Fluorescence characteristics of Schiff base-N,N/-bis(salicylidene) trans 1,2-diaminocyclohexane in the presence of bile acid host

Article abstract of DOI:10.1016/j.molliq.2015.08.043

Understanding the formation of bile acid micellar aggregates is of great importance due to its wide biological and pharmacological applications. In this paper, we study the fluorescence properties of Schiff base-N,N^/-bis(salicylidene) trans 1,2-diaminocyclohexane (H₂L) to understand micelle formation by aggregation of different important bile acids - cholic acid, deoxycholic acid, chenodeoxycholic acid and glycocholic acid by steady state and picosecond time-resolved fluorescence spectroscopy. The fluorescence band intensity was found to increase with concomitant blue shift with gradual addition of different bile acids. Several mean microscopic properties such as critical micelle concentration, polarity parameters and binding constant were estimated in the presence of bile acids. The increase in fluorescence quantum yields, fluorescence decay times and substantial decrease in nonradiative decay rate constants in bile acid micellar environment points to the restricted motion of the fluorophore inside the micellar subdomains.

Full text of DOI:10.1016/j.molliq.2015.08.043

Journal of Molecular Liquids 211 (2015) 1052–1059
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Fluorescence characteristics of Schiff base-N,N/-bis(salicylidene)
trans 1,2-diaminocyclohexane in the presence of bile acid host
⁎
Nayan Roy, Pradip C. Paul, T. Sanjoy Singh
Department of Chemistry, Assam University, Silchar, Assam 788 011, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 May 2015
Received in revised form 12 August 2015
Accepted 18 August 2015
Available online xxxx
Understanding the formation of bile acid micellar aggregates is of great importance due to its wide bio-
logical and pharmacological applications. In this paper, we study the fluorescence properties of Schiff
base-N,N/-bis(salicylidene) trans 1,2-diaminocyclohexane (H2L) to understand micelle formation by aggre-
gation of different important bile acids — cholic acid, deoxycholic acid, chenodeoxycholic acid and
glycocholic acid by steady state and picosecond time-resolved fluorescence spectroscopy. The fluorescence
band intensity was found to increase with concomitant blue shift with gradual addition of different bile
acids. Several mean microscopic properties such as critical micelle concentration, polarity parameters and
binding constant were estimated in the presence of bile acids. The increase in fluorescence quantum yields,
fluorescence decay times and substantial decrease in nonradiative decay rate constants in bile acid micellar
environment points to the restricted motion of the fluorophore inside the micellar subdomains.
© 2015 Elsevier B.V. All rights reserved.
Keywords:
Schiff base
Bile acid
Critical micelle concentration
Binding constant
Fluorescence decay
1. Introduction
different spectroscopy techniques like NMR, EPR and CD spectroscopy
[7–10], X-ray scattering [11], light dispersion and refractometry [12]
Bile acids (BAs) are steroid acids found predominantly in the bile of
mammals which are synthesized from cholesterol in the liver to make
up for a small daily loss during the enterohepatic circulation of bile
salts. The biosynthesis of bile acid is the principal route of cholesterol
consumption in the human body [1]. The one or more α-oriented hy-
droxyl groups of BAs are put on the concave surface (α-face) of the ste-
roid backbone and the methyl groups are positioned on the opposite
convex side (β-face). Free molecules of BAs, normally cylindrical shapes
of 20 Å long with a radius of about 3.5 Å, have a great surface activity and
inclination to the formation of large aggregates, owing to this difference
in orientation of hydrophilic and hydrophobic groups on the steroid ring
systems. Prior to secretion by the liver, they are conjugated with either
of the amino acids glycine or taurine. Conjugation further increases their
water solubility, preventing passive re-absorption once secreted into
small intestine. As a result, the concentration of bile acids in small intes-
tine can stay high enough to form micelles and solubilize lipids. Study of
formation of micelle of BAs is of great importance for understanding
their interaction with biological membranes [2], bile secretion [3],
cholesterol solubilization [4] and resveratrol [5] as well as for their
roles as promoters for transport of some drugs through the intestine
mucous membrane [6].
and also by various physical methods like heat of solution and surface
tension measurements [12,13]. In some of the recent publications,
Pártay et al. reported molecular dynamics (MD) computer simulation
method to study the aggregation process of the sodium salts of cholic
acid (CA) and deoxycholic acid (DCA) [14,15]. Such diversity of methods
used in the study of BAs further confirms the great importance and
existing interest in the study of these biological systems partly because
of their uniqueness in the structure when compared with that of the
conventional detergent monomers, which has a hydrophilic head
group and a long hydrophobic tail and, in part, due to their known im-
portance in biological functions. They are substantially different from
synthetic detergents and lipids as shown in regional polarity whereas
BAs possess “surface” polarity [16]. The diverse and unique properties
pertaining to both chemistry and biology arise from their structural
uniqueness. The biological activity of bile acids is mainly based on
their surfactant properties in their salt form. They may interact and
form mixed micelles with many water insoluble species. Due to differ-
ent chiral centers present in BAs, their carboxylic and hydroxyl groups
can be easily modified for much other application used in construction
of artificial receptors and supramolecular architectures. For example, it
has recently been reported that the promotory action of BAs in trans-
portation of drugs in biological system depends on their cmc values
[17]. So, determination of cmc values of different BA micellar systems
is of great importance for elucidating the mechanism of their action.
Nichifor et al. studied the aggregation behavior of BA modified dextran
using N-phenyl-1-naphthyl amine as a fluorescence probe and reported
that the cmc value depends on the nature of hydrophobic moiety and
The formation mechanism and structure of BA micelle as well as the
critical micelle concentration (cmc) values were investigated by using
⁎
Corresponding author.
E-mail addresses: takhelsingh@gmail.com, singhsanjoy2002@yahoo.co.in
(T. Sanjoy Singh).
http://dx.doi.org/10.1016/j.molliq.2015.08.043
0167-7322/© 2015 Elsevier B.V. All rights reserved.

N. Roy et al. / Journal of Molecular Liquids 211 (2015) 1052–1059
1053
the degree of substitution [18]. Fluorescence probes like fluorescein iso-
thiocyanate and also other polycyclic aromatic hydrocarbons having
wide range of aqueous solubility were used to determine the cmc
value of sodium taurocholate in water [19]. Matsuoka et al. used pyrene
as the fluorescence probe to determine the cmc of sodium salt of DCA
[20], whereas, Zhang et al. measured the fluorescence anisotropy of
1,6-diphenyl-1,3,5-hexatriene to study the micellization of CA salt
[21]. In our earlier paper, we also determine the cmc of different BAs
using intramolecular charge transfer (ICT) probe [22]. Such diversity
of different methods used to determine the formation mechanism and
structure of BAs as well as several mean microscopic properties like
cmc, polarity parameters and binding constant indicates its great impor-
tance and existing interest in research for further investigations.
Scheme II. General structure of bile acids (BAs) used in this work.
Schiff bases named after Hugo Schiff, are the compounds containing
azomethine group (−HC_N−) which play a significant importance in
chemistry especially in the development of Schiff base complexes, be-
cause they are potentially capable of forming stable complexes with dif-
ferent metal ions [23]. Schiff base leads to synthesis of various bioactive
compounds which shows a variety of biological activities including an-
tibacterial, antifungal, anticancer and herbicidal activities [24]. Howev-
er, fluorescence characteristics of Schiff base in different organized
media, which can compartmentalize solvents and solutes as well as se-
quester them from the bulk environment, are very rare in the literature.
So, interest in the binding of Schiff base ligand to different bile acid mi-
cellar media has been motivated to prompt the microenvironment of
the micellar systems as the fluorescence emission spectra of this Schiff
base (H2L) shows huge fluorescence maxima shift in homogenous buff-
er solution compared to nonpolar solvent like 1,4-dioxane. The extreme
sensitivity of this probe to polarity prompted us to study in a variety of
microheterogeneous medium like synthetic detergent where the polar-
ity decreases gradually from the bulk aqueous phase to the hydrocarbon
micellar core. Studies based on a completely different class of amphi-
philic compounds, like BAs and their salts, as an alternative to the syn-
thetic detergent or cyclodextrins to improve luminescence analysis
are scantly observed [25,26]. Hence, the aim of this work is to determine
several mean microscopic properties such as cmc, polarity parameters
and binding constant by the method of fluorescence spectroscopy. So,
in the present work, we report the fluorescence characteristic of H2L
(Scheme I) in presence of different bile acids — cholic acid (CA),
deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and glycocholic
acid (GCA) (Scheme II) by steady state and picosecond time-resolved
fluorescence spectroscopy. Here, several mean microscopic properties
such as critical micelle concentration, polarity parameters and binding
constant were estimated in the presence of bile acids and reported in
this paper. The increase in fluorescence quantum yields, fluorescence
decay times and substantial decrease in nonradiative decay rate con-
stants in bile acid micellar environment points to the restricted motion
of the fluorophore inside the micellar subdomains. The present work
can be extended to understand the aggregation behavior and structure
of the microenvironments of different biologically important heteroge-
neous systems like proteins, lipids and enzymes using H2L as fluores-
cent probe.
2. Materials and method
2.1. Materials
N,N/-Bis(salicylidene) trans 1,2-diaminocyclohexane (H2L) was de-
signed and synthesized by one step condensation of salicylaldehyde and
1,2-diaminocyclohexane in absolute ethanol (Scheme I) and character-
ized by FT-IR, 1H and 13C NMR and elemental analysis [27]. CA, DCA,
CDCA and GCA were all obtained from Sigma Aldrich Chemical Co. (Prod-
uct no. C1129, D2510, C9377 and G2878, respectively) and used as re-
ceived without further purification (Scheme II). The water used as
solvent in all the measurements was obtained from Elix10 water purifica-
tion system (Millipore India Pvt. Ltd.). All experiments were carried out at
room temperature (298 K). Bile acids were dissolved in Tris–HCl buffer
(pH 9.2). The ligand concentration (∼1.2 × 10−5 mol dm−3) was low
enough to avoid any aggregation and kept constant during the variation
of bile acids concentration.
2.2. Physical measurements
Steady-state absorption spectra were recorded on a Shimadzu UV-
1601PC absorption spectrophotometer and fluorescence spectra were
obtained in a PerkinElmer LS 45 spectrofluorimeter. Quartz cuvettes of
10 mm optical path length received from Perkin-Elmer, USA (part no.
B0831009) and Hellma, Germany (type 111-QS) were used for measur-
ing absorption and fluorescence spectra, respectively. In both fluores-
cence emission and excitation spectra measurements, 5 nm bandpass
was used in the excitation and emission side. The steady state anisotro-
py (r) can be determined by using the following equation [28]:
IVV − GIVH
IVV þ 2 GIVH
r ¼
ð1Þ
where IVV is the fluorescence intensity when both the excitation and
emission polarizers are oriented vertically and IVH is the fluorescence in-
tensity when the excitation polarizer is vertically and the emission
polarizer is horizontally oriented.
G is the correction factor for the sensitivity of the detector to the
polarization direction of the emission and is defined as:
IHV
IHH
G ¼
ð2Þ
Scheme I. Schematic diagram of N,N/-bis(salicylidene) trans 1,2-diaminocyclohexane
(H2L).
where IHV is fluorescence intensity with the excitation polarizer
horizontally and the emission polarizer vertically oriented, IHH is the

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N. Roy et al. / Journal of Molecular Liquids 211 (2015) 1052–1059
Table 1
fluorescence intensity with both the excitation and emission polarizers
oriented horizontally.
Fluorescence quantum yields (ϕf) were calculated by comparing the
total fluorescence intensity under the whole fluorescence spectral range
with that of a standard (ϕf = 0.546, quinine sulfate in 1 M sulfuric acid
[29]) using the following equation.
Fluorescence properties of H2L in homogeneous buffer solution of pH 9.2 and in presence
of different bile acids.
Environment λma ax/nma λmf ax/nmb Δνfwhm/cm−1c
ν
0,0/cm−1d ΔνSS/cm−1e
Water
CA
DCA
CDCA
GCA
325
322
328
325
324
494
422
410
425
419
4037
9871
6835
9239
9531
30,172
30,518
25,524
31,256
33,183
10,526
7359
6097
7239
6997
s
ꢀ
ꢁ2
Fi 1−10−A
ηi
ηs
ϕif ¼ ϕsf :
:
:
ð3Þ
a
Absorption maxima.
Fluorescence maxima.
Fluorescence spectral width at half maximum.
Intersection of normalized absorption and fluorescence emission spectra.
Stokes shift.
Fs
i
1−10−A
b
c
d
e
where F is the total fluorescence intensity under whole fluorescence
spectral curve, Ai and As are the optical densities of the sample and stan-
dard, respectively and ηi is the refractive index of the solvent at 298 K.
The fluorescence decay curves in homogeneous buffer solution of
pH 9.2 as well as in the presence of different bile acids — CA, DCA,
CDCA and GCA were obtained using LED based time-correlated single
photon counting (TCSPC) system obtained from Photon Technology In-
ternational (PTI). The instrument response function (IRF) was obtained
at 340 nm using a dilute colloidal suspension of dried non-dairy coffee
whitener. The half width of the IRF was ~100 ps. The samples were
excited at 340 nm and the fluorescence emission was collected at corre-
sponding emission wavelength. The number of counts in the peak chan-
nel was at least 10,000. In fluorescence lifetime measurements, the
emission was monitored at the magic angle (54.7°) to eliminate the
contribution from the decay of anisotropy.
As the fluorescence emission spectrum was strongly dependent on
the amount of BAs in solution, gradual addition of CA, DCA, CDCA and
GCA is associated with a blue shift in the fluorescence emission maxi-
mum along with increase in fluorescence quantum yield (Fig. 2) sug-
gesting that the environment around the probe gets perturbed in the
presence of different bile acids. In analogy with the results discussed
previously for H2L spectral properties in the presence of different micel-
lar solutions, a blue shift of the fluorescence emission maximum sug-
gests that the polarity of the micellar environment is less than the
polarity of the bulk water. While the fluorescence spectral blue shift
for H2L is about 6097 cm−1 in DCA compared to its position in homoge-
neous buffer solution, in CA, CDCA and GCA the shifts are 7359 cm−1
,
7239 cm−1 and 6997 cm−1, respectively. This indicates that the
micropolarity around the probe in these BAs is similar and appreciably
different from that in DCA. Apart from the shift in spectral position, fluo-
rescence intensity of H2L increases remarkably in the presence of all the
bile acid. This observation is true for all the bile acid studied here and
can be rationalized on the basis of the binding of the probe in a less
polar site within the micellar aggregates as compared to bulk aqueous
phase.
The changes of the emission profile of H2L with gradual addition of
different bile acids — CA, DCA, CDCA and GCA suggest its movement
from bulk aqueous phase to micellar environment. To understand the
rigidity of the microenvironment, the steady state fluorescence anisot-
ropy measurements were carried out which provide important infor-
mation regarding the binding and location of the probe molecule in
bile acids aggregated systems. The steady state anisotropy of H2L in
the presence of different BA systems is 0.17, 0.21, 0.14 and 0.19 respec-
tively for CA, DCA, CDCA and GCA. These fluorescence anisotropy values
are comparatively very high compared to free ligand (0.02). This obser-
vation clearly indicates that the probe molecule strongly interact with
different bile acids upon incorporation into the different binding sites.
The increase in anisotropy value suggests that the probe molecule
must feel restriction of its rotational motion on moving from the bulk
aqueous phase to micellar environment [30].
3. Results and discussion
3.1. Steady state spectral properties
The absorption spectrum of H2L in homogeneous buffer solution
of pH 9.2 shows a broad band with the maximum centered at around
320 nm. The fluorescence emission spectrum of H2L appears at
494 nm in homogeneous buffer solution. However, the absorption
maxima for the buffer solution of H2L were practically unaffected
by the presence of added bile acids. The fluorescence spectral posi-
tion is strongly dependent on the amount of bile acid in solution.
Here, the fluorescence emission maxima of H2L in different BAs mi-
cellar media are 422 nm, 410 nm, 425 nm and 419 nm in the case
of CA, DCA, CDCA and GCA, respectively and are shown in Fig. 1 and
the obtained values are reported in Table 1.
3.2. Determination of critical micelle concentration
The change in fluorescence emission maximum and intensity with
gradual addition of BAs (Fig. 2), can be attributed to the passage of the
probe (H2L) molecule from a highly polar aqueous phase to a relatively
nonpolar micellar phase. Surfactant molecules form aggregates at cmc
under certain environmental conditions and hence, the microenviron-
ment below and above critical micelle concentration are quite distinct.
At lower surfactant concentration, the change in fluorescence response
is not very prominent. However, after a certain micellar concentration,
both fluorescence emission position and intensity show a drastic
change. These sharp break points are conventionally assigned to critical
micelle concentration. Here, H2L has been used to estimate the cmc
values for different bile acids — CA, DCA, CDCA and GCA. The cmc values
Fig. 1. Fluorescence emission spectra of H2L in buffer solution (1), CDCA (2), CA (3), GCA
(4) and DCA (5) (λexc = 320 nm). The concentrations are in mM: (i) 25.9 (CA), (ii) 24.4
(DCA), (iii) 12.6 (CDCA) and (iv) 28.2 (GCA).

N. Roy et al. / Journal of Molecular Liquids 211 (2015) 1052–1059
1055
Fig. 2. Variation of H2L fluorescence spectra with increasing concentration of CA (i) and DCA (ii) (λexc = 320 nm). The concentration of bile acids is in mM: [CA] = 0.0, 1.4, 2.8, 4.2, 6.4, 8.9,
10.6, 12.3, 14.9, 17.9, 21.9 and 25.9 and [DCA] = 0.0, 1.8, 2.8, 4.2, 5.6, 7.6, 8.4, 9.8, 12.5, 17.3, 21.2 and 24.4. Inset shows the spectra in the presence and absence of bile acids.
were estimated from the maximum of the derivative plot of fluores-
cence intensity with BA concentration (Fig. 3) and the estimated cmc
values are 8.7 mM, 5.6 mM, 8.8 mM and 11.2 mM respectively for CA,
DCA, CDCA and GCA. These values were also reported in Table 2. As
can be seen from Table 2, the estimated cmc values obtained for dif-
ferent BAs from experimental data are in good agreement with the liter-
ature values obtained through other methods [31,32]. The close
agreement in the measured microscopic properties of micellar systems
in this study with the literature reported values obtained by using en-
tirely different type of probe indicates the insensitivity of the probe
molecule in these measurements.
The striking feature of BA aggregation scheme is that it is char-
acterized by at least two cmc values. According to the primary–
secondary micelle model of Small [33], at low concentrations, the
BAs form small primary micelles with a characteristic cmc value.
In this micelle, the BAs turn toward each other by their hydropho-
bic β-face. At the concentration range beyond second cmc, these
primary micelles attach together to form large secondary micelles
by hydrogen bonding interactions through their hydrophilic outer
surface. It is to be noted that the concentration range of BAs, used
in the present study, is limited to monitor the formation of the pri-
mary micelles only.
Fig. 3. Variation of H2L fluorescence intensity with different bile acids at specific wavelength for (i) CA, (ii) DCA, (iii) CDCA and (iv) GCA, respectively. Solid line with open circles (right
axis) represents spline fit of the derivative data in (i) CA, (ii) DCA, (iii) CDCD and (iv) GCA; the maximum of which indicates cmc for the micellization process.

1056
N. Roy et al. / Journal of Molecular Liquids 211 (2015) 1052–1059
Table 2
Critical micelle concentration (cmc), dielectric constant (ε) and ET(30) parameters as well
as binding constant (KS) obtained for different bile acids using H2L as a fluorescence probe
at pH 9.2.
Properties
Estimated values
CA
DCA
CDCA
GCA
cmc/mMa
8.7
5.6
8.8
11.2
[literature value]
[6.5–9.5]
[4.7]
[9.0]
[12.0]
Dielectric constantb (ε)
(i)
(ii)
40.6
15.4
23.6
1.94
39.0
18.6
35.7
12.1
ET(30) parametersc
(i)
48.7
42.8
48.1
47.0
(ii)
36.9
0.72
31.7
1.25
38.4
0.68
35.8
0.78
KSd, /102, M−1
(
error)
(0.01)
(0.02)
(0.02)
(0.01)
a
Estimated from the derivative plot of fluorescence intensity variation with bile acid
concentration.
b
Obtained from linear plots of (i) Stokes shift (Δνss) and (ii) fluorescence maxima (νf)
vs. ε.
c
Obtained from linear plots of (i) Stokes shift (Δνss) and (ii) fluorescence maxima (νf)
vs. ET(30).
d
Estimated from the slope of linear relation given in Eq. (14).
3.3. Polarity parameters
Difference in fluorescence properties of the probe in bile acid micel-
lar environment with those of the homogeneous solvents helps us to es-
timate the polarity of the microenvironment indicating the location of
the probe which is of great importance in the biological systems. How-
ever, it should be kept in mind that the polarity of different heteroge-
neous media is huge different from those of homogeneous medium.
Attention has been drawn to estimate the empirical polarity scale,
ET(30) using the following set of equations:
ꢂ
ꢃ
νf cm−1 ¼ ð28417:1 ꢀ 264:3Þ−ð127:9 ꢀ 6:5Þ ꢁ ET ð30Þ
ð4Þ
ð5Þ
ꢂ
ꢃ
ΔnSS cm−1 ¼ ð−3136:2 ꢀ 245:5Þ þ ð215:5 ꢀ 6:1Þ ꢁ ET ð30Þ
Fig. 4. Plots of fluorescence emission energy (νf) and Stoke's shift (ΔνSS) against (a) ET(30)
parameter and (b) dielectric constants (ε) of H2L.
where, fluorescence energy and Stokes shift are represented by νf and
ΔνSS, respectively. Similarly, dielectric constant (ε) for different BA sys-
tems was also evaluated using the following set of equations:
3.4. Estimation of the probe-micelle binding constant
The binding of a probe to micelles can be described by the following
equilibrium [35]:
ꢂ
ꢃ
νf cm−1 ¼ ð24490:3 ꢀ 225Þ−ð51:6 ꢀ 5:7Þ ꢁ ε
ð6Þ
ð7Þ
KS
Sa þ Dm ↔ Sm
ð8Þ
ꢂ
ꢃ
where, Sa and Sm denote the substrate concentrations expressed as
molarities in terms of total volume of solution in aqueous phase and
in micellar pseudophase.
ΔνSS cm−1 ¼ ð4337:5 ꢀ 301:3Þ þ ð74:4 ꢀ 8:3Þ ꢁ ε:
The equilibrium constant for the process (Eq. (8)), often termed as
binding constant, is given by
The estimated polarity parameters of H2L in different BA system are
reported in Table 2 and the corresponding plots are shown in Fig. 4. To
be best of our knowledge, there is no literature data available for the in-
terior polarity of BAs or their salts; so it was not possible to make any di-
rect comparison of the estimated parameters. However, in one of the
relatively recent report by Lee et al. [34], it was shown that the interior
polarity of the microaggregates formed from DCA modified chitosan is
substantially reduced when compared with bulk water. Similarly, in
our earlier paper [22], we also observed a decrease in polarity of the
BA systems when compared with bulk water phase. From Table 2, it is
shown clearly that the polarity of the microenvironment is in the
order of CDCA N CA N GCA N DCA which indicate more nonpolar environ-
ment in DCA compared to other BA systems, as also evidenced from the
fluorescence emission maxima obtained as described earlier.
½Smꢂ
KS
¼
:
ð9Þ
½Saꢂ½Dmꢂ
It is known that for the formation of primary micelle in BAs, the ag-
gregation number is very small and it shows rather weak dependence
on the surfactant concentration between the two cmc values. However,
in the vicinity of higher cmc value, the aggregation number increases
very sharply. For example, the mean aggregation number of the sodium
salt of both CA and DCA is about 2–5 within the concentration range of
50 mM; however, it changes abruptly to about 20 at ~100 mM surfac-
tant concentration [36].

N. Roy et al. / Journal of Molecular Liquids 211 (2015) 1052–1059
1057
The maximum surfactant concentration, for all the BAs used in this
study, is always within 30 mM range and the aggregation number for
the primary micelle formation can be assumed to be constant. With
this approximation, the total substrate concentration (St) and total
detergent concentration (Dt) can be written as [Sa] + [Sm] and
[Sm] + [Dm] + cmc, respectively.
interactions between the probe and the micelle [37,38]. On the basis
of this, time-resolved fluorescence studies were performed on H2L
under pre-micellar as well as fully primary micellized state in all differ-
ent bile acids and also in homogeneous buffer medium and the findings
are reported in Table 3. The homogeneous buffer solution of H2L fit very
well with single exponential decay. However, in micellar solutions, the
decay curves show contributions from more than one component. The
fluorescence decay curves were analyzed by non-linear least-square
iterative convolution method using Eq. (15) based on Lavenberg–
Marquardt [39] chi-sqare (χ2) minimization algorithm (Eq. (16))
The fraction of micellar associated substrate is defined as
½Smꢂ
f ¼
:
ð10Þ
½Stꢂ
X
Then from Eq. (9), one obtains
FðtÞ ¼
ai expð−t=tiÞ
ð15Þ
i
f
¼ KSf½Dtꢂ−½Stꢂ ꢁ f g−KS ꢁ cmc:
ð11Þ
1−f
αi is the associated pre-exponential factor corresponding to the decay
time τi.
Under the condition of [Sm] ≪ [Dt] and [Dt] ≫ cmc, the above equa-
tion can be approximated as
N
X
2
½yi−f ðxiÞꢂ
i¼1
χ2
¼
ð16Þ
f
¼ KS ꢁ ½Dtꢂ:
ð12Þ
N−P
1−f
N is the number of data points and P is the number of free para-
Experimentally, f can be calculated by steady state fluorescence
experiments in the presence and absence of micellar systems as follows:
meters in the fitting function. The reliability of fitting was checked by
numerical value of reduced chi-sqare (χ2) and also Durbin–Watson
(DW) parameter.
F−F0
Fm−F0
f ¼
ð13Þ
It is to be noted that in fully primary micellized condition, a three ex-
ponential function was needed to fit the experimental data as demon-
strated by visual inspection of the distribution of weighted residuals
with time (Fig. 6). However, as it is seen from Table 3 that the amplitude
of the short nanosecond component is larger compared to long nano-
second component in all different BAs. Multiexponential decay of fluo-
rescence is quite common and it is often difficult to mechanistically
assign the various components of the decay. The fluorescence lifetime
values of the probe in different BA micelles are clearly more than
those in homogeneous buffer solution. Even if we assume that the diffu-
sion of the probe molecule is rather slow in micellar medium, there is al-
ways a probability that the probe molecule in different microdomains of
BA micelles are excited simultaneously to give multiexponential fluo-
rescence decay. Instead of giving too much importance to individual
decay components, we define the mean fluorescence decay time of
the fluorophore inside different BA micelles using Eq. (17) to discuss
the fluorescence decay behavior.
where, F, F0 and Fm are the area under the whole fluorescence emission
spectra of the probe in surfactant, water and in fully micellized condi-
tion, respectively. Substituting the value of f in Eq. (12), one can write
F−F0
Fm−F
¼ KS ꢁ ½Dtꢂ:
ð14Þ
A plot of (F − F0) / (Fm − F) vs [Dt] gives a straight line, the slope of
which gives the value of the binding constant, Ks. The binding constant
value of H2L with different BA systems are 0.72 × 102 M−1
,
1.25 × 102 M−1, 0.68 × 102 M−1 and 0.78 × 102 M−1 respectively for
CA, DCA, CDCA and GCA. These values were also reported in Table 2
and some of the representation plots are shown in Fig. 5. Interestingly,
the binding constant of H2L with different BA systems are in the order
of DCA N GCA N CA N CDCA indicating a more nonpolar environment
of the probe in DCA compared to other BA systems, as also evidenced
from the fluorescence emission maxima obtained as described earlier.
X
bτN ¼
ai ꢁ τi
ð17Þ
i
3.5. Time-resolved fluorescence measurements
The calculated mean fluorescence decay values are 2.18 ns, 4.68 ns,
4.23 ns, 4.77 ns and 6.16 ns for buffer, CA, DCA, CDCA and GCA res-
pectively. These values were also listed in Table 3. It is interesting to
note that the mean fluorescence decay time in BAs micellar media are
Fluorescence lifetime of a fluorophore in a micellar solution serves
as a sensitive parameter for exploring the local environment around
the fluorophore. It also contributes to the understanding of different
Fig. 5. Variation of (F − F0) / (Fm − F) with total bile acids concentration [Dt] for CA (i) and GCA (ii). The binding constant value, Ks was calculated from the slope of the linear relation using
Eq. (14).

1058
N. Roy et al. / Journal of Molecular Liquids 211 (2015) 1052–1059
Table 3
Fluorescence quantum yield (ϕ), decay time (τ), mean fluorescence decay time (bτN), radiative (κr) and total nonradiative (κnr) decay constants of H2L in homogeneous buffer solution as
well as in presence of different bile acids.
Medium
ϕ/10−3
Fluorescence decay time (τ)
bτN
ns
κr
κnr
/107 s−1
/109 s−1
τ1/ns(α1)
τ2/ns(α2)
τ3/ns(α3)
Buffer
CA
DCA
CDCA
GCA
3.4
28.5
40.6
32.4
36.5
2.18(1.0)
–
–
2.18
4.68
4.23
4.77
6.16
0.15
0.61
0.96
0.68
0.59
0.46
0.21
0.23
0.20
0.16
1.63(0.48)
1.34(0.54)
1.48(0.52)
2.11(0.57)
5.04(0.38)
4.96(0.34)
5.84(0.36)
6.74(0.32)
14.2(0.14)
15.2(0.12)
15.8(0.12)
25.5(0.11)
about 2–3 times larger than the corresponding values in homogeneous
buffer solution. Fluorescence quantum yield (ϕ) calculated from the
area of the total fluorescence emission over the whole spectral range,
the radiative (κr) and nonradiative (κnr) decay rate constants calculated
from Eq. (18) are also listed in Table 3. The increase in fluorescence
quantum yields (ϕ), fluorescence decay times and substantial decrease
in nonradiative (κnr) decay rate constants in BAs micellar environment
points to the restricted motion of the fluorophore inside the micellar
subdomains. From Table 3, the fluorescence decay times increases in
the order of GCA N CDCA N CA N DCA and nonradiative decay rate con-
stants as DCA N CA N CDCA N GCA in BAs micellar environment which in-
dicates the restricted low-frequency motion of the fluorophore inside
the micellar subdomains as discussed earlier.
show marked differences in these environments. For example, the
shift in fluorescence maximum of H2L under fully micellized condition
of BA is about 6097 cm−1 from that in homogeneous buffer solution.
However, in case of synthetic detergents, the maximum shift was ob-
served in case of triton X-100 (TX-100); which is of the order of
7016 cm−1. These differences can be approximated due to the differ-
ence in micellar structure for BAs when compared with synthetic deter-
gents. In contrast to the spherical nature of the pseudo-particle formed
due to the self aggregation of linear surfactant molecules like SDS, CTAB
and TX-100, the BAs consist of a rigid steroid backbone giving rise to the
concave side with polar hydroxyl group (α-face) and the methyl groups
in the convex side (β-face). Aggregation of BAs in aqueous solution is
due to hydrophobic interaction of the apolar β-faces of steroid back-
bones with possibility of further aggregation through hydrogen bonding
in the α-faces. This unique arrangement based on facial amphiphilicity
renders a different aggregation pattern in BAs unlike the conventional
surfactants; where, the micellar structure is mostly approximated to
be spherical, originated from the mutual arrangement of the head and
tail groups with different hydrophobicity [40,41].
κr ¼ ϕ=bτN; κnr ¼ ð1−ϕÞ=bτN
ð18Þ
3.6. Comparison with binding of the probe in synthetic detergents
Although, the spectroscopic properties of H2L in the presence of BAs
show similar trend like blue shift with increase in fluorescence emission
intensity as in synthetic detergents, some of the calculated parameters
Again, the binding constant of H2L in the presence of BAs is compar-
atively very low compared to synthetic detergents like SDS, CTAB and
TX-100 as reported in Table 2. Here in case of synthetic detergents,
Fig. 6. Time-resolved fluorescence decay profile of H2L in the presence of different bile acids: CA (i), DCA (ii), CDCA (iii), and GCA (iv), respectively. IRF indicates instrument response func-
tion. The upper panels show the distribution of weighted residuals for three exponential fitting along with reduced chi-square (χ2) and Durbin–Watson (DW) parameters in each case.

N. Roy et al. / Journal of Molecular Liquids 211 (2015) 1052–1059
1059
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In this work, we have investigated the fluorescence properties of
N,N/-bis(salicylidene) trans 1,2-diaminocyclohexane (H2L) inside the
micellar environment and is found to be different in bile acid micellar
media when compared with homogeneous buffer solution. The blue
shift in fluorescence emission maxima, with considerable increase in
fluorescence band intensity as well as fluorescence lifetime value indi-
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micelle concentration obtained by using H2L as a probe in the presence
of different bile acids is in good agreement with literature reported
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Acknowledgments
Financial support through UGC-BSR Research Start-Up-Grant project
No. F.20-1/2012 (BSR)/20-1(2)(2012)(BSR) from the University Grants
Commission (UGC) and Start-Up Research Grant (Chemical Sciences)
project No. SB/FT/CS-064/2012 from the Science and Engineering Re-
search Board (SERB), Government of India was gratefully acknowledged
by Dr. T. Sanjoy Singh. The authors are indebted to Dr. S. Mitra and his
research scholars for their help in TCSPC measurements.
Glossary of acronyms
H2L: N,N/-bis(salicylidene) trans 1,2-diaminocyclohexane
BAs: bile acids
MD: molecular dynamics
CA: cholic acid
DCA: deoxycholic acid
CDCA: chenodeoxycholic acid
GCA: glycocholic acid
ICT: intramolecular charge transfer
SDS: sodium dodecyl sulfate
CTAB: cetyl trimethyl ammonium bromide
TX-100: Triton X-100
TCSPC: time-correlated single photon counting
PTI: photon technology international
IRF: instrument response function
cmc: critical micelle concentration
DW: Durbin–Watson
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