chapter 4 gemini imidazolium surfactants: synthesis and...
TRANSCRIPT
Chapter 4
Gemini Imidazolium Surfactants: Synthesis and
their Bio-Physiochemical Studies
Section 4.1: Synthesis and Characterization of gemini
imidazolium surfactants.
Section 4.2: Evaluation of Surface properties of gemini
imidazolium surfactants.
Section 4.3: Evaluation of Thermal stability of gemini
imidazolium surfactants by thermogravimetry analysis.
Section 4.4: Evaluation of DNA binding properties of gemini
imidazolium surfactants.
Section 4.5: Evaluation of Cytotoxicity of gemini imidazolium
surfactants.
Chapter-4
100
Introduction:
In recent years, new classes of amphiphilic molecules have emerged and have attracted the
attention of several industrial and academic research groups. One of these classes is the
gemini or dimeric surfactants, which are generally made up of two hydrocarbons chains and
two headgroups linked by a rigid or flexible spacer.84
These surfactants possess better
physicochemical properties such as lower critical micelle concentration (cmc) values, higher
solubilisation power, better wetting and foaming properties than the corresponding traditional
single-chain surfactants.85
In the past decade, the dicationic quaternary ammonium gemini surfactants have been
synthesized and studied extensively.86
In recent years apart from conventional quaternary
ammonium geminis several other new categories of gemini cationics i.e. pyridinium,22
imidazolium,23
piperidinum24
and pyrrolidinum25
have been synthesized and investigated for
their surface and biological properties. It has been demonstrated that an imidazolium moiety
containing amphiphile with low toxicity has a greater scope as a synthetic vectors for gene
delivery.87
We in the present work have synthesized a new series of gemini imidazolium surfactants (9-
13) by regioselective epoxy ring-opening reaction.
84
(a) Frindi, M.; Michels, B.; Levy, H.; Zana, R. Langmuir 1994, 10, 1140-1145. (b) Zana, R.; Benrraou, M.;
Rueff, M. Langmuir 1991, 7, 1072-1075.
85 Zana, R. Adv. Colloid Interface Sci. 2002, 97, 205-253.
86 (a) Fisicaro, E.; Compari, C.; Duce, E.; Donofrio, G.; Rozycka-Roszak, B.; Wozniak, E. Biochim. Biophys.
Acta. 2005, 1722, 224-233. (b) Gaucheron, J.; Wong, T.; Wong, K. F.; Maurer, N.; Cullis, P. R. Bioconjugate
Chem. 2002, 1, 671-675.
87 (a) Huang, Q. D.; Chen, H.; Zhou, L. H.; Huang, J.; Wu, J.; Yu, X. Q. Chem. Bio. Drug Des. 2008, 71, 224-
229. (b) Zhang, Y.; Chen, X.; Lan, J.; You, J.; Chen, L. Chem. Bio. Drug Des. 2009, 74, 282-288.
Gemini Imidazolium Surfactants: Synthesis and their Bio-Physiochemical Studies
101
Section 4.1: Synthesis and characterization of gemini imidazolium
surfactants.
Result and discussion:
Five new heterocyclic gemini imidazolium surfactants having hydroxy group have been
synthesized starting from 1,2-epoxydodecane and imidazole by energy saving and cost
effective green methodology. Initially stoichiometric ratio of 1,2-epoxydodecane (1) and
imidazole (2) were reacted in the presence of catalytic amount of zinc perchlorate to get 1-
(1H-imidazol-1-yl)dodecan-2-ol (3) which were subsequently reacted with various
dibromides (4-8) to get new hydroxy group containing gemini imidazolium surfactants (9-13;
Scheme – 4.1).
The structure of these gemini surfactants (9-13) have been established by 1H,
13C, DEPT, 2D
HETCOR and 2D COSY experiments by nuclear magnatic resonance.
Chapter-4
102
Figure 4.1: 13
C and 1H Chemical shifts in δ ppm of 3,3'-(butane-1,4-diyl)bis(1-(2-
hydroxydodecyl)-1H-imidazol-3-ium) bromide (10).
Figure 4.2: 1H spectra of 3,3'-(butane-1,4-diyl)bis(1-(2-hydroxydodecyl)-1H-imidazol-3-
ium) bromide (10).
Gemini Imidazolium Surfactants: Synthesis and their Bio-Physiochemical Studies
103
The structure revealing 13
C and 1H NMR chemical shifts (δ ppm) of the gemini surfactant
(10) have been shown in Figure 4.1. The methylene protons of carbon (i.e, -N-CH2-),
directly attached to ring nitrogen and adjacent to carbon attached to hydroxy group are non-
equivalent in nature and were observed as a pair of multiplets at δ 4.09-4.15 and δ 4.28-4.31
ppm; Ha and Hb respectively. The signal for protons attached to the C-atom bearing the
hydroxyl group (i.e, -CH-OH) appeared as a multiplet at δ 3.95 ppm. The protons attached to
spacer carbon (i.e, -N+-CH2-) appeared as multiplets at δ 4.44 ppm. The signals for
imidazolium protons attached to C-4 and C-5 (i.e, -+NCHCHN-) were observed between δ
7.45 ppm and δ 7.84 ppm as two independent singlets integrating for 2 protons each. The
signal for proton attached to C-2 of imidazolium ring (i.e., -+NCHN-) appeared in the range
of δ 9.46-9.47 as a distinct singlet.
Figure 4.3: 13
C spectra of 3,3'-(butane-1,4-diyl)bis(1-(2-hydroxydodecyl)-1H-imidazol-3-
ium) bromide (10).
13C NMR spectra depicted sp
3 carbon for terminal methyl at δ 14.14 ppm. The sp
2 hybridized
carbon (i.e., –CH2-N-), directly attached to the ring nitrogen was observed at δ 55.60. This
particular carbon (i.e., –CH2-N-) was identified on the bases of DEPT-135 spectra of the
molecule and appeared as a negative signal. The signal for spacer carbon (i.e, -N+-CH2-)
directly attached to the heterocyclic positively charged imidazolium nitrogen was observed at
δ 48.99 ppm. Carbon attached to hydroxyl group (i.e, -CH-OH) was observed at δ 69.34 ppm
as a positive signal in DEPT spectra. The imidazolium ring carbons C-4 and C-5 (i.e, -
Chapter-4
104
+NCHCHN-) were observed between δ 122.68-122.91 ppm and ring carbon C-2 (i.e., -
+NCHN-) was observed at δ 136.54 ppm.
Figure 4.4: 13
C/DEPT-135 spectra of3,3'-(butane-1,4-diyl)bis(1-(2-hydroxydodecyl)-1H-
imidazol-3-ium) bromide (10).
Figure 4.5: 2D HETCOR (1H-
13C) spectra of 3,3'-(butane-1,4-diyl)bis(1-(2-
hydroxydodecyl)-1H-imidazol-3-ium) bromide (10).
Gemini Imidazolium Surfactants: Synthesis and their Bio-Physiochemical Studies
105
The assignment of signals in 1H and
13C/DEPT-135 NMR spectra has been done on the basis
of 1H-
13C 2D HETCOR (Figure - 4.5) and
1H-
1H
2D COSY (Figure - 4.6) NMR spectra of
gemini surfactant (10). It can be clearly seen from the 1H-
13C
2D HETCOR NMR spectra of
gemini imidazolium surfactant 10 that the proton attached to C-2 of the imidazolium ring
was strongly deshielded and is attached to carbon at δ 136.54 (-+NCHN-). The methylene
protons of carbon directly attached to nitrogen of imidazolium ring adjacent to carbon
attached to hydroxy group (i.e. -NCHaHb-, observed at δ 55.60) are non-equivalent in nature
and each protons gives two independent signal. Similarly, the signal for methylene protons
directly attached to positively charged nitrogen of imidazolium ring (i.e. –N+CH2-, observed
at δ 55.60 in 13
C spectra) were found to give an independent signal as broad singlet in case of
gemini imidazolium surfactant 10 integrating for four protons.
Figure 4.6: 2D COSY (1H-
1H) spectra of 3,3'-(butane-1,4-diyl)bis(1-(2-hydroxydodecyl)-
1H-imidazol-3-ium) bromide (10).
The 1H-
1H
2D COSY NMR spectra of gemini imidazolium surfactant 10 (Figure - 4.6)
further provided comprehensive information about the structure of gemini imidazolium
surfactant which enabled us to solve the complicated structure with much ease. The point of
entry for solving the spectra was methine proton directly attached to carbon at δ 3.95 ppm.
Beginning from this centre at the diagonal and tracing either directly to left or directly down,
same results were evident as the spectrum is symmetrical and intersection point shows three
Chapter-4
106
cross peaks. By drawing lines through these cross peaks at right angle it was evident that the
methine proton was coupled with four adjacent protons. These protons are pairs of
nonequivalent methylene protons attached to carbon directly attached to nitrogen of
imidazolium ring and methylene protons adjacent to methine proton of alkyl chain length as
evident from 2D HETCOR spectra of the same molecule. Similarly, the methylene protons of
spacer attached to positively charged nitrogen of the imidazolium ring were found to couple
with adjacent methylene protons of the spacer units.
The formation of these gemini imidazolium surfactants have further been established by ESI-
MS (positive ion) mass spectroscopy. The parent ion peak for gemini surfactants have been
observed for mono positive ion, where direct loss of a bromide ion from the molecule led to
the formation of positively charged parent ion [(M-Br-)]
+. The [(M-Br
-)]
++1 and [(M-Br
-)]
++2
ions were also observed in each case. In case of surfactants 11 and 13 the peak corresponding
to the loss of both the bromide ions was also observed.
Experimental:
Materials and Methods: 1,2-Epoxydodecane, 1,3-dibromopropane, 1,4-dibromobutane, 1,5
dibromopentane, 1,6-dibromohexane, 1,8-dibromooctane, zinc perchlorate hexahydrate and
ethidium bromide were purchased from Sigma Aldrich, USA and were used without any
purification. Imidazole was purchased from Central Drug House (New Delhi, India).
Millipore water was used in all experiments.
Infrared (IR) spectra were recorded as a thin neat film on a Fourier transform infrared (FT-
IR) instrument (Model 8400s, Shimadzu, Kyoto, Japan). Mass spectra were recorded on
Waters Q-Tof Micromass equipment using ESI as ion source. 1H and
13C NMR spectra were
recorded either on AL-300 (JEOL, Japan) FT-NMR (300 MHz) system or a BRUKER
AVANCE II (Switzerland), FT- NMR (400 MHz) system as a solution in CDCl3, using
tetramethylsilane (TMS) as an internal standard.
Synthesis and Characterisation: In a typical procedure 1,2- Epoxydodecane (1; 11.04g,
60mmol), was reacted with imidazole (2; 4.08g, 60mmol) in the presence of a catalytic
amount of zinc perchlorate (Scheme - 4.1). After addition, the reaction was stirred for one
hour at 80 °C under solvent free condition. The progress of the reaction was monitored by
thin layer chromatography [Silica gel G coated (0.25mm thick) glass plates, using
hexane:ethyl acetate (in a ratio of 90:10 or 85:15) as the mobile phase; the spots were
Gemini Imidazolium Surfactants: Synthesis and their Bio-Physiochemical Studies
107
visualised by iodine]. The reaction was completed in one hour. The reaction mixture was
dissolved in 100ml of chloroform and filtered to recover the catalyst. The catalyst was reused
3-5 times without any loss in its activity. The chloroform layer was transferred to a separating
funnel and washed twice with water, followed by saturated solution of sodium chloride.
Chloroform was removed from the crude reaction mixture under reduced pressure in a rotary
flash evaporator at 40 °C. It was then allowed to cool. Purification of 1-(1H-imidazol-1-
yl)dodecan-2-ol (3, white crystalline solid, 13.11g, 87% yield) was done by recrystallization
in hexane. The 1-(1H-imidazol-1-yl)dodecan-2-ol (3; 1.512 g, 6mmol) was reacted with
various dibromides [1,3-dibromopropane (4; 0.606g, 3mmol), 1,4-dibromobutane (5; 0.648g,
3mmol), 1,5-dibromopentane (6; 0.690g, 3mmol), 1,6-dibromohexane (7; 0.732g, 3mmol)
and 1,8-dibromooctane (8; 0.816g, 3mmol)] at 80 °C for 30 minutes. The resulting crude
mixtures were cooled to 25 °C. The product was washed thrice with 50ml of diethyl ether and
cold precipitated in acetone to get respective gemini imidazolium surfactants (9-13). The
structures of all these products were confirmed by IR, NMR and mass spectroscopy.
1-(1H-imidazol-1-yl)dodecan-2-ol (3). White crystalline solid; 300 MHz 1H NMR (CDCl3,
TMS): δ (ppm) 0.86-0.89 (t, 3H, terminal CH3), 1.26-1.37 (br. s, 16H, chain CH2), 1.43-1.54
(m, 2H, CH2 α to CH-OH), 3.76-3.82 (dd, 2H, -CHaHb-N and -CH-OH), 3.91-3.97 (dd, 1H, -
CHaHb-N), 5.20 (br. s, 1H, OH), 6.84-6.88 (d, 2H, –NCHCHN-), 7.28-7.32 (s, 1H, –NCHN-).
75 MHz 13
C/DEPT-135 NMR (CDCl3): δ (ppm) 14.13 (+ve, terminal CH3), 22.69-34.58 (-
ve, CH2 chain), 53.65 (-ve, -CH2-N), 70.48 (+ve, -CH-OH), 119.71 (+ve, –NCHCHN-),
128.29 (+ve, –NCHCHN-), 137.46 (+ve, –NCHN-). IR (cm−1
) neat: 3408, 3230, 2919, 2850,
1739, 1647, 1563, 1459, 1350, 1237, 1108, 763. MS m/z (parent ions): 253 and 254
(M++1and M
++2).
3,3'-(Propane-1,3-diyl)bis(1-(2-hydroxydodecyl)-1H-imidazol-3-ium) bromide (9). White
paste; 300 MHz 1H NMR (CDCl3, TMS): δ (ppm) 0.86-0.90 (t, 6H, terminal 2 х CH3), 1.26
(br. s, 32H, chain 2 х CH2-chain), 1.51 (m, 4H, 2 х CH2 α to CH-OH), 2.57-2.69 (m, 4H, CH2
spacer and H2O molecule), 3.98 (m, 2H, 2 х -CH-OH), 4.07-4.14 (m, 2H, 2 х -CHaHb-N),
4.26-4.31 (m, 2H, 2 х -CHaHb-N), 4.57 (s, 4H, 2 х -CH2-N+), 4.84 (br. s, 2H, 2 х OH), 7.45
(s, 2H, -NCHCHN+), 7.95 (s, 2H, -NCHCHN
+), 9.51 (s, 2H, 2 х -NCHN
+). 75 MHz
13C/DEPT-135 NMR (CDCl3): δ (ppm) 13.98 (+ve, 2 х -CH3 (terminal)), 22.56-34.48 (-ve,
-CH2 chain length and spacer), 46.59 (-ve, 2 х -CH2-N+), 55.65 (-ve, 2 х -CH2-N), 69.12
(+ve, 2 х -CH-OH), 122.60-123.07 (+ve, 2 х -NCHCHN+), 136.58 (+ve, 2 х –NCHN
+). IR
Chapter-4
108
(cm−1
) neat: 3462, 3299, 2922, 2851, 1739, 1643, 1568, 1452, 1348, 1242, 1100, 747. MS
positive ions m/z (for C33H62BrN4O2+): 625.5 (Base peak), 626.4, 627.5, 628.5.
3,3'-(Butane-1,4-diyl)bis(1-(2-hydroxydodecyl)-1H-imidazol-3-ium) bromide (10). White
paste; 300 MHz 1H NMR (CDCl3, TMS): δ (ppm) 0.86-0.89 (t, 6H, terminal 2 х CH3),
1.25-1.29 (br. s, 32H, chain 2 х CH2), 1.49 (br. s, 4H, 2 х CH2 α to CH-OH), 2.04 (br. s, 4H,
CH2 spacer chain), 3.34 (s, 4H, 2 х H2O), 3.95 (br. s, 2H, 2 х -CH-OH), 4.09-4.15 9 (m, 2H,
2 х -CHaHb-N), 4.28-4.31 (m, 2H, -2 х CHaHb-N), 4.44 (s, 4H, 2 х -CH2-N+), 4.89 (br. s, 2H,
2 х OH), 7.45 (s, 2H, -2 х NCHCHN+), 7.84 (s, 2H, 2 х -NCHCHN
+), 9.46-9.47 (s, 2H, 2 х -
NCHN+). 75 MHz
13C/DEPT-135 NMR (CDCl3): δ (ppm) 14.14 (+ve, 2 х CH3 (terminal)),
22.71-34.58 (-ve, CH2 chain length and spacer), 48.99 (-ve, 2 х -CH2-N+), 55.60 [-ve, 2 х -
CH2-N], 69.34 [+ve, 2 х -CH-OH], 122.68-122.91 [+ve, 2 х -NCHCHN+)], 136.54 [+ve, 2 х -
NCHN+). IR (cm
−1) neat: 3400, 3262, 2912, 2886, 1746, 1662, 1567, 1461, 1356, 1240,
1112, 753. MS positive ions m/z (for C34H64BrN4O2+): 639.4 (Base peak), 640.4, 641.5,
642.5.
3,3'-(Pentane-1,5-diyl)bis(1-(2-hydroxydodecyl)-1H-imidazol-3-ium) bromide (11).
White paste; 300 MHz 1H NMR (CDCl3, TMS): δ (ppm) 0.86-0.89 (t, 6H, terminal 2 х
CH3), 1.20-1.35 (br. s, 32H, chain 2 х CH2), 1.41-1.52 (m, 6H, 2 х CH2 α to CH-OH and CH2
spacer chain), 1.99-2.02 (br. s, 4H, CH2 spacer chain), 2.95 (s, 2H, H2O), 3.99 (br. s, 2H, 2 х -
CH-OH), 4.15-4.22 (m, 2H, 2 х -CHaHb-N), 4.33-4.41 (m, 6H, 2 х -CHaHb-N and 2 х -CH2-
N+), 4.79-4.80 (br. s, 2H, 2 х OH), 7.48 (s, 2H, 2 х -NCHCHN
+), 7.77-7.79 (s, 2H, 2 х -
NCHCHN+), 9.69-9.72 (s, 2H, 2 х -NCHN
+). 75 MHz
13C/DEPT-135 NMR (CDCl3): δ
(ppm) 14.13 (+ve, 2 х CH3 (terminal)), 22.38-34.67 (-ve, CH2 chain length and spacer),
49.41 (-ve, 2 х -CH2-N+), 55.44 (-ve, 2 х -CH2-N), 69.23 (+ve, 2 х -CH-OH), 122.28-122.38
(+ve, 2 х -NCHCHN+), 122.99-123.06 [+ve, 2 х -NCHCHN
+), 136.77 [+ve, 2 х -NCHN
+).
IR (cm−1
) neat: 3387, 3253, 2919, 2877, 1748, 1657, 1575, 1437, 1329, 1208, 1147, 733.
MS positive ions m/z (for C35H66BrN4O2+): 653.4 (Base peak), 654.4, 655.4, 656.4, 573.5,
574.5.
3,3'-(Hexane-1,6-diyl)bis(1-(2-hydroxydodecyl)-1H-imidazol-3-ium) bromide (12). White
paste; 300 MHz 1H NMR (CDCl3, TMS): δ (ppm) 0.85-0.88 (t, 6H, terminal 2 х CH3), 1.25
(br. s, 36H, chain 2 х CH2 and CH2 spacer chain), 1.45-1.53 (m, 4H, 2 х CH2 α to CH-OH),
1.98 (m, 6H, CH2 spacer chain and H2O), 4.02 (m, 2H, 2 х -CHaHb-N), 4.29-4.37 (m, 8H, 2 х
-CH-OH, 2 х CHaHb-N and 2 х -CH2-N+), 4.73-4.78 (m, 2H, 2 х OH), 7.38 (s, 2H, 2 х -
Gemini Imidazolium Surfactants: Synthesis and their Bio-Physiochemical Studies
109
NCHCHN+), 7.57 (s, 2H, 2 х -NCHCHN
+), 9.83 (s, 2H, 2 х -NCHN
+). 75 MHz
13C/DEPT-
135 NMR (CDCl3): δ (ppm) 14.39 (+ve, 2 х CH3 (terminal)), 22.96-34.85 (-ve, CH2 chain
length and spacer), 49.77 (-ve, 2 х -CH2-N+), 55.67 (-ve, 2 х -CH2-N), 69.64 (+ve, 2 х -CH-
OH), 122.51 (+ve, 2 х -NCHCHN+), 123.33 (+ve, 2 х -NCHCHN
+), 136.99 (+ve, 2 х -
NCHN+). IR (cm
−1) neat: 3430, 3287, 2867, 2823, 1742, 1650, 1557, 1432, 1326, 1248,
1134, 787. MS positive ions m/z (for C36H68BrN4O2+): 667.4, 668.4, 669.4, 670.4, 587.5,
588.5, 202.2 (Base peak).
3,3'-(Octane-1,8-diyl)bis(1-(2-hydroxydodecyl)-1H-imidazol-3-ium) bromide (13). White
paste; 300 MHz 1H NMR (CDCl3, TMS): δ (ppm) 0.87-0.89 (t, 6H, terminal 2 х CH3),
1.26-1.52 (m, 44H, chain 2 х CH2, 2 х CH2 α to CH-OH and CH2 spacer chain), 1.96-2.04 (m,
8H, CH2 spacer chain and 2 х H2O), 4.00 (m, 2H, 2 х -CH-OH), 4.33-4.40 (m, 8H, 2 х -
CHaHb-N, 2 х CHaHb-N and 2 х -CH2-N+), 4.73 (br. s, 2H, 2 х OH), 7.45-7.51 (m, 4H, 2 х -
NCHCHN+), 9.83 (s, 2H, 2 х -NCHN
+). 75 MHz
13C/DEPT-135 NMR (CDCl3): δ (ppm)
14.03 (+ve, 2 х CH3 (terminal)), 22.58-34.39 (-ve, CH2 chain length and spacer), 49.72 (-ve,
2 х -CH2-N+), 55.26 (-ve, 2 х -CH2-N), 69.29 (+ve, 2 х -CH-OH), 121.77 (+ve, 2 х -
NCHCHN+), 123.14 (+ve, 2 х -NCHCHN
+), 136.71 (+ve, 2 х -NCHN
+). IR (cm
−1) neat:
3396, 3215, 2907, 2857, 1749, 1657, 1573, 1448, 1334, 1246, 1143, 744. MS positive ions
m/z (for C38H72BrN4O2+): 695.4, 696.5, 697.4, 698.5, 615.5, 616.5, 308.3 (Base peak).
Chapter-4
110
Section 4.2: Evaluation of surface properties of gemini imidazolium
surfactants.
Result and discussion:
a) Self-Aggregation Studies in Aqueous Solution: The surface properties of the gemini
imidazolium surfactants has been determined by surface tension measurements. Figure – 4.7
shows the surface tension (γ) versus log of concentration (C) plots for five gemini
imidazolium surfactants at 25 °C. The surface tension initially decreases with increasing
concentration of surfactants and then reaches a plateau region, indicating that micelles are
formed. The concentration corresponding to the break point is the critical micelle
concentration (cmc). The cmc values of these surfactants increases with the elongation of
spacer length. The cmc values as determined by surface tension were found to be lower than
that obtained by conductivity method; however, the trend of increasing cmc values with the
elongation of spacer length remained the same.
Rosen et al88
found different cmc values by two different techniques for a series of N-acyl-β-
alaninate gemini surfactants. Similar results have also been obtained by Pinazo et al89
for
arginine-based gemini surfactants and Esumi et al90
for trimeric surfactants. This behaviour
has also been discussed in detail by Fisicaro et al22d
for gemini pyridinium surfactants and
has been attributed to the formation of non-surface-active premicellar aggregates by
surfactants. Furthermore, the plot of Λ vs C0.5
also indicates the existence of such premicellar
aggregates for gemini imidazolium surfactants (9-13), where there is a significant difference
in the determination of the cmc values by surface tension and conductivity.
We have found very peculiar behaviour of the gemini surfactants (9-13) being reported in the
current study. It has been observed that the solution of the surfactant takes 30 min to stabilize
after being transferred from a volumetric flask to a thermostated vessel before the set of five
successive concordant readings can be recorded. Furthermore, the solution needs to be aged
for at least 24 h prior to evaluation at a constant temperature of 25 °C to get uniform results.
The initial sets of reading needs to be completely ignored and data obtained after the
stabilization of the surfactant solution for 30 min in a thermostated vessel is considered to be
accurate.
88
Tsubone, K.; Arakawa, Y.; Rosen, M. J. J. Colloid Interface Sci. 2003, 262, 516-524. 89
Pinazo, A.; Wen, X.; Perez, L.; Infante, M. R.; Franses, E. I. Langmuir 1999, 15, 3134-3142. 90
Yoshimura, T.; Yoshida, H.; Ohna, A.; Esumi, K. J. Colloid Interface Sci. 2003, 267, 167-172.
Gemini Imidazolium Surfactants: Synthesis and their Bio-Physiochemical Studies
111
The maximum surface excess concentration at the air/water interface91
Γmax, has been
calculated by applying the Gibbs adsorption isotherm (eq 4.1).
max1
2.30 logT
d
nRT d C
(4.1)
Here, γ denotes the surface tension, R is the gas constant, T is the absolute temperature, and
C is the surfactant concentration. Recent studies have been carried out by assuming that one
counterion is associated with the ionic headgroup, and value of n was taken to be 2.22c
The
value of n = 2 has been supported by the results obtained with neutron reflectivity studies.92
However, previous investigations on gemini imidazolium surfactants have been carried out
by assuming a value of n = 3, considering a divalent surfactant ion and two univalent
counterions.23d
Therefore, it becomes essential to calculate the value of Γmax by assuming the
value of n = 2 as well as n = 3.
25
30
35
40
45
50
55
60
-4.2 -4 -3.8 -3.6 -3.4 -3.2 -3 -2.8 -2.6
Gemini Surfactant 9Gemini Surfactant 10Gemini Surfactant 11Gemini Surfactant 12Gemini Surfactant 13
m
Nm
-1
LogC (mol/L)
Figure 4.7: Surface tension vs logC plot for gemini imidazolium surfactants.
The area occupied per surfactant molecule (Amin) at the air-water interface91a
has been
obtained by using eq 4.2
Amin = 1/N Γmax (4.2)
91
(a) Alami, E.; Beinert, G.; Marie, P.; Zana, R. Langmuir 1993, 9, 1465−1467. (b) Song, L. D.; Rosen, M. J.
Langmuir 1996, 12, 1149-1153. 92
Li, Z. X.; Dong, C. C.; Thomas, R. K. Langmuir 1999, 15, 4392-4396.
Chapter-4
112
Where N is Avogadro’s number and Amin is in nm2
(Table 4.1). Gemini imidazolium
surfactants (9-13) reported in the present studies have been found to be have lower Amin value
as compared to those of other gemini cationic surfactants,88,22d
including gemini imidazolium
surfactants23b,23d
reported previously. Because of the low Amin values, these new gemini
imidazolium surfactants have a greater tendency to form micelles instead of adsorbing at the
air-water interface. Unlike, the gemini pyridinium surfactants,22d
Amin values of these new
gemini imidazolium surfactants increase with increasing spacer length. Such a pattern of
increase in Amin values with increasing spacer length was also observed by Ao et al23d
for
gemini imidazolium surfactants and Zana et al91a
for gemini quaternary ammonium
surfactants.
Initially, lower Amin values were solely attributed to a tighter packing of the longer
hydrophobic chains at the interface.91b,93
However, recent studies by Fisicaro et al22d
revealed
that surfactant molecules having lower Amin value may have a greater tendency to form
premicellar aggregates instead of adsorpting at the air/water interface. A theoretical
explanation suggested that the dominant factor responsible for the variation in Amin values of
the surfactants is size of the hydrophilic headgroup and the solvation of the imidazolium
cation in water.94
The affinity to reduce surface tension (γcmc) and the ability to reduce the surface tension by 20
mNm-1
(C20) for these gemini imidazolium surfactants have also been calculated from the
plot of the decrease in surface tension versus the log of concentration (Table 4.1). The γcmc
values of these gemini surfactants were found to increase with increasing length of the spacer
units with the exception of gemini imidazolium surfactant 10. The trend of increasing surface
tension attained at the cmc for this series of gemini imidazolium surfactants can be explained
on the basis of cmc/c20 ratio observed for these surfactants. The ability of a particular
surfactant to reduce the surface tension depends upon the cmc/C20 ratio. Higher the observed
cmc/c20 ratio of a surfactant more is the tendency to reduce the surface tension of the system.1
Thus, gemini imidazolium surfactant 10 has the maximum ability to reduce surface tension of
the aqueous system in the series of gemini surfactants being reported.
The Gibbs free energy of micellization (ΔG°mic) has been calculated with the following
equation23b
93
Rosen, M. J.; Mathias, J. H.; Davenport, L. Langmuir 1999, 15, 7340−7346. 94
Anouti, M.; Jones, J.; Boisset, A.; Jacquemin, J.; Caillon-Caravanier, M.; Lemordant, D. J. Colloid Interface
Sci. 2009, 340, 104-111.
Gemini Imidazolium Surfactants: Synthesis and their Bio-Physiochemical Studies
113
ΔG°mic = RT (0.5 + β) ln Xcmc (4.3)
Where Xcmc is the molar fraction of the cmc and Xcmc = cmc/55.4, where cmc is in mols/L
and 55.4 comes from 1 L of water corresponding to 55.4 mols of water at 25 °C. β is the
degree of counterion binding to micelles (discussed later).
Similarly, the Gibbs free energy of adsorption (ΔG°ads) has been calculated with the
following equation:95
cmcads mic
πΔG° = ΔG°
Γ (4.4)
Here, πcmc denotes the surface pressure at the cmc (Πcmc = γo – γcmc, where γo and γcmc are the
surface tensions of water and the surfactant solution at the cmc, respectively).
Table 4.1: Surface Properties of Gemini imidazolium surfactants (9-13) as determined
by Surface Tension and Conductivity measurements.
Surfa
-ctant
CMCa
mM
CMCb
mM β
γ
mN/m
106Γmax
mol/m2
Amin
nm2
C20*10-4 CMCa/
C20
ΔG°mic
KJ/mol
ΔG°ads
KJ/mol
TK
(°C)
9 0.72 ± 0.01
1.37 ± 0.01
0.75 30.0 ±
0.3
2.53 ±
0.06 (1.69 ±
0.04)
0.65 ±
0.02 (0.98 ±
0.02)
1.23 5.8 -32.85 ±
0.22 -49.67 ±
0.57 22.0
10 0.76 ±
0.01
1.40 ±
0.01 0.70
28.1 ±
0.3
2.33 ± 0.09
(1.55 ±
0.06)
0.71 ± 0.03
(1.07 ±
0.04)
0.77 9.8 -31.63 ±
0.21
-50.63 ±
0.82 23.3
11 1.02 ±
0.01
1.47 ±
0.01 0.84
32.9 ±
0.4
2.29 ±
0.05
(1.52 ± 0.03)
0.72 ±
0.02
(1.09 ± 0.02)
1.90 5.3 -35.21 ±
0.22
-52.46 ±
0.56 20.5
12 1.07 ±
0.01
1.57 ±
0.01 0.74
35.2 ±
0.2
2.98 ±
0.02
(1.98 ± 0.02)
0.55 ±
0.01`
(0.83 ± 0.01)
3.63 2.9 -32.31 ±
0.19
-44.78 ±
0.20 28.6
13 1.14 ± 0.01
1.59 ± 0.01
0.70 37.6 ±
0.3
1.90 ±
0.02 (1.27 ±
0.02)
0.87 ±
0.01 (1.30 ±
0.02)
2.63 4.3 -31.14 ±
0.18 -49.45 ±
0.36 20.6
aCmc, from surface tension and
bCmc from conductivity; β, degree of counterion association; γcmc, the
surface tension at the cmc; Γmax, the maximum surface excess concentration; Amin, the area per molecule
at the interface; C20, the surfactant concentration required to reduce the surface tension of the solvent by
20 mN/m; ΔGºmic, Gibbs free energy of micellization; ΔGºads, Gibbs free energy of adsorption; Cmca/C20,
cmc from surface tension/C20; TK, Krafft point. The values in parentheses are for n = 3.
The results of the present study demonstrate a small energy gap between ΔG°mic and ΔG°ads
in individual gemini imidazolium surfactants. A recent study has shown that the smaller the
gap between these parameters, the greater the tendency of individual surfactants to aggregate
95
Yoshimura, T.; Ohna, A.; Esumi, K. Langmuir 2006, 22, 4643-4648.
Chapter-4
114
in solution rather than to adsorb at the air/water interface.96
A direct relation between the
calculated Amin value and the energy difference between ΔG°mic and ΔG°ads has been
observed for gemini imidazolium surfactants. The smaller the Amin value, the smaller the
energy difference between ΔG°mic and ΔG°ads. Similar results have also been observed by
Bhadani & Singh.76
b) Critical Micelle Concentration (cmc) and Degree of Counterion Binding: The cmc
values of the gemini imidazolium surfactants ‘9-13’ have also been evaluated by conductivity
method, and it has been observed that these values follow a similar trend of increasing cmc
values with increasing spacer length as observed by surface tension measurements. However,
the determined cmc values differ significantly by two different techniques. Compared to
gemini pyridinium surfactants reported earlier by Quagliotto et al22b
and Zhao et al,22c
the
cmc values of the gemini surfactants (9-13) increase with increasing spacer length. Such a
trend of increasing cmc values with increasing spacer length has also been observed
previously for the gemini quaternary ammonium surfactant91a
and gemini imidazolium
surfactants.23d
Another important parameter evaluated by a conductivity plot is the degree of counterion
binding (β) that signifies the ability of counterions to bind micelles. Gemini imidazolium
surfactants (9-13) show a degree of counterion binding of around 70-85% that is extremely
high for gemini cationic surfactants having a bromide counterion. Our recent studies have
shown that the degree of counterion binding (β) of gemini imidazolium surfactants76
is not
influenced by increases in the alkyl chain length and spacer units, and similar results have
also been observed in the present study.
0
100
200
300
400
500
180
200
220
240
260
280
300
320
340
0 0.5 1 1.5 2 2.5 3
0.5 1 1.5
(
S/c
m)
(S
cm2
mol-1)
C (mmol/L)
C 0.5
(mmol 0.5
L -0.5
)
cmc
C = 0.62 mM
C = 1.00 mM
Surfactant (9)
0
100
200
300
400
500
200
220
240
260
280
300
320
0 0.5 1 1.5 2 2.5
0.5 1 1.5
(S
cm2 m
ol -1)
(
S/c
m)
C (mmol/L)
C 0.5
(mmol 0.5
L -0.5
)
C = 0.59 mM
C = 0.82 mM
cmc
Gemini surfactant (10)
96
Yoshimura, T.; Bong, M.; Matsuoka, K.; Honda, C.; Endo, K. J. Colloid Interface Sci. 2009, 339, 230-235.
Gemini Imidazolium Surfactants: Synthesis and their Bio-Physiochemical Studies
115
0
100
200
300
400
500
200
240
280
320
360
0 0.5 1 1.5 2 2.5 3
0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
(
S/c
m)
(S
cm2
mol-1)
C (mmol/L)
C 0.5
(mmol 0.5
L -0.5
)
C = 0.62 mM
C = 1.08 mM
cmc
Gemini surfactant (11)
0
100
200
300
400
500
600
200
220
240
260
280
300
320
340
0 0.5 1 1.5 2 2.5 3
0.5 1 1.5
(
S/c
m)
(S
cm2 m
ol-1)
C (mmol/L)
C 0.5
(mmol 0.5
L -0.5
)
C = 0.68 mM
C = 1.23 mM
cmc
Gemini surfactant (12)
0
100
200
300
400
500
600
220
240
260
280
300
320
340
0 0.5 1 1.5 2 2.5
0.5 1 1.5
(S
/cm
)
(S cm
2 mol -1)
C (mmol/L)
C 0.5
(mmol 0.5
L -0.5
)
C = 0.68 mM
C = 1.18 mM
cmc
Gemini surfactant (13)
Figure 4.8: Specific conductivity vs concentration plot & molar conductivity vs C0.5
plot of gemini
imidazolium surfactants (9-13). The arrows indicate, from left to right, the onset of premicellar aggregate
formation and the concentrations at which the maximum and the cmc are attained, respectively. The
error estimate for the calculated value is ± 0.5%. Individual points shown with error bars represent the
mean value ± SEM.
The molar conductivity (Λ) data has been plotted against the square root of concentration
(C0.5
). From these plots (Figure 4.8) it is evident that these new gemini surfactants show
peculiar behaviour at low concentration. Gemini imidazolium surfactants (9-13) show the
occurrence of a maximum in these plots that would account for the formation of premicellar
aggregates.89,97
The existence of premicellar aggregates at low concentration has been
previously investigated by several research groups.88-90
Zana97
proposed dimer-type
premicellar aggregates, with their hydrophobic chains oriented with respect to each other,
leaving the two headgroups far apart from each other (at the edges of the dimer). Under these
conditions, the dimer is fully ionized and the conductivity of the dimer should be higher than
that of the surfactant monomers. Furthermore, Pinazo et al89
also evaluated this kind of
97
Zana, R. Colloid Interface Sci. 2002, 246, 182−190.
Chapter-4
116
behaviour and extended this discussion to the formation of oligomers, such as trimers,
tetramers, and so on. In Λ verues C0.5
plots for gemini surfactants (9-13), the arrows from left
to right indicate the onset of premicellar aggregate formation, the concentration at which the
maximum is attained, and the cmc as determined in a conductivity verues concentration plot.
At onset point, the surfactant monomers start to form premicellar aggregates, and because the
conductance of these premicellar aggregates is higher than that of surfactant monomers, they
should stay in the solution bulk and are not absorbed at the air-water interface. The maximum
in the molar conductivity plot probably came from the fact that as the concentration is further
increased oligomers larger than dimers start to form and may bind counterions (the β value is
found to be around 15-20% for oligomers), after which surfactants (9-13) form regular
micelles.
Krafft Points: The Krafft point of all of the gemini surfactants have been determined and
found to be less than 25 °C. Even though the Krafft temperature for a 1 wt% solution of
gemini surfactant (12) is around 28.6 °C, the stock solution of this surfactant at a
concentration of C = 10cmc showed no visible surfactant precipitate when stored at room
temperature for several weeks after being dissolved in water. No particular trend in the Krafft
point has been observed for the series of gemini imidazolium surfactants with respect to
increase in spacer units (Table 4.1). The krafft temperature was taken as the temperature
where the conductance versus temperature plot (T °C) showed a break (Figure 4.9). Break
usually coincided with the full clarification of the solution.
0
200
400
600
800
1000
1200
0 10 20 30 40 50
Gemini Surfactant 9Gemini Surfactant 10Gemini Surfactant 11Gemini Surfactant 12Gemini Surfactant 13
S/c
m)
T (C)
Figure 4.9: Plot of conductivity () versus temperature (T) for gemini surfactants (9-13).
The arrow indicates the krafft temperature taken from the plot.
Gemini Imidazolium Surfactants: Synthesis and their Bio-Physiochemical Studies
117
Experimental:
Surface tension measurements: The critical micelle concentration (cmc) and surface tension
attained at cmc were determined using a CSC (Central scientific Co., Inc., USA) Du Nouy
interfacial tensiometer with a platinum-iridium ring (circumference 5.992 cm) at 25.0 ± 0.1
°C. The tensiometer was calibrated using triply distilled water. Each of the surfactant
solutions was aged for 24 h prior to the determination of surface activity.98
For the
determination of cmc, an adequate quantity of a concentrated surfactant solution was added
into 20 ml of water in order to change the surfactant concentration from concentrations well
below the critical micelle concentration (cmc) to at least 2-3 times the cmc.
Conductivity Measurements: Conductivity was measured on a model EQ661 Equip-Tronics
auto temperature conductivity meter equipped with a conductivity cell. The aqueous solutions
were thermostated in the cell at 25.0 ± 0.1 °C. For the determination of cmc, an adequate
quantity of a concentrated surfactant solution was added into 25 ml of water in order to
change the surfactant concentration from concentrations well below the critical micelle
concentration (cmc) to at least 2-3 times the cmc. The degree of counterion binding (β) has
been calculated as (1-α), where α = Smicellar/Spremicellar (i.e., ratio of the slope after and before
cmc98
).
Krafft Point Measurements: The Krafft temperatures of gemini surfactants (9-13) were
determined using surfactant solutions of concentration 1 wt% (i.e., well above the cmc of the
investigated gemini surfactants) using the electrical conductivity method.22b
Each of the
surfactant was dissolved in water and then left in a refrigerator at a temperature of 1.5 °C for
1 day until precipitation occurred. The precipitated surfactant solution thus obtained was
introduced in the conductivity cell to measure the Krafft point.
98
Bordes, R.; Tropsch, J.; Holmberg, K. Langmuir 2010, 26, 3077-3083.
Chapter-4
118
Section 4.3: Evaluation of thermal stability of gemini imidazolium surfactants
by thermogravimetry analysis.
Result and discussion:
Gemini imidazolium surfactants (9-13) have been synthesized as their monohydrate or
dihydrate salts. The water of hydration of these gemini surfactants has been determined by
thermogravimetric analysis (TGA). The observed loss in weight due to the presence of water
molecules in the gemini surfactant corresponds to the signal in the 1H NMR having the exact
integration for water molecules. Thermal stability measurement shows that these gemini
surfactants are stable up to 310 °C. Figure 4.10(a) shows a characteristic curve for the
decomposition of the gemini surfactants as measured by thermal gravimetric analysis. The
onset temperature (Tonset) is the intersection of the baseline weight, either from the beginning
of the experiment and from the tangent of the weight versus temperature curve as
decomposition occurs. The starting temperature (Tstart) is the temperature at which the
decomposition of the sample begins (Figure 4.10(b)).99
The onset and starting temperatures
for the present gemini imidazolium surfactants are listed in Table 4.2.
0
20
40
60
80
100
50 100 150 200 250 300 350 400 450
Wei
gh
t (%
)
Temperature oC
(a)
75
80
85
90
95
100
0 50 100 150 200 250 300 350 400
Wei
gh
t (%
)
Temperature oC
Initial weight of
Surfactant = 19.308 mg
Percent weight loss
2.480 % (0.479 mg)
For loss of one
water moleculeStart temperature of
degradation (Tstart
)
Onset temperature of
degradation (Tonset
)
(b)
Figure 4.10: (a) TGA graph of Gemini surfactant (9) and (b) magnified TGA graph of surfactant (9)
indicating the loss of water molecules, with the start temperature of degradation and onset temperature
of degradation.
99
Fredlake, C. P.; Crosthwaite, J. M.; Hert, D. G.; Aki, S. N. V. K.; Brennecke, J. F. J. Chem. Eng. Data 2004,
49, 954-964.
Gemini Imidazolium Surfactants: Synthesis and their Bio-Physiochemical Studies
119
Thermal stability measurements designated that these new gemini surfactants have better
thermal stability. Gemini imidazolium surfactant 9 was found to be the most thermally stable
surfactant, having a Tstart of 285.5 °C and a Tonset of 308.8 °C whereas surfactant 13 was
found to be the least thermally stable surfactant, having a Tstart of 249.1 °C and a Tonset of
279.7 °C among the imidazolium geminis. Furthermore, it has also been found that thermal
stability of these gemini surfactants decreases with increasing spacer length.
Table 4.2: Onset and Starting Temperatures for the Thermal Decomposition of Gemini
Imidazolium Surfactants.
Temperature (°C) Surfactant (9) Surfactant (10) Surfactant (11) Surfactant (12) Surfactant (13)
Tonset 308.8 298.7 288.1 287.1 279.7
Tstart 285.5 274.4 273.3 269.1 249.1
Experimental:
Thermal stability Measurements: The thermal stability of the gemini surfactants was
measured with an SDT Q600 thermal gravimetric analyzer (TGA) using a nitrogen
atmosphere. Thermograms were recorded using a heating rate of 5 °C /min from 25 to 400
°C. The experiments were carried out on an alumina sample pan by using a nitrogen flow rate
of 100Ml/min. The water of hydration and thermal stability of the gemini imidazolium
surfactants (9-13) were determined from a TGA graph.
Chapter-4
120
Section 4.4: Evaluation of DNA binding properties of gemini imidazolium
surfactants.
Result and discussion:
a) Agarose Gel Electrophoresis: The DNA binding capability of gemini imidazolium
surfactants (9-13) and the reference, conventional quaternary ammonium gemini surfactant
12-2-12, have been investigated by agarose gel electrophoresis. It has been observed that all
gemini imidazolium surfactants were able to bind plasmid DNA at low concentration. All
gemini surfactants (9-13) were able to retard the migration of DNA towards positive
electrode at a concentration of 50 µM (Figure 4.11).
Figure 4.11: Agarose gel electrophoresis of pDNA and gemini surfactants at different concentrations.
The interaction of pDNA with imidazolium surfactants takes place at an even lower
concentration than 25 µM because these surfactants are able to replace ethidium bromide
from DNA in ethidium bromide exclusion experiments (discussed later) at a lower
concentration than 25 µM. However, effective binding occur at a concentration between 25 to
Gemini Imidazolium Surfactants: Synthesis and their Bio-Physiochemical Studies
121
50 µM because there is complete neutralization of the partial negative charge of pDNA via
the formation of a stable complex, which is evident from the retardation observed in gel
electrophoresis and the complete displacement of EB in exclusion experiments.
Because all of the imidazolium surfactants were able to bind pDNA to similar extents, it can
be concluded that the increase in the spacer length plays little if any role in their binding with
pDNA. The observed results were found to be in accordance with recent literature
reports.76,22e
This may be attributed to the fact that such molecules have a greater degree of
flexibility as compared to other gemini surfactants and can bind the oppositely charged sites
with ease.
b) Ethidium Bromide Exclusion Experiment: The DNA binding capability of gemini
imidazolium surfactants (9-13) has been further confirmed by EB exclusion experiments
using fluorescence spectroscopy. The fluorescence emission of EB is enhanced as a result of
intercalation between the DNA base pairs relative to that in water.100
The extent of binding of
a particular surfactant can be determined by its ability to displace EB from the DNA-EB
intercalated complex, hence causing a quenching in fluorescence intensity.101
Figure 4.12: Displacement of ethidium bromide from the pDNA-EB complex by gemini imidazolium
surfactants at different charge ratios.
100
(a) Lleres, D.; Clamme, J. P.; Dauty, E.; Blessing, T.; Krishnamoorthy, G.; Duportail, G.; Mely, Y. Langmuir
2002, 18, 10340-10347. (b) Rodriguez-Pulido, A.; Aicart, E.; Junquera, E. Langmuir 2009, 25, 4402-4411. 101
Barreleiro, P. C.; Lindman, B. J. Phys. Chem. B 2003, 107, 6208-6213.
Chapter-4
122
Figure 4.12 shows the tendency of gemini imidazolium surfactants (9-13) to displace EB
from DNA-EB intercalated complex with increasing N/P charge ratio. The results of the
experiment show that these gemini surfactants have an excellent binding capability. It has
been found that gemini imidazolium surfactant 9 has the maximum ability to displace EB
from DNA because it displaces about 82.31% of EB at an N/P charge ratio 2.0, wheares
80.97% EB was displaced by surfactant 11 at same N/P charge ratio. Gemini imidazolium
surfactants 10 and 12 have the weakest capability to displace EB from pDNA compared to
compounds in the same homologous series. Gemini surfactant 13 causes a significant
decrease in fluorescence intensity at a low N/P charge ratio of 1.0 to 1.25, but at a higher
charge ratio, the smallest displacement of EB from the DNA-EB complex is observed.
Therefore, it can be attributed that at a higher N/P charge ratio, DNA becomes saturated with
surfactant and the exclusion of EB no longer occurs for surfactant 13.
Experimental:
Materials and Methods: Agarose and Tris buffer were purchased from Sisco Research
Laboratory Pvt Ltd. (Mumbai, India). Plasmid DNA pUC 18 was purchased from Bangalore
GeNei (Bangalore, India). Millipore water was used in all experiments.
Agarose Gel Electrophoresis: pDNA (166 ng/well) and 10 μL of 12.5, 25, 50, and a 100 μM
gemini imidazolium surfactant (9-13) solution were loaded with 5 μL of glycerol into 1%
agarose gel containing 2 μL of ethidium bromide (0.5 mg/mL). Electrophoresis was carried
out at 100 V in Tris buffer for 30 min. The DNA band was visualized under UV
transillumination with an Alpha Imager HP (Alpha Innotech Corporation, U.S.). Photographs
were taken using the Alpha Imager.76
Ethidium bromide exclusion: A 2 μL solution of 0.25 mM of EB was mixed with 3 mL of
Millipore water, and the fluorescence spectra of water-EB were recorded in the absence of
pDNA and in the presence of pDNA (2 g) from 530 to 700 nm at an excitation wavelength
(λex) of 490 nm using a Perkin-Elmer LS 55 Fluorescence spectrophotometer. Fifteen
microliters of a 50 μM solution of gemini surfactants was added 12 times to a pDNA-EB
intercalated system to obtain 12 observations. The percentage of quenching observed from
the replacement of EB by cationic gemini surfactants from the pDNA upon interaction with
the cationic surfactants was calculated according to (I0 I)/(I0 IEB) x 100, where I0 and IEB
Gemini Imidazolium Surfactants: Synthesis and their Bio-Physiochemical Studies
123
are the fluorescence intensities of free and pDNA-bound EB, and I is the fluorescence
intensity in the presence of different amounts of surfactants.102
102
(a) Santhiya, D.; Maiti, S. J. Phys. Chem. B 2010, 114, 7602-7608. (b) Nisha, C. K.; Manorama, S. V.;
Ganguli, M.; Maiti, S.; Kizhakkedathu, J. N. Langmuir 2004, 20, 2386-2396.
Chapter-4
124
Section 4.5: Evaluation of cytotoxicity of gemini imidazolium surfactants.
Result and discussion:
Few reports are available regarding the cytotoxic effects of gemini imidazolium
surfactants.22e
The cytotoxicity of gemini imidazolium surfactants (9-13) has been assessed
on C6 glioma cells and compared to that of reference conventional quaternary ammonium
gemini surfactant 12-2-12. A recent report103
demonstrated a lower toxicity of the hydroxyl
group containing pyridinium surfactants compared to that of conventional cationic
surfactants. Similar results have been observed in the case of a hydroxyl group containing
gemini imidazolium surfactants because these molecules have been found to be less cytotoxic
than quaternary ammonium gemini surfactant 12-2-12. Most of the gemini surfactants have
been found to be less toxic than reference molecule 12-2-12, with the exception of gemini
surfactant 13. The presence of two hydroxyl groups in gemini imidazolium surfactants (9-13)
imparts polarity and is responsible for the increase in the hydrophilic character of the
molecules, which correspondingly reduces the toxicity of these surfactants. IC50 values of
gemini surfactants (9-13) are given in Figure 4.13.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 5 10 15 20 25 30
Surfactant 9 = 22.31Surfactant 10 = 25.43Surfactant 11 = 21.73Surfactant 12 = 20.76Surfactant 13 = 10.06
12-2-12 = 18.13
Ab
sorb
an
ce
Concentration (M)
IC 50
value of surfactants in M
Figure 4.13: Absorbance vs concentration (µM) of gemini surfactants for the determination of the IC50
value. The values represent the mean of IC50 of three different experiments done in triplicate.
103
Singh, S.; Bhadani, A.; Kataria, H.; Kaur, G.; Kamboj, R. Ind. Eng. Chem. Res. 2009, 48, 1673-1677.
Gemini Imidazolium Surfactants: Synthesis and their Bio-Physiochemical Studies
125
The toxicity of these gemini surfactants increases with increasing spacer length with the
exception of gemini imidazolium surfactant 10, which has been found to be least cytotoxic
among the series of gemini imidazolium surfactant synthesized in the present study. These
values indicate the micromolar concentration of gemini surfactants, which causes the death of
50% of the living cells. The most toxic geminis observed among gemini imidazolium
surfactants (9-13) is surfactant 13 with an IC50 value of 10.06 µM, whereas surfactant 10 with
an IC50 value 25.43 µM has been found to be the least toxic.
Experimental:
Materials and Methods: The MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide was purchased from SigmaAldrich, USA. Millipore water was used in all
experiments.
Cytotoxicity Assay: The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)-based cytotoxicity test was used to evaluate all of the Gemini surfactants, and the
tests were carried out on C6 glioma (cancerous brain cell line, passage number 65). Cells
were seeded in 96 well flat-bottomed microplates at a density of 5 104 per mL, 100 μL per
well, and were allowed to grow for 24 h. The compounds dissolved in double distilled water
were sterilized using Millipore filter (pore size 0.22 μm) and were added to the culture media
over a concentration range of 1-100 μM. The cytotoxicity of the compounds was assessed
after 24 h of exposure. The absorbance was read at 550 nm using a Muliskan PLUS plate
reader (Labsystem, Finland). The statistical analysis was performed using Sigma Stat 3.5.1
and Sigma Plot 11.0.50