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Covalently cross-linked sodium alginate beads
Food Hydrocolloids Vol.5 no.1I2 pp.119-123, 1991
Covalently cross-linked sodium alginate beads Sterker T.Moe, Gudmund Skjak-Brrek and Olav Smidsrod Division of Biotechnology, NTH, University of Trondheim, N-7034 TrondheimNTH, Norway
Introduction
Alginates, the glycuronans of (1~4) linked o-t-guluronate (G units) and [3-Dmannuronate (M units), have long been known for their gel-forming capabilities (1,2). It has also long been known that polysaccharides can be cross-linked by suitable agents (e.g. bifunctional epoxides or similar compounds) in the solid, aqueous or gel phase (3-6). Alginate can be cross-linked in the gel state by exchanging the water in the gel with ethanol or another solvent where the crosslinking agent (epiclorohydrin) is soluble. The cross-linking reaction is basecatalysed (7-9). Methods
Alginate from Laminaria hyperborea stipes (LF 10/60) was obtained from Protan AlS (Norway). It contained 68% G residues and had an average number of G residues in the GG-blocks (N O > l ) = 14.0. Composition and sequence parameters were determined by the method of Grasdalen et al. (1981) (10). Homogeneous calcium alginate gel beads were made according to Skjak-Bnek et al. (11,12). The water in the gel beads was exchanged with 96% ethanol, and the cross-linking reaction was allowed to proceed in a suspension containing 4.3 moll drn' epichlorohydrin, 0.14 mol/drrr' NaOH, 0.014 mol/dm' CaCl, and 60 vol% ethanol. The cross-linked gel beads were washed with water, and Ca 2 + removed by dialysis against 50 mM EDTA, pH 7 (9). The beads were dried by exchanging the water in the gel with ethanol followed by drying in an airstream. Practical applications
When dried, the beads are able to re-swell in water and aqueous solutions. This re-swelling is fast, and the rehydrated gel beads show no ten de nee to be 'sticky'. Gel beads with a swelling capacity of 100 VN d ry can easily be made with a high mechanical strength (Figure 1). The re-swelling is, as far as our experiments indicate, independent of the concentration of non-ionic solutes (Figure 2). The beads can be made with a low volume dependence upon the concentration of ionic solutes. The gel beads can be made with a lower volume dependence upon salt concentrations than traditional absorbents such as polyacrylic acid (Figure 3). They absorb water faster than other absorbents. Their high mechanical strength gives them the ability to absorb water which is contained in the space between the beads. The result is gel beads with a dry surface (9). 119
S.T.Moe, G.Skjak-Brrek and O.Smidsred
VIVdry 100
I
5
*--*----*---II,f-'---*
'1'
*I *
2
10 II
2
* Time after adding water, min. Fig. 1. Re-swelling of Na-alginate beads (swelling capacity 100 VN d r y ) in water.
10
o Distilled water + Glucose x Glycerol
*NeCI
* Na-galacturonate
•
ii' i i i I Ii" iii"" i' Ii" II"" ii' i i i Ii' ii' Ii"" Iii'"
o
1
2
5
'i' Ii i I 10
Time after adding water, minutes Fig. 2. Re-swelling of Na-alginate beads (swelling capacity 33 VNd r y ) in water and 0.1 mol/drrr' aqueous solutions.
Theoretical considerations In the classical treatments of swelling of polymer networks, the calculation of the
elastic contribution to osmotic pressure is based upon statistical thermodynamics and theory of probability. This contribution is proportional to the number of 120
Sodium alginate beads
V/Vrnax
0,1
*
A1gin!ltll. BWBIling cllpllCity33 V/V*'
*
PoIyacryI!ltlI
*
A1gin!ltll, swellingcapacity100 VIV*,
iii
10-6
10-5
,
iii
10""'
10-3
,
I
10-2
0,01
,
0,1
[NaCIl, moles/! Fig. 3. Volume dependence upon salt concentrations for Na-alginate and polyacrylate beads.
cross-links in the gel (13,14). Until recently, this approach has not been questioned (15). Even if the classical approach is correct, it still relies upon the assumption that the gel's elastic chains are Gaussian. Alginate oligomers with DP n <1000 are non-Gaussian (16), hence the cross-link density cannot be calculated using the classical expressions for osmotic pressure. Also, the cross-link density for the Na-alginate beads cannot be determined by consumption of reagent, spectroscopical methods or by the use of radiolabelled epichlorohydrin. When crosslinking polysaccharides with epichlorohydrin, a large amount of the cross-linking agent is consumed by side-reactions (17,18). The gel's cross-link density can be estimated if the following assumptions are made: (i) the network is fully extended and the end-to-end distance between cross-links equals the contour length; (ii) the distribution of cross-links is completely uniform (all elastic chains are of equal length); (iii) the network is ideal, i.e. there are no 'loose ends'. If these assumptions are valid, the amount of cross-links in the gel can be calculated when the amount of polysaccharide in one gel bead, the contour length of one monomer and the bead's maximum volume are known. The gel network will be fully stretched and the molecular structure will resemble Figure 4, where the distance between cross-links is denoted by I and the side of one unit cell containing 1.5 cross-links is denoted by s. The geometry of the structure gives the relationship:
L 1- y.9V3VMo . _ Lc = -c16Nmb ,I - N(v + 1) Nv where Mo is the molecular weight of one monomer, m the amount of polymer in 121
S.T.Moe, G.Skjak.Brrek and O.Smidsrlld
Fig. 4. Polymer network structure at maximum volume of gel. Thick line: polymer chain; circle: cross-link. Table I. Number of calculated repeating units between cross-links (n.) in some gel samples Sample
n.
VNo
1 2 3 4
13 19 26 28
2.6 5.1 10.3 11.5
nV
30
• •
•
20
•
10
0
5
10
15
Vmax
Vo Fig. 5. Calculated n v as a function of V maxNO for different gel samples.
the gel, b the contour length of one monomer and L G the total contour length of the polymer in the gel. The ratio lib, defined by n., is given by:
122
Sodium alginate beads
and is the average number of repeating units between cross-links and is a convenient way of expressing cross-link density in the gel (Table I; Figure 5). Acknowledgements This work was supported by a grant from Norges Tekniske Hogskoles Fond and by Protan Biopolymers NS. References I. 2. 3. 4. 5. 6.
Smidsrod.O . and Haug,A. (1968) Acta Chem . Scand. , 22, 1989-1997 . Smidred.O. , Haug,A. and Whittington ,S. (1972) Acta Chem . Scand. , 26, 2563-2566. Stanford,E.C.C. (1881) Brit. Pat. no . 142. Flodin ,P. (1962) Thesis , Uppsala . Porath.J. ; Janson ,J.-C. and Laas ,T. (1971) 1. Chromatogr . , 60,167-177. Birnbaum,S., Pendleton ,S.R. , Larsson,P.-O . and Mosbach,K. (1982) Biotechnol. Lett. 3,393400. 7. Ferrero,F. and Piccinini,N. (1982) Ann. Chim., 72, 73-82. 8. Ferrero,F., Campagna,P. and Piccinini,N. (1982) Chem. Eng. Comm. , 15, 197-206. 9. Skjak-Brrek.G. and Moe,S. (1990) Norwegian Patent Application no . 902386. 10. Grasdalen,H., Larsen,B . and Smidsred.O, (1981) Carbo Res., 89,179-191. II. Skjak-Brrek,G., Zanetti,F. and Paoletti ,S. (1988) Carbo Res., 185, 131-138. 12. Skjak-Brrek,G., Grasdalen,H . and Smidsred .O. (1989) Carbo Pol. , 10, 31-54. 13. Flory ,P.J . (1953) Principles of Polym er Chemistry. Oxford University Press, Ithaca, pp 402-593. 14. Treloar,L.R.G. (1975) The Physics of Rubb er Elasticity. Clarendon, Oxford . 15. Altenberger,A .R . and Dahler,J .S. (1990) J. Chem . Phys. , 92, 3100-3111. 16. Bailey,E ., Mitchell ,J.R . and Blanchard,J.M.V. (1977) Call. Pol. Sci. , 255, 856-860. 17. Kuniak,L. and Marchessault ,R .H . (1972) Die Starke , 24, 110-116. 18. Holmberg,L. (1983) Doctorial thesis, Swedish Universit y of Agr icultural Sciences, Uppsala.
Appendix b LG
m Mo N o >!
n. V Vo V max v I N
Contour length of one monomer Total contour length of the polymer in the gel Amount of polymer in the gel Molecular weight of one monomer Average number of G units in a G-block longer than two units Average number of repeating units between cross-links Volume of gel Volume of gel when found Maximum volume of gel Molar number of cross-links in the gel Contour length of the polymer chain between cross-links Avogadro'S number
123
Covalently cross-linked sodium alginate beads Sterker T.Moe, Gudmund Skjak-Brrek and Olav Smidsrod Division of Biotechnology, NTH, University of Trondheim, N-7034 TrondheimNTH, Norway
Introduction
Alginates, the glycuronans of (1~4) linked o-t-guluronate (G units) and [3-Dmannuronate (M units), have long been known for their gel-forming capabilities (1,2). It has also long been known that polysaccharides can be cross-linked by suitable agents (e.g. bifunctional epoxides or similar compounds) in the solid, aqueous or gel phase (3-6). Alginate can be cross-linked in the gel state by exchanging the water in the gel with ethanol or another solvent where the crosslinking agent (epiclorohydrin) is soluble. The cross-linking reaction is basecatalysed (7-9). Methods
Alginate from Laminaria hyperborea stipes (LF 10/60) was obtained from Protan AlS (Norway). It contained 68% G residues and had an average number of G residues in the GG-blocks (N O > l ) = 14.0. Composition and sequence parameters were determined by the method of Grasdalen et al. (1981) (10). Homogeneous calcium alginate gel beads were made according to Skjak-Bnek et al. (11,12). The water in the gel beads was exchanged with 96% ethanol, and the cross-linking reaction was allowed to proceed in a suspension containing 4.3 moll drn' epichlorohydrin, 0.14 mol/drrr' NaOH, 0.014 mol/dm' CaCl, and 60 vol% ethanol. The cross-linked gel beads were washed with water, and Ca 2 + removed by dialysis against 50 mM EDTA, pH 7 (9). The beads were dried by exchanging the water in the gel with ethanol followed by drying in an airstream. Practical applications
When dried, the beads are able to re-swell in water and aqueous solutions. This re-swelling is fast, and the rehydrated gel beads show no ten de nee to be 'sticky'. Gel beads with a swelling capacity of 100 VN d ry can easily be made with a high mechanical strength (Figure 1). The re-swelling is, as far as our experiments indicate, independent of the concentration of non-ionic solutes (Figure 2). The beads can be made with a low volume dependence upon the concentration of ionic solutes. The gel beads can be made with a lower volume dependence upon salt concentrations than traditional absorbents such as polyacrylic acid (Figure 3). They absorb water faster than other absorbents. Their high mechanical strength gives them the ability to absorb water which is contained in the space between the beads. The result is gel beads with a dry surface (9). 119
S.T.Moe, G.Skjak-Brrek and O.Smidsred
VIVdry 100
I
5
*--*----*---II,f-'---*
'1'
*I *
2
10 II
2
* Time after adding water, min. Fig. 1. Re-swelling of Na-alginate beads (swelling capacity 100 VN d r y ) in water.
10
o Distilled water + Glucose x Glycerol
*NeCI
* Na-galacturonate
•
ii' i i i I Ii" iii"" i' Ii" II"" ii' i i i Ii' ii' Ii"" Iii'"
o
1
2
5
'i' Ii i I 10
Time after adding water, minutes Fig. 2. Re-swelling of Na-alginate beads (swelling capacity 33 VNd r y ) in water and 0.1 mol/drrr' aqueous solutions.
Theoretical considerations In the classical treatments of swelling of polymer networks, the calculation of the
elastic contribution to osmotic pressure is based upon statistical thermodynamics and theory of probability. This contribution is proportional to the number of 120
Sodium alginate beads
V/Vrnax
0,1
*
A1gin!ltll. BWBIling cllpllCity33 V/V*'
*
PoIyacryI!ltlI
*
A1gin!ltll, swellingcapacity100 VIV*,
iii
10-6
10-5
,
iii
10""'
10-3
,
I
10-2
0,01
,
0,1
[NaCIl, moles/! Fig. 3. Volume dependence upon salt concentrations for Na-alginate and polyacrylate beads.
cross-links in the gel (13,14). Until recently, this approach has not been questioned (15). Even if the classical approach is correct, it still relies upon the assumption that the gel's elastic chains are Gaussian. Alginate oligomers with DP n <1000 are non-Gaussian (16), hence the cross-link density cannot be calculated using the classical expressions for osmotic pressure. Also, the cross-link density for the Na-alginate beads cannot be determined by consumption of reagent, spectroscopical methods or by the use of radiolabelled epichlorohydrin. When crosslinking polysaccharides with epichlorohydrin, a large amount of the cross-linking agent is consumed by side-reactions (17,18). The gel's cross-link density can be estimated if the following assumptions are made: (i) the network is fully extended and the end-to-end distance between cross-links equals the contour length; (ii) the distribution of cross-links is completely uniform (all elastic chains are of equal length); (iii) the network is ideal, i.e. there are no 'loose ends'. If these assumptions are valid, the amount of cross-links in the gel can be calculated when the amount of polysaccharide in one gel bead, the contour length of one monomer and the bead's maximum volume are known. The gel network will be fully stretched and the molecular structure will resemble Figure 4, where the distance between cross-links is denoted by I and the side of one unit cell containing 1.5 cross-links is denoted by s. The geometry of the structure gives the relationship:
L 1- y.9V3VMo . _ Lc = -c16Nmb ,I - N(v + 1) Nv where Mo is the molecular weight of one monomer, m the amount of polymer in 121
S.T.Moe, G.Skjak.Brrek and O.Smidsrlld
Fig. 4. Polymer network structure at maximum volume of gel. Thick line: polymer chain; circle: cross-link. Table I. Number of calculated repeating units between cross-links (n.) in some gel samples Sample
n.
VNo
1 2 3 4
13 19 26 28
2.6 5.1 10.3 11.5
nV
30
• •
•
20
•
10
0
5
10
15
Vmax
Vo Fig. 5. Calculated n v as a function of V maxNO for different gel samples.
the gel, b the contour length of one monomer and L G the total contour length of the polymer in the gel. The ratio lib, defined by n., is given by:
122
Sodium alginate beads
and is the average number of repeating units between cross-links and is a convenient way of expressing cross-link density in the gel (Table I; Figure 5). Acknowledgements This work was supported by a grant from Norges Tekniske Hogskoles Fond and by Protan Biopolymers NS. References I. 2. 3. 4. 5. 6.
Smidsrod.O . and Haug,A. (1968) Acta Chem . Scand. , 22, 1989-1997 . Smidred.O. , Haug,A. and Whittington ,S. (1972) Acta Chem . Scand. , 26, 2563-2566. Stanford,E.C.C. (1881) Brit. Pat. no . 142. Flodin ,P. (1962) Thesis , Uppsala . Porath.J. ; Janson ,J.-C. and Laas ,T. (1971) 1. Chromatogr . , 60,167-177. Birnbaum,S., Pendleton ,S.R. , Larsson,P.-O . and Mosbach,K. (1982) Biotechnol. Lett. 3,393400. 7. Ferrero,F. and Piccinini,N. (1982) Ann. Chim., 72, 73-82. 8. Ferrero,F., Campagna,P. and Piccinini,N. (1982) Chem. Eng. Comm. , 15, 197-206. 9. Skjak-Brrek.G. and Moe,S. (1990) Norwegian Patent Application no . 902386. 10. Grasdalen,H., Larsen,B . and Smidsred.O, (1981) Carbo Res., 89,179-191. II. Skjak-Brrek,G., Zanetti,F. and Paoletti ,S. (1988) Carbo Res., 185, 131-138. 12. Skjak-Brrek,G., Grasdalen,H . and Smidsred .O. (1989) Carbo Pol. , 10, 31-54. 13. Flory ,P.J . (1953) Principles of Polym er Chemistry. Oxford University Press, Ithaca, pp 402-593. 14. Treloar,L.R.G. (1975) The Physics of Rubb er Elasticity. Clarendon, Oxford . 15. Altenberger,A .R . and Dahler,J .S. (1990) J. Chem . Phys. , 92, 3100-3111. 16. Bailey,E ., Mitchell ,J.R . and Blanchard,J.M.V. (1977) Call. Pol. Sci. , 255, 856-860. 17. Kuniak,L. and Marchessault ,R .H . (1972) Die Starke , 24, 110-116. 18. Holmberg,L. (1983) Doctorial thesis, Swedish Universit y of Agr icultural Sciences, Uppsala.
Appendix b LG
m Mo N o >!
n. V Vo V max v I N
Contour length of one monomer Total contour length of the polymer in the gel Amount of polymer in the gel Molecular weight of one monomer Average number of G units in a G-block longer than two units Average number of repeating units between cross-links Volume of gel Volume of gel when found Maximum volume of gel Molar number of cross-links in the gel Contour length of the polymer chain between cross-links Avogadro'S number
123