COACERVATION AND PHASE BEHAVIOR OF AQUEOUS SOLUTIONS OF
OPPOSITELY CHARGED POLYELECTROLYTES
A Thesis
Presented to
The graduate faculty of the University of Akron
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
Shuyue Huang
May,2018
ABSTRACT
Complex coacervation is a liquid-liquid phase separation of a macromolecular solution composed of two oppositely charged polymers, leading to one dense polymer-
rich phase called complex coacervate and one solvent-rich phase. Complex coacervates
are capable of easily partitioning solutes within them based on relative affinities of
solute-water and solute-polyelectrolyte pairs, as the coacervate phase has low surface
tension with water, facilitating the transport of small molecules into the coacervate
phase. The uptake of small molecules is expected to influence the physicochemical
properties of the complex coacervate, including the hydrophobicity within coacervate
droplets, phase boundaries of coacervation and precipitation, solute uptake capacity, as
well as the coacervate rheological properties.
The phase separation, or complex coacervation, upon mixing of aqueous solutions
of oppositely charged polyelectrolytes, poly(diallyldimethylammonium chloride)
(PDAC) and poly(sodium 4-styrene sulfonate) (SPS) was investigated in the presence
of various concentrations of two different dyes, methylene blue (MB) or bromothymol
blue (BtB). Besides, partitioning of dyes, including methylene blue (MB), rhodamine
B (RhB), and bromothymol blue (BtB) into complex coacervates of branched
polyethylenimine and poly(sodium 4-styrenesulfonate) was studied.
Turbidity was used to show how various parameters affect phase behavior of
polyelectrolyte complexes. UV-vis spectroscopy was used to study the uptake ability of
small molecules into coacervate phase. Isothermal titration calorimetry (ITC) was
employed to study the intermolecular interaction of dyes revealing strong electrostatic,
π-π and cation-π interaction between polyelectrolyte and dye. Zeta potential was
applied to study the electrokinetic potential of the particles. Fluorescence measurement was used to study the hydrophobic interaction between dyes and coacervate droplets.
Dynamic rheological experiments were taken to study the impact of small molecules on the rheological behavior of coacervates.
ACKNOWLEDGEMENTS
I would like to express my sincere gratefulness to my advisor Professor Nicole S.
Zacharia for her constant guidance, encouragement and selfless support on my research.
It is a great honor and pleasure for me to be a member of her group and to work as a graduate student under her instruction.
I would like to extend my appreciation to Prof. Tianbo Liu and Prof. Xiong Gong for giving me permission to use their instruments in their labs. My studies would not be completed without their help. I would like to acknowledge all my group members, especially Mengmeng Zhao for her kind help and suggestions on my work.
I would like to thank Prof. Tianbo Liu and Prof. Kevin Cavicchi for being my committee members.
Lastly, I would express my deepest appreciation for my beloved my parents and grandparents for their support throughout all these years that I have studied abroad.
TABLE OF CONTENT
CHAPTER I ...... 1
1. Introduction ...... 1 1.1 Polyelectrolytes ...... 1 1.2 Polyelectrolyte complex and complex coacervation ...... 3 1.2.1 Applications of coacervation ...... 4
1.2.2 Factors influence PEC coacervation ...... 5
1.3 Turbidity measurement ...... 6 1.4 Zeta potential ...... 6 1.5 UV-VIS Spectroscopy ...... 7 23 1.6 Isothermal titration calorimetry ...... 9
CHAPTER II ...... 13
1. Introduction ...... 13
2. Experimental section ...... 16 2.1. Materials ...... 16 2. Turbidity measurement ...... 16 2.3. Zeta potential measurement ...... 18 2.4. Determining partition coefficient of dyes into coacervates ...... 19 2.5. Fluorescence spectroscopy ...... 20 2.6. Rheological measurements ...... 20 2.7. Isothermal titration calorimetry (ITC) ...... 21 2.8. Optical microscopy ...... 22 2.9. 1H NMR ...... 22
3. Results and discussion ...... 23 3.1. Solute partitioning into PDAC-SPS coacervates ...... 23 3.2. Effect of solutes on zeta potential of PDAC-SPS coacervates ...... 28
3.3. Isothermal titration calorimetry (ITC) ...... 30 3.4. Effect of accumulated MB on the hydrophobicity within PDAC-SPS ...... 32 3.5. Effect of solutes on PDAC-SPS phase behavior ...... 34 3.6. Effect of MB and BtB on water content of coacervates ...... 40 3.7. Effect of solutes on the rheological properties of complex ...... 41
4. Conclusion ...... 48
CHAPTER III ...... 50
1. Introduction ...... 50
2. Experimental section ...... 52 2.1 Materials ...... 52 2.4 UV-VIS measurement ...... 52 2.5 Isothermal Titration Calorimetry (ITC) ...... 53
3 Discussion and result ...... 54 3.1 Partition coefficient of dyes into complex coacervate ...... 54 3.3 Isothermal titration calorimetry (ITC) ...... 56
FUTURE WORK ...... 59
REFFERENCE ...... 60
LIST OF SCHEMES
1 The structure of PDAC, SPS, PAA and PEI ...... 3
2 This scheme describes the process of formation of coacervate. Mixture of
polycation and polyanion solution leads to a liquid-liquid phase separation
and the polymer-rich phase appearing as droplet is complex coacervate ...... 4
3 Electrostatic phenomenon in a solution for a charged particle. Graphical
description of the Zeta potential...... 7
4 Representative diagram of a typical power compensation ITC.24 ...... 11
5 Chemical Structures of PDAC, SPS, MB and BtB ...... 16
LIST OF TABLES
1 Detail information on the concentration of PDAC-MB and SPS-MB stock
solutions for the turbidity measurement...... 17
2 Detail information on the concentration of PDAC-BtB and SPS-BtB stock
solutions for the turbidity measurement...... 18
3 Concentration of MB and BtB in the PDAC-SPS system for the zeta potential
measurement ...... 19
4 Concentration of MB and BtB in the PDAC-SPS system for the partition
coefficient experiment ...... 20
5 Concentration of MB and BtB in the PDAC-SPS system for the preparation of
coacervate samples for rheological test ...... 21
6 The partition coefficients for MB, RhB and BtB into BPEI-SPS coacervate. pH
= 3.5.The coacervate solution was prepared with 40mM BPEI and 40mM SPS
stock solution in present of 0.5mM dyes. When the mixing ratio of BPEI/SPS
was 2/8, 3/7 and 4/6, the mixing order was BPEI to SPS. At other mixing ratios,
the mixing order was opposite...... 54
LIST OF FIGURES
21 1 Diagram of PDAC–PSS behaviour as a function of salt concentration ...... 6
2 Partition coefficient of (a) MB and (b) BtB into PDAC-SPS coacervates as a
function of various concentration of MB or BtB. PDAC-SPS coacervates were
prepared using PDAC (10 mM) and SPS (10 mM) stock solution with a mixing
ratio of PDAC:SPS=0.5 ...... 25
3 Zeta potential of PDAC-SPS coacervates with the addition various concentration
of MB or BtB PDAC-SPS coacervates were prepared using PDAC (10 mM) and
SPS (10 mM) stock solution with a mixing ratio of "PDAC:SPS=0.5" ...... 30
4 ITC data for the titration of 2.5 mM PDAC or SPS into 0.25 mM MB or BtB
solution...... 32
5 Fluorescence emission spectra of ANS sequestered in PDAC-SPS coacervate
dispersions with the addition of (a) 0 mM, (b) 0.1 mM, (c) 0.75 mM and (d) 1.0
mM MB recorded at room temperature. The coacervates were prepared with 10
mM PDAC and 10 mM SPS at a mixing ratio of PDAC:SPS = 0.5, with the
addition of 0.5 mM ANS ...... 34
6 Phase behavior of PDAC-SPS aqueous system as a function of polyelectrolyte
stoichiometry, total polymer concentration, and phase behavior of PDAC-SPS
aqueous system as a function of overall concentration of dye (b) MB or (c) BtB
and polyelectrolyte stoichiometry. Blue, red and black symbols represent clear
solution, coacervation and precipitation, respectively ...... 37
7 Frequency sweeps showing storage (G', solid) and loss (G'', open) modulus of
PDAC-SPS coacervates formed by mixing 5 mM PDAC stock solution and 5 mM
SPS stock solution at a stoichiometry of with the addition of
(a) various concentration of MB, and (b) PDACvarious ∶ SPS concentration = 0.75 of BtB ...... 45
8 Loss tangent (tan (δ)=G^''/G' of the PDAC-SPS coacervates formed with various
concentration of MB or BtB ...... 46
9 Plot of loss tangent (tan(δ)=G^''/G' of the PDAC-SPS coacervates as a function of
MB concentrations...... 47
10 UV-vis spectra of dyes (a, MB; b, RhB; c, BtB) at pH 3.5 and dyes in BPEI or
SPS aqueous solution ...... 56
11 ITC data for the titration of 2.5 mm BPEI or SPS into 0.25 mm dyes (MB, RhB
or BtB) respectively ...... 58
S1 UV-vis spectra of MB, MB-SPS, BtB and BtB-SPS aqueous solution at pH
3.0. The concentration of SPS is 20 mM. The concentration of dye is 0.01 mM.
...... 25
S2 UV-vis spectra of 0.01 × 10−3 M BtB at different pH, and 0.01 × 10−3 M BtB in
20 × 10−3 M PDAC aqueous solution at pH 3.0...... 26
S3 Partition coefficient of MB into PDAC-SPS coacervates as a function of molar
ratio of PDAC:SPS in the system. PDAC-SPS coacervates were prepared using
PDAC (10 mM) and SPS (10 mM) stock solution with various mixing ratio
(PDAC:SPS=0.067, 0.1, 0.2 and 0.5) at a fixed MB overall concentration of 0.01
mM...... 26
S4 PDAC:SPS molar ratio in the coacervate phase (PDAC/SPScoac) as a function of
overall PDAC:SPS mixing molar ratio (PDAC/SPSoverall) in the system,
measured using 1H NMR. PDAC-SPS coacervates were prepared using PDAC
(10 mM) and SPS (10 mM) stock solution with various mixing ratio
(PDAC:SPS=0.067, 0.1, 0.2 and 0.5) ...... 27
S5 Zeta potential of the PDAC-SPS coacervates as a function of PDAC:SPS molar
ratio in the system. PDAC-SPS coacervates were prepared using PDAC (10 mM)
and SPS (10 mM) stock solution with various mixing ratio (PDAC:SPS=0.067,
0.1, 0.2 and 0.5)...... 28
S6 Phase behavior of PDAC-SPS system without the addition of (a) salt or dye, and
with the addition of (b) 1 mM NaCl, (c) 0.1 mM MB, (d) 0.25 mM MB, (e)
0.5 mM MB, (f) 1 mM MB, (g) 0.1 mM BtB, and (h) 0.25 mM BtB. Blue, red and
black symbols represent clear solution, coacervation and precipitation,
respectively. The grey dashed lines represent the PDAC:SPS stoichiometry of 1.
...... 38
S7 Optical micrographs of (a) precipitates and (b) coacervates formed using 10
× 10−3 M PDAC and 10 × 10−3 M SPS with a ratio of 4:5 and 1:2, respectively.
The scale bar in these two optical micrographs is 50 µm...... 39
S8 Turbidity profile of PDAC-SPS system obtained using the same concentration of
PDAC and SPS aqueous solution (1, 5 and 10 mM), respectively, without the
addition of dyes. The transmittance (T) was recorded at 60 s after each PDAC
addition...... 40
S9 Water content of PDAC-SPS coacervates formed using prepared by mixing
PDAC (5 mM) and SPS (5 mM) with or without the addition of various
concentration of MB or BtB, at a stoichiometry of PDAC∶SPS=0.75 as a function
of dye concentration...... 41 CHAPTER I
1. Introduction
1.1 Polyelectrolytes
Polymer materials are widely used in all aspects of our lives. Rapid development
of polymer research contributes to amount of meaningful applications, such as in the
region of batteries1,2 and hydrogels3,4,5. In this thesis, the fundamental study and
promising use of polyelectrolytes complex coacervation was investigated.
Complex coacervation is a liquid-liquid phase separation that takes place when oppositely charged polymers mix together.6 Polyelectrolytes are commonly used in the research of complex coacervation. They are defined as linear macromolecule chains bearing a large number of chargeable groups and usually dissolved in polar solvent, generally water.7 In positively (or negatively) charged polyelectrolyte solution, a single species polymer with random polydispersity and one species of counter-ions which are small ions with oppositely charged sign to that of macromolecular charge are present.7
The charge of counter-ion and macromolecular must be in equivalent in order to reach
7 the condition of electroneutrality.
Depending upon the degree of charging, polyelectrolyte can be divided into two
types, including strong polyelectrolyte which completely dissociates in solution and
weak polyelectrolyte which is partially charged in solution. The structures of some
broadly used strong polyelectrolytes and weak polyelectrolytes are illustrated in the
scheme 1. Poly(diallyldimethylammonium chloride) (PDAC) and poly(sodium 4-
styrene sulfonate) (SPS) are strong polyelectrolytes. Poly(acrylic acid) (PAA) and
Polyethylenimine (PEI) are weak polyelectrolytes. Many physical properties of
1 polyelectrolyte solution are affected by charging degree. The extent of polyelectrolyte
dissociation necessarily affects the ionic strength of the solution. Ionic strength is a
characteristic of the polyelectrolyte as well as a measure of the ions concentration in
the solution. It is typically demonstrated as the average electrostatic interaction among
the polyelectrolyte's ions, indicating how effectively the charge on a particular ion is
shielded or stabilized by other ions (ionic atmosphere) in a polyelectrolyte.8,9 The
strength of these electrostatic interactions may be moderated by increasing the
concentration of added salt, which contributes to a screening effect by the small ions.
The screening effect actually is primarily the result of the interaction between the small
7 ions in the solution and charged chains.
Polyelectrolyte has many applications, majority of which are related to provide
surface charge to neutral particles leading to well-dispersion in aqueous solution. Due to specific properties, polyelectrolytes are often used as emulsifiers, conditioners,
Thickening agent, dispersant, suspension stabilizer, adhesive, soil conditioner, paper strengthening agent, fabric antistatic agent and so on.
2
Scheme 1 The structure of PDAC, SPS, PAA and PEI
1.2 Polyelectrolyte complex and complex coacervation
When oppositely charged polymers mixed together, these two kinds of polymer
will interact with each other to strongly associate or form complex (PEC), in solution.10
The polyelectrolyte complexes phenomenon was discovered and illustrated by
Bungenberg de Jong in the research of the phase behavior of water-soluble polymers
(weakly positively charged) and Arabic gum (weakly negatively charged).11 The PEC
formation is very fast and predominantly controlled by the diffusion of counterions.12
This process is entropy-driven, because the counterions are released and will not be
constrained by the polymer backbone chain any longer.12 During this process,
depending on ratio of charges, a dense phase with rich polymer separates from the dilute solution phase with poor polymer (aqueous phase), which is complex coacervation. The dense liquid phase is coacervate which is more viscous and concentrated than initial solution and has many other abundant unique properties. The physics of such charge
3 complexation is leading to new routes for the self-assembly of interesting macromolecular structures. In order to differentiate the complex coacervation from the simple coacervation of a single polymer, Bungenberg de Jong et al. named it “complex
11 13,14 coacervation”. The Scheme 2 describes the process of coacervate formation.
Scheme 2 This scheme describes the process of formation of coacervate. Mixture of
polycation and polyanion solution leads to a liquid-liquid phase separation and the polymer-rich phase appearing as droplet is complex coacervate
1.2.1 Applications of coacervation
Because of its unique properties, coacervation has a lot of practical applications.
According to the report form Xu Y et al., complex coacervation is effectively applied
for protein purification by selecting target protein without adverse effect on the protein activity.15 Another one kind of uses of coacervates is the environmentally friendly isolation of several compounds, which was reviewed via Melnyk A et al.16 In
biomaterials area, coacervation can also be applied in drug delivery. During the process of coacervation, the drug can be capsuled in droplets. Afterwards, the environmental conditions, including pH, temperature, ionic strength and charge density, alters and the flavor compounds can be released.17 What’s more, coacervates have some promising
18 applications in food industry.
4 1.2.2 Factors influence PEC coacervation
Polyelectrolyte complex coacervation is influenced by many factors. For
polymer properties, molecular weight, total polymer concentration and molecular
geometry; for environmental condition, ionic strength, temperature, mixing ratio and
14 pH are all factors influencing the coacervation of oppositely charged polyelectrolytes.
In this thesis, the effect of small molecules (dyes) on coacevation of PDAC and
SPS is studied. Some of the dyes are ionic in the aqueous solution, so we studied if the change of ionic strength significantly influences the coacervtaion of PDAC and SPS.
As reported by others, for the SPS/PDAC coacervation system in the present of salt, if the ionic strength increases, the screening effect will take place. The small counterions interact with polyelectrolyte chains and screen the PECs leading to the decrease of absolute value of zeta potential and precipitation. If the salt concentration is high enough, the small counterions can penetrate the PECs and continue screen resulting in the redissolution of PECs.19,20 Fig. 1 shows the diagram describing the boundary
between the stable colloidal dispersion and unstable aggregating precipitate as well as
the boundary between unstable aggregating precipitate and dissolved polymer chains
21 in aqueous solution.
5
Figure 1 Diagram of PDAC–PSS behaviour as a function of salt concentration21
Reprinted from Softer Matter, Copyright (2018), with permission from Royal Society
of Chemistry.
1.3 Turbidity measurement
During the process of complexation, three different phases are present, including
clear solution, coacervate and precipitate. Turbidity measurement has been widely
applied to determine the phase state of the mixed polycation and polyanion solution. At the beginning of the process, mixture leads to soluble complex without phase separation.
Only a single phase is present and the turbidity is constant. As the mixing ratio and many other conditions change, the value of turbidity will increase indicating a liquid- liquid phase separation (coacervation). When the value of the turbidity starts to decrease significantly, a kind of liquid-solid separation happens.
6 1.4 Zeta potential
Amount of significant properties of colloidal systems are depended on the particle charge. The potential distribution and charge determine the energy of interaction between particles. To investigate the particle charge and intermolecular electrostatic interactions in stable polyelectrolyte colloidal systems, a direct detection of the surface potential need to be attached, but in many practical circumstance it is difficult to
12 obtain.
Zeta potential is a kind of electrokinetic potential at the surface of the shear
between the fixed and moveable part of the double layer. Zeta potential measurement
provides us a strategy to illustrate the picture of charge and potential distribution across the entire interfacial region. Through measuring zeta potential, the particle charge and the ion distribution surrounding the particle can be analyzed. Scheme 322 demonstrated
the distribution of ions surrounding a charged particle in the solution.22
Scheme 3 Electrostatic phenomenon in a solution for a charged particle. Graphical description of the Zeta potential.22 Reprinted from Constr Build Mater, Copyright (2018), with permission from Elsevier. 7
1.5 UV-VIS Spectroscopy
UV-VIS spectroscopy is a kind of absorption spectroscopy and also one of the
classic physical methods for analyzing the structure of compounds. It has been widely
used to characterize the properties of polyelectrolytes, especially to determine the
23 concentration of the polyelectrolytes solution.
If the condition is assumed as monochromatic light and infinite dilution, which is
ideal condition, the intensity of transmitted light is affected by a change in the thickness of the absorbing material. Lanbert-Beer law gives an equation demonstrating the
23 relationship among them.
At a fixed length of the absorbing cell:
log(I /I)λ= λcl 0 ε c: The concentration of the absorbing species (moles/liter)
I0: The initial intensity of transmitted light through the column of the material
λ: The molar extinction coefficient (liters/ mole )
ε ∙ cm l : The cell length in centimeters
For polyelectrolytes, UV-VIS spectroscopy is of particular interest with respect to polyelectrolytes with chromophores groups, polyelectrolytes covalently labeled with
23 chromophores, and reaction between polyelectrolytes and chromophores.
8 The measurements can determine the concentration of polyelectrolytes in solution, characterize the conformation of polyelectrolytes as well as the changes of the
23 conformation and characterize the reaction between small molecules (dyes) and it.
For instance, as reported in previous research, the ability of sequestration of dyes
into pcomplex coacervates was studied by UV-VIS measurement. During the process of complex coacervation, small molecules, such as Methylene blue (MB),
Bromothymol Blue (BtB), rhodamine B (RhB) and acid red 1(AR1), in the solution are sequested into coacervates, because there may be electrostatic interaction, π-π staking and cation-π interaction between the small molecules and polyelectrolytes.24 After
centrifugation, the small molecules not sequestrated in the coacervate droplets are
leaved in the supernatant. The concentration of small molecules in supernantant can be obtained from the calculation with the data in UV-VIS spectroscopy by Lanbert-Beer equation. At the same time, the sequestration ratio of the small molecules is available.
Besides, the reaction between small molecules and polyelectrolytes can be
characterized via UV-VIS Spectroscopy. As is shown in the figure S1, for MB aqueous
solution without any polyelectrolyte, the λmax is at around 664 nm. However, when it comes to MB aqueous solution in the present of poly(sodium 4-styrene sulfonate) (SPS), the λmax shows a shift from 664 nm to 671 nm. This shift is a red shift representing a
change in absorbance to a longer wavelength which is equivalent to the lower photon
energy. This phenomenon characterizes the formation of π-π interaction between MB
25 and SPS.
26 1.6 Isothermal titration calorimetry
Isothermal titration calorimetry (ITC) is now commonly used to directly
9 characterize the thermodynamics of binding interactions between polymers and
compounds. In our works, ITC was applied to study the intermolecular interaction
between polyelectrolytes and small molecules.
There are two of significant questions of studying this intermolecular interaction.
The first one of them is how tightly does a small molecule bind to a specific interaction site of the polyelectrolyte and the second question is that how fast does the intereaction take place.
It is acknowledged that every physical change and chemical reaction is
accompanied with an enthalpy change. Here, an isothermal titration calorimeter (ITC)
is a measurement that detecting the changes in heat or enthalpy of the reaction. An
endothermic process and an exothermic process means taking up heat and giving up heat from the environment respectively. During these processes, the heat is equivalent to the enthalpy change in the reaction as well as the amount of reaction occurred. At the same time, the rate at which heat is exchanged with the surroundings is equal to the rate of the reaction and again the enthalpy change. In this circumstance, the amount of reaction happening and also the rate of reaction in the process can be obtained, which helps us deal with the two important questions mentioned above and the isothermal titration calorimeter is therefore an ideal instrument.
Calorimetric measurements can be taken by three different kinds of ways.
According to these three ways, the commercial instruments are divided into three types
as well. They are temperature change instruments, power compensation instruments,
and heat conduction instruments.
For power compensation instruments, the sample cell and reference cell is isothermal, which means the measurement are taken at a constant temperature. The 10 temperature controller, heater and reaction work together contributing to a constant
temperature environment. As the chemical reaction inputs heat into the cell, to cool
down the temperature, the temperature controller will control the heater and reduce the
providing of heat to accomplish the temperature constant. Therefore, the heating power mainly comes from two sources, including the reaction heat input and the controlled heater. The reaction heat input is compensated by the reduction of the heat input from the controlled heater, which remains at a constant level. Scheme 4. describe the major
26,27 features and mechanism of a typical power compensation ITC.
27 Scheme 4 Representative diagram of a typical power compensation ITC.
11 Major features of this type of instrument such as the reference and sample cells, syringe for adding titrant, and the adiabatic shield are noted in the figure. This diagram shows an oversimplification of how the power applied by the instrument to maintain constant temperature between the reference and sample cells is measured resulting in the instrument signal.
12
28 CHAPTER II
Reproduced from Huang, Shuyue, et al. "Effect of small molecules on the phase behavior and
coacervation of aqueous solutions of poly (diallyldimethylammonium chloride) and poly (sodium 4- styrene sulfonate)."Journal of colloid and interface science518 (2018): 216-224.
1. Introduction
It has been long known that the electrostatic interaction of oppositely charged
polyelectrolytes can lead to the formation of soluble complexes, or to phase separation
including liquid-liquid (coacervation) and liquid-solid (precipitation) separation, depending on factors such as charge density, ionic strength and polymer molecular weight.6,29,30,31 When complex coacervation occurs, a dense polymer-rich phase
(coacervate phase) and a very dilute polymer-deficient phase (aqueous phase), which exist in equilibrium, are formed.31,32,33 The coacervation process is generally considered to be entropically driven by the release of small counter ions, producing dispersed polyelectrolyte-rich liquid coacervate droplets.31,32,34 Complex coacervation has been
applied in various fields, including pharmaceutical delivery and food industries as
microencapsulates for drugs and flavors.35,17 For example, chitosan-based coacervates have been investigated for the delivery of proteins and vaccines, which provides insight for the development of coacervates for delivery of protein biologics and vaccines.36
Kohane et al. developed gelatin-gum Arabic complex coacervates for the encapsulation and thermally sensitive controlled release of flavor compounds, to improve the appeal
66 of frozen baked foods upon heating.
13 As the coacervate phase has low surface tension with water, it is capable of easily
partitioning solutes within it based on relative affinities between the solute and the
polyelectrolytes and water. For example, our previous study shows that complex
coacervates formed with oppositely charged polyelectrolytes have the ability to
efficiently sequester a cationic dye, methylene blue (MB), through both electrostatic
and p-p interactions.24 Another study on hydrogen-bonding complex coacervates
reveals that with the specific hydrogen bonding interaction between the solutes and
polymer, the uptake efficiency can be significantly increased. Additionally, by adjusting the hydrophobicity within coacervate droplets, the partition coefficient of solutes can be tuned.37 These preliminary studies may give insight into better designing polymeric
coacervate-based materials for drug delivery or personal care products.
It has been widely reported that the phase behavior of polyelectrolyte aqueous
solutions is highly dependent on many factors, i.e., molecular weight, polymer
concentration, stoichiometry of the two interacting polyelectrolytes, ionic strength,
charge density, as well as the temperature of the solution.38,6,39 For instance, it was
found that upon shortening the chain length of polyelectrolytes, or when there is a
significant deviation of the stoichiometric polycation/ polyanion ratio, the stability of
the complex coacervate phase was decreased.38 It has been reported that increases in
salt concentration can lead to transitions from precipitates to coacervates and finally to
polyelectrolyte solutions.38,40 The precipitate coacervate transition is regarded as the
result of successive replacement of ion pairs between oppositely charged
polyelectrolytes. Compared to the precipitate-coacervate transition, the
coacervatesolution transition occurs at relatively high salt concentrations, where
intermolecular electrostatic interactions have been significantly screened. However, there are few studies on how the sequestered solutes within coacervates would in turn
14 impact the phase behavior and coacervation of aqueous polyelectrolyte systems.
This study provides a more complete understanding of phase behavior and
complex coacervation of synthetic, strong polyelectrolytes in the presence of small
molecules, using poly(diallyldimethylammonium chloride) (PDAC) as the polycation
and poly(sodium 4-styrene sulfonate) (SPS) as the polyanion. The small molecules
partitioned into the coacervate in this work are an aromatic cationic water-soluble dye
(methylene blue or MB) as well as an aromatic non-ionic dye (bromothymol blue or
BtB) into the PDAC-SPS coacervates was studied. These various molecules can be seen in Scheme 1. The influence of the small molecules on the phase boundaries was examined and explained with a number of techniques, including spectrofluorimetry isothermal titration calorimetry (ITC) and rheology to examine the interaction between the dyes and polyelectrolytes, as well as the influence of these dyes on the physical properties of the coacervate. Although works studying how to encapsulate small molecules into coacervates are represented in the literature, there are few if any reports showing how the presence of these small molecules impact phase boundaries or other transitions such as precipitation and rheological properties of the coacervates themselves.
2. Experimental section
2.1. Materials
Poly(diallyldimethylammonium chloride) (PDAC, Mn=16000; PDI=1:4), SPS
( Mn=13000, PDI=1:6), and MB, BtB and ANS dyes were purchased from Sigma-
Aldrich. The molecular weight and polydispersity of PDAC and SPS were measured
by aqueous gel permeation chromatography (GPC) using polyethylene oxide standards.
Both the polycation PDAC and the polyanion SPS are strong polyelectrolytes and are 15 insensitive to pH. All water was dispensed from a Milli-Q water system at a resistivity
of 18.2 MX cm. All these materials were used as received without further purification.
Scheme 5 Chemical Structures of PDAC, SPS, MB and BtB.
2. Turbidity measurement
Turbidity was used to qualitatively measure the extent of coacervate formation as
a function of PDAC/SPS stoichiometry (PDAC/ SPS) at different total polyelectrolyte
concentration, with the addition of various amounts of MB or BtB. The detailed
information on the concentration of PDAC and SPS stock solutions with various
concentrations of added MB or BtB, namely PDAC-MB and SPSMB, or PDAC-BtB and SPS-BtB stock solutions, were listed in Tables 1 and 2. Turbidity measurements for the titration with the addition of MB were performed using a 2 cm path length fiber- optic colorimeter (Brinkmann PC 950) at a wavelength of 420 nm. Turbidity measurements for the titration with the addition of BtB were performed on an Agilent
8453 UV–vis Spectrophotometer at a wavelength of 600 nm, to avoid the absorbance of BtB at 420 nm. Turbidity was reported as 100 – T%, where T corresponds to the
16 transmittance. Titration of PDAC-dye stock solutions (1, 5, 10, 20, or 40 mM) into SPS- dye stock solutions (1, 5, 10, 20, or 40 mM) with matching polyelectrolyte and dye concentrations was done with stirring. The transmittance (T) was recorded at 60 s after each PDAC addition. All of the above polyelectrolyte concentrations are repeat unit concentrations.
Table 1 Detail information on the concentration of PDAC-MB and SPS-MB stock solutions for the turbidity measurement
PDAC-MB SPS-MB Stock Solution MB PDAC MB SPS 1 1 5 5 Concentration 0.1 10 0.1 10 (mM) 20 20 40 40 1 1 5 5 Concentration 0.25 10 0.25 10 (mM) 20 20 40 40 1 1 5 5 Concentration 0.5 10 0.5 10 (mM) 20 20 40 40
17 1 1 5 5 Concentration 1.0 10 1.0 10 (mM) 20 20 40 40
Table 2 Detail information on the concentration of PDAC-BtB and SPS-BtB stock solutions for the turbidity measurement.
PDAC-BtB SPS-BtB Stock Solution BtB PDAC BtB SPS
1 1 5 5 Concentration 0.1 10 0.1 10 (mM) 20 20
40 40
1 1
5 5 Concentration 0.25 10 0.25 10 (mM) 20 20 40 40
2.3. Zeta potential measurement
Zeta potential measurements were used as a way to approximate charge of the
coacervate particles in solution. Stock solutions of PDAC (10 mM) and SPS (10 mM)
with various concentrations of added MB or BtB (see Table 3) were prepared separately.
MB or BtB encapsulated PDAC-SPS coacervates were formed simply by mixing the dye added PDAC and SPS stock solutions with matching dye concentrations at a mixing ratio of PDAC:SPS = 0.5, keeping the total concentration of PDAC and SPS at 10 mM.
Stock solutions of PDAC (10 mM) and SPS (10 mM) without dye addition were prepared separately as well. Dye-free PDAC-SPS coacervate samples were prepared by mixing the PDAC and SPS stock solutions at various mixing ratios (PDAC:SPS = 0.067,
0.1, 0.2 and 0.5). The apparent zeta potentials of the as-prepared PDAC-SPS coacervates with various added dye concentrations or various PDAC:SPS molar ratios without dye addition were then estimated using a Brookhaven Instruments Zeta PALS 18 electrophoretic light scattering instrument (where the Smoluchowski model was used
to obtain apparent zeta potential values from the measured electrophoretic mobilities).
Table 3 Concentration of MB and BtB in the PDAC-SPS system for the zeta potential measurement.
Stock Solution MB BtB 0 0 0.01 0.01 Comcentration 0.02 0.02 (mM) 0.05 0.05 0.1 0.1
2.4. Determining partition coefficient of dyes into coacervates
Partition coefficients show the efficiency of a particular coacervate system to
encapsulate a dye. Stock solutions of PDAC (10 mM) and SPS (10 mM) were made
with the addition of various concentrations of MB or BtB, respectively (see Table 4).
PDAC-SPS coacervates with sequestered dyes were prepared by mixing the PDAC and
SPS stock solutions with a mixing ratio of PDAC: SPS = 0.5, to study the dye partition
coefficients as functions of dye concentration. Further, to examine how these partition
coefficients depend on the PDAC:SPS mixing ratio, stock solutions of PDAC (10 mM)
and SPS (10 mM) containing 0.01 mM MB were prepared. These solutions were mixed in various ratios to prepare additional batches of PDAC-SPS coacervates with sequestered MB and variable PDAC:SPS mixing ratios (PDAC:SPS = 0.067, 0.1, 0.2 and 0.5). After 24 h stirring, samples were centrifuged for 3 h at 8000 rpm (Allegra X-
30R Centrifuge, Beckman Coulter). After centrifugation, the supernatant was removed
using a micropipette, and the coacervate phase was left in the bottom of centrifuge tubes.
UV–vis measurements (Agilent 8453 spectrophotometer) were used to determine the dye concentration in the supernatant and to then infer it in the coacervate. The partition coefficient (K) was calculated as Eq. (1).
19
[ ] (1) [ ] 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝑖𝑖𝑛𝑛 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝐾𝐾 = 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝑖𝑖𝑖𝑖 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆
Table 4 Concentration of MB and BtB in the PDAC-SPS system for the partition coefficient experiment.
Stock Solution MB BtB 0.01 0.01 0.2 0.02 Concentration 0.25 0.05 (mM) 0.3 0.075 0.5 0.1
2.5. Fluorescence spectroscopy
Fluorescence spectrometry was used to measure polarity of the microenvironment within the coacervate. Steady state emission spectra of ANS within PDAC-SPS coacervate microdroplets with or without accumulated MB in coacervate were measured using a Horiba FluoroMax 4 spectrofluorometer with an excitation wavelength of 350 nm. Fluorescence emission was measured from 365 to 650 nm in quartz cuvettes for microdroplet dispersions prepared with 10 mM PDAC and 10 mM
SPS with a mixing ratio of PDAC:SPS = 0.5, 0.5 mM of added ANS and different (0,
0.1, 0.5 and 1 mM) concentrations of MB. All fluorescence was attributed to ANS within the microdroplets as ANS fluorescence was quenched in water.
2.6. Rheological measurements
20 The coacervate samples were prepared by mixing PDAC (5 mM) and SPS (5 mM) at a PDAC:SPS stoichiometry of 0.75 and various MB or BtB concentrations (see Table
5), and collected by centrifuging the mixtures at 8000 rpm for 3 h (Allegra X-30R
Centrifuge, Beckman Coulter). The dynamic rheological properties of the collected coacervates were characterized using a TA Instruments ARES-G2 rheometer in parallel plate geometry using an 8.00 mm diameter aluminium upper plate and 43.9 mm diameter aluminium lower plate along with a solvent trap. For dynamic rheological experiments, a constant strain amplitude of 0.4%, which was within the linear viscoelastic response region for all the collected coacervate samples, was used. The frequency was swept from 0.1 to 100 rad/s for these dynamic measurements.
Table 5Concentration of MB and BtB in the PDAC-SPS system for the preparation of coacervate samples for rheological test.
Stock Solution MB BtB 0 0 0.1 Comcentration 0.2 0.1 (mM) 0.4 0.2 0.5
2.7. Isothermal titration calorimetry (ITC)
ITC experiments were performed using a VP-ITC microcalorimeter (GE
Healthcare, USA). Each titration experiment involved 27 injections (10 lL) of 2.5 mM polyelectrolyte (either PDAC or SPS) solution at 5 min intervals into the sample cell containing 0.25 mM dye (MB or BtB) solution. Both the polyelectrolyte and dye solutions had the same pH of 3.00±0.02. The reference cell was filled with DI water, while the experimental temperature and stirring rate were fixed at 25 and 310 rpm, respectively. The heat of dilution, which was obtained through a control℃ experiment
21 where the PDAC or SPS were titrated into dye-free water at a matching pH, was later subtracted from the titration data to generate the final thermogram that reflected the enthalpic signature of the polyelectrolyte-dye binding.
2.8. Optical microscopy
An optical microscope (Olympus DP80) was used to obtain images of coacervate droplets. The coacervate mixture was centrifuged and then placed on a glass slide to image the droplets. Coacervate and precipitate samples were prepared by adding
10 mM PDAC solution into 10 mM SPS solution with a ratio of 1:4 and 1:1, respectively. Samples used for imaging were centrifuged for 15 min at 8000 rpm using a centrifuge (Allegra X-30R Centrifuge, Beckman Coulter) to achieve rapid sedimentation. After centrifugation, the supernatant was carefully removed by using a micro-pipette while the dense coacervate phase was left at the bottom of centrifuge tube, and transferred on to the glass slide to obtain optical microscope images. Similarly, optical microscope images of precipitate were obtained as well.
2.9. 1H NMR
1H NMR spectroscopy (Mercury 300 spectrometer) was used to measure the
PDAC:SPS ratios in the polyelectrolyte complex coacervates as follows: PDAC-SPS coacervate samples were prepared by mixing the PDAC (10 mM) and SPS (10 mM) stock solutions with various mixing ratios (PDAC:SPS = 0.067, 0.1, 0.2 and 0.5), and collected by centrifuging the mixtures at 8000 rpm for 3 h (Allegra X-30R Centrifuge,
Beckman Coulter). The collected coacervate samples were dried at 60 to remove water completely. The dried coacervate samples were then dissolved in ℃D2O with the addition of 2.5 M KBr.
22
3. Results and discussion
3.1. Solute partitioning into PDAC-SPS coacervates
The partitioning of the two dyes MB and BtB into PDAC-SPS coacervates was studied using UV–vis spectroscopy. Fig. 2 shows the partition coefficients of MB and
BtB into PDAC-SPS coacervates as a function of solute concentration, at a fixed mixing ratio of PDAC:SPS = 0.5. The partitioning of MB into the PDAC-SPS complex coacervate is significantly higher than that for BtB. This efficient uptake of MB likely
24 reflects the strong p-p interactions between MB and SPS as previously reported.
The UV–vis spectra of pure MB, MB with SPS, pure BtB and BtB with SPS
aqueous solution at pH 3.0 are shown in Fig. S1. The red shift in the UV–vis maximum absorbance wavelength (λmax) of MB in the aqueous solution with SPS indicates p-p
interaction between MB and SPS. However, there is no shift in λmax of BtB in the
aqueous solution with SPS, which suggests that there is no p-p interaction between BtB and SPS. In addition, as the concentration of MB increases from 0.01 to 0.5 mM, the partition coefficient of MB into the PDAC-SPS complex coacervate increases from approximately 120 to 210; even though the apparent zeta potential (which reflects the negative coacervate charge) is reduced from approximately -36 to -5 mV as the MB concentration in the system varies from 0 to 0.1 mM (Fig. 3). This trend indicates that the p-p stacking and possibly hydrophobic interactions are the main factors that contribute to the sequestration of MB, rather than electrostatic interactions. However,
PDAC-SPS coacervates show a reduced sequestration capacity for BtB compared to
23 that of MB, but there is also a trend of BtB partition coefficient increasing with BtB
concentration in the system. Specifically, the BtB partition coefficient into PDACSPS
coacervate increases from around 15 to 45, as the overall concentration of BtB varies
from 0.01 to 0.1 mM. This comparatively low sequestration of BtB into PDAC-SPS coacervate suggests that the affinity of BtB for the coacervate is not as strong as that of
MB. The partition coefficient representing MB concentration in the PDAC-SPS coacervate compared to its concentration in the water rich phase was also studied as a function of PDAC:SPS molar ratio (Fig. S3). As the PDAC:SPS mixing ratio increases from 0.067 to 0.5, the partition coefficient of MB decreases from approximately 139 to
117. The decrease in the MB partition coefficient of MB with the increase in
PDAC:SPS mixing ratio can be ascribed to the decreasing SPS content in the coacervate phase.
The composition of the as-prepared PDAC-SPS coacervates was determined using
1H NMR, as shown in Fig. S4. As the overall PDAC:SPS molar ratio in the system was increased from 0.067 to 0.5, the PDAC:SPS molar ratio in the coacervate phase increases from approximately 0.41 to 0.5 only, which does not vary as significantly as the overall molar ratio in the system. The zeta potential of the prepared PDAC-SPS coacervates with various overall PDAC:SPS ratio was measured as well (Fig. S5). The absolute zeta potential values of the PDAC-SPS coacervates decrease slightly as the overall PDAC:SPS molar ratio increases with a large range from 0.067 to 0.5, which corresponds to the slight variation of the coacervate composition obtained from 1H
NMR measurement.
24
Figure 2 Partition coefficient of (a) MB and (b) BtB into PDAC-SPS coacervates as a
function of various concentration of MB or BtB. PDAC-SPS coacervates were prepared using PDAC (10 mM) and SPS (10 mM) stock solution with a mixing ratio of
PDAC:SPS=0.5.
Figure S1 UV-vis spectra of MB, MB-SPS, BtB and BtB-SPS aqueous solution at pH
3.0. The concentration of SPS is 20 mM. The concentration of dye is 0.01 mM.
25
Figure S2 UV-vis spectra of 0.01 × 10−3 M BtB at different pH, and 0.01 × 10−3 M
BtB in 20 × 10−3 M PDAC aqueous solution at pH 3.0.
Figure S3 Partition coefficient of MB into PDAC-SPS coacervates as a function of molar ratio of PDAC:SPS in the system. PDAC-SPS coacervates were prepared using
PDAC (10 mM) and SPS (10 mM) stock solution with various mixing ratio
(PDAC:SPS=0.067, 0.1, 0.2 and 0.5) at a fixed MB overall concentration of 0.01 mM.
26
Figure S4 PDAC:SPS molar ratio in the coacervate phase (PDAC/SPScoac) as a
function of overall PDAC:SPS mixing molar ratio (PDAC/SPSoverall) in the system,
measured using 1H NMR. PDAC-SPS coacervates were prepared using PDAC (10 mM) and SPS (10 mM) stock solution with various mixing ratio (PDAC:SPS=0.067, 0.1, 0.2 and 0.5).
27
Figure S5 Zeta potential of the PDAC-SPS coacervates as a function of PDAC:SPS
molar ratio in the system. PDAC-SPS coacervates were prepared using PDAC (10 mM)
and SPS (10 mM) stock solution with various mixing ratio (PDAC:SPS=0.067, 0.1, 0.2 and 0.5).
3.2. Effect of solutes on zeta potential of PDAC-SPS coacervates
The apparent zeta potentials of the PDAC-SPS coacervates prepared with the
addition of various concentrations of MB or BtB were measured, as shown in Fig. 3.
An increase in MB concentration in the system results in a remarkable change in the
zeta potential value of the PDAC-SPS coacervates, varying from approximately -36 to
-5 mV as the concentration of MB increases from 0 to 0.1 mM. However, for the SPS-
PDAC coacervates with the addition of BtB, the variation in zeta potential with the addition of BtB is much slighter than that of MB, changing from -36 to -25 mV only.
These results are reasonable since the sequestered MB is positively charged while BtB is nonionic at the studied pH. Therefore, the dramatic decrease in the absolute zeta
28 potential value of PDAC-SPS coacervates with the addition of MB is because of its
positive charge. Additionally, even though the apparent zeta potential, which reflects
the negative charge of the coacervate, is reduced from approximately -36 to -5 mV as
the overall concentration in the system increases from 0 to 0.1 mM, the partition
coefficient of MB into PDAC-SPS coacervates still keeps increasing. This trend
indicates that the p-p stacking and possibly hydrophobic interactions are the main
factors that contribute to the sequestration of MB, rather than electrostatic interactions,
since the coacervate with low negative charge still uptake MB with a high efficiency.
The UV–vis spectra of BtB in aqueous solution at different pH values as well as
that for BtB in PDAC aqueous solution at pH = 3.0 are shown in Fig. S2. BtB acts as a
weak acid in aqueous solution (pKa = ~7.1),41 which can thus be in protonated or
deprotonated form, appearing yellow or blue respectively. The UV–vis peak at 433 nm is attributed to the protonated form of BtB, while the peak at 617 nm is assigned to the deprotonated form of BtB. However, since at the experimental pH value (pH = 3) BtB is nonionic in both pure aqueous solution and PDAC solution, it is unclear what the cause of the change in absolute zeta potential value of PDAC-SPS coacervate is as the concentration of BtB in the system increases. To better explain this phenomenon, ITC experiments were carried out to better understand the interaction between the dyes and polyelectrolytes used in this study.
29
Figure 3 Zeta potential of PDAC-SPS coacervates with the addition various
concentration of MB or BtB PDAC-SPS coacervates were prepared using PDAC (10 mM) and SPS (10 mM) stock solution with a mixing ratio of "PDAC:SPS=0.5" .
3.3. Isothermal titration calorimetry (ITC)
ITC was employed to study the intermolecular interaction of MB and BtB with
PDAC and SPS. ITC is able to show whether an association process occurs between
two molecules and allows for estimation of the intermolecular interaction strength
between the dyes and polyelectrolytes. Fig. 4 shows the ITC titration curves for the
titration of the polyelectrolyte (PDAC or SPS) solutions into dye (MB or BtB) solutions.
The ordinate axis represents the enthalpy changes due to the polyelectrolyte injection, and the abscissa is the corresponding composition of the system (polyelectrolyte monomer:dye ratio). Based on these results, there is no enthalpy change for the titration of PDAC into MB and SPS into BtB solution, indicating that there is no specific intermolecular interaction between MB and PDAC or BtB and SPS, or the interaction
30 is too weak to be detected by ITC. However, both the titration of SPS into MB and
PDAC into BtB shows a significant exothermic signal, which is an indication of a
substantial polyelectrolyte-dye interaction. In both dye-polymer systems, saturation
(where the exothermic binding heat diminishes to baseline levels) occurs near the 1:1 polyelectrolyte:dye ratio, suggesting that the binding sites for the MB and BtB dye molecules do not exceed one polyelectrolyte monomer unit in size. The titration of
PDAC into BtB has a less negative profile than that of SPS into MB, suggesting that
the interaction between MB and SPS is much stronger than that for BtB and PDAC.
However, as entropy plays a large role in the interactions here, it is important to also
note that this greater interaction strength is also supported by a more abrupt saturation transition,42 which is indicative of stronger binding and is consistent with the much
higher partition coefficient of MB into PDAC-SPS coacervate than that of BtB.
It is a little surprising to see such a strong association between PDAC and BtB
since at pH 3.0, BtB is non-ionized, as indicated in Fig. S2. It was reported that PDAC
is capable of forming cation-p interactions with aromatic molecules such as reduced
grapheme oxide, charged and uncharged lignin.43,44 Additionally, a careful observation
of the titration curve of SPS into MB (where the enthalpic signal increases in magnitude
up to the stoichiometric point) reveals that binding of MB to SPS occurs in two steps.
It has been reported that in aqueous solution, MB undergoes exothermic dimerization
and trimerization at MB concentration higher than 0.1 mM,45,46 with similar orders of
magnitude as the dye concentration in the ITC experiment. Addition of SPS into MB
aqueous solution may therefore result in destruction of the MB dimer and trimer, with
47 the formation of SPS-MB associations via p-p stacking and electrostatic interaction.
The breakup of dimer and trimer at low SPS:MB ratio could absorb heat and therefore
reduce the net exothermic signal.46 At higher SPS:MB ratios, the concentration of MB
31 that is unbound to SPS becomes lower, which corresponds to fewer MB aggregates in
solution. Thus, fewer MB aggregates likely break up at high SPS:MB ratios, and with
less heat absorbed by this unstacking process, the net exothermic signal increases.
Figure 4 ITC data for the titration of 2.5 mM PDAC or SPS into 0.25 mM MB or BtB
solution.
3.4. Effect of accumulated MB on the hydrophobicity within PDAC-SPS
coacervate droplets Generally, the partition coefficient of solutes into coacervates
decreases as the solution concentration in the system increases. For example, it was
reported that the encapsulation efficiency of BSA protein into polypeptide coacervates
decreases as the ratio of BSA to polypeptide increases.48 However, in our PDAC-SPS coacervate system with MB, the partition coefficient of MB was significantly increased as the concentration of MB in the system increases, which is unusual at first sight. An assumption that arises is that the accumulated MB within PDAC-SPS coacervates is capable of influencing the chemical environment of the coacervate and therefore further
32 uptake the MB molecules in aqueous solution into PDAC-SPS coacervate droplets,
resulting in such a high sequestration efficiency even at high MB concentrations.
To study how the accumulated MB can influence the environment within the
coacervate, fluorescence spectra of ANS sequestered within the PDAC-SPS coacervate
droplets with the presence of different concentration of MB in the system were
measured, as shown in Fig. 5. The photo-physical properties of are highly sensitive to
polarity and viscosity of the environment. As shown in Fig. 5, the fluorescence spectra
of ANS sequestered within PDACSPS coacervates with the addition of different
concentration of MB showed a broad emission band between 365 and 650 nm, which
indicates a combination of different micro-environments of varying polarities within
coacervate droplets. Deconvolution of the fluorescence spectra by Gaussian fitting
provides two specific emission peaks associated with two distinct microenvironments
of the coacervates. The peak located at ~470 nm was assigned to the nonpolar excited
state localized on the naphthalene ring of ANS, which represents a more hydrophobic
environment, which the other peak at ~530 nm was consistent with the emission from
the charge transfer state of ANS, which corresponds to a more hydrophilic environment.
As previously mentioned, the partition coefficient of MB into PDAC-SPS coacervates increases with the increasing concentration of MB. It is therefore no doubt that the sequestered amount of MB within the coacervates also increases with the overall MB concentration. As shown in Fig. 5, as the MB concentration increases from 0 to 1.0 mM, the relative intensity of emission peak at around 470 nm increases gradually, while the intensity of emission peak at approximately 530 nm attributed to the hydrophilicity or polarity of the environment within coacervate decreases to almost zero, indicating that the accumulated MB within coacervate droplets would increase the hydrophobicity of coacervate significantly. The enhanced hydrophobicity within PDAC-SPS coacervates
33 induced by the accumulated MB would in turn enable the coacervates to uptake more
MB, leading to a significantly increased MB partition coefficient with the increase of
MB concentration.
Figure 5 Fluorescence emission spectra of ANS sequestered in PDAC-SPS coacervate dispersions with the addition of (a) 0 mM, (b) 0.1 mM, (c) 0.75 mM and (d) 1.0 mM
MB recorded at room temperature. The coacervates were prepared with 10 mM
PDAC and 10 mM SPS at a mixing ratio of PDAC:SPS = 0.5, with the addition of 0.5 mM ANS.
3.5. Effect of solutes on PDAC-SPS phase behavior
34 The phase behavior of PDAC-SPS in the presence of added dye, with respect to
the overall dye concentration, PDAC/SPS mixing ratio, and the total polyelectrolyte
concentration, is illustrated and compared in a series of phase diagrams depicted in Figs.
5 and S6. Fig. S7 shows typical optical micrographs of precipitates and coacervates coexisting, or possibly aggregates of coacervate drops, prepared using PDAC and SPS aqueous solution. There are several characteristic features of the phase behavior of the
PDACSPS system with the addition of MB or BtB.
First of all, for all PDAC-SPS concentrations studied here, the single-phase
solution regime narrows with increasing MB or BtB concentration, indicating that the
presence of MB or BtB slightly favors the coacervation process. Additionally, the
broadening in the precipitation regime with increasing MB or BtB concentration
suggests that the presence of MB or BtB strongly favors precipitation. The increase in
ionic strength induced by the positively charged MB should not be the main factor that
impacts the phase behavior of the PDAC-SPS system, since the addition of 1 mM NaCl into the polyelectrolyte system has almost no impact on the phase diagram, as shown in Fig. S6. Increased hydrophobicity of coacervates with the addition of dyes is suggested as the main reason for the acceleration of coacervation. It has been reported that an increase in the hydrophobicity of polyelectrolytes increases the tendency towards coacervation despite the reduction in charge interactions between the polyelectrolytes.49 Additionally, the tendency towards precipitation was also enhanced as the concentration of MB or BtB in the system increases. There are multiple reasons for the increase in the tendency towards precipitation. Firstly, an increase in the MB or
BtB concentration of the system leads to a lower absolute value of zeta potential of the coacervates, resulting in weaker electrostatic repulsion between coacervates, which
might accelerate the precipitation. Secondly, the increased hydrophobicity of the
35 coacervates by the addition of dyes might also contribute to the enhancement of
tendency towards precipitation. Second, the total polyelectrolyte concentration has an
impact on the phase behavior of PDAC-SPS system as well. An increase in coacervate
formation as reflected by turbidity with increasing total polymer concentration was
observed for all MB and BtB concentrations. One example of the influence of
polyelectrolyte total concentration on the turbidity of PDAC-SPS system is shown in
Fig. S8. Additionally, a higher total polyelectrolyte concentration promotes coacervation and precipitation, as shown in Fig. S6. Mixtures with higher polymer concentrations have a higher number of oppositely charged sites available to interact, thus the coacervation and precipitation tend to occur earlier at a smaller PDAC to SPS ratio.
36
Figure 6 Phase behavior of PDAC-SPS aqueous system as a function of polyelectrolyte stoichiometry, total polymer concentration, and phase behavior of PDAC-SPS aqueous system as a function of overall concentration of dye (b) MB or (c) BtB and polyelectrolyte stoichiometry. Blue (●), red (●) and black (●) symbols represent clear solution, coacervation and precipitation, respectively.
37
Figure S6 Phase behavior of PDAC-SPS system without the addition of (a) salt or dye, and with the addition of (b) 1 mM NaCl, (c) 0.1 mM MB, (d) 0.25 mM MB, (e) 0.5 mM
MB, (f) 1 mM MB, (g) 0.1 mM BtB, and (h) 0.25 mM BtB. Blue, red and black symbols
38 represent clear solution, coacervation and precipitation, respectively. The grey dashed lines represent the PDAC:SPS stoichiometry of 1.
Figure S7 Optical micrographs of (a) precipitates and (b) coacervates formed using 10
× 10−3 M PDAC and 10 × 10−3 M SPS with a ratio of 4:5 and 1:2, respectively. The scale bar in these two optical micrographs is 50 µm.
39 Figure S8 Turbidity profile of PDAC-SPS system obtained using the same
concentration of PDAC and SPS aqueous solution (1, 5 and 10 mM), respectively,
without the addition of dyes. The transmittance (T) was recorded at 60 s after each
PDAC addition.
3.6. Effect of MB and BtB on water content of coacervates
Experimentally, the water content of ternary coacervate is determined simply by
weighing the complex coacervate before and after drying. Fig. S9 shows the water
content of PDAC-SPS coacervates as a function of concentration of MB or BtB in the
system. In general, an increase in the concentration of MB or BtB results in a decrease
in water content of the formed PDAC-SPS coacervates. To be specific, the water content of PDAC-SPS complex coacervate is reduced from approximately 86.9% to
64.6% as the concentration of MB increases from 0 to 0.5 mM. Similarly, the water content of PDAC-SPS coacervates is reduced from 86.9% to 76.0% with an increase in the concentration of BtB from 0 to 0.2 mM. This can be considered an indirect proof that the hydrophobicity of PDAC-SPS coacervates with the sequestered BtB might also be increased as the concentration of BtB increases.
40
Figure S9 Water content of PDAC-SPS coacervates formed using prepared by mixing
PDAC (5 mM) and SPS (5 mM) with or without the addition of various concentration of MB or BtB, at a stoichiometry of PDAC ∶ SPS=0.75 as a function of dye concentration.
3.7. Effect of solutes on the rheological properties of complex
Coacervates It has been reported that the rheological properties of polyelectrolyte
complexes and coacervates are influenced by various factors, including salt
concentration, polyelectrolyte stoichiometry and pH.4,3,50 Dynamic rheological
measurements were used to characterize the coacervate materials, as shown in Figs. 7
and 8. The frequency sweep was performed at a constant strain of 0.4%, which was
found to be in the linear regime. The viscoelastic behavior is highly dependent on the
addition of MB or BtB.
41 The presence of MB within the complex coacervates has a strong influence on the
rheological properties of PDAC-SPS coacervates. The frequency sweep data shows a decrease in the overall magnitude of the storage (G’) and loss (G”) moduli with increasing MB concentration (Fig. 7a), which is similar to the salt effect on the rheological properties of coacervates, although enhanced given the low overall concentration of dye.51,52,53 Furthermore, for the PDAC-SPS coacervate samples
prepared at high MB concentrations (0.4 and 0.5 mM), a frequency-dependent crossover between the storage and loss moduli can be observed, while at lower MB concentrations, the storage modulus was dominant over the entire frequency range, indicating the coacervate samples prepared at low MB concentrations are more solid-
like. For the coacervate samples prepared at high MB concentrations, at low frequencies the loss modulus dominates over the storage modulus, indicative of a viscoelastic liquid-like behavior, while at higher frequencies, a crossover in the two moduli was observed, followed by a region where solid-like behavior dominates. Additionally, as shown in Fig. 8a, tan( of PDAC-SPS coacervates formed at various concentration of
MB all exhibit a decreaseδ) with increasing frequency. At low MB concentrations (0, 0.1 and 0.2 mM) tan( is much lower than 1, indicative of solid-like feature of the coacervate. However,δ) for PDAC-SPS coacervates prepared at high MB concentrations
(0.4 and 0.5 mM), tan( is higher than 1, indicating a viscoelastic liquid-like behavior, while at high frequenciesδ) the tan( is lower than 1, with solid-like behavior dominating.
Plotting tan( data as a functionδ) of MB concentration instead of the frequency can help with visualizationδ) and elucidation of the relation between the crossover frequency and
MB concentration (Fig. 9). The crossover frequency increases as the concentration of
MB increases. The inverse of the crossover frequency (where tan( = 1) can be related to the longest relaxation time for the polymers in the sample.52 Here,δ) an observation of
42 increasing crossover frequency for increasing MB concentration corresponds to a
decrease in the time needed for polymer molecules to disentangle and relax. As
mentioned before, an increase in the MB concentration results in a decrease in water
content of the formed PDAC-SPS coacervates. If water is to be considered as a plasticizer for polyelectrolyte complexes,54,55,21 a lower water content should lead to a higher coacervate modulus. However, in this study, as the concentration of MB increases, though the water content is reduced, the overall magnitude of the storage (G’) and loss (G”) moduli decrease with increasing MB concentration. This unusual behavior indicates that the MB encapsulated within PDAC-SPS coacervates tends to efficiently impair the ionic bond between PDAC and SPS. The wt% of MB molecules in the PDAC-SPS coacervates is quite low, varying from 0.03% to 2.38% as the overall
MB concentration in the system increases from 0.01 to 0.5 mM, which indicates that the displacement of ionic bond pairs between the oppositely charged polyelectrolytes by the positively charged MB is limited. In addition to the displacement of ionic bond pairs by MB, the formation of ionic bond pairs between PDAC and SPS is further sterically hindered by the MB molecules which associates with SPS through both ionic and p-p interaction.
Similarly, with the increase in concentration of BtB in the coacervate, though the
water content of the PDAC-SPS coacervate decreases, there is still a decrease in the
overall magnitude of the storage (G’) and loss (G”) moduli and a slight increase in the
tan( ) (Figs.7b and 8b), indicating that the presence of BtB within PDAC-SPS coacervatesδ reduces the ionic crosslink density of the PDAC-SPS material, because of
the cation-p interaction between PDAC and BtB. The cation-p interaction is
electrostatic in nature, which can be conceptualized as the interaction of a positively
charged ion with the negative electrostatic potential surface of the p electrons above
43 and below the aromatic structure.56,57 Therefore, as with the MB case, the cation-π interaction between PDAC and BtB leads to a weaker interconnecting network of the
PDAC-SPS coacervates as the concentration of BtB increases.
44
Figure 7 Frequency sweeps showing storage (G', solid) and loss (G'', open) modulus of
PDAC-SPS coacervates formed by mixing 5 mM PDAC stock solution and 5 mM SPS stock solution at a stoichiometry of with the addition of (a) various concentration of MB, and (b) PDACvarious ∶concentration SPS = 0.75 of BtB.
45
Figure 8 Loss tangent (tan (δ)=G^''/G' of the PDAC-SPS coacervates formed with various concentration of MB or BtB.
46
Figure 9 Plot of loss tangent (tan(δ)=G^''/G' of the PDAC-SPS coacervates as a function of MB concentrations.
4. Conclusion
It has been widely reported that coacervates are capable of encapsulation of
various solutes, including small organic molecules, proteins, and
nanoparticles.24,37,48,58,59 Previous studies reveal that phase behavior of complex
coacervation is dependent on various factors, such as ionic strength, polymer molecular weight, pH, temperature, as well as the polymer concentration.6,38 However, there are
few studies on how the sequestered solutes within coacervates would in turn impact the phase behavior and coacervation of aqueous polyelectrolyte systems.
Presented here is a study of the sequestration of the small molecules MB and BtB
into PDAC-SPS complex coacervate, as well as the influence of the sequestered solutes on the phase behavior and properties of the coacervate. The absolute apparent zeta
47 potential values of the PDAC-SPS coacervates decrease as the concentration of MB or
BtB in the system increases. Hydrophobicity within the PDAC-SPS coacervate droplets can be increased simply by increasing the sequestered MB content within the complex coacervate. The phase behavior of PDAC-SPS system is highly dependent on the concentration of dyes as well as the total polyelectrolyte concentration. An increase in the MB or BtB concentration of the system tends to shift the window over which coacervation and precipitation occur to a wider range of stoichiometric ratios of oppositely charged polyelectrolyte. Additionally, for mixtures with higher total polyelectrolyte concentration, coacervation and precipitation tend to occur earlier at a smaller PDAC to SPS ratio. The accumulation of MB or BtB within PDAC-SPS coacervate phase has a strong influence on the rheological properties of the coacervates.
As the concentration of MB or BtB increases, there is a decrease in the overall magnitude of the storage (G’) and loss (G”) moduli and an increase in the tanedT, indicating that the presence of these two small molecules within PDAC-SPS coacervates can reduce the ionic bonds between PDAC and SPS. In conclusion, this work studies the encapsulation of solutes into coacervates as well as the influence of the sequestered solutes on the phase behavior of the polyelectrolyte solution, hydrophobicity within coacervate droplets, as well as the rheological properties of the coacervates, which may provide some guidance for design of solute-encapsulated coacervates with desired properties.
48
CHAPTER III
1. Introduction
When oppositely charged polymers mixed together in solution, these two kinds of
polymers will interact with each other to strongly associate or form Polyelectrolyte
complex (PEC).60 During this process, depending on ratio of charges, a dense polymer-
rich phase (coacervate) separates from the dilute solution phase (aqueous phase).30 The dense liquid phase is coacervate, which has abundant properties being used in practical applications, including industrial flocculants, coatings, drug delivery, food processing
17,24,48,35 and membranes.
One of the properties is that the liquid-liquid phase separation can sequestrate
small molecules into the coacervate. This phenomenon draws a lot of attention because of its promising use. Compartmentalization of the chemicals into coacervate leads to the creation of a specific environment for the chemicals, providing interactions such as electrostatic interaction, hydrogen bonding or hydrophobicity which might play an important part in chemical reaction rate, yield, as well as product selectivity. Besides, the sequestration of chemicals into the coacervate contributes to high concentration of reactants, which might also influence the chemical reaction process. Furthermore, these coacervate acts as nanoreactors having the microenvironments mimicking the protocell architecture and help us attain a better understanding of how protocell processes are
61,62 executed.
49 In this work, we explored the partitioning of some dyes in complex coacervates of
branched polyethylenimine (BPEI) and Poly(sodium 4-styrenesulfonate) (SPS).
Partition coefficient is the ratio of concentrations of a compound in a mixture of two
immiscible phases at equilibrium. This ratio is a measure of the difference in solubility
of the compound in these two phases and generally described as the concentration ratio of un-ionized species of compound.63 Here, the partition coefficients of three different
kinds of dyes into BPEI-SPS coacervates were obtained to study the uptake ability of
BPEI-SPS coacervate. UV-vis spectroscopy was employed to analyze the π – π
interactions between dyes and polyelectrolytes. Besides, Isothermal titration
calorimetry measurement was taken to further study the intermolecular interactions
between dyes and polyelectrolytes (BPEI or SPS).
2. Experimental section
2.1. Materials
Branched polyethylene imine (BPEI, weight-average molecular weight, Mw =
25000), Poly(sodium 4-styrenesulfonate) (PSS, Mw= 75000) were purchased from
Sigma-Aldrich. All the small molecules, including methylene blue (MB), rhodamine B
(RhB) and bromothymol blue (BtB) were purchased from Sigma-Aldrich. All water was dispensed from a Milli-Q water system at a resistivity of 18.2 MΩ cm. All these materials were used as received without further purification.
2.3. Preparation of Complex Coacervate droplets
Stock BPEI solutions of 40 mM and PSS solutions of 40 mM, were prepared at
pH = 3.5, 5.5 and 7.0. Complex coacervate droplets dispersions were prepared by
50 mixing stock BPEI stock solution with stock PSS solution together at different
BPEI/PSS mixing ratios in to opposite mixing order, under ultrasonic dispersion.
Polyelectrolyte concentration and ratio were with respect to functional group. To
prepare the complex coacervates containing different dyes, a certain volume of dye
stock solutions was added in the polyelectrolyte stock solutions to give a concentration of 0.01-0.5 mM for dyes, before mixing polyelectrolyte solutions.
2.4. UV-VIS measurement
The partition coefficients for solutes into BPEI-SPS coacervates were determined after stirring the coacervate solution for 24 hours. Dyes partition coefficient measurements were undertaken by separating the droplets from the aqueous suspensions by centrifugation at 8000 rpm for 2.5 h to produce a bulk coacervate phase
(Allegra X-30R, Beckman Coulter). UV−vis measurement (Agilent 8453) was used to determine either the dye content of the supernatant or the dye content of the coacervate via dissolution of the coacervate phase. The partition coefficient was calculated by
K=[solute in polymer rich phase]/[ solute in water rich phase].
2.5 Isothermal Titration Calorimetry (ITC)
Isothermal titration calorimetry experiments were performed on a Nano ITC
standard volume (TA Instruments). Each titration experiment involved 25 injections
(10 μL) of 2.5 mM polyelectrolyte (either SPS or BPEI) at 350s intervals into the sample cell (volume = 1 mL) containing 0.25 mM dye (MB，BtB or Rhb) solution.
Both titrant and titrate had same pH value of 3.5 . The reference cell was filled
with DI water; the experimental temperature and±0.02 stirring rate was fixed on 25 °C and
51 250 rpm, respectively. The heat of dilution which was obtained through a cotrol
eaperiment where the SPS or BPEI were titrated into dye-free water at a matching pH,
was later subtracted from the titration data to enerate the final thermogram that reflected the enthalpic signature of the polyelectrolyte-dye binding.
3. Discussion and result
3.1 Partition coefficient of dyes into complex coacervate
The ability of sequestrating small molecules into the BPEI-PSS coacervates was
studied by partition coefficient. Three different kinds of dyes including methylene blue
(MB, a cationic dye), rhodamine B (RhB, a zwitterionic dye), and bromothymol blue
(BtB, a weak acid dye) were used as the small molecules in this system. The partition coefficients (K) were determined at pH 3.5 and summarized in Table 6. to illustrate the
relative uptake ability of coacervate for these dyes.
BPEI:PSS K mixing ratio MB RhB BtB 2:8 7.5 23.5 39.7 3:7 12.3 28.4 50.7 4:6 25.8 49.0 57.0 5:5-6:4 - - - 7:3 3.5 157.3 378.1 8:2 2.8 135.5 444.2 9:1 3.3 102.2 619.9 Table 6The partition coefficients for MB, RhB and BtB into BPEI-SPS coacervate. pH = 3.5.The coacervate solution was prepared with 40mM BPEI and 40mM SPS stock solution in present of 0.5mM dyes. When the mixing ratio of BPEI/SPS was 2/8, 3/7 and 4/6, the mixing order was BPEI to SPS. At other mixing ratios, the mixing order was opposite.
52 As is shown in the Table 6, BPEI-SPS coacervates are able to sequestrate these
three kinds of dyes. At the same mixing ratio, KBtB>KRhB>KMB indicates that the uptake ability for BtB is better than that of RhB and MB. The sequestration of MB into BPEI-
SPS coacervate is weakest. As a cationic dye, MB is positively charged in the aqueous solution at pH=3.5, the electrostatic repulsion between it and the positively charged coacervate with excess BPEI leads to lower K values. While, at BPEI/PSS mixing ratios below 1, where the coacervate conformed with excess PSS is negatively charged, π–π interaction between MB and PSS, which is mentioned in the former chapter, drives stronger sequestration of MB. For BtB, based on the result in the Chapter II, when pH is lower than 5, there is no specific intermolecular interaction between BtB and SPS.
Therefore, the reaction between BPEI and BtB may be the dominant drive force of the sequestration. In Fig.10c, the slight red shift in the absorption peak of BtB is observed in the solution with the polycation BPEI indicating that there may be hydrogen bonding interaction between the sultone group of BtB, as an accepter, and amine/imine groups of BPEI, as a donor. This hydrogen bonding may be the main reason why BtB partitions into the coacervate significantly, especially under the excess BPEI circumstance that
BPEI/PSS mixing ratios are above 6/4. Between RhB and polyelectrolytes, the planar cationic xanthene ring of RhB enables the π–π interaction with PSS as well, which can be demonstrated by the red shift in the UV-vis absorption peak of RhB in the solution with PSS (Fig. 10b). The π–π interaction with PSS may be the major reason for the partitioning of RhB into BPEI-SPS coacervates. However, K is getting higher as the
BPEI/PSS mixing ratios are above 6/4, which indicates that there is another important factor resulting in the sequestration behavior.
53
Figure 10 UV-vis spectra of dyes (a, MB; b, RhB; c, BtB) at pH 3.5 and dyes in BPEI
or SPS aqueous solution
3.3. Isothermal titration calorimetry (ITC)
To further study the intermolecular interaction between dyes and polyelectrolytes,
ITC was employed. Fig. 11 shows the ITC titration curves for the titration of the polyelectrolyte (BPEI or SPS) solutions into dye (MB, BtB or RhB) solutions. As reported in former chapter, there is no enthalpy change for the titration of SPS into BtB illustrating that there is no specific interaction between SPS and BtB, or the intermolecular interaction is not strong enough to be detected. However, the other three titrations of SPS into MB, BPEI into BtB, SPS into RhB and BPEI into RhB show a
54 significant exothermic signal indicating a substantial polyelectrolyte-dye interaction in the binding strength order that SPS/MB>BPEI/BtB>SPS/RhB>BPEI/RhB>BPEI/MB.
The titration of SPS into MB has a strongest negative profile among these four titrations, suggesting that the interaction between MB and SPS is much stronger than that of other polyelectrolyte-dye interactions. While, the interaction strength between MB and BPEI is weakest, which means the π-π interaction as well as the electrostatic interaction are the main drive force portioning MB into coacervate. For RhB, its pKa is around 3.0,64
so this dye is not ionized at pH 3.5 in the solution. Besides, there is no shift for the absorption peak of RhB in the solution with the polycation BPEI. However, between
RhB and BPEI, there is a strong association, which may be caused by cation-π
interaction. The binding strength of SPS/MB is much stronger than that of BPEI/RhB
and SPS/RhB, but in Table 6, Obviously, RhB is sequestrated into the coacervate with higher K values than MB. The reason may be that there is also hydrophobic interaction between RhB and coacervate droplets. RhB has lower water solubility (ca. 15 mg/mL) than MB (ca. 35.5 mg/mL), so more RhB molecules are hydroponically stabilized within the coacervate. Significant enthalpy change indicates there is a strong interaction between BPEI and BtB and that might be hydrogen bonding interaction between the sultone group of BtB, as an accepter, and amine/imine groups of BPEI, as a donor.
Hydrogen bonding may be the main reason why BtB partitions into the coacervate significantly.
55
Figure 11 ITC data for the titration of 2.5 mm BPEI or SPS into 0.25 mm dyes (MB,
RhB or BtB) respectively.
56
FUTURE WORK
To further study the sequestration of those three dyes into BPEI/SPS coacervate and the reasons for the uptake ability, fluorescence measurements should be taken to study if there is hydrophobic interaction between dyes and coacervate droplets. BPEI-SPS coacervation has uptake ability for magnetite (Fe3O4) nanoparticles as well due to the
interplay of solubility, hydrophobic and electrostatic factors. In the future research, we
mean to study the sequestration of Fe3O4 nanoparticles and analyse the drive force of
the sequestration. As is reported brfore, the environment condition factors, including
PH and ionic strength of solution, which are closely related to the coacervation process, are taken into consideration. Their effects on the uptake ability of small molecules and nanoparticles into coacervates should be investigated next.
57
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