JOURNAL OF COLLOID AND INTERFACE SCIENCE 192, 83–93 (1997)
ARTICLE NO. CS974959
Principal Component Analysis of the Absorption Spectra of the Dye
Thiacyanine in the Presence of the Surfactant AOT: Precise
Identification of the Dye–Surfactant Aggregates
Anil Kumar Mandal and Medini Kanta Pal 1
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, 741235, India
Received December 16, 1996; accepted April 28, 1997
Spectral change of a cationic dye 3,3*-diethylthiacyanine iodide
(THIA) in the presence of an anionic surfactant AOT has been
presented. The THIA–AOT system exhibits blue-shifted metach-
romasia at AOT concentrations below its critical micellar concen-
tration (CMC) and is thought to be due to the aggregation of
electrostatically bound dye–AOT complex (DS). Metachromasia
is gradually reversed to the monomeric band (peak at 425 nm) by
AOT above its CMC. Principal component analysis (PCA)
method has been applied for spectral analysis; the results show
that the metachromatic peaks at 377 and 366 nm originate, re-
spectively, from trimer and hexamer of the dye associated with
AOT. From PCA, the molar absorption coefficient spectra of the
individual absorbing components and the equilibrium constants
for the systems, monomer ` trimer and hexamer ` monomer
below and above CMC of AOT, respectively, have also been ob-
tained. The micellar aggregation number of AOT obtained from
PCA is found to be 16 which is in good agreement with the litera-
ture value. q 1997 Academic Press
Key Words: anionic surfactant AOT; cationic dye thiacyanine;
principal component analysis method (PCA); resolution of the
observed spectra; metachromasia; metachromatic species and their
compositions; equilibrium constants and micellar aggregation
number.
INTRODUCTION
The interaction between cationic dyes and anionic poly-
mers has been the subject of very extended literature (1).
The spectroscopic feature of this interaction is the exhibition
of blue-shifted metachromasia or red shifted J-band in the
visible region, and these have been interpreted as due to
different modes of aggregation of the dye cations electrostat-
ically bound to the polyanions (2, 3). Similar spectral
changes of many dyes caused by surfactants have also been
observed (4–16), and these have also been interpreted as
due to aggregation of the dye molecules or dye–surfactant
ion pairs, or due to charge transfer (17) between dye and
1
To whom correspondence should be addressed.
83
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surfactant molecules. Though much attention has been paid
to the study of the dye–surfactant interaction at and around
the critical micellar concentrations (CMC) of the surfac-
tants, there are also interesting reports (4, 10–13) on the
dye–surfactant interaction below the CMC of surfactants.
Anyway, the progressive spectral change of a dye–surfactant
system at varying surfactant concentrations indicates that
none of the observed spectra can be assigned to a definite
composition of the dye–surfactant complex, and more than
one species of the complexes may have contributed to the
observed spectra even at a particular dye to surfactant ratio.
There are reports on the determination of the compositions
and molar absorption coefficients of dye–polyanion com-
plexes (18–21), but similar reports on the dye–surfactant
systems are scanty (4, 14). In this paper we report the spec-
trophotometric results and the application of the extended
principal component analysis (PCA) method (22) for the
determination of compositions and molar absorption coeffi-
cient spectra of the dye–surfactant complexes formed from
the interaction between a cationic dye, 3,3 *-diethylthiacya-
nine iodide (THIA), and an anionic surfactant, bis(2-ethyl-
hexyl) sodium sulfosuccinate, called aerosol-OT (AOT).
Extended PCA method is a very powerful tool for resolving
overlapping spectra as well as for determining the composi-
tions of the absorbing components in equilibrium (20, 21).
Probably, this is the first report on the application of PCA
method to the dye–surfactant system.
EXPERIMENTAL
The sodium salt of AOT from Sigma Chemical Co. and
THIA from Koch Light (UK) were used as received. A
stock solution of THIA (0.20 mM) was prepared by dissolv-
ing 2.33 mg dye in 5.0 mL methanol, making up to 25 mL
with water, and stored in the dark. Stock AOT solution (8.0
mM), and from it two other intermediate solutions (2.0 and
0.20 mM) were prepared in water. The absorption spectra
of the experimental solutions were recorded with a Shimadzu
spectrophotometer (UV-150-02 model) at laboratory tem-
0021-9797/97 $25.00
Copyright q 1997 by Academic Press
All rights of reproduction in any form reserved.
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84 MANDAL AND PAL
perature (257C) using matched pair cuvettes of 10.0 mm
pathlength. Absorption spectra were recorded within 10 min
of preparing the respective solutions. Appreciable adsorption
of THIA on glass surfaces necessitates some correction of
the dye concentration in the experimental solutions, particu-
larly in dilute aqueous AOT solutions. It has been found
that THIA perfectly obeys Beer’s law in 75% (v/v) ethanol
at least up to 10.0 mM and has the molar absorption coeffi-
cient value of 8.13 1 10 4 mol 01 cm2 at lmax (424 nm).
Thus, the more accurate concentration of the dye depicting
observed spectrum was estimated from the absorbance at
424 nm of 1.0 mL portion of the solution (pipetted from the
cuvette after recording each spectrum) diluted with 3.0 mL
of ethanol.
EXTENDED PCA METHOD (20, 22)
This method has been described in detail by Takatsuki
and Yamaoka (22); therefore, the concept of this method is
discussed here only briefly with some new idea. This method
needs a set of observed overlapping spectra, measured by
varying the initial concentration of one or more of the com-
ponents present in equilibrium system containing more than
one absorbing and one or more nonabsorbing components.
By this method not only the number of colored components,
but also their molar absorption coefficients and equilibrium
constant between either colored or the colored and colorless
components can be determined simultaneously. From the set
of, say m, overlapping spectra a data matrix d of the order
(m 1 n) is constructed, whose element dij (i Å 1 to m, j Å
1 to n) denotes the absorbance of the ith solution at the jth
wavelength. Now, according to Beer’s law for p absorbing
components in equilibrium,
d Å c 1 e, [1]
where c and e are concentration and molar absorption coef-
ficient matrices of the orders (m 1 p) and (p 1 n), respec-
tively. The element of the matrix c, say cij , represents the
equilibrium concentration of the jth component in the ith
solution (spectrum) and the element of e, say eij , represents
molar absorption coefficient of the ith component at the jth
wavelength. In most cases, c and e are unknown, and hence
the purpose of the application of the PCA method to chemi-
cal equilibrium for determining them simultaneously.
Now, if the individual rows of the matrix d are divided
by the respective initial or analytical concentration of an
absorbing component in m different mixtures, a second data
matrix ea , called apparent molar absorption coefficient ma-
trix of the particular component, is obtained. The diagonal-
ization (23) of the product ( e *a 1 e ), where e *a is the trans-
pose of ea , gives n eigenvalues ( l1 , l2 , . . . , ln ) and n
eigenvectors. The number of absorbing components repre-
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senting the observed spectra are correctly obtained from the
relative magnitudes of the eigenvalues. The following rela-
tion holds between the eigenvalues for the presence of p
absorbing components,
l1 § l2 § l3 § rrr § lp @ lp/1 § rrr § ln ú 0,
[1a]
i.e., there exists a large gap between the pth and (p / 1)th
eigenvalues. Thus, for p absorbing components only p eigen-
values become significant, and hence the corresponding ei-
genvectors are considered as the fundamental spectra and
common to the all m observed spectra. The matrices d and
e can then be expressed by the linear combinations of the p
eigenvectors as
dÅf1e [2]
ea Å l 1 e, [3]
where f and l are linear combination coefficient matrices of
the order (m 1 p) and e is the eigenvector matrix of the
order (p 1 n). Here f and l are unknown, which can be
calculated from the relations f Å d 1 e 01 and l Å ea 1 e 01 ,
respectively, provided that e is a square matrix. In most
cases e is not a square matrix, and in that case both sides
of Eqs. [2] and [3] are multiplied by e *, the transpose of
the matrix e, and then inverted as
d 1 e * Å f 1 (e 1 e * ) Å f 1 r, [4]
where r Å (e 1 e * ) is a square matrix. Therefore, f and l
can be calculated by the relations
f Å (d 1 e * ) 1 r 01 [5]
and
l Å ( ea 1 e * ) 1 r 01 . [6]
The matrix l has an important characteristic, that its row
elements satisfy a common equation. For example, when p
Å 2 the matrix l contains m rows and two columns; if the
elements of the first column are plotted against the corre-
sponding elements of the second column, a straight line is
obtained.
Now, the molar absorption coefficient e (Eq. [1]) should
also be related to the eigenvector matrix e by a transforma-
tion matrix t of the order (p 1 p) as
e Å t 1 e. [7]
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 PCA OF THIACYANINE ABSORPTION SPECTRA
From Eqs. [1], [2], and [7] it follows that
c Å f 1 t 01 . [8]
The matrix t is unknown which has to be determined for
determining e and c matrices. It is to be noted that the ele-
ments of the matrix t must satisfy the equation obtained from
those of the matrix l. This is because, if any one of the
rows of the data matrix ea is the molar absorption coefficient
spectrum of the particular component, the corresponding row
of the matrix l must also be a row of the desire matrix t.
The elements of the matrix t can be easily determined by
iteration using a computer program. In every iteration a rea-
sonable t matrix satisfying the equation derived from the
matrix l is constructed, which is used for the determination
of the matrices e and c. A possible equilibrium scheme
is then assumed for the system under investigation, and m
equilibrium constants corresponding to m experimental solu-
tions are computed using c matrix. If the system contains
some nonabsorbing components, they are also considered
for the equation of mass balance and their equilibrium con-
centrations can be calculated from their initial or analytical
concentrations and the matrix c. The m equilibrium constants
are then used to calculate the coefficient of variation S de-
fined by
m
S Å {(1/m) ∑ (ki 0 k) 2 }/k, [9]
iÅ1
where k denotes the average value of ki ’s. This coefficient
of variation S is used for monitoring the iteration. Only for
the correct t matrix and proper equilibrium scheme the value
of S will be minimized (zero or near to zero) as well as all
the elements of the matrices e and c will be positive. But
for incorrect t matrix or for improper equilibrium scheme,
either S value will not be minimized or some important
elements of e and/or c will become negative.
RESULTS AND DISCUSSION
Figure 1 summarizes the molar absorption coefficient
spectra of THIA in water (A) and in the presence of increas-
ing AOT concentration from 0.024 to 4.00 mM (B–Q); the
compositions of the solutions are given in Table 1. The
corrected concentrations of the dye in different solutions
were determined by the procedure indicated under Experi-
mental. At low AOT the dye concentrations become lower,
though initially the same amount of the stock dye was taken
in each solution, indicating higher adsorptivity of the dye
on the glasswares at lower AOT. This was confirmed by
dissolving the adsorbed dye on the test tubes in alcohol and
measuring the absorbance at 425 nm.
This dye alone in aqueous dilute solution exhibits a promi-
nent peak at 422 nm (24) with a vibronic shoulder around
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85
405 nm, as in spectrum A (Fig. 1). It is observed (Fig. 1)
that the absorbance at 422 nm rapidly decreases with increas-
ing AOT concentration, and progressively a sharp peak at
366 nm is developed. Beyond the CMC (2.40 mM) of AOT
(9) the absorbance at the metachromatic peak starts decreas-
ing with congruent rise in the absorbance at the monomeric
peak, slightly red shifted. The variations of the molar absorp-
tion coefficients at 422, 380, and 366 nm with increasing
AOT concentrations are summarized in Fig. 1a. It clearly
shows the rapid fall of absorbance of the monomer peak
with increasing AOT, followed by sharp rise again above
2.60 mM AOT where the absorbance at 366 nm starts falling.
Also the 366 nm peak rises in low AOT concentration region
with initial rise and fall of intensity at 380 nm. It is apparent
that the intensity of the 366 nm peak increases at the cost
of intensity at 380 nm at least up to 1.20 mM AOT, indicat-
ing that the species responsible for 366 nm peak is distinctly
different from the species showing the peak around 380 nm.
Similar spectral changes, i.e., exhibition of metachromasia
and its reversal to the monomeric band have also been ob-
served for pynacyanol–AOT (9) and pynacyanol–SDS (10)
systems. Metachromasia in THIA is also induced in the pres-
ence of polyanions (25) and SDS (communicated), but with
less prominent and less blue shifted peaks.
The shift of the lmax of a dye associated with aggregation
depends on the number of monomers aggregated and on
the geometry of aggregation (26). The planar dyes such as
acridine orange and methylene blue aggregate in stacked
manner and exhibit blue shifted metachromasia (2, 26),
where as nonplanar dyes such as pseudoisocyanine aggregate
in a staggered way and depict a red shift of the lmax (27,
28). The exhibition of the blue shifted metachromasia by
THIA in the presence of AOT below its CMC (Fig. 1),
indicates that THIA, a planar dye, can also form stacked
aggregates. THIA itself shows its metachromatic peak at 402
nm (24) due to dimeric aggregation at high dye concentra-
tion, but more blue shifted metachromasia by dilute THIA
in the presence of AOT, SDS, or polyanions (25) indicates
that the aggregation of THIA is facilitated by the presence of
these anions. In fact, the surfactant as well as dye molecules
having hydrophobic elements have their natural tendency
to be aggregated in aqueous medium. But the electrostatic
repulsion between the respective polar groups disfavors such
aggregation. It is well known that aggregation of dyes or
surfactants are facilitated by the presence of electrolytes
through the masking of repulsion by counter ion binding
(29). If a solution of an anionic surfactant is added to a
dilute solution of a cationic dye, even below CMC, the sur-
factant anions and the dye cations may act as counter ions,
thus reducing electrostatic repulsion standing in their way
of respective aggregation (9). Thus, below CMC of AOT,
electrostatically bound THIA–AOT complex or ion pair,
similar to the complex DS in Ref. (10), may form higher
aggregates resulting blue shifted metachromasia at 366 nm.
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86 MANDAL AND PAL

FIG. 1. (A) molar and (B–Q) apparent molar absorption coefficient (mol 01 cm2 ) spectra of THIA in the presence of varying AOT concentrations.
The compositions of the solutions are given in Table 1. (Inset) (1) Beer’s law plot of monomeric THIA at 422 nm and (2) that of trimeric THIA at
377 nm (concentrations of THIA are in monomeric units). The letters indicate the points corresponding to the spectra in the main figure.

Now, beyond the CMC of AOT the gradual reversal of met- the coexistence of two absorbing components in equilibrium.
achromasia to the monomeric band may be due to gradual Thus, one of the equilibria related to the spectra A to G
dissociation of the THIA–AOT aggregates followed by in- may involve THIA monomer, THIA–AOT aggregate (peak
corporation of the THIA–AOT monomers into the micelles around 380 nm), and nonabsorbing free AOT monomer.
(10), or due to the nucleation of the surfactant molecules The other equilibrium may involve the other THIA–AOT
onto the THIA–AOT aggregates with the formation of mi- aggregate (peak at 366 nm), AOT micelle containing THIA
celles followed by redistribution of dye molecules over the monomers, and free micellized AOT monomers; free AOT
micelles formed (15). monomers are not involved in the equilibrium, because their
It is to be noted further from Fig. 1 that the families of concentration above CMC would remain constant. It is to
the spectra A to G and L to Q pass through the isosbestic be mentioned here that in the presence of SDS or polyanions
points at 390 and 384.5 nm, respectively, indicating two (25) THIA depicts a broad peak around 380 nm and no peak
separate equilibria between absorbing components. It is well below 380 nm from which reversal of metachromasia starts.
known that the appearance of an isosbestic point indicates So, there is no doubt about the existence of the first equilib-
rium in AOT medium corresponding to the isosbestic point
TABLE 1 at 390 nm. Now, we proceed to determine the actual equilib-
Compositions of the Experimental Solutions of Fig. 1 rium schemes as well as the compositions of the THIA–
AOT aggregates and their molar absorption coefficients by
THIA concentration AOT concentration applying the PCA method.
Curve (mM) (mM) To determine how many dye molecules are aggregated
showing peak around 380 nm irrespective of AOT monomers
A 2.390 0.000
B 1.365 0.024 associated, we apply the PCA method to a different set of
C 1.658 0.033 spectra A to F, shown in Fig. 2, obtained by varying THIA
D 1.852 0.040 concentration at a fixed AOT concentration of 76.0 mM as
E 1.939 0.045 a medium. The spectrum A of the free dye is taken from
F 2.016 0.059
G 2.187 0.075
Fig. 1 and it has been used here as a basis spectrum for
H 2.337 0.120 PCA method application. The data matrix d for PCA is then
I 2.313 0.180 constructed, which has six rows corresponding to the spectra
J 2.509 0.300 A to F, each row contains 51 experimental optical density
K 2.657 0.800 data at 51 selected wavelengths from 350 to 500 nm at 3
L 2.706 1.800
M 2.706 2.400
nm intervals. The corresponding apparent molar absorption
N 2.706 2.800 coefficient matrix ea is obtained by dividing the rows of the
O 2.706 3.200 matrix d with the respective analytical dye concentrations.
P 2.706 3.600 The diagonalization of the product ( e *a 1 ea ) gives 51 eigen-
Q 2.706 4.000
vectors and 51 eigenvalues. Visual inspection of the eigen-

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 PCA OF THIACYANINE ABSORPTION SPECTRA
FIG. 1a. Variation of the molar absorption coefficients (mol 01 cm2 ) with AOT concentration at 422 nm (A), 380 nm (B), and 366 nm (C).
values shows that among the first four largest eigenvalues,
such as 9.8 1 10 10 , 1.14 1 10 10 , 6.0 1 10 6 , and 2.80 1
10 6 , only two are significant (see Eq. [1a]), because there
is a large gap between the second and third ones. This result
confirms the equilibrium coexistence of only two absorbing
species but not three, although it is apparent from the ob-
served spectra in Fig. 2 that there are three absorbing species
corresponding to the peaks around 380, 405, and 422 nm;
the reason behind it follows. The eigenvectors corresponding
to the significant eigenvalues are shown in Fig. 3, and they
have been used to construct the eigenvector matrix e of the
type (51 1 2). f and l matrices each of the type (6 1 2)
are then computed using the Eqs. [5] and [6], respectively.
The plot of the elements of the matrix l results a straight
line 1 as expected, shown in Fig. 4. As the spectrum A of
Fig. 2 is the molar absorption coefficient spectrum of THIA,
the first row of the matrix l must be that of the desire matrix
t of the type (2 1 2). The elements of the second row of
the matrix t are then determined by iteration assuming the
equilibrium scheme
[THIAm ]
m THIA ` THIAm; kÅ ,
[THIA] m
where m (an integer) is the stoichiometric coefficient. All
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87
the PCA criteria, indicated earlier, are simultaneously satis-
fied only for m Å 3 and for the second row elements of the
t matrix corresponding to the coordinates of the point X on
the line 1 (Fig. 4). The S value and the equilibrium constant
(Eq. [9]), thus obtained from PCA method for spectra B to
F (Fig. 2), are 0.023 and (3.16 { 0.12) 1 10 12 , respectively.
The question of equilibrium constant for spectrum A does
not arise, because it is the pure monomeric spectrum of the
dye. The metachromatic species showing peak around 380
nm, therefore, contains three THIA molecules (m Å 3). As
the matrix t is now known, the matrices c and e (Eqs. [7]
and [8]) are also known. The elements of the first and second
rows of e represent the molar absorption coefficients, in
monomeric units, of monomeric and trimeric THIA at se-
lected wavelengths. The plots of the first row elements of e
exactly fall on spectrum A (Fig. 2) as expected, and that of
the second row elements results in spectrum G shown in
Fig. 2. Spectrum G depicts two peaks at 377 and 440 nm
and a shoulder around 405 nm. They originate from the in-
phase and out-of-phase oscillations of the transition dipoles
of the three dye molecules in the aggregate; the strongest
blue shifted peak at 377 nm is due to the in-phase oscillations
of the three transition dipoles; and the other peak and shoul-
der are due to two other possible modes of out-of-phase
oscillations (30). Alternatively, this is also expected from
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88 MANDAL AND PAL

FIG. 2. (A) Molar absorption coefficient (mol 01 cm2 ) spectrum of 2.39 mM THIA in water; (B–F) apparent molar absorption coefficient (mol 01
cm2 ) spectra of 1.173, 1.612, 2.119, 2.677, and 3.036 mM THIA in the presence of 76.0 mM AOT; and (G) computed molar absorption coefficient
(mol 01 cm2 ) spectrum of trimeric THIA associated with AOT. (Inset) (1) Beer’s law plot of monomeric THIA at 422 nm and (2) that of trimeric THIA
at 377 nm (concentrations of THIA are in monomeric units). The letters indicate the points corresponding to the spectra in the main figure.

the exciton splitting theory (31, 32) that for N-mer dye
aggregates the N-fold degenerate excited states are split into
N separated sublevels due to the interaction between the
oscillating transition dipoles of the N dye molecules in the
aggregate. If the N dye molecules are stacked perfectly paral-
lel the optical transition from the ground state to the higher
of the split levels is intense, whereas that to the lower levels
are optically forbidden. The excited level associated with
the strong transition arises from the interaction between the
transition dipoles in the same phase (in-phase), and the
others, with the interactions in the opposite phases (out-of-
phases). Deviations from perfectly parallel stacking will also
allow the transitions to the lower excited states, but with less
intensities than to the upper excited state. Not necessarily all
the N-peaks will be shown up due to overlapping or feeble-
ness of the intensities. Thus, the depiction of two peaks by
trimeric THIA at 377 and 440 nm with e values of 3.20 1
10 4 and 6.4 1 10 3 mol 01 cm2 , respectively, and a shoulder
around 405 nm indicates that the stacking of THIA mono-
mers in the aggregate is not perfectly parallel, which is the
case, in general, for cyanine dyes (24). The prominent peak
around 405 nm in the observed spectra (Figs. 1 and 2)
are, therefore, due to joint contributions of the vibrational
transition of the monomer and one of the out-of-phase transi-
tions of the dye aggregate. It has been found that THIA in
SDS medium also forms trimeric aggregates showing peaks
at 380 nm (strong) and 435 nm (weak) with e values of
3.20 1 10 4 and 7.15 1 10 3 mol 01 cm2 , respectively, and a
shoulder around 403 nm.
The concentration matrix c, thus obtained, contains six
rows and two columns; the first and second column elements
represent the equilibrium concentrations (in monomeric
units) of the monomeric and trimeric THIA, respectively,
in the respective experimental solutions. Thus, the c matrix,
and the e values at 422 and 377 nm of monomeric and
trimeric THIA can be used for their Beer’s law verification.
They exactly obey Beer’s law as is evident from the plots
shown in the inset of Fig. 2.
For determining the number of AOT molecules associated
with the trimeric THIA, we apply the PCA method, in a
way similar to that above, to spectra A to G (Fig. 1) along
with spectrum G (Fig. 2) of the trimeric THIA. Here, both
the data matrices d and ea contain eight rows each having
51 absorbance and molar absorption coefficient data, respec-
tively, at 51 selected wavelengths from 350 to 500 nm at 3
nm intervals. It is to be mentioned here that the eighth row

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 PCA OF THIACYANINE ABSORPTION SPECTRA 89

FIG. 3. Variation of eigenvectors with wavelengths: (A, B) for spectra A to F in Fig. 2, (C, D) for spectra A to G (Fig. 1) and G (Fig. 2), and
(E, F) for spectra B to I in Fig. 6.

FIG. 4. Plot of li 1 versus li 2 : (1) for spectra A to F in Fig. 2; (2) for spectra A to G (Fig. 1) and G (Fig. 2); and (3) for spectra B to I in Fig. 6.

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90 MANDAL AND PAL
FIG. 5. (A–H) Absorption spectra of 5.117, 6.593, 8.069, 9.545, 10.873, 13.875, 15.301, and 18.352 mM THIA, respectively, in the presence of
4.00 mM AOT. (Inset) Beer’s law plot of aggregated THIA at the peak value of 366 nm; the concentrations and ODs are calculated by the procedure
indicated in the text.
of d matrix is obtained by multiplying the eighth row of ea
with an arbitrary concentration of 3.00 mM which does not
at all affect the results of PCA method, because the concen-
trations of the dye for this spectrum are not used for equilib-
rium constant calculation. The diagonalization of the prod-
uct ( e *a 1 ea ) yields 51 eigenvectors and 51 eigenvalues, of
which two eigenvalues are significant (the first four largest
eigenvalues are 1.53 1 10 11 , 1.40 1 10 10 , 2.0 1 10 7 , and
8.22 1 10 6 ). The eigenvectors for these two significant ei-
genvalues, shown in Fig. 3, are then used to construct e
matrix which in turn is used to compute the f and l matrices
(Eqs. [5] and [6]). The plot of the elements of l matrix of
the type (8 1 2) is shown in the Fig. 4. Now, the first and
eighth rows of the matrix ea represent the molar absorption
coefficients of the monomeric and trimeric THIA, the corre-
sponding rows of the matrix l therefore constitute the first
and second rows of the desire matrix t, and hence the concen-
tration matrix c (Eq. [8]) is known. Using this c matrix and
assuming the equilibrium scheme
3 THIA / n AOT ` THIA3AOTn;
[THIA3AOTn ]
kÅ ,
[THIA] 3 1 [AOT] n
where n is the stoichiometric coefficient, for each value of
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n a set of six equilibrium constants corresponding to spectra
B to G (Fig. 1) is obtained. Equation [9] provides minimum
S value only for n Å 3; the S value and the equilibrium
constant thus become 0.03 and (7.29 { 0.20) 1 10 24 , respec-
tively. The actual composition of the metachromatic species
showing peak at 377 nm is, therefore, THIA3AOT 3 . Now,
the concentration matrix c has been used for Beer’s law
verification for monomeric and trimeric THIA at 422 and
377 nm, respectively. They perfectly obey Beer’s law as
shown in the inset of Fig. 1.
Finally, we try to find out the composition of the metach-
romatic species showing sharp peak at 366 nm (Fig. 1). For
this we took a set of absorption spectra, shown in Fig. 5,
obtained by varying THIA concentration at a fixed AOT
concentration of 4.00 mM above its CMC. It is seen from
Fig. 5 that the absorbance at 425 nm (monomeric peak)
remains almost constant with progressive rise in absorbance
at 366 nm. From this result it is obvious that the number of
micelles containing THIA monomers is almost constant, and
that each micelle has a definite capacity of accommodating
a fixed number of monomeric dye molecules. It is also evi-
dent that the dye molecules, except associated with micelles,
form metachromatic aggregates probably with free AOT
molecules; this would lead to a decrease in the micelle con-
centration with increase in THIA concentration, because to
maintain the equilibrium between free AOT monomers and
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 PCA OF THIACYANINE ABSORPTION SPECTRA 91

FIG. 6. (B–I) Apparent molar absorption coefficient (mol 01 cm2 ) spectra of 3.69 mM THIA in the presence of 2.805, 2.913, 3.048, 3.258, 3.316,
3.504, 3.650, and 3.846 mM AOT; and (A, J) computed molar absorption coefficient (mol 01 cm2 ) spectra of hexameric and monomeric THIA,
respectively. (Inset) (1) Beer’s law plot of monomeric THIA at 425 nm and (2) that of hexameric THIA at 366 nm (concentrations of THIA are in
monomeric units). The letters indicate the points corresponding to the spectra in the main figure.

micelles. Since the AOT concentration is kept constant at
4.0 mM, and the dye concentration as well as the concentra-
tion of the metachromatic species is of the micromolar order,
the change in the micelle concentration with the formation
of metachromatic complex will be very small. Anyway, we
could not apply PCA method to the spectra in Fig. 5 for
determining the composition of the metachromatic species,
as the absorbance at 425 nm remains almost unchanged with
dye concentration; in this case PCA will give erroneous
results since PCA needs appreciable changes in the ab-
sorbances at all the peaks of the spectra (22). However,
these spectra have been used to evaluate the molar absorption
coefficient of the metachromatic species at 366 nm (see
below). For the determination of the composition we apply
the PCA method to another set of spectra B to I shown in
the Fig. 6, obtained by varying AOT concentration above
its CMC at a fixed THIA concentration of 3.69 mM. The dye
concentration of the stock and each experimental solution is
determined by the procedure mentioned under Experimental
and found that the dye concentration in each solution remains
unchanged, indicating no adsorption of dyes on the glass-
wares above the CMC of AOT. Here also both the d and ea
matrices contains eight rows and 51 columns. The diagonal-
ization of ( e *a 1 ea ) gives two significant eigenvalues (the
first four largest eigenvalues are 1.14 1 10 11 , 1.24 1 10 10 ,
1.01 1 10 6 , and 6.58 1 10 5 ) confirming the equilibrium
coexistence of two absorbing species. The eigenvector ma-
trix e corresponding to the significant eigenvalues, shown
in Fig. 3, are used to calculate f and l matrices. The elements
of l are plotted in Fig. 4. In this case all the elements of t
matrix are unknown; these are determined by iteration, as
before, considering the following equilibrium scheme simi-
lar to the equilibrium between J-aggregated porphyrin and
micellized monomeric porphyrin proposed by Barber et al.
(13):
THIAnAOTn / m AOT ` AOTm/nrnTHIA;
[THIA] n
kÅ ,
[AOT] m 1 [THIAnAOTn ]
where m and n are stoichiometric coefficients and hence m
/ n would be the micellar aggregation number of AOT.
Here, we assume that the ratio of THIA to AOT in the
metachromatic aggregate is 1:1. Since the micellized AOT
monomers are responsible for dissociating the metachro-

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92 MANDAL AND PAL
matic aggregate, their concentrations obtained by subtracting
the CMC (2.40 mM) from the total AOT concentrations
have been used for the equilibrium constant calculations in
PCA. Barber et al. (13) have probably also followed this
procedure for calculating micellized SDS monomer concen-
tration for determining micellar aggregation number of SDS.
This procedure has also been used by Sheu et al. (33) and
Huang (34) to see the variation of micellar aggregation num-
ber of AOT with AOT concentration above CMC. All the
PCA criteria are fulfilled for m Å 10 and n Å 6 and for the
elements of the first and second rows of t matrix correspond-
ing to the coordinates of points Y and Z on line 3 (Fig. 4).
The S value and the equilibrium constant come to be 0.01
and (1.43 { 0.01) 1 10 2 , respectively. The probable compo-
sition of the metachromatic aggregate is, therefore, THI-
A6AOT 6 and each micelle contains 16 AOT and 6 THIA
molecules. Based on SANS (small angle neutron scattering)
Sheu et al. (33) and Huang (34) have reported that the
minimum aggregation number of AOT micelles in aqueous
medium is 15, and that the aggregation number increases
linearly with the square root of monomer molefraction that
forms micelles. As per this linear variation the aggregation
number in our case would vary between 17 and 19, and in
average it should be 18 which is in good agreement with
the value 16 obtained from PCA method. Spectra A and J
in Fig. 6 are, respectively, the computed molar absorption
coefficient spectra (Eq. [7]) of THIA6AOT 6 and micellized
THIA monomers. Spectrum A shows a sharp peak at 366
nm with e value of 4.24 1 10 4 mol 01 cm2 and a weak broad
peak around 435 nm with a feeble shoulder around 395 nm.
According to the excitation splitting theory (31, 32), the
spectrum (A) should show six peaks including shoulders;
the others are not shown may be due to overlapping or
feebleness or forbidden of optical transitions. The inset of
Fig. 6 shows Beer’s law plots of monomeric and hexameric
THIA at 425 and 366 nm, respectively. The molar absorption
coefficient of THIA6AOT 6 at 425 nm is 9.8 1 10 2 mol 01
cm2 which is very small, and hence we can assume negligible
contribution of THIA6AOT 6 toward the observed optical
densities (OD) at 425 nm in Fig. 5. Now, we can determine
the molar absorption coefficient of hexameric THIA at 366
nm directly from the observed absorbance data in Fig. 5 as
described below, and it can be compared with the value
already obtained from PCA method. From ODs (Fig. 5) and
molar absorption coefficient ( e Å 7.26 1 10 4 mol 01 cm2 )
at 425 nm of the monomer (Fig. 6), the concentrations of
the monomers in the experimental solutions (Fig. 5) were
calculated. Then, subtracting the concentrations of the mono-
mers from the total analytical dye concentration, the concen-
tration of the aggregated dye in monomeric units were deter-
mined. Now the contribution of the monomers toward the
ODs at 366 nm were calculated from the concentrations of
the monomers and its e value of 2.6 1 10 3 mol 01 cm2 at
366 nm. Subtracting these ODs from the respective observed
AID JCIS 4959 / 6g2c$$$403 07-25-97 12:59:41
FIG. 7. Structures of THIA and AOT and two-dimensional speculative
models of THIA3AOT 3 , THIA6AOT 6 , and a segment of the micelle con-
taining THIA monomers (THIA6AOT 16 ).
ODs at 366 nm, the ODs of the aggregated THIA in the
individual solutions were determined. Thus we obtained
Beer’s law plot of the aggregated THIA as shown in the
inset of Fig. 5; from the slope e value comes to be 4.224 1
10 4 mol 01 cm2 which is in excellent agreement with the
value 4.24 1 10 4 mol 01 cm2 obtained from PCA method
application to the spectra in Fig. 6.
In conclusion, we can say that the expression made by
Gehlen et al. (15) about the redistribution of the dyes in the
surfactant micelles is translated into a quantitative model
containing 6 THIA molecules per micelle of AOT containing
16 AOT molecules. Application of PCA method quantita-
tively determines the compositions THIA6AOT 6 and THI-
A3AOT 3 to be precisely responsible for the observed metach-
romatic peaks at 366 and 377 nm at different compositions
of the experimental solutions. As THIA3AOT 3 and THI-
A6AOT 6 depict metachromasia and AOT can form spherical
micelles (33) in water near CMC, we propose the stacked
models for THIA3AOT 3 and THIA6AOT 6 , and a spherical
model for the micelle of AOT containing THIA monomers
(THIA6AOT 16 ). The corresponding two dimensional tenta-
tive speculative models are shown in Fig. 7.
The PCA method shows undoubtedly the composition of
the complex exhibiting peak at 425 nm in Fig. 6 (possibly
in Fig. 5 also) in the presence of excess AOT above CMC
as THIA6AOT 16 . A rough calculation shows that there are
excess micelles present free of dye, indicating that the dyes
are not distributed following poisson distribution law. Al-
though electrostatic attraction between dye cations and AOT
anions may be a good reason for this, the picture is not
totally clear yet. It will be interesting to verify whether THIA
is distributed according to poisson distribution among the
micelles of nonionic surfactant Triton X-100 or a cationic
surfactant cetylpyridinium chloride.
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 PCA OF THIACYANINE ABSORPTION SPECTRA
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