JOVRNAL OF
biochemical and
biophysical
methods
ELSEVIER
J. Biochem. Biophys. Methods 31 (19%) 145-150
Research article
Determination of surfactant critical micelle
concentration by a novel fluorescence
depolarization technique
Xichen Zhang avb,John K. Jackson a, Helen M. Burt a**
a Division of Pharmaceutics, Faculty of Pharmaceutical Sciences. University of British Columbia, 2146 East
Mall, Vancouver, BC V6T lZ3, Canada
b Angiogenesis Technologies, Vancouver, BC, Canada
Received 5 June 1995; accepted 28 June 1995
Abstract
A novel method using fluorescence depolarization to determine the critical micelle concentrations (CMC) of surfactants was developed. Fluorescence anisotropies of Triton X-100, sodium
dodecyl sulfate, and sodium cholate were measured using 1,6-diphenyl-1,3,5hexatriene
as a
fluorescence probe. Fluorescence anisotropy decreased with increasing surfactant concentrations
below the CMC and leveled off above the CMC. The depolarization method does not depend on
the concentration of DPH and is largely immune to light-scattering problems encountered in turbid
aqueous systems.
Keywords:
tant
Fluorescence
depolarization;
1,6-Diphenyl-
1,3,5-hexatriene;
Critical micelle concentration;
Surfac-
1. Introduction
Surfactants
are used in a wide variety of pharmaceutical applications such as in the
solubilization of hydrophobic drugs [l], and as additives in formulations including
creams, emulsions, microemulsions, and suspensions [2,3]. Micelles of amphipathic
diblock copolymer surfactant have been employed as colloidal carriers for anticancer
drugs [4,51.
* Corresponding
author. Fax: (1) (604) 822-3035.
0165-022X/96/$15.00
0 1996 Elsevier Science B.V. All rights reserved
SSDI 0165-022X(95)000321
146
A’. Zhung
et d/J.
Biochem.
Biophy.7.
Methods
31 f 19961
145-150
The critical micelle concentration
(CMC) of surfactants and polymeric surfactants
may be determined by measuring light-scattering
changes, hydrodynamic
and surface
tension properties, and by changes in absorbance or fluorescence upon dye solubilization
[6]. Pluorimetric determination of the CMC involves the use of the hydrophobic probe
1,6-diphenyl-I ,3,5_hexatriene (DPH) which shows an abrupt increase in fluorescence
intensity at the CMC since the formation of micelles offers a local hydrophobic
environment for DPH [7].
We hypothesized that when a surfactant concentration
increases to its CMC, an
abrupt change in the fluorescence depolarization of DPH may also occur. The depolarization of fluorescence is due to the rotation of the DPH molecules during the lifetime of
the excited state. The rotation of the DPH molecule is a function of the resistance
offered by the microenvironment
to the motion of the probe. Hence, at the CMC of a
surfactant solution there should be an abrupt change in the depolarization
of DPH
fluorescence due to the probe entering a different region of microviscosity within the
micelles.
Fluorescence depolarization can be described by fluorescence anisotropy (r), which
is defined as: r = (I,, - Ivh)/(lvv + 2 I”,,). where I,, and I,, are the emission intensities detected via a polarizer oriented parallel and vertical, respectively. to vertically
polarized monochromatic
excitation light. A smaller value of anisotropy indicates a
lower degree of depolarization, and therefore a less viscous environment [8].
Fluorescence anisotropy has been used to study the microviscosity of local microenvironments such as liposomes, cell membranes, and whole cells [8]. However, to our
knowledge, there are no reports of the use of fluorescence anisotropy determinations for
measuring CMCs. In this study, the validity of using anisotropy to determine the CMC
of various surfactants was evaluated.
2. Materials and methods
2.1. Materials
DPH, Triton X- 100 and sodium cholate were purchased from Sigma. Sodium dodecyl
sulfate (SDS) was from Bio-Rad. Tetrahydrofuran
(THF) was obtained from BDH
(Toronto).
2.2. Determination
ofjluorescence
intensity and anisotropy
A DPH stock solution of 10 mM in THF was prepared and a given volume of DPH
solution was added to aqueous surfactant solutions of various concentrations.
These
samples were equilibrated
overnight in a dark chamber [7]. A Shimadzu RF 540
spectrofluorophotometer
with a polarization
accessory was used to measure DPH
fluorescence. The wavelengths of excitation and emission were 355 nm and 428 nm,
respectively. The temperature was controlled at 25 or 37°C with a water bath circulator
(SLM Instruments).
X. Zhang et al./J.
Biochem. Biophys. Methods31 (1996) 145-150
147
3. Results and discussion
The fluorescence intensity of DPH in solutions of Triton X-100, SDS, and sodium
cholate increased rapidly with increasing surfactant concentration (Fig. 1). Abrupt
changes in fluorescence intensities were observed for Triton X-100 (Fig. la), SDS (Fig.
lb) and sodium cholate (Fig. lc) at concentrations of 0.28, 8 and 16.2 r&l, respectively.
Since DPH fluorescence intensity increases’ greatly above the CMC due to its incorporation into the hydrophobic interior of micelles, the abrupt change of the intensity
represents the value of CMC. The CMCs of Triton X-100, SDS, and sodium cholate
from fluorescence measurements were found to be 0.28, 8 and 16.2 mM at 25”C,
respectively. These values are generally in agreement with literature results (Table 1).
As the concentration of the surfactants increased, anisotropy values decreased and
then remained relatively constant as the surfactant concentration was increased further at
both 25 and 37°C (Fig. 2). The surfactant concentration corresponding to the change in
slope or the concentration at which anisotropy values became independent of concentration was taken to be the CMC of the surfactant. The decrease in anisotropy values as
surfactant concentrations are increased reflects a change in the environment of the DPH
probe. It is possible that at low surfactant concentrations the DPH molecules preferentially form aggregates in solution which act to impede rotational movement of the
probe resulting in high anisotropy values. Then as the surfactant concentration increases,
there may be a preferential association of DPH with surfactant molecules and a
corresponding breakdown of DPH aggregates leading to a decrease in anisotropy. At the
CMC, the DPH molecules become solubilized within the hydrophobic core of the
surfactant micelles, which possess a lower microviscosity. Above the CMC, there are no
further decreases in anisotropy since the environment of the DPH probe within the
micelle core does not change. Using the fluorescence anisotropy method, the CMCs of
Triton X-100, SDS, and sodium cholate were determined to be 0.24 mM, 8 mM, and 16
(0)
0
20
Concentration,
rodium
cholata
rb
mM
Fig. 1. Dependence of fluorescence intensity on surfactant concentration (25°C). (a) T&on X- 100; (b) SDS; (c)
sodium cholate. The DPH concentration was 5 pM.
148
X. Zhmg
Table I
Comparison
of measured
et d/J.
and published
Biochem. Biophys. Methods 31 (1996) 145-150
values of CMC at 25°C
CMC values (mM)
Surfactant
Triton X- 100
SDS
Sodium cholate
* From Chattopadhyay
Fluorescence
intensity
method
Fluorescence
anisotropy
method
Literature
values *
0.28
8
16.2
0.24
8
16
0.24-0.3
8.0 -8.2
13 -15
and London [7].
mM at 25°C respectively (Fig. 2). These values were similar to the CMCs determined
using the fluorescence
intensity method (Table 1). Lower anisotropy values were
generally obtained at the higher temperature, due to the increased fluidity and lower
microviscosity (Fig. 2).
One advantage of the depolarization method is that anisotropy is insensitive to the
DPH concentration since anisotropy values are dependent primarily on micelle solubilized DPH fluorescence
and are not affected significantly
by the number of DPH
molecules not associated with micelles as shown in Fig. 3. While fluorescence
anisotropies of DPH in Triton X-100 solutions were similar at DPH concentrations of 1,
5 and 10 PM (Fig. 3B), fluorescence intensity values at DPH concentration of 5 and 10
PM were markedly different to 1 PM DPH (Fig. 3A). Furthermore,
anisotropy
determinations
are relatively free of light-scattering
problems, which may be encountered in intensity determinations of slightly turbid suspensions [8].
4. Simplified
description
of the method and its applications
A novel method of determining
critical micelle concentration
using fluorescence
depolarization is described. Fluorescence anisotropy decreased with increasing surfactant
0.00
0.0
0.2
0.4
0.6
0
20
Concentration,
10
/--T
0
--_t_-_
10
20
2.0
mM
Fig. 2. Dependence of fluorescence anisotropy on surfactant concentration (open symbol: 25°C; closed symbol:
37°C). (a) Triton X-100; (b) SDS; (c) sodium cholate. The DPH concentration was 5 PM.
X. Zhang et al./J.
Biochem. Biophys. Merhods 31 (1996) 145-150
149
n
m
Concentration,
Fig. 3. Effect of DPH concentration
on fluorescence
%
intensity (A) and anisotropy
(B) of Triton X-100 (25°C).
concentration below the CMC and leveled off above the CMC. The surfactant concentration at which anisotropy values became independent of concentration was taken to be
the CMC of the surfactant. The depolarization method does not depend on the
concentration of DPH and is largely immune to light-scattering problems encountered in
turbid aqueous systems.
Acknowledgements
We are grateful for the financial support from Angiogenesis Technologies, Vancouver, BC, Canada.
150
X. Zhuq
et al./J.
Biochem. Biophys. Methods 31 (19961145-150
References
[I I Alkar-Gnyuksel,
H., Ramakrishnan,
S., Chai, H.B. and Pezzuto, J.M., A mixed micellar formulation
suitable for the parenteral administration of taxol, Pharmaceutical Res., I1 (1994) 206-212.
121 Junginger, H.E., Ointments and creams as colloidal drug delivery systems. In: Kreuter, J. (Ed.), Colloidal
Drug Delivery Systems. Marcel Dekker, New York, 1994, pp. l-30.
[3] Attwood, D., Microemulsions.
In Kreuter, J. (Ed.). Colloidal Drug Delivery Systems. Marcel Dekker, New
York, 1994, pp. 31-72.
(41 Kataoka, K., Kwon, G.S., Yokoyama, M., Okano, T. and Sakurai, Y., Block copolymer micelles as
vehicles for drug delivery, J. Controlled Release, 24 (1993) l19- 132.
[S] Kwon, G., Naito, M., Yokoyama, M., Okano, T., Sakurai, Y. and Kataoka, K., Micelles based on AB
block copolymers of polfiethylene
oxide) and poly(beta-benzyl
L-aspartate), Langmuir, 9 (1993) 945-949.
[6] Evans, D.F. and Wennetstrom,
H., The Colloidal Domain, Where Physics, Chemistry, Biology and
Technology Meet, Chapter 3, VCH, New York, 1994.
171 Chattopadhyaa,
A. and London, E., Fluorimetric determination of critical micelle concentration avoiding
interference from detergent charge, Anal. B&hem.,
139 (1984) 408-412.
[81 Burt, H.M. and Jackson, J.K., Monosodium mate monohydrate crystal induced changes in membrane
fluidity: a fluorescence polarization study, J. Rheumatol., 15 (1988) 1144-I 150.