Chapter 4 Gemini Imidazolium Surfactants: Synthesis and ...shodhganga.inflibnet.ac.in/bitstream/10603/23729/10... · Gemini Imidazolium Surfactants: Synthesis and their Bio-Physiochemical

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


Section 4.3: Evaluation of Thermal stability of gemini

imidazolium surfactants by thermogravimetry analysis.

Section 4.4: Evaluation of DNA binding properties of gemini


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).


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).


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


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 -


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.


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.


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.


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.


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


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


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


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.


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.


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.


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.


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


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


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.


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.


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

Documents

Chapter 4 Gemini Imidazolium Surfactants: Synthesis and ...shodhganga.inflibnet.ac.in/bitstream/10603/23729/10... · Gemini Imidazolium Surfactants: Synthesis and their Bio-Physiochemical