increasing
temperatures [39]. The impacts of temperature on CMC mainly reflect in
the following two aspects. On one side, the increase of temperature
could weaken the hydration of the hydrophilic groups, which is condu-
cive to the formation of micelles. On the other side, the structured water
molecules surrounding the hydrophobic tail would be broken down
with increasing the temperature, which is in disfavor of the formation
of micelles. These combined effects determine the variations of CMC.
Here, the CMC increase at a high temperature, indicating the influence
on hydrophobic interaction of tails plays a dominant role for (Mor)m-
P-m and (Mor)m-BP-m among the investigated temperatures.
o
The standard Gibbs free energy in the process of micellization ΔGmic
3.2. Thermodynamics of micellization
can be calculated by the following formula [25,40]:
o
The micellization thermodynamic parameters of surfactant solutions
ΔG ¼ RTð0:5 þ βÞ ln XCMC
ð5Þ
mic
can be determined by measuring the curves of conductivity at different
temperatures. In Fig. 4 and Fig. 5, there are two linear relationships be-
tween the conductivity and concentration for each temperature, and the
where XCMC = CMC/55.4 and 55.4 stems from 1 L of water correspond-
ing to 55.4 mol of water at 25 °C. The standard enthalpy change ΔHmic
o
Table 3
The values of CMC, β and thermodynamic parameters of (Mor)m-P-m and (Mor)m-BP-m at different temperatures.
Surfactant
T (K)
CMC(mmol/L)
β
ΔGmic (kJ/mol)
ΔGads (kJ/mol)
ΔHmic (kJ/mol)
-TΔSmic (kJ/mol)
(Mor)10-P-10
(Mor)12-P-12
(Mor)14-P-14
(Mor)16-P-16
(Mor)10-BP-10
(Mor)12-BP-12
(Mor)14-BP-14
(Mor)16-BP-16
288.15
10.411
10.504
10.768
11.020
11.284
2.159
2.254
2.228
2.317
2.349
0.330
0.360
0.370
0.380
0.400
0.119
0.126
0.129
0.137
0.139
5.530
5.560
5.804
5.830
6.160
0.900
0.910
0.930
0.942
0.964
0.334
0.353
0.358
0.381
0.388
0.089
0.091
0.091
0.092
0.092
0.3618
0.3581
0.3530
0.3509
0.3379
0.5716
0.5376
0.4871
0.4602
0.4565
0.4575
0.4322
0.3491
0.3286
0.3252
0.4591
0.3835
0.3814
0.3465
0.2107
0.5528
0.5525
0.5456
0.5409
0.5367
0.6351
0.5751
0.5412
0.5446
0.5071
0.5899
0.4592
0.4345
0.3386
0.3272
0.6492
0.5474
0.5050
0.4579
0.4340
−17.71
−17.93
−18.07
−18.28
−18.25
−26.06
−25.56
−24.71
−24.40
−24.67
−27.60
−27.14
−25.08
−24.83
−25.03
−29.99
−27.98
−28.34
−27.54
−23.48
−23.23
−23.62
−23.75
−24.03
−24.18
−29.99
−28.87
−28.38
−28.92
−28.28
−31.39
−27.97
−27.68
−25.13
−25.15
−36.73
−34.01
−33.17
−32.14
−31.84
−8.05
−8.81
−9.58
−10.4
−9.66
−9.12
−8.49
−7.88
−7.05
22.30
22.98
24.12
24.79
24.91
42.26
42.89
45.25
46.28
46.79
73.21
76.94
79.08
81.91
88.01
−16.30
−15.11
−13.63
−12.17
−10.53
15.64
18.53
20.82
22.24
24.71
82.63
88.18
91.67
96.63
99.92
61.65
66.77
70.27
74.05
77.17
293.15
298.15
303.15
308.15
−50.16
−11.2
288.15
−48.36
−48.54
−48.83
−49.18
−49.58
−69.65
−70.03
−70.33
−71.11
−71.82
−103.20
−104.92
−107.42
−109.45
−111.49
−6.93
293.15
298.15
303.15
308.15
−58.46
−54.62
−51.27
−50.97
−65.30
−62.99
−86.06
288.15
293.15
298.15
303.15
308.15
288.15
293.15
298.15
303.15
308.15
288.15
293.15
298.15
303.15
308.15
−8.51
−10.13
−11.86
−13.65
−45.62
−47.40
−49.20
−51.16
−52.99
−114.02
−116.15
−119.35
−121.76
−125.07
−98.38
−100.78
−103.44
−106.18
−109.02
288.15
293.15
298.15
303.15
308.15
288.15
293.15
298.15
303.15
308.15
288.15
293.15
298.15
303.15
308.15
6

L.-C. Zheng and Q.-X. Tong
Journal of Molecular Liquids 331 (2021) 115781
o
and standard entropy change ΔSmic of micellization can be obtained
through the following relations:
κ−κ0
C
Λ ¼
ð11Þ
ꢂ À
Áꢃ
o
∂
ΔG =T
∂
o
mic
ΔH
¼
ð6Þ
ð7Þ
mic
where K is the conductivity of the surfactant solution, K is the conduc-
tivity of secondary distilled water, and C is the concentration of surfac-
tant. Λ values of (Mor)16-P-16 (Fig. 6 d), (Mor)14-BP-14 (Fig. 7 c) and
Mor)16-BP-16 (Fig. 7 d) pass through a maximum while those of other
surfactants decline with the increase of C , proving the existence of
pre-micelles. Generally, there are pre-micelles in the aqueous solution
of gemini surfactants containing alkyl chains with at least 14 carbon
atoms due to the strong hydrophobic interaction between the long tails
0
ð1=TÞ
À
Á
o
mic
o
mic
o
mic
ΔS ¼ ΔH −ΔG
=T
(
0.5
Besides, the standard Gibbs free energy in the process of adsorption
ΔGads are from the following equation [41,42]:
o
o
o
ΔG ¼ ΔG −ΠCMC=Γmax
ð8Þ
ð9Þ
ads
mic
[
13,38,46–48].
ΠCMC ¼ γ −γ
0
CMC
3
.3. Micropolarity of aggregates
where ПCMC and γ
0
represent the surface pressure of surfactant at CMC
and the surface tension of water at 25 °C, respectively.
1 3
In the fluorescence emission spectrum, the ratio (I /I ) of fluores-
o
o
In Table 3, the values of ΔGads and ΔGmic are negative, illustrating the
adsorption and aggregation processes are spontaneous [25,42]. The ΔG
cence intensity at the first peak (373 nm) and the third peak
ic (384 nm) is sensitively dependent on the polarity of the microenviron-
ment in which pyrene is located [49,50]. When the surfactants form mi-
celles, pyrene will locate in the nonpolar region of micellar core, leading
m
-
o
of (Mor)m-P-m and (Mor)m-BP-m decrease with the increase of m,
indicating that the stronger aggregation ability with longer tails. The ab-
o
solute ΔGmic values of (Mor)m-BP-m are bigger than (Mor)m-P-m, sug-
1 3
to a sharp drop in I /I . Zara et al. [51] proposed that the concentration
gesting that the stronger aggregation trend of (Mor)m-BP-m in
aqueous. The negative values of ΔH om ic showing their micellization pro-
cess is exothermic.
corresponding to the midpoint of the descent curve between two plat-
forms can be regarded as the CMC. The CMC values measured by fluo-
−
3
−3
−4
rescence method are 6.75 × 10 , 1.058 × 10 , 2.36 × 10
and
−
5
−3
Gemini surfactants may form pre-micellar at a concentration lower
than CMC, which is a specific aggregation behavior different from tradi-
tional surfactants [28,43,44]. Zana et al. proposed that the existence of a
maximum value in the curve of molar conductivity versus the square
5.3 × 10
mol/L for (Mor)m-P-m ( Fig. 8a), and 2.614 × 10
,
−
4
−4
−5
6.94 × 10 , 1.85 × 10
and 5.1 × 10
mol/L for (Mor)m-BP-m
(Fig. 8b), corresponding to m = 10, 12, 14 and 16 respectively, which
is approximately consistent with the results obtained by surface tension
and conductivity method. Besides, the minimum I /I values of (Mor)m-
1 3
P-m and (Mor)m-BP-m are close, showing that the length of rigid
spacers has little effect on the micropolarity of aggregates.
0
.5
root of concentration (C ) is a typical sign of the formation of pre-
micelle [45]. The molar conductivity Λ can be calculated by the follow-
ing equation:
0.5
Fig. 6. Variation of the molar conductivity Λ with C for (Mor)m-P-m at different temperatures.
7

L.-C. Zheng and Q.-X. Tong
Journal of Molecular Liquids 331 (2021) 115781
0
.5
Fig. 7. Variation of the molar conductivity Λ with C for (Mor)m-BP-m at different temperatures.
3.4. Size and morphology of aggregates
form vesicles in the aqueous solution and the sizes of vesicles formed
by (Mor)m-BP-m are slightly larger. To further identify the aggregation
morphologies, we also carry out TEM experiments.
3.4.1. Dynamic light scattering (DLS)
The result obtained by dynamic light scattering (DLS) is the average
hydration diameter of the surfactant aggregates, which helps determine
the type of aggregates. In Fig. 9, the peaks at less than 1 nm in the curves
are caused by the interaction of multiple headgroups in gemini surfac-
tants and do not indicate the size of aggregates [52]. The hydration di-
ameters of (Mor)m-P-m aggregates are about 20–40 nm and
3
.4.2. Transmission electron microscopy (TEM)
Fig. 10 and Fig. 11 show that the synthesized surfactants form vesi-
cles with different sizes in aqueous solutions, which conform to the re-
sults from DLS. Compared with (Mor)m-P-m, (Mor)m-BP-m are
inclined to form larger vesicles. The possible reason is that the
biphenylene spacer is longer and more rigid than the phenylene spacer,
which prevents the tails from getting close to each other. Thus the tails
7
3
0–250 nm, and those of (Mor)m-BP-m aggregates are about
0–50 nm and 80–400 nm. The results show that these surfactants
Fig. 8. Pyrene fluorescence intensity ratio I
/I
1 3
vs C for (Mor)m-P-m (a) and (Mor)m-BP-m (b).
8

L.-C. Zheng and Q.-X. Tong
Journal of Molecular Liquids 331 (2021) 115781
Fig. 9. The size distributions of (Mor)m-P-m (a) and (Mor)m-BP-m (b) aggregates at 10 times CMC.
Fig. 10. TEM images of (Mor)m-P-m at a concentration of 10 times the CMC [(Mor)10-P-10 (a), (Mor)12-P-12 (b), (Mor)14-P-14 (c), (Mor)16-P-16 (d)].
9

L.-C. Zheng and Q.-X. Tong
Journal of Molecular Liquids 331 (2021) 115781
Fig. 11. TEM images of (Mor)m-BP-m at a concentration of 10 times the CMC [(Mor)10-BP-10 (a), (Mor)12-BP-12 (b), (Mor)14-BP-14 (c), (Mor)16-BP-16 (d)].
of (Mor)m-BP-m occupy more volumes in the aggregates, bringing
about the larger size.
The minimum inhibitory concentration (MIC) is one of the parame-
ters to measure the antibacterial property of an antimicrobial agent. The
lower the MIC is, the better the antibacterial property will be. The anti-
bacterial properties of target surfactants against Escherichia coli and Ba-
cillus subtilis were studied by the spread plate method. The MIC values
were investigated and shown in Table 4.
3.5. Thermaldynamic properties
The decomposition temperature (T
vealed by TGA. As shown in Fig. 12, all surfactants displayed excellent
thermal stability. T are recorded at 225, 237, 233, 239 °C for (Mor)m-
d
) of each target surfactant is re-
The MIC values of (Mor)m-P-m and (Mor)m-BP-m are smaller than
traditional bactericide 1227 for both bacteria, indicating the better anti-
bacterial performance of gemini surfactant. Compared with 1227, the
synthesized surfactants possess higher densities of positive charges
and hydrophobic chains, bringing about stronger electrostatic adsorp-
tion on the cell membrane of bacteria and more prominent hydrophobic
effects with the lipoid layers of cells [36,57]. The antibacterial activity of
the surfactant varies with the alkyl chain length and reaches the best
with an optimal length [58]. Since the MIC values of (Mor)m-P-m and
(Mor)m-BP-m exhibit a similar order with the change of hydrophobic
chain length, we take (Mor)m-P-m homologues as examples to discuss.
The cell membrane of bacteria is mainly composed of phospholipid bi-
layers and most phospholipid molecules contain 12 carbon atoms. The
physicochemical properties of surfactants containing 12 carbon atoms
are most similar to the lipid layer, and the energy needed to combine
them is minimum [36,58]. Therefore, (Mor)12-P-12 possesses the best
d
P-m, and 217, 202, 208, 216 °C for (Mor)m-BP-m, corresponding to
m = 10, 12, 14, 16 respectively, which exhibited better thermal stability
than the classic type surfactants m-s-m reported, such as 14‐2-14 (T
d
=
1
73.7 °C) and 16‐2-16 (T = 172.8 °C) [53].
d
3.6. Antibacterial property
The antibacterial mechanism [54–56] of cationic surfactants is gen-
erally considered as follows. For one thing, the headgroups of cationic
surfactant adsorb on the cell wall of bacteria, which brings about a steric
effect and changes the permeability of the cell wall. For another, alkane
chains insert into the lipid layer of cells by hydrophobic interaction,
leading to the deactivation of enzymes and the denaturation of the pro-
tein. As a result, the bacteria die.
10

L.-C. Zheng and Q.-X. Tong
Journal of Molecular Liquids 331 (2021) 115781
Fig. 12. TGA results of (Mor)m-P-m (a) and (Mor)m-BP-m (b).
among which (Mor)m-BP-m displayed a higher activity. Compounds
containing 12 carbon atoms in the tails exhibited optimal antibacterial
property among the synthesized gemini homologues.
Table 4
The MIC values of synthesized surfactants against E. coli and B. subtilis (mg/L).
surfactant
227
Escherichia coli
Bacillus subtilis
1
50 [57]
15
12
39
43
8
5
30
35
–
Author contributions
(
(
(
(
(
(
(
(
Mor)10-P-10
Mor)12-P-12
Mor)14-P-14
Mor)16-P-16
Mor)10-BP-10
Mor)12-BP-12
Mor)14-BP-14
Mor)16-BP-16
15
11
35
40
7
3
23
30
Qing-Xiao Tong: Funding acquisition; Project administration; Super-
vision; Writing - review & editing. Le-Chi Zheng: Data curation; Formal
analysis; Investigation; Methodology; Writing - original draft.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
antibacterial performance among the (Mor)m-P-m homologues. Com-
pared with (Mor)12-P-12, (Mor)10-P-10 has weaker hydrophobicity
because of the shorter tail, leading to the slightly inadequate binding
ability with the lipid layer. Thus, the antibacterial property of (Mor)
Acknowledgments
1
0-P-10 is a little inferior. The antibacterial performances of (Mor)14-
P-14 and (Mor)16-P-16 are relatively poor, which may be related to
the hydrophilic-hydrophobic balance of the surfactant molecules [59].
They are too hydrophobic to penetrate the cell membrane effectively.
Moreover, long tails will lead to poor water solubility [36,60], which
may be another reason for their low antibacterial activities.
This research was supported by the National Natural Science Foun-
dation of China (No. 51973107 and 51673113), the key project of De-
partment of Education of Guangdong Province (No. 2018KZDXM032)
and Guangdong Province Universities and Colleges Pearl River Scholar
Funded Scheme 2019(GDUPS 2019).
It is noteworthy that the MIC values of (Mor)m-BP-m are smaller
than (Mor)m-P-m for both bacteria, suggesting the better antibacterial
properties of (Mor)m-BP-m. It could be due to two reasons. For one
thing, the steric effect of biphenylene is more prominent than that of
phenylene, causing the lower permeability of the cell membrane. For
another, conformation changes of biphenylene strengthen the hydro-
phobic binding between the tails and the lipid layers of cells.
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