Synthesis, Surface and Antimicrobial Activities of Cationic Gemini Surfactants with Semi-Rigid Spacers

Article abstract of DOI:10.1007/s11743-015-1777-4

Four cationic gemini surfactants featuring semi-rigid spacers were synthesized via a two-step process. The surface-active properties of these surfactants were investigated through surface tension and electrical conductivity measurement. The thermodynamic parameters of micellization were evaluated from electrical conductivity measurements at temperatures ranging from 293 to 313 K. The aggregation behavior of these synthesized gemini surfactants in water were investigated using dynamic light scattering and transmission electron microscopy. Further, the antimicrobial activities of these synthesized gemini surfactants against both Gram-positive and Gram-negative bacteria were also investigated.

Full text of DOI:10.1007/s11743-015-1777-4

J Surfact Deterg
DOI 10.1007/s11743-015-1777-4
ORIGINAL ARTICLE
Synthesis, Surface and Antimicrobial Activities of Cationic
Gemini Surfactants with Semi-Rigid Spacers
Hai-lin Zhu1 Zhi-yong Hu1 Xue-mei Ma1 Jian-long Wang1 Duan-lin Cao1
•
•
•
•
Received: 5 June 2015 / Accepted: 7 December 2015
Ó AOCS 2016
Abstract Four cationic gemini surfactants featuring
semi-rigid spacers were synthesized via a two-step process.
The surface-active properties of these surfactants were
investigated through surface tension and electrical con-
ductivity measurement. The thermodynamic parameters of
micellization were evaluated from electrical conductivity
measurements at temperatures ranging from 293 to 313 K.
The aggregation behavior of these synthesized gemini
surfactants in water were investigated using dynamic light
scattering and transmission electron microscopy. Further,
the antimicrobial activities of these synthesized gemini
surfactants against both Gram-positive and Gram-negative
bacteria were also investigated.
wetting and foaming properties, than their conventional
counterparts [5–8]. Many studies have been focused on
new surfactants with tailored structures or functionalities in
order to obtain surfactants with specific physicochemical
properties for targeted applications.
The physicochemical properties of gemini surfactants
are primarily dependent on their molecular structures, such
as the charge of head groups, the length of the hydrophobic
tails and the nature of the spacers. The effect of the
hydrophobic chain length on the surface and bulk proper-
ties has been systematically investigated [9–11], whereas
the investigation of the spacer effect is less common. The
spacer connecting the two head groups can be flexible,
rigid, or semi-rigid [12]. Previous studies have shown that
the aggregation behavior of gemini surfacants is affected
by not only the length but also the rigidity of the spacer.
Additionally, various studies have reported the effects of
flexible [6, 8, 13] or rigid spacers [14–16] on the properties
of gemini surfactants, while relatively few studies have
reported on gemini surfactants with semi-rigid spacers [12,
17].
Keywords Gemini surfactants Á Semi-rigid spacer Á
Critical micelle concentration Á Thermodynamic
parameter Á Aggregation behavior Á Antimicrobial activity
Introduction
Gemini or dimeric surfactants have attracted attention
among several industrial and academic research groups [1–
4]. These surfactants, generally made up of two hydro-
carbon chains and two head groups linked by a spacer,
possess better physicochemical properties, such as lower
critical micelle concentration (CMC) values, higher solu-
bility power, unique rheological behavior, and enhanced
Currently, the most commonly studied gemini surfactant
type is alkanediyl-a-x-bis(alkyl dimethyl ammonium)
dibromide, which is referred to as m-s-m, where m and s
stand for the carbon atom number in the tail alkyl chain and
in the methylene spacer, respectively. Previous studies
have shown that 12-s-12 series surfactants with short
spacers (2 B s B 12) tend to form spherical or elongated
micelles, while 12-16-12 and 12-20-12 form vesicles [18].
Zhang et al. [17] studied the aggregation behavior of
two gemini surfactants, differing only in space rigidity,
1-dodecanaminium, N,N0-[[(2E)-1,4-dioxo-2-butene-1,4-
diyl] bis(oxy-2,1-ethanediyl)]bis[N,N- dimethyl-,bromide]
(12-fo-12) and 1-dodecanaminium, N,N0-[(1,4-dioxo-1,4-
& Hai-lin Zhu
zhuhailin99@126.com; zhuhailin@nuc.edu.cn
1
School of Chemical Engineering and Environment, North
University of China, Taiyuan 030051, People’s Republic of
China
butanediyl)bis(oxy-2,1-ethanediyl)]
bis[N,Ndimethyl-
123

J Surfact Deterg
O
,bromide] (12-su-12). They found that 12-su-12 with flex-
ible spacer formed solely micelles in aqueous solutions,
while 12-fo-12 featuring a semi-rigid spacer could form
micelles and vesicles, depending on the concentration. Zhu
et al. [12] found that gemini surfactants with semi-rigid
spacers self-assembled into the mixture of micelles and
vesicles at low surfactant concentration and formed vesi-
cles at high surfactant concentration, whereas gemini sur-
factants with long fully rigid spacers preferred to form
vesicles.
O
Cl
HO
Y OH +
Cl
(1)
Cl
Cl
Y
O
O
O
Cl
N
Cl
CnH2n+1N(CH3)2
O
O
CnH2n+1
N
CnH2n+1
Y
O
O
(2)
Cationic surfactants have been proven to have antimi-
crobial activities. Cationic gemini surfacants, which consist
of two symmetric quaternary ammonium groups, exhibit a
much better antimicrobial potency [19, 20]. The pathogenic
bacterial cell membrane is predominantly negatively
charged as compared with eukaryotic cells. Cationic sur-
factants have a unique ability to adsorb at the interface of
the cell membrane. The lipophilic component then diffuses
through the cell wall and the polar head binds to the cell
membrane, disrupting the cytoplasmic materials and killing
the bacteria. Although there are many reports discussing
the antimicrobial activity of cationic gemini surfactants,
their structure–activity relationship still remains elusive.
In this work, four cationic gemini surfactants, with the
spacer containing a rigid benzene ring and two ester
groups, were synthesized utilizing a two-step process. The
effects of different lengths of alkyl chain and spacer chain
on the aggregation and antimicrobial activities were sys-
tematically investigated.
I: n = 12, Y=
III: n = 12, Y=
, II: n = 14, Y=
, IV: n = 14, Y=
Scheme 1 The synthesized route of cationinc gemini surfactants
mixture, and then chloroacetyl chloride (0.25 mol) was
added dropwise after complete dissolution of the mixture.
The solution was next heated to 30 °C for 4 h. The mixture
was then filtered after the completion of the reaction. The
filtrate was concentrated under vacuum to obtain a brown
solid, which was then washed with absolute methanol to
obtain the resulting white solid (1).
This product (1) (10 mmol) was dissolved in 40 mL
anhydrous dioxane, then N,N-Dimethyldodecylamine or
N,N-Dimethyltetradecylamine (25 mmol) was added into
the mixture. The mixture was stirred under refluxing for
48 h. When the reaction was completed, the mixture was
cooled to room temperature and the white precipitate
obtained was collected by filtration. The crude product was
recrystallized from acetone at least three times to produce
the final products as a white solid (2).
The structure was confirmed by FT-IR, 1HNMR, ESI–MS
spectra. The FT-IR spectra were recorded in FTIR-8400S
instruments, and 1HNMR spectra were recorded in a chloro-
form (CDCl3) solution with a Bruker 300 MHz NMR Spec-
trometer, and ESI–MS spectra were recorded in a Fourier
Transform Ion Cyclotron Resonance Mass Spectrometer.
Experimental
Materials
N,N-Dimethyldodecylamine (97 %), N,N-Dimethylte-
tradecylamine (95 %), hydroquinone (99.5 %), 1,4-Ben-
zenedimethanol (99 %), chloroacetyl chloride (98 %),
triethylamine (98 %) were purchased from J&K Scientific
(Beijing) and used without further purification. All other
chemicals used were of reagent grade. Ultra-pure water
(ASINO, USA) was used to prepare the solutions for all
experiments.
Spectral Characteristics of Synthesized Surfactants
I: 1H NMR (CDCl3, ppm) 0.865(t,6H,2CH3), 1.240-
1.344(m,36H,2CH3(CH2)9), 1.817(m,4H,2CH3(CH2)9CH2),
3.625(s,12H,2 N(CH3)2), 3.846(m,4H,2CH3(CH2)9CH2
CH2), 5.977(s,4H,2 N(CH3)2CH2COO), 6.849(m,4H,
ArH). ESI–MS(positive): m/z 653.50289 [M-Cl]?,
309.26623 [M-2Cl]2?/2. FT-IR (KBr, cm-1): 2917, 2848,
1766, 1502, 1463, 1243, 1180, 1155, 833, 719.
Synthesis of the Surfactants Featuring Semi-Rigid
Spacers
The synthetic route is shown in Scheme 1.
II: 1H NMR (CDCl3, ppm) 0.852-0.893(t,6H,2CH3),
The synthesis route of the intermediate is similar to the
one reported in the literature [21]. Hydroquinone or 1,4-
Benzenedimethanol (0.1 mol) was added to 100 mL
anhydrous dioxane. Temperature was maintained at
0–5 °C. Triethylamine (0.3 mol) was added into the
1.248-1.349(m,44H,2CH3(CH2)11),
1.816(m,4H,2CH3
(CH2)11CH2), 3.636(s,12H,2 N(CH3)2), 3.853(m,4H,2CH3
(CH2)11CH2CH2), 6.005(s,4H,2 N(CH3)2CH2COO), 7.272
(m,4H,ArH). ESI–MS(positive): m/z 709.56446 [M-Cl]?,
123

J Surfact Deterg
337.29753 [M-2Cl]2?/2. FT-IR (KBr, cm-1): 2917, 2848,
1762, 1502, 1465, 1240, 1182, 1157, 835, 719.
Transmission Electron Microscopy (TEM)
III: 1H NMR (CDCl3, ppm) 0.874-0.916(t,6H,2CH3),
1.269-1.341(m,36H,2CH3(CH2)9), 1.720(m,4H,2CH3(CH2)9
CH2), 3.545(s,12H,2 N(CH3)2), 3.838(m,4H,2CH3(CH2)9
CH2CH2), 5.158(s,4H,2 N(CH3)2CH2COO), 5.330(m,4H,
CH2ArCH2), 7.346(m,4H,ArH). ESI–MS(positive): m/z
681.53316 [M-Cl]?, 323.28188 [M-2Cl]2?/2. FT-IR (KBr,
cm-1): 2917, 2850, 1747, 1517, 1465, 1241, 1191, 813, 719.
TEM measurements were performed using a JEM-1011
TEM instrument. The samples were prepared by the neg-
ative staining method. An amount of 2 % (wt) phospho-
tungstic acid was used as the staining agent. A drop of
surfactant solution was placed on a copper grid, and the
excess liquid was absorbed by a piece of filter paper. The
copper grid was then placed onto a drop of phosphotungstic
acid solution, and the sample was dried at room tempera-
ture before measurement.
1
IV: H NMR (CDCl3, ppm) 0.852-0.891(t,6H,2CH3),
1.247-1.323(m,44H,2CH3(CH2)11), 1.719(m,4H,2CH3
(CH2)11CH2), 3.531(s,12H,2 N(CH3)2), 3.814(m,4H,2CH3
(CH2)9CH2CH2), 5.125(s,4H,2 N(CH3)2CH2COO), 5.354
(m,4H,CH2ArCH2), 7.268-7.306(m,4H,ArH). ESI–MS
(positive): m/z 737.59576 [M-Cl]?, 351.31318 [M-2Cl]2?/2.
FT-IR (KBr, cm-1): 2915, 2848, 1747, 1519, 1467, 1243,
1189, 811, 719.
Determination of the Antimicrobial Activity
The antimicrobial activity of the synthesized surfactants
was tested against reference strains of Gram-positive
(Bacillus subtilis ATCC 6633) and Gram-negative
(Escherichia coli ATCC 8099). The organisms were
incubated by a streak culture method and then refrigerated.
The bacterial strains were put into incubator for 4 h for
activation, and were then scraped into sterile water by
inoculating loops. The organisms were shaken at 34 °C for
24 h to incubate them. The concentrations of the bacteria
suspensions were approximately 107 cfu/mL.
Surface Tension Measurements
The surface tensions of the aqueous surfactant solutions
were measured on a K100 automatic tensiometer (Kru¨ss,
Germany) with a du Nouy ring. Measurements were per-
formed at 20 0.1 °C until successive values agreed to
within 0.2 mN m-1
.
To assess the biocidal activity, a nutrient medium agar
was used, composed of (g L-1) 10.0 g peptone, 5.0 g beef
extract, 5.0 g sodium chloride and 20.0 g agar. The pH was
adjusted to 7.0–7.5. The agar medium was poured, while
hot, into a Petri dish (diameter 9 cm) after autoclaving.
The target gemini surfactants were dissolved in sterile
water after sterilization, and the solution was then added
into 10 mL bacteria suspensions and incubated by shaking
at 34 °C for 1 h. The test organisms were coated on the
Petri dish by the ten times dilution method and were put in
an incubator at 34 °C for 24 h. Bacteria suspensions
without surfactants were also incubated for comparison.
The antibacterial rate was calculated from the following
equation:
Krafft Temperature
The Krafft temperature(TK) was determined by heating the
surfactant solution until a clear solution was obtained [22]. All
solution concentrations of these surfactants were 1.0 wt% (at
least twice the CMC of the studied gemini surfactant).
Electrical Conductivity Measurements
The conductivity of the surfactant solutions was measured
by using a low-frequency conductivity analyzer (DDS-307;
Shanghai Precision & Scientific Instruments, China). A
super-thermostatic cistern (temperature fluctuation:
0.5 °C; SX-CH1015; Beijing Heng Odd Instruments,
China) was used to control the temperature. The uncertainty
I À II
gð%Þ ¼
 100
ð1Þ
I
where, I is the average clump count without surfactant and
II is the average clump count with surfactant. The lowest
concentration of the antibacterial agent with an antibacte-
rial rate above 90 % was determined to be the MIC.
of the measurement was found to be 0.01 lS cm-1
.
Dynamic Light Scattering (DLS) Measurements
DLS measurements were performed using the BrookHaven
90 Plus DLS instrument at 25 °C with 660 nm laser light,
and the light collection at 90°. All solutions were went
through ultrasonic treatment and filtered throught a 0.2-lm
Millipore filter into the cylindrical scattering cells before
the experiments. The data obtained in each case were the
average of 3 runs. The particle size was calculated from the
NNLS method.
Results and Discussion
Krafft Temperature
The Krafft temperatures (TK) were all determined by visual
observation of the clarification of 1.0 wt% aqueous
123

J Surfact Deterg
Table 1 Physico-chemical properties of synthesized surfactants
Surfactant
TKrafft (°C)
cCMC (mN m-1
)
CMCs (mmol L-1
)
pC20
Cmax (10-10 mol cm-2
)
Amin (nm2)
I
\0
\0
\0
\0
37.8
37.7
41.1
40.9
39a
0.65
0.10
0.58
0.09
16a
3.19
4.04
3.24
3.99
2.1a
2.9a
1.09
1.27
0.89
1.24
–
1.51
1.31
1.86
1.34
0.66b
0.62b
II
III
IV
DTAB
TTAB
a
37.8a
3.7a
–
Data taken from Ref. [24]
b
Data taken from Ref. [25]
concentration of the surfactant solution required to sup-
press surface tension by 20 mN m-1 (pC20) for the
surfactants.
Cmax, Amin, and pC20 were calculated by using the Gibbs
equations, as follows [19]:
ꢀ
ꢁ
À1
oc
Cmax
¼
;
ð2Þ
2:303nRT o log c
T;P
Amin ¼ ðNA Â CmaxÞÀ1 Â 1014;
c0 À 20 À cCMC
ð3Þ
ð4Þ
pC20
¼
À log c;
2:303nRTCmax
where R is the gas constant (8.314 J mol-1 K-1), T is the
absolute temperature, c is the surfactant concentration, NA
is Avogadro’s number (6.02 9 1023 mol-1), and c0 is the
surface tension of water. For a gemini surfactant, the value
of n is rather complex. The values n = 2 and 3 have been
used in many published documents. Nevertheless, if the
value used for n affects the way in which C and Amin vary,
then it has no bearing on the way in which C and Amin vary
with the tail carbon number. Therefore, the use of a dif-
ferent value of n (2 or 3) does not affect the conclusion
inferred from these variations [23]. Here, the value of n is
taken as 3.
Fig. 1 Dependence of surface tension on surfactant concentration at
20 °C
solutions of these surfactants [22]. It can be seen from
Table 1 that the Krafft points of the four synthesized
gemini surfactants featuring semi-rigid spacers are all
below 0 °C. Such low Krafft temperatures permit the use of
these surfactants in cold water.
Surface Activity
Table 1 shows the CMC values obtained for surfactants
I, II, III and IV were 0.65, 0.10, 0.58 and 0.09 mmol L-1
,
The surface tension measurement is a classical method of
studying the critical micelle concentration (CMC) of sur-
factants. The relationships between surface tensions (c) and
the concentrations of synthesized surfactants at 20 °C were
determined. Figure 1 shows that c first decreases with the
increase in the concentration of the surfactant and then
becomes almost constant after the concentration reaches a
certain value. There, the intersection point is taken as the
critical micelle concentration (CMC). Table 1 provides the
values of the CMC measured via the surface-tension
method (CMCs), the surface tension corresponding to
CMCs (cCMC), the maximum surface excess (Cmax), the
minimum surface area per surfactant (Amin), and the
respectively. Similar to conventional ionic surfactants, the
CMC values decreased with increasing alkyl chain length
from 12 to 14. This is attributed to the hydrophobicity of
surfactant molecules increasing with increasing the number
of methylene groups in the chains, which demonstrates the
increasing of the mutual repulsion between polar water
molecules and nonpolar hydrophobic chains. All the syn-
thesized gemini surfactants have lower CMC values than
their corresponding single-tail surfactant [24], suggesting
excellent micelle-forming ability in water. Among these
synthesized gemini surfactants, with an increase of the
spacer length by two methylene units, the CMC values
decrease slightly, while the cCMC values increase
123

J Surfact Deterg
obviously, showing slightly better micelle-forming ability
and weaker surface tension-lowering ability.
surface active. Increasing the length of hydrophobic chains,
pC20 value increases, showing a higher adsorption effi-
ciency. The pC20 values decrease slightly with the increase
in the number of methylene groups along the spacer.
The Amin value of the synthesized gemini surfactant is
larger than that of its corresponding single-tail surfactant
[25], suggesting that the presence of the spacer group
increases the value of Amin. This may be attributed to the
fact that the benzene ring is rigid and hydrophobic, and that
it prefers to lay on the surface when it is adsorbed at the
water–air interface. The Amin value increases with
increasing the spacer length. This is thought to be because
the spacer becomes more flexible and more hydrophobic
with increasing the two methylene units to the spacer,
resulting in weaker adsorption at the air–water interface.
The pC20 value measures the efficiency of the surfactant
in lowering surface tension [26]. The higher pC20 value
indicates that the surfactant adsorbs more efficiently at the
interface and reduces the interface tension more efficiently.
Comparing the pC20 values of these synthesized gemini
surfactants and their corresponding single-tail surfactants,
it is hypothesized that the synthesized surfactants are more
Thermodynamics of Micellization
The electrical conductivity (j) of the synthesized surfac-
tants in aqueous solution against c at different temperatures
is shown in Fig. 2. As the figure shows, electrical con-
ductivity increased with increasing the temperature and the
surfactant concentration. As the surfactant concentration
increased, electric conductivity increased rapidly, until a
certain concentration point where it became gradual. The
concentration at the break point was the CMC of the sur-
factant. Here, we used the Carpena method [27] in order to
determine the CMC values and the degree of counter ion
association (b). This method was based on the fitting of the
j–c curve to the integral of a Boltzmann-type sigmoidal
equation:
Fig. 2 Specific conductivity (j) versus surfactant concentration (c) at different temperature
123

J Surfact Deterg
ꢀ
ꢁ
1 þ eðcÀc Þ=Dc
D
micS0 ¼ ðDmicH0 À DmicG0Þ=T;
ð9Þ
0
jðcÞ ¼ jð0Þ þ A1c þ DcðA2 À A1Þ ln
;
1 þ eÀc =Dc
0
The thermodynamic parameters of micellization for syn-
thesized surfactants at different temperatures are listed in
Table 2. It is clear that, for synthesized gemini surfactants,
the free energies (DmicG0) of micellization are all negative,
indicating that the micellization processes are all sponta-
neous. With increasing alkyl chain length from 12 to 14,
the free energies of micellization (DmicG0) become more
negative, which implies that micellization is favored due to
the increase in the hydrophobic interactions between the
hydrophobic chains. When the spacer increases by two
methylene units, the free energies of micellization also
become more negative. This may be attributed to an
increase of hydrophobic interaction resulting in a stronger
tendency for aggregation. The values of DmicH0 were all
negative, which suggests that micellization was exothermic
at the experimental temperature, and the dispersion forces
play a significant role in the micellization process [29]. For
all the synthesized gemini surfactants, the values of the
standard entropy of micellization (TDmicS0) are larger than
the values of -DmicH0, which indicates that the micel-
lization is entropy-driven. With the increase of tempera-
ture, TDmicS0 decreases while -DmicH0 increases,
suggesting that the entropy-driven becomes weaker and the
enthalpy-driven becomes stronger, and that the enthalpy–
entropy compensation phenomenon may exist.
ð5Þ
ð6Þ
b were calculated from the following equation:
b ¼ 1 À A2=A1;
where j(0), A1, A2, Dc, and c0 are the values of j in pure
water, the pre-micellar slope, the post-micellar slope, the
width of the transition, and the central point of the transi-
tion region (CMC), respectively. The values of the CMC
and b in different temperatures enabled us to analyze the
thermodynamic energetics of micelle formation. For a
gemini surfactant, the free energy of micellization (DmicG0)
was given by Zana [28]:
D
micG0 ¼ RTð0:5 þ bÞ lnðCMCÞ;
ð7Þ
where CMC is in mole fraction units. The enthalpy of
micellization (DmicH0) can be determined by using the
Gibbs–Helmholtz equation:
ðoðDmicG0=TÞ
D
micH0 ¼ ÀT2
oT
ð8Þ
o ln CMC
oT
¼ ÀRT2ð0:5 þ bÞ
;
The standard entropy of micellization (DmicS0) is deter-
mined by using the following equation:
Table 2 Influence of
Surfactant
I
t
CMC
(mmol L-1
b
4
micG0
4
micH0
T4micS0
temperature on the parameters
of micellization for synthesized
surfactants
(°C)
)
(KJ mol-1
)
(KJ mol-1
)
(KJ mol-1
)
20
25
30
35
40
20
25
30
35
40
20
25
30
35
40
20
25
30
35
40
1.067
1.308
1.425
1.512
1.635
0.197
0.249
0.285
0.301
0.325
0.946
1.131
1.273
1.378
1.419
0.178
0.190
0.203
0.229
0.238
0.452
0.443
0.434
0.422
0.410
0.639
0.618
0.610
0.591
0.578
0.613
0.578
0.567
0.556
0.540
0.666
0.651
0.635
0.629
0.593
-25.19
-24.90
-24.88
-24.82
-24.71
-34.83
-34.12
-34.07
-33.88
-33.81
-29.78
-28.86
-28.72
-28.68
-28.63
-35.94
-35.90
-35.81
-35.86
-35.17
-10.34
-10.18
-10.85
-11.06
-11.28
-13.92
-14.13
-14.50
-14.73
-15.03
-12.09
-12.11
-12.39
-12.67
-12.89
-13.25
-13.53
-13.79
-14.17
-14.17
14.85
14.73
14.03
13.76
13.44
20.91
19.99
19.57
19.16
18.78
17.69
16.75
16.33
16.01
15.74
22.70
22.38
22.02
21.68
21.00
II
III
IV
123

J Surfact Deterg
for I and IV only large aggregates were found. As shown in
Fig. 4, the size of aggregates corresponded to the large size
distribution in the DLS plots as shown in Fig. 3a.
Antimicrobial Activity
The antimicrobial activities of these synthesized surfac-
tants were tested against reference strains of Bacillus
subtilis and Escherichia coli. Figure 5 shows the relation
curve of antibacterial rate and concentration of gemini
surfactants. From Fig. 5, one can see that these target
gemini surfactants exhibit antimicrobial activities against
both Gram-positive and Gram-negative bacteria, showing
broad bactericidal performance. Antibacterial rate increa-
ses with an increase in the concentration of the gemini
surfactants for any given bacteria. Bacterial cell surfaces
are usually negatively charged; cationic surfactants can
easily disturb bacterial membranes due to the positively
charged polar head. By increasing the concentration of the
surfactant, the positive charge density of the head group
increases, resulting in an increase in sterilization.
The MIC is defined as the lowest concentration of the
antibacterial agent where the antibacterial rate is above
90 %, which is used as a measurement of antibacterial
activity. The lower the MIC, the higher the antibacterial
activity of the reagent. Table 3 lists the MIC values for
synthesized gemini surfactants. Upon comparison with
benzalkonium chloride (1227) [30], these gemini surfac-
tants are more efficient biocides. Due to the existence of
spacers, these gemini surfactants contribute more positive
charge, thereby facilitating more electrostatic interaction at
the membrane, and exhibiting higher antibacterial activities
[31]. Electrostatic and hydrophobic interactions help to
facilitate adsorption onto cationic surfactants. The MIC
values decrease with the addition of two methylene units to
the spacer, which is in accordance with the surface activ-
ities. When comparing the MIC values of I, II and III, and
IV, one finds that the MIC value shows an increase with
increasing the alkyl chain length. This demonstrates that
antibacterial activities decrease when increasing the sur-
face activities of the reagents, which is opposite of the
hypothesis. It is well known that the antibacterial activities
are strongly dependent on the alkyl chain length, and that
there is an optimal chain length for the maximum
antibacterial activities [32]. Since most phospholipid
molecules in cells contain 12 carbon atoms, the surfactants
containing 12 carbon atoms are most similar to the phos-
pholipid molecule in physical and chemical properties, and
require minimal energy. Another strong explanation is that
the antibacterial activity depends on the hydrophilic–hy-
drophobic balance of the cationic surfactant. The
hydrophobic character increases with further increasing the
alkyl chain length (n [ 12), which may be too hydrophobic
Fig. 3 Hydrodynamic radius (Rh) distributions of the aggregates of
the surfactants. a Surfactants at 10 times the CMC, b surfactants at 10
times the CMC with 0.1 M Na Br addition
Size of Aggregation
The aggregation state of the four synthesized gemini surfac-
tants in aqueous solution was first studied by DLS. The con-
centrations were about 10 times the CMC, with the size
distribution by DLS shown in Fig. 3. From Fig. 3a, one can
see that there are obvious peaks with the hydrodynamic radius
(Rh) of about 50–200 nm, reflecting the typical size of large
aggregates. The effect of salt on the aggregation was also
investigated, and the results (Fig. 3b) show that the peaks with
a Rh value of 50–200 nm disappear, leaving micelles peaks
with a Rh value around 3 nm. This result agrees with the
observation by Zhang et al., for gemini surfactants with
diethylammonium headgroups and a diamido spacer [23].
To identify the morphologies of the aggregates, we
performed the TEM measurement. Figure 4 exhibits the
TEM images of the aggregates of the four synthesized
gemini surfactants. Through TEM observation, vesicles
were observed in the aqueous solutions of II and III, while
123

J Surfact Deterg
Fig. 4 TEM images of gemini
surfactants in aqueous solution
with 10 times the CMC
b Fig. 5 Bactericidal rate curve of four synthesized reagents.
a Scherichia coli, b Bacillus subtilis
Table 3 The MIC values of synthesized surfactants against Escher-
ichia coli and Bacillus subtilis (mg/L)
Microorganisms
I
II
III
IV
1227
50a
E. coli
18
20
39
34
17
11
26
22
B. subtilis
a
Data taken from Ref. [30]
to facilitate transport through the bacterial cell membrane
[33]. Additionally, further increasing the alkyl chain length
decreases the solubility of water, leading to lower
antibacterial activity.
Conclusion
The prepared gemini surfactants featuring semi-rigid
spacers are expected to have good surface activity. The
CMC values decrease with increases in the length of alkyl
chain of the surfactants, while cCMC values increase with
increases in the spacer length. The thermodynamic
parameters of micellization derived from electrical con-
ductivity indicate that the micellization is spontaneous and
123

J Surfact Deterg
Langmuir films of silver-nanoparticles at the air-water interface?
J Colloid Interface Sci 430:85–92
driven by entropy. It was found that these synthesized
gemini surfactants featuring a semi-rigid spacer formed
large aggregates in aqueous solution. The results of
antibacterial activity test show that these synthesized
gemini surfactants display excellent antimicrobial activities
both against B. subtilis and E. coli, and the antibacterial
rate increases with an increase in the concentration of the
gemini surfactants. Surfactants with the hydrophobic
chains containing 12 carbon atoms were found to have
higher activity among the synthesized gemini homologues.
14. Mivehi L, Bordes R, Holmberg K (2011) Adsorption of cationic
gemini surfactants at solid surfaces studied by QCM-D and SPR:
effect of the rigidity of the spacer. Langmuir 27:7549–7557
15. Zhou M, Liu HL, Yang HF, Liu XL, Zhang ZR, Hu Y (2006) Spon-
taneous crystallization at the air–water interface: an unusual feature of
gemini surfactant with a rigid spacer. Langmuir 22:10877–10879
16. Xie DH, Zhao JX (2013) Unique aggregation behavior of a car-
boxylate gemini surfactant with a long rigid spacer in aqueous
solution. Langmuir 29:545–553
17. Zhang ZG, Zheng PZ, Guo YM, Yang Y, Chen ZY, Wang XY,
An XQ, Shen WG (2012) The effect of the spacer rigidity on the
aggregation behavior of two ester-containing gemini surfactants.
J Colloid Interface Sci 379:64–71
Acknowledgments Authors would like to thank the National Nat-
ural Science Foundation of China (21406211) and the Natural Science
Foundation of North University of China for financial support.
18. Zana R (2002) Dimeric (gemini) surfactants: effect of the spacer
group on the association behavior in aqueous solution. J Colloid
Interface Sci 248:203–220
19. Kuperkar K, Modi J, Patel K (2012) Surface-active properties and
antimicrobial study of conventional cationic and synthesized
symmetrical gemini surfactants. J Surfactant Deterg 15:107–115
20. Guo SN, Sun XW, Zou QC, Zhang JZ, Ni H (2014) Antibacterial
activities of five cationic gemini surfactants with ethylene glycol
bisacetyl spacers. J Surfactant Deterg 17:1089–1097
References
1. Ren HC, Wang F, Zhang ZQ, Nie HH, Li N, Cui M (2015)
Synthesis, surface activity and aggregation behavior of gemini
imidazolium surfactants 1,3-bis(3-alkylimidazolium-1-yl) pro-
pane bromide. Colloid Surf A 467:1–8
21. Long DQ, Li DJ, Liu CY (2004) Synthesis of dihydroxybenzene-
bischloroacetates. Chin J Synth Chem 12:511–513
2. Wang MN, Han YC, Qiao FL, Wang YL (2015) Aggregation
behavior of a gemini surfactant with a tripeptide spacer. Soft
Matter 11:1517–1524
22. Davey TW, Ducker WA, Hayman AR, Simpson J (1998) Krafft
temperature depression in quaternary ammonium bromide sur-
factants. Langmuir 14:3210–3213
3. Tehrani-Bagha AR, Holmberg K (2010) Cationic ester-containing
gemini surfactants: physical–chemical properties. Langmuir
26:9276–9282
4. Hu DH, Guo XF, Jia LH (2013) Synthesis, surface active prop-
erties of novel gemini surfactants with amide groups and rigid
spacers. J Surfactant Deterg 16:913–919
5. Lin MQ, Hua Z, Ding B, Li MY (2015) Rheological properties of
quaternary ammonium gemini surfactants in aqueous solution.
J Surfactant Deterg 18:67–72
6. Zana R (2002) Dimeric and oligomeric surfactants. Behavior at
interfaces and in aqueous solution: a review. Adv Colloid Inter-
face Sci 97:205–253
23. Zhang Q, Gao ZN, Xu FX, Tai SX, Liu XG, Mo SB, Niu F (2012)
Surface tension and aggregation properties of novel cationic
gemini surfactants with diethylammonium headgroupsand a dia-
mido spacer. Langmuir 28:11979–11987
24. Abe Masahiko, Tsubone Kazuyki, Koike Takaaki, Tsuchiya Koji,
Takahiro Ohkubo H, Sakai H (2006) Polymerizable cationic
gemini surfactant. Langmuir 22:8293–8297
´
´
25. Rodrıguez A, Graciani MDM, Moreno-Vargas AJ, Moya ML
(2008) Mixtures of monomeric and dimeric surfactants:
hydrophobic chain length and Spacer group length effects on non
ideality. J Phys Chem B 112:11942–11949
26. Negm Nabel A, Tawfik Salah M (2014) Characterization, surface
properties and biological activity of some synthesized anionic
surfactants. J Ind Eng Chem 20:4463–4472
7. Tehrani-Bagha AR, Singh RG, Holmberg K (2012) Solubilization
of two organic dyes by cationic ester-containing gemini surfac-
tants. J Colloid Interface Sci 376:112–118
´
27. Carpena P, Aguiar J, Bernaola-Galvan P, Ruiz CC (2002) Prob-
8. Kuliszewska E, Brecker L (2014) Gemini surfactants foam for-
mation ability and foam stability depends on spacer length.
J Surfactant Deterg 17:951–957
lems associated with the treatment of conductivity—concentra-
tion data in surfactant solutions: simulations and experiments.
Langmuir 18:6054–6058
9. Zhu HL, Hu ZY, Wang JL, Cao DL (2015) Synthesis and prop-
erties of alkyl dibenzyl ether quaternary ammonium gemini sur-
factant. Tenside Surfactants Deterg 52:163–169
28. Zana R (1996) Critical micellization concentration of surfactants
in aqueous solution and free energy of micellization. Langmuir
12:1208–1211
10. Alimohammadi MH, Javadian S, Gharibi H, Tehrani-Bagha A,
Alavijeh MR, Kakaei K (2012) Aggregation behavior and inter-
micellar interactions of cationic gemini surfactants: effects of
alkyl chain, spacer lengths and temperature. J Chem Thermodyn
44:107–115
11. Li PX, Dong CC, Thomas RK, Penfold J, Wang Yilin (2011)
Neutron reflectometry of quaternary gemini surfactants as a
function of alkyl chain length: anomalies arising from ion asso-
ciation and premicellar aggregation. Lamgmuir 27:2575–2586
12. Zhu DY, Cheng F, Chen Y, Jiang SC (2012) Preparation, char-
acterization and properties of anionic gemini surfactants with
long rigid or semi-rigid spacers. Colloid Surf A 397:1–7
13. Datta S, Biswas J, Bhattacharya S (2014) How does spacer length
of imidazolium gemini surfactants control the fabrication of 2D-
29. Nusselder JJH, Engberts JBFN (1992) Toward a better under-
standing of the driving force for micelle formation and micellar
growth. J Collide Interface Sci 148:353–361
30. Tatsumi T, Imai Y, Kawaguchi K et al (2014) Antimicrobial
activity of cationic gemini surfactant containing an oxycarbonyl
group in the lipophilic portion against gram-positive and gram-
negative microorganisms. J Oleo Sci 63(2):137–140
31. Colomer A, Pinazo A, Manresa MA, Vinardell MP, Mitjans M,
´
Infante MR, Perez L (2011) Cationic surfactants derived from
lysine: effects of their structure and charge type on antimicrobial
and hemolytic activities. J Med Chem 54:989–1002
32. Kanazawa A, Ikeda T (2000) Multifunctional tetracoordinate
phosphorus species with high self-organizing ability. Coord
Chem Rev 198(1):117–131
123

J Surfact Deterg
received her Ph.D. from Lanzhou Institute of Chemical Physics of the
Chinese Academy of Sciences. Her research interests include
separation and analysis of natural products.
33. Zhong X, Guo JW, Fu SQ, Zhu DY, Peng JP (2014) Synthesis,
surface property and antimicrobial activity of cationic gemini
surfactants containing adamantane and amide groups. J Surfac-
tant Deterg 17:943–950
Jian-long Wang is currently a Professor at the School of Chemical
Engineering and Environment, North University of China. He
received his Ph.D. from Beijing Institute of Technology. His research
interests include green chemistry, supramolecules chemistry, and
organic synthesis.
Hai-lin Zhu is a lecturer at the School of Chemical Engineering and
the Environment, North University of China. Her research interests
mainly concern the synthesis and properties of special surfactants.
Zhi-yong Hu is an associate professor at the School of Chemical
Engineering and the Environment, North University of China. He
received his Ph.D. from Dalian University of Technology in Liaoning
province of the PR China. His research interests include synthesis and
application of green surfactants and chemically modified natural
polysaccharides.
Duanlin Cao is currently a Professor at the School of Chemical
Engineering and Environment, North University of China. He
received his M.Sc. in Organic Chemistry at Nanjing University of
Technology and his Ph.D. in Applied Chemistry at Tianjin University.
His research interests include green chemistry, supramolecules
chemistry, and organic synthesis.
Xue-mei Ma is an associate professor at the School of Chemical
Engineering and the Environment, North University of China. She
123