Article

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Determining the Critical Micelle Concentration of Surfactants by a

Simple and Fast Titration Method
Shengen Wu, Feiqing Liang, Danna Hu, Hao Li, Weijie Yang, and Qiuhua Zhu*
School of Pharmaceutical Sciences, Southern Medical University, 1838 Guangzhou Avenue North, Guangzhou 510515, China
*
S Supporting Information

ABSTRACT: Critical micelle concentration (CMC) is a

crucial parameter of widely used surfactants, and many
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methods have been developed for CMC determination.

However, the current methods for CMC determination,
such as conductive, surface tension, and fluorometric
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methods, are tedious and time- and sample-consuming

because a series of samples with different concentrations of
surfactants need to be prepared and measured. Although an
economical, simple, and fast titration method for CMC
determination (only one sample and several minutes are
needed) was reported using changes in the color/fluorescence
of ionic organic dyes, it has not been used in practical CMC
determination owing to the disadvantages of these dyes: very
narrow application range (only suitable for cationic or anionic surfactants) and difficult to identify titration end point, especially
using different concentrations (10−300 μM) for the same kind surfactants. Here a C6-unsubstituted tetrahydropyrimidine
(THP-T1) was found to possess unique and excellent characteristics in titrated surfactant solutions: above CMC, preferring to
dissolve in micelles and showing no emission, and not until near/at CMC, being released from micelles and instantly forming
aggregates with strong fluorescence. The fluorescence-turn-on change at CMC (titration end point) is so sensitive that it can be
clearly observed without comparison of blank and control of dye concentration, and the concentration (c′THP) of THP-T1 in
titrated solution at CMC is only about 1 μM for zwitterionic surfactants and 2.5 μM for other kinds of surfactants. The CMC
values determined by the THP-T1-based titration method are almost the same as those determined by the fluorometric method
using THP-T1 as probe. THP-T1 overcomes the disadvantages of reported dyes for CMC titration and realizes the economical,
simple and fast CMC titration of different kinds of surfactants for the first time.

S urfactants have diverse properties, such as dispersion,

solubilization, wetting, emulsification, foaming and corro-
sion resistance, which lead to their wide applications, for
instance in synthetic chemistry,1 protein separation,2 analytical
chemistry,3,4 multifunctional material chemistry5 and drug
delivery.6 Above a particular concentration called the critical
micelle concentration (CMC), they form thermodynamically
stable micelles and show sharp changes in properties. The
CMC value is usually influenced by environmental factors and
relates to suppliers. Therefore, the investigation and
application of surfactants necessitate the determination of
their CMC values.7−10 However, current methods for CMC
determination are tedious and time- and/or sample-consuming
because a series of surfactant solutions with different
concentrations below and above CMC must be prepared and
measured to obtain the relationship between surfactant
concentration and a certain parameter, from which the CMC
value is identified. The parameter is a physical property of
surfactant solutions, such as surface tension7,8,11,10 and electric
conductivity,11 or an optical property of probes in surfactant
solutions, such as absorbance,12 excited state lifetime,13
emission vibration fine structures,14−16,11 fluorescence inten-
sity,12,11 and wavelength.9 In 1946, Corrin et al. determined
the CMC values of anionic surfactants using cationic
pinacyanol chloride (Figure 1) as indicator (100−300 μM)
by a simple and fast titration method for the first time.17 Then
Corrin and Harkins determined the CMC values of cationic/
anionic surfactants using other anionic/cationic dyes (10−100
μM).18,19 However, the CMC titration method has not been
applied by others owing to the disadvantages of very narrow
application (a dye is only suitable for anionic or cationic
surfactants) and being difficult to identify changes in the color/
fluorescence of dyes at CMC, especially using different
concentrations for the same kind of surfactants. Developing a
fast and simple method for CMC determination of ionic and
nonionic surfactants is urgently needed in practical applica-
tions.
In 2011,20,21 we developed an efficient five-component
reaction for the synthesis of a new series of racemic C6-
unsubstituted tetrahydropyrimidines (THPs) and found that
THPs possess the usual aggregation-induced emission (AIE)
Received: October 11, 2019
Accepted: November 11, 2019
Published: November 11, 2019

© 2019 American Chemical Society 4259 DOI: 10.1021/acs.analchem.9b04638

Anal. Chem. 2020, 92, 4259−4265
Analytical Chemistry Article

Figure 1. Molecular structures of some reported ionic dyes for CMC titration of cationic or anionic surfactants17,18 and the structure of THP-T1
for CMC titration of different kinds of surfactants.

characteristic that was found in 200122 and has shown great titrated solution, that is, the concentrated SDS water/PBS
advantages in wide areas.23−25 In addition, THPs, possessing solution (50 mM) containing THP-T1 (about 1 and 2.5 μM
mixed through-bond and -space conjugation26 and efficient for zwitterionic and other kinds of surfactants at CMC,
heteroenantiomeric self-assembly,27 have the characteristics of respectively), was titrated using water/PBS. The titrated CMC
an unusual thermostimuli fluorescence response,28 no emission was calculated with the concentration (cS) of surfactant in
in surfactant micelles29,30 (conventional organic fluorophores initial titrated solution, the volume (Vs) of initial titrated
show increased fluorescent intensity owing to sequestration, solution, and the volume (Vtitr) of titrating solvent (here is
isolation from quenchers, and increased microviscosity of the water/PBS): CMC = (cS × Vs)/(Vs + Vtitr).
environment.31), and mechanofluorochromism.32 We devel-
oped THPs as a series of unique and clearly visible
fluorescence-turn-on (showing the strongest fluorescence at
■ RESULTS AND DISCUSSION
CMC Titration of Anionic SDS. The CMC titration of
CMC) probes for CMC determination of ionic surfactants29,30 SDS was first investigated. The CMC titration using THP-T1
as well as made high throughput CMC determination workable as indicator is based on its excellent characteristics: with the
using highly sensitive THPs.30 However, the THP-based CMC addition of water into the titrated solution, above CMC, it
determination is still conducted by the tedious and time- and prefers to dissolve in micelles and shows no emission; not until
sample-consuming fluorometric method. Can we realize near/at CMC, it is released partly/completely from micelles
simple, fast, and economical CMC titration using THPs and and instantly forms aggregates with strong fluorescence. The
resolve the thorny problem of tedious and time- and sample- fluorescence change of THP-T1 in the titration process is
consuming CMC determination? As an ideal indicator for shown in Scheme 1.
CMC titration, its change at CMC demands to be not only
clearly visible but also sensitive and prompt. Thus, clearly Scheme 1. CMC Titration Process of SDS Using THP-T1 as
visible fluorescence-turn-on change at CMC does not mean Probe and the Mechanism of the Fluorescence-Turn-on
that THPs can be used as ideal indicators for CMC titration. Change of THP-T1
After scanning the behaviors of 16 THPs reported in our
previous work30 in anionic sodium dodecyl sulfate (SDS) and
cationic cetyltrimethylammonium bromide (CTAB), we found
that only three of them meet the demands for ideal indicators.
We thought that other THPs were also only suitable for ionic
surfactants as the THP reported in our previous work.29
Pleasantly and unexpectedly, after further investigation, we
found that THP-T1 (Figure 1), one of the three THPs, can be
used as an excellent indicator for CMC titration of not only
ionic but also nonionic surfactants, such as anionic (SDS;
sodium dodecyl sulfonate, SDSN), cationic (CTAB; dodecyl-
dimethylbenzylammonium chloride, DDBAC; dodecyltrime-
thylammonium chloride, DTAC and cetyltrimethylammonium
chloride, CTAC), zwitterionic (dodecyl dimethyl betaine, BS-
12; dodecyl dimethyl hydroxyl sulfopropyl sulfobetaine, BHS-
12), and nonionic (polyethylene glycol tert-octylphenyl ether,
Triton X-100, Triton X-114) surfactants (the molecular
structures of these surfactants are shown in Figure S1).

■ EXPERIMENTAL SECTION
CMC Titration of Surfactants in Water/Phosphate
Buffer Saline (PBS). The CMC titration was conducted in a 5
mL bottle with a magnetic stirrer under UV light (365 nm)
(see Figure S2 and Movie in Supporting Information). The
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In terms of the THP-T1-based titration mechanism, the
sensitivity of THP-T1 to the titration end point (CMC) relates
to its concentration. Therefore, the suitable concentration of
THP-T1 in SDS titrated solution was screened. The titrated
CMC values are shown Table 1. When the concentration
(c′THP) of THP-T1 at CMC increases from 1.0 to 6.9 μM, the
DOI: 10.1021/acs.analchem.9b04638
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Table 1. Screening of the Amount of THP-T1 in SDS Titrated Solution (20 mM)a
entry VTHPb (μL) cTHPc (μM) VH2Od (mL) V′H2Oe (mL) c′THPf (μM) CMCg (mM) CMCh (mM)
1 3.0 3.0 2.34 ± 0.05 2.52 ± 0.04 0.9 5.68 ± 0.06 6.40/6.85i
2 3.3 3.3 2.10 ± 0.01 2.17 ± 0.01 1.0 6.31 ± 0.02
3 3.5 3.5 2.06 ± 0.01 2.14 ± 0.01 1.1 6.37 ± 0.01
4 4.0 4.0 2.03 ± 0.02 2.13 ± 0.02 1.3 6.39 ± 0.03 6.43/6.77
5 6.0 6.0 2.06 ± 0.02 2.14 ± 0.01 1.9 6.37 ± 0.02
6 7.5 7.5 2.06 ± 0.01 2.15 ± 0.01 2.4 6.35 ± 0.01
7 7.8 7.8 1.96 ± 0.01 2.05 ± 0.01 2.6 6.56 ± 0.01
8 8.0 8.0 1.95 ± 0.01 2.04 ± 0.01 2.6 6.58 ± 0.02 6.49/6.54
9 8.4 8.4 1.93 ± 0.01 2.01 ± 0.01 2.8 6.64 ± 0.02
10 9.0 9.0 1.93 ± 0.01 2.00 ± 0.01 3.0 6.68 ± 0.02
11 10 10 1.94 ± 0.02 2.03 ± 0.02 3.3 6.60 ± 0.04
12 18 18 1.90 ± 0.02 1.99 ± 0.01 6.0 6.69 ± 0.02 6.48/6.52
13 19 19 1.87 ± 0.02 1.96 ± 0.02 6.4 6.76 ± 0.05
14 20 20 1.76 ± 0.04 1.88 ± 0.03 6.9 6.94 ± 0.11
a
1 mL of concentrated SDS solution (20 mM) was used in initial titrated solution. bVolume of THP-T1 stock solution (1 mM) in concentrated
SDS solution. cConcentration of THP-T1 in initial SDS titrated solution. dVolume of water dropped into titrated solution near CMC (mean value
± standard deviation, three parallel titrations). eVolume of water dropped into titrated solution at CMC (mean value ± standard deviation, three
parallel titrations). fConcentration of THP-T1 in SDS titration-end-point solution. gCMC (mM) = cSDS × VSDS/(VSDS + V′H2O) = 20 × 1/(1 +
V′H2O). hCMC value determined from the samples kept for 0/30 min (the samples were prepared as our previous reported fluorometric method.29
i
CMC value determined from the samples for kept 10/30/60 min (Figure S4).

titrated CMC values (6.31−6.94 mM) of SDS are in the CMC
range (6.19−7.02 mM)30 determined by the fluorometric
method using THPs as fluorescent probes (Figure S3 shows
that, with c′THP down to 0.9 μM, the fluorescence of THP-T1
could still be observed clearly).
To confirm the suitable c′THP values, the CMC value of SDS
was determined by our reported fluorometric method29,30
using different concentrations of THP-T1 as probe. A series of
samples for fluorescence measurement were prepared by
diluting different volumes of concentrated SDS solution (20
mM) with different amount of THP-T1 (cTHP = 3/4/8/18
μM) into 5 mL. Figures S4−S7 show the excitation and
emission spectra of the samples kept for different times, and
the relationship between fluorescence intensity at the emission
peak of THP-T1 and the concentration of SDS. The CMC
values determined by the fluorometric method are listed in
Table 1. The CMC values of SDS determined from the
samples kept 0 min with an increase of cTHP from 3 to 18 μM
(entries 1, 4, 8 and 12) and from the samples kept 30 min with
an increase of cTHP from 8 to 18 μM (entries 8 and 12) are
almost the same (6.48 ± 0.05 mM), which prove that sample-
keeping time and THP-T1 have no influence on CMC.
Unexpectedly, the CMC values determined from the samples
kept 30 min with relatively lower cTHP (3 to 4 μM) (entries 1
and 4) are different (6.85 and 6.77 μM). After carefully
analyzing the factors influencing the line intersection
corresponding to CMC shown in Figures S4e, 5c−7c, we
found that the surfactant below and above CMC shows
different influences on the solubility of THP-T1 because it
exists as monomers below CMC but as monomers and micelles
above CMC. This means that surfactant will alter the line
intersection corresponding to CMC if the fluorescence
intensity of THP-T1, that is, the amount of THP-T1 in
aggregates, relates to its solubility; otherwise, it will not alter
the line intersection, that is, the CMC value. In the samples
just prepared, the equilibrium between precipitation and
dissolution of THP-T1 is not reached and hence the amount
of THP-T1 aggregates mainly depends on its aggregation rate
and concentration. In the samples kept for 30 min, the
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precipitation−dissolution equilibrium of THP-T1 is reached
(the same fluorescence intensity of THP-T1 in the samples
kept for 10 to 60 min in Figure S4e proves that the
precipitation−dissolution equilibrium is reached after samples
are kept 10 min), and hence the amount of THP-T1 in
aggregates relates to its concentration and solubility when
c′THP is not much higher than its solubility but only to its
concentration when c′THP is high enough to ignore its
solubility. It is clear that the amount of THP-T1 in aggregates
only in the samples kept 30 min with relatively lower cTHP
relates to its solubility, that is, is influenced by SDS, which well
explains why only the CMC values determined from the
samples kept 30 min with relatively lower cTHP (3 and 4 μM)
are different. Because the precipitation−dissolution equili-
brium of THP-T1 is not reached at titration end point (CMC),
the amount of THP-T1 in aggregates at CMC mainly depends
on its aggregation rate and concentration, and hence the
titrated CMC is not influenced by SDS. Therefore, for the
fluorescence method, the suitable c′THP is 0.9−6.0 μM when
determining samples immediately and 2.6−6.0 μM when
determining samples kept 30 min. For the titration method,
the suitable c′THP is 1.0−6.0 μM, at which the CMC values
(6.50 ± 0.15 mM) determined by titration are almost the same
as those (6.48 ± 0.05 mM) determined by the fluorescence
method. The experimental results in Figure S6c and S6e
indicate that the CMC value of SDS is not influenced by
DMSO when its concentration is ≤2%.
We also studied the influence of SDS concentration (cSDS) in
initial titrated solution on the titrated CMC value (Table S1).
The titrated CMC value is different and relatively low (5.40
and 5.74 mM) when cSDS = 10 and 15 mM (entries 1 and 2)
but almost keeps the same (6.60, 6.68, and 6.64 mM) when
cSDS increases from 20 to 30 and 35 mM (entries 3−5), which
indicates that the suitable cSDS is ≥20 mM.
CMC Titration of Cationic, Zwitterionic, and Nonionic
Surfactants and the Factors Influencing the Volume of
Water Added from Near CMC to CMC. The CMC titration
of cationic surfactant CTAB, zwitterionic surfactant BS-12, or
nonionic surfactant Triton X-100 is similar to that of anionic
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SDS. As shown in Scheme 2, upon addition of 4/2/8 drops of
water from near CMC to CMC, a sharp change in fluorescence
Scheme 2. CMC Titration Process of Different Kinds of
Surfactants CTAB, BS-12, and Triton X-100 Using THP-T1
as Fluorescence-Turn-on Probe
is clearly visible. For Triton X-100 with CMC values down to
0.28 mM, although eight drops of water were added from near
CMC to CMC, the concentration of Triton X-100 changes
slightly (0.30 to 0.28 mM). These results prove that THP-T1
is an excellent probe for CMC titration of different kinds of
surfactants, and the larger the determined CMC value, the less
the amount of water added from near CMC to CMC. This was
further proved by the CMC titration of cationic surfactants
DDBAC and DTAC with higher CMC values (6.84 and 14.59
mM, respectively) (sharp change in fluorescence upon addition
of only three/two drops of water from near CMC to CMC).
Although THP-T1 could not be used as a probe for CMC
titration of nonionic Tween-20 with very low CMC (0.029
mM) owing to the slow fluorescence change at CMC, it can
still be used as an excellent probe for CMC determination of
Tween-20 by the fluorescence method with c′THP down to 0.2
μM. The influences of THP-T1 and these surfactant
concentrations are similar to those of THP-T1 and SDS
concentrations on titrated CMC values. The screen of these
surfactant and THP-T1 concentrations (Tables S2−S12), the
CMC determination of these surfactants by the fluorometric
method using THP-T1 as probe (Figures S8−S18), and the
detailed discussion of these experimental results are displayed
in SI. The titrated CMC values of these surfactants and the
suitable concentration range of THP-T1 for different kinds of
surfactants are listed in Table S13.
From Scheme 2, it can be seen that the volume (ΔVH2O) of
water added from near CMC to CMC is different for different
surfactants (the volume of each drop of water here is about
0.043 mL). After careful analysis of the factors influencing
ΔVH2O, we found that the influential factors can be presented
by the equation, ΔVH2O = −Δcs × Vt‑1/CMC (the equation
deduction is in SI); here Δcs (=cs‑2 − cs‑1) is the change in the
surfactant concentration (cs) from near CMC (cs‑1) to CMC
(cs‑2) and Vt‑1 is the volume of titrated solution near CMC. As
an ideal CMC titration indicator, THP-T1 or other organic
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Article
dye is not released from micelles until near/at CMC. This
means that Δcs depends on the change in the concentration
(cmicelle) of the surfactant micelles from that (cmicelle‑1) near
CMC to that (cmicelle‑2 = 0) at CMC. The relationship between
Δcs and cmicelle‑1 can be expressed by the equation, −Δcs =
cmicelle‑1 × Nag‑1 (the equation deduction is in SI); here Nag‑1 is
the average aggregation number of the micelles near CMC.
Therefore, the influences of different factors on ΔVH2O can
also be represented by the equation, ΔVH2O = cmicelle‑1 × Nag‑1
× Vt‑1/CMC. As mentioned above, cmicelle‑1 depends on the
indicator concentration (cind‑1) near CMC; that is, the higher
the cind‑1, the higher the cmicelle‑1. The Nag value mainly depends
on surfactant property and concentration. Because the lower
the value of ΔVH2O, the more sensitive the fluorescence-turn-
on change at the titration end point (at CMC), from the
equation, one can see that Vt‑1/cind‑1 (cmicelle‑1) should be as low
as possible and that too large Nag‑1 or too low CMC is not
suitable for CMC titration. For example, for the above-
mentioned Tween-20 with a very low CMC (0.029 mM), its
CMC value could be determined by the fluorometric method
but not by the titration method. Although ΔVH2O is influenced
by four factors, ΔVH2O is only 0.08 to 0.35 mL for different
kinds of surfactant and hence −Δcs is only 0.02 to 0.2 mM
(Schemes 1 and 2) because c′THP is very low (only about 1 μM
for zwitterionic surfactants and 2.5 μM for other kinds of
surfactants; see General Concentration of THP-T1 for CMC
Determination), that is, cmicelle‑1 is very low. The Δcs value is
lower than standard deviation (±0.4 mM) for the CMC value
of SDS determined by the tedious and sample- and time-
consuming fluorometric method using pyrene as a probe.11
Determination of the Approximate CMC Values of
New Surfactants or Surfactants from Different Suppli-
ers Using THP-T1 as a Probe. For new surfactants or
surfactants from different suppliers (the CMC value of a
surfactant obtained from different commercial suppliers is
sometimes very different33), their approximate CMC values
can be determined simply by method II reported in our
previous paper,30 which is very useful to guide the preparation
of samples with suitable concentrations below/above CMC for
titration and fluorescence measurement. For example, we
determined the approximate CMC values of BS-12 (Figure 2),
Figure 2. Determination of the approximate CMC of BS-12. Different
concentrations of BS-12 solutions (from left to right: 1 mM to 5 mM)
with 1 μM of THP-T1. Samples were kept for 0 min and were
observed under a light with 365 nm.
Triton X-100, DDBAC, DTAC, and Tween-20 (Figures S19−
S22, respectively) before CMC titration and fluorescence
measurement. As shown in Figure 2, THP-T1 in BS-12
solutions shows no, weak, and strong emission at cBS‑12 > 4
mM, cBS‑12 = 3 mM, and cBS‑12 ≤2 mM, respectively, from
which it can be deduced that the approximate CMC value of
BS-12 is between 2 and 3 mM.
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Table 2. CMC Values of Different Kinds of Surfactants Determined by Titration, Fluorometric, and Conductive Methods
CMC (mM)
surfactant c′THPa (μM) titrationb fluorc pyrened literature conditione
SDS 2.5 6.56 ± 0.01 6.54 6.69 6.0218−8.112
CTAB 2.5 0.81 ± 0.01 0.81 0.80 0.70−1.211
BS-12 1 2.39 ± 0.01 2.56 2.20 1.2734−2.035
Triton X-100 2.5 0.29 ± 0.02 0.30 0.18 0.1913−0.312
DDBAC 2.5 8.63 ± 0.04 8.47 6.84 6.4036−8.8037 8.05
DTAC 2.5 16.27 ± 0.03 16.59 14.59 16.738−2234 16.10
Tween-20 0.2 0.029 0.027 0.01139−0.0358
a
Concentration of THP-T1 in surfactant solutions at CMC. bTitrated CMC mean value ± standard deviation (three parallel titrations). cCMC
value determined from the samples kept for 30 min by the fluorometric method using THP-T1 as a probe. dCMC value determined by the
fluorometric method using pyrene as a probe. eCMC value determined by the conductive method.

Table 3. Titrated CMC Values of Different Kinds of Surfactants in PBS

entry surfactant csa (mM) Vsb (mL) VTHPc (μL) cTHPd/μM VPBSe (mL) V′PBSf (mL) c′THPg (μM) CMCh (mM) CMCi (mM)
1 SDS 50 0.4 15 37.5 5.42 ± 0.02 5.62 ± 0.02 2.5 3.32 ± 0.01 6.35 ± 0.01
2 CTAB 10 0.4 17 42.5 6.30 ± 0.01 6.48 ± 0.02 2.5 0.58 ± 0.00 0.80 ± 0.01
3 DDBAC 50 0.6 15 25 5.12 ± 0.02 5.25 ± 0.02 2.6 5.13 ± 0.01 8.36 ± 0.04
4 DTAC 50 1 8 8 2.19 ± 0.01 2.34 ± 0.03 2.4 14.96 ± 0.14 16.43 ± 0.03
5 BS-12 50 0.2 4 20 3.92 ± 0.03 4.06 ± 0.02 0.9 2.35 ± 0.01 2.39 ± 0.01
6 Triton X-100 10 0.1 7.5 75 3.00 ± 0.02 3.19 ± 0.02 2.2 0.30 ± 0.01 0.28 ± 0.00
a
Concentration of surfactant in initial titrated solution. bVolume of initial titrated solution. cVolume of THP-T1 stock solution (1 mM) in initial
titrated solution. dConcentration of THP-T1 in initial surfactant titrated solution. eVolume of titrated PBS near titrating end point (at CMC)
(mean value ± standard deviation, three parallel titrations). fVolume of titrated PBS at titrating end point (at CMC) (mean value ± standard
deviation, three parallel titrations). gConcentration of THP-T1 in titration-end-point solution. hTitrated CMC mean value ± standard deviation in
PBS, CMC (mM) = cs × Vs/(Vs + V′PBS). iTitrated CMC mean value ± standard deviation in water (three parallel titrations).

Table 4. CMC Values of SDSN, CTAC, BHS-12, and Triton X-114 Determined by Titration Using General c′THP
surfactant csa (mM) Vsb (mL) VTHPc (μL) cTHPd (μM) VH2Oe (mL) V′H2Of (mL) c′THPg (μM) CMCh (mM) literature
SDSN 50 1 12.5 12.5 3.73 3.90 2.55 10.20 1040−10.8241
CTAC 50 0.1 12.5 125 4.60 4.75 2.58 1.03 1.0242−1.343
BHS-12 50 0.1 4 40 3.87 4.02 0.97 1.21 1.6544
Triton X-114 10 0.1 8.5 85 2.75 3.31 2.47 0.29 0.1845−0.3446
a
Concentration of surfactant in initial titrated solution. bVolume of initial titrated solution. cVolume of THP-T1 stock solution (1 mM) in initial
titrated solution. dConcentration of THP-T1 in initial titrated solution. eVolume of titrated H2O near titration end point (CMC) (mean value ±
standard deviation, three parallel titrations). fVolume of titrated H2O at titration end point (CMC) (mean value ± standard deviation, three parallel
titrations). gConcentration of THP-T1 in titration-end-point solution. hTitrated CMC mean value ± standard deviation in water, CMC (mM) = cs
× Vs/(Vs + V′H2O).

General Concentration of THP-T1 for CMC Determi-
nation and Comparison of the CMC Values Determined
by Titration and Other Methods. In practical applications,
general concentrations of surfactants and THP-T1 are needed.
According to the obtained results in Tables S1, S2, S4, S6, S9,
and S11, it can be deduced that the concentration cs of a
surfactant in the initial titrated solution is as large as possible
and the lowest general cs is 10 mM for surfactants with CMC <
1 mM and CMC + 30 mM for surfactants with CMC > 1 mM.
From Table S13, it can be seen that the general concentration
c′THP of THP-T1 at the titration end point (at CMC) is about
1 μM for zwitterionic surfactant BS-12 and 2.5 ± 0.3 μM for
other kinds of surfactants. As shown in Table 2, with general
c′THP, all the CMC values determined by titration are almost
the same as those determined by the fluorometric method with
6% relative errors for BS-12 and only 1−3% relative errors for
other kinds of surfactants. After study of the general c′THP for
determination of the approximate CMC values, it was found
that the general c′THP is 2.5 μM for surfactants with CMC > 0.1
mM and 0.2 μM for surfactants with CMC ≤ 0.1 mM.
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To assess THP-T1 as a CMC probe, the CMC values of the
surfactants listed in Table 2 were determined by the
fluorometric method using the widely used probe pyrene
(Figures S23−S29). As shown in Table 2, all the CMC values
of these surfactants determined using THP-T1 and pyrene as
probes are almost the same except those of DDBAC and
DTAC. In terms of the additive influence, the CMC values
determined by the conductivity method may be more accurate
than those by addition of probes. Therefore, the CMC values
of DDBAC and DTAC were determined by the conductive
method (Figure S30), which is similar to those determined
using THP-T1 as the probe but significantly higher than those
determined using pyrene as the probe (Table 2). These results
prove that THP-T1 is an excellent titration probe for fast and
simple CMC determination of different kinds of surfactants.
CMC Titration of Different Kinds of Surfactants in
PBS with General c′THP. For practical applications, the CMC
values of SDS, CTAB, BS-12, Triton X-100, DDBAC, and
DTAC in PBS, the most widely used buffer in biochemical
research, were determined by a simple and fast titration
method using general c′THP (about 1 μM for zwitterionic
DOI: 10.1021/acs.analchem.9b04638
Anal. Chem. 2020, 92, 4259−4265
Analytical Chemistry
surfactant and 2.5 μM for other kinds of surfactants) and PBS
as the titrating solution. All the CMC titrations in PBS could
be conducted successfully (Tables S14−S19) as those in pure
water. The titrated CMC values are listed in Table 3. The
CMC values of anionic and cationic surfactants in PBS are
lower than those in water (entries 1−4), but the CMC values
of zwitterionic and nonionic surfactant in PBS are consistent
with those in water (entries 5 and 6). Because PBS contains
several inorganic salts and the CMC values of zwitterionic and
nonionic surfactants in PBS are the same as those in water, it
can be deduced that the THP-T1-based CMC titration is not
influenced by inorganic salts. The lower CMC values of
cationic and anionic surfactants in PBS compared with those in
water are expected to originate from the influence of inorganic
salts because the CMC values of cationic and anionic
surfactants are sensitive to inorganic salts.19
CMC Titration of Other Surfactants with General
c′THP. For practical applications, we also determined the CMC
values of other surfactants (anionic SDSN, cationic CTAC,
zwitterionic BHS-12, and nonionic Triton X-114) using
general cs (10 mM for CMC = 0.1−1 mM and 50 mM for
CMC = 1−20 mM) and c′THP (about 1 μM for zwitterionic
surfactants and about 2.5 μM for other kinds of surfactants)
(Tables S20−S23). As shown in Table 4, the CMC values
determined by the titration method are similar to those
reported. These results further prove that the general
concentration of THP-T1 is suitable for different kinds of
surfactants. It is worth mentioning that if the change in the
fluorescence of THP-T1 about the CMC is too smooth, the
fluorescence of THP-T1 will appear before the titration end
point. For example, although THP-T1 can be used as a good
probe for CMC determination of zwitterionic 3-[(3-
cholamidopropyl)dimethylammonio]-1-propanesulfonate
(CHAPS) (CMC = 7.45 mM),30 the fluorescence of THP-T1
appears before the titration end point and hence leads to a
much higher CMC value because the change in the
fluorescence of THP-T1 about CMC is very smooth (Figure
S31).
■
CONCLUSION
We found that THP-T1 possesses unique and excellent
characteristics in titrated surfactant solutions (above CMC,
preferring to dissolve in micelles and showing no emission, and
not until near/at CMC, being released from micelles and
instantly forming aggregates with strong fluorescence) and can
be used as the following: (a) An excellent fluorescent indicator
for CMC titration of different kinds of surfactants in water and
PBS, with c′THP of about 1 μM for zwitterionic surfactants and
about 2.5 μM for other kinds of surfactants. THP-T1
overcomes the disadvantages of reported probes for CMC
titration, such as being only suitable for cationic/anionic
surfactants and being difficult to identify the fluorescence-turn-
off or color-conversion change at CMC and no general
concentration for the same kind surfactants, and enables simple
and fast CMC titration of different kinds of surfactants for the
first time. (b) A visible indicator for fast determination of
approximate CMC, which is very useful to quickly identify a
suitable concentration range for the precise CMC determi-
nation of newly developed/purchased surfactants or surfactants
in new environments. (c) An alternative to pyrene for CMC
determination of different kinds of surfactants by the
fluorescence method. The obvious advantages of THP-T1 as
a CMC probe are expected to make it a very useful probe in
4264
Article
wide applications and promote the application and inves-
tigation of surfactants. In fact, upon realization of CMC
titration for different kinds of surfactants, we no longer want to
determine CMC values by conventional methods, such as
fluorometric, conductive, and surface tension methods, because
the THP-T1-based titration method is so fast, simple, and
economical and the conventional methods are so tedious and
sample- and time-consuming. Development of a simple
instrument for CMC determination based on the excellent
characteristics of THP-T1 is underway in our group.
■ ASSOCIATED CONTENT
* Supporting Information
S
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.analchem.9b04638.
Tables S1−S22, Figures S1−S31, and 1H NMR spectra
of THP-T1 (PDF)
CMC titration of SDS (MP4)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: zhuqh@smu.edu.cn.
ORCID
Qiuhua Zhu: 0000-0003-3731-4487
Author Contributions
The manuscript was written through contributions of all
authors.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We are very grateful for financial support from the Science and
the Special Funds for Scientific and Technological Innovation
and Cultivation of Guangdong University Students
(pdjh2019b0102).
■
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4265 DOI: 10.1021/acs.analchem.9b04638

Anal. Chem. 2020, 92, 4259−4265