Determination of Critic al Mic elle Conc entration

Values Using Capillary Elec trophoresis

Instrumentation
Alejandro Cifuentes,

J ose L. Bernal,

and J ose C. Diez-Masa*

,
Laboratory of Analytical Chemistry, University of Valladolid, Paseo de la Magdalena s/n, 47011 Valladolid, Spain, and
Institute of Organic Chemistry (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
Thepurposeofthisworkistodemonstratetheusefulness
of capillary electrophoresis (CE) instrumentation for
determiningvaluesofcritical micelleconcentration(cmc)
ofsurfactants. TheapproachessentiallyconsistsofaCE
version of thetraditional method of measuringvalues of
cmcbyconductivity. Namely, thedifferentconductivities
of ionic surfactants in solution depending on their ag-
gregation state, i.e., as monomers or micelles, and the
effect on theelectrical current as usuallymeasured in a
CE apparatusareemployedtodeterminethecmcvalues.
Thecmc of sodiumdodecyl sulfate(SDS) and cetyltrim-
ethylammoniumbromide (CTAB) is obtained in several
mediasuchaswater, aqueoussolutionscontainingsalts,
organosalinesolutions, andaqueoussolutionscontaining
-cyclodextrin. Thecmcvaluesfor SDSandCTAB under
these conditions are in good agreement with those re-
portedintheliterature. Advantagesanddrawbacksofthis
procedureas well as its implications in micellar electro-
kineticchromatographyarediscussed. Fromour results,
it is deduced that the present method can be used with
highconfidencetodeterminevalues of cmc inafast and
easyway.
In the early 1930s, Bury and co-workers
1,2
established the term
critical micelle concentration (cmc), defining it as a concentra-
tion range below which surfactant is in solution as a monomer
and above which practically all additional surfactant added to the
solution forms micelles. As mentioned by Mukerjee and Mysels,
3
cmc is probably the simplest way of describing the colloid and
surface behavior of a surfactant solute. Moreover, this value
determines the industrial usefulness and biological activity of
detergents as well as some other interesting surfactant features
like solute-solvent and solute-solute interactions.
Directly related to the solute-solvent interactions in micellar
mediais the application of surfactants in separation science.
4
That
is, the different associations or solubilizations of analytes by
aqueous micelles are employed for their separation in, e.g., liquid
chromatography, liquid-liquid extractions, cloud point extractions,
membrane techniques, etc. In 1984, Terabe and co-workers
5
introduced a new separation mode of capillary electrophoresis
(CE) using surfactants. This new CE mode, called micellar
electrokinetic chromatography (MEKC), allows the separation of
analytes without charge under the influence of an electric field,
while improving the solubility of highly hydrophobic substances
in the separation buffer. Both effects are the result of the use of
surfactants, mainly sodium dodecyl sulfate (SDS), which are added
to the separation buffer at concentrations higher than their cmc.
As described in the literature,
6-10
MEKC has found numerous
applications in the separation of drugs, metabolites, and other
small molecules and also in the field of biopolymers.
In MEKC, as well as in any other separation technique using
amphiphilic molecules, agood knowledge of the cmc value of the
surfactant employed in the actual separation media is mandatory
to optimize and even to carry out the analysis. Moreover, it has
to be stressed that cmc values depend on the separation condi-
tions, e.g., ionic strength, temperature, additives, etc.
In order to determine such values, a large variety of methods
have been traditionally employed,
3
e.g., conductivity, light scat-
tering, surface tension, spectrophotometry, etc. More recently,
other new methods, such as speed of sound
11
or NMR,
12
have
been shown to be useful for this type of determination.
MEKC has been also applied to the determination of cmc
values.
13-15
In some works, plots of capacity factor, k, versus
concentration of surfactant have been employed,
13,14
obtaining the
cmc value by extrapolating to k ) 0. However, this method does
not seem to be reliable enough to obtain cmc values, as mentioned
by some authors.
13
The other MEKC procedure is based on the
variation of the effective electrophoretic mobility of a neutral
compound with the surfactant concentration,
15
which, in turn, is
related to the cmc value. However, it is well known that the use
of an indicator dye which is included into the micelle can alter
the cmc value to some extent.
3
In fact, cmc values measured using
* Corresponding author. Fax: 34-1-5644853. E-mail: diez-masa@pinar1.csic.es.

University of Valladolid.

Institute of Organic Chemistry (CSIC).

(1) Davies, D. G.; Bury, C. R. J. J. Chem. Soc. 1930, 2263-2270.
(2) Grindley, J.; Bury, C. R. J. J. Chem. Soc. 1929, 679-684.
(3) Mukerjee, P.; Mysels, K. J. Critical Micelle Concentration of Aqueous
Surfactan Systems; National Bureau of Standards: Washington, DC, 1970.
(4) Armstrong, D. W. Sep. Purif. Methods 1985, 14, 213-304.
(5) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem.
1984, 56, 111-113.
(6) Kuhr, W. G. Anal. Chem. 1990, 62, 403R-414R.
(7) Kuhr, W. G.; Monnig, C. A. Anal. Chem. 1992, 64, 389R-407R.
(8) Monnig, C. A.; Kennedy, R. T. Anal. Chem. 1994, 66, 280R-314R.
(9) St. Claire, R. L. Anal. Chem. 1996, 68, 569R-586R.
(10) Matsubara, N.; Terabe, S. In Capillary Electrophoresis in Analytical Bio-
technology; Righetti, P. G., Ed.; CRC Press: Boca Raton, FL, 1996; pp 155-
182.
(11) Junquera, E.; Tardajos, G.; Aicart, E. Langmuir 1993, 9, 1213-1219.
(12) Lee, Y. S.; Woo, K. W. J. Colloid InterfaceSci. 1995, 169, 34-38.
(13) Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1985, 57, 834-841.
(14) Strasters, J. K.; Khaledi, M. G. Anal. Chem. 1991, 63, 2503-2508.
(15) Jacquier, J. C.; Desbene, P. L. J. Chromatogr. A 1995, 718, 167-175.
Anal. Chem. 1997, 69, 4271-4274
S0003-2700(97)00696-3 CCC: $14.00 1997 American Chemical Society Analytical Chemistry, Vol. 69, No. 20, October 15, 1997 4271
such a procedure are systematically lower than those obtained
by other methods.
3
Probably due to the aforementioned problems, we have
observed through literature that authors using CE apparatus use
other instrumentation, e.g., surface tension apparatus,
16
conduc-
timetric titration,
17,18
etc., to carry out determinations of cmc values
when needed. However, according to Tickle et al.,
19
it seems
possible to determine in an easy way the cmc values of detergents
by plotting the electrical current values, as measured by a CE
instrument, versus the surfactant concentrations at agiven electric
field. In this work, we discuss, validate, and extend the usefulness
of this CE approach.
EXPERIMENTAL SECTION
Instrumentation. Measurements were carried out using a
P/ ACE 5000 HPCE (Beckman Instruments Inc., Fullerton, CA)
electrophoresis apparatus controlled by a Pentium 100 MHz
personal computer. Two fused silica capillaries (Polymicro
Technologies Inc., Phoenix, AZ) were employed. One, with 100
m i.d., 360 m o.d., and 27 cm of total length, was used to
determine the cmc of cetyltrimethylammonium bromide (CTAB)
in water. For all the other experiments, a capillary with 75 m
i.d., 360 m o.d., and 47 cm of total length was employed. The
temperature of the capillary was maintained at 25 C. The
electrical current data were collected and analyzed using System
Gold software from Beckman running on the Pentium 100 MHz
computer. As stated in the apparatus specifications provided by
the manufacturer, the minimum detectable variation of the electric
current was 0.1 A.
Reagents. Sodium dodecyl sulfate (SDS), sodium chloride,
and borax (sodium tetraborate decahydrate) were from E. Merck
(Darmstadt, Germany). Acetonitrile was from Scharlau (Barce-
lona, Spain). Cetyltrimethylammonium bromide(CTAB) was from
EGA-Chemie (Steinheim, Germany), and -cyclodextrin was from
Fluka (Madrid, Spain).
Procedures. A 50 mM SDS solution was freshly prepared
every day by dissolving the required amount of surfactant in
Milli-Q water (Millipore Corp., Bedford, MA). Likewise, aqueous
solutions of 5 mM CTAB, 20 mM -cyclodextrin, 40 mM borax,
and 60 mM sodium chloride in Milli-Q water were also prepared.
Final solutions were obtained by conveniently mixing and diluting
these solutions.
Cmc values were determined for both surfactants in the
following media: CTAB in water, SDSin water, SDSin a 30 mM
NaCl aqueous solution, SDSin a 10 mM -cyclodextrin aqueous
solution, SDS in a 20 mM borax (pH 9.2) aqueous solution, and
SDSin a 5 mM borax solution containing water-acetonitrile (85:
15 v/ v). In Table 1, voltages and capillaries dimensions employed
are indicated for each case. These experimental conditions were
chosen in order to obtain an adequate value of electric current,
as will be discussed below. All determinations were done at 25
C. At least nine different concentrations of surfactant were
monitored to determine the cmc value in each set of given
conditions.
Prior to measurement of the electric current, the capillary was
consecutively rinsed with water and the new surfactant solution
for 0.5 min. The high voltage was applied, and after waiting 1
min for equilibration of the CE system, the electric current was
measured.
THEORY
It is well known that micellation of surfactants in aqueous
mediaoccurs due to the fact that the reduction of the hydrocarbon-
water interface is energetically favored. The critical micelle
concentration at which aggregation takes place reflects that the
hydrophobic interaction between the hydrocarbonaceous moieties
of the surfactant molecules is balanced by the hydration and
electrostatic repulsive effects of hydrophilic head groups.
20
Thus,
hydrophobic forces control the formation of micelles, while
electrostatic ones limit the maximum size (aggregation number)
that micelles can reach under determined conditions. This
equilibrium can be explained through a scheme similar to that
used by Evans
21
in 1956. Assuming that the micelle is composed
of nionic monomers or amphiphiles (S
-
) and it carries mco-ions
(e.g., Na
+
) induced within the micelle, at concentration not greatly
above the cmc, we can write
(16) Piera, E.; Erra, P.; Infante, M. R. J. Chromatogr. A 1997, 757, 275-280.
(17) Saitoh, K.; Kiyohara, C.; Suzuki, N. J. HighResolut. Chromatogr. 1991, 14,
245-248.
(18) Terabe, S.; Katsura, T.; Okada, Y.; Ishihama, Y.; Otsuka, K. J. Microcolumn
Sep. 1993, 5, 23-33.
(19) Tickle, D. C.; Okafo, G. N.; Camilleri, P.; Jones, R. F. D.; Kirby, A. J. Anal.
Chem. 1994, 66, 4121-4126.
(20) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological
Membranes; Wiley: New York, 1980.
(21) Evans, H. C. J. Chem. Soc. 1956, 579-586.
Table 1. Cmc Values Determined by CE and Compared with Those Found in the Literature
cmc
a
(mM)
CE conditions
CE results from
least-squares fitting
b
CE literature refs
SDSin water 75 m i.d., 47 cm; 20 kV 1170c+ 0.90, r
2
) 0.9996 8.3 8.1-8.4 3, 32
530.5c+ 6.21, r
2
) 0.996
CTAB in water 100 m i.d., 27 cm; 20 kV 6200c+ 0.45, r
2
) 0.9997 0.93 0.90-0.98 3, 33
1500c+ 4.80, r
2
) 0.998
SDS+ 20 mM borax 75 m i.d., 47 cm; 15 kV 577.1c+ 43.55, r
2
) 0.975 3.1 2.6-3.2 17, 28
411c+ 44.06, r
2
) 0.995
SDS+ 10 mM -cyclodextrin 75 m i.d., 47 cm; 20 kV 1159.1c+ 0.08, r
2
) 0.9996 14.8 14.0-16.0 25-27
505c+ 9.73, r
2
) 0.9993
SDS+ 30 mM NaCl 75 m i.d., 47 cm; 20 kV 950c+ 52.13, r
2
) 0.9991 3.6 2.9-3.7 3, 28
350c+ 54.30, r
2
) 1
SDS+ 5 mM borax + 15%acetonitrile 75 m i.d., 47 cm; 20 kV 1566.1c+ 18.26, r
2
) 0.9999 7.3 7.7 28
1367.9c+ 19.71, r
2
) 0.9992
a
All determinations were done at 25 C.
b
r is the correlation coefficient of the equation i ) Bc + A, where i is the electric current (A) and c
the surfactant concentration (mol/ L). The first equation applies for c < cmc and the second one for c > cmc, respectively.
4272 Analytical Chemistry, Vol. 69, No. 20, October 15, 1997
This equilibrium shows that, at concentration of surfactant c
>cmc, amphiphiles are mainly in amicellar form, i.e., nmolecules
of surfactant plus mco-ions will aggregate to form amicelle, while
at c< cmc, surfactant will be in amonomeric form, moving freely
in solution in a way similar to that of co-ions at these low
concentrations.
On the other hand, in a CE instrument, applying a voltage V
on a capillary of radius r and total length l, filled with a surfactant
solution, gives an electric current I according to Ohms law:
22
where
Na
+,
S
-, and
mic
are the specific conductivities of the co-
ion, amphiphile, and micelle, respectively. It can be easily
deduced from the equilibrium shown above that, at c< cmc, the
main contribution to the overall conductivity of the solution comes
from
Na
+ and
S
-, while at c> cmc, the main contribution comes
from
mic
, with alownumber of co-ions, e.g., sodium, moving freely
in solution at these high concentrations.
21
The observed decrease
in conductivity of solutions of ionic surfactants above the cmc is
explained through both inclusion within the micelle of ions of
charge opposite (co-ions) to that of amphiphiles
21
and the increase
in resistance to migration of the micelle caused by the ionic
atmosphere of co-ions surrounding this ordered structure.
23
Thus, in CE, by plotting electric current against different
surfactant concentrations at a given voltage, experimental points
must fit into two lines whose slopes should be different depending
on the range of cconsidered.
19
That is, the two slopes correspond
to the monomeric and micellar states of the surfactant. By
employing simple mathematical procedures as usually applied for
the determination of cmc in other methods,
3
it will be possible to
calculate such avalue. In our case, the cmc values were calculated
from the intersection point of two straight lines, whose equations
were calculated by the linear least-squares method.
RESULTS AND DISCUSSION
In Figure 1A, the plot of the electric current values obtained
for different concentrations of SDSin water at 25 C is shown. As
expected, experimental points fit into two straight lines of different
slope whose values are given in Table 1, i.e., 1170 at c < cmc
versus 530.5 at c > cmc. As can be seen in Figure 1A, this
variation of slope takes place at a SDSconcentration of 8 mM.
Such variation is originated by the conductivity change brought
about by the micelle formation, that concentration (or range of
concentrations
3
) being the cmc value. In order to give a more
precise value of cmc, the procedure explained under Theory was
applied. The experimental data appearing too close to the cmc
value, indicated by filled squares in Figure 1, were not included
in the cmc calculations, as recommended by Mukerjee and
Mysels.
3
The intersection point of the two straight lines, i.e., the
cmc values of SDS in water at 25 C, gave a c value equal to 8.3
mM by this procedure. As can be deduced from Table 1, this
value is very close to those found in the literature, i.e., from 8.1
to 8.4 mM, depending on the authors, which seems to indicate
the usefulness of the present procedure.
Similar representation was obtained when the electric current
was plotted versus different concentrations of CTAB in water at
25 C, and these results are also given in Table 1. In this case,
the intersection point of the two straight lines gave a cmc value
equal to 0.93 mM, which is in good agreement with those values
found in the literature under the same conditions, i.e., 0.90-0.98
mM. The versatility of this CE procedure can be easily under-
stood with the CTAB example, as is discussed next. For
determining the cmc of CTAB, the capillary of 75 m i.d. and 47
cm length used in the previous case was substituted by one of
100 m i.d. and 27 cm of length. This was done because CTAB
solutions were of very low concentration, ranging from 0.2 to 1.6
mM. Their low conductivity, together with the high electrical
resistance of the 75 m i.d. capillary employed, resulted in a very
low electric current. Namely, under an electric field of 42 kV/ m,
avariation in the CTAB concentration from 1.2 to 1.6 mM induced
a current variation as small as 0.2 A. Such a value is too close
to the minimum current variation detected by the CE instrument
used, i.e., 0.1 A. Moreover, by applying the highest electric field
provided by the instrument, 64 kV/ m under these conditions, the
(22) Foret, F.; Krivankova, L.; Bocek, P. In CapillaryZoneElectrophoresis; Radola,
B. J., Ed.; VCH: Weinheim, 1993; pp 7-15.
(23) Hunter, T. J. Zeta Potential in Colloid Science: Principleand Applications;
Academic Press: London, 1981; pp 98-112.
nNaSf nNa
+
+ nS
-
a (S
n
Na
m
)
(n-m)-
+ (n - m)Na
+
V )
l
r
2
(
Na
+
+
S
-
+
mic
)
I
Figure 1. Plots of electric current vs concentration of SDS under
different conditions. Fused silica capillary: 75 m i.d. and 47 cm
length. Temperature was kept at 25 C. Conditions: (A) SDS in
water; current measured at 20 kV. (B) SDS + 10 mM -cyclodextrin;
current measured at 20 kV. (C) SDS + 5 mM borax + 15%
acetonitrile; current measured at 25 kV.
Analytical Chemistry, Vol. 69, No. 20, October 15, 1997 4273
variation of electric current observed was only 0.3 A. These
two low values of electric current reduced substantially the
sensitivity of the method since concentration steps smaller than
0.3-0.4 mM CTAB were avoided. Therefore, in order to increase
the sensitivity of our system, a shorter capillary with 100 m i.d.
was employed. This capillary allowed us to work with smaller
concentration steps. Namely, for the same increase in concentra-
tion as above (1.2-1.6 mM) and the same electric field of 42 kV/
m, a variation of 0.6 A was measured.
Besides the possibility to manipulate the sensitivity of this
method by choosing the adequate capillary size, the high voltage
applied can also be selected to improve the sensitivity of the
method. However, in this case, systematic errors can arise from
the increase in temperature of the solvent due to the Joule effect
if heat is not properly dissipated.
24
In our case, to overcome this
problem, the power generated in the capillary was always kept
below 2 W/ m, even when, according to the instruments specifica-
tions, it was able to dissipate up to 5 W/ m.
Figure 1B shows the plot obtained when the electric current
was represented against aqueous solutions containing different
concentrations of SDSplus 10 mM -cyclodextrin. The cmc value
of SDScalculated at the intersection of the two straight lines given
in Table 1 was 14.8 mM. This value also agrees quite well with
those found in the literature, i.e., 14.0-16.0 mM. Further, by
comparing the cmc value obtained, 14.8 mM, with that for SDS
in water, 8.3 mM, it can be deduced that -cyclodextrin shifts the
cme of SDS to higher values. This effect has already been
studied,
11,25-27
and it is explained through the inclusion of SDS
monomers into the cavity of -cyclodextrin following an ap-
proximately 1:1 stoichiometry. Therefore, this procedure would
allow one to determine the cmc of the surfactant in MEKCbuffers
in some enantiomeric separations where surfactant and cyclodex-
trin are contained in the buffer. This knowledge can help to
optimize the separation conditions, as well as to carry out CE
thermodynamic studies
18
or any other investigation
3,16
for which
the cmc value is required.
When the electric current was measured for solutions contain-
ing different concentrations of SDS plus 5 mM borax and 15%
acetonitrile, the plot shown in Figure 1C was obtained. As can
be seen, under these conditions, the change in slope is not as
abrupt as that observed in the other two cases, i.e., 1566.1 versus
1367.9, as given in Table 1. This is probably due to the denaturing
effect of acetonitrile on micelles, which has been demonstrated
to bring about the generation of small surfactant-solvent mixed
aggregates.
28
This effect is typically explained through the
multiple equilibria model,
29
also called the stepwise aggregation
model.
30
Therefore, the change in conductivity or electric current
is lower than that observed for the previous cases. Although,
under these conditions, to refer to cmc value is not very rigorous,
the cmc value was calculated for comparative purposes. From
Figure 1C, a value of 7.3 mM was obtained. This value is slightly
lower than SDSin water and similar to the value of 7.7 mM found
in the literature for the same conditions.
We have also included in Table 1 the results obtained for the
determination of the cmc of SDSin aqueous solutions containing
20 mM borax (pH 9.2) or 30 mM NaCl. As can be seen, under
these conditions, there is also a good agreement between the CE
results and those reported in the literature, which seems to
corroborate the applicability of this method for cmc determina-
tions.
Further, some other possibilities derived from the use of aCE
instrument to determine cmc values should be mentioned. For
instance, compared to traditional conductimetry measurements,
the CE procedure would allow the study in an easy way of the
influence of temperature on cmc values based on the good
temperature control provided by the CE instruments. Moreover,
it can also permit determination of cmc values in an unattended
mode, while the volume of surfactant required (and additives if
any) for each measurement is much smaller than that normally
needed in traditional conductimetry. Besides, unlike other CE
procedures,
13-15
the present method for determining cmc values
does not require dyes nor separations. However, although the
method involving electrical conductance is the preferred method
for determining the cmc, and far more accurate results have been
reported using this method than any other,
31
the method is limited
to ionic surfactants. It also presents limitations for measurements
carried out in highly conductive solutions.
ACKNOWLEDGMENT
This work was supported by the Commission of the European
Communities (Training and Mobility of Researchers, Contract No.
ERBFMBICT950003) and by aDGICYT project (PB94-02818-C02-
C02). The authors thank Dr. F. Ortega, Physical Chemistry
Department, Complutense University (Madrid, Spain), for fruitful
discussion.
Received for review July 1, 1997. Accepted July 10,
1997.
X
AC970696N
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X
Abstract published in AdvanceACS Abstracts, September 1, 1997.
4274 Analytical Chemistry, Vol. 69, No. 20, October 15, 1997