Professional Documents
Culture Documents
6.1 Introduction
Novel and smart biomateriali; can be made through inter and intra-molecular
complexation of bio-polyelectrolytes. Coacervates are a new class of polymeric
materials formed via electrostatic interactions between complementary_polyelectrolytes
or a polyelectrolyte and colloid pair [1-6]. Such materials have found applications in
protein purification [7], drug and enzyme immobilization [8] and in trapping organic
plumes [9]. A variety of coacervates have been made in the past by suitably tuning and
enhancing the electrostatic interactions between pairs of polyions. In these transitions,
the screened electrostatic interactions existing between oppositely charged
polyelectrolytes yield soluble intermolecular complexes that precede coacervation.
During such a process, the homogeneous solution undergoes a liquid-liquid phase
separation with the polymer-rich phase compartmentalizing itself into a dense state
withdrawn from the bulk of the solvent, called the supernatant that co-exists
thermodynamically with the coacervate. The solvent present in the concentrated phase
largely constitutes the solvation liquid. The supernatant is a very dilute polyelectrolyte
solution.
This phenomenon has been conceptualized in various models. In Veis model [10] the
aggregates are referred to as symmetrical aggregate polymer (SAP) whereas Tainaka's
revised model [11] refers to these as asymmetrically aggregated polymers (AAP). In
Nakajima-Sato model [12] the solute-solvent interactions was incorporated in to the
earlier proposed Overbeek-Voorn model [13] through Flory-Huggins prescriptions.
Regardless, it is accepted that the coacervate phase owes its origin to the formation of
intermolecular soluble aggregates. Tainaka model [11] assumes Gaussian distribution
112
of segments in AAP aggregates independent of their size and that all the counter-ions
are bound' to the AAP aggregates. It should be realized that when two oppositely
charged segments join together, some amount of counter ion is always released in to the
solvent, there by increasing the entropy of the solution. This will assist the process to
move towards coacervation. A more rigorous model describing coacervation [14] and
its conceptualization in the free-energy landscape formalism was proposed recently
[15].
Thermal experiments were carried out using a Labsys TG-DSCI6 differential scanning
calorimeter instrument manufactured by Setaram, France. Samples (typically =50 mg)
were heated in aluminum cups sealed with lids at a controlled rate of 1°C/min up to a
maximum temperature of 100°C starting from room temperature (20°C).
113
6.2 Results and Discussions
(6.1)
Where, ~ defines is the correlation length of the concentration fluctuations or mesh size
of the network and Ir (0) is related to the cross-link density and longitudinal osmotic
modulus. Experiments carried out in the semi dilute regime of polymer solutions have
shown deviations from the Ornstein-Zernike function.
Long wavelength concentration fluctuations in these systems often gives rise to excess
scattering in the low-q region of the SANS data. The origin of these fluctuations
remains debatable, and a clear cause that generates such excess scattering is yet to be
established. However, Koberstein et al. [28] have suggested long-range random
inhomogeneities with correlation length many times larger than the radius of gyration
114
of the dissolved polymer to cause this excess scattering. If the spatial scale of density
fluctuations due to the presence of inhomogeneities is large compared to the correlation
length ~, then the two contributions can be treated separately and added to give the total
structure factor as
Where SL(q) is the Ornstein-Zernike (O-Z) function, and the Debye-Bueche (D-B)
S ( ) - lexCO)
ex q - Cl+q2f)2
(6.3)
where IexCO) is the extrapolated structure factor at zero wave vector. It is possible to
Study low-q domain of the structure factor provided a high instrumental resolution
SANS spectrometers is used to collect data. We have examined neutron scattering data
1 15
10
0 0.01 M
0 0.02M
b. 0.05M
<> 0.1 M
,.....,
m0.1
0-
0.01
0.001
0.01 0.1
q I(A Or 1
Figure 6.1: Plot of static structure factor, S( q) versus scattering wave' vector, q for
agar-gelatin coacervates prepared in D20 detennined from SANS measurements
perfonned at 20°e. Notice the invariance of scattering profiles with ionic strength of
the coacervate samples.
A least-squares fit of the structure factor data in the low-q range, 0.018 kl nm- I :s q :s
0.072 kl (see Figure 6.2), to the D-B function yields size of the heterogeneities
(s=220± 20 A) present inside the coacervate phase. Secondly, the measured correlation
length (Fig. 2) detennined by fitting high-q region data to O-Z function gave, S =12±2
A much smaller than the persistence length of gelatin, which is ~ 25 A-I. For gelatin'
coacervates, gels and sols the correlation length values reported [21] are 12 A, 26 A and
26 A respectively (see Table-I)_ Size of heterogeneities reported for such systems were
~ 200 A which is comparable to the size of the heterogeneities, s=220 ± 30 A reported
now. Thus, it can be inferred that the simple and complex coacervates of gelatin bear
identical structural signatures. On the other hand, agar gels are associated with
contrastingly different length scales; the correlation length and heterogeneity size are
59 A and 700 A respectively [29]. These values remained unchanged as the
116
concentration of CaCh added to these gels was varied from 0.01 to 1M. The agar-
gelatin samples did not exhibit any such dependence either.
,'~- .
o O.01M
O.02M
O.05M
O.1M
-1
q2/(AO r1
Figure 6.2: Plot of reciprocal of static structure factor, l/S(q) versus scattering wave
vector, q for agar-gelatin coacervates prepared in D20 determined from SANS
measurements; data is from Figure 1. Low-q data fitted to Debye-Bueche and high-q
fitted to Ornstein-Zernike function. This defines a clear q-cut off.
storage modulus (G') with temperature (T) gave unambiguous values for two melting
transition temperatures and the data are shown in Figure 6.3 for various salt
concentrations. Let us compare these temperatures with the thermal properties of agar
[30] and gelatin gels [31]. Agar has a gelation temperature ~ 40°C and melting
temperature ~ 85°C. On the other hand, gelatin gels at ~ 30°C. The data shown in
Figure 6.3, thus, represents a linear combination of the thermal behaviour of agar and
117
gelatin gels. One can also argue that such behaviour owes it origin to the presence of
gel-like (physical) network structures inside the coacervate phase. Agar and gelatin gels
are known to comprise of hydrogen bond stabilized double [32] and triple helix [33]
units that constitute the three dimensional interconnected network. The gelatin gels are
rubbery and since, these molecules are rich in glycine, proline and hydroxyproline
residues, inside the triple helix, the individual molecules feature in poly(L-proline II)
(trans) helix conformation in contrast, agar gels are composed of micro-domains of
polymer-rich spinodal phases that imparts it a non-rubbery structure [18]. The shear
induced flow behaviour of coacervate samples were quite revealing. The shear rate
dependent viscosity data of these samples is plotted in Figure 6.4 which reveals the
non-Newtonian yield.
C X
I- 0.10 0.8 I-
l! "'0
:::--.
(!) 0.6 (!)
"'0 0.05 "'0
0.4
0.2
0.00
0.0
>
10 20 30 40 50 60 70 80 90
TO/C
Figure 6.3: Plot of the first derivative of isochronal storage modulus in a temperature
sweep experiment. Notice the near invariance of transition temperatures with ionic
strength of the samples. It appears that the strong associative interactions present inside
the dense medium are not effected by screening.
I 18
3r-----------------------~~~~~,
o O.01M
k o O.02M
11=110 (dy/dtr
Ll O.05M
2 <) O.1M
k=1.2
- - fitting
-1
-2 -1 o 1 2
(dy/dt)/s-1
Figure 6.4: Plot of viscosity versus shear rate for samples prepared with different ionic
strengths. The Carreau model provided excellent fitting to the data within experimental
error. For clarity fitting is shown for one sample only having salt concentration
=0.05M. The exponent, k changed by less than 10% as the salt concentration increased
from 0.01 to O.1M.
In addition shear thinning behaviour was exhibited by these samples. The data
presented in Figure 6.4 were fitted to Carreau model expression [34]
(6.4)
with k = 1.2±0.2 independent of ionic strength. In fact, k reveals the viscous response
of the samples to applied shear: k=O gives Newtonian, k<O indicates shear-thickening
and k>O implies shear-thinning behaviour. Thus, shear-thinning features are clearly
manifested in these samples. It is interesting to note that alike the SANS results, the
rheology data did not exhibit any ionic strength dependence. Alcohol induced simple
coacervates of gelatin formed due to self-charge neutralization has been shown to
exhibit non-Newtonian behaviour in the past [21). Thus, the agar-gelatin coacervate has
rheological features not very different from that of gelatin coacervate indicating that the
119
Carreau model can be universally applied to describe such material. In contrast, the gels
of agar and gelatin have been shown to exhibit viscoelastic response (see Table-I).
120
for SANS experiments). The values listed are representative. The transition
temperatures are associated with an uncertainty of ±30C and the same with
characteristic sizes is ±15% of the listed data, typically.
3: ref[21], b: ref[31], C: ref [29], d: ref [30] and e: ref[41], f: ref [36], g: ref [35]
121
2
~ -2
<-
3:
0
LL -4
.....
co
Q)
I -6
-8
-10
0 20 40 60 80 100 120
TPC
Figure 6.5: DSC thennograms for agar-gelatin coacervates prepared in water. The rate
of heating was 1DC Imin. The initial concentration of each of these biopolymers was
0.1 % w/v. Notice absence of any endothenn at the higher end of temperature. A single
endothenn was located close to 30DC invariant of ionic strength of the coacervate
samples.
6.3 Conclusions
Agar-gelatin coacervate samples were probed by three techniques in order to detennine
the micro-structure of this phase. The experimental results reveal that the coacervate
phase is a heterogeneous viscous material. The polymer-rich phase comprises
physically crosslinked networks of agar and gelatin molecules. There is finite
possibility of existence of asymmetric double helices of agar coexisting with triple
helices of gelatin. The presence of inter-penetrating networks of these biopolymers can
not be claimed with certainty at this stage. However, it will be appropriate to make a
physical comparison between agar-gelatin complex coacervate with agar, and gelatin
gels, and gelatin coacervates.Such a comparison has been provided in Table-I. The
agar-gelatin coacervate has been found to retain the thennal properties of its
122
constituents to a remarkable extent. This indicates the presence of physica11y entangled
networks of agar and gelatin in the coacervate phase. The viscoelastic response of this
phase to external stress is dependent on the specific nature and density of these
cross links. Thus, detennination of the degree of helicity present in the coacervate phase
wi11 be of significant importance. Coacervates, like gels, are biphasic in nature
comprising the solvated polymer and solvent in various structural fonns. Thus, the
exact detennination of the amount of interstitial and free water present in this material
is required to be evaluated which wi11 provide a better understanding of the thennal and
temporal stability of this material.
123
6.4 References
[1] H.G. Bungenberg de Jong; In Colloid Science; Vol-IT; Ed.; Kruyt, H. R.,Elsevier,
New York, 1949.
[2] K.Kaibara, T.Okazaki, H.B. Bohidar and P.L.Dubin; Biomacromolecules, 1,100,
2000.
[3] H.B. Bohidar, P.L. Dubin, P. Majhi, C.Tribet and W. Jaeger; Biomacromolecules, 6,
1573,2005.
[4] B. Mohanty and H.B. Bohidar; Biomacromolecules, 4, 1080,2003.
[5] E. Kokufuta; Prog. Polym. Sci, 17, 647, 1992.
[6] A Eisenberg and F.E. Bailey; Coulombic Interactions in Macromolecular Systems,
Ed.; ACS Vol. 302, 1986.
[7] H.L Hinze and D.W.Armstrong; Ordered Media in Chemical Separation, ACS
Vol. 342, 1987.
[8] J. Xia, K.W. Mattison, V. Romano, P.,L. Dubin and B. Muhoberac; Biopolymers,
41,359,1997.
[9] Y.Wang, J. Banziger,G. Filipelli and P.L. Dubin; Environ. Sci. Tech., 35,
2608,2001.
[10] (a) A Veis and J. Cohen; J. Phys. Chem.,78, 6238,1956 (b) AVeis.; J. Phys.
Chem.,65, 1960,1963 (c) A Veis; J. Phys. Chem., 61, 1798,1961 and (d) A. Veis
and C. Aranyi; J. Phys. Chem., 64, 1203,1960.
[11] K.Tainaka; Biopolymers, 19, 1289,1980; J. Physics. Soc. Japan, 46,1899,1979.
[12] ANakajima and H. Sato; Biopolymers, 10, 1345,1972.
[13] J. Overbeek and MJ. Voom; J. Cell. Compo Physiol., 49, supp-l, 7,1957.
[14] AGupta and H.B. Bohidar; Phys. Rev. E.,72, 011507,2005.
[15J AGupta, Reena and H.B. Bohidar; J. Chem. Phys.,125, 054904,2006.
[16] S.S. Singh and H.B. Bohidar; Int. J. BioI. Macromolecules, 41,185,2007.
[17J J.S. Craigie, C. Leigh; in Hellebust and J.S. Craigie, editors Hand Book of
Phycological Methods, Cambridge Univ. Press; Cambridge; 109-131,1978.
[18J E.Pines and W. Prins; Macromolecules, 6,888,1972.
124
[19] J.P.Busnel, E.R. Morris and S.B. Ross-Murphy; Int. J. BioI. Macromol.,
11,119,1989 and J.P.Busnel and S.B. Ross-Murphy; Int. J. BioI. Macromol.,
10,121,1988.
[20] A. Veis; The Macromolecular Chemistry of Gelatin, Academic Press, New York,
1964.
[21] B. Mohanty, V.K. Aswal, 1. Kohlbrecher and H.B. Bohidar; J Plym. Sci: Part B,
44, 1653,2006.
[22] P.S. Goyal, V.K. Aswal and J.V. Joshi; Current Sci., 79, 947,2000.
[23] P.Thiyagaranjan, J.E. Epperson, R.K. Crawford, J.M. Carpenter, T.E. Klippert and
D.G. Wozniak; J Appl. Crstyllogr.,30, 280,1997.
[24] G.L.Squires; Thermal Neutron Scattering, Cambridge University Press:
Cambridge, 1987.
[25] L.A.Feigin and D.I.Svergun; Structure Analysis by Small-Angle Neutron X-Ray
Scattering and Neutron Scattering; Plenum Press: New York, 1987.
[26] P. Debye and A.M. BuchE; J Appl. Phys., 20, 518,1949.
[27] P.G. de Gennes; Scaling Concepts in Polymer Physics, 2nd Ed.; Cornell University
Press: Ithaca, New York, 1985.
[28J1.T. Koberstein, C. Picot and H. Benoit; Polymer,26,673,1985.
[29] Nicolas, Fatin-Rouge, KJ.Wilkinson and J. Buffle; J Phys. Chem. B,110,20133,
2006.
[30] K. Prasad, A.K. Siddhanta, A. K. Rakshit, A. Bhattacharya and P.K. Ghosh; In(l. J
Bioi. Macromolecules, 35,135,2005.
[31] H.B.Bohidar and S.S. Jena; J Chem. Phys., 98, 8970,1993.
[32] M. Djabourov, A.H. Clark, D.W. Rowlands and S.B. Ross-Murphy;
Macromolecules, 22, 180,1989 ..
[33] M. Djabourov, J. Leblond and P. Papon; J Phys. France,49,319,1988 and ibid
49,433.
[34] H.A. Barnes; A handbook of Elementary Rheology, University of Wales Press,
Wales, England, 2000.
[35] M. Watase, A.H. Nishinari, A.K. Clark and S.B. Ross-Murphy; Macromolecules,
22,1196,1989.
125
[36] S. Chattetjee and H.B. Bohidar; Int. 1. Bioi. Macromolecules, 35,81,2005.
[37] N.Toyoda, M. Takenaka, S. Saito and T. Hashimoto; Polymer, 42, 9193,2001.
[38] T.J. Hashimoto; Polymer Sci: Part B: Polymer Phys., 42,3027,2004.
[39] A. Onuki and T. Taniguchi; J Chem Phys., 106,5761,1997. ,<:,
126