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A small angle neutron scattering study of the thicknesses of vesicle bilayersformed from mixtures of alkyl sulfates and cationic bolaform surfactants†

Frank Pierce Hubbard Jr. and Nicholas Lawrence Abbott*

Received 3rd February 2008, Accepted 11th July 2008

First published as an Advance Article on the web 10th September 2008

DOI: 10.1039/b801981a

Past studies have established that the thickness of a vesicle bilayer formed from a mixture of

conventional anionic and cationic surfactants is determined by a delicate balance of factors, including

electrostatic interactions, van der Waals forces, and chain packing constraints. This complex balance of

factors means that the bilayer thickness is not easily predicted from knowledge of the structure of

conventional surfactants. In this paper, we report the synthesis of a family of rigid, bolaform

surfactants with the structure bis(trimethylammoniumalkyloxy)azobenzene dibromide, where the alkyl

group was butyl, hexyl, or octyl. We used small angle neutron scattering and quasi-elastic light

scattering to characterize the microstructures formed when the cationic bolaform surfactants were

mixed with either sodium dodecylsulfate or sodium tetradecylsulfate in aqueous solutions. Small angle

neutron scattering spectra interpreted using form factor fits indicate that the three rigid, bolaform

surfactants span the bilayers of vesicles formed by these surfactant mixtures, thus constraining the

bilayer thicknesses to the end-to-end lengths of the bolaform surfactants. We conclude that use of rigid,

bolaform surfactants provides a simple means to design vesicles from anionic and cationic surfactants

that form spontaneously and have predictable bilayer thicknesses. These results suggest some simple

rules that enable the rational design of surfactant-based nanostructures.

Introduction

The study of aqueous mixtures of cationic and anionic surfac-

tants has led to the discovery that a wide range of microstruc-

tures can form in solution.1–15 Rod-like micelles and vesicles are

examples of two microstructures that have attracted particular

attention due to the solution properties that accompany these

microstructures, such as elevated solution viscosity for rod-like

micelles16 and the ability to encapsulate small volumes of solvent

with vesicles.17 Despite the number of studies dedicated to

investigations of cationic and anionic surfactant mixtures,1–15 the

relationship between the type of aggregate microstructure

formed in solution and the architecture of the surfactants is not

fully understood. Packing parameter arguments, which are

capable of providing insights into the type of microstructure

found in solutions that contain a single surfactant,18,19 are not

readily applied to surfactant mixtures. For vesicles, in particular,

the packing of surfactants into the two leaves of the bilayer is

a complex phenomenon.18,19 While models have been developed

that have shown agreement with experimental results for a few

systems,20–22 the impact of surfactant architecture on vesicle size

and bilayer thickness is still not easily predicted. Data presented

in Fig. 1 illustrate this point by comparing experimentally

measured vesicle bilayer thicknesses to the molecular lengths of

three binary mixtures of anionic and cationic surfactants that

Department of Chemical and Biological Engineering, University ofWisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706-1691,USA. E-mail: abbott@engr.wisc.edu; Fax: +1-608-262-5434; Tel: +1-608-265-5278

† Electronic supplementary information (ESI) available: Determinationof the uncertainty in the bilayer thickness measured by SANS. SeeDOI: 10.1039/b801981a

This journal is ª The Royal Society of Chemistry 2008

form vesicles. Inspection of Fig. 1 reveals no simple relationship

between bilayer thickness and surfactant molecular lengths (see

below for additional discussion).9,10,20,23,24

Recently, we reported that mixtures of sodium dode-

cylsulfate (SDS) and a rigid, bolaform surfactant, bis(-

trimethylammoniumhexyloxy)azobenzene dibromide (BTHA),

form vesicles in aqueous solution.25 A complementary study

confirmed these findings by combining small angle neutron

scattering (SANS) results with cryo-transmission electron

microscopy (cryo-TEM).26 When characterizing the microstruc-

ture of the vesicles formed from BTHA and SDS, we observed

Fig. 1 Comparison of the measured vesicle bilayer thickness to the

molecular length of surfactant for vesicles formed from mixtures of

cetyltrimethylammonium bromide (CTAB) or cetyltrimethylammonium

tosylate (CTAT) and various anionic surfactants (sodium dode-

cylbenzenesulfonate (SDBS), sodium octylsulfate (SOS), sodium per-

fluoroctanoate (FC7) and sodium perfluorohaxanoate (FC5)).

Soft Matter, 2008, 4, 2225–2231 | 2225

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that the bilayer thickness of the vesicles was similar to the end-to-

end length of BTHA. This observation led us to speculate that

BTHA spanned the bilayer of these vesicles. We also found that

the bilayer thickness was not strongly dependent on the surfac-

tant mixing ratio (15–90 mol% BTHA) or total surfactant

concentration (0.1–1.0 wt% surfactant).27 These observations

led us to hypothesize that manipulation of the length of rigid,

bolaform surfactants with architectures similar to BTHA may

provide a simple means to design the bilayer thicknesses of

vesicles.

The study reported in this paper was designed to test the above

hypothesis regarding control over bilayer thickness of vesicles

formed by anionic and cationic surfactants. We synthesized two

homologues of BTHA with different alkyl spacers and studied

the self-assembly of these surfactants in aqueous mixtures with

two different anionic surfactants. The molecular structure of the

surfactants used in this work is presented in Fig. 2. The lengths of

the bolaform surfactants were altered by changing the alkyl

spacers between the central azobenzene moieties and the head

groups. The bolaform surfactant series studied here is termed

BTxA, where x refers to the length of the alkyl spacer, which

varies between 4 (BTBA), 6 (BTHA), or 8 carbons (BTOA). We

also mixed each of the BTxA surfactants with two alkyl sulfate

surfactants having different chain lengths (termed SxS, where

x ¼ 12 or 14 carbons). This latter experiment was performed to

determine the effect of the length of the anionic surfactant on the

bilayer thickness of the vesicles formed in aqueous solution.

As mentioned above, the class of surfactant mixtures described

in this report has been characterized previously by quasi-elastic

light scattering (QLS), SANS, and cryo-TEM, confirming that

vesicles form in solution.25–28 Because the study reported here

sought to determine the dependence of vesicle bilayer thickness

on surfactant molecular architecture, and because SANS has

been established by a series of prior studies to be an accurate

Fig. 2 Molecular structures of the

2226 | Soft Matter, 2008, 4, 2225–2231

method for determining vesicle bilayer thicknesses,23 our study

focused on the use of SANS. We complemented our SANS

measurements with measurements of hydrodynamic size using

QLS in order to identify trends in vesicle size that accompany

changes in the molecular structure of the surfactants. Whereas

previous studies of azobenzene-based surfactants have reported

on the sensitivity of these systems to illumination,25,27,28 we

employ azobenzene here to create rigid, bolaform surfactants.

Several studies of rigid, bolaform surfactants have been reported

previously in the literature.14,29–35 However, in those past studies,

either vesicle formation was not reported,29,30,32,35 or the authors

did not investigate the relationship between the length of the

bolaform surfactant and the thickness of vesicle bilayers formed

by the surfactants.14,31,34

Experimental section

Materials

All reagents were obtained from Aldrich (Milwaukee, WI). D2O

(99.9% deuteration) was obtained from Cambridge Isotope

Laboratories (Andover, MA). Sodium dodecylsulfate (SDS) and

sodium tetradecylsulfate (STS) were recrystallized three times in

ethanol prior to use. The bolaform surfactants BTBA, BTHA,

and BTOA were synthesized as described below:

(a) 4,40-Dihydroxyazobenzene. 4,40-Dihydroxyazobenzene

was synthesized according to the literature.36 This method

resulted in a better yield (34%) than other approaches (<10%)

reported in the past.25,28

(b) 4,40-Dibromoalkyloxyazobenzene (alkyl ¼ butyl, hexyl, or

octyl). 4,40-Dihydroxyazobenzene was dissolved in ethanol

(30 mL g�1 of compound) and the solution was heated to reflux.

surfactants used in this work.

This journal is ª The Royal Society of Chemistry 2008

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Three molar equivalents of KOH were then added, followed

by addition of 10 molar equivalents of a,u-dibromoalkane. The

resulting mixture was then stirred at reflux for 4 h. After cooling,

a precipitate formed, which was filtered and washed with

ethanol. The crude product was then dissolved in toluene,

filtered while hot, and then recrystallized. Typical yields were

54–67%. 1H NMR (CDCl3, 300 MHz, ppm): d 1.5 (m,

-OCH2CH2(CH2)nCH2CH2Br; 0H for BTBA, 8H for BTHA,

16H for BTOA), 1.9 (m, -OCH2CH2(CH2)nCH2CH2Br; 8H), 3.4

(t, -CH2CH2Br; 4H), 4.0 (t, -OCH2CH2-; 4H), 6.9 (d, phenyl ring,

4Ha), 7.8 (d, phenyl ring, 4Hb). Note that in the above, n ¼ 0 for

BTBA, 2 for BTHA, and 4 for BTOA.

(c) 4,40-Bis(trimethylammoniumalkyloxy)azobenzene dibro-

mide (alkyl ¼ butyl, hexyl, or octyl). The dibromoalkylox-

yazobenzene compounds were dissolved separately in toluene

(15 mL toluene g�1 of compound) and heated to 80 �C. Four

molar equivalents of trimethylamine were added, and the

resulting mixture was then stirred for 96 h. As the reaction

proceeded, precipitate formed. After reaction, the mixture was

cooled and the precipitates filtered. The crude products were then

dissolved in ethanol, filtered while hot, and then recrystallized

three times. Typical yields were 62–90%. 1H NMR (DMSO,

300 MHz, ppm): d 1.3 (m, -OCH2CH2(CH2)nCH2CH2N(CH3)3;

0H for BTBA, 8H for BTHA, 16H for BTOA), 1.7 (m,

-OCH2CH2(CH2)nCH2CH2N(CH3)3; 8H), 3.0 (s, -N(CH3)3;

18H), 3.3 (m, -CH2CH2N(CH3)3; 4H), 4.0 (t, -OCH2CH2-; 4H),

7.1 (d, phenyl ring, 4Ha), 7.8 (d, phenyl ring, 4Hb). Note that in

the above, n ¼ 0 for BTBA, 2 for BTHA, and 4 for BTOA. ESI-

MS: m/z 221.3 [M � 2Br]2+, 523.6 [M � Br]+ for BTBA. m/z 249.2

[M � 2Br]2+, 577.2 [M � Br]+ for BTHA. m/z 277.4 [M � 2Br]2+,

633.7 [M � Br]+ for BTOA.

Methods

Stock solutions of either BTxA or SxS were prepared by

weighing the appropriate mass of surfactant into a scintillation

vial and then adding deionized and distilled H2O (18.2 MU cm,

Millipore, Billerica, MA) or D2O. Surfactant mixtures were

prepared by mixing stock solutions filtered through a 0.22 mm

Millex�-GV filter. For QLS measurements, surfactant mixtures

were prepared in 12 mm diameter borosilicate test tubes (Fisher,

Atlanta, GA) that were cleaned with piranha solution (70%

H2SO4, 30% H2O2) prior to use. WARNING: piranha solution

should be handled with extreme caution; in some circumstances

(most probably when it has been mixed with significant quantities

of an oxidizable organic material) it has detonated unexpectedly.

The test tubes were sealed with polyethylene caps (Andwin

Scientific, Addison, IL). Prior to use, the caps were soaked in

a SDS solution overnight, rinsed thoroughly with water and

ethanol, and dried under vacuum. Care was taken such that the

surfactant solution to be examined never came into contact with

the caps. After sealing, samples were placed in a circulating water

bath maintained at 25 �C for one week prior to measurement.

The bath was covered with aluminium foil to prevent exposure of

the samples to light. For SANS measurements, solutions were

prepared in small scintillation vials and sealed with Teflon screw

caps prior to measurement. These samples were also equilibrated

in the dark at 25 �C for one week prior to measurement.

This journal is ª The Royal Society of Chemistry 2008

Quasi-elastic light scattering

Quasi-elastic light scattering measurements were conducted

using a Brookhaven light scattering apparatus (Brookhaven

Instruments, Holtsville, NY), comprising a BI-9000AT digital

autocorrelator, a BI-200SM goniometer, and a 25 mW laser

(637 nm, Coherent Radius 635-25). The detector angle was set to

90� and the autocorrelation curves were analyzed using the

method of cumulants.37 This method provides the average decay

rate, hti ¼ hDTiq2, where hDTi is the average translational

diffusion coefficient and q is the magnitude of the scattering

vector. The normalized, relative variance is calculated as,

v ¼ (ht2i � hti2)/hti2. hDTi is related to the hydrodynamic

diameter according to the Stokes–Einstein equation.17

Measurements at other angles (70�–130�) confirmed that the

autocorrelation functions corresponded to center of mass diffu-

sion of the aggregates in solution.

Small angle neutron scattering

Small angle neutron scattering measurements were performed

using the NG3 instrument at National Institute of Standards and

Technology (NIST) in Gaithersburg, MD. The wavelengths of

the neutrons were on average 6 A, with a spread in wavelength,

Dl/l, of 14%. Data were collected with the detector set at two

positions: 1.9 m and 13.17 m from the sample. By offsetting the

detector 25 cm from center, these distances covered q-ranges of

0.02–0.35 A�1 and 0.0035–0.05 A�1, respectively, where q is the

magnitude of the scattering vector. During measurement,

samples were held in quartz cells with a path length of 2.0 mm

and placed in a sample chamber thermostatted at 25.0 � 0.1 �C.

To ensure good statistics, at least 5 � 105 detector counts were

collected for each sample at each distance. The data were cor-

rected for detector efficiency, background radiation, empty cell

scattering, and incoherent scattering to calculate the scattered

intensity on an absolute scale. These procedures were performed

using a computer program provided by NIST that runs on IGOR

Pro (Wavemetrics, Lake Owego, OR).38

We used Guinier analysis and form factor modeling to inter-

pret SANS spectra. Both of these techniques have been discussed

extensively in the literature20,25,39–42 and are only summarized

here. The scattered intensity, I(q), is related to the differential

scattering cross section, dS(q)/dU, by:

IðqÞ ¼ðRðqÞdSðqÞ

dUdq (1)

where R(q) is the resolution function, accounting for instru-

mental smearing effects such as wavelength spread, imperfect

collimation, and finite slit width/length effects.

The differential scattering cross section can be expressed in

terms of a form factor, P(q), and a structure factor, S(q). For

samples identified by Guinier analysis to be consistent with

vesicles, we used a polydisperse core–shell model for P(q), in

which the vesicles have a polydisperse core radius and a constant

shell thickness. As reported below, the solutions used in our

study were dilute (0.1 wt% total surfactant), hence we set S(q) to

unity. The goodness of fit of the form factor to our experimental

data provides further support for this assumption. The differ-

ential scattering cross section was thus evaluated as:42

Soft Matter, 2008, 4, 2225–2231 | 2227

Fig. 3 SANS data obtained for mixtures of BTxA and SxS. Data sets

are offset by indicated factors for clarity. (A) BTxA–SDS mixtures, (B)

BTxA–STS mixtures.

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dSðqÞdU

¼ n

ðN

0

GðrcÞP2ðqrcÞdrc (2)

where rc is the core radius, G(rc) is the distribution of core radii,

and P2(qrc) is the form factor for a single vesicle. The distribution

of vesicle sizes was modeled as a Schultz distribution,25,42

GðrcÞ ¼rc

z

GðZ þ 1Þ

�Z þ 1

�rc

�Zþ1

exp

��rc

�rc

ðZ þ 1Þ�

(3)

where �rc is the mean core radius, and Z is related to the variance

of the core radii, s2, by:

1

Z þ 1¼ s2

�rc2

(4)

The form factor for a single vesicle is:

PðqrcÞ ¼4p

q3ðrv � rsÞf½sinqðrc þ tÞ � sinqrc�

� ½qðrc þ tÞcosqðrc þ tÞ � qrccosqrc�g (5)

where t is the shell thickness. Thus, the fit parameters in this

model are the core radius and polydispersity (defined as s/�rc), the

shell thickness, and the volume fraction of aggregates. For

the SANS data reported here, the error bars in the data are

smaller than the symbols used in the plots, except in the high-q

region (q > 0.2 A�1), where the background due to scattering

from the solvent was large compared to coherent scattering from

aggregates. The error bars for the bilayer thickness values

reported in this paper were obtained by retaining the values for

the vesicle size and polydispersity from the optimized fit, and

then recalculating the scattered intensity with a different bilayer

thickness until the calculated result fell just outside of the range

of the data. Using this approach, we were able to estimate the

bilayer thickness to within 0.15 nm.

Results and discussion

Characterization of vesicle bilayer thickness

We have reported previously that the fully extended, end-to-end

length of BTHA is 2.8 nm; a value obtained by using known

values for bond lengths and bond angles.27 This length corre-

sponds to the distance from the nitrogen atom of one trime-

thylammonium head group to the nitrogen atom of the

opposite head group of the surfactant. The length of the

surfactant was defined in this manner because coherent scat-

tering of neutrons results from contrast in the scattering length

density between the deuterated solvent and the vesicle bilayer.43

It is generally accepted that for aggregates formed from

surfactants and surfactant mixtures, H2O (or in the case of

SANS, D2O) penetrates to some extent into the head group

region of an aggregate.20 Penetration of D2O into the head

group region will decrease the contrast, thus reducing the

apparent bilayer thickness of a vesicle measured by SANS.

Using the definition of length described above, we calculated

the end-to-end lengths for each of the bolaform surfactants

used in this study to be 2.3 nm for BTBA, 2.8 nm for BTHA,

and 3.3 nm for BTOA.

2228 | Soft Matter, 2008, 4, 2225–2231

As mentioned in the Introduction, we determined previously

that the measured bilayer thickness of vesicles formed by BTHA

and SDS does not depend strongly on either the mixing ratio of

the surfactants or the total surfactant concentration.27 Thus, we

report our findings here for a single solution composition:

0.1 wt% total surfactant, 15 mol% BTxA, and 85 mol% SxS,

a composition that was demonstrated previously to result in

vesicle formation for solutions containing BTHA and SDS. The

SANS spectra obtained for all of the samples are presented in

Fig. 3. Inspection of Fig. 3A reveals that the scattering from each

of the binary mixtures of SDS and BTxA at low q (q < 0.03 A�1)

follows a slope of �2, consistent with a bilayer geometry.39 The

absence of lamellar ‘‘clouds’’ or ‘‘wisps’’ that have been reported

for other surfactant mixtures suggests that these bilayer struc-

tures are in fact vesicles.10,11 The scattering curves from the

BTBA–SDS and the BTHA–SDS samples display few features

other than the �2 slope. In contrast, the scattering spectrum

recorded for the BTOA–SDS sample displays a minimum at a q

value of approximately 0.013 A�1. Past studies have demon-

strated that the size and polydispersity of vesicles formed in

solution determines whether or not minima are seen in the SANS

spectra.44 As we demonstrate in the next section (Characteriza-

tion of vesicle size), the difference in size of the vesicles formed in

solution can explain why a minimum is observed for the mixture

containing BTOA and not for mixtures of the other bolaform

surfactants. Fig. 3B, which shows the SANS spectra obtained

from mixtures of STS and BTxA, is similar to Fig. 3A: the

BTBA–STS and the BTHA–STS samples closely follow a �2

slope for the q range investigated, whereas the BTOA–STS

sample shows a slight minimum at a q value of approximately

0.019 A�1.

This journal is ª The Royal Society of Chemistry 2008

Table 1 Quasi-elastic light scattering results and parameter estimates obtained from form factor fits to the SANS data presented in Fig. 3

SampleQLS diameter/nm(H2O/D2O) Form factor diameter/nm Form factor polydispersity Bilayer thickness/nm O(c2/N)

BTBA–SDS 433/448 196 � 10 0.29 � 0.03 2.4 � 0.15 1.5BTBA–STS O(mm) 202 � 11 0.15 � 0.01 2.5 � 0.15 1.4BTHA–SDS 202/212 196 � 9 0.14 � 0.01 2.8 � 0.15 1.9BTHA–STS 221/231 262 � 12 0.38 � 0.03 2.9 � 0.15 2.7BTOA–SDS 109/138 44 � 3 0.26 � 0.02 3.3 � 0.15 1.6BTOA–STS 49/57 20 � 2 0.54 � 0.04 3.1 � 0.15 2.0

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We fit the SANS data presented in Fig. 3 with the polydisperse

core–shell model described in the Methods section. Our principal

objective in this report was to determine the bilayer thickness.

We predicted that the vesicle bilayers characterized in this study

would range from �2 nm to �3 nm, and thus would require

a measurement with a precision better than 0.5 nm. Although

cryo-TEM is a useful method for characterizing the micro-

structure of surfactant solutions, it is not well suited for deter-

mination of vesicle bilayer thicknesses.23 The fits to the SANS

data are drawn as solid lines in Fig. 3. The goodness of fit of the

model to the data is excellent over the entire q range, consistent

with the presence of vesicles for all samples. The parameter

estimates obtained from the fits are displayed in Table 1 along

with values of reduced c2 (to indicate goodness of fit). We focus

first on the values for the vesicle bilayer thickness that emerge

from the fit of the form factor model to the SANS data.

Inspection of Table 1 reveals that for each of the bolaform

surfactants, substituting STS for SDS resulted in a change in

bilayer thickness of 0.2 nm or less for all mixtures. As noted in

the Methods section, the form factor fits allow estimation of the

bilayer thickness to within 0.15 nm, indicating that the difference

in length upon substitution of SDS with STS for each of the

bolaform surfactant mixtures is comparable to or within

the uncertainty of the measurement.

To facilitate interpretation of the data in Table 1, the measured

bilayer thickness for each sample has been plotted along with the

length of the bolaform surfactant in Fig. 4. The lengths of SDS

and STS are also indicated as horizontal lines in the plot.

Inspection of Fig. 4 reveals that for mixtures of BTxA and SDS,

Fig. 4 Comparison of the measured vesicle bilayer thickness to the

molecular length of surfactant for vesicles formed from mixtures of

BTxA and SxS. The molecular lengths of SDS and STS are indicated with

horizontal lines.

This journal is ª The Royal Society of Chemistry 2008

there is good agreement between the measured bilayer thickness

and the molecular length of the BTxA surfactant, as defined

above: 2.4 nm (bilayer thickness) vs. 2.3 nm (surfactant length)

for BTBA, 2.8 nm vs. 2.8 nm for BTHA, and 3.3 nm vs. 3.3 nm

for BTOA. For mixtures of BTxA and STS, the agreement is also

good: 2.5 nm vs. 2.3 nm for BTBA, 2.9 nm vs 2.8 nm for BTHA,

and 3.1 nm vs. 3.3 nm for BTOA. Flexible bolaform surfactants,

specifically surfactants with a saturated alkyl chain and head

groups at either end, can form looped conformations in surfac-

tant aggregates.45–47 In this study, the rigid azobenzene group

(trans conformation) appears to prevent looped conformations,

and thus promotes a bilayer spanning conformation. Hence, the

lengths of the rigid, bolaform surfactants appear to largely

dictate the thicknesses of the bilayers of vesicles formed in

solution.

It is also insightful to compare the result presented in Fig. 4

to results shown in Fig. 1 for bilayer thicknesses of

conventional cationic–anionic surfactant mixtures. The

cationic surfactant used to obtain the data in Fig. 1 was either

cetyltrimethylammonium bromide (CTAB),9,10,20,23,24 or cetyl-

trimethylammonium toyslate (CTAT),48 and the anionic surfac-

tants were either sodium dodecylbenzenesulfonate (SDBS),

sodium octylsulfate (SOS),20 or sodium perfluorooctanoate

(FC7).23 In each of these three systems, SANS was used to

measure bilayer thicknesses at a total concentration of surfactant

of 1.0–2.0 wt%, and a mixing ratio of cationic to anionic

surfactant of 7 : 3 (CTAT–SDBS), 3 : 7 (CTAB–SOS) or 2 : 8

(CTAB–FC7) by weight. We note that SOS and FC7 are

approximately the same length, but chemically different (FC7 is

perfluorinated). Also, SOS and FC7 are straight-chain, but

SDBS was of the ‘‘hard’’ type, and contained some branches.

Inspection of Fig. 1 reveals that for the CTAB–SOS system, the

measured bilayer thickness is close to the extended, end-to-end

length of CTAB: 2.2 nm vs. 2.0 nm. However, the agreement is

likely to be fortuitous, as the measured bilayer thickness

increases to 2.9 nm upon substitution of a different anionic

surfactant of similar length (FC7). Comparing these results with

CTAT–SDBS, it is clear that branched chains or bulky coun-

terions can also affect the thickness of vesicle bilayers for single-

tailed surfactant systems. The results presented in Fig. 1 indicate

that for conventional surfactant mixtures, there is no obvious

relationship between either of the surfactant lengths and the

vesicle bilayer thickness. Examination of other surfactant

systems that form vesicles, where the bilayer thickness was

reported, yields similar conclusions.13,48,49 The trends reported in

Fig. 4 for vesicles prepared using rigid bolaform surfactants thus

represent qualitatively different behaviors as compared to those

seen previously when using conventional surfactant mixtures.

Soft Matter, 2008, 4, 2225–2231 | 2229

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Characterization of vesicle size

While the investigation reported in this paper was designed

primarily to test the hypothesis discussed above regarding control

of the bilayer thickness of vesicles, in the course of analyzing our

SANS data we observed features in the spectra that suggested

trends in the sizes of the vesicles formed in solution. To provide

further insight into those trends, we performed QLS measure-

ments. We note here that the sizes of the vesicles were measured

after one week of equilibration at 25 �C. The results are presented

in Table 1. The QLS measurements were performed in both H2O

and D2O, and the similarity in aggregate sizes in the two solvents

confirmed that isotopic substitution of the solvent had a minimal

effect on the aggregates formed in these systems.25 The QLS

measurements of vesicles formed by BTBA and BTHA indicate

that the vesicles are sufficiently large that SANS does not lead to

accurate estimates of size. For example, based on the vesicle sizes

obtained from QLS, minima in the SANS spectra would be

expected at q-values of approximately 0.0029 A�1 for BTHA–

SDS and 0.0015 A�1 for mixtures containing BTBA–SDS. The

NG3 instrument used in this study has a qmin of approximately

0.0034 A�1 in its standard configuration. We also note that our

estimates of vesicle size depend on the method of measurement

(QLS or SANS), with QLS measurements being larger than

estimates of size based on form factor fits (see, for example, the

data in Table 1 corresponding to BTOA). This situation is

common in polydisperse systems because QLS results are

strongly biased towards the larger aggregates in solution, even if

they are present in small proportions.13,25,42 This observation

results from the fact that QLS reports the ratio of the fourth to

the third moment of the number average size distribution,50

whereas SANS reflects the true number average distribution.

We make two observations regarding the measurements of size

presented in Table 1. First, inspection of Table 1 reveals that the

hydrodynamic diameter (as measured by QLS) of the vesicles

ranges from 50 nm (with BTOA) to over a micrometre (with

BTBA) with decrease in the length of the bolaform surfactant.

With the exception of the BTBA–STS sample, vesicles that

formed were determined to be stable in solution for at least two

months. After a period of 14+ days, we observed the BTBA–STS

sample (in H2O and in D2O) to form two coexisting phases:

a colorless, surfactant-lean phase that contained very few, if any,

aggregates (upper phase), and a yellowish, turbid phase that

contained aggregates that were a few micrometres in size (lower

phase), according to QLS measurements. Second, we observed

that an increase in the length of the anionic surfactant had an

effect on vesicle size that depended on the length of the cationic

surfactant. Replacement of SDS by STS resulted in (i) a decrease

in the vesicle size (from 109 nm to 49 nm) for mixtures with

BTOA, (ii) essentially no change in vesicle size for mixtures with

BTHA, and (iii) an increase in vesicle size (from 433 nm to

a micrometre) for mixtures with BTBA. We note again that the

vesicles formed by BTBA and STS are not equilibrium structures.

Although the molecular-level origins of the relationship observed

between vesicle size and surfactant length has not yet been

elucidated, clear trends in vesicle size exist within this family of

surfactant pairs suggesting that mixtures of anionic surfactants

and cationic bolaform surfactants may provide a simple means to

engineer vesicles of desired sizes as well as bilayer thicknesses.

2230 | Soft Matter, 2008, 4, 2225–2231

Formation of vesicles using bolaform surfactants

For all surfactant pairs studied in our experiments, the length of

the anionic surfactant is equal to or greater than half of the

length of the bolaform surfactant (see Fig. 4). All of these systems

formed vesicles. We also prepared aqueous mixtures of BTxA

with sodium decylsulfate, and observed precipitation in all

samples within one day of equilibration at 25 �C. In this latter

case, the fully extended length of the anionic surfactant is less

than half the length of the bolaform surfactant. As described in

more detail below, this result suggests that vesicles form in

mixtures of anionic surfactants and rigid, cationic, bolaform

surfactants only when the length of the anionic surfactant is at

least half of the length of the bolaform surfactant. This result

contrasts with systems containing mixtures of single-tailed

surfactants (see Fig. 1) in which vesicle formation is possible

when the length of the anionic surfactant is less than half of that

of the cationic surfactant.

The packing of amphiphilic molecules into self-assembled

structures is often discussed in terms of the well-known packing

parameter formula given as:19

P ¼ vt

ahlt(6)

where vt and lt are the volume and length of the hydrocarbon tail

of a surfactant, respectively, and ah is the area occupied by the

head group of the surfactant.18,19 According to the packing

parameter formula, vesicles are the favored microstructure when

the packing parameter has a value ½ < P < 1.18,19 We caution,

however, that this result does not obviously apply to vesicles

formed from rigid, bolaform surfactants. In particular, we note

that this range of values for the packing parameter is derived

under the assumption that the packing constraints of the outer

leaf of the bilayer dictate the microstructure of the system. In

contrast, the inner leaf of the bilayer of the vesicle is assumed to

adopt a head group area that results in a near optimal packing.18

When dealing with rigid, bolaform surfactants that span

the bilayer of a vesicle (as inferred here from the dependence of the

bilayer thickness on the length of the bolaform surfactant), the

packing of the inner and outer leaves of the bilayer are coupled.

Specifically, the use of a rigid, bolaform surfactant constrains the

number of cationic head groups on the outer surface of the vesicle

to be equal to the number of cationic head groups on the inner

surface of the vesicle. This constraint likely underlies the obser-

vation of precipitation when each BTxA surfactant was mixed

with sodium decylsulfate (see above). In this case, because the

length of the anionic surfactant was less than half the length of the

rigid, bolaform surfactant, formation of vesicles was likely not

favored because the rigid bolaform surfactant would have to tilt in

order to prevent the presence of voids (hydrocarbon-free regions)

within the bilayer. Such a constraint does not exist in conventional

surfactant systems where interdigitation of tails can occur to

create thin bilayers, and different densities of cationic and anionic

groups can exist on the inner and outer leaves of the bilayer.

Conclusion

In this paper, we report that use of rigid bolaform surfactants

provides a simple means to design vesicles that have predictable

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bilayer thicknesses from anionic and cationic surfactants. We

synthesized a family of rigid, bolaform surfactants with the

structure bis(trimethylammoniumalkyloxy)azobenzene dibro-

mide, where the alkyl group was butyl, hexyl, or octyl, and mixed

these cationic surfactants with either sodium dodecylsulfate or

sodium tetradecylsulfate in aqueous solutions. By using SANS,

we observed that these mixtures formed vesicles with bilayer

thicknesses that correlated closely with the lengths of the three

cationic, bolaform surfactants, and were independent of the

length of the anionic surfactant. This result suggests that the

rigid, bolaform surfactants span the bilayers of these vesicles,

thus constraining the bilayer thickness to the length of the

bolaform surfactant. The formation of vesicles was observed to

be favorable only when the length of the anionic surfactant was

at least half of the length of the rigid, bolaform, cationic

surfactant. This result is also consistent with the bolaform

surfactant spanning the bilayer, and is in contrast to mixtures of

single-headed surfactants where vesicle formation is possible

even when the length of one surfactant is less than half the length

of the other surfactant. Although the dependence of vesicle size

on length of the bolaform surfactants appears to be complex, we

found that the vesicle size determined by QLS tends to decrease

as the length of the bolaform surfactant in the mixture is

increased. The origin of this trend is not understood. Overall, our

results suggest that rigid, bolaform surfactants provide a means

to prepare vesicles from mixtures of anionic and cationic

surfactants that possess bilayer thicknesses that can be predicted

based on the length of the bolaform surfactant.

Acknowledgements

This work utilized facilities supported in part by the NSF under

Agreement No. DMR-0454672. FPH and NLA acknowledge the

support of the National Institute of Standards and Technology,

U.S. Department of Commerce, in providing the neutron

research facilities used in this work. Additional support for this

work was also provided by NSF through CTS-0553760, CBET-

0754921 and DMR-0602570. Financial support from the donors

to the Petroleum Research Fund is gratefully acknowledged.

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