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Elsevier Editorial System(tm) for Carbohydrate Polymers Manuscript Draft Manuscript Number: CARBPOL-D-10-00507R1 Title: "Synergistic interaction of Locust Bean Gum and Xanthan investigated by rheology and light scattering" Article Type: Research Paper Keywords: Polysaccharides; Synergistic interactions; Hydrogels; Rheology; Light Scattering; Gel point. Corresponding Author: Dr. Tonmmasina Coviello, Corresponding Author's Institution: University of Rome First Author: Chiara Sandolo, Dr. Order of Authors: Chiara Sandolo, Dr.; Donatella Bulone, Dr.; Maria R. Mangione; Silvia Margheritelli; Chiara Di Meo, Dr.; Franco Alhaique, Prof.; Pietro Matricardi, Dr.; Tonmmasina Coviello Abstract: The use of synergistic interactions between polysaccharides can represent an important approach for the preparation of physical networks with a wide range of industrial applications, such as in the food and biomedical fields. Locust Bean Gum and Xanthan, two biocompatible polymers, are able to form gels when mixed together. A detailed study was carried out on three samples, prepared at different weight ratio and two temperatures, by means of light scattering and rheological techniques. The solid-like character of each sample was evaluated by measurements of the viscoelastic spectrum and intensity autocorrelation function. The contribution of fast and slow fluctuations to the static light scattered intensity was also evaluated at different angles. The power law exponents for the sol-gel transitions were estimated together with the fractal dimensions. The scattering results were found in good agreement with the mechanical ones in revealing a strong dependence of the structural properties of the different samples on their preparation conditions. Indeed, results show that a fine tuning of the properties of the mixed gels is possible through the change of the temperature preparation and/or the polymer weight ratio.
Dear Editor
please find the revised version of the paper entitled “Synergistic interaction of Locust Bean Gum
and Xanthan investigated by rheology and light scattering” by Chiara Sandolo et al., that was
submitted for publication on Carbohydrate Polymers.
We are grateful to the referees for their comments and suggestions that helped us to improve the
manuscript.
Separately, we report the answers to the reviewers. Text variations are written in blue colour.
Looking forward to hear from you for further information, thank you in advance for the attention.
Yours sincerely,
Tommasina Coviello
Rome, May 20, 2010
Cover Letter
Answers to the reviewers (corrections in the text are in blue colour)
Reviewer #1: The manuscript contains an interesting comparison between experimental data which
were obtained with different techniques and converge to similar conclusions about the structural
effects of blending ratio and modes in LBG-xanthan mixtures, and this is not a frequent result. The
paper is well written and clear and does not need corrections. The only observation concerns the
number of figures: Figure 3 seems to be redundant and could be eliminated without any
consequence for the offered explanations and comments.
Fig. 3 is now omitted.
Reviewer #2: General: The abbreviation 'Xanth' for xanthan seems unnecessary. I recommend just
using 'xanthan'.
Xanthan is now used throughout the text.
p.2, l. 61: Tm depends strongly on the ionic strength (45C only under certain conditions), and the
content of pyruvate and acetate. True 'unentangled' random coils exist even well above Tm.
The reviewer is correct: for this reason the “hot samples” were prepared at a temperature (75 °C)
well above Tm in water. The corresponding sentence was changed and implemented.
p. 4. Since Mw of xanthan was obtained by LS, what were the estimates of Rg and A2? Should be
included for the sake of completeness
The Rg and the A2 values are now added in the text.
p. 4. l.113. 'Hot gelation': Were the samples cooled to RT after 15 min at 75C? Cooling rate?
The following explanation was added in the text: The “hot samples”, after heating at 75 °C for 15
minutes, were left to slowly cool down in the switched off thermostatted bath, until room
temperature was reached.
p.3-4: Please specify the temperature used for NMR analysis
The information was added.
p.4. It should be pointed out that the autoclaving of xanthan necessarily leads to a order-disorder
transition, and the reverse upon cooling. Thus, the xanthan is a renatured type rather than the native
type, as these may in fact differ (perfect double-strands are not necessarily formed)
The following explanation was added:
“It must be pointed out that the Xanthan chains undergo an order-disorder transition during
autoclaving and therefore a renaturated state (where not necessarily perfect double-strands are
formed) should be considered in the subsequent interactions with the LBG chains.”
Response to Reviewers
Fig 1: The graphical quality is unsatisfactory (unless this is related to problems reading the pdf file
on my Mac)
Probably the graphical quality depends on Mac (we verified quality on the pdf version).
P. 11, l. 270-270: This sentence sounds a bit unclear to me. Please rephrase/clarify.
The sentence was changed:
“Furthermore, the usual evolution with time of the two moduli (and correspondingly of the
mechanical spectra) observed for a chemical cross-linking reaction, is detected here by changing
the weight ratio between the two polymers (Fig. 1B).”
p. 11, l. 272: 'Random coil' only during heat treatment, presumably renaturated after cooling (and
during rheological measurements). Needs to be stated clearly.
The sentence was changed:
“A quite different situation occurs for the “hot samples”: at 75 °C LBG keeps its random coil
conformation (present at room temperature) but also Xanthan is dispersed as single chains.
Actually, Xanthan is not allowed to renaturate during cooling, because it is implied in the network
formation with the LBG chains.”
1
Synergistic interaction of Locust Bean Gum and Xanthan investigated by 1
rheology and light scattering 2
3
Chiara Sandoloa, Donatella Bulone
b, Maria R. Mangione
b, Silvia Margheritelli
a, Chiara Di Meo
a, 4
Franco Alhaiquea, Pietro Matricardi
a, Tommasina Coviello
a,* 5
6 a
Department of Drug Chemistry and Technologies, Faculty of Pharmacy, “Sapienza”, University 7
of Rome, 00185 Rome, Italy 8 b
Institute of Biophysics at Palermo Italian National Research Council Via Ugo La Malfa, 153 9
90146 Palermo (Italy)
10
11
12
13
* Corresponding author. Tel.: +39 06 49913557; fax: +39 06 49913133. 14
E-mail address: [email protected] (T. Coviello). 15
16
17
18
19
ABSTRACT 20
21 The use of synergistic interactions between polysaccharides can represent an important approach for 22
the preparation of physical networks with a wide range of industrial applications, such as in the 23
food and biomedical fields. Locust Bean Gum and Xanthan, two biocompatible polymers, are able 24
to form gels when mixed together. A detailed study was carried out on three samples, prepared at 25
different weight ratio and two temperatures, by means of light scattering and rheological 26
techniques. The solid-like character of each sample was evaluated by measurements of the 27
viscoelastic spectrum and intensity autocorrelation function. The contribution of fast and slow 28
fluctuations to the static light scattered intensity was also evaluated at different angles. The power 29
law exponents for the sol-gel transitions were estimated together with the fractal dimensions. The 30
scattering results were found in good agreement with the mechanical ones in revealing a strong 31
dependence of the structural properties of the different samples on their preparation conditions. 32
Indeed, results show that a fine tuning of the properties of the mixed gels is possible through the 33
change of the temperature preparation and/or the polymer weight ratio. 34
35
36
37
Keywords: Polysaccharides; Synergistic interactions; Hydrogels; Rheology; Light Scattering; Gel 38
point. 39
*ManuscriptClick here to view linked References
2
1. Introduction 40 41
Binary mixtures of certain polysaccharides often exhibit unexpected synergistic interactions, e.g. 42
the mixture may gel under conditions where the individual components are non-gelling. Striking 43
example of this behaviour occurs when the bacterial polysaccharide Xanthan gum (Scheme 1a) is 44
mixed with some plant galactomannans (Morris, 1990). It is well known that the synergistic 45
gelation occurs by direct binding between the two polymers and not by thermodynamic 46
incompatibility. Furthermore, X-ray diffraction pattern obtained from fibres prepared from the 47
synergistic gel, are new patterns and not the simple sum of the diffraction patterns of the component 48
polysaccharides (Cairns, Miles, Morris, & Brownsey, 1988). In particular, Xanthan and Locust 49
Bean Gum (LBG) (Scheme 1b), when mixed together, give a network whose strength depends on 50
the temperature preparation and on the weight ratio between the two components. A wide debate is 51
still open in elucidating the actual mechanism leading to the gel formation and, during the past 52
decades, several models have been proposed (Lundin, & Hermansson, 1995; Richter, Boyko, 53
Matzker, & Schröter, 2004; Richter, Brand, & Berger, 2005; Wang, Y. J., Wang, Z., & Sun, 2002; 54
Zhan, Ridout, Brownsey, & Morris, 1993). It is acquired that the presence of the acetyl substituents 55
on the Xanthan chains (Wang Y. J., Wang Z., & Sun, 2002; Ojinnaka, Brownsey, Morris, E. R., & 56
Morris, V. J., 1998) as well as the branching degree of the galactomannan are crucial for gel 57
formation (an increase of the number of galactose units hinders gel formation (Dea, Clark, & 58
McCleary,1986) ). 59
The possibility to modulate the mechanical properties of the gel by varying the relative amount of 60
the two polymers and taking also into account the different conformation assumed by the Xanthan 61
chains in distilled water at 25 °C (described in terms of a double-helix ordered structure) and at T > 62
45 °C (random coil conformation) (Coviello, Kajiwara, Burchard, Dentini, & Crescenzi, 1986, 63
Hacche, Washington, & Brant, 1987) appears to be particularly interesting. The temperature at 64
which the helix-coil transition occurs, Tm, deeply depends on several factors (ionic strength, content 65
of pyruvate and acetate minor components) and therefore for the “hot samples” preparation a rather 66
3
high temperature, T=75 °C, was adopted. In the present work a comparison among three samples 67
prepared at different weight ratio and two temperatures is reported: rheological characterization and 68
light scattering techniques were applied. Morphological features of the samples, acquired by means 69
of SEM spectroscopy, are also given. 70
The importance of a study on this binary mixture is also supported by the fact that these polymers 71
(both individually and mixed together) are already widely used in food industry (Dolz, Hernández, 72
Delegido, Alfaro, & Muňoz, 2007; Mandala, Kapetanakou, & Kostaropoulos, 2008; Pedersen,1980; 73
Sahin, & Ozdemir, 2004; Ramírez, Barrera, Morales, & Vázquez, 2002; Secouard, Malhiac, Grisel, 74
& Decroix, 2003). Furthermore, it must be pointed out that the single polymers are already used as 75
excipients in tablet formulations, and the LBG/Xanthan gels have been proposed in pharmaceutical 76
applications for slow release purposes (Baichwal, 2004; Fadden, Kulkarni, & Sorg, 2004; Mannion, 77
Melia, Mitchell, Harding, & Green, 1991; Rolf, 2003; Sugden, K. 1989; Ughini, Andreazza, Ganter, 78
& Bresolin, 2004; Venkataraju, Gowda, Rajesh, & Shivakumar, 2008;). The biocompatibility of the 79
two polysaccharides, together with their peculiar synergic behaviour, can still offer new interesting 80
applications. In this sense, even more significant appears to be any attempt to elucidate the 81
molecular structure and the spectroscopic characteristics of these mixed gels in order to improve 82
and to better modulate their potential uses. 83
84
2. Materials and Methods 85
2.1. Materials 86
Locust Bean Gum (LBG), from Ceratonia Campestris, was provided by Carbomer (USA). The 87
ratio between Mannose and Galactose, was estimated by means of 1H NMR (carried out at 70 °C 88
with a Brucker AVANCE AQS 600 spectrometer, operating at 600.13 MHz) and an M/G value of 89
3.4 was found. The molecular weight (Mw) of LBG, 5.0x105, was estimated from the usual Zimm 90
plot analysis of static light scattering measurements carried out at 25 °C in 0.1 M NaCl aqueous 91
4
solutions of sterilized LBG. The estimated radius of gyration was Rg = 102 nm and the second virial 92
coefficient A2 = -3.21 x 10-4
cm3mol/g
2. Procedure details are given in the literature (Coviello, 93
Kajiwara, Burchard, Dentini, & Crescenzi, 1986). 94
Xanthan Gum, from Xanthomonas campestris, was provided by Fluka (Italy). For each repeating 95
unit, 1.6 acetate and 2.7 pyruvate groups were estimated by 1H NMR measurements (carried out at 96
85 °C with a Brucker AVANCE AQS 600 spectrometer, operating at 600.13 MHz). The molecular 97
weight (Mw) of Xanthan, 1.25x106, was obtained by means of static light scattering measurements, 98
carried out at 25 °C in 0.1 M NaCl aqueous solutions of sterilized Xanthan, and reported according 99
to the classical Zimm plot representation. The estimated radius of gyration was Rg = 140 nm and the 100
second virial coefficient A2 = +6.09 x 10-4
cm3mol/g
2. 101
The two polysaccharides were used after sterilization, centrifugation and purification. A given 102
amount of Xanthan, sodium salt, (polymer concentration, cp = 0.5% w/V) was dissolved in distilled 103
water, under magnetic and mechanical stirring, at room temperature for 48 h, while LBG (cp = 0.5% 104
w/V) was dispersed in distilled water, under magnetic and mechanical stirring, at 80 °C for 24 h and 105
at room temperature for 24 additional hours (Kok, Hill, & Mitchell, 1999). The obtained solutions 106
were autoclaved at 121 °C for 20 minutes and then centrifuged at 8.000 rpm for 15 minutes at 25 107
°C. The supernatant solutions (the polymer weight loss was 15 % in the case of Xanthan and 30 108
% for LBG) were then exhaustively dialyzed at 4 °C against distilled water using dialysis 109
membranes with a cut-off 12,000–14,000. In order to convert the Xanthan polymer in the sodium 110
form, NaOH 0.2 N was added to the dialyzed solution up to pH = 7.0. Finally, the samples were 111
freeze-dried and stored in a desiccator until use. 112
All other products and reagents were of analytical grade. 113
114
2.2. LBG/Xanthan hydrogel preparations 115
116
5
The LBG/Xanthan hydrogels were prepared by mixing appropriate amounts of LBG and 117
Xanthan solutions (cp = 0.125 % w/V) previously autoclaved at 121 °C for 20 min and filtered 118
through cellulose nitrate membranes with pore sizes of 8 and 1.2 μm in sequence. It must be pointed 119
out that the Xanthan chains undergo an order-disorder transition during autoclaving and therefore a 120
renaturated state (where not necessarily perfect double-strands are formed) should be considered in 121
the subsequent interactions with the LBG chains. Different aliquots of the two polymer solutions 122
were mixed for 15 minutes at 75 °C (“hot gelation”) (where the Xanthan chains adopt a coil 123
conformation (T > Tm)) or at room temperature (“cold gelation”) (where the Xanthan chains are in 124
double helix conformation (T < Tm)), leading to samples with final different weight ratios. The “hot 125
samples”, after heating at 75 °C for 15 minutes, were left to slowly cool down in the switched off 126
thermostatted bath, until room temperature was reached. Three samples were prepared, namely 1:1 127
(50% of both, LBG and Xanthan solutions), 1:3 (25% of LBG solution and 75% of Xanthan 128
solution), and 1:9 (10% of LBG solution and 90% of Xanthan solution). 129
130
2.3. Rheological characterization 131
The rheological characterization of the LBG/Xanthan gels was performed by means of a 132
controlled stress rheometer, Haake Rheo-Stress RS300 model, with a Thermo Haake DC50 water 133
bath; a grained plate-plate device (Haake PP35 TI: diameter = 35 mm; gap between plates = 1mm) 134
was used in order to reduce the extent of the wall slippage phenomena (Lapasin, & Pricl, 1995). 135
Rheological properties were studied with oscillatory experiments; mechanical spectra were recorded 136
in the frequency range 0.001–10 Hz. The linear viscoelastic region was assessed, at 1 Hz, by stress 137
sweep experiments; a constant deformations, γ = 0.01, was used. All samples were analysed at 25 ± 138
1 °C, one day and 6 days after the mixing of the two polymer solutions. 139
140
2.4. Light Scattering 141
6
142
Each sample was placed into a thermostatted cell compartment of a Brookhaven Instruments 143
BI200-SM goniometer. The temperature was controlled to within 0.1 °C using a thermostatted 144
recirculating bath. The light scattered intensity and time autocorrelation function (TCF) were 145
measured by using a Brookhaven BI-9000 correlator and a 100 mW Ar laser (Melles Griot) tuned at 146
= 514.5 nm or a 50 mW He-Ne tuned at = 632.8 nm. Measurements were taken at different 147
scattering vector q = n0-1
sin(/2), where n is the refractive index of the solution, 0 is the 148
wavelength of the incident light, and is the scattering angle. To overcome the non-ergodicity, 149
typical of gelled systems (Pusey, & van Megen, 1989; Rodd, Dunstan, Boger, Schmidt, & 150
Burchard, 2001), the measurements were carried out by collecting the signal over different regions 151
of the specimen, automatically scanned by means of a motor-driven cell holder. In dynamic light 152
scattering (DLS) experiments the correlator operated in the multi- mode. About 150 153
measurements, lasting 7 min each, were taken over different sample positions; the intensity 154
autocorrelation function was then obtained by their sum normalized by the square of the total 155
photon number. The average scattered intensity factor and its dependence on q-vector were obtained 156
by collecting the signal over large scattering volumes while the samples were continuously rotating. 157
Static light scattering data were corrected for the background scattering of the solvent and 158
normalized by using toluene as calibration liquid. The static light scattered intensity was also 159
studied in terms of fluctuant and static components (Manno, Bulone, Martorana, & San Biagio, 160
2004; Barthès, Bulone, Manno, Martorana, & San Biagio, 2007). For this purpose about 700 161
intensity values were collected over different sample positions on a scattering volume 162
corresponding to a single speckle. The integration time (TI) for a single measurement was 1 s. 163
164
2.6. Data Analysis 165
166
7
Data distribution of the scattered intensity was analyzed with an expression (Manno, 167
Bulone, Martorana, & San Biagio, 2004; Barthès, Bulone, Manno, Martorana, & San Biagio, 2007) 168
derived by extending the Pusey and van Megen theory for non-ergodic media (Pusey, & van 169
Megen, 1989) to the case of no completely frozen-in systems. Indeed, the core of this expression is 170
the ratio between the coherence time of the scattering radiation and the integration time TI of the 171
measurement of the scattered intensity. As in the Pusey and van Megen approach, it was assumed 172
that the total scattered field E is the sum of a fast-fluctuating field EF due to small-size objects with 173
an average intensity IF and a coherence time F <TI, and a slowly decaying field ES due to large-size 174
slowly diffusing objects, with an average intensity IS and a coherence time S>TI. By taking into 175
account the ratio N = F /TI, it was shown that the total time-integrated intensity is given by two 176
statistically independent contributions: an exponentially slow contribution with average 177
NIII FSNG /2 , and a Gaussian distributed fast contribution with average NIII FFG /2 178
and variance NI G /22 . The expression for intensity distribution function is: 179
180
C
II
IIerf
I
IIIerf
I
II
IIP
NGNG
NGG
NG
NGG
NG
G
GTI
2exp
2
1
222exp
1)(
222
(1) 181
182
where dtexerfx
t
0
2
2)( is the error function and C1 is a normalization constant: 183
21
2
1
GI
erfC . 184
The expression is correct to the first order in F/TI and allows to distinguish between a fast 185
fluctuating and a slowly relaxing contribution even when their time scales are not completely 186
separated. 187
2.6. Scanning electron microscopy (SEM) 188
8
The SEM images were obtained using a FEI Quanta 400 FEG apparatus. Freeze-dried 189
hydrogels were mounted on appropriate stubs and examined under vacuum (50 Pa), with no need of 190
the gold coating technique, at an accelerating voltage of 15 KV. All images were acquired digitally 191
using xTmicroscope Control software and at 800x magnification. 192
193
3. Results and Discussion 194
195
3.1. Rheological investigations 196
197
The mechanical spectra of the LBG/Xanthan samples, prepared at different weight ratios in 198
“hot” and “cold conditions” recorded at 25 °C one day after and 6 days after the mixing of the two 199
polymer solutions (in the meanwhile the samples were kept at a constant temperature of 25 °C), are 200
shown in Fig. 1. 201
It is well known that both polymers, alone, are not capable to form gels, while, when mixed in 202
appropriate amounts, a three-dimensional network is formed. It is evident that, when the polymer 203
solutions are mixed in “cold conditions” the degree of entanglements and the number of effective 204
physical cross-links is deeply dependent on the weight ratio considered (see Fig. 1). If Xanthan is 205
the main component of the mixture (1:9), then the typical solution behaviour is observed: the loss 206
modulus is always higher than the storage modulus and both show a strong dependence on the 207
applied frequency. If the amount of Xanthan is reduced, but still predominant in comparison with 208
LBG (1:3), the characteristic gel point is observed. At the gel point the G’ and G” moduli overlap 209
and increase together, showing the typical power law behaviour (see below). Further decrease of the 210
Xanthan percentage in the mixture (1:1) leads to the formation of a gel, i.e., in the whole range of 211
explored frequencies (ca. four decades), G’ is always higher than G”. It must be underlined that, for 212
an appropriate comparison with LS experiments, the used polymer concentration is quite low (cp = 213
0.125 %). As a consequence the formed gel is a weak network: the difference between G’ and G” 214
9
values is less than a factor ten. Furthermore, the complete gel formation does not occur 215
instantaneously: consequently the time evolution of the moduli was followed and the measurements 216
were carried out also after six days (see Fig. 2A, B and C). For the 1:9 and 1:1 ratios an increase of 217
G’ is observed. For the 1:1 ratio the frequency dependence remains the same but the difference 218
between the two moduli increases with time indicating an increase of the number of the physically 219
active bonds. A similar behaviour is detected for the 1:9 ratio: the frequency dependence does not 220
change with time, the G” values are almost the same at 1 and 6 days while an increase with time is 221
observed for G’. Nevertheless this increase does not change the nature of the system that shows 222
always a solution behaviour (G’<G”). 223
On the other side, when the two polymer solutions are mixed in “hot conditions”, remarkable 224
differences are registered in the mechanical spectra (Fig. 1C and D and Fig. 2D, E and F). All 225
absolute values of the moduli are higher than those of the “cold samples” indicating that, when 226
Xanthan is in a disordered conformation, a more entangled network with a higher number of 227
effective cross-links can be formed in the presence of LBG. (see Fig. 2). For the sample with the 1:1 228
weight ratio, after one day, a gel is already formed, although a slight dependence from the 229
frequency can be observed for both moduli (Fig. 2D). After 6 days, an almost independence on the 230
frequency is monitored (Fig. 2D): this is an indication that the network evolves and more effective 231
physical cross-links are formed with time, which contribute to the elastic modulus. 232
Also for the network prepared at 1:9 ratio in “hot conditions”, an evolution is observed (Fig. 2F); 233
but, while for the “hot sample” a gel point occurs already after one day (Fig. 2F) (similar spectra 234
were registered for the 1:3 “cold sample”, Fig. 2B) the corresponding 1:9 “cold sample” still shows 235
mechanical spectra typical of solutions after 6 days (Fig. 2C). Clearly in the 1:9 “hot sample” the 236
Xanthan chains, being in the disordered conformation, are more capable to form physical 237
entanglements with the LBG chains in comparison with the 1:9 “cold sample” where the solution 238
behaviour is always observed (Fig. 2C). When a lower amount of Xanthan is present (1:3 ratio), for 239
10
the “hot samples” (Fig. 2E) the evolution of a very weak gel is monitored with time, while, for the 240
“cold samples”, a gel point transition is always observed (Fig. 2B). 241
Summarizing: the variations with time of the moduli are more significant in “hot samples” (Fig. 2D, 242
E and F). For the “cold samples” weak gel behaviour is monitored for the 1:1 ratio while gel 243
behaviour is monitored for the corresponding “hot samples” (Fig. 2A and D). The 1:9 ratio “hot 244
samples” show a gel point transition with a negligible evolution with time while, for the “cold 245
samples”, the corresponding spectra are those typical of solutions (Fig. 2C and F). For the 1:3 ratio 246
the “hot samples” are weak gels while the “cold samples” are at the gel point (Fig. 2B and E). 247
The 1:1 ratio is the most effective leading to a gel for both temperature preparation conditions 248
(tough weak gel is formed in the case of the “cold samples”). The difference detected between the 249
1:3 (gel point for the “cold samples”) and 1:9 ratios (gel point for the “hot samples”), indicates the 250
crucial role played by the conformation of the Xanthan chains. When a high amount of Xanthan 251
chains is present in double helix conformation (1:9, “cold samples”), a predominant liquid-like 252
behaviour is observed. On the other side, when the Xanthan chains are in the disordered 253
conformation, also for a high amount of the polysaccharide (1:9, “hot samples”), a gel point 254
transition is monitored. 255
It is interesting to point out that the critical exponents for gelation, n, are very close to the 256
theoretical one 0.5 (Chambon, Petrovic, MacKnight, & Winter, 1986) for both 1:3, “cold samples” 257
and 1:9 , “hot samples” (Table 1), i.e. for those samples showing a gel-point behaviour. 258
Actually such theoretical 0.5 value was firstly hypothesized by Winter and Chambon (Winter, & 259
Chambon,1986) as the universal parameter for the sol-gel transition, nevertheless later works 260
clarified that there is no universal value for n that can assume any value between 0 and 1, depending 261
on several parameters, such as cross-linker amount, polymer concentration, molecular weight, and 262
the specific pre-gel history (Coviello, & Burchard, 1992; Grisel, & Muller, 1998; Hsu, & Jamieson, 263
1993; Matricardi, Dentini, Crescenzi, 1993; Michon, Cuvelier, & Launay, 1993; Richter, Boyko, & 264
Schröter, 2004; Richter, Boyko, Matzker, & Schröter, 2004; Sandolo, Matricardi, Alhaique, & 265
11
Coviello, 2009; Winter, Izuka, & De Rosa, 1994). Usually, in a cross-linking reaction, just beyond 266
the gel point, G' levels off as the frequency decreases, and G" is proportional to . At later stages of 267
the reaction, the equilibrium modulus increases and G' and G" depart from each other. This is 268
actually what we observe for our “cold samples” at different weight ratios: the solution behaviour 269
for the 1:9, the gel point behaviour for the 1:3 and the gel behaviour for the 1:1 samples (Fig. 1B). 270
In the case of “hot samples” the solution behaviour is not detectable but gel point, weak gel and gel 271
behaviours are monitored for the 1:9, for the 1:3 and for 1:1 samples, respectively (Fig. 1D). 272
The critical exponent value of 0.5 detected for the two cases above reported (1:9 “hot samples” and 273
1:3 “cold samples”) suggests that, in accordance with the critical exponent found for stoichiometric 274
chemical crosslinkings, the networks are based on “one to one” interactions between the LBG and 275
Xanthan chains. In the case of the 1:9 “cold sample” there are actually too few LBG chains able to 276
build up a three-dimensional network (Fig. 2C). As LBG increases (1:3 “cold sample”) a gel point 277
is observed indicating that a sufficient number of LBG chains can interact with Xanthan (Fig. 2B). 278
Finally, for the 1:1 “cold sample” a real weak gel is obtained (Fig. 2A). In conclusion it can be 279
assumed that, for the “cold samples”, the network is mainly dominated by LBG and the network 280
formation is mediated by the addition of an appropriate amount of Xanthan chains. Furthermore, the 281
usual evolution with time of the two moduli (and correspondingly of the mechanical spectra) 282
observed for a chemical cross-linking reaction, is detected here by changing the weight ratio 283
between the two polymers (Fig. 1B). 284
A quite different situation occurs for the “hot samples”: at 75 °C LBG keeps its random coil 285
conformation (present at room temperature) but also Xanthan is dispersed as single chains. 286
Actually, Xanthan is not allowed to renaturate during cooling, because it is implied in the network 287
formation with the LBG chains. In this case no solution behaviour is ever detected: the 1:9 “hot 288
sample” is already at the gel point (Fig. 2F), thus showing that the different conformation assumed 289
by Xanthan plays a crucial role in the building up of the network. For the other ratios, 1:3 and 1:1, 290
weak gel and gel behaviours are monitored (Fig. 2E and 2D). 291
12
For the LBG/Xanthan cross-linking reaction it is also possible to assume an analogy between the 292
weight ratio and the reaction time and to report the loss tangent (tan ) as a function of the weight 293
ratio (instead of the reaction time). In fact, according to the definition of the gel point the loss 294
tangent is given (Kramers-Kronig relation) by tan = tan (n/2) = G"/G' (Ferry, 1980), indicating 295
that tan , at the sol-gel transition, is independent on and its value depends only on n. Fig. 3A 296
shows the results obtained by dynamic frequency sweep measurements for some selected ratios 297
(1:9, 1:3 and 1:1 “cold samples”) by plotting tan measured at various frequencies as a function of 298
the LBG weight ratio. According to the Winter-Chambon criterion (Winter, Morganelli, & 299
Chambon, 1988) the weight ratio at which tan curves converge is defined as the gel point. For the 300
samples, prepared in “cold conditions”, this value is practically coincident with the 1:3 weight ratio 301
and is equal to 1. The frequency-dependent measurement at the gel point should exhibit the same 302
slope for G’ and G”, as shown in Fig. 1B and reported in Table 1. Thus, for the LBG/Xanthan 303
weight ratios corresponding to the gel point, 1:3 “cold sample” and 1:9 “hot sample”, tan ≈ 1 (and 304
therefore n ≈ 0.5), as shown in Fig. 3B, for both systems, in the whole explored frequency domain. 305
Furthermore, the power law observed at the gel point implies a selfsimilar structure of the network 306
(Muthukumar,1985; Vilgis, & Winter, 1988); thus, it can be assumed that also the LBG/Xanthan 307
networks assume a selfsimilar structure when the gel point is detected (1:3 “cold sample” and 1:9 308
“hot sample”), and such connectivity can be related to the fractal dimension, df. In fact, according to 309
an important, and still valid work (Muthukumar, 1989), in the case that the excluded volume effect 310
is fully screened the following relation holds for a polydisperse system: f
f
dd
dddn
22
22, where 311
d is the space dimension (i.e. 3). For the tested systems (see Table 1) at the gel point (1:3 “cold 312
sample” and “1:9 “hot sample”) n ≈ 0.5 and the corresponding df value is ≈ 2, the same of an 313
isolated linear chain of infinite length under conditions (Muller, Gérard, Dugand, Rempp, & 314
Gnanou, 1991). This df value could be surprising for a chemical cross-linking reaction where at the 315
gel point the polymer is a highly branched macromolecule, but it can be reasonable for a physical 316
13
gel. In this case the critical state is not due to the simultaneous chemical reaction of many 317
monomers, but to the physical interactions among chains of quite long contour length (order of 318
hundred of nm). As a consequence the build up of peculiar structures is observed, as those typical of 319
biological networks that rarely can be strictly identified within the theoretical frameworks of 320
electrical analogue or tree-models. 321
322
3.2. Light Scattering investigations 323
324
325
The intensity autocorrelation functions (TCF) of LBG/Xanthan samples prepared at the three 326
different weight ratios in “hot” and “cold conditions”, were acquired 6 days after the preparation. 327
DLS measurements were carried out by collecting the signal on different regions of each sample to 328
take into account the non-ergodicity of gel systems. Results are shown in Fig. 4. Samples with a 329
higher content of LBG display higher values of a no-decay component, which is an indication of the 330
presence of a higher fraction of scattered light coming from scatterers unable to relax over the 331
applied experimental time scale. For a given weight ratio of LBG/Xanthan, “hot samples” appear 332
always more dynamically frozen than the cold ones. By comparing the time dependence of TCFs it 333
can be noted that the dynamics of samples 1:1, either “hot” or “cold”, and 1:3 “hot”, appears to be 334
almost entirely frozen, whereas the autocorrelation function of sample 1:9 “cold” displays a liquid-335
like character. Furthermore, samples 1:3 “cold” and 1:9 “hot” follow a power-law dependence, g(2) 336
t-
, with an exponent of 0.48 and 0.44, respectively. This power-law behavior is usually taken as a 337
mark of the gelation threshold (Shibayama, & Norisuye, 2002). The same two samples were seen to 338
be at the gelation point with a critical exponent close to 0.5 by rheological results (see the 339
“Rheological investigations” section). Although a relation between critical exponents obtained by 340
DLS and rheology is predicted by the Doi and Onuki theory (Richter, 2007), the critical exponents 341
obtained by means of light scattering measurements were, in the present study, lower than those 342
derived by rheology, as already observed by other researchers for several different systems (Richter, 343
2007). 344
14
The dynamic properties of the samples were also investigated by looking at the statistical properties 345
of the scattered light. Indeed, gel systems are characterized by the presence of very slow 346
fluctuations in the average intensity reflecting the reduced speckle mobility (Rodd, Dunstan, Boger, 347
Schmidt, & Burchard, 2001) and due to either dynamical or structural network inhomogeneities 348
(Richert, 2002). For each scattering angle the intensity from single speckles was collected with an 349
integration time of 1 s over different sample positions. Data were then normalized by their mean 350
value and the data distribution was fitted to the convolution of a Gaussian with an exponential 351
function (see Eq. (1)), which allowed to distinguish the fast fluctuating and slowly relaxing 352
contribution. The fraction of the fast contribution to the scattered light at different q-vector values is 353
shown in Fig 5A. A lower fraction of the fast relaxing contribution, XF, is observed at higher LBG 354
content, with the “hot samples” being more frozen than the “cold” ones, consistently with the DLS 355
results. The decrease of XF with q lowering implies that density fluctuations occurring over larger 356
length scale are more hindered. This was observed in all samples, including LBG 1:9 “cold”, that 357
appears liquid in the range of several ten nanometers. 358
Information on the structural properties can be provided by the dependence of the scattered light 359
intensity on q wave-vector. Indeed, for a system of molecules in solution, the q-dependence of the 360
scattered light is given by: 361
I(q) P(q) S(q) 362
where P(q) is the form factor of the single molecule and S(q) is the structure factor giving the 363
intensity contribution due to the spatial correlation between molecules in solution. In the case of 364
fractal aggregates, S(q) follows a power-law (Thill, Lambert, Moustier, Ginestet, Audic, & Bottero, 365
2000) of the form fdqqS
)( . The law is valid only over a length scale smaller than the aggregate 366
dimension (R) and larger than the dimension of the primary particles assembled in the aggregate 367
(r0), where P(q) 1. This leads to the well known relation fdqqI
)( with r0 << 1/q << R often 368
used for determining the fractal dimension. The q-dependence of the scattered intensities for 369
15
LBG/Xanthan samples was obtained by averaging the signal over different regions of the sample. 370
Data for each sample, normalized for the weight average molecular weight, are shown in Fig. 5 B. 371
Except for the absolute value, all samples display a very similar I(q) profile, despite their different 372
dynamical behavior. A sign of curvature can be envied in S(q) profile at about 6x104 cm
-1 373
(corresponding to 170 nm) that could signal the approaching to some characteristic length of the 374
system. At higher q values, I(q) follows a power-law with a slope of 2.4 ± 0.2. This value of fractal 375
dimension indicates a self-similar and quite compact structure, somehow larger, but anyhow 376
comparable, than that obtained from rheology and DLS for samples 1:3 “cold” and 1:9 “hot”. 377
However, sample polydispersity may severely affect the accuracy of the fractal dimension 378
determination by means of this type of data, since aggregates of different size can contribute to 379
scattered intensity with a different form factor. The similarity of I(q) profile for the different 380
samples suggests that the structure is almost the same, at least on the length-scale ranging from two 381
hundred down to few nanometers. A further comment can be made on the variation of the intensity 382
absolute value for the different samples. Actually, we observe that the intensity increases with the 383
increase of LBG content in the samples and the increase is even higher for those systems behaving 384
as “strong” gels, although the average spatial coordination inside the aggregates does not 385
appreciably change, suggesting the formation of ticker or denser junction regions inside the same 386
type of aggregates. 387
388
3.3. Scanning electron microscopy characterization 389
390
The SEM micrographs, taken six days after preparation on freeze-dried samples, support the 391
considerations above discussed. In Fig. 6 the photographs of the two single polymers (LBG and 392
Xanthan) and of the two samples prepared at low and high temperature with a weight ratio of 1:1 393
are shown. The last two systems are both gels, as indicated by the frequency spectra and by the 394
TCFs, but the different strength of the networks, estimated with the absolute values of the elastic 395
16
moduli and by the decay behaviour of the two TCFs, is clearly evidenced by the different texture 396
observed in the pictures. 397
It is interesting to appreciate the variations between the initial aspect of the single polymers, and the 398
final situations, when the synergistic effects, deeply influenced by the preparation temperature, 399
change dramatically also the macroscopic morphology of the mixed systems. 400
401 402
4. Conclusions 403 404
The LBG/Xanthan system leads to different types of network depending on the ratio of the two 405
polymers and on the preparation temperature. In particular, for the 1:1 ratio a gel is always 406
obtained; for the 1:3 ratio a weak gel is observed in “hot conditions” while a gel point is detected in 407
“cold conditions”; for the 1:9 ratio the gel point is monitored in “hot conditions” while the solution 408
is present in “cold conditions”. These results indicate how a fine tuning of the properties of the 409
mixed gels is possible through the variation of the preparation temperature and/or the weight ratio 410
between the two polymers. 411
Interesting is the qualitative agreement between the two approaches: the TCFs of 1:1 samples (“hot” 412
and “cold” samples) do not relax in the explored time window and similarly the corresponding 413
mechanical spectra are those of a gel in the whole explored frequency domain. Also for the other 414
two weight ratios a correspondence between the rheology and the DLS can be appreciated. 415
Furthermore, looking at the values of Table 1, it is possible to see the good accordance between the 416
fractal dimensions, obtained with the rheology approach, and the DLS technique for the two 417
systems that show sol-gel transition in the mechanical spectra and power law behaviour in DLS. 418
The slight discrepancy between the two critical exponents is reasonable: the theory mainly deals 419
with chemical network formation while in the present case physical interactions lead to the gel: 420
furthermore, the light scattering is a non perturbative technique while a periodic deformation is 421
applied in rheology partly disturbing the crosslinking process with a possible shift in the 422
determination of the infinite cluster formation and detection. 423
17
424
Acknowledgements 425
Financial supports from FIRB, Fondo per gli Investimenti della Ricerca di Base, Research Program: 426
Ricerca e Sviluppo del Farmaco (CHEM-PROFARMA-NET), grant no. RBPR05NWWC_003 is 427
acknowledged. 428
429
430
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562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
23
595
Legends to the figures 596
597
Scheme1. Repeating units of LBG (left) and Xanthan (right). 598
599
Fig. 1. Comparison among the mechanical spectra of LBG/Xanthan samples prepared at different 600
weight ratio (1:1, 1:3 and 1:9), prepared at two different temperatures (A, B : T=25 °C; C, D: T= 75 601
°C) and recorded at 25 °C one day (A and C) and six days (B and D) after the preparation. 602
603
Fig. 2. Comparison among the mechanical spectra of LBG/Xanthan recorded at 25 °C one day and 604
six days after the preparation at different weight ratio. 605
“Cold samples”: A: 1:1; B: 1:3 and C: 1:9. “Hot samples”: D: 1:1; E: 1:3 and F: 1:9. 606
607
Fig. 3. A) tan of LBG/Xanthan samples prepared in “cold conditions” and recorded at 25 °C six 608
days after the preparation at different frequencies as a function of the content of LBG (e.g., LBG = 609
0.50 corresponds to 1:1 weight ratio). B) tan of LBG/Xanthan samples prepared in “hot” and 610
“cold conditions” as a function of frequency for the 1:3 and 1:9 ratios. 611
612
Fig. 4. Intensity autocorrelation functions of LBG/Xanthan samples obtained 6 days after their 613
preparation. 614
615
Fig. 5. Fraction of the fast relaxing contribution to scattered light vs. wave-vector q estimated for 616
the LBG/Xanthan studied at different weight ratio (A). Log-log plot of the average scattered light 617
intensity, measured for the LBG/Xanthan at different weight ratio, vs. q-vector. Data were 618
normalized by toluene scattering and the weight average molecular weight (B). 619
620
Fig. 6. Scanning electron micrograph of surface morphology of freeze-dried samples of LBG, 621
Xanthan (top), and LBG/Xanthan 1:1 (bottom) prepared at 75 and 25 °C (magnification 800x). 622
623
Table 1
C. Sandolo et al.
Table 1
Apparent exponents relative to the two moduli, n’ and n”, evaluated for two weight ratios, power
law exponents from the TCFs and the corresponding fractal dimensions, df.
------------------------------------------------------------------------------------------------------------------------
Rheology Light Scattering
------------------------------------------------------------------------------------------------------------------------
“cold samples” “hot samples” “cold samples” “hot samples”
1:3 1:9 1:3 1:9
------------------------------------------------------------------------------------------------------------------------
time (days) n’ n” n’ n”
------------------------------------------------------------------------------------------------------------------------
1 0.56 0.54 0.53 0.59
6 0.47 0.43 0.50 0.56 0.48 0.44
time (days) df (n’) df (n”) df (n’) df (n”) df df
------------------------------------------------------------------------------------------------------------------------
1 1.92 1.95 1.96 1.89
6 2.04 2.08 2.00 1.93 2.08 2.27
------------------------------------------------------------------------------------------------------------------------
Table 1
Scheme 1
C. Sandolo et al.
Locust Bean Gum Xanthan
O
O OH
HO
OO
HOOH
O
HO
O
O
HO
HO
O
O
-O2C
O
HOOH
-O2C
OO HO
OH
O
O
O
OHO
HO
O
HO
O
HO
HOOH
O
OHO
HO
O
O
O
HO
HOOH
O
OH
HO
OH
OH
Scheme 1
Figure 1
C. Sandolo et al.
1 day, cold
0.001
0.01
0.1
1
10
0.001 0.01 0.1 1 10f (Hz)
G', G'' (Pa)
G' 1:1
G'' 1:1
G' 1:3
G'' 1:3
G' 1:9
G'' 1:9
A 6 days, cold
0.001
0.01
0.1
1
10
0.001 0.01 0.1 1 10f (Hz)
G', G'' (Pa)
G' 1:1
G'' 1:1
G' 1:3
G'' 1:3
G' 1:9
G'' 1:9
B
1day, hot
0.01
0.1
1
10
100
0.001 0.01 0.1 1 10f (Hz)
G', G'' (Pa)
1:1 G'1:1 G''1:3 G'1:3 G"1:9 G'1:9 G"
C 6 days, hot
0.01
0.1
1
10
100
0.001 0.01 0.1 1 10f (Hz)
G', G'' (Pa)
1:3 G'1:3 G''1:1 G'1:1 G"1:9 G'1:9 G"
D
Figure 1
Figure 2
C. Sandolo et al.
1:1 cold sample
0.1
1
10
0.001 0.01 0.1 1 10f (Hz)
G', G'' (Pa)
G' (1:1) 6 days
G'' (1:1) 6 days
G' (1:1) 1 day
G'' (1:1) 1 day
A
1:3 cold sample
0.01
0.1
1
10
0.001 0.01 0.1 1 10f (Hz)
G', G''(Pa)
G' (1:3) 6 days
G'' (1:3) 6 days
G' (1:3) 1 day
G'' (1:3) 1 day
B
1:9 cold sample
0.001
0.01
0.1
1
0.001 0.01 0.1 1 10f (Hz)
G', G'' (Pa)
G' (1:9) 6 days
G'' (1:9) 6 days
G' (1:9) 1 day
G'' (1:9) 1 day
C
1:1 hot sample
0.1
1
10
100
0.001 0.01 0.1 1 10f (Hz)
G', G''(Pa)
G', 6 days G'', 6 days
G', 1 day G", 1 day
D
1:3 hot sample
0.1
1
10
0.001 0.01 0.1 1 10f (Hz)
G',G'' (Pa)
G', 1 day G'', 1 day
G', 6 days G'', 6 days
E
1:9 hot sample
0.01
0.1
1
0.001 0.01 0.1 1f (Hz)
G',G''(Pa)
G', 1 day G'', 1 day
G', 6 days G'', 6 days
F
Figure 2
Figure 3
C. Sandolo et al.
6 days, cold
0
1
2
3
4
5
6
7
8
9
0.0 0.1 0.2 0.3 0.4 0.5 LBG (%)
tan
1 Hz
0.63 Hz
0.40 Hz
0.25 Hz
0.16 Hz
0.1 Hz
0.0631 Hz
0.0398 Hz
A
B
0
1
2
3
4
0.001 0.01 0.1 1 10f (Hz)
tan1:3 cold 6 days
1:9 hot 6 days
1:3 cold 1 day
1:9 hot 1 day
Figure 3
Figure 4
C. Sandolo et al.
(s)
100 101 102 103 104 105 106 107
g(2
) ()
-1
0.01
0.1
1
1:1 hot
1:1 cold
1:3 hot
1:3 cold
1:9 hot
1:9 cold
Figure 4
Figure 5
C. Sandolo et al.
q (cm-1)
105 106
I N (
arb
. un.)
101
102
103
104
105
q(cm-1)
1e+5 2e+5 3e+5
XF
0.0
0.2
0.4
0.6
0.8
1.0
1:1 hot
1:1 cold
1:3 hot
1:3 cold
1:9 hot
1:9 cold
A B
Figure 5
Figure 6
C. Sandolo et al.
LBG Xanth
LBG/Xanth 1:1 hot sample LBG/Xanth 1:1 cold sample
Figure 6