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Thermodynamics of Multi-Stage Self-Assembly of pHSensitive Gradient Copolymers in Aqueous Solutions.

Journal:
Manuscript ID
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Complete List of Authors:

Macromolecules
ma-2015-02228e
Article
08-Oct-2015
Cernochova, Zulfiya; Institute of Macromolecular Chemistry AS CR, v.v.i.,
Bogomolova, Anna; Institute of Macromolecular Chemistry, Supromolecular
Structures
Borisova, Olga; Moscow State University, Polymer Science
Cernoch, Peter; Institute of Macromolecular Chemistry, Academy of
Sciences of the Czech Republic - Supramolecular Polymer Systems
Filippov, Sergey; Institute of Macromolecular Chemistry,
Billon, Laurent; IPREM EPCP UMR 5254, Chemistry
Borisov, Oleg; University of Pau, Laboratory of Polymer Materials
Stepanek, Petr; Institute of Macromolecular Chemistry, Academy of
Sciences of the Czech Republic - Supramolecular Polymer Systems

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Thermodynamics of Multi-Stage Self-Assembly of pH-Sensitive Gradient Copolymers in

Aqueous Solutions.
Zulfiya ernochova, Anna Bogomolovaa, Olga V. Borisovab, Peter ernocha, Sergey K.
Filippova, Laurent Billonc, Oleg V. Borisovc,d, Petr tpneka
a

Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovskho nm. 2, CZ-162 06 Praha 6

Czech Republic

Department of Polymer Science, Moscow State University, Leninskie Gory, Moscow 119191,
Russia

UPPA, CNRS UMR 5254 IPREM Equipe de Physique et Chimie des Polymres, Pau, France
d

Saint-Petersburg State Polytechnic University, 195251 St. Petersburg, Russia

Abstract
The self-assembly thermodynamics of pH-sensitive di-block and tri-block gradient copolymers of
acrylic acid and styrene was studied for the first time using isothermal titration calorimetry (ITC)
and dynamic light scattering (DLS) performed at varying pH. We were able to monitor each step
of micellization as a function of decreasing pH. The growth of micelles is a multi-stage process
that is pH dependent with several exothermic and endothermic components. The first step of
protonation of the acrylic acid monomer units was accompanied mainly by conformational
changes and the beginning of self-assembly. In the second stage of self-assembly, the micelles
become larger and the number of micelles becomes smaller. While solution acidity increases, the

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isothermal calorimetry data show a broad deep minimum corresponding to an exothermic process
attributed to increase in the size of hydrophobic domains and an increase in the structures
hydrophobicity. The minor change in heat capacity (Cp) confirms the structural changes during
this exothermic process. The exothermic process terminates the deionization of acrylic acid. The
pH-dependence of the -potential of the block gradient copolymer micelles exhibits a plateau in
the regime corresponding to pH-controlled variation of the micellar dimensions. The onset of
micelle formation and the solubility of the gradient copolymers were found to be dependent on
the length of the gradient block.

Introduction
New types of recently synthesized amphiphilic block copolymers with novel properties attract
strong interest of material scientists and chemical engineers. The ideal prospective block
copolymer in certain modern technological and industrial applications should form
nanostructures capable to respond quickly to external physical or chemical stimuli1. Moreover,
these desirable properties can often be fine-tuned by molecular design of the polymers, and be
combined with prolonged structural stability in otherwise unsuitable environments and narrowed
response ranges to varied external conditions. Typical control parameters of interest include
temperature, pH, ionic strength, magnetic field, light and others. By varying chemical nature of
monomer units constituting polymer chains, polymer materials with a wide range of functional
properties can be created.
The spontaneous assembly of block copolymer in selective solvents gives rise to different
nanostructures, including spherical micelles27, cylinders8, vesicles9,10, worm-like micelles11 and
others, depending on the molecular weight of polymer blocks. pH-sensitive amphiphilic block
2

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copolymers capable of pH-triggered assembly in aqueous environments have a broad range of

prospective applications in industry and medicine and are intensively studied by numerous
research groups24,12,13. Amphiphilic gradient and block-gradient copolymers represent a new
class of responsive polymers with very special properties

1421

. Gradient copolymers were found

to form aggregates of variable size and demonstrated a temperature-dependent cloud point19.

Self-assembly of block gradient copolymers of acrylic acid (AA) and styrene (S) was studied
experimentally2,3 by dynamic light scattering (DLS) recently. It was demonstrated that in aqueous
solutions above pH 7 the copolymer molecules are dispersed molecularly; they are stabilized by
electrostatic repulsion between the ionized macromolecules. As the pH decreases, upon
protonation of AA function, the Coloumbic interactions decrease, and the hydrophobic
interactions become more important. In the case of di-block gradient copolymers, P(S-grad-AA)b-PAA the spherical micelles growth was observed as acidity increases up to pH~4. The
association was found to be reversible, that is molecular solution of the copolymers was
recovered upon subsequent increase in pH. This conclusion was in a line with the results of
small-angle neutron scattering (SANS) experiments. The latter showed a reversible pH-triggered
appearance of a wide correlation peak that was described using superposition of a form-factor for
polydisperse spheres and a Percus-Yevick structure factor2. In contrast to di-block gradient
copolymers, the tri-block gradient copolymers3, P(S-grad-AA)-b-PAA-b-P(S-grad-AA), form
flower-like micelles in dilute aqueous solution, and a reversible pH-triggered sol-gel transition
was observed in semi-dilute solution at pH 6.4-6.5.
There is a remarkable difference in the relationships of micelle formation in solutions of block
copolymers and block-gradient copolymers of AA and S with the same net composition: while
the block copolymers associate irreversibly and form frozen micelles
3

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, which cannot be re-

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arranged by variation in environmental conditions (pH, ionic strength, etc.), the micelles of block
gradient copolymers re-dissolve completely when the pH increases. The assembly of block
copolymers occurs highly cooperatively and leads to fairly monodisperse micelles. On the
contrary, the association of block-gradient copolymer proceeds progressively upon lowering of
pH and the resulting copolymer aggregates demonstrate high polydispersity2,3, that points to low
cooperativity of the process.
In the present article we aim to unravel mechanisms and driving forces of the reversible selfassembly in aqueous solutions of di- and tri-block gradient copolymers of S and AA and study of
thermodynamics of the assembly by using Isothermal Titration Calorimetry (ITC). The main
driving

force

for

association

of

amphiphilic

polyelectrolytes

are

the

hydrophobic

interactions23,24,25. The equilibrium aggregation number in self-assembled structures of

amphiphilic ionic block copolymers is controlled by the balance between the hydrophobic
attraction of associating blocks and Coulomb repulsions between charged polyelectrolyte blocks.
In the case of pH sensitive polyelectrolyte blocks (weak polyacid or polybase) the degree of
ionization of the copolymers and the strength of the electrostatic repulsions depends not only on
the solution pH, but also on their aggregation state and ionic strength of the solution

26,27

. The

main relationships of self-assembly of amphiphilic ionic diblock copolymers are discussed in the
recent review

28

. Thermodynamic studies of AA and S copolymers and of the mixtures of

PAA/surfactant have been published by a number of authors24,29,30. These studies found that
PAA-b-PS (with PAA/PS ratios in the range of 0.08-0.33) copolymers in N,N-dimethyl
formamide/water solutions form spherical crew-cut micelles with a large PS core and with a thin
PAA corona. A closed association model was applied to describe the micellization process. The
standard changes in Gibbs free energy, entropy and enthalpy (G, S and H, respectively)
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were found to be negative under these experimental conditions, which indicated that S was
unfavorable and that a decrease in standard enthalpy was solely responsible for micelle
formation. However, it was also found that this enthalpy-driven process could be transformed
into an entropy driven by increasing the water content in the mixture of solvents at constant
concentration of polymer or increasing the PS block length29.The strength of interactions could
be altered and/or monitored by addition of a surfactant or extra ions. The formation of PAA
complexes with sodium dodecyl sulfate in an aqueous environment was investigated using
isothermal titration calorimetry(ITC), an ion-selective electrode and DLS30. Interactions between
PAA and SDS were shown to occur at a degree of neutralization () of PAA lower than 0.2. The
cooperative binding of SDS to the non-charged segments of PAA is driven by hydrophobic
interactions. When PAA molecules are ionized, there was electrostatic repulsion between the
polyelectrolyte and SDS; thus, binding was hindered. The enthalpy change30 for this interaction
was positive, and the entropy change was negative. Interaction of poly(sodium acrylate)
(NaPAA) and poly(sodium styrene sulfonate) (NaPSS) with water over a wide range of
concentrations was observed by ITC24. Comparison of the dilution curves between several lowmolecular-weight salts revealed that the specific structure of the styrene hydrophobic backbone
of NaPSS yields a more exothermic dilution isotherm than NaSO4. Additionally, the binding of
Ca2+ ions to PAA was found to be a spontaneous process that was highly endothermic; i.e., the
binding is solely driven by entropy. This result suggests that the driving energy source for the
binding of multivalent ions onto polyelectrolytes is liberation of water molecules from the
hydration shells of the components, not Coulombic interactions24.
Hence, ITC is an extremely powerful method for the study of thermodynamic characteristics of
self-assembly processes for a broad spectrum of amphiphilic systems 24,3138. The rest of the paper
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is organized as follows: We start with presenting DLS-titration data for dilute solutions of di- and
tri-block gradient copolymers which provide a useful guide line for analysis and interpretation of
the ITC spectra. The results of the electrophoretic mobility measurements in micellar solution
presented towards the end of the paper complement the insights obtained from ITC and DLS
experiments. Finally we outline our conclusions.

Results and Discussion

Synthesis
The synthesis of the block gradient copolymers was performed, as described in previous
publications2,3, in dioxane at 120 0C in the presence of alkoxyamine (Blocbuilder) as an initiator
and slight excess of nitroxide SG1 (9%) to avoid side reactions and to ensure a good control of
the chains growth. The Nitroxide Mediated Polymerization (NMP) of acrylic acid was performed
first during four hours and then an amount of styrene equal to the residual acrylic acid present in
solution was added and the reaction was maintained for two more hours. In order to obtain
bifunctional macroinitiator for symmetric triblock copolymer we used dialkoxyamine as an
initiator for NMP of the (central) acrylic acid block. The apparent reactivity ratios of acrylic acid
and styrene monomers in the case of use of SG1-terminated PAA as a macroinitiator ensures the
gradient structure of the terminal copolymer block3. This tendency to form gradient structure is
slightly reinforced by the continuous addition of styrene, which promotes incorporation of the
styrene monomers during polymerization of acrylic acid. During the synthesis of the copolymers,
aliquots were removed and analyzed by 1H NMR in order to determine the monomer conversion
and the composition of the chain as a function of time, which confirmed the gradient structure of
the copolymers. Further details of the synthesis are presented in our previous publications2,3 The

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molecular characteristics of resulting di- and tri-block gradient copolymers are collected in Table
1.
Scheme 3.Chemical structure of gradient di-block and tri-block copolymers. SG1 is a residue of
the initiator.

Table 1.Molecular characteristics of the copolymers.

molar fraction of groups

number of units
Copolymer

Total MNa,

S groups

g/mole,

in polymer, %

AA groups
hydrophilic

gradient block

blockb

AA
groupsc

D1

14000

46

24

100

27

73

D2

17000

61

39

70

36

64

D3

21000

80

80

90

32

68

T1

12000

20

100

28

72

S groups

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T2

18000

26

19

105

27

73

T3

17000

24

140

23

77

average molecular weight and b degree of polymerization of PAA and the gradient blocks determined by NMR,

molar fraction of styrene and acrylic acid in the final copolymers determined by NMR

Dynamic Light Scattering

The results of the DLS titration measurements of aqueous solutions of di- and tri-block gradient
copolymers with concentration of 0.1 w% are presented in Figures 1a and 1b, respectively. At
high pH values, the copolymers are molecularly dispersed in the solution, the apparent
hydrodynamic radii of the solute species are virtually independent of pH. However, upon
approaching pH=7.5-7.0, the size of the particles exhibit steep increase up to the value of
approximately 7-8 nm. We ascribe this transition as the onset of cooperative association of the
copolymer molecules and particles emerging in the solution can be identified as micelles.
Subsequent decrease in pH leads to moderate increase in the hydrodynamic size of the micelles.
This trend is explained by an interplay of two opposite effects caused by a decrease in pH: on one
hand, the aggregation number and the size of the hydrophobic core of the aggregates increase, on
the other hand a decrease in the degree of ionization leads to progressive shrinkage of the PAA
coronae of the micelles. This quasi-plateau regime corresponding to pH-driven growth of the
micelles occurs between pH=7.0-6.0 in the D1 and D2 solutions (Fig.1 a)) and at pH=6.0-5.0 in
the D3 copolymer solution and is followed by a rapid growth in the apparent hydrodynamic
radius of the solute particles at smaller pH. This range of the DLS titration curves corresponds to
formation of larger aggregates and eventually to macroscopic phase separation in the block
copolymer solution. This conclusion is supported by the analysis of the small angle neutron
scattering (SANS) data obtained for D1 copolymer in ref.2 (see SI for details). As follows from
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the detailed treatment of the SANS data, in the range of pH=6-7 the number of molecules in an
aggregate is approximately 100, that is compatible with the core/corona micellar structure. As pH
is lowered further to pH~5, the number of molecules per aggregate sharply increases up to >103
that provides evidence of formation of irregular aggregates at the edge of phase separation in the
solution.

a)

b)

Figure 1.pH dependence of the hydrodynamic radii (RH) measured in di-block gradient
copolymers solutions (a), and tri-block gradient copolymers solutions (b).
The DLS titration curves for the tri-block copolymers T1 and T2 exhibit similar trends as the
analogous curves for diblock gradient copolymers. We remind the reader that in this case
association is expected to lead to formation of flower-like micelles in dilute solution. The
micellization transition and a conspicuous quasi-plateau in the RH vs pH curve occur between pH
values of 6.5 and 5.5 for T1 and T2 copolymers. For the most hydrophilic T3 copolymer the
onset of micelle formation occurs at pH 5.9 and the quasi-plateau region extends below pH=4.0.
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Remarkably, an abrupt growth of the size of the triblock gradient copolymer aggregates at low
pH preceding phase separation is governed not only by hydrophobic forces (as in the diblock
gradient copolymer solution), but also by entropic bridging attraction between flower-like
micelles that is not counterbalanced by weakened electrostatic repulsions.
We remark that in previous publications2,3 the DLS data were evaluated using Malvern DTS
software giving number-based particles distribution. The algorithm of this method takes into
account fractions of prevailing number of objects, small particles, e.g. usually individual
macromolecules. For the case when association in the solution takes place, it is expected that this
algorithm is not accurate enough since the sizes of micellar structures may vary in a broad range
of values. Number-based particle distribution will therefore preferentially account for the
particles of smaller sizes. The previous DLS-titration measurements on block gradient copolymer
solutions have shown that number-based fitting did not provide exact determination of micelle
formation onset. The obtained sizes of particles were smaller compared to the DLS-natural
intensity-based distribution. The intensity-based Repes algorithm39 used for obtaining results
presented in Figure 1 provides better control over the Laplace inverse transformation procedure
and therefore better resolution of the pH-dependence of the dimensions of the block gradient
copolymers aggregates. In particular, this enabled us to specify the pH threshold corresponding to
the onset of micellization of the block gradient copolymers and to observe the pH-induced
growth of aggregatively stable micelles preceding macroscopic phase separation in the solution.
Electrophoretic mobility
The electrophoretic mobility titration measurements were performed for the D3 and T2 block
gradient copolymers. As follows from DLS titration data presented in Figure 1, these copolymers

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exhibit similar scenario of pH-controlled self-assembly though with a shifted onset of the micelle
formation. In Figure 7(a,b), the zeta-potential pH-dependent data are presented for the D3 and T2
copolymers. A decrease in pH leads to protonation of the AA groups and thus to a decrease in the
magnitude of negative charge of the copolymer molecules. An interplay of this decrease in the
degree of ionization with the concomitant change in the aggregation state (structure and size of
the aggregates) leads to complex patterns in the zeta-potential versus pH dependence.

a)

b)

Figure 7. Zeta-potential values as a function of pH for the di-block gradient copolymer D3 (a),
and the tri-block gradient copolymer T2 (b) in solution with polymer concentration 0.1 w%.
Initial values of zeta-potential at pH values of about 11-10 for both the di-block and tri-block
copolymer are similar and equal approximately to -20 -25 mV. In this pH-range the copolymers
are molecularly dissolved and form the unimer solution. Molecularly dispersed copolymers
assume the shape of unimolecular globule formed by (partially collapsed) amphiphilic gradient

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block linked to extended polyelectrolyte block. From the beginning of the titration down to pH
value around 8.0, the zeta-potential varies between -40 mV and -20 mV.
According to the DLS titration data, cooperative association in the D3 copolymer solution begins
at pH7.2 and is followed by slow increase in the micellar size upon subsequent decrease in pH.
Correspondingly, in the pH range from 7.5 to 4.7, a virtually constant zeta-potential value is
observed. This behaviour of the zeta-potential can be explained by localization of the majority of
the counterions inside the PAA corona domains of the micelles. The net (residual) charge of the
micelle with arbitrary aggregation number is proportional to the extension of the corona that
results in constant zeta-potential26-28. BelowpH4.7, a sharp change towards a neutral point
occurs. Approaching zeta-potential value of zero means that repulsion between growing particles
decreases significantly, and the solution approaches precipitation threshold.
The behavior of the zeta-potential as a function of pH for the tri-block copolymer T2 is more
complex. At high pH values and down to pH7.0, the pH where micellization begins according to
the DLS data, oscillations of the zeta-potential between -10 mV and -32 mV occur. When the pH
decreases down to 7.0 the zeta potential reaches a plateau value which is maintained between pH
6.9 and 4.8. In this pH range, we observed by DLS micellar aggregates with the size weakly
increasing upon a decrease in pH. According to the DLS data, further sharp particle growth in the
T2 copolymer solution occurs at pH below 4.6. At this pH, zeta potential of the aggregates adopts
a new constant value of -2 mV until the end of titration at pH 3.7. Such a small and stable zetapotential value at low pH is remarkable for a solution of the T2 copolymer and might be related
to the bridging mechanism of the particle growth.

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Isothermal Titration Calorimetry

The ITC experiment was performed in order to investigate the self-assembly thermodynamics of
the block gradient copolymers upon decreasing pH, to characterize the self-assembly process step
by step and to elucidate the prevailing driving force at each particular stage (pH range) of the
assembly. We also tried to assess the effects of the polymer architecture and the length of the
gradient block on the association process of the copolymer molecules.
The experimental data were treated according to the following scheme: first, a manual titration of
the experimental solution was conducted to obtain the dependence of pH on the volume of added
acid V(HCl) data; second, an automated ITC titration generated the dependence of the heat Q for
injection number i, Q(i) as a function of the amount of added acid (HCl). The results of these two
experiments were combined to determine the final dependence of Q(i) on pH. Thus, because the
polymer was initially deprotonated (high pH), the graphs must be read in the direction of
decreasing pH.
The important note here is that the ITC experiment with hydrochloric acid as a titrant must be
performed with great care, and the analysis must be conducted very meticulously. In acid
titrations, there are several simultaneous contributions to heat effect for the i injection that can be
written as the following:
(1)
where Qnet is the heat of the neutralization of excess sodium hydroxide by hydrochloric acid in
the aqueous solution, Qprot is the heat of the reaction between our target polymer and
hydrochloric acid, Qconf is the heat of conformational changes in the polymer molecule and the
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surrounding medium as a result of protonation, Qhydroph is the heat of any hydrophobic

interactions in the gradient block of the molecule and Qdil is the heat of dilution of the
hydrochloric acid. It is possible to eliminate some of these heats using a blank titration. Thus, the
dilution experiment for hydrochloric acid into an aqueous solution was performed to exclude the
dilution contributed heat from the general consideration. One can also minimize the Qnet value by
decreasing the pH value to pH=7, the neutralization point of base-acid equilibrium. However, the
DLS data show that the onset of assembly in some cases occurs at pH=7.5. Therefore, we cannot
exclude the neutralization effect completely, but we can conduct measurements in the region of
pH 4-8 and consider the neutralization effects in the region of pH 7-8 only. Thus, we considered
the effects of protonation, conformational changes and hydrophobic interactions as the most
significant.
Here, it is important to note that we distinguish two terms: exothermic/endothermic reactions and
exothermic/endothermic processes. The first corresponds to the type of reaction, i.e., whether it
releases or absorbs heat, and the second refers to the changes in the enthalpy during the
experiment. The titration isotherms of di-block gradient copolymers are presented in Figure 2 as a
function of pH. Measurements in this plot were performed at 25 C. As was mentioned, the plot
must be read in the direction of decreasing pH. There are three distinguishable regions in the ITC
curve and three consecutive processes: an endothermic process from pH 8 to5.6, followed by an
exothermic process from pH 5.6 to 4.5, and finally, another endothermic process from pH 4.5 to 4
that merges with the zero mark on the scale of heat energy. The first endothermic process
sometimes displays two steps, and we will try to elucidate the reasons for this behavior later on.

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Figure 2. ITC curves of titration of the di-block gradient copolymers.

We took an attempt to perform a consecutive comparison of the results from the ITC and DLS
experiments at every region. That strategy allows us not only to characterize each particular
region on the ITC curve but also to get a new inside into the process of polymeric micelles
formation. There is a clear correlation between position of extreme points observed in the
isotherm and value of characteristic points visible from the DLS data. Thus, the first
endothermic process corresponds to the transition from species with a radius of approximately
4-5 nm to the 7.5-nm particles, which could be interpreted as the association of individual
macromolecules into polymeric micelles. One can try to assess the contributions that can result in
the endothermic effect of association process. The protonation of the acrylic acid groups on
polymeric molecule results in conformation changes. The initially expanded charged blocks,
which are typically characterized by a nearly rod-like conformation, undergo the partial coiling
during the protonation reaction on acrylic groups. The change in conformation state of acrylic
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block results in shift of hydrophobic/hydrophilic balance, and hydrophobic interactions between

the gradient blocks of different molecules become more pronounced. Thus, we can assume that
four reactions contribute to the first endothermic process (neutralization of NaOH, protonation of
the acrylic group and, as a result, conformational changes in the macromolecule and hydrophobic
interaction between gradient blocks). The heat (enthalpy) of each particular reaction mentioned
above can be assessed. The neutralization reaction between NaOH and HCl is an exothermic
reaction with Hnet= - 13.6 kcal/mol. The protonation reaction is also associated with an
exothermic reaction. Moreover, there is a common point of view that hydrophobic interactions
have an endothermic nature. To make clear the heat effect that might be associated with the next
contribution (conformational changes), an experiment with the PAA and PMA homopolymers
was performed (Figure 3).

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Figure 3. ITC titration curves for PMA (concentration 0.25 w%, black open circles) and PAA
(concentration 0.01 w%, red open circles).
These experiments show that the endothermic effect is observed during the whole titration
process of PAA and most of titration process in PMA. Since we know that protonation of PAA is
accompanied with conformational changes of the polyacrylic acid molecule and the structural
rearrangements of the surrounding medium, we can attribute the endothermic effect of reaction to
the structural rearrangement in system in general. Interestingly, the effect of the structural
changes of the system is significant and should be observed in the second stage of titration.
As it was mentioned previously, the first process shows a two-step tendency in some isotherms.
The chemical structure of the copolymers studied here corresponds to the presence of acrylic acid
groups in both the hydrophilic and gradient blocks. One can assume that being surrounded by
styrene groups might affect the protonation constant of the acrylic acid groups in the gradient
block. Thus, there are two different protonation constants that could explain the behavior
reflected in the ITC data. To verify that statement the calculation of degree of ionization was
performed. The degree of ionization was calculated from the experimental data according to the
following equation:

(2)
Where [] represents the molar concentration of the indicated components. At pH~5
approximately 50% of the acrylic groups are protonated. It is known that the second process of
the titration, having a lower reaction constant, can start only when the first process is completely
finished. It is also clear that the encirclement of the acrylic groups in the gradient block by
styrene increases the rate of protonation. Thus, the two-step tendency of some isotherms cannot
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be explained by protonation alone. Moreover, as mentioned above, the protonation reaction must
be accompanied by a release of heat energy. There are only two types of reactions that are
endothermic: hydrophobic interactions and structural changes in the system. It seems that the
appearance of two steps is a consequence of a combination of these two reactions. In addition,
there is a correlation between the length of the gradient block and the occurrence of two steps.
The longer the gradient block is, the more stepwise the process is.
Now, one can try to characterize the first endothermic process quantitatively. According to eq. 1,
there are two parallel processes and two contributions where hydrochloric acid is involved: the
neutralization reaction and protonation of the polymeric molecules. Because the neutralization
reaction (reaction between strong acid and strong base) occurs much faster than the protonation
of the acrylic groups of the polymer (reaction between strong acid and weak base), the former
reaction has priority over the latter. Consequently, the heat of the neutralization reaction has to
occur at the beginning of the titration where the pH is still high. Because we cannot distinguish
these two contributions from the ITC data and we do not know the precise concentration of
NaOH in the solution (and, therefore, the exact amount of HCl needed to neutralize the excess
NaOH), it is hard to draw quantitative conclusions from the first endothermic process given by

where nHCl is the total molar amount of HCl injected and Hnet is the enthalpy of the
neutralization reaction. However, if we suppose to a first approximation that the end of the
neutralization reaction correlates with the crossing of the experimental ITC curve through the
zero heat, comparison of the apparent values of heat energy for the different polymers becomes
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possible (Table 2). Interestingly, the most hydrophilic polymer, D1, shows the highest apparent
heat energy value in Figure 4. In terms of the endothermic process, this result could mean that
much more additional energy must be applied to the system to promote the transition from single
polymer molecules to micelles or in other words there should be an additional contribution
(release of hydrating water molecules or general rearrangement in the system) that could help to
overcome the negative effect of transformation from single polymer chain to micelle. Here, it is
worth to note that ITC curves depict the heat effect of reaction, the enthalpy contribution, not the
entropy one.

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Figure 4 (a, b). ITC curves for the di-block copolymers after subtracting the dilution heat.

First endothermic process

Second exothermic process

H, kJ/mol

Cp, kJ/mol K

H, kJ/mol

S, J/mol K

G, kJ/mol

Cp, kJ/mol K

D1

2.4

105

-3.3

65.5

-22.8

-75.5

D3

1.2

110

-2.8

71.9

-24.3

-72.7

T1

2.1

105

-2.3

76.3

-25.1

-83.7

Table 2.Thermodynamic parameters and heat capacity changes for the di- and tri-block gradient
copolymers.

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The second exothermic process was observed in the pH range of 4.5 5.6. According to the
DLS data, this process corresponds to a sharp increase in the particle size, and a phase separation
is achieved at the end of this region. Interestingly, this process occurs even when the protonation
of the acrylic groups is not completed. Nevertheless, the ITC data show the existence of only one
process in this region, since no tendency for multiple steps was observed. By looking back at the
possible components of our titration process, two of them could be excluded: the dilution effects
and the heat of neutralization. We know that neutralization reaction has to be completed when
reaching pH 7. Thus, in this region, we are dealing with only one system, the
polymer/hydrochloric acid interactions (including the effects of protonation, hydrophobic
interactions and conformational (structural) changes related to the increasing number of
micelles). This fact makes quantitative analysis of the process possible. We used a simple One
Set of Identical Sites model for our fitting. A detailed explanation of this model can be found
elsewhere40. This model describes the process with several thermodynamic parameters, enthalpy,
H, entropy, S, and Gibbs energy, G. The results of this fit are presented in Table 2. It is
apparent from the table that the second process is driven by entropy. The positive value of
entropy is usually associated with the release of ions or water initially bound to the
macromolecules. At the same time, negative enthalpy value can also been found as a
characteristic of the second process for all polymers. Comparing values of the Gibbs free energy
change for exothermic process we can conclude, that data in Table 2 show that the second
process is more favourable for the diblock copolymer D3 and less favourable for the most
hydrophilic polymer D1. However, the gain in entropy, which is also highest for diblock D3
copolymer, makes the process feasible for both polymers.

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A third process is sometimes visible on the isotherms. The ITC experiment performed on PMA
(Figure 3) displays a similar behavior as gradient block copolymers at the low pH values. One
can assume that this process could be related to some rearrangement of polymer molecules within
the micelles. One possible combination of the different reactions and their heats are presented in
Scheme 1.

Scheme 2. Graphic scheme of the sub-reactions and overall processes occurring during the ITC
titrations and their relative heats.

ITC measurements at several temperatures were made to better understand structural changes in
the vicinity of the precipitation point. The Q(i) vs pH curves are available in the supplementary
materials. The apparent values of heat energy from the first process and H values from the fit of
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the second process were used in the analysis. The analysis was performed over the temperature
range from 288.15 to 323.15 K. The consecutive values of heat capacity were calculated from the
slope of the H(T) curves (Figure 5 a, b). A significant drop in the heat capacity was observed
when the polymer system undergoes the transformation from state with endothermic effect
(endothermic process) to the state with exothermic one (exothermic process). Such decrease in
the heat capacity parameter is usually associated with structural changes when large system
energy is required for the formation of microstructures or with increase in the structures
hydrophobicity

25,4345

. We have shown already before that the conformational changes in the

copolymer molecule and the surrounding medium are a significant part of the second process.
Moreover, the growth of styrene domains in the micellar core has been observed previously over
the same pH range.2,3 It is logical to assume that there is a certain rearrangement of polymer
molecules within the micelles and micellar core that results in a significant change of heat
capacity value of our systems. The changes in heat capacity are compiled in Table 2.

a)

b)

Figure 5. Enthalpy changes for the di- and tri- block gradient copolymers calculated for the major
endothermic process a) and calculated for the exothermic process at lower pH values b).

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The ITC experiment for the tri-block copolymers was also performed and the results are
presented in Figure 6a, b, c.

a)

b)

Figure 6. ITC curves for tri-block gradient copolymers T1, T2 and T3 (a), and fitting data for T1
tri-block gradient copolymer (b)
These tri-block copolymers consist of a central acrylic acid block with gradient blocks at each
end of the macromolecule. Such polymers were shown to form flower-like micelles in dilute
aqueous solution.3 The isotherms for tri-block gradient copolymers can be analyzed in a similar
manner as the di-block gradient copolymer data. Remarkable difference is exhibited only by the
T3 triblock copolymer. The beginning of the second process in the T3 tri-block copolymer is
shifted to a pH of 7.2. This difference can be explained by the relatively highest hydrophilicity of
T3 among all of the polymers and, in particular, among the tri-block copolymers.

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Important information can also be gained from NMR data.3 The mole fractions of styrene and
acrylic acid units, respectively, in the polymers are 0.32 and 0.68 for T1, 0.28 and 0.72 for T2,
and 0.23 and 0.77 for T3. Thus, the content of styrene groups is highest for T1 and the
hydrophilic T3 has the highest number of acrylic acid groups. In Figure 6b the ITC curves for the
T1 copolymer are presented. The parameters calculated from the experimental curves are
presented in Table 2. A comparison of the thermodynamic parameters of the tri-block T1
copolymer with those of the di-block D1 and D3 copolymers reveals several features. For the
second process, the tri-block polymer has the lowest enthalpy and the highest entropy, and these
changes are expected to be favourable for the overall process.
One of the most important parameters to characterize in acidic/basic systems is pK. In the case of
PAA, the apparent (effective) pK value depends on chain length and usually lies in the range of
5.2 to 4.8. At those values, 50% of the acrylic groups are protonated. In the Q(i) vs pH curve, we
observe a deep minimum in this region, which was previously attributed by us to the structural
changes in the micelle cores related to the growth of the styrene-rich domains and the
simultaneous dehydration processes. This fact and a significant change in RH as measured by
DLS experiments, permit the assumption that the effective pK value of PAA block in our
polymer lies precisely in the same pH region 4.5 - 6. It is known that in the block gradient
copolymers, the effective pK is shifted according to the amount of AA units and the length of the
gradient part of the polymer chain. Association of block-gradient copolymers into micelles
suppresses ionization of the AA monomer units26,27, that is, leads to slightly higher values of
apparent pK.

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Conclusion
In this paper, we studied the thermodynamics of the pH-triggered self-assembly of di- and
triblock gradient copolymers made of AA and S in aqueous solutions. To unravel the
relationships of the self-assembly in this system we used combination of DLS and zeta-potential
titration experiments and made the main emphasis of the ITC measurements.
In contrast to earlier DLS studies we performed here fitting of the raw DLS data using intensitybased size distribution that enabled us to successfully observe the micelle formation in details. In
particular, we were able to localize the pH range corresponding to the onset of cooperative
association (micellization) in the solution of block-gradient copolymers. The width of the
transition range from unimers to micelles is about one pH unit, which is in a line with low
cooperativity of association. The latter feature is explained by small surface tension at the
enriched by AA units interface between the core and hydrated coronal domain of the micelles.
Furthermore, the DLS data enabled us to observe the growth of micelles upon lowering of pH
followed by a sharp increase in the apparent hydrodynamic radius of the solute particles. We
ascribe this second transition to the macroscopic phase separation in the solution.
The electrophoretic mobility measurements revealed appearance of the quasi-plateau regime in
the pH dependence of the zeta-potential in the range of pH corresponding to the micellar solution.
This result is consistent with theoretical predictions26-28 concerning localization of counterions in
the coronae of osmotic micelles in solutions of low ionic strength.
The ITC measurements revealed that the copolymers with large gradient blocks demonstrated
two clear discernible processes during the endothermic stage of the titrations as a function of pH.
The fitting procedure allowed us to determine the heats H of the reactions occurring during
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these processes. The first process was attributed to neutralization of the hydrophilic segments of
the macromolecules. The second process was attributed to micelle formation. To distinguish the
influence of the acrylic acid groups and the hydrophobic styrene groups on the self-assembly
process, ITC titrations of homopolymers of PAA and PMA were performed. These experiments
confirmed that because of the absence of hydrophobic groups in PAA it does not undergo intraor intermolecular self-organization. The Q(i) function showed a strong endothermic peak that
returned to the baseline as the acrylic acid groups of PAA were protonated. The aqueous
solutions of PMA formed supramolecular structures. This ITC experiment revealed a broad peak,
which showed a significant minimum in the Q(i) function. This minimum was similar to those
found for the gradient block copolymers of AA and S. The study on the minor changes of Q(i)
yielded an equilibrium constant and H of the processes of the block gradient copolymers
assembly. The heat capacity changes determined in the ITC experiments confirmed previous
findings2,3 that at pH values below the micelle formation threshold, the growth of hydrophobic
domains (presumably the micellar cores) occurs. Minor changes in the heat capacity indicated
that reorganization inside the core is accompanied by a dehydration process. Thermodynamic
parameters obtained from the analysis of the endothermic peak associated with both micelle
formation and electrostatic interactions. From the analysis of the exothermic process associated
with structural changes in the core we found that the free energy values of the first and second
processes are characterized by large changes in enthalpy and entropy, respectively.

Experimental section
Materials.

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Polyacrylic acid homopolymer with a molecular weight of 8000 g/mole was purchased from
Sigma Aldrich. Polymethacrylic acid homopolymer with a molecular weight of 100000 g/mole
was purchased from PolySciences, Inc., Warrington, PA. The homopolymers were used as
received. For ITC measurements water solutions with 0.01 w% of PAA and 0.25 w% of PMA
were prepared. Water was deionized using a Millipore Milli-Q water purification set-up and
filtered through a 0.2-m PVDF syringe filter before use. Solutions were prepared by dissolving
the appropriate amount of polymer in an aqueous NaOH 0.1 M solution. The molecular
characteristics of the di-block and tri-block gradient copolymers are presented in Table 1.
Isothermal Titration Calorimetry (ITC). Microcalorimetry experiments were performed using
a MicroCal 200 isothermal titration calorimeter. The experiments were performed using injection
volumes varying between 1 and 2 L of a 0.15 M hydrochloric acid solution into the sample cell,
which contained 200 L of a polymer solution (50 mg/mL) or water. The volume of titrant
solution was 40 L. The ionic strength generated in the system due to added acid remained far
below characteristic threshold2 necessary to affect the self-assembly. The syringe was used as a
stirrer. The syringe rotation speed was 1000 rpm. To achieve concentration equilibrium, the delay
between measurements was 5 min. Measurements were conducted at several temperatures
between 15 and 55 C. The data were analyzed using Microcal ORIGIN software. The
experimental enthalpy was obtained by integrating the raw data. To account for dilution effects,
the data of solvent titration was subtracted from the experimental data. The integrated molar
enthalpy change per injection was obtained by dividing the experimentally measured enthalpy by
the number of moles of acid added.
Dynamic Light Scattering (DLS). The pH dependence of the particles hydrodynamic radius,
RH, and scattering intensity, IS, were measured at a scattering angle of = 173 using a
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ZetasizerNano ZS instrument, model ZEN3600 (Malvern Instruments, U.K.). Measurements

were repeated three times at each pH. Correlation functions g2(t) were analyzed by a regularized
inverse Laplace transformation by the REPES algorithm39, which provided distributions A(), of

2
relaxation times according to g (t ) = 1 + A(t ) exp( t )d ,where is an instrumental
2

factor. For the diffusion of nanoparticles in liquid, the hydrodynamic radius RH can be
determined

using

the

Stokes-Einstein

2 1
equation D = (q ) ,

q = (4n 0 ) sin( 2 ) D = kT 6RH , where k is the Boltzmann constant, n the refractive index
and the viscosity of solvent, D is the diffusion coefficient, q is the scattering vector, o is the
wavelength and the scattering angle.
Zeta-Potential Measurements. Electrophoretic mobility Ub was measured using the same
ZetasizerNano-ZS instrument used for the DLS measurements. The DTS software was used to
compute the zeta-potential by the Henry equation:

where r is the dielectric constant of the sample, is a dynamic viscosity (Pa s), and is the zetapotential (V); f(ka) is Henrys function, which is calculated by the approximation of
Smoluchowski (f(ka) = 1.5) for water solutions of a spherical particles with moderate ionic
strength.
Acknowledgement Financial support provided by the Czech Ministry of Education, Youth and
Sports (grant # MSMT KONTAKT II LH14079) is gratefully acknowledged. This work was
partially supported by Russian Science Foundation grant # 14-33-00003.

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Supporting information
ITC curves of titration of di- and tri-block gradient copolymers at different temperatures (SI 1
SI 3). Results of fit of the small angle neutron scattering experiments on the basis of PercusYevick approximation for the structure factor and formfactor of polydisperse spheres. This
material is available free of charge via the Internet at http://pubs.acs.org.

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For Table of Contents use only

Thermodynamics of Multi-Stage Self-Assembly of pH-Sensitive Gradient Copolymersin
Aqueous Solutions.
Zulfiya ernochov, Anna Bogomolova, Olga V.Borisova, Peter ernoch, Sergey K. Filippov,
Laurent Billon, Oleg V. Borisov, Petr tpnek

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