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Swelling of agarose gel and its related changes
Food Hydrocolloids vol.I no.4 pp.3l7-325, 1987
Swelling of agarose gel and its related changes A.Hayashi and T.Kanzaki Department of Chemistry, Ritsumeikan University, Tojiin, Kita-ku, Kyoto 603, Japan Abstract. Swelling and its related changes in agarose gels were investigated. The behavior of agarose gel was different to that of synthetic polymer gels: the agarose gel did not show any swelling or shrinkage in water. The gel shrank only a little in acetone and in ether. The shrunken gel did not recover its original volume again with re-treatment in water. It was found that agarose solution showed a retrogradation-like reaction similar to amylose solution. These anomalous phenomena including syneresis were interpreted based upon the liquid phase separation mechanism for the agarose gelation proposed by us.
Introduction
Organic polymer gels are known to swell easily within a certain limited extent in an appropriate solvent. The swelling behavior of the polymer gels was studied theoretically by Flory (1). Recently, Tanaka et at. (2) found a reversible, discontinuous volume change in ionic polymer gels and they explained this phenomenon in terms of mean field theory based on the extension of Flory's formula for the free energy of gels. In these cases, the gels usually consist of three-dimensional networks of randomly-coiled molecular chains crosslinked with a covalent bond. On the other hand, some natural polymer gels, for example, agar gel and bean-curd (tofu), are hardly swollen in water although water is a good solvent for both polymers. Agarose gel is one such non-swelling polymer gel. Agarose is a gelling fraction of agar polysaccharides. The mechanism of gelation has been investigated by many workers. According to Rees' proposal (3), the agarose chain is almost helical in the gel and the junction zones of the network are caused by the formation of intermolecular double helices. In our laboratory, the gelling process of agarose was studied by fluorescence polarization and we found that the network structure resulted from a liquid phase separation of rod-like, helical segments in agarose molecular chains (4-6). In any event, the agarose molecular chains in the gel are not in the randomly-coiled conformation seen in the usual synthetic polymer gels but in the rod-like, helical form. The agarose gel is thermo-reversible and the gelling behavior is not a first order transition. The melting temperature of the gel is much higher than the gelling temperature, that is, the agarose gel shows a distinct hysteresis phenomenon. When the agarose gel is placed in cool air, water droplets are released by the gel and are seen on the surface with little shrinkage in the volume. The phenomenon is called the syneresis of the gel. As mentioned above, agarose gels show apparently different behavior from synthetic polymer gels. These characteristic features have not always been interpreted satisfactorily. In this paper, these phenomena are studied and the results examined on the basis of our model of the mechanism of gelation in the agarose system.
© IRL Press Limited, Oxford, England
317
A.Hayashi and T.Kanzaki
Materials and methods
Sample gels The sample of agarose was 'Agarose GP-36' (Nakarai Chemicals Ltd, Kyoto, Japan), which is a purified agarose similar to 'Agarose A-37' used in the preceding works (4-6). The swollen agarose powder in water was dissolved on a water bath at 90°C, and then gelled for 3 h in an incubator at the same temperature as the standing temperature for swelling, shrinking or syneresis. The gel was cut off into a cube of -1 em".
Swelling in water and in saturated moisture The gel used for the swelling test over a long period contained 0.05% methylparaben. The addition of methylparaben did not cause any observable changes in the physical properties of the gel. The gel cube was weighed and then immersed in water containing 0.05 % methylparaben at a constant temperature. At a certain time interval, the gel was weighed. At the same time, the gel was placed in a desiccator saturated with moisture and weighed similarly.
Shrinkage of gel in acetone and in ether The gel was immersed in an acetone - water mixture and the change in length of a side was measured after certain time intervals. The solvent was changed every day and the volume change was calculated from the change in the length. Drying in air and observation of syneresis The gel was placed on a glass plate in an incubator with or without air blowing. At a certain time interval, the gel was weighed and the syneresis was checked at the same time.
Retrogradation-like phenomenon in agarose solution The experimental procedure was similar to that in our preceding work on amylose (7,8), in which we showed that the retrogradation of amylose proceeds according to Avrarni' s equation (10). The onset ofthe change in the solution phase was checked qualitatively with the change in light scattering. The light scattering was measured by a Hitachi Fluorescence Spectrophotometer 650-lOS at a wave length of 500 nm. The quantitative analysis of the reaction was made as follows: at a certain interval, a pre-determined volume of the solution was centrifuged at 10 000 r.p.m. for 15 min, then the concentration of agarose in the supernatant solution was determined by the phenol- H 2S04 method (9). Results Experiments were carried out on two concentrations of agarose gel, 1 and 2 g/dl, and at several temperatures. When the trend of the results was similar in a series of experiments, only one of the results is given as a typical example. Figure 1 shows the swelling of agarose gel in water. The concentration of agarose was 2 gldl, and the temperature was 100e. The gel did not show any weight or volume change within 50 days. Similar results were obtained in other concentrations and at 318
Swelling of agarose gel and its related changes
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other temperatures below 30°C. There was no weight change observed under conditions of saturated moisture either. Figure 2 shows the shrinkage of the gel in an acetone-water mixture, after immersion for 10 days. The concentration of agarose was 2 g/dl and the temperature was 20 o e. It was found that the volume of gel slightly decreases at the higher concentrations of acetone. The volume change of agarose gel is, however, less than 20 % even in pure acetone. The shrunken gel in acetone was immersed again in water or in ether . The volume recovered - 5 % in water and decreased again - 5 % in ether. The shrunken 319
A.Hayashi and T .Kanzaki
o
50
-0---(
100
o
10
20
30
t (hours) Fig. 3. Weight changes of agarose gel (2.0 g/dl) in blowing air at 5 (0) and 20°C (6).
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Fig. 4. Changes in reduced intensity of light scattering of agarose solution (0.08 g/dl) at 12 (6), 21 (0), and 25°C (e).
gel in ether was immersed in acetone and then in water but the recovered volume was only - 5 %. If the sample size was reduced and if the solvent, acetone, was changed more frequently in order to exchange water in the gel for pure acetone quickly, the volume change became smaller. When the gel was cut up into pieces of 1 x 0.1 x 0.1 em and the acetone was changed every 20 min for several hours, the decrease in the volume was only - 3 % after 3 days. This gel did not show any observable decrease in volume with succeeding treatment in ether. Figure 3 shows the results of air drying a 1 gldl gel at 5 and 20°C with air blowing. 320
Swelling of agarose gel and its related changes
1
o
-1
-2
o
2
3
In t (ainutes) Fig. 5. Retrogradation-like reaction of agarose solution (0.08 d/gl) at 25°C.
Syneresis was observed, in this case, only at the bottom of the gel where the gel was in contact with the glass plate. The rate of drying is faster at higher temperatures. Syneresis could not be estimated quantitatively; however, it was evident that it depends upon the experimental conditions. Figure 4 shows the change in light scattering of the agarose solution with time. The sample solution contained 0.08 gldl of agarose and 0.1 M NaCl. It is shown that the retrogradation-like reaction proceeds fairly quickly and faster at lower temperatures. Figure 5 shows the result of the quantitative analysis of the retrogradation-like phenomenon in the agarose solution . 0 is the fraction of the unprecipitated agarose and corresponds to the fraction of the uncrystallized matrix in the Avrami' s crystallization formula (10), In 0 = -kf'. The exponent of Avrami 's equation is found to be n = 1.25 from the slope of the line in Figure 5. The shape and the size of the precipitated fine particles at the bottom of the centrifuge tube were spheres of - 10 JLm in diameter at the initial stage of the reaction. At the latter stages these particles become larger in size and irregular in shape and show a weak polarization under a polarizing microscope. These results show that a retrogradation-like phenomenon occurs in the agarose solution similar to that in amylose solutions. Discussion We have previously proposed a model for agarose gels (4-6) which is as follows. The agarose macromolecule is composed of an alternating chain of helical segments and kink segments. Gelation occurs below the helix temperature and the relationship between gelation temperature and agarose concentration is similar to the phase diagram for rod-like polymers given by Flory (11), when it is assumed that the free energy parameter X is proportional to the reciprocal of temperature because such a relation is known to exist in an aqueous solution of hydrophilic polymers. From this similarity 321
A.Hayashi and T.Kanzaki
x
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--- - . . --.0
.
ill
/
0.. __0__ . _ .I ~:
I
...-----I
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/
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Fig. 6. Schematic phase diagram for agarosc solution. 1. Random coil. II. Anisotropic, helix. II' Isotropic, helix . II " Two phases: isotropic, helix (v2') and anisotropic, helix (v2") ' III . 2 phases: isotropic, helix (c a ) and anisotropic , helix (cb) ' A . Helix point. B. Phase separation point (gelation point).
ra ndom coi I
II aniso. II ' iso ,
lI! coacervate (Vb, ll ) + dil.solu . (V., Il ')
r etrograded aggreg ate
II· iso . + an is o, Fig . 7. Schematic representation for phase changes in dilute agarose solution.
and other experimental results, we believe that the gel results from a phase separation of the rod-like, helical segments in the agarose macromolecule. Figure 6 shows the phase diagram for the agarose solution. The right half of the diagram is drawn from an assumption based upon Flory's diagram. The scale of the horizontal axis (concentration) of the right half is reduced arbitrarily compared with the left half obtained by experiments. The information of each phase separated by lines is given in the footnote of the figure. When a hot solution of PI (concentration: c, temperature : TI , random coil) is cooled to reach to the helix point, P2 (T2 ) , the segments wind up into rod-like helices. These helices separate into two phases at the phase separation point, P3 (T3) . At a point, P (concentration: c, temperature : T ), the solution consists of the dilute solution phase (concentration: ca , isotropic) and the concentrated solution phase (concentration: Cb, anisotropic). The ratio of both phases is given by (Cb - c):(c - cJ . The schematic 322
Swelling of agarose gel and its related changes
presentation of this process is shown in Figure 7. At the point, P, the fine particles of the concentrated solution phase (coacervate) are dispersed in the dilute solutionphase. Some of the helical segments of the molecule in the dilute solution phase are likely to be included in the coacervate particles. At a concentration higher than a critical value, the polymer molecule in the dilute solution phase acts as an intercoacervate junction. The coacervate particles are thus connected three-dimensionally, and the solution loses its fluidity to form a gel. The remarkable feature of our model is that the gel consists of a two-phase structure composed of a heterogeneous solution. An equilibrium exists between the dilute solution phase and the coacervate at least at the gel setting point. If the equilibrium still remains after the gelation, the ratio of both phases in the gel must change based upon the phase diagram when the gel is immersed in water. As a result, the gel must swell and then must dissolve in water. When the temperature is raised over the gelation point, the gel must also melt into a homogeneous solution. The transformation from the coacervate to the dilute solution phase is, however, disturbed by the crystallization of helical segments in the solution phases, that is, the equilibrium between both phases no longer exists in the gel. Evidence for the crystallization is shown in the retrogradation-like behavior. In the preceding work for amylose retrogradation (7), it was found that the retrogradation is caused by crystallization of helical segments. In the case of a dilute agarose solution such as 0.08 g/dl, if the crystallization of helical segments occurs, the segments become tied up into bundles (see Figure 7). As a result, the pendant coacervate particles of these segments join to form an aggregate. This is retrogradation showing precipitation of aggregates from the very dilute solution unable to make gel. The exponent of the Avrami's equation, n, for agarose is 1.25, somewhat smaller than the 2.0 for amylose reported by us (7). In the course of the preceding work for amylose, we sometimes experienced that n had a value of - 1.0 if the solution contained some undissolved crystals or some impurities such as dust particles. Colwell also reported a similar value for the retrogradation of a concentrated solution of amylose (12). The agarose sample solution used here was used without any filtration, hence it seems probable that it may have contained some impurities which could act as nuclei for crystallization. When a similar kind of crystallization proceeds in the network of the gel, there appears to be no observable changes directly. However, it may cause a thermo-hysteresis of the gel. The gelation point is the phase separation temperature and the melting point of the gel is the melting temperature of the crystal in the gel. This crystallization occurs as soon as the helix is formed in the solution as shown by the 'scanning curve' (partial hysteresis curve) in the preceding paper (5). Thus, crystallization prevents the gel from swelling as a result of the two-phase structure. The existence of the retrogradation-like phenomenon seems reasonable because amylose and agarose have a similar gelation mechanism (13). The possibility of swelling of the three-dimensional network structure is the same as for synthetic polymer gels. The Tanaka's formula for swelling and collapse of the gel (2) consists of four terms, entropy and enthalpy of mixing, excess rubber elasticity of network and osmotic pressure of ion. The effects of ionic groups and enthalpy are negligible, because we used a pure agarose sample at the same temperature for gel setting and swelling. The entropy term always increases swelling and the rubber elasticity 323
A.Hayashi and T.Kanzaki
reduces it. At this point, we cannot deal with them quantitatively. As mentioned above, agarose is almost in a double helical form in the gel and the helical segments tend to crystallize. Therefore, the molecules in the dilute solution phase seem to make a hard and brittle network in the gel. This hard network will result in a strong elasticity against the swelling effect in water. In other words, the entropy-elasticity resulting from flexible molecular chains is not expected to exist to a greater extent in the agarose gel aged for some while. This is a qualitative interpretation for the fact that the agarose gel does not swell in water as shown in Figure 1. Acetone is a non-solvent for agarose and is used for the purification of agarose as a precipitant from aqueous solution. We might expect, therefore, that the gel would shrink quickly on addition of acetone. However, the agarose gel keeps> 80% of the original volume even in pure acetone, as shown in Figure 2, and , moreover, the gel still keeps - 80 % of the original volume in ether. The original volume cannot be recovered. This means that the shrinkage of the gel is again not due to the entropyelasticity. Under such conditions, the affinity of solvent molecules for polymer segments decreases and the cohesive force between polymer segments increases. This situation may result in a collapse of the network structure thereby shrinking the gel. The network of the gel is fairly hard and brittle; hence, it is difficult to recover the original structure again after its collapse . When pure acetone is used to shrink the gel, water molecules within the matrix are gradually exchanged for acetone molecules by diffusion. On removal of water molecules, the cohesion between polymer segments may increase as a result of hydrogen bonding. It is well known that an increase in hydrogen bonding is often observed in the drying of an aqueous solution of hydrophilic polymers. In order to reduce the time for water molecules to exchange, an experiment was carried out ona small piece of gel changing the acetone quickly and frequently. The results showed that the agarose gel was almost able to retain its original volume. The most probable reason for syneresis seems to be that the agarose gel contains a large proportion of solution phase of much lower concentration (c a) than the total agarose concentration (c). Synthetic polymer gels usually have a homogeneous inner structure and when their surface dries to yield a higher concentration, the concentration within the gel changes to restore the equilibrium. This continuous adjustment of the inner solution concentration may prevent shrinkage. In addition the higher concentration in the surface layer means a stronger network and, therefore, solvent from within the gel shows little tendency to diffuse through cracks in the surface. When an agarose gel shrinks on rapid drying, forces exerted on the surface by the solution inside cause cracks to form. Syneresis thus occurs as a result of the inner dilute solution or solvent migrating through these cracks in the dried surface. The reason why the syneresis is often observed at low temperatures may be due to the slow evaporation of water. Several anomalous properties of the agarose gel in this paper are thus interpreted based on the two-phase structure of the gel proposed by us. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research, to which the authors ' thanks are due.
324
Swelling of agarose gel and its related changes
References 1. F1ory,P.J. (1953) Principles of Polymer Chemistry. Cornell University Press, New York. 2. Tanaka,T., Fillmore,D., Sun,S.T., Nishio,I., Swislow.G. and Shah,A. (1980) Phys. Rev. Lett., 45,1636. 3. Arnott,S., Fulmer.A,; Scott,W.E., Dea,I.C.M., Moorhouse.R. and Rees,D.A. (1974) J. Mol. Biol., 90,269. 4. Hayashi,A., Kinoshita.K. and Kuwano,M. (1977) Polym. J., 9, 219. 5. Hayashi.A., Kinoshita,K., Kuwano,M. and Nose,A. (1978) Polym. J., 10, 485. 6. Hayashi,A., Kinoshita.K. and Yasueda,S. (1980) Polym. J., 12,447. 7. Hayashi,A., Kotani.K, and Cho,C. (1984) Agric. Biol. Chem., 48, 949. 8. Hayashi,A., Kinoshita,K. and Kotani,Y. (1983) Agric. Biol. Chem., 47,1705. 9. Dubois,M., Gilles,K.A., Hamilton,J.K., Revers,P.A. and Smith,F. (1956) Anal. Chem., 28, 350. 10. Avrami,M. (1941) J. Chem. Phys., 9, 177. 11. Flory,PJ. (1961) J. Polym. Sci., 49, 105. 12. Colwell,K.H., Axford,D.W.E., Chaberline,N. and Elton,G.H.A. (1969) J. Sci. Fd. Agric., 20, 550. 13. Hayashi,A., Kinoshita,K. and Miyake,Y. (1983) Agric. Biol. Chem., 47, 1699.
325
Swelling of agarose gel and its related changes A.Hayashi and T.Kanzaki Department of Chemistry, Ritsumeikan University, Tojiin, Kita-ku, Kyoto 603, Japan Abstract. Swelling and its related changes in agarose gels were investigated. The behavior of agarose gel was different to that of synthetic polymer gels: the agarose gel did not show any swelling or shrinkage in water. The gel shrank only a little in acetone and in ether. The shrunken gel did not recover its original volume again with re-treatment in water. It was found that agarose solution showed a retrogradation-like reaction similar to amylose solution. These anomalous phenomena including syneresis were interpreted based upon the liquid phase separation mechanism for the agarose gelation proposed by us.
Introduction
Organic polymer gels are known to swell easily within a certain limited extent in an appropriate solvent. The swelling behavior of the polymer gels was studied theoretically by Flory (1). Recently, Tanaka et at. (2) found a reversible, discontinuous volume change in ionic polymer gels and they explained this phenomenon in terms of mean field theory based on the extension of Flory's formula for the free energy of gels. In these cases, the gels usually consist of three-dimensional networks of randomly-coiled molecular chains crosslinked with a covalent bond. On the other hand, some natural polymer gels, for example, agar gel and bean-curd (tofu), are hardly swollen in water although water is a good solvent for both polymers. Agarose gel is one such non-swelling polymer gel. Agarose is a gelling fraction of agar polysaccharides. The mechanism of gelation has been investigated by many workers. According to Rees' proposal (3), the agarose chain is almost helical in the gel and the junction zones of the network are caused by the formation of intermolecular double helices. In our laboratory, the gelling process of agarose was studied by fluorescence polarization and we found that the network structure resulted from a liquid phase separation of rod-like, helical segments in agarose molecular chains (4-6). In any event, the agarose molecular chains in the gel are not in the randomly-coiled conformation seen in the usual synthetic polymer gels but in the rod-like, helical form. The agarose gel is thermo-reversible and the gelling behavior is not a first order transition. The melting temperature of the gel is much higher than the gelling temperature, that is, the agarose gel shows a distinct hysteresis phenomenon. When the agarose gel is placed in cool air, water droplets are released by the gel and are seen on the surface with little shrinkage in the volume. The phenomenon is called the syneresis of the gel. As mentioned above, agarose gels show apparently different behavior from synthetic polymer gels. These characteristic features have not always been interpreted satisfactorily. In this paper, these phenomena are studied and the results examined on the basis of our model of the mechanism of gelation in the agarose system.
© IRL Press Limited, Oxford, England
317
A.Hayashi and T.Kanzaki
Materials and methods
Sample gels The sample of agarose was 'Agarose GP-36' (Nakarai Chemicals Ltd, Kyoto, Japan), which is a purified agarose similar to 'Agarose A-37' used in the preceding works (4-6). The swollen agarose powder in water was dissolved on a water bath at 90°C, and then gelled for 3 h in an incubator at the same temperature as the standing temperature for swelling, shrinking or syneresis. The gel was cut off into a cube of -1 em".
Swelling in water and in saturated moisture The gel used for the swelling test over a long period contained 0.05% methylparaben. The addition of methylparaben did not cause any observable changes in the physical properties of the gel. The gel cube was weighed and then immersed in water containing 0.05 % methylparaben at a constant temperature. At a certain time interval, the gel was weighed. At the same time, the gel was placed in a desiccator saturated with moisture and weighed similarly.
Shrinkage of gel in acetone and in ether The gel was immersed in an acetone - water mixture and the change in length of a side was measured after certain time intervals. The solvent was changed every day and the volume change was calculated from the change in the length. Drying in air and observation of syneresis The gel was placed on a glass plate in an incubator with or without air blowing. At a certain time interval, the gel was weighed and the syneresis was checked at the same time.
Retrogradation-like phenomenon in agarose solution The experimental procedure was similar to that in our preceding work on amylose (7,8), in which we showed that the retrogradation of amylose proceeds according to Avrarni' s equation (10). The onset ofthe change in the solution phase was checked qualitatively with the change in light scattering. The light scattering was measured by a Hitachi Fluorescence Spectrophotometer 650-lOS at a wave length of 500 nm. The quantitative analysis of the reaction was made as follows: at a certain interval, a pre-determined volume of the solution was centrifuged at 10 000 r.p.m. for 15 min, then the concentration of agarose in the supernatant solution was determined by the phenol- H 2S04 method (9). Results Experiments were carried out on two concentrations of agarose gel, 1 and 2 g/dl, and at several temperatures. When the trend of the results was similar in a series of experiments, only one of the results is given as a typical example. Figure 1 shows the swelling of agarose gel in water. The concentration of agarose was 2 gldl, and the temperature was 100e. The gel did not show any weight or volume change within 50 days. Similar results were obtained in other concentrations and at 318
Swelling of agarose gel and its related changes
o
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00 ., ..c u ..., ..c C
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00
~
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o
30
60
t (days) Fig. 1. Weight changes of agarose gel (2.0 g/dl) in water at lOoe.
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Fig. 2. Volume changes of agarose gel (2.0 g/dl) in acetone-water mixture at 20 o e.
other temperatures below 30°C. There was no weight change observed under conditions of saturated moisture either. Figure 2 shows the shrinkage of the gel in an acetone-water mixture, after immersion for 10 days. The concentration of agarose was 2 g/dl and the temperature was 20 o e. It was found that the volume of gel slightly decreases at the higher concentrations of acetone. The volume change of agarose gel is, however, less than 20 % even in pure acetone. The shrunken gel in acetone was immersed again in water or in ether . The volume recovered - 5 % in water and decreased again - 5 % in ether. The shrunken 319
A.Hayashi and T .Kanzaki
o
50
-0---(
100
o
10
20
30
t (hours) Fig. 3. Weight changes of agarose gel (2.0 g/dl) in blowing air at 5 (0) and 20°C (6).
100
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200
300
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Fig. 4. Changes in reduced intensity of light scattering of agarose solution (0.08 g/dl) at 12 (6), 21 (0), and 25°C (e).
gel in ether was immersed in acetone and then in water but the recovered volume was only - 5 %. If the sample size was reduced and if the solvent, acetone, was changed more frequently in order to exchange water in the gel for pure acetone quickly, the volume change became smaller. When the gel was cut up into pieces of 1 x 0.1 x 0.1 em and the acetone was changed every 20 min for several hours, the decrease in the volume was only - 3 % after 3 days. This gel did not show any observable decrease in volume with succeeding treatment in ether. Figure 3 shows the results of air drying a 1 gldl gel at 5 and 20°C with air blowing. 320
Swelling of agarose gel and its related changes
1
o
-1
-2
o
2
3
In t (ainutes) Fig. 5. Retrogradation-like reaction of agarose solution (0.08 d/gl) at 25°C.
Syneresis was observed, in this case, only at the bottom of the gel where the gel was in contact with the glass plate. The rate of drying is faster at higher temperatures. Syneresis could not be estimated quantitatively; however, it was evident that it depends upon the experimental conditions. Figure 4 shows the change in light scattering of the agarose solution with time. The sample solution contained 0.08 gldl of agarose and 0.1 M NaCl. It is shown that the retrogradation-like reaction proceeds fairly quickly and faster at lower temperatures. Figure 5 shows the result of the quantitative analysis of the retrogradation-like phenomenon in the agarose solution . 0 is the fraction of the unprecipitated agarose and corresponds to the fraction of the uncrystallized matrix in the Avrami' s crystallization formula (10), In 0 = -kf'. The exponent of Avrami 's equation is found to be n = 1.25 from the slope of the line in Figure 5. The shape and the size of the precipitated fine particles at the bottom of the centrifuge tube were spheres of - 10 JLm in diameter at the initial stage of the reaction. At the latter stages these particles become larger in size and irregular in shape and show a weak polarization under a polarizing microscope. These results show that a retrogradation-like phenomenon occurs in the agarose solution similar to that in amylose solutions. Discussion We have previously proposed a model for agarose gels (4-6) which is as follows. The agarose macromolecule is composed of an alternating chain of helical segments and kink segments. Gelation occurs below the helix temperature and the relationship between gelation temperature and agarose concentration is similar to the phase diagram for rod-like polymers given by Flory (11), when it is assumed that the free energy parameter X is proportional to the reciprocal of temperature because such a relation is known to exist in an aqueous solution of hydrophilic polymers. From this similarity 321
A.Hayashi and T.Kanzaki
x
r---~----.\·H-------,
P
T-
--- - . . --.0
.
ill
/
0.. __0__ . _ .I ~:
I
...-----I
~.,.
: P3 (T3)
/
-B'"
Fig. 6. Schematic phase diagram for agarosc solution. 1. Random coil. II. Anisotropic, helix. II' Isotropic, helix . II " Two phases: isotropic, helix (v2') and anisotropic, helix (v2") ' III . 2 phases: isotropic, helix (c a ) and anisotropic , helix (cb) ' A . Helix point. B. Phase separation point (gelation point).
ra ndom coi I
II aniso. II ' iso ,
lI! coacervate (Vb, ll ) + dil.solu . (V., Il ')
r etrograded aggreg ate
II· iso . + an is o, Fig . 7. Schematic representation for phase changes in dilute agarose solution.
and other experimental results, we believe that the gel results from a phase separation of the rod-like, helical segments in the agarose macromolecule. Figure 6 shows the phase diagram for the agarose solution. The right half of the diagram is drawn from an assumption based upon Flory's diagram. The scale of the horizontal axis (concentration) of the right half is reduced arbitrarily compared with the left half obtained by experiments. The information of each phase separated by lines is given in the footnote of the figure. When a hot solution of PI (concentration: c, temperature : TI , random coil) is cooled to reach to the helix point, P2 (T2 ) , the segments wind up into rod-like helices. These helices separate into two phases at the phase separation point, P3 (T3) . At a point, P (concentration: c, temperature : T ), the solution consists of the dilute solution phase (concentration: ca , isotropic) and the concentrated solution phase (concentration: Cb, anisotropic). The ratio of both phases is given by (Cb - c):(c - cJ . The schematic 322
Swelling of agarose gel and its related changes
presentation of this process is shown in Figure 7. At the point, P, the fine particles of the concentrated solution phase (coacervate) are dispersed in the dilute solutionphase. Some of the helical segments of the molecule in the dilute solution phase are likely to be included in the coacervate particles. At a concentration higher than a critical value, the polymer molecule in the dilute solution phase acts as an intercoacervate junction. The coacervate particles are thus connected three-dimensionally, and the solution loses its fluidity to form a gel. The remarkable feature of our model is that the gel consists of a two-phase structure composed of a heterogeneous solution. An equilibrium exists between the dilute solution phase and the coacervate at least at the gel setting point. If the equilibrium still remains after the gelation, the ratio of both phases in the gel must change based upon the phase diagram when the gel is immersed in water. As a result, the gel must swell and then must dissolve in water. When the temperature is raised over the gelation point, the gel must also melt into a homogeneous solution. The transformation from the coacervate to the dilute solution phase is, however, disturbed by the crystallization of helical segments in the solution phases, that is, the equilibrium between both phases no longer exists in the gel. Evidence for the crystallization is shown in the retrogradation-like behavior. In the preceding work for amylose retrogradation (7), it was found that the retrogradation is caused by crystallization of helical segments. In the case of a dilute agarose solution such as 0.08 g/dl, if the crystallization of helical segments occurs, the segments become tied up into bundles (see Figure 7). As a result, the pendant coacervate particles of these segments join to form an aggregate. This is retrogradation showing precipitation of aggregates from the very dilute solution unable to make gel. The exponent of the Avrami's equation, n, for agarose is 1.25, somewhat smaller than the 2.0 for amylose reported by us (7). In the course of the preceding work for amylose, we sometimes experienced that n had a value of - 1.0 if the solution contained some undissolved crystals or some impurities such as dust particles. Colwell also reported a similar value for the retrogradation of a concentrated solution of amylose (12). The agarose sample solution used here was used without any filtration, hence it seems probable that it may have contained some impurities which could act as nuclei for crystallization. When a similar kind of crystallization proceeds in the network of the gel, there appears to be no observable changes directly. However, it may cause a thermo-hysteresis of the gel. The gelation point is the phase separation temperature and the melting point of the gel is the melting temperature of the crystal in the gel. This crystallization occurs as soon as the helix is formed in the solution as shown by the 'scanning curve' (partial hysteresis curve) in the preceding paper (5). Thus, crystallization prevents the gel from swelling as a result of the two-phase structure. The existence of the retrogradation-like phenomenon seems reasonable because amylose and agarose have a similar gelation mechanism (13). The possibility of swelling of the three-dimensional network structure is the same as for synthetic polymer gels. The Tanaka's formula for swelling and collapse of the gel (2) consists of four terms, entropy and enthalpy of mixing, excess rubber elasticity of network and osmotic pressure of ion. The effects of ionic groups and enthalpy are negligible, because we used a pure agarose sample at the same temperature for gel setting and swelling. The entropy term always increases swelling and the rubber elasticity 323
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reduces it. At this point, we cannot deal with them quantitatively. As mentioned above, agarose is almost in a double helical form in the gel and the helical segments tend to crystallize. Therefore, the molecules in the dilute solution phase seem to make a hard and brittle network in the gel. This hard network will result in a strong elasticity against the swelling effect in water. In other words, the entropy-elasticity resulting from flexible molecular chains is not expected to exist to a greater extent in the agarose gel aged for some while. This is a qualitative interpretation for the fact that the agarose gel does not swell in water as shown in Figure 1. Acetone is a non-solvent for agarose and is used for the purification of agarose as a precipitant from aqueous solution. We might expect, therefore, that the gel would shrink quickly on addition of acetone. However, the agarose gel keeps> 80% of the original volume even in pure acetone, as shown in Figure 2, and , moreover, the gel still keeps - 80 % of the original volume in ether. The original volume cannot be recovered. This means that the shrinkage of the gel is again not due to the entropyelasticity. Under such conditions, the affinity of solvent molecules for polymer segments decreases and the cohesive force between polymer segments increases. This situation may result in a collapse of the network structure thereby shrinking the gel. The network of the gel is fairly hard and brittle; hence, it is difficult to recover the original structure again after its collapse . When pure acetone is used to shrink the gel, water molecules within the matrix are gradually exchanged for acetone molecules by diffusion. On removal of water molecules, the cohesion between polymer segments may increase as a result of hydrogen bonding. It is well known that an increase in hydrogen bonding is often observed in the drying of an aqueous solution of hydrophilic polymers. In order to reduce the time for water molecules to exchange, an experiment was carried out ona small piece of gel changing the acetone quickly and frequently. The results showed that the agarose gel was almost able to retain its original volume. The most probable reason for syneresis seems to be that the agarose gel contains a large proportion of solution phase of much lower concentration (c a) than the total agarose concentration (c). Synthetic polymer gels usually have a homogeneous inner structure and when their surface dries to yield a higher concentration, the concentration within the gel changes to restore the equilibrium. This continuous adjustment of the inner solution concentration may prevent shrinkage. In addition the higher concentration in the surface layer means a stronger network and, therefore, solvent from within the gel shows little tendency to diffuse through cracks in the surface. When an agarose gel shrinks on rapid drying, forces exerted on the surface by the solution inside cause cracks to form. Syneresis thus occurs as a result of the inner dilute solution or solvent migrating through these cracks in the dried surface. The reason why the syneresis is often observed at low temperatures may be due to the slow evaporation of water. Several anomalous properties of the agarose gel in this paper are thus interpreted based on the two-phase structure of the gel proposed by us. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research, to which the authors ' thanks are due.
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References 1. F1ory,P.J. (1953) Principles of Polymer Chemistry. Cornell University Press, New York. 2. Tanaka,T., Fillmore,D., Sun,S.T., Nishio,I., Swislow.G. and Shah,A. (1980) Phys. Rev. Lett., 45,1636. 3. Arnott,S., Fulmer.A,; Scott,W.E., Dea,I.C.M., Moorhouse.R. and Rees,D.A. (1974) J. Mol. Biol., 90,269. 4. Hayashi,A., Kinoshita.K. and Kuwano,M. (1977) Polym. J., 9, 219. 5. Hayashi.A., Kinoshita,K., Kuwano,M. and Nose,A. (1978) Polym. J., 10, 485. 6. Hayashi,A., Kinoshita.K. and Yasueda,S. (1980) Polym. J., 12,447. 7. Hayashi,A., Kotani.K, and Cho,C. (1984) Agric. Biol. Chem., 48, 949. 8. Hayashi,A., Kinoshita,K. and Kotani,Y. (1983) Agric. Biol. Chem., 47,1705. 9. Dubois,M., Gilles,K.A., Hamilton,J.K., Revers,P.A. and Smith,F. (1956) Anal. Chem., 28, 350. 10. Avrami,M. (1941) J. Chem. Phys., 9, 177. 11. Flory,PJ. (1961) J. Polym. Sci., 49, 105. 12. Colwell,K.H., Axford,D.W.E., Chaberline,N. and Elton,G.H.A. (1969) J. Sci. Fd. Agric., 20, 550. 13. Hayashi,A., Kinoshita,K. and Miyake,Y. (1983) Agric. Biol. Chem., 47, 1699.
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